COMPOSITIONS ET PROCEDES DE SEQUENCAGE PAR PAIRES

COMPOSITIONS AND METHODS FOR PAIRWISE SEQUENCING

Abrégé français

La présente invention fournit des compositions et des procédés utilisant les compositions pour effectuer un séquençage par paires et pour générer des molécules matrices de concaténation pour le séquençage par paires. Les concatémères peuvent être générés à l'aide d'une réaction d'amplification en cercle roulant réalisée soit sur le support, soit en solution, puis distribuée sur un support. La réaction d'amplification par cercle roulant génère des concatémères contenant des copies en tandem d'une séquence d'intérêt et d'au moins une séquence d'adaptateur universelle. Une augmentation du nombre de copies en tandem dans un concatémère donné augmente le nombre de sites le long du concatémère pour s'hybrider à de multiples amorces de séquençage servant de sites d'initiation multiples pour des réactions de séquençage catalysées par polymérase. Lorsque la réaction de séquençage utilise des nucléotides marqués de manière détectable et/ou des molécules multivalentes marquées de manière détectable (par exemple, ayant des unités nucléotidiques), les signaux émis par les nucléotides ou les unités nucléotidiques qui participent aux réactions de séquençage en parallèle le long du concatémère produisent une intensité de signal accrue pour chaque concatémère.

Abrégé anglais

The present disclosure provides compositions and methods that employ the compositions for conducting pairwise sequencing and for generating concatemer template molecules for pairwise sequencing. The concatemers can be generated using a rolling circle amplification reaction which is conducted either on-support, or conducted in-solution and then distributed onto a support. The rolling circle amplification reaction generates concatemers containing tandem copies of a sequence of interest and at least one universal adaptor sequence. An increase in the number of tandem copies in a given concatemer increases the number of sites along the concatemer for hybridizing to multiple sequencing primers which serve as multiple initiation sites for polymerase-catalyzed sequencing reactions. When the sequencing reaction employs detectably labeled nucleotides and/or detectably labeled multivalent molecules (e.g., having nucleotide units), the signals emitted by the nucleotides or nucleotide units that participate in the parallel sequencing reactions along the concatemer yields an increased signal intensity for each concatemer.

Revendications

358
What is claimed:
1. A method for pairwise sequencing, comprising:
a) providing a plurality of immobilized single stranded nucleic acid
concatemer
template molecules each comprising at least one nucleotide having a scissile
moiety that can be cleaved to generate an abasic site in the concatemer
template
molecule, wherein individual concatemer template molecules in the plurality
are
immobilized to a first surface primer that is immobilized to a support, and
wherein the immobilized first surface primer lacks a nucleotide having a
scissile
moiety;
b) sequencing the plurality of immobilized concatemer template molecules
thereby
generating a plurality of extended forward sequencing primer strands, wherein
individual immobilized concatemer template molecules have two or more
extended forward sequencing primer strands hybridized thereon;
c) retaining the plurality of immobilized concatemer template molecules and
replacing the plurality of extended forward sequencing primer strands with a
plurality of forward extension strands that are hybridized to the retained
immobilized single stranded nucleic acid concatemer template molecules by
conducting a primer extension reaction;
d) removing the retained immobilized concatemer template molecules by
generating
abasic sites in the immobilized single stranded concatemer template molecules
at
the nucleotide(s) having the scissile moiety and generating gaps at the abasic
sites
to generate a plurality of gap-containing single stranded nucleic acid
concatemer
template molecules while retaining the plurality of forward extension strands
and
retaining the plurality of immobilized surface primers; and
e) sequencing the plurality of retained forward extension strands thereby
generating
a plurality of extended reverse sequencing primer strands, wherein individual
retained forward extension strands have two or more extended reverse
sequencing
primer strands hybridized thereon.

359
2. The method of claim 1, wherein individual concatemer template molecules
in the plurality
are covalently joined to an immobilized first surface primer.
3. The method of claim 1, wherein individual concatemer template molecules
in the plurality
are hybridized to an immobilized first surface primer.
4. The method of claim 1, wherein individual immobilized concatemer
template molecules in
the plurality comprise two or more copies of a sequence of interest, and
wherein the
individual immobilized concatemer template molecules further comprise any one
or any
combination of two or more of
(i) two or more copies of a universal binding sequence for a soluble
forward sequencing primer,
(ii) two or more copies of a universal binding sequence for a soluble
reverse sequencing prirner,
(iii) two or rnore copies of a universal binding sequence for an
irnmobilized first surface primer,
(iv) two or rnore copies of a universal binding sequence for an
irnmobilized second surface primer,
(v) two or rnore copies of a universal binding sequence for a first soluble
amplification primer,
(vi) two or rnore copies of a universal binding sequence for a second
soluble amplification primer,
(vii) two or more copies of a universal binding sequence for a soluble
compaction oligonucleotide,
(viii) two or more copies of a sample barcode sequence and/or
(ix) two or more copies of a unique molecular index sequence.
5. The method of claim 1, wherein the sequencing of step (b) comprises
hybridizing a
plurality of soluble forward sequencing primers to the plurality of
immobilized concaterner
template molecules and conducting one or more sequencing reactions.

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6. The method of claim 1, wherein the sequencing of step (e) cornprises
hybridizing a
plurality of soluble reverse sequencing primers to the plurality of
immobilized concatemer
template molecules and conducting one or rnore sequencing reactions.
7. The method of claim 4, wherein the support further comprises a plurality
of immobilized
second surface primers that lack a nucleotide havine a scissile moiety.
8. The method of claim 7, wherein at least one copy of the universal
binding sequence for the
immobilized second surface primer in the individual concatemer template
molecules is
hybridized to an immobilized second surface primer.
9. The method of claim 7, wherein the plurality of immobilized second
surface primers have
3' OH extendible ends.
1(. The method of claim 7, wherein the plurality of immobilized second
surface primers have
3' non-extendible ends.
11. The method of claim 10, wherein the 3' non-extendible end comprises a
phosphate group, a
dideoxycytidine group, an inverted dT, or an amino group.
12. A method for pairwise sequencing, comprisina:
a) providing a support having a plurality of a first surface primer
immobilized
thereon wherein each of the first surface primers have a 3' extendible end and
lack a nucleotide having a scissile moiety;
b) generating a plurality of immobilized single stranded nucleic acid
concatemer
template molecules by hybridizing a plurality of single-stranded circular
nucleic
acid library molecules to the plurality of immobilized first surface primers
and
conducting a rolling circle amplification reaction with a plurality of a
strand
displacing polymerase, and a plurality of nucleotides which include dATP,
dCTP,
dGTP, dTTP and a nucleotide having a scissile moiety that can be cleaved to
generate an abasic site, thereby generating a plurality of immobilized single
stranded nucleic acid concatemer templale molecules having at. least one

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nucleotide with a scissile moiety, wherein individual single stranded nucleic
acid
concatemer template molecules are covalently joined to an immobilized first
surface primer,
c) sequencing the plurality of immobilized concatemer template molecules
thereby
generating a plurality of extended forward sequencing primer strands, wherein
individual immobilized concatemer template molecules have two or more
extended forward sequencing primer strands hybridized thereon;
d) retaining the plurality of immobilized concatemer template molecules and
replacing the plurality of extended forward sequencing primer strands with a
plurality of forward extension strands that are hybridized to the retained
immobilized single stranded nucleic acid concatemer template molecules by
conducting a primer extension reaction;
e) removing the retained immobilized concatemer template molecules by
generating
abasic sites in the immobilized single stranded concatemer template molecules
at
the nucleotide(s) having the scissile moiety and generating gaps at the abasic
sites
to generate a plurality of gap-containing single stranded nucleic acid
concatemer
template molecules while retaining the plurality of forward extension strands
and
retaining the plurality of immobilized first surface primers; and
0 sequencing the plurality of retained forward extension strands
thereby generating
a plurality of extended reverse sequencing primer strands, wherein individual
forward extension strands have two or more extended reverse sequencing primer
strands hybridized thereon.
13. The
method of claim 12, wherein each of the single stranded circular nucleic acid
library
molecules in the plurality comprises a sequence of interest and wherein the
individual
library molecules further comprise any one or any combination of two or more
of
a universal binding sequence for a soluble forward sequencing primer,
(ii) a universal binding sequence for a soluble reverse sequencing primer,
(iii) a universal binding sequence for an immobilized first surface primer,
(iv) a universal binding sequence for an immobilized second surface
primer,
. .

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(v) a universal binding sequence for a first soluble amplification primer,
(vi) a universal binding sequence for a second soluble amplification
primer,
(vii) a universal binding sequence for a soluble compaction oligonucleotide,
(viii) a sample barcode sequence and/or
(ix) a unique molecular index sequence.
14. The method of claim 12, wherein individual immobilized single stranded
nucleic acid.
concatemer template molecules generated by the rolling circle amplification
reaction
comprise two or more copies of a sequence of interest and wherein the
individual
immobilized concatemer template molecules further comprise anv one or any
combination
of two or more of
two or more copies of a universal binding sequence for a soluble
forward sequencing prim.er,
(ii) two or more copies of a universal binding sequence for a
soluble
reverse sequencing primer,
(ih) two or more copies of a universal binding sequence for an
immobilized first surface primer,
(iv) two or more copies of a universal binding sequence for an
immobilized second surface primer,
(v) two or more copies of a universal binding sequence for a first soluble
amplification primer,
(vi) two or more copies of a universal binding sequence for a second
soluble amplification primer,
(vii) two or more copies of a universal binding sequence for a soluble
compaction oligonucleotide,
(-Oil) two or more copies of a sample barcode sequence and/or
(ix) two or more copies of a unique molecular index sequence.

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15. The method of claim 12, wherein the sequencing of step (c) comprises
hybridizing a
plurality of soluble forward sequencing primers to the plurality of
immobilized concatemer
template molecules and conducting one or rnore sequencing reactions.
16. The method of claim 12, wherein the sequencing of step (1) comprises
hybridizing a
plurality of soluble reverse sequencing primers to the plurality of
immobilized concatemer
template molecules and conducting one or more sequencing reactions.
17. The method of claim 14, wherein the support further comprises a
plurality of immobilized
second surface primers that lack a nucleotide having a scissile moiety.
18. The method of claim 17, wherein at least one copy of the universal
binding sequence for
the immobilized second surface primer in the individual concatemer ternplate
rnolecules is
hybridized to an immobilized second surface primer.
19. The method of claim 17, wherein the plurality of immobilized second
surface primers have
3' OH extendible ends.
20. The method of claim 17, wherein the plurality of immobilized second
surface primers have
3' non-extendible ends.
21. The method of claim 20, wherein the 3' non-extendible end comprises a
phosphate group, a
dideoxycytidine group, an inverted dT, or an amino group.
22. A method for pairwise sequencing, comprising:
a) contacting in-solution a plurality of single-stranded circular
nucleic acid library
molecules to a plurality of first soluble amplification primers, a plurality
of a
strand displacing polymerase, and a plurality of nucleotides which include
dATP,
dCTP, dGIP, dTTP and a nucleotide having a scissile moiety that can be cleaved
to generate an abasic site, under a condition suitable to form a plurality of
library-
primer duplexes and suitable for conducting a rolling circle amplification

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reaction, thereby generating a plurality of single stranded nucleic acid
concatemers having at least one nucleotide with a scissile moiety;
b) distributing the rolling circle amplification reaction onto a support
having a
plurality of the first surface primers immobilized thereon, under a condition
suitable for hybridizing one or more portions of individual single stranded
concatemers to one or more immobilized first surface primers, wherein each of
the first surface primers lack a nucleotide having a scissile moiety;
c) continuing the rolling circle amplification reaction on the support to
generate a
plurality of immobilized concatemer template molecules;
d) sequencing the plurality of immobilized concatemer template molecules
thereby
generating a plurality of extended forward sequencing primer strands wherein
individual immobilized concatemer template molecules have two or more
extended forward sequencing primer strands hybridized thereon;
e) retaining the plurality of immobilized concatemer template molecules and
replacing the plurality of extended forward sequencing primer strands with a
plurality of forward extension strands that are hybridized to the retained
immobilized single stranded nucleic acid concatemer template molecules by
conducting a primer extension reaction;
0 removing the retaimi immobilized concatemer template molecules by
generating
abasic sites in the immobilized single stranded concatemer template molecules
at
the nucleotide(s) having the scissile moiety and generating gaps at the abasic
sites
to generate a plurality of gap-containing single stranded nucleic acid
concatemer
template molecules while retaining the plurality of forward extension strands
and
retaining the plurality of immobilized first surface primers; and
g) sequencing the plurality of retained forward extension strands thereby
generating
a plurality of extended reverse sequencing primer strands wherein individual
forward extension strands have two or more extended reverse sequencing primer
strands hybridized thereon.
. . .

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23. The method of claim 22, wherein each of the single stranded circular
nucleic acid library
molecules in the plurality comprises a sequence of interest and wherein the
individual
library molecules further comprise any one or any combination of two or more
of
a universal binding sequence for a soluble forward sequencing primer,
(ii) a universal binding sequence for a soluble reverse sequencing primer,
(iii) a universal binding sequence for an immobilized first surface
prirner,
(iv) a universal binding sequence for an irnrnobilized second surface
primer,
(v) a universal binding sequence for a first soluble amplification prirner,
(vi) a universal binding sequence for a second soluble amplification
prirner,
(vii) a universal binding sequence for a soluble compaction oligonucleotide,
(viii) a sample barcode sequence and/or
(ix) a unique molecular index sequence.
24. The method of claim 22, wherein individual immobilized single stranded
nucleic acid
concatemer template molecules generated by the rolling circle amplification
reaction
comprise two or more copies of a sequence of interest and wherein the
individual
immobilized concatemer template molecules further comprise any one or any
combination
of two or more of
(i) two or more copies of a universal binding sequence for a soluble
forward sequencing primer,
(ii) two or more copies of a universal binding sequence for a soluble
reverse sequencing primer,
(iii) two or more copies of a universal binding sequence for an
immobilized first surface primer,
(iv) two or more copies of a universal binding sequence for an
immobilized second surface primer,
(v) two or more copies of a universal binding sequence for a first soluble
amplification primer,

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(vi) two or more copies of a universal binding sequence for a second
soluble amplification primer,
(vii) two or more copies of a universal binding sequence for a soluble
compaction oligonucleotide,
(viii) two or more copies of a sample barcode sequence and/or
(ix) two or more copies of a unique molecular index sequence.
25. The method of claim 22, wherein the sequencing of step (d) comprises
hybridizing a
plurality of soluble forward sequencing primers to the plurality of
immobilized concatemer
template molecules and conducting one or more sequencing reactions.
26. The method of claim 22, wherein the sequencing of step (g) comprises
hybridizing a
plurality of soluble reverse sequencing primers to the plurality of
immobilized concatemer
template molecules and conducting one or more sequencing reactions,
27. The method of claim 24, wherein the support further comprises a
plurality of immobilized
second surface primers that lack a nucleotide having a scissile moiety.
28. The method of claim 27, wherein at least one copy of the universal
binding sequence for
the immobilized second surface primer in the individual concatemer template
molecules is
hybridized to an immobilized second surface primer.
29. The method of claim 27, wherein the plurality of immobilized second
surface primers have
3' 011 extendible ends.
30. The method of claim 27, wherein the plurality of immobilized second
surface primers have
3' non-extendible ends.
31. The method of claim 30, wherein the 3' non-extendible end comprises a
phosphate group, a
dideoxycytidine group, an inverted dT, or an amino group,
32. A method for pairwise sequencing, comprising:

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a) providing a support having a plurality of a first surface primer
immobilized
thereon wherein individual first surface primers in the plurality comprise a
first
portion (SPI-A) and a second portion (SP1-B), and the individual first surface
primers comprising a 3' extendible end and lacking a nucleotide having a
scissile
moiety that can be cleaved to generate an abasic site in the first surface
primer;
b) contacting the plurality of the first surface primers with a plurality
of single
stranded linear nucleic acid library molecules, each library molecule having
at the
5' end a universal sequence (SP1-A') that binds the first portion of the
immobilized first surface primer, and the library molecules each having at the
3'
end a universal sequence (SP1-B') that binds the second portion of the
immobilized first surface primer, wherein the contacting is conducted under a
condition suitable for hybridizing individual library molecules to an
immobilized
first surface primer to form a circularized library molecule having a gap or
nick
between the 5' and 3' ends of the circularized library molecule;
c) enzymatically closing the gap or nick thereby forming individual
covalently
closed circular molecules that are hybridized to an immobilized first surface
primer;
d) generating a plurality of immobilized single stranded nucleic acid
concatemer
template molecules by conducting a rolling circle amplification reaction with
a
plurality of a strand displacing polymerase, and a plurality of nucleotides
which
include dATP, dCTP, dGTP, dTTP and a nucleotide having a scissile moiety that
can be cleaved to generate an abasic site, thereby generating a plurality of
immobilized single stranded nucleic acid concatemer template molecules having
at least one nucleotide with a scissile moiety, wherein individual single
stranded
nucleic acid concatemer template molecules are covalently joined to an
immobilized first surface primer;
e) sequencing the plurality of immobilized concatemer template molecules
thereby
generating a plurality of extended forward sequencing primer strands, wherein
individual immobilized concatemer template molecules have two or more
extended forward sequencing primer strands hybridized thereon;

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f) retaining the plurality of immobilized concatemer template molecules and
replacing the plurality of extended forward sequencing primer strands with a
plurality of forward extension strands that are hybridized to the retained
inunobilized single stranded nucleic acid concatemer ternplate rnolecules by
conducting a prirner extension reaction;
g) removing the retained immobilized concatemer template molecules by
generating
abasic sites in the immobilized single stranded concatemer template molecules
at
the nucleotide(s) having the scissile moiety and generating gaps at the abasic
sites
to generate a plurality of gap-containing single stranded nucleic acid
concatemer
template molecules while retaining the plurality of forward extension strands
and.
retaining the plurality of immobilized first surface primers; and.
h) sequencing the plurality of retained forward extension strands thereby
generating
a plurality of extended reverse sequencing primer strands, wherein individual
forward extension strands have two or more extended reverse sequencing primer
strands hybridized thereon.
33. The method of claim 32, wherein individual linear library molecules in
the plurality
comprise a sequence of interest and the library molecules further comprise any
one or any
combination of two or more of:
a universal binding sequence for a soluble forward sequencing primer,
(ii) a universal binding sequence for a soluble reverse sequencing primer,
(iii) a universal binding sequence for a first portion of an immobilized
first
surface primer (SRI-A),
(iv) a universal binding sequence for a second portion of an immobilized
first surface primer (SPI -B),
(v) a universal binding sequence for an immobilized second surface
primer,
(vi) a universal binding sequence for a first soluble amplification primer,
(vii) a universal binding sequence for a second soluble amplification
primer,
(viii) a universal binding sequence for a soluble compaction oligonucleotide,

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(ix) a sample barcode sequence and/or
(x) a unique molecular index sequence.
34. The method of claim 32, wherein individual immobilized single stranded
nucleic acid
concatemer template molecules generated by the rolling circle amplification
reaction
comprise two or more copies of a sequence of interest and wherein the
individual
immobilized concatemer template molecules further comprise any one or any
combination
of two or more of
two or more copies of a universal binding sequence for a soluble
forward sequencing primer,
(ii) two or more copies of a universal binding sequence for a soluble
reverse sequencing primer,
(iii) two or more copies of a universal binding sequence for a first
portion
of an immobilized first surface primer (SP1-A),
(iv) two or more copies of a universal binding sequence for a second
portion of an immobilized first surface primer (SP1 43),
(v) two or more copies of a universal binding sequence for an
immobilized second surface primer,
(vi) two or more copies of a universal binding sequence for a first soluble
amplification primer,
(vii) two or more copies of a universal binding sequence for a second
soluble amplification primer,
(viii) two or more copies of a universal binding sequence for a soluble
compaction oligonucleotide,
(ix) two or more copies of a sample barcode sequence and/or
(x) two or more copies of a unique molecular index sequence.
35. The method of claim 32, wherein the sequencing of step (e) cornprises
hybridizing a
plurality of soluble forward sequencing primers to the plurality of
immobilized concatemer
template molecules and conducting one or more sequencing reactions.

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36. The method of claim 32, wherein the sequencing of step (h) comprises
hybridizing a
plurality of soluble reverse sequencing primers to the plurality of
immobilized concatemer
template molecules and conducting one or rnore sequencing reactions.
37. The method of claim 32, wherein the support further comprises a
plurality of immobilized
second surface primers that lack a nucleotide havine a scissile moiety.
38. The method of claim 34, wherein at least one copy of the universal
binding sequence for
the immobilized second surface primer in the individual concatemer template
molecules is
hybridized to an immobilized second surface primer.
39. The method of claim 34, wherein the plurality of immobilized second
surface primers have
3' OH extendible ends.
40. The method of claim 34, wherein the plurality of immobilized second
surface primers have
3' non-extendible ends.
41. The method of claim 40, wherein the 3' non-extendible end comprises a
phosphate group, a
dideoxycytidine group, an inverted dT, or an amino group.
42. The method of claim 32, wherein the closing the gap in the circularized
library molecule
comprises conducting a polymerase-catalyzed gap fill-in reaction using the
immobilized
first surface primer as a template molecule, and ligating the nick to form a
covalently
closed circular molecule, wherein individual covalently closed circular
molecules are
hybridized to an immobilized first surface primer.
43. The m.ethod of claim 32, wherein the closing the nick in the
circularized library molecule
comprises conducting a ligation reaction to form a covalently closed circular
molecule, and
wherein individual covalently closed circular molecules are hybridized to an
immobilized
first surface primer.
44. A method for pairwise sequencing, comprising:

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a) providing a plurality of immobilized single stranded nucleic acid
concatemer
template molecules each lacking a scissile moiety that can be cleaved to
generate
an abasic site in the concatemer template molecule, wherein individual
concatemer template molecules in the plurality are immobilized to a first
surface
primer that is immobilized to a support, and wherein the immobilized first
surface
primer lacks a nucleotide having a scissile moiety;
b) sequencing the plurality of immobilized concatemer template molecules
thereby
generating a plurality of extended forward sequencing primer strands, wherein
individual immobilized concatemer template molecules have two or more
extended forward sequencing primer strands hybridized thereon;
c) retaining the plurality of immobilized concatemer template molecules and
replacing the plurality of extended forward sequencing primer strands with a
plurality of forward extension strands by conducting a primer extension
reaction
with a plurality of soluble amplification primers and a plurality of strand-
displacing polymerases to generate a plurality of forward extension strands
and a
plurality of partially displaced forward extension strands that are hybridized
to the
immobilized concatemer template molecules to form a plurality of immobilized
amplicons, and the primer extension reaction generates a plurality of detached
forward extension strands (e.g., that are not hybridized to the immobilized
concatemer template molecules); and
d) sequencing the plurality of immobilized partially displaced forward
extension
strands thereby generating a first plurality of extended reverse sequencing
primer
strands, and sequencing the plurality of immobilized detached forward
extension
strands thereby generating a second plurality of extended reverse sequencing
primer strands, wherein individual immobilized partially displaced forward
extension strands have two or more extended reverse sequencing primer strands
hybridized thereon, and wherein in individual immobilized detached forward
extension strands have two or more extended reverse sequencing primer strands
hybridized thereon.

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45. The method of claim 44, wherein individual concaterner template
molecules in the plurality
are covalently joined to an inunobilized first surface primer.
46. The method of claim 44, wherein individual concatemer template
molecules in the plurality
are hybridized to an immobilized first surface prirner.
47. The method of claim 44, wherein individual immobilized concatemer
template molecules
in the plurality comprise two or more copies of a sequence of interest, and
wherein the
individual immobilized concatemer template molecules further comprise any one
or any
combination of two or more of
two or more copies of a universal binding sequence for a soluble
forward sequencing primer,
(ii) two or more copies of a universal binding sequence for a
soluble
reverse sequencing primer,
(ih) two or more copies of a universal binding sequence for an
immobilized first surface primer,
(iv) two or more copies of a universal binding sequence for an
immobilized second surface primer,
(v) two or more copies of a universal binding sequence for a first soluble
amplification primer,
(vi) two or more copies of a universal binding sequence for a second
soluble amplification primer,
(vii) two or more copies of a universal binding sequence for a soluble
compaction oligonucleotide,
(-Oil) two or more copies of a sample barcode sequence and/or
(ix) two or more copies of a unique molecular index sequence.
48. The method of claim 44, wherein the sequencing of step (b) comprises
hybridizing a
plurality of soluble forward sequencing primers to the plurality of
immobilized concatemer
template molecules and conducting one or rnore sequencing reactions.

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49. The method of claim 44, wherein the sequencing of step (d) comprises
hybridizing a
plurality of soluble reverse sequencing primers to the plurality of
immobilized partially
displaced forward extension strands and the plurality of immobilized detached
extended
forward sequencing primer strands, and conducting one or more sequencing
reactions.
50. The method of claim 47, wherein the support further comprises a
plurality of immobilized
second surface primers that lack a nucleotide having a scissile moiety.
51. The method of claim 50, wherein at least one copy of the universal
binding sequence for
the immobilized second surface primer in the individual concatemer template
molecules is
hybridized to an immobilized second surface primer.
52. The method of claim 50, wherein the plurality of immobilized second
surface primers have
3' OH extendible ends.
53. The method of claim 50, wherein the plurality of immobilized second
surface primers have
3' non-extendible ends.
54. The method of claim 53, wherein the 3' non-extendible end comprises a
phosphate group, a
dideoxycytidine group, an inverted dT, or an amino group.
55. A method for pairwise sequencing, comprising:
a) providing a support having a plurality of a first surface primer
immobilized
thereon wherein each of the first surface primers have a 3' extendible end and
lack a nucleotide having a scissile moiety;
b) generating a plurality of immobilized single stranded nucleic acid
concatemer
template molecules by hybridizing a plurality of single-stranded circular
nucleic
acid library molecules to the plurality of immobilized first surface primers
and
conducting a rolling circle amplification reaction with a plurality of a
strand
displacing polymerase, and a plurality of nucleotides )Nhich lack a nucleotide
having a scissile moiety that can be cleaved to generate an abasic site,
thereby
generating a plurality of immobilized single stranded nucleic acid concatemer

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template molecules, wherein individual single stranded nucleic acid concatemer
template molecules are covalently joined to an immobilized first surface
primer;
c) sequencing the plurality of immobilized concatemer template molecules
thereby
generating a plurality of extended forward sequencing primer strands, wherein
individual immobilized concatemer template molecules have two or more
extended forward sequencing primer strands hybridized thereon;
d) retaining the plurality of immobilized concatemer template molecules and
replacing the plurality of extended forward sequencing primer strands with a
plurality of forward extension strands by conducting a primer extension
reaction
with a plurality of soluble amplification primers and a plurality of strand-
displacing polymerases to generate a plurality of forward extension strands
and a
plurality of partially displaced forward extension strands that are hybridized
to the
immobilized concatemer template molecules to form a plurality of immobilized
amplicons, and the primer extension reaction generates a plurality of detached
forward extension strands (e.g., that are not hybridized to the immobilized
concatemer template molecules); and
e) sequencing the plurality of immobilized partially displaced forward
extension
strands thereby generating a first plurality of extended reverse sequencing
primer
strands, and sequencing the plurality of immobilized detached forward
extension
strands thereby generating a second plurality of extended reverse sequencing
primer strands, wherein individual immobilized partially displaced forward
extension strands have two or more extended reverse sequencing primer strands
hybridized thereon, and wherein in individual immobilized detached forward
extension strands have two or more extended reverse sequencing primer strands
hybridized thereon.
56. The
method of claim 55, wherein each of the single stranded circular nucleic acid
library
molecules in the plurality comprises a sequence of interest, and wherein the
individual
library molecules further comprise any one or any combination of two or more
of
a universal binding sequence for a soluble forward sequencing primer,
(ii) a universal binding sequence for a soluble reverse
sequencing primer,

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(iii) a universal binding sequence for an immobilized first surface
printer,
(iv) a universal binding sequence for an immobilized second surface
primer,
(v) a universal binding sequence for a first soluble amplification primer,
(vi) a universal binding sequence for a second soluble amplification
primer,
(vii) a universal binding sequence for a soluble compaction oligonucleotide,
(viii) a sample barcode sequence and/or
(ix) a unique molecular index sequence.
57. The method of claim 55, wherein individual immobilized single stranded
nucleic acid
concatemer template molecules generated by the rolling circle amplification
reaction
comprise two or more copies of a sequence of interest, wherein the individual
immobilized
concatemer template molecules further comprise any one or any combinafion of
two or
more of
two or more copies of a universal binding sequence for a soluble
forward sequencing primer,
two or more copies of a universal binding sequence for a soluble
reverse sequencing primer,
(iii) two or more copies of a universal binding sequence for an
immobilized first surface printer,
(iv) two or more copies of a universal binding sequence for an
immobilized second surface primer,
(v) two or more copies of a universal binding sequence for a first soluble
amplification primer,
(vi) two or more copies of a universal binding sequence for a second
soluble amplification primer,
(vii) two or more copies of a universal binding sequence for a soluble
compaction oligonucleotide,
(viii) two or more copies of a sample barcode sequence andlor
(ix) two or more copies of a unique molecular index sequence.

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58. The method of claim 55, wherein the sequencing of step (c) comprises
hybridizing a
plurality of soluble forward sequencing prirners to the plurality of
immobilized concatemer
template molecules and conducting one or more sequencing reactions.
59. The method of claim 55, wherein the sequencing of step (e) comprises
hybridizing a
plurality of soluble reverse sequencing primers to the plurality of
immobilized partially
displaced forward extension strands and the plurality of irnmobilized detached
extended
forward sequencing primer strands, and conducting one or more sequencing
reactions.
60. The method of claim 57, wherein the support further comprises a
plurality of immobilized
second surface primers that lack a nucleotide havine a scissile moiety.
I. The
method of claim 60, wherein at least one copy of the universal binding
sequence for
the immobilized second surface primer in the individual concatemer template
molecules is
hybridized to an immobilized second surface primer.
62. The method of claim 60, wherein the plurality of immobilized second
surface prirners have
3' OH extendible ends.
63. The method of clairn 60, wherein the plurality of immobilized second
surface primers have
3' non-extendible ends.
64. The method of claim (3, wherein the 3' non-extendible end comprises a
phosphate group, a
dideoxycytidine group, an inverted dT, or an amino group.
65. A method for pairwise sequencing, comprising:
a) contacting in-solution a plurality of single-stranded circular
nucleic acid library
molecules to a plurality of first soluble amplification primers, a plurality
of a
strand displacing polym.erase, and a plurality of nucleotides which lacks a
nucleotide having a scissile moiety that can be cleaved to generate an ahasic
site,
under a condition suitable to form a plurality of libraiy-primer duplexes and

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suitable for conducting a rolling circle amplification reaction, thereby
generating
a plurality of single stranded nucleic acid concatemers;
b) distributing the rolling circle amplification reaction onto a support
having a
plurality of the first surface primers immobilized thereon, under a condition
suitable for hybridizing one or more portions of individual single stranded
concatemers to one or more immobilized first surface primers, wherein each of
the first surface primers lack a nucleotide having a scissile moiety;
c) continuing the rolling circle amplification reaction on the support to
generate a
plurality of immobilized concatemer template molecules;
d) sequencing the plurality of immobilized concatemer template molecules
thereby
generating a plurality of extended forward sequencing primer strands wherein
individual immobilized concatemer template molecules have two or more
extended forward sequencing primer strands hybridized thereon;
e) retaining the plurality of immobilized concatemer template molecules and
replacing the plurality of extended forward sequencing primer strands with a
plurality of forward extension strands by conducting a prirner extension
reaction
with a plurality of a second soluble amplification prirner and a plurality of
strand-
displacing polymerases to generate a plurality of forward extension strands
and a
plurality of partially displaced forward extension strands that are hybridized
to the
immobilized concatemer ternplate rnolecules to form a plurality of
imrnobilized
arnplicons, and the primer extension reaction generates a plurality of
detached
forward extension strands (e.g., that are not hybridized to the immobilized
concatemer template molecules); and
f) sequencing the plurality of immobilized partially displaced forward
extension
strands thereby generating a first plurality of extended reverse sequencing
primer
strands, and sequencing the plurality of immobilized detached forward
extension
strands thereby generating a second plurality of extended reverse sequencing
primer strands, wherein individual immobilized partially displaced forward
extension strands have two or more extended reverse sequencing primer strands
hybridized thereon, and wherein in individual immobilized detached forward

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extension strands have two or more extended reverse sequencing primer strands
hybridized thereon.
66. The method of claim 65, wherein each of the single stranded circular
nucleic acid library
molecules in the plurality comprises a sequence of interest, and wherein the
individual
library molecules further comprise any one or any combination of two or more
of
(i) a universal binding sequence for a soluble forward sequencing primer,
(ii) a universal binding sequence for a soluble reverse sequencing primer,
(iii) a universal binding sequence for an immobilized first surface primer,
(iv) a universal binding sequence for an immobilized second surface
prirner,
(v) a universal binding sequence for a first soluble amplification primer,
(vi) a universal binding sequence for a second soluble amplification
primer,
(vii) a universal binding sequence for a soluble compaction oligonucleotide,
(viii) a sample barcode sequence and/or
(ix) a unique molecular index sequence.
67. The method of claim 65, wherein individual immobilized single stranded
nucleic acid
concatemer template molecules generated by the rolling circle amplification
reaction
comprise two or more copies of a sequence of interest, and wherein the
individual
immobilized concatemer template molecules further comprise any one or any
combination
of two or more of
two or more copies of a universal binding sequence for a soluble
forward sequencing printer,
(ii) two or more copies of a universal binding sequence for a soluble
reverse sequencing prirn.er,
(iii) two or more copies of a universal binding sequence for art
immobilized first surface primer,
(iv) two or more copies of a universal binding sequence for an
immobilized second surface primer,

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(v) two or more copies of a universal binding sequence for a first soluble
amplification primer,
(vi) two or more copies of a universal binding sequence for a second
soluble amplification primer,
(vii) two or more copies of a universal binding sequence for a soluble
compaction oligonucleotide,
(viii) two or more copies of a sample barcode sequence andlor
(ix) two or more copies of a unique molecular index sequence.
68. The method of claim 65, wherein the sequencing of step (d) comprises
hybridizing a
plurality of soluble forward sequencing primers to the plurality of
immobilized concatemer
template molecules and conducting one or more sequencing reactions.
69. The method of claim 65, wherein the sequencing of step (0 cornprises
hybridizing a
plurality of soluble reverse sequencing primers to the plurality of
immobilized partially
displaced forward extension strands and the plurality of immobilized detached
extended
forward sequencing primer strands, and conducting one or more sequencing
reactions.
70. The method of claim 65, wherein the support further comprises a
plurality of immobilized
second surface primers that lack a nucleotide having a scissile moiety.
71. The method of claim 70, wherein at least one copy of the universal
binding sequence for
the immobilized second surface primer in the individual concatemer template
molecules is
hybridized to an immobilized second surface primer.
72. The method of claim 70, wherein the plurality of immobilized second
surface primers have
3' OH extendible ends.
73. The method of claim 70, wherein the plurality of immobilized second
surface primers have
3' non-extendible ends.

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74. The method of clairn 73, wherein the 3' non-extendible end comprises a
phosphate group, a
dideoxycytidine group, an inverted dT, or an amino group.
75. A method for pairwise sequencing, comprising:
a) providing a plurality of immobilized single stranded nucleic acid
concatemer
template molecules each comprising at least one nucleotide having a scissile
moiety that can be cleaved to generate an a basic site in the concatemer
template
molecule, wherein individual concatemer ternplate molecules in the plurality
are
immobilized to a first surface prirner that is immobilized to a support,
wherein the
immobilized first surface prirners include a nucleotide having a scissile
moiety,
wherein the support further comprises a plurality of immobilized second
surface
primers which lack a nucleotide having a scissile moiety and have an
extendible
terminal 3'0H group, and wherein the immobilized concatemer template
molecule comprises two or more copies of a universal binding sequence for an
immobilized second surface primer (wherein the support comprises an excess of
immobilized first and second surface primers compared to the number of
immobilized concatemer template molecules);
b) sequencing the plurality of immobilized concatemer template molecules
with a
plurality of soluble forward sequencing primers thereby generating a plurality
of
extended forward sequencing primer strands, wherein individual immobilized
concaterner template molecules have two or more extended forward sequencin.g
primer strands hybridized thereon;
c) removing the extended forward sequencing primer strands and retaining
the
immobilized concatemer template molecules;
d) gen.erating a first plurality of immobilized forward extension strands
by
hybridizing at least one portion of individual immobilized concatemer template
molecules to a second surface primer and conducting a primer extension
reaction
from the second surface prirners that are hybridized to a portion of the
immobilized concatemer template molecule to generate a plurality of forward
extension strands having a sequence that is complementary to at least a
portion of

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the immobilized concatemer template molecules and are covalently joined to an
immobilized second surface primer;
e) contacting the plurality of immobilized concatemer template molecules
and the
plurality of immobilized forward extension strands with a relaxing solution
which
comprises at least one chaotropic agent;
f) dissociating the at least one portion of the immobilized concatemer
template
molecules from the immobilized second surface primers and retaining the
immobilized forward extension strands, and re-hybridizing at least one portion
of
the immobilized concatemer template molecules to one of the immobilized second
surface primers that are not covalently joined to a forward extension strand,
wherein the dissociating and re-associating comprises a temperature rarnp-up,
a
temperature plateau, and temperature ramp-down, and washing the relaxing
solution from the support;
g) contacting the re-hybridized immobilized concatemer template molecules
with an
amplification solution and conducting a primer extension reaction from the
second surface primers that are re-hybridized to a portion of the immobilized
concatemer template molecules to generate a plurality of newly synthesized
forward extension strands having a sequence that is complementary to at least
a
portion of the immobilized concatemer template molecules and are covalently
joined to an immobilized second suiface primer;
h) repeating steps (e) ¨ (g) at least once;
i) removing the retained immobilized concatemer template molecules by
generating
abasic sites in the immobilized single stranded concatemer template molecules
and the immobilized first surface primers at the nucleotide(s) having the
scissile
moiety and generating gaps at the abasic sites thereby generating a plurality
of
gap-containing nucleic acid molecules while retaining the plurality of
immobilized forward extension strands and retaining the plurality of
immobilized
second surface primers; and
j) sequencing the plurality of retained immobilized forward extension
strands with a
plurality of soluble reverse sequencing primers thereby generating a plurality
of
extended reverse sequencing primer strands.
. .

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76. The method of claitn 75, wherein individual concatemer template
molecules in the plurality
are coyalently joined to an immobilized first surface primer.
77. The method of claim 75, wherein individual concaterner template
molecules in the plurality
are hybridized to an immobilized first surface primer.
78. The method of claim 75, wherein individual immobilized conoatemer
template molecules
in the plurality comprise two or more copies of a sequence of interest, and
wherein the
individual immobilized concatemer template molecules further comprise any one
or any
combination of two or more of
two or more copies of a universal binding sequence for a soluble
forward sequencing primer,
(ii) two or more copies of a universal binding sequence for a soluble
reverse sequencing primer,
(iii) two or more copies of a universal binding sequence for an
immobilized first surface primer,
(iv) two or more copies of a universal binding sequence for an.
immobilized second surface primer,
(v) two or more copies of a universal binding sequence for a first soluble
amplification primer,
(vi) two or more copies of a universal binding sequence for a second
soluble amplification primer,
(vii) two or more copies of a universal binding sequence for a soluble
compaction oligonucleotide,
(viii) two or more copies of a sample barcode sequence andlor
(ix) two or more copies of a unique tnolecular index sequence.
79. A method for pairwise sequencing, comprising:
a) providing a support having a plurality of first and second surface
primers
immobilized thereon, wherein the first surface primers have a scissile moiety
that

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can be cleaved to generate an abasic site, and wherein the second surface
primers
lack a nucleotide having a scissile moiety and the second surface prirners
have an
extendible terminal 3'0H group;
b) generating a plurality of immobilized single stranded nucleic acid
concatemer
template rnolecules by hybridizing a plurality of single-stranded circular
nucleic
acid library molecules to the plurality of immobilized first surface primers
and
conducting a rolling circle amplification reaction with a plurality of a
strand
displacing polymerase, and a plurality of nucleotides which include dATP,
dCTP,
dGTP, dTTP and a plurality of nucleotides having a scissile moiety that can be
cleaved to generate an abasic site, thereby generating a plurality of
immobilized
single stranded nucleic acid concatemer template rnolecules having at least
one
nucleotide with a scissile moiety, wherein individual single stranded nucleic
acid
concatemer template molecules are covalently joined to an immobilized first
surface primer;
c) sequencing the plurality of immobilized concatemer template molecules
with a
plurality of soluble forward sequencing primers thereby generating a plurality
of
extended forward sequencing primer strands, wherein individual immobilized
concaterner template molecules have two or more extended forward sequencing
primer strands hybridized thereon;
d) removing the extended forward sequencing primer strands and retaining
the
immobilized concatemer template molecules;
e) generating a first plurality of immobilized forward extension strands by
hybridizing at least one portion of individual immobilized concaterner
template
molecules to a second surface primer and conducting a prirner extension
reaction
frorn the second surface primers that are hybridized to a portion of the
immobilized concatemer template molecule to generate a plurality of forward
extension strands having a sequence that is complementaiy to at least a
portion of
the immobilized concatemer template molecules and are covalently joined to an
immobilized second surface primer;

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f) contacting the plurality of immobilized concatemer template molecules
and the
plurality of immobilized forward extension strands with a relaxing solution
which
comprises at least one chaotropic agent;
g) dissociating the at least one portion of the immobilized concatemer
template
molecules from the immobilized second surface primers and retaining the
immobilized forward extension strands, and re-hybridizing at least one portion
of
the immobilized concatemer template molecules to one of the immobilized second
surface primers that are not covalently joined to a forward extension strand,
wherein the dissociating and re-associating comprises a temperature rarnp-up,
a
temperature plateau, and temperature ramp-down, and washing the relaxing
solution from the support;
h) contacting the re-hybridized immobilized concatemer template molecules
with an
amplification solution and conducting a primer extension reaction from the
second surface primers that are re-hybridized to a portion of the immobilized
concatemer template molecules to generate a plurality of newly synthesized
forward extension strands having a sequence that is complementary to at least
a
portion of the immobilized concatemer template molecules and are covalently
joined to an immobilized second suiface primer;
i) repeating steps (f) ¨ (h) at least once;
j) removing the retained immobilized concatemer template molecules by
generating
abasic sites in the immobilized single stranded concatemer template molecules
and the immobilized first surface primers at the nucleotide(s) having the
scissile
moiety and generating gaps at the abasic sites to generate a plurality of gap-
containing nucleic acid molecules while retaining the plurality of immobilized
forward extension strands and retaining the plurality of immobilized second
surface primers; and
k) sequencing the plurality of retained immobilized forward extension
strands with a
plurality of soluble reverse sequencing primers thereby generating a plurality
of
extended reverse sequencing primer strands.
. .

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80. The
method of claim 79, wherein each of the single stranded circular nucleic acid
library
molecules in the plurality comprises a sequence of interest and wherein the
individual
library molecules further comprise any one or any combination of two or more
of
a universal binding sequence for a soluble forward sequencing primer,
(ii) a universal binding sequence for a soluble reverse sequencing primer,
(iii) a universal binding sequence for an immobilized first surface primer,
(iv) a universal binding sequence for an irnmobilized second surface
primer,
(v) a universal binding sequence for a first soluble amplification primer,
(vi) a universal binding sequence for a second soluble amplification
prirner,
(vii) a universal binding sequence for a soluble compaction oligonucleotide,
(viii) a sample barcode sequence and/or
(ix) a unique molecular index sequence.
81. The
method of claim 79, wherein individual immobilized concatemer template
molecules
in the plurality comprise two or more copies of a sequence of interest, and
wherein the
individual immobilized concatemer template molecules further comprise an.y one
or any
combination of two or more of
two or more copies of a universal binding sequence for a soluble
forward sequencing primer,
(ii) two or more copies of a universal binding sequence for a soluble
reverse sequencing prirn.er,
(iii) two or more copies of a universal binding sequence for an
immobilized first surface printer,
(iv) two or more copies of a universal binding sequence for an
immobilized second surface primer,
(v) two or more copies of a universal binding sequence for a first soluble
amplification primer,
(vi) two or more copies of a universal binding sequence for a second
soluble amplification primer,

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(vii) two or more copies of a universal binding sequence for a soluble
compaction oligonucleotide,
(viii) two or more copies of a sarnple barcode sequence andlor
(ix) two or more copies of a unique molecular index sequence.
82. A method for pairwise sequencing, comprising:
a) contacting in-solution a plurality of single-stranded circular nucleic
acid library
molecules to a plurality of first soluble arnplification prirners, a plurality
of a
strand displacing polymerase, and a plurality of nucleotides which include
dATP,
KTP, dGTP, dTTP and a plurality of nucleotides having a scissile moiety that
can be cleaved to generate an abasic site, under a condition suitable to form
a
plurality of library-prirner duplexes and suitable for conducting a rolling
circle
amplification reaction, thereby generating a plurality of single stranded
nucleic
acid concatemers having at least one nucleotide with a scissile moiety;
b) distributing the rolling circle amplification reaction onto a support
having a
plurahty of the first surface primers immobilized thereon, under a condition
suitable for hybridizing one or more portions of individual single stranded
concaterners to one or more immobilized first surface primers, wherein the
immobilized first surface primers include a nucleotide having a scissile
moiety,
wherein the support further comprises a plurality of immobilized second
surface
primers which lack a nucleotide having a scissile moiety and have an
extendible
terminal 3'0H group;
c) continuing the rolling circle amplification reaction on the support in
the presence
of a plurality of nucleotides which include a plurality of nucleotides having
a
scissile moiety to generate a plurality of immobilized concatemer template
molecules;
d) sequencing the plurality of immobilized concatemer template molecules
with a
plurality of soluble forward sequencing primers thereby generating a plurality
of
extended forward sequencing primer strands, wherein individual immobilized
concaterner template molecules have two or more extended forward sequencing
prirner strands hybridized thereon;

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e) removing the extended forward sequencing primer strands and retaining
the
immobilized concatemer template molecules;
f) generating a first plurality of immobilized forward extension strands by
hybridizing at least one portion of individual immobilized concatemer template
molecules to a second surface primer and conducting a primer extension
reaction
from the second surface primers that are hybridized to a portion of the
immobilized concatemer template molecule to generate a plurality of forward
extension strands having a sequence that is complementary to at least a
portion of
the immobilized concatemer template molecules and are covalently joined to an
immobilized second surface primer;
g) contacting the plurality of immobilized concatemer template molecules
and the
plurality of immobilized forward extension strands with a relaxing solution
which
comprises at least one chaotropic agent;
h) dissociating the at least one portion of the immobilized concatemer
template
molecules from the immobilized second surface primers and retaining the
immobilized forward extension strands, and re-hybridizing at least one portion
of
the immobilized concatemer template molecules to one of the immobilized second
surface primers that are not covalently joined to a forward extension strand,
wherein the dissociating and re-associating comprises a temperature ramp-up, a
temperature plateau, and temperature ramp-down, and washing the relaxing
solution from the support;
i) contacting the re-hybridized immobilized concatemer template molecules
with an
amplification solution and conducting a primer extension reaction from the
second surface primers that are re-hybridized to a portion of the immobilized
concatemer template molecules to generate a plurality of newly synthesized
forward extension strands having a sequence that is complementary to at least
a
portion of the immobilized concatemer template molecules and are covalently
joined to an immobilized second surface primer;
j) repeating steps (g) (i) at least once;
k) removing the retained immobilized concatemer template molecules by
generating
abasic sites in the immobilized single stranded concatemer template molecules

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and the immobilized first surface prirners at the nucleotide(s) having the
scissile
rnoiety and generating gaps at the abasic sites to generate a plurality of gap-
containing nucleic acid molecules while retaining the plurality of immobilized
forward extension strands and retaining the plurality of immobilized second
surface primers; and
1) sequencing the plurality of retained imrnobilized forward extension
strands with a
plurality of soluble reverse sequencing primers thereby generating a plurality
of
extended reverse sequencing primer strands.
83. The
rnethod of claim 82, wherein each of the single stranded circular nucleic acid
library
molecules in the plurality comprises a sequence of interest and wherein the
individual
library rnolecules further comprise any one or any combination of two or more
of
(i) a universal binding sequence for a soluble forward sequencing prirner,
(ii) a universal binding sequence for a soluble reverse sequencing primer,
(iii) a universal binding sequence for an immobilized first surface primer,
(iv) a universal binding sequence for an immobilized second surface
primer,
(v) a universal binding sequence for a first soluble amplification primer,
(vi) a universal binding sequence for a second soluble amplification
primer,
(vii) a universal binding sequence for a soluble compaction oligonucleotide,
(viii) a sample barcode sequence and/or
(ix) a unique molecular index sequence.
84. The
method of claim 82, wherein individual immobilized concatemer template
molecules
in the plurality comprise two or more copies of a sequence of interest, and
wherein the
individual immobilized concaterner template molecules further comprise any one
or any
cotnbi nation of two or more of
(i) two or more copies of a universal binding sequence for a
soluble
forward sequencing primer,

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(ii) two or more copies of a universal binding sequence for a soluble
reverse sequencing primer,
(iii) two or more copies of a universal binding sequence for an
immobilized first surface printer,
(iv) two or more copies of a universal binding sequence for an
immobilized second surface primer,
(v) two or more copies of a universal binding sequence for a first soluble
amplification primer,
(vi) two or more copies of a universal binding sequence for a second
soluble amplification printer,
(vii) two or more copies of a universal binding sequence for a soluble
compaction oligonucleotide,
(viii) two or more copies of a sample barcode sequence andlor
(ix) two or more copies of a unique molecular index sequence.
85. The method of claim I, 12, 22, 32, 44, 55, 65, 75, 79 or 82, wherein
the support comprises
a planar substrate which comprises glass, fused-silica, silicon, a polymer
(e.g., polystyrene
(PS), macroporous polystyrene (MWS), polymethylmethacrylate (PMMA),
polycarbonate
(PC), polypropylene (PP), polyethylene (PE), high density polyethylene (HDPE),
cyclic
olefin polymers (COP), cyclic olefin copolymers (COC), polyethylene
terephthalate
(PET)), or any combination thereof.
86. The method of claim I, 12, 22, 32, 44, 55, 65, 75, 79 or 82, wherein
the support comprises
at least one hydrophilic polymer coating having a water contact angle of no
more than 45
degrees, and wherein at least one of the hydrophilic polymer coatings
comprising branched
hydrophilic polymer haying at least 4 branches,
87. The method of claim 1, 12, 22, 32, 44, 55, 65, 75, 79 or 82, wherein
the 5' end of the
plurality of first surface primers are immobilized to the support or
immobilized to a coating
on the support.

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88. The method of claim 1, 12, 22, 32, 44, 55, 65, 75, 79 or 82, wherein
the plurality of first
surface primers comprise modified oligonucleotide rnolecules having 2-10
phosphorothioate linkages at their 5' ends to confer resistance to nuclease
degradation.
89. The method of claim 7, 17, 27, 37, 50, 60 or 70, wherein the 5' end of
the plurality of
second surface primers are immobilized to the support or immobilized to a
coating on the
support.
90. The method of claim 7, 17, 27, 37, 50, 60 or 70, wherein the plurality
of second surface
prirners cornprise modified oligonucleotide rnolecules having 2-10
phosphorothioate
linkages at their 5' ends to confer resistance to nuclease degradation.
91. The method of claim 1, 12, 22, 32, 75, 79 or 82, wherein the
imtnobilized concatemer
template molecules comprise at least one nucleotide having a scissile moiety
which
comprises uridine, 8-oxo-7,8-dihydrogunine, or deoxyinosine.
92. The method of claim 1, 12, 22, 32, 75, 79 or 82, wherein the
nucleotides with a scissile
moiety are located at randomly distributed positions in individual immobilized
concatemer
template molecules in the plurality.
93. The method of claim 1, 12, 22, 32, 75, 79 or 82, wherein 0.01 - 30% of
the thymidine
nucleotides in the individual immobilized concatemer template molecules are
replaced
with uridine.
94. The method of claim 1, 12, 22, 32, 75, 79 or 82, wherein 0.01 - 30% of
the guanosine
nucleotides in the individual immobilized concatemer template molecules are
replaced
with 8-oxo-7,8-dihydrogunine or deoxyinosine.
95. The method of claim 5, 15, 25, 35, 48, 58, 68, 75, 79 or 82 wherein the
soluble forward
sequencing primer comprises a 3' OH extendible end and lacks a nucleotide
having a.
scissile moiety.

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96. The method of claim 6, 16, 26, 36, 49, 59, 69, 75, 79 or 82, wherein
the soluble reverse
sequencing primer comprises a 3' OH extendible end and lacks a nucleotide
having a
scissile moiety.
97. The method of claim 4, 13, 22, 23, 33, 47, 56, 65 or 66, wherein the
first soluble
amplification primer comprises a 3' OH extendible end and lacks a nucleotide
having a
scissile moiety.
98. The method of claim 4, 13, 23, 33, 47, 56 or 66, wherein the second
soluble amplification
prirner comprises a 3' OH extendible end and lacks a nucleotide having a
scissile rnoiety.
99. The method of claim I. wherein the forward sequencing of step (b)
cornprises, or the
method of claim 12 wherein the forward sequencing of step (c) comprises, or
the method of
claim 22 wherein the forward sequencing of step (d) comprises , or the method
of claim 32
wherein the forward sequencing of step (e) comprises, or the method of claim
44 wherein
the forward sequencing of step (b) comprises, or the method of clairn 55
wherein the
forward sequencing of step (c) comprises, or the method of claim 65 wherein
the forward
sequencing of step (d) comprises, or the method of claim 75 wherein the
forward
sequencing of step (b), or the method of claim 79 wherein the forward
sequencing of step
(c) comprises, or the method of claim 82 wherein the forward sequencing of
step (d)
comprises:
a) contacting a plurality of sequencing polymerases to (i) a plurality of
immobilized
concatemer template molecules and (ii) a plurality of the soluble forward
sequencing primers, wherein the contacting is conducted under a condition
suitable to form a plurality of complexed polymerases each comprising a
sequencing polym.erase bound to a nucleic acid duplex wherein the nucleic acid
duplex comprises a immobilized concatemer template molecule hybridized to a
soluble forward sequencing primer;
b) contacting the plurality of complexed sequencing polymerases with a
plurality of
nucleotides under a condition suitable for binding at least one nucleotide to
a
complexed sequencing polymerase, wherein the plurality of nucleotides

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comprises at least one nucleotide analog labeled with a fluorophore and having
a
removable chain terminating moiety at the sugar 3' position;
c) incorporating at least one nucleotide into the 3' end of the hybridized
forward
sequencing primers thereby generating a plurality of nascent extended forward
sequencing primers; and
d) detecting the incorporated nucleotide and identifying the nucleo-base of
the
incorporated nucleotide.
100. The method of claim 1 wherein the reverse sequencing of step (e)
comprises, or the method
of claim 12 wherein the reverse sequencing of step (f) comprises, or the
method of claim 22
wherein the reverse sequencing of step (g) comprises, or the method of claim
32 wherein
the reverse sequencing of step (h) comprises, or the method of claim 75
wherein the reverse
sequencing of step (j), or the method of claim 79 wherein the reverse
sequencing of step (k)
comprises, or the method of claim 82 wherein the reverse sequencing of step
(1) comprises:
a) contacting a plurality of sequencing polymerases to (i) a plurality of
the retained
forward extension strands and (ii) a plurality of the soluble reverse
sequen.cing
primers, wherein the contactin.g is con.ducted under a condition suitable to
form. a
plurality of complexed polym.erases each cornprising a sequen.cing polyrnerase
bound to a nucleic acid duplex wherein the n.ucleic acid duplex comprises a
retained forward extension stran.d hybridized to a soluble reverse sequencin.g
primer;
b) contacting the plurality of complexed sequencing polyrnerases with a
plurality of
nucleotides under a condition suitable for binding at least one nucleotide to
a
complexed sequencing polymerase, wherein the plurality of nucleotides
comprises at least one nucleotide analog labeled with a fluorophore and having
a
removable chain terminating moiety at the sugar 3' position;
c) incorporating at least one nucleotide into the 3' end of the hybridized
reverse
sequencing primers thereby generating a plurality of nascent extended reverse
sequencing primers; and
d) detecting the incorporated nucleotide and identifying the nucleo-base of
the
incorporated nucleotide.

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101. The method of claim 100, wherein the reverse sequencing of step (a)
comprises hybridizing
the plurality of soluble reverse sequencing primers to the plurality of the
retained forward
extension strands in the presence of a high efficiency hybridization buffer
which
comprises:
(i) a first polar aprotic solvent which comprises acetonitrile at 25-50% by
yolurne
of the hybridization buffer;
(ii) a second polar aprotic solvent which cornprises formamide at 5-10% by
volume of the hybridization buffer;
(iii) a pH buffering systern which comprises 2-(N-morpholino)ethanesulfonic
acid
(WS) at a pH of 5-6.5; and
(iv) a crowding agent which cornprises polyethylene glycol (PEG) at 5-35% by
volume of the hybridization buffer.
102, The method of claim 44 wherein the reverse sequencing of step (d)
comprises, or the
method of claim 55 wherein the reverse sequencing of step (e) cornprises, or
the method of
claim 65 wherein the reverse sequencing of step (f) comprises:
a) contacting a plurality of sequencing polymerases to (i) a plurality of
the
immobilized partially displaced forward extension strands, (ii) a plurality of
plurality of immobilized detached extended forward sequencing primer strands,
and (iii) a plurality of the soluble reverse sequencing primers, wherein the
contacting is conducted under a condition suitable to form a plurality of
cornplexed polyrnerases each comprising a sequencing poly merase bound to a
nucleic acid duplex wherein the nucleic acid duplex cornprises a soluble
reverse
sequencing primer hybridized to an immobilized partially displaced forward
extension strand or an immobilized detached extended forward sequencing
printer
strand;
b) contacting the plurality of complexed sequencing polymerases with a
plurality of
nucleotides under a condition suitable for binding at least one nucleotide to
a
complexed sequencing polymerase, wherein the plurality of nucleotides

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comprises at least one nucleotide analog labeled with a fluorophore and having
a
removable chain terminating moiety at the sugar 3' position;
c) incorporating at least one nucleotide into the 3' end of the hybridized
reverse
sequencing primers thereby generating a plurality of nascent extended reverse
sequencing primers; and
d) detecting the incorporated nucleotide and identifying the nucleo-base of
the
incorporated nucleotide.
103. The method of claim 102, wherein the reverse sequencing of step (a)
comprises hybridizing
the plurality of soluble reverse sequencing primers to the plurality of the
retained forward
extension strands in the presence of a high efficiency hybridization buffer
which
comprises:
(i) a first polar aprotic solvent which comprises acetonitrile at 25-50 A
by volume
of the hybridization buffer;
(ii) a second polar aprotic solvent which comprises formamide at 5-10% by
volume
of the hybridization buffer;
(iii) a pH buffering system which comprises 2-(N-morpholino)ethanesulfonic
acid
(MES) at a pH of 5-6.5; and
(iv) a crowding agent which comprises polyethylene glycol (PEG) at 5-35% by
volume of the hybridization buffer.
104. The method of claim 1 wherein the forward sequencing of step (b) and the
reverse
sequencing of step (e) comprises, or the method of claim 12 wherein the
forward
sequencing of step (c) and the reverse sequencing of step (f) comprises, or
the method of
claim 22 wherein the forward sequencing of step (d) and the reverse sequencing
of step (g)
comprises, or the method of claim 32 wherein the forward sequencing of step
(e) and the
reverse sequencing of step (h) comprises, or the method of claim 44 wherein
the forward
sequencing of step (b) and the reverse sequencing of step (d) comprises, or
the method of
claim 55 wherein the forward sequencing of step (c) and the reverse sequencing
of step (e)
comprises, or the method of claim 65 wherein the forward sequencing of step
(d) and the
reverse sequence of step (f), comprises, or the method of claim 75 wherein the
forward
. . .

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sequencing of step (b) and the reverse sequence of step (j), comprises, or the
method of
claim 79 wherein the forward sequencing of step (c) and the reverse sequence
of step (k),
comprises, or the method of claim 82 wherein the forward sequencing of step
(d) and the
reverse sequence of step (1), comprises:
1) conducting a sequencing reaction at a position on the template molecule
using
multivalent molecules which bind but do not incorporate;
2) conducting a sequencing reaction at the same position on the template
molecule
using nucleotides with incorporation; and
3) repeating steps (a) and (b) at the next position on the template
molecule.
105. The method of claim 1 wherein the forward sequencing of step (b) and the
reverse
sequencing of step (e) comprises, or the method of claim 12 wherein the
forward
sequencing of step (c) and the reverse sequencing of step (f) comprises, or
the method of
claim 22 wherein the forward sequencing of step (d) and the reverse sequencing
of step (g)
comprises, or the method of claim 32 wherein the forward sequencing of step
(e) and the
reverse sequencing of step (h) comprises, or the method of claim 75 wherein
the forward
sequencing of step (b) and the reverse sequence of step (j), comprises, or the
method of
claim 79 wherein the forward sequencing of step (c) and the reverse sequence
of step (k),
comprises, or the method of claim 82 wherein the forward sequencing of step
(d) and the
reverse sequence of step (1), comprises:
a) contacting a plurality of a first sequencing polymerase to (i) a
plurality of nucleic
acid template molecules and (ii) a plurality of soluble sequencing primers,
wherein the contacting is conducted under a condition suitable to form a
plurality
of first complexed polymerases each comprising a first sequencing polymerase
bound to a nucleic acid duplex wherein the nucleic acid duplex comprises the
nucleic acid template molecule hybridized to the sequencing primer, wherein
(1)
the plurality of nucleic acid template molecules comprise a plurality of the
immobilized concatemer template molecules and the plurality of soluble primers
comprise a plurality of the soluble forward sequencing primers, or wherein (2)
the
plurality of nucleic acid template molecules comprise a plurality of the
retained
. .

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forward extension strands and the plurality of soluble sequencing primers
comprise a plurality of the soluble reverse sequencing primers;
b) contacting the plurality of first cornplexed polymerases with a
plurality of
detectably labeled multivalent molecules to form a plurality of multivalent-
complexed polymerases, under a condition suitable for binding complementary
nucleotide units of the multivalent molecules to at least two of the plurality
of first
complexed polymerases thereby forming a plurality of multivalent-complexed
polymerases, and the condition inhibits incorporation of the complementary
nucleotide units into the sequencing primers of the plurality of multivalent-
complexed polyrnerases, wherein individual multivalent molecules in the
plurality
of multivalent molecules comprise a core attached to multiple nucleotide arms
and each nucleotide arm is attached to a nucleotide unit;
c) detecting the plurality of rnultivalent-complexed polymerases and
d) identifying the nucleo-base of the complementary nucleotide units that
are bound
to the plurality of first complexed polymerases in the plurality of
multivalent-
complexed polymerases, thereby determining the sequence of the nucleic acid
template.
106, The method of claim 105, wherein the reverse sequencing of step (e) in
claim 1, or the
reverse sequencing of step (f) in claim 12 comprises, or the reverse
sequencing of step (g)
in claim 22 comprises, or the reverse sequencing of step (h ín clairn 32
comprises, or the
reverse sequencing of step (j) in claim 75, or the reverse sequencing of step
(k) in claim 79
cornprises, or the reverse sequencing of step (I) in claim 82 comprises:
hybridizing the plurality of soluble reverse sequencing primers to the
plurality of the
retained forward extension strands in the presence of a high efficiency
hybridization buffer
which comprises:
(i) a first polar aprotic solvent which comprises acetonitrile at 25-50% by
volume
of the hybridization buffer;
(ii) a second polar aprotic solvent which comprises formamide at 5-10% by
volume of the hybridization buffer;

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(iii) a pfl buffering system which comprises 2-(N-morpholino)ethanesulfonic
acid
(MES) at a pH of 5-6.5; and
(iv) a crowding agent which comprises polyethylene glycol (PEG) at 5-35% by
volurne of the hybridization buffer.
107. The method of claim 105, further comprising:
e) dissociating the plurality of multivalent-complexed polymerases and
removing the
plurality of first sequencing polymerases and their bound multivalent
molecules,
and retaining the plurality of nucleic acid duplexes;
0 contacting the plurality of the retained nucleic acid duplexes of
step (e) with a
plurality of second sequencing polymerases, wherein the contacting is
conducted.
under a condition suitable for binding the plurality of second sequencing
polymerases to the plurality of the retained nucleic acid duplexes, thereby
forming
a plurality of second complexed polymerases each comprising a second
sequencing polymerase bound to a retained nucleic acid duplex;
g) contacting the plurality of second complexed polymerases with a
plurality of
nucleotides, wherein the contacting is conducted under a condition suitable
for
binding complementary nucleotides from. the plurality of nucleotides to at
least
two of the second complexed polymerases of step (0 thereby forming a plurality
of nucleotide-complexed polymerases and the condition is suitable for
promoting
incorporation of the bound complementary nucleotides into the sequencing
primers of the nucleotide-complexed polymerases;
h) detecting the complementary nucleotides which are incorporated into the
sequencing primers of the nucleotide-complexed polymerases; and
i) identifying the nucleo-bases of the complementary nucleotides which are
incorporated into the sequencing primers of the nucleotide-complexed
polymerases.
108, The method of claim 105, further comprising: forming at least one avidity
complex in step
(b), the method comprising:

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a) binding a first sequencing primer, a first sequencing polymerase, and a
first
multivalent molecule to a first portion of a nucleic acid template molecule
thereby
forming a first binding complex, wherein a first nucleotide unit of the first
multivalent molecule binds to the first sequencing polymerase; and
b) binding a second sequencing primer, a second sequencing polymerase, and
the
first multivalent molecule to a second portion of the same nucleic acid
template
molecule thereby forming a second binding complex, wherein a second nucleotide
unit of the second multivalent molecule binds to the second sequencing
polymerase, wherein the first and second binding complexes which include the
same multivalent molecule forms an avidity complex.
109. The method of claim 108, wherein (i) the first sequencing primer
comprises a soluble
forward sequencing primer and the nucleic acid template molecule comprises an
immobilized concatemer template molecule, (ii) the secon.d sequencing primer
comprises a
soluble forward sequen.cing prirner and the n.ucleic acid template molecule
cornprises the
same immobilized concaterner template molecule, and (iii) the first and second
sequen.cing
primers have the sam.e sequence.
110. The method of claim. 108, wherein (i) the first sequencing primer
cornprises a soluble
reverse sequencing primer and the nucleic acid template molecule com.prises a
retained
forward extension strand, (ii) the second sequencing primer com.prises a
soluble reverse
sequencing primer and the nucleic acid template molecule comprises the same
retained
forward extension strand, and (iii) the first and second sequencing primers
have the same
sequence.
111. The method of claim 108, further comprising: ft-irming at least one
avidity complex in step
(b), the method comprising:
a) contacting a plurality of first sequencing polymerases and a
plurality of second
sequencing primers with different portions of a nucleic acid template molecule
to
form at least first and second complexed polymerases on the sarne nucleic acid
template molecule;
. . .

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b) contacting a plurality of multivalent molecules to the at least first
and second
complexed polymerases on the same nucleic acid template molecule, under
conditions suitable to bind a single multivalent molecule from the plurality
to the
first and second complexed polymerases, wherein at least a first nucleotide
unit of
the single multivalent molecule is bound to the first complexed polymerase
which
includes a first sequencing primer hybridized to a first portion of the
nucleic acid
template molecule thereby forming a first binding complex, and wherein at
least a
second nucleotide unit of the single multivalent molecule is bound to the
second
complexed polymerase which includes a second sequencing primer hybridized to
a second portion of the sarne nucleic acid template molecule thereby forming a
second binding complex, wherein the contacting is conducted under a condition
suitable to inhibit polymerase-catalyzed incorporation of the bound first and
second nucleotide units in the first and second binding complexes, and wherein
the first and second binding complexes which are bound to the same multivalent
molecule forms an avidity complex;
c) detecting the first and second binding complexes on the same nucleic
acid
template molecule, and
d) identifying the first nucleotide unit in the first binding complex
thereby
determining the sequence of the first portion of the nucleic acid template
molecule, and identifying the second nucleotide unit in the second binding
complex thereby determining the sequence of the second portion of the same
nucleic acid template molecule.
112. The method of claim 111, wherein (i) the plurality of first sequencing
primers comprise a
plurality of first soluble forward sequencing primers and the nucleic acid
template
molecule comprises an immobilized concatemer template molecule, (ii) the
plurality of
second sequencing primers comprise a plurality of second soluble forward
sequencing
primers and the nucleic acid template molecule comprises the same immobilized
concatemer template molecule, and (iii) the plurality of first and second
sequencing primers
have the same sequence.
. . .

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113. The method of claim 111, wherein (i) the plurality of first sequencing
primers comprises a
plurality of first soluble reverse sequencing primer and the nucleic acid
template molecule
comprises a retained forward extension strand, (ii) the plurality of second
sequencing
primers comprise a plurality of second soluble reverse sequencing primers and
the nucleic
acid template molecule comprises the same retained forward extension strand,
and (iii) the
plurality of first and second sequencing primers have the same sequence.
114. The method of claim 44 wherein the forward sequencing of step (b) and the
reverse
sequencing of step (d) comprises, or the method of claim 44 wherein the
forward
sequencing of step (c) and the reverse sequencing of step (e) comprises, or
the method of
claim 65 wherein the forward sequencing of step (d) and the reverse sequencing
of step (f)
comprises:
a) contacting a plurality of a first sequencing polymerase to (i) a
plurality of nucleic
acid ternplate rnolecules and (ii) a plurality of soluble sequencing primers,
wherein the contacting is conducted under a condition suitable to form a
plurality
of first complexed polymerases each comprising a first sequencing polymerase
bound to a nucleic acid duplex wherein the nucleic acid duplex comprises the
nucleic acid ternplate molecule hybridized to the soluble sequencing primer,
wherein (1) the plurality of nucleic acid template rnolecules comprise a
plurality
of the immobilized concatemer template molecules and the plurality of
sequencing prirners cornprise a plurality of the soluble forward sequencing
primers, or wherein (2) the plurality of nucleic acid template molecules
comprise
a plurality of immobilized partially displaced forward extension strands and
the
plurality of sequencing primers comprise a plurality of the soluble reverse
sequencing primers, or wherein (3) the plurality of nucleic acid template
molecules comprise a plurality of immobilized detached extended forward
sequencing primer strands and the plurality of sequencing primers comprise a
plurality of the soluble reverse sequencing primers;
b) contacting the plurality of first complexed polymerases with a plurality
of
detectably labeled multivalent molecules to form a plurality of multivalent-
complexed polymerases, under a condition suitable for binding complementary
. .

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nucleotide units of the multivalent molecules to at least two of the plurality
of first
complexed polyrnerases thereby forming a plurality of multivalent-complexed
polytnerases, and the condition inhibits incorporation of the complementary
nucleotide units into the sequencing primers of the plurality of multivalent-
complexed polytnerases, wherein individual tnultivalent molecules in the
plurality
of multivalent molecules cornprise a core attached to multiple nucleotide arms
and each nucleotide arm is attached to a nucleotide unit;
c) detecting the plurality of multivalent-complexed polymerases; and
d) identifying the nucleo-base of the complementary nucleotide units that
are bound
to the plurality of first complexed polymerases in the plurality of
multivalent-
complexed polymerases, thereby determining the sequence of the nucleic acid
template.
115. The method of claim 114, wherein the reverse sequencing of step (d) of
claim 44
comprises, or the reverse sequencing of step (e) of claim 55 comprises, the
reverse
sequencing of step (f) of claim 65 comprises: hybridizing the plurality of
soluble reverse
sequencing primers to the plurality of immobilized parfially displaced forward
extension
strands or the plurality of immobilized detached extended forward sequencing
primer
strands in the presence of a high efficiency hybridization buffer which
comprises:
a first polar aprotic solvent which comprises acetonitrile at 25-50% by volume
of the hybridimtion buffer;
(ii) a second polar aprotic solvent which comprises formamide at 5-10% by
volume of the hybridization buffer;
(iii) a pH buffering system which comprises 2-(N-morpholino)ethanesulfonic
acid
(MES) at a pH of 5-6.5; and
(iv) a crowding agent which comprises polyethylene glycol (n-3G) at 5-35%
by
volume of the hybridization buffer.
116, The method of claim l 14, further cotnprising:

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e) dissociating the plurality of multivalent-complexed polymerases and
retnoving the
plurality of first sequencing polymerases and their bound multivalent
molecules,
and retaining the plurality of nucleic acid duplexes;
contacting the plurality of the retained nucleic acid duplexes of step (e)
with a
plurality of second sequencing polymerases, wherein the contacting is
conducted
under a condition suitable for binding the plurality of second sequencing
polyrnerases to the plurality of the retained nucleic acid duplexes, thereby
forming
a plurality of second complexed polyrnerases each comprising a second
sequencing polymerase bound to a retained nucleic acid duplex;
g) contacting the plurality of second cornplexed polymerases with a
plurality of
nucleotides, wherein the contacting is conducted under a condition suitable
for
binding complementary nucleotides from the plurality of nucleotides to at
least
two of the second complexed polymerases of step (f) thereby forrning a
plurality
of nucleotide-complexed polymerases and the condition is suitable for
promoting
incorporation of the bound complementary nucleotides into the sequencing
primers of the nucleotide-com.plexed polymerases;
h) detecting the complementary nucleotides which are incorporated into the
sequencin.g primers of the nucleotide-complexed polymerases; and
i) identifying the nucleo-bases of the complementary nucleotides which are
incorporated into the sequencing primers of the nucleotide-complexed
polymerases.
117, The method of claim 114, further comprising: forming at least one avidity
complex in step
(b), the method comprising:
a) binding a first sequencing primer, a first sequencing polymerase, and a
first
multivalent molecule to a first portion of a nucleic acid template molecule
thereby
forming a first binding complex, wherein a first nucleotide unit of the first
multivalent molecule binds to the first sequencing polymerase; and
b) binding a second sequencing primer, a second sequencing polymerase, and
the
first multivalent rnolecule to a second portion of the same nucleic acid
template
rnolecule thereby forming a second binding complex, wherein a second
nucleotide

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unit of the second multivalent molecule binds to the second sequencing
polymerase, wherein the first and second binding complexes which include the
same multivalent molecule forms an avidity complex.
118. The method of claim 117, wherein (i) the first sequencing primer
comprises a soluble
forward sequencing primer and the nucleic acid template molecule comprises an
immobilized concatemer template molecule, (ii) the second sequencing primer
comprises a
soluble forward sequencing primer and the nucleic acid template molecule
comprises the
same immobilized concatemer template molecule, and (iii) the first and second
sequencing
primers have the sarne sequence.
119. The method of claim 117, wherein (i) the first sequencing primer
comprises a soluble
reverse sequencing primer and the nucleic acid template molecule comprises an
immobilized partially displaced forward extension strand, (ii) the second
sequencing primer
comprises a soluble reverse sequencing primer and the nucleic acid template
molecule
comprises the sarne immobilized partially displaced forward extension strand,
and (iii) the
first and second sequencing primers have the same sequence.
120. The method of claim 117, wherein (i) the first sequencing primer
comprises a soluble
reverse sequencing primer and the nucleic acid template molecule comprises an
immobilized detached extended forward sequencing primer strand, (ii) the
second
sequencing primer comprises a soluble reverse sequencing primer and the
nucleic acid
template molecule comprises the same immobilized detached extended forward
sequencing
primer strand, and (iii) the first and second sequencing primers have the same
sequence.
121. The method of claim 117, further comprising: f*()rming at least one
avidity complex in step
(b), the method comprising:
a) contacting a plurality of first sequencing polymerases and a
plurality of second
sequencing primers with different portions of a nucleic acid template molecule
to
form at least first and second complexed polymerases on the same nucleic acid
template molecule;

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b) contacting a plurality of multivalent molecules to the at least first
and second
complexed polymerases on the same nucleic acid template molecule, under
conditions suitable to bind a single multivalent molecule from the plurality
to the
first and second complexed polymerases, wherein at least a first nucleotide
unit of
the single multivalent molecule is bound to the first complexed polymerase
which
includes a first sequencing primer hybridized to a first portion of the
nucleic acid
template molecule thereby forming a first binding complex, and wherein at
least a
second nucleotide unit of the single multivalent molecule is bound to the
second
complexed polymerase which includes a second sequencing primer hybridized to
a second portion of the sarne nucleic acid template molecule thereby forming a
second binding complex, wherein the contacting is conducted under a condition
suitable to inhibit polymerase-catalyzed incorporation of the bound first and
second nucleotide units in the first and second binding complexes, and wherein
the first and second binding complexes which are bound to the same multivalent
molecule forms an avidity complex;
c) detecting the first and second binding complexes on the same nucleic
acid
template molecule, and
d) identifying the first nucleotide unit in the first binding complex
thereby
determining the sequence of the first portion of the nucleic acid template
molecule, and identifying the second nucleotide unit in the second binding
complex thereby determining the sequence of the second portion of the same
nucleic acid template molecule.
122. The method of claim 121, wherein (i) the plurality of first sequencing
primers comprise a
plurality of first soluble forward sequencing primers and the nucleic acid
template
molecule comprises an immobilized concatemer template molecule, (ii) the
plurality of
second sequencing primers comprise a plurality of second soluble forward
sequencing
primers and the nucleic acid template molecule comprises the same immobilized
concatemer template molecule, and (iii) the plurality of first and second
sequencing primers
have the same sequence.
. .

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123. The method of claim 121, wherein (0 the plurality of first sequencing
primers comprises a
plurality of first soluble reverse sequencing printer and the nucleic acid
template molecule
cotnprises an immobilized partially displaced forward extension strand, (.ii)
the plurality of
second sequencing primers comprise a plurality of second soluble reverse
sequencing
primers and the nucleic acid template molecule comprises the same immobilized
partially
displaced forward extension strand, and (hi) the plurality of first and second
sequencing
primers have the same sequence.
124. The method of claim 121, wherein 0) the plurality of first sequencing
primers comprises a
plurality of first soluble reverse sequencing printer and the nucleic acid
template molecule
comprises an immobilized detached extended forward sequencing primer strands,
(ii) the
plurality of second sequencing primers comprise a plurality of second soluble
reverse
sequencing primers and the nucleic acid template molecule comprises the same
immobilized detached extended forward sequencing primer strands, and (iii) the
plurality
of first and second sequencing primers have the same sequence.
125. The method of claim 99, 100, 102, 107 or 116, wherein. individual
nucleotides in the
plurality of nucleotides comprise an aromatic base, a five carbon sugar, and 1-
10 phosphate
groups, wherein the aromatic base of the nucleotide comprises adenine,
guanine, cytosine,
thymine or uracil.
126. The method of claim 125, wherein the plurality of nucleotides comprises
one type of
nucleotide selected from a group consisting of dATP, dGTP, dCTP and dTTP.
127, The method of claim 125, wherein the plurality of nucleotides comprises a
mixture of any
combination of two or more types of nucleotides selected from a group
consisting of dATP,
dGIP, dCTP andlor dT __ FP.
128, The method of claim 125, wherein at least one of the nucleotides in the
plurality of
nucleotides comprises a fluorescently-laheled nucleotide.

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129. The method of claim 125, wherein at least one of the plurality of
nucleotides lacks a
fluorophore label.
130. The method of claim 99, 100, 102, 107 or 116, wherein at least one of the
nucleotides in
the plurality of nucleotides comprises a chain terminating moiety attached to
3'-OH sugar
position via cleavable moiety, and wherein the chain terminating moiety
comprises an alkyl
group, alkenyl group, alkynyl group, allyl group, aryl group, benzyl group,
azide group,
amine group, amide group, keto group, isocyanate group, phosphate group, thio
group,
disulfide group, carbonate group, urea group, or silyl group.
131. The method of claim 130, wherein:
the chain terrninating moieties alkyl, alkenyl, alkynyl and allyl are
cleavable/removable with tetrakis(triphenylphosphine)palladiurn(0) (Pd(PPh3)4)
with piperidine, or with 2,3-Dichloro-5,6-dicyano-1,4-benzo-quinone (DDQ);
(ii) the chain terminating moieties aryl and benzyl are
cleavable/rernovable with H2
Pd/C;
(iii) the chain terminating moieties amine, amide, keto, isocyanate,
phosphate, thio,
disulfide are cleavable/removable with a thiol reagent which comprises beta-
mercaptoethanol or dithiothritol (DTT);
(iv) the chain terminating moieties amine, amide, keto, isocyanate,
phosphate, thio,
disulfide are cleavable/removable with a phosphine reagent which comprises
Tris(2-carboxyethyl)phosphine (TCEP), bis-sulfo triphenyl phosphine (BS-TPP),
or Tri(hydroxyproyl)phosphine (THPP);
(v) the chain terminating moieties amine, amide, keto, isocyanate,
phosphate, thio,
disulfide are cleavable/removable with 4-dimethylaminopyridine (4-DMAP);
(vi) the chain terminating moiety carbonate is cleavable/removable with
potassium
carbonate (K2CO3) in MeOH, with triethylamine in pyridine, or with Zn in
acetic
acid (AcOH); and
(vii) the chain terminating moieties urea and silyl are cleavable with
tetrabutylammonium fluoride, pyridine-HF, with ammonium fluoride, or with
triethylarnine trihydrofluoride.
. .

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132. The method of claim 130, wherein at least one of the nucleotides in the
plurality of
nucleotides comprises a chain tertninating moiety attached to 3'-011 sugar
position via
cleavable moiety, and wherein the chain terminating moiety comprises a 3' 0-
azido or a 3'
O-azidotnethyl group.
133. The method of claim 130, wherein:
(i) the chain terminating moieties 3' O-azido and 3' O-azidomethyl group
are
cleavable/removable with a phosphine compound which comprise a derivatized
phosphine moiety, derivatized tri-aryl phosphine moiety, Tris(2-
carboxyethyl)phosphine (TCEP), bis-sulfo triphenyl phosphine (BS-TPP) or
Tri(hydroxyproyi)phosphine (THPP); and
(ii) the chain terminating moieties 3' O-azido and 3' O-azidomethyl group are
cleavable/removable with 4-dimethylaminopyridine (4-111AP),
134. The method of claim 104, 105, 108, 111, 114,117 or 121, wherein.
individual multivalent
molecules in the plurality of multivalent molecules comprises (a.) a core; and
(b) a plurality
of nucleotide arms which conlprise (i) a core attachnlent moiety, (ii) a
spacer comprising a.
PEG moiety, (iii) a linker, and (iv) a nucleotide unit, wh.erein the core is
attached to the
plurality of nucleotide arms via their core attachment moiety, wherein the
spacer is
attached to the linker, and w.herein the linker is attached to the nucleotide
unit.
135. The method of claim I 34, wherein the core comprises an avidin-type
moiety and the core
attachment moiety comprises biotin,
136. The method of claim 134, wherein the linker comprises an aliphatic chain
having 2-6
subunits or an oligo ethylene glycol chain having 2-6 subunits.
137, The method of claim I 34, wherein the linker further comprises an
aromatic moiety.
138, The method of claim I 34, wherein the nucleotide unit comprises an
aromatic base, a five
carbon sugar and 1-10 phosphate groups.

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139. The method of claim 134, wherein the linker is attached to the nucleotide
unit through the
base.
140. The method of clairn 134, wherein the plurality of nucleotide arms
attached to the core
have the same type of a nucleotide unit, and wherein the types of nucleotide
unit is selected
frorn a group consisting of dATP, dGTP, dCTP, dTTP and dUTP.
141. The method of claim 134, wherein the plurality of multivalent molecules
comprise one type
of a rnultivalent molecule wherein each multivalent rnolecule in the plurality
has the same
type of nucleotide unit selected from a group consisting of dATP, dGTP, dCTP,
dTIP and
dUTP.
142. The method of claim 134, wherein the plurality of tnultivalent molecules
cotnprise a
mixture of any combination of two or more types of multivalent molecules each
type
having nucleotide units selected from a group consisting of dATP, dGTP, dCTP,
dTTP and
dUTP.
143. The method of claim 134, wherein the plurality of rnultivalent molecules
are fluorescently-
labeled multivalent tnolecules.
144. The method of claim 134, wherein
the core of individual fluorescently-labeled rnultivalent molecules is
attached to a
fluorophore which corresponds to the nucleotide units that are attached to the
nucleotide arms;
(ii) at least one of the nucleotide arms cornprises a linker that is
attached to a
fluorophore which corresponds to the nucleotide units that are attached to the
nucleotide arms; and/or
(iii) at least one of the nucleotide arms comprises a nucleotide unit that
is attached to a
fluorophore which corresponds to the nucleotide units that are attached to the
nucleotide arms.

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145. The method of claim 134, wherein the plurality of multivalent molecules
lack a
fluorophore.
146. The method of claim 134, wherein at least one of the multivalent
molecules in the plurality
of multivalent molecules comprises nucleotide units having a chain terminating
moiety
attached to the sugar position via a cleavable moiety, and wherein the
chain
terminating moiety comprises an alkyl group, alkenyl group, alkynyl group,
allyl group,
aryl group, benzyl group, azide group, amine group, amide group, keto group,
isocyanate
group, phosphate group, thio group, disulfide group, carbonate group, urea
group, or silyl
group.
147. The method of claim 146, wherein:
(i) the chain terminating moieties alkyl, alkenyl, alkynyl and allyl are
cleavable/removable with tetrakis(triphenylphosphine)palladium(0) (pd(PPh3)4)
with piperidine, or with 2,3-Dichloro-5,6-dicyano-1,4-benzo-quinone (DDQ);
(ii) the chain terminating moieties aryl and benzyl are cleavable/removable
with H2
Pd/C;
(iii) the chain terminating moieties amine, amide, keto, isocyanate,
phosphate, thio,
disulfide are cleavable/removable with a thiol reagent which comprises beta-
rriercaptoethanol or dithiothritol (DTT);
(iv) the chain terminating moieties amine, amide, keto, isocyanate,
phosphate, thio,
disulfide are cleavable/rernovable with a phosphine reagent which comprises
Tris(2-carboxyethyl)phosphine (TCEP), bis-sulfo triphenyl phosphine (BS-TPP),
or Tri(hydroxyproyl)phosphine (IIIPP);
(v) the chain terminating moieties amine, amide, keto, isocyanate,
phosphate, thio,
disulfide are cleavable/rernovable with 4-dimethylaminopyridine (4-DMAP);
(vi) the chain terminating moiety carbonate is cleavablelremovable with
potassiurn
carbonate (K2CO3) in MeOH, with triethylamine in pyridine, or with Zn in
acetic
acid (AcOH); and
. .

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(vii) the chain terminating moieties urea and silyi are cleavable with
tetrabutylammonium fluoride, pyridine-HF, with anunonium fluoride, or with
triethylamine trihydrofluoride.
148. The method of claim 146, wherein at least one of the multivalent
molecules in the plurality
of multivalent molecules comprises nucleotide units having a chain terminating
rnoiety
attached to the 3'-OH sugar position via a cleavable moiety, and wherein the
chain
terrninating moiety comprises a 3' O-azido or 3' 0-azidomethyl group.
149. The method of claim 146, wherein:
(i) the chain terminating rnoieties 3' O-azido and 3' 0-azidomethyl group
are
cleavable/removable with a phosphine cornpound which comprise a derivatized
tri-alkyl phosphine moiety, derivatized tri-aryl phosphine moiety, Tris(2-
earboxyethyl)phosphine (TCEP), bis-sulfo triphenyl phosphine (BS-TPP) or
Tri(hydroxyproyl)phosphine (THPP); and
(ii) the chain terminating moieties 3' O-azido and 3' 0-azidomethyl are
cleavable/removable with 4-dimethylaminopyridine (4-DMAP).
150, The method of claim 99, 100 or 102, wherein the plurality of sequencing
polymerases in
step (a) comprises a recombinant wild type DNA polymerase, and wherein the
plurality of
nucleotides in step (b) comprises fluorescently-labeled nucleotides having a
removable
chain terminating moiety at the 3' sugar position.
151. The method of claim 99, 100 or 102, wherein the plurality of sequencing
polymerases in
step (a) comprises a mutant DNA polymerase, and wherein the plurality of
nucleotides in
step (b) comprises fluorescently-labeled nucleotides having a removable chain
terminating
moiety at the 3' sugar position.
152, The method of claim 105 or 114, wherein the plurality of first sequencing
polymerases of
step (a) comprise a recombinant wild type DNA polymerase.

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153. The method of claim 105 or 114, wherein the plurality of first sequencing
polymerases of
step (a) comprise mutant DNA polymerase.
154. The method of claim 107 or 116, wherein the plurality of second
sequencing polymerases
of step (f) comprise recombinant wild type DNA polymerase, and wherein the
plurality of
nucleotides in step (b) comprises fluorescently-labeled nucleotides having a
removable
chain terminating moiety at the 3' sugar position.
155. The method of claim 107 or 116, wherein the plurality of second
sequencing polymerases
of step (f) comprise mutant DNA polymerase, and wherein the plurality of
nucleotides in
step (b) comprises fluorescently-labeled nucleotides having a removable chain
terminating
moiety at the 3' sugar position.
156. The method of claim 1 at step (c), or the method of claim 12 step (d), or
the method of
claim 22 step (e), or the method of clairn 32 at step (f), wherein the
replacing the plurality
of extended forward sequencing primer strands with a plurality of forward
extension
strands that are hybridized to the retained immobilized single stranded
nucleic acid
concatemer template molecules by conducting a primer extension reaction
comprises:
(i) contacting at least one extended forward sequencing prirner strand
with a plurality
of strand displacing polymerases and a plurality of nucleotides and in the
absence
of soluble amplification primers, under a condition suitable to conduct a
strand
displacing primer extension reaction using the at least one extended forward
sequencing primers strand to initiate the primer extension reaction thereby
generating a forward extension strand that is covalently joined to the
extended
forward sequencing primers strand, wherein the forward extension strand is
hybridized to the immobilized concatemer template molecule.
157. The method of claim 1 at step (c), or the method of claim 12 step (d), or
the method of
claim 22 step (e), or the method of claim 32 at step (f), wherein the
replacing the plurality
of extended forward sequencing primer strands with a plurality of forward
extension
strands that are hybridized to the retained immobilized single stranded
nucleic acid

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concatemer template molecules by conducting a primer extension reaction
comprises
removing the plurality of extended forward sequencing primer strands by:
(i) contacting the plurality of extended forward sequencing primer strands
with a 5'
to 3' double-stranded DNA exonuclease;
(ii) contacting the plurality of extended forward sequencing primer strands
with a
denaturation reagent comprising any combination of formamide, acetonitrile,
guanidinium chloride and/or a pH buffering agent; or
(iii) contacting the plurality of extended forward sequencing primer strands
with 100%
formamide.
158. The method of claim 1 at step (c), or the method of claim 12 step (d), or
the method of
claim 22 step (e), or the method of claim 32 at step (0, wherein the replacing
the plurality
of extended forward sequencing primer strands with a plurality of forward
extension
strands that are hybridized to the retained immobilized single stranded
nucleic acid
concatemer template molecules by conducting a primer extension reaction
comprises:
(i) removing the plurality of extended forward sequencing primer strands
while
retaining the immobilized concatemer template molecules; and
(ii) contacting the plurality of retained immobilized concatemer molecules
with a
second plurality of soluble forward sequencing primers, a plurality of
nucleotides
and a plurality of primer extension polymerases, under a condition suitable to
hybridize the plurality of soluble forward sequencing primers to the plurality
of
retained immobilized concatemer template molecules and suitable for conducting
polymerase-catalyzed primer extension reactions thereby generating a plurality
of
forward extension strands,
wherein the plurality of nucleotides comprise dATP, dGTP, dCTP and
dTTP but lacks dUTP,
wherein in the plurality of primer extension polymerases are tolerant of
uridine-containing template strands, and
wherein the soluble sequencing primers hybridize with the forward
sequencing primer binding sequence in the retained immobilized
concatemer molecules.

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159. The method of claitn 158, wherein the contacting cotnprises: contacting
the plurality of
retained immobilized concatemer molecules with the plurality of soluble
forward
sequencing primers in the presence of a high efficiency hybridization buffer
which
comprises:
(i) a first polar aprotic solvent which comprises acetonitrile at 25-50% by
yolurne
of the hybridization buffer;
(ii) a second polar aprotic solvent which cornprises formarnide at 5-10% by
volume of the hybridization buffer;
(iii) a pH buffering systern which comprises 2-(N-morpholino)ethanesulfonic
acid
(NIFS) at a pH of 5-6.5; and
(iv) a crowding agent which cornprises polyethylene glycol (PEG) at 5-35% by
volume of the hybridization buffer.
160, The method of claim 1 at step (c), or the method of claim 12 step (d), or
the method of
claim 22 step (e), or the method of claim. 32 at step (), wherein the
replacing the plurality
of extended forward sequencing primer strands with a plurality of forward
extension
strands that are hybridized to the retained imrnobilized single stranded
nucleic acid
concatemer template molecules by conducting a primer extension reaction
comprises:
(i) removing the plurality of extended forward sequencing primer strand
while
retaining the immobilized concatemer template molecules; and
(it) contacting the plurality of retained immobilized concatemer molecules
with a
plurality of soluble amplification primers, a. plurality of nucleotides and a
plurality
of primer extension polymerases, under a condition suitable to hybridize the
plurality of soluble amplification primers to the plurality of retained
immobilized
concaterner template molecules and suitable for conducting polymerase-
catalyzed
primer extension reactions thereby generating a pl urality of forward
extension
strands, wherein the soluble amplification primers hybridize with the soluble
amplification primer binding sequence in the retained immobilized concatemer
molecules,

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wherein the plurality of nucleotides cotnprise dATP, dGTP, dCTP and
&FIT but lacks dUTP,
wherein in the plurality of primer extension polyrnerases are tolerant of
uridine-containing template strands, and
wherein the soluble sequencing primers hybridize with the forward
sequencing primer binding sequence in the retained immobilized
concatemer molecules.
161. The method of claim 160 wherein the contacting comprises: contacting the
plurality of
retained immobilized concatemer molecules with the plurality of soluble
amplification
primers in the presence of a high efficiency hybridization buffer which
comprises:
(i) a first polar aprotic solvent which comprises acetonitrile at 25-50% by
volume
of the hybridization buffer;
(ii) a second polar aprotic solvent which comprises formamide at 5-10% by
volume of the hybridization buffer;
(iii) a pH buffering system which comprises 2-(N-morpholino)ethanesulfonic
acid.
(MES) at a pH of 5-6.5; and
(iv) a crowding agent which comprises polyethylene glycol (PEG) at 5-35% by
volume of the hybridization buffer.
162. The method of claim. 156, 158 or 160, further comprising: contacting the
plurality of
retained immobilized con.caterner molecules with a plurality of soluble
compaction
ol igonucleotides.
163. The method of claim 44 at step (c), or the method of claim 55 at step
(d), or the method of
claim 65 at step (e), wherein replacing the plurality of extended forward
sequencing prirner
strands comprises:
contacting at least one extended forward sequencing primer strand with a
plurality
of strand displacing polymerases and a plurality of nucleotides and in the
absence
of soluble amplification primers, under a condition suitable to conduct a
strand
displacing primer extension reaction using the at least one extended forward

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sequencing primer strand to initiate the primer extension reaction thereby
generating a plurality of forward extension strands, a plurality of partially
displaced extended forward sequencing strands and a plurality of detached
extended forward sequencing primer strands.
164. The method of claim 44 at step (c), or the method of claim 55 at step
(d), or the method of
claim 65 at step (e), wherein replacing the plurality of extended forward
sequencing primer
strands comprises: comprises removing the plurality of extended forward
sequencing
primer strands by:
(i) contacting the plurality of extended forward sequencing primer strands
with a 5'
to 3' double-stranded DNA exonuclease;
(ii) contacting the plurality of extended forward sequencing primer strands
with a
denaturation reagent comprising any combination of formamide, acetonitrile,
guanidinium chloride and/or a pH buffering agent; or
(iii) contacting the plurality of extended forward sequencing primer strands
with 100%
forrnamide.
165. The method of claim 44 at step (c), or the method of claim 55 at step
(d), or the method of
claim 65 at step (e), wherein replacing the plurality of extended forward
sequencing primer
strands comprises:
(i) removing the plurality of extended forward sequencing primer strands
while
retaining the immobilized concatemer template molecules; and
(ii) contacting the plurality of retained immobilized concatemer molecules
with a
second plurality of soluble forward sequencing primers, a plurality of
nucleotides
and a plurality of strand displacing polymerases, under a condition suitable
to
hybridize the plurality of soluble forward sequencing primers to the plurality
of
retained immobilized concatemer template molecules and suitable for conducting
polymerase-catalyzed strand displacing reactions thereby generating a
plurality of
forward extension strands and a plurality of partially displaced extended
forward
sequencing strands that are hybridized to the immobilized concatemer template
molecules to form a plurality of immobilized amplicons, and the primer
extension

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reaction generates a plurality of detached extended forward sequencing primer
strands (e.g., that are not hybridized to the inunobilized concatemer template
molecules),
wherein the plurality of nucleotides cornprise dATP, dGTP, dCTP and
dTTP but lacks dUTP, and
wherein the soluble forward sequencing prirners hybridize with the
forward sequencing prirner binding sequence in the retained
irnrnobilized concaterner rnolecules.
166. The rnethod of clairn 165, wherein the contacting cornprises: contacting
the plurality of
retained immobilized concaterner rnolecules with the plurality of soluble
forward
sequencing primers in the presence of a high efficiency hybridization buffer
which
comprises:
(i) a first polar aprotic solvent which comprises acetonitrile at 25-50% by
volume
of the hybridimtion buffer;
(ii) a second polar aprotic solvent which comprises formamide at 5-10% by
volume of the hybridization buffer;
(iii) a pH buffering systern. which comprises 2-(N-morpholino)ethanesulfonic
acid.
(MES) at a pH of 5-6.5; and
(iv) a crowding agent which comprises polyethylene glycol (PEG) at 5-35% by
volume of the hybridization buffer.
167. The method of claim 44 at step (c), or the method of claim 55 at step
(d), or -the method of
claim 65 at step (e), wherein replacing -the plurality of extended forward
sequencing primer
strands comprises:
(i) removing the plurality of extended forward sequencing prirner strand
while
retaining the immobilized concatemer template molecules; and
(ii) contacting the plurality of retained immobilized concaterner molecules
with a
plurahty of soluble amplification primers, a plurality of nucleotides and a
plurality of strand displacing polymerases, under a condition suitable to
hybridize the plurality of soluble amplification primers to the plurality of

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retained immobilized concatemer template molecules and suitable for conducting
polymerase-catalyzed strand displacing reactions thereby generating a
plurality
of forward extension strands and a plurality of partially displaced extended
forward sequencing strands that are hybridized to the immobilized concatemer
template molecules to form a plurality of immobilized amplicons, and the
primer
extension reaction generates a plurality of detached extended forward
sequencing
primer strands (e.g., that are not hybridized to the immobilized concatemer
template molecules),
wherein the plurality of nucleotides comprise dATP, dGTP, dCTP and
&MT but lacks dUTP,
wherein the soluble amplification primers hybridize with the soluble
amplification primer binding sequence in the retained immobilized
concatemer molecules.
168. The method of claim 167, wherein the contacting comprises: contacting the
plurality of
retained immobilized concatemer molecules with the plurality of soluble
amplification
primers in the presence of a high efficiency hybridization buffer which
comprises:
(i) a first polar aprotic solvent which comprises acetonitrile at 25-50% by
volume
of the hybridization buffer;
(ii) a second polar aprotic solvent which comprises formamide at 5-10% by
volume of the hybridization buffer;
(iii) a pl-I buffering system which comprises 2-(1=1-morpho1ino)ethanesu1fonic
acid
(WS) at a pH of 5-6.5; and
(iv) a crowding agent which comprises polyethylene glycol (PEG) at 5-35% by
volume of the hybridization buffer.
169. The method of claim 1, 12, 22, 32, 75, 79 or 82, wherein the at least one
of the retained
immobilized concatemer template molecules includes one or more nucleotides
having a
scissile moiety, and wherein the scissile moiety comprises uridine or 8-oxo-
7,8-
dihydroguanine, or deoxyinosine.

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170. The method of claim 169, wherein the retained immobilized concatemer
template molecule
comprises one or more uridines, and wherein the generating the abasic sites at
the uridines
comprises contacting the retained immobilized concatemer template molecule
with uracil
DNA glycosylase (UDG).
171. The method of claim 169, wherein the retained immobilized concatemer
template molecule
comprises one or more 8oxoG, and wherein the generating the abasic sites at
the 8oxoG
comprises contacting the retained immobilized concatemer template molecule
with an Fpg
enzyme (formamidopyrimidine DNA glycosylase).
172. The method of claim 169, wherein the retained immobilized concatemer
template molecule
comprises one or more deoxyinosine, and wherein the generating the abasic
sites at the
deoxyinosine comprises contacting the retained immobilized concatemer template
molecule with an AlkA glycosylase enzyme.
173. The method of claim 170, 171 or 172, further comprising generating a gap
at the abasic
sites to generate at least one gap-containing concatemer template molecule,
which
comprises: contacting the retained immobilized template molecules containing
one or more
abasic sites with an endonuclease IV, AP lyase (e.g., DNA-apurinic lyase or
DNA-
apyrimidinic lyase), F'PG glycosylase/AP lyase and/or endo VIII glycosylase/AP
lyase.
174. The method of claim 150, wherein the immobilized concatemer template
molecules
comprise 0.1 ¨ 30% uridine, and wherein the plurality of wild type sequencing
polymerases
yield an error rate of incorporating dUTP of at least 0.1X compared to an
error rate of
incorporating dTTP.
175. The method of claim 151, wherein the immobilized concatemer template
molecules
comprise 0.1 ¨ 30% uridine, and wherein the plurality of mutant sequencing
polymerases
yield an error rate of incorporating dUTP of at least 0.1X compared to an
error rate of
incorporating dTTP.

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176. The method of claim 154, wherein the inunobilized concaterner template
molecules
comprise 0.1 --- 300/o uridine, and wherein the plurality of wild type
sequencing polymerases
yield an error rate of incorporating dUTP of at least 0.1X compared to an
error rate of
incorporating dITP.
177. The method of claim 155, wherein the immobilized concatemer template
molecules
comprise 0.1 ¨ 30% uridine, and wherein the plurality of mutant sequencing
polymerases
yield an error rate of incorporating dtiTP of at least 0.1X compared to an
error rate of
incorporating dTTP.
178. The method of claim 1, 12, 22, 32, 44, 55, 65, 75, 79 or 82, wherein the
ratio of a first base
fluorescent signal of R2 (e.g., reverse sequencing) to a first base
fluorescent signal of RI
(e.g., forward sequencing) is at least 0.7 for sequencing using 1, 2, 3 or 4
dyes colors.
179. The method of claim 12, wherein the rolling circle amplification of step
(b) comprises a
plurality of compaction oligonucleotides andlor hexamine to generate
immobilized
concatemer template molecules having a more compact size andlor shape compared
to a
rolling circle amplification reaction in the absence of compaction
oligonucleotides andlor
hexarnine.
180. The method of claim 12, wherein the primer extension reaction of step (d)
comprises a
plurality of compaction oligonucleotides andlor hexamine to generate a
plurality of forward
extension strands having a more compact size and/or shape compared to a primer
extension
reaction in the absence of compaction oligonucleotides and/or hexamine.
181. The method of claim 22, wherein the roiling circle amplification of step
(a), (b) andlor (c)
comprises a plurality of compaction oligonucleotides and/or hexamine to
generate
concatemer molecules having a more compact size and/or shape compared to a
rolling
circle amplification reaction in the absence of compaction oligonucleotides
and/or
hexamine.

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182. The method of claim 22, wherein the prirner extension reaction of step
(e) comprises a
plurality of compaction oligonucieotides and/or hexarnine to generate a
plurality of forward
extension strands having a more compact size and/or shape compared to a
printer extension
reaction in the absence of compaction oligonucleotides and/or hexamine.
183. The method of claim 32, wherein the rolling circle amplification of step
(d) comprises a
plurality of compaction oligonucleotides and/or hexamine to generate
immobilized
concatemer template molecules having a more compact size and/or shape compared
to a
rolling circle amplification reaction in the absence of compaction
oligonucleotides and/or
hexamine.
184. The method of claim 32, wherein the primer extension reaction of step (f)
comprises a
plurality of compaction oligonucleotides and/or hexamine to generate a
plurality of forward
extension strands having a more compact size andlor shape compared to a primer
extension
reaction in the absence of compaction oligonucleotides andlor hexamine.
185. The method of claim 44, wherein the primer extension reaction of step (c)
comprises a
plurality of compaction oligonucleotides and/or hexamine to generate a
plurality of primer
extension products having a more compact size and/or shape compared to a
primer
extension reaction in the absence of compaction oligonucleotides and/or
hexamine, wherein
the plurality of primer extension products include a plurality of forward
extension strands,
a plurality of partially displaced extended forward sequencing strands and a
plurality of
detached extended forward sequencing primer strands.
186, The method of claim 55, wherein the rolling circle amplification of step
(b) comprises a
plurality of compaction oligonucleotides and/or hexatnine to generate
immobilized
concatemer template molecules having a more compact size and/or shape compared
to a
rolling circle amplification reaction in the absence of compaction
oligonucleotides and/or
hexamine,
187, The method of claim 55, wherein the primer extension reaction of step (d)
comprises a
plurality of compaction oligonucleotides and/or hexatnine to generate a
pturality of primer

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extension products having a more compact size and/or shape compared to a
primer
extension reaction in the absence of compaction oligonucleotides andlor
hexamine, wherein
the plurality of primer extension products include a plurality of forward
extension strands,
a plurality of partially displaced extended forward sequencing strands and a
plurality of
detached extended forward sequencing primer strands.
188. The method of claim 65, wherein the rolling circle arnplification of
steps (a), (b) andlor (c)
comprises a plurality of compaction oligonucleotides and/or hexarnine to
generate a
plurality of concatemer molecules having a rnore compact size andlor shape
cornpared to a.
rolling circle arnplification reaction in the absence of compaction
oligonucleotides andlor
hexamine.
189. The method of claim 65, wherein the primer extension reaction of step (e)
cornprises a
plurality of compaction oligonucleotides andlor hexarnine to generate a
plurality of prirner
extension products having a more compact size and/or shape compared to a
primer
extension reaction in the absence of compaction oligonucleotides andlor
hexarnine, wherein
the plurality of primer extension products include a plurality of forward
extension strands,
a plurality of partially displaced extended forward sequencing strands and a
plurality of
detached extended forward sequencing primer strands.
190, The method of claim 179, 181, 183, 186 or _188, wherein the plurality of
immobilized
concatemer template molecules or the plurality of immobilized concatemer
molecules have
FWHM (full width half maximum) of no more than about 5 um.
191. The method of claim I 80, 182 or 184, wherein the plurality of forward
extension strand
have FWI-IM (full width half maximum) of no more than about 5 gm.
192. The method of claim 185, 18'7 or I 89, wherein the plurality of primer
extension products
have FWEIM (full width half maximum) of no more than about 5 tun.
193, A method for pairwise sequencing, comprising:

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a) providing a plurality of single stranded nucleic acid concaterner
tetnplate
rnolecules inunobilized to a support;
b) sequencing the plurality of inunobilized concatemer template molecules
with a
first plurality of sequencing polymerases, a plurality of soluble forward
sequencMg primers and a first plurality of rnultivalent molecules, thereby
generating a plurality of extended forward sequencing primer strands;
c) retaining the plurality of immobilized concatemer template molecules
and.
replacing the plurality of extended forward sequencing primer strands with a.
plurality of forward extension strands that are hybridized to the retained
immobilized concatemer template molecules by conducting a primer extension
reaction;
d) removing the retained immobilized concatemer template molecules while
retaining the plurality of forward extension strands and retaining the
plurality of
immobilized surface prinlers; and
e) sequencing the plurality of retained forward extension strands with a
second
plurality of sequencing polymerases, a plurality of soluble reverse sequencing
prinlers and a second plurality of multivalent molecules,
wherein individual multivalent molecules in the first plurality of multivalent
molecules of step (b) and in the second plurality of multivalent molecules of
step (e) comprise (i) a core; and (ii) a plurality of nucleotide arms which
comprise a core attachment moiety, a spacer, a linker, and a nucleotide unit,
wherein the core is attached to the plurality of nucleotide arrns via their
core
attachment moiety, wherein the spacer is attached to the linker, wherein the
linker is attached to the nucleotide unit
19,4, The method of claim 193, wherein a nucleotide unit of an individual
multivalent molecule
of step (b) binds a first polymerase which is bound to a nucleic acid duplex
comprising an
immobilized concatemer template molecule hybridized to a forward sequencing
primer.

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195. The rnethod of claim 193, wherein a nucleotide unit of an individual
multivalent tnolecule
of step (e) binds a second polytnerase which is bound to a nucleic acid duplex
cornprising a
retained forward extension strand hybridized to a reverse sequencing primer.
196. The rnethod of claim 193, wherein the core cornprises streptavidin and
the core attachtnent
moiety cornprises biotin.
197. The rnethod of clairn 193, wherein in the spacer comprises a polyethylene
glycol (PEG)
moiety.
198. The method of claim 193, wherein the linker cornprises an aliphatic chain
having 2-6
subunits or an oligo ethylene glycol chain having 2-6 subunits.
199. The method of claim 193, wherein the plurality of nucleotide arms
attached to the core
have the sam.e type of a nucleotide unit, and wherein the types of nucleotide
unit is selected
from a group consisting of dA __ dGTP, dCTP, dTTP and dljTP.
200. The method of claim 193, wherein the first plurality of multivalent
molecules of step (b)
and the second plurality of multivalent molecules of step (e) comprise one
type of a
multivalent molecule wherein each multivalent molecule in the plurality has
the sarne type
of nucleotide unit selected from a group consisting of dA dGTP, dCTP, dTTP
and
dUTP.
201. The method of claim 193, wherein the first plurality of multivalent
molecules of step (b)
and the second plurality of multivalent molecules of step (0) comprises a
mixture of any
cotnbination of two or tnore types of multivalent molecules each type having
nucleotide
units selected from a group consisting of dATP, dGTP, dC[P, dTTP and dUTP.
202. The method of claim 193, wherein at least one multivalent molecule in the
first plurality of
multivalent rnolecules of step (b) is labeled with a fluorophore, and wherein
at least one

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multivalent molecule in the second plurality of multivalent molecules of step
(e) is labeled
with a fluorophore.
203. The method of claim 193, wherein individual multivalent molecules are
attached to a
fluorophore that corresponds to the nucleotide units that are attached to the
nucleotide arms
of a given multivalent molecule.
204. The method of claim 193, wherein the sequencing of step (b), comprises:
a) contacting the first plurality of sequencing polymerases to (i) the
plurality of
immobilized concatemer template molecules and (ii) the plurality of soluble
forward sequencing primers, wherein the contacting is conducted under a
condition suitable to form a plurality of first complexed polymerases each
comprising a first sequencing polymerase bound to a nucleic acid duplex which
comprises an irnmobilized concaterner template molecule hybridized to a
soluble
forward sequencing primer;
b) contacting the plurality of first complexed polymerases with a plurality
of
fluorophore-labeled multivalent molecules to form a plurality of binding
complexes, wherein the contacting is conducted under a condition suitable for
binding complementaiy nucleotide units of the multivalent molecules to at
least
two of the plurality of first corriplexed polyrnerases thereby forming a
plurality of
binding complexes, and the condition inhibits incorporation of the
complementary
nucleotide units into the forward sequencing primers;
c) detecting the plurality of binding complexes; and
d) identifying the nucleo-base of the complementary nucleotide units that
are bound
to the plurality of first complexed polymerases, thereby determining the
sequence
of the immobilized coneaterner template molecules.
205. The method of clairn 204, wherein individual binding complexes in the
plurality comprise a
first sequencing polymerase bound to a multivalent molecule, wherein the
binding
complexes exhibit a persistence time of greater than 0.5 seconds.

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206. The method of claim 193, further comprising: forrning at least one
avidity complex in step
(b), the method comprising:
a) binding a first soluble forward sequencing primer, a first forward
sequencing
poiymerase, and a first multivalent molecule to a first portion of a first
concatemer template molecule thereby forming a first binding complex, wherein
a
first nucleotide unit of the first multivalent molecule binds to the first
sequencing
polymerase; and
b) binding a second forward sequencing primer, a second forward sequencing
polymerase, and the first multivalent rnolecule to a second portion of the
sarne
first concatemer template rnolecule thereby forming a second binding cornplex,
wherein a second nucleotide unit of the second multivalent rnolecule binds to
the
second sequencing polymerase, and wherein the first and second binding
complexes which include the same multivalent molecule forms an avidity
complex.
207. The method of claim 193, wherein the plurality of immobilized concatemer
template
molecules comprise at least one nucleotide having a scissile moiety that can
be cleaved to
generate an abasic site in the concatemer template molecule, wherein the at
least one
nucleotide having a scissile moiety in the immobilized concatemer template
molecules
which comprises uridine, 8-oxo-7,8-dihydrogunine, or deoxyinosine.
208. The method of claim 193, wherein the plurality of immobilized concatemer
template
molecules lack a nucleotide having a scissile moiety that can be cleaved to
generate an
abasic site in the concatemer template molecule, wherein the immobilized
concatemer
template molecules lack a nucleotide having a scissile moiety which comprises
uridine, 8-
oxo-7,8-dihydrogunine, or deoxyinosine.
209. The method of claim 193, wherein individual concatemer template molecules
in the
plurality are covalently joined to the immobilized surface primer.

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210. The method of claim 193, wherein individual concaterner tetnplate
molecules in the
plurality are hybridized to the imtnobilized surface prirner.
211. The method of claitn 193, wherein individual concatemer tnolecules in the
plurality are
immobilized to a surface primer which is innnobilized to the support, wherein
the
immobilized surface primer lacks a nucleotide having a scissile moiety, and
wherein the
nucleotide having a scissile moiety comprises uridine, 8-oxo-7,8-
dihydrogunine, or
deoxyinosine.
212. A method for pairwise sequencing, comprising:
a) providing a plurality of immobilized single stranded nucleic acid
concatemer
template molecules each comprising at least one nucleotide having a scissile
moiety that can be cleaved to generate an abasic site in the concatemer
template
molecule, wherein individual concatemer template molecules in the plurality
are
immobilized to a surface primer that is immobilized to a support, wherein the
immobilized surface primer lacks a nucleotide having a scissile moiety, and
wherein the nucleotide having a scissile moiety comprises uridine;
b) sequencing the plurality of immobilized concatemer template molecules
with a
first plurality of sequencing polymerases, a plurality of soluble forward
sequencing primers and a first plurality of multivalent molecules, thereby
generating a plurality of extended forward sequencing primer strands, wherein
individual immobilized concatemer template molecules have two or more
extended forward sequencing primer strands hybridized thereon;
c) retaining the plurality of immobilized concatemer template molecules and
replacing the plurality of extended forward sequencing primer strands with a
plurality of forward extension strands that are hybridized to the retained
immobilized concatemer template tnolecules by conducting a primer extension
reaction;
d) removing the retained immobilized con.catemer template molecules by
generating
abasic sites in the itnmobilized concatemer template rnolecules at the
nucleotide(s) having the scissile tnoiety and generating gaps at the abasic
sites to

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generate a plurality of gap-containing concatemer template molecules while
retaining the plurality of forward extension strands and retaining the
plurality of
immobilized surface primers; and
e) sequencing the plurality of retained forward extension strands with
a second
plurality of sequencing polymerases, a plurality of soluble reverse sequencing
primers and a second plurality of multivalent molecules, thereby generating a
plurality of extended reverse sequencing primer strands, wherein individual
retained forward extension strands have two or more extended reverse
sequencing
primer strands hybridized thereon,
wherein individual multivalent molecules in the first plurality of
multivalent molecules of step (b) and in the second plurality of multivalent
molecules of step (e) comprise (i) a core; and (ii) a plurality of nucleotide
arms which comprise a core attachment moiety, a spacer, a linker, and a
nucleotide unit, wherein the core is attached to the plurality of nucleotide
arms via their core attachment moiety, wherein the spacer is attached to
the linker, wherein the linker is attached to the nucleotide unit.
213. The method of claim 212, wherein a nucleotide unit of an individual
multivalent molecule
of step (b) binds a first polymerase which is bound to a nucleic acid duplex
comprising an
immobilized concatemer template molecule hybridized to a forward sequencing
primer.
214. The method of claim 212, wherein a nucleotide unit of an individual
multivalent molecule
of step (e) binds a second polymerase which is bound to a nucleic acid duplex
comprising a
retained forward extension strand hybridized to a reverse sequencing primer.
215. The method of claim 212, wherein the core comprises streptavidin and the
core attachment
moiety comprises biotin.
216. The method of claim 212, wherein in the spacer comprises a polyethylene
glycol (PEG)
moiety, and wherein the linker comprises an aliphatic chain having 2-6
subunits or an oligo
ethylene glycol chain having 2-6 subunits.

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217. The method of claitn 212, wherein the plurality of nucleotide arrns
attached to the core
have the sarne type of a nucleotide unit, and wherein the types of nucleotide
unit is selected
from a group consisting of dATP, dGTP, dCIP, d'ITP and dUTP.
218. The rnethod of clairn 212, wherein the first plurality of multivalent
molecules of step (b)
and the second plurality of multivalent molecules of step (e) cornprises one
type of a
multivalent molecule wherein each multivalent molecule in the plurality has
the same type
of nucleotide unit selected frorn a group consisting of dATP, dGTP, dCTP, dTTP
and
dUTP.
219. The rnethod of clairn 212, wherein the first plurality of multivalent
molecules of step (b)
and the second plurality of multivalent molecules of step (e) cornprises a
mixture of any
combination of two or more types of multivalent molecules each type having
nucleotide
units selected from a group consisting of dATP, dGTP, dCIP, dTTP and dljTP.
220. The method of claim 212, wherein at least one multivalent molecule in the
first plurality of
multivalent molecules of step (b) is labeled with a fluorophore, and wherein
at least one
multivalent molecule in the second plurality of multivalent molecules of step
(e) is labeled
with a fluorophore.
221. The method of claim 212, wherein the generating the abasic sites at the
uridines at step (d)
comprises contacting the immobilized concatemer template molecules with uracil
DNA
glycosylase (UDG).
222. The method of clairn 212, wherein generating the plurality of gap-
containing single
stranded nucleic acid concatemer ternplate molecules of step (d) comprises
contacting the
retained immobilized template molecules containing one or more abasic sites
with art
endonuclease Iv, AP lyase (e.g., DNA-apurinic lyase or DNA-apyrimidinic
lyase), FPG-
glycosylase/AP lyase and/or endo VIII glycosylase/AP lyase.

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223. A method for pairwise sequencing, comprising:
a) providing a plurality of single stranded nucleic acid concatemer
template
molecules immobilized to a support, wherein individual concatemer template
molecules in the plurality are immobilized to a surface primer where the
surface
primer is immobilized to the support;
b) sequencing the plurality of immobilized concatemer template molecules
with a
first plurality of sequencing polymerases, a plurality of soluble forward
sequencing primers and a first plurality of multivalent molecules, thereby
generating a plurality of extended forward sequencing primer strands;
c) retaining the plurality of immobilized concatemer template molecules and
replacing the plurality of extended forward sequencing primer strands with a
plurality of forward extension strands that are hybridized to the retained
immobilized single stranded nucleic acid concatemer template molecules by
conducting a primer extension reaction;
d) removing the retaimxi immobilized concatemer template molecules while
retaining the plurality of forward extension strands and retaining the
plurality of
immobilized surface primers; and
e) sequencing the plurality of retained forward extension strands with a
second
plurality of sequencing polymerases, a plurality of soluble reverse sequencing
primers and a second plurality of multivalent molecules,
wherein the support comprises at least one hydrophilic polymer coating layer
and a
plurality of surface primers immobilized to the at least one hydrophilic
polymer coating
layer, and wherein the at least one hydrophilic polymer coating layer has a
water contact
angle of no more than 45 degrees.
224. The method of claim 223, wherein the at least one hydrophilic polymer
coating layer
comprises a molecule selected from a group consisting of polyethylene glycol
(PEG),
poly(vinyl alcohol) (PVA), poly(vinyl pyridine), poly(vinyl pyrrolidone)
(PVP),
poly(acrylic acid) (PAA), polyacrylamide, poly(N-isopropylacrylamide)
(PNIPAM),
poly(methyl methaciylate) (PMA), poly(2-hydroxylethyl methacrylate) (PHEMA),
. .

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poly(oligo(ethylene glycol) methyl ether methactylate) (POEGMA), polyglutamic
acid
(PGA), poly-glucoside, streptavidin, and dextran.
225. The method of claim 223, wherein the at least one hydrophilic polyrner
coating layer
comprises polyethylene glycol (PEG).
226. The method of claim 223, wherein the at least one hydrophilic polymer
coating layer
cornprises polyrner molecules haying a rnolecular weight of at least 1000
Daltons.
227. The rnethod of clairn 223, wherein the at least one hydrophilic polyrner
coating layer
comprises branched polymer molecules haying 4-8 branches.
228. The method of claim 223, wherein the support comprises one hydrophilic
polymer coating
layer and a plurality of surface primers at a surface density of least l000/
m2.
229. The method of clairn 223, wherein the support comprises:
a) a first coating layer comprising a first monolayer of hydrophilic
polymer
molecules tethered to the support;
b) a second coating layer comprising a second monolayer of hydrophilic
polymer
molecules tethered to the first monolayer; and
c) a third coating layer comprising a third monolayer of hydrophilic
polymer
molecules tethered to the second monolayer, and wherein the hydrophilic
polymer
molecules of the first layer, second layer or third layer comprise branched
polymer layers.
230. The method of claim 223, wherein the surface primers are immobilized to
the hydrophilic
polymer molecules of the second rnonolayer or third rnonolayer, and the
surface primers
are distributed at a plurality of depths throughout the second layer or the
third layer.

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231. The method of claim 223, wherein one or more of the at least one
hydrophilic polyrner
coating layers comprise a plurality of surface primers at a surface density of
least
1000/ m2.
232. The method of claim 223, wherein one or more of the at least one
hydrophilic polyrner
coating layers cornprise a plurality of surface primers at a surface density
of 1000 ¨ 15,000
per pill2.
233. The method of claim 223, wherein the hydrophilic polymer coating layer on
the support
exhibits a contrast-to-noise (CNR) ratio of at least 20 when a fluorescent
image of the
support is obtained by contacting the support with a fluorescently-labeled
nucleotide (Cy3-
labeled nucleotide) and acquiring a fluorescence image using an inverted
fluorescence
microscope and a camera under non-signal saturating conditions while the
support is
immersed in a buffer.
234. The method of claim 223, wherein the support comprises glass or plastic.
235. The method of claim 223, wherein the support is configured on a flowcell,
or an interior of
a capillary lumen.
236. The method of claim 223, wherein the plurality of immobilized single
stranded concatemer
template molecules comprise at least one nucleotide having a scissile moiety
that can be
cleaved to generate an abasic site in the concatemer template molecule,
wherein the at least
one nucleotide haying a scissile moiety in the immobilized concatetner
template molecules
which comprises uridine, 8-oxo-7,8-dihydrogunine, or deoxyinosine.
237. The method of claim 223, wherein the plurality of immobilized single
stranded concatemer
template tnolecules lack a nucleotide having a scissile moiety that can be
cleaved to
generate an abasic site in the concatemer template molecule,, wherein the
immobilized
concatemer template rnolecules lack a nucleotide having a scissile moiety
which cotnprises
uridine, 8-oxo-7,8-dihydrogunine, or deoxyinosine.

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238. The method of claim 223, wherein individual concatemer template molecules
in the
plurality are covalently joined to an immobilized surface primer.
239. The method of claim 223, wherein individual concatemer template molecules
in the
plurality are hybridized to an immobilized surface primer.
240. The method of claim 223, wherein the concatemer template molecules
comprise clonally
amplified nucleic acid molecules.
241. The method of claim 223, wherein the immobilized surface primer lacks a
nucleotide
having a scissile moiety, and wherein the nucleotide having a scissile moiety
comprises
uridine, 8-oxo-7,8-dihydrogunine, or deoxyinosine.
242. A method for pairwise sequencing, comprising:
a) providing a plurality of immobilized single stranded nucleic acid
concatemer
template molecules each comprising at least one nucleotide having a scissile
moiety that can be cleaved to generate an abasic site in the concatemer
template
molecule, wherein individual concatemer template molecules in the plurality
are
immobilized to a surface primer that is immobilized to a support, wherein the
immobilized surface primer lacks a nucleotide having a scissile moiety, and
wherein the nucleotide having a scissile moiety in the concatemer template
molecules comprise uridine;
b) sequencing the plurality of immobilized concatemer template molecules
with a
first plurality of sequencing polymerases, a plurality of soluble forward
sequencing primers and a first plurality of multivalent molecules, thereby
generating a plurality of extended forward sequencing primer strands, wherein
individual immobilized concatemer template molecules have two or more
extended forward sequencing primer strands hybridized thereon;
c) retaining the plurality of immobilized concatemer template molecules and
replacing the plurality of extended forward sequencing primer strands with a

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plurality of forward extension strands that are hybridized to the retained
immobilized single stranded nucleic acid concatemer template molecules by
conducting a primer extension reaction;
d) removing the retained immobilized concatemer template molecules by
generating
abasic sites in the immobilized single stranded concatemer template molecules
by
contacting the retained immobilized concatemer template molecules with uracil
DNA glycosylase (UDG), and generating gaps at the abasic sites by contacting
the abasic sites with an endonuclease IV, AP lyase (e.g., DNA-apurinic lyase
or
DNA-apyrimidinic lyase), FPG glycosylase/AP lyase and/or endo VIII
glycosylase/AP lyase to generate a plurality of gap-containing single stranded
nucleic acid concatemer template molecules while retaining the plurality of
forward extension strands and retaining the plurality of immobilized surface
primers; and
e) sequencing the plurality of retained forward exten.sion strands with a
second
plurality of sequencing polymerases, a plurality of soluble reverse sequencing
prim.ers and a secon.d plurality of m.ultivalent molecules, thereby generating
a
plurality of exten.ded reverse sequencing primer strands, wherein in.dividual
retained forward extension stran.ds have two or more extended reverse
sequencin.g
primer strands hybridized thereon,
wherein the support com.prises at least one hydrophilic polymer coating
layer and a plurality of surface primers imrnobilized to the at least one
hydrophilic polymer coating layer, and wherein the at least one
hydrophilic polymer coating layer has a water contact angle of no more
than 45 degrees.
243. The method of claim 242, wherein the at least one hydrophilic polymer
coating layer
comprises a molecule selected from a group consisting of polyethylene glycol
(PEG),
poly(vinyl alcohol) (pVA), poly(vinyl pyridine), poly(vinyl pyrrolidone)
(PVP),
poly(acrylic acid) (PAA), polyacrylamide, poly(N-isopropylacrylamide)
(PNIPAM),
poly(methyl methacrylate) (PMA), poly(2-hydroxylethyl methacrylate) (PHEMA),
. .

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poly(oligo(ethylene glycol) methyl ether rnethacrylate) (POEGMA), polyglutamic
acid
(PGA), poly-lysine, poly-glucoside, streptavidin, and dextran.
244. The method of claim 242, wherein the at least one hydrophilic polyrner
coating layer
comprises polymer molecules having a molecular weight of at least 1000
Dattons.
245. The method of claim 242, wherein the at least one hydrophilic polymer
coating layer
comprises branched polymer molecules having 4-8 branches.
246. The method of claim 242, wherein the support comprises one hydrophilic
polymer coating
layer and a plurality of surface primers at a surface density of least
1000/pm2.
247. The method of claim 242, wherein the support comprises:
a) a first coating layer comprising a first monolayer of hydrophilic
polymer
molecules tethered to the support;
b) a second coating layer comprising a second monolayer of hydrophilic
polymer
molecules tethered to the first monolayer; and
c) a third coating layer comprising a third monolayer of hydrophilic
polymer
molecules tethered to the second monolayer, and wherein the hydrophilic
polymer
molecules of the first layer, second layer or third layer comprise branched
polymer layers.
248. The method of claim 242, wherein on.e or more of the at least one
hydrophilic polytner
coating layers comprise a plurality of surface primers at a surface density of
least
1000/um2.
249. The method of claim 242, wherein the support exhibits a contrast-to-noise
(CNR) ratio of at
least 20 when a fluorescent image of the support is obtained by contacting the
support with
a fluorescently-labeled nucleotide (Cy3-labeled nucleotide) and acquiring a
fluorescence
image using an inverted fluorescence microscope and a camera under non-signal
saturating
conditions while the support is immersed in a buffer.

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250. The method of claim 242, wherein the support comprises glass or plastic.
251. The method of claim 242, wherein individual concatemer template molecules
in the
plurality are covalently joined to an immobilized surface primer or the
individual
concatemer template molecules in the plurality are hybridized to an
immobilized surface
primer.
252. A method for pairwise sequencing, comprising:
a) providing a plurality of immobilized single stranded nucleic acid
concatemer
template molecules, wherein individual concatemer template molecules in the
plurality are immobilized to a first surface primer that is immobilized to a
support,
wherein the immobilized first surface primers lack uridine, and wherein at
least
one of the immobilized concatemer template molecules in the plurality
comprises
a uridine-containing concatemer template molecule having up to 30% of
thymidines replaced with uridine;
b) sequencing the plurality of immobilized concatemer template molecules
with a
plurality of soluble forward sequencing primers and (i) a plurality of a first
sequencing polymerase and a plurality of multivalent molecules and (ii) a
plurality of a second sequencing polymerase and a plurality of nucleotide
analogs,
thereby generating a plurality of extended forward sequencing primer strands,
wherein individual immobilized concatemer template molecules have two or more
extended forward sequencing primer strands hybridized thereon;
c) removing the plurality of extended forward sequencing primer strands
while
retaining the immobilized concatemer template molecules, and conducting a
primer extension reaction with a plurality of soluble extension primers, a
plurality
of nucleotides and a plurality of primer extension polymerases, thereby
generating
a plurality of forward extension strands that are hybridized to the retained
immobilized concatemer template molecules;
d) removing the retained immobilized concatemer template molecules by
generating
abasic sites in the immobilized concatemer template molecules at the

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nucleotide(s) having the scissile moiety and generating gaps at the abasic
sites to
generate a plurality of gap-containing concatemer template molecules while
retaining the plurality of forward extension strands and retaining the
plurality of
immobilized surface primers; and
e) sequencing the plurality of retained forward extension strands with
a plurality of
soluble reverse sequencing primers and (i) a plurality of a first sequencing
polymerase and a plurality of multivalent molecules and (ii) a plurality of a
second sequencing polyrnerase and a plurality of nucleotide analogs, thereby
generating a plurality of extended reverse sequencing primer strands, wherein
individual retained forward extension strands have two or more extended
reverse
sequencing primer strands hybridized thereon,
wherein individual multivalent molecules of steps (b) and (e) comprise (1)
a core; and (2) a plurality of nucleotide arms which comprise (i) a core
attachment moiety, (ii) a spacer, (iii) a linker, and (iv) a nucleotide unit,
wherein the core is attached to the plurality of nucleotide arms via their
core attachment moiety, wherein the spacer is attached to the linker, and
wherein the linker is attached to the nucleotide unit.
253. The method of claim 252, wherein individual concatemer template molecules
in the
plurality are covalently joined to an immobilized first suiface primer.
254. The method of claim 252, wherein individual concatemer template molecules
in the
plurality are hybridized to an immobilized first surface primer.
255. The method of claim 252, wherein at least one of the plurality of
immobilized concatemer
template molecules lack a uridine.
256. The method of claim 252, wherein the immobilized concatemer template
tnolecules
comprise two or more copies of the sequence of interest, and two or more
copies of a
universal binding sequence for a soluble amplification primer, and wherein the
plurality of
. .

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soluble extension primers of step (c) comprise a plurality of soluble
amplification primers
that hybridize to the universal binding sequence for the soluble amplification
primer.
257. The method of claim 252, wherein the itnmobilized concatemer template
rnolecules
comprise two or more copies of the sequence of interest, and two or copies of
a universal
binding sequence for a soluble forward sequencing prirner, and wherein the
plurality of
soluble extension prirners of step (c) cornprise a plurality of soluble
forward sequencing
primers that hybridize to the universal binding sequence for the soluble
forward sequencing
primer.
258. The method of claim 252, wherein the plurality of first sequencing
polymerases in steps (b)
and (e) bind a concatemer template molecule, a soluble sequencing prirner and
a
multivalent molecule to form a binding complex which exhibits a persistence
tirne of
greater than 0.5 seconds, wherein the nucleic acid template molecule comprises
an
immobilized concatemer template molecule or a retained forward extension
strand, and
wherein the soluble sequencing primer comprises a soluble forward sequencing
primer or a
soluble reverse sequencing primer.
259. The method of claim 252, wherein the core of the multivalent molecules
comprises
streptavidin and the core attachment moiety comprise biotin.
260. The method of claim 252, wherein in the spacer of the multivalent
molecules comprises a
polyethylene glycol (PEG) moiety,
261. The method of claim 252, wherein the linker of the multivalent molecules
comprises an
aliphatic chain having 2-6 subunits or an oligo ethylene glycol chain having 2-
6 subunits.
262. The method of clairn 252, wherein the plurality of nucleotide arms
attached to the core of
the multivalent molecules have the satne type of a nucleotide unit, and
wherein the types of
nucleotide unit is selected frotn a group consisting of dATP, dGTP, dCIP, drIP
and
dUTP.

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263. The method of claim 252, wherein the plurality of multivalent molecules
of step (b) and (e)
comprise one type of a multivalent molecule wherein each multivalent molecule
in the
plurality has the sarne type of nucleotide unit selected from a group
consisting of dATP,
dGTP, dCTP, &FIT and dUTP.
264. The method of claim 252, wherein the plurality of multivalent molecules
of step (b) and (0
comprise a rnixture of any cornbination of tWO Or rnore types of multivalent
molecules each
type having nucleotide units selected from a group consisting of dATP, dGTP,
dCTP,
dTIP and dt_TIT.
265. The rnethod of clairn 252, wherein the plurality of multivalent
rnolecules of step (b) and (e)
comprise fluorophore-labeled multivalent rnolecules.
266. The method of claim 252, wherein the nucleotide analog of step (b)
comprises a removable
chain terminating moiety at the 3' sugar group, and wherein the nucleotide
analog of step
(e) comprises a removable chain terminating moiety at the 3' sugar group,
wherein the
removable chain terminating moiety comprises an alkyl group, alkenyl group,
alkynyl
group, allyl group, aryl group, henzyl group, azide group, azido group, 0-
azidornethyl
group, amine group, amide group, keto group, isocyanate group, phosphate
group, thio
group, disulfide group, carbonate group, urea group, or silyl group, and
wherein the
removable chain terrninating moiety is cleavable with a chemical compound to
generate an
extendible 3'011. moiety on the sugar group.
267, The method of claim 252, wherein the plurality of nucleotide analogs of
steps (h) and (c)
comprise one type of nucleotide selected from a group consisting of dATP,
dGTP, dCTP,
dTTP and dU FP.
268, The method of claim 252, wherein the plurality of nucleotide analogs of
steps (h) and (e)
comprise a mixture of any combination of two or rnore types of nucleotides
selected from a
group consisting of dATP, dGTP, dCTP, dTTP and dUTP.

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269. The method of claim 252, wherein the plurality of nucleotide analogs of
steps (b) and (e)
cotnprise at least one fluorophore-iabeled nucleotide analog.
270. The method of claim 252, wherein the sequencing of step (b), comprises:
a) contacting the plurality of a first sequencing polymerase to (i) a
plurality of
nucleic acid ternplate molecules which comprise a plurality of irnrnobilized
concaterner template molecules or a plurality of retained forward extension
strands, and (ii) the plurality of soluble sequencing prirners which cornprise
a
plurality of soluble forward sequencing primers or a plurality of soluble
reverse
sequencing primers, wherein the contacting is conducted under a condition
suitable to form a plurality of first complexed polymerases each cornprising a
first
sequencing polymerase bound to a nucleic acid duplex wherein the nucleic acid
duplex comprises a nucleic acid template molecule hybridized to a soluble
sequencing primer;
b) contacting the plurality of first complexed polymerases with a plurality
of
fluorophore-labeled multivalent molecules to form a plurality of multivalent-
complexed polymerases, wherein the contacting is conducted under a condition
suitable for binding complementary nucleotide units of the multivalent
molecules
to at least two of the plurality of first complexed polymerases thereby
forming a
plurality of multivalent-complexed polymerases, and the condition inhibits
incorporation of the complementary nucleotide units into the hybridized
sequencing primers of the plurality of multivalent-complexed polymerases;
c) detecting the plurality of multivalent-complexed polymerases; and
d) identifying the nucleo-base of the complementary nucleotide units that
are bound
to the plurality of first complexed polymerases in the plurality of
multivalent-
complexed polymerases, thereby determining the sequence of the nucleic acid
template.
271. The method of claim 270, further cornprising:

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e) dissociating the plurality of multivalent-complexed polymerases by
removing the
plurality of first sequencing polyrnerases and their bound multivalent
molecules,
and retaining the plurality of nucleic acid duplexes;
0 contacting the plurality of the retained nucleic acid duplexes of
step (e) with a
plurality of a second sequencing polymerase, wherein the contacting is
conducted
under a condition suitable for binding the plurality of second sequencing
polyrnerases to the plurality of the retained nucleic acid duplexes, thereby
forrning
a plurality of second complexed polyrnerases each comprising a second
sequencing polymerase bound to a retained nucleic acid duplex;
g) contacting the plurality of second cornplexed polymerases with a
plurality of
nucleotides, wherein the contacting is conducted under a condition suitable
for
binding complementary nucleotides to at least two of the second complexed
polyrnerases of step (0 thereby forming a plurality of nucleotide-complexed
polymerases and the condition is suitable for promoting incorporation of the
bound complementary nucleotides into the hybridized sequencing primers of the
nucleotide-complexed polymerases thereby generating a plurality of extended
sequencing primer strands wherein the plurality of extended sequencing primer
strands comprise a plurality of extended forward sequencing primer strands or
a.
plurality of extended reverse sequencing primer strands.
272. The method of claim 252, wherein the generating the abasic sites at the
uridines of the
immobilized concatetner template molecules of step (d) comprises contacting
the
immobilized concaterner template molecule with uracil DNA glycosylase (UDG).
273, The method of claim 252, wherein generating the plurality of gap-
containing concatemer
template molecules of step (d) comprises contacting the retained immobilized
template
molecules containing one or inore abasic sites with an endonuclease IV, AP
lyase (e.g.,
DNA-apurinic lyase or DNA-apyrimidinic lyase), FPG glycosylase/AP lyase and/or
endo
VIII glycosylase/AP lyase.

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274. The method of claim 252, wherein the support comprises at least one
hydrophilic polymer
coating layer and a plurality of surface primers immobilized to the at least
one hydrophilic
polymer coating layer, and wherein the at least one hydrophilic polymer
coating layer has a
water contact angle of no more than 45 degrees.
275. The method of claim 274, wherein the at least one hydrophilic polymer
coating layer
comprises a molecule selected from a group consisting of polyethylene glycol
(PEG),
poly(vinyl alcohol) (PVA), poly(vinyl pyridine), poly(vinyl pyrrolidone)
(PVP),
poly(acrylic acid) (PAA), polyacrylamide, poly(N-isopropylacrylamide)
(PNIPAM),
poly(methyl methacrylate) (PMA), poly(2-hydroxylethyl methacrylate) (PHEMA),
poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA), polyglutamic
acid
(PGA), poly-lysine, poly-glucoside, streptavidin, and dextran.
276. The method of claim 275, wherein the at least on.e hydrophilic polymer
coating layer
comprises polym.er molecules havin.g a rnolecular weight of at least 1000
Daltons.
277. The m.ethod of claim 275, wherein the at least one hydrophilic polymer
coating layer
comprises branched polymer molecules having 4-8 branches.
278. The rnethod of clairn 275, wherein the support cornprises:
a) a first coating layer cornprisin.g a first mon.olayer of hydrophilic
polymer
molecules tethered to the support;
b) a second coating layer comprising a second monolayer of hydrophilic
polymer
molecules tethered to the first monolayer; and
c) a third coating layer comprising a third monolayer of hydrophilic
polymer
molecules tethered to the second monolayer, and wherein the hydrophilic
polymer
molecules of the first layer, second layer or third layer comprise branched
polymer layers.
. . .

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279. The method of claim 278, wherein the surface primers are immobilized to
the hydrophilic
polymer molecules of the second monolayer or third monolayer, and the surface
primers
are distributed at a plurality of depths throughout the second layer or the
third layer.
280. The method of claim 275, wherein one or more of the at least one
hydrophilic polymer
coating layers comprise a plurality of surface primers at a surface density of
least
100011.1m2.
281. A method for pairwise sequencing, comprising:
a) providing a support having a plurality of surface primers immobilized
thereon
wherein the surface primers comprise a 3' extendible end and lack a nucleotide
having a scissile moiety that can be cleaved to generate an abasic site in the
surface primers;
b) generating a plurality of immobilized single stranded nucleic acid
concatemer
template molecules by hybridizing a plurality of single-stranded circular
nucleic
acid library molecules to the plurality of immobilized surface primers and
conducting a rolling circle amplification reaction with a plurality of a
strand
displacing polymerase and a plurality of nucleotides, thereby generating a
plurality of immobilized single stranded nucleic acid concatemer ternplate
molecules, wherein individual single stranded nucleic acid concaterner
template
molecules are covalently joined to an immobilized surface primer, and wherein
the plurality of nucleotides lacks a nucleotide having a scissile moiety that
can be
cleaved to generate an abasic site in the concatemer template molecules;
c) sequencing the plurality of immobilized concatemer template molecules
with a
plurality of soluble forward sequencing primers, thereby generating a
plurality of
extended forward sequencing primer strands, wherein individual immobilized
concatemer template molecules have two or more extended forward sequencing
primer strands hybridized thereon;
d) retaining the plurality of immobilized concatemer template molecules and
replacing the plurality of extended forward sequencing primer strands with a
plurality of forward extension strands by conducting a primer extension
reaction

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with a plurality of soluble amplification primers and a plurality of strand-
displacing polymerases to generate a plurality of forward extension strands
and a
plurality of partially displaced forward extension strands wherein the forward
extension strands and the partially displaced forward extension strands are
hybridized to the immobilized concatemer template molecules to form a
plurality
of immobilized amplicons; and
e) sequencing the plurality of immobilized partially displaced forward
extension
strands with a plurality of soluble reverse sequencing primers, thereby
generating
a plurality of extended reverse sequencing primer strands, wherein individual
immobilized partially displaced forward extension strands have two or more
extended reverse sequencing primer strands hybridized thereon, and wherein the
immobilized partially displaced forward extension strands remain hybridized to
the retained immobilized concatemer template molecules during sequencing of
step (e).
282. The method of claim 281, wherein each of the single stranded circular
nucleic acid library
molecules in the plurality comprises a sequence of interest, and wherein the
individual
library molecules further comprise any one or any combination of two or more
of
a universal binding sequence for a soluble forward sequencing primer,
(ii) a universal binding sequence for a soluble reverse sequencing primer,
(iii) a universal binding sequence for an immobilized surface primer,
(iv) a universal binding sequence for a first soluble amplification primer,
(v) a universal binding sequence for a second soluble amplification primer,
(vi) a universal binding sequence for a soluble compaction oligonucleotide,
(vii) a sample barcode sequence and/or
(viii) a unique molecular index sequence.
283. The method of claim 281, wherein individual immobilized single stranded
nucleic acid
concatemer template molecules generated by the rolling circle amplification
reaction
comprise two or more copies of a sequence of interest, wherein the individual
irninobilized

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concatemer template molecules further comprise any one or any combination of
two or
more of
two or more copies of a universal binding sequence for a soluble forward
sequencing primer,
(ii) two or more copies of a universal binding sequence for a soluble
reverse
sequencing primer,
(iii) two or more copies of a universal binding sequence for an immobilized
surface primer,
(iv) two or more copies of a universal binding sequence for a first soluble
amplification primer,
(v) two or more copies of a universal binding sequence for a second soluble
amplification primer,
(vi) two or more copies of a universal binding sequence for a soluble
compaction oligonucleotide,
(vii) two or more copies of a sample barcode sequence and/or
(viii) two or more copies of a unique molecular index sequence.
284. The method of claim 281, wherein replacing the plurality of extended
forward sequencing
primer strands of step (d) comprises:
(i) removing the plurality of extended forward sequencing primer strands
while retaining the immobilized concatemer template molecules; and
(ii) contacting the plurality of retained immobilized concatemer molecules
with the plurality of soluble amplification primers, a plurality of
nucleotides and a plurality of strand displacing polymerases, under a
condition suitable to hybridize the plurality of soluble ainplification
primers to the plurality of retained immobilized concatemer template
molecules and suitable for conducting polymerase-catalyzed strand
displacing reactions thereby generating a plurality of forward extension
strands and a plurality of partially displaced extended forward sequencing
strands that are hybridized to the immobilized concatemer template
molecules to form a plurality of immobilized amplicons.
. .

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285. The method of claim 284, wherein the strand displacing polymerase
comprises phi29 DNA
polymerase, large fragment of Bst DNA polymerase, large fragment of Bsu DNA
polymerase (exo-), Bca DNA polymerase (exo-), Klenow fragment of E. coli DNA
polymerase, T5 polymerase, M-MuLV reverse transcriptase, HIV viral reverse
transcriptase, Deep Vent DNA polymerase and KOD DNA polymerase.
286. The method of claim 281 wherein the forward sequencing of step (c)
comprises:
a) contacting a plurality of sequencing polymerases and a plurality of the
soluble
forward sequencing primers to a plurality of immobilized concatemer template
molecules, wherein the contacting is conducted under a condition suitable to
form
a plurality of complexed polymerases each comprising a sequencing polymerase
bound to a nucleic acid duplex wherein the nucleic acid duplex comprises an
immobilized concatemer template molecule hybridized to a soluble forward
sequencing primer;
b) contacting the plurality of complexed sequencing polymerases with a
plurality of
nucleotides under a condition suitable for binding at least one nucleotide to
a
complexed sequencing polymerase, wherein the plurality of nucleotides
comprises at least one nucleotide analog labeled with a fluorophore and having
a
removable chain terminating moiety at the sugar 3' position;
c) incorporating at least one nucleotide into the 3' end of the hybridized
forward
sequencing primers thereby generating a plurality of nascent extended forward
sequencing primers; and
d) detecting the incorporated nucleotide and identifying the nucleo-base of
the
incorporated nucleotide.
287. The method of claim 286, wherein at least one of the nucleotides in the
plurality of
nucleotides of step (b) comprises a removable chain terminating moiety
attached to the 3'
carbon position of the sugar group, wherein the removable chain terminating
moiety
comprises an alkyl group, alkenyl group, alkynyl group, allyl group, aryl
group, benzyl
group, azide group, azido group, O-azidomethyl group, amine group, amide
group, keto

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group, isocyanate group, phosphate group, thio group, disulfide group,
carbonate group,
urea group, or silyl group, and wherein the removable chain terminating moiety
is
cleavable with a chemical compound to generate an extendible 3'0H moiety on
the sugar
group.
288. The method of claim 281, wherein the reverse sequencing of step (e)
comprises:
a) contacting a plurality of sequencing polymerases and a plurality of the
soluble
reverse sequencing primers to the plurality of the immobilized partially
displaced
forward extension strands, wherein the contacting is conducted under a
condition
suitable to form a plurality of complexed polymerases each comprising a
sequencing polymerase bound to a nucleic acid duplex wherein the nucleic acid
duplex comprises a soluble reverse sequencing primer hybridized to an
immobilized partially displaced forward extension strand;
b) contacting the plurality of complexed sequencing polymerases with a
plurality of
nucleotides under a condition suitable for binding at least one nucleotide to
a
complexed sequencing polymerase, wherein the plurality of nucleotides
comprises at least one nucleotide analog labeled with a fluorophore and having
a
removable chain terminating moiety at the sugar 3' position;
c) incorporating at least one nucleotide into the 3' end of the hybridized
reverse
sequencing primers thereby generating a plurality of nascent extended reverse
sequencing primers; and
d) detecting the incorporated nucleotide and identifying the nucleo-base of
the
incorporated nucleotide.
289. The method of claim 288, wherein at least one of the nucleotides in the
plurality of
nucleotides of step (h) comprises a removable chain terminating moiety
attached to the 3'
carbon position of the sugar group, wherein the removable chain terminating
moiety
comprises an alkyl group, alkenyl group, alkynyl group, allyl group, aryl
group, benzyl
group, azide group, azido group, 0-azidomethyl group, amine group, amide
group, keto
group, isocyanate group, phosphate group, thio group, disulfide group,
carbonate group,
urea group, or silyl group, and wherein the removable chain terminating moiety
is
. .

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cleavable with a chemical compound to generate an extendible 3'0H moiety on
the sugar
group.
290. The method of claim 281, wherein the forward sequencing of step (b) and
the reverse
sequencing of step (e) comprise:
a) contacting a plurality of a first sequencing polymerase and a plurality
of soluble
sequencing primers to a plurality of nucleic acid template molecules, wherein
the
contacting is conducted under a condition suitable to form a plurality of
first
complexed polymerases each comprising a first sequencing polyrnerase bound to
a nucleic acid duplex which comprises the nucleic acid template molecule
hybridized to the soluble sequencing primer, wherein (1) the plurality of
nucleic
acid template molecules comprise a plurality of the immobilized concatemer
template molecules and the plurality of sequencing primers comprise a
plurality
of the soluble forward sequencing primers, or wherein (2) the plurality of
nucleic
acid template molecules comprise a plurality of immobilized partially
displaced
forward extension strands and the plurality of sequencing primers comprise a
plurality of the soluble reverse sequencing primers;
b) contacting the plurality of first complexed polymerases with a plurality
of
detectably labeled multivalent molecules to form a plurality of multivalent-
complexed polymerases, wherein individual multivalent molecules in the
plurality
of multivalent molecules comprise a core attached to multiple nucleotide arms
and each nucleotide arm is attached to a nucleotide unit, wherein the
contacting is
performed under a condition suitable for binding complementary nucleotide
units
of the multivalent molecules to at least two of the plurality of first
complexed
polymerases thereby forming a plurality of multivalent-complexed polymerases,
and the condition inhibits incorporation of the complementary nucleotide units
into the sequencing primers of the plurality of multivalent-complexed
polyrnerases;
c) detecting the plurality of multivalent-complexed polymerases; and
d) identifying the nucleo-base of the complementary nucleotide units that
are bound
to the plurality of first complexed polymerases in the plurality of
multivalent-
...

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complexed polymerases, thereby determining the sequence of the nucleic acid
template.
291. The method of claim 290, further comprising:
e) dissociating the plurality of multivalent-complexed polymerases and
removing the
plurality of first sequencing polymerases and their bound multivalent
molecules,
and retaining the plurality of nucleic acid duplexes;
0 contacting the plurality of the retained nucleic acid duplexes of
step (e) with a
plurality of second sequencing polyrnerases, wherein the contacting is
conducted
under a condition suitable for binding the plurality of second sequencing
polyrnerases to the plurality of the retained nucleic acid duplexes, thereby
forrning
a plurality of second complexed polyrnerases;
g) contacting the plurality of second complexed polymerases with a
plurality of
nucleotides under a condition suitable for binding complementary nucleotides
from the plurality of nucleotides to at least two of the second complexed
polymerases of step (f) thereby forming a plurality of nucleotide-complexed
polymerases and the condition is suitable for promoting incorporation of the
bound complementary nucleotides into the sequencing primers of the nucleotide-
complexed polymerases.
292. The method of claim 291, further comprising:
h) detecting the complementary nucleotides which are incorporated into
the
sequencing primers of the nucleotide-complexed polymerases.
293. The method of claim 291, further comprising:
h) detecting the complementary nucleotides which are incorporated into
the
sequencing primers of the nucleotide-complexed polymerases; and
i) identifying the nucleo-bases of the complementary nucleotides which
are
incorporated into the sequencing printers of the nucleotide-complexed
polytnerases.

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294. The method of claim 290, further comprising: forming at least one avidity
complex in step
(b), the method comprising:
a) binding a first sequencing primer, a first sequencing polymerase, and a
first
multivalent molecule to a first portion of a nucleic acid template molecule
thereby
forming a first binding complex, wherein a first nucleotide unit of the first
multivalent molecule binds to the first sequencing polymerase; and
b) binding a second sequencing primer, a second sequencing polymerase, and
the
first multivalent molecule to a second portion of the same nucleic acid
template
molecule thereby forming a second binding complex, wherein a second nucleotide
unit of the second multivalent molecule binds to the second sequencing
polymerase, wherein the first and second binding complexes which include the
same multivalent molecule forms an avidity complex.
295. The method of claim 294, wherein (i) the first sequencing primer
comprises a soluble
forward sequencing prirner and the nucleic acid template molecule comprises an
immobilized concatemer template molecule, (ii) the second sequencing primer
comprises a
soluble forward sequencing prirner and the nucleic acid template molecule
comprises the
same immobilized concaterner template molecule, and (iii) the first and second
sequencing
primers have the same sequence.
296. The method of claim 294, wherein (i) the first sequencing primer
cornprises a soluble
reverse sequencing primer and the nucleic acid template molecule comprises an
immobilized partially displaced forward extension strand, (ii) the second
sequencing primer
comprises a soluble reverse sequencing primer and the nucleic acid template
molecule
comprises the same immobilized partially displaced forward extension strand,
and (iii) the
first and second sequencing primers have the same sequence.
297. The method of claim 290, wherein individual multivalent molecules in the
plurality of
multivalent molecules comprises (a) a core; and (b) a plurality of nucleotide
arms which
comprise (i) a core attachment moiety, (ii) a spacer comprising a PEG moiety,
(iii) a linker,
and (iv) a nucleotide unit, wherein the core is attached to the plurality of
nucleotide arrns

WO 2022/266470 PCT/US2022/034038
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via their core attachment moiety, wherein the spacer is attached to the
linker, and wherein
the linker is attached to the nucleotide unit.
298. The method of claim 297, wherein
(i) the core comprises streptavidin and the core attachment moiety
comprises
biotin;
(ii) the linker comprises an aliphatic chain having 2-6 subunits or an oligo
ethylene glycol chain having 2-6 subunits; and
(iii) the nucleotide unit comprises an arornatic base, a five carbon sugar and
1-10
phosphate groups.
299. The method of claim 297, wherein the plurality of nucleotide arms
attached to the core
have the same type of a nucleotide unit, and wherein the types of nucleotide
unit is selected
from a group consisting of dA FP, dGTP, dCTP, dTTP and dljTP.
300. The method of claim 297, wherein the plurality of multivalent molecules
comprise one type
of a multivalent molecule wherein each multivalent molecule in the plurality
has the sarne
type of nucleotide unit selected from a group consisting of dATP, dG FP,
dCTP, dTTP and
dUTP,
301, The method of claim 297, wherein the plurality of multivalent molecules
comprise a
mixture of any combination of two or more types of multivalent molecules each
type
having nucleotide units selected from a group consisting of dATP, dGTP, dCTP,
dTTP and
dUTP.
302. The method of claim 290, wherein the plurality of detectably labeled
multivalent molecules
comprise fluorescently-labeled multivalent molecules,
303. The method of claim 281, wherein the support comprises at least one
hydrophilic polymer
coating layer and a plurality of surface primers immobilized to the at least
one hydrophilic

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polymer coating layer, and wherein the at least one hydrophilic polymer
coating layer has a
water contact angle of no more than 45 degrees.
304. The method of claim 303, wherein the at least one hydrophilic polymer
coating layer
comprises a molecule selected from a group consisting of polyethylene glycol
(PEG),
poly(vinyi alcohol) (PVA), poly(vinyl pyridine), poly(vinyl pyrrolidone)
(PVP),
poly(acrylic acid) (PAA), polyacrylamide, poly(N-isopropylacrylamide)
(PNIPAM),
poly(methylmethacrylate) (MIA), poly(2-hydroxylethyl methacrylate) (PHEMA),
poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA), polyglutamic
acid.
(PGA), poly-lysine, poly-glucoside, streptavidin, and dextran.
305. The method of claim 303, wherein the at least one hydrophilic polymer
coating layer
cornprises polymer molecules having a molecular weight of at least 1000
Daltons.
306, The method of claim 303, wherein the at least one of the hydrophilic
polymer coating layer
comprises branched hydrophilic polymer molecules haying 4-8 branches.
307. The method of claim 303, wherein the at least one hydrophilic polymer
coating layer
comprises a plurality of surface primers at a surface density of least
1000/um2,
308, The method of claim 303, wherein the support comprises:
a) a first coating layer comprising a first monolayer of hydrophilic
polymer
molecules tethered to the support;
1)) a second coating layer comprising a second rnonolayer of
hydrophilic polyrner
molecules tethered to the first monolayer; and
c) a third coating layer comprising a third monolayer of hydrophilic
polymer
molecules tethered to the second monolayer, and wherein the hydrophilic
polymer
molecules of the first. layer, second layer or third layer comprise branched
polymer layers,
wherein the surface primers are immobilized to the hydrophilic polymer
rnolecules of the second monolayer or third monolayer, and the surface primers

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are distributed at a plurality of depths throughout the second layer or the
third
layer.
309. The method of claim 303, wherein the hydrophilic coating layer on the
support exhibits a
contrast-to-noise (CNR) ratio of at least 20 when a fluorescent image of the
support is
obtained by contacting the support with a fluorescently-labeled nucleotide
(Cy3-labeled
nucleotide) and acquiring a fluorescence image using an inverted fluorescence
microscope
and a camera under non-signal saturating conditions while the support is
irnrnersed in a
buffer.

Description

DEMANDE OU BREVET VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.
CECI EST LE TOME 1 DE 3
CONTENANT LES PAGES 1 A 166
NOTE : Pour les tomes additionels, veuillez contacter le Bureau canadien des
brevets
JUMBO APPLICATIONS/PATENTS
THIS SECTION OF THE APPLICATION/PATENT CONTAINS MORE THAN ONE
VOLUME
THIS IS VOLUME 1 OF 3
CONTAINING PAGES 1 TO 166
NOTE: For additional volumes, please contact the Canadian Patent Office
NOM DU FICHIER / FILE NAME:
NOTE POUR LE TOME / VOLUME NOTE:

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COMPOSITIONS AND METHODS FOR PAIRWISE SEQUENCING
100011 Throughout this application various publications, patents, and/or
patent applications are
referenced. The disclosures of the publications, patents andlor patent
applications are hereby
incorporated by reference in their entireties into this application in order
to more fully describe
the state of the art to which this disclosure pertains.
CROSS-REFERENCE TO RELATED APPLICATIONS
[0002] This application claims the benefit of U.S, Provisional Patent
Application No.
63/212,059, filed June 17, 2021; U.S. Patent Application No. 17/377,284, filed
July 15, 2021,
now issued as U.S. Patent No. 11,220,707; U.S. Patent Application No.
17/377,285, filed July
15, 2021, now issued as U.S. Patent No. 11,236,388; U.S. Patent Application
No, 17/377,279,
filed July 15, 2021; U.S. Patent Application No. 17/377,283, filed July 15,
2021; U.S. Patent
Application No. 17/521,239, filed November 8,2021; U.S. Patent Application No.
17/554,396,
filed December 17, 2021, the contents of each of which are incorporated by
reference herein in
their entireties.
TECHNICAL HELD
[00031 The present disclosure provides compositions and methods that employ
the
compositions for conducting pairwise sequencing and for generating concatemer
template
molecules for pairwise sequencing.
BACKGROUND OF THE INVENTION
100041 Polynucleotide sequencing technology has applications in biomedical
research and
healthcare settings. Improved methods of polynucleotide require enhanced
surface chemistry, on-
support polynucleotide amplification, and base calling. Currently, these
elements produce
barriers in existing sequencing technology that result in limits in throughput
and poor signal-to-
noise ratio, and ultimately to increased costs associated with polynucleotide
sequencing.
[00051 There exists a need for new polynucleotide sequencing methods with
improved
surface chemistry, on-support amplification, and base calling. The present
disclosure provides
methods and compositions to improve sequencing of polynucleotides,

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SUMMARY OF THE INVENTION
[0006i The present disclosure provides a method for pairwise sequencing,
comprising: a)
providing a plurality of immobilized single stranded nucleic acid concatemer
template molecules
each comprising at least one nucleotide having a scissile moiety that can be
cleaved to generate
an abasic site in the concatemer template molecule, wherein individual
concatemer template
molecules in the plurality are immobilized to a first surface primer that is
immobilized to a
support, and wherein the immobilized first surface primer lacks a nucleotide
having a scissile
moiety; b) sequencing the plurality of immobilized concatemer template
molecules thereby
generating a plurality of extended forward sequencing primer strands, wherein
individual
immobilized concatemer template molecules have two or more extended forward
sequencing
primer strands hybridized thereon; c) retaining the plurality of immobilized
concatemer template
molecules and replacing the plurality of extended forward sequencing primer
strands with a
plurality of forward extension strands that are hybridized to the retained
immobilized single
stranded nucleic acid concatemer template molecules by conducting a primer
extension reaction;
d) removing the retained immobilized concatemer template molecules by
generating abasic sites
in the immobilized single stranded concatemer template molecules at the
nucleotide(s) having
the scissile moiety and generating gaps at the abasic sites to generate a
plurality of gap-
containing single stranded nucleic acid concatemer template molecules while
retaining the
plurality of forward extension strands and retaining the plurality of
immobilized surface primers;
and e) sequencing the plurality of retained forward extension strands thereby
generating a
plurality of extended reverse sequencing primer strands, wherein individual
retained forward
extension strands have two or more extended reverse sequencing primer strands
hybridized
thereon.
[0007] in some embodiments, the individual concatemer template molecules in
the plurality
are covalently joined to an immobilized first surface primer. In some
embodiments, the
individual concatemer template molecules in the plurality are hybridized to an
immobilized first
surface primer. In some embodiments, the individual immobilized concatemer
template
molecules in the plurality comprise two or more copies of a sequence of
interest, and wherein the
individual immobilized concatemer template molecules further comprise any one
or any
combination of two or more of (i) two or more copies of a universal binding
sequence for a

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soluble forward sequencing primer, (ii) two or more copies of a universal
binding sequence for a
soluble reverse sequencing primer, (iii) two or more copies of a universal
binding sequence for
an immobilized first surface primer, (iv) two or more copies of a universal
binding sequence for
an immobilized second surface primer, (v) two or more copies of a universal
binding sequence
for a first soluble amplification primer, (vi) two or more copies of a
universal binding sequence
for a second soluble amplification primer, (vii) two or more copies of a
universal binding
sequence for a soluble compaction oligonucleotide, (viii) two or more copies
of a sample
barcode sequence and/or (ix) two or more copies of a unique molecular index
sequence.
100081 In some embodiments, the sequencing of step (b) comprises
hybridizing a plurality of
soluble forward sequencing primers to the plurality of immobilized concatemer
template
molecules and conducting one or more sequencing reactions. In some
embodiments, the
sequencing of step (e) comprises hybridizing a plurality of soluble reverse
sequencing primers to
the plurality of immobilized concatemer template molecules and conducting one
or more
sequencing reactions.
[00091 In some embodiments, the support further comprises a plurality of
immobilized
second surface primers that lack a nucleotide having a scissile moiety. In
some embodiments, at
least one copy of the universal binding sequence for the immobilized second
surface primer in
the individual concatemer template molecules is hybridized to an immobilized
second surface
primer. In some embodiments, the plurality of immobilized second surface
primers have 3' OH
extendible ends. In some embodiments, the plurality of immobilized second
surface primers have
3' non-extendible ends. In some embodiments, the 3' non-extendible end
comprises a phosphate
group, a dideoxycytidine group, an inverted di, or an amino group.
[00101 The present disclosure also provides a method for pairwise
sequencing, comprising:
a) providing a support having a plurality of a first surface primer
immobilized thereon wherein
each of the first surface primers have a 3' extendible end and lack a
nucleotide having a scissile
moiety; b) generating a plurality of immobilized single stranded nucleic acid
concatemer
template molecules by hybridizing a plurality of single-stranded circular
nucleic acid library
molecules to the plurality of immobilized first surface primers and conducting
a rolling circle
amplification reaction with a plurality of a strand displacing polymerase, and
a plurality of
nucleotides which include dATP, dCTP, cliff?, drIP and a nucleotide having a
scissile moiety
that can be cleaved to generate an abasic site, thereby generating a plurality
of immobilized

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single stranded nucleic acid concatemer template molecules having at least one
nucleotide with a
scissile moiety, wherein individual single stranded nucleic acid concatemer
template molecules
are covalently joined to an immobilized first surface primer, c) sequencing
the plurality of
immobilized concatemer template molecules thereby generating a plurality of
extended forward
sequencing primer strands, wherein individual immobilized concatemer template
molecules have
two or more extended forward sequencing primer strands hybridized thereon; d)
retaining the
plurality of immobilized concatemer template molecules and replacing the
plurality of extended
forward sequencing primer strands with a plurality of forward extension
strands that are
hybridized to the retained immobilized single stranded nucleic acid concatemer
template
molecules by conducting a primer extension reaction; e) removing the retained
immobilized
concatemer template molecules by generating abasic sites in the immobilized
single stranded
concatemer template molecules at the nucleotide(s) having the scissile moiety
and generating
gaps at the abasic sites to generate a plurality of gap-containing single
stranded nucleic acid
concatemer template molecules while retaining the plurality of forward
extension strands and
retaining the plurality of immobilized first surface primers; and 0 sequencing
the plurality of
retained forward extension strands thereby generating a plurality of extended
reverse sequencing
primer strands, wherein individual forward extension strands have two or more
extended reverse
sequencing primer strands hybridized thereon.
[00111 In some embodiments, each of the single stranded circular nucleic
acid library
molecules in the plurality comprises a sequence of interest and wherein the
individual library
molecules further comprise any one or any combination of two or more of (i) a
universal binding
sequence for a soluble forward sequencing primer, (ii) a universal binding
sequence for a soluble
reverse sequencing primer, (iii) a universal binding sequence for an
immobilized first surface
primer, (iv) a universal binding sequence for an immobilized second surface
primer, (v) a
universal binding sequence for a first soluble amplification primer, (vi) a
universal binding
sequence for a second soluble amplification primer, (vii) a universal binding
sequence for a
soluble compaction oligonucleotide, (viii) a sample barcode sequence and/or
(ix) a unique
molecular index sequence.
[00121 in some embodiments, the individual immobilized single stranded
nucleic acid
concatemer template molecules generated by the rolling circle amplification
reaction comprise
two or more copies of a sequence of interest and wherein the individual
immobilized concatemer

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template molecules further comprise any one or any combination of two or more
of (i) two or
more copies of a universal binding sequence for a soluble forward sequencing
primer, (ii) two or
more copies of a universal binding sequence for a soluble reverse sequencing
primer, (iii) two or
more copies of a universal binding sequence for an immobilized first surface
primer, (iv) two or
more copies of a universal binding sequence for an immobilized second surface
primer, (v) two
or more copies of a universal binding sequence for a first soluble
amplification primer, (vi) two
or more copies of a universal bindinQ sequence for a second soluble
amplification primer, (vii)
two or more copies of a universal binding sequence for a soluble compaction
oliganucleotide,
(viii) two or more copies of a sample barcode sequence and/or (ix) two or more
copies of a
unique molecular index sequence.
[00131 In some embodiments, the sequencing of step (c) comprises
hybridizing a plurality of
soluble forward sequencing primers to the plurality of immobilized concatemer
template
molecules and conducting one or more sequencing reactions. In some
embodiments, the
sequencing of step (0 comprises hybridizing a plurality of soluble reverse
sequencing primers to
the plurality of immobilized concatemer template molecules and conducting one
or more
sequencing reactions,
[0014] In some embodiments, the support further comprises a plurality of
immobilized
second surface primers that lack a nucleotide having a scissile moiety. In
some embodiments, at
least one copy of the universal binding sequence for the immobilized second
surface primer in
the individual concatemer template molecules is hybridized to an immobilized
second surface
primer. In some embodiments, the plurality of immobilized second surface
primers have 3' OH
extendible ends. In some embodiments, the plurality of immobilized second
surface primers have
3' non-extendible ends. In some embodiments, the 3' non-extendible end
comprises a phosphate
group, a dideoxycytidine group, an inverted dT, or an amino group.
[0015] The present disclosure also provides a method for pairwi.se
sequencing, comprising:
a) contacting in-solution a plurality of single-stranded circular nucleic acid
library molecules to a
plurality of first soluble amplification primers, a plurality of a strand
displacing polymerase, and
a plurality of nucleotides which include dATP, dCTP, dGTP, dTTP and a
nucleotide having a
scissile moiety that can be cleaved to generate an abasic site, under a
condition suitable to form a
plurality of library-primer duplexes and suitable for conducting a rolling
circle amplification
reaction, thereby generating a plurality of single stranded nucleic acid
concatemers having at

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least one nucleotide with a scissile moiety; b) distributing the rolling
circle amplification reaction
onto a support having a plurality of the first surface primers immobilized
thereon, under a
condition suitable for hybridizing one or more portions of individual single
stranded concatemers
to one or more immobilized first surface primers, wherein each of the first
surface primers lack a
nucleotide having a scissile moiety; c) continuing the rolling circle
amplification reaction on the
support to generate a plurality of immobilized concatemer template molecules;
d) sequencing the
plurality of immobilized concatemer template molecules thereby generating a
plurality of
extended forward sequencing primer strands wherein individual immobilized
concatemer
template molecules have two or more extended forward sequencing primer strands
hybridized
thereon; e) retaining the plurality of immobilized concatemer template
molecules and replacing
the plurality of extended forward sequencing primer strands with a plurality
of forward extension
strands that are hybridized to the retained immobilized single stranded
nucleic acid concatemer
template molecules by conducting a primer extension reaction; f) removing the
retained
immobilized concatemer template molecules by generating abasic sites in the
immobilized single
stranded concatemer template molecules at the nucleotide(s) having the
scissile moiety and
generating gaps at the abasic sites to generate a plurality of gap-containing
single stranded
nucleic acid concatemer template molecules while retaining the plurality of
forward extension
strands and retaining the plurality of immobilized first surface primers; and
g) sequencing the
plurality of retained forward extension strands thereby generating a plurality
of extended reverse
sequencing primer strands wherein individual forward extension strands have
two or more
extended reverse sequencing primer strands hybridized thereon.
[0016] In some embodiments, each of the single stranded circular nucleic
acid library
molecules in the plurality comprises a sequence of interest and wherein the
individual library
molecules further comprise any one or any combination of two or more of (i) a
universal binding
sequence for a soluble forward sequencing primer, (ii) a universal binding
sequence for a soluble
reverse sequencing primer, (iii) a universal binding sequence for an
immobilized first surface
primer, (iv) a universal binding sequence for an immobilized second surface
primer, (v) a
universal binding sequence for a first soluble amplification primer, (vi) a
universal binding
sequence for a second soluble amplification primer, (vii) a universal binding
sequence for a
soluble compaction oligonucleotide, (viii) a sample barcode sequence and/or
(ix) a unique
molecular index sequence.

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[0017] In some embodiments, individual immobilized single stranded nucleic
acid
concatemer template molecules generated by the rolling circle amplification
reaction comprise
two or more copies of a sequence of interest and wherein the individual
immobilized concatemer
template molecules further comprise any one or any combination of two or more
of (i) two or
more copies of a universal binding sequence for a soluble forward sequencing
primer, (ii) two or
more copies of a universal binding sequence for a soluble reverse sequencing
primer, (iii) two or
more copies of a universal binding sequence for an immobilized first surface
primer, (iv) two or
more copies of a universal binding sequence for an immobilized second surface
primer, (v) two
or more copies of a universal binding sequence for a first soluble
amplification primer, (vi) two
or more copies of a universal binding sequence for a second soluble
amplification primer, (vii)
two or more copies of a universal binding sequence for a soluble compaction
oligonucleotide,
(viii) two or more copies of a sample barcode sequence and/or (ix) two or more
copies of a
unique molecular index sequence.
[0018] In some embodiments, the sequencing of step (d) comprises
hybridizing a plurality of
soluble forward sequencing primers to the plurality of immobilized concatemer
template
molecules and conducting one or more sequencing reactions. In some
embodiments, the
sequencing of step (g) comprises hybridizing a plurality of soluble reverse
sequencing primers to
the plurality of immobilized concatemer template molecules and conducting one
or more
sequencing reactions.
[0019] In some embodiments, the support further comprises a plurality of
immobilized
second surface primers that lack a nucleotide having a scissile moiety, In
some embodiments, at
least one copy of the universal binding sequence for the immobilized second
surface primer in
the individual concatemer template molecules is hybridized to an immobilized
second surface
primer. In some embodiments, the plurality of immobilized second surface
primers have 3' OH
extendible ends. In some embodiments, the plurality of immobilized second
surface primers have
3' non-extendible ends, In some embodiments, the 3' non-extendible end
comprises a phosphate
group, a dideoxycytidine group, an inverted dT, or an amino group.
[0020] The present disclosure provides a method for pairwise sequencing,
comprising: a)
providing a support having a plurality of a first surface primer immobilized
thereon wherein
individual first surface primers in the plurality comprise a first portion
(SP1-A) and a second
portion (S.P1-B), and the individual first surface primers comprising a 3'
extendible end and

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lacking a nucleotide having a scissile moiety that can be cleaved to generate
an abasic site in the
first surface primer; b) contacting the plurality of the first surface primers
with a plurality of
single stranded linear nucleic acid library molecules, each library molecule
having at the 5' end a
universal sequence (SPI-A') that binds the first portion of the immobilized
first surface primer,
and the library molecules each having at the 3' end a universal sequence (SP1-
B') that binds the
second portion of the immobilized first surface primer, wherein the contacting
is conducted
under a condition suitable for hybridizing individual library molecules to an
immobilized first
surface primer to form a circularized library molecule having a gap or nick
between the 5' and 3'
ends of the circularized library molecule; c) enzymatically closing the gap or
nick thereby
forming individual covalently closed circular molecules that are hybridized to
an immobilized
first surface primer; d) generating a plurality of immobilized single stranded
nucleic acid
concatemer template molecules by conducting a rolling circle amplification
reaction with a
plurality of a strand displacing polymerase, and a plurality of nucleotides
which include dATP,
dCTP, dGTP, dTTP and a nucleotide having a scissile moiety that can be cleaved
to generate an
abasic site, thereby generating a plurality of immobilized single stranded
nucleic acid
concatemer template molecules having at least one nucleotide with a scissile
moiety, wherein
individual single stranded nucleic acid concatemer template molecules are
covalently joined to
an immobilized first surface primer; e) sequencing the plurality of
immobilized concatemer
template molecules thereby generating a plurality of extended forward
sequencing primer
strands, wherein individual immobilized concatemer template molecules have two
or more
extended forward sequencing primer strands hybridized thereon; 0 retaining the
plurality of
immobilized concatemer template molecules and replacing the plurality of
extended forward
sequencing primer strands with a plurality of forward extension strands that
are hybridized to the
retained immobilized single stranded nucleic acid concatemer template
molecules by conducting
a primer extension reaction; g) removing the retained immobilized concatemer
template
molecules by generating abasic sites in the immobilized single stranded
concatemer template
molecules at the nucleotide(s) having the scissile moiety and generating gaps
at the abasic sites
to generate a plurality of gap-containing single stranded nucleic acid
concatemer template
molecules while retaining the plurality of forward extension strands and
retaining the plurality of
immobilized first surface primers; and h) sequencing the plurality of retained
forward extension
strands thereby generating a plurality of extended reverse sequencing primer
strands, wherein

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individual forward extension strands have two or more extended reverse
sequencing primer
strands hybridized thereon.
[0021] In some embodiments, individual linear library molecules in the
plurality comprise a
sequence of interest and the library molecules further comprise any one or any
combination of
two or more of: (i) a universal binding sequence for a soluble forward
sequencing primer, (ii) a
universal binding sequence for a soluble reverse sequencing primer, (iii) a
universal binding
sequence for a first portion of an immobilized first surface primer (SPI-A),
(iv) a universal
binding sequence for a second portion of an immobilized first surface primer
(SPI-B), (v) a
universal binding sequence for an immobilized second surface primer, (vi) a
universal binding
sequence for a first soluble amplification primer, (vii) a universal binding
sequence for a second
soluble amplification primer, (viii) a universal binding sequence for a
soluble compaction
oligonucleotide, (ix) a sample barcode sequence and/or (x) a unique molecular
index sequence.
[0022] In some embodiments, individual immobilized single stranded nucleic
acid
concatemer template molecules generated by the rolling circle amplification
reaction comprise
two or more copies of a sequence of interest and wherein the individual
immobilized concatemer
template molecules further comprise any one or any combination of two or more
of (i) two or
more copies of a universal binding sequence for a soluble forward sequencing
primer, (ii) two or
more copies of a universal binding sequence for a soluble reverse sequencing
primer, (iii) two or
more copies of a universal binding sequence for a first portion of an
immobilized first surface
primer (SPI -A), (iv) two or more copies of a universal binding sequence for a
second portion of
an immobilized first surface primer (SPI-B), (v) two or more copies of a
universal binding
sequence for an immobilized second surface primer, (vi) two or more copies of
a universal
binding sequence for a first soluble amplification primer, (vii) two or more
copies of a universal
binding sequence for a second soluble amplification primer, (viii) two or more
copies of a
universal binding sequence for a soluble compaction oligonucleotide, (ix) two
or more copies of
a sample barcode sequence and/or (x) two or more copies of a unique molecular
index sequence.
[0023] In some embodiments, the sequencing of step (e) comprises
hybridizing a plurality of
soluble forward sequencing primers to the plurality of immobilized concatemer
template
molecules and conducting one or more sequencing reactions. En som.e
embodiments, the
sequencing of step (h) comprises hybridizing a plurality of soluble reverse
sequencing primers to

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the plurality of immobilized concatemer template molecules and conducting one
or more
sequencing reactions.
[00241 In some embodiments, the support further comprises a plurality of
immobilized
second surface primers that lack a nucleotide having a scissile moiety. In
some embodiments, at
least one copy of the universal binding sequence for the immobilized second
surface primer in
the individual concatemer template molecules is hybridized to an immobilized
second surface
primer. In some embodiments, the plurality of immobilized second surface
primers have 3' OH
extendible ends. In some embodiments, the plurality of immobilized second
surface primers have
3' non-extendible ends. In some embodiments, the 3' non-extendible end
comprises a phosphate
group, a dideoxycytidine group, an inverted dl, or an amino group.
10025] In some embodiments, the closing the gap in the circularized library
molecule
comprises conducting a polymerase-catalyzed gap fill-in reaction using the
immobilized first
surface primer as a template molecule, and ligating the nick to form a
covalently closed circular
molecule, wherein individual covalently closed circular molecules are
hybridized to an
immobilized first surface primer. In some embodiments, the closing the nick in
the circularized
library molecule comprises conducting a ligation reaction to form a covalently
closed circular
molecule, and wherein individual covalently closed circular molecules are
hybridized to an
immobilized first surface primer.
[00261 The present disclosure provides a method for pairwise sequencing,
comprising: a)
providing a plurality of immobilized single stranded nucleic acid concatemer
template molecules
each lacking a scissile moiety that can be cleaved to generate an abasic site
in the concatemer
template molecule, wherein individual concatemer template molecules in the
plurality are
immobilized to a first surface primer that is immobilized to a support, and
wherein the
immobilized first surface primer lacks a nucleotide having a scissile moiety;
b) sequencing the
plurality of immobilized concatemer template molecules thereby generating a
plurality of
extended forward sequencing primer strands, wherein individual immobilized
concatemer
template molecules have two or more extended forward sequencing primer strands
hybridized
thereon; c) retaining the plurality of immobilized concatemer template
molecules and replacing
the plurality of extended forward sequencing primer strands with a plurality
of forward extension
strands by conducting a primer extension reaction with a plurality of soluble
amplification
primers and a plurality of strand-displacing polymerases to generate a
plurality of forward

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extension strands and a plurality of partially displaced forward extension
strands that are
hybridized to the immobilized concatemer template molecules to form a
plurality of immobilized
amplicons, and the primer extension reaction generates a plurality of detached
forward extension
strands (e.g., that are not hybridized to the immobilized concatemer template
molecules); and d)
sequencing the plurality of immobilized partially displaced forward extension
strands thereby
generating a first plurality of extended reverse sequencing primer strands,
and sequencing the
plurality of immobilized detached forward extension strands thereby generating
a second
plurality of extended reverse sequencing primer strands, wherein individual
immobilized
partially displaced forward extension strands have two or more extended
reverse sequencing
primer strands hybridized thereon, and wherein in individual immobilized
detached forward
extension strands have two or more extended reverse sequencing primer strands
hybridized
thereon.
10027] In some embodiments, individual concatemer template molecules in the
plurality are
covalently joined to an immobilized first surface primer. In some embodiments,
individual
concatemer template molecules in the plurality are hybridized to an
immobilized first surface
primer. In some embodiments, individual immobilized concatemer template
molecules in the
plurality comprise two or more copies of a sequence of interest, and wherein
the individual
immobilized concatemer template molecules further comprise any one or any
combination of
two or more of (i) two or more copies of a universal binding sequence for a
soluble forward
sequencing primer, (ii) two or more copies of a universal binding sequence for
a soluble reverse
sequencing primer, (iii) two or more copies of a universal binding sequence
for an immobilized
first surface primer, (iv) two or more copies of a universal binding sequence
for an immobilized
second surface primer, (v) two or more copies of a universal binding sequence
for a first soluble
amplification primer, (vi) two or more copies of a universal binding sequence
for a second
soluble amplification primer, (vii) two or more copies of a universal binding
sequence for a
soluble compaction oligonucleotide, (viii) two or more copies of a sample
barcode sequence
and/or (ix) two or more copies of a unique molecular index sequence.
[0028] In some embodiments, the sequencing of step (b) comprises
hybridizing a plurality of
soluble forward sequencing primers to the plurality of immobilized concatemer
template
molecules and conducting one or more sequencing reactions. In some
embodiments, the
sequencing of step (d) comprises hybridizing a plurality of soluble reverse
sequencing primers to

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the plurality of immobilized partially displaced forward extension strands and
the plurality of
immobilized detached extended forward sequencing primer strands, and
conducting one or more
sequencing reactions.
[0029] In some embodiments, the support further comprises a plurality of
immobilized
second surface primers that lack a nucleotide having a scissile moiety. In
some embodiments, at
least one copy of the universal binding sequence for the immobilized second
surface primer in
the individual concatemer template molecules is hybridized to an immobilized
second surface
primer. In some embodiments, the plurality of immobilized second surface
primers have 3' OH
extendible ends. In some embodiments, the plurality of immobilized second
surface primers have
3' non-extendible ends. In some embodiments, the 3' non-extendible end
comprises a phosphate
group, a dideoxycytidine group, an inverted dl, or an amino group.
[0030] The present disclosure also provides a method for pairwise
sequencing, comprising:
a) providing a support having a plurality of a first surface primer
immobilized thereon wherein
each of the first surface primers have a 3' extendible end and lack a
nucleotide having a scissile
moiety; b) generating a plurality of immobilized single stranded nucleic acid
concatemer
template molecules by hybridizing a plurality of single-stranded circular
nucleic acid library
molecules to the plurality of immobilized first surface primers and conducting
a rolling circle
amplification reaction with a plurality of a strand displacing polymerase, and
a plurality of
nucleotides which lack a nucleotide having a scissile moiety that can be
cleaved to generate an
abasic site, thereby generating a plurality of immobilized single stranded
nucleic acid
concatemer template molecules, wherein individual single stranded nucleic acid
concatemer
template molecules are covalently joined to an immobilized first surface
primer; c) sequencing
the plurality of immobilized concatemer template molecules thereby generating
a plurality of
extended forward sequencing primer strands, wherein individual immobilized
concatemer
template molecules have two or more extended forward sequencing primer strands
hybridized
thereon; d) retaining the plurality of immobilized concatemer template
molecules and replacing
the plurality of extended forward sequencing primer strands with a plurality
of forward extension
strands by conducting a primer extension reaction with a plurality of soluble
amplification
primers and a plurality of strand-displacing polymerases to generate a
plurality of forward
extension strands and a plurality of partially displaced forward extension
strands that are
hybridized to the immobilized concatemer template molecules to form a
plurality of immobilized

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amplicons, and the primer extension reaction generates a plurality of detached
forward extension
strands (e.g., that are not hybridized to the immobilized concatemer template
molecules); and e)
sequencing the plurality of immobilized partially displaced forward extension
strands thereby
generating a first plurality of extended reverse sequencing primer strands,
and sequencing the
plurality of immobilized detached forward extension strands thereby generating
a second
plurality of extended reverse sequencing primer strands, wherein individual
immobilized
partially displaced forward extension strands have two or more extended
reverse sequencing
primer strands hybridized thereon, and wherein in individual immobilized
detached forward
extension strands have two or more extended reverse sequencing primer strands
hybridized
thereon.
100311 In some embodiments, each of the single stranded circular nucleic
acid library
molecules in the plurality comprises a sequence of interest, and wherein the
individual library
molecules further comprise any one or any combination of two or more of (i) a
universal binding
sequence for a soluble forward sequencing primer, (ii) a universal binding
sequence for a soluble
reverse sequencing primer, (iii) a universal binding sequence for an
immobilized first surface
primer, (iv) a universal binding sequence for an immobilized second surface
primer, (v) a
universal binding sequence for a first soluble amplification primer, (vi) a
universal binding
sequence for a second soluble amplification primer, (vii) a universal binding
sequence for a
soluble compaction oligonucleotide, (viii) a sample barcode sequence and/or
(ix) a unique
molecular index sequence.
[00321 In some embodiments, individual immobilized single stranded nucleic
acid
concatemer template molecules generated by the rolling circle amplification
reaction comprise
two or more copies of a sequence of interest, wherein the individual
immobilized concatemer
template molecules further comprise any one or any combination of two or more
of (i) two or
more copies of a universal binding sequence for a soluble forward sequencing
primer, (ii) two or
more copies of a universal binding sequence for a soluble reverse sequencing
primer, (iii) two or
more copies of a universal binding sequence for an immobilized first surface
primer, (iv) two or
more copies of a universal binding sequence for an immobilized second surface
primer, (v) two
or more copies of a universal binding sequence for a first soluble
amplification primer, (vi) two
or more copies of a universal binding sequence for a second soluble
amplification primer, (vii)
two or more copies of a universal binding sequence for a soluble compaction
oligonucleotide,

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Orli. two or more copies of a sample barcode sequence and/or (ix) two or more
copies of a
unique molecular index sequence.
[00331 In some embodiments, the sequencing of step (c) comprises
hybridizing a plurality of
soluble forward sequencing primers to the plurality of immobilized concatemer
template
molecules and conducting one or more sequencing reactions. In some
embodiments, the
sequencing of step (e) comprises hybridizing a plurality of soluble reverse
sequencing primers to
the plurality of immobilized partially displaced forward extension strands and
the plurality of
immobilized detached extended forward sequencing primer strands, and
conducting one or more
sequencing reactions.
100341 In some embodiments, the support further comprises a plurality of
immobilized
second surface primers that lack a nucleotide having a scissile moiety. In
some embodiments, the
at least one copy of the universal binding sequence for the immobilized second
surface primer in
the individual concatemer template molecules is hybridized to an immobilized
second surface
primer. In some embodiments, the plurality of immobilized second surface
primers have 3' OH
extendible ends. In some embodiments, the plurality of immobilized second
surface primers have
3' non-extendible ends. In some embodiments, the 3' non-extendible end
comprises a phosphate
group, a dideoxycytidine group, an inverted dT, or an amino group.
[00351 The present disclosure also provides a method for pairwise
sequencing, comprising:
a) contacting in-solution a plurality of single-stranded circular nucleic acid
library molecules to a
plurality of first soluble amplification primers, a plurality of a strand
displacing polymerase, and
a plurality of nucleotides which lacks a nucleotide having a scissile moiety
that can be cleaved to
generate an abasic site, under a condition suitable to form a plurality of
library-primer duplexes
and suitable for conducting a rolling circle amplification reaction, thereby
generating a plurality
of single stranded nucleic acid concatemers; b) distributing the rolling
circle amplification
reaction onto a support having a plurality of the first surface primers
immobilized thereon, under
a condition suitable for hybridizing one or more portions of individual single
stranded
concatemers to one or more immobilized first surface primers, wherein each of
the first surface
primers lack a nucleotide having a scissile moiety; c) continuing the rolling
circle amplification
reaction on the support to generate a plurality of immobilized concatemer
template molecules; d)
sequencing the plurality of immobilized concatemer template molecules thereby
generating a
plurality of extended forward sequencing primer strands wherein individual
immobilized

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concatemer template molecules have two or more extended forward sequencing
primer strands
hybridized thereon; e) retaining the plurality of immobilized concatemer
template molecules and
replacing the plurality of extended forward sequencing primer strands with a
plurality of forward
extension strands by conducting a primer extension reaction with a plurality
of a second soluble
amplification primer and a plurality of strand-displacing polymerases to
generate a plurality of
forward extension strands and a plurality of partially displaced forward
extension strands that are
hybridized to the immobilized concatemer template molecules to form a
plurality of immobilized
amplicons, and the primer extension reaction generates a plurality of detached
forward extension
strands (e.g., that are not hybridized to the immobilized concatemer template
molecules); and 0
sequencing the plurality of immobilized partially displaced forward extension
strands thereby
generating a first plurality of extended reverse sequencing primer strands,
and sequencing the
plurality of immobilized detached forward extension strands thereby generating
a second
plurality of extended reverse sequencing primer strands, wherein individual
immobilized
partially displaced forward extension strands have two or more extended
reverse sequencing
primer strands hybridized thereon, and wherein in individual immobilized
detached forward
extension strands have two or more extended reverse sequencing primer strands
hybridized
thereon.
[0036] In some embodiments, each of the single stranded circular nucleic
acid library
molecules in the plurality comprises a sequence of interest, and wherein the
individual library
molecules further comprise any one or any combination of two or more of (i) a
universal binding
sequence for a soluble forward sequencing primer, (ii) a universal binding
sequence for a
soluble reverse sequencing primer, (iii) a universal binding sequence for an
immobilized first
surface primer, (iv) a universal binding sequence for an immobilized second
surface primer, (v) a
universal binding sequence for a first soluble amplification primer, (vi) a
universal binding
sequence for a second soluble amplification primer, (vii) a universal binding
sequence for a
soluble compaction oligonucleotide, (viii) a sample barcode sequence and/or
(ix) a unique
molecular index sequence.
[0037] In some embodiments, individual immobilized single stranded nucleic
acid
concatemer template molecules generated by the rolling circle amplification
reaction comprise
two or more copies of a sequence of interest, and wherein the individual
immobilized
concatemer template molecules further comprise any one or any combination of
two or more of

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16
(i) two or more copies of a universal binding sequence for a soluble forward
sequencing primer,
(ii) two or more copies of a universal binding sequence for a soluble reverse
sequencing primer,
(iii) two or more copies of a universal binding sequence for an immobilized
first surface primer,
(iv) two or more copies of a universal binding sequence for an immobilized
second surface
primer, (v) two or more copies of a universal binding sequence for a first
soluble amplification
primer, (vi) two or more copies of a universal binding sequence for a second
soluble
amplification primer, (vii) two or more copies of a universal binding sequence
for a soluble
compaction oligonucleotide, (viii) two or more copies of a sample barcode
sequence and/or (ix)
two or more copies of a unique molecular index sequence.
100381 In some embodiments, the sequencing of step (d) comprises
hybridizing a plurality of
soluble forward sequencing primers to the plurality of immobilized concatemer
template
molecules and conducting one or more sequencing reactions. In some
embodiments, the
sequencing of step (f) comprises hybridizing a plurality of soluble reverse
sequencing primers to
the plurality of immobilized partially displaced forward extension strands and
the plurality of
immobilized detached extended forward sequencing primer strands, and
conducting one or more
sequencing reactions.
[00391 In some embodiments, the support further comprises a plurality of
immobilized
second surface primers that lack a nucleotide having a scissile moiety. In
some embodiments, at
least one copy of the universal binding sequence for the immobilized second
surface primer in
the individual concatemer template molecules is hybridized to an immobilized
second surface
primer. In some embodiments, the plurality of immobilized second surface
primers have 3' OH
extendible ends. In some embodiments, the plurality of immobilized second
surface primers have
3' non-extendible ends. In some embodiments, the 3' non-extendible end
comprises a phosphate
group, a dideoxycytidine group, an inverted di, or an amino group.
[00401 The present disclosure also provides a method for pairwise
sequencing, comprising:
a) providing a plurality of immobilized single stranded nucleic acid
concatemer template
molecules each comprising at least one nucleotide having a scissile moiety
that can be cleaved to
generate an abasic site in the concatemer template molecule, wherein
individual concatemer
template molecules in the plurality are immobilized to a first surface primer
that is immobilized
to a support, wherein the immobilized first surface primers include a
nucleotide having a scissile
moiety, wherein the support further comprises a plurality of immobilized
second surface primers

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17
which lack a nucleotide having a scissile moiety and have an extendible
terminal 3'0H group,
and wherein the immobilized concatemer template molecule comprises two or more
copies of a
universal binding sequence for an immobilized second surface primer (wherein
the support
comprises an excess of immobilized first and second surface primers compared
to the number of
immobilized concatemer template molecules); b) sequencing the plurality of
immobilized
concatemer template molecules with a plurality of soluble forward sequencing
primers thereby
generating a plurality of extended forward sequencing primer strands, wherein
individual
immobilized concatemer template molecules have two or more extended forward
sequencing
primer strands hybridized thereon; c) removing the extended forward sequencing
primer strands
and retaining the immobilized concatemer template molecules; d) generating a
first plurality of
immobilized forward extension strands by hybridizing at least one portion of
individual
immobilized concatemer template molecules to a second surface primer and
conducting a primer
extension reaction from the second surface primers that are hybridized to a
portion of the
immobilized concatemer template molecule to generate a plurality of forward
extension strands
having a sequence that is complementary to at least a portion of the
immobilized concatemer
template molecules and are covalently joined to an immobilized second surface
primer; e)
contacting the plurality of immobilized concatemer template molecules and the
plurality of
immobilized forward extension strands with a relaxing solution which comprises
at least one
chaotropic agent; 0 dissociating the at least one portion of the immobilized
concatemer template
molecules from the immobilized second surface primers and retaining the
immobilized forward
extension strands, and re-hybridizing at least one portion of the immobilized
concatemer
template molecules to one of the immobilized second surface primers that are
not covalently
joined to a forward extension strand, wherein the dissociating and re-
associating comprises a
temperature ramp-up, a temperature plateau, and temperature ramp-down, and
washing the
relaxing solution from the support; g) contacting the re-hybridized
immobilized concatemer
template molecules with an amplification solution and conducting a primer
extension reaction
from the second surface primers that are re-hybridized to a portion of the
immobilized
concatemer template molecules to generate a plurality of newly synthesized
forward extension
strands having a sequence that is complementary to at least a portion of the
immobilized
concatemer template molecules and are covalently joined to an immobilized
second surface
primer; h) repeating steps (e) ¨ (g) at least once; i) removing the retained
immobilized

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18
concatemer template molecules by generating abasic sites in the immobilized
single stranded
concatemer template molecules and the immobilized first surface primers at the
nucleotide(s)
having the scissile moiety and generating gaps at the abasic sites thereby
generating a plurality of
gap-containing nucleic acid molecules while retaining the plurality of
immobilized forward
extension strands and retaining the plurality of immobilized second surface
primers; and j)
sequencing the plurality of retained immobilized forward extension strands
with a plurality of
soluble reverse sequencing primers thereby generating a plurality of extended
reverse sequencing
primer strands.
[0041] In some embodiments, individual concatemer template molecules in the
plurality are
covalently joined to an immobilized first surface primer. In some embodiments,
individual
concatemer template molecules in the plurality are hybridized to an
immobilized first surface
primer. In some embodiments, individual immobilized concatemer template
molecules in the
plurality comprise two or more copies of a sequence of interest, and wherein
the individual
immobilized concatemer template molecules further comprise any one or any
combination of
two or more of (i) two or more copies of a universal binding sequence for a
soluble forward
sequencing primer, (ii) two or more copies of a universal binding sequence for
a soluble reverse
sequencing primer, (iii) two or more copies of a universal binding sequence
for an immobilized
first surface primer, (iv) two or more copies of a universal binding sequence
for an immobilized
second surface primer, (v) two or more copies of a universal binding sequence
for a first soluble
amplification primer, (vi) two or more copies of a universal binding sequence
for a second
soluble amplification primer, (vii) two or more copies of a universal binding
sequence for a
soluble compaction oligonucleotide, (viii) two or more copies of a sample
barcode sequence
and/or (ix) two or more copies of a unique molecular index sequence.
[0042] The present disclosure also provides a method for pairwise
sequencing, comprising:
a) providing a support having a plurality of first and second surface primers
immobilized
thereon, wherein the first surface primers have a scissile moiety that can be
cleaved to generate
an abasic site, and wherein the second surface primers lack a nucleotide
having a scissile moiety
and the second surface primers have an extendible terminal 3'01I group; b)
generating a plurality
of immobilized single stranded nucleic acid concatemer template molecules by
hybridizing a
plurality of single-stranded circular nucleic acid library molecules to the
plurality of immobilized
first surface primers and conducting a rolling circle amplification reaction
with a plurality of a

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19
strand displacing polymerase, and a plurality of nucleotides which include
dATP, dCTP, dG'FP,
d'ITP and a plurality of nucleotides having a scissile moiety that can be
cleaved to generate an
abasic site, thereby generating a plurality of immobilized single stranded
nucleic acid
concatemer template molecules having at least one nucleotide with a scissile
moiety, wherein
individual single stranded nucleic acid concatemer template molecules are
covalently joined to
an immobilized first surface primer; c) sequencing the plurality of
immobilized concatemer
template molecules with a plurality of soluble forward sequencing primers
thereby generating a
plurality of extended forward sequencing primer strands, wherein individual
immobilized
concatemer template molecules have two or more extended forward sequencing
primer strands
hybridized thereon; d) removing the extended forward sequencing primer strands
and retaining
the immobilized concatemer template molecules; e) generating a first plurality
of immobilized
forward extension strands by hybridizing at least one portion of individual
immobilized
concatemer template molecules to a second surface primer and conducting a
primer extension
reaction from the second surface primers that are hybridized to a portion of
the immobilized
concatemer template molecule to generate a plurality of forward extension
strands having a
sequence that is complementary to at least a portion of the immobilized
concatemer template
molecules and are covalently joined to an immobilized second surface primer; 0
contacting the
plurality of immobilized concatemer template molecules and the plurality of
immobilized
forward extension strands with a relaxing solution which comprises at least
one chaotropic agent;
g) dissociating the at least one portion of the immobilized concatemer
template molecules from
the immobilized second surface primers and retaining the immobilized forward
extension
strands, and re-hybridizing at least one portion of the immobilized concatemer
template
molecules to one of the immobilized second surface primers that are not
covalently joined to a
forward extension strand, wherein the dissociating and re-associating
comprises a temperature
ramp-up, a temperature plateau, and temperature ramp-down, and washing the
relaxing solution
from the support; h) contacting the re-hybridized immobilized concatemer
template molecules
with an amplification solution and conducting a primer extension reaction from
the second
surface primers that are re-hybridized to a portion of the immobilized
concatemer template
molecules to generate a plurality of newly synthesized forward extension
strands having a
sequence that is complementary to at least a portion of the immobilized
concatemer template
molecules and are covalently joined to an immobilized second surface primer;
i) repeating steps

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(f) (h) at least once; j) removing the retained immobilized concatemer
template molecules by
generating abasic sites in the immobilized single stranded concatemer template
molecules and
the immobilized first surface primers at the nucleotide(s) having the scissile
moiety and
generating gaps at the abasic sites to generate a plurality of gap-containing
nucleic acid
molecules while retaining the plurality of immobilized forward extension
strands and retaining
the plurality of immobilized second surface primers; and k) sequencing the
plurality of retained
immobilized forward extension strands with a plurality of soluble reverse
sequencing primers
thereby generating a plurality of extended reverse sequencing primer strands.
[00431 In some embodiments, each of the single stranded circular nucleic
acid library
molecules in the plurality comprises a sequence of interest and wherein the
individual library
molecules further comprise any one or any combination of two or more of (i) a
universal binding
sequence for a soluble forward sequencing primer, (ii) a universal binding
sequence for a soluble
reverse sequencing primer, (iii) a universal binding sequence for an
immobilized first surface
primer, (iv) a universal binding sequence for an immobilized second surface
primer, (v) a
universal binding sequence for a first soluble amplification primer, (vi) a
universal binding
sequence for a second soluble amplification primer, (vii) a universal binding
sequence for a
soluble compaction oligonucleotide, (viii) a sample barcode sequence and/or
(ix) a unique
molecular index sequence.
[00441 In some embodiments, individual immobilized concatemer template
molecules in the
plurality comprise two or more copies of a sequence of interest, and wherein
the individual
immobilized concatemer template molecules further comprise any one or any
combination of
two or more of (i) two or more copies of a universal binding sequence for a
soluble forward
sequencing primer, (ii) two or more copies of a universal binding sequence for
a soluble reverse
sequencing primer, (iii) two or more copies of a universal binding sequence
for an immobilized
first surface primer, (iv) two or more copies of a universal binding sequence
for an immobilized
second surface primer, (v) two or more copies of a universal binding sequence
for a first soluble
amplification primer, (vi) two or more copies of a universal binding sequence
for a second
soluble amplification primer, (vii) two or more copies of a universal binding
sequence for a
soluble compaction oligonucleotide, (viii) two or more copies of a sample
barcode sequence
and/or (ix) two or more copies of a unique molecular index sequence.

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21
100451 The
present disclosure also provides a method for pairwise sequencing, comprising:
a) contacting in-solution a plurality of single-stranded circular nucleic acid
library molecules to a
plurality of first soluble amplification primers, a plurality of a strand
displacing polymerase, and
a plurality of nucleotides which include dATP, dCIP, dGTP, dilIP and a
plurality of nucleotides
having a scissile moiety that can be cleaved to generate an abasic site, under
a condition suitable
to form a plurality of library-primer duplexes and suitable for conducting a
rolling circle
amplification reaction, thereby generating a plurality of single stranded
nucleic acid concatemers
having at least one nucleotide with a scissile moiety; b) distributing the
rolling circle
amplification reaction onto a support having a plurality of the first surface
primers immobilized
thereon, under a condition suitable for hybridizing one or more portions of
individual single
stranded concatemers to one or more immobilized first surface primers, wherein
the immobilized
first surface primers include a nucleotide having a scissile moiety, wherein
the support further
comprises a plurality of immobilized second surface primers which lack a
nucleotide having a
scissile moiety and have an extendible terminal 3'0H group; c) continuing the
rolling circle
amplification reaction on the support in the presence of a plurality of
nucleotides which include a
plurality of nucleotides having a scissile moiety to generate a plurality of
immobilized
concatemer template molecules; d) sequencing the plurality of immobilized
concatemer template
molecules with a plurality of soluble forward sequencing primers thereby
generating a plurality
of extended forward sequencing primer strands, wherein individual immobilized
concatemer
template molecules have two or more extended forward sequencing primer strands
hybridized
thereon; e) removing the extended forward sequencing primer strands and
retaining the
immobilized concatemer template molecules; t) generating a first plurality of
immobilized
forward extension strands by hybridizing at least one portion of individual
immobilized
concatemer template molecules to a second surface primer and conducting a
primer extension
reaction from the second surface primers that are hybridized to a portion of
the immobilized
concatemer template molecule to generate a plurality of forward extension
strands having a
sequence that is complementary to at least a portion of the immobilized
concatemer template
molecules and are covalently joined to an immobilized second surface primer;
g) contacting the
plurality of immobilized concatemer template molecules and the plurality of
immobilized
forward extension strands with a relaxing solution which comprises at least
one chaotropic agent;
h) dissociating the at least one portion of the immobilized concatemer
template molecules from

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22
the immobilized second surface primers and retaining the immobilized forward
extension
strands, and re-hybridizing at least one portion of the immobilized concatemer
template
molecules to one of the immobilized second surface primers that are not
covalently joined to a
forward extension strand, wherein the dissociating and re-associating
comprises a temperature
ramp-up, a temperature plateau, and temperature ramp-down, and washing the
relaxing solution
from the support; i) contacting the re-hybridized immobilized concatemer
template molecules
with an amplification solution and conducting a primer extension reaction from
the second
surface primers that are re-hybridized to a portion of the immobilized
concatemer template
molecules to generate a plurality of newly synthesized forward extension
strands having a
sequence that is complementary to at least a portion of the immobilized
concatemer template
molecules and are covalently joined to an immobilized second surface primer;
j) repeating steps
(g) ¨ (i) at least once; k) removing the retained immobilized concatemer
template molecules by
generating abasic sites in the immobilized single stranded concatemer template
molecules and
the immobilized first surface primers at the nucleotide(s) having the scissile
moiety and
generating gaps at the abasic sites to generate a plurality of gap-containing
nucleic acid
molecules while retaining the plurality of immobilized forward extension
strands and retaining
the plurality of immobilized second surface primers; and 1) sequencing the
plurality of retained
immobilized forward extension strands with a plurality of soluble reverse
sequencing primers
thereby generating a plurality of extended reverse sequencing primer strands.
[0046] In some embodiments, each of the single stranded circular nucleic
acid library
molecules in the plurality comprises a sequence of interest and wherein the
individual library
molecules further comprise any one or any combination of two or more of (i) a
universal binding
sequence for a soluble forward sequencing primer, (ii) a universal binding
sequence for a soluble
reverse sequencing primer, (iii) a universal binding sequence for an
immobilized first surface
primer, (iv) a universal binding sequence for an immobilized second surface
primer, (v) a
universal binding sequence for a first soluble amplification primer, (vi) a
universal binding
sequence for a second soluble amplification primer, (vii) a universal binding
sequence for a
soluble compaction oligonucleotide, (viii) a sample barcode sequence and/or
(ix) a unique
molecular index sequence.
[0047i In some embodiments, individual immobilized concatemer template
molecules in the
plurality comprise two or more copies of a sequence of interest, and wherein
the individual
. .

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23
immobilized concatemer template molecules further comprise any one or any
combination of
two or more of (i) two or more copies of a universal binding sequence for a
soluble forward
sequencing primer, (ii) two or more copies of a universal binding sequence for
a soluble reverse
sequencing primer, (iii) two or more copies of a universal binding sequence
for an immobilized
first surface primer, (iv) two or more copies of a universal binding sequence
for an immobilized
second surface primer, (v) two or more copies of a universal binding sequence
for a first soluble
amplification primer, (vi) two or more copies of a universal binding sequence
for a second
soluble amplification primer, (vii) two or more copies of a universal binding
sequence for a
soluble compaction oligonucleotide, (viii) two or more copies of a sample
barcode sequence
and/or (ix) two or more copies of a unique molecular index sequence.
[00481 In any of the foregoing or related embodiments, the support
comprises a planar
substrate which comprises glass, fused-silica, silicon, a polymer (e.g.,
polystyrene (PS),
macroporous polystyrene (MPPS), polymethylmethaci-ylate (PMNLA,),
polycarbonate (PC),
polypropylene (PP), polyethylene (PE), high density polyethylene (HDPE),
cyclic olefin
polymers (COP), cyclic olefin copolymers (COC), polyethylene terephthalate
(PET)), or any
combination thereof.
[00491 In any of the foregoing or related embodiments, the support
comprises at least one
hydrophilic polymer coating having a water contact angle of no more than 45
degrees, and
wherein at least one of the hydrophilic polymer coatings comprising branched
hydrophilic
polymer having at least 4 branches.
[00501 In any of the foregoing or related embodiments, the 5' end of the
plurality of first
surface primers are immobilized to the support or immobilized to a coating on
the support, In
any of the foregoing or related embodiments, the plurality of first surface
primers comprise
modified oligonucleotide molecules having 2-10 phosphorothioate linkages at
their 5' ends to
confer resistance to nuclease degradation.
[0051] In any of the foregoing or related embodiments, the 5' end of the
plurality of second
surface primers are immobilized to the support or immobilized to a coating on
the support. In
some embodiments, the plurality of second surface primers comprise modified
oligonucleotide
molecules having 2-10 phosphorothioate linkages at their 5' ends to confer
resistance to nuclease
degradation.

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24
[0052] In any of the foregoing or related embodiments, the immobilized
concatemer template
molecules comprise at least one nucleotide having a scissile moiety which
comprises uridine, 8-
oxo-7,8-dihydrogunine, or deoxyinosine.
[00531 In any of the foregoing or related embodiments, the nucleotides with
a scissile moiety
are located at randomly distributed positions in individual immobilized
concatemer template
molecules in the plurality.
[0054] In any of the foregoing or related embodiments, 0.01 ¨ 30% of the
thymidine
nucleotides in the individual immobilized concatemer template molecules are
replaced with
uridine. in any of the foregoing or related embodiments, 0.01 ¨ 30% of the
guanosine nucleotides
in the individual immobilized concatemer template molecules are replaced with
8-oxo-7,8-
dihydrogunine or deoxyinosine.
100551 In any of the foregoing or related embodiments, the soluble forward
sequencing
primer comprises a 3' OH extendible end and lacks a nucleotide having a
scissile moiety. In any
of the foregoing or related embodiments, the soluble reverse sequencing primer
comprises a 3'
OH extendible end and lacks a nucleotide having a scissile moiety.
100561 In any of the foregoing or related embodiments, the first soluble
amplification primer
comprises a 3' OH extendible end and lacks a nucleotide having a scissile
moiety. In any of the
foregoing or related embodiments, the second soluble amplification primer
comprises a 3' OH
extendible end and lacks a nucleotide having a scissile moiety.
100571 In any of the foregoing or related embodiments, the forward
sequencing step
comprises: a) contacting a plurality of sequencing polymerases to (i) a
plurality of immobilized
concatemer template molecules and (ii) a plurality of the soluble forward
sequencing primers,
wherein the contacting is conducted under a condition suitable to form a
plurality of complexed
polymerases each comprising a sequencing polymerase bound to a nucleic acid
duplex wherein
the nucleic acid duplex comprises a immobilized concatemer template molecule
hybridized to a
soluble forward sequencing primer; b) contacting the plurality of complexed
sequencing
polymerases with a plurality of nucleotides under a condition suitable for
binding at least one
nucleotide to a complexed sequencing polym.erase, wherein the plurality of
nucleotides
comprises at least one nucleotide analog labeled with a fluorophore and having
a removable
chain terminating moiety at the sugar 3' position; c) incorporating at least
one nucleotide into the
3' end of the hybridized forward sequencing primers thereby generating a
plurality of nascent

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extended forward sequencing primers; and d) detecting the incorporated
nucleotide and
identifying the nucleo-base of the incorporated nucleotide.
[0058] In any of the foregoing or related embodiments, the reverse
sequencing step
comprises: a) contacting a plurality of sequencing polymerases to (i) a
plurality of the retained
forward extension strands and (ii) a plurality of the soluble reverse
sequencing primers, wherein
the contacting is conducted under a condition suitable to form a plurality of
complexed
polymerases each comprising a sequencing polymerase bound to a nucleic acid
duplex wherein
the nucleic acid duplex comprises a retained forward extension strand
hybridized to a soluble
reverse sequencing primer; b) contacting the plurality of complexed sequencing
polymerases
with a plurality of nucleotides under a condition suitable for binding at
least one nucleotide to a
complexed sequencing polymerase, wherein the plurality of nucleotides
comprises at least one
nucleotide analog labeled with a fluorophore and having a removable chain
terminating moiety
at the sugar 3' position; c) incorporating at least one nucleotide into the 3'
end of the hybridized.
reverse sequencing primers thereby generating a plurality of nascent extended
reverse
sequencing primers; and d) detecting the incorporated nucleotide and
identifying the nucleo-base
of the incorporated nucleotide.
[00591 In some embodiments, the reverse sequencing of step (a) comprises
hybridizing the
plurality of soluble reverse sequencing primers to the plurality of the
retained forward extension
strands in the presence of a high. efficiency hybridization buffer which
comprises: (i) a first polar
aprotic solvent which comprises acetonitri le at 25-50% by volume of the
hybridization buffer;
(ii) a second polar aprotic solvent which comprises formamide at 5-10% by
volume of the
hybridization buffer; (iii) a pH buffering system which comprises 2-(N-
morpholino)ethanesulfonic acid (MES) at a of 5-6.5; and (iv) a crowding
agent which
comprises polyethylene glycol (PEG) at 5-35% by volume of the hybridization
buffer.
[0060] In some embodiments, the reverse sequencing step comprises: a)
contacting a
plurality of sequencing polymerases to (i) a plurality of the immobilized
partially displaced
forward extension strands, (ii) a plurality of plurality of immobilized
detached extended forward
sequencing primer strands, and (iii) a plurality of the soluble reverse
sequencing primers,
wherein the contacting is conducted under a condition suitable to form a
plurality of complexed
polymerases each comprising a sequencing polymerase bound to a nucleic acid
duplex wherein
the nucleic acid duplex comprises a soluble reverse sequencing primer
hybridized to an

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immobilized partially displaced forward extension strand or an immobilized
detached extended
forward sequencing primer strand; b) contacting the plurality of complexed
sequencing
polymerases with a plurality of nucleotides under a condition suitable for
binding at least one
nucleotide to a complexed sequencing polymerase, wherein the plurality of
nucleotides
comprises at least one nucleotide analog labeled with a fluorophore and having
a removable
chain terminating moiety at the sugar 3' position; c) incorporating at least
one nucleotide into the
3' end of the hybridized reverse sequencing primers thereby generating a
plurality of nascent
extended reverse sequencing primers; and d) detecting the incorporated
nucleotide and
identifying the nucleo-base of the incorporated nucleotide.
100611 In some embodiments, the reverse sequencing of step a) comprises
hybridizing the
plurality of soluble reverse sequencing primers to the plurality of the
retained forward extension
strands in the presence of a high efficiency hybridization buffer which
comprises: (i) a first polar
aprotic solvent which comprises acetonitrile at 25-50% by volume of the
hybridization buffer;
(ii) a second polar aprotic solvent which comprises formamide at 5-10% by
volume of the
hybridization buffer; (iii) a pH buffering system which comprises 2-(N-
morpholino)ethanesulfonic acid (MES) at a pH of 5-6.5; and (iv) a crowding
agent which
comprises polyethylene glycol (PEG) at 5-35% by volume of the hybridization
butler.
[00621 In any of the foregoing or related embodiments, the forward
sequencing step and the
reverse sequencing step comprises: 1) conducting a sequencing reaction at a
position on the
template molecule using multivalent molecules which bind but do not
incorporate; 2) conducting
a sequencing reaction at the same position on the template molecule using
nucleotides with
incorporation; and 3) repeating steps a) and b) at the next position on the
template molecule.
[00631 In any of the foregoing or related embodiments, the forward
sequencing step and the
reverse sequencing step comprises: a) contacting a plurality of a first
sequencing polymerase to
(i) a plurality of nucleic acid template molecules and (ii) a plurality of
soluble sequencing
primers, wherein the contacting is conducted under a condition suitable to
form a plurality of
first complexed polymerases each comprising a first sequencing polymerase
bound to a nucleic
acid duplex wherein the nucleic acid duplex comprises the nucleic acid
template molecule
hybridized to the sequencing primer, wherein (1) the plurality of nucleic acid
template molecules
comprise a plurality of the immobilized concatemer template molecules and the
plurality of
soluble primers comprise a plurality of the soluble forward sequencing
primers, or wherein (2)

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27
the plurality of nucleic acid template molecules comprise a plurality of the
retained forward
extension strands and the plurality of soluble sequencing primers comprise a
plurality of the
soluble reverse sequencing primers; b) contacting the plurality of first
complexed polymerases
with a plurality of detectably labeled multivalent molecules to form a
plurality of multivalent-
complexed polymerases, under a condition suitable for binding complementary
nucleotide units
of the multivalent molecules to at least two of the plurality of first
complexed polymerases
thereby forming a plurality of multivalent-complexed polymerases, and the
condition inhibits
incorporation of the complementary nucleotide units into the sequencing
primers of the plurality
of multivalent-complexed polymerases, wherein individual multivalent molecules
in the plurality
of multivalent molecules comprise a core attached to multiple nucleotide arms
and each
nucleotide arm is attached to a nucleotide unit; c) detecting the plurality of
multivalent-
complexed polymerases; and d) identifying the nucleo-base of the complementary
nucleotide
units that are bound to the plurality of first complexed polymerases in the
plurality of
multivalent-complexed polymerases, thereby determining the sequence of the
nucleic acid
template.
100641 In some embodiments, the reverse sequencing of step comprises:
hybridizing the
plurality of soluble reverse sequencing primers to the plurality of the
retained forward extension
strands in the presence of a high efficiency hybridization buffer which
comprises: (i) a first polar
aprotic solvent which comprises acetonitrile at 25-50% by volume of the
hybridization buffer;
(ii) a second polar aprotic solvent which comprises formamide at 5-10% by
volume of the
hybridization buffer; (iii) a pH buffering system which comprises 2-(N-
morpholino)ethanesulfonic acid (MES) at a pH of 5-6.5; and (iv) a crowding
agent which
comprises polyethylene glycol (PEG) at 5-35% by volume of the hybridization
buffer.
[00651 In some embodiments, the method further comprises: e) dissociating
the plurality of
multivalent-complexed polymerases and removing the plurality of first
sequencing polymerases
and their bound multivalent molecules, and retaining the plurality of nucleic
acid duplexes; t)
contacting the plurality of the retained nucleic acid duplexes of step (e)
with a plurality of second
sequencing polymerases, wherein the contacting is conducted under a condition
suitable for
binding the plurality of second sequencing polymerases to the plurality of the
retained nucleic
acid duplexes, thereby forming a plurality of second complexed polymerases
each comprising a
second sequencing polymerase bound to a retained nucleic acid duplex; g)
contacting the

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plurality of second complexed polymerases with a plurality of nucleotides,
wherein the
contacting is conducted under a condition suitable for binding complementary
nucleotides from
the plurality of nucleotides to at least two of the second complexed
polymerases of step (f)
thereby forming a plurality of nucleotide-complexed polymerases and the
condition is suitable
for promoting incorporation of the bound complementary nucleotides into the
sequencing
primers of the nucleotide-complexed polymerases; h) detecting the
complementary nucleotides
which are incorporated into the sequencing primers of the nucleotide-complexed
polymerases;
and d) identifying the nucleo-bases of the complementary nucleotides which are
incorporated
into the sequencing primers of the nucleotide-complexed polymerases.
100661 In some embodiments, the method further comprises: forming at least
one avidity
complex in step (b), the method comprising: a) binding a first sequencing
primer, a first
sequencing polymerase, and a first multivalent molecule to a first portion of
a nucleic acid
template molecule thereby forming a first binding complex, wherein a first
nucleotide unit of the
first multivalent molecule binds to the first sequencing polymerase; and b)
binding a second
sequencing primer, a second sequencing polymerase, and the first multivalent
molecule to a
second portion of the same nucleic acid template molecule thereby forming a
second binding
complex, wherein a second nucleotide unit of the second multivalent molecule
binds to the
second sequencing polymerase, wherein the first and second binding complexes
which include
the same multivalent molecule forms an avidity complex.
[00671 In some embodiments, (i) the first sequencing primer comprises a
soluble forward
sequencing primer and the nucleic acid template molecule comprises an
immobilized concatemer
template molecule, (ii) the second sequencing primer comprises a soluble
forward sequencing
primer and the nucleic acid template molecule comprises the same immobilized
concatemer
template molecule, and (iii) the first and second sequencing primers have the
same sequence.
[00681 In some embodiments, wherein (i) the first sequencing primer
comprises a soluble
reverse sequencing primer and the nucleic acid template molecule comprises a
retained forward
extension strand, (ii) the second sequencing primer comprises a soluble
reverse sequencing
primer and the nucleic acid template molecule comprises the same retained
forward extension
strand, and (iii) the first and second sequencing primers have the same
sequence.
[00691 In some embodiments, the method further comprises: forming at least
one avidity
complex in step (b), the method comprising: a) contacting a plurality of first
sequencing
. .

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polymerases and a plurality of second sequencing primers with different
portions of a nucleic
acid template molecule to form at least first and second complexed polymerases
on the same
nucleic acid template molecule; b) contacting a plurality of multivalent
molecules to the at least
first and second complexed polymerases on the same nucleic acid template
molecule, under
conditions suitable to bind a single multivalent molecule from the plurality
to the first and
second complexed polymerases, wherein at least a first nucleotide unit of the
single multivalent
molecule is bound to the first complexed polymerase which includes a first
sequencing primer
hybridized to a first portion of the nucleic acid template molecule thereby
forming a first binding
complex, and wherein at least a second nucleotide unit of the single
multivalent molecule is
bound to the second complexed polymerase which includes a second sequencing
primer
hybridized to a second portion of the same nucleic acid template molecule
thereby forming a
second binding complex, wherein the contacting is conducted under a condition
suitable to
inhibit polymerase-catalyzed incorporation of the bound first and second
nucleotide units in the
first and second binding complexes, and wherein the first and second binding
complexes which
are bound to the same multivalent molecule forms an avidity complex; c)
detecting the first and
second binding complexes on the same nucleic acid template molecule, and d)
identifying the
first nucleotide unit in the first binding complex thereby determining the
sequence of the first
portion of the nucleic acid template molecule, and identifying the second
nucleotide unit in the
second binding complex thereby determining the sequence of the second portion
of the same
nucleic acid template molecule.
[00701 In some embodiments, (i) the plurality of first sequencing primers
comprise a
plurality of first soluble forward sequencing primers and the nucleic acid
template molecule
comprises an immobilized concatemer template molecule, (ii) the plurality of
second sequencing
primers comprise a plurality of second soluble forward sequencing primers and
the nucleic acid
template molecule comprises the same immobilized concatemer template molecule,
and (iii) the
plurality of first and second sequencing primers have the same sequence.
[0071] in some embodiments, (i) the plurality of first sequencing primers
comprises a
plurality of first soluble reverse sequencing primer and the nucleic acid
template molecule
comprises a retained forward extension strand, (ii) the plurality of second
sequencing primers
comprise a plurality of second soluble reverse sequencing primers and the
nucleic acid template
. .

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molecule comprises the same retained forward extension strand, and (iii) the
plurality of first and
second sequencing primers have the same sequence.
[0072] In any of the foregoing or related embodiments, the forward
sequencing step and the
reverse sequencing step comprises: a) contacting a plurality of a first
sequencing polymerase to
(i) a plurality of nucleic acid template molecules and (ii) a plurality of
soluble sequencing
primers, wherein the contacting is conducted under a condition suitable to
form a plurality of
first complexed polymerases each comprising a first sequencing polymerase
bound to a nucleic
acid duplex wherein the nucleic acid duplex comprises the nucleic acid
template molecule
hybridized to the soluble sequencing primer, wherein (I) the plurality of
nucleic acid template
molecules comprise a plurality of the immobilized concatemer template
molecules and the
plurality of sequencing primers comprise a plurality of the soluble forward
sequencing primers,
or wherein (2) the plurality of nucleic acid template molecules comprise a
plurality of
immobilized partially displaced forward extension strands and the plurality of
sequencing
primers comprise a plurality of the soluble reverse sequencing primers, or
wherein (3) the
plurality of nucleic acid template molecules comprise a plurality of
immobilized detached
extended forward sequencing primer strands and the plurality of sequencing
primers comprise a.
plurality of the soluble reverse sequencing primers b) contacting the
plurality of first complexed
polymerases with a plurality of detectably labeled multivalent molecules to
form a plurality of
multivalent-complexed polymerases, under a condition suitable for binding
complementary
nucleotide units of the multivalent molecules to at least two of the plurality
of first complexed
polymerases thereby forming a plurality of multivalent-complexed polymerases,
and the
condition inhibits incorporation of the complementary nucleotide units into
the sequencing
primers of the plurality of multi valent-complexed polymerases, wherein
individual multivalent
molecules in the plurality of multivalent molecules comprise a core attached
to multiple
nucleotide arm.s and each nucleotide arm is attached to a nucleotide unit; c)
detecting the
plurality of multivalent-complexed polymerases; and d) identifying the nucleo-
base of the
complementary nucleotide units that are bound to the plurality of first
complexed polymerases in
the plurality of multivalent-complexed polymerases, thereby determining the
sequence of the
nucleic acid template.
[0073] In any of the foregoing or related embodiments, the reverse
sequencing step
comprises: hybridizing the plurality of soluble reverse sequencing primers to
the plurality of

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immobilized partially displaced forward extension strands or the plurality of
immobilized
detached extended forward sequencing primer strands in the presence of a high
efficiency
hybridization buffer which comprises: (i) a first polar aprotic solvent which
comprises
acetonitrile at 25-50% by volume of the hybridization buffer; (ii) a second
polar aprotic solvent
which comprises formamide at 5-10% by volume of the hybridization buffer;
(iii) a pH buffering
system which comprises 2-(N-morpholino)ethanesulfonic acid (NIES) at a pH of 5-
6.5; and (iv) a
crowding agent which comprises polyethylene glycol (PEG) at 5-35% by volume of
the
hybridization buffer.
[0074] In some embodiments, the method further comprises: e) dissociating
the plurality of
multivalent-complexed polymerases and removing the plurality of first
sequencing polymerases
and their bound multivalent molecules, and retaining the plurality of nucleic
acid duplexes; f)
contacting the plurality of the retained nucleic acid duplexes of step (e)
with a plurality of second
sequencing polymerases, wherein the contacting is conducted under a condition
suitable for
binding the plurality of second sequencing polymerases to the plurality of the
retained nucleic
acid duplexes, thereby forming a plurality of second complexed polymerases
each comprising a
second sequencing polymerase bound to a retained nucleic acid duplex; g)
contacting the
plurality of second complexed polymerases with a plurality of nucleotides,
wherein the
contacting is conducted under a condition suitable for binding complementary
nucleotides from
the plurality of nucleotides to at least two of the second complexed
polymerases of step (f)
thereby forming a plurality of nucleotide-complexed polymerases and the
condition is suitable
for promoting incorporation of the bound complementary nucleotides into the
sequencing
primers of the nucleotide-complexed polymerases; h) detecting the
complementary nucleotides
which are incorporated into the sequencing primers of the nucleotide-complexed
polymerases;
and i) identifying the nucleo-bases of the complementary nucleotides which are
incorporated into
the sequencing primers of the nucleotide-complexed polymerases.
[0075] In some embodiments, the method further comprises: forming at least
one avidity
complex in. step (b), the method comprising: a) binding a first sequencing
primer, a first
sequencing polymerase, and a first multivalent molecule to a first portion of
a nucleic acid
template molecule thereby forming a first binding complex, wherein a first
nucleotide unit of the
first multivalent molecule binds to the first sequencing polymerase; and b)
binding a second
sequencing primer, a second sequencing polymerase, and the first multivalent
molecule to a

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second portion of the same nucleic acid template molecule thereby forming a
second binding
complex, wherein a second nucleotide unit of the second multivalent molecule
binds to the
second sequencing polymerase, wherein the first and second binding complexes
which include
the same multivalent molecule forms an avidity complex.
[00761 In some embodiments, (i) the first sequencing primer comprises a
soluble forward
sequencing primer and the nucleic acid template molecule comprises an
immobilized concatemer
template molecule, (ii) the second sequencing primer comprises a soluble
forward sequencing
primer and the nucleic acid template molecule comprises the same immobilized
concatemer
template molecule, and (iii) the first and second sequencing primers have the
same sequence. In
some embodiments, (i) the first sequencing primer comprises a soluble reverse
sequencing
primer and the nucleic acid template molecule comprises an immobilized
partially displaced
forward extension strand, (ii) the second sequencing primer comprises a
soluble reverse
sequencing primer and the nucleic acid template molecule comprises the same
immobilized
partially displaced forward extension strand, and (iii) the first and second
sequencing primers
have the same sequence. In some embodiments, (i) the first sequencing primer
comprises a
soluble reverse sequencing primer and the nucleic acid template molecule
comprises an
immobilized detached extended forward sequencing primer strand, (ii) the
second sequencing
primer comprises a soluble reverse sequencing primer and the nucleic acid
template molecule
comprises the same immobilized detached extended forward sequencing primer
strand, and (iii)
the first and second sequencing primers have the same sequence.
[00771 In some embodiments, the method further comprises: forming at least
one avidity
complex in step (b), the method comprising: a) contacting a plurality of first
sequencing
polymerases and a plurality of second sequencing primers with different
portions of a nucleic
acid template molecule to form at least first and second complexed polymerases
on the same
nucleic acid template molecule; b) contacting a plurality of multivalent
molecules to the at least
first and second complexed polymerases on the same nucleic acid template
molecule, under
conditions suitable to bind a single multivalent molecule from the plurality
to the first and
second complexed polymerases, wherein at least a first nucleotide unit of the
single multivalent
molecule is bound to the first complexed polymerase which includes a first
sequencing primer
hybridized to a first portion of the nucleic acid template molecule thereby
forming a first binding
complex, and wherein at least a second nucleotide unit of the single
multivalent molecule is

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bound to the second complexed polytnerase which includes a second sequencing
primer
hybridized to a second portion of the same nucleic acid template molecule
thereby forming a
second binding complex, wherein the contacting is conducted under a condition
suitable to
inhibit polymerase-catalyzed incorporation of the bound first and second
nucleotide units in the
first and second binding complexes, and wherein the first and second binding
complexes which
are bound to the same multivalent molecule forms an avidity complex; c)
detecting the first and
second binding complexes on the same nucleic acid template molecule, and d)
identifying the
first nucleotide unit in the first binding complex thereby determining the
sequence of the first
portion of the nucleic acid template molecule, and identifying the second
nucleotide unit in the
second binding complex thereby determining the sequence of the second portion
of the same
nucleic acid template molecule.
10078] In some embodiments, (i) the plurality of first sequencing primers
comprise a
plurality of first soluble forward sequencing primers and the nucleic acid
template molecule
comprises an immobilized concatetner template molecule, (ii) the plurality of
second sequencing
primers comprise a plurality of second soluble forward sequencing primers and
the nucleic acid
template molecule comprises the same immobilized concatemer template molecule,
and (in) the
plurality of first and second sequencing primers have the same sequence. In
some embodiments,
(i) the plurality of first sequencing primers comprises a plurality of first
soluble reverse
sequencing primer and the nucleic acid template molecule comprises an
immobilized partially
displaced forward extension strand, (ii) the plurality of second sequencing
primers comprise a
plurality of second soluble reverse sequencing primers and the nucleic acid
template molecule
comprises the same immobilized partially displaced forward extension strand,
and (di) the
plurality of first and second sequencing primers have the sam.e sequence. In
some embodiments,
(i) the plurality of first sequencing primers comprises a plurality of first
soluble reverse
sequencing primer and the nucleic acid template molecule comprises an
immobilized detached
extended forward sequencing primer strands, (ii) the plurality of second
sequencing primers
comprise a plurality of second soluble reverse sequencing primers and the
nucleic acid template
molecule comprises the same immobilized detached extended forward sequencing
primer
strands, and (iii) the plurality of first and second sequencing primers have
the same sequence.
[0079] In any of the foregoing or related embodiments, individual
nucleotides in the plurality
of nucleotides comprise an aromatic base, a five carbon sugar, and 1-10
phosphate groups,

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wherein the aromatic base of the nucleotide comprises adenine, guanine,
cytosine, thymine or
uracil. In some embodiments, the plurality of nucleotides comprises one type
of nucleotide
selected from a group consisting of dATP, cliff?, dCTP and MT. In some
embodiments, the
plurality of nucleotides comprises a mixture of any combination of two or more
types of
nucleotides selected from a group consisting of dATP, dGTP, dCTP and/or MP. hi
some
embodiments, at least one of the nucleotides in the plurality of nucleotides
comprises a
fluorescently-labeled nucleotide. In some embodiments, at least one of the
plurality of
nucleotides lacks a fluorophore label.
10080] In any of the foregoing or related embodiments, at least one of the
nucleotides in the
plurality of nucleotides comprises a chain terminating moiety attached to 3'-
OH sugar position
via cleavable moiety, and wherein the chain terminating moiety comprises an
alkyl group,
alkenyl group, alkynyl group, allyl group, aryl group, benzyl group, azide
group, amine group,
amide group, keto group, isocyanate group, phosphate group, thio group,
disulfide group,
carbonate group, urea group, or sily1 group.
PM In some embodiments, the chain terminating moieties alkyl, alkenyl,
alkynyl and ally]
are cleavable/removable with tetrakis(triphenylphosphine)palladium(0)
(Pd(PPh3)4) with
piperidine, or with 2,3-Dichloro-5,6-dicyano-1,4-benai-quinone (DDQ); (i) the
chain
terminating moieties aryl and benzyl are cleavable/removable with H2 Pd/C;
(ii) the chain
terminating moieties amine, amide, keto, isocyanate, phosphate, thio,
disulfide are
cleavable/removable with a thiol reagent which comprises beta-mercaptoethanol
or dithiothritol
(DTT); (iii) the chain terminating moieties amine, amide, keto, isocyanate,
phosphate, thio,
disulfide are cleavable/removable with a phosphine reagent which comprises
Tris(2-
carboxyethyl)phosphine (TCEP), bis-sulfo triphenyl phosphine (BS-TPP), or
Tri(hydroxyproyl)phosphine (II-IPP); (iv) the chain terminating moieties
amine, amide, keto,
isocyanate, phosphate, thio, disulfide are cleavable/removable with 4-
dimethylaminopyridine (4-
DMAP); (v) the chain terminating moiety carbonate is cleavable/removable with
potassium
carbonate (K2CO3) in Me011, with triethylamine in pyridine, or with Zn in
acetic acid (Ac011);
and (vi) the chain terminating moieties urea and silyl are cleavable with
tetrabutOammonium
fluoride, pyridine-IIF, with ammonium fluoride, or with triethylamine
trihydrofluoride.
[0082i In some embodiments, at least one of the nucleotides in the
plurality of nucleotides
comprises a chain terminating moiety attached to 3'-OH sugar position via
cleavable moiety, and

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wherein the chain terminating moiety comprises a 3' 0-azido or a 3' 0-
azidomethyl group. In
some embodiments, (i) the chain terminating moieties 3' 0-azido and 3' 0-
azidomethyl group
are cleavable/removable with a phosphine compound which comprise a derivatized
tri-alkyl
phosphine moiety, derivatized tri-aryl phosphine moiety, Tris(2-
carboxyethyl)phosphine
(TCEP), his-sulfo triphenyl phosphine (13S-TPP) or Tri(hydroxyproyl)phosphine
(TFIPP); and
(ii) the chain terminating moieties 3' 0-azido and 3' 0-azidomethyl group are
cleavable/removable with 4-dimethylaminopyridine (4-DMAP).
100831 In any of the foregoing or related embodiments, individual
multivalent molecules in
the plurality of multivalent molecules comprises (a) a core; and (b) a
plurality of nucleotide arms
which comprise (i) a core attachment moiety, (ii) a spacer comprising a PEG
moiety, (iii) a
linker, and (iv) a nucleotide unit, wherein the core is attached to the
plurality of nucleotide arms
via their core attachment moiety, wherein the spacer is attached to the
linker, and wherein the
linker is attached to the nucleotide unit.
10084] In some embodiments, the core comprises an avidin-type moiety and
the core
attachment moiety comprises biotin. In some embodiments, the linker comprises
an aliphatic
chain having 2-6 subunits or an oligo ethylene glycol chain having 2-6
subunits. In some
embodiments, the linker further comprises an aromatic moiety. In some
embodiments, the
nucleotide unit comprises an aromatic base, a five carbon sugar and 1-10
phosphate groups. In
some embodiments, the linker is attached to the nucleotide unit through the
base.
10085] In some embodiments, the plurality of nucleotide arms attached to
the core have the
same type of a nucleotide unit, and wherein the types of nucleotide unit is
selected from a group
consisting of dATP, dGTP, WTI), dTTP and d'UTP. In some embodiments, the
plurality of
multivalent molecules comprise one type of a multivalent molecule wherein each
multivalent
molecule in the plurality has the same type of nucleotide unit selected from a
group consisting of
dATP, dGTP, dCTP, dITP and dUS['P. In some embodiments, the plurality of
multivalent
molecules comprise a mixture of any combination of two or more types of
multivalent molecules
each type having nucleotide units selected from a group consisting of dATP,
dGTP, dCTP, dITP
and/or &TR.
[0086] In some embodiments, the plurality of multivalent molecules are
fluorescently-
labeled multivalent molecules. In some embodiments, (i) the core of individual
fluorescently-
labeled multivalent molecules is attached to a ftuorophore which corresponds
to the nucleotide

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units that are attached to the nucleotide arms; (ii) at least one of the
nucleotide arms comprises a
linker that is attached to a fluorophore which corresponds to the nucleotide
units that are attached
to the nucleotide arms; and/or (iii) at least one of the nucleotide arms
comprises a nucleotide unit
that is attached to a fluorophore which corresponds to the nucleotide units
that are attached to the
nucleotide arms.
[00871 In some embodiments, the plurality of multivalent molecules lack a
fluorophore.
10088] In some embodiments, at least one of the multivalent molecules in
the plurality of
multivalent molecules comprises nucleotide units having a chain terminating
moiety attached to
the 3'-OH sugar position via a cleavable moiety, and wherein the chain
terminating moiety
comprises an alkyl group, alkenyl group, alkynyl group, allyl group, aryl
group, benzyl group,
azide group, amine group, amide group, keto group, isocyanate group, phosphate
group, thio
group, disulfide group, carbonate group, urea group, or silyl group.
10089] In some embodiments, (i) the chain terminating moieties alkyl,
alkenyl, alkynyl and
ally' are cleavable/removable with tetrakis(triphenylphosphine)palladium(0)
(Pd(PPh3)4) with
piperidine, or with 2,3-Dichloro-5,6-dicyano-1,4-benzo-quinone (DDQ); (ii) the
chain
terminating moieties aryl and benzyl are cleavable/removable with H2 Pd/C;
(iii) the chain
terminating moieties amine, amide, keto, isocyanate, phosphate, thio,
disulfide are
cleavable/removable with a thiol reagent which comprises beta-mercaptoethanol
or dithiothritol
(DTT); (iv) the chain terminating moieties amine, amide, keto, isocyanate,
phosphate, thio,
disulfide are cleavable/removable with a phosphine reagent which comprises
Tris(2-
carboxyethyl)phosphine (TCEP), bis-sulfo triphenyl phosphine (BS-TPP), or
Tri(hydroxyproyl)phosphine (v) the chain terminating moieties amine, amide,
keto,
isocyanate, phosphate, thio, disulfide are cleavable/removable with 4-
dimethylaminopyridine (4-
DMAP); (vi) the chain terminating moiety carbonate is cleavable/removable with
potassium
carbonate (K2CO3) in Me011, with triethylamine in pyridine, or with Zn in
acetic acid (Ac011);
and (vii) the chain terminating moieties urea and silyl are cleavable with
tetrabutylammonium
fluoride, pyridine-IEF, with ammonium fluoride, or with triethylamine
trihydrofluoride.
[0090] In some embodiments, at least one of the multivalent molecules in
the plurality of
multivalent molecules comprises nucleotide units having a chain terminating
moiety attached to
the 3'-OH sugar position via a cleavable moiety, and wherein the chain
terminating moiety
comprises a 3' 0-azido or 3' 0-azidomethyl group.

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[0091i In some embodiments, (i) the chain terminating moieties 3' 0-azido
and 3' 0-
azidomethyl group are cleavable/removable with a phosphine compound which
comprise a
derivatized tri-alkyl phosphine moiety, derivatized tri-aryl phosphine moiety,
Tris(2-
carboxyethyl)phosphine (TCEP), bis-sulfo triphenyl phosphine (BS-11)P) or
Tri(hydroxyproyl)phosphine (THPP); and (ii) the chain terminating moieties 3'
0-azido and 3'
0-azidomethyl are cleavable/removable with 4-dimethylaminopyridine (4-DMAP).
10092] In some embodiments, the plurality of sequencing polymerases in step
(a) comprises
a recombinant wild type DNA polymerase, and the plurality of nucleotides in
step (b) comprises
fluorescently-labeled nucleotides having a removable chain terminating moiety
at the 3' sugar
position.
10093] In some embodiments, the plurality of sequencing polymerases in step
(a) comprises
a mutant DNA polymerase, and the plurality of nucleotides in step (b)
comprises fluorescently-
labeled nucleotides having a removable chain terminating moiety at the 3'
sugar position.
[0094] In some embodiments, the plurality of first sequencing polymerases
of step (a)
comprise a recombinant wild type DNA. polymerase. In some embodiments, the
plurality of first
sequencing polymerases of step (a) comprise mutant DNA polymerase.
[0095] In some embodiments, the plurality of second sequencing polymerases
of step (f)
comprise recombinant wild type DNA polymerase, and the plurality of
nucleotides in step (b)
comprises fluorescently-labeled nucleotides having a removable chain
terminating moiety at the
3' sugar position.
[0096] In some embodiments, the plurality of second sequencing polymerases
of step (f)
comprise mutant DNA polymerase, and the plurality of nucleotides in step (b)
comprises
fluorescently-labeled nucleotides having a removable chain terminating moiety
at the 3' sugar
position.
[0097] In any of the foregoing or related embodiments, the replacing the
plurality of
extended forward sequencing primer strands with a plurality of forward
extension strands that
are hybridized to the retained immobilized single stranded nucleic acid
concatemer template
molecules by conducting a primer extension reaction comprises: (i) contacting
at least one
extended forward sequencing primer strand with a plurality of strand
displacing polymerases and
a plurality of nucleotides and in the absence of soluble amplification
primers, under a condition
suitable to conduct a strand displacing primer extension reaction using the at
least one extended

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forward sequencing primers strand to initiate the primer extension reaction
thereby generating a
forward extension strand that is covalently joined to the extended forward
sequencing primers
strand, wherein the forward extension strand is hybridized to the immobilized
concatemer
template molecule.
[00981 In any of the foregoing or related embodiments, the replacing the
plurality of
extended forward sequencing primer strands with a plurality of forward
extension strands that
are hybridized to the retained immobilized single stranded nucleic acid
concatemer template
molecules by conducting a primer extension reaction comprises removing the
plurality of
extended forward sequencing primer strands by: (i) contacting the plurality of
extended forward
sequencing primer strands with a 5' to 3' double-stranded DNA exonuclease;
(ii) contacting the
plurality of extended forward sequencing primer strands with a denaturation
reagent comprising
any combination of formamide, acetonitrile, guanidiniurn chloride and/or a pH
buffering agent;
or (iii) contacting the plurality of extended forward sequencing primer
strands with 100%
formamide.
[00991 In any of the foregoing or related embodiments, the replacing the
plurality of
extended forward sequencing primer strands with a plurality of forward
extension strands that
are hybridized to the retained immobilized single stranded nucleic acid
concatemer template
molecules by conducting a primer extension reaction comprises: (i) removing
the plurality of
extended forward sequencing primer strands while retaining the immobilized
concatemer
template molecules; and (ii) contacting the plurality of retained immobilized
concatemer
molecules with a second plurality of soluble forward sequencing primers, a
plurality of
nucleotides and a plurality of primer extension polymerases, under a condition
suitable to
hybridize the plurality of soluble forward sequencing primers to the plurality
of retained
immobilized concatemer template molecules and suitable for conducting
polymerase-catalyzed
primer extension reactions thereby generating a plurality of forward extension
strands, wherein
the plurality of nucleotides comprise dATP, dGTP, dCTP and dTIP but lacks
dUTP, wherein in
the plurality of primer extension polymerases are tolerant of uridine-
containing template strands,
and wherein the soluble sequencing primers hybridize with the forward
sequencing primer
binding sequence in the retained immobilized concatemer molecules.
[00100j In some embodiments, the contacting comprises: contacting the
plurality of retained
immobilized concatemer molecules with the plurality of soluble forward
sequencing primers in

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the presence of a high efficiency hybridization buffer which comprises: (i) a
first polar aprotic
solvent which comprises acetonitrile at 25-50% by volume of the hybridization
buffer; (ii) a
second polar aprotic solvent which comprises formamide at 5-10% by volume of
the
hybridization buffer; (iii) a pH buffering system which comprises 2-(N-
morpholino)ethanesulfonic acid (MES) at a pH of 5-6.5; and (iv) a crowding
agent which
comprises polyethylene glycol (PEG) at 5-35% by volume of the hybridization
buffer.
100101] In any of the foregoing or related embodiments, the replacing the
plurality of
extended forward sequencing primer strands with a plurality of forward
extension strands that
are hybridized to the retained immobilized single stranded nucleic acid
concatemer template
molecules by conducting a primer extension reaction comprises: (i) removing
the plurality of
extended forward sequencing primer strand while retaining the immobilized
concatemer template
molecules; and (ii) contacting the plurality of retained immobilized
concatemer molecules with a
plurality of soluble amplification primers, a plurality of nucleotides and a
plurality of primer
extension polymerases, under a condition suitable to hybridize the plurality
of soluble
amplification primers to the plurality of retained immobilized concatemer
template molecules
and suitable for conducting polymerase-catalyzed primer extension reactions
thereby generating
a plurality of forward extension strands, wherein the soluble amplification
primers hybridize
with the soluble amplification primer binding sequence in the retained
immobilized concatemer
molecules, wherein the plurality of nucleotides comprise dATP, dGTP, dCTP and
dTTP but
lacks dUTP, wherein in the plurality of primer extension polymerases are
tolerant of uridine-
containing template strands, and wherein the soluble sequencing primers
hybridize with the
forward sequencing primer binding sequence in the retained immobilized
concatemer molecules.
[00102] In some embodiments, the contacting comprises: contacting the
plurality of retained
immobilized concatemer molecules with the plurality of soluble amplification
primers in the
presence of a high efficiency hybridization buffer which comprises: (i) a
first polar aprotic
solvent which comprises acetonitrile at 25-50% by volume of the hybridization
buffer; (ii) a
second polar aprotic solvent which comprises formamide at 5-10% by volume of
the
hybridization buffer; (iii) a pH buffering system which comprises 2-(N-
morpholino)ethanesulfonic acid (MES) at a pH of 5-6.5; and (iv) a crowding
agent which
comprises polyethylene glycol (PEG) at 5-35% by volume of the hybridization
buffer.

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[00103] In some embodiments, the method further comprises: contacting the
plurality of
retained immobilized concatemer molecules with a plurality of soluble
compaction
oligonucleotides.
[00104] In any of the foregoing or related embodiments, the replacing the
plurality of
extended forward sequencing primer strands comprises: (i) contacting at least
one extended
forward sequencing primer strand with a plurality of strand displacing
polymerases and a
plurality of nucleotides and in the absence of soluble amplification primers,
under a condition
suitable to conduct a strand displacing primer extension reaction using the at
least one extended
forward sequencing primer strand to initiate the primer extension reaction
thereby generating a
plurality of forward extension strands, a plurality of partially displaced
extended forward
sequencing strands and a plurality of detached extended forward sequencing
primer strands.
[00105] In any of the foregoing or related embodiments, replacing the
plurality of extended
forward sequencing primer strands comprises: comprises removing the plurality
of extended
forward sequencing primer strands by: (i) contacting the plurality of extended
forward
sequencing primer strands with a 5' to 3' double-stranded DNA exonuclease;
(ii) contacting the
plurality of extended forward sequencing primer strands with a denaturation
reagent comprising
any combination of formamide, acetonitrile, guanidinium chloride and/or a pH
buffering agent;
or (iii) contacting the plurality of extended forward sequencing primer
strands with 100%
formamide.
[00106] In any of the foregoing or related embodiments, the replacing the
plurality of
extended forward sequencing primer strands comprises: (i) removing the
plurality of extended
forward sequencing primer strands while retaining the immobilized concatemer
template
molecules; and (ii) contacting the plurality of retained immobilized
concatemer molecules with a
second plurality of soluble forward sequencing primers, a plurality of
nucleotides and a plurality
of strand displacing polymerases, under a condition suitable to hybridize the
plurality of soluble
forward sequencing primers to the plurality of retained immobilized concatemer
template
molecules and suitable for conducting polymerase-catalyzed strand displacing
reactions thereby
generating a plurality of forward extension strands and a plurality of
partially displaced extended
forward sequencing strands that are hybridized to the immobilized concatemer
template
molecules to form a plurality of immobilized amplicons, and the primer
extension reaction
generates a plurality of detached extended forward sequencing primer strands
(e.g., that are not

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hybridized to the immobilized concatemer template molecules), wherein the
plurality of
nucleotides comprise dATP, dGTP, dCTP and dTTP but lacks dUTP, and wherein the
soluble
forward sequencing primers hybridize with the forward sequencing primer
binding sequence in
the retained immobilized concatemer molecules.
i00107i In some embodiments, the contacting comprises: contacting the
plurality of retained
immobilized concatemer molecules with the plurality of soluble forward
sequencing primers in
the presence of a high efficiency hybridization buffer which comprises: (i) a
first polar aprotic
solvent which comprises acetonitrile at 25-50% by volume of the hybridization
buffer; (ii) a
second polar aprotic solvent which comprises formamide at 5-10% by volume of
the
hybridization buffer; (iii) a pH buffering system which comprises 2-(N-
morpholino)ethanesulfonic acid (MES) at a pH of 5-6.5; and (iv) a crowding
agent which
comprises polyethylene glycol (PEG) at 5-35% by volume of the hybridization
buffer.
100108] In any of the foregoing or related embodiments, the replacing the
plurality of
extended forward sequencing primer strands comprises: (i) removing the
plurality of extended
forward sequencing primer strand while retaining the immobilized concatemer
template
molecules; and (ii) contacting the plurality of retained immobilized
concatemer molecules with a
plurality of soluble amplification primers, a plurality of nucleotides and a
plurality of strand
displacing polymerases, under a condition suitable to hybridize the plurality
of soluble
amplification primers to the plurality of retained immobilized concatemer
template molecules
and suitable for conducting polymerase-catalyzed strand displacing reactions
thereby generating
a plurality of forward extension strands and a plurality of partially
displaced extended forward
sequencing strands that are hybridized to the immobilized concatemer template
molecules to
form a plurality of immobilized amplicons, and the primer extension reaction
generates a
plurality of detached extended forward sequencing primer strands (e.g., that
are not hybridized to
the immobilized concatemer template molecules), wherein the plurality of
nucleotides comprise
dATP, dGT.P, dCTP and dTTP but lacks d'UTP, wherein the soluble amplification
primers
hybridize with the soluble amplification primer binding sequence in the
retained immobilized
concatemer molecules.
[00109] In some embodiments, the contacting comprises: contacting the
plurality of retained
immobilized concatemer molecules with the plurality of soluble amplification
primers in the
presence of a high efficiency hybridization buffer which comprises: (i) a
first polar aprotic

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solvent which comprises acetonitrile at 25-50% by volume of the hybridization
buffer; (ii) a
second polar aprotic solvent which comprises formamide at 5-10% by volume of
the
hybridization buffer; (iii) a pH buffering system which comprises 2-(N-
morphohno)ethanesulfonic acid (NIES) at a pH of 5-6.5; and (iv) a crowding
agent which
comprises polyethylene glycol (PEG) at 5-35% by volume of the hybridization
buffer.
1001101 In any of the foregoing or related embodiments, the at least one of
the retained
immobilized concatemer template molecules includes one or more nucleotides
having a scissile
moiety, and wherein the scissile moiety comprises uridine or 8-oxo-7,8-
dihydroguanine, or
deoxyinosine. In any of the foregoing or related embodiments, the retained
immobilized
concatemer template molecule comprises one or more uridines, and wherein the
generating the
abasic sites at the uridines comprises contacting the retained immobilized
concatemer template
molecule with uracil DNA glycosylase (UDG). In any of the foregoing or related
embodiments,
the retained immobilized concatemer template molecule comprises one or more
8oxoG, and.
wherein the generating the abasic sites at the 8oxoG comprises contacting the
retained
immobilized concatemer template molecule with an Fpg enzyme
(formamidopyrimidine DNA
glycosylase). In any of the foregoing or related embodiments, the retained
immobilized
concatemer template molecule comprises one or more deoxyinosine, and wherein
the generating
the abasic sites at the deoxyinosine comprises contacting. the retained
immobilized concatemer
template molecule with an AlkA. glycosylase enzyme.
1001.1.1.1 In any of the foregoing or related embodiments, the method
further comprises
generating a gap at the abasic sites to generate at least one gap-containing
concatemer template
molecule, which comprises: contacting the retained immobilized template
molecules containing
one or more abasic sites with an endonuclease IV, AP lyase (e.g., DNA-apurinic
lyase or DNA-
apyrimidinic lyase), FPG glycosylase/AP lyase and/or endo VIII glycosylase/AP
lyase.
[00112] In any of the foregoing or related embodiments, the immobilized
concatetner template
molecules comprise 0.1 --- 30% uridine, and wherein the plurality of wild type
sequencing
polymerases yield an error rate of incorporating dUIP of at least 0.1X
compared to an error rate
of incorporating MP. In any of the foregoing or related embodiments, the
immobilized
concaterner template molecules comprise 0.1 -- 30% uridine, and wherein the
plurality of mutant
sequencing polymerases yield an error rate of incorporating dUTP of at least
0.1X compared to
an error rate of incorporating MP. In any of the foregoing or related
embodiments, the

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immobilized concatemer template molecules comprise 0.1 ---- 30% uridine, and
wherein the
plurality of wild type sequencing polymerases yield an error rate of
incorporating dUTP of at
least 0.1X compared to an error rate of incorporating dTTP. In any of the
foregoing or related
embodiments, the immobilized concatemer template molecules comprise 0.1 ¨ 30%
uridine, and
wherein the plurality of mutant sequencing polymerases yield an error rate of
incorporating
dUTP of at least 0.1X compared to an error rate of incorporating d'ITP.
[001131 In
any of the foregoing or related embodiments, the ratio of a first base
fluorescent
signal of R2 (e.g., reverse sequencing) to a first base fluorescent signal of
RI (e.g., forward.
sequencing) is at least 0.7 for sequencing using 1, 2, 3 or 4 dyes colors.
1001141 In
any of the foregoing or related embodiments, the rolling circle amplification
step
comprises a plurality of compaction oligonucleotides and/or hexamine to
generate immobilized
concatemer template molecules having a more compact size and/or shape compared
to a rolling
circle amplification reaction in the absence of compaction oligonucleotides
and/or hexamine.
10011.51 In any of the foregoing or related embodiments, the primer extension
reaction of step
comprises a plurality of compaction oligonucleotides and/or hexamine to
generate a plurality of
forward extension strands having a more compact size and/or shape compared to
a primer
extension reaction in the absence of compaction oligonucleotides and/or
hexamine.
1001.1.61 In
any of the foregoing or related embodiments, the rolling circle amplification
step
comprises a plurality of compaction oligonucleotides and/or hexamine to
generate concatemer
molecules having a more compact size and/or shape compared to a rolling circle
amplification
reaction in the absence of compaction oligonucleotides and/or hexamine.
100117] In any of the foregoing or related embodiments, the primer extension
reaction step
comprises a plurality of compaction oligonucleotides and/or hexamine to
generate a plurality of
forward extension strands having a more compact size and/or shape compared to
a primer
extension reaction in the absence of compaction oligonucleotides and/or
hexamine.
[00118] In
any of the foregoing or related embodiments, the rolling circle amplification
step
comprises a plurality of compaction oligonucleotides and/or hexamine to
generate immobilized
concatemer template molecules having a more compact size and/or shape compared
to a rolling
circle amplification reaction in the absence of compaction oligonucleotides
and/or hexamine.
[001191 In any of the foregoing or related embodiments, the primer extension
reaction step
comprises a plurality of compaction oligonucleotides and/or hexamine to
generate a plurality of

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forward extension strands having a more compact size and/or shape compared to
a primer
extension reaction in the absence of compaction oligonucleotides and/or
hexamine.
[00120i In any of the foregoing or related embodiments, the primer extension
reaction step
comprises a plurality of compaction oligonucleotides and/or hexamine to
generate a plurality of
primer extension products having a more compact size and/or shape compared to
a primer
extension reaction in the absence of compaction oligonucleotides and/or
hexamine, wherein the
plurality of primer extension products include a plurality of forward
extension strands, a plurality
of partially displaced extended forward sequencing strands and a plurality of
detached extended
forward sequencing primer strands.
1001211 In any of the foregoing or related embodiments, the rolling circle
amplification step
comprises a plurality of compaction oligonucleotides and/or hexamine to
generate immobilized
concatemer template molecules having a more compact size and/or shape compared
to a rolling
circle amplification reaction in the absence of compaction oligonucleotides
and/or hexamine.
[00122] In any of the foregoing or related embodiments, the primer extension
reaction step
comprises a plurality of compaction oligonucleotides and/or hexamine to
generate a plurality of
primer extension products having a more compact size and/or shape compared to
a primer
extension reaction in the absence of compaction oligonucleotides and/or
hexamine, wherein the
plurality of primer extension products include a plurality of forward
extension strands, a plurality
of partially displaced extended forward sequencing strands and a plurality of
detached extended
forward sequencing primer strands.
[00123] In any of the foregoing or related embodiments, the rolling circle
amplification step
comprises a plurality of compaction oligonucleotides and/or hexamine to
generate a plurality of
concatemer molecules having a more compact size and/or shape compared to a
rolling circle
amplification reaction in the absence of compaction oligonucleotides and/or
hexamine.
[00124] In any of the foregoing or related embodiments, the primer extension
reaction step
comprises a plurality of compaction oligonucleotides and/or hexamine to
generate a plurality of
primer extension products having a more compact size and/or shape compared to
a primer
extension reaction in the absence of compaction oligonucleotides and/or
hexamine, wherein the
plurality of primer extension products include a plurality of forward
extension strands, a plurality
of partially displaced extended forward sequencing strands and a plurality of
detached extended
forward sequencing primer strands.

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[00125j In any of the foregoing or related embodiments, the plurality of
immobilized
concatemer template molecules or the plurality of immobilized concatemer
molecules have
FWHM (full width half maximum) of no more than about 5 gm. In any of the
foregoing or
related embodiments, the plurality of forward extension strand have FWHM (full
width half
maximum) of no more than about 5 gm. In any of the foregoing or related
embodiments, the
plurality of primer extension products have FWHM (full width half maximum) of
no more than
about 5 ium.
DESCRIPTION OF THE DRAWENGS
[00126j The novel features of the invention are set forth with particularity
in the appended
claims. A better understanding of the features and advantages of the present
invention will be
obtained by reference to the following detailed description that sets forth
illustrative embodiments,
in which the principles of the invention are utilized, and the accompanying
drawings of which:
100127] Figure 1 is a schematic showing an exemplary single stranded nucleic
acid
concatemer template molecule immobilized to an immobilized first surface
primer. The
immobilized concatemer template molecule comprises at least one nucleotide
having a scissile
moiety that can be cleaved to generate an abasic site in the immobilized
concatemer template
molecule. In some embodiments, the immobilized concatemer template molecule
can be
generated by conducting an on-support rolling circle amplification reaction.
The arrangement of
the various primer binding sequences is for illustration purposes. The skilled
artisan will
appreciate that many other arrangements are possible. Figures 2-12 show the
workflow of
pairwise sequencing the immobilized concatem.er template molecule depicted in
Figure 1.
[00128] Figure 2 is a schem.atic showing an exemplary forward sequencing
reaction
conducted on the immobilized concatemer template molecule shown in Figure 1.
The forward
sequencing reaction can be conducted with a plurality of soluble forward
sequencing primers and
generates a plurality of extended forward sequencing primer strands. The
immobilized
concatemer template molecule can have two or more extended forward sequencing
primer
strands hybridized thereon.
[001.29] Figure 3 is a schematic showing an exemplary method for replacing the
extended
forward sequencing primer strands by conducting a primer extension reaction
with a strand

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displacing polymerase in the absence of a soluble primer thereby generating a
forward extension
strand.
[001301 Figure 4 is a schematic showing an exemplary method for replacing the
extended
forward sequencing primer strands by conducting a primer extension reaction
with a soluble
forward sequencing primer thereby generating a forward extension strand.
1001311 Figure 5 is a schematic showing an exemplary method for replacing the
extended
forward sequencing primer strands by conducting a primer extension reaction
with a soluble
amplification primer thereby generating a forward extension strand.
1001321 Figure 6 is a schematic showing an exemplary method for generating
abasic sites in
the immobilized single stranded concatemer template molecules at the
nucleotides having the
scissile moiety and generating gaps at the abasic sites to generate a
plurality of gap-containing
concatemer template molecules while retaining the plurality of forward
extension strands and
retaining the plurality of immobilized first surface primers. The forward
extension strand can be
generated by the method depicted in Figures 3 or 4.
[001331 Figure 7 is a schematic showing an exemplary retained forward
extension strand after
removal of the gap-containing concatemer template molecule as shown in Figure
6.
[001341 Figure 8 is a schematic showing an exemplary is a schematic showing an
exemplary
method for generating abasic sites in the immobilized single stranded
concatemer template
molecules at the nucleotides having the scissile moiety and generating gaps at
the abasic sites to
generate a plurality of gap-containing concatemer template molecules while
retaining the
plurality of forward extension strands and retaining the plurality of
immobilized first surface
primers. The forward extension strand can be generated by the method depicted
in Figure 5.
[001351 Figure 9 is a schematic showing an exemplary retained forward
extension strand after
removal of the gap-containing concatemer template molecule as shown in Figure
8.
[00136] Figure 10 is a schematic showing an exemplary reverse sequencing
reaction
conducted on the retained forward extension strand shown in Figure 7. The
reverse sequencing
reaction can be conducted with a plurality of soluble reverse sequencing
primers. The retained
forward extension strand can have two or more extended reverse sequencing
primer strands
hybridized thereon. The extended reverse sequencing primer strands are not
hybridized to the
first surface primer, or covalently joined to the first surface primer.
Therefore, the extended
reverse sequencing primer strands are not immobilized to the support. For the
sake of simplicity,

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Figures 1-10 show an exemplary immobilized concatemer molecule with one copy
of the
sequence of interest and various universal primer binding sites. The skilled
artisan will
appreciate that the immobilized concatemer molecule can include two or more
tandem copies
containing the sequence of interest and various universal primer binding
sites.
[001371 Figure 11 is a schematic showing an exemplary reverse sequencing
reaction
conducted on the retained forward extension strand shown in Figure 9. The
retained forward
extension strand can have two or more extended reverse sequencing primer
strands hybridized
thereon. The extended reverse sequencing primer strands are not hybridized to
the first surface
primer, or covalently joined to the first surface primer. Therefore, the
extended reverse
sequencing primer strands are not immobilized to the support. For the sake of
simplicity, Figures
1-11 show an exemplary immobilized concatemer molecule with one copy of the
sequence of
interest and various universal primer binding sites. The skilled artisan will
appreciate that the
immobilized concatemer molecule can include two or more tandem copies
containing the
sequence of interest and various universal primer binding sites.
[001381 Figure 12 is a schematic showing an exemplary support having a first
and second
surface primers immobilized thereon. A. portion of the immobilized concatemer
template
molecule shown in Figure 1 is hybridized to the immobilized second surface
primer. The
immobilized concatemer template molecule has two or more copies of a universal
binding
sequence for an immobilized second surface primer. The portion of the
immobilized concatemer
template molecule that includes the universal binding sequence for an
immobilized second
surface primer can hybridize to the immobilized second surface primer.
[00139] Figure 13 is a schematic showing an exemplary single stranded nucleic
acid
concatemer template molecule immobilized to an immobilized first surface
primer. The
immobilized concatemer template molecule comprises at least one nucleotide
having a scissile
moiety that can be cleaved to generate an abasic site in the immobilized
concatemer template
molecule. In some embodiments, the immobilized concatemer template molecule
can be
generated by conducting an in-solution rolling circle amplification reaction
and distributing the
rolling circle amplification reaction onto the support. The arrangement of the
various primer
binding sequences is for illustration purposes. The skilled artisan will
appreciate that many other
arrangements are possible. Figures 14-25 show the workflow of pairwise
sequencing the
immobilized concatemer template molecule depicted in Figure 13.

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[001401 Figure 14 is a schematic showing an exemplary forward sequencing
reaction
conducted on the immobilized concatemer template molecule shown in Figure 13.
The forward
sequencing reaction can be conducted with a plurality of soluble forward
sequencing primers.
The immobilized concatemer template molecule can have two or more extended
forward
sequencing primer strands hybridized thereon.
1001411 Figure 15 is a schematic showing an exemplary method for replacing the
extended
forward sequencing primer strands by conducting a primer extension reaction
with a strand
displacing polymerase in the absence of a soluble primer.
1001421 Figure 16 is a schematic showing an exemplary method for replacing the
extended
forward sequencing primer strands by conducting a primer extension reaction
with a soluble
forward sequencing primer.
1001431 Figure 17 is a schematic showing an exemplary method for replacing the
extended
forward sequencing primer strands by conducting a primer extension reaction
with a soluble
amplification primer.
[00144] Figure 18 is a schematic showing an exemplary method for generating
abasic sites in
the immobilized single stranded concatemer template molecules at the
nucleotides having the
scissile moiety and generating gaps at the abasic sites to generate a
plurality of gap-containing
concatemer template molecules while retaining the plurality of forward
extension strands and
retaining the plurality of immobilized first surface primers. The forward
extension strand can be
generated by the method depicted in Figures 15 or 16.
[001451 Figure 19 is a schematic showing an exemplary retained forward
extension strand
after removal of the gap-containing concatemer template molecule as shown in
Figure 18.
[00146] Figure 20 is a schematic showing an exemplary method for generating
abasic sites in
the immobilized single stranded concatemer template molecules at the
nucleotides having the
scissile moiety and generating gaps at the abasic sites to generate a
plurality of gap-containing
concatemer template molecules while retaining the plurality of forward
extension strands and
retaining the plurality of immobilized first surface primers. The forward
extension strand can be
generated by the method depicted in Figure 17.
[00147] Figure 21 is a schematic showing an exemplary retained forward
extension strand
after removal of the gap-containing concatemer template molecule as shown in
Figure 20.

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[00148j Figure 22 is a schematic showing an exemplary reverse sequencing
reaction
conducted on the retained forward extension strand shown in Figure 19. The
reverse sequencing
reaction can be conducted with a plurality of soluble reverse sequencing
primers. The retained
forward extension strand depicted in Figure 22 is a concatemer molecule that
can include two or
more tandem copies of the sequence of interest and various primer binding
sites. Such a
concatemer molecule can have two or more extended reverse sequencing primer
strands
hybridized thereon. The extended reverse sequencing primer strands are not
hybridized to the
first surface primer, or covalently joined to the first surface primer.
Therefore, the extended
reverse sequencing primer strands are not immobilized to the support. For the
sake of simplicity,
Figures 13-23 show an exemplary immobilized concatemer molecule with one copy
of the
sequence of interest and various universal primer binding sites. The skilled
artisan will
appreciate that the immobilized concatemer molecule can include two or more
tandem copies
containing the sequence of interest and various universal primer binding
sites.
[00149] Figure 23 is a schematic showing an exemplary reverse sequencing
reaction
conducted on the retained forward extension strand shown in Figure 21. The
retained forward
extension strand can have two or more extended reverse sequencing primer
strands hybridized
thereon. The retained forward extension strand depicted in Figure 23 is a
concatemer molecule
that includes two or more tandem copies of the sequence of interest and
various primer binding
sites. Such a concatemer molecule can have two or more extended reverse
sequencing primer
strands hybridized thereon. The extended reverse sequencing primer strands are
not hybridized to
the first surface primer, or covalently joined to the first surface primer.
Therefore, the extended
reverse sequencing primer strands are not immobilized to the support. For the
sake of simplicity,
Figures 13-23 show an exemplary immobilized concatemer molecule with two
tandem copies
containing the sequence of interest and various universal primer binding
sites. The skilled artisan
will appreciate that the immobilized concatemer molecule can include three or
more tandem
copies containing the sequence of interest and various universal primer
binding sites.
[00150] Figure 24 is a schematic showing an exemplary support having a first
and second
surface primers immobilized thereon. A portion of the immobilized concatemer
template
molecule shown in Figure 13 is hybridized to the immobilized second surface
primer. The
immobilized concatemer template molecule has two or more copies of a universal
binding
sequence for an immobilized second surface primer. The portion of the
immobilized concatemer

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template molecule that includes the universal binding sequence for an
immobilized second
surface primer can hybridize to the immobilized second surface primer.
[001511 Figure 25 is a schematic showing an exemplary support having a first
surface primer
immobilized thereon, which in some embodiments, can be used to conduct an on-
support
pairwise sequencing workflow.
1001521 Figure 26 is a schematic showing an exemplary on-support rolling
circle
amplification reaction using a nucleic acid circular library molecule, the
immobilized first
surface primer shown in Figure 25, and a mixture of nucleotides including
nucleotides having a
scissile moiety that can be cleaved to generate an abasic site. The rolling
circle amplification
reaction generates an immobilized single stranded nucleic acid concatemer
template molecule
having at least one nucleotide with a scissile moiety which can be cleaved to
generate an abasic
site in the immobilized concatemer template molecule. The arrangement of the
various primer
binding sequences in the nucleic acid circular library molecule is for
illustration purposes. The
skilled artisan will appreciate that many other arrangements are possible.
Figures 26-37 show the
workflow of pairwise sequencing the immobilized concatemer template molecule
depicted in
Figure 26.
[001531 Figure 27 is a schematic showing an exemplary forward sequencing
reaction
conducted on the immobilized concatemer template molecule shown in Figure 26.
The forward
sequencing reaction can be conducted with a plurality of soluble forward
sequencing primers and
generates a plurality of extended forward sequencing primer strands. The
immobilized
concatemer template molecule can have two or more extended forward sequencing
primer
strands hybridized thereon.
[00154] Figure 28 is a schematic showing an exemplary method for replacing the
extended
forward sequencing primer strands by conducting a primer extension reaction
with a strand
displacing polymerase in the absence of a soluble primer thereby generating a
forward extension
strand.
[00155] Figure 29 is a schematic showing an exemplary method for replacing the
extended
forward sequencing primer strands by conducting a primer extension reaction
with a soluble
forward sequencing primer thereby generating a forward extension strand.

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[00156] Figure 30 is a schematic showing an exemplary method for replacing the
extended
forward sequencing primer strands by conducting a primer extension reaction
with a soluble
amplification primer thereby generating a forward extension strand.
[00157] Figure 31 is a schematic showing an exemplary method for generating
abasic sites in
the immobilized single stranded concatemer template molecules at the
nucleotides having the
scissile moiety and generating gaps at the abasic sites to generate a
plurality of gap-containing
concatemer template molecules while retaining the plurality of forward
extension strands and
retaining the plurality of immobilized first surface primers. The forward
extension strand can be
generated by the method depicted in Figures 28 or 29.
[00158] Figure 32 is a schematic showing an exemplary retained forward
extension strand
after removal of the gap-containing concatemer template molecule as shown in
Figure 31.
[00159] Figure 33 is a schematic showing an exemplary method for generating
abasic sites in
the immobilized single stranded concatemer template molecules at the
nucleotides having the
scissile moiety and generating gaps at the abasic sites to generate a
plurality of gap-containing
concatemer template molecules while retaining the plurality of forward
extension strands and
retaining the plurality of immobilized first surface primers. The forward
extension strand can be
generated by the method depicted in Figure 30.
[00160] Figure 34 is a schematic showing an exemplary retained forward
extension strand
after removal of the gap-containing concatemer template molecule as shown in
Figure 33.
[00161] Figure 35 is a schematic showing an exemplary reverse sequencing
reaction
conducted on the retained forward extension strand shown in Figure 32. The
reverse sequencing
reaction can be conducted with a plurality of soluble reverse sequencing
primers. The retained
forward extension strand can have two or more extended reverse sequencing
primer strands
hybridized thereon. The extended reverse sequencing primer strands are not
hybridized to the
first surface primer, or covalently joined to the first surface primer.
Therefore, the extended
reverse sequencing primer strands are not immobilized to the support. For the
sake of simplicity,
Figures 26-36 show an exemplary immobilized concatemer molecule with one copy
of the
sequence of interest and various universal primer binding sites. The skilled
artisan will
appreciate that the immobilized concatemer molecule can include two or more
tandem copies
containing the sequence of interest and various universal primer binding
sites.

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[00162j Figure 36 is a schematic showing an exemplary reverse sequencing
reaction
conducted on the retained forward extension strand shown in Figure 34. The
retained forward
extension strand can have two or more extended reverse sequencing primer
strands hybridized
thereon. The extended reverse sequencing primer strands are not hybridized to
the first surface
primer, or covalently joined to the first surface primer. Therefore, the
extended reverse
sequencing primer strands are not immobilized to the support. For the sake of
simplicity, Figures
26-36 show an exemplary immobilized concatemer molecule with one copy of the
sequence of
interest and various universal primer binding sites. The skilled artisan will
appreciate that the
immobilized concatemer molecule can include two or more tandem copies
containing the
sequence of interest and various universal primer binding sites.
100163] Figure 37 is a schematic showing an exemplary support having a first
and second
surface primers immobilized thereon. A portion of the immobilized concatemer
template
molecule shown in Figure 26 is hybridized to the immobilized second surface
primer. The
immobilized concatemer template molecule has two or more copies of a universal
binding
sequence for an immobilized second surface primer. The portion of the
immobilized concatemer
template molecule that includes the universal binding sequence for an
immobilized second
surface primer can hybridize to the immobilized second surface primer.
[00164] Figure 38 is a schematic showing an exemplary in-solution rolling
circle
amplification reaction using a nucleic acid circular library molecule, a
soluble first amplification
primer, and a mixture of nucleotides including nucleotides having a scissile
moiety that can be
cleaved to generate an abasic site. The rolling circle amplification reaction
generates in solution
single stranded nucleic acid concatemer molecules having at least one
nucleotide with a scissile
moiety which can be cleaved to generate an abasic site in the concatemer
molecule. The
arrangement of the various primer binding sequences in the nucleic acid
circular library molecule
is for illustration purposes. The skilled artisan will appreciate that many
other arrangements are
possible. Figures 38-52 show the workflow of pairwise sequencing the
concatemer molecule
depicted in Figure 38.
[001.65] Figure 39 is a schematic showing an exemplary method comprising
distributing the
rolling circle amplification reaction depicted in Figure 38 onto a support
having a first surface
primer immobilized thereon. The concatemer molecule can hybridize to the
immobilized first
surface primer.

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[001661 Figure 40 is a schematic showing an exemplary method which depicts the
rolling
circle amplification reaction continuing on the support thereby generating an
immobilized
concatemer template molecule which includes at least one nucleotide with a
scissile moiety
which can be cleaved to generate an abasic site in the immobilized concatemer
template
molecule.
1001671 Figure 41 is a schematic showing an exemplary immobilized concatemer
template
molecule generated by the method depicted in Figure 40.
1001681 Figure 42 is a schematic showing an exemplary forward sequencing
reaction
conducted on the immobilized concatemer template molecule shown in Figure 41.
The forward
sequencing reaction can be conducted with a plurality of soluble forward
sequencing primers.
The immobilized concatemer template molecule can have two or more extended
forward
sequencing primer strands hybridized thereon.
1001691 Figure 43 is a schematic showing an exemplary method for replacing the
extended
forward sequencing primer strands by conducting a primer extension reaction
with a strand
displacing polymerase in the absence of a soluble primer.
[001701 Figure 44 is a schematic showing an exemplary method for replacing the
extended
forward sequencing primer strands by conducting a primer extension reaction
with a soluble
forward sequencing primer.
[001711 Figure 45 is a schematic showing an exemplary method for replacing the
extended
forward sequencing primer strands by conducting a primer extension reaction
with a soluble
amplification primer.
[00172] Figure 46 is a schematic showing an exemplary method for generating
abasic sites in
the immobilized single stranded concatemer template molecules at the
nucleotides having the
scissile moiety and generating gaps at the abasic sites to generate a
plurality of gap-containing
concatemer template molecules while retaining the plurality of forward
extension strands and
retaining the plurality of immobilized first surface primers. The forward
extension strand can be
generated by the method depicted in Figure 43 or 44.
[00173] Figure 47 is a schematic showing an exemplary retained forward
extension strand
after removal of the gap-containing concatemer template molecule as shown in
Figure 46.
[001741 Figure 48 is a schematic showing an exemplary method for generating
abasic sites in
the immobilized single stranded concatemer template molecules at the
nucleotides having the

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scissile moiety and generating gaps at the abasic sites to generate a
plurality of gap-containing
concatemer template molecules while retaining the plurality of forward
extension strands and
retaining the plurality of immobilized first surface primers. The forward
extension strand can be
generated by the method depicted in Figure 45.
[00175j Figure 49 is a schematic showing an exemplary retained forward
extension strand
after removal of the gap-containing concatemer template molecule as shown in
Figure 48.
100176] Figure 50 is a schematic showing an exemplary reverse sequencing
reaction
conducted on the retained forward extension strand shown in Figure 47. The
reverse sequencing
reaction can be conducted with a plurality of soluble reverse sequencing
primers. The retained
forward extension strand depicted in Figure 50 is a concatemer molecule that
can include two or
more tandem copies of the sequence of interest and various primer binding
sites. Such a
concatemer molecule can have two or more extended reverse sequencing primer
strands
hybridized thereon. The extended reverse sequencing primer strands are not
hybridized to the
first surface primer, or covalently joined to the first surface primer.
Therefore, the extended
reverse sequencing primer strands are not immobilized to the support. For the
sake of simplicity,
Figures 41-50 show an exemplary immobilized concatemer molecule with one copy
of the
sequence of interest and various universal primer binding sites. The skilled
artisan will
appreciate that the immobilized concatemer molecule can include two or more
tandem copies
containing the sequence of interest and various universal primer binding
sites.
[00177] Figure 51 is a schematic showing an exemplary reverse sequencing
reaction
conducted on the retained forward extension strand shown in Figure 49. The
retained forward
extension strand can have two or more extended reverse sequencing primer
strands hybridized
thereon. The retained forward extension strand depicted in Figure 51 is a
concatemer molecule
that includes two or more tandem copies of the sequence of interest and
various primer binding
sites. Such a concatemer molecule can have two or more extended reverse
sequencing primer
strands hybridized thereon. The extended reverse sequencing primer strands are
not hybridized to
the first surface primer, or covalently joined to the first surface primer.
Therefore, the extended
reverse sequencing primer strands are not immobilized to the support. For the
sake of simplicity,
Figures 41-51 show an exemplary immobilized concatemer molecule with two
tandem copies
containing the sequence of interest and various universal primer binding
sites. The skilled artisan

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will appreciate that the immobilized concatemer molecule can include three or
more tandem
copies containing the sequence of interest and various universal primer
binding sites.
[001781 Figure 52 is a schematic showing an exemplary support having a first
and second
surface primers immobilized thereon. A portion of the immobilized concatemer
template
molecule shown in Figure 41 is hybridized to the immobilized second surface
primer. The
immobilized concatemer template molecule has two or more copies of a universal
binding
sequence for an immobilized second surface primer. The portion of the
immobilized concatemer
template molecule that includes the universal binding sequence for an
immobilized second
surface primer can hybridize to the immobilized second surface primer.
1001791 Figure 53 is schematic showing a linear single stranded library
molecule (left top
schematic) hybridizing with a double stranded splint molecule (left bottom
schematic) to
generate a circular library molecule with two gaps (right schematic). The
splint molecule
comprises a first splint strand (long strand) hybridized to a second splint
strand (short strand).
The first splint strand comprises a left sequence that hybridizes with a
sequence on one end of
the linear single stranded library molecule, and a right sequence that
hybridizes with a sequence
on the other end of the linear single stranded library molecule. The interior
portion of the first
splint strand hybridizes to the second splint strand.
[001801 Figure 54 is a schematic showing the circular library molecule (left
schematic) which
is shown in Figure 53 undergoing a ligation reaction to generate a single
stranded covalently
closed circular molecule which is hybridized to the first splint strand
(center schematic). The
single stranded covalently closed circular molecule is subjected to a rolling
circle amplification
reaction using the 3' end of the first splint strand to initiate the RCA
reaction (right schematic).
[001811 Figure 55 is a schematic showing an exemplary support having a first
surface primer
immobilized thereon, which in some embodiments, can be used to conduct an on-
support ligation
reaction for a pairwise sequencing workflow. Figures 55-72 show the workflow
of on-support
ligation and pairwise sequencing.
[001821 'Figure 56 is a schematic showing an exemplary single stranded linear
library
molecule comprising a sequence of interest and various universal adaptor
sequences for primer
binding sites. The arrangement of the various universal adaptor sequences in
this schematic is for
illustration purposes. The skilled artisan will appreciate that many other
arrangements, and
combinations of universal adaptor sequences, are possible.

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[00183] Figure 57 is a schematic showing an exemplary single stranded linear
library
molecule hybridized to an immobilized first surface primer to form a
circularized library
molecule having an asymmetrically positioned gap or nick.
[00184] Figure 58 (left) is a schematic showing an exemplary single stranded
linear library
molecule hybridized to an immobilized first surface primer to form a
circularized library
molecule having an asymmetrically positioned gap or nick. Figure 58 (right) is
a schematic
showing an exemplary single stranded linear library molecule hybridized to an
immobilized first
surface primer to form a circularized library molecule having a symmetrically
positioned gap or
nick. The schematics shown in Figures 57 and 58 represent several embodiments
of a
circularized library molecule comprising a single stranded linear library
molecule hybridized to
an immobilized first surface primer.
[00185] Figure 59 is a schematic showing an exemplary covalently closed
circular library
molecule generated by covalently closing the gap or nick.
[00186] Figure 60 (left) is a schematic showing an exemplary covalently closed
circular
library molecule generated by covalently closing the gap or nick. Figure 60
(right) is a
schematic showing an exemplary covalently closed circular library molecule
generated by
covalently closing the gap or nick. The schematics shown in Figures 57 and 58
represent several
embodiments of a covalently closed circular library molecule hybridized to an
immobilized first
surface primer.
[00187] Figure 61 is a schematic showing an exemplary on-support rolling
circle
amplification reaction using a covalently closed circular library molecule,
the immobilized first
surface primer shown in Figure 55, and a mixture of nucleotides including
nucleotides having a
scissile moiety that can be cleaved to generate an abasic site. The rolling
circle amplification
reaction generates an immobilized single stranded nucleic acid concatemer
template molecule
having at least one nucleotide with a scissile moiety which can be cleaved to
generate an abasic
site in the immobilized concatemer template molecule.
[00188] 'Figure 62 is a schematic showing an exemplary forward sequencing
reaction
conducted on the immobilized concatemer template molecule shown in Figure 61.
The forward
sequencing reaction can be conducted with a plurality of soluble forward
sequencing primers and
generates a plurality of extended forward sequencing primer strands. The
immobilized

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concatemer template molecule can have two or more extended forward sequencing
primer
strands hybridized thereon.
[00189i Figure 63 is a schematic showing an exemplary method for replacing the
extended
forward sequencing primer strands by conducting a primer extension reaction
with a strand
displacing polymerase in the absence of a soluble primer thereby generating a
forward extension
strand.
100190] Figure 64 is a schematic showing an exemplary method for replacing the
extended
forward sequencing primer strands by conducting a primer extension reaction
with a soluble
forward sequencing primer thereby generating a forward extension strand.
1001911 Figure 65 is a schematic showing an exemplary method for replacing the
extended
forward sequencing primer strands by conducting a primer extension reaction
with a soluble
amplification primer thereby generating a forward extension strand.
100192] Figure 66 is a schematic showing an exemplary method for generating
abasic sites in
the immobilized single stranded concatemer template molecules at the
nucleotides having the
scissile moiety and generating gaps at the abasic sites to generate a
plurality of gap-containing
concatemer template molecules while retaining the plurality of forward
extension strands and
retaining the plurality of immobilized first surface primers. The forward
extension strand can be
generated by the method depicted in Figures 63 or 64.
1001931 Figure 67 is a schematic showing an exemplary retained forward
extension strand
after removal of the gap-containing concatemer template molecule as shown in
Figure 66,
1001941 Figure 68 is a schematic showing an exemplary method for generating
abasic sites in
the immobilized single stranded concatemer template molecules at the
nucleotides having the
scissile moiety and generating gaps at the abasic sites to generate a
plurality of gap-containing
concatemer template molecules while retaining the plurality of forward
extension strands and
retaining the plurality of immobilized first surface primers. The forward
extension strand can be
generated by the method depicted in Figure 65.
[00195] Figure 69 is a schematic showing an exemplary retained forward
extension strand
after removal of the gap-containing concatemer template molecule as shown in
Figure 68.
[00196] Figure 70 is a schematic showing an exemplary reverse sequencing
reaction
conducted on the retained forward extension strand shown in Figure 67. The
reverse sequencing
reaction can be conducted with a plurality of soluble reverse sequencing
primers. The retained

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forward extension strand can have two or more extended reverse sequencing
primer strands
hybridized thereon. The extended reverse sequencing primer strands are not
hybridized to the
first surface primer, or covalently joined to the first surface primer.
Therefore, the extended
reverse sequencing primer strands are not immobilized to the support.
1001971 Figure 71 is a schematic showing an exemplary reverse sequencing
reaction
conducted on the retained forward extension strand shown in Figure 69. The
retained forward
extension strand can have two or more extended reverse sequencing primer
strands hybridized
thereon. The extended reverse sequencing primer strands are not hybridized to
the first surface
primer, or covalently joined to the first surface primer. Therefore, the
extended reverse
sequencing primer strands are not immobilized to the support.
100198] Figure 72 is a schematic showing an exemplary support having a first
and second
surface primers immobilized thereon. A portion of the immobilized concatemer
template
molecule shown in Figure 61 is hybridized to the immobilized second surface
primer. The
immobilized concatemer template molecule has two or more copies of a universal
binding
sequence for an immobilized second surface primer. The portion of the
immobilized concatemer
template molecule that includes the universal binding sequence for an
immobilized second
surface primer can hybridize to the immobilized second surface primer.
[00199] Figure 73 is a schematic showing an exemplary single stranded nucleic
acid
concatemer template molecule immobilized to an immobilized first surface
primer. In some
embodiments, the immobilized concatemer template molecule can be generated by
conducting an
on-support rolling circle amplification reaction. The arrangement of the
various primer binding
sequences is for illustration purposes. The skilled artisan will appreciate
that many other
arrangements are possible. Figures 73-79 show the workflow of pairwise
sequencing the
immobilized concatemer template molecule depicted in Figure 73.
[00200] Figure 74 is a schematic showing an exemplary forward sequencing
reaction
conducted on the immobilized concatemer template molecule shown in Figure 73.
The forward
sequencing reaction can be conducted with a plurality of soluble forward
sequencing primers and
generates a plurality of extended forward sequencing primer strands. The
immobilized
concatemer template molecule can have two or more extended forward sequencing
primer
strands hybridized thereon.

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[002011 Figure 75 is a schematic showing an exemplary method for replacing the
extended
forward sequencing primer strands by conducting a primer extension reaction
with soluble
amplification primers and strand displacing polymerases in the presence of
compaction
oligonucleotides, thereby generating a forward extension strand and a
partially displaced forward
extension strand which are hybridized to the immobilized concatemer template
molecule thereby
forming an immobilized amplicon.
1002021 Figure 76 is a schematic showing a continuation of the exemplary
strand displacing
method shown in Figure 75, where the polymerase-catalyzed strand displacing
reaction generates
a forward extension strand and a partially displaced forward extension strand
which are
hybridized to the immobilized concatemer template molecule, and a detached
forward extension
strand which is not hybridized to the immobilized concatemer template
molecule.
1002031 Figure 77 is a schematic showing an exemplary hybridization complex
comprising a
forward extension strand and a partially displaced forward extension strand
which are hybridized
to the immobilized concatemer template molecule, and an immobilized detached
forward
extension strand which is hybridized to the partially displaced forward
extension strand.
[00204] Figure 78 is a schematic showing an exemplary reverse sequencing
reaction
conducted on the hybridization complex shown in Figure 77. The reverse
sequencing reaction
can be conducted with a plurality of soluble reverse sequencing primers on the
partially
displaced forward extension strand and the immobilized detached forward
extension strand. The
reverse sequencing reaction generates extended reverse sequencing primer
strands. For the sake
of simplicity, Figure 78 shows one copy of an extended reverse sequencing
primer strand on the
partially displaced forward extension strand, and one copy of an extended
reverse sequencing
primer strand on the immobilized detached forward extension strand. The
skilled artisan will
appreciate that the partially displaced forward extension strand and the
immobilized detached
forward extension strand can include two or more extended reverse sequencing
primer strands
hybridized thereon.
[002051 Figure 79 is a schematic showing an exemplary support having a first
and second
surface primers immobilized thereon. A portion of the immobilized concatemer
template
molecule shown in Figure 73 is hybridized to the immobilized second surface
primer. The
immobilized concatemer template molecule has two or more copies of a universal
binding
sequence for an immobilized second surface primer. The portion of the
immobilized concatemer

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template molecule that includes the universal binding sequence for an
immobilized second
surface primer can hybridize to the immobilized second surface primer.
1002061 Figure 80 is a schematic showing an exemplary single stranded nucleic
acid
concatemer template molecule immobilized to an immobilized first surface
primer. In some
embodiments, the immobilized concatemer template molecule can be generated by
conducting an
in-solution rolling circle amplification reaction and distributing the rolling
circle amplification
reaction onto the support. The arrangement of the various primer binding
sequences is for
illustration purposes. The skilled artisan will appreciate that many other
arrangements are
possible. Figures 80-86 show the workflow of pairwise sequencing the
immobilized concatemer
template molecule depicted in Figure 80.
1002071 Figure 81 is a schematic showing an exemplary forward sequencing
reaction
conducted on the immobilized concatemer template molecule shown in Figure 80.
The forward
sequencing reaction can be conducted with a plurality of soluble forward
sequencing primers.
The immobilized concatemer template molecule can have two or more extended
forward
sequencing primer strands hybridized thereon.
[00208] Figure 82 is a schematic showing an exemplary method for replacing the
extended
forward sequencing primer strands by conducting a primer extension reaction
with soluble
amplification primers and strand displacing polymerases in the presence of
compaction
oligonucleotides, thereby generating a forward extension strand and a
partially displaced forward
extension strand which are hybridized to the immobilized concatemer template
molecule thereby
forming an immobilized amplicon.
[00209] Figure 83 is a schematic showing a continuation of the exemplary
strand displacing
method shown in Figure 82, where the polymerase-catalyzed strand displacing
reaction generates
a forward extension strand and a partially displaced forward extension strand
which are
hybridized to the immobilized concatemer template molecule, and a detached
forward extension
strand which is not hybridized to the immobilized concatemer template
molecule.
[00210] Figure 84 is a schematic showing an exemplary hybridization complex
comprising a
forward extension strand and a partially displaced forward extension strand
which are hybridized
to the immobilized concatemer template molecule, and an immobilized detached
forward
extension strand which is hybridized to the partially displaced forward
extension strand.

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[002111 Figure 85 is a schematic showing an exemplary reverse sequencing
reaction
conducted on the hybridization complex shown in Figure 84. The reverse
sequencing reaction
can be conducted with a plurality of soluble reverse sequencing primers on the
partially
displaced forward extension strand and the immobilized detached forward
extension strand. The
reverse sequencing reaction generates extended reverse sequencing primer
strands. For the sake
of simplicity, Figure 85 shows one copy of an extended reverse sequencing
primer strand on the
partially displaced forward extension strand, and one copy of an extended
reverse sequencing
primer strand on the immobilized detached forward extension strand. The
skilled artisan will
appreciate that the partially displaced forward extension strand and the
immobilized detached
forward extension strand can include two or more extended reverse sequencing
primer strands
hybridized thereon.
1002121 Figure 86 is a schematic showing an exemplary support having a first
and second
surface primers immobilized thereon. A portion of the immobilized concatemer
template
molecule shown in Figure 80 is hybridized to the immobilized second surface
primer. The
immobilized concatemer template molecule has two or more copies of a universal
binding
sequence for an immobilized second surface primer. The portion of the
immobilized concatemer
template molecule that includes the universal binding sequence for an
immobilized second
surface primer can hybridize to the immobilized second surface primer.
[002131 Figure 87 is a schematic showing an exemplary support having a first
surface primer
immobilized thereon, which in some embodiments, can be used to conduct an on-
support
pairwise sequencing workflow.
[00214] Figure 88 is a schematic showing an exemplary on-support rolling
circle
amplification reaction using a nucleic acid circular library molecule, the
immobilized first
surface primer shown in Figure 87. The rolling circle amplification reaction
generates an
immobilized single stranded nucleic acid concatemer template molecule. The
arrangement of the
various primer binding sequences in the nucleic acid circular library molecule
is for illustration
purposes. The skilled artisan will appreciate that many other arrangements are
possible. Figures
87-94 show the workflow of pairwise sequencing the immobilized concatemer
template
molecule depicted in Figure 87.
1002151 Figure 89 is a schematic showing an exemplary single stranded nucleic
acid
concatemer template molecule immobilized to an immobilized first surface
primer.

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[00216j Figure 90 is a schematic showing an exemplary forward sequencing
reaction
conducted on the immobilized concatemer template molecule shown in Figure 89.
The forward
sequencing reaction can be conducted with a plurality of soluble forward
sequencing primers and
generates a plurality of extended forward sequencing primer strands. The
immobilized
concatemer template molecule can have two or more extended forward sequencing
primer
strands hybridized thereon.
100217] Figure 91 is a schematic showing an exemplary method for replacing the
extended
forward sequencing primer strands by conducting a primer extension reaction
with soluble
amplification primers and strand displacing polymerases in the presence of
compaction
oligonucleotides, thereby generating a forward extension strand and a
partially displaced forward
extension strand which are hybridized to the immobilized concatemer template
molecule thereby
forming an immobilized amplicon.
100218] Figure 92 is a schematic showing a continuation of the exemplary
strand displacing
method shown in Figure 9L where the polymerase-catalyzed strand displacing
reaction generates
a forward extension strand and a partially displaced forward extension strand
which are
hybridized to the immobilized concatemer template molecule, and a detached
forward extension
strand which is not hybridized to the immobilized concatemer template
molecule.
[00219] Figure 93 is a schematic showing an exemplary hybridization complex
comprising a
forward extension strand and a partially displaced forward extension strand
which are hybridized
to the immobilized concatemer template molecule, and an immobilized detached
forward
extension strand which is hybridized to the partially displaced forward
extension strand.
[00220] Figure 94 is a schematic showing an exemplary reverse sequencing
reaction
conducted on the hybridization complex shown in Figure 93. The reverse
sequencing reaction
can be conducted with a plurality of soluble reverse sequencing primers on the
partially
displaced forward extension strand and the immobilized detached forward
extension strand. The
reverse sequencing reaction generates extended reverse sequencing primer
strands. For the sake
of simplicity, Figure 94 shows one copy of an extended reverse sequencing
primer strand on the
partially displaced forward extension strand, and one copy of an extended
reverse sequencing
primer strand on the immobilized detached forward extension strand. The
skilled artisan will
appreciate that the partially displaced forward extension strand and the
immobilized detached

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forward extension strand can include two or more extended reverse sequencing
primer strands
hybridized thereon.
[00221j Figure 95 is a schematic showing an exemplary in-solution rolling
circle
amplification reaction using a nucleic acid circular library molecule, a
soluble first amplification
primer, and a mixture of nucleotides. The rolling circle amplification
reaction generates in
solution single stranded nucleic acid concatemer molecules. The arrangement of
the various
primer binding sequences in the nucleic acid circular library molecule is for
illustration purposes.
The skilled artisan will appreciate that many other arrangements are possible.
Figures 95-103
show the workflow of pairwise sequencing the concatemer molecule depicted in
Figure 96.
1002221 Figure 96 is a schematic showing an exemplary method comprising
distributing the
rolling circle amplification reaction depicted in Figure 95 onto a support
having a first surface
primer immobilized thereon. The concatemer molecule can hybridize to the
immobilized first
surface primer.
[00223] Figure 97 is a schematic showing an exemplary method which depicts the
rolling
circle amplification reaction continuing on the support thereby generating an
immobilized
concatemer template molecule.
[00224] Figure 98 is a schematic showing an exemplary single stranded nucleic
acid
concatemer template molecule immobilized to an immobilized first surface
primer.
[00225] Figure 99 is a schematic showing an exemplary forward sequencing
reaction
conducted on the immobilized concatemer template molecule shown in Figure 98.
The forward
sequencing reaction can be conducted with a plurality of soluble forward
sequencing primers.
The immobilized concatemer template molecule can have two or more extended
forward
sequencing primer strands hybridized thereon.
[00226] Figure 100 is a schematic showing an exemplary method for replacing
the extended
forward sequencing primer strands by conducting a primer extension reaction
with soluble
amplification primers and strand displacing polymerases in the presence of
compaction
oligonucleotides, thereby generating a forward extension strand and a
partially displaced forward
extension strand which are hybridized to the immobilized concatemer template
molecule thereby
forming an immobilized amplicon.
[00227j Figure 101 is a schematic showing a continuation of the exemplary
strand displacing
method shown in Figure 100, where the polymerase-catalyzed strand displacing
reaction

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generates a forward extension strand and a partially displaced forward
extension strand which
are hybridized to the immobilized concatemer template molecule, and a detached
forward
extension strand which is not hybridized to the immobilized concatemer
template molecule.
[00228] Figure 102 is a schematic showing an exemplary hybridization complex
comprising a
forward extension strand and a partially displaced forward extension strand
which are hybridized
to the immobilized concatemer template molecule, and an immobilized detached
forward
extension strand which is hybridized to the partially displaced forward
extension strand.
[00229] Figure 103 is a schematic showing an exemplary reverse sequencing
reaction
conducted on the hybridization complex shown in Figure 102. The reverse
sequencing reaction
can be conducted with a plurality of soluble reverse sequencing primers on the
partially
displaced forward extension strand and the immobilized detached forward
extension strand. The
reverse sequencing reaction generates extended reverse sequencing primer
strands. For the sake
of simplicity, Figure 103 shows one copy of an extended reverse sequencing
primer strand on the
partially displaced forward extension strand, and one copy of an extended
reverse sequencing
primer strand on the immobilized detached forward extension strand. The
skilled artisan will
appreciate that the partially displaced forward extension strand and the
immobilized detached
forward extension strand can include two or more extended reverse sequencing
primer strands
hybridized thereon.
[00230] Figure 104 is a schematic of various exemplary configurations of
multivalent
molecules. Left: schematics of multivalent molecules having a starburst or
helter-skelter
configuration. Center: a schematic of a multivalent molecule having a
dendrimer configuration.
Right: a schematic of multiple multivalent molecules formed by reacting
streptavidin with 4-arm
or 8-arm PEG-NHS with biotin and dNIPs. Nucleotide units are designated 'N',
biotin is
designated 13', and streptavidin is designated 'SA'.
[00231] Figure 105 is a schematic of an exemplary multivalent molecules
comprising a
generic core attached to a plurality of nucleotide-arms.
[00232] Figure 106 is a schematic of an exemplary multivalent molecule
comprising a
dendrimer core attached to a plurality of nucleotide-arms.
[00233] Figure 107 shows a schematic of an exemplary multivalent molecule
comprising a
core attached to a plurality of nucleotide-arms, where the nucleotide arms
comprise biotin,
spacer, linker and a nucleotide unit

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[002341 Figure 108 is a schematic of an exemplary nucleotide-arm comprising a
core
attachment moiety, spacer, linker and nucleotide unit.
[002351 Figure 109 shows the chemical structure of an exemplary spacer, and
the chemical
structures of various exemplary linkers, including an 11-atom Linker, 16-atom
Linker, 23-atom
Linker and an N3 Linker.
1002361 Figure 110 shows the chemical structures of various exemplary linkers,
including
Linkers 1-9.
1002371 Figure 111 shows the chemical structures of various exemplary linkers
joined/attached to nucleotide units.
1002381 Figure 112 shows the chemical structures of various exemplary linkers
joined/attached to nucleotide units.
1002391 Figure 113 shows the chemical structures of various exemplary linkers
joined/attached to nucleotide units.
[002401 Figure 11.4 shows the chemical structure of an. exemplary nucleotide-
arm. In this
example, the nucleotide unit is connected to the linker via a propargyl amine
attachment at the 5
position of a pyrimidine base or the 7 position of a purine base. This
nucleotide-arm shows an
exemplary biotinylated nucleotide-arm.
[00241.1 Figure 11.5 is an exemplary schematic illustration of one embodiment
of the low
binding support comprising a glass substrate and alternating layers of
hydrophilic coatings which
are covalently or non-covalently adhered to the glass, and which further
comprises chemically-
reactive functional groups that serve as attachment sites for oligonucleotide
primers (e.g. capture
oligonucleotides and circularization oligonucleotides). in an alternative
embodiment, the support
can be made of any material such as glass, plastic or a polymer material.
[002421 Figure 116A is a schematic of a guanine tetrad (e.g., G-tetrad).
[002431 Figure 116B is a schematic of an intramolecular G-quadruplex
structure.
[00244] Figure 11.7 is a schematic of an exemplary single cycle showing
flowing in a nucleic
acid relaxing buffer with temperature ramp-up and ramp-down, a washing step,
and flowing in a
flexing amplification buffer containing a strand-displacing DNA polymerase
with temperature
ramp-up and MBA incubation and ramp-down. One or more cycles can be conducted
of the
flowing in a flexing amplification buffer containing a strand-displacing DNA
polymerase with
temperature ramp-up and MBA amplification and ramp-down.

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[00245] Figure 118 (left) is a graph showing the error rate from RI sequencing
reads of
template molecules having various levels of uracil. Figure 118 (right) is a
graph showing the
phasing rate from RI sequencing reads of template molecules having various
levels of uracil.
The data shows that sequencing template molecules having lower levels of
incorporated uracil
yield lower error rates and phasing rates. The level of uracil in the template
molecules also
affects the intensity ratio of R2/R1 reads.
[00246] Figure 119 is a graph showing increased ratio of signal intensity
for R2/R1
sequencing reads when the sequencing workflow employs a cleaving reagent that
includes a.
compound that reduces photo-damage to nucleic acids. Lanes 1, 3, 5 and 7 show
the IC/R1
signal intensity using different cleaving reagent formulations without a
compound that reduces
photo-damage. Lanes 2, 4, 6 and 8 show the R2/R1 signal intensity using
corresponding cleaving
reagent formulations that include a compound that reduces photo-damage.
DETAILED DESCRIPTION
Definitions
1002471 The headings provided herein are not limitations of the various
aspects of the
disclosure, which aspects can be understood by reference to the specification
as a whole.
1002481 Unless defined otherwise, technical and scientific terms used herein
have meanings
that are commonly understood by those of ordinary skill in the art unless
defined otherwise.
Generally, terminologies pertaining to techniques of molecular biology,
nucleic acid chemistry,
protein chemistry, genetics, microbiology, transgenic cell production, and
hybridization
described herein are those well-known and commonly used in the art. Techniques
and procedures
described herein are generally performed according to conventional methods
well known in the
art and as described in various general and more specific references that are
cited and discussed
-throughout the instant specification. For example, see Sambrook et al.,
Molecular Cloning: A
Laboratory Manual (Third ed., Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y,
2000). See also Ausubel et al., Current Protocols in Molecular Biology, Greene
Publishing
Associates (1992). The nomenclatures utilized in connection with, and the
laboratory procedures
and techniques described herein are those well-known and commonly used in the
art,

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[00249] Unless otherwise required by context herein, singular terms shall
include pluralities
and plural terms shall include the singular. Singular forms "a", "an" and
"the", and singular use
of any word, include plural referents unless expressly and unequivocally
limited on one referent.
[00250] It is understood the use of the alternative term (e.g., "or") is
taken to mean either one
or both or any combination thereof of the alternatives.
1002511 The term "and/or" used herein is to be taken mean specific disclosure
of each of the
specified features or components with or without the other. For example, the
term "and/or" as
used in a phrase such as "A and/or B" herein is intended to include: "A and
B"; "A or B"; "A"
(A alone); and "B" (B alone). In a similar manner, the term "and/or" as used
in a phrase such as
"A, B, and/or C" is intended to encompass each of the following aspects: "A,
B, and C"; "A, B,
or C"; "A or C"; "A or B"; "B or C"; "A and B"; "B and C"; "A and C"; "A" (A
alone); "B" (B
alone); and "C" (C alone).
[002521 As used herein and in the appended claims, term "comprising",
"including",
"having" and "containing", and their grammatical variants, as used herein are
intended to be non-
limiting so that one item or multiple items in a list do not exclude other
items that can be
substituted or added to the listed items. It is understood that wherever
aspects are described
herein with the language "comprising," otherwise analogous aspects described
in terms of
consisting of' and/or "consisting essentially of' are also provided.
[00253] As used herein, the terms "about" and "approximately" refer to a value
or
composition that is within an acceptable error range for the particular value
or composition as
determined by one of ordinary skill in the art, which will depend in part on
how the value or
composition is measured or determined, i.e., the limitations of the
measurement system. For
example, "about" or "approximately" can mean within one or more than one
standard deviation
per the practice in the art. Alternatively, "about" or "approximately" can
mean a range of up to
10% (i.e., +10%) or more depending on the limitations of the measurement
system. For example,
about 5 mg can include any number between 4.5 mg and 5.5 mg. Furthermore,
particularly with
respect to biological systems or processes, the terms can mean up to an order
of magnitude or up
to 5-fold of a value. When particular values or compositions are provided in
the instant
disclosure, unless otherwise stated, the meaning of "about" or "approximately"
should he
assumed to be within an acceptable error range for that particular value or
composition. Also,

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where ranges and/or subranges of values are provided, the ranges and/or
subranges can include
the endpoints of the ranges and/or subranges.
[00254j The term "biological sample" refers to a single cell, a plurality of
cells, a tissue, an
organ, an organism, or section of any of these biological samples. The
biological sample can be
extracted (e.g., biopsied) from an organism, or obtained from a cell culture
grown in liquid or in
a culture dish. The biological sample comprises a sample that is fresh,
frozen, fresh frozen, or
archived (e.g., formalin-fixed paraffin-embedded; FFPE). The biological sample
can be
embedded in a wax, resin, epoxy or agar. The biological sample can be fixed,
for example in any
one or any combination of two or more of acetone, ethanol, methanol,
formaldehyde,
pamfonnaldehyde-Triton or glutaraldehyde. The biological sample can be
sectioned or non-
sectioned. The biological sample can be stained, de-stained or non-stained.
1002551 The nucleic acids of interest can be extracted from biological samples
using any of a
number of techniques known to those of skill in the art. For example, a
typical DNA extraction
procedure comprises (i) collection of the cell sample or tissue sample from
which DNA is to be
extracted, (ii) disruption of cell membranes (i.e., cell lysis) to release DNA
and other
cytoplasmic components, (iii) treatment of the lysed sample with a
concentrated salt solution to
precipitate proteins, lipids, and RNA., followed by centrifugation to separate
out the precipitated
proteins, lipids, and RNA, and (iv) purification of DNA from the supernatant
to remove
detergents, proteins, salts, or other reagents used during the cell membrane
lysis. A variety of
suitable commercial nucleic acid extraction and purification kits are
consistent with the
disclosure herein. Examples include, but are not limited to, the QIAamp kits
(for isolation of
genomic DNA from human samples) and DNAeasy kits (for isolation of genomic DNA
from
animal or plant samples) from Qiagen (Germantown, MD), or the Maxwell and
ReliaPrepTM
series of kits from Promega (Madison, WI).
[00256] The terms "nucleic acid", "polynucleotide" and "oligonucleotide" and
other related
terms used herein are used interchangeably and refer to polymers of
nucleotides and are not
limited to any particular length. Nucleic acids include recombinant and
chemically-synthesized
forms. Nucleic acids can be isolated. Nucleic acids include DNA molecules
(e.g., cDNA or
genomic DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNA generated
using
nucleotide analogs (e.g., peptide nucleic acids (PNA) and non-naturally
occurring nucleotide
analogs), and chimeric forms containing DNA and RNA. Nucleic acids can be
single-stranded

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or double-stranded. Nucleic acids comprise polymers of nucleotides, where the
nucleotides
include natural or non-natural bases and/or sugars. Nucleic acids comprise
naturally-occurring
internucleosidic linkages, for example phosphdiester linkages. Nucleic acids
can lack a
phosphate group. Nucleic acids comprise non-natural internucleoside linkages,
including
phosphorothioate, phosphorothiolate, or peptide nucleic acid (MA) linkages. In
some
embodiments, nucleic acids comprise a one type of polynucleotides or a mixture
of two or more
different types of polynucleotides.
1002571 The term "universal sequence", "universal adaptor sequences" and
related terms
refers to a sequence in a nucleic acid molecule that is common among two or
more
polynucleotide molecules. For example, adaptors having the same universal
sequence can be
joined to a plurality of polynucleotides so that the population of co-joined
molecules carry the
same universal adaptor sequence. Examples of universal adaptor sequences
include an
amplification primer sequence, a sequencing primer sequence or a capture
primer sequence (e.g.,
soluble or support-immobilized capture primers).
[002581 The term. "operably linked" and "operably joined" or related terms
as used herein
refers to juxtaposition of components, The juxtapositioned components can be
linked together
covalently. For example, two nucleic acid components can be enzymatically
li.gated together
where the linkage that joins together the two components comprises
phosphodiester linkage. A
first and second nucleic acid component can be linked together, where the
first nucleic acid
component can confer a function on a second nucleic acid component. For
example, linkage
between a primer binding sequence and a sequence of interest forms a nucleic
acid library
molecule having a portion that can bind to a primer. In another example, a
transgene (e.g., a
nucleic acid encoding a polypeptide or a nucleic acid sequence of interest)
can be ligated to a
vector where the linkage permits expression or functioning of the transgene
sequence contained
in the vector. In some embodiments, a transgene is operably linked to a host
cell regulatory
sequence (e.g., a promoter sequence) that affects expression of the transgene.
In some
embodiments, the vector comprises at least one host cell regulatory sequence,
including a
promoter sequence, enhancer, transcription and/or translation initiation
sequence, transcription
and/or translation termination sequence, polypeptide secretion signal
sequences, and the like. In
some embodiments, the host cell regulatory sequence controls expression of the
level, timing
and/or location of the transgene.

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[00259] The terms "linked", "joined", "attached", "appended" and variants
thereof comprise
any type of fusion, bond, adherence or association between any combination of
compounds or
molecules that is of sufficient stability to withstand use in the particular
procedure. The
procedure can include but are not limited to: nucleotide binding; nucleotide
incorporation; de-
blocking (e.g., removal of chain-terminating moiety); washing; removing;
flowing; detecting;
imaging and/or identifying. Such linkage can comprise, for example, covalent,
ionic, hydrogen,
dipole-dipole, hydrophilic, hydrophobic, or affinity bonding, bonds or
associations involving van
der Waals forces, mechanical bonding, and the like. In some embodiments, such
linkage occurs
intramolecularly, for example linking together the ends of a single-stranded
or double-stranded
linear nucleic acid molecule to form a circular molecule. In some embodiments,
such linkage
can occur between a combination of different molecules, or between a molecule
and a non-
molecule, including but not limited to: linkage between a nucleic acid
molecule and a solid
surface; linkage between a protein and a detectable reporter moiety; linkage
between a
nucleotide and detectable reporter moiety; and the like. Soule examples of
linkages can be found,
for example, in Hermanson, G.., "Bioconjugate Techniques", Second Edition
(2008); Asla.m, M.,
Dent, A., "Bioconjugation: Protein Coupling Techniques for the Biomedical
Sciences", London:
Macmillan(1998); Aslam, M., Dent, A.., "Bioconjugation: Protein Coupling
Techniques for the
Biomedical Sciences", London: Macmillan (1.998).
[002601 The term "adaptor" and related terms refers to oligonucleotides
that can be operably
linked (appended) to a target polynucleotide, where the adaptor confers a
function to the co-
joined adaptor-target molecule. Adaptors comprise DNA., RNA, chimeric DNA/RNA,
or analogs
thereof. Adaptors can include at least one ribonucleoside residue. Adaptors
can be single-
stranded, double-stranded, or have single-stranded and/or double-stranded
portions. Adaptors can
be configured to be linear, stem-looped, hairpin, or Y-shaped forms. Adaptors
can be any length,
including 4-100 nucleotides or longer. Adaptors can have blunt ends, overhang
ends, or a
combination of bath. Overhang ends include 5' overhang and 3' overhang ends.
The 5' end of a
single-stranded adaptor, or one strand of a double-stranded adaptor, can have
a 5' phosphate
group or lack a 5' phosphate group. Adaptors can include a 5' tail that does
not hybridize to a
target polynucleotide (e.g., tailed adaptor), or adaptors can be non-tailed.
An adaptor can include
a sequence that is complementary to at least a portion of a primer, such as an
amplification
primer, a sequencing primer, or a capture primer (e.g., soluble or immobilized
capture primers).

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Adaptors can include a random sequence or degenerate sequence. Adaptors can
include at least
one inosine residue. Adaptors can include at least one phosphorothioate,
phosphorothiolate
and/or phosphoramidate linkage. Adaptors can include a barcode sequence which
can be used to
distinguish polynucleotides (e.g., insert sequences) from different sample
sources in a multiplex
assay. Adaptors can include a unique identification sequence (e.g., unique
molecular index,
UMI; or a unique molecular tag) that can be used to uniquely identify a
nucleic acid molecule to
which the adaptor is appended. In some embodiments, a unique identification
sequence can be
used to increase error correction and accuracy, reduce the rate of false-
positive variant calls
and/or increase sensitivity of variant detection. Adaptors can include at
least one restriction
enzyme recognition sequence, including any one or any combination of two or
more selected
from a group consisting of type I, type II, type III, type IV, type Hs or type
IIB.
1002611 The term "nucleic acid template", "template polynucleotide", "nucleic
acid target"
"target polynucleotide", "template strand" and other variations refer to a
nucleic acid strand that
serves as the basis nucleic acid molecule for any of the analysis methods
describe herein (e.g.,
primer extension, amplifying and/or sequencing). The template nucleic acid can
be single-
stranded or double-stranded, or the template nucleic acid can have single-
stranded or double-
stranded portions. The template nucleic acid can be obtained from a naturally-
occurring source,
recombinant form, or chemically synthesized to include any type of nucleic
acid analog. The
template nucleic acid can be linear, circular, or other forms. The template
nucleic acids can
include an insert region having an insert sequence which is also known as a
sequence of interest.
The template nucleic acids can also include at least one adaptor sequence. The
template nucleic
acid can be a concatemer having two or tandem copies of a sequence of interest
and at least one
adaptor sequence. The insert region can be isolated in any form, including
chromosomal,
genomic, organellar (e.g., mitochondrial, chloroplast or ribosomal),
recombinant molecules,
cloned, amplified, cDNA, RNA such as precursor mRNA or mRNA, oligonucleotides,
whole
genomic DNA, obtained from fresh frozen paraffin embedded tissue, needle
biopsies, circulating
tumor cells, cell free circulating DNA, or any type of nucleic acid library.
The insert region can
be isolated from any source including from organisms such as prokaryotes,
eukaiyotes (e.g.,
humans, plants and animals), fungus, viruses cells, tissues, normal or
diseased cells or tissues,
body fluids including blood, urine, serum, lymph, tumor, saliva, anal and
vaginal secretions,
amniotic samples, perspiration, semen, environmental samples, culture samples,
or synthesized

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nucleic acid molecules prepared using recombinant molecular biology or
chemical synthesis
methods. The insert region can be isolated from any organ, including head,
neck, brain, breast,
ovary, cervix, colon, rectum, endometrium, gallbladder, intestines, bladder,
prostate, testicles,
liver, lung, kidney, esophagus, pancreas, thyroid, pituitary, thymus, skin,
heart, larynx, or other
organs. The template nucleic acid can be subjected to nucleic acid analysis,
including sequencing
and composition analysis.
[00262] The term "polymerase" and its variants, as used herein, comprises an
enzyme
comprising a domain that binds a nucleotide (or nucleoside) where the
polymerase can form a
complex having a template nucleic acid and a complementary nucleotide. The
polymerase can
have one or more activities including, but not limited to, base analog
detection activities, DNA
polymerization activity, reverse transcriptase activity, DNA binding, strand
displacement
activity, and nucleotide binding and recognition. A polymerase can be any
enzyme that can
catalyze polymerization of nucleotides (including analogs thereof) into a
nucleic acid strand.
Typically but not necessarily such nucleotide polymerization can occur in a
template-dependent
fashion. Typically, a polymerase comprises one or more active sites at which
nucleotide binding
and/or catalysis of nucleotide polymerization can occur. In some embodiments,
a polymerase
includes other enzymatic activities, such as for example, 3' to 5' exonuclease
activity or 5' to 3'
exonuclease activity. In some embodiments, a polymerase has strand displacing
activity. A
polymerase can include without limitation naturally occurring polymerases and
any subunits and
truncations thereof, mutant polymerases, variant polymerases, recombinant,
fusion or otherwise
engineered polymerases, chemically modified polymerases, synthetic molecules
or assemblies,
and any analogs, derivatives or fragments thereof that retain the ability to
catalyze nucleotide
polymerization (e.g., catalytically active fragment). The polymerase includes
catalytically
inactive polymerases, catalytically active polymerases, reverse
transcriptases, and other enzymes
comprising a nucleotide binding domain. In some embodiments, a polymerase can
be isolated
from a cell, or generated using recombinant DNA technology or chemical
synthesis methods. In
some embodiments, a polymerase can be expressed in prokaryote, eukaryote,
viral, or phage
organisms. In some embodiments, a polymerase can be post-translationally
modified proteins or
fragments thereof. A polymerase can be derived from a prokaryote, eukaryote,
virus or phage. A
polymerase comprises DNA-directed DNA polymerase and RNA-directed DNA
polymerase.

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[00263j The term "strand displacing" refers to the ability of a polymerase to
locally separate
strands of double-stranded nucleic acids and synthesize a new strand in a
template-based manner.
Strand displacing polymerases displace a complementary strand from a template
strand and
catalyze new strand synthesis. Strand displacing polymerases include
mesophilic and
thermophilic polymerases. Strand displacing polymerases include wild type
enzymes, and
variants including exonuclease minus mutants, mutant versions, chimeric
enzymes and truncated
enzymes. Examples of strand displacing polymerases include phi29 DNA
polymerase, large
fragment of Bst DNA polymerase, large fragment of Bsu DNA polymerase (exo-),
Bca DNA
polymerase (exo-), Klenow fragment of E. coli DNA polymerase, T5 polymerase, M-
MuLV
reverse transcriptase, HIV viral reverse transcriptase, Deep Vent DNA
polymerase and KOD
DNA polymerase. The phi29 DNA polymerase can be wild type phi29 DNA polymerase
(e.g.,
MagniPhi from Expedeon), or variant EquiPhi29 DNA polymerase (e.g., from
Thermo Fisher
Scientific), or chimeric QualiPhi DNA polymerase (e.g., from 4basebio).
[00264] As used herein, the term "DNA. primase-polymerase" and related terms
refers to
enzymes having activities of a DNA polymerase and an RNA primase. A DNA
primase-
polymerase enzyme can utilize deoxyribonucleotide triphosphates to synthesize
a DNA primer
on a single-stranded DNA template in a template-sequence dependent manner, and
can extend
the primer strand via nucleotide polymerization (e.g., primer extension), in
the presence of a
catalytic divalent cation (e.g., magnesium and/or manganese). The DNA primase-
polymerase
include enzymes that are members of DnaG-like primases (e.g., bacteria) and
AEP-like primases
(Archaea and Eukaiyotes). An exemplary DNA primase-polymerase enzyme is Tth
PrimPol
from Thermus= thermophilus HB27.
[00265] As used herein, the term "fidelity" refers to the accuracy of DNA
polymerization by
template-dependent DNA polymerase. The fidelity of a DNA polymerase is
typically measured
by the error rate (the frequency of incorporating an inaccurate nucleotide,
i.e., a nucleotide that is
not complementary to the template nucleotide). The accuracy or fidelity of DNA
polymerization
is maintained by both the polymerase activity and the 3'-5' exonuclease
activity of a DNA
polymerase.
[00266] As used herein, the term "binding complex" refers to a complex formed
by binding
together a nucleic acid duplex, a polymerase, and a free nucleotide or a
nucleotide unit of a
multivalent molecule, where the nucleic acid duplex comprises a nucleic acid
template molecule

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hybridized to a nucleic acid primer. In the binding complex, the free
nucleotide or nucleotide
unit may or may not be bound to the 3 end of the nucleic acid primer at a
position that is
opposite a complementary nucleotide in the nucleic acid template molecule. A
"ternary,
complex" is an example of a binding complex which is formed by binding
together a nucleic acid
duplex, a polymerase, and a free nucleotide or nucleotide unit of a
multivalent molecule, where
the free nucleotide or nucleotide unit is bound to the 3' end of the nucleic
acid primer (as part of
the nucleic acid duplex) at a position that is opposite a complementary
nucleotide in the nucleic
acid template molecule.
[002671 The term "persistence time" and related terms refers to the length
of time that a
binding complex remains stable without dissociation of any of the components,
where the
components of the binding complex include a nucleic acid template and nucleic
acid primer, a
polymerase, a nucleotide unit of a multivalent molecule or a free (e.g.,
unconjugated) nucleotide.
The nucleotide unit or the free nucleotide can be complementary or non-
complementary to a
nucleotide residue in the template molecule. The nucleotide unit or the free
nucleotide can bind
to the 3' end of the nucleic acid primer at a position that is opposite a
complementary nucleotide
residue in the nucleic acid template molecule. The persistence time is
indicative of the stability
of the binding complex and strength of the binding interactions, Persistence
time can be
measured by observing the onset and/or duration of a binding complex, such as
by observing a
signal from a labeled component of the binding complex. For example, a labeled
nucleotide or a
labeled reagent comprising one or more nucleotides may be present in a binding
complex, thus
allowing the signal from the label to be detected during the persistence time
of the binding
complex. One exemplary label is a fluorescent label. The binding complex
(e.g., ternary
complex) remains stable until subjected to a condition that causes
dissociation of interactions
between any of the polymerase, template molecule, primer and/or the nucleotide
unit or the
nucleotide. For example, a dissociating condition comprises contacting the
binding complex with
any one or any combination of a detergent, EDTA and/or water.
[00268] The term "primer" and related terms used herein refers to an
oligonucleotide that is
capable of hybridizing with a DNA and/or RNA polynucleotide template to form a
duplex
molecule. Primers comprise natural nucleotides and/or nucleotide analogs.
Primers can be
recombinant nucleic acid molecules. Primers may have any length, but typically
range from 4-50
nucleotides. A typical primer comprises a 5' end and 3' end. The 3' end of the
primer can

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include a 3 OH moiety which serves as a nucleotide polymerization initiation
site in a
polymerase-catalyzed primer extension reaction. Alternatively, the 3' end of
the primer can lack
a 3' OH moietyõ or can include a terminal 3' blocking group that inhibits
nucleotide
polymerization in a polymerase-catalyzed reaction. Any one nucleotide, or more
than one
nucleotide, along the length of the primer can be labeled with a detectable
reporter moiety. A
primer can be in solution (e.g., a soluble primer) or can be immobilized to a
support (e.g., a
capture primer).
1002691 When used in reference to nucleic acid molecules, the terms
"hybridize" or
"hybridizing" or "hybridization" or other related terms refers to hydrogen
bonding between two
different nucleic acids to form a duplex nucleic acid. Hybridization also
includes hydrogen
bonding between two different regions of a single nucleic acid molecule to
form a self-
hybridizing molecule having a duplex region. Hybridization can comprise Watson-
Crick or
Hoogstein binding to form a duplex double-stranded nucleic acid, or a double-
stranded region
within a nucleic acid molecule. The double-stranded nucleic acid, or the two
different regions of
a single nucleic acid, may be wholly complementary, or partially
complementary.
Complementary nucleic acid strands need not hybridize with each other across
their entire
length The complementary base pairing can be the standard A-T or C-G base
pairing, or can be
other forms of base-pa.iring interactions. Duplex nucleic acids can include
mismatched base-
paired nucleotides,
1002701 When used in reference to nucleic acids, the terms "extend",
"extending", "extension"
and other variants, refers to incorporation of one or more nucleotides into a
nucleic acid
molecule. Nucleotide incorporation. comprises polymerization of one or more
nucleotides into
the terminal 3' OH end of a nucleic acid strand (e.g., a nucleic acid primer),
resulting in
extension of the nucleic acid strand (e.g., extended primer). Nucleotide
incorporation can be
conducted with natural nucleotides and/or nucleotide analogs. Typically, but
not necessarily,
nucleotide incorporation occurs in a template-dependent fashion. Any suitable
method of
extending a nucleic acid molecule may be used, including primer extension
catalyzed by a DNA
polymerase or RNA polymerase.
[00271] In some embodiments, any of the amplification primer sequences,
sequencing primer
sequences, capture primer sequences (capture oligonucleotides), target capture
sequences,
circularization anchor sequences, sample barcode sequences, spatial barcode
sequences, or

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anchor region sequences can be about 3-50 nucleotides in length, or about 5-40
nucleotides in
length, or about 5-25 nucleotides in length.
[00272] The term "nucleotides" and related terms refers to a molecule
comprising an aromatic
base, a five carbon sugar (e.g., ribose or deoxyribose), and at least one
phosphate group.
Canonical or non-canonical nucleotides are consistent with use of the term.
The phosphate in
some embodiments comprises a monophosphate, diphosphate, or triphosphate, or
corresponding
phosphate analog. The term "nucleoside" refers to a molecule comprising an
aromatic base and
a sugar. Nucleotides and nucleosides can be non-labeled or labeled with a
detectable reporter
moiety.
100273] Nucleotides (and nucleosides) typically comprise a hetero cyclic
base including
substituted or unsubstituted nitrogen-containing parent heteroaromatic ring
which are commonly
found in nucleic acids, including naturally-occurring, substituted, modified,
or engineered
variants, or analogs of the same. The base of a nucleotide (or nucleoside) is
capable of forming
Watson-Crick and/or Hoogstein hydrogen bonds with an appropriate complementary
base.
Exemplary bases include, but are not limited to, purines and pyrimidines such
as: 2-aminopurine,
2,6-diaminopurine, adenine (A), ethenoadenine, W-A2-isopenten.yladenine (6iA),
N6-A2-
isopentenyl-2-methylthioa.denine (2ms6iA), N6-methyladenine, guanine (G),
isoguanin.e, N2-
dimethylguanine (dInG), 7-meth.ylguanine (7mG), 2-thiopyrimidi.ne, 6-
thioguanin.e (6sG),
hypoxanthin.e and 06-methylguanine; 7-deaza-purin.es such as 7-deazaadenine (7-
deaza-A) and
7-deazagua.nine (7-deaza-G); pyrimidines such as cytosine (C), 5-
propynylcytosine, isocytosi.ne,
thymine (T), zlathiothymine (4sT), 5,6-dihydrothymine, alarnethylthymine,
uracil (U), 4-
thiouracil (4sIJ) and 5,6-dihydroura.cil (dihydrouracil; D); indoles such as
nitroindole and 4-
methylindole; pyrroles such as nitropyrrole; nebula.rine; inosines;
hydroxymethylcytosines; 5-
methycytosin.es; base (Y); as well as methylated, glycosylated, and a.cylated
base moieties; and
the like. Additional exemplary bases can be found in Fasman, 1989, in
"Practical Handbook of
Biochemistry and Molecular Biology", pp. 385-394, CRC Press, Boca Raton, Fla.
[00274] Nucleotides (and nucleosides) typically comprise a sugar moiety, such
as carbocyclic
moiety (Ferraro and Gotor 2000 Chem. Rev. 100: 4319-48), acyclic moieties
(Martinez, et al.,
1999 Nucleic Acids Research 27: 1271-1274; Martinez, et al., 1997 Bioorganic &
Medicinal
Chemistry Letters vol. 7: 3013-3016), and other sugar moieties (Joeng, et al.,
1993 J. Med.
Chem. 36: 2627-2638; Kim, et al., 1993 J. Med. Chem. 36: 30-7; .Escheninosser
1999 Science

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284:2118-2124; and U.S. Pat. No. 5,558,991). The sugar moiety comprises:
ribosyl; 2'-
deoxyribosyl; 31-deoxyribosyl; 2',3'-dideoxyribosyl; 2',3'-
didehydrodideoxyribosyl; 2'-
alkoxyribosyl; 2'-azidoribosyl; 21-aminoribosy1; 2'-fluororibosyl; 2'-
mercaptoriboxyl; 2'-
alkylthioribosyl; 3'-alkoxyribosyl; 3'-azidoribosyl; 3'-aminoribosyl; 3'-
fluororibosyl; 3'-
mercaptoriboxyl; 31-alkylthioribosyl carbocyclic; acyclic or other modified
sugars.
1002751 In some embodiments, nucleotides comprise a chain of one, two or three
phosphorus
atoms where the chain is typically attached to the 5' carbon of the sugar
moiety via an ester or
phosphoramide linkage. In some embodiments, the nucleotide is an analog having
a phosphorus
chain in which the phosphorus atoms are linked together with intervening 0, S.
NI-I, methylene
or ethylene. In some embodiments, the phosphorus atoms in the chain include
substituted side
groups including 0, S or B113. In some embodiments, the chain includes
phosphate groups
substituted with analogs including phosphoramidate, phosphorothioate,
phosphordithioate, and
0-methylphosphoroamidite groups.
1002761 The term "reporter moiety", "reporter moieties" or related terms
refers to a compound
that generates, or causes to generate, a detectable signal, A reporter moiety
is sometimes called a
"label". Any suitable reporter moiety may be used, including luminescent,
photoluminescent,
electroluminescent, bioluminescent, chemiluminescent, fluorescent,
phosphorescent,
chromophore, radioisotope, electrochemical, mass spectrometry, Raman, hapten,
affinity tag,
atom, or an enzyme. A reporter moiety generates a detectable signal resulting
from a chemical or
physical change (e.g., heat, light, electrical, pH, salt concentration,
enzymatic activity, or
proximity events). A proximity event includes two reporter moieties
approaching each other, or
associating with each other, or binding each other. It is well known to one
skilled in the art to
select reporter moieties so that each absorbs excitation radiation and/or
emits -fluorescence at a
wavelength distinguishable from the other reporter moieties to permit
monitoring the presence of
different reporter moieties in the sam.e reaction or in different reactions.
Two or more different
reporter moieties can be selected having spectrally distinct emission
profiles, or having minimal
overlapping spectral emission profiles. Reporter moieties can be linked (e.g.,
operably linked) to
nucleotides, nucleosides, nucleic acids, enzymes (e.g., polym.erases or
reverse transcriptases), or
support (e.g., surfaces).
[00277] A reporter moiety (or label) comprises a fluorescent label or a
fluorophore.
Exemplary fluorescent moieties which may serve as fluorescent labels or
fluorophores include,

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but are not limited to fluorescein and fluorescein derivatives such as
carboxyfluorescein,
tetrachlorofluorescein, hexachlorofluorescein, carboxynapthofluorescein,
fluorescein
isothiocyanate, NHS-fluorescein, iodoacetamidofluorescein, fluorescein
maleimide, SAMSA-
fluorescein, fluorescein thiosemicarbazide, carbohydrazinomethylthioacetyl-
amino fluorescein,
rhodamine and rhodamine derivatives such as TRFIC, T.MR, lissamine rhodamine,
Texas Red,
rhodamine B, rhodamine 6G, rhodamine 10, NHS-rhodamine, TMR-iodoacetamide,
lissarnine
rhodamine B sulfonyl chloride, lissamine rhodamine B sulfonyl hydrazine, Texas
Red sulfonyl
chloride, Texas Red hydrazide, coumarin and coumarin derivatives such as AMCA,
AMCA-
NHS, A_MCA-sulfo-NHS, AMCA-HPDP, DCIA, AMCE-hydrazide, BODIPY and derivatives
such as BODIPY FL C3-SE, BODIPY 530/550 C3, BODIPY 530/550 C3-SE, BODIPY
530/550
C3 hydrazide, BODIPY 493/503 C3 hydrazide, BOD1PY FL C3 hydrazide, BODIPY FL
IA,
BODIPY 530/551 IA, Br-BODIPY 493/503, Cascade Blue and derivatives such as
Cascade Blue
acetyl azide, Cascade Blue cadaverine, Cascade Blue ethylenediainine, Cascade
Blue hydrazide,
Lucifer Yellow and derivatives such as Lucifer Yellow iodoa.cetamide, Lucifer
Yellow CH,
cyanine and derivatives such as indolium based cyanine dyes, ben.zo-indolium
based cyanine
dyes, pyridium based cyanine dyes, thiozolium based cyanine dyes, quinolinium
based cyanine
dyes, imidazolium based cyanine dyes, Cy 3, Cy5, lanthanide chelates and
derivatives such as
BCPDA, TBP, TMT, BHHCT, BCOT, Europium chelates, Terbium chelates, Alexa Fluor
dyes,
DyLight dyes, Atto dyes, LightCycler Red dyes, CAL Flour dyes, JOE and
derivatives thereof,
Oregon Green dyes, WelIRED dyes, IRD dyes, phycoerythrin and phycobilin dyes,
Malachite
green, stilbene, DEG dyes, NR dyes, near-infrared dyes and others known in the
art such as those
described in Haugland, Molecular Probes Handbook, (Eugene, Oreg.) 6th Edition;
Lakowicz,
Principles of Fluorescence Spectroscopy, 2nd Ed., Plenum Press New York
(1999), or
Hermanson, Bioconjugate Techniques, 2nd Edition, or derivatives thereof, or
any combination
thereof Cyanine dyes may exist in either sulfonated or non-sulfonated forms,
and consist of two
indolenin, benzo-indolium, pyridium, thiozolium, and/or quinolinium groups
separated by a
polymethine bridge between. two nitrogen atoms. Commercially available cyanine
fluorophores
include, for example, Cy3, (which may comprise 116-(2,5-dioxopyrrolidin-l-
yloxy)-6-
oxoh.exyl]-2-(3- {146-(2,5-dioxopyrrol idin-1 -yloxy)-6-oxohexyli-3,3-dimethy1-
1,3-dihydro-2H-
indo1-2-ylidenel prop-1 -en-1. -y1)-3,3-dimethy1-3H- indolium or 1-[6-(2,5-
dioxopyrrolidin-l-
yloxy)-6-oxohexylj-2-(3- {1- [6-(2,5-dioxopyrrolid in-l-yloxy)-6-oxohexy 11-
3,3-d imethy1-5 -s ulfo-

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1,3-dihydro-2H-indol-2-ylidenel prop-1. -en-l-y1)-3,3-dimethyl-317I-indolium-5-
sulfonate), Cy5
(which may comprise 1-(6-((2,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-2-41E,3E)-
5-4E)-1-(6-
((2,5-dioxopyrrolidin-1-y1)oxy)-6-oxohexyl)-3,3-dimethyl-5-indolin-2-
ylidene)penta-1,3-dien-1-
y1)-3,3-dimethyl-31-I-indol-1-ium or 1-(6-((2,5-dioxopyrrolidin-1-ypoxy)-6-
oxohexyl)-2-
4 1.E,3E,)-54(E)-1-(6-((2,5-dioxopyrrolidin-l-ypoxy)-6-oxohexyl)-3,3-dimethyl-
5-sulfoindoi in-
2-ylidene)penta-1,3-dien-l-y1)-3,3-dimethyl-3H-indol-l-ium-5-sulfonate), and
Cy7 (which may
comprise 1-(5-carboxvpenty1)-2-1(1E,3E,5E,7Z)-7-(1-ethyl-1,3-dihydro-2H-indol-
2-
ylidene)hepta-1,3,5-trien-1-01-3H-indolium or 1-(5-carboxypent0)-2-
[(1E,3E,5E,7Z)-7-(1-
ethyl-5-sulfo-1,3-dihydro-2H-indo1-2-ylidene)hepta-1,3,5-trien-1-01-3H-
indolium-5-sulfonate),
where "Cy" stands for 'cyanine', and the first digit identifies the number of
carbon atoms
between two indolenine groups. Cy2 which is an oxazole derivative rather than
indolenin, and
the benzo-derivatized Cy3.5, Cy5.5 and Cy7.5 are exceptions to this rule.
[002781 In some embodiments, the reporter moiety can be a FRET pair, such that
multiple
classifications can be performed under a single excitation and imaging step.
As used herein,
FRET may comprise excitation exchange (Forster) transfers, or electron-
exchange (Dexter)
transfers.
[002791 The term "support" as used herein refers to a substrate that is
designed for deposition
of biological molecules or biological samples for assays and/or analyses.
Examples of biological
molecules to be deposited onto a support include nucleic acids (e.g., DNA,
RNA.), polypeptides,
saccharides, lipids, a single cell or multiple cells. Examples of biological
samples include but are
not limited to saliva, phlegm, mucus, blood, plasma, serum, urine, stool,
sweat, tears and fluids
from. tissues or organs.
[00280] In some embodiments, the support is solid, semi-solid, or a
combination of both. In
some embodiments, the support is porous, semi-porous, non-porous, or any
combination of
porosity. In some embodiments, the support can be substantially planar,
concave, convex, or any
combination thereof. In some embodiments, the support can be cylindrical, for
example
comprising a capillary or interior surface of a capillary.
1002811 In some embodiments, the surface of the support can be substantially
smooth. In
some embodiments, the support can be regularly or irregularly textured,
including bumps,
etched, pores, three-dimensional scaffolds, or any combination thereof.

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[00282] In some embodiments, the support comprises a bead having any shape,
including
spherical, hemi-spherical, cylindrical, barrel-shaped, toroidal, disc-shaped,
rod-like, conical,
triangular, cubical, polygonal, tubular or wire-like.
[00283] The support can be fabricated from any material, including but not
limited to glass,
fused-silica, silicon, a polymer (e.g., polystyrene (PS), macroporous
polystyrene (MPPS),
polymethylmethacrylate (PMMA), polycarbonate (PC), polypropylene (PP),
polyethylene (PE),
high density polyethylene (HDPE), cyclic olefin polymers (COP), cyclic olefin
copolymers
(COC), polyethylene terephthalate (PET)), or any combination thereof. Various
compositions of
both glass and plastic substrates are contemplated.
1002841 The support can have a plurality (e.g., two or more) of nucleic
acid templates
immobilized thereon. The plurality of immobilized nucleic acid templates have
the same
sequence or have different sequences. in some embodiments, individual nucleic
acid template
molecules in the plurality of nucleic acid templates are immobilized to a
different site on the
support. In some embodiments, two or more individual nucleic acid template
molecules in the
plurality of nucleic acid templates are immobilized to a site on the support.
1002851 The term "array" refers to a support comprising a plurality of
sites located at pre-
determined locations on the support to form an array of sites. The sites can
be discrete and
separated by interstitial regions. In some embodiments, the pre-determined
sites on the support
can be arranged in one dimension in a row or a column, or arranged in two
dimensions in rows
and columns. In some embodiments, the plurality of pre-determined sites is
arranged on the
support in an organized fashion. In some embodiments, the plurality of pre-
determined sites is
arranged in any organized pattern, including rectilinear, hexagonal patterns,
grid patterns,
patterns having reflective symmetry, patterns having rotational symmetry, or
the like The pitch
between different pairs of sites can be that same or can vary In sonic
embodiments, the support
comprises at least 102 sites, at least 103 sites, at least 104 sites, at least
105 sites, at least 106 sites,
at least 107 sites, at least 108 sites, at least 109 sites, at least 1019
sites, at least 1011 sites, at least
1012 sites, at least 1013 sites, at least 1014 sites, at least 1015 sites, or
more, where the sites are
located at pre-determined locations on the support. In sonic embodiments, a
plurality of pre-
determined sites on the support (e.g., 102 -- 015 sites or more) are
immobilized with nucleic acid
templates to form a nucleic acid template array. In some embodiments, the
nucleic acid templates
that are immobilized at a plurality of pre-determined sites by hybridization
to immobilized

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surface capture primers, or the nucleic acid templates are covalently attached
to the surface
capture primer. In some embodiments, the nucleic acid templates that are
immobilized at a
plurality of pre-determined sites, for example immobilized at 102 - 10" sites
or more. In some
embodiments, the immobilized nucleic acid templates are clonally-amplified to
generate
immobilized nucleic acid polonies at the plurality of pre-determined sites. In
some
embodiments, individual immobilized nucleic acid polonies comprise single-
stranded or double-
stranded concatemers.
1002861 In some embodiments, a support comprising a plurality of sites located
at random
locations on the support is referred to herein as a support having randomly
located sites thereon.
The location of the randomly located sites on the support are not pre-
determined. The plurality of
randomly-located sites is arranged on the support in a disordered and/or
unpredictable fashion. In
some embodiments, the support comprises at least 102 sites, at least 103
sites, at least 104 sites, at
least 105 sites, at least 106 sites, at least 107 sites, at least 108 sites,
at least 109 sites, at least 1010
sites, at least 10" sites, at least 1012 sites, at least 1013 sites, at least
10" sites, at least 10" sites,
or more, where the sites are randomly located on the support. In some
embodiments, a plurality
of randomly located sites on the support (e.g., 102 - 10" sites or more) are
immobilized with
nucleic acid templates to form a support immobilized with nucleic acid
templates. In some
embodiments, the nucleic acid templates that are immobilized at a plurality of
randomly located
sites by hybridization to immobilized surface capture primers, or the nucleic
acid templates are
covalently attached to the surface capture primer. In some embodiments, the
nucleic acid
templates that are immobilized at a plurality of randomly located sites, for
example immobilized
at 102 10" sites or more. In some embodiments, the immobilized nucleic acid
templates are
clonally-amplified to generate immobilized nucleic acid polonies at the
plurality of randomly
located sites. In some embodiments, individual immobilized nucleic acid
polonies comprise
single-stranded or double-stranded concatemers.
[00287] When used in reference to a low binding surface coating, one or more
layers of a
multi-layered surface coating may comprise a branched polymer or may be
linear. Examples of
suitable branched polymers include, but are not limited to, branched PEG;
branched poly(vinyl
alcohol) (branched PVA), branched poly(vinyl pyridine), branched poly(vinyl
pyrrolidone)
(branched PVP), branched), poly(acrylic acid) (branched PAA), branched
polyacrylamide,
branched poly(N-isopropylacrylamide) (branched PNIPAM), branched poly(methyl

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methacrylate) (branched PMA), branched poly(2-hydroxylethyl methacrylate)
(branched
PHEMA), branched poly(oligo(ethylene glycol) methyl ether methacrylate)
(branched
POEGMA), branched polyglutamic acid (branched P(IA), branched poly-lysine,
branched poly-
glucoside, and dextran.
[00288] In some embodiments, the branched polymers used to create one or more
layers of
any of the multi-layered surfaces disclosed herein may comprise at least 4
branches, at least 5
branches, at least 6 branches, at least 7 branches, at least 8 branches, at
least 9 branches, at least
branches, at least 12 branches, at least 14 branches, at least 16 branches, at
least 18 branches,
at least 20 branches, at least 22 branches, at least 24 branches, at least 26
branches, at least 28
branches, at least 30 branches, at least 32 branches, at least 34 branches, at
least 36 branches, at
least 38 branches, or at least 40 branched.
1002891 Linear, branched, or multi-branched polymers used to create one or
more layers of
any of the multi-layered surfaces disclosed herein may have a molecular weight
of at least 500,
at least 1,000, at least 2,000, at least 3,000, at least 4,000, at least
5,000, at least 10,000, at least
15,000, at least 20,000, at least 25,000, at least 30,000, at least 35,000, at
least 40,000, at least
45,000, or at least 50,000 daltons,
[002901 In some embodiments, e.g., wherein at least one layer of a multi-
layered surface
comprises a branched polymer, the number of covalent bonds between a branched
polymer
molecule of the layer being deposited and molecules of the previous layer may
range from about
one covalent linkage per molecule and about 32 covalent linkages per molecule.
In some
embodiments, the number of covalent bonds between a branched polymer molecule
of the new
layer and molecules of the previous layer may be at least 1, at least 2, at
least 3, at least 4, at least
5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12,
at least 14, at least 16, at least
18, at least 20, at least 22, at least 24, at least 26, at least 28, at least
30, or at least 32 covalent
linkages per molecule.
[00291] Any reactive functional groups that remain following the coupling
of a material layer
to the surface may optionally be blocked by coupling a small, inert molecule
using a high yield
coupling chemistry. For example, in the case that amine coupling chemistry is
used to attach a
new material layer to the previous one, any residual amine groups may
subsequently be
acetylated or deactivated by coupling with a small amino acid such as glycine.

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[00292] The number of layers of low non-specific binding material, e.g., a
hydrophilic
polymer material, deposited on the surface, may range from I. to about 10. In
some
embodiments, the number of layers is at least 1, at least 2, at least 3, at
least 4, at least 5, at least
6, at least 7, at least 8, at least 9, or at least 10. In some embodiments,
the number of layers may
be at most 10, at most 9, at most 8, at most 7, at most 6, at most 5, at most
4, at most 3, at most 2,
or at most 1. Any of the lower and upper values described in this paragraph
may be combined to
form a range included within the present disclosure, for example, in some
embodiments the
number of lavers may range from about 2 to about 4. In some embodiments, all
of the layers
may comprise the same material. In some embodiments, each layer may comprise a
different
material. In some embodiments, the plurality of layers may comprise a
plurality of materials. In
some embodiments at least one layer may comprise a branched polymer. In some
embodiment,
all of the layers may comprise a branched polymer.
[0029.3] One or more layers of low non-specific binding material may in some
cases be
deposited on and/or conjugated to the substrate surface using a polar probe
solvent, a polar or
polar aprotic solvent, a non.polar solvent, or any combination thereof. In
some embodiments the
solvent used for layer deposition and/or coupling may comprise an alcohol
(e.g., methanol,
ethanol, propanol, etc.), another organic solvent (e.g., a.cetonitrile,
dirnethyl sulfoxide (DMS0),
dimethyl form.a.mide (min, etc.), water, an aqueous buffer solution (e.g.,
phosphate buffer,
phosphate buffered saline, 3(. -morpholin.o)propanesulfonic acid (MOPS),
etc.), or any
combination thereof. In some embodiments, an organic component of the solvent
mixture used
may comprise at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,
55%, 60%,
70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the total, with the balance made
up of water
or an aqueous buffer solution. In some embodiments, an aqueous component of
the solvent
mixture used may comprise at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,
45%, 50%,
55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the total, with the
balance made
up of an organic solvent. The pH of the solvent mixture used may be less than
6, about 6, 6.5, 7,
7.5, 8, 8.5, 9, or greater than pH 9.
[00294j The term "branched polymer" and related terms refers to a polymer
having a plurality
of functional groups that help conjugate a biologically active molecule such
as a nucleotide, and
the functional group can be either on the side chain of the polymer or
directly attaches to a
central core or central backbone of the polymer. The branched polymer can have
linear backbone

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with one or more functional groups coming off the backbone for conjugation.
The branched
polymer can also be a polymer haying one or more sidechains, wherein the side
chain has a site
suitable for conjugation. Examples of the functional group include but are
limited to hydroxyl,
ester, amine, carbonate, acetal, aldehyde, aldehyde hydrate, alkenyl,
acrylate, methacrylate,
acrylamide, active sulfone, hydrazide, thiol, alkanoic acid, acid halide,
isocyanate,
isothiocyanate, maleirnide, vinylsulfone, dithiopyridine, vinylpyridine,
iodoacetamide, epoxide,
glyoxal, dione, mesylate, tosylate, and tresylate.
1002951 When used in reference to immobilized nucleic acids, the term
"immobilized" and
related terms refer to nucleic acid molecules that are attached to a support
through covalent bond.
or non-covalent interaction, or attached to a coating on the support, or
buried within a matrix
formed by a coating on the support, where the nucleic acid molecules include
surface capture
primers, nucleic acid template molecules and extension products of capture
primers. Extension
products of capture primers includes nucleic acid concatemers that can form
nucleic acid
polonies,
[002961 In some embodiments, one or more nucleic acid templates are
immobilized on the
support, for example immobilized at the sites on the support. In some
embodiments, the one or
more nucleic acid templates are clonally-amplified. In some embodiments, the
one or more
nucleic acid templates are clonally-amplified off the support (e.g., in-
solution) and then
deposited onto the support and immobilized on the support. In some
embodiments, the clonal
amplification reaction of the one or more nucleic acid templates is conducted
on the support
resulting in immobilization on the support. In some embodiments, the one or
more nucleic acid
templates are clonally-amplified (e.g., in solution or on the support) using a
nucleic acid
amplification reaction, including any one or any combination of: polymerase
chain reaction
(PCR), multiple displacement amplification (AMA), transcription-mediated
amplification
(TMA), nucleic acid sequence-based amplification (NASBA), strand displacement
amplification
(SDA), real-time SDA., bridge amplification, isothermal bridge amplification,
rolling circle
amplification (RCA), circle-to-circle amplification, heli.case-dependent
amplification,
recombinase-dependent amplification, and/or single-stranded binding (SSB)
protein-dependent
amplification.
[00297] The term "surface primer", "surface capture primer" and related
terms refers to
single-stranded oligonucleotides that are immobilized to a support and
comprise a sequence that

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can hybridize to at least a portion of a nucleic acid template molecule.
Surface primers can be
used to immobilize template molecules to a support via hybridization. Surface
primers can be
immobilized to a support in a manner that resists primer removal during
flowing, washing,
aspirating, and changes in temperature, pH, salts, chemical and/or enzymatic
conditions.
Typically, but not necessarily, the 5' end of a surface primer can be
immobilized to a support.
Alternatively, an interior portion or the 3' end of a surface primer can be
immobilized to a
support.
1002981 The surface primers comprise DNA, RNA, or analogs thereof. 'The
surface primers
can include a combination of DNA and RNA. The sequence of surface primers can
be wholly
complementary or partially complementary along their length to at least a
portion of the nucleic
acid template molecule (e.g., linear or circular template molecules). A
support can include a
plurality of immobilized surface primers having the same sequence, or having
two or more
different sequences. Surface primers can be any length, for example 4-50
nucleotides, or 50-100
nucleotides, or 100-1.50 nucleotides, or longer lengths,
[00299] A surface primer can include a terminal 3' nucleotide having a sugar
3' OH moiety
which. is extendible for nucleotide polymerization (e.g., polymerase catalyzed
polymerization). A
surface primer can include a terminal 3' nucleotide having a moiety that
blocks polymerase-
catalyzed extension, A surface primer can include a terminal 3' nucleotide
having the 3' sugar
position linked to a chain-terminating moiety that inhibits nucleotide
polymerization, The 3'
chain-terminating moiety can be removed (e.g., de-blocked) to convert the 3'
end to an
extendible 3' OH end using a de-blocking agent. Examples of chain terminating
moieties include
alkyl group, alkenyl group, alkynyl group, allyl group, aryl group, benzyl
group, azide group,
amine group, amide group, keto group, isocyanate group, phosphate group, thio
group, disulfide
group, carbonate group, urea group, or say] group. Azide type chain
terminating moieties
including azide, azido and azidom ethyl groups. Examples of de-blocking agents
include a
phosphine compound, such as Tris(2-carboxyethyl)phosphine (TCEP) and bis-sulfo
triphenyl
phosphine (BS-TPP), for chain-terminatin.g groups azide, azido and
a.zidomethyl groups.
Examples of de-blocking agents include
tetrakis(triphenylphosphine)palladium(0) (Pd(PP113)4)
with piperidine, or with 2,3-Dichloro-5,6-dicyano-1,4-benzo-quinone (DDQ), for
chain-
terminating groups alkyl, alkenyl, alkynyl and allyi. Examples of a de-
blocking agent includes
Pd/C for chain-terminating groups aryl and benzyl. Examples of de-blocking
agents include

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86
phosphine, beta-mercaptoethanol or dithiothritol (DTI), for chain-terminating
groups amine,
amide, keto, isocyanate, phosphate, thio and disulfide. Examples of de-
blocking agents include
potassium carbonate (K2CO3) in Me0H, triethylamine in pyridine, and Zn in
acetic acid (AcOH),
for carbonate chain-terminating groups. Examples of de-blocking agents include
tetrabutylammonium fluoride, pyridine-HF, with ammonium fluoride, and
triethylamine
trihydrofluoride, for chain-terminating groups urea and silyl.
100300] In some embodiment, the plurality of immobilized surface capture
primers on the
support are in fluid communication with each other to permit flowing a
solution of reagents (e.g.,
linear or circular nucleic acid template molecules, soluble primers, enzymes,
nucleotides,
divalent cations, buffers, reagents and the like) onto the support so that the
plurality of
immobilized surface capture primers on the support can be essentially
simultaneously reacted
with the reagents in a massively parallel manner. In some embodiments, the
fluid communication
of the plurality of immobilized surface capture primers can be used to conduct
nucleic acid
amplification reactions (e.g., RCA, MDA., PCR and bridge amplification)
essentially
simultaneously on the plurality of immobilized surface capture primers.
[00301] In some embodiment, the plurality of immobilized single stranded
nucleic acid
concatemer template molecules on the support are in fluid communication with
each other to
permit flowing a solution of reagents (e.g., soluble primers, enzymes,
nucleotides, divalent
cations, buffers, reagents and the like) onto the support so that the
plurality of immobilized
concatemer template molecules on the support can be essentially simultaneously
reacted with the
reagents in a massively parallel manner. In some embodiments, the fluid
communication of the
plurality of immobilized single stranded nucleic acid concatemer template
molecules can be used
to conduct nucleotide binding assays and/or conduct nucleotide polymerization
reactions (e.g.,
primer extension or sequencing) essentially simultaneously on the plurality of
immobilized
single stranded nucleic acid concatemer template molecules, and optionally to
conduct detection
and imaging for massively parallel sequencing.
[003021 When used in reference to nucleic acids, the terms "amplify",
"amplifying",
"amplification", and other related terms include producing multiple copies of
an original
polynucleotide template molecule, where the copies comprise a sequence that is
complementary
to the template sequence, or the copies comprise a sequence that is the same
as the template
sequence. In some embodiments, the copies comprise a sequence that is
substantially identical to

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a template sequence, or is substantially identical to a sequence that is
complementary to the
template sequence.
[00303i The present disclosure provides various pH buffering agents. The full
name of the pH
buffering agents is listed herein.
The term "'Fris" refers to a pH buffering agent Tris(hydroxymethyl)-
aminomethane. The term
"Tris-HCI" refers to a pH buffering agent Tris(hydroxymethyp-aminomethane
hydrochloride.
The term "Tricine" refers to a pH buffering agent N4tris(hydroxyrnethyl)
inethAglycine. The term "Bicine" refers to a pH buffering agent N,N-bis(2-
hydrox-yethyl)glycine. The term "Bis-Tris propane" refers to a pH buffering
agent 1,3
Bis[tris(hydroxymethyl)methylamino]propane. The term "HEPES" refers to a pH
buffering agent
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid. The term "MES" refers to a
pH buffering
agent 2-(N-morpholino)ethanesulfonic acid). The term "MOPS" refers to a pH
buffering agent 3-
(N-morpholino)propanesulfonic acid. The term "MOPSO" refers to a pH buffering
agent 3-(N-
rnerpholino)-2-hydroxyprepanesulfonic acid. The term "BES" refers to a pH
buffering agent
N,N-bis(2-hydroxyethyl)-2-aininoethanesulfonic acid. The term. "TES" refers to
a pH buffering
agent 2-[(2-Hydroxy-1,1bis(hydroxymethyl)
ethypaminoiethanesulfonic acid). The term "CAPS" refers to a pH buffering
agent 3-
(cyclohexylainine)-1-propanesuninic acid. The term. "TAPS" refers to a pH
buffering agent N-
[Tris(hydroxyrnetbArnethyl]-3-amino propane sulfonic acid. The term "TAPSO"
refers to a pH
buffering agent N4Tris(hydroxyinethyl)netny1.1-3-annno-2-
hyldroxypropansulfonic acid. The
term "ACES" refers to a pH buffering agent N-(2-A.cetamido)-2-
aminoethanesulfonic acid. The
term "PIPES" refers to a pH buffering agent piperazine-1,4-bis(2-ethanesulfmne
acid.
Introduction
[00304] The present disclosure provides compositions and methods that employ
the
compositions for conducting pairwise sequencing and for generating concatem.er
template
molecules for pairwise sequencing.
[00305] Pairwise sequencing comprises obtaining a first sequencing read of a
first region of a
first nucleic acid strand (e.g., sense strand), and obtaining a second
sequencing read of a second
region of a second nucleic acid strand that is complementary to the first
stand (e.g., anti-sense
strand), wherein the first and second strands correspond to two complementary
strands of the
same double stranded template molecule. The first sequencing read of the first
sequenced region

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and the second sequencing read of the second sequenced region can having
overlapping
sequences which correspond to complementary sequences from the first and
second strands of
the double stranded template molecule. The first and second sequencing reads
can be aligned so
that the overlapping sequencing reads can yield sequence information of a
paired region in the
original double stranded nucleic acid source (e.g., a paired region in the
genome), and the
accuracy of the sequence information can be ascertained from the first and
second sequencing
reads with a high level of confidence. The first sequencing read of the first
sequenced region and.
the second sequencing read of the second sequenced region do not necessarily
have overlapping
sequences in which case sequence information of a paired region in the
original double stranded
nucleic acid source cannot be ascertained with a high level of confidence. The
first and second.
sequencing reads can initiate at one end of their respective template
molecules, or can initiate at
an internal position.
[003061 The compositions and methods for pairwise sequencing described herein
offers
several advantages which improves the quality of the sequencing data,
including increased signal
intensity which improves base call accuracy. The pairwise sequencing methods
also saves time
by obviating the need to prepare separate nucleic acid libraries each
corresponding to the sense
and anti-sense strands of the double stranded template molecule having the
sequence of interest.
Additionally, the pairwise sequencing methods generate and sequence the sense
and anti-sense
strands directly on the support/substrate used to conduct the sequencing
reactions.
1003071 The present disclosure provides pairwise sequencing methods that
employ a support
having a plurality of surface primers immobilized thereon. The immobilized
surface primers are
in fluid communication with each other to permit flowing various solutions of
linear or circular
nucleic acid template molecules, soluble primers, enzymes, nucleotides,
divalent cations, buffers,
reagents, and the like, onto the support so that the plurality of immobilized
surface primers (arid
products generated from the immobilized surface primers) react with the
solutions in a massively
parallel manner.
[00308] The
present disclosure provides pairwise sequencing methods comprising the steps:
(a) providing a plurality of single stranded nucleic acid concatemer template
molecules
immobilized to a support; (h) sequencing the plurality of immobilized
concatemer template
molecules with a first plurality of sequencing polymerases, a plurality of
soluble forward
sequencing primers and a first plurality of multivalent molecules, thereby
generating a plurality

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89
of extended forward sequencing primer strands; (c) retaining the plurality of
immobilized
concatemer template molecules and replacing the plurality of extended forward
sequencing
primer strands with a plurality of forward extension strands that are
hybridized to the retained
immobilized concatemer template molecules by conducting a primer extension
reaction; (d)
removing the retained immobilized concatemer template molecules while
retaining the plurality
of forward extension strands; and (e) sequencing the plurality of retained
forward extension
strands with a second plurality of sequencing polymerases, a plurality of
soluble reverse
sequencing primers and a second plurality of multivalent molecules. In some
embodiments,
individual concatemer template molecules in the plurality are immobilized to a
surface primer
where the surface primer is immobilized to the support. In some embodiments,
individual
concatemer template molecules are covalently joined to a surface primer, or
individual
concatemer template molecules are hybridized to a surface primer. In some
embodiments, the
immobilized surface primer includes or lacks a nucleotide having a scissile
moiety that can be
cleaved to generate an abasic site in the surface primer. In some embodiments,
the plurality of
concatemer template molecules comprise at least one nucleotide having a
scissile moiety that can
be cleaved to generate an abasic site in the concatemer template molecule. In
some
embodiments, the plurality of concatemer template molecules lack a nucleotide
having a scissile
moiety. Exemplary nucleotides having a scissile moiety (e.g., in the surface
primer or the
concatemer template molecule) include uridine, 8-oxo-7,8-dihydrogunine and
deoxyinosine.
[00309] In some embodiments, pairwise sequencing methods include a rolling
circle
amplification reaction which is conducted on-support by distributing a
plurality of single
stranded circular library molecules onto the support having a plurality of
surface primers
immobilized thereon. Individual surface primers are designed to capture, via
hybridization, a
single circular library molecule. The rolling circle amplification reaction
can be conducted on the
support. In some embodiments, for the on-support RCA reaction, a solution of
single stranded
circular library molecules is flowed onto the support so that individual
circular molecules are
captured via hybridization to individual surface primers. Individual circular
library molecules
include at least a sequence of interest and a universal surface primer binding
site, and optionally
include universal sequencing primer binding sites, universal amplification
primer binding site, an
additional surface primer binding site, and a sample barcode and/or a
molecular index. A single
immobilized surface primer will capture a single circular library molecule and
the rolling circle
. .

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amplification reaction generates a single stranded linear concatemer that is
covalently linked to
the immobilized surface primer by employing the terminal 3' end of the surface
primer as a
primer extension initiation site. Thus, individual concatemer molecules are
immobilized to the
support as concatemers that are covalently linked to an immobilized surface
primer. The single
stranded concatemer includes multiple tandem copies of the sequence of
interest and the
universal sequencing primer binding sites. A single surface primer will
capture a single circular
library molecule and generate a single concatemer molecule.
1003101 In some embodiments, pairwise sequencing methods include a rolling
circle
amplification reaction which is conducted in-solution to generate a plurality
of concatemers
which are distributed onto the support having a plurality of surface primers
immobilized thereon.
Individual surface primers are designed to capture, via hybridization, a
single concatemer having
complementary sequences of the circular library molecules. The rolling circle
amplification
reaction can continue on the support. In some embodiments, for the in-solution
RCA reaction, a
plurality of single stranded circular library molecules are subjected to a
rolling circle
amplification reaction in a reaction vessel. Individual circular library
molecules include at least a
sequence of interest and a universal surface primer binding site, and
optionally include universal
sequencing primer binding sites, universal amplification primer binding site,
an additional
surface primer binding site, and a sample barcode and/or a molecular index.
The RCA reaction
can be conducted for a very short period of time or can be conducted for
longer periods of time,
to generate a plurality of concatemers hybridized to their respective circular
library molecules
which are then distributed onto the support having a plurality of surface
primers immobilized
thereon. A solution of concatemer molecules is flowed onto the support so that
individual
concatemer molecules are captured via hybridization to individual surface
primers. Individual
concatemer molecules include at least a sequence of interest, universal
surface primer binding
site(s), universal sequencing primer binding sites, and optionally a sample
barcode and/or a
molecular index. A single immobilized surface primer will capture a single
concatemer molecule
and the rolling circle amplification reaction (now on the support) continues
thereby extending the
single stranded concatemer that is hybridized to the immobilized surface
primer. Thus, individual
concatemer molecules are immobilized to the support as concatemers that are
hybridized to an
immobilized surface primer. The single stranded concatemer includes multiple
tandem copies of
the sequence of interest and the universal sequencing primer binding sites. A
single surface
. .

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primer will capture a single concatemer molecule and generate a single
extended concatemer
molecule.
[00311] The rolling circle amplification reaction, conducted either by in-
solution or on-
support, will generate concatemers that are immobilized to the support
immobilized
concatemers offer several advantages compared to non-concatemer molecules. The
number of
tandem copies in the concatemer is tunable by controlling the time,
temperature and
concentration of reagents of the in-solution or on-support rolling circle
amplification reaction.
The concatemer can self-collapse into a compact nucleic acid nanoball.
inclusion of one or more
compaction oligonucleotides during the RCA reaction can further compact the
size and/or shape
of the nanoball. An increase in the number of tandem copies in a given
concatemer increases the
number of sites along the concatemer for hybridizing to multiple sequencing
primers which serve
as multiple initiation sites for polymerase-catalyzed sequencing reactions.
When the sequencing
reaction employs detectably labeled nucleotides and/or detectably labeled
multivalent molecules
(e.g., baying nucleotide units), the signals emitted by the nucleotides or
nucleotide units that
participate in the parallel sequencing reactions along the concatemer yields
an increased signal
intensity for each concatemer, Multiple portions of a given concatemer can be
simultaneously
sequenced. Furthermore, a plurality of binding complexes can form along a
particular
concatemer molecule, each binding complex comprising a sequencing polymerase
bound to a
multivalent molecule wherein the plurality of binding complexes remain stable
without
dissociation resulting in increased persistence time which increases signal
intensity and reduces
imaging time,
[00312] The level of sequencing accuracy can be further improved by obtaining
partially or
wholly overlapping sequencing reads from both sense and anti-sense strands,
and aligning the
sequencing reads which provides redundant sequencing data.
[00313] Thus, the pairwise sequencing compositions and methods described
herein provide
improved sequencing data quality in a massively parallel manner,
Methods for Pairwise Sequencing ¨ Generating Ahasic Sites
[003141 The present disclosure provides nairwise sequencing methods,
comprising step (a):
providing a plurality of immobilized single stranded nucleic acid concatemer
template molecules
each comprising at least one nucleotide having a scissile moiety, wherein
individual concatemer
template molecules in the plurality are immobilized to a first surface primer
that is immobilized

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to a support, and wherein the immobilized first surface primer lacks a
nucleotide having a
scissile moiety. In some embodiments, the support comprises a plurality of
first surface primers.
in some embodiments, the support lacks a plurality of second surface primers.
In some
embodiments, the support comprises a plurality of first and second surface
primers.
[00315] In some embodiments, individual immobilized concatemer template
molecules are
covalently joined to an immobilized surface primer (e.g., an immobilized first
surface primer)
(Figure 1). In an alternative embodiment, individual immobilized concatemer
template molecules
are hybridized to an immobilized surface primer (e.g., an immobilized first
surface primer)
(Figure 13).
1003161 In some embodiments, individual concatemer template molecules in the
plurality
comprise two or more copies of a sequence of interest, and wherein the
individual immobilized
concatemer template molecules further comprise any one or any combination of
two or more of:
(i) two or more copies of a universal binding sequence for a soluble forward
sequencing primer,
(ii) two or more copies of a universal binding sequence for a soluble reverse
sequencing primer,
(iii) two or more copies of a universal binding sequence for an immobilized
first surface primer,
(iv) two or more copies of a universal binding sequence for an immobilized
second surface
primer, (v) two or more copies of a universal binding sequence for a first
soluble amplification
primer, (vi) two or more copies of a universal binding sequence for a second
soluble
amplification primer, (vii) two or more copies of a universal binding sequence
for a soluble
compaction oligonucleotide, (viii) two or more copies of a sample barcode
sequence and/or (ix)
two or more copies of a unique molecular index sequence,
[00317] In some embodiments, the universal binding sequence (or a
complementary sequence
thereof) for the forward sequencing primer can hybridize to at least a portion
of the forward
sequencing primer. In some embodiments, the universal binding sequence (or a
complementary
sequence thereof) for the reverse sequencing primer can hybridize to at least
a portion of the
reverse sequencing primer. In some embodiments, the universal binding sequence
(or a
complementary sequence thereof) for the immobilized first surface primer can
hybridize to at
least a portion of the immobilized first surface primer. In some embodiments,
the universal
binding sequence (or a complementary sequence thereof) for the immobilized
second surface
primer can hybridize to at least a portion of the immobilized second surface
primer. In some
embodiments, the universal binding sequence (or a complementary sequence
thereof) for the first

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soluble amplification primer can hybridize to at least a portion of the first
soluble amplification
primer. In some embodiments, the universal binding sequence (or a
complementary sequence
thereof) for the second soluble amplification primer can hybridize to at least
a portion of the
second soluble amplification primer. In some embodiments, the universal
binding sequence (or a
complementary sequence thereof) for the soluble compaction oligonucleotide can
hybridize to at
least a portion of the soluble compaction oligonucleotide.
100318] In some embodiments, the scissile moiety in the immobilized concatemer
template
molecules of step (a) can be converted into abasic sites in the immobilized
concatemer template
molecules. In some embodiments, the scissile moiety in the immobilized
concatemer template
molecules comprises uridine, 8-oxo-7,8-dihydroguanine (e.g., 8oxoG) or
deoxyinosine. In the
concatemer template molecules, the uridine can be converted to an abasic site
using uracil DNA
glycosylase (UDG), the 8oxoG can be converted to an abasic site using FPG
glycosylase, and the
deoxyinosine can be converted to an abasic site using AlkA glycosylase. In
some embodiments,
the immobilized concatemer template molecules include 1-20, 20-40, 40-60, 60-
80, 80400, or a
higher number of nucleotides with a scissile moiety. In some embodiments,
about 0.1-1%, or
about 1-5%, or about 5-10%, or about 10-20%, or about 20-30% or a higher
percent of the dITP
in the immobilized concatemer template molecules are replaced with nucleotides
having a
scissile moiety. In some embodiments, the nucleotides having a scissile moiety
are distributed at
random positions along individual immobilized concatemer template molecules.
In some
embodiments, the nucleotides having a scissile moiety are distributed at
different positions in the
different immobilized concatemer template molecules.
[00319] In some embodiments, the immobilized first surface primers comprise
single stranded
oligonucleotides comprising DNA, RNA or a combination of DNA and RNA. The
immobilized
first surface primers can be immobilized to the support or immobilized to a
coating on the
support. The immobilized first surface primers can be embedded and attached
(coupled) to the
coating on the support. In some embodiments, the 5' end of the immobilized
first surface primers
are immobilized to a support or immobilized to a coating on the support.
Alternatively, an
interior portion or the 3' end of the immobilized first surface primers can be
immobilized to a
support or immobilized to a coating on the support. The support comprises a
plurality of
immobilized first surface primers having the same sequence. The immobilized
first surface
primers can be any length, for example 4-50 nucleotides, or 50-100
nucleotides, or 100-150

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nucleotides, or longer lengths. In some embodiments, the 3' terminal end of
the immobilized first
surface primers comprise an extendible 3' OH moiety. In some embodiments, the
3' terminal end
of the immobilized first surface primers comprise a 3' non-extendible moiety.
[003201 In some embodiments, the plurality of immobilized first surface
primers comprise at
least one phosphorothioate diester bond at their 5' ends which can render the
first surface
primers resistant to exonuclease degradation. In some embodiments, the
plurality of immobilized
first surface primers comprise 2-5 or more consecutive phosphorothioate
diester bonds at their 5'
ends. In some embodiments, the plurality of immobilized first surface primers
comprise at least
one ribonucleotide and/or at least one 2'-0-methyl or 2'-0-methoxyethyl (MOE)
nucleotide
which can render the first surface primers resistant to exonuclease
degradation.
100321] In some embodiments, the immobilized first surface primers comprise at
least one
locked nucleic acid (LNA) which comprises a methylene bridge bond between a 2'
oxygen and
4' carbon of the pentose ring. Immobilized first surface primers that include
at least one LNA
can be resistant to nuclease digestions and can exhibit increased melting
temperature when
hybridized to the forward extension strand.
[00322] In some embodiments, the immobilized concatemer template molecules
further
comprise two or more copies of a universal binding sequence (or complementary
sequence
thereof) for an immobilized second surface primer having a sequence that
differs from the first
immobilized surface primer. The immobilized second surface primers of step (a)
comprise single
stranded oligonucleotides comprising DNA, RNA or a combination of DNA and RNA.
The
immobilized second surface primers can be immobilized to the support or
immobilized to a
coating on the support. The immobilized second surface primers can be embedded
and attached
(coupled) to the coating on the support In some embodiments, the 5' end of the
immobilized
second surface primers are immobilized to a support or immobilized to a
coating on the support.
Alternatively, an interior portion or the 3' end of the immobilized second
surface primers can be
immobilized to a support or immobilized to a coating on the support. The
support comprises a
plurality of immobilized second surface primers having the same sequence. The
immobilized
second surface primers can be any length, for example 4-50 nucleotides, or 50-
100 nucleotides,
or 100-150 nucleotides, or longer lengths.
[00323j In some embodiments, the 3' terminal end of the immobilized second
surface primers
comprise an extendible 3' OH moiety. In some embodiments, the 3' terminal end
of the

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immobilized second surface primers comprise a 3' non-extendible moiety. In
some
embodiments, the 3' terminal end of the immobilized second surface primers
comprise a moiety
that blocks primer extension (e.g., non-extendible terminal 3' end), such as
for example a
phosphate group, a dideoxycytidine group, an inverted dT, or an amino group.
The immobilized
second surface primers are not extendible in a primer extension reaction. The
immobilized
second surface primers lack a nucleotide having a scissile moiety.
100324] In some embodiments, the plurality of immobilized second surface
primers comprise
at least one phosphorothioate diester bond at their 5' ends which can render
the second surface
primers resistant to exonuclease degradation. In some embodiments, the
plurality of immobilized
second surface primers comprise 2-5 or more consecutive phosphorothioate
diester bonds at their
5' ends. In some embodiments, the plurality of immobilized second surface
primers comprise at
least one ribonucleotide and/or at least one 2'-O-methyl or 2'-0-methoxyethyl
(MOE) nucleotide
which can render the second surface primers resistant to exonuclease
degradation.
[00325] In some embodiments, individual immobilized single stranded nucleic
acid
concatemer template molecule are joined or immobilized to an immobilized first
surface primer,
and at least one portion of the individual concatemer template molecule is
hybridized to an
immobilized second surface primer. The immobilized second surface primers
serve to pin down
a portion of the immobilized concatemer template molecules to the support (see
Figures 12 and
24).
[00326] In some embodiments, the support comprises about 102 ¨ 10" immobilized
first
surface primers per mm2. In some embodiments, the support comprises about 102
¨ 10"
immobilized second surface primers per mm2. In some embodiments, the support
comprises
about 102 10" immobilized first surface primers and immobilized second surface
primers per
mm2.
[00327] The immobilized surface primers (e.g., first and second surface
primers) are in fluid
communication with each other to permit flowing various solutions of linear or
circular nucleic
acid template molecules, soluble primers, enzymes, nucleotides, divalent
cations, buffers,
reagents, and the like, onto the support so that the plurality of immobilized
surface primers (and
the primer extension products generated from the immobilized surface primers)
react with the
solutions in a massively parallel manner.

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[00328j In some embodiments, the pairwise sequencing method further comprises
step (b):
sequencing the plurality of immobilized concatemer template molecules thereby
generating a
plurality of extended forward sequencing primer strands. The sequencing of
step (b) comprises
contacting the plurality of immobilized concatemer template molecules with a
plurality of
soluble forward sequencing primers under a condition suitable to hybridize at
least one forward
sequencing primer to at least one of the forward sequencing primer binding
sites/sequences of
the immobilized concatemer template molecules, and conducting forward
sequencing reactions
using one or more types of sequencing polymerases, a plurality of nucleotides
and/or multivalent
molecules, and the hybridized first forward sequencing primers. The forward
sequencing
reactions can generate a plurality of extended forward sequencing primer
strands. In some
embodiments, individual immobilized concatemer template molecules have
multiple copies of
the forward sequencing primer binding sites, wherein each forward sequencing
primer binding
site is capable of hybridizing to a first forward sequencing primer.
Individual forward sequencing
primer binding sites in a given immobilized concatemer template molecule can
be hybridized to
a forward sequencing primer and can undergo a sequencing reaction. Individual
immobilized
concatemer template molecules can undergo two or more sequence reactions,
where each
sequencing reaction is initiated from a first forward sequencing primer that
is hybridized to a
forward sequencing primer binding site (e.g., see Figures 2 and 14). In some
embodiments, the
soluble forward sequencing primers comprise 3' OH extendible ends. In some
embodiments, the
soluble forward sequencing primers comprise a 3' blocking moiety which can be
removed to
generate a 3' OH extendible end. In some embodiments, the soluble forward
sequencing primers
lack a nucleotide having a scissile moiety. In some embodiments, the
sequencing reactions
comprise a plurality of nucleotides (or analogs thereof) labeled with a
detectable reporter moiety.
In some embodiments, the sequencing reaction comprise a plurality of
multivalent molecules
having a plurality of nucleotide units attached to a core, where the
multivalent molecules are
labeled with a detectable reporter moiety. In some embodiments, the core is
labeled with a
detectable reporter moiety. In some embodiments, at least one linker and/or at
least one
nucleotide unit of a nucleotide arm is labeled with a detectable reporter
moiety. In some
embodiments, the detectable reporter moiety comprises a fluorophore. An
exemplary nucleotide
arm is shown in Figure 108, and exemplary multivalent molecules are shown in
Figures 104-107.

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[00329j In some embodiments, the pairwise sequencing method further comprises
step (c):
retaining the plurality of immobilized concatemer template molecules and
replacing the plurality
of extended forward sequencing primer strands with a plurality of forward
extension strands that
are hybridized to the retained immobilized single stranded nucleic acid
concatemer template
molecules. The plurality of extended forward sequencing primer strands can be
removed and
replaced with a plurality of forward extension strands by conducting a primer
extension reaction
(see Figures 3-5, and Figures 15-17).
1003301 In some embodiments, step (c) comprises contacting at least one
extended forward
sequencing primer strand with a plurality of strand displacing polymerases and
a plurality of
nucleotides and in the absence of soluble amplification primers, under a
condition suitable to
conduct a strand displacing primer extension reaction using the at least one
extended forward
sequencing primers strand to initiate the primer extension reaction thereby
generating a forward
extension strand that is covalently joined to the extended forward sequencing
primers strand,
wherein the forward extension strand is hybridized to the immobilized
concatemer template
molecule. For example, one of the extended forward sequencing primer strands
can serve as a
primer for the strand displacing polymerase. The strand displacing polymerase
can extend the
extended forward sequencing primer strand, and displace downstream extended
forward
sequencing primer strands while synthesizing an extended strand that replaces
the downstream
extended forward sequencing primer strands (Figures 3 and 15). The newly
extended strand is
covalently joined to an extended forward sequencing primer strand. The
immobilized concatemer
template molecules are retained.
[00331] The primer extension reaction can optionally include a plurality of
compaction
oligonucleotides and/or hexamine (e.g., cobalt hexamine III) to generate
forward extension
strands. Individual forward extension strands can collapse into a nanoball
having a more compact
size and/or shape compared to a nanoball generated from a primer extension
reaction conducted
without compaction oligonucleotides and/or hexamine (e.g., cobalt hexamine
III). Inclusion of
compaction oligonucleotides and/or hexamine (e.g., cobalt hexamine III) in the
primer extension
reaction can improve FWHM (full width half maximum) of a spot image of the
nanoball. The
spot image can be represented as a Gaussian spot and the size can be measured
as a FWHM. A
smaller spot size as indicated by a smaller FWHM typically correlates with an
improved image
of the spot. In some embodiments, the FWHM of a nanoball spot can be about 10
um or smaller.

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i00332i Examples of strand displacing polymerases include phi29 DNA
polymerase, large
fragment of Bst DNA polymerase, large fragment of Bsu DNA polymerase (exo-),
Bca DNA
polymerase (exo-), Klenow fragment of E. coli DNA polymerase, T5 polymerase, M-
MuLV
reverse transcriptase, HIV viral reverse transcriptase, Deep Vent DNA
polymerase and KOD
DNA polymerase. The phi29 DNA polymerase can be wild type phi29 DNA polymerase
(e.g.,
MagniPhi from Expedeon), or variant EquiPhi29 DNA polymerase (e.g., from
Thermo Fisher
Scientific), or chimeric QualiPhi DNA polymerase (e.g., from 4basebio).
1003331 In some embodiments, step (c) comprises: (i) removing the plurality of
extended
forward sequencing primer strand while retaining the immobilized concatemer
template
molecules; and (ii) contacting the plurality of retained immobilized
concatemer molecules with a
plurality of soluble forward sequencing primers (e.g., a second plurality of
soluble forward
sequencing primers), a plurality of nucleotides (e.g., a second plurality of
nucleotides) and a
plurality of primer extension polymerases, under a condition suitable to
hybridize the plurality of
soluble forward sequencing primers to the plurality of retained immobilized
concatemer template
molecules and suitable for conducting polymerase-catalyzed primer extension
reactions thereby
generating a plurality of forward extension strands, wherein the soluble
sequencing primers
hybridize with the forward sequencing primer binding sequence in the retained
immobilized
concatemer molecules (Figures 4 and 16). The primer extension reaction can
optionally include
a plurality of compaction oligonucleotides and/or hexamine (e.g., cobalt
hexamine Ill) to
generate forward extension strands. Individual forward extension strands can
collapse into a
nanoball having a more compact size and/or shape compared to a nanoball
generated from a
primer extension reaction conducted without compaction oligonucleotides and/or
hexamine (e.g.,
cobalt hexamine R. Inclusion of compaction oligonucleotides and/or hexamine
(e.g., cobalt
hexamine III) in the primer extension reaction can improve FWEIM (full width
half maximum)
of a spot image of the nanoball. The spot image can be represented as a
Gaussian spot and the
size can be measured as a FWTIM. A smaller spot size as indicated by a smaller
FWIIM typically
correlates with an improved image of the spot. In some embodiments, the FWHM
of a nanoball
spot can be about 10 gm or smaller.
[00334] In some embodiments, in step (c), the condition suitable to hybridize
the plurality of
soluble forward sequencing primers to the plurality of retained immobilized
single stranded
nucleic acid concatemer template molecules comprises hybridizing retained
immobilized
. .

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concatemer template molecules with the soluble primers in the presence of a
primer extension
polymerase, a plurality of nucleotides, and a high efficiency hybridization
buffer. In some
embodiment, the high efficiency hybridization buffer comprises: (i) a first
polar aprotic solvent
having a dielectric constant that is no greater than 40 and having a polarity
index of 4-9; (ii) a
second polar aprotic solvent having a dielectric constant that is no greater
than 115 and is present
in the hybridization buffer formulation in an amount effective to denature
double-stranded
nucleic acids; (iii) a pH buffer system that maintains the pH of the
hybridization buffer
formulation in a range of about 4-8; and (iv) a crowding agent in an amount
sufficient to enhance
or facilitate molecular crowding. In some embodiments, the high efficiency
hybridization buffer
comprises: (i) the first polar aprotic solvent comprises acetonitrile at 25-
50% by volume of the
hybridization buffer; (ii) the second polar aprotic solvent comprises
formamide at 5-10% by
volume of the hybridization buffer; (iii) the pH buffer system comprises 2-(N-
morpholino)ethanesulfonic acid (MES) at a pH of 5-6.5; and (iv) the crowding
agent comprises
polyethylene glycol (PEG) at 5-35% by volume of the hybridization buffer. In
some
embodiments, the high efficiency hybridization buffer further comprises
betaine.
1003351 In some embodiments, step (c) comprises: (i) removing the plurality of
extended
forward sequencing primer strand while retaining the immobilized concatemer
template
molecules; and (ii) contacting the plurality of retained immobilized
concatemer molecules with a
plurality of soluble amplification primers, a plurality of nucleotides (e.g.,
a second plurality of
nucleotides) and a plurality of primer extension polymerases, under a
condition suitable to
hybridize the plurality of soluble amplification primers to the plurality of
retained immobilized
concatemer template molecules and suitable for conducting polymerase-catalyzed
primer
extension reactions thereby generating a plurality of forward extension
strands, wherein the
soluble amplification primers hybridize with the soluble amplification primer
binding sequence
in the retained immobilized concatemer molecules (Figures 5 and 17). The
primer extension
reaction can optionally include a plurality of compaction oligonucleotides
and/or hexamine (e.g.,
cobalt hexamine Hp to generate forward extension strands. Individual forward
extension strands
can collapse into a nanoball having a more compact size and/or shape compared
to a nanoball
generated from a primer extension reaction conducted without compaction
oligonucleotides
and/or hexamine (e.g., cobalt hexamine III). Inclusion of compaction
oligonucleotides and/or
hexamine (e.g., cobalt hexamine III) in the primer extension reaction can
improve FWHM (full
. .

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width half maximum) of a spot image of the nanoball. The spot image can be
represented as a
Gaussian spot and the size can be measured as a FWHM. A smaller spot size as
indicated by a
smaller FWHM typically correlates with an improved image of the spot. In some
embodiments,
the FWHM of a nanoball spot can be about 10 gm or smaller.
[00336j In some embodiments, in step (c), the condition suitable to hybridize
the plurality of
soluble amplification primers to the plurality of retained immobilized single
stranded nucleic
acid concatemer template molecules comprises hybridizing retained immobilized
concatemer
template molecules with the soluble primers in the presence of a primer
extension polymerase, a
plurality of nucleotides, and a high efficiency hybridization buffer. In some
embodiment, the
high efficiency hybridization buffer comprises: (i) a first polar aprotic
solvent having a dielectric
constant that is no greater than 40 and having a polarity index of 4-9; (ii) a
second polar aprotic
solvent having a dielectric constant that is no greater than 115 and is
present in the hybridization
buffer formulation in an amount effective to denature double-stranded nucleic
acids; (iii) a pH
buffer system that maintains the pH of the hybridization buffer formulation in
a range of about 4-
8; and (iv) a crowding agent in an amount sufficient to enhance or facilitate
molecular crowding.
In some embodiments, the high efficiency hybridization butler comprises: (i)
the first polar
aprotic solvent comprises acetonitrile at 25-50% by volume of the
hybridization buffer; (ii) the
second polar aprotic solvent comprises formamide at 5-10% by volume of the
hybridization
buffer; (iii) the pH buffer system comprises 24N-morpholino)ethanesulfonic
acid (ME.S) at a pH
of 5-6.5; and (iv) the crowding agent comprises polyethylene glycol (PEG) at 5-
35% by volume
of the hybridization buffer. In some embodiments, the high efficiency
hybridization buffer
further comprises betaine.
[00337] In some embodiments, in step (c), the plurality of extended forward
sequencing
primer strands can be removed using an enzyme or a chemical reagent. For
example, the
plurality of extended forward sequencing primer strands can be enzymatically
degraded using a
5' to 3' double-stranded DNA exonuclease, including17 exonuclease (e.g., from
New England
Biolabs, catalog 4 M0263S). In some embodiments, the plurality of extended
forward
sequencing primer strands can be removed with a temperature that favors
nucleic acid
denaturation.
1003381 In some embodiments, in step (c), a denaturation reagent can be used
to remove the
plurality of extended forward sequencing primer strands, wherein the
denaturation reagent
. .

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comprises any one or any combination of compounds such as formamide,
acetonitrile,
guanidinium chloride and/or a pH buffering agent (e.g., Tris-HC1, NIES,
ITIEPES, MOPS, or the
like). Optionally, the denaturation reagent can further comprise PEG
100339] In some embodiments, in step (c), the plurality of extended forward
sequencing
primer strands can be removed using an elevated temperature (e.g., heat) with
or without a
nucleic acid denaturation reagent. The plurality of extended forward
sequencing primer strands
can be subjected to a temperature of about 45-50 C, or about 50-60 C, or
about 60-70 C, or
about 70-80 C, or about 80-90 C, or about 90-95 C, or higher temperature.
[003401 In some embodiments, in step (c), the plurality of extended forward
sequencing
primer strands can be removed using 100% formamide at a temperature of about
65 C for about
3 minutes, and washing with a reagent comprising about 50 rnIVI NaCI or
equivalent ionic
strength and having a pH of about 6.5 ¨ 8.5.
[003411 In some embodiments, the primer extension polymerase of step (c)
comprises a high
fidelity polymerase. In some embodiments, the primer extension polymerase of
step (c)
comprises a DNA polymerase capable of catalyzing a primer extension reaction
using a ura.cil-
containing template molecule (e.g., a uracil-tolerant polymerase). Exemplary
polymerases
include, but are not limited to, Q5U Hot Start high-fidelity DNA polymerase
(e.g., catalog #
M0515S from New England Biolabs), Taq DNA polymerase, One Taq DNA polymerase
(e.g.,
mixture of Taq and Deep Vent DNA pol.ymerases, catalog #M0480S from New
England
Biolabs), LongA.mp Taq DNA polymerase (e.g., catalog #M03235 from New England
Biolabs),
Epimark Hot Start Taq DNA polymerase (e.g., catalog #M0490S from New England
Biolabs),
-Bst DNA polymerase (e.g., large fragment, catalog #M0275S from New England
Biolabs), Bsu
DNA polymerase (e.g., large fragment, catalog #M0330S from New England
Biolabs), Phi29
DNA polymerase (e.g., catalog # M02695 from New England Biolabs), K col/ DNA
polymerase
(e.g., catalog # M02095 from New England Biolabs), Therminator DNA polymerase
(e.g.,
catalog #M0261S from New England Biolabs), Vent DNA polym.erase and Deep Vent
DNA
polymerase.
[00342] The pairwise methods described herein can provide increased accuracy
in a
downstream sequencing reaction because step (c) replaces the extended forward
sequencing
primer strands that were generated in step (b) with forward extension strands
having reduced
base errors. The extended forward sequencing primer strands are generated in
step (b) and may

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or may not contain erroneously incorporated nucleotides due to polymerase-
catalyzed mis-paired
bases. When step (c) is conducted with a high fidelity DNA polymerase, the
resulting forward
extension strands may have reduced base errors compared to the extended
forward sequencing
primer strands. The forward extension strands will be used as a nucleic acid
template for a
downstream sequencing step (e.g., see step (e) below). Thus, step (c) can
increase the sequencing
accuracy of the downstream step (e) and therefore increase the overall
sequencing accuracy of
the pairwise sequencing workflow.
1003431 In some embodiments, the pairwise sequencing method further comprises
step (d):
removing the retained immobilized concatemer template molecules by generating
abasic sites in
the immobilized single stranded concatemer template molecules at the
nucleotide(s) having the
scissile moiety and generating gaps at the abasic sites to generate a
plurality of gap-containing
single stranded nucleic acid concatemer template molecules while retaining the
plurality of
forward extension strands and retaining the plurality of immobilized surface
primers (Figures 6
and 18).
[003441 The abasic sites are generated on the retained concatemer template
strands that
contain nucleotides having scissile moieties. In some embodiments, the
scissile moieties in the
retained concatemer template molecules comprises uridine, 8-oxo-7,8-
dihydrogua.nine (e.g.,
8oxoG) or deonrinosine. The abasic sites can be removed to generate a
plurality of single
stranded nucleic acid template molecules having gaps while retaining the
plurality of forward
extension strands. The abasic sites can be generated by contacting the
immobilized concatemer
template molecules with an enzyme that removes the nucleo-base at the
nucleotide having the
scissile moiety, The uracil in the retained concatemer template strands can be
converted to an
abasic site using uracil DNA glycosylase (UDG). The 8oxoG in the retained
concatemer
template strands can be converted to an abasic site using FPG glycosylase. The
deoxyinosine in
the retained concatemer template strands can be converted to an abasic site
using AlkA
glycosylase.
[00345] In some embodiments, in step (d), the gaps can be generated by
contacting the abasic
sites in the immobilized concatemer template molecules with an enzyme or a
mixture of enzymes
having lyase activity that breaks the phosphodiester backbone at the 5' and 3'
sides of the abasic
site to release the base-free deoxyribose and generate a gap (Figures 6 and
l8). The abasic sites
can be removed using AP lyase, Endo IV endonuclease, FPG glycosylase/AP lyase,
Endo VIII

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glycosylaselAP lyase. In some embodiments, generating the abasic sites and
removal of the
abasic sites to generate gaps can be achieved using a mixture of uracil DNA
glycosylase and
DNA glycosylase-lyase endonuclease V111, for example USER (Uracil-Specific
Excision
Reagent Enzyme from New England Biolabs) or thermolabile USER (also from New
England
.Biolabs).
1003461 In some embodiments, in step (d), the plurality of gap-containing
template molecules
can be removed using an enzyme, chemical and/or heat. After the gap-removal
procedure, the
plurality of retained forward extension strands (e.g., see Figures 7 and 9,
and Figures 19 and 21).
is hybridized to the retained immobilized surface primers
1003471 For example, the plurality of gap-containing template molecules can be
enzymatically
degraded using a 5' to 3' double-stranded DNA exonuclease, including T7
exonuclease (e.g.,
from New England Biolabs, catalog # M0263S). When a 5' to 3' double-stranded
DNA
exonuclease is used for removing gap-containing template molecules, then the
plurality of
soluble amplification primers in step (c) can comprise at least one
phosphoroth.ioate diester bond
at their 5' ends which can render the soluble amplification primers resistant
to exonuclease
degradation, In some embodiments, the plurality of soluble amplification
primers in step (c)
comprise 2-5 or more consecutive phosphorothioate diester bonds at their 5'
ends. In some
embodiments, the plurality soluble amplification primers in step (c) comprise
at least one
ribonucleotide and/or at least one 2'-0-methyl or 2'-0-methoxyethyl (MOE)
nucleotide which
can render the forward sequencing primers resistant to exonuclease
degradation.
[00348] In some embodiments, the plurality of gap-containing template
molecules can be
removed using a chemical reagent that favors nucleic acid denaturation. The
denaturation reagent
can include any one or any combination of compounds such as formamide,
acetonitrile,
gua.nidinium chloride and/or a buffering agent (e.g., Tris-HCI, MES, flEPES,
or the like).
[00349] In some embodiments, the plurality of gap-containing template
molecules can be
removed using an elevated temperature (e.g., heat) with or without a nucleic
acid denaturation
reagent. The gap-containing template molecules can be subjected to a
temperature of about 45-50
or about 50-60 C, or about 60-70 C, or about 70-80 'C., or about 80-90 'C,
or about 90-95
or higher temperature.
[00350] In some embodiments, the plurality of gap-containing template
molecules can be
removed using 100% formamide at a temperature of about 65 C for about 3
minutes, and

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washing with a reagent comprising about 50 mM NaCI or equivalent ionic
strength and having a
pH of about 6.5 8.5.
[003511 In some embodiments, the pairwise sequencing method further comprises
step (e):
sequencing the plurality of retained forward extension strands thereby
generating a plurality of
extended reverse sequencing primer strands. In some embodiments, the
sequencing of step (e)
comprises contacting the plurality of retained forward extension strands with
a plurality of
soluble reverse sequencing primers under a condition suitable to hybridize the
reverse
sequencing primers to the reverse sequencing primer binding site of the
retained forward
extension strands, and by conducting sequencing reactions using the hybridized
reverse
sequencing primers wherein the forward sequencing reactions generates a
plurality of extended
reverse sequencing primer strands (Figures 10 and 11, and Figures 22 and 23).
The extended
reverse sequencing primer strands are hybridized to the retained forward
extension strand. The
retained forward extension strand is hybridized to the first surface primer.
The extended reverse
sequencing primer strands are not hybridized to the first surface primer, or
covalently joined to
the first surface primer. Therefore, the extended reverse sequencing primer
strands are not
immobilized to the support.
[003521 For the sake of simplicity, Figures 7 and 9 show exemplary retained
forward
extension strands each having one copy of the sequence of interest and various
universal primer
binding sites. The skilled artisan will appreciate that the retained forward
extension strand can
include two or more tandem copies containing the sequence of interest and
various universal
primer binding sites. Therefore, the reverse sequencing reaction can generate
a plurality of
extended reverse sequencing primer strands hybridized to the same retained
forward extension
strand.
[00353] In some embodiments, in step (e), the condition suitable to hybridize
the reverse
sequencing primers to the reverse sequencing primer binding sequences of the
retained forward
extension strands comprises contacting the plurality of soluble reverse
sequencing primers and
the retained forward extension strands with a high efficiency hybridization
buffer. In some
embodiments, the high efficiency hybridization buffer comprises: (i) a first
polar aprotic solvent
having a dielectric constant that is no greater than 40 and having a polarity
index of 4-9; (ii) a
second polar aprotic solvent having a dielectric constant that is no greater
than 115 and is present
in the hybridization buffer formulation in an amount effective to denature
double-stranded
. .

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nucleic acids; (iii) a pH buffer system that maintains the pH of the
hybridization buffer
formulation in a range of about 4-8; and (iv) a crowding agent in an amount
sufficient to enhance
or facilitate molecular crowding. In some embodiments, the high efficiency
hybridization buffer
comprises: (1) the first polar aprotic solvent comprises acetonitrile at 25-
50% by volume of the
hybridization buffer; (ii) the second polar aprotic solvent comprises
formamide at 5-10% by
volume of the hybridization buffer; (iii) the pH buffer system comprises 2-(N-
morpholino)ethanesulfonic acid (MES) at a pH of 5-6.5; and (iv) the crowding
agent comprises
polyethylene glycol (PEG) at 5-35% by volume of the hybridization buffer. In
some
embodiments, the high efficiency hybridization buffer further comprises
betaine.
1003541 In an alternative embodiment, the sequencing of step (e) comprises
using the
immobilized surface primer as a sequencing primer and conducting sequencing
reactions to
generate a plurality of reverse sequencing strands.
100355] In some embodiments, the reverse sequencing reactions of step (e)
comprises
contacting the plurality of soluble reverse sequencing primers with the
reverse sequencing primer
binding sequences of the retained forward extension strands, one or more types
of sequencing
polymerases, and a plurality of nucleotides or a plurality of multivalent
molecules. In some
embodiments, the soluble reverse sequencing primers comprise 3' OH extendible
ends. In some
embodiments, the soluble reverse sequencing primers comprise a 3' blocking
moiety which can
be removed to generate a 3' OH extendible end. In some embodiments, the
soluble reverse
sequencing primers lack a nucleotide having a scissile moiety. The sequencing
reactions that
employ nucleotides and/or multivalent molecules is described in more detail
below. The reverse
sequencing reactions can generate a plurality of extended reverse sequencing
primer strands. In
some embodiments, individual retained forward extension strands have multiple
copies of the
reverse sequencing primer binding sequences/sites, wherein each reverse
sequencing primer
binding site is capable of hybridizing to a reverse sequencing primer.
Individual reverse
sequencing primer binding sites in a given retained forward extension strand
can be hybridized to
a reverse sequencing primer and can undergo a sequencing reaction. Thus, an
individual retained
forward extension strand can undergo two or more sequence reactions, where
each sequencing
reaction is initiated from a reverse sequencing primer that is hybridized to a
reverse sequencing
primer binding site (e.g., see Figures 10 and 11, and Figures 22 and 23). In
some embodiments,
the sequencing reactions comprise a plurality of nucleotides (or analogs
thereof) labeled with a

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detectable reporter moiety. In some embodiments, the sequencing reaction
comprise a plurality
of multivalent molecules having nucleotide units, where the multivalent
molecules are labeled
with a detectable reporter moiety. In some embodiments, the detectable
reporter moiety
comprises a fluorophore.
[003561 In some embodiments, at least one washing step can be conducted after
any of steps
(a) ¨ (e). The washing step can be conducted with a wash buffer comprising a
pH buffering
agent, a metal chelating agent, a salt, and a detergent.
1003571 In some embodiments, the pH buffering compound in the wash buffer
comprises any
one or any combination of two or more of Tris, Tris-HC1, Tricine, Bicine, Bis-
Tris propane,
HEPES, IVIES, MOPS, MOPSO, BES, TES, CAPS, TAPS, TAPSO, ACES, PIPES,
ethanolamine (a.k.a 2-amino methanol; MEA), a citrate compound, a citrate
mixture, NaOH
and/or KOH. In some embodiments, the pH buffering agent can be present in the
wash buffer at
a concentration of about 1-100 inM, or about 10-50 mM, or about 10-25 mM. In
some
embodiments, the pH of the pH buffering agent which is present in any of the
reagents described
here in can be adjusted to a pH of about 4-9, or a pH of about 5-9, or a pH of
about 5-8.
1003581 In some embodiments, the metal chelating agent in the wash buffer
comprises EDTA
(ethylenediaminetetraacetic acid), EGTA (ethylene glycol tetraacetic acid),
HEDTA
(hydroxyethylethylenediaminetriacetic acid), DPTA (diethylene triarnine
pentaacetic acid), NTA
(N,N-bis(carboxyinethyl)glycine), citrate anhydrous, sodium citrate, calcium
citrate, ammonium
citrate, ammonium bicitrate, citric acid, potassium citrate, or magnesium
citrate. In some
embodiments, the wash buffer comprises a chelating agent at a concentration of
about 0.01 ¨ 50
mM, or about 0.1 20 mM, or about 0.2 10 mM.
[003591 In some embodiments, the salt in the wash buffer comprises NaC1, KCI,
NI-12SO4 or
potassium glutamate. In some embodiments, the detergent comprises an ionic
detergent such as
SDS (sodium dodecyl sulfate). The wash buffer can include a monovalent salt at
a concentration
of about 25-500 mM, or about 50-250 mM, or about 100-200 mM.
[00360] In some embodiments, the detergent in the wash buffer comprises a non-
ionic
detergent such as Triton X-100, Tween 20, Tween 80 or Nonidet P-40. In some
embodiments,
the detergent comprises a zwitterionic detergent such as CHAPS (34(3-
cholamidopropyl)dimethylammonio)-1-propanesulfonate) or N-Dodecyl-N,N-dimethy1-
3-
amonio-1-propanesulfate (DetX). In some embodiments, the detergent comprises
LDS (lithium
. . .

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dodecyl sulfate), sodium taurodeoxycholate, sodium taurochoiate, sodium
glycocholate, sodium
deoxycholate or sodium cholate. in some embodiments, the detergent is included
in the wash
buffer at a concentration of about 0.01-0.05%, or about 0.05-0.1%, or about
0.1-0.15%, or about
0.15-0.2%, or about 0.2-0.25%.
On Support RCA and Pairwise Sequencing --- Generating Ahasic Sites
[00361] The present disclosure provides pairwise sequencing methods,
comprising step (a):
providing a support having a plurality of surface primers (e.g., a plurality
of first surface primers)
immobilized thereon wherein each of the surface primers have a 3' OH
extendible end and lack a
nucleotide having a scissile moiety (Figure 25). For example, the surface
primers lack uridine, 8-
oxo-7,8-dihydroguanine (e.g., 8oxoG) and deoxyinosine. In some embodiments,
the support
comprises a plurality of first surface primers. In some embodiments, the
support lacks a plurality
of second surface primers. In some embodiments, the support comprises a
plurality of first and
second surface primers.
[003621 In some embodiments, the immobilized first surface primers comprise
single stranded
oligonucleotides comprising DNA, RNA or a combination of DNA and RNA. The
first surface
primers comprise a sequence that is wholly complementary or partially
complementary along
their lengths to at least a portion of a nucleic acid library molecule (e.g.,
linear or circular library
molecules). The first surface primers can include a terminal 3' nucleotide
having a sugar 3' OH
moiety which is extendible for nucleotide polymerization (e.g., polymerase
catalyzed
polymerization).
[003631 The immobilized first surface primers can be immobilized to the
support or
immobilized to a coating on the support. The immobilized first surface primers
can be embedded
and attached (coupled) to the coating on the support. In some embodiments, the
5' end of the
immobilized first surface primers are immobilized to a support or immobilized
to a coating on
the support. Alternatively, an interior portion or the 3' end of the
immobilized first surface
primers can be immobilized to a support or immobilized to a coating on the
support. The support
comprises a plurality of immobilized first surface primers having the same
sequence. The
immobilized first surface primers can be any length, for example 4-50
nucleotides, or 50-100
nucleotides, or 100-150 nucleotides, or longer lengths.
[00364] In some embodiments, the plurality of immobilized first surface
primers comprise at
least one phosphorothioate diester bond at their 5' ends which can render the
first surface

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primers resistant to exonuclease degradation. In some embodiments, the
plurality of immobilized
first surface primers comprise 2-5 or more consecutive phosphorothioate
diester bonds at their 5'
ends. In some embodiments, the plurality of immobilized first surface primers
comprise at least
one ribonucleotide and/or at least one 2'-0-methyl or 2'-O-methoxyethyl (MOE)
nucleotide
which can render the first surface primers resistant to exonuclease
degradation.
1003651 In some embodiments, the immobilized first surface primers comprise at
least one
locked nucleic acid (LNA) which comprises a methylene bridge bond between a 2'
oxygen and
4' carbon of the pentose ring. Immobilized first surface primers that include
at least one LNA
can be resistant to nuclease digestions and can exhibit increased melting
temperature when
hybridized to the forward extension strand.
100366) In some embodiments, the support further comprises a plurality of a
second surface
primer immobilized thereon (Figure 37). The second surface primers have a
sequence that differs
from the first immobilized surface primer. The immobilized second surface
primers of step (a)
comprise single stranded oligonucleotides comprising DNA, RNA or a combination
of DNA and
RNA. The second surface primers comprise a sequence that is wholly
complementary or partially
complementary along their lengths to at least a portion of an immobilized
single stranded
concatemer template molecule. The immobilized second surface primers can be
immobilized to
the support or immobilized to a coating on the support. The immobilized second
surface primers
can be embedded and attached (coupled) to the coating on the support. In some
embodiments,
the 5' end of the immobilized second surface primers are immobilized to a
support or
immobilized to a coating on the support. Alternatively, an interior portion or
the 3' end of the
immobilized second surface primers can be immobilized to a support or
immobilized to a coating
on the support. The support comprises a plurality of immobilized second
surface primers having
the same sequence. The immobilized second surface primers can be any length,
for example 4-50
nucleotides, or 50-100 nucleotides, or 100-150 nucleotides, or longer lengths.
In some
embodiments, the 3' terminal end of the immobilized second surface primers
comprise an
extendible 3' OH moiety. In some embodiments, the 3' terminal end of the
immobilized second
surface primers comprise a 3' non-extendible moiety. The 3' terminal end of
the immobilized
second surface primers comprise a moiety that blocks primer extension, such as
for example a
phosphate group, a dideoxycytidine group, an inverted dT, or an amino group.
The immobilized
. .

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second surface primers are not extendible in a primer extension reaction. The
immobilized
second surface primers lack a nucleotide having a scissile moiety.
[003671 In some embodiments, the plurality of immobilized second surface
primers comprise
at least one phosphorothioate diester bond at their 5' ends which can render
the second surface
primers resistant to exonuclease degradation. In some embodiments, the
plurality of immobilized
second surface primers comprise 2-5 or more consecutive phosphorothioate
diester bonds at their
5' ends. In some embodiments, the plurality of immobilized second surface
primers comprise at
least one ribonucleotide and/or at least one 2'-O-methyl or 2'-0-methoxyethyl
(MOE) nucleotide
which can render the second surface primers resistant to exonuclease
degradation.
1003681 In some embodiments, individual immobilized single stranded nucleic
acid
concatemer template molecule are covalently joined to an immobilized first
surface primer, and
at least one portion of the individual concatemer template molecule is
hybridized to an
immobilized second surface primer (Figure 37). The immobilized second surface
primers serve
to pin down a portion of the immobilized concatemer template molecules to the
support. The
immobilized concatemer template molecule has two or more copies of a universal
binding
sequence for an immobilized second surface primer. The portion of the
immobilized concatemer
template molecule that includes the universal binding sequence for an
immobilized second
surface primer can hybridize to the immobilized second surface primer. In some
embodiments,
the second surface primers include a terminal 3' blocking group that renders
them non-
extendible. In some embodiments, the second surface primers have terminal 3'
extendible ends.
[003691 In some embodiments, the support comprises about 102 ¨ 1015
immobilized first
surface primers per mm2. In some embodiments, the support comprises about 102
1015
immobilized second surface primers per mm2. In some embodiments, the support
comprises
about 102 --- 1015 immobilized first surface primers and immobilized second
surface primers per
mm2.
[003701 The immobilized surface primers (e.g., first and second surface
primers) are in fluid
communication with each other to permit flowing various solutions of linear or
circular nucleic
acid template molecules, soluble primers, enzymes, nucleotides, divalent
cations, buffers,
reagents, and the like, onto the support so that the plurality of immobilized
surface primers (and
the primer extension products generated from the immobilized surface primers)
react with the
solutions in a massively parallel manner.
. .

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[00371j In some embodiments, the pairwise sequencing method further comprises
step (b):
generating a plurality of immobilized single stranded nucleic acid concatemer
template
molecules wherein individual single stranded nucleic acid concatemer template
molecules are
joined (e.g., covalently joined) to an immobilized surface primer (e.g., an
immobilized first
surface primer), by hybridizing a plurality of single-stranded circular
nucleic acid library
molecules to the plurality of immobilized first surface primers and conducting
a rolling circle
amplification reaction with a plurality of a strand displacing polymerase, and
a plurality of
nucleotides which include dATP, dCTP, dGTP, dTTP and a nucleotide having a
scissile moiety,
thereby generating a plurality of immobilized single stranded nucleic acid
concatemer template
molecules (Figure 26). In some embodiments, the rolling circle amplification
reaction can be
conducted in the presence, or in the absence, of a plurality of compaction
oligonucleotides.
1003721 In some embodiments, the single-stranded circular nucleic acid library
molecules
comprise covalently closed circular molecules. In some embodiments, the single-
stranded
circular nucleic acid library molecules can be removed from the concatemer
template molecules
with at least one washing step which is conducted under a condition suitable
to retain the single
stranded nucleic acid concatemer template molecules where individual
concatemer template
molecules are operably joined to an immobilized first surface primer.
[00373] In some embodiments, each of the single stranded circular nucleic acid
library
molecules in the plurality comprise a sequence of interest, and wherein the
individual
immobilized concatemer template molecules further comprise any one or any
combination of
two or more of (i) a universal binding sequence (or complementary sequence
thereof) for a
soluble forward sequencing primer, (ii) a universal binding sequence (or
complementary
sequence thereof) for a soluble reverse sequencing primer, (iii) a universal
binding sequence (or
complementary sequence thereof) for an immobilized first surface primer, (iv)
a universal
binding sequence (or complementary sequence thereof) for an immobilized second
surface
primer, (v) a universal binding sequence (or complementary sequence thereof)
for a first soluble
amplification primer, (vi) a universal binding sequence (or complementary
sequence thereof) for
a second soluble amplification primer, (vii) a universal binding sequence (or
complementary
sequence thereof) for a soluble compaction oligonucleotide, (viii) a sample
barcode sequence
and/or (ix) a unique molecular index sequence.

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[00374] In some embodiments, the universal binding sequence (or a
complementary sequence
thereof) for the forward sequencing primer can hybridize to at least a portion
of the forward
sequencing primer. In some embodiments, the universal binding sequence (or a
complementary
sequence thereof) for the reverse sequencing primer can hybridize to at least
a portion of the
reverse sequencing primer. In some embodiments, the universal binding sequence
(or a
complementary sequence thereof) for the immobilized first surface primer can
hybridize to at
least a portion of the immobilized first surface primer. In some embodiments,
the universal
binding sequence (or a complementary sequence thereof) for the immobilized
second surface
primer can hybridize to at least a portion of the immobilized second surface
primer. In some
embodiments, the universal binding sequence (or a complementary sequence
thereof) for the first
soluble amplification primer can hybridize to at least a portion of the first
soluble amplification
primer. In some embodiments, the universal binding sequence (or a
complementary sequence
thereof) for the second soluble amplification primer can hybridize to at least
a portion of the
second soluble amplification primer. In some embodiments, the universal
binding sequence (or a
complementary sequence thereof) for the soluble compaction oligonucleotide can
hybridize to at
least a portion of the soluble compaction oligonucleotide.
[003751 In some embodiments, the rolling circle amplification reaction of
step (b) generates a
plurality of immobilized single stranded nucleic acid concatemer template
molecules each
comprising a concatemer having at least one nucleotide having a scissile
moiety and two or more
copies of a sequence of interest, and wherein the immobilized concatemer
template molecules
further comprise any one or any combination of two or more of: (i) two or more
copies of a.
universal binding sequence (or a complementary sequence thereof) for a soluble
forward
sequencing primer, (ii) two or more copies of a universal binding sequence (or
a complementary
sequence thereof) for a soluble reverse sequencing primer, (iii) two or more
copies of a universal
binding sequence (or a complementary sequence thereof) for an immobilized
first surface primer,
(iv) two or more copies of a universal binding sequence (or a complementary
sequence thereof)
for an immobilized second surface primer, (v) two or more copies of a
universal binding
sequence (or a complementary sequence thereof) for a first soluble
amplification primer, (vi) two
or more copies of a universal binding sequence (or a complementary sequence
thereof) for a
second soluble amplification primer, (vii) two or more copies of a universal
binding sequence (or
a complementary sequence thereof) for a soluble compaction oligonucleotide,
(viii) two or more

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copies of a sample barcode sequence and/or (ix) two or more copies of a unique
molecular index
sequence.
[00376] In some embodiments, the plurality of immobilized single stranded
nucleic acid
concatemer template molecules that are generated by the rolling circle
amplification reaction of
step (b) further comprise two or more copies of a universal binding sequence
(or complementary
sequence thereof) for immobilized second sequence surface primers. In some
embodiments;
individual immobilized single stranded nucleic acid concatemer template
molecule are joined
(e.g., covalently joined) to an immobilized first surface primer, and at least
one portion of the
individual concatemer template molecule is hybridized to an immobilized second
surface primer.
The immobilized second surface primers serve to pin down a portion of the
immobilized
concatemer template molecules to the support (see Figure 37). In some
embodiments, the second
surface primers include a terminal 3' blocking group that renders them non-
extendible.
[00.377] The rolling circle amplification reaction of step (b) can be
conducted with a
nucleotide mixture containing dATP, dC IF, dGIP, dTTP and a nucleotide
having a scissile
moiety to generate immobilized concatemer template molecules which includes at
least one
nucleotide having a scissile moiety. The scissile moieties in the immobilized
concatemer
template molecules can be converted into abasic sites. In some embodiments, in
the nucleotide
mixture, the nucleotide having the scissile moiety comprises uridin.e, 8-oxo-
7,8-dihydroguanine
(e.g., 8oxoG) or deoxyinosine. In the immobilized concatemer template
molecules, the uridine
can be converted to an abasic site using uracil DNA glycosylase (UDG), the
8oxoG can be
converted to an abasic site using FPG glycosylase, and the deoxyin.osin.e can
be converted to an
abasic site using AlkA. glycosylase.
[00378] In some embodiments, the nucleotide mixture can include an amount of
dUTP so that
a target percent of the thy mi dine in the resulting concatemer molecules are
replaced with dUTP.
For example, when 30% of dTTP in the concatemer molecules are to be replaced
with dUTP
(e.g., 30% is the target percent) then the nucleotide mixture can contain 7.5%
dUTP (e.g., 30/4 =
7.5%), 17.5% dTTP, and 25% each for dATP, dCTP and dGTP. The target percent of
dTTP to be
replaced by dUTP can be about 0.1-1%, or about 1-5%, or about 5-10%, or about
10-20%, or
about 20-30%, or about 30-45%, or about 45-50%, or a higher percent of the
dTTP in the
immobilized concatemer template molecules are replaced with nucleotides having
a scissile
moiety.

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1003791 In some embodiments, the nucleotide mixture can include an amount of
deoxyinosine
so that a target percent of the guanosine in the resulting concatetner
molecules are replaced with
deoxyinosine. For example, when 30% of dGTP in the concatemer molecules are to
be replaced
with deoxyinosine (e.g., 30% is the target percent) then the nucleotide
mixture can contain 7.5%
deoxyinosine (e.g., 30/4 = 7.5%), 17.5% dGTP, and 25% each for dATP, dCIP and
&FUR The
target percent of dGIP to be replaced by deoxyinosine can be about 0.1-1%, or
about 1-5%, or
about 5-10%, or about 10-20%, or about 20-30% or about 30-45%, or about 45-
50%, or a
higher percent of the dGTP in the immobilized concatemer template molecules
are replaced with
nucleotides having a scissile moiety.
1003801 In some embodiments, the nucleotide mixture can include an amount of
8oxoG so
that a target percent of the guanosine in the resulting concatemer molecules
are replaced with
8oxoG. For example, when 30% of dGTP in the concatemer molecules are to be
replaced with
8oxoG (e.g., 30% is the target percent) then the nucleotide mixture can
contain 7.5% 8oxoG
(e.g., 30/4 = 7.5%), 17.5% dGTP, and 25% each for dATP, dCTP and dTTP. The
target percent
of dGTP to be replaced by 8oxoG can be about 0.1-1%, or about 1-5%, or about 5-
10%, or about
10-20%, or about 20-30%, or about 30-45%, or about 45-50%, or a higher percent
of the dGTP
in the immobilized concatemer template molecules are replaced with nucleotides
haying a
scissile moiety.
[003811 In some embodiments, the rolling circle amplification reaction.
generates immobilized
concatemer template molecules with incorporated nucleotides having a scissile
moiety that are
distributed at random positions along individual immobilized concatemer
template molecules. In
some embodiments, the nucleotides havin.g a scissile moiety are distributed at
different positions
in the different immobilized concatemer template molecules.
1003821 In some embodiments, the pairwise sequencing method further comprises
step (c):
sequencing the plurality of immobilized concatemer template molecules thereby
generating a
plurality of extended forward sequencing primer strands. The sequencing of
step (c) comprises
contacting the plurality of immobilized concatemer template molecules with a
plurality of
soluble forward sequencing primers under a condition suitable to hybridize at
least one forward
sequencing primer to at least one of the forward sequencing primer binding
sites/sequences of
the immobilized concatemer template molecules, and conducting forward
sequencing reactions
using one or more types of sequencing polymerases, a plurality of nucleotides
and/or multivalent

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molecules, and the hybridized first forward sequencing primers (Figure 27). In
some
embodiments, the soluble forward sequencing primers comprise 3' OH extendible
ends. In some
embodiments, the soluble forward sequencing primers comprise a 3' blocking
moiety which can
be removed to generate a 3' OH extendible end. In some embodiments, the
soluble forward
sequencing primers lack a nucleotide having a scissile moiety. The forward
sequencing reactions
can generate a plurality of extended forward sequencing primer strands. In
some embodiments,
individual immobilized concatemer template molecules have multiple copies of
the forward
sequencing primer binding sites, wherein each forward sequencing primer
binding site is capable
of hybridizing to a first forward sequencing primer. Individual forward
sequencing primer
binding sites in a given immobilized concatemer template molecule can be
hybridized to a
forward sequencing primer and can undergo a sequencing reaction. Individual
immobilized
concatemer template molecules can undergo two or more sequence reactions,
where each
sequencing reaction is initiated from a first forward sequencing primer that
is hybridized to a
forward sequencing primer binding site (e.g., see Figure 27). In some
embodiments, the
sequencing reactions comprise a plurality of nucleotides (or analogs thereof)
labeled with a
detectable reporter moiety. In some embodiments, the sequencing reaction
comprise a plurality
of multivalent molecules having a plurality of nucleotide units attached to a
core, where the
multivalent molecules are labeled with a detectable reporter moiety. In some
embodiments, the
core is labeled with a detectable reporter moiety. In some embodiments, at
least one linker and/or
at least one nucleotide unit of a nucleotide arm is labeled with a detectable
reporter moiety. In
some embodiments, the detectable reporter moiety comprises a fluorophore. An
exemplary
nucleotide arm is shown in Figure 108, and exemplary multivalent molecules are
shown in
Figures 104-107.
[00383] In some embodiments, the pairwise sequencing method further comprises
step (d):
retaining the plurality of immobilized concatemer template molecules and
replacing the plurality
of extended forward sequencing primer strands with a plurality of forward
extension strands that
are hybridized to the retained immobilized single stranded nucleic acid
concatemer template
molecules. The plurality of extended forward sequencing primer strands can be
removed and
replaced with a plurality of forward extension strands by conducting a primer
extension reaction
(see Figures 28-30).

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in some embodiments, step (d) comprises contacting at least one extended
forward sequencing
primer strand with a plurality of strand displacing polymerases and a
plurality of nucleotides and
in the absence of soluble amplification primers, under a condition suitable to
conduct a strand
displacing primer extension reaction using the at least one extended forward
sequencing primers
strand to initiate the primer extension reaction thereby generating a forward
extension strand that
is covalently joined to the extended forward sequencing primers strand,
wherein the forward
extension strand is hybridized to the immobilized concatemer template molecule
(Figure 28). For
example, one of the extended forward sequencing primer strands can serve as a
primer for the
strand displacing polymerase. The strand displacing polymerase can extend the
extended forward
sequencing primer strand, and displace downstream extended forward sequencing
primer strands
while synthesizing an extended strand that replaces the downstream extended
forward
sequencing primer strands. The newly extended strand is covalently joined to
an extended
forward sequencing primer strand. The immobilized concatemer template
molecules are retained.
The primer extension reaction can optionally include a plurality of compaction
oligonucleotides
and/or hexamine (e.g., cobalt hexamine 111) to generate forward extension
strands. Individual
forward extension strands can collapse into a nanoball having a more compact
size and/or shape
compared to a nanoball generated from a primer extension reaction conducted
without
compaction oligonucleotides and/or hexamine (e.g., cobalt hexamine ITT).
Inclusion of
compaction oligonucleotides and/or hexamine (e.g., cobalt hexamine 111) in the
primer extension
reaction can improve FWHM (full width half maximum) of a spot image of the
nanoball. The
spot image can be represented as a Gaussian spot and the size can be measured
as a FWHM. A
smaller spot size as indicated by a smaller FWHM typically correlates with an
improved image
of the spot. In some embodiments, the FWEIM of a nanoball spot can be about 10
um or smaller.
[00384] Examples of strand displacing polymerases include phi29 DNA
polymerase, large
fragment of Bst DNA polymerase, large fragment of Bsu DNA polymerase (exo-),
Bca DNA
polymerase (exo-), Klenow fragment of E. coli DNA polymerase, T5 polymerase, M-
MuL,V
reverse transcriptase, HIV viral reverse transcriptase, Deep Vent DNA
polymerase and KOD
DNA polymerase. The phi29 DNA polymerase can be wild type phi29 DNA polymerase
(e.g.,
MagniPhi from Expedeon), or variant EquiPhi29 DNA polymerase (e.g., from
Thermo Fisher
Scientific), or chimeric QualiPhi DNA polymerase (e.g., from 4basebio).

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[00385j In some embodiments, step (d) comprises: (i) removing the plurality of
extended
forward sequencing primer strand while retaining the immobilized concatemer
template
molecules; and (ii) contacting the plurality of retained immobilized
concatemer molecules with a
plurality of soluble forward sequencing primers (e.g., a second plurality of
soluble forward
sequencing primers), a plurality of nucleotides (e.g., a second plurality of
nucleotides) and a
plurality of primer extension polymerases, under a condition suitable to
hybridize the plurality of
soluble forward sequencing primers to the plurality of retained immobilized
concatemer template
molecules and suitable for conducting polymerase-catalyzed primer extension
reactions thereby
generating a plurality of forward extension strands, wherein the soluble
sequencing primers
hybridize with the forward sequencing primer binding sequence in the retained
immobilized
concatemer molecules (Figure 29). The primer extension reaction can optionally
include a
plurality of compaction oligonucleotides and/or hexamine (e.g., cobalt
hexamine Ill) to generate
forward extension strands. Individual forward extension strands can collapse
into a nanoball
having a more compact size and/or shape compared to a nanoball generated from
a primer
extension reaction conducted without compaction oligonucleotides and/or
hexamine (e.g., cobalt
hexamine III). Inclusion of compaction oligonucleotides and/or hexamine (e.g.,
cobalt hexamine
Ill) in the primer extension reaction can improve FWHM (full width half
maximum) of a spot
image of the nanoball. The spot image can be represented as a Gaussian spot
and the size can be
measured as a FWHM. A smaller spot size as indicated by a smaller FWHM
typically correlates
with an improved image of the spot. In some embodiments, the FWHM of a
nanoball spot can be
about 10 pm or smaller.
[00386] In some embodiments, in step (d), the condition suitable to hybridize
the plurality of
soluble forward sequencing primers to the plurality of retained immobilized
single stranded
nucleic acid concatemer template molecules comprises hybridizing retained
immobilized
concatemer template molecules with the soluble primers in the presence of a
primer extension
polymerase, a plurality of nucleotides, and a high efficiency hybridization
buffer. In some
embodiment, the high efficiency hybridization buffer comprises: (i) a first
polar aprotic solvent
having a dielectric constant that is no greater than 40 and having a polarity
index of 4-9; (ii) a
second polar aprotic solvent having a dielectric constant that is no greater
than 115 and is present
in the hybridization buffer formulation in an amount effective to denature
double-stranded
nucleic acids; (iii) a pH buffer system that maintains the pH of the
hybridization buffer

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formulation in a range of about 4-8; and (iv) a crowding agent in an amount
sufficient to enhance
or facilitate molecular crowding. In some embodiments, the high efficiency
hybridization buffer
comprises: (i) the first polar aprotic solvent comprises acetonitrile at 25-
50% by volume of the
hybridization buffer; (ii) the second polar aprotic solvent comprises
formamide at 5-10% by
volume of the hybridization buffer; (iii) the pH buffer system comprises 2-(N-
morphotino)ethanesulfonic acid (VIES) at a p1-1 of 5-6.5; and (iv) the
crowding agent comprises
polyethylene glycol (PEG) at 5-35% by volume of the hybridization buffer. In
some
embodiments, the high efficiency hybridization buffer further comprises
betaine.
[00.3871 In some embodiments, step (d) comprises: (1) removing the
plurality of extended
forward sequencing primer strand while retaining the immobilized concatemer
template
molecules; and (ii) contacting the plurality of retained immobilized
concatemer molecules with a
plurality of soluble amplification primers, a plurality of nucleotides (e.g.,
a second plurality of
nucleotides) and a plurality of primer extension polymerases, under a
condition suitable to
hybridize the plurality of soluble amplification primers to the plurality of
retained immobilized
concatemer template molecules and suitable for conducting polymerase-catalyzed
primer
extension reactions thereby generating a plurality of forward extension
strands, wherein the
soluble amplification primers hybridize with the soluble amplification primer
binding sequence
in the retained immobilized concatemer molecules (Figure 30), The primer
extension reaction
can optionally include a plurality of compaction oligonucleotides and/or
hexamine (e.g., cobalt
hexamine Ill) to generate forward extension strands. Individual forward
extension strands can
collapse into a nanoball having a more compact size and/or shape compared to a
nanoball
generated from a primer extension reaction conducted without compaction
oligonucleotides
and/or hexamine (e.g., cobalt hexamine Inclusion of compaction
oligonucleotides and/or
hexatnine (e.g., cobalt hexamine III) in the primer extension reaction can
improve FWHM (full
width half maximum) of a spot image of the nanoba.11. The spat image can be
represented as a
Gaussian spot and the size can be measured as a Mil:NI A smaller spot size as
indicated by a
smaller FWFIM typically correlates with an improved image of the spot. In some
embodiments,
the FWFIM of a na.noball spot can be about 10 pun or smaller.
[00388] In some embodiments, in step (d), the condition suitable to
hybridize the plurality of
soluble amplification primers to the plurality of retained immobilized single
stranded nucleic
acid concatemer template molecules comprises hybridizing retained immobilized
concatemer

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template molecules with the soluble primers in the presence of a primer
extension polymerase, a
plurality of nucleotides, and a high efficiency hybridization buffer. In some
embodiment, the
high efficiency hybridization buffer comprises: (i) a first polar aprotic
solvent having a dielectric
constant that is no greater than 40 and having a polarity index of 4-9; (ii) a
second polar aprotic
solvent having a dielectric constant that is no greater than 115 and is
present in the hybridization
buffer formulation in an amount effective to denature double-stranded nucleic
acids; (iii) a pH
buffer system that maintains the pH of the hybridization buffer formulation in
a range of about 4-
8; and (iv) a crowding agent in an amount sufficient to enhance or facilitate
molecular crowding.
In some embodiments, the high efficiency hybridization buffer comprises: (i)
the first polar
aprotic solvent comprises acetonitrile at 25-50% by volume of the
hybridization buffer; (ii) the
second polar aprotic solvent comprises formamide at 5-10% by volume of the
hybridization
buffer; (iii) the pH buffer system comprises 2-(N-morpholino)ethanesulfonic
acid (MES) at a pH
of 5-6.5; and (iv) the crowding agent comprises polyethylene glycol (PEG) at 5-
35% by volume
of the hybridization buffer. In some embodiments, the high efficiency
hybridization buffer
further comprises betaine.
[00389] In some embodiments, in step (d), the plurality of extended forward
sequencing
primer strands can be removed using an enzyme or a chemical reagent. For
example, the
plurality of extended forward sequencing primer strands can be enzymatically
degraded using a
5' to 3' double-stranded DNA exonuclease, including T7 exonuclease (e.g., from
New England
Biolabs, catalog M0263S). In some embodiments, the plurality of extended
forward
sequencing primer strands can be removed with a temperature that favors
nucleic acid
denaturation.
[00390] In some embodiments, in step (d), a denaturation reagent can be used
to remove the
plurality of extended forward sequencing primer strands, wherein the
denaturation reagent
comprises any one or any combination of compounds such as formamide,
acetonitrile,
guanidinium chloride and/or a buffering agent (e.g., Tris-HCI, MES, HEPES, or
the like).
[00391] In some embodiments, in step (d), the plurality of extended forward
sequencing
primer strands can be removed using an elevated temperature (e.g., heat) with
or without a
nucleic acid denaturation reagent. The plurality of extended forward
sequencing primer strands
can be subjected to a temperature of about 45-50 C, or about 50-60 "C, or
about 60-70 "C, or
about 70-80 C, or about 80-90 C, or about 90-95 "C, or higher temperature.

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[00392j In some embodiments, in step (d), the plurality of extended forward
sequencing
primer strands can be removed using 100% formamide at a temperature of about
65 'V for about
3 minutes, and washing with a reagent comprising about 50 mM NaCI or
equivalent ionic
strength and having a pH of about 6.5 - 8.5.
1.00393i In some embodiments, the primer extension polymerase of step (d)
comprises a high
fidelity polymerase. In some embodiments, the primer extension polymerase of
step (d)
comprises a DNA polymerase capable of catalyzing a primer extension reaction
using a uracil-
containing template molecule (e.g., a uracil-tolerant polymerase). Exemplary
polymerases
include, but are not limited to, Q5U Hot Start high-fidelity DNA polymerase
(e.g., catalog #
M0515S from New England Biolabs), Taq DNA polymerase, One Taq DNA polymerase
(e.g.,
mixture of Taq and Deep Vent DNA polymerases, catalog #M0480S from New England
Biolabs), LongAmp Taq DNA polymerase (e.g., catalog #M0323S from New England
Biolabs),
Epimark Hot Start Taq DNA polymerase (e.g., catalog #M0490S from New England
Biolabs),
Bst DNA polymerase (e.g., large fragment, catalog #M0275S from New England
Biolabs), Bsu
DNA polymerase (e.g., large fragment, catalog #M0330S from New England
Biolabs), Phi29
DNA polymerase (e.g., catalog # M02695 from New England Biolabs), E. coil DNA
polymerase
(e.g., catalog # M0209S from New England Biolabs), Therminator DNA polymerase
(e.g.,
catalog 4M0261S from New England Biolabs), Vent DNA polymerase and Deep Vent
DNA
polymerase.
[00394] The pairvvise methods described herein can provide increased accuracy
in a
downstream sequencing reaction because step (d) replaces the extended forward
sequencing
primer strands that were generated in step (c) with forward extension strands
having reduced
base errors. The extended forward sequencing primer strands are generated in
step (c) and may
or may not contain erroneously incorporated nucleotides due to polymerase-
catalyzed mis-paired
bases. When step (d) is conducted with a high fidelity DNA polymerase, the
resulting forward
extension strands may have reduced base errors compared to the extended
forward sequencing
primer strands. The forward extension strands will be used as a nucleic acid
template for a
downstream sequencing step (e.g., see step (f) below). Thus, step (d) can
increase the sequencing
accuracy of the downstream step (f) and therefore increase the overall
sequencing accuracy of
the pairwise sequencing workflow.

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[00395] In some embodiments, the pairwise sequencing method further comprises
step (e):
removing the retained immobilized concatemer template molecules by generating
abasic sites in
the immobilized single stranded concatemer template molecules at the
nucleotide(s) having the
scissile moiety and generating gaps at the abasic sites to generate a
plurality of gap-containing
single stranded nucleic acid concatemer template molecules while retaining the
plurality of
forward extension strands and retaining the plurality of immobilized surface
primers (Figures 31
and 33).
1003961 The abasic sites are generated on the retained concatemer template
strands that
contain nucleotides having scissile moieties. In some embodiments, the
scissile moieties in the
retained concatemer template molecules comprises uridine, 8-oxo-78-
dihydroguanine (e.g.,
8oxoG) or deoxyinosine. The abasic sites can be removed to generate a
plurality of single
stranded nucleic acid template molecules having gaps while retaining the
plurality of forward
extension strands. The abasic sites can be generated by contacting the
immobilized concatemer
template molecules with an enzyme that removes the nucleo-base at the
nucleotide having the
scissile moiety. The uracil in the retained concatemer template strands can be
converted to an
abasic site using uracil DNA. glycosylase (UDG). The 8oxoG in the retained
concatemer
template strands can be converted to an abasic site using FPG glycosylase. The
deoxyin.osin.e in
the retained concatemer template strands can be converted to an abasic site
using AlkA
glycosylase.
1003971 In some embodiments, in step (e), the gaps can be generated by
contacting the abasic
sites in the immobilized concatemer template molecules with an enzyme or a
mixture of enzymes
having lyase activity that. breaks the phosphodiester backbone at the 5' and
3' sides of the abasic
site to release the base-free deoxyribose and generate a gap (Figures 31 and
33). The abasic sites
can be removed using AP lyase, Endo IV endonuclease, FPG glycosylaselAP lyase,
Endo VIII
glycosylase/AP lyase. In some embodiments, generating the abasic sites and
removal of the
abasic sites to generate gaps can be achieved using a mixture of uracil DNA
glycosylase and
DNA glycosylase-lyase endonuclease VIII, for example USER (Uracil-Specific
Excision
Reagent. Enzyme from New England Biolabs) or thermolabile USER (also from New
England
Biolabs).
[00398] In some embodiments, in step (e), the plurality of gap-containing
template molecules
can be removed using an enzyme, chemical compound and/or heat. After the gap-
removal

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procedure, the plurality of retained forward extension strands are hybridized
to the retained
immobilized surface primers (figures 32 and 34).
[00399] For example, the plurality of gap-containing template molecules can
be enzymatically
degraded using a 5' to 3' double-stranded DNA exonuclease, including 17
exonuclease (e.g.,
from New England Biolabs, catalog #1140263S). When a 5' to 3' double-stranded
DNA
exonuclease is used for removing gap-containing template molecules, then the
plurality of
soluble amplification primers in step (e) can comprise at least one
phosphorothioate diester bond
at their 5' ends which can render the soluble amplification primers resistant
to exonuclease
degradation. In some embodiments, the plurality of soluble amplification
primers in step (d)
comprise 2-5 or more consecutive phosphorothioate diester bonds at their 5'
ends. In some
embodiments, the plurality soluble amplification primers in step (d) comprise
at least one
ribonucleotide and/or at least one 2'41'1-methyl or 2'-0-methoxyethyl (MOE)
nucleotide which
can render the forward sequencing primers resistant to exonuclease
degradation.
100400] In some embodiments, the plurality of gap-containing template
molecules can be
removed using a chemical reagent that favors nucleic acid denaturation. The
denaturation reagent
can include any one or any combination of compounds such as formamide,
acetonitrile,
guanidinium chloride and/or a buffering agent (e.g., Tris-HCI, MES,HEPES, or
the like).
1004011 In some embodiments, the plurality of gap-containing template
molecules can be
removed using an elevated temperature (e.g., heat) with or without a nucleic
acid denaturation
reagent. The gap-containing template molecules can be subjected to a
temperature of about 45-50
or about 50-60 C, or about 60-70 C, or about 70-80 C, or about 80-90 C, or
about 90-95
or higher temperature.
[00402] In some embodiments, the plurality of gap-containing template
molecules can be
removed using 100% forma.mide at a temperature of about 65 "C for about 3
minutes, and
washing with a reagent comprising about 50 mM NaC1 or equivalent ionic
strength and having a
PH of about 6.5 --- 8.5.
[00403] In some embodiments, the pairwise sequencing method further comprises
step (f):
sequencing the plurality of retained forward extension strands thereby
generating a plurality of
extended reverse sequencing primer strands, in some embodiments, the
sequencing of step (f)
comprises contacting the plurality of retained forward extension strands with
a plurality of
soluble reverse sequencing primers under a condition suitable to hybridize the
reverse

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sequencing primers to the reverse sequencing primer binding site of the
retained forward
extension strands, and by conducting sequencing reactions using the hybridized
reverse
sequencing primers wherein the forward sequencing reactions generates a
plurality of extended
reverse sequencing primer strands (Figures 35 and 36). The extended reverse
sequencing primer
strands are hybridized to the retained forward extension strand. The retained
forward extension
strand is hybridized to the first surface primer. The extended reverse
sequencing primer strands
are not hybridized to the first surface primer, or covalently joined to the
first surface primer.
Therefore, the extended reverse sequencing primer strands are not immobilized
to the support.
[00404] For the sake of simplicity, Figures 32 and 34 show exemplary retained
forward
extension strands each having one copy of the sequence of interest and various
universal primer
binding sites. The skilled artisan will appreciate that the retained forward
extension strand can
include two or more tandem copies containing the sequence of interest and
various universal
primer binding sites. Therefore, the reverse sequencing reaction can generate
a plurality of
extended reverse sequencing primer strands hybridized to the same retained
forward extension
strand.
[00405] In some embodiments, in step (f), the condition suitable to hybridize
the reverse
sequencing primers to the reverse sequencing primer binding sequences of the
retained forward
extension strands comprises contacting the plurality of soluble reverse
sequencing primers and
the retained forward extension strands with a high efficiency hybridization
buffer. In some
embodiments, the high efficiency hybridization buffer comprises: (i) a first
polar aprotic solvent
having a dielectric constant that is no greater than 40 and having a polarity
index of 4-9; (ii) a
second polar aprotic solvent having a dielectric constant that is no greater
than 115 and is present
in the hybridization buffer formulation in an amount effective to denature
double-stranded
nucleic acids; (iii) a pH buffer system that maintains the pH of the
hybridization buffer
formulation in a range of about 4-8; and (iv) a crowding agent in an amount
sufficient to enhance
or facilitate molecular crowding. In some embodiments, the high efficiency
hybridization buffer
comprises: (1) the first polar aprotic solvent comprises acetonitrile at 25-
50% by volume of the
hybridization buffer; (ii) the second polar aprotic solvent comprises
formamide at 5-10% by
volume of the hybridization buffer; (iii) the pH buffer system comprises 2-(N-
morpholino)ethanesulfonic acid (MES) at a pH of 5-6.5; and (iv) the crowding
agent comprises

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polyethylene glycol (PEG) at 5-35% by volume of the hybridization buffer. In
some
embodiments, the high efficiency hybridization buffer further comprises
betaine.
[004061 In an alternative embodiment, the sequencing of step (f) comprises
using the
immobilized surface primer as a sequencing primer and conducting sequencing
reactions to
generate a plurality of reverse sequencing strands.
1004071 In some embodiments, the reverse sequencing reactions of step (f)
comprises
contacting the plurality of reverse sequencing primers with the reverse
sequencing primer
binding sequences of the retained forward extension strands, one or more types
of sequencing
polymerases, and a plurality of nucleotides and/or a plurality of multivalent
molecules. In some
embodiments, the soluble reverse sequencing primers comprise 3' OH extendible
ends. In some
embodiments, the soluble reverse sequencing primers comprise a 3' blocking
moiety which can
be removed to generate a 3' OH extendible end. In some embodiments, the
soluble reverse
sequencing primers lack a nucleotide having a scissile moiety. The sequencing
reactions that
employ nucleotides and/or multivalent molecules is described in more detail
below. The reverse
sequencing reactions can generate a plurality of extended reverse sequencing
primer strands. In
some embodiments, individual retained forward extension strands have multiple
copies of the
reverse sequencing primer binding sequences/sites, wherein each reverse
sequencing primer
binding site is capable of hybridizing to a reverse sequencing primer.
Individual reverse
sequencing primer binding sites in a given retained forward extension strand
can be hybridized to
a reverse sequencing primer and can undergo a sequencing reaction. Thus, an
individual retained
forward extension strand can undergo two or more sequence reactions, where
each sequencing
reaction is initiated from a reverse sequencing primer that is hybridized to a
reverse sequencing
primer binding site (e.g., see Figures 35 and 36). In some embodiments, the
sequencing reactions
comprise a plurality of nucleotides (or analogs thereof) labeled with a
detectable reporter moiety.
In some embodiments, the sequencing reaction comprise a plurality of
multivalent molecules
having nucleotide units, where the multivalent molecules are labeled with a
detectable reporter
moiety. In some embodiments, the detectable reporter moiety comprises a
fluorophore.
[004081 In some embodiments, at least one washing step can be conducted after
any of steps
(a) --- (1). The washing step can be conducted with a wash buffer comprising a
pH buffering
agent, a metal chelating agent, a salt, and a detergent.

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100409j In some embodiments, the pH buffering compound in the wash buffer
comprises any
one or any combination of two or more of iris, Tris-HCI, Tricine, Bicine, Bis-
Tris propane,
HEPES, MES, MOPS, MOPSO, BES, TES, CAPS, TAPS, TAPSO, ACES, PIPES,
ethanolamine (a.k.a 2-amino methanol; MEA), a citrate compound, a citrate
mixture, NaOH
and/or KOH. In some embodiments, the pH buffering agent can be present in the
wash buffer at
a concentration of about 1-100 mM, or about 10-50 rnM, or about 10-25 mM. In
some
embodiments, the pH of the pH buffering agent which is present in any of the
reagents described
here in can be adjusted to a pH of about 4-9, or a pH of about 5-9, or a pH of
about 5-8.
100410) In some embodiments, the metal chelating agent in the wash buffer
comprises EDTA
(ethylenediaminetetraacetic acid), EGTA (ethylene glycol tetraacetic acid),
HEDTA
(hydrox-yethylethylenecliaminetriacetic acid), 'DMA (diethylene triamine
pentaacetic acid), NIA
(N,N-bis(carboxymethyl)glycine), citrate anhydrous, sodium citrate, calcium
citrate, ammonium
citrate, ammonium bicitrate, citric acid, potassium citrate, or magnesium
citrate. In some
embodiments, the wash buffer comprises a chelating agent at a concentration of
about 0.01 ¨ 50
mM, or about 0.1 ¨20 mM, or about 0.2 ¨ 10 mM.
[0041.11 In some embodiments, the salt in the wash buffer comprises NaCl, KCI,
NH2SO4 or
potassium glutamate. In some embodiments, the detergent comprises an ionic
detergent such as
SDS (sodium dodecyl sulfate). The wash buffer can include a monovalent salt at
a concentration
of about 25-500 mM., or about 50-250 mM, or about 100-200 mM.
[0041.2] In some embodiments, the detergent in the wash buffer comprises a non-
ionic
detergent such as Triton X-100, Tween 20, Tween 80 or Nonidet P-40. In some
embodiments,
the detergent comprises a zwitterionic detergent such as CHAPS (34(3-
cholamidopropyl)dimethylammonio]-1-propanesulfonate) or N-Dodecyl-N;N-dimethy1-
3-
amonio-1 -propanesulfate (DetX). In some embodiments, the detergent comprises
LDS (lithium
dodecyl sulfate), sodium taurodeoxycholate, sodium taurocholate, sodium
glycocholate, sodium
deoxycholate or sodium cholate. In some embodiments, the detergent is included
in the wash
buffer at a concentration of about 0.01-0.05%, or about 0.05-0.1%, or about
0.1-0.15%, or about
0.15-0 2%, or about 0.2-0 25%.
In Solution RCA and Pairwise Sequencing Generating Abasic Sites
[0041.3] The present disclosure provides pairvvise sequencing methods,
comprising step (a):
contacting in-solution a plurality of single-stranded circular nucleic acid
library molecules to a

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plurality of soluble first amplification primers, a plurality of a strand
displacing polymerase, and
a plurality of nucleotides which include dATP, dCTP, dGIP, dTTP and a
nucleotide having a
scissile moiety, under a condition suitable to form a plurality of library-
primer duplexes and
suitable for conducting a rolling circle amplification reaction, thereby
generating a plurality of
single stranded nucleic acid concatemers having at least one nucleotide with a
scissile moiety
(Figure 38). In some embodiments, the soluble first amplification primer
comprises a sequence
that selectively hybridizes to a universal binding sequence in the circular
nucleic acid library
molecules, such as for example a universal binding sequence (or a
complementary sequence
thereof) for the first soluble amplification primer. Alternatively, the
soluble first amplification
primer comprises a random sequence that binds non-selectively to a sequence in
the circular
nucleic acid library molecules.
1004141 In some embodiments, individual single stranded circular nucleic acid
library
molecules in the plurality comprises a sequence of interest and wherein the
individual library
molecules further comprise any one or any combination of two or more of (i) a
universal binding
sequence (or a complementary sequence thereof) for a soluble forward
sequencing primer, (ii) a
universal binding sequence (or a complementary sequence thereof) for a soluble
reverse
sequencing primer, (iii) a universal binding sequence (or a complementary
sequence thereof) for
an immobilized first surface primer, (iv) a universal binding sequence (or a
complementary
sequence thereof) for an immobilized second surface primer, (v) a universal
binding sequence (or
a complementary sequence thereof) for a first soluble amplification primer,
(vi) a universal
binding sequence (or a complementary sequence thereof) for a second soluble
amplification
primer, (vii) a universal binding sequence (or a complementary sequence
thereof) for a soluble
compaction oligonucleotide, (viii) a sample barcode sequence and/or (ix) a
unique molecular
index sequence. In some embodiments, the single-stranded circular nucleic acid
library
molecules comprise covalently closed circular molecules.
[0041.5] In some embodiments, the rolling circle amplification reaction of
step (a) generates a
plurality of single stranded nucleic acid concatemer molecules in solution,
comprising a
concatemer having at least one nucleotide having a scissile moiety. In some
embodiments,
individual concatemer template molecules in the plurality comprise two or more
copies of a
sequence of interest, and wherein the individual immobilized concatemer
template molecules
further comprise any one or any combination of two or more of: (i) two or more
copies of a

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universal binding sequence for a soluble forward sequencing primer, (ii) two
or more copies of a
universal binding sequence for a soluble reverse sequencing primer, (iii) two
or more copies of a
universal binding sequence for an immobilized first surface primer, (iv) two
or more copies of a
universal binding sequence for an immobilized second surface primer, (v) two
or more copies of
a universal binding sequence for a first soluble amplification primer, (vi)
two or more copies of a
universal binding sequence for a second soluble amplification primer, (vii)
two or more copies of
a universal binding sequence for a soluble compaction oligonucleotide, (viii)
two or more copies
of a sample 'barcode sequence and/or (ix) two or more copies of a unique
molecular index
sequence.
1004161 In some embodiments, the universal binding sequence (or a
complementary sequence
thereof) for the forward sequencing primer can hybridize to at least a portion
of the forward
sequencing primer. In some embodiments, the universal binding sequence (or a
complementary
sequence thereof) for the reverse sequencing primer can hybridize to at least
a portion of the
reverse sequencing primer. In some embodiments, the universal binding sequence
(or a
complementary sequence thereof) for the immobilized first surface primer can
hybridize to at
least a portion of the immobilized first surface primer. In some embodiments,
the universal
binding sequence (or a complementary sequence thereof) for the immobilized
second surface
primer can hybridize to at least a portion of the immobilized second surface
primer. In some
embodiments, the universal binding sequence (or a complementary sequence
thereof) for the first
soluble amplification primer can hybridize to at least a portion of the first
soluble amplification
primer. In some embodiments, the universal binding sequence (or a
complementary sequence
thereof) for the second soluble amplification primer can hybridize to at least
a portion of the
second soluble amplification primer. In some embodiments, the universal
binding sequence (or a
complementary sequence thereof) for the soluble compaction oligonucleotide can
hybridize to at
least a portion of the soluble compaction oligonucleotide.
[00417] The in-solution rolling circle amplification reaction of step (a)
can be conducted with
a nucleotide mixture containing dATP, dCTP, dGIP, dTTP and a nucleotide having
a scissile
moiety to generate the concatemer molecules which includes at least one
nucleotide having a
scissile moiety. The scissile moieties in the concatemer molecules can be
converted into ahasic
sites. In some embodiments, in the nucleotide mixture, the nucleotide having
the scissile moiety
comprises uridine, 8-oxo-7,8-dihydroguanine (e.g., 8oxoG) or deoxyinosine. In
the concatemer

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molecules, the uridine can be converted to an abasic site using uracil DNA
glycosylase (UDG),
the 8oxoG can be converted to an abasic site using FPG glycosylase, and the
deoxyinosine can
be converted to an abasic site using AlkA glycosylase.
10041M In some embodiments, the nucleotide mixture can include an amount of
dUTP so that
a target percent of the thymidine in the resulting concatemer molecules are
replaced with dUTP.
For example, when 30% of dTTP in the concatemer molecules are to be replaced
with dUTP
(e.g., 30% is the target percent) then the nucleotide mixture can contain 7.5%
dUTP (e.g., 30/4 =
7.5%), 17.5% dTTP, and 25% each for dATP, dCIP and dGTP. 'The target percent
of dTTP to be
replaced by dUTP can be about 0.1-1%, or about 1-5%, or about 5-10%, or about
10-20%, or
about 20-30%, or about 30-45%, or about 45-50%, or a higher percent of the
dTTP in the
concatemer molecules are replaced with nucleotides having a scissile moiety.
1004191 In some embodiments, the nucleotide mixture can include an amount of
deoxyinosine
so that a target percent of the guanosine in the resulting concatemer
molecules are replaced with
deoxyinosine. For example, when 30% of dGTP in the concatemer molecules are to
be replaced
with deoxyinosine (e.g., 30% is the target percent) then the nucleotide
mixture can contain 7.5%
deoxyinosine (e.g., 30/4 = 7.5%), 17.5% dGTP, and 25% each for dATP, dCTP and
dTTP. The
target percent of dG ___________________________________________________ FP to
be replaced by deoxyinosine can be about 0.1-1%, or about 1-5%, or
about 5-10%, or about 10-20%, or about 20-30%, or about 30-45%, or about 45-
50%, or a
higher percent of the dGTP in the concatemer molecules are replaced with
nucleotides having a
scissile moiety.
1004201 In some embodiments, the nucleotide mixture can include an amount of
8oxoG so
that a target percent of the guanosine in the resulting concatemer molecules
are replaced with
8oxoG. For example, when 30% of dGTP in the concatemer molecules are to be
replaced with
8oxoG (e.g., 30% is the target percent) then the nucleotide mixture can
contain 7.5% 8oxoG
(e.g., 30/4 = 7.5%), 17.5% dGTP. and 25% each for dATP, dCIP and dTTP, The
target percent
of dGTP to be replaced by 8oxoG can be about 0.1-1%, or about 1-5%, or about 5-
10%, or about
10-20%, or about 20-30%, or about 30-45%, or about 45-50%, or a higher percent
of the dGTP
in the concatemer molecules are replaced with nucleotides having a scissile
moiety.
1004211 In
some embodiments, the in-solution rolling circle amplification reaction
generates
concatemer molecules with incorporated nucleotides having a scissile moiety
that are distributed
at random positions along individual immobilized concatemer template
molecules. In some

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embodiments, the nucleotides having a scissile moiety are distributed at
different positions in the
different concatemer molecules.
[00422j In some embodiments, the pairwise sequencing method further comprises
step (b):
distributing the rolling circle amplification reaction from step (a) onto a
support having a
plurality of the first surface primers immobilized thereon, under a condition
suitable for
hybridizing one or more portions of individual single stranded concatemers to
one or more
immobilized first surface primers (Figure 39). In some embodiments, the
immobilized first
surface primers have terminal 3' group that are non-extendible. In some
embodiments, the 3'
terminal end of the immobilized first surface primers comprise a moiety that
blocks primer
extension, such as for example a phosphate group, a dideoxycytidine group, an
inverted dT, or an
amino group. In some embodiments, the immobilized first surface primer have an
extendible
3'0H end. In some embodiments, the immobilized first surface primers lack a
nucleotide having
a scissile moiety. The concatemers are immobilized to the support by
hybridization to the
immobilized first surface primers. In some embodiments, the support comprises
a plurality of
first surface primers. In some embodiments, the support lacks a plurality of
second surface
primers. In some embodiments, the support comprises a plurality of first and
second surface
primers.
[00423] In some embodiments, the pairwise sequencing method further comprises
step (c):
continuing the rolling circle amplification reaction on the support to
generate a plurality of
extended concatemer template molecules that are immobilized via hybridization
to the
immobilized first surface primers (Figure 40). The on-support RCA reaction can
be conducted
with a plurality of a strand displacing polymerase, and a plurality of
nucleotides which include
dATP, dCTP, dGTP, dTTP and a nucleotide having a scissile moiety, under a
condition suitable
to generate a plurality of extended concatemers having at least one nucleotide
with a scissile
moiety (Figure 41). In some embodiments, the rolling circle amplification
reaction on the
support can be conducted in the presence, or in the absence, of a plurality of
compaction
oligonucleotides.
[00424] In some embodiments, the on-support rolling circle amplification
reaction generates
immobilized concatemer template molecules with incorporated nucleotides having
a scissile
moiety that are distributed at random positions along individual immobilized
concatemer

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template molecules. in some embodiments, the nucleotides having a scissile
moiety are
distributed at different positions in the different immobilized conca.temer
template molecules.
[00425] In some embodiments, the immobilized first surface primers comprise
single stranded
oligonucleotides comprising DNA, RNA or a combination of DNA and RNA. The
first surface
primers comprise a sequence that is wholly complementary or partially
complementary along
their lengths to at least a portion of the concatemer molecules. In some
embodiments, the first
surface primers can lack a terminal 3' OH extendible end which renders the
first surface primers
non-extendible. In some embodiments, the first surface primers include a
terminal 3' OH group
which is extendible for nucleotide polymerization (c.a., polymerase catalyzed
polymerization).
The immobilized first surface primers can be immobilized to the support or
immobilized to a.
coating on the support. The immobilized first surface primers can be embedded
and attached
(coupled) to the coating on the support. in some embodiments, the 5' end of
the immobilized
first surface primers are immobilized to a support or immobilized to a coating
on the support.
Alternatively, an interior portion or the 3' end of the immobilized first
surface primers can be
immobilized to a support or immobilized to a coating on the support. The
support comprises a
plurality of immobilized first surface primers having the same sequence. The
immobilized first
surface primers can be any length, for example 4-50 nucleotides, or 50-100
nucleotides, or 100-
150 nucleotides, or longer lengths.
[004261 In some embodiments, the plurality of immobilized first surface
primers comprise 3'
extendible ends. In some embodiments, the 3' terminal end of the immobilized
first surface
primers comprise a moiety that blocks primer extension, such as for example a
phosphate group,
dideoxycytidine group, an inverted dT, or an amino group. In some embodiments,
the
immobilized first surface primers are not extendible in a primer extension
reaction. The
immobilized first surface primers lack a nucleotide having a scissile moiety.
[00427] In some embodiments, the plurality of immobilized first surface
primers comprise at
least one phosphorothioate diester bond at their 5' ends which can render the
first surface
primers resistant to exonuclease degradation. In some embodiments, the
plurality of immobilized
first surface primers comprise 2-5 or more consecutive phosphorothioate
diester bonds at their 5'
ends. In sonic embodiments, the plurality of immobilized first surface primers
comprise at least
one ribonucleotide and/or at least one 2'-0-tnethyl or 2'-0-methoxyethy1 (MOE)
nucleotide
which can render the first surface primers resistant to exonuclease
degradation.

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[004281 In some embodiments, the immobilized first surface primers comprise at
least one
locked nucleic acid (LNA) which comprises a methylene bridge bond between a 2'
oxygen and
4' carbon of the pentose ring. Immobilized first surface primers that include
at least one LNA
can be resistant to nuclease digestions and can exhibit increased melting
temperature when
hybridized to the concatemer template molecules.
1004291 In some embodiments, the support further comprises a plurality of a
second surface
primer immobilized thereon (Figure 52). The second surface primers have a
sequence that differs
from the first immobilized surface primer. The immobilized second surface
primers comprise
single stranded oligonucleotides comprising DNA, RNA or a combination of DNA
and RNA.
The second surface primers comprise a sequence that is wholly complementary or
partially
complementary along their lengths to at least a portion of a concatemer
molecule. The
immobilized second surface primers can be immobilized to the support or
immobilized to a
coating on the support. The immobilized second surface primers can be embedded
and attached
(coupled) to the coating on the support. In some embodiments, the 5' end of
the immobilized
second surface primers are immobilized to a support or immobilized to a
coating on the support.
Alternatively, an interior portion or the 3' end of the immobilized second
surface primers can be
immobilized to a support or immobilized to a coating on the support. The
support comprises a
plurality of immobilized second surface primers having the same sequence. The
immobilized
second surface primers can be any length, for example 4-50 nucleotides, or 50-
100 nucleotides,
or 100-150 nucleotides, or longer lengths.
[004301 In some embodiments, the 3' terminal end of the immobilized second
surface primers
comprise an extendible 3' OH moiety. In some embodiments, the 3' terminal end
of the
immobilized second surface primers comprise a 3' non-extendible moiety. The 3'
terminal end of
the immobilized second surface primers comprise a moiety that blocks primer
extension, such as
for example a phosphate group, a dideoxycytidine group, an inverted dT, or an
amino group. The
immobilized second surface primers are not extendible in a primer extension
reaction. The
immobilized second surface primers lack a nucleotide having a scissile moiety.
[00431] In some embodiments, the plurality of immobilized second surface
primers comprise
at least one phosphorothioate diester bond at their 5' ends which can render
the second surface
primers resistant to exonuclease degradation. In some embodiments, the
plurality of immobilized
second surface primers comprise 2-5 or more consecutive phosphorothioate
diester bonds at their

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5' ends. In some embodiments, the plurality of immobilized second surface
primers comprise at
least one ribonucleotide and/or at least one 2'-0-methyl or 2'-0-methoxyethyl
(MOE) nucleotide
which can render the second surface primers resistant to exonuclease
degradation.
[004321 In some embodiments, individual immobilized single stranded nucleic
acid
concatemer template molecule are hybridized to an immobilized first surface
primer, and at least
one portion of the individual concatemer template molecule is hybridized to an
immobilized
second surface primer (Figure 52). The immobilized second surface primers
serve to pin down a
portion of the immobilized concatemer template molecules to the support. The
immobilized
concatemer template molecule has two or more copies of a universal binding
sequence for an
immobilized second surface primer. The portion of the immobilized concatemer
template
molecule that includes the universal binding sequence for an immobilized
second surface primer
can hybridize to the immobilized second surface primer. In some embodiments,
the second
surface primers include a terminal 3' blocking group that renders them non-
extendible. In some
embodiments, the second surface primers have terminal 3' extendible ends.
[00433] In some embodiments, the support comprises about 102 ¨ 1015
immobilized first
surface primers per mm2. In some embodiments, the support comprises about 102
¨ 1015
immobilized second surface primers per mm2. In some embodiments, the support
comprises
about 102 ¨ 1015 immobilized first surface primers and immobilized second
surface primers per
MM2 .
[00434] The immobilized surface primers (e.g., first and second surface
primers) are in fluid
communication with each other to permit flowing various solutions of linear or
circular nucleic
acid template molecules, soluble primers, enzymes, nucleotides, divalent
cations, buffers,
reagents, and the like, onto the support so that the plurality of immobilized
surface primers react
with the solutions in a massively parallel manner.
[00435] In some embodiments, the pairwise sequencing method further comprises
step (d):
sequencing the plurality of immobilized concatemer template molecules thereby
generating a
plurality of extended forward sequencing primer strands. The sequencing of
step (d) comprises
contacting the plurality of immobilized concatemer template molecules with a
plurality of
soluble forward sequencing primers under a condition suitable to hybridize at
least one forward
sequencing primer to at least one of the forward sequencing primer binding
sites/sequences of
the immobilized concatemer template molecules, and conducting forward
sequencing reactions

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using one or more types of sequencing polymerases, a plurality of nucleotides
and/or multivalent
molecules, and the hybridized first forward sequencing primers. In some
embodiments, the
soluble forward sequencing primers comprise 3' OH extendible ends. In some
embodiments, the
soluble forward sequencing primers comprise a 3' blocking moiety which can be
removed to
generate a 3' OH extendible end. In some embodiments, the soluble forward
sequencing primers
lack a nucleotide having a scissile moiety. The forward sequencing reactions
can generate a
plurality of extended forward sequencing primer strands (Figure 42). In some
embodiments,
individual immobilized concatemer template molecules have multiple copies of
the forward
sequencing primer binding sites, wherein each forward sequencing primer
binding site is capable
of hybridizing to a first forward sequencing primer. Individual forward
sequencing primer
binding sites in a given immobilized concatemer template molecule can be
hybridized to a
forward sequencing primer and can undergo a sequencing reaction. Individual
immobilized
concatemer template molecules can undergo two or more sequence reactions,
where each
sequencing reaction is initiated from a first forward sequencing primer that
is hybridized to a
forward sequencing primer binding site (e.g., see Figure 42). In some
embodiments, the
sequencing reactions comprise a plurality of nucleotides (or analogs thereof)
labeled with a
detectable reporter moiety. In some embodiments, the sequencing reaction
comprise a plurality
of multivalent molecules having a plurality of nucleotide units attached to a
core, where the
multivalent molecules are labeled with a detectable reporter moiety. In some
embodiments, the
core is labeled with a detectable reporter moiety. In some embodiments, at
least one linker and/or
at least one nucleotide unit of a nucleotide arm is labeled with a detectable
reporter moiety. In
some embodiments, the detectable reporter moiety comprises a fluorophore. An
exemplary
nucleotide arm is shown in Figure 108, and exemplary multivalent molecules are
shown in
Figures 104-107.
[00436] In some embodiments, the pairwise sequencing method further comprises
step (e):
retaining the plurality of immobilized concatemer template molecules and
replacing the plurality
of extended forward sequencing primer strands with a plurality of forward
extension strands that
are hybridized to the retained immobilized single stranded nucleic acid
concatemer template
molecules. The plurality of extended forward sequencing primer strands can be
removed and
replaced with a plurality of forward extension strands by conducting a primer
extension reaction
(See Figures 43-45).

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[004371 In some embodiments, step (e) comprises contacting at least one
extended forward
sequencing primer strand with a plurality of strand displacing polymerases and
a plurality of
nucleotides and in the absence of soluble amplification primers, under a
condition suitable to
conduct a strand displacing primer extension reaction using the at least one
extended forward
sequencing primers strand to initiate the primer extension reaction thereby
generating a forward
extension strand that is covalently joined to the extended forward sequencing
primers strand,
wherein the forward extension strand is hybridized to the immobilized
concatemer template
molecule (Figure 43). For example, one of the extended forward sequencing
primer strands can
serve as a primer for the strand displacing polymerase. The strand displacing
polymerase can
extend the extended forward sequencing primer strand, and displace downstream
extended
forward sequencing primer strands while synthesizing an extended strand that
replaces the
downstream extended forward sequencing primer strands. The newly extended
strand is
covalently joined to an extended forward sequencing primer strand. The
immobilized concatemer
template molecules are retained. The primer extension reaction can optionally
include a plurality
of compaction oligonucleotides and/or hexamine (e.g., cobalt hexamine III) to
generate forward
extension strands. Individual forward extension strands can collapse into a
nanoball having a
more compact size and/or shape compared to a nanoball generated from a primer
extension
reaction conducted without compaction oligonucleotides and/or hexamine (e.g.,
cobalt hexamine
1:11). Inclusion of compaction oligonucleotides and/or hexamine (e.g., cobalt
hexamine Ill) in the
primer extension reaction can improve FWHM (full width half maximum) of a spot
image of the
nanoball. The spot image can be represented as a Gaussian spot and the size
can be measured as
a FWHM. A smaller spot size as indicated by a smaller FWHM typically
correlates with an
improved image of the spot. In some embodiments, the FV,THM of a nanoball spot
can be about
gm or smaller.
[004381 Examples of strand displacing polymerases include phi29 DNA
polymerase, large
fragment of Bst DNA polymerase, large fragment of Bsu DNA polymerase (exo-),
Bca DNA
polymerase (exo-), Klenow fragment of E. coli DNA polymerase, T5 polymerase, M-
MuLV
reverse transcriptase, HIV viral reverse transcriptase, Deep Vent DNA
polymerase and KOD
DNA polymerase. The phi29 DNA polymerase can be wild type phi29 DNA polymerase
(e.g.,
MagniPhi from Expedeon), or variant EquiPhi29 DNA polymerase (e.g., from
Thermo Fisher
Scientific), or chimeric QualiPhi DNA polymerase (e.g., from 4basebio).

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[00439j In some embodiments, step (e) comprises: (i) removing the plurality of
extended
forward sequencing primer strand while retaining the immobilized concatemer
template
molecules; and (ii) contacting the plurality of retained immobilized
concatemer molecules with a
plurality of soluble forward sequencing primers (e.g., a second plurality of
soluble forward
sequencing primers), a plurality of nucleotides (e.g., a second plurality of
nucleotides) and a
plurality of primer extension polymerases, under a condition suitable to
hybridize the plurality of
soluble forward sequencing primers to the plurality of retained immobilized
concatemer template
molecules and suitable for conducting polymerase-catalyzed primer extension
reactions thereby
generating a plurality of forward extension strands, wherein the soluble
sequencing primers
hybridize with the forward sequencing primer binding sequence in the retained
immobilized
concatemer molecules (Figure 44). The primer extension reaction can optionally
include a
plurality of compaction oligonucleotides and/or hexamine (e.g., cobalt
hexamine III) to generate
forward extension strands. Individual forward extension strands can collapse
into a nanoball
having a more compact size and/or shape compared to a nanoball generated from
a primer
extension reaction conducted without compaction oligonucleotides and/or
hexamine (e.g., cobalt
hexamine III). Inclusion of compaction oligonucleotides and/or hexamine (e.g.,
cobalt hexamine
Ill) in the primer extension reaction can improve FWHM (full width half
maximum) of a spot
image of the nanoball. The spot image can be represented as a Gaussian spot
and the size can be
measured as a FWHM. A smaller spot size as indicated by a smaller FWHM
typically correlates
with an improved image of the spot. In some embodiments, the FWHM of a
nanoball spot can be
about 10 pm or smaller.
[00440] In some embodiments, in step (e), the condition suitable to hybridize
the plurality of
soluble forward sequencing primers to the plurality of retained immobilized
single stranded
nucleic acid concatemer template molecules comprises hybridizing retained
immobilized
concatemer template molecules with the soluble primers in the presence of a
primer extension
polymerase, a plurality of nucleotides, and a high efficiency hybridization
buffer. In some
embodiment, the high efficiency hybridization buffer comprises: (i) a first
polar aprotic solvent
having a dielectric constant that is no greater than 40 and having a polarity
index of 4-9; (ii) a
second polar aprotic solvent having a dielectric constant that is no greater
than 115 and is present
in the hybridization buffer formulation in an amount effective to denature
double-stranded
nucleic acids; (iii) a pH buffer system that maintains the pH of the
hybridization buffer

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formulation in a range of about 4-8; and (iv) a crowding agent in an amount
sufficient to enhance
or facilitate molecular crowding. In some embodiments, the high efficiency
hybridization buffer
comprises: (i) the first polar aprotic solvent comprises acetonitrile at 25-
50% by volume of the
hybridization buffer; (ii) the second polar aprotic solvent comprises
formamide at 5-10% by
volume of the hybridization buffer; (iii) the pH buffer system comprises 2-(N-
morpholino)ethanesulfonic acid (VMS) at a p1-1 of 5-6.5; and (iv) the crowding
agent comprises
polyethylene glycol (PEG) at 5-35% by volume of the hybridization buffer. In
some
embodiments, the high efficiency hybridization buffer further comprises
betaine.
[004411 In some embodiments, step (e) comprises: (1) removing the plurality
of extended
forward sequencing primer strand while retaining the immobilized concatemer
template
molecules; and (ii) contacting the plurality of retained immobilized
concatemer molecules with a
plurality of soluble amplification primers, a plurality of nucleotides (e.g.,
a second plurality of
nucleotides) and a plurality of primer extension polymerases, under a
condition suitable to
hybridize the plurality of soluble amplification primers to the plurality of
retained immobilized
concatemer template molecules and suitable for conducting polymerase-catalyzed
primer
extension reactions thereby generating a plurality of forward extension
strands, wherein the
soluble amplification primers hybridize with the soluble amplification primer
binding sequence
in the retained immobilized concatemer molecules (Figure 45), The primer
extension reaction
can optionally include a plurality of compaction oligonucleotides and/or
hexamine (e.g., cobalt
hexamine Ill) to generate forward extension strands. Individual forward
extension strands can
collapse into a nanoball having a more compact size and/or shape compared to a
nanoball
generated from a primer extension reaction conducted without compaction
oligonucleotides
and/or hexamine (e.g., cobalt hexamine Inclusion of compaction
oligonucleotides and/or
hexamine (e.g., cobalt hexamine III) in the primer extension reaction can
improve FWHM (full
width half maximum) of a spot image of the nanoball. The spat image can be
represented as a
Gaussian spot and the size can be measured as a Mil:NI A smaller spot size as
indicated by a
smaller FWFIM typically correlates with an improved image of the spot. In some
embodiments,
the FWFIM of a nanoball spot can be about 10 mn or smaller.
[00442] In some embodiments, in step (e), the condition suitable to
hybridize the plurality of
soluble amplification primers to the plurality of retained immobilized single
stranded nucleic
acid concatemer template molecules comprises hybridizing retained immobilized
concatemer

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template molecules with the soluble primers in the presence of a primer
extension polymerase, a
plurality of nucleotides, and a high efficiency hybridization buffer. In some
embodiment, the
high efficiency hybridization buffer comprises: (i) a first polar aprotic
solvent having a dielectric
constant that is no greater than 40 and having a polarity index of 4-9; (ii) a
second polar aprotic
solvent having a dielectric constant that is no greater than 115 and is
present in the hybridization
buffer formulation in an amount effective to denature double-stranded nucleic
acids; (iii) a pH
buffer system that maintains the pH of the hybridization buffer formulation in
a range of about 4-
8; and (iv) a crowding agent in an amount sufficient to enhance or facilitate
molecular crowding.
In some embodiments, the high efficiency hybridization buffer comprises: (i)
the first polar
aprotic solvent comprises acetonitrile at 25-50% by volume of the
hybridization buffer; (ii) the
second polar aprotic solvent comprises formamide at 5-10% by volume of the
hybridization
buffer; (iii) the pH buffer system comprises 2-(1V-morpholino)ethanesulfonic
acid (MES) at a pH
of 5-6.5; and (iv) the crowding agent comprises polyethylene glycol (PEG) at 5-
35% by volume
of the hybridization buffer. In some embodiments, the high efficiency
hybridization buffer
further comprises betaine.
100443] In some embodiments, in step (e), the plurality of extended forward
sequencing
primer strands can be removed using an enzyme or a chemical reagent. For
example, the
plurality of extended forward sequencing primer strands can be enzymatically
degraded using a
5' to 3' double-stranded DNA. exonuclease, including T7 exonuclease (e.g.,
from New England
Biolabs, catalog # M0263S). In some embodiments, the plurality of extended
forward
sequencing primer strands can be removed with a temperature that favors
nucleic acid
denaturation.
[00444] In some embodiments, in step (e), a denaturation reagent can be used
to remove the
plurality of extended forward sequencing primer strands, wherein the
denaturation reagent
comprises any one or any combination of compounds such as fonna.mide,
acetonitrile,
guanidinium chloride and/or a buffering agent (e.g., Tris-HCI, MES, HEPES, or
the like).
[00445] In some embodiments, in step (e), the plurality of extended forward
sequencing
primer strands can be removed using an elevated temperature (e.g., heat) with
or without a
nucleic acid denaturation reagent. The plurality of extended forward
sequencing primer strands
can be subjected to a temperature of about 45-50 C., or about 50-60 "C, or
about 60-70 "C, or
about 70-80 C, or about 80-90 C, or about 90-95 "C, or higher temperature.

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[00446] In some embodiments, in step (e), the plurality of extended forward
sequencing
primer strands can be removed using 100% formamide at a temperature of about
65 'C for about
3 minutes, and washing with a reagent comprising about 50 triM NaCI or
equivalent ionic
strength and having a pH of about 6.5 ¨ 8.5.
[00447] In some embodiments, the primer extension polymerase of step (e)
comprises a high
fidelity polymerase. in some embodiments, the primer extension polymerase of
step (e)
comprises a DNA polymerase capable of catalyzing a primer extension reaction
using a uracil-
containing template molecule (e.g., a uracil-tolerant polymerase). Exemplary
polymerases
include, but are not limited to, Q5U Hot Start high-fidelity DNA polymerase
(e.g., catalog #
1\40515S from New England Biolabs), Taq DNA polymerase, One Taq DNA polymerase
(e.g.,
mixture of Taq and Deep Vent DNA polymerases, catalog #1140480S from New
England
Biolabs), LongAmp Taq DNA polymerase (e.g., catalog #M0323S from New England
Biolabs),
Epimark Hot Start Taq DNA polymerase (e.g., catalog #M.04905 from New England
Biolabs);
Bst DNA polymerase (e.g., large fragment, catalog #M0275S from New England
Biolabs), Bsu
DNA polymerase (e.g., large fragment, catalog #M0330S from New England
Biolabs), Phi29
DNA polymerase (e.g., catalog # M0269S from New England Biolabs), E. col/ DNA
polymerase
(e.g., catalog # M0209S from New England Biolabs), Therminator DNA polymerase
(e.g.,
catalog 41\40261S from New England Biolabs), Vent DNA polymerase and Deep Vent
DNA.
polymerase.
1004481 The pairwise methods described herein can provide increased accuracy
in a.
downstream sequencing reaction because step (e) replaces the extended forward
sequencing
primer strands that were generated in step (d) with forward extension strands
having reduced
base errors. The extended forward sequencing primer strands are generated in
step (d) and may
or may not contain erroneously incorporated nucleotides due to polymerase-
catalyzed mis-paired
bases. When step (e) is conducted with a high fidelity DNA polymerase, the
resulting forward
extension strands may have reduced base errors compared to the extended
forward sequencing
primer strands. The forward extension strands will be used as a nucleic acid
template for a
downstream sequencing step (e.g., see step (f) below). Thus, step (e) can
increase the sequencing
accuracy of the downstream step (g) and therefore increase the overall
sequencing accuracy of
the pairwise sequencing workflow.

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[00449j In some embodiments, the pairwise sequencing method further comprises
step (I):
removing the retained immobilized concatemer template molecules by generating
abasic sites in
the immobilized single stranded concatemer template molecules at the
nucleotide(s) having the
scissile moiety and generating gaps at the abasic sites to generate a
plurality of gap-containing
single stranded nucleic acid concatemer template molecules while retaining the
plurality of
forward extension strands and retaining the plurality of immobilized surface
primers (Figures 46
and 48).
[004501 The abasic sites are generated on the retained concatemer template
strands that
contain nucleotides having scissile moieties. In some embodiments, the
scissile moieties in the
retained concatemer template molecules comprises uridine, 8-oxo-7,8-
dihydroguanine (e.g.,
8oxoG) or deoxyinosine. The abasic sites can be removed to generate a
plurality of single
stranded nucleic acid template molecules having gaps while retaining the
plurality of forward
extension strands. The abasic sites can be generated by contacting the
immobilized concatemer
template molecules with an enzyme that removes the nucleo-base at the
nucleotide having the
scissile moiety. The uracil in the retained concatemer template strands can be
converted to an
abasic site using uracil DNA glycosylase (UDG). The 8oxoG in the retained
concatemer
template strands can be converted to an abasic site using FPG glycosylase. The
deoxyinosine in
the retained concatemer template strands can be converted to an abasic site
using AlkA
glycosylase.
[00451] In some embodiments, in step (f), the gaps can be generated by
contacting the abasic
sites in the immobilized concatemer template molecules with an enzyme or a
mixture of enzymes
having lyase activity that breaks the phosphodiester backbone at the 5' and 3'
sides of the abasic
site to release the base-free deoxyribose and generate a gap (Figures 46 and
48). The abasic sites
can be removed using AP lyase, Endo TV endonuclease, FPG glycosylase/AP lyase,
Endo VIII
glycosylase/AP lyase. In some embodiments, generating the abasic sites and
removal of the
abasic sites to generate gaps can be achieved using a mixture of uracil DNA
glycosylase and
DNA glycosylase-lyase endonuclease VIII, for example USER (Uracil-Specific
Excision
Reagent Enzyme from New England Biolabs) or thermolabile USER (also from New
England
Biolabs).
[00452j In some embodiments, in step (f), the plurality of gap-containing
template molecules
can be removed using an enzyme, chemical compound and/or heat. After the gap-
removal

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procedure, the plurality of retained forward extension strands can be
hybridized to the retained
immobilized surface primers (figures 47 and 49).
[00453] For example, the plurality of gap-containing template molecules can
be enzymatically
degraded using a 5' to 3' double-stranded DNA exonuclease, including 17
exonuclease (e.g.,
from New England .Biolabs, catalog # M0263S). When a 5' to 3' double-stranded
DNA
exonuclease is used for removing gap-containing template molecules, then the
plurality of
soluble amplification primers in step (e) can comprise at least one
phosphorothioate &ester bond
at their 5' ends which can render the soluble amplification primers resistant
to exonuclease
degradation. In some embodiments, the plurality of soluble amplification
primers in step (e)
comprise 2-5 or more consecutive phosphorothioate diester bonds at their 5'
ends. In some
embodiments, the plurality soluble amplification primers in step (e) comprise
at least one
ribonucleotide and/or at least one 2'41'1-methyl or 2'-0-methoxyethyl (MOE)
nucleotide which
can render the forward sequencing primers resistant to exonuclease
degradation.
100454] In some embodiments, the plurality of gap-containing template
molecules can be
removed using a chemical reagent that favors nucleic acid denaturation. The
denaturation reagent
can include any one or any combination of compounds such as formamide,
acetonitrile,
guanidinium chloride and/or a buffering agent (e.g., Tris-HCI, MES, HEPES, or
the like).
100455] In some embodiments, the plurality of gap-containing template
molecules can be
removed using an elevated temperature (e.g., heat) with or without a nucleic
acid denaturation
reagent. The gap-containing template molecules can be subjected to a
temperature of about 45-50
or about 50-60 C, or about 60-70 C, or about 70-80 C, or about 80-90 C, or
about 90-95
c17, or higher temperature.
[00456] In some embodiments, the plurality of gap-containing template
molecules can be
removed using 100% forma.mide at a temperature of about 65 'V for about 3
minutes, and
washing with a reagent comprising about 50 mM MO or equivalent ionic strength
and having a
PH of about 6.5 --- 8.5.
[00457] In some embodiments, the pairwise sequencing method further comprises
step (g):
sequencing the plurality of retained forward extension strands thereby
generating a plurality of
extended reverse sequencing primer strands. In some embodiments, the
sequencing of step (g)
comprises contacting the plurality of retained forward extension strands with
a plurality of
soluble reverse sequencing primers under a condition suitable to hybridize the
reverse

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sequencing primers to the reverse sequencing primer binding site of the
retained forward
extension strands, and by conducting sequencing reactions using the hybridized
reverse
sequencing primers wherein the forward sequencing reactions generates a
plurality of extended
reverse sequencing primer strands (Figures 50 and 51). The extended reverse
sequencing primer
strands are hybridized to the retained forward extension strand. The retained
forward extension
strand is hybridized to the first surface primer. The extended reverse
sequencing primer strands
are not hybridized to the first surface primer, or covalently joined to the
first surface primer.
Therefore, the extended reverse sequencing primer strands are not immobilized
to the support.
[004581 For the sake of simplicity, Figures 47 and 49 show exemplary retained
forward
extension strands each having either (i) one copy of the sequence of interest
and various
universal primer binding sites (Figure 47) or (ii) two tandem copies of the
sequence of interest
and various universal primer binding sites (Figure 49). The skilled artisan
will appreciate that the
retained forward extension strand can include two, three, four or many more
tandem copies
containing the sequence of interest and various universal primer binding
sites. Therefore, the
reverse sequencing reaction can generate a plurality of extended reverse
sequencing primer
strands hybridized to the same retained forward extension strand.
[004591 In some embodiments, in step (g), the condition suitable to
hybridize the reverse
sequencing primers to the reverse sequencing primer binding sequences of the
retained forward
extension strands comprises contacting the plurality of soluble reverse
sequencing primers and
the retained forward extension strands with a high efficiency hybridization
buffer, In. some
embodiments, the high efficiency hybridization buffer comprises: (i) a first
polar aprotic solvent
having a dielectric constant that is no greater than 40 and having a polarity
index of 4-9; (ii) a
second polar aprotic solvent having a dielectric constant that is n.o greater
than 115 and is present
in the hybridization buffer formulation in an amount effective to denature
double-stranded
nucleic acids; (iii) a pH buffer system that inaintain.s the of the
hybridization buffer
formulation in a range of about 4-8; and (iv) a crowding agent in an amount
sufficient to enhance
or facilitate molecular crowding. In some embodiments, the high efficiency
hybridization buffer
comprises: (i) the first polar aprotic solvent comprises acetortitrile at 25-
50% by volume of the
hybridization buffer; (ii) the second polar aprotic solvent comprises
fonnamide at 540% by
volume of the hybridization buffer; (iii) the pH buffer system comprises 2-(N-
morpholino)ethanesulfonic acid (MES) at a of 5-6.5; and (iv) the crowding
agent comprises

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polyethylene glycol (PEG) at 5-35% by volume of the hybridization buffer. In
some
embodiments, the high efficiency hybridization buffer further comprises
betaine.
[00460) In an alternative embodiment, the sequencing of step (g) comprises
using the
immobilized surface primer as a sequencing primer and conducting sequencing
reactions to
generate a plurality of reverse sequencing strands.
1004611 In some embodiments, the reverse sequencing reactions of step (g)
comprises
contacting the plurality of reverse sequencing primers with the reverse
sequencing primer
binding sequences of the retained forward extension strands, one or more types
of sequencing
polymerases, and a plurality of nucleotides and/or a plurality of multivalent
molecules. In some
embodiments, the soluble reverse sequencing primers comprise 3' OH extendible
ends. In some
embodiments, the soluble reverse sequencing primers comprise a 3' blocking
moiety which can
be removed to generate a 3' OH extendible end. In some embodiments, the
soluble reverse
sequencing primers lack a nucleotide having a scissile moiety. The sequencing
reactions that
employ nucleotides and/or multivalent molecules is described in more detail
below. The reverse
sequencing reactions can generate a plurality of extended reverse sequencing
primer strands. In
some embodiments, individual retained forward extension strands have multiple
copies of the
reverse sequencing primer binding sequences/sites, wherein each reverse
sequencing primer
binding site is capable of hybridizing to a reverse sequencing primer.
Individual reverse
sequencing primer binding sites in a given retained forward extension strand
can be hybridized to
a reverse sequencing primer and can undergo a sequencing reaction. Thus, an
individual retained
forward extension strand can undergo two or more sequence reactions, where
each sequencing
reaction is initiated from a reverse sequencing primer that is hybridized to a
reverse sequencing
primer binding site (Figures 50 and 51). In some embodiments, the sequencing
reactions
comprise a plurality of nucleotides (or analogs thereof) labeled with a
detectable reporter moiety.
In some embodiments, the sequencing reaction comprise a plurality of
multivalent molecules
having nucleotide units, where the multivalent molecules are labeled with a
detectable reporter
moiety. In some embodiments, the detectable reporter moiety comprises a
fluorophore.
[00462] In some embodiments, at least one washing step can be conducted after
any of steps
(a) --- (g). The washing step can be conducted with a wash buffer comprising a
pH buffering
agent, a metal chelating agent, a salt, and a detergent.

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[00463j In some embodiments, the pH buffering compound in the wash buffer
comprises any
one or any combination of two or more of iris, Tris-HC1, Tricine, Bicine, Bis-
Tris propane,
HEPES, MES, MOPS, MOPSO, BES, TES, CAPS, TAPS, TAPSO, ACES, PIPES,
ethanolamine (a.k.a 2-amino methanol; MEA), a citrate compound, a citrate
mixture, NaOH
and/or KOH. In some embodiments, the pH buffering agent can be present in the
wash buffer at
a concentration of about 1-100 mM, or about 10-50 rnM, or about 10-25 mM. In
some
embodiments, the pH of the pH buffering agent which is present in any of the
reagents described
here in can be adjusted to a pH of about 4-9, or a pH of about 5-9, or a pH of
about 5-8.
100464] In some embodiments, the metal chelating agent in the wash buffer
comprises EDTA
(ethylenediaminetetraacetic acid), EGTA (ethylene glycol tetraacetic acid),
HEDTA
(hydrox-yethylethylenecliaminetriacetic acid), 'DMA (diethylene triamine
pentaacetic acid), NIA
(N,N-bis(carboxymethyl)glycine), citrate anhydrous, sodium citrate, calcium
citrate, ammonium
citrate, ammonium bicitrate, citric acid, potassium citrate, or magnesium
citrate. In some
embodiments, the wash buffer comprises a chelating agent at a concentration of
about 0.01 ¨ 50
mM, or about 0.1 ¨20 mM, or about 0.2 ¨ 10 mM.
[00465] In some embodiments, the salt in the wash buffer comprises NaCl, KCl,
NH2SO4 or
potassium glutamate. In some embodiments, the detergent comprises an ionic
detergent such as
SDS (sodium dodecyl sulfate). The wash buffer can include a monovalent salt at
a concentration
of about 25-500 mM., or about 50-250 mM, or about 100-200 mM.
[00466] In some embodiments, the detergent in the wash buffer comprises a non-
ionic
detergent such as Triton X-100, Tween 20, Tween 80 or Nonidet P-40. In some
embodiments,
the detergent comprises a zwitterionic detergent such as CHAPS (34(3-
cholamidopropyl)dimethylammonio]-1-propanesulfonate) or N-Dodecyl-N;N-dimethy1-
3-
amonio-1 -propanesulfate (DetX). In some embodiments, the detergent comprises
LDS (lithium
dodecyl sulfate), sodium taurodeoxycholate, sodium taurocholate, sodium
glycocholate, sodium
deoxycholate or sodium cholate. In some embodiments, the detergent is included
in the wash
buffer at a concentration of about 0.01-0.05%, or about 0.05-0.1%, or about
0.1-0.15%, or about
0.15-0 2%, or about 0.2-0 25%.
On Support Ligation and RCA and Pairwise Sequencing
[00467] The present disclosure provides pairvvise sequencing methods,
comprising step (a):
providing a support having a plurality of surface primers (e.g., a plurality
of first surface primers)

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immobilized thereon, wherein individual first surface primers in the plurality
comprise a first
portion (SPI-A) and a second portion (SPI-B), and the individual first surface
primers
comprising a 3' extendible end and lacking a nucleotide having a scissile
moiety that can be
cleaved to generate an abasic site in the first surface primer. In some
embodiments, the
immobilized first surface primers lack a nucleotide having a scissile moiety
(Figure 55). For
example, the surface primers lack uridine, 8-oxo-7,8-dihydroguanine (e.g.,
8oxoG) and
deoxyinosine. In some embodiments, the first and second portions (SPI-A and
SPI -B) of the
first surface primers have the same or different lengths. The first portion
(SPI-A) of the first
surface primers can be about 4-50 nucleotides, or 50-100 nucleotides, or 100-
150 nucleotides, or
longer lengths. The second portion (SPI-B) of the first surface primers can be
about 4-50
nucleotides, or 50-100 nucleotides, or 100-150 nucleotides, or longer lengths.
In some
embodiments, the first and second portions (SPI -A and SPI -B) of the
immobilized first surface
primers have the same or different sequences. In some embodiments, the support
comprises a
plurality of first surface primers. In some embodiments, the support lacks a
plurality of second
surface primers. In some embodiments, the support comprises a plurality of
first and second
surface primers.
[004681 In some embodiments, the immobilized first surface primers comprise
single stranded
oligonucleotides comprising DNA, RNA or a combination of DNA and RNA. The
first surface
primers comprise a sequence that is wholly complementary or partially
complementary along
their lengths to at least a portion of a nucleic acid library molecule (e.g.,
linear or circular library
molecules). The first surface primers can include a terminal 3' nucleotide
having a sugar 3' OH
moiety which is extendible for nucleotide polymerization (e.g., polymerase
catalyzed
polymerization).
[00469] The immobilized first surface primers can be immobilized to the
support or
immobilized to a coating on the support. The immobilized first surface primers
can be embedded
and attached (coupled) to the coating on the support. In some embodiments, the
5' end of the
immobilized first surface primers are immobilized to a support or immobilized
to a coating on
the support. Alternatively, an interior portion or the 3' end of the
immobilized first surface
primers can be immobilized to a support or immobilized to a coating on the
support. The support
comprises a plurality of immobilized first surface primers having the same
sequence. The

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immobilized first surface primers can be any length, for example 4-50
nucleotides, or 50-100
nucleotides, or 100-150 nucleotides, or longer lengths.
[00470] In some embodiments, the plurality of immobilized first surface
primers comprise at
least one phosphorothioate diester bond at their 5' ends which can render the
first surface
primers resistant to exonuclease degradation. In some embodiments, the
plurality of immobilized
first surface primers comprise 2-5 or more consecutive phosphorothioate
diester bonds at their 5'
ends. In some embodiments, the plurality of immobilized first surface primers
comprise at least
one ribonucleotide and/or at least one 2'-0-methyl or 2'-0-methoxyethyl (MOE)
nucleotide
which can render the first surface primers resistant to exonuclease
degradation.
[00471] In some embodiments, the immobilized first surface primers comprise at
least one
locked nucleic acid (LNA) which comprises a methylene bridge bond between a 2'
oxygen and
4' carbon of the pentose ring. Immobilized first surface primers that include
at least one LNA
can be resistant to nuclease digestions and can exhibit increased melting
temperature when
hybridized to the forward extension strand.
[00472] In some embodiments, the support further comprises a plurality of a
second surface
primer immobilized thereon (Figure 72). The second surface primers have a
sequence that differs
from the first immobilized surface primer. The immobilized second surface
primers of step (a)
comprise single stranded oligonucleotides comprising DNA, RNA or a combination
of DNA and
RNA. The second surface primers comprise a sequence that is wholly
complementary or partially
complementary along their lengths to at least a portion of an immobilized
single stranded
concatemer template molecule. The immobilized second surface primers can be
immobilized to
the support or immobilized to a coating on the support. The immobilized second
surface primers
can be embedded and attached (coupled) to the coating on the support. In some
embodiments,
the 5' end of the immobilized second surface primers are immobilized to a
support or
immobilized to a coating on the support. Alternatively, an interior portion or
the 3' end of the
immobilized second surface primers can be immobilized to a support or
immobilized to a coating
on the support. The support comprises a plurality of immobilized second
surface primers having
the same sequence. The immobilized second surface primers can be any length,
for example 4-50
nucleotides, or 50-100 nucleotides, or 100-150 nucleotides, or longer lengths.
In some
embodiments, the 3' terminal end of the immobilized second surface primers
comprise an
extendible 3' OH moiety. In some embodiments, the 3' terminal end of the
immobilized second
. .

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surface primers comprise a 3' non-extendible moiety. The 3' terminal end of
the immobilized
second surface primers comprise a moiety that blocks primer extension, such as
for example a
phosphate group, a dideoxycytidine group, an inverted dT, or an amino group.
The immobilized
second surface primers are not extendible in a primer extension reaction. The
immobilized
second surface primers lack a nucleotide having a scissile moiety.
1004731 In some embodiments, the plurality of immobilized second surface
primers comprise
at least one phosphorothioate diester bond at their 5' ends which can render
the second surface
primers resistant to exonuclease degradation. In some embodiments, the
plurality of immobilized
second surface primers comprise 2-5 or more consecutive phosphorothioate
diester bonds at their
5' ends. In some embodiments, the plurality of immobilized second surface
primers comprise at
least one ribonucleotide and/or at least one 2'-0-methyl or 2'-O-methoxyethyl
(MOE) nucleotide
which can render the second surface primers resistant to exonuclease
degradation.
100474] In some embodiments, individual immobilized single stranded nucleic
acid
concatemer template molecule are covalently joined to an immobilized first
surface primer, and
at least one portion of the individual concatemer template molecule is
hybridized to an
immobilized second surface primer (Figure 72). The immobilized second surface
primers serve
to pin down a portion of the immobilized concatemer template molecules to the
support. The
immobilized concatemer template molecule has two or more copies of a universal
binding
sequence for an immobilized second surface primer. The portion of the
immobilized concatemer
template molecule that includes the universal binding sequence for an
immobilized second
surface primer can hybridize to the immobilized second surface primer. In some
embodiments,
the second surface primers include a terminal 3' blocking group that renders
them non-
extendible. In some embodiments, the second surface primers have terminal 3'
extendible ends.
[00475] In some embodiments, the support comprises about 102 --- 10'5
immobilized first
surface primers per mm2. In some embodiments, the support comprises about 102 -
-- 1015
immobilized second surface primers per mm2. In some embodiments, the support
comprises
about 102-- IV immobilized first surface primers and immobilized second
surface primers per
mm2.
[00476] The immobilized surface primers (e.g., first and second surface
primers) are in fluid
communication with each other to permit flowing various solutions of linear or
circular nucleic
acid template molecules, soluble primers, enzymes, nucleotides, divalent
cations, buffers,

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reagents, and the like, onto the support so that the plurality of immobilized
surface primers (and
the primer extension products generated from the immobilized surface primers)
react with the
solutions in a massively parallel manner.
1004771 In some embodiments, the pairwise sequencing method further comprises
step (b):
contacting the plurality of the first surface primers with a plurality of
single stranded linear
nucleic acid library molecules each library molecule having 5' and 3' ends.
The contacting is
conducted under a condition suitable for hybridizing individual library
molecules to an
immobilized first surface primer to form a circularized library molecule
having a gap or nick
between the 5' and 3' ends of the circularized library molecule (Figures 57
and 58).
1004781 In some embodiments, the position of the gap or nick in the
circularized library
molecules can be asymmetrical or symmetrical relative to the duplex formed by
hybridizing the
5' and 3' ends of the linear library molecule to the immobilized first surface
primers. For
example, Figure 57 shows an asymmetrical positioned gap or nick. Figure 58
(left) shows an
asymmetrical positioned gap or nick. Figure 58 (right) shows a symmetrical
positioned gap or
nick. An asymmetrical or symmetrical positioned gap/nick can be generated by
adjusting the
length of the first portion (SP1-A.) and the second portion (SPI-B) in the
immobilized first
surface primers.
1004791 In some embodiments, individual library molecules in the plurality
comprise a
sequence of interest and the library molecules further comprise any one or any
combination of
two or more of: (i) a universal binding sequence (or complementary sequence
thereof) for a
soluble forward sequencing primer; (ii) a universal binding sequence (or
complementary
sequence thereof.) for a soluble reverse sequencing primer; (iii) a universal
binding sequence (or
complementary sequence thereof) for a first portion of an immobilized first
surface primer (SP1-
A); (iv) a universal binding sequence (or complementary sequence thereof) for
a second portion
of an immobilized first surface primer (SPI -B); (v) a universal binding
sequence (or
complementary sequence thereof) for an immobilized second surface primer; (vi)
a universal
binding sequence (or complementary sequence thereof) for a first soluble
amplification primer;
(vii) a universal binding sequence (or complementary sequence thereof) for a
second soluble
amplification primer; (viii) a universal binding sequence (or complementary
sequence thereof)
for a soluble compaction oligonucleotide; (ix) a sample -barcode sequence
and/or (x) a unique

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molecular index sequence. An exemplary single stranded linear library molecule
is shown in
Figure 56.
[00480I In some embodiments, the universal binding sequence for a first
portion of an
immobilized first surface primer (e.g., SP1-A') in the linear library molecule
can hybridize to the
first portion of the immobilized first surface primer (SP1-A). In some
embodiments, the
universal binding sequence for a second portion of an immobilized first
surface primer (e.g.,
SP1-B') in the linear library molecule can hybridize to the second portion of
the immobilized
first surface primer (SP1-B). In some embodiments, the immobilized first
surface primers
comprise a first portion (SP1-A) and a second portion (SP1-B) which hybridize
to SP1-A' and
SP1-B' in the linear library molecule, and the first surface primers serve as
a nucleic acid splint
molecule for circularizing the linear library molecules.
1004811 In some embodiments, the pairwise sequencing method further comprises
step (c):
enzymatically closing the gap or nick thereby forming individual single
stranded covalently
closed circular molecules that are hybridized to an immobilized first surface
primer (Figure 59,
Figure 60 (left) and Figure 60 (right)).
[00482] In some embodiments, the gap in the circularized library molecule is
closed by
conducting a polymerase-catalyzed gap fill-in reaction using the 3' extendible
end of the library
molecule as an initiation site for the polymerase-catalyzed fill-in reaction
and using the
immobilized first surface primer as a template molecule thereby forming
circularized molecule
having a nick. The nick is closed by conducting an enzymatic ligation reaction
to form a single
stranded covalently closed circular molecule, wherein individual covalently
closed circular
molecules are hybridized to an immobilized first surface primer. In some
embodiments, the gap
fill-in reaction can be conducting with a plurality of nucleotides and a
polymerase that lacks 5' to
3' strand displacement activity. The polymerase comprises E coli DNA
polymerase I, Klenow
fragment of E. coli DNA polymerase I, 17 DNA polymerase, or T4 DNA polymerase.
In some
embodiments, the ligation reaction can be conducted using a DNA ligase which
comprises a T3,
T4, T7 or Taq DNA ligase.
[00483] In some embodiments, the nick in the circularized library molecule is
closed by
conducting a ligase-catalyzed ligation reaction to form a single stranded
covalently closed
circular molecule, wherein individual covalently closed circular molecules are
hybridized to an

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immobilized first surface primer. In some embodiments, the ligase enzyme
comprises T3, T4, T7
or Tag DNA ligase.
[00484] In some embodiments, the pairwise sequencing method further comprises
step (d):
generating a plurality of immobilized single stranded nucleic acid concatemer
template
molecules by conducting a rolling circle amplification reaction with a
plurality of a strand
displacing polymerase, and a plurality of nucleotides which include dATP,
deTP, dGTP, DTP
and a nucleotide having a scissile moiety that can be cleaved to generate an
abasic site, thereby
generating a plurality of immobilized single stranded nucleic acid concatemer
template
molecules having at least one nucleotide with a scissile moiety; wherein
individual single
stranded nucleic acid concatemer template molecules are coyalently joined to
an immobilized
first surface primer (Figure 61). In some embodiments, the rolling circle
amplification reaction
can be conducted in the presence, or in the absence, of a plurality of a
plurality of compaction
oligonucleotides.
1004851 In some embodiments, the single-stranded circular nucleic acid
library molecules can
be removed from the concatemer template molecules with at least one washing
step which is
conducted under a condition suitable to retain the single stranded nucleic
acid concatemer
template molecules where individual concatemer template molecules are operably
joined to an
immobilized first surface primer.
[004861 In some embodiments, individual immobilized concatemer template
molecules
generated by the rolling circle amplification reaction comprise two or more
copies of a sequence
of interest and wherein the individual immobilized concatemer template
molecules further
comprise any one or any combination of two or more of: (i.) two or more copies
of a universal
binding sequence for a soluble forward sequencing primer; (ii) two or more
copies of a universal
binding sequence for a soluble reverse sequencing primer; (iii) two or more
copies of a universal
binding sequence for a first portion of an immobilized first surface primer
(SP1.-A); (iv) two or
more copies of a universal binding sequence for a second portion of an
immobilized first surface
primer (SP1-B); (v) two or more copies of a universal binding sequence for an
immobilized
second surface primer; (vi) two or more copies of a universal binding sequence
for a first soluble
amplification primer (vii) two or more copies of a universal binding sequence
for a second
soluble amplification primer; (viii) two or more copies of a universal binding
sequence for a

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soluble compaction oligonucleotide; (ix) two or more copies of a sample
barcode sequence
and/or (x) two or more copies of a unique molecular index sequence.
[00487] In some embodiments, the plurality of immobilized single stranded
nucleic acid
concatemer template molecules that are generated by the rolling circle
amplification reaction of
step (d) further comprise two or more copies of a universal binding sequence
(or complementary
sequence thereof) for immobilized second sequence surface primers. In some
embodiments,
individual immobilized single stranded nucleic acid concatemer template
molecule are joined
(e.g., covalently joined) to an immobilized first surface primer, and at least
one portion of the
individual concatemer template molecule is hybridized to an immobilized second
surface primer.
The immobilized second surface primers serve to pin down a portion of the
immobilized
concatemer template molecules to the support (see Figure 72). In some
embodiments, the second
surface primers include a terminal 3' blocking group that renders them non-
extendible.
[00488] The rolling circle amplification reaction of step (d) can be
conducted with a.
nucleotide mixture containing dATP, dC IF, dGIP, dTTP and a nucleotide
having a scissile
moiety to generate immobilized concatemer template molecules which includes at
least one
nucleotide having a scissile moiety. The scissile moieties in the immobilized
concatemer
template molecules can be converted into abasic sites. In some embodiments, in
the nucleotide
mixture, the nucleotide having the scissile moiety comprises uridin.e, 8-oxo-
7,8-dihydroguanin.e
(e.g., 8oxoG) or deoxyinosine. In the immobilized concatemer template
molecules, the uridine
can be converted to an abasic site using uracil DNA glycosylase (UDG), the
8oxoG can be
converted to an abasic site using FPG glycosylase, and the deoxyin.osin.e can
be converted to an
abasic site using AlkA. glycosylase.
[00489] In some embodiments, the nucleotide mixture can include an amount of
dUTP so that
a target percent of the thymidine in the resulting concatemer molecules are
replaced with dUTP.
For example, when 30% of dTTP in the concatemer molecules are to be replaced
with dUTP
(e.g., 30% is the target percent) then the nucleotide mixture can contain 7.5%
dUTP (e.g., 30/4 =
7.5%), 17.5% dTTP, and 25% each for dATP, dCTP and dGTP. The target percent of
dTTP to be
replaced by dUTP can be about 0.1-1%, or about 1-5%, or about 5-10%, or about
10-20%, or
about 20-30%, or about 30-45%, or about 45-50%, or a higher percent of the
dTTP in the
immobilized concatemer template molecules are replaced with nucleotides having
a scissile
moiety.

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[00490] In some embodiments, the nucleotide mixture can include an amount of
deoxyinosine
so that a target percent of the guanosine in the resulting concatemer
molecules are replaced with
deoxyinosine. For example, when 30% of dGTP in the concatemer molecules are to
be replaced
with deoxyinosine (e.g., 30% is the target percent) then the nucleotide
mixture can contain 7.5%
deoxyinosine (e.g., 30/4 = 7.5%), 17.5% dGTP, and 25% each for dATP, dCIP and
dTTP. The
target percent of dGIP to be replaced by deoxyinosine can be about 0.1-1%, or
about 1-5%, or
about 5-10%, or about 10-20%, or about 20-30% or about 30-45%, or about 45-
50%, or a
higher percent of the dGTP in the immobilized concatemer template molecules
are replaced with
nucleotides having a scissile moiety.
1004911 In some embodiments, the nucleotide mixture can include an amount of
8oxoG so
that a target percent of the guanosine in the resulting concatemer molecules
are replaced with
8oxoG. For example, when 30% of dGTP in the concatemer molecules are to be
replaced with
8oxoG (e.g., 30% is the target percent) then the nucleotide mixture can
contain 7.5% 8oxoG
(e.g., 30/4 = 7.5%), 17.5% dGTP, and 25% each for dATP, dCTP and dTTP. The
target percent
of dGTP to be replaced by 8oxoG can be about 0.1-1%, or about 1-5%, or about 5-
10%, or about
10-20%, or about 20-30%, or about 30-45%, or about 45-50%, or a higher percent
of the dGTP
in the immobilized concatemer template molecules are replaced with nucleotides
baying a
scissile moiety.
[00492] In some embodiments, the rolling circle amplification reaction.
generates immobilized
concatemer template molecules with incorporated nucleotides having a scissile
moiety that are
distributed at random positions along individual immobilized concatemer
template molecules. In
some embodiments, the nucleotides havin.g a scissile moiety are distributed at
different positions
in the different immobilized concatemer template molecules.
[00493] In some embodiments, the pairwise sequencing method further comprises
step (e):
sequencing the plurality of immobilized concatemer template molecules thereby
generating a
plurality of extended forward sequencing primer strands. The sequencing of
step (e) comprises
contacting the plurality of immobilized concatemer template molecules with a
plurality of
soluble forward sequencing primers under a condition suitable to hybridize at
least one forward
sequencing primer to at least one of the forward sequencing primer binding
sites/sequences of
the immobilized concatemer template molecules, and conducting forward
sequencing reactions
using one or more types of sequencing polymerases, a plurality of nucleotides
and/or multivalent

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molecules, and the hybridized first forward sequencing primers (Figure 62). In
some
embodiments, the soluble forward sequencing primers comprise 3' OH extendible
ends. In some
embodiments, the soluble forward sequencing primers comprise a 3' blocking
moiety which can
be removed to generate a 3' OH extendible end. In some embodiments, the
soluble forward
sequencing primers lack a nucleotide having a scissile moiety. The forward
sequencing reactions
can generate a plurality of extended forward sequencing primer strands. In
some embodiments,
individual immobilized concatemer template molecules have multiple copies of
the forward
sequencing primer binding sites, wherein each forward sequencing primer
binding site is capable
of hybridizing to a first forward sequencing primer. Individual forward
sequencing primer
binding sites in a given immobilized concatemer template molecule can be
hybridized to a
forward sequencing primer and can undergo a sequencing reaction. Individual
immobilized
concatemer template molecules can undergo two or more sequence reactions,
where each
sequencing reaction is initiated from a first forward sequencing primer that
is hybridized to a
forward sequencing primer binding site (e.g., see Figure 62). In some
embodiments, the
sequencing reactions comprise a plurality of nucleotides (or analogs thereof)
labeled with a
detectable reporter moiety. In some embodiments, the sequencing reaction
comprise a plurality
of multivalent molecules having a plurality of nucleotide units attached to a
core, where the
multivalent molecules are labeled with a detectable reporter moiety. In some
embodiments, the
core is labeled with a detectable reporter moiety. In some embodiments, at
least one linker and/or
at least one nucleotide unit of a nucleotide arm is labeled with a detectable
reporter moiety. In
some embodiments, the detectable reporter moiety comprises a fluorophore. An
exemplary
nucleotide arm is shown in Figure 108, and exemplary multivalent molecules are
shown in
Figures 104-107.
[00494] In some embodiments, the pairwise sequencing method further comprises
step (1):
retaining the plurality of immobilized concatemer template molecules and
replacing the plurality
of extended forward sequencing primer strands with a plurality of forward
extension strands that
are hybridized to the retained immobilized single stranded nucleic acid
concatemer template
molecules. The plurality of extended forward sequencing primer strands can be
removed and
replaced with a plurality of forward extension strands by conducting a primer
extension reaction
(see Figures 63- 65).

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[00495] In some embodiments, step (f) comprises contacting at least one
extended forward
sequencing primer strand with a plurality of strand displacing polymerases and
a plurality of
nucleotides and in the absence of soluble amplification primers, under a
condition suitable to
conduct a strand displacing primer extension reaction using the at least one
extended forward
sequencing primers strand to initiate the primer extension reaction thereby
generating a forward
extension strand that is coyalently joined to the extended forward sequencing
primers strand,
wherein the forward extension strand is hybridized to the immobilized
concatemer template
molecule (Figure 63). For example, one of the extended forward sequencing
primer strands can
serve as a primer for the strand displacing polymerase. The strand displacing
polymerase can
extend the extended forward sequencing primer strand, and displace downstream
extended
forward sequencing primer strands while synthesizing an extended strand that
replaces the
downstream extended forward sequencing primer strands. The newly extended
strand is
covalently joined to an extended forward sequencing primer strand. The
immobilized concatemer
template molecules are retained. The primer extension reaction can optionally
include a plurality
of compaction oligonucleotides and/or hexamine (e.g., cobalt hexamine III) to
generate forward
extension strands. Individual forward extension strands can collapse into a
nanoball having a
more compact size and/or shape compared to a nanoball generated from a primer
extension
reaction conducted without compaction oligonucleotides and/or hexamine (e.g.,
cobalt hexamine
III). Inclusion of compaction oligonucleotides and/or hexamine (e.g., cobalt
hexamine in the
primer extension reaction can improve FWFINI (full width half maximum) of a
spot image of the
nanoball. The spot image can be represented as a Gaussian spot and the size
can be measured as
FWHIVI. A smaller spot size as indicated by a smaller FWIIM typically
correlates with an
improved image of the spot. In some embodiments, the FWITIM of a taa,noball
spot can be about
um or smaller,
[00496]
Examples of strand displacing polym.erases include phi29 DNA polymerase, large
fragment of Bst DNA polymerase, large fragment of -Bsu DNA polymerase (exo-),
Bca, DNA
polymerase (ex0-), Klenow fragment of E. coli DNA polymerase, T5 polymerase, M-
Mti-LN
reverse transcriptase, HIV viral reverse transcriptase, Deep Vent DNA
polymerase and KOD
DNA polymerase. The phi29 DNA polym.erase can be wild type phi29 DNA
polymerase
MagniPin from Expedeon), or variant EquiPhi29 DNA polymerase (e.g., from
Thermo Fisher
Scientific), or chimeric QualiPhi DNA polymerase (e.g., from 4basebio).

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[00497j In some embodiments, step (f) comprises: (i) removing the plurality of
extended
forward sequencing primer strand while retaining the immobilized concatemer
template
molecules; and (ii) contacting the plurality of retained immobilized
concatemer molecules with a
plurality of soluble forward sequencing primers (e.g., a second plurality of
soluble forward
sequencing primers), a plurality of nucleotides (e.g., a second plurality of
nucleotides) and a
plurality of primer extension polymerases, under a condition suitable to
hybridize the plurality of
soluble forward sequencing primers to the plurality of retained immobilized
concatemer template
molecules and suitable for conducting polymerase-catalyzed primer extension
reactions thereby
generating a plurality of forward extension strands, wherein the soluble
sequencing primers
hybridize with the forward sequencing primer binding sequence in the retained
immobilized
concatemer molecules (Figure 64). The primer extension reaction can optionally
include a
plurality of compaction oligonucleotides and/or hexamine (e.g., cobalt
hexamine Ea) to generate
forward extension strands. Individual forward extension strands can collapse
into a nanoball
having a more compact size and/or shape compared to a nanoball generated from
a primer
extension reaction conducted without compaction oligonucleotides and/or
hexamine (e.g., cobalt
hexamine III). Inclusion of compaction oligonucleotides and/or hexamine (e.g.,
cobalt hexamine
HI) in the primer extension reaction can improve FWHM (full width half
maximum) of a spot
image of the nanoball. The spot image can be represented as a Gaussian spot
and the size can be
measured as a FWHM. A smaller spot size as indicated by a smaller FWHM
typically correlates
with an improved image of the spot. In some embodiments, the FWHM of a
nanoball spot can be
about 10 pm or smaller.
[00498] In some embodiments, in step (f), the condition suitable to hybridize
the plurality of
soluble forward sequencing primers to the plurality of retained immobilized
single stranded
nucleic acid concatemer template molecules comprises hybridizing retained
immobilized
concatemer template molecules with the soluble primers in the presence of a
primer extension
polymerase, a plurality of nucleotides, and a high efficiency hybridization
buffer. In some
embodiment, the high efficiency hybridization buffer comprises: (i) a first
polar aprotic solvent
having a dielectric constant that is no greater than 40 and having a polarity
index of 4-9; (ii) a
second polar aprotic solvent having a dielectric constant that is no greater
than 115 and is present
in the hybridization buffer formulation in an amount effective to denature
double-stranded
nucleic acids; (iii) a pH buffer system that maintains the pH of the
hybridization buffer

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formulation in a range of about 4-8; and (iv) a crowding agent in an amount
sufficient to enhance
or facilitate molecular crowding. In some embodiments, the high efficiency
hybridization buffer
comprises: (i) the first polar aprotic solvent comprises acetonitrile at 25-
50% by volume of the
hybridization buffer; (ii) the second polar aprotic solvent comprises
formamide at 5-10% by
volume of the hybridization buffer; (iii) the pH buffer system comprises 2-(N-
morpholino)ethanesulfonic acid (IVIES) at a p1-1 of 5-6.5; and (iv) the
crowding agent comprises
polyethylene glycol (PEG) at 5-35% by volume of the hybridization buffer. In
some
embodiments, the high efficiency hybridization buffer further comprises
betaine.
[004991 In some embodiments, step (f) comprises: (i) removing the plurality
of extended.
forward sequencing primer strand while retaining the immobilized concatemer
template
molecules; and (ii) contacting the plurality of retained immobilized
concatemer molecules with a
plurality of soluble amplification primers, a plurality of nucleotides (e.g.,
a second plurality of
nucleotides) and a plurality of primer extension polymerases, under a
condition suitable to
hybridize the plurality of soluble amplification primers to the plurality of
retained immobilized
concatemer template molecules and suitable for conducting polymerase-catalyzed
primer
extension reactions thereby generating a plurality of forward extension
strands, wherein the
soluble amplification primers hybridize with the soluble amplification primer
binding sequence
in the retained immobilized concatemer molecules (Figure 65), The primer
extension reaction
can optionally include a plurality of compaction oligonucleotides and/or
hexamine (e.g., cobalt
hexamine Ill) to generate forward extension strands. Individual forward
extension strands can
collapse into a nanoball having a more compact size and/or shape compared to a
nanoball
generated from a primer extension reaction conducted without compaction
oligonucleotides
and/or hexamine (e.g., cobalt hexamine Inclusion of compaction
oligonucleotides and/or
hexamine (e.g., cobalt hexamine III) in the primer extension reaction can
improve 17WI-IM (full
width half maximum) of a spot image of the nanoball. The spat image can be
represented as a
Gaussian spot and the size can be measured as a Mil:M. A smaller spot size as
indicated by a
smaller FWFIM typically correlates with an improved image of the spot. In some
embodiments,
the FWFIM of a nanoball spot can be about 10 pun or smaller.
[00500] In some embodiments, in step (f), the condition suitable to
hybridize the plurality of
soluble amplification primers to the plurality of retained immobilized single
stranded nucleic
acid concatemer template molecules comprises hybridizing retained immobilized
concatemer

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template molecules with the soluble primers in the presence of a primer
extension polymerase, a
plurality of nucleotides, and a high efficiency hybridization buffer. In some
embodiment, the
high efficiency hybridization buffer comprises: (i) a first polar aprotic
solvent having a dielectric
constant that is no greater than 40 and having a polarity index of 4-9; (ii) a
second polar aprotic
solvent having a dielectric constant that is no greater than 115 and is
present in the hybridization
buffer formulation in an amount effective to denature double-stranded nucleic
acids; (iii) a pH
buffer system that maintains the pH of the hybridization buffer formulation in
a range of about 4-
8; and (iv) a crowding agent in an amount sufficient to enhance or facilitate
molecular crowding.
In some embodiments, the high efficiency hybridization buffer comprises: (i)
the first polar
aprotic solvent comprises acetonitrile at 25-50% by volume of the
hybridization buffer; (ii) the
second polar aprotic solvent comprises formamide at 5-10% by volume of the
hybridization
buffer; (iii) the pH buffer system comprises 2-(N-morpholino)ethanesulfonic
acid (MES) at a pH
of 5-6.5; and (iv) the crowding agent comprises polyethylene glycol (PEG) at 5-
35% by volume
of the hybridization buffer. In some embodiments, the high efficiency
hybridization buffer
further comprises betaine.
[00501] In some embodiments, in step (f), the plurality of extended forward
sequencing
primer strands can be removed using an enzyme or a chemical reagent. For
example, the
plurality of extended forward sequencing primer strands can be enzymatically
degraded using a
5' to 3' double-stranded DNA exonuclease, including T7 exonuclease (e.g., from
New England
Biolabs, catalog M0263S). In some embodiments, the plurality of extended
forward
sequencing primer strands can be removed with a temperature that favors
nucleic acid
denaturation.
[00502] In some embodiments, in step (0, a denaturation reagent can be used to
remove the
plurality of extended forward sequencing primer strands, wherein the
denaturation reagent
comprises any one or any combination of compounds such as formamide,
acetonitrile,
guanidinium chloride and/or a buffering agent (e.g., Tris-HCI, MES, HEPES, or
the like).
[00503] In some embodiments, in step (0, the plurality of extended forward
sequencing
primer strands can be removed using an elevated temperature (e.g., heat) with
or without a
nucleic acid denaturation reagent. The plurality of extended forward
sequencing primer strands
can be subjected to a temperature of about 45-50 C, or about 50-60 "C, or
about 60-70 "C, or
about 70-80 C, or about 80-90 C, or about 90-95 "C, or higher temperature.

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[00504] In some embodiments, in step (f), the plurality of extended forward
sequencing
primer strands can be removed using 100% formamide at a temperature of about
65 C for about
3 minutes, and washing with a reagent comprising about 50 inNiNaCI or
equivalent ionic
strength and having a pH of about 6.5 ¨ 8.5.
[00505] In some embodiments, the primer extension polymerase of step (f)
comprises a high
fidelity polymerase. in some embodiments, the primer extension polymerase of
step (d)
comprises a DNA polymerase capable of catalyzing a primer extension reaction
using a uracil-
containing template molecule (e.g., a uracil-tolerant polymerase). Exemplary
polymerases
include, but are not limited to, Q5U Hot Start high-fidelity DNA polymerase
(e.g., catalog #
M0515S from New England Biolabs), Tag DNA polymerase, One Taq DNA polymerase
(e.g.,
mixture of Taq and Deep Vent DNA polymerases, catalog #1140480S from New
England
Biolabs), LongAmp Tag DNA polymerase (e.g., catalog #M0323S from New England
Biolabs),
Epimark Hot Start Taq DNA polymerase (e.g., catalog #M0490S from New England
Biolabs),
Bst DNA polymerase (e.g., large fragment, catalog #M0275S from New England
Biolabs), Bsu
DNA polymerase (e.g., large fragment, catalog #M0330S from New England
Biolabs), Phi29
DNA polymerase (e.g., catalog # M0269S from New England Biolabs), K coh DNA
polymerase
(e.g., catalog # M0209S from New England Biolabs), Thermina.tor DNA polymerase
(e.g.,
catalog #M0261S from New England Biolabs), Vent DNA polymerase and Deep Vent
DNA
polymerase.
1005061 The pairwise methods described herein can provide increased accuracy
in a.
downstream sequencing reaction because step (f) replaces the extended forward
sequencing
primer strands that were generated in step (e) with forward extension strands
having reduced
base errors. The extended forward sequencing primer strands are generated in
step (e) and may
or may not contain, erroneously incorporated nucleotides due to polymerase-
catalyzed mis-paired
bases. When step (e) is conducted with a high fidelity DNA polymerase, the
resulting forward
extension strands may have reduced base errors compared to the extended
forward sequencing
primer strands. The forward extension strands will be used as a nucleic acid
template for a
downstream sequencing step (e.g., see step (h) below). Thus, step (f) can
increase the sequencing
accuracy of the downstream step (II) and therefore increase the overall
sequencing accuracy of
the pairwise sequencing workflow.

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[00507] In some embodiments, the pairwise sequencing method further comprises
step (g):
removing the retained immobilized concatemer template molecules by generating
abasic sites in
the immobilized single stranded concatemer template molecules at the
nucleotide(s) having the
scissile moiety and generating gaps at the abasic sites to generate a
plurality of gap-containing
single stranded nucleic acid concatemer template molecules while retaining the
plurality of
forward extension strands and retaining the plurality of immobilized surface
primers (Figures 66
and 67, and Figures 68 and 69).
1005081 The abasic sites are generated on the retained concatemer template
strands that
contain nucleotides having scissile moieties. In some embodiments, the
scissile moieties in the
retained concatemer template molecules comprises uridine, 8-oxo-7,8-
dihydroguanine (e.g.,
8oxoG) or deoxyinosine. The abasic sites can be removed to generate a
plurality of single
stranded nucleic acid template molecules having gaps while retaining the
plurality of forward
extension strands. The abasic sites can be generated by contacting the
immobilized concatemer
template molecules with an enzyme that removes the nucleo-base at the
nucleotide having the
scissile moiety. The uracil in the retained concatemer template strands can be
converted to an
abasic site using uracil DNA. glycosylase (UDG). The 8oxoG in the retained
concatemer
template strands can be converted to an abasic site using FPG glycosylase. The
deoxyin.osin.e in
the retained concatemer template strands can be converted to an abasic site
using AlkA
glycosylase.
1005091 In some embodiments, in step (g), the gaps can be generated by
contacting the abasic
sites in the immobilized concatemer template molecules with an enzyme or a
mixture of enzymes
having lyase activity that breaks the phosphodiester backbone at the 5' and 3'
sides of the abasic
site to release the base-free deoxyribose and generate a gap (Figures 66 and
68). The abasic sites
can be removed using AP lyase, Endo IV endonuelease, FPG glycosylase/AP lyase,
Endo VIII
glycosylase/AP lyase. In some embodiments, generating the abasic sites and
removal of the
abasic sites to generate gaps can be achieved using a mixture of uracil DNA
glycosylase and
DNA glycosylase-lyase endonuclease VIII, for example USER. (Uracil-Specific
Excision
Reagent. Enzyme from New England Biolabs) or thermolabile USER (also from New
England
Biolabs).
[00510] In some embodiments, in step (g), the plurality of gap-containing
template molecules
can be removed using an enzyme, chemical compound and/or heat. After the gap-
removal

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procedure, the plurality of retained forward extension strands are hybridized
to the retained
immobilized surface primers (figures 67 and 69).
[00511] For example, the plurality of gap-containing template molecules can
be enzymatically
degraded using a 5' to 3' double-stranded DNA exonuclease, including 17
exonuclease (e.g.,
from New England .Biolabs, catalog # M0263S). When a 5' to 3' double-stranded
DNA
exonuclease is used for removing gap-containing template molecules, then the
plurality of
soluble amplification primers in step (f) can comprise at least one
phosphorothioate diester bond.
at their 5' ends which can render the soluble amplification primers resistant
to exonuclease
degradation. In some embodiments, the plurality of soluble amplification
primers in step (0
comprise 2-5 or more consecutive phosphorothioate diester bonds at their 5'
ends. In some
embodiments, the plurality soluble amplification primers in step (0 comprise
at least one
ribonucleotide and/or at least one 2'41'1-methyl or 2'-0-methoxyethyl (MOE)
nucleotide which
can render the forward sequencing primers resistant to exonuclease
degradation.
100512] In some embodiments, the plurality of gap-containing template
molecules can be
removed using a chemical reagent that favors nucleic acid denaturation. The
denaturation reagent
can include any one or any combination of compounds such as formamide,
acetonitrile,
guanidinium chloride and/or a buffering agent (e.g., Tris-HCI, MES, ITEPES, or
the like).
100513] In some embodiments, the plurality of gap-containing template
molecules can be
removed using an elevated temperature (e.g., heat) with or without a nucleic
acid denaturation
reagent. The gap-containing template molecules can be subjected to a
temperature of about 45-50
or about 50-60 C, or about 60-70 C, or about 70-80 C, or about 80-90 C, or
about 90-95
c17, or higher temperature.
[00514] In some embodiments, the plurality of gap-containing template
molecules can be
removed using 100% forma.mide at a temperature of about 65 'V for about 3
minutes, and
washing with a reagent comprising about 50 mM MO or equivalent ionic strength
and having a
PH of about 6.5 --- 8.5.
[00515] In some embodiments, the pairwise sequencing method further comprises
step (h):
sequencing the plurality of retained forward extension strands thereby
generating a plurality of
extended reverse sequencing primer strands. In some embodiments, the
sequencing of step (h)
comprises contacting the plurality of retained forward extension strands with
a plurality of
soluble reverse sequencing primers under a condition suitable to hybridize the
reverse

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sequencing primers to the reverse sequencing primer binding site of the
retained forward
extension strands, and by conducting sequencing reactions using the hybridized
reverse
sequencing primers wherein the forward sequencing reactions generates a
plurality of extended
reverse sequencing primer strands (Figures 70 and 71). The extended reverse
sequencing primer
strands are hybridized to the retained forward extension strand. The retained
forward extension
strand is hybridized to the first surface primer. The extended reverse
sequencing primer strands
are not hybridized to the first surface primer, or covalently joined to the
first surface primer.
Therefore, the extended reverse sequencing primer strands are not immobilized
to the support.
[00516] In some embodiments, in step (h), the condition suitable to
hybridize the reverse
sequencing primers to the reverse sequencing primer binding sequences of the
retained forward.
extension strands comprises contacting the plurality of soluble reverse
sequencing primers and
the retained forward extension strands with a high efficiency hybridization
buffer. In some
embodiments, the high efficiency hybridization buffer comprises: (i) a first
polar aprotic solvent
having a dielectric constant that is no greater than 40 and having a polarity
index of 4-9; (ii) a
second polar aprotic solvent having a dielectric constant that is no greater
than 115 and is present
in the hybridization buffer formulation in an amount effective to denature
double-stranded
nucleic acids; (iii) a pH buffer system that maintains the pH of the
hybridization buffer
formulation in a range of about 4-8; and (iv) a crowding agent in an amount
sufficient to enhance
or facilitate molecular crowding. In some embodiments, the high efficiency
hybridization buffer
comprises: (i) the first polar aprotic solvent comprises acetonitrile at 25-
50% by volume of the
hybridization buffer; (ii) the second polar aprotic solvent comprises
forniamide at 5-10% by
volume of the hybridization buffer; (iii) the pH buffer system comprises 2-(N-
morpholino)eihanesul fonic acid (MES) at a pH of 5-6.5; and (iv) the crowding
agent comprises
polyethylene glycol (PEG) at 5-35% by volume of the hybridization buffer. In
some
embodiments, the high efficiency hybridization buffer further comprises
betain.e.
[00517] In an alternative embodiment, the sequencing of step (h) comprises
using the
immobilized surface primer as a sequencing primer and conducting sequencing
reactions to
generate a plurality of reverse sequencing strands.
[00518] In some embodiments, the reverse sequencing reactions of step (h)
comprises
contacting the plurality of reverse sequencing primers with the reverse
sequencing primer
binding sequences of the retained forward extension strands, one or more types
of sequencing

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polymerases, and a plurality of nucleotides and/or a plurality of multivalent
molecules. In some
embodiments, the soluble reverse sequencing primers comprise 3' OH extendible
ends. In some
embodiments, the soluble reverse sequencing primers comprise a 3' blocking
moiety which can
be removed to generate a 3' OH extendible end. In some embodiments, the
soluble reverse
sequencing primers lack a nucleotide having a scissile moiety. The sequencing
reactions that
employ nucleotides and/or multivalent molecules is described in more detail
below. The reverse
sequencing reactions can generate a plurality of extended reverse sequencing
primer strands. In
some embodiments, individual retained forward extension strands have multiple
copies of the
reverse sequencing primer binding sequences/sites, wherein each reverse
sequencing primer
binding site is capable of hybridizing to a reverse sequencing primer.
Individual reverse
sequencing primer binding sites in a given retained forward extension strand
can be hybridized to
a reverse sequencing primer and can undergo a sequencing reaction. Thus, an
individual retained
forward extension strand can undergo two or more sequence reactions, where
each sequencing
reaction is initiated from a reverse sequencing primer that is hybridized to a
reverse sequencing
primer binding site (e.g., see Figures 70 and 71). In some embodiments, the
sequencing reactions
comprise a plurality of nucleotides (or analogs thereof) labeled with a
detectable reporter moiety.
In some embodiments, the sequencing reaction comprise a plurality of
multivalent molecules
having nucleotide units, where the multivalent molecules are labeled with a
detectable reporter
moiety. In some embodiments, the detectable reporter moiety comprises a
fluorophore.
[00519] In some embodiments, at least one washing step can be conducted after
any of steps
(a) ¨ (h). The washing step can be conducted with a wash buffer comprising a
pH buffering
agent, a metal chelating agent, a salt, and a detergent.
[00520] In some embodiments, the pH buffering compound in the wash buffer
comprises any
one or any combination of two or more of Tris, Tris-HC1, Tricine, Bicine, Bis-
Tris propane,
HEPES, MES, MOPS, MOPSO, BES, TES, CAPS, TAPS, TAPSO, ACES, PIPES,
ethanolamine (a.k.a 2-amino methanol; MEA), a citrate compound, a citrate
mixture, NaOH
and/or KOH. In some embodiments, the pH buffering agent can be present in the
wash buffer at
a concentration of about 1-100 mM, or about 10-50 mM, or about 10-25 mM. In
some
embodiments, the pH of the pH buffering agent which is present in any of the
reagents described
here in can be adjusted to a pH of about 4-9, or a pH of about 5-9, or a pH of
about 5-8.

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[005211 In some embodiments, the metal chelating agent in the wash buffer
comprises ED'FA
(ethylenaliaminetetraacetic acid), EG'FA (ethylene glycol tetraacetic acid),
HEDTA
(hydroxyethylethylenediaminetriacetic acid), DPIA (diethylene triamine
pentaacetic acid), NTA
(N,N-bis(carboxyrnethyl)glycine), citrate anhydrous, sodium citrate, calcium
citrate, ammonium
citrate, ammonium bicitrate, citric acid, potassium citrate, or magnesium
citrate. In some
embodiments, the wash buffer comprises a chelating agent at a concentration of
about 0.01 ¨ 50
inM., or about 0.1 ¨20 inM., or about 0.2 ¨ 10 inM.
1005221 In some embodiments, the salt in the wash buffer comprises NaCI, KCI,
NH2SO4 or
potassium glutamate. In some embodiments, the detergent comprises an ionic
detergent such as
SDS (sodium dodecyl sulfate). The wash buffer can include a monovalent salt at
a concentration
of about 25-500 rriM, or about 50-250 rnM, or about 100-200 mM.
1005231 In some embodiments, the detergent in the wash buffer comprises a non-
ionic
detergent such as Triton X-100, Tween 20, Tvveen 80 or Nonidet P-40. In some
embodiments,
the detergent comprises a zwitterionic detergent such as CHAPS (3-[(3-
cholamidopropyl)dimethylammonio]-1-propanesulfonate) or N-Dodecyl-./V,N-
dimethyl-3-
amonio-l-propanesulfate (DetX). In some embodiments, the detergent comprises
LDS (lithium
dodecyl sulfate), sodium taurodeoxycholate, sodium taumcholate, sodium
glycocholate., sodium
deoxycholate or sodium cholate. In some embodiments, the detergent is included
in the wash
buffer at a concentration of about 0.01-0.05%, or about 0.05-0 1%, or about
0.1-0 15%, or about
0.15-0.2%, or about 0.2-0.25%.
Methods for Pairwise Sequencing ¨ Lacking A.basic Sites
[005241 The present disclosure provides pairwise sequencing methods,
comprising step (a):
providing a plurality of immobilized single stranded nucleic acid concatemer
template molecules
each lacking a scissile moiety that can be clawed to generate an abasic site
in the concatemer
template molecule, wherein individual concatemer template molecules in the
plurality are
immobilized to a first surface primer that is immobilized to a support, and
wherein the
immobilized first surface primer lacks a nucleotide having a scissile moiety.
In some
embodiments, the support comprises a plurality of first surface primers. In
some embodiments,
the support lacks a plurality of second surface primers. In some embodiments,
the support
comprises a plurality of first and second surface primers. Exemplary
nucleotides having a scissile
moiety include uridine, 8-oxo-7,8-dihydroguanine (e.g., 8oxoG) and
deoxyinosine.

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[00525] In some embodiments, individual immobilized concatemer template
molecules are
covalently joined to an immobilized surface primer (e.g., an immobilized first
surface primer)
(Figure 73). In an alternative embodiment, individual immobilized concatemer
template
molecules are hybridized to an immobilized surface primer (e.g., an
immobilized first surface
primer) (Figure 80).
1005261 In some embodiments, individual concatemer template molecules in the
plurality
comprise two or more copies of a sequence of interest, and wherein the
individual immobilized
concatemer template molecules further comprise any one or any combination of
two or more of:
(i) two or more copies of a universal binding sequence for a soluble forward
sequencing primer,
(ii) two or more copies of a universal binding sequence for a soluble reverse
sequencing primer,
(iii) two or more copies of a universal binding sequence for an immobilized
first surface primer,
(iv) two or more copies of a universal binding sequence for an immobilized
second surface
primer, (v) two or more copies of a universal binding sequence for a first
soluble amplification
primer, (vi) two or more copies of a universal binding sequence for a second
soluble
amplification primer, (vii) two or more copies of a universal binding sequence
for a soluble
compaction oligonucleotide, (viii) two or more copies of a sample barcode
sequence and/or (ix)
two or more copies of a unique molecular index sequence,
1005271 In some embodiments, individual concatemer template molecules in the
plurality
comprise two or more copies of a sequence of interest and two or more copies
of a universal
binding sequence for a soluble compaction oligonucleotide, and wherein the
individual
immobilized concatemer template molecules further comprise any one or any
combination of
two or more of: (i) two or more copies of a universal binding sequence for a
soluble forward
sequencing primer, (ii) two or more copies of a universal binding sequence for
a soluble reverse
sequencing primer, (iii) two or more copies of a universal binding sequence
for an immobilized
first surface primer, (iv) two or more copies of a universal binding sequence
for an immobilized
second surface primer, (v) two or more copies of a universal binding sequence
for a first soluble
amplification primer, (vi) two or more copies of a universal binding sequence
for a second
soluble amplification primer, (vii) two or more copies of a sample barcode
sequence and/or (viii)
two or more copies of a unique molecular index sequence,
[00528] In some embodiments, the universal binding sequence (or a
complementary sequence
thereof) for the forward sequencing primer can hybridize to at least a portion
of the forward

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sequencing primer. In some embodiments, the universal binding sequence (or a
complementary
sequence thereof) for the reverse sequencing primer can hybridize to at least
a portion of the
reverse sequencing primer. In some embodiments, the universal binding sequence
(or a
complementary sequence thereof) for the immobilized first surface primer can
hybridize to at
least a portion of the immobilized first surface primer. In some embodiments,
the universal
binding sequence (or a complementary sequence thereof) for the immobilized
second surface
primer can hybridize to at least a portion of the immobilized second surface
primer. In some
embodiments, the universal binding sequence (or a complementary sequence
thereof) for the first
soluble amplification primer can hybridize to at least a portion of the first
soluble amplification
primer. In some embodiments, the universal binding sequence (or a
complementary sequence
thereof) for the second soluble amplification primer can hybridize to at least
a portion of the
second soluble amplification primer. In some embodiments, the universal
binding sequence (or a
complementary sequence thereof) for the soluble compaction oligonucleotide can
hybridize to at
least a portion of the soluble compaction oligonucleotide.
[005291 In some embodiments, the immobilized first surface primers comprise
single stranded
oligonucleotides comprising DNA, RNA or a combination of DNA and RNA. The
immobilized
first surface primers can be immobilized to the support or immobilized to a
coating on the
support. The immobilized first surface primers can be embedded and attached
(coupled) to the
coating on the support. In some embodiments, the 5' end of the immobilized
first surface primers
are immobilized to a support or immobilized to a coating on the support.
Alternatively, an
interior portion or the 3' end of the immobilized first surface primers can be
immobilized to a
support or immobilized to a coating on the support. The support comprises a
plurality of
immobilized first surface primers having the same sequence. The immobilized
first surface
primers can be any length, for example 4-50 nucleotides, or 50-100
nucleotides, or 100-150
nucleotides, or longer lengths. In some embodiments, the 3' terminal end of
the immobilized first
surface primers comprise an. extendible 3' OH moiety. In some embodiments, the
3' terminal end
of the immobilized first surface primers comprise a 3' non-extendible moiety.
1005301 In some embodiments, the plurality of immobilized first surface
primers comprise at
least one phosphorothioate diester bond at their 5' ends which can render the
first surface
primers resistant to exonuclea.se degradation. In some embodiments, the
plurality of immobilized
first surface primers comprise 2-5 or more consecutive phosphorothioate
diester bonds at their 5'

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ends. In some embodiments, the plurality of immobilized first surface primers
comprise at least
one ribonucleotide and/or at least one 2' -0-methyl or 2'-0-tnethoxyethyl
(MOE) nucleotide
which can render the first surface primers resistant to exonuclease
degradation.
[005311 In some embodiments, the immobilized first surface primers comprise at
least one
locked nucleic acid (LNA) which comprises a methylene bridge bond between a 2'
oxygen and
4' carbon of the pentose ring. Immobilized first surface primers that include
at least one LNA
can be resistant to nuclease digestions and can exhibit increased melting
temperature when
hybridized to the forward extension strand.
[005.321 In some embodiments, the immobilized concatemer template molecules
further
comprise two or more copies of a universal binding sequence (or complementary
sequence
thereof) for an immobilized second surface primer having a sequence that
differs from the first
immobilized surface primer. The immobilized second surface primers of step (a)
comprise single
stranded oligonucleotides comprising DNA, RNA or a combination of DNA and RNA.
The
immobilized second surface primers can be immobilized to the support or
immobilized to a
coating on the support. The immobilized second surface primers can be embedded
and attached
(coupled) to the coating on the support. In some embodiments, the 5' end of
the immobilized
second surface primers are immobilized to a support or immobilized to a
coating on the support.
Alternatively, an interior portion or the 3' end of the immobilized second
surface primers can be
immobilized to a support or immobilized to a coating on the support. The
support comprises a
plurality of immobilized second surface primers having the same sequence. The
immobilized
second surface primers can be any length, for example 4-50 nucleotides, or 50-
100 nucleotides,
or 100-150 nucleotides, or longer lengths.
[00533] In some embodiments, the 3' terminal end of the immobilized second
surface primers
comprise an extendible 3' OH moiety, In some embodiments, the 3' terminal end
of the
immobilized second surface primers comprise a 3' non-extendible moiety. In
some
embodiments, the 3' terminal end of the immobilized second surface primers
comprise a moiety
that blocks primer extension (e.g., non-extendible terminal 3' end), such as
for example a
phosphate group, a dideoxycytidine group, an inverted dT, or an amino group,
The immobilized
second surface primers are not extendible in a primer extension reaction. The
immobilized
second surface primers lack a nucleotide having a scissile moiety.

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[00534j In some embodiments, the plurality of immobilized second surface
primers comprise
at least one phosphorothioate diester bond at their 5' ends which can render
the second surface
primers resistant to exonuclease degradation. In some embodiments, the
plurality of immobilized
second surface primers comprise 2-5 or more consecutive phosphorothioate
diester bonds at their
5' ends. In some embodiments, the plurality of immobilized second surface
primers comprise at
least one ribonucleotide and/or at least one 2'-O-methyl or 2'-0-methoxyethyl
(MOE) nucleotide
which can render the second surface primers resistant to exonuclease
degradation.
1005351 In some embodiments, individual immobilized single stranded nucleic
acid
concatemer template molecules are joined or immobilized to an immobilized
first surface primer,
and at least one portion of the individual concatemer template molecule is
hybridized to an
immobilized second surface primer. The immobilized second surface primers
serve to pin down
a portion of the immobilized concatemer template molecules to the support (see
Figures 79 and
86).
[00536] In some embodiments, the support comprises about 102 ¨ 1015
immobilized first
surface primers per mm2. In some embodiments, the support comprises about 102
¨ 10'
immobilized second surface primers per mm2. In some embodiments, the support
comprises
about 102 ¨ 1015 immobilized first surface primers and immobilized second
surface primers per
mm2.
[00537] The immobilized surface primers (e.g., first and second surface
primers) are in fluid
communication with each other to permit flowing various solutions of linear or
circular nucleic
acid template molecules, soluble primers, enzymes, nucleotides, divalent
cations, buffers,
reagents, and the like, onto the support so that the plurality of immobilized
surface primers (and
the primer extension products generated from the immobilized surface primers)
react with the
solutions in a massively parallel manner.
[00538] In some embodiments, the pairwise sequencing method further comprises
step (b):
sequencing the plurality of immobilized concatemer template molecules thereby
generating a
plurality of extended forward sequencing primer strands. The sequencing of
step (b) comprises
contacting the plurality of immobilized concatemer template molecules with a
plurality of
soluble forward sequencing primers under a condition suitable to hybridize at
least one forward
sequencing primer to at least one of the forward sequencing primer binding
sites/sequences of
the immobilized concatemer template molecules, and conducting forward
sequencing reactions

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using one or more types of sequencing polymerases, a plurality of nucleotides
and/or multivalent
molecules, and the hybridized first forward sequencing primers. In some
embodiments, the
soluble forward sequencing primers comprise 3' OH extendible ends. In some
embodiments, the
soluble forward sequencing primers comprise a 3' blocking moiety which can be
removed to
generate a 3' OH extendible end. In some embodiments, the soluble forward
sequencing primers
lack a nucleotide having a scissile moiety. The forward sequencing reactions
can generate a
plurality of extended forward sequencing primer strands. In some embodiments,
individual
immobilized concatemer template molecules have multiple copies of the forward
sequencing
primer binding sites, wherein each forward sequencing primer binding site is
capable of
hybridizing to a first forward sequencing primer. Individual forward
sequencing primer binding
sites in a given immobilized concatemer template molecule can be hybridized to
a forward
sequencing primer and can undergo a sequencing reaction. Individual
immobilized concatemer
template molecules can undergo two or more sequence reactions, where each
sequencing
reaction is initiated from a first forward sequencing primer that is
hybridized to a forward
sequencing primer binding site (e.g., see Figures 74 and 81). In some
embodiments, the
sequencing reactions comprise a plurality of nucleotides (or analogs thereof)
labeled with a
detectable reporter moiety. In some embodiments, the sequencing reaction
comprise a plurality
of multivalent molecules having a plurality of nucleotide units attached to a
core, where the
multivalent molecules are labeled with a detectable reporter moiety. In some
embodiments, the
core is labeled with a detectable reporter moiety. In some embodiments, at
least one linker and/or
at least one nucleotide unit of a nucleotide arm is labeled with a detectable
reporter moiety. In
some embodiments, the detectable reporter moiety comprises a fluorophore. An
exemplary
nucleotide arm is shown in Figure 108, and exemplary multivalent molecules are
shown in
Figures 104-107.
[00539] In some embodiments, the pairwise sequencing method further comprises
step (c):
retaining the plurality of immobilized concatemer template molecules and
replacing the plurality
of extended forward sequencing primer strands with a plurality of forward
extension strands by
conducting a primer extension reaction. The extended forward sequencing primer
strands can be
removed from the retained immobilized concatemer template molecules. The
retained
immobilized concatemer template molecule can be hybridized to a plurality of
soluble
amplification or sequencing primers and subjected to a primer extension
reaction. The primer
. .

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