Note: Descriptions are shown in the official language in which they were submitted.
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Concurrent sequencing of forward and reverse complement strands on
concatenated polynucleotides for methylation detection
Field of the Invention
The invention relates to methods of detecting modified cytosines in nucleic
acid
sequences.
Background of the Invention
Modified cytosines, including 5-methylcytosine (5mC), are a well-studied
epigenetic
modification that play fundamental roles in human development and disease. Its
genome-wide distribution differs between tissue types, and between healthy and
diseased states. In recent years, 5mC has also gained prominence as a tool for
clinical
diagnostics: its distribution in cell-free DNA (cfDNA) ¨ obtained from a
liquid biopsy ¨
can be used for the tissue-specific prediction of early-stage cancer.
As a result, there has been an intense focus on developing methods for mapping
modified cytosines at single base resolution, with minimal loss of sample
polynucleotide
quantity, quality, and complexity.
However, there remains a need to develop new methods for detecting modified
cytosines, and in particular methods that enable quick and accurate detection
of modified
cytosines.
Summary of the Invention
According to an aspect of the present invention, there is provided a method of
preparing
at least one polynucleotide sequence for detection of modified cytosines,
comprising:
synthesising at least one polynucleotide sequence comprising a first
portion and a second portion,
wherein the at least one polynucleotide sequence comprises portions of
a double-stranded nucleic acid template, and the first portion comprises a
forward
strand of the template, and the second portion comprises a reverse complement
strand of the template; or wherein the first portion comprises a reverse
strand of
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the template, and the second portion comprises a forward complement strand of
the template,
wherein the template is generated from a target polynucleotide to be
sequenced via complementary base pairing, and wherein the target
polynucleotide has been pre-treated using a conversion reagent,
wherein the conversion reagent is configured to convert a modified
cytosine to thymine or a nucleobase which is read as thymine/uracil, and/or
wherein the conversion reagent is configured to convert an unmodified cytosine
to uracil or a nucleobase which is read as thymine/uracil.
In one embodiment, the target polynucleotide has been pre-treated using a
conversion
reagent configured to convert a modified cytosine to thymine or a nucleobase
which is
read as thymine/uracil.
In another embodiment, the target polynucleotide has been pre-treated using a
conversion reagent configured to convert an unmodified cytosine to uracil or a
nucleobase which is read as thymine/uracil.
In one embodiment, the conversion agent comprises a chemical agent and/or an
enzyme.
In one example, the chemical agent comprises a boron-based reducing agent.
In a further example, the boron-based reducing agent is an amine-borane
compound or
an azine-borane compound.
In one embodiment, the boron-based reducing agent is selected from the group
consisting of pyridine borane, 2-picoline borane, t-butylamine borane, ammonia
borane,
ethylenediamine borane and dimethylamine borane.
In one embodiment, the chemical agent comprises sulfite; such as bisulfite or
sodium
bisulfite.
In one embodiment, the enzyme comprises a cytidine deaminase.
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In one embodiment, the cytidine deaminase is a wild-type cytidine deaminase or
a
mutant cytidine deaminase. In one example, the cytidine deaminase is a mutant
cytidine
deaminase.
In one example, the cytidine deaminase is a member of the AID subfamily, the
APOBEC1
subfamily, the APOBEC2 subfamily, the APOBEC3A subfamily, the APOBEC3B
subfamily, the APOBEC3C subfamily, the APOBEC3D subfamily, the APOBEC3F
subfamily, the APOBEC3G subfamily, the APOBEC3H subfamily, or the APOBEC4
subfamily; in one embodiment, the cytidine deaminase is a member of the
APOBEC3A
subfamily.
In one aspect, the cytidine deaminase comprises amino acid substitution
mutations at
positions functionally equivalent to (Tyr/Phe)130 and Tyr132 in a wild-type
APOBEC3A
protein.
In one embodiment, the (Tyr/Phe)130 is Tyr130, and the wild-type APOBEC3A
protein
is SEQ ID NO. 32.
In one embodiment, the substitution mutation at the position functionally
equivalent to
Tyr130 comprises Ala, Val or Trp.
In one aspect, the substitution mutation at the position functionally
equivalent to Tyr132
comprises a mutation to His, Arg, Gln or Lys.
In one embodiment, the mutant cytidine deaminase comprises a ZDD motif H4P/AN]-
E-
X[23-281- P-C-X[2-41-C (SEQ ID NO. 67).
In one embodiment, the mutant cytidine deaminase is a member of the APOBEC3A
subfamily and comprises a ZDD motif HXEX24SW(S/T)PCX[2_4]CX6FX8LX5R(UDYX[8_
ii]LX2LX[io]M (SEQ ID NO. 68).
In one embodiment, the mutant cytidine deaminase converts 5-methylcytosine to
thymine by deamination at a greater rate than conversion rate of cytosine to
uracil by
deamination; wherein the rate may be at least 100-fold greater.
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In one embodiment, the target polynucleotide is treated with a further agent
prior to
treatment with the conversion reagent.
In one embodiment, the further agent is configured to convert a modified
cytosine to
another modified cytosine.
In one embodiment, the further agent configured to convert a modified cytosine
to
another modified cytosine comprises a chemical agent and/or an enzyme.
In another embodiment, the further agent configured to convert a modified
cytosine to
another modified cytosine comprises an oxidising agent; such as a metal-based
oxidising
agent; for example, a transition metal-based oxidising agent; or such as a
ruthenium-
based oxidising agent.
In another embodiment, the further agent configured to convert a modified
cytosine to
another modified cytosine comprises a reducing agent; such as a Group III-
based
reducing agent; for example, a boron-based reducing agent.
In one aspect, the further agent configured to convert a modified cytosine to
another
modified cytosine comprises a ten-eleven translocation (TET) methylcytosine
dioxygenase; for example, wherein the TET methylcytosine dioxygenase may be a
member of the TETI subfamily, the TET2 subfamily, or the TET3 subfamily.
In one example, the further agent is configured to reduce/prevent deamination
of a
particular modified cytosine.
In one embodiment, the further agent configured to reduce/prevent deamination
of a
particular modified cytosine comprises a chemical agent and/or an enzyme.
In one embodiment, the further agent configured to reduce/prevent deamination
of a
particular modified cytosine comprises a glycosyltransferase; such as a [3-
glucosyltransferase.
In another embodiment, the further agent configured to reduce/prevent
deamination of a
particular modified cytosine comprises a hydroxylamine or a hydrazine.
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In one embodiment, the modified cytosine is selected from the group consisting
of: 5-
methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine and 5-
carboxylcytosine.
In one embodiment, the forward strand of the template is not identical to the
reverse
5 complement strand of the template.
In one embodiment, the forward strand comprises a guanine base at a first
position, and
wherein the reverse complement strand comprises an adenine base at a second
position
corresponding to the same position number as the first position; or wherein
the forward
strand comprises an adenine base at a first position, and wherein the reverse
complement strand comprises a guanine base at a second position corresponding
to the
same position number as the first position.
In one embodiment, the method further comprises a step of preparing the first
portion
and the second portion for concurrent sequencing.
In one embodiment, the method comprises simultaneously contacting first
sequencing
primer binding sites located after a 3'-end of the first portions with first
primers and
second sequencing primer binding sites located after a 3'-end of the second
portions
with second primers.
In one embodiment, a proportion of first portions is capable of generating a
first signal
and a proportion of second portions is capable of generating a second signal,
wherein
an intensity of the first signal is substantially the same as an intensity of
the second
signal.
In one embodiment, the method further comprises a step of selectively
processing the at
least one polynucleotide sequence comprising the first portion and the second
portion,
such that a proportion of first portions are capable of generating a first
signal and a
proportion of second portions are capable of generating a second signal,
wherein the
selective processing causes an intensity of the first signal to be greater
than an intensity
of the second signal.
In one example, a concentration of the first portions capable of generating
the first signal
is greater than a concentration of the second portions capable of generating
the second
signal.
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In one aspect, a ratio between the concentration of the first portions capable
of
generating the first signal and the concentration of the second portions
capable of
generating the second signal is between 1.25:1 to 5:1, or between 1.5:1 to
3:1, or about
2:1.
In one embodiment, selective processing comprises preparing for selective
sequencing
or conducting selective sequencing.
In one embodiment, selectively processing comprises contacting first
sequencing primer
binding sites located after a 3'-end of the first portions with first primers
and contacting
second sequencing primer binding sites located after a 3'-end of the second
portions
with second primers, wherein the second primers comprises a mixture of blocked
second
primers and unblocked second primers.
In one embodiment, the blocked second primer comprises a blocking group at a
3' end
of the blocked second primer.
In one aspect, the blocking group is selected from the group consisting of: a
hairpin loop,
a deoxynucleotide, a deoxyribonucleotide, a hydrogen atom instead of a 3'-OH
group, a
phosphate group, a phosphorothioate group, a propyl spacer, a modification
blocking the
3'-hydroxyl group, or an inverted nucleobase.
In one embodiment, the blocked second primer comprises a sequence as defined
in SEQ
ID NO. 11 to 16 or a variant or fragment thereof and/or the unblocked second
primer
comprises a sequence as defined in SEQ ID NO. 11 to 14 or a variant or
fragment
thereof.
In one embodiment, the first signal and the second signal are spatially
unresolved.
In one example, the at least one polynucleotide sequence comprising the first
portion
and the second portion is/are attached to a solid support, wherein the solid
support may
be a flow cell.
In another example, the at least one polynucleotide sequence comprising the
first portion
and the second portion forms a cluster on the solid support.
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In one embodiment, the cluster is formed by bridge amplification.
In one embodiment, the at least one polynucleotide sequence comprising the
first portion
and the second portion forms a monoclonal cluster.
In one embodiment, the solid support comprises at least one first immobilised
primer and
at least one second immobilised primer.
In one embodiment, the first immobilised primer comprises a sequence as
defined in
SEQ ID NO. 1 or 5, or a variant or fragment thereof; and the second
immobilised primer
comprises a sequence as defined in SEQ ID NO. 2, or a variant or fragment
thereof.
In one embodiment, each polynucleotide sequence comprising the first portion
and the
second portion is attached to a first immobilised primer.
In one embodiment, each polynucleotide sequence comprising the first portion
and the
second portion further comprises a second adaptor sequence, wherein the second
adaptor sequence is substantially complementary to the second immobilised
primer.
In one embodiment, the step of synthesising the at least one polynucleotide
sequence
comprising a first portion and a second portion comprises:
synthesising a first precursor polynucleotide fragment comprising a
complement of the first portion and a hybridisation complement sequence,
synthesising a second precursor polynucleotide fragment comprising a
second portion and a hybridisation sequence,
annealing the hybridisation complement sequence of the first precursor
polynucleotide fragment with the hybridisation sequence on the second
precursor
polynucleotide fragment to form a hybridised adduct,
synthesising a first precursor polynucleotide sequence by extending the
first precursor polynucleotide fragment to form a complement of the second
portion, and
synthesising the at least one polynucleotide sequence by forming a
complement of the first precursor polynucleotide sequence.
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In one embodiment, the first precursor polynucleotide fragment comprises a
first
sequencing primer binding site complement.
In one embodiment, the first sequencing primer binding site complement is
located
before a 5'-end of the complement of the first portion, such as immediately
before the 5'-
end of the complement of the first portion.
In one embodiment, the first precursor polynucleotide fragment comprises a
second
adaptor complement sequence.
In one embodiment, the second adaptor complement sequence is located before a
5'-
end of the complement of the first portion.
In one embodiment, the first precursor polynucleotide fragment comprises a
first
sequencing primer binding site complement and a second adaptor complement
sequence.
In one embodiment, the first sequencing primer binding site complement is
located
before a 5'-end of the complement of the first portion, and wherein the second
adaptor
complement sequence is located before a 5'-end of the first sequencing primer
binding
site complement.
In one embodiment, the first precursor polynucleotide fragment comprises a
second
sequencing primer binding site complement.
In one embodiment, the hybridisation sequence complement comprises the second
sequencing primer binding site complement.
In one embodiment, the second precursor polynucleotide fragment comprises a
first
adaptor complement sequence.
In one embodiment, the method further comprises concurrently sequencing
nucleobases
in the first portion and the second portion.
In one embodiment, the first portion is at least 25 base pairs and the second
portion is
at least 25 base pairs.
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According to another aspect of the present invention, there is provided a
method of
sequencing at least one polynucleotide sequence to detect modified cytosines,
comprising:
preparing at least one polynucleotide sequence for detection of modified
cytosines using a method as described herein;
concurrently sequencing nucleobases in the first portion and the second
portion; and
identifying modified cytosines by detecting differences when comparing a
sequence output from the first portion with a sequence output from the second
portion.
In one embodiment, the step of concurrently sequencing nucleobases comprises
performing sequencing-by-synthesis or sequencing-by-ligation.
In one embodiment, the step of preparing the at least one polynucleotide
sequence
comprises using a method as described herein; and wherein the step of
concurrent
sequencing nucleobases in the first portion and the second portion is based on
the
intensity of the first signal and the intensity of the second signal.
In one example, the method further comprises a step of conducting paired-end
reads.
In one embodiment, the step of concurrently sequencing nucleobases comprises:
(a) obtaining first intensity data comprising a combined intensity of a
first
signal component obtained based upon a respective first nucleobase at the
first portion
and a second signal component obtained based upon a respective second
nucleobase
at the second portion, wherein the first and second signal components are
obtained
simultaneously;
(b) obtaining second intensity data comprising a combined intensity of a
third
signal component obtained based upon the respective first nucleobase at the
first portion
and a fourth signal component obtained based upon the respective second
nucleobase
at the second portion, wherein the third and fourth signal components are
obtained
simultaneously;
(c) selecting one of a plurality of classifications based on the first and
the
second intensity data, wherein each classification represents a possible
combination of
respective first and second nucleobases; and
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(d) based on the selected classification, base calling
the respective first and
second nucleobases.
In a further embodiment, selecting the classification based on the first and
second
5 intensity data comprises selecting the classification based on the
combined intensity of
the first and second signal components and the combined intensity of the third
and fourth
signal components.
In one embodiment, the plurality of classifications comprises sixteen
classifications, each
10 classification representing one of sixteen unique combinations of first
and second
nucleobases.
In one embodiment, the first signal component, second signal component, third
signal
component and fourth signal component are generated based on light emissions
associated with the respective nucleobase.
In one embodiment, the light emissions are detected by a sensor, wherein the
sensor is
configured to provide a single output based upon the first and second signals.
In one embodiment, the sensor comprises a single sensing element.
In one embodiment, the method further comprises repeating steps (a) to (d) for
each of
a plurality of base calling cycles.
In one embodiment, the step of concurrently sequencing nucleobases comprises:
(a) obtaining first intensity data comprising a combined intensity of a
first
signal component obtained based upon a respective first nucleobase at the
first portion
and a second signal component obtained based upon a respective second
nucleobase
at the second portion, wherein the first and second signal components are
obtained
simultaneously;
(b) obtaining second intensity data comprising a combined intensity of a
third
signal component obtained based upon the respective first nucleobase at the
first portion
and a fourth signal component obtained based upon the respective second
nucleobase
at the second portion, wherein the third and fourth signal components are
obtained
simultaneously;
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(c) selecting one of a plurality of classifications based on the first and
the
second intensity data, wherein each classification of the plurality of
classifications
represents one or more possible combinations of respective first and second
nucleobases, and wherein at least one classification of the plurality of
classifications
represents more than one possible combination of respective first and second
nucleobases; and
(d) based on the selected classification, determining sequence information
from the first portion and the second portion.
In one embodiment, selecting the classification based on the first and second
intensity
data comprises selecting the classification based on the combined intensity of
the first
and second signal components and the combined intensity of the third and
fourth signal
components.
In one embodiment, when based on a nucleobase of the same identity, an
intensity of
the first signal component is substantially the same as an intensity of the
second signal
component and an intensity of the third signal component is substantially the
same as
an intensity of the fourth signal component.
In one embodiment, the plurality of classifications consists of a
predetermined number
of classifications.
In one embodiment, the plurality of classifications comprises:
one or more classifications representing matching first and second
nucleobases; and
one or more classifications representing mismatching first and second
nucleobases, and
wherein determining sequence information of the first portion and second
portion comprises:
in response to selecting a classification representing matching first
and second nucleobases, determining a match between the first and
second nucleobases; or
in response to selecting a classification representing mismatching
first and second nucleobases, determining a mismatch between the first
and second nucleobases.
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In one embodiment, determining sequence information of the first portion and
the second
portion comprises, in response to selecting a classification representing a
match
between the first and second nucleobases, base calling the first and second
nucleobases.
In one embodiment, determining sequence information of the first portion and
the second
portion comprises, based on the selected classification, determining that the
second
portion is modified relative to the first portion at a location associated
with the first and
second nucleobases.
In one embodiment, the first signal component, second signal component, third
signal
component and fourth signal component are generated based on light emissions
associated with the respective nucleobase.
In one embodiment, the light emissions are detected by a sensor, wherein the
sensor is
configured to provide a single output based upon the first and second signals.
In one embodiment, the sensor comprises a single sensing element.
In one embodiment, the method further comprises repeating steps (a) to (d) for
each of
a plurality of base calling cycles.
According to another aspect of the present invention, there is provided a kit
comprising
instructions for preparing at least one polynucleotide sequence for detection
of modified
cytosines as described herein, and/or for sequencing at least one
polynucleotide
sequence to detect modified cytosines as described herein.
According to another aspect of the present invention, there is provided a data
processing
device comprising means for carrying out a method as described herein.
In one aspect, the data processing device is a polynucleotide sequencer.
According to another aspect of the present invention, there is provided a
computer
program product comprising instructions which, when the program is executed by
a
processor, cause the processor to carry out a method as described herein.
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According to another aspect of the present invention, there is provided a
computer-
readable storage medium comprising instructions which, when executed by a
processor,
cause the processor to carry out a method as described herein.
According to another aspect of the present invention, there is provided a
computer-
readable data carrier having stored thereon a computer program product as
described
herein.
According to another aspect of the present invention, there is provided a data
carrier
signal carrying a computer program product as described herein.
Description of the Drawings
Features of examples of the present disclosure will become apparent by
reference to the
following detailed description and drawings, in which like reference numerals
correspond
to similar, though perhaps not identical, components. For the sake of brevity,
reference
numerals or features having a previously described function may or may not be
described in connection with other drawings in which they appear.
Figure 1 shows a forward strand, reverse strand, forward complement strand,
and
reverse complement strand of a polynucleotide molecule.
Figure 2 shows the preparation of a concatenated polynucleotide sequence
comprising
a first portion and a second portion using a tandem insert method, comprising
(A)
preparation of a desired first (forked) adaptor and second (forked) adaptor
from three
oligos; (B) different types of first (forked) adaptors and second (forked)
adaptors that do
not anneal to each other due to the presence of a third oligo on at least one
of the first
(forked) adaptor and/or the second (forked) adaptor; (C) ligation of the
template
polynucleotide strand and adaptors generates three products, with the desired
product
containing both types of adaptor being produced at a proportion of 50%; (D)
synthesis of
concatenated strands from the desired product; and (E) completion of the
synthesis of
the concatenated strands from the desired product.
Figure 3 shows an example of a concatenated polynucleotide sequence comprising
a
first portion and a second portion, as well as terminal and internal adaptor
sequences.
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Figure 4 shows an example of a concatenated polynucleotide sequence comprising
a
first portion and a second portion, as well as terminal and internal adaptor
sequences.
Figure 5 shows a typical solid support.
Figure 6 shows the stages of bridge amplification for concatenated
polynucleotide
sequences and the generation of an amplified cluster, comprising (A) a
concatenated
library strand hybridising to a immobilised primer; (B) generation of a
template strand
from the library strand; (C) dehybridisation and washing away the library
strand; (D)
generation of a template complement strand from the template strand via bridge
amplification and dehybridisation of the sequence bridge; (E) further
amplification to
provide a plurality of template and template complement strands; and (F)
cleavage of
one set of the template and template complement strands.
Figure 7 shows the detection of nucleobases using 4-channel, 2-channel and 1-
channel
chemistry.
Figure 8 shows a method of selective sequencing.
Figure 9 is a plot showing graphical representations of sixteen distributions
of signals
generated by polynucleotide sequences according to one embodiment
Figure 10 is a flow diagram showing a method for base calling according to one
embodiment.
Figure 11 is a plot showing graphical representations of nine distributions of
signals
generated by polynucleotide sequences according to one embodiment.
Figure 12 shows the effect of unmodified cytosine to uracil conversion
treatment of a
double-stranded polynucleotide, and a scatter plot showing the resulting
distributions of
signals generated by polynucleotide sequences.
Figure 13 shows the effect of modified cytosine to thymine conversion
treatment of a
double-stranded polynucleotide, and a scatter plot showing the resulting
distributions of
signals generated by polynucleotide sequences.
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Figure 14 shows alternative signal distributions using a different dye-
encoding scheme.
Figure 15 shows alternative signal distributions using a different dye-
encoding scheme.
5 Figure 16 shows alternative signal distributions using a different dye-
encoding scheme.
Figure 17 is a flow diagram showing a method for determining sequence
information
according to one embodiment.
10 Figure 18A shows the effect of pre-treatment of library strands using C
to U conversion
on bases in template strands. Figure 18B shows the effect of pre-treatment of
library
strands using mC to T conversion on bases in template strands.
Figure 19A shows 9 QaM analysis conducted on the signals obtained from the
custom
15 second hyb run of Example 1. The x-axis shows signal intensity from a
"red" wavelength
channel, whilst the y-axis shows signal intensity from a "green" wavelength
channel. G
is not associated with any dyes and as such appears contributes no intensity
for both
"red" and "green" channels. C is associated with a "red" dye and as such
contributes
intensity to the "red" channel, but not the "green" channel. T is associated
with a "green"
dye and as such contributes intensity to the "green" channel, but not the "red
channel. A
is associated with both a "red" dye and a "green" dye, and as such contributes
intensity
to both the "red" channel and "green" channel. Since the template comprises
forward
and reverse complement strands that are sequenced simultaneously, most of the
readout will generate (G,G) read (bottom left corner), (CC) read (bottom right
corner),
(T,T) read (top left corner), and (A,A) read (top right corner) clouds.
However, a central
cloud corresponding to (C,T) or (T,C) reads corresponds with the presence of
modified
cytosines. Figure 19B shows sequence data generated from two different primers
used
(HYB2'-ME and HP10) in the custom second hyb run of Example 1. Mismatches
between
the two sequences allow identification of modified cytosines. For example, 5-
mC present
in the original forward strand of the target polynucleotide is read as T in
the HP10 read,
whereas C present in the original reverse complement strand of the target
polynucleotide
(corresponding to the same position as 5-mC in the original forward strand of
the target
polynucleotide) is read as C in the HYB2'-ME read.
Detailed Description of the Invention
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All patents, patent applications, and other publications referred to herein,
including all
sequences disclosed within these references, are expressly incorporated herein
by
reference, to the same extent as if each individual publication, patent or
patent
application was specifically and individually indicated to be incorporated by
reference.
All documents cited are, in relevant part, incorporated herein by reference in
their
entireties for the purposes indicated by the context of their citation herein.
However, the
citation of any document is not to be construed as an admission that it is
prior art with
respect to the present disclosure.
The present invention can be used in sequencing, in particular concurrent
sequencing.
Methodologies applicable to the present invention have been described in WO
08/041002, WO 07/052006, WO 98/44151, WO 00/18957, WO 02/06456, WO
07/107710, W005/068656, US 13/661,524 and US 2012/0316086, the contents of
which
are herein incorporated by reference. Further information can be found in US
20060024681, US 20060292611, WO 06/110855, WO 06/135342, WO 03/074734,
W007/010252, WO 07/091077, WO 00/179553, WO 98/44152 and WO 2022/087150,
the contents of which are herein incorporated by reference.
As used herein, the term "variant" refers to a variant polypeptide sequence or
part of the
polypeptide sequence that retains desired function of the full non-variant
sequence. For
example, a desired function of the immobilised primer retains the ability to
bind (i.a
hybridise) to a target sequence.
As used in any aspect described herein, a "variant" has at least 25%, 26%,
27%, 28%,
29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%,
44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%,
59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%,
74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% overall
sequence identity to the non-variant nucleic acid sequence. The sequence
identity of a
variant can be determined using any number of sequence alignment programs
known in
the art. As an example, Emboss Stretcher from the EMBL-EBI may be used:
https://www.ebi.ac.uk/Tools/psa/emboss stretcher/ (using default parameters:
pair
output format, Matrix = BLOSUM62, Gap open = 1, Gap extend = 1 for proteins;
pair
output format, Matrix = DNAfull, Gap open = 16, Gap extend = 4 for
nucleotides).
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As used herein, the term "fragment" refers to a functionally active series of
consecutive
nucleic acids from a longer nucleic acid sequence. The fragment may be at
least 99%,
at least 95%, at least 90%, at least 80%, at least 70%, at least 60%, at least
50%, at
least 40% or at least 30% the length of the longer nucleic acid sequence. A
fragment as
used herein may also retain the ability to bind (i.e. hybridise) to a target
sequence.
Sequencing generally comprises four fundamental steps: 1) library preparation
to form a
plurality of target polynucleotides for identification; 2) cluster generation
to form an array
of amplified template polynucleotides; 3) sequencing the cluster array of
amplified
template polynucleotides; and 4) data analysis to identify characteristics of
the target
polynucleotides from the amplified template polynucleotide sequences. These
steps are
described in greater detail below.
Library strands and template terminology
For a given double-stranded polynucleotide sequence 100 to be identified, the
polynucleotide sequence 100 comprises a forward strand of the sequence 101 and
a
reverse strand of the sequence 102. See Figure 1.
When the polynucleotide sequence 100 is replicated (e.g. using a DNA/RNA
polymerase), complementary versions of the forward strand 101 of the sequence
100
and the reverse strand 102 of the sequence 100 are generated. Thus,
replication of the
polynucleotide sequence 100 provides a double-stranded polynucleotide sequence
100a
that comprises a forward strand of the sequence 101 and a forward complement
strand
of the sequence 101', and a double-stranded polynucleotide sequence 100b that
comprises a reverse strand of the sequence 102 and a reverse complement strand
of
the sequence 102'.
The term "template" may be used to describe a complementary version of the
double-
stranded polynucleotide sequence 100. As such, the "template" comprises a
forward
complement strand of the sequence 101' and a reverse complement strand of the
sequence 102'. Thus, by using the forward complement strand of the sequence
101' as
a template for complementary base pairing, a sequencing process (e.g. a
sequencing-
by-synthesis or a sequencing-by-ligation process) reproduces information that
was
present in the original forward strand of the sequence 101. Similarly, by
using the reverse
complement strand of the sequence 102' as a template for complementary base
pairing,
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a sequencing process (e.g. a sequencing-by-synthesis or a sequencing-by-
ligation
process) reproduces information that was present in the original reverse
strand of the
sequence 102.
The two strands in the template may also be referred to as a forward strand of
the
template 101' and a reverse strand of the template 102'. The complement of the
forward
strand of the template 101' is termed the forward complement strand of the
template 101,
whilst the complement of the reverse strand of the template 102' is termed the
reverse
complement strand of the template 102.
Generally, where forward strand, reverse strand, forward complement strand,
and
reverse complement strand are used herein without qualifying whether they are
with
respect to the original polynucleotide sequence 100 or with respect to the
"template",
these terms may be interpreted as referring to the "template".
Language for original polynucleotide Corresponding language for the
sequence 100 "template"
Forward strand of the sequence 101 Forward complement strand
of the
template 101 (sometimes referred to
herein as forward complement strand
101)
Reverse strand of the sequence 102 Reverse complement strand
of the
template 102 (sometimes referred to
herein as reverse complement strand
102)
Forward complement strand of the Forward strand of the template 101'
sequence 101' (sometimes referred to
herein as forward
strand 101')
Reverse complement strand of the Reverse strand of the template 102'
sequence 102' (sometimes referred to
herein as reverse
strand 102')
Library preparation
Library preparation is the first step in any high-throughput sequencing
platform. These
libraries allow templates to be generated via complementary base pairing that
can
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subsequently be clustered and amplified. During library preparation, nucleic
acid
sequences, for example genomic DNA sample, or cDNA or RNA sample, is converted
into a sequencing library, which can then be sequenced. By way of example with
a DNA
sample, the first step in library preparation is random fragmentation of the
DNA sample.
Sample DNA is first fragmented and the fragments of a specific size (typically
200-500
bp, but can be larger) are ligated, sub-cloned or "inserted" in-between two
oligo adaptors
(adaptor sequences). The original sample DNA fragments are referred to as
"inserts".
The target polynucleotides may advantageously also be size-fractionated prior
to
modification with the adaptor sequences.
As described herein, typically the templates to be generated from the
libraries may
include a concatenated polynucleotide sequence comprising a first portion and
a second
portion. Generating these templates from particular libraries may be performed
according to methods known to persons of skill in the art. However, some
example
approaches of preparing libraries suitable for generation of such templates
are described
below.
In some embodiments, the library may be prepared by using a tandem insert
method
described in more detail in e.g. WO 2022/087150, which is incorporated herein
by
reference. This procedure may be used, for example, for preparing templates
comprising
concatenated polynucleotide sequences comprising a first portion and a second
portion,
wherein the first portion is a forward strand of the template, and the second
portion is a
reverse complement strand of the template (or alternatively, wherein the first
portion is a
reverse strand of the template, and the second portion is a forward complement
strand
of the template). Such libraries may also be referred to as cross-tandem
inserts. A
representative process for conducting a tandem insert method is shown in
Figure 2A to
2E.
The processes described above in relation to tandem insert methods generate
libraries
that have concatenated polynucleotides.
Thus, one strand of a concatenated polynucleotide within a polynucleotide
library may
comprise, in a 5' to 3' direction, a second primer-binding complement sequence
302 (e.g.
P7), a first terminal sequencing primer binding site complement 303' (e.g. B15-
ME; or if
ME is not present, then B15), a first insert sequence 401, a hybridisation
complement
sequence 403 (e.g. ME'-HYB2-ME; or if ME' and ME are not present, then HYB2),
a
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second insert sequence 402, a second terminal sequencing primer binding site
304 (e.g.
ME'-A14'; or if ME' is not present, then A14'), and a first primer-binding
sequence 301'
(e.g. P5') (Figures 3 and 4 ¨ bottom strand).
5 Although not shown in Figures 3 and 4, the strand may further comprise
one or more
index sequences. As such, a first index sequence (e.g. i7) may be provided
between the
second primer-binding complement sequence 302 (e.g. P7) and the first terminal
sequencing primer binding site complement 303' (e.g. B15-ME; or if ME is not
present,
then B15). Separately, or in addition, a second index complement sequence
(e.g. i5')
10 may be provided between the second terminal sequencing primer binding
site 304 (e.g.
ME'-A14') and the first primer-binding sequence 301' (e.g. P5'). Thus, in some
embodiments, one strand of a polynucleotide within a polynucleotide library
may
comprise, in a 5' to 3' direction, a second primer-binding complement sequence
302 (e.g.
P7), a first index sequence (e.g. i7), a first terminal sequencing primer
binding site
15 complement 303' (e.g. B15-ME; or if ME is not present, then B15), a
first insert sequence
401, a hybridisation complement sequence 403 (e.g. ME'-HYB2-ME; or if ME' and
ME
are not present, then HYB2), a second insert sequence 402, a second terminal
sequencing primer binding site 304 (e.g. ME'-A14'; or if ME' is not present,
then A14'), a
second index complement sequence (e.g. i5'), and a first primer-binding
sequence 301'
20 (e.g. P5')
Another strand of a concatenated polynucleotide within a polynucleotide
library may
comprise, in a 5' to 3' direction, a first primer-binding complement sequence
301 (e.g.
P5), a second terminal sequencing primer binding site complement 304' (e.g.
A14-ME;
or if ME is not present, then A14), a second insert complement sequence 402',
a
hybridisation sequence 403' (e.g. ME'-HYB2'-ME; or if ME' and ME are not
present, then
HYB2'), a first insert complement sequence 401', a first terminal sequencing
primer
binding site 303 (e.g. ME'-B15'; or if ME' is not present, then B15'), and a
second primer-
binding sequence 302' (e.g. P7') (Figures 3 and 4 ¨ top strand).
Although not shown in Figures 3 and 4, the another strand may further comprise
one or
more index sequences. As such, a second index sequence (e.g. i5) may be
provided
between the first primer-binding complement sequence 301 (e.g. P5) and the
second
terminal sequencing primer binding site complement 304' (e.g. A14-ME; or if ME
is not
present, then A14). Separately, or in addition, a first index complement
sequence (e.g.
i7') may be provided between the first terminal sequencing primer binding site
303 (e.g.
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ME'-615'; or if ME' is not present, then B15') and the second primer-binding
sequence
302' (e.g. P7'). Thus, in some embodiments, another strand of a polynucleotide
within a
polynucleotide library may comprise, in a 5' to 3' direction, a first primer-
binding
complement sequence 301 (e.g. P5), a second index sequence (e.g. i5), a second
terminal sequencing primer binding site complement 304' (e.g. A14-ME; or if ME
is not
present, then A14).), a second insert complement sequence 402', a
hybridisation
sequence 403' (e.g. ME'-HYB2'-ME; or if ME' and ME are not present, then
HYB2'), a
first insert complement sequence 401', a first terminal sequencing primer
binding site
303 (e.g. ME'-615'; or if ME' is not present, then B15'), a first index
complement
sequence (e.g. i7'), and a second primer-binding sequence 302' (e.g. P7').
As described herein, the first insert sequence 401 and the second insert
sequence 402
may comprise different types of library sequences.
In one embodiment, the first insert sequence 401 may comprise a forward strand
of the
sequence 101, and the second insert sequence may comprise a reverse complement
strand of the sequence 102' (or the first insert sequence 401 may comprise a
reverse
strand of the sequence 102, and the second insert sequence 402 may comprise a
forward complement strand of the sequence 101'), for example where the library
is
prepared using a tandem insert method.
As will be understood by the skilled person, a double-stranded nucleic acid
will typically
be formed from two complementary polynucleotide strands comprised of
deoxyribonucleotides or ribonucleotides joined by phosphodiester bonds, but
may
additionally include one or more ribonucleotides and/or non-nucleotide
chemical
moieties and/or non-naturally occurring nucleotides and/or non-naturally
occurring
backbone linkages. In particular, the double-stranded nucleic acid may include
non-
nucleotide chemical moieties, e.g. linkers or spacers, at the 5 end of one or
both strands.
By way of non-limiting example, the double-stranded nucleic acid may include
methylated nucleotides, uracil bases, phosphorothioate groups, peptide
conjugates etc.
Such non-DNA or non-natural modifications may be included in order to confer
some
desirable property to the nucleic acid, for example to enable covalent, non-
covalent or
metal-coordination attachment to a solid support, or to act as spacers to
position the site
of cleavage an optimal distance from the solid support. A single stranded
nucleic acid
consists of one such polynucleotide strand. Where a polynucleotide strand is
only
partially hybridised to a complementary strand ¨ for example, a long
polynucleotide
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strand hybridised to a short nucleotide primer ¨ it may still be referred to
herein as a
single stranded nucleic acid.
A sequence comprising at least a primer-binding sequence (a primer-binding
sequence
and a sequencing primer binding site, or a combination of a primer-binding
sequence,
an index sequence and a sequencing primer binding site) may be referred to
herein as
an adaptor sequence, and an insert (or inserts in concatenated strands) is
flanked by a
5' adaptor sequence and a 3' adaptor sequence. The primer-binding sequence may
also
comprise a sequencing primer for the index read.
As used herein, an "adaptor" refers to a sequence that comprises a short
sequence-
specific oligonucleotide that is ligated to the 5 and 3' ends of each DNA (or
RNA)
fragment in a sequencing library as part of library preparation. The adaptor
sequence
may further comprise non-peptide linkers.
In a further embodiment, the P5' and P7' primer-binding sequences are
complementary
to short primer sequences (or lawn primers) present on the surface of a flow
cell. Binding
of P5' and P7' to their complements (P5 and P7) on ¨ for example ¨ the surface
of the
flow cell, permits nucleic acid amplification. As used herein " denotes the
complementary strand.
The primer-binding sequences in the adaptor which permit hybridisation to
amplification
primers (e.g. lawn primers) will typically be around 20-40 nucleotides in
length, although
the invention is not limited to sequences of this length. The precise identity
of the
amplification primers (e.g. lawn primers), and hence the cognate sequences in
the
adaptors, are generally not material to the invention, as long as the primer-
binding
sequences are able to interact with the amplification primers in order to
direct PCR
amplification. The sequence of the amplification primers may be specific for a
particular
target nucleic acid that it is desired to amplify, but in other embodiments
these sequences
may be "universal" primer sequences which enable amplification of any target
nucleic
acid of known or unknown sequence which has been modified to enable
amplification
with the universal primers. The criteria for design of PCR primers are
generally well
known to those of ordinary skill in the art.
The index sequences (also known as a barcode or tag sequence) are unique short
DNA
(or RNA) sequences that are added to each DNA (or RNA) fragment during library
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preparation. The unique sequences allow many libraries to be pooled together
and
sequenced simultaneously. Sequencing reads from pooled libraries are
identified and
sorted computationally, based on their barcodes, before final data analysis.
Library
multiplexing is also a useful technique when working with small genomes or
targeting
genomic regions of interest. Multiplexing with barcodes can exponentially
increase the
number of samples analysed in a single run, without drastically increasing run
cost or
run time. Examples of tag sequences are found in W005/068656, whose contents
are
incorporated herein by reference in their entirety. The tag can be read at the
end of the
first read, or equally at the end of the second read, for example using a
sequencing
primer complementary to the strand marked P7. The invention is not limited by
the
number of reads per cluster, for example two reads per cluster: three or more
reads per
cluster are obtainable simply by dehybridising a first extended sequencing
primer, and
rehybridising a second primer before or after a cluster repopulation/strand
resynthesis
step. Methods of preparing suitable samples for indexing are described in, for
example
WO 2008/093098, which is incorporated herein by reference. Single or dual
indexing
may also be used. With single indexing, up to 48 unique 6-base indexes can be
used to
generate up to 48 uniquely tagged libraries. With dual indexing, up to 24
unique 8-base
Index 1 sequences and up to 16 unique 8-base Index 2 sequences can be used in
combination to generate up to 384 uniquely tagged libraries. Pairs of indexes
can also
be used such that every i5 index and every i7 index are used only one time.
With these
unique dual indexes, it is possible to identify and filter indexed hopped
reads, providing
even higher confidence in multiplexed samples.
The sequencing primer binding sites are sequencing and/or index primer binding
sites
and indicate the starting point of the sequencing read. During the sequencing
process,
a sequencing primer anneals (i.e. hybridises) to at least a portion of the
sequencing
primer binding site on the template strand. The polymerase enzyme binds to
this site and
incorporates complementary nucleotides base by base into the growing opposite
strand.
In concatenated strands, the hybridisation sequence (or the hybridisation
sequence
complement) may comprise an internal sequencing primer binding site. In other
words,
an internal sequencing primer binding site may form part of the hybridisation
sequence.
For example, ME'-HYB2 (or ME'-HYB2') may act as an internal sequencing primer
binding site to which a sequencing primer can bind. Alternatively, the
hybridisation
sequence may be an internal sequencing primer binding site. For example, HYB2
(or
HYB2') may act as an internal sequencing primer binding site to which a
sequencing
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primer can bind. Accordingly, we may refer to the hybridisation site herein as
comprising
a second sequencing primer binding site, or as a second sequencing primer
binding site.
The target polynucleotide (or in some embodiments, the polynucleotide library)
is pre-
treated to allow sequencing of modified cytosines. Such methods are described
in further
detail herein.
Cluster generation and amplification
Once a double stranded nucleic acid library is formed, typically, the library
has previously
been subjected to denaturing conditions to provide single stranded nucleic
acids.
Suitable denaturing conditions will be apparent to the skilled reader with
reference to
standard molecular biology protocols (Sambrook et al., 2001, Molecular
Cloning, A
Laboratory Manual, 4th Ed, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor
Laboratory Press, NY; Current Protocols, eds Ausubel et al). In one
embodiment,
chemical denaturation may be used.
Following denaturation, a single-stranded library may be contacted in free
solution onto
a solid support comprising surface capture moieties (for example P5 and P7
lawn
primers).
Thus, embodiments of the present invention may be performed on a solid support
200,
such as a flowcell. However, in alternative embodiments, seeding and
clustering can be
conducted off-flowcell using other types of solid support.
The solid support 200 may comprise a substrate 204. See Figure 5. The
substrate 204
comprises at least one well 203 (e.g. a nanowell), and typically comprises a
plurality of
wells 203 (e.g. a plurality of nanowells).
In one embodiment, the solid support comprises at least one first immobilised
primer and
at least one second immobilised primer.
Thus, each well 203 may comprise at least one first immobilised primer 201,
and typically
may comprise a plurality of first immobilised primers 201. In addition, each
well 203 may
comprise at least one second immobilised primer 202, and typically may
comprise a
plurality of second immobilised primers 202. Thus, each well 203 may comprise
at least
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one first immobilised primer 201 and at least one second immobilised primer
202, and
typically may comprise a plurality of first immobilised primers 201 and a
plurality of
second immobilised primers 202.
5 The first immobilised primer 201 may be attached via a 5'-end of its
polynucleotide chain
to the solid support 200. When extension occurs from first immobilised primer
201, the
extension may be in a direction away from the solid support 200.
The second immobilised primer 202 may be attached via a 5'-end of its
polynucleotide
10 chain to the solid support 200. When extension occurs from second
immobilised primer
202, the extension may be in a direction away from the solid support 200.
The first immobilised primer 201 may be different to the second immobilised
primer 202
and/or a complement of the second immobilised primer 202. The second
immobilised
15 primer 202 may be different to the first immobilised primer 201 and/or a
complement of
the first immobilised primer 201.
The (or each of the) first immobilised primer(s) 201 may comprise a sequence
as defined
in SEQ ID NO. 1 0r5, or a variant or fragment thereof. The second immobilised
primer(s)
20 202 may comprise a sequence as defined in SEQ ID NO. 2, or a variant or
fragment
thereof.
By way of brief example, following attachment of the P5 and P7 primers to the
solid
support, the solid support may be contacted with the template to be amplified
under
25 conditions which permit hybridisation (or annealing ¨ such terms may be
used
interchangeably) between the template and the immobilised primers. The
template is
usually added in free solution under suitable hybridisation conditions, which
will be
apparent to the skilled reader. Typically, hybridisation conditions are, for
example,
5xSSC at 40 C. However, other temperatures may be used during hybridisation,
for
example about 50 C to about 75 C, about 55 C to about 70 C, or about 60 C to
about
65 C. Solid-phase amplification can then proceed. The first step of the
amplification is a
primer extension step in which nucleotides are added to the 3 end of the
immobilised
primer using the template to produce a fully extended complementary strand.
The
template is then typically washed off the solid support. The complementary
strand will
include at its 3' end a primer-binding sequence (i.e. either P5' or P7') which
is capable
of bridging to the second primer molecule immobilised on the solid support and
binding.
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Further rounds of amplification (analogous to a standard PCR reaction) leads
to the
formation of clusters or colonies of template molecules bound to the solid
support. This
is called clustering.
Thus, solid-phase amplification by either a method analogous to that of WO
98/44151 or
that of WO 00/18957 (the contents of which are incorporated herein in their
entirety by
reference) will result in production of a clustered array comprised of
colonies of "bridged"
amplification products. This process is known as bridge amplification. Both
strands of
the amplification products will be immobilised on the solid support at or near
the 5 end,
this attachment being derived from the original attachment of the
amplification primers.
Typically, the amplification products within each colony will be derived from
amplification
of a single template molecule. Other amplification procedures may be used, and
will be
known to the skilled person. For example, amplification may be isothermal
amplification
using a strand displacement polymerase; or may be exclusion amplification as
described
in WO 2013/188582. Further information on amplification can be found in WO
02/06456
and WO 07/107710, the contents of which are incorporated herein in their
entirety by
reference.
Through such approaches, a cluster of template molecules is formed, comprising
copies
of a template strand and copies of the complement of the template strand.
In some cases, to facilitate sequencing, one set of strands (either the
original template
strands or the complement strands thereof) may be removed from the solid
support
leaving either the original template strands or the complement strands.
Suitable methods
for removing such strands are described in more detail in application number
WO
07/010251, the contents of which are incorporated herein by reference in their
entirety.
The steps of cluster generation and amplification for templates including a
concatenated
polynucleotide sequence comprising a first portion and a second portion are
illustrated
below and in Figure 6.
In cases where single (concatenated) polynucleotide strands are used, each
polynucleotide sequence may be attached (via the 5'-end of the (concatenated)
polynucleotide sequence) to a first immobilised primer. Each polynucleotide
sequence
may comprise a second adaptor sequence, wherein the second adaptor comprises a
portion which is substantially complementary to the second immobilised primer
(or is
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substantially complementary to the second immobilised primer). The second
adaptor
sequence may be at a 3'-end of the (concatenated) polynucleotide sequence.
In an embodiment, a solution comprising a polynucleotide library prepared by a
tandem
insert method as described above may be flowed across a flowcell.
A particular concatenated polynucleotide strand from the polynucleotide
library to be
sequenced comprising, in a 5' to 3' direction, a second primer-binding
complement
sequence 302 (e.g. P7), a first terminal sequencing primer binding site
complement 303'
(e.g. B15-ME), a first insert sequence 401, a hybridisation complement
sequence 403
(e.g. ME'-HYB2-ME), a second insert sequence 402, a second terminal sequencing
primer binding site 304 (e.g. ME'-A14'), and a first primer-binding sequence
301' (e.g.
P5'), may anneal (via the first primer-binding sequence 301') to the first
immobilised
primer 201 (e.g. P5 lawn primer) located within a particular well 203 (Figure
6A).
The polynucleotide library may comprise other concatenated polynucleotide
strands with
different first insert sequences 401 and second insert sequences 402. Such
other
polynucleotide strands may anneal to corresponding first immobilised primers
201 (e.g.
P5 lawn primers) in different wells 203, thus enabling parallel processing of
the various
different concatenated strands within the polynucleotide library.
A new polynucleotide strand may then be synthesised, extending from the first
immobilised primer 201 (e.g. P5 lawn primer) in a direction away from the
substrate 204.
By using complementary base-pairing, this generates a template strand
comprising, in a
5' to 3' direction, the first immobilised primer 201 (e.g. P5 lawn primer)
which is attached
to the solid support 200, a second terminal sequencing primer binding site
complement
304' (e.g. A14-ME; or if ME is not present, then A14), a second insert
complement
sequence 402' (which represents a type of "second portion"), a hybridisation
sequence
403' (which comprises a type of "second sequencing primer binding site") (e.g.
ME'-
HYB2'-ME; or if ME' and ME are not present, then HYB2'), a first insert
complement
sequence 401' (which represents a type of "first portion"), a first terminal
sequencing
primer binding site 303 (which represents a type of "first sequencing primer
binding site")
(e.g. ME'-B15'; or if ME' is not present, then B15'), and a second primer-
binding
sequence 302' (e.g. P7') (Figure 6B). Such a process may utilise a polymerase,
such as
a DNA or RNA polymerase.
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If the polynucleotides in the library comprise index sequences, then
corresponding index
sequences are also produced in the template.
The concatenated polynucleotide strand from the polynucleotide library may
then be
dehybridised and washed away, leaving a template strand attached to the first
immobilised primer 201 (e.g. P5 lawn primer) (Figure 6C).
The second primer-binding sequence 302' (e.g. P7') on the template strand may
then
anneal to a second immobilised primer 202 (e.g. P7 lawn primer) located within
the well
203. This forms a "bridge".
A new polynucleotide strand may then be synthesised by bridge amplification,
extending
from the second immobilised primer 202 (e.g. P7 lawn primer) (initially) in a
direction
away from the substrate 204. By using complementary base-pairing, this
generates a
template strand comprising, in a 5' to 3' direction, the second immobilised
primer 202
(e.g. P7 lawn primer) which is attached to the solid support 200, a first
terminal
sequencing primer binding site complement 303' (e.g. B15-ME; or if ME is not
present,
then B15), a first insert sequence 401, a hybridisation complement sequence
403 (e.g.
ME'-HYB2-ME; or if ME' and ME are not present, then HYB2), a second insert
sequence
402, a second terminal sequencing primer binding site 304 (e.g. ME'-A14'; or
if ME' is
not present, then A14'), and a first primer-binding sequence 301' (e.g. P5').
Again, such
a process may utilise a polymerase, such as a DNA or RNA polymerase.
The strand attached to the second immobilised primer 202 (e.g. P7 lawn primer)
may
then be dehybridised from the strand attached to the first immobilised primer
201 (e.g.
P5 lawn primer) (Figure 6D).
A subsequent bridge amplification cycle can then lead to amplification of the
strand
attached to the first immobilised primer 201 (e.g. P5 lawn primer) and the
strand attached
to the second immobilised primer 202 (e.g. P7 lawn primer). The second primer-
binding
sequence 302' (e.g. P7') on the template strand attached to the first
immobilised primer
201 (e.g. P5 lawn primer) may then anneal to another second immobilised primer
202
(e.g. P7 lawn primer) located within the well 203. In a similar fashion, the
first primer-
binding sequence 301' (e.g. P5') on the template strand attached to the second
immobilised primer 202 (e.g. P7 lawn primer) may then anneal to another first
immobilised primer 201 (e.g. P5 lawn primer) located within the well 203.
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Completion of bridge amplification and dehybridisation may then provide an
amplified
cluster, thus providing a plurality of concatenated polynucleotide sequences
comprising
a first insert complement sequence 401' (i.e. "first portions") and a second
insert
complement sequence 402' (i.e. second portions"), as well as a plurality of
concatenated
polynucleotide sequences comprising a first insert sequence 401 and a second
insert
sequence 402 (Figure 6E).
If desired, further bridge amplification cycles may be conducted to increase
the number
of polynucleotide sequences within the well 203.
In one example, before sequencing, one group of strands (either the group of
template
polynucleotides, or the group of template complement polynucleotides thereof)
is
removed from the solid support to form a (monoclonal) cluster, leaving either
the
templates or the template complements (Figure 6F).
Sequencing
As described herein, the template provides information (e.g. identification of
the genetic
sequence, identification of epigenetic modifications) on the original target
polynucleotide
sequence. For example, a sequencing process (e.g a sequencing-by-synthesis or
sequencing-by-ligation process) may reproduce information that was present in
the
original target polynucleotide sequence, by using complementary base pairing.
In one embodiment, sequencing may be carried out using any suitable
"sequencing-by-
synthesis" technique, wherein nucleotides are added successively in cycles to
the free
3 hydroxyl group, resulting in synthesis of a polynucleotide chain in the 5'
to 3' direction.
The nature of the nucleotide added may be determined after each addition. One
particular sequencing method relies on the use of modified nucleotides that
can act as
reversible chain terminators. Such reversible chain terminators comprise
removable 3'
blocking groups. Once such a modified nucleotide has been incorporated into
the
growing polynucleotide chain complementary to the region of the template being
sequenced there is no free 3'-OH group available to direct further sequence
extension
and therefore the polymerase cannot add further nucleotides. Once the nature
of the
base incorporated into the growing chain has been determined, the 3' block may
be
removed to allow addition of the next successive nucleotide. By ordering the
products
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derived using these modified nucleotides it is possible to deduce the DNA
sequence of
the DNA template. Such reactions can be done in a single experiment if each of
the
modified nucleotides has attached thereto a different label, known to
correspond to the
particular base, to facilitate discrimination between the bases added at each
5 incorporation step. Suitable labels are described in PCT application
PCT/GB2007/001770, the contents of which are incorporated herein by reference
in their
entirety. Alternatively, a separate reaction may be carried out containing
each of the
modified nucleotides added individually.
10 The modified nucleotides may carry a label to facilitate their
detection. Such a label may
be configured to emit a signal, such as an electromagnetic signal, or a
(visible) light
signal.
In a particular embodiment, the label is a fluorescent label (e.g. a dye).
Thus, such a
15 label may be configured to emit an electromagnetic signal, or a
(visible) light signal. One
method for detecting the fluorescently labelled nucleotides comprises using
laser light of
a wavelength specific for the labelled nucleotides, or the use of other
suitable sources of
illumination. The fluorescence from the label on an incorporated nucleotide
may be
detected by a CCD camera or other suitable detection means. Suitable detection
means
20 are described in PCT/US2007/007991, the contents of which are
incorporated herein by
reference in their entirety_
However, the detectable label need not be a fluorescent label. Any label can
be used
which allows the detection of the incorporation of the nucleotide into the DNA
sequence.
Each cycle may involve simultaneous delivery of four different nucleotide
types to the
array of template molecules. Alternatively, different nucleotide types can be
added
sequentially and an image of the array of template molecules can be obtained
between
each addition step.
In some embodiments, each nucleotide type may have a (spectrally) distinct
label. In
other words, four channels may be used to detect four nucleobases (also known
as 4-
channel chemistry) (Figure 7 ¨ left). For example, a first nucleotide type
(e.g. A) may
include a first label (e.g. configured to emit a first wavelength, such as red
light), a second
nucleotide type (e.g. G) may include a second label (e.g. configured to emit a
second
wavelength, such as blue light), a third nucleotide type (e.g. T) may include
a third label
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(e.g. configured to emit a third wavelength, such as green light), and a
fourth nucleotide
type (e.g. C) may include a fourth label (e.g. configured to emit a fourth
wavelength, such
as yellow light). Four images can then be obtained, each using a detection
channel that
is selective for one of the four different labels. For example, the first
nucleotide type (e.g.
A) may be detected in a first channel (e.g. configured to detect the first
wavelength, such
as red light), the second nucleotide type (e.g. G) may be detected in a second
channel
(e.g. configured to detect the second wavelength, such as blue light), the
third nucleotide
type (e.g. T) may be detected in a third channel (e.g. configured to detect
the third
wavelength, such as green light), and the fourth nucleotide type (e.g. C) may
be detected
in a fourth channel (e.g. configured to detect the fourth wavelength, such as
yellow light).
Although specific pairings of bases to signal types (e.g. wavelengths) are
described
above, different signal types (e.g. wavelengths) and/or permutations may also
be used.
In some embodiments, detection of each nucleotide type may be conducted using
fewer
than four different labels. For example, sequencing-by-synthesis may be
performed
using methods and systems described in US 2013/0079232, which is incorporated
herein
by reference.
Thus, in some embodiments, two channels may be used to detect four nucleobases
(also
known as 2-channel chemistry) (Figure 7 ¨ middle). For example, a first
nucleotide type
(e.g. A) may include a first label (e.g. configured to emit a first
wavelength, such as green
light) and a second label (e.g. configured to emit a second wavelength, such
as red light),
a second nucleotide type (e.g. G) may not include the first label and may not
include the
second label, a third nucleotide type (e.g. T) may include the first label
(e.g. configured
to emit the first wavelength, such as green light) and may not include the
second label,
and a fourth nucleotide type (e.g. C) may not include the first label and may
include the
second label (e.g. configured to emit the second wavelength, such as red
light). Two
images can then be obtained, using detection channels for the first label and
the second
label. For example, the first nucleotide type (e.g. A) may be detected in both
a first
channel (e.g. configured to detect the first wavelength, such as red light)
and a second
channel (e.g. configured to detect the second wavelength, such as green
light), the
second nucleotide type (e.g. G) may not be detected in the first channel and
may not be
detected in the second channel, the third nucleotide type (e.g. T) may be
detected in the
first channel (e.g. configured to detect the first wavelength, such as red
light) and may
not be detected in the second channel, and the fourth nucleotide type (e.g. C)
may not
be detected in the first channel and may be detected in the second channel
(e.g.
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configured to detect the second wavelength, such as green light). Although
specific
pairings of bases to signal types (e.g. wavelengths) and/or combinations of
channels are
described above, different signal types (e.g. wavelengths) and/or permutations
may also
be used.
In some embodiments, one channel may be used to detect four nucleobases (also
known
as 1-channel chemistry) (Figure 7 ¨ right). For example, a first nucleotide
type (e.g. A)
may include a cleavable label (e.g. configured to emit a wavelength, such as
green light),
a second nucleotide type (e.g. G) may not include a label, a third nucleotide
type (e.g.
T) may include a non-cleavable label (e.g. configured to emit the wavelength,
such as
green light), and a fourth nucleotide type (e.g. C) may include a label-
accepting site
which does not include the label. A first image can then be obtained, and a
subsequent
treatment carried out to cleave the label attached to the first nucleotide
type, and to attach
the label to the label-accepting site on the fourth nucleotide type. A second
image may
then be obtained. For example, the first nucleotide type (e.g. A) may be
detected in a
channel (e.g. configured to detect the wavelength, such as green light) in the
first image
and not detected in the channel in the second image, the second nucleotide
type (e.g.
G) may not be detected in the channel in the first image and may not be
detected in the
channel in the second image, the third nucleotide type (e.g. T) may be
detected in the
channel (e.g. configured to detect the wavelength, such as green light) in the
first image
and may be detected in the channel (e.g configured to detect the wavelength,
such as
green light) in the second image, and the fourth nucleotide type (e.g. C) may
not be
detected in the channel in the first image and may be detected in the channel
in the
second image (e.g. configured to detect the wavelength, such as green light).
Although
specific pairings of bases to signal types (e.g. wavelengths) and/or
combinations of
images are described above, different signal types (e.g. wavelengths), images
and/or
permutations may also be used.
In one embodiment, the sequencing process comprises a first sequencing read
and
second sequencing read. The first sequencing read and the second sequencing
read
may be conducted concurrently. In other words, the first sequencing read and
the second
sequencing read may be conducted at the same time.
The first sequencing read may comprise the binding of a first sequencing
primer (also
known as a read 1 sequencing primer) to the first sequencing primer binding
site (e.g.
first terminal sequencing primer binding site 303 in templates including a
concatenated
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polynucleotide sequence comprising a first portion and a second portion). The
second
sequencing read may comprise the binding of a second sequencing primer (also
known
as a read 2 sequencing primer) to the second sequencing primer binding site
(e.g. a
portion of hybridisation sequence 403' in templates including a concatenated
polynucleotide sequence comprising a first portion and a second portion).
This leads to sequencing of the first portion (e.g. first insert complement
sequence 401'
in templates including a concatenated polynucleotide sequence comprising a
first portion
and a second portion) and the second portion (e.g. second insert complement
sequence
402' in templates including a concatenated polynucleotide sequence comprising
a first
portion and a second portion).
Alternative methods of sequencing include sequencing by ligation, for example
as
described in US 6,306,597 or WO 06/084132, the contents of which are
incorporated
herein by reference.
The methods for sequencing described above generally relate to conducting non-
selective sequencing. However, methods of the present invention relating to
selective
processing may comprise conducting selective sequencing, which is described in
further
detail below under selective processing.
Selective processing methods
In some embodiments, selective processing methods may be used to generate
signals
of different intensities. Accordingly, in some embodiments, the method may
comprise
selectively processing at least one polynucleotide sequence comprising a first
portion
and a second portion, such that a proportion of first portions are capable of
generating a
first signal and a proportion of second portions are capable of generating a
second
signal, wherein the selective processing causes an intensity of the first
signal to be
greater than an intensity of the second signal.
The method may comprise selectively processing a plurality of polynucleotide
sequences
each comprising a first portion and a second portion, such that a proportion
of first
portions are capable of generating a first signal and a proportion of second
portions are
capable of generating a second signal, wherein the selective processing causes
an
intensity of the first signal to be greater than an intensity of the second
signal.
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By "selective processing" is meant here performing an action that changes
relative
properties of the first portion and the second portion in the at least one
polynucleotide
sequence comprising a first portion and a second portion (or the plurality of
polynucleotide sequences each comprising a first portion and a second
portion), so that
the intensity of the first signal is greater than the intensity of the second
signal. The
property may be, for example, a concentration of first portions capable of
generating the
first signal relative to a concentration of second portions capable of
generating the
second signal. The action may include, for example, conducting selective
sequencing,
or preparing for selective sequencing.
In one embodiment, the selective processing results in the concentration of
the first
portions capable of generating the first signal being greater than the
concentration of the
second portions capable of generating the second signal. In other words, the
method of
the invention results in an altered ratio of R1: R2 molecules, such as within
a single cluster
or a single well.
In one embodiment, the ratio may be between 1.25:1 to 5:1, between 1.5:1 to
3:1, or
about 2:1.
Selective processing may refer to conducting selective sequencing.
Alternatively,
selective processing may refer to preparing for selective sequencing. As shown
in Figure
8, in one example, selective sequencing may be achieved using a mixture of
unblocked
and blocked sequencing primers.
Where the method of the invention involves a single (concatenated)
polynucleotide
strand with a first and second portion, the single (concatenated)
polynucleotide strand
may comprise a first sequencing primer binding site and a second sequencing
primer
binding site, where the first sequencing primer binding site and second
sequencing
primer binding site are of a different sequence to each other and bind
different
sequencing primers.
In one embodiment, binding of first sequencing primers to the first sequencing
primer
site generates a first signal and binding of second sequencing primers to the
second
sequencing primer site generates a second signal, where the intensity of the
first signal
is greater than the intensity of the second signal. This may be applied to
embodiments
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where the single (concatenated) polynucleotide strand comprises a first
sequencing
primer binding site and a second sequencing primer binding site. This is
achieved using
a mixed population of blocked and unblocked second sequencing primers that
bind the
second sequencing primer site. Any ratio of blocked:unblocked second primers
can be
5 used that generates a second signal that is of a lower intensity than the
first signal, for
example, the ratio of blocked:unblocked primers may be: 20:80 to 80:20, or 1:2
to 2:1.
In one embodiment, a ratio of 50:50 of blocked:unblocked second primers is
used, which
in turn generates a second signal that is around 50% of the intensity of the
first signal.
The first and second sequencing primers may be added to the flow cell at the
same time,
or separately but sequentially.
By "blocked" is meant that the sequencing primer comprises a blocking group at
a 3' end
of the sequencing primer. Suitable blocking groups include a hairpin loop
(e.g. a
polynucleotide attached to the 3'-end, comprising in a 5' to 3' direction, a
cleavable site
such as a nucleotide comprising uracil, a loop portion, and a complement
portion,
wherein the complement portion is substantially complementary to all or a
portion of the
immobilised primer), a deoxynucleotide, a deoxyribonucleotide, a hydrogen atom
instead
of a 3'-OH group, a phosphate group, a phosphorothioate group, a propyl spacer
(e.g. -
0-(CH2)3-0H instead of a 3'-OH group), a modification blocking the 3'-hydroxyl
group
(e.g. hydroxyl protecting groups, such as silyl ether groups (e.g.
trimethylsilyl, triethylsilyl,
triisopropylsilyl, t-butyl(dimethyl)silyl, t-butyl(diphenyl)sily1), ether
groups (e.g. benzyl,
ally!, t-butyl, methoxymethyl (MOM), 2-methoxyethoxymethyl (M EM),
tetrahydropyranyl),
or acyl groups (e.g. acetyl, benzoyl)), or an inverted nucleobase. However,
the blocking
group may be any modification that prevents extension (i.e. elongation) of the
primer by
a polymerase.
The sequence of the sequencing primers and the sequence primer binding sites
are not
material to the methods of the invention, as long as the sequencing primers
are able to
bind to the sequence primer binding site to enable amplification and
sequencing of the
regions to be identified.
In one embodiment, the first sequencing primer binding site may be selected
from ME'-
A14' (as defined in SEQ ID NO. 17 or a variant or fragment thereof), A14' (as
defined in
SEQ ID NO. 18 or a variant or fragment thereof), ME'-B15' (as defined in SEQ
ID NO.
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19 or a variant or fragment thereof) and B15' (as defined in SEQ ID NO. 20 or
a variant
or fragment thereof); and the second sequencing primer binding site may be
selected
from ME'-HYB2 (as defined in SEQ ID NO. 21 or a variant or fragment thereof),
HYB2
(as defined in SEQ ID NO. 11 or a variant or fragment thereof), ME'-HYB2' (as
defined
in SEQ ID NO. 22 or a variant or fragment thereof) and HYB2' (as defined in
SEQ ID NO.
13 or a variant or fragment thereof).
In another embodiment, the first sequencing primer binding site is ME'-615'
(as defined
in SEQ ID NO. 19 or a variant or fragment thereof), and the second sequencing
primer
binding site is ME'-HYB2' (as defined in SEQ ID NO. 22 or a variant or
fragment thereof).
Alternatively, the first sequencing primer binding site is B15' (as defined in
SEQ ID NO.
or a variant or fragment thereof), and the second sequencing primer binding
site is
HYB2' (as defined in SEQ ID NO. 13 or a variant or fragment thereof). The
first and
second sequencing primer sites may be located after (e.g. immediately after) a
3'-end of
15 the first and second portions to be identified.
In another embodiment, the first sequencing primer binding site is ME'-A14'
(as defined
in SEQ ID NO. 17 or a variant or fragment thereof), and the second sequencing
primer
binding site is ME'-HYB2 (as defined in SEQ ID NO. 21 or a variant or fragment
thereof).
20 Alternatively, the first sequencing primer binding site may be A14' (as
defined in SEQ ID
NO. 18 or a variant or fragment thereof) and the second sequencing primer
binding site
may be HYB2 (as defined in SEQ ID NO. 11 or a variant or fragment thereof).
The first
and second sequencing primer sites may be located after (e.g. immediately
after) a 3'-
end of the first and second portions to be identified.
In one example, the sequencing primer (which may be referred to herein as the
second
sequencing primer) comprises or consists of a sequence as defined in SEQ ID
NO. 11
to 16, or a variant or fragment thereof. The sequencing primer may further
comprise a 3'
blocking group as described above to create a blocked sequencing primer.
Alternatively,
the primer comprises a 3'-OH group. Such a primer is unblocked and can be
elongated
with a polymerase.
In one embodiment, the unblocked and blocked second sequencing primers are
present
in the sequencing composition in equal concentrations. That is, the ratio of
blocked:unblocked second sequencing primers is around 50:50. The sequencing
composition may further comprise at least one additional (first) sequencing
primer. This
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additional sequencing primer may be selected from A14-ME (as defined in SEQ ID
NO.
9 or a variant or fragment thereof), A14 (as defined in SEQ ID NO. 7 or a
variant or
fragment thereof), B15-ME (as defined in SEQ ID NO. 10 or a variant or
fragment thereof)
and B15 (as defined in SEQ ID NO. 8 or a variant or fragment thereof). In one
embodiment, the sequencing composition comprises blocked second sequencing
primers, unblocked second sequencing primers and at least one first sequencing
primer,
wherein the first sequencing primer is A14, or B15, or is both A14 and B15.
As shown in Figure 8, selective sequencing may be conducted on the amplified
(monoclonal) cluster shown in Figure 6F. A plurality of first sequencing
primers 501 are
added. These first sequencing primers 501 (e.g. B15-ME; or if ME is not
present, then
B15) anneal to the first terminal sequencing primer binding site 303 (which
represents a
type of "first sequencing primer binding site") (e.g. ME'-B15'; or if ME' is
not present, then
B15'). A plurality of second unblocked sequencing primers 502a and a plurality
of second
blocked sequencing primers 502b are added, either at the same time as the
first
sequencing primers 501, or sequentially (e.g. prior to or after addition of
first sequencing
primers 501). These second unblocked sequencing primers 502a (e.g. HYB2-ME; or
if
ME is not present, then HYB2) and second blocked sequencing primers 502b (e.g.
blocked HYB2-ME; or if ME is not present, then blocked HYB2) anneal to an
internal
sequencing primer binding site in the hybridisation sequence 403' (which
represents a
type of "second sequencing primer binding site") (e.g. ME'-HYB2'; or if ME' is
not present,
then HYB2'). This then allows the first insert complement sequences 401' (i.e.
"first
portions") to be sequenced and the second insert complement sequences 402'
(i.e.
"second portions") to be sequenced, wherein a greater proportion of first
insert
complement sequences 401' are sequenced (grey arrow) compared to a proportion
of
second insert complement sequences 402' (black arrow).
Although Figure 8 shows selective sequencing being conducted on a template
strand
attached to first immobilised primer 201, in some embodiments the (monoclonal)
cluster
may instead have template strands attached to second immobilised primer 202.
In such
a case, the first sequencing primers may instead correspond to A14-ME (or if
ME is not
present, then A14), and the second unblocked sequencing primers may instead
correspond to HYB2'-ME (or if ME is not present, then HYB2') and second
blocked
sequencing primers may instead correspond to blocked HYB2'-ME (or if ME is not
present, then blocked HYB2').
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In yet other embodiments, the positioning of first sequencing primers and
second
sequencing primers may be swapped. In other words, the first sequencing
binding
primers may anneal instead to the internal sequencing primer binding site, and
the
second sequencing binding primers may anneal instead to the terminal
sequencing
primer binding site.
Figure 8 shows concurrent sequencing of a concatenated strand according to the
above
method. As shown in Figure 8, a polynucleotide strand with a first portion
(insert) and
second portion (insert) can be accurately and simultaneously sequenced by a
selective
sequencing method that uses a mixture of unblocked and blocked sequencing
primers
as described above.
Data analysis using 16 QaM
Figure 9 is a scatter plot showing an example of sixteen distributions of
signals generated
by polynucleotide sequences disclosed herein.
The scatter plot of Figure 9 shows sixteen distributions (or bins) of
intensity values from
the combination of a brighter signal (i.e. a first signal as described herein)
and a dimmer
signal (i.e. a second signal as described herein); the two signals may be co-
localized
and may not be optically resolved as described above. The intensity values
shown in
Figure 9 may be up to a scale or normalisation factor; the units of the
intensity values
may be arbitrary or relative (i.e., representing the ratio of the actual
intensity to a
reference intensity). The sum of the brighter signal generated by the first
portions and
the dimmer signal generated by the second portions results in a combined
signal. The
combined signal may be captured by a first optical channel and a second
optical channel.
Since the brighter signal may be A, T, C or G, and the dimmer signal may be A,
T, C or
G, there are sixteen possibilities for the combined signal, corresponding to
sixteen
distinguishable patterns when optically captured. That is, each of the sixteen
possibilities
corresponds to a bin shown in Figure 9. The computer system can map the
combined
signal generated into one of the sixteen bins, and thus determine the added
nucleobase
at the first portion and the added nucleobase at the second portion,
respectively.
For example, when the combined signal is mapped to bin 1612 for a base calling
cycle,
the computer processor base calls both the added nucleobase at the first
portion and the
added nucleobase at the second portion as C. Mien the combined signal is
mapped to
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bin 1614 for the base calling cycle, the processor base calls the added
nucleobase at
the first portion as C and the added nucleobase at the second portion as T.
When the
combined signal is mapped to bin 1616 for the base calling cycle, the
processor base
calls the added nucleobase at the first portion as C and the added nucleobase
at the
second portion as G. When the combined signal is mapped to bin 1618 for the
base
calling cycle, the processor base calls the added nucleobase at the first
portion as C and
the added nucleobase at the second portion as A.
When the combined signal is mapped to bin 1622 for the base calling cycle, the
processor base calls the added nucleobase at the first portion as T and the
added
nucleobase at the second portion as C. When the combined signal is mapped to
bin
1624 for the base calling cycle, the processor base calls both the added
nucleobase at
the first portion and the added nucleobase at the second portion as T. When
the
combined signal is mapped to bin 1626 for the base calling cycle, the
processor base
calls the added nucleobase at the first portion as T and the added nucleobase
at the
second portion as G. When the combined signal is mapped to bin 1628 for the
base
calling cycle, the processor base calls the added nucleobase at the first
portion as T and
the added nucleobase at the second portion as A.
When the combined signal is mapped to bin 1632 for the base calling cycle, the
processor base calls the added nucleobase at the first portion as G and the
added
nucleobase at the second portion as C. When the combined signal is mapped to
bin
1634 for the base calling cycle, the processor base calls the added nucleobase
at the
first portion as G and the added nucleobase at the second portion as T. When
the
combined signal is mapped to bin 1636 for the base calling cycle, the
processor base
calls both the added nucleobase at the first portion and the added nucleobase
at the
second portion as G. When the combined signal is mapped to bin 1638 for the
base
calling cycle, the processor base calls the added nucleobase at the first
portion as G and
the added nucleobase at the second portion as A.
When the combined signal is mapped to bin 1642 for the base calling cycle, the
processor base calls the added nucleobase at the first portion as A and the
added
nucleobase at the second portion as C. When the combined signal is mapped to
bin
1644 for the base calling cycle, the processor base calls the added nucleobase
at the
first portion as A and the added nucleobase at the second portion as T. When
the
combined signal is mapped to bin 1646 for the base calling cycle, the
processor base
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calls the added nucleobase at the first portion as A and the added nucleobase
at the
second portion as G. When the combined signal is mapped to bin 1648 for the
base
calling cycle, the processor base calls both the added nucleobase at the first
portion and
the added nucleobase at the second portion as A.
5
In this particular example, T is configured to emit a signal in both the IMAGE
1 channel
and the IMAGE 2 channel, A is configured to emit a signal in the IMAGE 1
channel only,
C is configured to emit a signal in the IMAGE 2 channel only, and G does not
emit a
signal in either channel. However, different permutations of nucleobases can
be used to
10 achieve the same effect by performing dye swaps. For example, A
may be configured to
emit a signal in both the IMAGE 1 channel and the IMAGE 2 channel, T may be
configured to emit a signal in the IMAGE 1 channel only, C may be configured
to emit a
signal in the IMAGE 2 channel only, and G may be configured to not emit a
signal in
either channel.
Further details regarding performing base-calling based on a scatter plot
having sixteen
bins may be found in U.S. Patent Application Publication No. 2019/0212294, the
disclosure of which is incorporated herein by reference.
Figure 10 is a flow diagram showing a method 1700 of base calling according to
the
present disclosure. The described method allows for simultaneous sequencing of
two (or
more) portions (e.g. the first portion and the second portion) in a single
sequencing run
from a single combined signal obtained from the first portion and the second
portion,
thus requiring less sequencing reagent consumption and faster generation of
data from
both the first portion and the second portion. Further, the simplified method
may reduce
the number of workflow steps while producing the same yield as compared to
existing
next-generation sequencing methods. Thus, the simplified method may result in
reduced
sequencing runtime.
As shown in Figure 10, the disclosed method 1700 may start from block 1701.
The
method may then move to block 1710.
At block 1710, intensity data is obtained. The intensity data includes first
intensity data
and second intensity data. The first intensity data comprises a combined
intensity of a
first signal component obtained based upon a respective first nucleobase of
the first
portion and a second signal component obtained based upon a respective second
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nucleobase of the second portion. Similarly, the second intensity data
comprises a
combined intensity of a third signal component obtained based upon the
respective first
nucleobase of the first portion and a fourth signal component obtained based
upon the
respective second nucleobase of the second portion.
As such, the first portion is capable of generating a first signal comprising
a first signal
component and a third signal component. The second portion is capable of
generating a
second signal comprising a second signal component and a fourth signal
component.
As described above, the first portion and the second portion may be arranged
on the
solid support such that signals from the first portion and the second portion
are detected
by a single sensing portion and/or may comprise a single cluster such that
first signals
and second signals from each of the respective first portions and second
portions cannot
be spatially resolved.
In one example, obtaining the intensity data comprises selecting intensity
data that
corresponds to two (or more) different portions (e.g. the first portion and
the second
portion). In one example, intensity data is selected based upon a chastity
score. A
chastity score may be calculated as the ratio of the brightest base intensity
divided by
the sum of the brightest and second brightest base intensities. The desired
chastity score
may be different depending upon the expected intensity ratio of the light
emissions
associated with the different portions. As described above, it may be desired
to produce
clusters comprising the first portion and the second portion, which give rise
to signals in
a ratio of 2:1. In one example, high-quality data corresponding to two
portions with an
intensity ratio of 2:1 may have a chastity score of around 0.8 to 0.9.
After the intensity data has been obtained, the method may proceed to block
1720. In
this step, one of a plurality of classifications is selected based on the
intensity data. Each
classification represents a possible combination of respective first and
second
nucleobases. In one example, the plurality of classifications comprises
sixteen
classifications as shown in Figure 9, each representing a unique combination
of first and
second nucleobases. Where there are two portions, there are sixteen possible
combinations of first and second nucleobases. Selecting the classification
based on the
first and second intensity data comprises selecting the classification based
on the
combined intensity of the first and second signal components and the combined
intensity
of the third and fourth signal components.
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The method may then proceed to block 1730, where the respective first and
second
nucleobases are base called based on the classification selected in block
1720. The
signals generated during a cycle of a sequencing are indicative of the
identity of the
nucleobase(s) added during sequencing (e.g. using sequencing-by-synthesis). It
will be
appreciated that there is a direct correspondence between the identity of the
nucleobases that are incorporated and the identity of the complementary base
at the
corresponding position of the template sequence bound to the solid support.
Therefore,
any references herein to the base calling of respective nucleobases at the two
portions
encompasses the base calling of nucleobases hybridised to the template
sequences
and, alternatively or additionally, the identification of the corresponding
nucleobases of
the template sequences. The method may then end at block 1740.
Data analysis using 9 QaM
For two portions of polynucleotide sequences (e.g. a first portion and a
second portion
as described herein), there are sixteen possible combinations of nucleobases
at any
given position (i.e., an A in the first portion and an A in the second
portion, an A in the
first portion and a T in the second portion, and so on). When the same
nucleobase is
present at a given position in both portions, the light emissions associated
with each
target sequence during the relevant base calling cycle will be characteristic
of the same
nucleobase. In effect, the two portions behave as a single portion, and the
identity of the
bases at that position are uniquely callable.
However, when a nucleobase of the first portion is different from a nucleobase
at a
corresponding position of the second portion, the signals associated with each
portion in
the relevant base calling cycle will be characteristic of different
nucleobases. In one
embodiment, the first signal coming from the first portion have substantially
the same
intensity as the second signal coming from the second portion. The two signals
may also
be co-localised, and may not be spatially and/or optically resolved.
Therefore, when
different nucleobases are present at corresponding positions of the two
portions, the
identity of the nucleobases cannot be uniquely called from the combined signal
alone.
However, useful sequencing information can still be determined from these
signals.
The scatter plot of Figure 11 shows nine distributions (or bins) of intensity
values from
the combination of two co-localised signals of substantially equal intensity.
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The intensity values shown in Figure 11 may be up to a scale or normalisation
factor; the
units of the intensity values may be arbitrary or relative (i.e., representing
the ratio of the
actual intensity to a reference intensity). The sum of the first signal
generated from the
first portion and the second signal generated from the second portion results
in a
combined signal. The combined signal may be captured by a first optical
channel and a
second optical channel. The computer system can map the combined signal
generated
into one of the nine bins, and thus determine sequence information relating to
the added
nucleobase at the first portion and the added nucleobase at the second
portion.
Bins are selected based upon the combined intensity of the signals originating
from each
target sequence during the base calling cycle. For example, bin 1803 may be
selected
following the detection of a high-intensity (or "on/on") signal in the first
channel and a
high-intensity signal in the second channel. Bin 1806 may be selected
following the
detection of a high-intensity signal in the first channel and an intermediate-
intensity
("on/off" or "off/on") signal in the second channel. Bin 1809 may be selected
following
the detection of a high-intensity signal in the first channel and a low-
intensity or zero-
intensity ("off/off") signal in the second channel. Bin 1802 may be selected
following the
detection of an intermediate-intensity signal in the first channel and a high-
intensity signal
in the second channel. Bin 1805 may be selected following the detection of an
intermediate-intensity signal in the first channel and an intermediate-
intensity signal in
the second channel. Bin 1808 may be selected following the detection of an
intermediate-
intensity signal in the first channel and a low-intensity or zero-intensity
signal in the
second channel. Bin 1801 may be selected following the detection of a low-
intensity
signal in the first channel and a high-intensity signal in the second channel.
Bin 1804
may be selected following the detection of a low-intensity or zero-intensity
signal in the
first channel and an intermediate-intensity signal in the second channel. Bin
1807 may
be selected following the detection of a low-intensity or zero-intensity
signal in the first
channel and a low-intensity signal in the second channel.
Four of the nine bins represent matches between respective nucleobases of the
two
portions sensed during the cycle (bins 1801, 1803, 1807, and 1809). In
response to
mapping the combined signal to a bin representing a match, the computer
processor
may detect a match between the first portion and the second portion at the
sensed
position. In response to mapping the combined signal to a bin representing a
match, the
computer processor may base call the respective nucleobases. For example, when
the
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combined signal is mapped to bin 1801 for a base calling cycle, the computer
processor
base calls both the added nucleobase at the first portion and the added
nucleobase at
the second portion as T. When the combined signal is mapped to bin 1803 for
the base
calling cycle, the processor base calls both the added nucleobase at the first
portion and
the added nucleobase at the second portion as A. When the combined signal is
mapped
to bin 1807 for the base calling cycle, the processor base calls both the
added
nucleobase at the first portion and the added nucleobase at the second portion
as G.
When the combined signal is mapped to bin 1809 for the base calling cycle, the
processor base calls both the added nucleobase at the first portion and the
added
nucleobase at the second portion as C.
The remaining five bins are "ambiguous". That is to say that these bins each
represent
more than one possible combination of first and second nucleobases. Bins 1802,
1804,
1806, and 1808 each represent two possible combinations of first and second
nucleobases. Bin 1805, meanwhile, represents four possible combinations.
Nevertheless, mapping the combined signal to an ambiguous bin may still allow
for
sequencing information to be determined. For example, bins 1802, 1804, 1805,
1806,
and 1808 represent mismatches between respective nucleobases of the two
portions
sensed during the cycle. Therefore, in response to mapping the combined signal
to a bin
representing a mismatch, the computer processor may detect a mismatch between
the
first portion and the second portion at the sensed position.
The remaining five bins are "ambiguous". That is to say that these bins each
represent
more than one possible combination of first and second nucleobases. Bins 1802,
1804,
1806, and 1808 each represent two possible combinations of first and second
nucleobases. Bin 1805, meanwhile, represents four possible combinations.
Nevertheless, mapping the combined signal to an ambiguous bin may still allow
for
sequencing information to be determined. For example, bins 1802, 1804, 1805,
1806,
and 1808 represent mismatches between respective nucleobases of the two
portions
sensed during the cycle. Therefore, in response to mapping the combined signal
to a bin
representing a mismatch, the computer processor may detect a mismatch between
the
first portion and the second portion at the sensed position.
In this particular example, A is configured to emit a signal in both the first
channel and
the second channel, C is configured to emit a signal in the first channel
only, T is
configured to emit a signal in the second channel only, and G does not emit a
signal in
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either channel. However, different permutations of nucleobases can be used to
achieve
the same effect by performing dye swaps. For example, A may be configured to
emit a
signal in both the first channel and the second channel, T may be configured
to emit a
signal in the first channel only, C may be configured to emit a signal in the
second
5 channel only, and G may be configured to not emit a signal in either
channel.
The number of classifications which may be selected based upon the combined
signal
intensities may be predetermined, for example based on the number of portions
expected to be present in the nucleic acid cluster. Whilst Figure 11 shows a
set of nine
10 possible classifications, the number of classifications may be greater
or smaller.
In addition to identifying matches and mismatches, the mapping of the combined
signal
to each of the different bins (e.g in combination with additional knowledge,
such as the
library preparation methods used) can provide additional information about the
first
15 portion and the second portion, or about sequences from which the first
portion and the
second portion were derived. For example, given the nucleic acid material
input and the
processing methods used to generate the nucleic acid clusters, the first
portion and the
second portion may be expected to be identical at a given position. In this
case, the
mapping of the combined signal to a bin representing a mismatch may be
indicative of
20 an error introduced during library preparation. In addition, the first
portion and the second
portion may be expected to be different, for example due to deliberate
sequence
modifications introduced during library preparation to detect modified
cytosines.
As mentioned herein, the library preparation may involve treatment with a
conversion
25 agent. In cases where the conversion reagent is configured to convert an
unmodified
cytosine to uracil or a nucleobase which is read as thymine/uracil, the
correspondence
between bases in the original polynucleotide and in the converted strands is
shown in
Figure 12, alongside a scatter plot showing potential resulting distributions
for the
combined signal intensities resulting from the simultaneous sequencing of the
target
30 sequences. An A-T or T-A base pair in the original molecule will result
in a match (A/A
or TIT) at the corresponding position of the forward and reverse complement
strands of
the library. An mC-G or G-mC base pair in the library will also result in a
match (GIG or
C/C) at the corresponding position of the forward and reverse complement
strands of the
library. For a C-G base pair, however, the conversion of unmodified cytosine
to uracil (or
35 a nucleobase which is read as thymine/uracil) in the forward strand of
the library ("top"
strand) will result in a T at the corresponding position of the forward strand
of the library.
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Meanwhile, the corresponding position on the reverse complement strand of the
library
("bottom" strand) will be occupied by C. Alternatively, for a G-C base pair,
the conversion
of unmodified cytosine to uracil (or a nucleobase which is read as
thymine/uracil) in the
reverse strand of the library ("bottom" strand) will result in an A at the
corresponding
position of the reverse complement strand of the library. Meanwhile, the
corresponding
position of the forward strand of the library ("top" strand) will be occupied
by G. Therefore,
in response to mapping the combined signal to the distribution representing
GIG or C/C,
the presence of a modified cytosine can be determined at the corresponding
position in
the original polynucleotide.
In other cases where the conversion reagent is configured to convert a
modified cytosine
to thymine or a nucleobase which is read as thymine/uracil, Figure 13 shows
the
correspondence between bases in the original polynucleotide and in the
converted
strands, alongside a scatter plot showing potential resulting distributions
for the
combined signal intensities resulting from the simultaneous sequencing of the
target
sequences. An A-T or T-A base pair in the library will result in a match (A/A
or T/T) at
the corresponding position of the forward and reverse complement strands of
the library.
A C-G or G-C base pair in the library will also result in a match (GIG or C/C)
at the
corresponding position of the forward and reverse complement strands of the
library. For
a mC-G base pair, however, the conversion of 5-methylcytosine to thymine in
the forward
strand of the library ("top" strand) will result in a T at the corresponding
position of the
forward strand of the library. Meanwhile, the corresponding position on the
reverse
complement strand of the library ("bottom" strand) will be occupied by C.
Alternatively,
the conversion of 5-methylcytosine to thymine in the reverse strand of the
library
("bottom" strand) will result in an A at the corresponding position of the
reverse
complement strand of the library. Meanwhile, the corresponding position of the
forward
strand of the library ("top" strand) will be occupied by G. Therefore, in
response to
mapping the combined signal to the distribution representing an A/G, G/A, TIC,
or C/T
mismatch, the presence of a modified cytosine can be determined at the
corresponding
position in the original polynucleotide.
Figure 14 represents the distributions resulting from the use of an
alternative dye-
encoding scheme following use of a conversion reagent configured to convert an
unmodified cytosine to uracil or a nucleobase which is read as thymine/uracil,
and Figure
15 represents the distributions resulting from the use of an alternative dye-
encoding
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scheme following use of a conversion reagent configured to convert a modified
cytosine
to thymine or a nucleobase which is read as thymine/uracil.
Figure 16 represents yet another distribution resulting from the use of an
alternative dye-
encoding scheme following use of a conversion reagent configured to convert a
modified
cytosine to thymine or a nucleobase which is read as thymine/uracil. In this
case,
modified cytosines fall within a central bin.
In the present example, for each base pair in the original double-stranded DNA
molecule,
it may be assumed that there are six possibilities: A-T, T-A, C-G, G-C, mC-G
and G-mC.
As shown in Figures 12 to 15, each of these possibilities is uniquely
represented by one
of the plurality of classifications. According to the present methods, it is
therefore
possible to determine both the sequence and "methylation" status (i.e presence
of
modified cytosines) of a double-stranded polynucleotide in a single sequencing
run.
In addition to determining "methylation" status, it may also be possible to
identify library
preparation/sequencing errors. Using the dye-encoding scheme shown in Figures
12
and 13, the central column of distributions is indicative of such errors.
Using the dye
encoding scheme shown in Figures 14 and 15, the central row of distributions
is
indicative of such errors.
The dye-encoding scheme may be optimised to allow for different combinations
of first
and second nucleobases to be resolved. This may be particularly useful where
sequence
modifications of a known type have been introduced into the first portions and
the second
portions. For example, where sequence modifications have been introduced that
result
in the conversion of unmodified cytosines to uracil or nucleobases which is
read as
thymine/uracil, or the conversion of modified cytosines to thymine or
nucleobases which
are read as thymine/uracil, the dye-encoding scheme may be selected such that
the
resulting combination of first and second nucleobases do not fall within the
central bin
(which represents four different nucleobase combinations).
In the case of conversion of modified cytosines to thymine (or nucleobases
which are
read as thymine/uracil), a TIC or G/A mismatch between the forward and reverse
complement strands is indicative of the presence of a mC-G or G-mC base pair
at the
corresponding position of the library. The dye-encoding scheme may therefore
be
designed such that these mismatches may be resolved from other possible
combinations
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of nucleobases. This may be achieved by detecting light emissions from A and T
bases
in a first illumination cycle, and from C and T bases in a second illumination
cycle. In
another example, light emissions may be detected from C and G bases in a first
illumination cycle, and from C and T bases in a second illumination cycle. In
another
example, light emissions may be detected from C and A bases in a first
illumination cycle,
and from C and G bases in a second illumination cycle.
In the case of unmodified cytosines to uracil (or nucleobases which is read as
thymine/uracil), a C/C or GIG match between the forward and reverse complement
strands is indicative of the presence of a mC-G or G-mC base pair at the
corresponding
position of the library. In this case, a mC-G or G-mC base pair will always be
resolvable.
However, the dye-encoding scheme can still be designed to optimise the
resolution
between unmodified bases
Figure 17 is a flow diagram showing a method 1900 of determining sequence
information
according to the present disclosure. The described method allows for the
determination
of sequence information from two (or more) portions (e.g. the first portion
and the second
portion) in a single sequencing run from a single combined signal obtained
from the first
portion and the second portion.
As shown in Figure 17, the disclosed method 1900 may start from block 1901.
The
method may then move to block 1910.
At block 1910, intensity data is obtained. The intensity data includes first
intensity data
and second intensity data. The first intensity data comprises a combined
intensity of a
first signal component obtained based upon a respective first nucleobase of
the first
portion and a second signal component obtained based upon a respective second
nucleobase of the second portion. Similarly, the second intensity data
comprises a
combined intensity of a third signal component obtained based upon the
respective first
nucleobase of the first portion and a fourth signal component obtained based
upon the
respective second nucleobase of the second portion.
As such, the first portion is capable of generating a first signal comprising
a first signal
component and a third signal component. The second portion is capable of
generating a
second signal comprising a second signal component and a fourth signal
component.
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As described above, the first portion and the second portion may be arranged
on the
solid support such that signals from the first portion and the second portion
are detected
by a single sensing portion and/or may comprise a single cluster such that
first signals
and second signals from each of the respective first portions and second
portions cannot
be spatially resolved.
In one example, obtaining the intensity data comprises selecting intensity
data, for
example based upon a chastity score. A chastity score may be calculated as the
ratio of
the brightest base intensity divided by the sum of the brightest and second
brightest base
intensities. In one example, high-quality data corresponding to two portions
with a
substantially equal intensity ratio may have a chastity score of around 0.8 to
0.9, for
example 0.89-0.9.
After the intensity data has been obtained, the method may proceed to block
1920. In
this step, one of a plurality of classifications is selected based on the
intensity data. Each
classification represents one or more possible combinations of respective
first and
second nucleobases, and at least one classification of the plurality of
classifications
represents more than one possible combination of respective first and second
nucleobases. In one example, the plurality of classifications comprises nine
classifications as shown in Figure 11. Selecting the classification based on
the first and
second intensity data comprises selecting the classification based on the
combined
intensity of the first and second signal components and the combined intensity
of the
third and fourth signal components.
The method may then proceed to block 1930, where sequence information of the
respective first and second nucleobases is determined based on the
classification
selected in block 1920. The signals generated during a cycle of a sequencing
are
indicative of the identity of the nucleobase(s) added during sequencing (e.g.
using
sequencing-by-synthesis). For example, it may be determined that there is a
match or a
mismatch between the respective first and second nucleobases. Where it is
determined
that there is a match between the first and second respective nucleobases, the
nucleobases may be base called. Whether there is a match or a mismatch,
additional or
alternative information may be obtained, as described above. It will be
appreciated that
there is a direct correspondence between the identity of the nucleobases that
are
incorporated and the identity of the complementary base at the corresponding
position
of the template sequence bound to the solid support. Therefore, any references
herein
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to the base calling of respective nucleobases at the two portions encompasses
the base
calling of nucleobases hybridised to the template sequences and, alternatively
or
additionally, the identification of the corresponding nucleobases of the
template
sequences. The method may then end at block 1940.
5
Sequencing of modified cytosines
The present invention is directed to methods of preparing a polynucleotide
strand for
detection of modified cytosines, such that where the strand comprises two
portions (in
10 other words, a concatenated polynucleotide sequence comprising a
first portion and a
second portion) to be identified, the first portion comprising a forward
strand and the
second portion comprising a reverse complement strand (or the first portion
comprising
a reverse strand and the second portion comprising a forward complement
strand), such
portions can be identified concurrently, thus facilitating the detection of
modified
15 cytosines. Advantageously, the methods of the present invention
allow a decrease in the
amount of time taken to detect modified cytosines.
In some embodiments, selective processing methods may be used when preparing
the
templates. This leads to further advantages, as it also becomes possible to
identify which
20 strand of the original library that the modified cytosine was on,
whilst maintaining
reductions in time taken to detect modified cytosines.
Accordingly, we describe a method of preparing at least one polynucleotide
sequence
for detection of modified cytosines, comprising:
25 synthesising at least one polynucleotide sequence comprising a first
portion and a second portion,
wherein the at least one polynucleotide sequence comprises portions of
a double-stranded nucleic acid template, and the first portion comprises a
forward
strand of the template, and the second portion comprises a reverse complement
30 strand of the template; or wherein the first portion comprises a
reverse strand of
the template, and the second portion comprises a forward complement strand of
the template,
wherein the template is generated from a (double-stranded) target
polynucleotide to be sequenced via complementary base pairing, and wherein
35 the target polynucleotide has been pre-treated using a
conversion reagent,
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wherein the conversion reagent is configured to convert a modified
cytosine to thymine or a nucleobase which is read as thymine/uracil, and/or
wherein the conversion reagent is configured to convert an unmodified cytosine
to uracil or a nucleobase which is read as thymine/uracil.
As described herein, the at least one polynucleotide sequence comprises
portions of a
double-stranded nucleic acid template, and the first portion may comprise (or
be) the
forward strand of a polynucleotide sequence (e.g. forward strand of a
template), and the
second portion may comprise (or be) the reverse complement strand of the
polynucleotide sequence (e.g. reverse complement strand of the template) (in
effect, a
reverse complement strand may be considered a "copy" of the forward strand).
Alternatively, the first portion may comprise (or be) the reverse strand of a
polynucleotide
sequence (e.g reverse strand of a template), and the second portion may
comprise (or
be) the forward complement strand of the polynucleotide sequence (e.g. forward
complement strand of the template) (in effect, a forward complement may be
considered
a "copy" of the reverse strand).
The first portion may be derived from a forward strand of a target
polynucleotide to be
sequenced, and the second portion may be derived from a reverse complement
strand
of the target polynucleotide to be sequenced; or the first portion may be
derived from a
reverse strand of a target polynucleotide to be sequenced, and the second
portion may
be derived from a forward complement strand of the target polynucleotide to be
sequenced.
The template is generated from a (double-stranded) target polynucleotide to be
sequenced via complementary base pairing. The (double-stranded) target
polynucleotide may be one (double-stranded) polynucleotide present in a
polynucleotide
library to be sequenced. As such, the template allows sequence information to
be
obtained for that particular polynucleotide.
The method may further comprise a step of preparing the first portion and the
second
portion for concurrent sequencing.
For example, the method may comprise simultaneously contacting first
sequencing
primer binding sites located after a 3'-end of the first portions with first
primers and
second sequencing primer binding sites located after a 3'-end of the second
portions
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with second primers. Thus, the first portions and second portions are primed
for
concurrent sequencing.
In some embodiments, a proportion of first portions may be capable of
generating a first
signal and a proportion of second portions may be capable of generating a
second signal,
wherein an intensity of the first signal is substantially the same as an
intensity of the
second signal.
In other embodiments (e.g. where selective processing methods are used as
described
herein), a proportion of first portions may be capable of generating a first
signal and a
proportion of second portions may be capable of generating a second signal,
wherein an
intensity of the first signal is substantially the same as an intensity of the
second signal.
The first signal and the second signal may be spatially unresolved (e.g.
generated from
the same region or substantially overlapping regions).
Further aspects relating to selective processing methods (e.g. conducting
selective
sequencing or preparing for selective sequencing) have already been described
herein
and apply to the methods of preparing at least one polynucleotide sequence for
detection
of modified cytosines as described herein.
The first portion may be referred to herein as read 1 (R1). The second portion
may be
referred to herein as read 2 (R2).
In one embodiment, the first portion is at least 25 or at least 50 base pairs
and the second
portion is at least 25 base pairs or at least 50 base pairs.
The single (concatenated) polynucleotide strand may be attached to a solid
support. In
one embodiment, this solid support is a flow cell. In one example, the
polynucleotide
strand is attached to the solid support in a single well of the solid support.
The polynucleotide strand or strands may form or be part of a cluster on the
solid support.
As used herein, the term "cluster" may refer to a clonal group of template
polynucleotides
(e.g. DNA or RNA) bound within a single well of a solid support (e.g. flow
cell). As such,
a cluster may refer to the population of polynucleotide molecules within a
well that are
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then sequenced. A "cluster" may contain a sufficient number of copies of
template
polynucleotides such that the cluster is able to output a signal (e.g. a light
signal) that
allows sequencing reads to be performed on the cluster. A "cluster" may
comprise, for
example, about 500 to about 2000 copies, about 600 to about 1800 copies, about
700 to
about 1600 copies, about 800 to 1400 copies, about 900 to 1200 copies, or
about 1000
copies of template polynucleotides.
A cluster may be formed by bridge amplification, as described above.
Where the method of the invention involves a single polynucleotide strand with
a first
and second portion, before sequencing one group of strands (either the group
of
template polynucleotides, or the group of template complement polynucleotides
thereof)
may be removed from the solid support, leaving either the templates or the
template
complements, as explained above. Such a cluster may be considered to be a
"monoclonal" cluster.
By "monoclonal" cluster is meant that the population of polynucleotide
sequences that
are then sequenced (as the next step) are substantially the same ¨ i.e. copies
of the
same sequence. As such, a "monoclonal" cluster may refer to the population of
single
polynucleotide molecules within a well that are then sequenced. A "monoclonal"
cluster
may contain a sufficient number of copies of a single template polynucleotide
(or copies
of a single template complement polynucleotide) such that the cluster is able
to output a
signal (e.g. a light signal) that allows sequencing reads to be performed on
the
"monoclonal" cluster. A "monoclonal" cluster may comprise, for example, about
500 to
about 2000 copies, about 600 to about 1800 copies, about 700 to about 1600
copies,
about 800 to 1400 copies, about 900 to 1200 copies, or about 1000 copies of a
single
template polynucleotide (or copies of a single template complement
polynucleotide). The
copies of the single template polynucleotide (and/or single template
complement
polynucleotides) may comprise at least about 50%, at least about 60%, at least
about
70%, at least about 80%, at least about 90%, or about 95%, 98%, 99% or 100% of
all
polynucleotides within a single well of the flow cell, and thus providing a
substantially
monoclonal "cluster".
The at least one polynucleotide sequence comprising a first portion and a
second portion
may be prepared using a tandem insert method as described herein. Accordingly,
in one
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embodiment, the step of synthesising the at least one polynucleotide sequence
comprising a first portion and a second portion may comprise:
synthesising a first precursor polynucleotide fragment comprising a
complement of the first portion and a hybridisation complement sequence,
synthesising a second precursor polynucleotide fragment comprising a
second portion and a hybridisation sequence,
annealing the hybridisation complement sequence of the first precursor
polynucleotide fragment with the hybridisation sequence on the second
precursor
polynucleotide fragment to form a hybridised adduct,
synthesising a first precursor polynucleotide sequence by extending the
first precursor polynucleotide fragment to form a complement of the second
portion, and
synthesising the at least one polynucleotide sequence by forming a
complement of the first precursor polynucleotide sequence.
In one embodiment, the first precursor polynucleotide fragment may comprise a
first
sequencing primer binding site complement.
In one embodiment, the first sequencing primer binding site complement may be
located
before a 5'-end of the complement of the first portion, such as immediately
before the 5'-
end of the complement of the first portion.
In one embodiment, the first precursor polynucleotide fragment may comprise a
second
adaptor complement sequence.
In one embodiment, the second adaptor complement sequence may be located
before
a 5'-end of the complement of the first portion.
In one embodiment, the first precursor polynucleotide fragment may comprise a
first
sequencing primer binding site complement and a second adaptor complement
sequence.
In one example, the first sequencing primer binding site complement may be
located
before a 5'-end of the complement of the first portion, and wherein the second
adaptor
complement sequence may be located before a 5'-end of the first sequencing
primer
binding site complement.
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In one embodiment, the first precursor polynucleotide fragment may comprise a
second
sequencing primer binding site complement.
5 In one embodiment, the hybridisation sequence complement may comprise the
second
sequencing primer binding site complement.
In one embodiment, the second precursor polynucleotide fragment may comprise a
first
adaptor complement sequence.
In some embodiments, the method may further comprise a step of concurrently
sequencing nucleobases in the first portion and the second portion.
The target polynucleotide (or in some embodiments, the polynucleotide library)
has been
pre-treated using a conversion reagent. In some embodiments, the method of
preparing
at least one polynucleotide sequence for detection of modified cytosines may
include a
step of treating the target polynucleotide using a conversion agent. Figure 18
shows the
effect of the pre-treatment of the target polynucleotide of various conversion
agents on
the bases in the resulting template strands.
The conversion reagent is configured to convert a modified cytosine to thymine
or a
nucleobase which is read as thymine/uracil, and/or is configured to convert an
unmodified cytosine to uracil or a nucleobase which is read as thymine/uracil.
As used herein, the term "modified cytosine" may refer to any one or more of 5-
methylcytosine (5-mC), 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (54C)
and
5-carboxylcytosine (5-caC):
NH2 NH2 0 NH2 0 NH2
HON HN HO).11 N
5-methylcytosine 5-hydroxymethylcytosine
5-formylcytosine 5-carboxylcytosine
(5-mC) (5-hmC) (5-fC) (5-caC)
wherein the wavy line indicates an attachment point of the modified cytosine
to the
polynucleotide.
As used herein, the term "unmodified cytosine" refers to cytosine (C):
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NH2
0
cytosine
(C)
wherein the wavy line indicates an attachment point of the unmodified cytosine
to the
polynucleotide.
As used herein, the term "conversion reagent configured to convert a modified
cytosine
to thymine or a nucleobase which is read as thymine/uracil" may refer to a
reagent which
converts one or more modified cytosines (e.g. 5-methylcytosine, 5-
hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine) to thymine
(i.e. would
base pair with adenine), or to an equivalent nucleobase which would base pair
with
adenine. The conversion may comprise a deamination reaction converting the
modified
cytosine to thymine or nucleobase which is read as thymine/uracil.
As used herein, the term "conversion reagent configured to convert an
unmodified
cytosine to uracil or a nucleobase which is read as thymine/uracil" may refer
to a reagent
which converts one or more unmodified cytosines to uracil (i.e. would base
pair with
adenine), or to an equivalent nucleobase which would base pair with adenine.
The
conversion may comprise a deamination reaction converting the unmodified
cytosine to
uracil or nucleobase which is read as thymine/uracil.
In general, if modified cytosines were present in the target polynucleotide to
be
sequenced, the forward strand of the template will then not be identical to
the reverse
complement strand of the template as a result of treatment of the target
polynucleotide
with the conversion agent (alternatively, the reverse strand of the template
will then not
be identical to the forward complement strand of the template as a result of
treatment of
the target polynucleotide with the conversion agent). However, if modified
cytosines were
not present in the target polynucleotide to be sequenced, the forward strand
of the
template will then be (substantially) identical to the reverse complement
strand of the
template despite treatment of the target polynucleotide with the conversion
agent
(alternatively, the reverse strand of the template will then be
(substantially) identical to
the forward complement strand of the template despite treatment of the target
polynucleotide with the conversion agent). As such, mismatches between the
forward
strand of the template and the reverse complement strand of the template allow
the
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detection of modified cytosines (alternatively, mismatches between the reverse
strand of
the template and the forward complement strand of the template allow detection
of
modified cytosines).
Where the forward strand (or reverse strand) of the template is not identical
to the reverse
complement strand (or forward complement strand) of the template as a result
of
treatment with the conversion agent, the forward strand (or reverse strand) of
the
template may comprise a guanine base at a first position, which leads to a
basecall of C
for the original target polynucleotide; and wherein the reverse complement
strand (or
forward complement strand) of the template may comprise an adenine base at a
second
position corresponding to the same position number as the first position,
which leads to
a basecall of T for the original target polynucleotide. The adenine base at
the second
position within the template may have been generated as a result of conversion
of
modified cytosines in the target polynucleotide to thymine, or to an
equivalent
nucleobase which would base pair with adenine; or may have been generated as a
result
of conversion of unmodified cytosines in the target polynucleotide to uracil,
or to an
equivalent nucleobase which would base pair with adenine. In particular, the
adenine
base at the second position within the template may have been generated as a
result of
conversion of unmodified cytosines in the target polynucleotide to uracil, or
to an
equivalent nucleobase which would base pair with adenine.
In other cases, the forward strand (or reverse strand) of the template
comprises an
adenine base at a first position, which leads to a basecall of T for the
original target
polynucleotide; and wherein the reverse complement strand (or forward
complement
strand) of the template comprises a guanine base at a second position
corresponding to
the same position number as the first position, which leads to a basecall of C
for the
original target polynucleotide. Similarly, the adenine base at the first
position within the
template may have been generated as a result of conversion of modified
cytosines in the
target polynucleotide to thymine, or to an equivalent nucleobase which would
base pair
with adenine; or may have been generated as a result of conversion of
unmodified
cytosines in the target polynucleotide to uracil, or to an equivalent
nucleobase which
would base pair with adenine. In particular, the adenine base at the first
position within
the template may have been generated as a result of conversion of modified
cytosines
in the target polynucleotide to thymine, or to an equivalent nucleobase which
would base
pair with adenine.
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In some embodiments, the conversion reagent configured to convert a modified
cytosine
to thymine or a nucleobase which is read as thymine/uracil may further be
configured to
be selective for converting one or more modified cytosines (e.g. 5-
methylcytosine, 5-
hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine) over
converting
unmodified cytosine. The selectivity may be measured by comparing reaction
parameters (e.g. deamination reaction parameters) of the conversion of a
particular
modified cytosine to thymine or equivalent nucleobase which is read as
thymine/uracil,
with corresponding reaction parameters (e.g. deamination reaction parameters)
of the
conversion of unmodified cytosine to uracil or nucleobase which is read as
thymine/uracil. For example, reaction parameters such as rate of reaction or
yield may
be compared. In the case of rate of reaction, a rate of a reaction (e.g.
deamination) of
the particular modified cytosine to thymine or nucleobase which is read as
thymine/uracil
may be greater (e.g at least 2 times greater, at least 5 times greater, at
least 10 times
greater, at least 20 times greater, at least 50 times greater, or at least 100
times greater)
than a corresponding rate of a reaction (e.g. deamination) of the unmodified
cytosine to
uracil or nucleobase which is read as thymine/uracil. In the case of yield, a
yield of a
reaction (e.g. deamination) of the particular modified cytosine to thymine or
nucleobase
which is read as thymine/uracil may be greater (e.g. at least 2 times greater,
at least 5
times greater, at least 10 times greater, at least 20 times greater, at least
50 times
greater, or at least 100 times greater) than a corresponding yield of a
reaction (e.g.
deamination) of the unmodified cytosine to uracil or nucleobase which is read
as
thymine/uracil.
In some embodiments, the conversion reagent configured to convert an
unmodified
cytosine to uracil or a nucleobase which is read as thymine/uracil may further
be
configured to be selective for converting unmodified cytosine over converting
one or
more modified cytosines (e.g. 5-methylcytosine, 5-hydroxymethylcytosine, 5-
formylcytosine and 5-carboxylcytosine). The selectivity may be measured by
comparing
reaction parameters (e.g. deamination reaction parameters) of the conversion
of
unmodified cytosine to uracil or nucleobase which is read as thymine/uracil,
with
corresponding reaction parameters (e.g. deamination reaction parameters) of
the
conversion of a particular modified cytosine to thymine or nucleobase which is
read as
thymine/uracil. For example, reaction parameters such as rate of reaction or
yield may
be compared. In the case of rate of reaction, a rate of a reaction (e.g.
deamination) of
the unmodified cytosine to uracil or nucleobase which is read as
thymine/uracil may be
greater (e.g. at least 2 times greater, at least 5 times greater, at least 10
times greater,
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at least 20 times greater, at least 50 times greater, or at least 100 times
greater) than a
rate of a reaction (e.g. deamination) of the particular modified cytosine to
uracil or the
nucleobase which is read as thymine/uracil. In the case of yield, a yield of a
reaction (e.g.
deamination) the unmodified cytosine to uracil or nucleobase which is read as
thymine/uracil may be greater (e.g. at least 2 times greater, at least 5 times
greater, at
least 10 times greater, at least 20 times greater, at least 50 times greater,
or at least 100
times greater) than a corresponding yield of a reaction (e.g. deamination) of
the particular
modified cytosine to uracil or the nucleobase which is read as thymine/uracil.
In some embodiments, the conversion agent may comprise a chemical agent and/or
an
enzyme.
In some embodiments, the chemical agent may comprise a boron-based reducing
agent.
In one embodiment, the boron-based reducing agent is an amine-borane compound
or
an azine-borane compound (wherein the term "azine" refers to a nitrogenous
heterocyclic
compound comprising a 6-membered aromatic ring). Non-limiting examples of
amine-
borane compounds include compounds such as t-butylamine borane, ammonia
borane,
ethylenediamine borane and dimethylamine borane. Non-limiting examples of
azine-
borane compounds include compounds such as pyridine borane and 2-picoline
borane.
In general, boron-based reducing agents are able to convert 5-formylcytosine
and 5-
carboxylcytosine to dihydrouracil (i.e. a nucleobase which is read as
thymine/uracil). The
reaction proceeds by reduction of the internal C=C bond of 5-formylcytosine or
5-
carboxylcytosine, deamination, and then decarboxylation to form dihydrouracil
(illustrated below using 5-carboxylcytosine):
O NH2 o NH2
HO N HO
N.,L00
0 0 0
HONH NH
This process is selective for a particular type of modified cytosine (5-
carboxylcytosine)
and does not convert unmodified cytosine. Where distinction between other
modified
cytosines and unmodified cytosines is desired (or even between different types
of
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modified cytosines), treatment with further agents as described herein prior
to treatment
with the boron-based reducing agent may provide such distinction. In
particular, boron-
based reducing agents may be combined with ten-eleven translocation (TET)
methylcytosine dioxygenases, 13-glucosyltransferases, oxidising agents, oximes
and/or
5 hydrazones as described herein.
In some embodiments, the chemical agent may comprise sulfite. The sulfite may
be
present in a partially acid/salt form (e.g. as bisulfite ions), or be present
in a salt form
(e.g. as sulfite ions). In cases where the sulfite is present in a salt form,
the sulfite may
10 comprise a cation (not including H ). For example, the cation may be
selected from
"metal cations" or "non-metal cations". Metal cations may include alkali metal
ions (e.g.
lithium, sodium, potassium, rubidium or caesium ions). Non-metal cations may
include
ammonium salts (e.g alkylammonium salts) or phosphonium salts (e.g.
alkylphosphonium salts). The term "sulfite" also encompasses "metabisulfite",
which
15 dissolves in aqueous solution to form bisulfite.
In general, sulfite (e.g. bisulfite) is able to convert unmodified cytosine to
uracil. The
reaction proceeds via conjugate addition of sulfite to the internal C=C of
unmodified
cytosine, deamination, and then elimination of sulfite to reform the internal
C=C bond to
20 form uracil:
NH2 NH2
s.'N 0
0 0
-)L NH }Is, NH
N 0
This process is selective for unmodified cytosine over certain types of
modified cytosine
(5-methylcytosine and 5-hydroxymethylcytosine). However, 5-formylcytosine and
5-
25 carboxylcytosine are converted to their equivalent deaminated versions.
Where
distinction between different types of modified cytosines (e.g. 5-
formylcytosine and 5-
carboxylcytosine) is desired, treatment with further agents as described
herein prior to
treatment with the sulfite may provide such distinction. In particular, the
sulfite may be
combined with ten-eleven translocation (TET) methylcytosine dioxygenases,
30 glucosyltransferases, oxidising agents and/or reducing agents as
described herein.
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In some embodiments, the enzyme may comprise a cytidine deaminase.
As used herein, the term "cytidine deaminase" may refer to an enzyme which is
able to
catalyse the following reaction:
NH2 0
R =Jk..N1 R J-L,NH
0
wherein R is hydrogen, methyl, hydroxymethyl, formyl or carboxyl, and wherein
the wavy
line indicates an attachment point to a polynucleotide.
In one embodiment, the cytidine deaminase is a wild-type cytidine deaminase or
a
mutant cytidine deaminase. In one example, the cytidine deaminase is a mutant
cytidine
deaminase.
In some embodiments, the cytidine deaminase is a member of the APOBEC protein
family. In one embodiment, the cytidine deaminase is a member of the AID
subfamily,
the APOBEC1 subfamily, the APOBEC2 subfamily, the APOBEC3 subfamily (e.g. the
APOBEC3A subfamily, the APOBEC3B subfamily, the APOBEC3C subfamily, the
APOBEC3D subfamily, the APOBEC3F subfamily, the APOBEC3G subfamily, or the
APOBEC3H subfamily), or the APOBEC4 subfamily; in one embodiment, the cytidine
deaminase is a member of the APOBEC3A subfamily.
In general, cytidine deanninases are able to catalyse the deamination of all
modified
cytosines (particularly 5-methylcytosine, 5-hydroxymethylcytosine and 5-
formylcytosine)
to their equivalent deaminated versions (i.e. nucleobases which are read as
thymine/uracil), as well as catalysing the deamination of unmodified cytosines
to uracil.
Nevertheless, rates of reaction may differ depending on the type of modified
cytosine;
for example, wild-type APOBEC3A catalyses the deamination of unmodified
cytosine
and 5-methylcytosine relatively efficiently, whereas deamination of 5-
hydroxymethylcytosine is -5000-fold slower relative to unmodified cytosine,
deamination
of 5-formylcytosine is -3700-fold slower relative to unmodified cytosine, and
deamination
of 5-carboxylcytosine is >20000-fold slower relative to unmodified cytosine.
Where
distinction between modified cytosines and unmodified cytosines is desired (or
even
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between different types of modified cytosines), treatment with further agents
as
described herein prior to treatment with the cytidine deaminase may provide
such
distinction. In particular, the cytidine deaminase may be combined with ten-
eleven
translocation (TET) methylcytosine dioxygenases and/or 13-glucosyltransferases
as
described herein. Alternatively, or in addition, particular cytidine
deaminases (e.g. mutant
cytidine deaminases) may be chosen which have higher affinities for modified
cytosines
as substrates over unmodified cytosines, or vice versa.
The APOBEC protein family is a member of the large cytidine deaminase
superfamily
that contains a canonical zinc-dependent deaminase (ZDD) signature motif
embedded
within a core cytidine deaminase fold. This fold includes a five-stranded
mixed beta (b)-
sheet surrounded by six alpha (a)-helices with the order al-b1-b2-a2-b3-a3-b4-
a4-b5-
a5-a6 (Salter et al., Trends Biochem Sci. 2016 41(7):578-594.
doi:10.1016/j.tibs.2016.05.001; Salter et al., Trends Biochem. Sci.
2018,43(8):606-622
doi.org/10.1016/j.tibs.2018.04.013). Each cytidine deaminase domain core
structure of
APOBEC proteins contains a highly conserved spatial arrangement of the
catalytic
centre residues of a zinc-binding motif H4P/ANFE-X[23_28]-P-C-X[2_4]-C (SEQ ID
NO. 67)
(referred to herein as the ZDD motif, where X is any amino acid, and the
subscript range
of numbers after X refers to the number of amino acids) (Salter et al., Trends
Biochem
Sci. 2016 41(7):578-594. doi:10.1016/j.tibs.2016.05.001). Without intending to
be limited
by theory, the H and two C residues coordinate a Zn atom, and the E residue
polarises
a water molecule near the Zn-atom for catalysis (Chen et al., 2021, Viruses,
13:497,
doi.org/10.3390/v13030497).
Some members of the APOBEC protein family, e.g., the AID subfamily, the
APOBEC1
subfamily, the APOBEC2 subfamily, the APOBEC3A subfamily, the APOBEC3C
subfamily, the APOBEC3H subfamily, and the APOBEC4 subfamily, include one copy
of
the ZDD motif. Other members of the APOBEC protein family, e.g., the APOBEC3B
subfamily, the APOBEC3D subfamily, the APOBEC3F subfamily, and the APOBEC3G
subfamily, include two copies of the ZDD motif, but often only the C-terminal
copy is
active (Salter et al., Trends Biochem Sci. 2016 41(7):578-594.
doi:10.1016/j.tibs.2016.05.001). Thus, a mutant cytidine deaminase disclosed
herein
includes one or two ZDD motifs. In one embodiment, a mutant cytidine deaminase
based
on a member of the APOBEC3A subfamily includes the following ZDD motif:
HXEX24SW(S/T)PCX[24]CX6FX8LX5R(LJOYX[8_11]LX2LX[10]M (SEQ ID NO. 68) (where X
is
any amino acid, and the subscript number or range of numbers after X refers to
the
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number of amino acids) (Salter et al., Trends Biochem Sci. 2016 41(7):578-594.
doi:10.1016/j.tibs.2016.05.001).
Non-limiting examples of wild-type cytidine deaminases in the APOBEC protein
family
are shown in the table below (from UniProt, database of protein sequence and
functional
information, available at uniprot.org; or GenBank, collection of nucleotide
sequences and
their protein translations, available at ncbi.nlm.nih.gov/protein/):
APOBEC protein Non-limiting examples
AID UniProt: Q9GZX7 (SEQ ID NO. 23);
UniProt: G3QLD2 (SEQ ID NO. 24);
Uniprot Q9VVVE0 (SEQ ID NO. 25)
APOBEC1 UniProt: P41238 (SEQ ID NO. 26);
NCB! XP_030856728.1 (SEQ ID NO. 27);
Uniprot P51908 (SEQ ID NO. 28)
APOBEC2 UniProt: Q9Y235 (SEQ ID NO. 29);
Uniprot G3SGN8 (SEQ ID NO. 30);
Uniprot Q9VVV35 (SEQ ID NO. 31)
APOBEC3A UniProt: P31941(SEQ ID NO. 32);
GenBank: XP_045219544.1 (SEQ ID NO. 33);
GenBank: AER45717.1 (SEQ ID NO. 34);
GenBank: XP_003264816.1 (SEQ ID NO. 35);
GenBank: PNI48846.1 (SEQ ID NO. 36);
GenBank: AD085886.1 (SEQ ID NO. 37)
APOBEC3B UniProt: Q9UH17 (SEQ ID NO. 38);
Uniprot G3QV16 (SEQ ID NO. 39);
Uniprot F6M3K5 (SEQ ID NO. 40)
APOBEC3C UniProt: Q9NRW3 (SEQ ID NO. 41);
Uniprot Q694B5 (SEQ ID NO. 42);
Uniprot BOLVV74 (SEQ ID NO. 43)
APOBEC3D UniProt: Q96AK3 (SEQ ID NO. 44);
NCB! NP_001332895.1 (SEQ ID NO. 45);
NCB! NP_001332931.1 (SEQ ID NO. 46)
APOBEC3F UniProt: 08IUX4 (SEQ ID NO. 47);
Uniprot G3RD21 (SEQ ID NO. 48);
Uniprot Q1GOZ6 (SEQ ID NO. 49)
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APOBEC3G UniProt: Q9HC16 (SEQ ID NO. 50);
Uniprot Q694C1 (SEQ ID NO. 51);
Uniprot U5NDB3 (SEQ ID NO. 52)
APOBEC3H UniProt: Q6NTF7 (SEQ ID NO. 53);
Uniprot B7TOU7 (SEQ ID NO. 54);
Uniprot Q19Q52 (SEQ ID NO. 55)
APOBEC4 UniProt: Q8WVV27(SEQ ID NO. 56);
NCB! XP_004028087.1 (SEQ ID NO. 57);
Uniprot Q497M3 (SEQ ID NO. 58)
In one embodiment, the mutant cytidine deaminase may comprise amino acid
substitution mutations at positions functionally equivalent to (Tyr/Phe)130
and Tyr132 in
a wild-type APOBEC3A protein. Such mutant cytidine deaminases are described in
further detail in US Provisional Application 63/328,444, which is incorporated
herein by
reference. By "functionally equivalent" it is meant that the mutant cytidine
deaminase has
the amino acid substitution at the amino acid position in a reference (wild-
type) cytidine
deaminase that has the same functional role in both the reference (wild-type)
cytidine
deaminase and the mutant cytidine deaminase.
In one embodiment, the (Tyr/Phe)130 may be Tyr130, and the wild-type APOBEC3A
protein may be SEQ ID NO. 32.
In some embodiments, the mutant cytidine deaminase may convert 5-
methylcytosine to
thymine by deamination at a greater rate than conversion rate of cytosine to
uracil by
deamination; wherein the rate may be at least 100-fold greater.
In one embodiment, the substitution mutation at the position functionally
equivalent to
Tyr130 may comprise Ala, Val or Trp.
In one embodiment, the substitution mutation at the position functionally
equivalent to
Tyr132 may comprise a mutation to His, Arg, Gln or Lys.
In one embodiment, the mutant cytidine deaminase may comprise a ZDD motif H-
[P/A/V]-E-X[23_28]-P-C-X[2_4]-C (SEQ ID NO. 67).
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In one embodiment, the mutant cytidine deaminase may be a member of the
APOBEC3A
subfamily and may comprise a ZDD motif HXEX24SW(S/T)PCX[2_4]CX6FX8LX5R(UDYX[8_
ii]I_X2LX[io]M (SEQ ID NO. 68).
5 In some embodiments, the target polynucleotide may be treated with a
further agent prior
to treatment with the conversion reagent.
In one embodiment, the further agent may be configured to convert a modified
cytosine
(e.g. one of 5-methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine and 5-
10 carboxylcytosine) to another modified cytosine (e.g. another one of 5-
methylcytosine, 5-
hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine).
For example, the further agent may be configured to convert 5-methylcytosine
to 5-
hydroxymethylcytosine. In the same or other embodiments, the further agent may
be
15 configured to convert 5-hydroxymethylcytosine to 5-formylcytosine. In
the same or other
embodiments, the further agent may be configured to convert 5-formylcytosine
to 5-
carboxylcytosine. In some embodiments, the further agent may be configured to
convert
5-methylcytosine to 5-hydroxymethylcytosine, 5-hydroxymethylcytosine to 5-
formylcytosine, and 5-formylcytosine to 5-carboxylcytosine.
20 In other embodiments, the further agent may be configured to convert 5-
formylcytosine
to 5-hydroxymethylcytosine.
In another embodiment, the further agent configured to convert a modified
cytosine (e.g.
one of 5-methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine and 5-
25 carboxylcytosine) to another modified cytosine (e.g. another (different)
one of 5-
methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine and 5-
carboxylcytosine)
may comprise a chemical agent and/or an enzyme.
The further agent configured to convert a modified cytosine to another
modified cytosine
30 may be a chemical agent; for example, an oxidising agent; such as a
metal-based
oxidising agent; such as a transition metal-based oxidising agent; such as a
ruthenium-
based oxidising agent. The oxidising agent may be configured to convert 5-
hydroxymethylcytosine to 5-formylcytosine. Non-limiting examples of the
oxidising agent
include ruthenate (e.g. potassium ruthenate, K2Ru04), or perruthenate (e.g.
potassium
35 perruthenate, KRu04).
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The further agent configured to convert a modified cytosine to another
modified cytosine
may be a chemical agent; for example, a reducing agent; such as a Group III-
based
reducing agent; for example, a boron-based reducing agent. The oxidising agent
may be
configured to convert 5-formylcytosine to 5-hydroxymethylcytosine. Non-
limiting
examples of the reducing agent include borohydride (e.g. sodium borohydride,
lithium
borohydride), or triacetoxyborohydride (e.g. sodium triacetoxyborohydride).
The further agent configured to convert a modified cytosine to another
modified cytosine
may be an enzyme; such as a ten-eleven translocation (TET) methylcytosine
dioxygenase; wherein the TET methylcytosine dioxygenase may be a member of the
TET1 subfamily, the TET2 subfamily, or the TET3 subfamily. The enzyme may be
configured to convert 5-methylcytosine to 5-hydroxymethylcytosine, 5-
hydroxymethylcytosine to 5-formylcytosine, and 5-formylcytosine to 5-
carboxylcytosine.
Non-limiting examples of the TET methylcytosine dioxygenase include:
TET protein Non-limiting examples
TETI UniProt: Q8NFU7 (SEQ ID NO. 59)
UniProt: Q3URK3 (SEQ ID NO. 60)
TET2 UniProt: Q6N021 (SEQ ID NO. 61)
UniProt: 04JK59 (SEQ ID NO. 62)
TET3 UniProt: 043151 (SEQ ID NO. 63)
UniProt: Q8BG87 (SEQ ID NO. 64)
In one embodiment, the further agent may be configured to reduce/prevent
deamination
of a particular modified cytosine (e.g. one of 5-methylcytosine, 5-
hydroxymethylcytosine,
5-formylcytosine and 5-carboxylcytosine). Such a further agent configured to
reduce/prevent deamination of a particular modified cytosine may be used in
combination with a further agent configured to convert a modified cytosine to
another
modified cytosine.
For example, the further agent may be configured to convert 5-
hydroxymethylcytosine to
a 5-hydroxymethylcytosine analogue bearing a hydroxyl protecting group. The 5-
hydroxymethylcytosine analogue bearing the hydroxyl protecting group may be
resistant
to oxidation to form 5-formylcytosine. Non-limiting examples of hydroxyl
protecting
groups include sugar groups (e.g. glycosyl), silyl ether groups (e.g.
trimethylsilyl,
triethylsilyl, triisopropylsilyl, t-butyl(dimethyl)silyl, t-
butyl(diphenyl)sily1), ether groups
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(e.g. benzyl, ally!, t-butyl, methoxymethyl (MOM), 2-methoxyethoxymethyl
(MEM),
tetrahydropyranyl), or acyl groups (e.g. acetyl, benzoyl).
In other embodiments, the further agent may be configured to convert 5-
formylcytosine
to a 5-formylcytosine analogue bearing an oxime or a hydrazone group. The 5-
formylcytosine analogue bearing the oxime or hydrazone group may be resistant
to
oxidation to form 5-carboxylcytosine.
In one embodiment, the further agent configured to reduce/prevent deamination
of a
particular modified cytosine (e.g. one of 5-methylcytosine, 5-
hydroxymethylcytosine, 5-
formylcytosine and 5-carboxylcytosine) may comprise a chemical agent and/or an
enzyme.
The further agent configured to reduce/prevent deamination of a particular
modified
cytosine may be an enzyme; for example, a glycosyltransferase (e.g. a-
glucosyltransferase or p-glucosyltransferase); such as a p-
glucosyltransferase. Such a
further agent may be configured to convert 5-hydroxymethylcytosine to a 5-
hydroxymethylcytosine analogue bearing a hydroxyl protecting group, wherein
the
hydroxyl protecting group is glycosyl. A non-limiting example of the enzyme
includes 14-
f3GT, for example as supplied by New England BioLabs (catalog # M0357S,
M0357L) or
by ThermoFisher Scientific (catalog # E00831); further non-limiting examples
of
glycosyltransferases include:
Glucosyltransferase Non-limiting examples
a-glucosyltransferase UniProt: P04519 (SEQ ID NO. 65)
p-glucosyltransferase UniProt: P04547 (SEQ ID NO. 66)
The further agent configured to reduce/prevent deamination of a particular
modified
cytosine may be a chemical agent; such as a hydroxylamine or a hydrazine. Such
a
further agent may be configured to convert 5-formylcytosine to a 5-
formylcytosine
analogue bearing an oxime or a hydrazone group. Non-limiting examples of
hydroxylamines include 0-alkylhydroxylamines (e.g. 0-methylhydroxylamine, 0-
ethylhydroxylamine), 0-arylhydroxylamines (e.g. 0-phenylhydroxylamine). Non-
limiting
examples of hydrazines include acylhydrazides (e.g. acethydrazide,
benzhydrazide),
alkylsulfonylhydrazides (e.g. methylsulfonylhydrazide), or
arylsulfonylhydrazides (e.g.
benzenesulfonylhydrazide, p-toluenesulfonylhydrazide).
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Specific methods of modified cytosine sequencing using conversion agents
(optionally
combined with further agents) are further illustrated below. However, the type
of
conversion agents and/or further agents are not limited thereto.
BS-seq
Bisulfite sequencing (BS-seq) involves using bisulfite as the conversion
agent. This
process is described in Frommer et al. (Proc. Natl. Acad. Sci. U.S.A., 1992,
89, pp. 1827-
1831), which is incorporated herein by reference. This process converts
unmodified
cytosines in the target polynucleotide to uracil, as well as 5-formylcytosine
and 5-
carboxylcytosine to deaminated analogues, but does not convert 5-
methylcytosine and
5-hydroxymethylcytosine. Accordingly, BS-seq allows identification of the
modified
cytosines 5-mC and 5-hmC by reading them as C; whereas unmodified C, 5-fC and
5-
caC are converted to nucleobases which are read as T/U.
OxBS-seq
Oxidative bisulfite sequencing (oxBS-seq) involves using potassium
perruthenate as the
further agent and bisulfite as the conversion agent. This process is described
in Booth
et al. (Science, 2012, 336, pp. 934-937), which is incorporated herein by
reference.
Potassium perruthenate causes oxidation of 5-hydroxymethylcytosine in the
target
polynucleotide to 5-formylcytosine. Subsequent treatment with bisulfite
converts
unmodified cytosines in the target polynucleotide to uracil, as well as 5-
formylcytosine
(including residues that used to be 5-hydroxymethylcytosine) and 5-
carboxylcytosine to
deaminated analogues, but does not convert 5-methylcytosine. Accordingly, oxBS-
seq
allows identification of the modified cytosine 5-mC by reading them as C;
whereas
unmodified C, 5-hmC, 5-fC and 5-caC are converted to nucleobases which are
read as
T/U.
RedBS-seq
Reduced bisulfite sequencing (redBS-seq) involves using sodium borohydride as
the
further agent and bisulfite as the conversion agent. This process is described
in Booth
et al. (Nat. Chem., 2014, 6, pp. 435-440), which is incorporated herein by
reference.
Sodium borohydride causes reduction of 5-formylcytosine in the target
polynucleotide to
5-hydroxymethylcytosine. Subsequent treatment with bisulfite converts
unmodified
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cytosines in the target polynucleotide to uracil, as well as 5-
carboxylcytosine to its
deaminated analogue, but does not convert 5-hydroxymethylcytosine (including
residues
that used to be 5-formylcytosine) and 5-methylcytosine. Accordingly, redBS-seq
allows
identification of the modified cytosines 5-mC, 5-hmC and 5-fC by reading them
as C;
whereas unmodified C and 5-caC are converted to nucleobases which are read as
T/U.
TAB-seq
TET-assisted bisulfite sequencing (TAB-seq) involves using a T4 bacteriophage
glucosyltransferase and a TET1 enzyme as the further agents and bisulfite as
the
conversion agent. This process is described in Yu et al. (Cell, 2012, 149, pp.
1368-1380),
which is incorporated herein by reference. The T4 bacteriophage p-
glucosyltransferase
converts 5-hydroxymethylcytosine in the target polynucleotide to p-glucosy1-5-
hydroxymethylcytosine, which prevents oxidation. TET1 enzyme causes oxidation
of 5-
methylcytosine and 5-formylcytosine in the target polynucleotide to 5-
carboxylcytosine.
Subsequent treatment with bisulfite converts unmodified cytosines in the
target
polynucleotide to uracil, as well as 5-carboxylcytosine (including residues
that used to
be 5-methylcytosine and 5-formylcytosine) to its deaminated analogue, but does
not
convert 3-glucosy1-5-hydroxymethylcytosine. Accordingly, TAB-seq allows
identification
of the modified cytosine 5-hmC (as the protected glycosyl residue) by reading
it as C;
whereas unmodified C, 5-mC, 5-fC and 5-caC are converted to nucleobases which
are
read as T/U.
ACE-seq
APOBEC-coupled epigenetic sequencing (ACE-seq) involves using a T4
bacteriophage
p-glucosyltransferase as a further agent and APOBEC3A as the conversion agent.
This
process is described in Schutsky et al. (Nat. Biotechnol., 2018, 36, pp. 1083-
1090), which
is incorporated herein by reference. The T4 bacteriophage p-
glucosyltransferase
converts 5-hydroxymethylcytosine in the target polynucleotide to p-glucosy1-5-
hydroxymethylcytosine, which prevents oxidation. Subsequent treatment with
APOBEC3A converts unmodified cytosines in the target polynucleotide to uracil,
as well
as 5-methylcytosine to its deaminated analogue. 5-formylcytosine is also able
to convert
to its deaminated analogue, but reacts slower relative to unmodified cytosine
and 5-
methylcytosine. 5-carboxylcytosine is also able to convert to its deaminated
analogue,
but reacts far slower than unmodified cytosine and 5-methylcytosine, and
slower than 5-
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formylcytosine. Accordingly, ACE-seq allows identification of the modified
cytosine 5-
hmC (as the protected glycosyl residue) by reading it as C; whereas unmodified
C and
5-mC are converted to nucleobases which are read as T/U; 5-fC is converted to
a
nucleobase which is read as T/U to a limited extent; 5-caC is converted to a
nucleobase
5 which is read as T/U to a more limited extent.
EM-seq
Enzymatic Methyl sequencing (EM-seq) involves using T4 bacteriophage
10 glucosyltransferase and a TET2 enzyme as the further agents and APOBEC3A
as the
conversion agent. This process is described in Vaisvila et al. (Genome Res.
2021, 31,
pp. 1280-1289), US 10,619,200 B2 and US 9,121,061 B2, which are incorporated
herein
by reference. The T4 bacteriophage p-glucosyltransferase converts 5-
hydroxymethylcytosine in the target polynucleotide to 3-glucosy1-5-
15 hydroxymethylcytosine, which prevents oxidation. The TET2 enzyme causes
oxidation
of 5-methylcytosine in the target polynucleotide to 5-hydroxymethylcytosine,
which in
turn is converted to 3-glucosy1-5-hydroxymethylcytosine by the T4
bacteriophage
glucosyltransferase. The TET2 enzyme also causes oxidation of 5-formylcytosine
in the
target polynucleotide to 5-carboxylcytosine. Subsequent treatment with
APOBEC3A
20 converts unmodified cytosines in the target polynucleotide to uracil, as
well as 5-
carboxylcytosine (including residues that used to be 5-formylcytosine) to a
limited extent.
Accordingly, EM-seq allows identification of the modified cytosines 5-mC and 5-
hmC (as
protected glycosyl residues) by reading them as C; whereas unmodified C is
converted
to U; 5fC and 5-caC are converted to nucleobases which are read as T/U to a
limited
25 extent.
Modified APOBEC
Modified APOBEC sequencing involves using a mutant APOBEC3A enzyme as the
30 conversion agent, which is described in more detail in the Reference
Examples 1 to 4
below. This process is described in US Provisional Application 63/328,444,
which is
incorporated herein by reference.
TAPS
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TET-assisted pyridine borane sequencing (TAPS) involves using a TET1 enzyme as
the
further agent and pyridine borane as the conversion agent. This process is
described in
Liu et al. (Nature Biotechnology, 2019, 37, pp. 424-429), which is
incorporated herein by
reference. The TET1 enzyme causes oxidation of 5-methylcytosine, 5-
hydroxymethylcytosine and 5-formylcytosine in the target polynucleotide to 5-
carboxylcytosine. Subsequent treatment with pyridine borane converts 5-
carboxylcytosine (including residues that used to be 5-methylcytosine, 5-
hydroxymethylcytosine and 5-formylcytosine) to dihydrouracil, but does not
convert
unmodified cytosine. Accordingly, TAPS allows identification of the modified
cytosines
5-mC, 5-hmC, 5-fC and 5-caC by reading them as T/U; whereas unmodified
cytosine is
read as C.
TAPS/3
TET-assisted pyridine borane sequencing with p-glucosyltransferase blocking
(TAPS13)
involves using a T4 p-glucosyltransferase and a TET1 enzyme as the further
agents, and
pyridine borane as the conversion agent. This process is described in Liu et
al. (Nature
Communications, 2021, 12, 618), which is incorporated herein by reference. The
T4 p-
glucosyltransferase converts 5-hydroxymethylcytosine in the target
polynucleotide to 3-
glucosy1-5-hydroxymethylcytosine, which prevents oxidation. The TET1 enzyme
causes
oxidation of 5-methylcytosine and 5-formylcytosine in the target
polynucleotide to 5-
carboxylcytosine. Subsequent treatment with pyridine borane converts 5-
carboxylcytosine (including residues that used to be 5-methylcytosine and 5-
formylcytosine) to dihydrouracil, but does not convert unmodified cytosine or
p-glucosyl-
5-hydroxymethylcytosine. Accordingly, TAPS p allows identification of the
modified
cytosines 5-mC, 5-fC and 5-caC by reading them as T/U; whereas unmodified
cytosine
and 5-hmC are read as C.
CAPS
Chemical-assisted pyridine borane sequencing (CAPS) involves using a potassium
ruthenate (K2Ru04) as the further agent and 2-picoline borane as the
conversion agent.
This process is described in Liu et al. (Nature Communications, 2021, 12,
618), which is
incorporated herein by reference. Potassium ruthenate causes oxidation of 5-
hydroxymethylcytosine in the target polynucleotide to 5-formylcytosine.
Subsequent
treatment with 2-picoline borane converts 5-formylcytosine (including residues
that used
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to be 5-hydroxymethylcytosine) and 5-carboxylcytosine to dihydrouracil, but
does not
convert unmodified cytosine or 5-methylcytosine. Accordingly, CAPS allows
identification of the modified cytosines 5-hmC, 5-fC and 5-caC by reading them
as T/U;
whereas unmodified cytosine and 5-mC are read as C.
PS
Pyridine borane sequencing (PS) involves using pyridine borane as the
conversion
agent. This process is described in Liu et al. (Nature Communications, 2021,
12, 618),
which is incorporated herein by reference. Treatment with pyridine borane
converts 5-
formylcytosine and 5-carboxylcytosine to dihydrouracil, but does not convert
unmodified
cytosine, 5-methylcytosine or 5-hydroxymethylcytosine. Accordingly, PS allows
identification of the modified cytosines 5-fC and 5-caC by reading them as
T/U; whereas
unmodified cytosine, 5-mC and 5-hmC are read as C.
PS-c
Pyridine borane sequencing for 5-caC (PS-c) involves using 0-
ethylhydroxylamine as
the further agent and pyridine borane as the conversion agent. This process is
described
in Liu et al. (Nature Communications, 2021, 12, 618), which is incorporated
herein by
reference. The 0-ethylhydroxylamine converts 5-formylcytosine to an oxime
derivative,
which prevents 5-formylcytosine from converting to dihydrouracil. Subsequent
treatment
with pyridine borane converts 5-carboxylcytosine to dihydrouracil, but does
not convert
unmodified cytosine, 5-methylcytosine, 5-hydroxycytosine or the oxime
derivative of 5-
formylcytosine. Accordingly, PS-c allows identification of the modified
cytosine 5-caC by
reading it as T/U; whereas unmodified cytosine, 5-mC, 5-hmC and 5-fC are read
as C.
Methods of sequencing
Also described herein is a method of sequencing at least one polynucleotide
sequence
to detect modified cytosines, comprising:
preparing at least one polynucleotide sequence for detection of modified
cytosines using a method as described herein;
concurrently sequencing nucleobases in the first portion and the second
portion; and
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identifying modified cytosines by detecting differences when comparing a
sequence output from the first portion with a sequence output from the second
portion.
In one embodiment, sequencing is performed by sequencing-by-synthesis or
sequencing-by-ligation.
In one embodiment, the step of preparing the at least one polynucleotide
sequence
comprises using a selective processing method as described herein; and wherein
the
step of concurrent sequencing nucleobases in the first portion and the second
portion is
based on the intensity of the first signal and the intensity of the second
signal.
In one embodiment, the method may further comprise a step of conducting paired-
end
reads.
In some embodiments, where the method comprises a step of selectively
processing the
at least one polynucleotide sequence comprising the first portion and the
second portion,
such that a proportion of first portions are capable of generating a first
signal and a
proportion of second portions are capable of generating a second signal,
wherein the
selective processing causes an intensity of the first signal to be greater
than an intensity
of the second signal, the data may be analysed using 16 QAM as mentioned
herein.
Accordingly, the step of concurrently sequencing nucleobases may comprise:
(a) obtaining first intensity data comprising a combined intensity of a
first
signal component obtained based upon a respective first nucleobase at the
first portion
and a second signal component obtained based upon a respective second
nucleobase
at the second portion, wherein the first and second signal components are
obtained
simultaneously;
(b) obtaining second intensity data comprising a combined intensity of a
third
signal component obtained based upon the respective first nucleobase at the
first portion
and a fourth signal component obtained based upon the respective second
nucleobase
at the second portion, wherein the third and fourth signal components are
obtained
simultaneously;
(c) selecting one of a plurality of classifications based on the first and
the
second intensity data, wherein each classification represents a possible
combination of
respective first and second nucleobases; and
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(d) based on the selected classification, base calling
the respective first and
second nucleobases.
In one embodiment, selecting the classification based on the first and second
intensity
data may comprise selecting the classification based on the combined intensity
of the
first and second signal components and the combined intensity of the third and
fourth
signal components.
In one embodiment, the plurality of classifications may comprise sixteen
classifications,
each classification representing one of sixteen unique combinations of first
and second
nucleobases.
In one embodiment, the first signal component, second signal component, third
signal
component and fourth signal component may be generated based on light
emissions
associated with the respective nucleobase.
In one embodiment, the light emissions may be detected by a sensor, wherein
the sensor
is configured to provide a single output based upon the first and second
signals.
In one example, the sensor may comprise a single sensing element.
In one embodiment, the method may further comprise repeating steps (a) to (d)
for each
of a plurality of base calling cycles.
In some embodiments, where a proportion of first portions is capable of
generating a first
signal and a proportion of second portions is capable of generating a second
signal,
wherein an intensity of the first signal is substantially the same as an
intensity of the
second signal, the data may be analysed using 9 QAM as mentioned herein.
Accordingly, the step of concurrently sequencing nucleobases may comprise:
(a) obtaining first intensity data comprising a combined
intensity of a first
signal component obtained based upon a respective first nucleobase at the
first portion
and a second signal component obtained based upon a respective second
nucleobase
at the second portion, wherein the first and second signal components are
obtained
simultaneously;
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(b)
obtaining second intensity data comprising a combined intensity of a
third
signal component obtained based upon the respective first nucleobase at the
first portion
and a fourth signal component obtained based upon the respective second
nucleobase
at the second portion, wherein the third and fourth signal components are
obtained
5 simultaneously;
(C)
selecting one of a plurality of classifications based on the first and
the
second intensity data, wherein each classification of the plurality of
classifications
represents one or more possible combinations of respective first and second
nucleobases, and wherein at least one classification of the plurality of
classifications
10
represents more than one possible combination of respective first and second
nucleobases; and
(d)
based on the selected classification, determining sequence information
from the first portion and the second portion_
15 In
one embodiment, selecting the classification based on the first and second
intensity
data may comprise selecting the classification based on the combined intensity
of the
first and second signal components and the combined intensity of the third and
fourth
signal components.
20 In
one embodiment, when based on a nucleobase of the same identity, an intensity
of
the first signal component may be substantially the same as an intensity of
the second
signal component and an intensity of the third signal component is
substantially the same
as an intensity of the fourth signal component.
25 In
one embodiment, the plurality of classifications may consist of a
predetermined
number of classifications.
In one embodiment, the plurality of classifications may comprise:
one or more classifications representing matching first and second
30 nucleobases; and
one or more classifications representing mismatching first and second
nucleobases, and
wherein determining sequence information of the first portion and second
portion comprises:
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in response to selecting a classification representing matching first
and second nucleobases, determining a match between the first and
second nucleobases; or
in response to selecting a classification representing mismatching
first and second nucleobases, determining a mismatch between the first
and second nucleobases.
In one embodiment, determining sequence information of the first portion and
the second
portion may comprise, in response to selecting a classification representing a
match
between the first and second nucleobases, base calling the first and second
nucleobases.
In another embodiment, determining sequence information of the first portion
and the
second portion may comprise, based on the selected classification, determining
that the
second portion is modified relative to the first portion at a location
associated with the
first and second nucleobases.
In one embodiment, the first signal component, second signal component, third
signal
component and fourth signal component may be generated based on light
emissions
associated with the respective nucleobase.
In one embodiment, the light emissions may be detected by a sensor, wherein
the sensor
is configured to provide a single output based upon the first and second
signals.
In one embodiment, the sensor may comprise a single sensing element.
In one embodiment, the method may further comprise repeating steps (a) to (d)
for each
of a plurality of base calling cycles.
Kits
Methods as described herein may be performed by a user physically. In other
words, a
user may themselves conduct the methods of preparing at least one
polynucleotide
sequence for detection of modified cytosines as described herein, and as such
the
methods as described herein may not need to be computer-implemented.
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In another aspect of the invention, there is provided a kit comprising
instructions for
preparing at least one polynucleotide sequence for detection of modified
cytosines as
described herein, and/or for sequencing at least one polynucleotide sequence
to detect
modified cytosines as described herein.
In one embodiment, the kit may further comprise a sequencing primer comprising
or
consisting of a sequence selected from SEQ ID NO. 7 to 16 or a variant or
fragment
thereof.
In one embodiment, the kit may comprise a sequencing composition comprising a
sequencing primer selected from SEQ ID NO. 7 to 10 or a variant or fragment
thereof,
and a sequencing primer selected from SEQ ID NO. 11 to 16 or a variant or
fragment
thereof.
Computer programs and products
In other embodiments, methods as described herein may be performed by a
computer.
In other words, a computer may contain instructions to conduct the methods of
preparing
at least one polynucleotide sequence for detection of modified cytosines as
described
herein, and as such the methods as described herein may be computer-
implemented.
Accordingly, in another aspect of the invention, there is provided a data
processing
device comprising means for carrying out the methods as described herein.
The data processing device may be a polynucleotide sequencer.
The data processing device may comprise reagents used for synthesis methods as
described herein.
The data processing device may comprise a solid support, such as a flow cell.
In another aspect of the invention, there is provided a computer program
product
comprising instructions which, when the program is executed by a processor,
cause the
processor to carry out the methods as described herein.
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In another aspect of the invention, there is provided a computer-readable
storage
medium comprising instructions which, when executed by a processor, cause the
processor to carry out the methods as described herein.
In another aspect of the invention, there is provided a computer-readable data
carrier
having stored thereon the computer program product as described herein.
In another aspect of the invention, there is provided a data carrier signal
carrying the
computer program product as described herein.
The various illustrative imaging or data processing techniques described in
connection
with the embodiments disclosed herein can be implemented as electronic
hardware,
computer software, or combinations of both. To illustrate this
interchangeability of
hardware and software, various illustrative components, blocks, modules, and
steps
have been described above generally in terms of their functionality. Whether
such
functionality is implemented as hardware or software depends upon the
particular
application and design constraints imposed on the overall system. The
described
functionality can be implemented in varying ways for each particular
application, but such
implementation decisions should not be interpreted as causing a departure from
the
scope of the disclosure.
The various illustrative detection systems described in connection with the
embodiments
disclosed herein can be implemented or performed by a machine, such as a
processor
configured with specific instructions, a digital signal processor (DSP), an
application
specific integrated circuit (ASIC), a field programmable gate array (FPGA) or
other
programmable logic device, discrete gate or transistor logic, discrete
hardware
components, or any combination thereof designed to perform the functions
described
herein. A processor can be a microprocessor, but in the alternative, the
processor can
be a controller, microcontroller, or state machine, combinations of the same,
or the like.
A processor can also be implemented as a combination of computing devices,
e.g., a
combination of a DSP and a microprocessor, a plurality of microprocessors, one
or more
microprocessors in conjunction with a DSP core, or any other such
configuration. For
example, systems described herein may be implemented using a discrete memory
chip,
a portion of memory in a microprocessor, flash, EPROM, or other types of
memory.
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The elements of a method, process, or algorithm described in connection with
the
embodiments disclosed herein can be embodied directly in hardware, in a
software
module executed by a processor, or in a combination of the two. A software
module can
reside in RAM memory, flash memory, ROM memory, EPROM memory, EEPROM
memory, registers, hard disk, a removable disk, a CD-ROM, or any other form of
computer-readable storage medium known in the art. An exemplary storage medium
can
be coupled to the processor such that the processor can read information from,
and write
information to, the storage medium. In the alternative, the storage medium can
be
integral to the processor. The processor and the storage medium can reside in
an ASIC.
A software module can comprise computer-executable instructions which cause a
hardware processor to execute the computer-executable instructions.
Computer-executable instructions may be stored in a (transitory or non-
transitory)
computer readable storage medium (e.g., memory, storage system, etc.) storing
code,
or computer readable instructions.
Additional Notes
The embodiments described herein are exemplary. Modifications, rearrangements,
substitute processes, etc. may be made to these embodiments and still be
encompassed
within the teachings set forth herein. One or more of the steps, processes, or
methods
described herein may be carried out by one or more processing and/or digital
devices,
suitably programmed.
Conditional language used herein, such as, among others, "can," "might,"
"may," "e.g.,"
and the like, unless specifically stated otherwise, or otherwise understood
within the
context as used, is generally intended to convey that certain embodiments
include, while
other embodiments do not include, certain features, elements and/or states.
Thus, such
conditional language is not generally intended to imply that features,
elements and/or
states are in any way required for one or more embodiments or that one or more
embodiments necessarily include logic for deciding, with or without author
input or
prompting, whether these features, elements and/or states are included or are
to be
performed in any particular embodiment. The terms "comprising," "including,"
"having,"
"involving," and the like are synonymous and are used inclusively, in an open-
ended
fashion, and do not exclude additional elements, features, acts, operations,
and so forth.
Also, the term "or' is used in its inclusive sense (and not in its exclusive
sense) so that
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when used, for example, to connect a list of elements, the term "or" means
one, some,
or all of the elements in the list. The term "comprising" may be considered to
encompass
"consisting".
5 Disjunctive language such as the phrase "at least one of X, Y or Z,"
unless specifically
stated otherwise, is otherwise understood with the context as used in general
to present
that an item, term, etc., may be either X, Y or Z, or any combination thereof
(e.g., X, Y
and/or Z). Thus, such disjunctive language is not generally intended to, and
should not,
imply that certain embodiments require at least one of X, at least one of Y or
at least one
10 of Z to each be present.
The terms "about" or "approximate" and the like are synonymous and are used to
indicate
that the value modified by the term has an understood range associated with
it, where
the range can be 20%, 15%, 10%, 5%, or 1%. The term "substantially" is
used to
15 indicate that a result (e.g., measurement value) is close to a targeted
value, where close
can mean, for example, the result is within 80% of the value, within 90% of
the value,
within 95% of the value, or within 99% of the value. The term "partially" is
used to indicate
that an effect is only in part or to a limited extent.
20 Unless otherwise explicitly stated, articles such as "a" or "an" should
generally be
interpreted to include one or more described items. Accordingly, phrases such
as "a
device configured to" or "a device to" are intended to include one or more
recited devices.
Such one or more recited devices can also be collectively configured to carry
out the
stated recitations. For example, "a processor to carry out recitations A, B
and C" can
25 include a first processor configured to carry out recitation A working
in conjunction with
a second processor configured to carry out recitations B and C.
While the above detailed description has shown, described, and pointed out
novel
features as applied to illustrative embodiments, it will be understood that
various
30 omissions, substitutions, and changes in the form and details of the
devices or algorithms
illustrated can be made without departing from the spirit of the disclosure.
As will be
recognized, certain embodiments described herein can be embodied within a form
that
does not provide all of the features and benefits set forth herein, as some
features can
be used or practiced separately from others. All changes which come within the
meaning
35 and range of equivalency of the claims are to be embraced within their
scope.
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It should be appreciated that all combinations of the foregoing concepts
(provided such
concepts are not mutually inconsistent) are contemplated as being part of the
inventive
subject matter disclosed herein. In particular, all combinations of claimed
subject matter
appearing at the end of this disclosure are contemplated as being part of the
inventive
subject matter disclosed herein.
The present invention will now be described by way of the following non-
limiting
examples.
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Examples
Reference Examples 1 to 4 ¨ Purification and deaminase activity of mutant
APOBEC3A
proteins
Swal assay method
This assay was adapted and modified from Schutsky et al., Nucleic Acid
Research, 45,
7655-7665, 2017. doi: 10.1093/nar/gkx345. Modifications to Schutsky et al.
included the
following. Instead of performing DNA precipitation and redissolving the DNA
substrate
into Swal compatible buffer, 1 pL of the altered cytidine deaminase
APOBEC3A(Y130A)
deamination reaction mixture was aliquoted into 9 pL Swal compatible buffer
for
restriction enzyme digestion for our gel assay. Appropriate controls were
performed to
determine the Swal restriction enzyme digestion efficiency was not compromised
by the
APOBEC reaction buffer. Instead of introducing 1.5-fold excess complementary
strand
prior to overnight Swal restriction enzyme digestion, 3-fold excess
complementary strand
was introduced. Instead of running the pre-heated 20% acrylamide/Tris-Borate-
EDTA
(TBE)/urea gel reported by Schutsky et al., the gel run was performed at room
temperature with a 15% acrylamide/Tris-Borate-EDTA (TBE)/urea gel and observed
good resolution between cut (deaminated) and uncut (unreacted) oligo
substrates.
Reference Example 1 ¨ Purification of APOBEC3A(Y130X) mutant proteins
The impact of all possible amino acid substitutions at position 130 of
APOBEC3A on the
deaminase activity of this enzyme was systematically assessed. To this end, 19
different
His-tagged APOBEC3A constructs were cloned, each encoding a different amino
acid at
position 130 relative to the wild type tyrosine. The corresponding proteins
were
expressed in BL21(DE3) cells, purified using Ni-NTA agarose beads, and
desalted/concentrated using spin columns to storage buffer (50mM Tris pH 7.5,
200mM
NaCI, 5%(v/v) glycerol, 0.01% (v/v) Tween-20, 0.5mM DTT). This yielded
APOBEC3A(Y130X) mutant protein preparations with 80-85% purity, as judged by
SOS-
PAGE analysis.
Reference Example 2 ¨ DNA deaminase activity of APOBEC3A(Y130X) mutant
proteins
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The deaminase activity of all purified APOBEC3A(Y130X) proteins was then
analysed
using the Swal assay, with a 37 C/2 hour reaction time and NEB APOBEC3A as
positive
control. 10-20 pM final concentration of Y130X recombinant enzymes were
incubated
with oLB1609 (C oligo, top panel) and oLB1612 (5mC oligo, bottom panel) at 37
C for 2
hours. NEB APOBEC3A enzyme was purchased from NEBNext0 Enzymatic Methyl-seq
Kit (Catalog it E7120). Wild type APOBEC3A deaminated 5mC and C substrates to
completion, consistent with previous literature. Different mutants exhibited a
wide range
of reactivities towards 5mC and C substrates, with some showing preference
towards
either substrate. Remarkably, APOBEC3A(Y130A) (first box) deaminated 5mC
substrates almost completely (94.2%), while it deaminated the corresponding C
substrate to a minor extent (29.4%). Other mutants, such as APOBEC3A(Y130P)
and
APOBEC3A(Y130T), also exhibited more complete deamination of the 5mC than C
substrate, albeit to a lesser extent than APOBEC3A(Y130A). In contrast,
APOBEC3A(Y130L) (second box) deaminated approximately half of the C substrate
(56%), but almost none of the 5mC substrate (6.8%). The deaminase activity of
all
APOBEC3A(Y130X) mutants is quantified and summarised in the table below:
% c % 5mC
Protein % C %
5mC
deamination deamination Protein
deamination deamination
NEB APOBEC 92 94.9
NEB APOBEC 99.3
95.3
Y130A 29.4 94.2
Y130V 11.6
15
Y130G 3.7 19.1
Y130D 0.8
2.3
Y130L 56 6.8
Y130E 0
2.9
Y130F 84.6 94.3
Y130S 53.5
95.4
Y1301 8.1 8.1
Y130C 88.2
96.2
Y1301-1 83.4 95.8
Y130W 97.6
71.6
Y130Q 1.6 14.6
Y130P 0.2
22.3
Y130M 37 55.4
Y13OR 0.6
0.8
Y130N 3.6 9.1
Y130T 8
28.1
Y130K 0.3 0.9
Because these Swal assays were performed as a single endpoint measurement (2
hour),
it could be possible that the respective deamination reactions had already
saturated. A
time course analysis of APOBEC3A(Y130A) deaminase activity was therefore
performed. The extent of C and 5mC deamination was monitored at 0, 5, 10, 30,
60 and
120 minutes by incubation of -10-20 pM of APOBEC(Y130A) with 500nM C and 5mC
oligonucleotide substrate. A greater difference in the extent of 5mC versus C
deamination was observed at t 30 min.
SUBSTITUTE SHEET (RULE 26)
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The kinetics of deamination by wild type APOBEC3A and mutant APOBEC3A(Y130A)
were quantitatively compared. The initial deamination reaction velocity was
measured at
a range of DNA substrate concentrations and used to construct Michaelis-Menten
curves
for 5mC and C substrates, respectively. The resulting Km and Kcat values were
then
derived from these data. The catalytic efficiency of APOBEC3A(Y130A) was -100-
fold
higher on 5mC than C substrates corroborating the endpoint Swal assays shown
above.
Reference Example 3 - Purification of APOBEC3A(Y130A-Y132H) double mutant
protein
APOBEC3A(Y130A-Y132H) protein was expressed in BL21(DE3) cells, purified using
Ni-NTA agarose beads, and desalted/concentrated using spin columns to storage
buffer
(50mM Tris pH 7.5, 200mM NaCI, 5%(v/v) glycerol, 0.01% (v/v) Tween-20, 0.5mM
DTT).
This yielded APOBEC3A(Y130A-Y132H) mutant protein preparations with 90-95%
purity, as judged by SDS-PAGE analysis.
Reference Example 4- DNA deaminase activity of APOBEC3A(Y130A-Y132H) double
mutant protein
The deaminase activity of purified APOBEC3A(Y130A-Y132H) double mutant protein
was then analyzed using the Swal assay, with a 37 C/ 2 hour reaction time and
NEB
APOBEC3A as positive control. The conditions used were the same as described
in
Reference Example 2 with the exception that the Swal assay used reaction
conditions
of 40 mM sodium acetate pH 5.2, 37 C for 1 hour to 16 hours. The DNA
substrates are
shown below:
5' GAGGTGTATGGTTGTACTAAT / 5mC /ACT / 5mC / CT GGA/ 5mC / GAATCTTAA/ 5mC/ACAA/
5mC / G
TGCAG/ 5mC/ CAAA/ 5mC/ GCTT / 5mC/ GC/ 5mC /ACGG/ 5mC /AACGT G/ 5mC/ GGACT /
5mC/ GT CG/
5mC/ CTTA/ 5mC /AAT CG/ 5mC/ GCAGGT/ 5mC /ACGTT GAAGAT GAGGAT G- 3'
GAGGTGTATGGTTGTAG/5mC/GCAAATCGTAAAA/5mC/GCAAAGCGAAAAC/5mC/GCAAACCGTAAA
C/5mC/GAAAAGCGCTTGAAGATGAGGATG
GAGGTGTATGGTTGTAG/5mC/GGAAAACGGAAAT/5mC/GGAAAACGTAAAG/5mC/GTAAATCGGAAA
G/ 5 mC / GAAAAG C G GT T GAAGAT GAG GAT G
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GAGGTGTATGGTTGTAA/5mC/GTAAACCGCAAAC/5mC/GGAAAACGAAAAT/5mC/GCAAACCGAAAA
C/5mC/GTAAAACGCTTGAAGATGAGGATG
GAGGTGTATGGTTGTAA/5mC/GAAAACCGGAAAT/5mC/GAAAAGCGTAAAT/5mC/GTAAATCGCAAA
5 A/ 5 mC / GGAAAT C GAT T GAAGAT GAG GAT G
After the deaminase reaction the deaminated oligo substrates were PCR-
amplified,
sequenced, and the number of C and 5mC deamination events per read were
counted.
APOBEC3A(Y130A-Y132H) exhibited higher levels of deamination at all methylated
10 sites compared to unmethylated sites. This was consistent across both
CpG and non-
CpG contexts, and was robust to variation in reaction time. The difference in
deamination
level between methylated and unmethylated sites was markedly higher for
APOBEC3A(Y130A-Y132H) than APOBEC3A(Y130A),
indicating that
APOBEC3A(Y130A-Y132H) achieves better discrimination of methylated sites than
15 APOBEC3A(Y130A). In addition, APOBEC3A(Y130A-Y132H) deaminated
methylated
sites more efficiently than unmethylated sites across all xCpGx motifs.
Example 1 ¨ Methylation analysis on methylated pUC19 sample using 9 QaM
20 Oligo sequences:
For transposon annealing (underline indicates ME' or ME):
ME'-HYB2 (SEQ ID NO. 21)
/ 5 P h o s /CTGTCTCTTATACACATCTGAGTAAGTGGAGAGATAGGAAGG
25 ME'-HYB2' (SEQ ID NO. 22)
/5Phos/CTGTCTCTTATACACATCTCCTTCCTATCTCTTCCACTTACTC
Biotin-A14-ME (SEQ ID NO. 9)
Bi o in- T C GT C GGCAGC GT CAGAT GT GTATAAGAGACAG
Biotin-B15-ME (SEQ ID NO. 10)
30 Biotin- GT CT C GT GGGCT CGGAGAT GT GTATAAGAGACAG
Sequencing oligos (underline indicates ME):
HYB2-ME (SEQ ID NO. 12)
GAGTAAGTGGAAGAGATAGGAAGGAGATGTGTATAAGAGACAG
35 HYB2'-ME (SEQ ID NO. 14)
CCTTCCTATCTCTTCCACTTACTCAGATGTGTATAAGAGACAG
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Preparation of forked adaptors:
1. 5p1 of 200pM stock of biotin-A14-ME oligo was combined
with 10p1 of 100pM
stock of ME'-HYB2 oligo. 2p1 of 10x TEN Annealing buffer (Illumina) and 3p1
of IDTE buffer (Illumina) was added ("A14" transposome mixture).
2. Separately, 5p1 of 200pM stock of biotin-B15-ME oligo was combined with
10p1 of 100pM stock of ME'-HYB2' oligo. 2p1 of 10x TEN Annealing buffer
(Illumina) and 3p1 of IDTE buffer (Illumina) was added ("B15" transposome
mixture) with 10x TEN and IDTE buffers.
3. Each mixture was heated to 95C for 30s followed by a slow cool (0.10/s
ramp
rate) to 100.
4. 2p1 of each annealed mixture was combined with 46p1 of Standard Storage
Buffer (contains 50% glycerol, Illumina) and 2p1 of Tn5 transposase (-90pM
stock).
5. Each mixture was mixed and incubated overnight at 370. Following the
incubation step, the two separately prepared transposome complexes were
combined together by adding 50p1 of each to another 100p1 of Standard
Storage Buffer to give 200p1 of 1pM transposome mix.
Loading of forked adaptors onto beads:
1. 200p1 of MyOne Ti Streptavidin beads (Thermofisher) were washed twice
with 200p1 Tagmentation Wash buffer (TVVB, Illumina).
2. Beads were resuspended in 960plof TVVB and 40plof 1pM
transposome mix
from step 5 of "Preparation of forked adaptors" was added.
3. Beads were mixed on a rotator for 30mins to lhr at room
temperature.
4. Beads were put on a magnet and beads were washed twice with TVVB.
5. Beads were resuspended in original volume (200p1) of
BLT Storage Buffer
(Illumina). The BLTs were stored at 40 until needed.
Tagmentation:
1. 10p1 BLT (bead linked transposomes) from step 5 of "Loading of forked
adaptors
onto beads" were combined with 10Ong DNA in 30p1(pUC19 methylated control
DNA) and 10p1 of TB1 (5x Tag buffer, Illumina).
2. The combination was mixed and incubated at 550 for 5min, followed by a hold
step at 100.
3. 10p1 ST2 Stop buffer was added and mixed.
4. The mixture was incubated at room temp for 5mins.
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5. The tubes were transferred to a magnet.
6. The beads were washed twice with 100p1 Tagmentation Wash buffer (TWB,
7. The beads were resuspended in 50p1 of ELM (Extension Ligation Mix,
IIlumina).
8. The mixture was incubated at 37C for 5mins, then 50C for 5mins, followed by
a
hold step at 100.
Hybridisation and extension on beads:
1. The tubes from step 8 of "Tagmentation" were placed on a magnet until the
BLT
beads pelleted.
2. The beads were washed once with 200p1 of Tagmentation Wash Buffer (TWB,
3. The beads were washed once with 200p1 of 0.1N NaOH ¨ the beads were left to
sit in 0.1N NaOH for 30s during this wash step.
4. Beads were washed once with 200p1 of TWB.
5. Bears were resuspended in 100p1 of HT1 (Hybridisation Buffer, IIlumina).
6. Beads were heated in HT1 to 700 for 30s followed by a slow cool (0.1C/s)
down
to 100.
7. Beads were washed twice with 200p1 of TWB.
8. Beads were resuspended in 100plof PAM (Patterned Amplification Mix,
IIlumina)
supplemented with 50mM KCI.
9. Beads were heated in PAM to 50C for 5mins, then 60C for 5mins.
10. Beads were washed twice with 200p1 of TWB.
11. Beads were resuspended in 50p1 of RSB (Resuspension Buffer, IIlumina).
Methylation analysis conversion method:
1. The following TET master mix (TET MM) was prepared and kept on ice:
lx (pi) 4.5x
(p1)
Water 9.00 40.50
Reconstituted TET2 Reaction Buffer (NEB
EM-seq kit) 10 45
Oxidation Supplement (NEB EM-seq kit) 1 4.5
DTT (NEB EM-seq kit) 1 4.5
TET2 (NEB EM-seq kit) 4 18
Total 25 112.5
2. On ice, 25p1 of TET MM was added to 20plof adaptor-ligated DNA in the form
of
BLTs in RSB (from step 11 of "Hybridisation and extension on beads").
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3. The mixture was vortexed and centrifuged briefly.
4. 500mM of Fe(II) solution (NEB EM-seq kit) was freshly prepared and diluted
by
adding 1p1 to 1249p1 of water.
5. 5p1 of the diluted Fe(II) solution was added to the 45 pl of adaptor-
ligated DNA
with TET MM prepared in step 2.
6. The mixture was vortexed (or pipette mixed 10x), centrifuged briefly,
incubated
for lhr at 37C, then put on ice.
7. 1p1 of Stop reagent was added, vortexed (or pipette mixed 10x), and
incubated
at 37C for 30 mins.
8. The beads were washed once with 100p1 Wash buffer, and then resuspended in
35p1 water.
9. In a PCR tube, the 35p1 of TET-oxidised DNA from step 8 was combined with
10p1 of sodium acetate / acetic acid buffer (pH 4.3) and 5 pl of 1 M pyridine
borane. The mixture was incubated overnight at 400.
10. The beads were washed twice with 100p1 Wash buffer, then resuspended in 20
pl of RSB.
11. The 20u1 of beads+DNA in RSB from step 10 was combined with 25p1 of Q5U
Mastermix (NEB) and 5p1 of UDI primers (Unique Dual Index primers, IIlumina).
12. The mixture was amplified by PCR: cycling procedure ¨ 980 for 30s followed
by
3 cycles of (980 10s, 620 30s, 650 3min), then 6 cycles of (98C 10s, 620 30s,
65C 30s), 65C for 5 mins and then hold at 4C.
13. PCR products were analysed by TapeStation D1000 (Agilent), and then
subjected to a further SPRI clean-up before quantification using a Qubit Broad
Range dsDNA assay kit (Thermofisher).
Sequencing:
Sequencing was conducted on the MiniSeq. Standard clustering on the MiniSeq
and a
standard first hyb was conducted for the 1s! 36 cycles of sequencing.
A custom second hyb was used from the "Cust3" position of the reagent
cartridge. This
primer hyb maintains a higher temperature (60C) than normal during the post-
hyb wash
(which usually drops to 400). This higher temperature was to ensure that the
right
sequencing primers hyb to the right places on the cluster strands.
The primer mix for this custom hyb was HP10 R1 primer mix (Illumina) spiked
with 0.5pM
each of HYB2'-ME and HYB2-ME primers. These primers are all unblocked and
allow
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concurrent sequencing of both the first portion and the second portion, and so
generate
the 9 QaM signal during sequencing. The converted library was loaded onto the
MiniSeq
cartridge at 1pM final concentration. The MiniSeq was set up to save 3 tiles
of images
per cycle, for later off-line analysis. The 9 QaM results are shown in Figure
19A, where
modified cytosines can be identified by a characteristic central cloud in the
plot (indicated
by circled region). The actual genetic sequences are shown in Figure 19B,
where
modified cytosines can be assigned to cases where a C-T mismatch is observed
between the HYB2'-ME read and the HP10 read.
Overall, these results (in particular the custom second hyb results) show that
methylation
analysis (in this case, all 5-mC, 5-hmC, 5-fC and 5-caC, as a result of TAPS
analysis)
can be conducted on polynucleotide sequences to identify modified cytosines.
In
particular, by enabling concurrent sequencing of the forward and reverse
complement
strands of the template (or reverse and forward complement strands of the
template),
modified cytosines can be identified quickly and accurately.
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SEQUENCE LISTING
(Underlined sequences are ME or ME' sequences)
SEQ ID NO. 1: P5 sequence
5
AATGATACGGCGACCACCGAGATCTACAC
SEQ ID NO. 2: P7 sequence
10 CAAGCAGAAGACGGCATACGAGAT
SEQ ID NO. 3: P5' sequence (complementary to P5)
GIGTAGATCTCGGIGGICGCCGTATCATT
SEQ ID NO. 4: P7' sequence (complementary to P7)
ATCTCGTATGCCGTCTTCTGCTTG
SEQ ID NO. 5: Alternative P5 sequence
AATGATACGGCGACCGA
SEQ ID NO. 6: Alternative P5' sequence (complementary to alternative P5
sequence)
TCGGTCGCCGTATCATT
SEQ ID NO. 7: A14
TCGTCGGCAGCGTC
SEQ ID NO. 8: B15
GTCTCGTGGGCTCGG
SEQ ID NO. 9: A14-ME
TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
SEQ ID NO. 10: B15-ME
GICTCGTGGGCTCGGAGATGIGTATAAGAGACAG
SEQ ID NO. 11: HYB2
GAGTAAGTGGAAGAGATAGGAAGG
SEQ ID NO. 12: HYB2-ME
GAGTAAGT GGAAGAGATAGGAAGGAGAT GT GTATAAGAGACAG
SEQ ID NO. 13: HYB2'
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CCT TCCTAT CT CT TCCACT TACT C
SEQ ID NO. 14: HYB2'-ME
CCTICCIATCTCTICCACTTACTCAGATGIGTATAAGAGACAG
SEQ ID NO. 15: HYB2'-block
CCTTCCTATCTCTTCCACTTACT- 3 ' pr opano 1
SEQ ID NO. 16: HYB2'-ME-block
CCT TCCTATCTCTTCCACTTACTCAGATGTGTATAAGAGACAG- 3 propano 1
SEQ ID NO. 17: ME'-A14'
CIGICICTTATACACATCTGACGCTGCCGACGA
SEQ ID NO. 18: A14'
GACGCTGCCGACGA
SEQ ID NO. 19: ME'-B15'
CTGTCTCTTATACACATCTCCGAGCCCACGAGAC
SEQ ID NO. 20: B15'
C CGAGCC CAC GAGAC
SEQ ID NO. 21: ME'-HYB2
CTGTCTCTTATACACATCTGAGTAAGTGGAAGAGATAGGAAGG
SEQ ID NO. 22: ME'-HYB2'
CTGTCTCTTATACACATCTCCT TCCTAT CT CT TCCACT TACT C
SEQ ID NO. 23: UniProt Q9GZX7
MDSLLMNRRKFLYQFKNVRWAKGRRETYLCYVVKRRDSAT SFSLDFGYLRNKNGCHVELLFLRY
I SDWDLDPGRCY RVTW FT SWS PCY DCARHVAD FLRGNPNL SLRI FT ARLY FCEDRKAEPEGLRR
LHRAGVQ IAIMT FKDY FYCWNT FVENHERT FKAWEGLHENSVRLSRQLRRILLPLYEVDDLRDA
FRTLGL
SEQ ID NO. 24: UniProt G3QLD2
MDSLLMNRRKFLYQFKNVRWAKGRRETYLCYVVKRRDSAT SFSLDFGYLRNKNGCHVELLFLRY
I SDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRGNPNLSLRI FTARLY FCEDRKAEPEGLRR
LHRAGVQ IAIMT FKENHERT FKAWEGLHENSVRLSRQLRRILLPLYEVDDLRDAFRILGL
SEQ ID NO. 25: Uniprot Q9VVVE0
CA 03223669 2023- 12- 20
WO 2023/175040
PCT/EP2023/056668
92
MDSLLMKQKKFLY H FKNVRWAKGRHETYLCYVVKRRDSAT SC SLDFGHLRNKSGCHVELL FLRY
I SDWDLDPGRCYRVTWFTSWSPCYDCARHVAE FLRWNPNLSLRI FTARLY FCEDRKAEPEGLRR
LHRAGVQ IGIMT FKDY FYCWNT FVENRERT FKAWEGLHENSVRLTRQLRRILLPLYEVDDLRDA
FRMLGF
SEQ ID NO. 26: UniProt P41238
MT SEKGP STGDPTLRRRIE PWE FDVFY DPRELRKEACLLY E I KWGMSRKI WRS SGKNTINHVEV
NFIKKFT SERDFHPSMSCS ITWFLSWSPCWEC SQAI RE FL SRHPGVTLVI YVARL FWHMDQQNR
QGLRDLVNSGVT IQ IMRASEYYHCWRNFVNY P PGDEAHWPQY PPLWMMLYALELHC I IL SL PPC
LKI SRRWQNHLT F FRLHLQNCHYQT I PPHILLATGL I HP SVAWR
SEQ ID NO. 27: NCB! XP_030856728.1
MSRKIWRSSGKNTINHVEVNE KKFT SERHFHPS SCS ITWFLSWS PCWEC SQAI RE FL SQHPG
VTLVIYVARLFWHMDQQNRQGLRDLVNSGVT I Q IMRAS EYYHCWRN FVNY PPGDEAHWPQY PPL
WMMLYALELHC I ILSLPPCLKISRRWQNHLT FFRLHLQNCHYQT I P PH ILLATGL I HP SVAWR
SEQ ID NO. 28: Uniprot P51908
MSSETGPVAVDPILRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSVWRHTSQNTSNHVEV
NFLEKFTTERY FRPNTRCS ITWFLSWSPCGECSRAITE FL SRHPYVTL FIY IARLYHHTDQRNR
QGLRDL I SSGVT IQ IMT EQEYCYCWRNFVNY P PSNEAYWPRY PHLWVKLYVLELYC I ILGLPPC
LKILRRKQPQLT F FT ITLQTCHYQRI PPHLLWATGLK
SEQ ID NO. 29: UniProt Q9Y235
MAQKEEAAVAT EAASQNGEDLENLDDPEKLKEL I EL PP FE IVTGERLPANFFKFQ FRNVEY SSG
RNKT FLCYVVEAQGKGGQVQASRGYLEDEHAAAHAEEAFFNT IL PAFDPALRYNVTWYVS S S PC
AACADRI IKTLSKTKNLRLLILVGRLFMWEEPEIQAALKKLKEAGCKLRIMKPQDFEYVWQNFV
EQEEGESKAFQPWEDIQENFLYYEEKLADILK
SEQ ID NO. 30: Uniprot G3SGN8
MAQKE EAAAAT EAAAAT EAASQNGE DLENLDDPE KLKEL I EL PP FE IVTGERL PANE FKFQ FRN
VEY SSGRNKT FLCYVVEAQGKGGQVQASRGYLEDEHAA_AHAERAFFNT IL PAFDPALRYNVTWY
VSSSPCAACADRI IKTLSKTKNLRLL ILVGRL FMWEEPEIQAALKKLKEAGCKLRIMKPQDFEY
VWQNFVEQEEGESKAFQPWEDIQENFLYYEEKLADILK
SEQ ID NO. 31: Uniprot 09VVV35
MAQKE EAAEAAAPASQNGDDLENLE DPE KLKEL DL PP FE IVTGVRLPVN FFKFQ FRNVEY SSG
RNKT FLCYVVEVQSKGGQAQATQGYLEDEHAGAHAEEAFFNT IL PAFDPALKYNVTWYVS S S PC
AACADRI LKTL SKTKNLRLL LVSRL FMWE E PEVQAALKKLKEAGCKLRIMKPQD FEY IWQNFV
EQEEGESKA.FEPWEDIQENFLYYEEKLA.DILK
SEQ ID NO. 32: UniProt P31941
MEAS PASGPRHLMDPH I FT SN FNNG I GRHKTYLCYEVE RLDNGT SVKMDQHRG FLHNQAKNLLC
GFYGRHAELRFLDLVPSLQLDPAQ I Y RVTW FI SWSPCFSWGCAGEVRAFLQENTHVRLRI FAAR
I YDYDPLYKEALQMLRDAGAQVS IMTYDEFKHCWDT FVDHQGCP FQPWDGLDEHSQALSGRLRA.
I LQNQGN
SEQ ID NO. 33: GenBank XP_045219544.1
CA 03223669 2023- 12- 20
WO 2023/175040
PCT/EP2023/056668
93
MDGS PAS RPRHLMDPNT FT FN FNNDL SVRGRHQT YLCY EVERLDNGTWVPMDE RRGFLHNKAKN
VPCGDYGCHVELRFLCEVP SWQLDPAQT YRVTWF I SWS PC FRKGCAGQVRAFLQENKHVRLRI F
AARIYDYDPRYQEALRTLRDAGAQVS IMTYEE FKHCWDT FVDRQGRPFQPWDGLDEHSQALSGR
LRAILQNQGN
SEQ ID NO. 34: GenBank AER45717.1
MEASPASGPRHLMDPCVFT SN FNNG I RWHKT YLCY EVERLDNGTWVKMDQHRG FL HNQARNPLY
GLDGRHAELRFLGLL PYWQLDPAQ I Y RVTW FI SWS PC FSWGCARQVRAFLQENTHVRLRI FAR
I YDYDPLYKEALQMLRDAGAQVS IMTYDE FEYCWNT FVDHQGCP FQPWDGLEEHSQALSGKLQA
I LLNQGN
SEQ ID NO. 35: GenBank XP_003264816.1
MEAS PAS RPGHLMDPQVFT SN FNNG I RWHKTYLCYEVE RLDNGTWVKMDQHRG FL HNQAKNL FC
GFYGRHAELCFLDLVPSLQLDPAQTYRVTWFI SWS PC FSWGCAEQVRAFLQENTHVRLRL FAAR
I YDYDPLYKEALQMLRGAGAQVS IMTYHE FKHCWDT FVDHQGRP FQPWDGLEEHSQALSGRLQA
I LQNQGN
SEQ ID NO. 36: GenBank PNI48846.1
T EAS PASGPRHLMDPH I FT SN FNNG IGRRKT YLCY EVERLDNGT SVKMDQHRG FL HNQAKNLLC
G FYGRHAELC FLDLVPSLQLDPAQ I Y RVTW FI SWS PC FSWGCAGQVRAFLQENTHVRLRI FAR
I YDYDPLYKEALQMLRDAGAQVS IMTYDE FKHCWDT FVDHQGCP FQPWDGLEEHSQALSGRLRA
I LQNQGN
SEQ ID NO. 37: GenBank AD085886.1
VEAS PASGPRHLMDPH I FT SNFNNVI GRHKTYLCY EVERLDNGTWVKMDQHRG FL HNQAKNLLC
G FYGRHAELRFLDLVPSLQLDPAQ I Y RVTW FI SWS PC FSWGCAGQVRAFLQENTHVRLH I FAR
I YDYDPLYKEALQMLRDAGAQVS IMTYDE FKHCWDT FVDHQGCP FQPWDGLEEHSQALSGRLRA
I LQNQGN
SEQ ID NO. 38: UniProt Q9UH17
MNPQ I RNPMERMY RDT FYDNFENEP ILYGRSYTWLCYEVKIKRGRSNLLWDTGVFRGQVY FKPQ
Y HAEMC FL SWFCGNQLPAY KC FQ ITWFVSWTPCPDCVAKLAE FL SE HPNVTLT I SAARLY Y YWE
RDYRRALCRLSQAGARVT IMDYEE FAYCWENFVYNEGQQFMPWYKFDENYAFLHRTLKE I LRYL
MDPDT FT FNFNNDPLVLRRRQTYLCYEVERLDNGTWVLMDQHMGFLCNEAKNLLCGFYGRHAEL
RFLDLVPSLQLDPAQ IYRVTWFI SWS PC FSWGCAGEVRAFLQENTHVRLRI FAARIYDYDPLYK
EALQMLRDAGAQVS IMTYDE FEYCWDT FVYRQGCPFQPWDGLEEHSQALSGRLRAILQNQGN
SEQ ID NO. 39: Uniprot G3QV16
MNPQ I RNPMERMY RGT FYNNFENEP ILYGRSYNWLCYEVKIKRGRSNLLWNTGVFRGQMYSQPE
HHAEMC FL SWFCGNQLPAY KC FQ ITWFVSWTPCPDCVAKLAE FLAEYPNVTLT I STARLY Y YWE
RDYRRALCRLSQAGARMKIMDYEECAYCWENFVYKEGQQFMPWYKFDENYAFLHHTLKE I LRHL
MDPDT FT FN FNNDPLVLRRHQTYLCY EVERLDNGTWVLMDRHMG FLCNEAKNLLCGFYGRHAEL
RFLDLVPSLQLDPAQ IYRVTWFI SWS PC FSWGCAGQVCE FLQENTHMRLRI FAARIYDYDPLYK
KALQMLRDAGAQVS IMTYDE FKHCWDT FVYRQGCPFQPWDGLEEHSQALSGRLQAILQNQGN
SEQ ID NO. 40: Uniprot F6M3K5
CA 03223669 2023- 12- 20
WO 2023/175040
PCT/EP2023/056668
94
MNPQI RNPMERMY RRT FNYNFENEP ILYGRSYTWLCYEVKIRKDPSKLPWDTGVERGQMYSKPE
HHAEMCFLSWFCGNQLPAHKRFQ ITWFVSWTPCPDCVAKVAE FLAEYPNVTLT I SAARLYYYWE
TDYRRALCRLRQAGARVKIMDYEE FAYCWENFVYNEDQSFMPWYKFDDNYAFLHHKLKE I LRHL
MDPDT FT SNFNNDLSVLGRHQTYLCYEVERLDNGTWVPMDQHWGFLCNQAKNVPRGDYGCHAEL
C FLDQVS SWQLDPAQTYRVTWFI SWS PC FSWGCADQVYAFLQENT HVRLRI FAARIYDYNPLYQ
EALRTLRDAGAQVSIMTYDE FEYCWDT FVDRQGRPFQPWDGLDEHSQALSGRLRA ILQNQGN
SEQ ID NO. 41: UniProt Q9NRW3
MNPQ I RNPMKAMY PGT FY FQFKNLWEANDRNETWLC FTVEGI KRRSVVSWKTGVFRNQVDS ET H
CHAERC FLSWFCDDI LS PNTKYQVIWYT SWSPCPDCAGEVAE FLARHSNVNLT I FTARLYY FQY
PCYQEGLRSLSQEGVAVE IMDYEDFKYCWENFVYNDNE PFKPWKGLKTNFRLLKRRLRE SLQ
SEQ ID NO. 42: Uniprot Q694B5
MNPQ I RNPMKAMY PGT FY FQFKNLWEANDRNETWLC FTVEGI KRRSVVSWKTGVFRNQVDS ET H
CHAERCFLSWFCDDILS PNTNYQVTWYT SWSPCPECAGEVAE FLARHSNVNLT I FTARLYY FQD
TDYQEGLRSLSQEGVAVKIMDYKDFKYCWENFVYNDDE PFKPWKGLKYNFRFLKRRLQE ILE
SEQ ID NO. 43: Uniprot BOLVV74
MNPQ I RNPMKAMDPGT FY FQ FKNLWEANNRNETWLC FIVE VI KQHSTVSWETGVFRNQVDLET H
CHAERCFLSWFCEDILS PNTDYQVIWYT SWSPCLDCAGEVAKFLARHNNVMLT IYTARLYY SQY
PNYQQGLRSLSEKGVSVKIMDYEDFKYCWEKFVYDDGE PFKPWKGLKT SFRFLKRRLRE ILQ
SEQ ID NO. 44: UniProt Q96AK3
MNPQ I RNPMERMY RDT FYDNFENEP ILYGRSYTWLCYEVKIKRGRSNLLWDTGVFRGPVLPKRQ
SNHRQEVY FRFENHAEMCFLSWFCGNRLPANRRFQ ITWFVSWNPCLPCVVKVT KFLAEH PNVTL
T SAARLYYYRDRDWRWVLLRLHKAGARVKIMDY ED FAYCWENFVCNEGQ P FMPWYKFDDNYAS
LHRTLKE ILRNPME AMY PHI FY FHFKNLLKACGRNE SWLC FTMEVT KHHSAVFRKRGVFRNQVD
PET HCHAERC FLSWFCDDI LS PNTNYEVTWYT SWSPCPECAGEVAE FLARHSNVNLT I FTARLC
Y FWDTDYQEGLCSLSQEGASVKIMGYKDFVSCWKNFVYSDDEP FKPWKGLQINFRLLKRRLRE I
LQ
SEQ ID NO. 45: NCB! NP_001332895.1
MNPQ I RNPMERMY RRT FYNHFENEP ILYGRSYTWLCYEVKIKRGCSNL IWDTGVFRGPVLPKLQ
SNHRQEVY FQ FENHAEMC F FSWFCGNRL PANRRFQ I TW FVSWNPCL PCVVKVT KFLAEH PNVTL
T I SAARLYYYQDREWRRVLRRLHKAGARVKIMDYKDFAHCWEN FVYNEGQQ FMPWYKFDDNYAS
LHRTLKE ILRNPMEAMY PHVFY FHFENLLKACGRNE SWLC FTVDVTEHHPPVSWERGVFRNPVD
PET HCHAERC FLSWFCDDI LS PNTNYQVTWYT SWSPCPECAREVAE FLARHSNVKLT I FTARLY
HFWNTDYQEGLCSLSQEGASVKIMSYKDFVSCWKNFVY SDDE PFKPWKGLKTNFRLLKTMLRE I
LQ
SEQ ID NO. 46: NCB! NP_001332931.1
MNPQ I RNPMERMY RRT FNYNFENEP ILYGRSYTWLCYEVKIRKDPSKLPWDTGVFRGQVY FQPQ
YHAEMCFLSWFCGNQLPAYKRFQ ITWFVSWNPCPDCVAKVTE FLAEHPNVTLT I S VARLYYYRG
KDWRRALCRLHQAGARVKIMDYEE FAYCWENFVYNEGQSFMPWDKFDDNYAFLHHKLKE I LRNP
MKAMY PHT FY FHFENLQKAYGRNETWLCFAVE I I KQH STVPWKTGVFRNQVDPET HCHAERC FL
SWFCDNILSPKKNYQVIWY I SWS PCPECAGEVAE FLAT HSNVKLT I YTARLYY FWDTDYQEGLR
SLSEEGASMEIMGYEDFKYCWENFVYNDGEPFKPWKGINTNFRFLERRLWKILQ
CA 03223669 2023- 12- 20
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SEQ ID NO. 47: UniProt Q8IUX4
MKPHFRNTVERMY RDT FSYNFYNRP ILSRRNTVWLCYEVKTKGPSRPRLDAKI FRGQVY SQPEH
HAEMC FL SW FCGNQLPAY KC FQ ITWFVSWT PC PDCVAKLAE FLAE HPNVTLT I SAARLYYYWER
5 DYRRALCRLSQAGARVKIMDDEE FAYCWENFVYSEGQP FMPWYKFDDNYAFLHRTLKE I LRNPM
EAMY PH I FY FH FKNLRKAYGRNE SWLC FTMEVVKHH S PVSWKRGVFRNQVDPETHCHAE RC FL S
W FCDD IL S PNTNY EVTWYT SWSPCPECAGEVAEFLARHSNVNLT I FTARLYY FWDTDYQEGLRS
LSQEGASVE IMGYKDFIKYCWENFVYNDDEP FKPWKGLKYNFL FLDSKLQE ILE
10 SEQ ID NO. 48: Uniprot G3RD21
MKPQ FRNTVERMY RGT FSYNFNNRP ILSRRNTVWLCYEVKIKGPSRPPLDAKI FRGQVY FQPQY
HAEMC FL SW FCGNQL PAYKC FQ I TC FVSWT PC PDCVAKLAE FLAEH PNVTLT I SAARLYYYWER
DYRRALRRLRQAGARVKIMDDEE FAYCWENFVY SEGQPFMPWHKFDDNYAFLHRTLKE ILRNPM
15 EAMY PH I FY FH FKNLLKAYGRNE SWLC FTMEVI KHH S PVSWKRGVFRNQVDS ETHCHAE
RC FL S
W FCDD IL S PNTNYQVTWYT SWSPCPECAGEVAEFLARHSNVNLT I FTARLYY FWDTDYQEGLRS
LNQEGASVKIMGYKDFKYCWENFVYNDDEPFKPWKGLKYNFL FLDSKLQE ILE
SEQ ID NO. 49: Uniprot Q1GOZ6
MQPQYRNTVERMYRGT FFYNFNNRP I LS RRNTVWLCYEVKTRGP SMPTWDTKI FRGQVY SKPEH
HAEMC FL SRFCGNQL PAYKRFQ I TW FVSWT PC PDCVAKVAE FLAEH PNVTLT I SAARLYYYWET
DYRRALCRLRQAGARVKIMDYEE FAYCWENFVYNEGQS FMPWDKFDDNYAFLHHKLKE I LRNPM
EAT Y PHI FY FH FKNLRKAYGRNETWLC FTME I IKQH STVSWETCVFRNQVDPE SRCHAE RC FL S
W FCED IL S PNT DYQVTWYT SWSPCLDCAGEVAEFLARHSNVKLAI FAARLYY FWDTHYQQGLRS
LSEKGASVE IMGYKDFKYCWENFVYNGDEPFKPWKGLKYNFL FLDSKLQE ILE
SEQ ID NO. 50: UniProt Q9HC16
MKPHFRNTVERMY RDT FSYNFYNRP ILSRRNTVWLCYEVKTKGPSRPPLDAKI FRGQVY SELKY
HPEMRFFHWFSKWRKLHRDQEYEVTWY I SWSPCTKCTRDMAT FLAEDPKVTLT I FVARLYY FWD
PDYQEALRSLCQKRDGPRATMKIMNY DE FQHCWSKFVY SQRELFEPWNNLPKYY ILLHIMLGE I
LRH SMDP PT FT FNFNNEPWVRGRHETYLCYEVERMHNDTWVLLNQRRGFLCNQAPHKHGFLEGR
HAELCFLDVIP FWKLDLDQDY RVTC FT SWS PC FSCAQEMAKF I SKNKHVSLC I FTARIYDDQGR
CQEGLRTLAEAGAKI S IMTY SE FKHCWDT FVDHQGCP FQPWDGLDEHSQDLSGRLRAILQNQEN
SEQ ID NO. 51: Uniprot Q694C1
MTPQ FRNTVERMY RDT FSYNFNNRP ILSRRNTVWLCYEVKIKDPSRPPLDAKI FRGQVY SELKY
HPEMRFFHWFSKWRKLHRDQEYEVTWY I SWSPCTKCTRNVAT FLAEDPKVTLT I FVARLYY FWD
QDYQEALRSLCQKRDGFRATMKIMNY DE FQHCWSKFVY SQREL FE FWNNL FKYYMLLH IMLGE I
LRHSMDPPT FT SNFNNEHWVRGRHETYLCY EVERLHNDTWVLLNQRRGFLCNQAPHKHG FLEGR
HAELCFLDVIP FWKLDLHQDY RVTC FT SWS PC FSCAQEMAKF I SNKKHVSLC I FAARIYDDQGR
CQEGLRTLAEAGAKI S IMTY SE FKHCWDT FVYHQGCPFQFWDGLEEHSQALSGRLQAILQNQGN
SEQ ID NO. 52: Uniprot U5NDB3
MNPQ I RNMVE PMDPRT FVSN FNNRP I LSGLNTVWLCCEVKT KDPSGP PLDAKI FQGKVLRSKAK
Y HPEMRFLQWFREWRQLHHDQEY KVTINYVSWS PCTRCANSVAT FLAKDPKVTLT I FVARLYY FW
KPNYQQALRILCQKRDGPHATMKIMNYNEFQDCWNKFVDGRGKP FKPWNNLPKHYTLLQATLGE
LLRHLMDPGT FT SNFNNKPWVSGQHETYLCYKVERLHNDTWVPLNQHRGFLRNQAPNIHGFPKG
RHAELC FLDL I P FWKLDGQQYRVTC FT SWS PC FSCAQEMAKFI SNNEHVSLC I FAARIYDDQGR
YQEGLRTLHRDGAKIAMMNY SE FEYCWDT FVDCQGCPFQPWDGLDEHSQALSERLRAILQNQGN
CA 03223669 2023- 12- 20
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PCT/EP2023/056668
96
SEQ ID NO. 53: UniProt Q6NTF7
MALLTAETFRLQFNNKRRLRRPYYPRKALLCYQLTPQNGSTPTRGY FENKKKCHAE ICF INE I K
SMGLDETQCYQVTCYLTWS PCSSCAWELVDFI KAHDHLNLGI FASRLYYHWCKPQQKGLRLLCG
SQVPVEVMGFPE FADCWENFVDHEKPLS FNPYKMLEELDKNSRAIKRRLERIKIPGVRAQGRYM
DILCDAEV
SEQ ID NO. 54: Uniprot B7TOU7
MALLTAETFRLQFNNKLRLRRPYYRRKTLLCYQLTPQNGSMPTRGY FKNKKKCHAE ICF INE I K
SMGLDETQCYQVICYLTWSPCSSCAWKLVDFIKAHDHLNLRI FASRLYYHWCKRQQEGLRLLCG
SQVPVEVMGFPEFADCWENFVDHEKPLSFDPSKMLEELDKNSQAIKRRLERIKSRSVDVLENGL
RSLQLGPVT PS SSRSNSR
SEQ ID NO. 55: Uniprot Q19Q52
MALLTAKT FSLQFNNKRRVNKPYYPRKALLCYQLT PQNGST PTRGHLKNKKKDHAE I RF INKI K
SMGLDETQCYQVTCYLTWSPCPSCAGELVDFIKAHRHLNLRI FASRLYYHWRPNYQEGLLLLCG
SQVPVEVMGLPE FTDCWENFVDHKE P PS FNPSEKLEELDKNSQAIKRRLERIKSRSVDVLENGL
RSLQLGPVT PS SS IRNSR
SEQ ID NO. 56: UniProt Q8VVVV27
MEP IY EEYLANHGT IVKPYYWLS FSLDCSNCPYH IRTGEEARVSLT E FCQ I FGFPYGTT FPQTK
HLT FY ELKT SSGSLVQKGHAS SCTGNY I HPESML FEMNGYLDSAIYNNDS IRH I ILY SNNS PCN
EANHCCI SKMYNFLITYPGITLS IY FSQLY HT EMDFPASAWNREALRSLASLWPRVVLS P I SGG
IWHSVLHS F I SGVSGSHVFQP ILTGRALADRHNAYE INAITGVKPY FT DVLLQTKRNPNTKAQE
ALE SY PLNNAFPGQ F FQMP SGQLQPNLP PDLRAPVVFVLVPLRDLP PMHMGQNPNKPRN IVRHL
NMPQMSFQETKDLGRLPTGRSVE IVE I TEQ FAS S KEADEKKKKKGKK
SEQ ID NO. 57: NCB! XP_004028087.1
MEP IY EEYLANHGT IVKPYYWLS FSLDCSNCPYH IRTGEEARVSLT E FCQ I FGFPYGTT FPQTK
HLT FY ELKT SSGSLVQKGHAS SCTGNY I HPESML FEMNGYLDSAIYNNDS I RH I ILY SNNS PCN
EANHCCI SKMYNFLITYPGITLS IY FSQLY HT EMDFPASAWNREALRSLASLWPRVVLS P I SGG
IWHSVLHS F I SGVSGSHVFQP ILTGRALADRHNAYE INAITGVKPY FT DVLLQTKRNPNTKAQE
ALE SY PLNNAFPGQS FQMPSGQLQPNLPPDVRAPVVFVLVPLRDLPPMHMGQNPNKPRNIVRHL
NMPQMSFQETEDLGRLPTGRSVE IVE IT ERFAS S KEADEKKKKKKGKK
SEQ ID NO. 58: Uniprot Q497M3
MEPLYEE ILTQGGTIVKPYYWLSLSLGCTNCPYHIRTGEEARVPYTEFHQTFGFPWSTYPQTKH
LT FYELRSS SKNL IQKGLASNCTGSHNHPEAMLFEKNGYLDAVI FHNSNI RH I ILYSNNSPCNE
AKHCC SKMYNFLMNYPEVTL SVFFSQLYHTEKQ FPT SAWNRKALQ SLASLWPQVIL SP ICGGL
WHAILEKFVSNISGSTVPQPFIAGRILADRYNTYE INS I IAAKPY FTDGLLSRQKENQNREAWA
AFEKHPLGSAAPAQRQPTRGQDPRT PAVLMLVSNRDLP P I HVGST PQKPRTVVRHLNMLQL SS F
KVKDVKKPPSGRPVEEVEVMKESARSQKANKKNRSQWKKQTLVIKPRICRLLER
SEQ ID NO. 59: UniProt Q8NFU7
MSRSRHARP SRLVRKEDVNKKKKNSQLRKTTKGANKNVASVKIL S PGKLKQL I QE RDVKKKTE P
KPPVPVRSLLTRAGAARMNLDRTEVL FQNPESLTCNGFTMALRSTSLSRRLSQPPLVVAKSKKV
PLSKGLEKQHDCDYKILPALGVKHSENDSVPMQDTQVLPDIETL IGVQNPSLLKGKSQETTQFW
SQRVEDSKINI PT HSGPAAE ILPGPLEGTRCGEGL FSEETLNDT SGSPKMFAQDTVCAPFPQRA
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TPKVTSQGNPSIQLEELGSRVESLKLSDSYLDPIKSEHDCYPTSSLNKVIPDLNLRNCLALGGS
TSPTSVIKFLLAGSKQATLGAKPDHQEAFEATANQQEVSDTTSFLGQAFGAIPHQWELPGADPV
HGEALGETPDLPEIPGAIPVQGEVFGTILDQQETLGMSGSVVPDLPVFLPVPPNPIATFNAPSK
WPEPQSTVSYGLAVQGAIQILPLGSGHTPQSSSNSEKNSLPPVMAISNVENEKQVHISFLPANT
QGFPLAPERGLFHASLGIAQLSQAGPSKSDRGSSQVSVISTVHVVNTTVVIMPVPMVSTSSSSY
TILLPTLEKKKRKRCGVCEPCQQKTNCGECTYCKNRKNSHQICKKRKCEELKKKPSVVVPLEVI
KENKRPQREKKPKVLKADFDNKPVNGPKSESMDYSRCGHGEEQKLELNPHTVENVTKNEDSMTG
IEVEKWTQNKKSQLTDHVKGDFSANVPEAEKSKNSEVDKKRTKSPKLFVQTVRNGIKHVHCLPA
ETNVSFKKFNIEEFGKTLENNSYKFLKDTANHKNAMSSVATOMSCDHLKGRSNVLVFQQPG.FNC
SSIPHSSHSIINHHASIHNEGDQPKTPENIPSKEPKDGSPVQPSLLSLMKDRRLTLEQVVAIEA
LTQLSEAPSENSSPSKSEKDEESEQRTASLLNSCKAILYTVRKDLQDPNLQGEPPKLNHCPSLE
KQSSCNTVVFNGQTTTLSNSHINSATNQASTKSHEYSKVINSLSLFIPKSNSSKIDINKSIAQG
IITLDNCSNDLHQLPPRNNEVEYCNQLLDSSKKLDSDDLSCQDATHTQIEEDVATQLTQLASII
KINYIKPEDKKVESTPTSLVTCNVQQKYNQEKGTIQQKPPSSVHNNHGSSLTKQKNPTQKKTKS
TPSRDRRKKKPTVVSYQENDRQKWEKLSYMYGTICDIWIASKFQNFGQFCPHDFPTVFGKISSS
TKIWKPLAQTRSIMQPKTVFPFLTQIKLQRYPESAEEKVKVEPLDSLSLFHLKTESNGKAFTDK
AYNSQVQLTVNANQKAHPLTQPSSPPNQCANVMAGDDQIRFQQVVKEQLMHQRLPTLPGISHET
PLPESALTLRNVNVVCSGGITVVSTKSEEEVCSSSFGTSEFSTVDSAQKNFNDYAMNFFTNPTK
NLVSITKDSELPTCSCLDRVIQKDKGPYYTHLGAGPSVAAVREIMENRYGQKGNAIRIEIVVYT
GKEGKSSHGCPIAKWVLRRSSDEEKVLCLVRQRTGHHCPTAVMVVLIMVWDGIPLPMADRLYTE
LTENLKSYNGHPTDRRCTLNENRICTCQGIDPETCGASFSFGCSWSMYENGCKFGRSPSPRRFR
IDPSSPLHEKNLEDNLQSLATRLAPIYKQYAPVAYQNQVEYENVARECRLGSKEGRPFSGVTAC
LDFCAHPHRDIHNMNNGSTVVCILTREDNRSLGVIPQDEQLHVLPLYKLSDTDEFGSKEGMEAK
IKSGAIEVLAPRRKKRTCFTQPVPRSGKKRAAMMTEVLAHKIRAVEKKPIPRIKRKNNSITTNN
SKPSSLPTLGSNTETVQPEVKSETEPHFILKSSDNTKTYSLMPSAPHPVKEASPGFSWSPKTAS
ATPAPLKNDATASCGFSERSSTPHCIMPSGRLSGANAAAADGPGISQLGEVAPLPTLSAPVMEP
LINSEPSTGVTEPLTPHQPNHQPSFLTSPQDLASSPMEEDEQHSEADEPPSDEPLSDDPLSPAE
EKLPHIDEYWSDSEHIFLDANIGGVAIAPAHGSVLIECARRELHATTPVEHPNRNHPIRLSLVF
YQHKNLNKPQHGFELNKIKFEAKEAKNKKMKASEQKDQAANEGPEQSSEVNELNQIPSHKALTL
THDNVVTVSPYALTHVAGPYNHWV
SEQ ID NO. 60: UniProt Q3URK3
MSRSRPAKPSKSVETKLQKKKDIQMKTKTSKQAVRHGASAKAVNPGKPKQLIKRRDGKKETEDK
TPTPAPSFLTRAGAARMNRDRNQVLFQNPDSLTCNGFTMALRRTSLSWRLSQRPVVTPKPKKVP
PSKKQCTHNIQDEPGVKHSENDSVPSQHATVSPGTENGEQNRCLVEGESQEITQSCPVFEERIE
DTQSCISASGNLEAEISWPLEGTHCEELLSHQTSDNECTSPQECAPLPQRSTSEVISQKNTSNQ
LADLSSQVESIKLSDPSPNPTGSDHNGFPDSSFRIVPELDLKTCMPLDESVYPTALIRFILAGS
QPDVFDTKPQEKTLITTPEQVGSHPNQVLDATSVLGQAFSTLPLQWGFSGANLVQVEALGKGSD
SPEDLGAITMLNQQETVAMDMDRNATPDLPIFLPKPPNTVATYSSPLLGPEPHSSTSCGLEVQG
ATPILTLDSGHTPQLPPNPESSSVPLVIAANGTRAEKQFGTSLFPAVPQGFTVAAENEVQHAPL
DLTQGSQAAPSKLEGEISRVSITGSADVKATAMSMPVTQASTSSPPCNSTPPMVERRKRKACGV
CEPCQQKANCGECTYCKNRKNSHQICKKRKCEVLKKKPEATSQAQVTKENKRPQREKKPKVLKT
DFNNKPVNGPKSESMDCSRRGHGEEEQRLDLITHPLENVRKNAGGMTGIEVEKWAPNKKSHLAE
GQVKGSCDANLTGVENPQPSEDDKQQTNPSPTFAQTIRNGMKNVHCLPTDTHLPLNKLNHEEFS
KALGNNSSKLLTDPSNCKDAMSVITSGGECDHLKGPRNTLLFQKPGLNCRSGAEPTIFNNHPNT
HSAGSRPHPPEKVPNKEPKDGSPVQPSLLSLMKDRRLTLEQVVAIEALTQLSEAPSESSSPSKP
EKDEEAHQKTASLLNSCKAILHSVRKDLQDPNVQGKGLHHDTVVFNGQNRTFKSPDSFATNQAL
IKSQGYPSSPTAEKKGAAGGRAPFDGFENSHPLPIESHNLENCSQVLSCDQNLSSHDPSCQDAP
YSQIEEDVAAQLTQLASTINHINAEVRNAESTPESLVAKNTKQKHSQEKRMVHQKPPSSTQTKP
SVPSAKPKKAQKKARATPHANKRKKKPPARSSQENDQKKQEQLAIEYSKMHDIWMSSKFQRFGQ
SSPRSFEWLLRNIFVFNQILEPVTQSKTPSQHNELFPFINQIKFTRNPELAKEKVEVEFSDSLF
TCQFKIESGGQTFAEPADNSQGQPMVSVNQEAHPLPQSPPSNQCANIMAGAAQTQFHLGAQENL
VHQIPPFTLFGTSPDTLLPDPASILREGEVLHFDGITVVTEKREAQTSSNGPLGPTTDSAQSEF
KESIMDLLSKPAKNLIAGLKEQEAAPCDCDGGTQKEKGPYYTHLGAGPSVAAVRELMETRFGQK
CA 03223669 2023- 12- 20
WO 2023/175040
PCT/EP2023/056668
98
GKAIRIEKIVETGKEGKSSQGCPVAKWVIRRSGPEEKLICLVRERVDHHCSTAVIVVLILLWEG
IPRLMADRLYKELTENLRSYSGHPTDRRCTLNKKRICTCQGIDPKTCGASFSFGCSWSMYENGC
KFGRSENPRKFRLAPNYPLHEKQLEKNLQELATVLAPLYKQMALWAYQNQVEYEEVAGDCRLGN
EEGRPFSGVTCCMDFCAHSHKDIHNMHNGSTVVCTLIRADGRDTNCPEDEQLHVLPLYRLADTD
EFGSVEGMKAKIKSGAIQVNGPTRKRRLRFTEPVPRCGKRAKMKQNHNKSGSHNTKSFSSASST
SHLVKDESTDFCPLQASSAETSTCTYSKTASGGFAETSSILHCIMPSGAHSGANAAAGECTGTV
QPAEVAAHPHQSLPTADSPVHAEPLTSPSEQLTSNQSNQQLPLLSNSQKLASCQVEDERHPEAD
EPQHPEDDNLPQLDEFWSDSEEIYADPSEGGVAIAPIHGSVLIECARKELHATTSLRSPKRGVP
FRVSLVFYQHKSLNKPNH.GFDINKIKCKCKKVTKKKPADRECPDVSPEANLSH.QIPSRVASTL1
RDNVVTVSPYSLTHVAGPYNRWV
SEQ ID NO. 61: UniProt Q6N021
MEQDRINHVEGNRLSPFLIPSPPICQTEPLATKLQNGSPLPERAHPEVNGDTKWHSEKSYYGIP
CMKGSQNSRVSPDFTQESRGYSKCLQNGGIKRTVSEPSLSGLLQIKKLKQDQKANGERRNEGVS
QERNPGESSQPNVSDLSDKKESVSSVAQENAVEDFTSFSTHNCSGPENPELQILNEQEGKSANY
HDKNIVLLKNKAVLMPNGATVSASSVEHTHGELLEKTLSQYYPDCVSIAVQKTTSHINAINSQA
TNELSCEITHPSHTSGQINSAQTSNSELPPKPAAVVSEACDADDADNASKLAAMLNICSFQKPE
QLQQQKSVFEICPSPAENNIQGTTKLASGEEFCSGSSSNLQAPGGSSERYLKQNEMNGAYFKQS
SVETKDSFSATTIPPPPSQLLLSPPPPLPQVPQLPSEGKSTLNGGVLEEHHHYPNQSNTTLLRE
VKIEGKPEAPPSQSPNPSTHVCSPSPMLSERPQNNCVNRNDIQTAGTMTVPLCSEKTRPMSEHL
KHNPPIEGSSGELQDNCQQLMRNKEQEILKGRDKEQTRDLVPPTQHYLKPGWIELKAPREHQAE
SHLKRNEASLPSILQYQPNLSNQMTSKQYTGNSNMPGGLPRQAYTQKTTQLEHKSQMYQVEMNQ
C'QSQC,TVDQHLQFQKPSHQVHFSKTDHLPKAHVQSLCC,TRFHFQQRADSQTEKLMSPVLKQHLN
QQASETEPFSNSHLLQHKPHKQAAQTQPSQSSHLPQNQQQQQKLQIKNKEEILQTFPHPQSNND
QQREGSFFGQTKVEECFHGENQYSKSSEFETHNVQMGLEEVQNINRRNSPYSQTMKSSACKIQV
SCSNNTHLVSENKEQTTHPELFAGNKTQNLHHMQYFPNNVIPKQDLLHRCFQEQEQKSQQASVL
QGYKNRNQDMSGQQAAQLAQQRYLIHNHANVFPVPDQGGSHTQTPPQKDTQKHAALRWHLLQKQ
EQQQTQQPQTESCHSQMHRPIKVEPGCKPHACMHTAPPENKTWKKVTKQENPPASCDNVQQKSI
IETMEQHLKQFHAKSLFDHKALTLKSQKQVKVEMSGPVTVLTRQTTAAELDSHTPALEQQTTSS
ENTPTHRTAASVLNNFIESPSNLLDTPIKNLLDTPVNTQYDFPSCRCVEQIIENDEGPFYTHLG
AGPNVAAIREIMEERFGQKGKAIRIERVIYIGKEGKSSQGCPIAKWVVRRSSSEEKLLCLVRER
AGHTCEAAVIVILILVWEGIPLSLADKLYSELTETLRKYGTLTNRRCALNEERTCACQGLDPET
CGASFSFGCSWSMYYNGCKFARSKIPRKFKLLGDDPKEEEKLESHLQNLSTLMAPTYKKLAPDA
YNNQIEYEHRAPECRLGLKEGRPFSGVTACLDFCAHAHRDLHNMQNGSTLVCILTREDNREFGG
KPEDEQLHVLPLYKVSDVDEFGSVEAQEEKKRSGAIQVLSSFRRKVRMLAEPVKTCRQRKLEAK
KAAAEKLSSLENSSNKNEKEKSAPSRTKQTENASQAKQLAELLRLSGPVMQQSQQPQPLQKQPP
QPQQQQRPQQQQPHHPQTESVNSYSASGSTNPYMRRPNPVSPYPNSSHTSDIYGSTSPMNFYST
SSQAAGSYLNSSNPMNPYPGLLNQNTQYPSYQCNGNLSVDNCSPYLGSYSPQSQPMDLYRYPSQ
DPLSKLSLPPIHTLYQPRFGNSQSFTSKYLGYGNQNMQGDGFSSCTIRPNVHHVGKLPPYPTHE
MDGHFMGATSRLPPNLSNPNMDYKNGEHHSPSHIIHNYSAAPGMENSSLHALHLQNKENDMLSH
TANGLSKMLPALNHDRTACVQGGLHKLSDANGQEKQPLALVQGVASGAEDNDEVWSDSEQSFLD
PDIGGVAVAPTHGSILIECAKRELHATTPLKNPNRNHPTRISLVEYQHKSMNEPKHGLALWEAK
MAEKAREKEEECEKYGPDYVPQKSHGKKVKREPAEPHETSEPTYLRFIKSLAERTMSVITDSTV
TTSPYAFTRVTGPYNRYI
SEQ ID NO. 62: UniProt Q4JK59
MEQDRITHAEGTRLSPFLIAPPSPISHTEPLAVELQNGSPLAERPHPEVNGDTKWQSSQSCYGI
SHMKGSQSSHESPHEDRGYSRCLQNGGIKRTVSEPSLSGLHPNKILKLDQKAKGESNIFEESQE
RNHGKSSRQPNVSGLSDNGEPVISTTQESSGADAFPTRNYNGVEIQVLNEQEGEKGRSVTLLKN
KIVLMPNGATVSAHSEENTRGELLEKTQCYPDCVSIAVQSTASHVNTPSSQAAIELSHEIPQPS
LTSAQINFSQTSSLQLPPEPAAMVTKACDADNASKPAIVPGTCPFQKAEHQQKSALDIGPSRAE
NKTIQGSMELFAEEYYPSSDRNLQASHGSSEQYSKQKETNGAYFRQSSKFPKDSISPTTVTPPS
CA 03223669 2023- 12- 20
WO 2023/175040
PCT/EP2023/056668
99
QSLLAPRLVLQPPLEGKGALNDVALEEHHDYPNRSNRILLREGKIDHQPKTSSSQSLNPSVHTP
NPPLMLPEQHQNDCGSPSPEKSRKMSEYLMYYLPNHGHSGGLQEHSQYLMGHREQEIPKDANGK
QTQGSVQAAPGWIELKAPNLHEALHQTKRKDISLHSVLHSQTGEWNQMSSKQSIGNVNMPGGFQ
RLPYLQKTAQPEQKAQMYQVQVNQGPSPGMGDQHLQFQKALYQECIPRTDPSSEAHPQAPSVPQ
YHFQQRVNPSSDKHLSQQATETQRLSGELQHTPQTQASQTPASQNSNFPQICQQQQQQQLQRKN
KEQMPQTFSHLQGSNDKQREGSCFGQIKVEESECVGNQYSKSSNFQTHNNTQGGLEQVQNINKN
FPYSKILTPNSSNLQILPSNDTHPACEREQALHPVGSKTSNLQNMQYFPNNVTPNQDVHRCFQE
QAQKPQQASSLQGLKDRSQGESPAPPAEAAQQRYLVHNEAKALPVPEQGGSQTQTPPQKDTQKH
AALRWLLLQKQEQQQTQQSQPGHNQMLRPIKTEPVSKPSSYRYPLSPPQENMSSRIKQEISSPS
RDNGQPKSIIETMEQHLKQFQLKSLCDYKALTLKSQKHVKVPTDIQAAESENHARAAEPQATKS
TDCSVLDDVSESDTPGEQSQNGKCEGCNPDKDEAPYYTHLGAGPDVAAIRTLMEERYGEKGKAI
RIEKVIYIGKEGKSSQGCFIAKWVYRRSSEEEKLLCLVRVRPNHTCETAVMVIAIMLWDGIFKL
LASELYSELTDILGKCGICTNRRCSQNETRNCCCQGENTETCGASFSFGCSWSMYYNGCKFARS
KKPRKFRLHGAEPKEEERLGSHLQNLATVIAPIYKKLAPDAYNNQVEFEHQAPDCCLGLKEGRP
FSGVTACLDFSAHSHRDQQNMPNGSTVVVTLNREDNREVGAKPFDEQFHVLPMYIIAPFDEFGS
TEGQEKKIRMGSIEVLQSFRRRRVIRIGELFKSCKKKAEFKKAKTKKAARKRSSLENCSSRTEK
GKSSSHTKLMENASHMKQMTAQPQLSGPVIRQPPTLQRHLQQGQRPQQFQPPQFQPQTTPQPQF
QPQHIMPGNSQSVGSHCSGSTSVYTRQPTPHSPYPSSAHTSDIYGDTNHVNFYPTSSHASGSYL
NPSNYMNPYLGLLNQNNQYAPFPYNGSVPVDNGSPFLGSYSPQAQSRDLHRYPNQDHLTNQNLP
PIHTLHQQTFGDSPSKYLSYGNQNMQRDAFTTNSTLKPNVHHLATFSPYPTPKMDSHFMGAASR
SPYSHPHTDYKTSEHHLPSHTIYSYTAAASGSSSSHAFHNKENDNIANGLSRVLPGFNHDRTAS
AQELLYSLTGSSQEKQPEVSGQDAAAVQEIEYWSDSEHNFQDPCIGGVAIAPTHGSILIECAKC
EVHATTKVNDPDRNHPTRISLVLYRHKNLFLPKHCLALWEAKMAEKARKEEECGKNGSDHVSQK
NHGKQEKREPTGPQEPSYLRFIQSLAENTGSVITDSTVITSPYAFTQVTGPYNTEV
SEQ ID NO. 63: UniProt 043151
MSQFQVPLAVQPDLPGLYDFPQRQVMVGSFPGSGLSMAGSESQLRGGGDGRKKRKRCGTCEPCR
RLENCGACTSCTNRRTHQICKLRKCEVLKKKVGLLKEVEIKAGEGAGPWGQGAAVKTGSELSPV
DGPVPGQMDSGPVYHGDSRQLSASGVPVNGAREPAGPSLLGTGGPWRVDQKPDWEAAPGPAHTA
RLEDAHDLVAFSAVAEAVSSYGALSTRLYETFNREMSREAGNNSRGPRFGFEGCSAGSEDLDTL
QTALALARHGMHPPNCNCDGPECFDYLEWLEGKIKSVVMEGGEERPRLFGPLPFGEAGLPAPST
RPLLSSEVPQISPQEGLPLSQSALSIAKEKNISLQTAIAIEALTQLSSALPQPSHSTPQASCPL
FEALSPFAFFRSFQSYLRAFSWFVVFPEEHSSFAFDSSAFFPATFRTEFFEAWGIDTPFATPRS
SWPMPRPSPDPMAELEQLLGSASDYIQSVFKRPEALPTKPKVKVEAPSSSPAPAPSPVLQREAP
TPSSEPDTHQKAQTALQQHLHHKRSLFLEQVHDTSFPAPSEPSAPGWWPPPSSPVPRLPDRPPK
EKKKKLPTPAGGPVGTEKAAPGIKPSVRKPIQIKKSRPREAQPLFPPVRQIVLEGLRSPASQEV
QAHPPAPLPASQGSAVPLPPEPSLALFAPSPSRDSLLPPTQEMRSPSPMTALQPGSTGPLPPAD
DKLEELIRQFEAEFGDSFGLPGPPSVPIQDPENQQTCLPAPESPFATRSPKQIKIESSGAVTVL
STTCFHSEEGGQEATPTKAENPLTPTLSGFLESPLKYLDTPTKSLLDTPAKRAQAEFPTCDCVE
QIVEKDEGPYYTHLGSGPTVASIRELMEERYGEKGKAIRIEKVIYIGKEGKSSRGCPIAKWVIR
RHTLEEKLLCLVRHRAGHHCQNAVIVILILAWEGIPRSLGDTLYQELTDTLRKYGNPTSRRCGL
NDDRICACQGKDPNTCGASFSFGCSWSMYENGCKYARSKTPRKFRLAGDNPKEEEVLRKSFQDL
ATEVAPLYKRLAPQAYQNQVINEEIAIDCRLGLKEGRPFAGVTACMDFCAHAHKDQHNLYNGCT
VVCILTKEDNRCVGKIPEDEQLHVLPLYKMANTDEFGSEENQNAKVGSGAIQVLTAFPREVRRL
PEPAKSCRQRQLEARKAAAEKKKIQKEKLSTPEKIKQEALELAGITSDPGLSLKGGLSQQGLKP
SLKVEPQNHESSFKYSGNAVVESYSVLGNCRPSDPYSMNSVYSYHSYYAQPSLTSVNGEHSKYA
LPSFSYYCFPSSNPVFPSQFLCPCAWCHSCSSCSFEKKPDLHALHNSLSPAYCCAEFAFLPSQA
VPTDAHHPTPHHQQPAYPGPKEYLLFKAPLLHSVSRDPSPFAQSSNCYNRSIKQEPVD2LTQAE
PVPRDAGKMGKTPLSEVSQNGGPSHLWGQYSGGPSMSPKRINGVGGSWGVFSSGESPAIVPDKL
SSFGASCLAPSHFIDGQWGLFPGEGQQAASHSGGRLRGKPWSPCKFGNSTSALAGPSLTEKPWA
LGAGDENSALEGSFGFQDELWNFMKGEEGRIPAAGASQLDRANQSFGLFLGSSEELFGALESEE
KLWDFFSLEEGFAEEPPSKGAVKEEKGGGGAEEEEEELWSDSEHNFLDENIGGVAVAPAHGSIL
IECARRELHATTFLKKPNRCHPTRISLVEYQHKNLNQPNHGLALWEAKMKQLAERARARQEEAA
CA 03223669 2023- 12- 20
WO 2023/175040
PCT/EP2023/056668
100
RLGLGQQ EAKLYGKKRKWGGT VVAE PQQ KS KKGVVPT RQALAVPTD SAVT VS S YAYT KVTG PY S
RW I
SEQ ID NO. 64: UniProt Q8BG87
MSQ FQVPLAVQPDLSGLYDFPQGQVMVGGFQGPGLPMAGSETQLRGGGDGRKKRKRCGTCDPCR
RLENCGSCT SCTNRRTHQ I CKLRKCEVLKKKAGLLKEVE I NAREGTGPWAQGATVKTGS EL S PV
DGPVPGQMDSGPVYHGDSRQL ST SGAPVNGAREPAGPGLLGAAGPWRVDQKPDWEAASGPTHAA
RLEDAHDLVAFSAVAEAVS SYGALSTRLY ET FNREMSREAGSNGRGPRPESCSEGSEDLDTLQT
ALALARHGMKP PNCTCDGPEC PD FLEWLEGKI KSMAMEGGQGRPRL PGAL PP S EAGL PAPSTRP
PLL SSEVPQVP PLEGLPLSQSAL S IAKEKNI SLQTAIAIEALTQLS SALPQP SHST SQASC PL P
EAL SP SAP FRS PQ SYLRAP SWPVVP PEEHP S FAPDS PAFP PAT PRPE FSEAWGIDT P PAT
PRNS
WPVPRPS PDPMAELEQLLGSASDY IQSVFKRPEAL PT KPKVKVEAPS SS PAPVPS P I SQREAPL
LSSEPDTHQKAQTALQQHLHHKRNL FLEQAQDAS FPI STE PQAPGWWAPPGS PAPRP PDKP PKE
KKKKP PT PAGGPVGAEKTT PG I KT SVRKP I Q I KKSRSRDMQPL FLPVRQ I VLEGLKPQASEGQA
PLPAQLSVP PPASQGAASQ SCAT PLT PE PSLAL FAP SP SGDSLL PPTQEMRS P SPMVALQSGST
GGPLP PADDKLEEL RQ FEAE FGDS FGLPGPPSVP IQEPENQSTCLPAPES P FAT RS PKKI KI E
SSGAVTVLSTICEHSEEGGQEAT PT KAENPLT PTLSGELE SPLKYLDT PT KSLLDT PAKKAQSE
FPTCDCVEQIVEKDEGPYYTHLGSGPTVAS IRELMEDRYGEKGKAI RI EKVI YTGKEGKSSRGC
P IAKWVIRRHTLEEKLLCLVRHRAGHHCQNAVIVIL ILAWEGIPRSLGDTLYQELTDTLRKYGN
E'TSRRCGLNDDRICACQGKDE'NTCGAS FS FGCSWSMY FNGCKYARSKT PRKFRLTGDNPKE EEV
LRNSFQDLATEVAPLYKRLAPQAYQNQVTNEDVAIDCRLGLKEGRP FSGVTACMDFCAHAHKDQ
HNLYNGCTVVCILTKEDNRCVGQ I PE DEQLHVLPLY KMASTDE FGS EENQNAKVS SGAI QVLTA
FPREVRRLPEPAKSCRQRQLEARKAAAEKKKLQKEKLSTPEKIKQEALELAGVITDPGLSLKGG
L SQQSLKPSLKVE PQNH FS S FKY SGNAVVESYSVLGSCRPSDPYSMSSVYSYHSRYAQPGLASV
NGFHSKYTL PS FGYYGFPS SNPVFP SQ FLGPSAWGHGGSGGS FEKKPDLHALHNSLNPAYGGAE
FAELPGQAVAT DNHHP I PHHQQPAY PGPKEYLLPKVPQLHPASRDP SP FAQSSSCYNRS IKQEP
I DPLTQAES I PRDSAKMSRT PLPEASQNGGPSHLWGQY SGGPSMSPKRINSVGGNWGVEPPGES
PT IVPDKLNSFGASCLT PSHFPESQWGL FTGEGQQSAPHAGARLRGKPWSPCKFGNGTSALTGP
SLT EKPWGMGTGD FNPALKGGPG FQDKLWNPVKVEEGRI PT PGANPLDKAWQAFGMPLS SNEKL
FGALKSEEELWDP FSLEEGTAEE PP SHGVVEEEKSGPTVEEDEEELWSDSEHNFLDENIGGVAV
APAHC S IL I ECARRELHATT PLKKPNRCHPTRI SLVFYQHKNLNQPNHGLALWEAKMKQLAERA
RQRQEEAARLGLGQQEAKLYGKKRKWGGAMVAEPQHKEKKGAI PT RQALAMPT DSAVTVS SYAY
T KVTGPY SRW I
SEQ ID NO. 65: UniProt P04519
MRIC I FMARGLEGCGVT KFSLEQRDW FI KNGHEVTLVYAKDKS FTRT S SHDHKS FS I PVILAKE
Y DKALKLVNDCDIL I INSVPATSVQEAT INNYKKLLDNIKPS IRVVVYQHDHSVLSLRRNLGLE
ETVRRADVI FS HS DNGD FNKVLMKEWY PETVSL FDD I EEAPTVYN FQ PPMD IVKVRSTYWKDVS
INMNINRW IGRTTTWKGFYQMFDFHEKFLKPAGKSTVMEGLERSPAF IAIKEKGI PYEYYGNR
E IDKMNLAPNQPAQILDCY INSEMLERMSKSGFGYQLSKLNQKYLQRSLEYTHLELGACGT I PV
FWKSTGENLKFRVDNT PLT SHDSGI IWFDENDME ST FERIKELSSDRALYDREREKAYE FLYQH
QDSSFCFKEQFDIITK
SEQ ID NO. 66: UniProt P04547
MKIAI INMGNNVINFKTVPSSET IYL FKVI SEMGLNVDI I SLKNGVYT KS FDEVDVNDY DRL IV
VNS S INF FGGKPNLAIL SAQKFMAKY KSKI YYL FTDIRLP FSQSWPNVKNRPWAYLYTEEELL I
KSP IKVI SQGINLDIAKAAHKKVDNVIE FEY FP I EQYKIHMNDFQL SKPIKKILDVI YGGS FRS
GQRESKMVE FL FDTGLN I E FEGNAREKQEKNPKYPWTKAPVFIGKI PMNMVSEKNSQAIAAL I I
GDKNYNDNFITLRVWETMASDAVML I DEE EDT KHRI INDARFYVNNRAEL IDRVNELKHSDVLR
KEMLS IQHDILNKTRAKKAEWQDAFKKAIDL
CA 03223669 2023- 12- 20
WO 2023/175040
PCT/EP2023/056668
101
SEQ ID NO. 67: ZDD motif
H- [ P /A/ V] J z-z1 -C
SEQ ID NO. 68: ZDD motif
FixsX24sw (s/i) PCX [2-4 ] CX6FXBLX5R (L/ I YX [8-11] LX2LX [10]1/1
SEQ ID NO. 69
GAGGTGTATGGTTGTACTAAT/5mC/ACT/5mC/CTGGA/5mC/GAATCTTAA/5mC/ACAA/5
mC/GTGCAG/5mC/CAAA/5mC/GCTT/5mC/GC/5mC/ACGG/5mC/AACGTG/5mC/GGACT
/5mC/GTCG/5mC/CTTA/5mC/AATCG/5mC/GCAGGT/5mC/ACGTTGAAGATGAGGATG
SEQ ID NO. 70
GAGGTGTATGGTTGTAG/5mC/GCAAATCGTAAAA/5mC/GCAAAGCGAAAAC/5mC/GCAAAC
CGTAAAC/5mC/GAAAAGCGCTTGAAGATGAGGATG
SEQ ID NO. 71
GAGGIGTATGGTTGTAG/5mC/GGAAAACGGAAAT/5mC/GGAAAACGTAAAG/5mC/GTAAAT
CGGAAAG/5mC/GAAAAGCGGTTGAAGATGAGGATG
SEQ ID NO. 72
GAGGIGTATGGTTGTAA/5mC/GTAAACCGCAAAC/5mC/GGAAAACGAAAAT/5mC/GCAAAC
CGAAAAC/5mC/GTAAAACGCTTGAAGATGAGGATG
SEQ ID NO. 73
GAGGTGTATGGTTGTAA/5mC/GAAAACCGGAAAT/5mC/GAAAAGCGTAAAT/5mC/GTAAAT
CGCAAAA/5mC/GGAAATCGATTGAAGATGAGGATG
CA 03223669 2023- 12- 20
