Note: Descriptions are shown in the official language in which they were submitted.
BLLN-Po5-PCT
HOMOLOGOUS GENOMIC REGIONS FOR CHARACTERIZATION
ASSOCIATED WITH BIOLOGICAL TARGETS
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S. Patent
Application No.
16/055,889, filed on 06-AUG-2018, which claims the benefit of U.S. Provisional
Application serial number 62/541,555, filed on 0:I-AUG-2017, both of which are
incorporated herein in their entirety by this reference.
TECHNICAL FIELD
[0002] This disclosure relates generally to the field of genomics.
REFERENCE TO SEQUENCE LISTING SUBMITTED VIA EFS-WEB AS ASCII TEXT
FILE
[0003] The Sequence Listing in file "BLLN-Po5-US-SEQ-LST-
PatentIn_ST25.txt"
created on 19-DEC-2018, 5,422 bytes, machine format IBM-PC, MS-Windows
operating
system, in accordance with 37 C.F.R. 1.821- to 1.825, is hereby
incorporated by
reference in its entirety for all purposes.
BRIEF DESCRIPTION OF THE FIGURES
[0004] FIGURES 1A-1C include flowchart representations of variations of
an
embodiment of a method;
[0005] FIGURE 2 includes a schematic representation (chr 21 target
sequence =
portion of SEQ ID NO: 8; chr 21 target-associated molecule sequence = portion
of SEQ
ID NO: 9; chr 18 reference sequence = portion of SEQ ID NO: 10; chr 18
reference-
associated molecule sequence = portion of SEQ ID NO: 11) of a variation of an
embodiment of a method;
[0006] FIGURE 3 includes a schematic representation of a variation of an
embodiment of a method;
[0007] FIGURE 4 includes a specific example of results from using spike-
in
molecules for facilitating diagnosis of trisomy 21;
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[0008] FIGURE 5 includes a schematic representation of facilitating
diagnosis of
sickle cell disease in a variation of an embodiment of a method;
[0009] FIGURE 6 includes a schematic representation of facilitating
diagnosis of
rare variant-associated condition in a variation of an embodiment of a method;
[0010] FIGURES 7A-7B include specific examples of target-associated
molecules
(hg19 = SEQ ID NO: 1; CCL31 spk = SEQ ID NO: 2) and applying the target-
associated
molecules for detecting copy number variants;
[0011] FIGURE 8 includes a specific example of a plasmid;
[0012] FIGURES 9A-9B include specific examples of target-associated
molecules
(hp() = SEQ ID NO: 3; HbA_spk = SEQ ID NO: 4; HbS_spk = SEQ ID NO: 5) and
applying the target-associated molecules for detecting single nucleotide
polymorphisms;
[0013] FIGURE 10 includes a schematic representation of facilitating
characterization associated with a plurality of targets, in a variation of an
embodiment of
a method;
[0014] FIGURE ii includes specific examples of determining and generating
target-associated molecules and reference-associated molecules (target
sequence = SEQ
ID NO: 8; target-associated molecule sequence = SEQ ID NO: 9);
[0015] FIGURE 12 includes specific examples of target-associated
molecules and
reference-associated molecules (chr21:17197217+17197359 Target = SEQ ID NO: 6;
chr21:17197217+17197359 Spike-in = SEQ ID NO: 7; chr21:34950645 34950764
Target
SEQ ID NO: 8; chr21:34950645+34950764 Spike-in = SEQ ID NO: 9;
chri8:216483+216603 Target = SEQ ID NO: 10; chi-18:216483+216603 Spike-in =
SEQ
ID NO: ii; chri8:74561484+74561606 Target = SEQ ID NO: 12;
Chr18:74561484+74561606 Spike-in = SEQ ID NO: 13; 6.118:12340277+12340405
Target = SEQ ID NO: 14; chri8:12340277+12340405 Spike-in = SEQ ID NO: 15);
[0016] FIGURE 13 includes a flowchart representation of variations of an
embodiment of a method;
[0017] FIGURE 14 includes a flowchart representation of variations
associated
with spinal muscular atrophy condition characterization, of an embodiment of a
method;
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[0018] FIGURE 15A includes a specific example of a graph representation
of reads
of SMNi, SMN2, and regions of homology, such as where two of a set of
potential primer
pairs (e.g., ten primer pairs, etc.) are characterized, such as where c49_5
compares SMNi
and SMN2 to a region of homology in chromosome 9, such as where c49_6 compares
SMNi and SMN2 to a region of homology in chromosome 4, and such as where the
DNA
sample has an expected ratio of 0.4:0.2:0.4 for region_of homology:SMN1:SMN2,
and
such as where the expected ratio is measured with high accuracy and precision;
[0019] FIGURE 15B includes a specific example of a graph representation
of
determined SMNi copy number, such as for facilitating diagnosis and/or
suitable
characterization of a spinal muscular atrophy condition.
[0020] FIGURE 16 includes a flowchart representation of variations
associated
with alpha-thalassemia condition characterization, of an embodiment of a
method;
[0021] FIGURE 17 includes a specific example of a graph representation of
an
abundance metric ratio between an abundance metric for HBA1 and an abundance
metric
for HBA2, such as for use in facilitating diagnosis of one or more alpha-
thalassemia
conditions;
[0022] FIGURE 18 includes a flowchart representation of variations
associated
with trisomy 21 condition characterization, of an embodiment of a method;
[0023] FIGURE 19A includes a specific example of using target-associated
molecules and reference-associated molecules;
[0024] FIGURE 19B includes a specific example of using a homologous
native
genomic region;
[0025] FIGURE 20 includes a flowchart representation of variations
associated
with alpha-thalassemia condition characterization, of an embodiment of a
method;
[0026] FIGURE 21 includes a specific example of a graph representation
indicating
different abundance metrics for different polymorphic alleles, such as for use
in diagnosis
of one or more alpha-thalassemia conditions;
[0027] FIGURE 22 includes a flowchart representation of variations
associated
with alpha-thalassemia condition characterization, of an embodiment of a
method;
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[0028] FIGURE 23 includes a specific example of a flowchart indicating
alpha-
thalassemia-associated assays to perform based on maternal genotype, such as
where
lines are labeled with the number of cases for every 100,000 births, such as
where
parentheticals indicate percent of percent of individual based on the carrier
test, or for
the specific maternal genotype, such as where other maternal genotype alleles
can
additionally or alternatively be used.
DESCRIPTION OF THE EMBODIMENTS
[0029] The following description of the embodiments (e.g., including
variations of
embodiments, examples of embodiments, specific examples of embodiments, other
suitable variants, etc.) is not intended to be limited to these embodiments,
but rather to
enable any person skilled in the art to make and use.
1. Overview.
[0030] As shown in FIGURE 13, embodiments of a method loo (e.g., for
facilitating
characterization of one or more conditions, such as one or more medical
conditions, such
as one or more genetic disorders, such as from one or more biological samples,
etc.) can
include: generating a co-amplified mixture %So based on co-amplifying a set of
nucleic
acid molecules (e.g., cell-free nucleic acids such as cell-free DNA and/or
cell-free RNA,
etc.) from the biological sample (e.g., maternal sample, etc.), wherein the
set of nucleic
acid molecules includes: a genomic region of interest associated with the
medical
condition; and a homologous native genomic region with partial sequence
similarity to
the genomic region of interest (e.g., where the homologous native genomic
region
corresponds to the only instance, or one of a small number of instances, of
such homology
to the genomic region of interest; etc.); sequencing the co-amplified mixture
S182;
determining an abundance metric (e.g., read count; any suitable type of
abundance metric
described herein; etc.) for the genomic region of interest and an abundance
metric (e.g.,
read count; any suitable type of abundance metric described herein; etc.) for
the
homologous native genomic region S184; and/or facilitating the
characterization (e.g.,
prenatal diagnosis for a fetus; etc.) of the medical condition S186 based on
the abundance
metric (e.g., read count; etc.) for the genomic region of interest and the
abundance metric
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(e.g., read count; etc.) for the homologous native genomic region (e.g., based
on relative
copy numbers for the genomic region of interest and the homologous native
genomic
regions, determined from the abundance metrics; etc.).
[0031] In
a specific example, as shown in FIGURE 13, the method loo (e.g., for
facilitating prenatal diagnosis of a genetic disorder from a maternal sample
associated
with a pregnant woman; etc.) can include: determining a first genomic region
of interest
S17o (e.g., from a set of genomic regions of interest; etc.) associated with
the genetic
disorder; determining a first homologous native genomic region S172 (e.g.,
from a set of
homologous native genomic regions including homologous native genomic regions
with
partial sequence similarity to genomic regions of interest from the set of
genomic regions
of interest; etc.); determining a first primer pair S174 (e.g., from a set of
primer pairs
designed for co-amplification of genomic regions of interest and corresponding
homologous native genomic regions; etc.) for co-amplification of the first
genomic region
of interest and the first homologous native genomic region, based on the
partial sequence
similarity between the first homologous native genomic region and the first
genomic
region of interest; generating a co-amplified mixture Si8o from co-amplifying,
based on
the first primer pair (and/or the set of primer pairs; etc.), a set of nucleic
acid molecules
(e.g., cell-free nucleic acids such as cell-free DNA and/or cell-free RNA,
etc.) including
the first genomic region of interest (and/or the set of genomic regions of
interest; etc.)
and the first homologous native genomic region (and/or the set of homologous
native
genomic regions; etc.), where the set of nucleic acid molecules are from the
maternal
sample; sequencing the co-amplified mixture S182; determining an abundance
metric
(e.g., read count; any suitable type of abundance metric described herein;
etc.) for the first
genomic region of interest (and/or the set of genomic regions of interest;
etc.) and an
abundance metric (e.g., read count; any suitable type of abundance metric
described
herein; etc.) for the first homologous native genomic region (and/or the set
of
homologous native genomic regions; etc.) S184 based on sequence dissimilarity
between
the first genomic region of interest and the first homologous native genomic
region
(and/or sequence dissimilarity between any suitable pairs of genomic region of
interest
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and homologous native genomic region; etc.); and/or facilitating the prenatal
diagnosis
of the genetic disorder Si86 based on the abundance metric (e.g., read count;
etc.) for the
first genomic region of interest relative to the abundance metric (e.g., read
count; etc.) for
the first homologous native genomic region (and/or on the abundance metrics
for the set
of genomic regions of interest and/or for the set of homologous native genomic
regions;
etc.).
[0032] Embodiments of the method ioo and/or system 200 can function to
facilitate characterization (e.g., diagnosis) of one or more genetic disorders
(and/or other
suitable conditions), such as single gene disorders (e.g., alpha-thalassernia
conditions,
spinal muscular atrophy conditions; etc.), chromosomal abnormalities (e.g.,
trisomy
conditions such as trisomy 21 condition; etc.), and/or any other suitable
conditions (e.g.,
described herein, etc.). In specific examples, native genomic regions
homologous to
genomic regions of interest (e.g., where a homologous native genomic region
corresponds
to the only instance, or one of a small number of instances, of such homology
to the
corresponding genomic region of interest; etc.) can be identified and/or used
to reduce
amplification biases by facilitating co-amplification of the homologous native
genomic
regions and the corresponding genomic regions of interest (e.g., using primer
pairs
designed for shared sequence regions of the homologous native genomic regions
and the
corresponding genomic regions of interest; etc.), such as in relation to
circulating free
DNA (e.g., used for liquid biopsies, oncology, etc.) of a natural size
distribution from
sheared apoptotic processes.
[0033] Genomic regions of interest (and/or homologous native genomic
regions)
can include any one or more target-associated regions (e.g., described
herein). Genomic
regions of interest (and/or homologous native genomic regions) can be
associated with
(e.g., include a genomic region of; etc.) any suitable biological targets
(e.g., described
herein), such as biological targets associated with one or more spinal
muscular atrophy
conditions, alpha-thalassemia conditions, trisomy 21 conditions, and/or any
suitable
conditions described herein.
[0034] A homologous native genomic region preferably corresponds to the
only
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instance, or one of a small number of instances, of such homology (e.g.,
satisfying
identification criteria for homologous native genomic regions; etc.) to the
corresponding
genomic region of interest; but can alternatively correspond to one of any
number of
native genomic regions homologous to the corresponding genomic region of
interest. The
number of native genomic regions homologous to the corresponding genomic
region of
interest can be used in determining reference ratios for comparison to
observed ratios
between the homologous native genomic region and the corresponding genomic
region of
interest, such as for facilitating characterization (e.g., diagnosis) of one
or more genetic
disorders and/or suitable conditions. In a specific example, the method loo
can include
determining a reference ratio for the genomic region of interest relative to
the
homologous native genomic region, based on a number of genomic regions
homologous
to the genomic region of interest, where facilitating the characterization of
the medical
condition can include: determining an observed ratio based on the abundance
metric
(e.g., read count; etc.) for the genomic region of interest relative to the
abundance metric
(e.g., read count; etc.) for the homologous native genomic region; and/or
comparing the
observed ratio to the reference ratio.
[0035]
Determining homologous native genomic regions is preferably based on one
or more identification criteria including any one or more of: amplicon size
criteria (e.g.,
resulting in amplicons at or below 200 base pair size when using cell free
DNA; at or below
loo base pair size; amplicon size criteria of at, below, or above any suitable
number of
base pair size; etc.); homology threshold criteria (e.g., 80-95% homology);
sequence
region criteria (e.g., 10-30 nucleotides and/or any suitable number of
nucleotides of
perfect complementarity for primer binding sequence regions; sequence
dissimilarity
regions between primer binding sequence regions; etc.); number of homology
native
genomic regions (e.g., a homologous native genomic region corresponding to the
only
instance, or one of a small number of instances, of such homolog to the
corresponding
genomic region of interest; etc.); and/or any other suitable criteria. In a
specific example,
the primer pair corresponds to a first primer binding sequence region and a
second
primer binding sequence region; the genomic region of interest and the
homologous
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native genomic region each include the first primer binding sequence region
and the
second primer binding sequence region; the homologous native genomic region
further
includes a sequence dissimilarity region with sequence dissimilarity to a
corresponding
sequence region of the genomic region of interest; and the sequence
dissimilarity region
is located between the first primer binding sequence region and the second
primer
binding sequence region of the homologous native genomic region. In a specific
example,
the partial sequence similarity between the genomic region of interest and the
homologous native genomic region satisfies a homology threshold of at least
80%
homology, but any suitable homology threshold value can be used.
[0036] Determining homologous native genomic regions can include
performing
any one or more of: BLAST operations, Burrows-Wheeler transforms, and/or any
suitable
approaches for identifying homology between genomic regions.
[0037] Any suitable number of genomic regions of interest, homologous
native
genomic regions, corresponding primer pairs (e.g., for corresponding co-
amplification;
etc.) can be determined and/or used. In a specific example, the set of nucleic
acid
molecules (e.g., to be amplified; from the biological sample; cell-free
nucleic acids such
as cell-free DNA and/or cell-free RNA; etc.) includes: a set of genomic
regions of interest
including the genomic region of interest, and a set of homologous native
genomic regions
including the homologous native genomic regions; and where facilitating the
characterization of the medical condition includes facilitating the
characterization of the
medical condition based on abundance metric ratios (e.g., read count ratios;
etc.) between
abundance metrics (e.g., read counts; etc.) for the set of genomic regions of
interest and
abundance metrics (e.g., read counts; etc.) for the set of homologous native
genomic
regions. In a specific example, the set of nucleic acid molecules (e.g., to be
amplified; from
the biological sample; cell-free nucleic acids such as cell-free DNA and/or
cell-free RNA;
etc.) includes first nucleic acid molecules and second nucleic acid molecules,
where the
first nucleic acid molecules include the genomic region of interest, where the
second
nucleic acid molecules include the homologous native genomic region, and where
co-
amplifying a set of nucleic acid molecules includes co-amplifying the first
and the second
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nucleic acid molecules based on a primer pair designed based on the partial
sequence
similarity between the genomic region of interest and the homologous native
genomic
region.
[0038] Abundance metrics (e.g., for genomic regions of interest; for
homologous
native genomic regions; etc.) can include read counts and/or any suitable
types of
abundance metrics described herein (e.g., in relation to Section 2.4, etc.),
and/or can be
determined in any suitable manner described herein (e.g., in relation to
Section 2.4, etc.).
[0039] Abundance metrics can include one or more intensity metrics. In an
example, the abundance metric for the genomic region of interest includes an
intensity
metric for the genomic region of interest, where the abundance metric for the
homologous
native genomic region includes an intensity metric for the homologous native
genomic
region, and where determining the abundance metric for the genomic region of
interest
and the abundance metric for the homologous native genomic region includes
determining the intensity metric for the genomic region of interest and the
intensity
metric for the homologous native genomic region based on sequence
dissimilarity
between the genomic region of interest and the homologous native genomic
region.
[0040] In variations, the abundance metric can include an intensity
metric
including a peak intensity metric (e.g., maximum intensity for the peak;
overall intensity
for the peak; etc.) and/or suitable output metric associated with Sanger
sequencing (e.g.,
associated with a chromatogram-related output from Sanger sequencing) and/or
other
suitable types of sequencing and/or abundance quantification. In a specific
example, a
peak intensity metric for a base of the genomic region of interest (e.g., at a
position of
sequence dissimilarity to the homologous native genomic region, etc.) can be
compared
to a peak intensity metric for a base of the homologous native genomic region
(e.g., at the
position of sequence dissimilarity to the genomic region of interest; etc.).
In a specific
example, a set of peak intensity metrics for a set of bases of the genomic
region of interest
can be compared to a set of peak intensity metrics for a set of bases of the
homologous
native genomic regions (e.g., ratios of peak intensity metrics for pairs of
bases at positions
of sequence dissimilarity between the genomic region of interest and the
homologous
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native genomic region; etc.). However, peak intensity metrics can be
associated with any
suitable type of sequencing, and can be configured in any suitable manner.
[0041] In variations, the abundance metric can include an intensity
metric and/or
suitable output metrics associated with digital PCR, real-time PCR, and/or
other suitable
types of abundance quantification. In a specific example, intensity metrics
can include
fluorescence metrics, such as indicating binary readouts (e.g., "o" or "1",
where such
readouts can indicate abundance of a genomic region of interest, of a
homologous native
genomic region; etc.). In a specific example, intensity metrics can include
relative
fluorescence metrics, such as for abundance determination. Additionally or
alternatively,
intensity metrics can be configured in any suitable manner. However,
comparison of
metrics between genomic regions of interest and homologous native genomic
regions,
and/or use of target-associated molecules and/or reference-associated
molecules can be
performed in any suitable manner.
[0042] As shown in FIGURES 14, 15A-15B, and 16, any suitable portions of
embodiments of the method loo and/or system 200 can be performed for one or
more
single gene disorders. In a specific example, the genetic disorder includes a
single gene
disorder; the first genomic region of interest is associated with a gene
associated with the
single gene disorder; and facilitating the prenatal diagnosis of the genetic
disorder
includes facilitating the prenatal diagnosis of the single gene disorder based
on the
abundance metric (e.g., read count; etc.) for the first genomic region of
interest associated
with the gene relative to the abundance metric (e.g., read count; etc.) for
the first
homologous native genomic region. In a specific example, as shown in FIGURE
16, the
single gene disorder includes an alpha-thalassemia condition; the gene
includes HBAi
gene, where the first genomic region of interest is from the HBAi gene; the
first
homologous native genomic region is from HBA2 gene; facilitating the prenatal
diagnosis
of the genetic disorder includes facilitating the prenatal diagnosis of the
alpha-
thalassemia condition based on the abundance metric (e.g., read count; etc.)
for the first
genomic region of interest from the HBAI gene relative to the abundance metric
(e.g.,
read count; etc.) for the first homologous native genomic region from the HBA2
gene. In
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a specific example, as shown in FIGURE 17, an abundance metric ratio between
an
abundance metric for HBAI and an abundance metric for HBA2 can be used in
facilitating
diagnosis of one or more alpha-thalassemia conditions.
[0043] In
a specific example, as shown in FIGURE 14, the single gene disorder
includes a spinal muscular atrophy condition; the genomic region of interest
is from
SMNi gene; the genomic region of interest includes at least one of (e.g., in
relation to
GRCh37 genome version coordinates) clir5:70238181-70238382, chr5:70247763-
70248428, and chr5:70247785-70248019; and the homologous native genomic region
includes at least one of (e.g., in relation to GRCh37 genome version
coordinates)
chr9 :20331713-20331910 (e.g., homologous to
chr5:70238181-70238382),
chr9:20332293-20332902 (e.g., homologous to chr5:70247763-70248428), and
chr4:183948110-183948725 (e.g., homologous to chr5:70247785-70248019), such as
where primer pairs can be designed for co-amplification of pairs of genomic
region of
interest and corresponding homologous native genomic region. In a specific
example, the
single gene disorder includes a spinal muscular atrophy condition; the gene
includes
SMNi gene, where the first genomic region of interest is from the SMNi gene;
the first
homologous native genomic region is from SMN2 gene; and facilitating the
prenatal
diagnosis of the genetic disorder includes facilitating the prenatal diagnosis
of the spinal
muscular atrophy condition based on the abundance metric (e.g., read count;
etc.) for the
first genomic region of interest from the SMNi gene relative to the abundance
metric (e.g.,
read count; etc.) for the first homologous native genomic region from the SMN2
gene. In
a specific example, the method loo can include determining a second homologous
native
genomic region (e.g., as shown in FIGURE 15A, a region of homology in
chromosome 9
and/or a region of homology in chromosome 4; etc.) with partial sequence
similarity to
the first genomic region of interest from the SMNi gene, where facilitating
the prenatal
diagnosis of the genetic disorder includes facilitating the prenatal diagnosis
of the spinal
muscular atrophy condition based on the abundance metric (e.g., read count;
etc.) for the
first genomic region of interest from the SMNi gene, the abundance metric
(e.g., read
count; etc.) for the first homologous native genomic region from the SMN2
gene, and an
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abundance metric (e.g., read count; any suitable type of abundance metric
described
herein; etc.) for the second homologous native genomic region. In a specific
example, as
shown in FIGURE 15B, the method loo can include determining SMNi copy number
(e.g.,
using approaches described herein, etc.), such as for facilitating diagnosis
and/or suitable
characterization of a spinal muscular atrophy condition.
[0044] As
shown in FIGURE 18, any suitable portions of embodiments of the
method loo and/or system 200 can be performed for one or more chromosomal
abnormalities (e.g., trisomy conditions; etc.). In a specific example, the
genetic disorder
includes a chromosomal abnormality, where the first genomic region of interest
is
associated with a first chromosome associated with the chromosomal
abnormality, where
the first homologous native genomic region is associated with a second
chromosome, and
where facilitating the prenatal diagnosis of the genetic disorder includes
facilitating the
prenatal diagnosis of the chromosomal abnormality based on the abundance
metric (e.g.,
read count; etc.) for the first genomic region of interest associated with the
first
chromosome relative to the abundance metric (e.g., read count; etc.) for the
first
homologous native genomic region associated with the second chromosome. In a
specific
example, the chromosomal abnormality includes a trisomy 21 condition, where
the first
genomic region of interest is associated with the first chromosome including
chromosome
21, and where facilitating the prenatal diagnosis of the genetic disorder
includes
facilitating the prenatal diagnosis of the trisomy 21 condition based on the
abundance
metric (e.g., read count; etc.) for the first genomic region of interest
associated with
chromosome 21 relative to the abundance metric (e.g., read count; etc.) for
the first
homologous native genomic region associated with the second chromosome. In a
specific
example, the first genomic region of interest is associated with a first loci
of chromosome
21, and where the method further includes: determining a second genomic region
of
interest associated with a second loci of chromosome 21; determining a second
homologous native genomic region with partial sequence similarity to the
second genomic
region of interest; determining a second primer pair for co-amplification of
the second
genomic region of interest and the second homologous native genomic region,
where
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generating the co-amplified mixture includes co-amplifying, based on the first
and the
second primer pairs, a set of nucleic acid molecules (e.g., cell-free nucleic
acids such as
cell-free DNA and/or cell-free RNA, etc.) including the first genomic region,
the first
homologous native genomic region, the second genomic region, and the second
homologous native genomic region; determining an abundance metric (e.g., read
count;
any suitable type of abundance metric described herein; etc.) for the second
genomic
region of interest and an abundance metric (e.g., read count; any suitable
type of
abundance metric described herein; etc.) for the second homologous native
genomic
region based on sequence dissimilarity between the second genomic region of
interest and
the second homologous native genomic region; and facilitating the prenatal
diagnosis of
the trisomy 21 condition based on the abundance metric (e.g., read count;
etc.) for the
first genomic region of interest relative to the abundance metric (e.g., read
count; etc.) for
the first homologous native genomic region, and based on the abundance metric
(e.g.,
read count; etc.) for the second genomic region of interest relative to the
abundance
metric (e.g., read count; etc.) for the second homologous native genomic
region.
[0045]
Determining a genomic region of interest S170 and/or determining a
homologous native genomic region S172, and/or determining primers S174 can
additionally or alternatively include and/or be performed in any suitable
manner
analogous to that described in relation to Section 2.1, S110, Section 2.2,
S120, and/or to
any suitable portion described herein; and/or can be performed in any suitable
manner.
Generating an amplified mixture Si8o (e.g., a co-amplified mixture) can
additionally or
alternatively include and/or be performed in any suitable manner analogous to
that
described in relation to Section 2.3, S13o, and/or to any suitable portion
described herein;
and/or can be performed in any suitable manner. Determining abundance metrics
(e.g.,
read counts; any suitable type of abundance metrics described herein; etc.),
relative copy
numbers, count ratios, other ratios, and/or other suitable abundance metrics
5184 can
additionally or alternatively include and/or be performed in any suitable
manner
analogous to that described in relation to Section 2.4, S14o, and/or to any
suitable portion
described herein; and/or can be performed in any suitable manner. Facilitating
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characterization of one or more genetic disorders S186 (e.g., alpha-
thalassemia
conditions; spinal muscular atrophy conditions; trisomy 21 conditions; etc.)
can
additionally or alternatively include and/or be performed in any suitable
manner
analogous to that described in relation to Section 2.5, S15o, and/or to any
suitable portion
described herein; and/or can be performed in any suitable manner.
[0046] In variations, the method ioo can include performance of and/or
use of any
suitable combinations of approaches (e.g., described herein) including and/or
associated
with genomic regions of interest, homologous native genomic regions, target-
associated
molecules, and/or reference-associated molecules, which can function to use
genomic
regions of interest and homologous native genomic regions in combination with
target-
associated molecules (e.g., described herein; etc.) and reference-associated
molecules
(e.g., described herein; etc.). In specific examples, using such approaches
can improve
upon (e.g., correct for) capture efficiency and amplification efficiency. In a
specific
example, using genomic regions of interest with corresponding homologous
native
genomic regions can improve upon capture efficiency, such as by co-amplifying
cell-free
nucleic acids with shared and/or similar length distributions (e.g., natural
length
distributions resulting from apoptotic processes; with similar ends; etc.) for
facilitating
similar binding (e.g., similar capture efficiency; etc.) of primers (e.g.,
primer pairs
targeting regions of sequence similarity between the genomic region of
interest and
corresponding homologous native genomic region; etc.). In a specific example,
as shown
in FIGURE 19B, a xi/yi abundance metric ratio can correspond to a genomic
region of
interest (e.g., xi) and homologous native genomic region (e.g., yi; not having
copy
number variation; etc.). In a variation, optimizing amplification conditions
(e.g., PCR
conditions such as annealing time, such as for facilitating similar
amplification rates for
every PCR cycle; etc.) can improve amplification efficiency associated with
use of genomic
regions of interest and homologous native genomic regions.
[0047] In a specific example, using target-associated molecules and
reference-
associated molecules can improve upon amplification efficiency, such as where
such
molecules can be synthetically generated to have defined base pair differences
(e.g.,
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relative corresponding biological targets and references; etc.) optimized for
reducing
amplification bias (e.g., through co-amplification of the target-associated
molecules and
corresponding genomic region of interest; through co-amplification of the
reference-
associated molecules and corresponding homologous native genomic region;
etc.). In a
specific example, as shown in FIGURE 19A, a target-associated abundance ratio
(e.g.,
xi/xi') can be used with a reference-associated abundance ratio (e.g., y-
C/y1), such as
where xi/xi' * yf/yi = xi/yi due to xi' = yf (e.g., by designing use at the
same abundance,
such as placing both target-associated and reference-associated (i.e.,
homologous-region-
associated) synthetic molecules on same plasmid to ensure in ratio of xi' and
yf prior to
co-amplification as described herein; etc.). In a variation, optimizing
capture efficiency
can include shearing (and/or otherwise causing) target-associated molecules
and/or
reference-associated molecules to have a similar size distribution to
corresponding
endogenous molecules.
[0048] In
examples, target-associated molecules can be associated with genomic
regions of interest (and/or suitable biological targets described herein;
etc.), and/or
reference-associated molecules can be associated with homologous native
genomic
regions (and/or suitable references described herein; etc.). In a specific
example, the
method can include any one or more of: co-amplifying (e.g., in a same or
different co-
amplified mixture as a co-amplified mixture generated from co-amplifying a
genomic
region of interest and homologous native genomic region; etc.) a set of target-
associated
molecules and first nucleic acid molecules from the cell-free nucleic acids
(and/or suitable
components of a biological sample such as a maternal sample; etc.), where the
set of
target-associated molecules can include: target-associated regions with
sequence
similarity to the genomic region(s) of interest; and target variation regions
with sequence
dissimilarity to a sequence region of the genomic region(s) of interest; co-
amplifying (e.g.,
in a same or different co-amplified mixture as a co-amplified mixture
generated from co-
amplifying a genomic region of interest and homologous native genomic region;
in a same
or different co-amplified mixture as a co-amplified mixture generated from co-
amplifying
target-associated molecules and nucleic acid molecules including the genomic
region of
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interest; etc.) a set of reference-associated molecules and second nucleic
acid molecules
from the cell-free nucleic acids, where the set of reference-associated
molecules can
include: reference-associated regions with sequence similarity to homologous
native
genomic region(s); and reference variation regions with sequence dissimilarity
to a
sequence region of the homologous native genomic region(s); determining one or
more
abundance metrics (e.g., read counts; intensity metrics; etc.) for the set of
target-
associated molecules; determining one or more abundance metrics (e.g., read
counts;
intensity metrics; etc.) for the set of reference-associated molecules; and/or
facilitating
one or more characterizations (e.g., diagnoses) of the medical condition
(e.g., genetic
disorders) based on the abundance metric for the genomic region of interest,
the
abundance metric for the homologous native genomic region, the abundance
metric for
the set of target-associated molecules, and/or the abundance metric for the
set of
reference-associated molecules. In a specific example, synthetic molecules for
both target
(e.g., SMNi, etc.) and reference (e.g., homologous region on Chromosome 4 or 9
homologous to SMNi, with defined base-pair differences to SMN1 and its
homologous
region, etc.) can be designed and added to a circulating free DNA sample
purified from
the blood of a pregnant woman. In a specific example, addition of synthetic
molecules can
correct for biases due to amplification while using primers that co-amplify
SMNi and its
homologous region can correct for capture efficiency biases that can stem from
differences in size distribution of cell free DNA and synthetic molecules. In
particular,
after co-amplification of al sets of molecules (e.g., 4 sets) and sequencing,
the abundance
ratio of SMNi and reference can be calculated by normalizing the ratio of
abundance
metrics for SMNi and Chromosome 4/9, by the ratio of abundance metrics for
target-
associated and reference-associated synthetic molecules. In a specific example
where the
pregnant woman has previously been determined to be a carrier for SMN1, and
sequence
data can be used to distinguish SMNi and SMN2 sequences, a statistically
significant
decrease from 0.50 for SMN1 to the reference ratio would indicate a spinal
muscular
atrophy disorder in the fetus.
[0049]
However, combining use of genomic regions of interest, homologous native
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genomic regions, target-associated molecules, and/or reference-associated
molecules can
be performed in any suitable manner.
[0050] However, any suitable portions (e.g., of embodiments) associated
with use
of homologous native genomic regions can be performed in any suitable manner.
[0051] Additionally or alternatively, embodiments of the method 100
(e.g., for
facilitating prenatal characterization of an alpha-thalassemia condition from
a maternal
sample from a mother; etc.) can include: generating an amplified mixture from
amplifying a set of nucleic acid molecules from the maternal sample based on a
set of
primers designed for amplification of a genomic region of interest associated
with at least
one of HBAi gene and HBA2 gene (and/or any suitable gene associated with an
alpha-
thalassemia condition; etc.); sequencing the amplified mixture; determining an
abundance metric (e.g., read count; any suitable type of abundance metric
described
herein; etc.) for the genomic region of interest; and/or facilitating the
prenatal
characterization (and/or other suitable characterization) of the alpha-
thalassemia
condition based on the abundance metric (e.g., read count; etc.) for the
genomic region
of interest.
[0052] In a variation (e.g., for a polymorphic allele assay), as shown in
FIGURE 20,
the set of primers can include primers designed for amplification of a set of
genomic
regions of interest corresponding to polymorphic alleles associated with the
at least one
of HBAI gene and HBA2 gene (e.g., an HBAI-HBA2 genomic region), where the
polymorphic alleles are within a SEA deletion (e.g., bases 16:215396-234699,
in relation
to GRCh37 genome reference build coordinates), where the polymorphic alleles
include
at least one of (e.g., within the SEA deletion region) rs2858935,
1.5397817350, r51203833,
rs2541675, rs2974771, rs57397665, r53020596, rs2541669, r52238369, m1639532,
rs2858942, rm05364, rs11863726, rs3062451, r528673162, and rs3859139 (e.g., as
shown in FIGURE 21, amplifying or co-amplifying one or more of these
polymorphic
alleles and determining presence or absence of the polymorphic allele from
sequencing
information and associated abundance metrics for use in diagnosis of an alpha-
thalassemia condition; etc.), where the set of genomic regions of interest
includes the
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genomic region of interest, and/or where the method can include: determining
an allele
characterization based on abundance metrics (e.g., read counts; etc.) for the
set of
genomic regions of interest; determining an absence or presence of a father-
associated
deletion corresponding to the at least one of the HMI gene and the HBA2 gene
based on
a comparison between the allele characterization and a mother allele
characterization for
the mother; and/or facilitating the prenatal characterization of the alpha-
thalassemia
condition based on the absence or presence of the father-associated deletion.
Presence of
the father-associated deletion can indicate an alpha-thalassemia condition,
whereas
absence of the father-associated deletion can indicate a lack of the alpha-
thalassemia
condition. Any suitable number of genomic regions of interest (e.g., 12
different regions,
corresponding to 16 polymorphic alleles and/or single nucleotide
polymorphisms; etc.)
associated with alleles corresponding to HBAI gene and/or HBA2 gene can be
used. In a
specific example, the comparison between the allele characterization (e.g.,
for the fetus)
and the mother allele characterization can include a determination of whether
the allele
characterization includes one or more alleles not belonging to the mother,
thereby
indicating inheritance from the father and lack of a father-associated
deletion.
[0053] In
a variation (e.g., for a Constant Spring assay, etc.), as shown in FIGURE
22, the genomic region of interest can include a point mutation (e.g.,
Constant Spring
mutation, etc.) in the HBA2 gene, where the method can include: adding, to the
maternal
sample, a set of quality control template (QCT) molecules including: target-
associated
regions with sequence similarity to a target sequence region of the genomic
region of
interest, and variation regions with sequence dissimilarity to a sequence
region of the
genomic region of interest; and where facilitating the prenatal
characterization of the
alpha-thalassemia condition includes facilitating the prenatal
characterization of the
alpha-thalassemia condition based on an absolute count for the genomic region
of interest
determined from the abundance metric (e.g., read count; etc.) for the genomic
region of
interest and an abundance metric (e.g., read count; any suitable type of
abundance metric
described herein; etc.) for the set of QCT molecules. QCT molecules (and/or
components
of QCT molecules, such as target-associated regions and/or variation regions;
etc.) and/or
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portions of embodiments of the method 100 and/or system zoo relating to QCT
molecules can include and/or be analogous to that described in U.S.
Application No.
16/056,254 filed 06-AUG-2018, which is incorporated in its entirety by this
reference.
[0054] Additionally or alternatively, embodiments of the method loo
and/or
system 200 can function to facilitate the diagnosis (and/or other suitable
characterizations) of one or more alpha-thalassemia conditions, such as based
on
characterization of one or more associated genomic regions of interest in
relation to a
biological sample (e.g., a maternal sample, such as for facilitating diagnosis
and/or other
suitable characterizations for a fetus).
[0055] Additionally or alternatively, as shown in FIGURE 23, embodiments
of the
method 100 can include determining (and/or performing) one or more assays
(and/or
suitable portions of embodiments of the method loo) to perform based on
maternal
genotype (e.g., in relation to alleles associated with one or more alpha-
thalassemia
conditions; etc.), such as for facilitating characterization (e.g., diagnosis,
etc.) of one or
more alpha-thalassemia conditions; etc.).
[0056] Generating an amplified mixture (e.g., for facilitating
characterization of an
alpha-thalassemia condition) can additionally or alternatively include and/or
be
performed in any suitable marmer analogous to that described in relation to
Section 2.3,
S130, and/or to any suitable portion described herein; and/or can be performed
in any
suitable manner. Determining read counts and/or suitable abundance metrics
(e.g., for
facilitating characterization of an alpha-thalassemia condition) can
additionally or
alternatively include and/or be performed in any suitable manner analogous to
that
described in relation to Section 2.4, S140, and/or to any suitable portion
described herein;
and/or can be performed in any suitable manner. Facilitating characterization
of one or
more alpha-thalassemia conditions can additionally or alternatively include
and/or be
performed in any suitable manner analogous to that described in relation to
Section 2.5,
S15o, and/or to any suitable portion described herein; and/or can be performed
in any
suitable manner.
[0057] However, any suitable portions (e.g., of embodiments) associated
with
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alpha-thalassemia condition characterization can be performed in any suitable
manner.
[0058] As shown in FIGURES 1A-1C, 2-3, and 5-6, embodiments of a method
loo
(e.g., for facilitating characterization of one or more conditions, such as
one or more
medical conditions, such as one or more genetic disorders, etc.) can include:
generating a
set of target-associated molecules (e.g., associated with one or more target
molecules
associated with the one or more conditions; etc.) Silo; generating a set of
reference-
associated molecules (e.g., associated with one or more reference molecules;
etc.) S120;
facilitating generation of at least one spike-in mixture (e.g., one or more
spike-in
mixtures; one or more mixtures generated based on spiking-in target-associated
molecules and/or reference-associated molecules) S13o, such as based on
processing the
set of target-associated molecules and the set of reference-associated
molecules with a
biological sample (e.g., a biological sample including target molecules and/or
reference
molecules; a biological sample from a user; etc.), such as where the spike-in
mixtures can
enable increased accuracy (e.g., through minimization of amplification biases,
such as
through generation of the spike-in mixtures from co-amplification; etc.) in
abundance
determination (e.g., for facilitating the characterization of the one or more
conditions;
etc.); determining one or more abundance metrics (e.g., a comparison between a
target-
associated abundance metric such as a target-associated count ratio, and a
reference-
associated abundance metric such as a reference-associated count ratio; a
target-
associated count metric relative to a reference-associated count metric), such
as based on
an analysis (e.g., sequencing operation, etc.) of the at least one spike-in
mixture (e.g.,
based on sequence reads from sequencing the at least one spike-in mixtures;
etc.) S14o;
and/or facilitating the characterization of the one or more conditions based
on the one or
more abundance metrics Si5o.
[0059] Additionally or alternatively, embodiments of the method loo can
include
facilitating treatment Si6o (e.g., of the one or more conditions based on the
one or more
abundance metrics, etc.) and/or any other suitable process.
[0060] In a specific example, the method loo can include: generating a
set of
target-associated nucleic acids (e.g., a target-associated spike-in), where
nucleic acids of
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the set of target-associated nucleic acids include target-associated sequences
(e.g., a
nucleotide sequence matching a target sequence region of a target molecule in
the
biological sample, such as a target molecule corresponding to a biological
target
associated with a medical condition; etc.) associated with a target chromosome
(and/or
other suitable biological target; etc.) (e.g., chromosome 21, where different
sets of target-
associated nucleic acids can be generated, such as where each set can
correspond to a
different loci of chromosome 21 and/or can include target-associated regions
including
nucleic acid sequences matching a target sequence region for the corresponding
loci; etc.),
and include variation regions (e.g., including a variation sequence with one
or more
mutations, polymorphisms, and/or modifications to a target sequence
identifying
chromosome 21, etc.); generating a set of reference nucleic acids (e.g., a
reference spike-
in), where nucleic acids of the set of reference-associated nucleic acids
include reference-
associated sequences associated with a reference chromosome (and/or other
suitable
biological reference) (e.g., chromosome 18, where different sets of reference-
associated
nucleic acids can be generated, such as where each set can correspond to a
different loci
of chromosome 18 and/or can include reference-associated regions including
nucleic acid
sequences matching a reference sequence region for the corresponding loci;
etc.), and can
include variation regions (e.g., including a variation sequence with one or
more
mutations, polymorphisms, and/or modifications to a reference sequence
identifying
chromosome 18, etc.); combining the set of target-associated nucleic acids and
the set of
reference-associated nucleic acids with a biological sample (e.g., using equal
abundances
of the set of target-associated nucleic acids and the set of reference-
associated nucleic
acids; where the biological sample includes a blood sample from a pregnant
female; etc.);
amplifying the set of target-associated nucleic acids and target nucleic acids
(e.g.,
endogenous DNA molecules identifying chromosome 21) from the biological sample
based on a set of target-associated primers (e.g., targeting a sequence shared
by the
target-associated nucleic acids and the target nucleic acids); amplifying the
set of
reference-associated nucleic acids and reference nucleic acids (e.g.,
endogenous DNA
molecules identifying chromosome 18) from the biological sample based on a set
of
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reference-associated primers (e.g., targeting a sequence shared by the
reference-
associated nucleic acids and the reference nucleic acids); determining a
target-associated
count ratio between a first count of target nucleic acids including the target
sequence (e.g.,
a sequence read count for target molecules corresponding to the biological
target; etc.)
and a second count of target-associated nucleic acids (e.g., a sequence read
count
corresponding to the spike-in molecules), where individual count ratios
associated with
different target sequences (e.g., corresponding to different loci of
chromosome 21) can be
combined to determine an overall count ratio; determining a reference-
associated count
ratio between a first count of reference nucleic acids including the reference
sequence
(e.g., a sequence read count for reference molecules corresponding to the
biological
reference; etc.) and a second count of reference-associated nucleic acids
(e.g., a count of
the spike-in reference molecules), where individual reference-associated count
ratios
associated with different reference sequences (e.g., corresponding to
different loci of
chromosome 18) can be combined to determine an overall reference-associated
count
ratio; and/or characterizing (e.g., detecting; diagnosing; etc.) one or more
medical
conditions (e.g., Down syndrome; etc.) for a user (e.g., the user providing
the biological
sample; etc.) based on a comparison between the target-associated count ratio
and the
reference-associated count ratio (e.g., when the target-associated count ratio
corresponding to chromosome 21 exceeds the reference-associated count ratio
corresponding to chromosome 18 beyond a statistically significant threshold
amount,
etc.).
[0061] In
a specific example, the method loo (e.g., for facilitating prenatal
diagnosis of a genetic disorder from a maternal sample associated with a
pregnant
woman, etc.) can include generating a set of target-associated molecules
(e.g., target-
associated nucleic acid molecules; etc.) including target-associated regions
with sequence
similarity to a target sequence region of an biological target (e.g., HbS
mutated
hemoglobin; etc.) associated with the genetic disorder (e.g., sickle cell
disease; etc.); and
target variation regions with sequence dissimilarity to a sequence region
(e.g., a sequence
region adjacent in sequence position to the target sequence region; a sequence
region
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proximal in sequence position to the target sequence region; etc.) of the
biological target;
generating a set of reference-associated molecules including reference-
associated regions
with sequence similarity to a reference sequence region of an endogenous
reference
molecule (e.g., HbA normal hemoglobin; etc.); and reference variation regions
with
sequence dissimilarity to a sequence region of the endogenous reference
molecule;
generating a first spike-in mixture based on amplifying the set of target-
associated
molecules and first nucleic acid molecules from the maternal sample (e.g.,
using primers
targeting sequences corresponding to the sequence similarity between the
target-
associated regions and the target sequence regions, such as for facilitating
co-
amplification; through polymerase chain reaction (PCR) with the primers;
etc.), where
the first nucleic acid molecules (e.g., nucleic acids; nucleic acid fragments;
fetal nucleic
acid molecules; nucleic acid molecules from the mother; etc.) include the
target sequence
region; generating a second spike-in mixture (e.g. via a separate sample
processing
container and set of sample processing operations from co-amplification of the
set of
target-associated molecules and the first nucleic acid molecules; via the same
sample
processing container and set of sample processing operations as the co-
amplification of
the set of target-associated molecules and the first nucleic acid molecules;
where
amplification operations can be performed in the same container for first,
second, and/or
any suitable co-amplification using the same amplification operations, in
separate
containers using separate containers; where any suitable number of containers
can be
used for any suitable number of mixtures; such as where the first and the
second spike-in
mixtures and/or any suitable mixtures are in the same or different containers;
etc.) based
on amplifying the set of reference-associated molecules and second nucleic
acid
molecules (e.g., nucleic acids; nucleic acid fragments; fetal nucleic acid
molecules; nucleic
acid molecules from the mother; etc.) from the maternal sample, where the
second nucleic
acid molecules include the reference sequence region; sequencing (e.g., via
high-
throughput sequencing, etc.) the first and the second spike-in mixtures (e.g.,
in a single
container; in different containers; in a plurality of containers; etc.) to
determine a read
count for the endogenous biological target (e.g., sequence read count for
sequences
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including the target sequence region; sequence read count for target molecules
corresponding to the biological target; etc.), a read count for the set of
target-associated
molecules (e.g., sequence read count for sequences corresponding to the target-
associated
molecules; sequence read count for target-associated molecules; etc.), a read
count for the
endogenous reference molecule (e.g., sequence read count for sequences
including the
reference sequence region; etc.), and a read count for the set of reference-
associated
molecules (e.g., sequence read count for sequences corresponding to the
reference-
associated molecules; etc.); determining a target-associated count ratio based
on the read
count for the biological target and the read count for the set of target-
associated molecules
(e.g., target-associated count ratio of sequence read count for endogenous HbS
to
sequence read count for HbS spike-in molecules; etc.); determining a reference-
associated count ratio based on the read count for the endogenous reference
molecule and
the read count for the set of reference-associated molecules (e.g., reference-
associated
count ratio of sequence read count for endogenous HbA to sequence read count
for HbA
spike-in molecules; etc.); and/or facilitating the prenatal diagnosis of the
genetic disorder
based on a comparison between the target-associated count ratio and the
reference-
associated count ratio.
[0062] In
a specific example, the method 100 (e.g., for facilitating characterization
of a medical condition from a biological sample, etc.) can include generating
a set of
target-associated molecules including target-associated regions with sequence
similarity
to a target sequence region of a biological target (e.g., where the set of
target-associated
molecules can additionally or alternatively include target variation regions
with sequence
dissimilarity to a sequence region of the biological target; etc.); generating
a set of
reference-associated molecules including reference-associated regions with
sequence
similarity to a reference sequence region of a biological reference (e.g.,
where the set of
reference-associated molecules can additionally or alternatively include
reference
variation regions with sequence dissimilarity to a sequence region of the
biological
reference; etc.); facilitating generation of at least one spike-in mixture,
where the
generation of the at least one spike-in mixture (e.g., one or more spike-in
mixtures; etc.)
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includes amplification of the set of target-associated molecules, the set of
reference-
associated molecules, first nucleic acid molecules from the biological sample,
and second
nucleic acid molecules from the biological sample (e.g., co-amplification of
the set of
target-associated molecules and the first nucleic acid molecules, such as with
a first set of
primers targeting the set of target-associated molecules and the first nucleic
acid
molecules, such as based on the sequence similarity; co-amplification of the
first set of
reference-associated molecules and the second nucleic acid molecules, such as
in the
same or different sample compartments using same, similar, or different sample
processing operations, such as with a second set of primers targeting the set
of reference-
associated molecules and the second nucleic acid molecules, such as based on
the
sequence similarity; etc.), where the first nucleic acid molecules are
associated with (e.g.,
include; etc.) the target sequence region (and/or the sequence regions to
which the target
variation regions include sequence dissimilarity; etc.), and where the second
nucleic acid
molecules are associated with (e.g., include; etc.) the reference sequence
region (and/or
the sequence regions to which the reference variation regions include sequence
dissimilarity; etc.); determining at least one abundance metric associated
with the
biological target, the set of target-associated molecules, the biological
reference, and the
set of reference-associated molecules, based on sequence reads from sequencing
of the at
least one spike-in mixture (e.g., determining a count for the biological
target, a count for
the set of target-associated molecules, a count for the biological reference,
and a count for
the set of reference-associated molecules, based on sequencing of the at least
one spike-
in mixture; determining a target-associated count ratio and a reference-
associated count
ratio based on the count for the biological target, the count for the first
set of target-
associated molecules, the count for the biological reference, and the count
for the first set
of reference-associated molecules; etc.); and/or facilitating the
characterization of the
medical condition based on the at least one abundance metric (e.g., based on
the target-
associated count ratio and/or the reference-associated count ratio; etc.).
[0063]
Embodiments of the method loo and/or system 200 can function to
improve accuracy of determining abundance metrics associated with one or more
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biological targets (e.g., enabling accurate comparisons of abundance
measurements for
molecules including sequences across multiple loci, such as enabling accuracy
associated
with coefficient of variation of less than o.1% and/or any suitable accuracy;
etc.).
Embodiments of the method 100 and/or system 200 can additionally or
alternatively
function to leverage the abundance metrics to facilitate characterization
(e.g., detect;
diagnose; analyze; providing information regarding; provide parameters used in
types of
characterization such as diagnosis; improve accuracy regarding diagnosis;
etc.) and/or
facilitate treatment (e.g., through treatment determination, treatment
evaluation and
modification over time, treatment recommendation, provision, administration,
etc.) of
one or more conditions (e.g., medical conditions such as one or more
chromosomal
abnormalities and/or single gene disorders; such as an aneuploidy-associated
condition,
where characterization can require highly accurate abundance determination;
etc.), such
as in relation to noninvasive prenatal testing (NIPT).
[0064]
Embodiments can additionally or alternatively function to detect, quantify,
and/or otherwise characterize breakpoints (e.g., quantitatively detecting
target sequences
including small deletions and/or insertions, such as in relation to detecting
beta-
thalassemia 619 bp-deletion; such as in relation to NIPT; etc.). In a specific
example, the
method wo can include: synthesizing target-associated spike-in molecules
including
target-associated sequences differing (e.g., by a small number of base pairs)
from the
target sequences (e.g., a sequence associated with beta-thalassemia 619 bp-
deletion; a
sequence associated with a different genetic abnormality; etc.) for the
biological sample;
synthesizing reference-associated spike-in molecules including reference-
associated
sequences differing (e.g., by a small number of base pairs) from the reference
sequences
(e.g., a sequence without the beta-thalassemia 619 bp-deletion; a sequence
without the
genetic abnormality; etc.) for the biological sample; determining abundance
ratio metrics
respectively for the target (e.g., endogenous to spike-in ratio) and the
reference (e.g.,
endogenous to spike-in ratio), such as through performing processing
operations (e.g.,
amplification, sequencing, etc.) described herein; and/or comparing the
abundance ratio
metrics for detecting a condition associated with the target (e.g.,
thalassemia, etc.).
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[0065] Embodiments can additionally or alternatively function to detect,
quantify,
and/or otherwise characterize molecules of a particular locus (e.g., for
determining an
initial abundance metric for a particular locus in a biological sample such as
in single-
gene NIPT, where the initial abundance metric can be compared to final
abundance
metrics for evaluating statistical confidence; etc.). In a specific example
(e.g., in relation
to inheriting sickle cell disease), the method loo can include: synthesizing
target-
associated spike-in molecules including target-associated sequences differing
(e.g., by a
small number of base pairs) from the target sequences (e.g., a sequence at a
locus
associated with sickle cell disease; a sequence associated with beta-
thalassemia 619 bp-
deletion; a sequence associated with a different genetic abnormality; etc.)
for the
biological sample; processing the target-associated spike-in molecules (e.g.,
of known
abundance) with target molecules from a biological sample (e.g., performing
amplification, sequencing, etc.); determining one or more abundance metrics
for the
target (e.g., number of molecules in the biological sample for the target
locus, etc.) based
on processing the abundance ratio metric (e.g., endogenous to spike-in) with
the known
abundance metric of the spike-in molecules (e.g., multiplying the abundance
ratio by the
known number of spike-in molecules); and/or facilitating characterization of
the one or
more conditions (e.g., status of disease state; etc.) based on processing the
one or more
abundance metrics with outputs from approaches for determining fetal fraction
of
molecules (e.g., determining proportion of molecules belonging to mother
versus fetus).
However, embodiments can include any suitable functionality.
[0066] Embodiments of the method loo and/or system 200 can be used in
association with one or more conditions (e.g., in association with
characterizing,
diagnosing, treating, and/or performing processes related to one or more
conditions;
etc.), where the conditions can include and/or otherwise be associated with
one or more
of: NIPT (e.g., in relation to genetic screening for presence of chromosomal
abnormalities
including aneuploidy, such as trisomy 21 or Down syndrome, trisomy 18 or
Edwards
syndrome, trisomy 13 or Patau syndrome, sex chromosome aneuploidies such as
Turner
syndrome, other suitable aneuploidies; chromosomal abnormalities including
DiGeorge
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syndrome; in relation to genetic screening for single gene disorders such as
spinal
muscular atrophy conditions and/or alpha-thalassemia conditions; rare variant-
associated conditions; etc.); other prenatal testing; aneuploidy analysis
and/or other
suitable analysis outside of a prenatal context; spinal muscular atrophy
conditions; alpha-
thalassemia conditions; genetic disorders (e.g., single gene disorders
including sickle cell
disease and/or rare variant-associated conditions; chromosomal abnormalities;
disorders associated with gene amplification; gene deletion; partial
chromosomal
abnormalities; 22(111.2 deletion syndrome or DiGeorge syndrome; Charcot-Marie-
Tooth
syndrome, cystic fibrosis, Huntington's disease; Duchenne muscular dystrophy;
hemophilia, thalassemia; rare variant-associated conditions etc.), other
conditions
associated with chromosome abnormalities (e.g., additional, missing, irregular
chromosomal DNA, etc.), rare variant-associated conditions, cancer (e.g.,
through
analyses associated with any suitable oncogenes, cancer biomarkers, and/or
other cancer-
associated targets; through analyses associated with liquid biopsies), and/or
any other
suitable conditions. Conditions can additionally or alternatively include:
psychiatric and
behavioral conditions (e.g., a psychological disorder; depression; psychosis;
etc.);
communication-related conditions (e.g., expressive language disorder;
stuttering;
phonological disorder; autism disorder; voice conditions; hearing conditions;
eye
conditions; etc.); sleep-related conditions (e.g., insomnia, sleep apnea;
etc.);
cardiovascular-related conditions (e.g., coronary artery disease; high blood
pressure;
etc.); metabolic-related conditions (e.g., diabetes, etc.), rheumatoid-related
conditions
(e.g., arthritis, etc.); weight-related conditions (e.g., obesity, etc.); pain-
related
conditions; endocrine-related conditions; genetic-related conditions; chronic
disease;
and/or any other suitable type of conditions.
[0067]
Embodiments of the method loo and/or system 200 can additionally or
alternatively transform entities (e.g., biological samples, targets,
references, synthesized
molecules, users, sample handling systems, computational systems, etc.) into
different
states or things. For example, the method loo can include synthesizing spike-
in molecules
(e.g., target-associated molecules, reference-associated molecules) including
variation
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regions to process alongside (e.g., amplify with) target molecules and/or
reference
molecules for transformation into forms suitable for accurate abundance
determination
while minimizing amplification bias. Such processes can enable previously
=performable characterizations (e.g., of medical conditions; etc.) and/or
treatment
evaluations (e.g., through facilitating improved accuracy for meaningful
quantification
and comparisons of spike-in molecules, target molecules, and/or reference
molecules,
such as associated with sequences across different loci, etc.). However,
portions of
embodiments of the method loo and/or system 200 can provide any other suitable
benefits, such as in the context of using non-generalized systems and/or
performing
unconventional processes.
[0068]
Sequencing and/or sequencing-related technologies (e.g., in relation to
S13o and/or S14o) associated with one or more portions of embodiments of the
method
loo and/or system 200 (e.g., in relation to sequencing associated with genomic
regions
of interest and/or homologous native genomic regions; etc.)can include high
throughput
sequencing, which can include and/or be associated with any one or more of:
NGS, NGS-
associated technologies, massively parallel signature sequencing, Polony
sequencing, 454
pyrosequencing, Illumina sequencing, SOLID sequencing, Ion Torrent
semiconductor
sequencing, DNA nanoball sequencing, Heliscope single molecule sequencing,
Single
molecule real time (SMRT) sequencing, Nanopore DNA sequencing, any generation
number of sequencing technologies (e.g., second-generation sequencing
technologies,
third-generation sequencing technologies, fourth-generation sequencing
technologies,
etc.), amplicon-associated sequencing (e.g., targeted amplicon sequencing),
met agenome-associated sequencing, sequencing-by-synthesis, tunneling currents
sequencing, sequencing by hybridization, mass spectrometry sequencing,
microscopy-
based techniques, and/or any suitable technologies related to high throughput
sequencing. Additionally or alternatively, sequencing and/or sequencing-
related
technologies can include and/or apply any suitable sequencing technologies
(e.g., Sanger
sequencing, capillary sequencing, any suitable sequencing technologies, etc.).
Additionally or alternatively, any suitable portions of embodiments of the
method 100
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and/or system 200 can be performed with, include, and/or otherwise be
associated with
(e.g., generating target-associated molecules and/or reference-associated
molecules for;
determining abundance metrics based upon corresponding outputs; etc.) any
suitable
abundance determination techniques (e.g., for measuring relative abundance of
different
DNA sequences; sequence-specific abundance determination techniques; etc.),
including
any one or more of: microarrays, fluorescence in situ hybridization (FISH)
probes, and/or
any suitable techniques. In examples, a large number (and/or any suitable
number of
spike-ins, such as target-associated molecules and/or reference-associated
molecules,
can be designed, generated, and/or otherwise processed with single-nucleotide
polymorphisms relative to a large number (and/or any suitable number) of
target
sequences in a way that the polymorphisms can be detected by one or more
microarrays.
A microarray can then be used to detect the abundance of each spike-in to each
target. In
a specific example, since all spike-ins can be added at equimolar
concentration, any
significant differences at different target regions will indicate a difference
in abundance
of that target region. These differences, aggregated over multiple adjacent
target
sequences, can then be used to characterize microdeletions, microinsertions,
copy
number variations, and/or chromosomal abnormalities both for prenatal
diagnostics and
for liquid biopsies (and/or for any suitable conditions). The aggregation
calculations can
be performed by any mathematical averaging techniques, including but not
limited to
local weighting, local regression, Kernel smoothing, and Hidden Markov Models,
and/or
using any suitable analytical techniques described herein. However, any
suitable portions
of embodiments of the method roo and/or system 200 can be performed with,
include,
and/or otherwise be associated with any suitable abundance determination
techniques in
any suitable manner.
[0069]
Additionally or alternatively, data described herein (e.g., abundance
metrics; characterizations; models; ratios; identifiers; read depths; sequence
reads;
molecule designs such as target-associated molecule designs, reference-
associated
molecule designs, primer designs, experiment designs; etc.) can be associated
with any
suitable temporal indicators (e.g., seconds, minutes, hours, days, weeks, time
periods,
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time points, timestamps, etc.) including one or more: temporal indicators
indicating
when the data was collected, determined, transmitted, received, and/or
otherwise
processed; temporal indicators providing context to content described by the
data, such
as temporal indicators indicating different stages of spike-in mixture
generation and/or
suitable sequencing library preparation and/or sequencing; changes in temporal
indicators (e.g., data over time; change in data; data patterns; data trends;
data
extrapolation and/or other prediction; etc.); and/ or any other suitable
indicators related
to time.
[0070] Additionally or alternatively, parameters, metrics, inputs,
outputs, and/or
other suitable data described herein can be associated with value types
including any one
or more of: scores, binary values, classifications, confidence levels,
identifiers (e.g.,
sample identifiers, molecule identifiers for any suitable molecules described
herein, etc.),
values along a spectrum, and/or any other suitable types of values. Any
suitable types of
data described herein can be used as inputs, generated as outputs, and/or
manipulated in
any suitable manner for any suitable components associated with embodiments of
the
method loo and/or system 200.
[0071] One or more instances and/or portions of embodiments of the method
loo
and/or processes described herein can be performed asynchronously (e.g.,
sequentially),
concurrently (e.g., in parallel; concurrently processing biological samples in
a multiplex,
automated manner, such as to generated one or more spike-in mixtures;
concurrently
computationally processing sequence reads to improve system processing
ability, such as
for determining one or more abundance metrics and/or facilitating one or more
characterizations; etc.), in temporal relation to a trigger event, and/or in
any other
suitable order at any suitable time and frequency by and/or using one or more
instances
of embodiments of the system 200, components, and/or entities described
herein.
[0072] Embodiments of the system 200 can include a sample handling
network
configured to generate molecules (e.g., target-associated molecules, reference-
associated
molecules), process biological samples, facilitate generation of spike-in
mixtures (and/or
suitable sequencing libraries; etc.) and/or perform other suitable processes;
a sequencing
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system configured to sequence processed genetic material from spike-in
mixtures; a
computing system (e.g., remote computing system, local computing system, etc.)
configured to analyze the sequences, to determine abundance metrics, to
facilitate
characterizations, and/or perform suitable computational processes; and/or any
other
suitable components. However, the method loo and system 200 can be configured
in any
suitable manner.
2.1 Generating target-associated molecules.
[0073] Embodiments of the method 100 can include generating one or more
target-
associated molecules Silo, which can function to synthesize one or more
molecules
sharing one or more characteristics (e.g., sequence characteristics,
functional
characteristics, structural characteristics, evolutionary characteristics,
etc.) with one or
more targets (e.g., biological targets; etc.), which can facilitate similar
sample processing
parameters (e.g., amplification parameters, etc.) to reduce bias (e.g.,
amplification bias,
such as through co-amplification with nucleic acid molecules from the
biological sample
and including one or more target sequence regions of the one or more
biological targets,
etc.) and to improve accuracy during downstream processing.
[0074] Target-associated molecules preferably include target-associated
regions
(e.g., each target-associated molecule including one or more target-associated
regions;
etc.). For example, a target-associated molecules can include a target-
associated region
with sequence similarity (e.g., full sequence similarity; sequence similarity
greater than a
threshold percentage and/or amount; etc.) to a target sequence region of a
biological
target associated with the medical condition.
[0075] Target-associated regions (and/or the target-associated molecules)
are
preferably associated with (e.g., sharing nucleotide sequences with; sharing
sets of bases
with a target sequence at corresponding positions; able to be processed with;
able to be
amplified with, such as through co-amplification; able to be targeted by the
same primers;
complementary to; targeting; digitally associated with in a computing system;
etc.) one
or more biological targets and/or target molecules (e.g., target molecules
corresponding
to biological targets; target molecules including target sequence regions of
biological
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targets; etc.). Biological targets (e.g., target markers; corresponding to,
causing,
contributing to, therapeutic in relation to, correlated with, and/or otherwise
associated
with one or more medical conditions; targets of interest; known or identified
targets;
unknown or previously unidentified targets; etc.) can include any one or more
of target
sequence regions (e.g., sequences identifying a chromosome; sequences
indicative of a
condition; sequences that are invariant across a population and/or any
suitable set of
subjects; conserved sequences; sequences including mutations, polymorphisms;
nucleotide sequences; amino acid sequences; etc.), genes (e.g., associated
with one or
more single gene disorders, etc.), loci, chromosomes (e.g., associated with
one or more
chromosomal abnormalities; etc.) proteins (e.g., serum proteins, antibodies,
etc.),
peptides, carbohydrates, lipids, nucleic acids (e.g., extracellular RNA,
microRNA,
messenger RNA, where abundance determination for RNA targets can include
suitable
reverse transcriptase operations, etc.), cells (e.g., whole cells, etc.),
metabolites, natural
products, genetic predisposition biomarkers, diagnostic biomarkers, prognostic
biomarkers, predictive biomarkers, other molecular biomarkers, gene expression
markers, imaging biomarkers, and/or other suitable targets. Targets are
preferably
associated with conditions described herein, and can additionally or
alternatively be
associated with one or more conditions including: symptoms, causes, diseases,
disorders,
and/or any other suitable aspects associated with conditions. In an example,
as shown in
FIGURE ii, target-associated molecules can include nucleotide sequences
identical to one
or more regions of a target sequence of a target molecule (e.g., identifying
chromosome
21), where primers can concurrently target both the target-associated
molecules and the
target molecules by targeting the identical regions (e.g., for facilitating co-
amplification,
such as to reduce amplification bias, etc.). In another example, target-
associated
molecules can include sequences with any suitable sequence identity to target
molecule
sequences, where any number and/or type of primers can be used in concurrently
or
separately targeting the target-associated molecules and target molecules.
However,
targets (e.g., biological targets, etc.) can be configured in any suitable
manner.
Additionally or alternatively, target-associated molecules (e.g., target-
associated regions
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of target-associated molecules; etc.) can share any suitable characteristics
(e.g.,
components, etc.) with biological targets (e.g., with target molecules
corresponding to
biological targets; etc.), such as to facilitate similar sample processing
parameters to be
able to subsequently generate meaningful comparisons between abundance metrics
for
the target-associated molecules and the target molecules. However, target-
associated
molecules can be configured in any suitable manner.
[0076]
Target-associated molecules preferably include target variation regions
(e.g., variation regions of target-associated molecules; each target-
associated molecule
including one or more variation regions; etc.), where a variation region can
include
different characteristics from the characteristics of the target molecule.
Variation regions
preferably include one or more variations (e.g., single nucleotide variations,
etc.), such as
variations that can enable a corresponding target-associated molecule (e.g.,
the target-
associated molecule including the variation region; etc.) to proceed through
sample
processing operations in a similar manner to the corresponding target
molecules (e.g.,
nucleic acids including a target sequence region of a biological target;
etc.), while
facilitating differentiation of the target-associated molecules from the
target molecules
(e.g., during post-processing of sequence reads for the one or more spike-in
mixtures,
where sequence reads including the variation region can be mapped to the
target-
associated molecules as opposed to the biological target; etc.). Such
differentiation can
facilitate determination of different corresponding abundance metrics that can
be
meaningful compared (e.g., where the initial abundance, such as the number of
molecules
and/or concentration, of the set of target-associated molecules can be known
prior to
generating the spike-in mixture, etc.). In an example, the variation region
can include a
sequence variation region including a nucleotide sequence differing from a
sequence
region of the target molecule. In a specific example, as shown in FIGURES 7A
and 9A,
variation regions can include one or more deletions (e.g., 5-base pair
deletion relative a
sequence region of "tgagt" of the biological target, as shown in FIGURE 7A; 5-
base pair
deletion relative a sequence region of "aatgt" of the biological target such
as HbS, as
shown in FIGURE 9A; etc.) and/or insertions (e.g., 5-base pair insertion of
"tgagt", as
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shown in FIGURE 7A; 5-base pair insertion of "aatgt" relative the biological
target such
as HbS, as shown in FIGURE 9A, etc.) relative to a sequence region (e.g.,
sequence region
of hgv9 and/or any suitable genome references, corresponding to CCL3L1, as
shown in
FIGURE 7A; corresponding to hemoglobin, as shown in FIGURE 9A; etc.).
Variation
regions can be designed in coordination with the target-associated regions to
facilitate
appropriate sequence dissimilarity and sequence similarity, respectively. In a
specific
example, as shown in FIGURE ii, the target-associated molecule can include a
nucleotide
sequence variation region differing from the corresponding target nucleotide
sequence by
ro bases (e.g., where the target sequence includes a "aacggtattt" region
(portion of SEQ
ID NO: 8) and where the variation region includes a "tctatatagg" region
(portion of SEQ
ID NO: 9) at corresponding positions, etc.). Sequence variation regions can
differ by
target sequences by any suitable number and type of bases, at any suitable
positions (e.g.,
sequential positions, non-sequential), across any suitable loci, for any
suitable
chromosome and/or other target, and/or can differ from target sequences in any
suitable
manner. Sequence variation regions can include any one or more of
substitutions,
insertions, deletions, any suitable mutation types, and/or any suitable
modifications (e.g.,
relative one or more sequence regions of a biological target; etc.). For
example, target
variation regions can include a target variation region including at least one
of a first
substitution, a first insertion, and a first deletion, relative to the
sequence region of the
biological target, and reference variation regions can include a reference
variation region
including at least one of a second substitution, a second insertion, and a
second deletion,
relative to the sequence region of the biological reference.
[0077] Additionally or alternatively, variation regions can include non-
sequence
variation regions, with functional, structural, evolutionary, and/or other
suitable
characteristics that are different from the characteristics of the one or more
target
molecules (e.g., of any suitable type, etc.). However, variation regions can
be configured
in any suitable manner, and target-associated molecules can include any
suitable
nucleotide sequence regions.
[0078] In a specific example, as shown in FIGURE 7A, target-associated
molecules
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(e.g., spike-in DNA CCL3L1 gene, etc.) can include a sequence (e.g., including
one or more
target-associated regions and target variation regions; as shown in the bottom
alignment
of FIGURE 7A; etc.) including engineered indels that enable differentiation
between
sequencing products derived from human DNA (e.g., from a biological sample,
etc.) and
target-associated molecules (e.g., synthetic spike-in DNA, etc.), such as
where the top
alignment, shown in FIGURE 7A, is the expected CCL3L1 amplicon after PCR using
forward primer=5'-GGGTCCAGAAATACGTCAGT-3' (SEQ ID NO: 16) and reverse
primer=5'-CATGTFCCCAAGGCTCAG-3' (SEQ ID NO: 17) based on the hg19 human
genome reference assembly. In a specific example (e.g., validating usage of
target-
associated molecules, such as for characterizing copy number variation; etc.),
as shown
in FIGURE 7B, copy number of CCL3L1 (C-C Motif Chemoldne Ligand 3 Like 1) can
be
measured in HapMap samples using spike-ins, where results can be improved over
reported CCL3L1 Copy number measurements assayed by ddPCR (e.g., NA18573=1,
NA18501=3, NA18537=6, NA19239=9-10), where 40 ng of genomic DNA can be used in
a PCR reaction with PCR primers specific to CCL3L1, and 30,000 copies of
CCL3L1 spike-
in DNA is added; and after PCR amplification, the "Ref. Ratio" of genomic DNA
to spike-
in DNA can be measured by DNA sequencing, where NA185o1 has been reported to
have
3 copies of the CCL3L1 gene, and where CCL3L1 copy number was calculated for
NA18573, NA18537, and NA19239 by normalizing their respective Ref. Ratios to
the
NA185o1 Ref. Ratio and multiplying by 3.
[0079] In
a variation, target-associated molecules can include one or more
sequencing molecules (e.g., sequencing regions, etc.) configured to aid in the
operation of
sequencing systems. Sequencing molecules can include sequencing primers (e.g.,
Universal PCR primers, Sequencing Primer 1, Sequencing Primer 2 and/or other
suitable
sequence molecules associated with Illumina sequencing systems), adapter
sequences,
and/or other suitable components associated with any suitable sequencing
systems.
Additionally or alternatively, any suitable components described herein (e.g.,
primer
molecules used during amplification operations in generating the spike-in
mixture) can
include and/or can otherwise be associated with sequencing molecules. However,
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sequencing molecules can be configured in any suitable manner.
[0080] The target-associated molecules (and/or other suitable components
described herein, such as reference-associated molecules, components of spike-
in
mixtures, etc.) can be of any suitable size (e.g., 80-150 base pairs in
length, including one
or more variation regions of to base pairs each or to base pairs total; sizes
selected based
on suitability for different conditions and/or applications described herein;
etc.). The set
of target-associated molecules can include any number of target-associated
molecules
associated with any suitable number of targets (e.g., any number of target
sequences
associated with any number of chromosomes; biological targets; etc.),
biological samples
(e.g., concurrently synthesizing a batch of molecules for use with samples
across multiple
users, to improve efficiency of the sample handling system; etc.),conditions
(e.g., set of
target-associated molecules associated with biological targets associated with
different
conditions; etc.), and/or other suitable aspects.
[0081] In variations, generating target-associated molecules can include
generating different types of target-associated molecules (e.g., including
different target-
associated regions, different variation regions, different sequence molecules,
etc.), such
as sets of target-associated molecules (e.g., each set corresponding to a
different type of
target-associated molecules; etc.). Target-associated molecules can include
sets of target-
associated molecules (e.g., a plurality of different sets, etc.), each set
including a different
target-associated region associated with (e.g., with sequence similarity to;
etc.) a different
target sequence region (e.g., different target sequence regions of a same
biological target
such as a chromosome; different target sequence regions of different
biological targets
such as different genes; etc.), which can facilitate different pairs of a
target-associated
region type (e.g., corresponding to a specific target-associated region
sequence; etc.) and
a target sequence region type (e.g., corresponding to a specific target
sequence of a
biological target; etc.), such as to determine corresponding abundance metrics
such as
individual count ratios (e.g., corresponding to the different pairs; such as
individual count
ratios corresponding to different loci of a chromosome biological target;
etc.), which can
be used in determining an overall abundance metric with increased accuracy
through, for
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example, averaging and/or performing any suitable combination operations with
the
individual count ratios.
[0082] For example, the method loo can include generating a first set of
target-
associated molecules including first target-associated regions with sequence
similarity to
a first target sequence region of a first biological target; generating a
second set of target-
associated molecules including second target-associated regions with sequence
similarity
to a second target sequence region (e.g., of the first biological target; of a
second biological
target; etc.); determining a first target-associated count ratio associated
with the first set
of target-associated molecules and the first target sequence region (e.g.,
ratio of sequence
read count for the first set of target-associated molecules and sequence read
count for the
target molecules including the first target sequence region; etc.);
determining a second
target-associated count ratio associated with the second set of target-
associated
molecules and the second target sequence region (e.g., ratio of sequence read
count for
the second set of target-associated molecules and sequence read count for the
target
molecules including the second target sequence region; etc.), such as where
facilitating
characterization of the medical condition can include facilitating
characterization of the
medical condition based on the first target-associated count ratio and the
second target-
associated count ratio (and/or one or more reference-associated count ratios.
[0083] In a specific example, different sets of target-associated
molecules can be
associated with different target sequences across different loci. In a
specific example, each
set can be associated with a different locus for the same chromosome (e.g., a
first, second,
third, and fourth locus for chromosome 21), where a sequence of a target-
associated
molecule of a given set can include a sequence region shared by the locus
corresponding
to the set, and can include a sequence variation region differing (e.g., by 10
bases) from
the sequence for the locus. In a specific example, as shown in FIGURE 12, a
first set of
target-associated molecules can be associated with a first locus of chromosome
21, and a
second set of target-associated molecules can be associated with a second
locus of
chromosome 21.
[0084] Any number of sets of target-associated molecules and/or any
number of
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types of target-associated molecules can be generated and/or associated with
any suitable
number of biological targets. In an example, selecting different target-
associated
molecule sets can be based on accuracy requirements for a given condition
and/or
application (e.g., selecting a number of sets leading to a corresponding
suitable number
of individual count ratios to be used in achieving a target accuracy for
diagnosing Down
syndrome), but can be selected based on any suitable criteria (e.g., parameter
to be
optimized). However, generating different sets of target-associated molecules
can be
performed in any suitable manner.
[0085]
Generating target-associated molecules can include determining target
sequence regions (e.g., target sequences, etc.), which can function to select
target
sequence regions upon which the generation of target-associated molecules can
be based.
Determining target sequences can be based on: one or more conditions (e.g.,
selecting
target sequences identifying chromosome 21 for facilitating Down syndrome
diagnosis,
etc.), amplification parameters (e.g., selecting target sequences of a
particular length,
nucleotide sequence, and/or other parameter for optimizing amplification
specificity,
such as in relation to primer specificity for the target sequences in relation
to PCR
amplification, etc.), sequencing parameters (e.g., selecting target sequences
for reducing
cost, improving accuracy, and/or for other suitable optimizations in relation
to
sequencing systems and/or operations, etc.), other sample processing
parameters, and/or
other suitable criteria. In an example, determining target sequences can
include
computationally searching a database (e.g., DNA database, genome database,
gene
expression database, phenotype database, RNA database, protein databases,
etc.) to
generate a target sequence candidate list; and filtering the target sequence
candidate list
based on criteria described herein, and/or any suitable criteria. In a
specific example, as
shown in FIGURE ii, determining targeting sequences can include extracting a
target
sequence candidate list (e.g., based on exome pull down; merge into chunks of
a suitable
number of base pairs; etc.); filtering out candidates including defined types
of mutations
and/or polymorphisms (e.g., filtering out candidates associated with common
single
nucleotide polymorphisms to obtain candidates with relative invariance across
subjects
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of a population, etc.); identifying primers for the remaining candidates
(e.g., with a
Primer-BLAST for 80-150 bp amplicons); and determining candidate regions that
are
suitable for variation in generating a variation region of target-associated
molecule (e.g.,
through scrambling bases at positions of Forward Primer+[11,20)). However,
determining target sequences can be performed in any suitable manner.
[0086] Generating the target-associated molecules can include
synthesizing the
molecules through performing any one or more of: plasmid-based nucleic acid
synthesis,
other artificial gene synthesis techniques, phosphoramidite approaches, post-
synthetic
processing, purification (e.g., using high-performance liquid chromatography
or other
chromatography approaches, desalting, washing, centrifuging, etc.),
amplification
techniques (e.g., PCR, etc.), ta: :ing techniques (e.g., molecular ta: ing
techniques,
fluorescent tagging techniques, particle labeling techniques, etc.), molecule
cloning
techniques, and/or any suitable sample processing technique.
[0087] In variations, generating target-associated molecules can be based
on a
desired abundance (e.g., determined based on condition, sample, sequencing
parameters,
sample processing parameters, etc.), such as an abundance ratio (e.g., ratio
of target-
associated molecule abundance to reference-associated molecule abundance;
stoichiometric ratio; concentration ratio; molecule ratio; ratio of any
suitable abundance
type; etc.). For example, the method leo can include determining an abundance
ratio for
the set of target-associated molecules and the set of reference-associated
molecules (e.g.,
based on the medical condition such as a rare variant-associated condition;
etc.),
generating the set of target-associated molecules based on the abundance ratio
(e.g.,
according to a determined stoichiometric ratio between the target-associated
molecules
and the reference-associated molecules; etc.); generating the set of reference-
associated
molecules based on the abundance ratio; and/or determining at least one
abundance
metric (e.g., associated with the biological target, the set of target-
associated molecules,
the biological reference, the set of reference-associated molecules, etc.)
based on the
abundance ratio (and/or sequence reads from sequencing corresponding one or
more
spike-in mixtures; etc.) and/or any other suitable data. In an example,
generating the
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target-associated molecules can include generating the set of target-
associated molecules
at a first abundance at least substantially similar (e.g., stoichiometrically
equal or
substantially equal ratios; substantially similar concentrations; etc.) to a
second
abundance of the generated set of reference-associated molecules. However,
generating
target-associated molecules (and/or reference-associated molecules) at desired
abundances can be performed in any suitable manner.
[0088] In
a variation, as shown in FIGURE 8 and ii, synthesizing the molecules
can include generating one or more plasmids. The plasmids preferably include
the one or
more target-associated molecules (e.g., target-associated regions of target-
associated
molecules; variation regions of target-associated molecules; any suitable
regions of
target-associated molecules; etc.) and/or the one or more reference-associated
molecules
(e.g., any suitable regions of reference-associated molecules; etc.), where
including both
the target-associated molecules and the reference-associated molecules can
facilitate
generation of target-associated molecules and reference-associated molecules
of same or
substantially similar abundance (e.g., same molar ratios) such as for use in
generating
one or more spike-in mixtures. In an example, as shown in FIGURES 2 and 3, the
method
loo can include generating a plasmid including: different types of target-
associated
molecules (e.g., each type corresponding to a different loci for chromosome
21, etc.) and
different types of reference-associated molecules (e.g., each type
corresponding to a
different loci for chromosome 18, etc.). In an example (e.g., such as where a
set of target-
associated molecules is generated at a first abundance at least substantially
similar to a
second abundance of a generated set of reference-associated molecules; etc.),
the method
too can include generating at least one plasmid including target-associated
regions (e.g.,
of target-associated molecules; etc.), target variation regions (e.g., of
target-associated
molecules; etc.), reference-associated regions (e.g., of reference-associated
molecules;
etc.), and reference variation regions (e.g., of reference-associated
molecules; etc.),
generating the set of target-associated molecules (e.g., at the first
abundance; etc.) based
on processing of the at least one plasmid; and/or generating the set of
reference-
associated molecules (e.g., at the second abundance; etc.) based on the
processing of the
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at least one plasmid.
[0089] Additionally or alternatively, the plasmids can include one or
more: cut sites
(e.g., HindIII, EcoRI, Xhol, BamHI, Pstl, etc.), origin of replication sites
(e.g., pUCig ORI,
other pUC sites, etc.), multiple cloning sites, selectable markers (e.g. ICanR
for kanamycin
resistance; resistance associated with ampicillin, chloramphenicol,
tetracycline; etc.),
reporter markers, backbone, and/or any suitable components. The plasmids can
be of any
suitable length (e.g., fewer than 10 kilobases; greater than 10 kilobases;
etc.), and different
sets of target-associated molecules and/or reference-associated molecules can
be
distributed across different plasmids in any suitable manner (e.g., a first
plasmid
including the different sets of target-associated molecules; a second plasmid
including the
different sets of reference-associated molecules; etc.). However, leveraging
plasmids
and/or other suitable techniques to generate any suitable components (e.g., at
any
suitable abundance) described herein can be performed in any suitable manner.
Additionally or alternatively, any suitable number of molecules and/or types
of molecules
can be generated at any suitable time and frequency. However, generating
target-
associated molecules S110 can be performed in any suitable manner.
2.2 Generating reference-associated molecules.
[0090] Embodiments of the method loo can include generating one or more
reference-associated molecules S120, which can function to synthesize one or
more
molecules sharing one or more characteristics with one or more biological
references
(e.g., reference molecules corresponding to the one or more biological
references; etc.),
which can facilitate similar amplification parameters and/or other sample
processing
parameters during processing of the reference-associated molecules and
reference
molecules (e.g., nucleic acids including one or more reference sequence
regions; etc.).
Reference-associated molecules are preferably associated with one or more
references
(e.g., biological references, etc.), such as references facilitating abundance
metric
comparisons to abundance metrics for target molecules and/or target-associated
molecules (e.g., comparisons between reference-associated count ratios and
target-
associated count ratios; etc.). For example, as shown in FIGURE 12, the set of
reference-
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associated molecules can be associated with a chromosomal biological reference
(e.g.,
chromosome 18).
[0091] Reference-associated molecules can include any one or more of
reference-
associated regions (e.g., with sequence similarity to a reference sequence
region of a
biological reference; etc.); reference variation regions (e.g., variation
regions of reference-
associated molecules; with sequence dissimilarity to a sequence region of the
biological
reference; etc.); sequencing molecules; and/or any other suitable regions. In
a specific
example, reference-associated molecules can include nucleotide sequences
shared with
reference sequence regions, and can include a sequence variation region
differing from
the reference sequence (e.g., by io base pairs). Additionally or
alternatively, references
(e.g., biological references; etc.) can include any suitable targets (e.g.,
biological targets;
described herein; etc.); can be associated with any suitable biological
targets (e.g.,
wildtype version of a mutation associated with a biological target; etc.);
and/or can
include any suitable similarity and/or difference from targets.
[0092] In a variation, generating the reference-associated molecules can
include
selecting reference sequences associated with one or more conditions (e.g., a
different
condition from a condition associated with the target sequences), which can
enable
concurrent screening of a plurality of conditions (e.g., through performing a
single
instance of an embodiment of the method loo; through performing any suitable
portions
of embodiments of the method loo). In a specific example, the method loo can
include
selecting target sequences identifying a first chromosome (e.g., chromosome 21
for
characterizing trisomy 21, etc.); and selecting reference sequences
identifying a second
chromosome (e.g., chromosome 18 for characterizing trisomy 18, etc.).
Additionally or
alternatively, applying embodiments of the method loo to characterize and/or
treat
multiple conditions can be performed in any suitable manner.
[0093] Reference-associated molecules can be configured in any suitable
manner
analogous to target-associated molecules (e.g., any suitable size, type,
regions, such as
analogous to size, type, regions of target-associated region; etc.). In an
example, example,
as shown in FIGURE 9A, a reference-associated molecule can include one or more
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reference-associated regions with sequence similarity to a reference sequence
region (e.g.,
of a biological reference, such as HbA, etc.); and one or more reference
variation regions
with sequence dissimilarity to a sequence region (e.g., of the biological
reference; a 5-base
pair deletion relative the sequence region, as shown in FIGURE 9A; a 5-base
pair insertion
such as "tcaga" relative the sequence region, as shown in FIGURE 9A; etc.). In
a specific
example, as shown in FIGURE 9A, reference-associated molecules (e.g.,
"HbA_spk"; HbA
spike-in DNA; etc.) can include a sequence including one or more reference-
associated
regions and reference variation regions (e.g., as shown in the middle
alignment of
FIGURE 9A; etc.), where the top alignment is the expected HBB (hemoglobin
beta)
amplicon after PCR using forward primer=5'-GCAGTAACGGCAGACTICTCCA-3' (SEQ
ID NO: 18) and reverse primer=5'-AAGTCAGGGCAGAGCCATCTA-3' (SEQ ID NO: 19)
based on the hg19 human genome reference assembly, and where the bottom
alignment
includes a sequence of target-associated molecules (e.g., "HbS_spk"; HbS spike-
in DNA;
etc.) respectively, and where PCR primers can include a phosphorothioate bond
at 3'
terminal nucleotide bond. In a specific example (e.g., validating usage of
target-associated
molecules and/or reference-associated molecules, such as for single gene
disorders
and/or rare variant-conditions, such as for detecting single nucleotide
polymorphisms
(SNPs); etc.), as shown in FIGURE 9B, measurement of HbS allele fraction from
NA12892
(HbAA), NA18853 (HbAS), NA19239 (HbAS), and NA16265 HbSS) can be used to
illustrate application of target-associated molecules and/or reference-
associated
molecules. However, reference-associated molecules can be configured in any
suitable
manner.
[0094]
Generating reference-associated molecules S120 can be performed in any
suitable manner analogous to generating target-associated molecules Silo
(e.g.,
generating reference-associated molecules including reference-associated
regions and/or
reference variation regions in a marmer analogous to generating target-
associated
molecules including target-associated regions and/or target variation regions;
determining reference sequences; synthesizing using any suitable sample
processing
technique, synthesizing using plasmids; etc.), and/or can be performed in any
suitable
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manner.
2.3 Facilitating Generation of a Spike-In Mixture.
[0095] Embodiments of the method io o can include facilitating generation
of one
or more spike-in mixtures S13o (e.g., based on processing the set of target-
associated
molecules and the set of reference-associated molecules with one or more
biological
samples from a user, etc.), which can function to amplify (e.g., under similar
amplification
parameters), perform pre-processing upon (e.g., sample preparation, lysis,
bead-based
processes, other purification and/or nucleic acid extraction techniques,
etc.), and/or
otherwise process the target-associated molecules, reference-associated
molecules,
components of the biological sample (e.g., nucleic acid molecules; etc.),
and/or other
suitable components into a form (e.g., one or more mixtures; etc.) suitable
for subsequent
analysis (e.g., sequencing; etc.) and/or abundance metric determination.
[0096] Facilitating generation of the spike-in mixtures can include any
one or more
of: preparing and/or providing components for generation of the spike-in
mixtures (e.g.,
providing one or more sets of target-associated molecules and/or one or more
sets of
reference-associated molecules to an entity for generation by the entity of
the at least one
spike-in mixtures with a biological sample obtained by the entity; etc.);
generating the
spike-in mixtures (e.g., performing the actual generation of the spike-in
mixtures; etc.);
guiding (e.g., instructing; etc.) one or more entities in generation of the
one or more spike-
in mixtures;) and/or performing any suitable processes for facilitating
generation of the
one or more spike-in mixtures.
[0097] Collected biological samples (e.g., collected using sample
containers
provided to users in sample collection kits; collected by other entities
generating the
spike-in mixtures; etc.) can include any one or more of: blood, plasma, serum,
tissue,
biopsies, sweat, urine, feces, semen, vaginal discharges, tears, interstitial
fluid, other body
fluid, and/or any other suitable samples (e.g., associated with a human user,
animal,
object such as food, microorganisms, etc.). In examples, such as for NIPT,
biological
samples can include one or more maternal samples. Biological samples
preferably include
target molecules (e.g., nucleic acid molecules including one or more target
sequence
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regions; etc.) and/or reference molecules (e.g., nucleic acid molecules
including one or
more reference sequence regions; etc.), such as where the target molecules can
be
amplified with the target-associated molecules under similar parameters; where
the
reference molecules can be amplified with the reference-associated molecules
under
similar parameters; etc.). Additionally or alternatively, biological samples
can include
components from multiple users (e.g., a blood sample including nucleic acids
from a
mother and nucleic acids from the mother's unborn baby, where the nucleic acid
mixture
can be indicative of an abnormal abundance of chromosome 18, etc.), components
collected across multiple time periods, and/or components varying across any
suitable
condition, such that generating spike-in mixture(s) can be performed for any
suitable
number and type of entities.
[0098]
Facilitating generation of one or more spike-in mixtures preferably includes
combining target-associated molecules with one or more target molecules from
the
biological sample (and/or combining target-associated molecules with molecules
potentially including target sequence regions, such as where a biological
sample may lack
target molecules and/or associated target sequence regions; etc.); and/or
combining
reference-associated molecules with one or more reference molecules from the
biological
sample. Combining can include one or more of: combining each of the molecules
into a
single mixture (e.g., including the target-associated molecules, target
molecules,
reference-associated molecules, reference molecules; etc.); subsampling a
biological
sample (e.g., a pre-processed sample) for a first and a second mixture, where
target-
associated molecules can be spiked into the first mixture (e.g., which
includes target
molecules), and reference-associated molecules can be spiked into the second
mixture
(e.g., which includes reference molecules); subsampling the pre-processed
biological
sample into a plurality of mixtures, each corresponding to a different set of
target-
associated molecules (e.g., corresponding to different target loci for a
target chromosome,
etc.) and/or a different set of reference-associated molecules (e.g.,
corresponding to
different loci for a reference chromosome, etc.); and/or any other suitable
approach to
combining the molecules. Additionally or alternatively, separate mixtures can
be
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generated for each type of molecule (e.g., without combining different types
of molecules).
Combining molecules preferably includes using an abundance of target-
associated
molecules that is the same or substantially similar to the abundance of
reference-
associated molecules. Further, combining molecules preferably includes using
the same
or substantially similar abundances across different sets of target-associated
molecules
(e.g., associated with different loci), and across different sets of reference-
associated
molecules. Alternatively, any suitable abundances for different molecule types
can be
used.
[0099] In a variation, combining molecules can include modifying (e.g.,
during pre-
processing) abundances of the target-associated molecules, the reference-
associated
molecules, and/or other suitable components. For example, modifying abundances
can
be based on one or more desired abundances (e.g., a desired abundance ratio,
such as
determined based on a medical condition, associated probabilities, etc.). For
example,
modifying abundances of molecules can include measuring initial abundances of
the
molecules (e.g., abundance of the target-associated or reference-associated
DNA
molecules extracted from plasmid-based synthesis); and modifying the
abundances (e.g.,
through dilution, amplification, etc.) based on expected abundances of target
molecules
and/or reference molecules (e.g., expected count for endogenous target
molecules and
endogenous reference molecules in the biological sample, etc.). In another
variation,
generating spike-in mixtures can omit modification (e.g., during pre-
processing) of
abundances (e.g., where the abundance results for a first instance of an
embodiment of
the method loo can be used in determining a correction factor to be used in
subsequent
instances of the embodiment of the method loo; etc.). However, combining
molecules
can be performed in any suitable manner.
[00100] Generating the spike-in mixture preferably includes amplifying
(e.g., co-
amplifying, etc.) the target-associated molecules with the target molecules,
and
amplifying (e.g., co-amplifying, etc.) the reference-associated molecules with
the
reference molecules. Amplification can include performing any one or more of:
PCR-
based techniques (e.g., solid-phase PCR, RT-PCR, qPCR, multiplex PCR,
touchdown PCR,
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nanoPCR, nested PCR, hot start PCR, etc.), helicase-dependent amplification
(HDA), loop
mediated isothermal amplification (LAMP), self-sustained sequence replication
(3SR),
nucleic acid sequence based amplification (NASBA), strand displacement
amplification
(SDA), rolling circle amplification (RCA), ligase chain reaction (LCR), and/or
any other
suitable amplification techniques and/or associated protocols (e.g., protocols
for
minimizing amplification bottlenecking). In an example, generating a spike-in
mixture
can include performing a plurality of PCR rounds to amplify the target-
associated
molecules with the target molecules (e.g., using primers targeting a sequence
shared by
both the target-associated molecules and the target molecules), and to amplify
the
reference-associated molecules with the reference molecules (e.g., using
primers
targeting a sequence shared by both the reference-associated molecules and the
reference
molecules). In a specific example, the amount of amplification (e.g., number
of PCR
rounds, cycles, etc.) can be performed according to results of validation
experiments (e.g.,
during primer selection and validation, stopping PCR reactions at different
amplification
cycles and visualizing products by gel electrophoresis to determine adequacy
of
amplification for conditions and/or applications described herein, such as
sufficient
amplification for next-gen sequencing while minimizing saturation to
facilitate
preservation of ratios; etc.). In specific examples, generating spike-in
mixtures can
include subsampling the biological sample into different subsamples designated
for
different pairs of a target molecule type (or reference molecule type) and a
target-
associated molecule type (or reference-associated molecule type), each pair
corresponding to a different loci (e.g., of chromosome 21 or chromosome 18,
etc.) and/or
different target; and amplifying the different subsamples (e.g., through sets
of PCR
rounds) by using primers specific to the pair corresponding to the subsample.
Additionally or alternatively, target molecules and target-associated
molecules for
multiple pairs of a target molecule type (e.g., associated with a plurality of
different
targets, etc.) may be amplified in the same tube (and/or any suitable number
of tubes),
such as through multiplex PCR, which can facilitate conserving a precious
sample; an
amplified target molecule and target-associated molecule pair may then be
selectively
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sequenced via a sequencing oligonucleotide that is specific to the target
pair. In this or
other examples, subsampling and/or other sample modification operations can be
performed in any suitable order.
[00101] In
variations, as shown in FIGURE in, generation of spike-in mixtures can
be associated with (e.g., tailored for, adapted to; etc.) a plurality of
target sequence regions
(e.g., corresponding to a large number of target sequence regions; a large
number of
biological targets; etc.). For example, a target sequence region can be from a
set of target
sequence regions; a set of target-associated molecules can be from sets of
target-
associated molecules (e.g., different sets of target-associated molecules;
etc.) associated
with different target sequence regions from the set of target sequence
regions, where
generation of at least one spike-in mixture can include preamplification with
the
biological sample based on a set of non-specific primers, to generate a pre-
amplified
sample; and/or subsampling of the pre-amplified sample to facilitate target
sequence
region-specific co-amplification based on sets of specific primers associated
with the set
of target sequence regions and the sets of target-associated molecules (e.g.,
where
different sets of primer types can be used for different co-amplification
operations
between different target sequence regions and different corresponding sets of
target-
associated molecules; etc.). For example, as shown in FIGURE 10, optional
preamplification PCR that is not allele-specific can be performed before
dividing the
resultant pre-amplified DNA (e.g., including components of the biological
sample; target-
associated molecules; reference-associated molecules; etc.) into multiple
downstream
allele-specific PCRs (e.g., for facilitating co-amplification; for
facilitating amplification
bias reduction; etc.), such as by designing and/or applying PCR primers that
are outside
the one or more target polymorphisms, where the preamplification PCR can
include
multiplex PCR using multiple primer pairs if multiple alleles spanning
multiple loci are
to be measured, and where pre-amplification can enable the pre-amplified
product to be
allocated into multiple subsequent allele-specific PCR reactions without
diluting out rare
variants (e.g., in relation to characterization of one or more rare variant-
associated
conditions; etc.). In examples, target-associated molecules and/or reference-
associated
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molecules (e.g., spike-in DNA, etc.) can be added either before pre-
amplification PCR
(e.g., as shown in FIGURE io), and/or the target-associated molecules and/or
reference-
associated molecules spike-in DNA can be added after pre-amplification and
before
sample division into allele-specific PCR. However addition of target-
associated molecules
and/or reference-associated molecules (e.g., in relation to different portions
of generation
of one or more spike-in mixtures; etc.) can be performed at any suitable time
and
frequency. Additionally or alternatively, any suitable sample processing
operations can
be performed in any suitable sequence and/or frequency for facilitating
generation of one
or more spike-in mixtures.
[00102] Additionally or alternatively, target molecules can be amplified
independently from the target-associated molecules, and reference molecules
can be
amplified independently from reference-associated molecules. However,
amplifying
molecules in relation to generating one or more spike-in mixtures can be
performed in
any suitable manner (e.g., where primers can be configured in any suitable
manner, etc.),
and generating one or more spike-in mixtures Siso can be performed in any
suitable
manner.
2.4 Determining an abundance metric.
[00103] Embodiments of the method loo can include determining an abundance
metric S14o (e.g., for one or more biological targets based on an analysis of
the one or
more spike-in mixtures, etc.), which can function to accurately determine
abundance
metrics (e.g., count metrics such as sequence read count, absolute molecule
count, etc.)
such as for use in characterizing one or more conditions (e.g., based on
comparison of
abundance metrics; based on abundance metrics that can be compared across
target-
associated molecules, reference-associated molecules, biological targets,
biological
references; such as for detecting an elevated abundance of chromosome 21 in
relation to
a reference chromosome in a blood sample of a pregnant female, etc.). Analyses
of one or
more spike-in mixtures (e.g., for facilitating determination of one or more
abundance
metrics; etc.) can include one or more of: sequencing of the spike-in mixture
(and/or a
processed form of the spike-in mixture),. such as using any suitable
sequencing
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technologies (e.g., described herein, etc.); computationally processing the
sequence read
results (e.g., mapping sequence reads to sequences associated with target
molecules,
target-associated molecules, reference molecules, reference-associated
molecules, and/or
other suitable molecules, to determine corresponding abundances); and/or any
other
suitable processes. Computational processing (e.g., of the sequence reads
results; etc.),
determining abundance metrics, facilitating characterization of one or more
conditions,
and/or suitable portions of embodiments of the method loo (e.g., facilitating
treatment,
etc.) can include any one or more of: performing pattern recognition on data,
performing
statistical estimation on data (e.g. ordinary least squares regression, non-
negative least
squares regression, principal components analysis, ridge regression, etc.),
fusing data
from multiple sources, combination of values (e.g., averaging values, etc.),
compression,
conversion (e.g., digital-to-analog conversion, analog-to-digital conversion),
wave
modulation, normalization, deconvolving (e.g., Fourier deconvolution; Gaussian
function-based deconvolution; Lucy-Richardson deconvolution etc.), extracting
features,
updating, ranking, validating, filtering (e.g., for baseline correction, data
cropping, etc.),
noise reduction, smoothing, filling (e.g., gap filling), aligning, model
fitting, windowing,
clipping, transformations, mathematical operations (e.g., derivatives, moving
averages,
summing, subtracting, multiplying, dividing, etc.), multiplexing,
demultiplexing,
interpolating, extrapolating, clustering, other signal processing operations,
other image
processing operations, visualizing, and/or any other suitable processing
operations.
[00104]
Abundance metrics can include any one or more of counts (e.g., sequence
read count; absolute molecule count; counts of target-associated molecules;
counts for
biological targets, such as for target molecules corresponding to the
biological targets;
counts for reference-associated molecules; counts for biological references,
such as for
reference molecules corresponding to the biological references; etc.); ratios
(e.g., a target-
associated count ratio of a count for a biological target to a count for
target-associated
molecules; a reference-associated count ratio of a count for a biological
reference to a
count for reference-associated molecules; ratios with any suitable numerator
and
denominator associated with counts and/or other suitable abundance metrics;
etc.);
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individual abundance metrics (e.g., individual abundance metrics such as
individual
counts for pairs of target-associated region type and target sequence region
type;
individual counts for individual samples; individual abundance metrics such as
individual
counts for different types of molecules, targets, references, described
herein; etc.); overall
abundance metrics (e.g., based on individual abundance metrics; overall target-
associated count ratios; overall reference-associated count ratios; etc.);
relative
abundances; absolute abundances; and/or other suitable abundance metrics.
Abundance
metrics associated with target molecules and/or biological targets (e.g., a
target-
associated count ratio) can preferably be compared to abundance metrics
associated with
reference molecules and/or biological references (e.g., a reference-associated
count
ratio), which can facilitate relative abundance analyses (e.g., in screening
for conditions
associated with aneuploidy; for suitable comparisons usable in
characterization of one or
more conditions; etc.).
[00105] In
a variation, determining an abundance metric can include determining
an overall count ratio from a plurality of individual count ratios, which can
increase the
accuracy of the count ratio. For example, as shown in FIGURE 3, determining
abundance
metrics can include determining an overall target-associated count ratio from
averaging
individual count ratios calculated for different pairs of target-associated
molecule type
and target sequence region type (e.g., corresponding to different loci of a
target
chromosome) (and/or target molecule type, biological target type, etc.);
determining an
overall reference-associated count ratio from averaging individual count
ratios calculated
for different pairs of reference-associated molecule type and reference
sequence region
type (e.g., corresponding to different loci of a reference chromosome) (and/or
reference
molecule type, biological reference type, etc.); and/or comparing the overall
target-
associated count ratio to the overall reference-associated count ratio (e.g.,
in facilitating
characterization of one or more conditions, etc.). For example determining an
overall
target-associated count ratio can be based on combination of a first target-
associated
count ratio (e.g., determined based on a count for first target-associated
molecules and a
count for first target molecules including a first target sequence region;
etc.) and a second
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target-associated count ratio (e.g., determined based on a count for second
target-
associated molecules and a count for second target molecules including a
second target
sequence region; etc.), such as where facilitating characterization of the
medical condition
can based on the overall target-associated count ratio (and/or one or more
reference-
associated count ratios, such as one or more overall reference-associated
count ratios;
etc.).
[00106] Additionally or alternatively, determining overall abundance
metrics from
individual abundance metrics (and/or suitable portions of embodiments of the
method
oo) can leverage any suitable statistical approach (e.g., averaging, median,
etc.), and/or
can be performed in any suitable manner. In another variation, abundance
metrics can
be determined over time (e.g., for different biological samples collected over
time; by
performing multiple instances of embodiments of the method roo over time;
etc.), such
as where the series of abundance metrics can be analyzed in facilitating
characterizations
of one or more conditions (e.g., monitoring chromosome 21 abundance over
different
stages of the pregnancy, and processing the set of data to diagnose Down
syndrome; etc.),
treatments, and/or other suitable information. In another variation,
determining an
abundance metric can include applying an abundance determination model
including any
one or more of: probabilistic properties, heuristic properties, deterministic
properties,
and/or any other suitable properties. Additionally or alternatively,
determining overall
abundance metrics can be performed in any suitable manner. However,
determining
abundance metrics S14o can be performed in any suitable manner.
2.5 Facilitating Characterization of a Condition.
[00107] Embodiments of the method 100 can include facilitating
characterization of
one or more conditions S15o (e.g., medical conditions such as genetic
disorders; based on
one or more abundance metric; etc.), which can function to detect, diagnose,
analyze,
determine characterizations for, aid one or more care providers in relation
to, provide
data (e.g., parameters; etc.) regarding; and/or otherwise facilitate
characterization of one
or more conditions. Characterizations can include any one or more of:
diagnoses, risk
assessments, causes (e.g., identification of user behaviors, demographics,
medical
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history, genetics, and/or other suitable aspects contributing to the
condition), and/or
other suitable information informative of the one or more conditions. In
variations, one
or more characterizations can be used in any one or more of: determining a
treatment,
informing users, informing care providers (e.g., guiding care provider in
diagnoses; etc.),
and/or performing any suitable operations. Facilitating one or more
characterizations is
preferably based on comparisons of count ratios (e.g., a comparison of a
target-associated
count ratio against a reference-associated count ratios), but can additionally
or
alternatively be based on any number and/or type of abundance metrics (e.g.,
any suitable
analytical techniques applied to the abundance metrics; etc.). In an example,
as shown in
FIGURE 2, a comparison between a count ratio for chromosome 21 and a count
ratio for
chromosome 18 (e.g., for a biological sample from a pregnant female) can
indicate
outcomes of: elevated relative abundance of chromosome 21 (e.g., with
statistical
significance indicating diagnosis of Down syndrome), an elevated relative
abundance of
chromosome 18 (e.g., with statistical significance indicating diagnosis of
Edwards
syndrome), no elevation of either chromosome, and/or other suitable outcomes.
In an
example, the method 100 can include determining abundance metrics for a first
population of subjects exhibiting the condition and for a second population of
subjects
not exhibiting the condition; determining a set of reference abundance metrics
(e.g., for
a reference model, such as a machine learning model, generated with the
abundance
metrics and supplementary features regarding the populations of subjects)
based on the
abundance metrics; and facilitating characterization of a condition for a
current subject
based on comparing abundance metrics for the subject to the reference
abundance
metrics (e.g., inputting the subject's abundance metrics and associated
supplementary
features regarding the subject into the reference model, etc.).
[00108] In
examples, as shown in FIGURES 2-3, a medical condition can include
one or more genetic disorders including one or more chromosomal abnormalities,
where
the target sequence region (e.g., of a biological target, such as chromosome
21, etc.) is
associated with a first chromosome associated with the chromosomal
abnormality, and
where facilitating characterization of the medical condition (e.g., prenatal
diagnosis of the
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genetic disorder, etc.) includes facilitating the prenatal diagnosis of the
chromosomal
abnormality (e.g., based on a comparison between a target-associated count
ratio and a
reference-associated count ratio; based on any suitable abundance metrics;
etc.). In an
example, the one or more chromosomal abnormalities can include at least one of
a copy
number variation condition and a trisomy condition, and where facilitating the
prenatal
diagnosis of the genetic disorder can include facilitating the prenatal
diagnosis of the copy
number variation condition and the trisomy condition (e.g., based on the
comparison
between the target-associated count ratio and the reference-associated count
ratio; based
on any suitable abundance metrics; etc.). In an example, the one or more
chromosomal
abnormalities can include a trisomy 21 condition, where the target sequence
region is
associated with the first chromosome (e.g., chromosome 21, etc.) where the
reference
sequence region (e.g., of a biological reference, etc.) is associated with a
second
chromosome (e.g., chromosome 18; any suitable chromosomes; etc.), and/or where
facilitating the prenatal diagnosis of the chromosomal abnormality can include
facilitating the prenatal diagnosis of the trisomy 21 condition (and/or
trisomy 18
condition; etc.) (e.g., based on the comparison between the target-associated
count ratio
and the reference-associated count ratio; based on any suitable abundance
metrics; etc.).
In an example (e.g., including a plurality of sets of target-associated
molecules and sets of
reference-associated molecules; etc.), the medical condition can include one
or more
chromosomal abnormalities; where the first target sequence region (e.g., with
sequence
similarity to first target-associated regions of a first set of target-
associated molecules;
etc.) corresponds to a first loci of a first chromosome; where the second
target sequence
region (e.g., with sequence similarity to second target-associated regions of
a second set
of target-associated molecules; etc.) corresponds to a second loci of the
first chromosome;
where the first reference sequence region (e.g., with sequence similarity to
first reference-
associated regions of a first set of reference-associated molecules; etc.)
corresponds to a
first loci of a second chromosome; where generating a second of reference-
associated
molecules can include generating a second set of reference-associated
molecules
including second reference-associated regions with sequence similarity to a
second
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reference sequence region corresponding to a second loci of the second
chromosome;
where determining abundance metrics can include determining a second reference-
associated count ratio associated with the second set of reference-associated
molecules
and the second reference sequence region (e.g., a reference-associated count
ratio of a
sequence read count for reference molecules including the second reference
sequence
region, to a sequence read count for the second set of reference-associated
molecules;
etc.); and/or where facilitating characterization of the medical condition can
include
facilitating characterization of the chromosomal abnormality based on the
first target-
associated count ratio (e.g., associated with the first set of target-
associated molecules
and the first target sequence region; etc.), the second target-associated
count ratio (e.g.,
associated with the second set of target-associated molecules and the second
target
sequence region; etc.), the first reference-associated count ratio (e.g.,
associated with the
first set of reference-associated molecules and the first reference sequence
region; etc.),
and the second reference-associated count ratio (and/or any suitable abundance
metrics;
etc.). In an example, the first chromosome includes chromosome 21; the second
chromosome includes chromosome 18; and where facilitating characterization of
the
medical condition includes facilitating characterization of a trisomy 21
condition and a
trisomy 18 condition based on the first target-associated count ratio, the
second target-
associated count ratio, the first reference-associated count ratio, and the
second
reference-associated count ratio (and/or any suitable abundance metrics;
etc.). In a
specific example (e.g., illustrating non-invasive prenatal testing of trisomy
21 using DNA
sequencing of spike-in molecules; etc.), as shown in FIGURE 8, twenty-six
types of target-
associated molecules and/or reference-associated molecules (e.g., twenty-six
spike-in
sequences; etc.) can be designed to co-amplify with associated targets (e.g.,
associated
target sequence regions) located on either chromosome 21 or chromosome 18 in a
multiplex PCR; where o% or 20% Trisomy 21 (T21) affected DNA is added to 33n
of
normal human DNA and synthetic spike-in plasmid (e.g., as shown in FIGURE 4);
where
the human DNA and synthetic spike-in DNA are co-amplified using common primer
pairs
in a multiplex PCR reaction and prepared for DNA sequencing (e.g., on any
suitable
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sequencing technologies; on the Illumina Miseq; etc.); where spike-in
corrected read
counts can enable NIPT for T21; where, to compensate for the variance
associated with
technical replicates in PCR, a spike-in corrected T21 risk score can be
calculated using
(R2hf. mNk)/(RiviR) . ;
`8-
where RsPk is the number of reads originating from spike-in
sequences (e.g., in relation to chromosome 21 or chromosome 18), and Rhg is
the number
of reads originating from human DNA (e.g., in relation to chromosome 21 or
chromosome
18); and where, by applying the correction factor, a significant difference
between T21
affected and unaffected samples can be observed (e.g., p = 0.0025).
[00109]
In an example, a medical condition can include one or more genetic
disorders including one or more single gene disorders, where the target
sequence region
(e.g., of a biological target, such as a gene corresponding to the single gene
disorder, etc.)
includes a mutation associated with the single gene disorder, where the
reference
sequence region (e.g., of a biological reference, such as the gene
corresponding to the
single gene disorder; etc.) lacks the mutation, and where facilitating
characterization of
the medical condition (e.g., facilitating the prenatal diagnosis of the
genetic disorder; etc.)
=
includes facilitating the prenatal diagnosis of the single gene disorder
(e.g., based on the
comparison between the target-associated count ratio and the reference-
associated count
ratio; based on any suitable abundance metrics; etc.). In an example, a
medical condition
can include one or more single gene disorders including at least one of a
cancer condition
and sickle cell disease, where the target sequence region can include a
mutation
associated with the at least one of the cancer condition and the sickle cell
disease, where
a reference sequence region lacks the mutation, and/or where facilitating the
characterization of the medical condition can include facilitating the
characterization of
the at least one of the cancer condition and the sickle cell disease (e.g.,
based on the target-
associated count ratio and the reference-associated count ratio; based on any
suitable
abundance metrics; etc.). In a specific example, as shown in FIGURE 5, the
method loo
can include sample processing operations and computational processes tailored
to
facilitating characterization of sickle cell disease (e.g., where I-lbS
mutation is expected to
be present for 40-6096 of the allele fraction, such as in the context of NIPT;
etc.); such as
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where stoichiometrically equal amounts of target-associated molecules (e.g.,
"HbS_SPK")
and reference-associated molecules (e.g., "HbA SPK") can be added to a
biological
sample; where the resulting mixture can be divided evenly between two or more
PCR
reactions (e.g., a first PCR reaction for co-amplification of target-
associated molecules
and nucleic acids including the target sequence region; a second PCR reaction
for co-
amplification of reference-associated molecules and nucleic acids including
the reference
sequence region; etc.); where first abundance metrics including a target-
associated count
ratio (e.g., IlbS:HbS_SPK ratio) and a reference-associated count ratio (e.g.,
HbA:HbA SPK ratio) can be calculated, such as based on sequence reads from
sequencing the product of the allele-specific PCR reactions; where second
abundance
metrics including allele fractions (e.g., for HbA and HbS) can be calculated
based on the
first abundance metrics; and where a characterization (e.g., diagnoses of
fetal sickle cell
disease (SCD) can be facilitated (e.g., determined) based on the first
abundance metrics
and/or second abundance metrics (e.g., based on a comparison of HbA and HbS
abundances; etc.).
[00110] In an example, a medical condition can include at least one of a
chromosomal abnormality and a single gene disorder, where the target sequence
region
is associated with at least one of a first chromosome (e.g., associated with
the
chromosomal abnormality, etc.) and a mutation (e.g., associated with the
single gene
disorder, etc.), where the reference sequence region is associated with at
least one of a
second chromosome and a lack of the mutation, and where facilitating
characterization
of the medical condition includes facilitating characterization of the at
least one of the
chromosomal abnormality and the single gene disorder (e.g., based on one or
more
abundance metrics; etc.). However, facilitating characterization of
chromosomal
abnormalities and/or single gene disorders can be performed in any suitable
manner.
[00111] In an example, as shown in FIGURE 6, the medical condition can
include a
rare variant-associated condition, such as where a determined abundance ratio
can be
used in facilitating downstream processes. In an example, the abundance ratio
corresponds to an abundance of the set of reference-associated molecules
(e.g., associated
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with wildtype; etc.) that is greater than an abundance of the set of target-
associated
molecules (e.g., associated with the rare variant; etc.), such as to account
for the relative
frequency between the rare variant and wildtype (e.g., adding a greater
abundance of
reference-associated molecules to account for the greater expected frequency
of wildtype
molecules; etc.), where generation of the at least one spike-in mixture can
include:
allocation of a first abundance of the biological sample for co-amplification
of the set of
target-associated molecules and the first nucleic acid molecules (e.g.,
including the target
sequence region associated with the rare variant-associated condition; etc.);
and
allocation of a second abundance of the biological sample for co-amplification
of the set
of reference-associated molecules and the second nucleic acid molecules (e.g.,
including
a reference sequence region associated with wildtype; etc.), where the first
abundance of
the biological sample is greater than the second abundance of the biological
sample (e.g.,
to account for the low expected frequency of the rare variant, for
facilitating sufficient
amplification; etc.); and/or where facilitating characterization of the
medical condition
includes facilitating characterization of the rare variant-associated
condition (e.g., based
on one or more abundance metric; etc.). In a specific example, as shown in
FIGURE 6,
the G12D mutation is expected to be present at very low frequency (e.g., <10%
allele
fraction), where portions of embodiments of the method loo can be applied to
optimize
for sensitive detection of G12D DNA (and/or other suitable rare variant-
associated
conditions and/or other suitable conditions; etc.), such as including any one
or more of:
adding reference-associated molecules and target-associated molecules at
determined
abundance ratios (e.g., adding KRAS_WT_SPK:ICRAS_G12D_SPK at a loa
stoichiometry, indicated by the smaller lettering for KRAS_G12D_SPK, as shown
in
FIGURE 6; at abundance ratios accounting for the relative frequency of the
rare variant
to wildtype; etc.); using a greater amount of the sample for the rare variant-
specific PCR
(e.g., G12D-specific PCR, etc.) than the wildtype-specific PCR (e.g., KRAS_WT
specific
PCR, etc.); and/or loading a greater amount of the rare variant-associated
product (e.g.,
product of the G12D Specific PCR; etc.) into the sequencer compared to
wildtype-specific
PCR product (e.g., product from ICRAS_WT specific PCR; etc.); where the
measurement
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from the G12D PCR is the <endogenous G12D>: <Gi2D_SPK> ratio, and the
measurement from the WT PCR is the <endogenous WT KRAS>:<WT_SPK> ratio; and
given the added 10:1
WT_SPK:G12D_SPK, if measuring <endogenous
G12D>/<G12D_SPK> = 1, and <endogenous WT KRAS4 <WT_SPK> = 1, then a
resulting calculation can determine that 1*1/(1*1 + i*w) = 9.1% of the
circulating DNA is
ICRAS_G12D. In another example (e.g., example B), if measuring <endogenous
G12D>:<G12D_SPK> = al, and <endogenous WT KRAS>:<WT_SPK> = 1, then a
resulting calculating can determine the KRAS_G12D allele frequency (AF) to be
43.1 * 1/
(0.3.*1 + eio) = 0.99%. In examples, applying such approaches (e.g., for
facilitating
characterization of rare variant-associated conditions; etc.) can overcome
limitations of
sequencing instruments. In a specific example, if the sequencing error is 196,
then
difficulty arises in distinguishing whether measuring a 1% allele fraction is
due to the
presence of a true variant or due to sequencing error, such as where the limit
of detection
of the sequencer would then be 1% allele fraction. Target-associated molecules
and/or
reference-associated molecules (e.g., spike-ins, etc.) can act as an internal
control to
measure the allele frequency, such as in example B, the lowest allele fraction
sequenced
is <endogenous G12D>:<G12D_SPK> = 10%, which is above the sequencing limit of
detection of 1%; however, in examples, portions of embodiments of the method
100 can
be applied to calculate Cr12D to WT allele fraction as 0.99%, which is at the
limit of
detection of the sequencer, where introducing unbalanced stoichiometry of
WT_SPK and
G12D_SPK can improve the G12D signal above the noise floor of the DNA
sequencer.
However, facilitating characterizations of rare variant associated conditions
can be
performed in any suitable manner.
[00112] In
variations, facilitating one or more characterizations can be based on one
or more fetal fraction measurements (and/or any other suitable data, such as
one or more
abundance metrics; etc.). For example, facilitating prenatal diagnosis can
include
facilitating the prenatal diagnosis of one or more genetic disorders based on
one or more
fetal fraction measurements and/or one or more abundance metrics (e.g., one or
more
target-associated count ratios, one or more reference-associated count ratios;
etc.).
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However, facilitating characterizations based on fetal fraction measurements
can be
performed in any suitable manner.
[00113]
Facilitating characterization of one or more conditions and/or any other
suitable portions of embodiments of the method loo (e.g., determining
abundance
metrics; etc.) can include applying one or more artificial intelligence
approaches (e.g.,
machine learning approaches, etc.) including any one or more of: supervised
learning
(e.g., using logistic regression, using back propagation neural networks,
using random
forests, decision trees, etc.), unsupervised learning (e.g., using an Apriori
algorithm, using
K-means clustering), semi-supervised learning, a deep learning algorithm
(e.g., neural
networks, a restricted Boltzmann machine, a deep belief network method, a
convolutional
neural network method, a recurrent neural network method, stacked auto-encoder
method, etc.), reinforcement learning (e.g., using a Q-learning algorithm,
using temporal
difference learning), a regression algorithm (e.g., ordinary least squares,
logistic
regression, stepwise regression, multivariate adaptive regression splines,
locally
estimated scatterplot smoothing, etc.), an instance-based method (e.g., k-
nearest
neighbor, learning vector quantization, self-organizing map, etc.), a
regularization
method (e.g., ridge regression, least absolute shrinkage and selection
operator, elastic net,
etc.), a decision tree learning method (e.g., classification and regression
tree, iterative
dichotomiser 3, C4.5, chi-squared automatic interaction detection, decision
stump,
random forest, multivariate adaptive regression splines, gradient boosting
machines,
etc.), a Bayesian method (e.g., naive Bayes, averaged one-dependence
estimators,
Bayesian belief network, etc.), a kernel method (e.g., a support vector
machine, a radial
basis function, a linear discriminate analysis, etc.), a clustering method
(e.g., k-means
clustering, expectation maximization, etc.), an associated rule learning
algorithm (e.g., an
Apriori algorithm, an Eclat algorithm, etc.), an artificial neural network
model (e.g., a
Perceptron method, a back-propagation method, a Hopfield network method, a
self-
organizing map method, a learning vector quantization method, etc.), a
dimensionality
reduction method (e.g., principal component analysis, partial lest squares
regression,
Sammon mapping, multidimensional scaling, projection pursuit, etc.), an
ensemble
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method (e.g., boosting, bootstrapped aggregation, AdaBoost, stacked
generalization,
gradient boosting machine method, random forest method, etc.), and/or any
suitable
artificial intelligence approach.
[00114] However, facilitating characterization of the one or more
conditions S15o
can be performed in any suitable manner.
2.6 Facilitating treatment.
[00115] Embodiments of the method loo can additionally or alternatively
include
facilitating treatment Si60 (e.g., based on one or more abundance metrics;
based on one
or more characterizations of one or more conditions; etc.), which can function
to leverage
abundance data to determine, provide, administer, promote, recommend, and/or
otherwise facilitate treatment provision (e.g. personalized treatment
provision, etc.) for
one or more conditions. Facilitating treatment can include applying any
suitable
techniques associated with analyzing abundance metrics (e.g., for facilitating
one or more
characterizations; using similar or different statistical operations or
algorithms; using the
same or different abundance metrics, supplementary data, other suitable data;
etc.).
Treatments can include any one or more of: therapeutic compositions (e.g.,
pregnancy-
related compositions, medication-based treatments, probiotic-based treatments,
topical-
based treatments, etc.), surgical treatments, medical device-based treatments,
health-
related notifications (e.g., transmitted to the subject, to a care provider,
etc.) including
condition-related and/or treatment-related information derived based on the
abundance
data; diet-related treatments; cognitive/behavioral treatments; physical
therapies;
clinical-related treatments (e.g., telemedicine, scheduling a care provider
appointment,
etc.); alternative medicine-based treatments; environmental-based treatments;
and/or
any other suitable type of treatments. However, facilitating treatment Sr6o
can be
performed in any suitable manner.
[00116] However, embodiments of the method loo can be performed in any
suitable
manner.
[00117] Embodiments of the method ioo and/or system 200 can include every
combination and permutation of the various system components and the various
method
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processes, including any variants (e.g., embodiments, variations, examples,
specific
examples, figures, etc.), where portions of embodiments of the method locos
and/or
processes described herein can be performed asynchronously (e.g.,
sequentially),
concurrently (e.g., in parallel), or in any other suitable order by and/or
using one or more
instances, elements, components of, and/or other aspects of the system 200
and/or other
entities described herein.
[00118] Any of the variants described herein (e.g., embodiments,
variations,
examples, specific examples, figures, etc.) and/or any portion of the variants
described
herein can be additionally or alternatively combined, aggregated, excluded,
used,
performed serially, performed in parallel, and/or otherwise applied.
[00119] Portions of embodiments of the method loo and/or system 200 can be
embodied and/or implemented at least in part as a machine configured to
receive a
computer-readable medium storing computer-readable instructions. The
instructions
can be executed by computer-executable components that can be integrated with
embodiments of the system 200. The computer-readable medium can be stored on
any
suitable computer-readable media such as RAMs, ROMs, flash memory, EEPROMs,
optical devices (CD or DVD), hard drives, floppy drives, or any suitable
device. The
computer-executable component can be a general or application specific
processor, but
any suitable dedicated hardware or hardware/firmware combination device can
alternatively or additionally execute the instructions.
[00120] As a person skilled in the art will recognize from the previous
detailed
description and from the figures and claims, modifications and changes can be
made to
embodiments of the method 100, system 200, and/or variants without departing
from
the scope defined in the claims.
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CLAIMS
We Claim:
1. A method for facilitating prenatal diagnosis of a genetic disorder from a
maternal
sample associated with a pregnant woman, the method comprising:
= determining a first genomic region of interest associated with the
genetic disorder;
= determining a first homologous native genomic region with partial
sequence
similarity to the first genomic region of interest;
= determining a first primer pair for co-amplification of the first genomic
region of
interest and the first homologous native genomic region, based on the partial
sequence similarity between the first homologous native genomic region and the
first genomic region of interest;
= generating a co-amplified mixture from co-amplifying, based on the first
primer
pair, a set of nucleic acid molecules comprising the first genomic region of
interest
and the first homologous native genomic region, wherein the set of nucleic
acid
molecules are from the maternal sample;
= sequencing the co-amplified mixture;
= determining an abundance metric for the first genomic region of interest
and an
abundance metric for the first homologous native genomic region based on
sequence dissimilarity between the first genomic region of interest and the
first
homologous native genomic region; and
= facilitating the prenatal diagnosis of the genetic disorder based on the
abundance
metric for the first genomic region of interest relative to the abundance
metric for
the first homologous native genomic region.
2. The method of Claim 1,
= wherein the genetic disorder comprises a single gene disorder,
= wherein the first genomic region of interest is associated with a gene
associated
with the single gene disorder, and
= wherein facilitating the prenatal diagnosis of the genetic disorder
comprises
facilitating the prenatal diagnosis of the single gene disorder based on the
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abundance metric for the first genomic region of interest associated with the
gene
relative to the abundance metric for the first homologous native genomic
region.
3. The method of Claim 2,
= wherein the single gene disorder comprises an alpha-thalassemia
condition,
= wherein the gene comprises HBAI gene, wherein the first genomic region of
interest is from the HBA1 gene,
= wherein the first homologous native genomic region is from HBA2 gene, and
= wherein facilitating the prenatal diagnosis of the genetic disorder
comprises
facilitating the prenatal diagnosis of the alpha-thalassemia condition based
on the
abundance metric for the first genomic region of interest from the HBAI gene
relative to the abundance metric for the first homologous native genomic
region
from the HBA2 gene.
4. The method of Claim 2,
= wherein the single gene disorder comprises a spinal muscular atrophy
condition,
= wherein the gene comprises SMNi gene, wherein the first genomic region of
interest is from the SMNi gene,
= wherein the first homologous native genomic region is from SMN2 gene, and
= wherein facilitating the prenatal diagnosis of the genetic disorder
comprises
facilitating the prenatal diagnosis of the spinal muscular atrophy condition
based
on the abundance metric for the first genomic region of interest from the SMNi
gene relative to the abundance metric for the first homologous native genomic
region from the SMN2 gene.
5. The method of Claim 4, further comprising:
= determining a second homologous native genomic region with partial
sequence
similarity to the first genomic region of interest from the SMNi gene,
= wherein facilitating the prenatal diagnosis of the genetic disorder
comprises
facilitating the prenatal diagnosis of the spinal muscular atrophy condition
based
on the abundance metric for the first genomic region of interest from the SMNi
gene, the abundance metric for the first homologous native genomic region from
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the SMN2 gene, and an abundance metric for the second homologous native
genomic region.
6. The method of Claim 1,
= wherein the genetic disorder comprises a chromosomal abnormality,
= wherein the first genomic region of interest is associated with a first
chromosome
associated with the chromosomal abnormality,
= wherein the first homologous native genomic region is associated with a
second
chromosome, and
= wherein facilitating the prenatal diagnosis of the genetic disorder
comprises
facilitating the prenatal diagnosis of the chromosomal abnormality based on
the
abundance metric for the first genomic region of interest associated with the
first
chromosome relative to the abundance metric for the first homologous native
genomic region associated with the second chromosome.
7. The method of Claim 6,
. wherein the chromosomal abnormality comprises a trisomy 21 condition,
= wherein the first genomic region of interest is associated with the first
chromosome
comprising chromosome 21, and
. wherein facilitating the prenatal diagnosis of the genetic disorder
comprises
facilitating the prenatal diagnosis of the trisomy 21 condition based on the
abundance metric for the first genomic region of interest associated with
chromosome 21 relative to the abundance metric for the first homologous native
genomic region associated with the second chromosome.
8. The method of Claim 7, wherein the first genomic region of interest is
associated with
a first loci of chromosome 21, and wherein the method further comprises:
= determining a second genomic region of interest associated with a second
loci of
chromosome 21;
= determining a second homologous native genomic region with partial
sequence
similarity to the second genomic region of interest;
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= determining a second primer pair for co-amplification of the second
genomic
region of interest and the second homologous native genomic region, wherein
generating the co-amplified mixture comprises co-amplifying, based on the
first
and the second primer pairs, a set of nucleic acid molecules comprising the
first
genomic region, the first homologous native genomic region, the second genomic
region, and the second homologous native genomic region;
= determining an abundance metric for the second genomic region of interest
and
an abundance metric for the second homologous native genomic region based on
sequence dissimilarity between the second genomic region of interest and the
second homologous native genomic region; and
= facilitating the prenatal diagnosis of the trisomy 21 condition based on
the
abundance metric for the first genomic region of interest relative to the
abundance
metric for the first homologous native genomic region, and based on the
abundance metric for the second genomic region of interest relative to the
abundance metric for the second homologous native genomic region.
9. A method for facilitating characterization of a medical condition from cell-
free nucleic
acids, the method comprising:
= generating a co-amplified mixture based on co-amplifying cell-free
nucleic acids
comprising:
= a genomic region of interest associated with the medical condition; and
= a homologous native genomic region with partial sequence similarity to
the
genomic region of interest;
= sequencing the co-amplified mixture;
= determining an abundance metric for the genomic region of interest and an
abundance metric for the homologous native genomic region; and
= facilitating the characterization of the medical condition based on the
abundance
metric for the genomic region of interest and the abundance metric for the
homologous native genomic region.
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10. The method of Claim 9,
= wherein the cell-free nucleic acids comprise first nucleic acid molecules
and second
nucleic acid molecules,
= wherein the first nucleic acid molecules comprise the genomic region of
interest,
. wherein the second nucleic acid molecules comprise the homologous native
genomic region, and
. wherein co-amplifying a set of nucleic acid molecules comprises co-
amplifying the
first and the second nucleic acid molecules based on a primer pair designed
based
on the partial sequence similarity between the genomic region of interest and
the
homologous native genomic region.
The method of Claim 10,
. wherein the primer pair corresponds to a first primer binding sequence
region and
a second primer binding sequence region,
. wherein the genomic region of interest and the homologous native genomic
region
each comprise the first primer binding sequence region and the second primer
binding sequence region,
. wherein the homologous native genomic region further comprises a sequence
dissimilarity region with sequence dissimilarity to a corresponding sequence
region of the genomic region of interest, and
= wherein the sequence dissimilarity region is located between the first
primer
binding sequence region and the second primer binding sequence region of the
homologous native genomic region.
12. The method of Claim 9, further comprising:
= determining a reference ratio for the genomic region of interest relative
to the
homologous native genomic region, based on a number of genomic regions
homologous to the genomic region of interest,
. wherein facilitating the characterization of the medical condition
comprises:
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= determining an observed ratio based on the abundance metric for the
genomic region of interest relative to the abundance metric for the
homologous native genomic region; and
= comparing the observed ratio to the reference ratio.
13. The method of Claim 9, wherein the partial sequence similarity between the
genomic
region of interest and the homologous native genomic region satisfies a
homology
threshold of at least 80% homology.
14. The method of Claim 9,
= wherein the cell-free nucleic acids comprise:
= a set of genomic regions of interest comprising the genomic region of
interest, and
= a set of homologous native genomic regions comprising the homologous
native genomic regions; and
= wherein facilitating the characterization of the medical condition
comprises
facilitating the characterization of the medical condition based on abundance
metric ratios between abundance metrics for the set of genomic regions of
interest
and abundance metrics for the set of homologous native genomic regions.
15. The method of Claim 9,
= wherein the single gene disorder comprises a spinal muscular atrophy
condition,
= wherein the genomic region of interest is from SMN1 gene,
= wherein the genomic region of interest comprises at least one of
chr5:70238181-
70238382, chr5:70247763-70248428, and chr5:70247785-70248019, and
= wherein the homologous native genomic region comprises at least one of
chr9:20331713-20331910, chr9:20332293-20332902, and chr4:183948110-
183948725.
16. The method of Claim 9,
= wherein the medical condition comprises an alpha-thalassemia condition,
= wherein the genomic region of interest is from HBM. gene,
= wherein the homologous native genomic region is from HBA2 gene, and
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= wherein facilitating the characterization of the medical condition
comprises
facilitating the characterization of the alpha-thalassemia condition based on
the
abundance metric for the genomic region of interest from the HMI gene relative
to the abundance metric for the homologous native genomic region from the HBA2
gene.
17. The method of Claim 9,
= wherein the medical condition comprises a trisomy 21 condition,
= wherein the genomic region of interest is associated with the first
chromosome
comprising chromosome 21, and
= wherein facilitating the characterization of the medical condition
comprises
facilitating the characterization of the trisomy 21 condition based on the
abundance metric for the genomic region of interest associated with chromosome
21 relative to the abundance metric for the homologous native genomic region
associated a second chromosome.
18. The method of Claim 9, further comprising:
= co-amplifying a set of target-associated molecules and first nucleic acid
molecules
from the cell-free nucleic acids, wherein the set of target-associated
molecules
comprises:
= target-associated regions with sequence similarity to the genomic region
of
interest; and
= target variation regions with sequence dissimilarity to a sequence region
of
the genomic region of interest;
= co-amplifying a set of reference-associated molecules and second nucleic
acid
molecules from the cell-free nucleic acids, wherein the set of reference-
associated
molecules comprises:
= reference-associated regions with sequence similarity to homologous
native
genomic region; and
= reference variation regions with sequence dissimilarity to a sequence
region
of the homologous native genomic region;
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= determining an abundance metric for the set of target-associated
molecules; and
= determining an abundance metric for the set of reference-associated
molecules,
= wherein facilitating the characterization of the medical condition
comprises
facilitating the characterization of the medical condition based on the
abundance
metric for the genomic region of interest, the abundance metric for the
homologous native genomic region, the abundance metric for the set of target-
associated molecules, and the abundance metric for the set of reference-
associated
molecules.
19. The method of Claim 9, wherein the abundance metric for the genomic region
of
interest comprises an intensity metric for the genomic region of interest,
wherein the
abundance metric for the homologous native genomic region comprises an
intensity
metric for the homologous native genomic region, and wherein determining the
abundance metric for the genomic region of interest and the abundance metric
for the
homologous native genomic region comprises determining the intensity metric
for the
genomic region of interest and the intensity metric for the homologous native
genomic
region based on sequence dissimilarity between the genomic region of interest
and the
homologous native genomic region.
20.A method for facilitating prenatal characterization of an alpha-thalassemia
condition
from a maternal sample from a mother, the method comprising:
= generating an amplified mixture from amplifying a set of nucleic acid
molecules
from the maternal sample based on a set of primers designed for amplification
of
a genomic region of interest associated with at least one of HBAI gene and
HBA2
gene;
= sequencing the amplified mixture;
= determining an abundance metric for the genomic region of interest; and
= facilitating the prenatal characterization of the alpha-thalassemia
condition based
on the abundance metric for the genomic region of interest.
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21. The method of Claim 20, wherein the set of primers comprises primers
designed for
amplification of a set of genomic regions of interest corresponding to
polymorphic
alleles associated with the at least one of HBA1 gene and HBA2 gene, wherein
the
polymorphic alleles are within a SEA deletion, wherein the polymorphic alleles
comprise at least one of rs2858935, rs397817350, rs1203833, r52541675,
rs2974774
rs57397665, rs3020596, rs2541669, r52238369, r511639532, r52858942, rs1005364,
rs11863726, rs3062451, rs28673162, and rs3859139, wherein the set of genomic
regions of interest comprises the genomic region of interest, and wherein the
method
further comprises:
= determining an allele characterization based on abundance metrics for the
set of
genomic regions of interest;
= determining an absence or presence of a father-associated deletion
corresponding
to the at least one of the HBA1 gene and the HBA2 gene based on a comparison
between the allele characterization and a mother allele characterization for
the
mother; and
= facilitating the prenatal characterization of the alpha-thalassemia
condition based
on the absence or presence of the father-associated deletion.
22.The method of Claim 20, wherein the genomic region of interest comprises a
point
mutation in the HBA2 gene, wherein the method further comprises:
= adding, to the maternal sample, a set of quality control template (QCT)
molecules
comprising:
= target-associated regions with sequence similarity to a target sequence
region of the genomic region of interest, and
= variation regions with sequence dissimilarity to a sequence region of the
genomic region of interest; and
= wherein facilitating the prenatal characterization of the alpha-
thalassemia
condition comprises facilitating the prenatal characterization of the alpha-
thalassemia condition based on an absolute count for the genomic region of
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interest determined from the abundance metric for the genomic region of
interest
and an abundance metric for the set of QCT molecules.
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ABSTRACT
Embodiments of a method and/or system can include generating a co-amplified
mixture
based on co-amplifying a set of nucleic acid molecules (e.g., cell-free
nucleic acids, etc.)
from the biological sample, wherein the set of nucleic acid molecules includes
a genomic
region of interest associated with the medical condition; and a homologous
native
genomic region with partial sequence similarity to the genomic region of
interest;
sequencing the co-amplified mixture; determining an abundance metric for the
genomic
region of interest and an abundance metric for the homologous native genomic
region;
and/or facilitating the characterization of the medical condition based on the
abundance
metric for the genomic region of interest and the abundance metric for the
homologous
native genomic region.
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