MULTIMODAL ASSAY FOR DETECTING NUCLEIC ACID ABERRATIONS
First Claim
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1. A method for determining a nucleotide sequence for one or more target nucleic acids of interest in a subject comprising:
- a) obtaining a nucleic acid sample isolated from a subject;
b) adding an anchor sequence to one of the 3′
or 5′
end of a plurality of nucleic acids from the sample in step a) to create an anchor product;
c) hybridizing an anchor primer to the anchor product of step b), wherein the anchor primer is substantially complementary to the anchor sequence from step b), and hybridizing a genome-informed primer, which is substantially complementary to a repeat sequence in the nucleic acid, to produce a plurality of replicons, wherein the anchor sequence and the repeat sequence flank a gap region in the plurality of target nucleic acid sequences of interest;
d) sequencing a plurality of amplicons that are amplified from the replicons in step c) to determine the nucleotide sequence of one or more target nucleic acids.
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Abstract
The invention provides methods for determining whether a subject is predisposed to the disease or condition, or for diagnosing a disease or condition, or for detecting the state of a disease or condition, by detecting nucleic acid fragment size patterns, copy number variations, mutational landscape, genomic instability, methylation status, and combinations thereof in a subject. The invention further provides methods for selecting nucleic acid molecules for use in the methods of the invention.
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Citations
96 Claims
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1. A method for determining a nucleotide sequence for one or more target nucleic acids of interest in a subject comprising:
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a) obtaining a nucleic acid sample isolated from a subject; b) adding an anchor sequence to one of the 3′
or 5′
end of a plurality of nucleic acids from the sample in step a) to create an anchor product;c) hybridizing an anchor primer to the anchor product of step b), wherein the anchor primer is substantially complementary to the anchor sequence from step b), and hybridizing a genome-informed primer, which is substantially complementary to a repeat sequence in the nucleic acid, to produce a plurality of replicons, wherein the anchor sequence and the repeat sequence flank a gap region in the plurality of target nucleic acid sequences of interest; d) sequencing a plurality of amplicons that are amplified from the replicons in step c) to determine the nucleotide sequence of one or more target nucleic acids. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59)
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60. A method of detecting copy number variation in a subject, comprising:
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a) obtaining a nucleic acid sample isolated from a subject; b) adding an anchor sequence to one of the 3′
or the 5′
end of a plurality of nucleic acid sequences from the sample in step a);c) capturing a plurality of target sequences of interest in the nucleic acid sample obtained in step a) by using one or more populations of molecular inversion probes (MIPs) to produce a plurality of replicons, wherein each of the MIPs in the population of MIPs comprises in sequence the following components; anchor arm—
polynucleotide linker—
genome-informed arm;wherein the anchor arm in each of the MIPs is substantially complementary to the anchor sequence from step b), and the genome-informed arm in each of the MIPs is substantially complementary to a repeat sequence in the nucleic acid, such that the anchor sequence and the repeat sequence flank a unique gap region in the plurality of target sequences of interest; d) sequencing a plurality of MIP amplicons that are amplified from the replicons obtained in step c); e) determining a number of a first population of amplicons of the plurality of amplicons provided in step d) based on the number of unique amplicon sequences; f) determining a number of each of a second population of amplicons of the plurality of amplicons provided in step d) based on the number of unique amplicon sequences; g) determining, for each target sequence of interest from which the first population of amplicons was produced, a site capture metric based at least in part on the number of capture events determined in step e); h) identifying a first subset of the site capture metrics determined in step g) that satisfy at least one criterion; i) determining, for each target sequence of interest from which the second population of amplicons was produced, a site capture metric based at least in part on the number of capture events determined in step f); j) identifying a second subset of the site capture metrics determined in step i) that satisfy the at least one criterion; k) normalizing a first measure determined from the first subset of site capture metrics identified in step h) by a second measure determined from the second subset of site capture metrics identified in step j) to obtain a test ratio; l) comparing the test ratio to a plurality of reference ratios that are computed based on reference nucleic acid samples isolated from reference subjects without a copy number variation at the target sequences of interest; and m) determining, based on the comparing in step l), whether a copy number variation is present at the target sequences of interest in a subject. - View Dependent Claims (61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78)
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79. A method of determining the methylation status of one or more nucleic acid fragments in a subject comprising:
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a) obtaining a nucleic acid sample isolated from a subject; b) performing bisulfite conversion of the nucleic acid sample; c) adding an anchor sequence to the bisulfite-converted nucleic acid of step b); d) capturing a plurality of target sequences of interest in the nucleic acid sample obtained in step a) by using one or more populations of molecular inversion probes (MIPs) to produce a plurality of replicons, wherein each of the MIPs in the population of MIPs comprises in sequence the following components; anchor arm—
polynucleotide linker—
genome-informed arm;wherein the anchor arm in each of the MIPs is substantially complementary to the anchor sequence from step c), and the genome-informed arm in each of the MIPs is substantially complementary to a repeat sequence in the nucleic acid, such that the anchor sequence and the repeat sequence flank a unique gap region in the plurality of target sequences of interest; e) sequencing a plurality of MIP amplicons that are amplified from the replicons obtained in step d); and f) determining the number of occurrences of cytosine nucleotides at each corresponding known CpG site within the MIP amplicons sequenced at step e), wherein the methylation status is determined based on the number of occurrences of cytosine nucleotides at each corresponding known CpG site. - View Dependent Claims (80, 81, 82, 83, 84)
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85. A method for characterizing one or nucleic acids of interest from a subject, comprising:
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a) obtaining a nucleic acid sample isolated from a subject; b) adding clip sequences to the 3′ and
5′
ends of each of a plurality of target nucleic acids from the sample in step a) to create a clip product, wherein the two clip sequences flank a gap region in the target nucleic acid sequence of interest;c) hybridizing a capture probe comprising two clip binding arms to the clip product of step b), wherein the two clip binding arms are on opposite ends of the same capture probe, and wherein each clip binding arm is substantially complementary one of the clip sequences from step b); d) using a ligation/extension mixture to extend and ligate the gap region between the two clip binding arms to form a single-stranded circular nucleic acid molecule; and e) analyzing a plurality of amplicons that are amplified from single-stranded circular nucleic acid molecules of step d) to characterize the one or more nucleic acids of interest. - View Dependent Claims (86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96)
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Specification