Systems and methods to detect rare mutations and copy number variation
First Claim
1. A method of detecting a somatic genetic mutation in double-stranded cell-free deoxyribonucleic acid (cfDNA) from a blood sample for cancer testing in a human subject, comprising:
- (a) converting between 100 and 100,000 haploid human genome equivalents of cfDNA derived from the blood sample into non-uniquely tagged parent polynucleotides, wherein the converting comprises ligating the cfDNA with a plurality of molecular barcodes, wherein a given molecular barcode from among the plurality of molecular barcodes is a member of a set of molecular barcodes having between 2 and 1,000 different molecular barcode sequences with a length of at least 5 nucleotides, wherein a molecule of the cfDNA is ligated to a molecular barcode at both ends of the molecule of the cfDNA;
(b) amplifying a plurality of the non-uniquely tagged parent polynucleotides to produce corresponding amplified progeny polynucleotides;
(c) sequencing at least a subset of the amplified progeny polynucleotides to produce sequence reads;
(d) mapping a plurality of the sequence reads to a human reference sequence to produce mapped sequence reads; and
(e) generating a set of consensus sequences from some or all of the mapped sequence reads to detect the somatic genetic mutation, wherein generating a given consensus sequence of the set of consensus sequences is based on information from at least the molecular barcodes, wherein a consensus sequence within the set of consensus sequences corresponds to a unique cfDNA molecule from among the non-uniquely tagged parent polynucleotides, and wherein the somatic genetic mutation comprises a single nucleotide variation (SNV), a copy number variation (CNV), an indel, or a gene fusion.
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Abstract
The present disclosure provides a system and method for the detection of rare mutations and copy number variations in cell free polynucleotides. Generally, the systems and methods comprise sample preparation, or the extraction and isolation of cell free polynucleotide sequences from a bodily fluid; subsequent sequencing of cell free polynucleotides by techniques known in the art; and application of bioinformatics tools to detect rare mutations and copy number variations as compared to a reference. The systems and methods also may contain a database or collection of different rare mutations or copy number variation profiles of different diseases, to be used as additional references in aiding detection of rare mutations, copy number variation profiling or general genetic profiling of a disease.
308 Citations
26 Claims
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1. A method of detecting a somatic genetic mutation in double-stranded cell-free deoxyribonucleic acid (cfDNA) from a blood sample for cancer testing in a human subject, comprising:
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(a) converting between 100 and 100,000 haploid human genome equivalents of cfDNA derived from the blood sample into non-uniquely tagged parent polynucleotides, wherein the converting comprises ligating the cfDNA with a plurality of molecular barcodes, wherein a given molecular barcode from among the plurality of molecular barcodes is a member of a set of molecular barcodes having between 2 and 1,000 different molecular barcode sequences with a length of at least 5 nucleotides, wherein a molecule of the cfDNA is ligated to a molecular barcode at both ends of the molecule of the cfDNA; (b) amplifying a plurality of the non-uniquely tagged parent polynucleotides to produce corresponding amplified progeny polynucleotides; (c) sequencing at least a subset of the amplified progeny polynucleotides to produce sequence reads; (d) mapping a plurality of the sequence reads to a human reference sequence to produce mapped sequence reads; and (e) generating a set of consensus sequences from some or all of the mapped sequence reads to detect the somatic genetic mutation, wherein generating a given consensus sequence of the set of consensus sequences is based on information from at least the molecular barcodes, wherein a consensus sequence within the set of consensus sequences corresponds to a unique cfDNA molecule from among the non-uniquely tagged parent polynucleotides, and wherein the somatic genetic mutation comprises a single nucleotide variation (SNV), a copy number variation (CNV), an indel, or a gene fusion. - 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)
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Specification