Systems and methods to detect rare mutations and copy number variation
DCFirst Claim
1. A method for detecting somatic genetic variants of cell-free deoxyribonucleic acid (DNA) in a human subject, the method comprising:
- a) obtaining 10 to 100 nanograms (ng) of double-stranded cell-free DNA from a blood sample from the human subject;
b) ligating adapters comprising molecular barcodes to ends of a plurality of molecules of the double-stranded cell-free DNA to produce tagged parent polynucleotides, wherein the molecular barcodes together constitute a set of 5-100 molecular barcodes from 5-20 nucleotides in length;
c) amplifying a plurality of the tagged parent polynucleotides to produce progeny polynucleotides with associated molecular barcodes;
d) selectively enriching the progeny polynucleotides for target regions associated with cancer, whereby enriched progeny polynucleotides are generated;
e) sequencing a portion of the enriched progeny polynucleotides to produce sequencing reads of the progeny polynucleotides with associated molecular barcodes;
f) aligning mappable portions of the sequencing reads to a human reference genome;
g) grouping a plurality of the sequencing reads into families based on the sequence information of the molecular barcodes and the beginning and end base positions of the mapped portion of the progeny polynucleotides; and
h) detecting, from among a plurality of the families, the presence or absence of one or more somatic genetic variants comprising a single nucleotide variant (SNV), a copy number variation (CNV), an insertion or deletion (indel), a gene fusion, or any combination thereof.
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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.
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Citations
28 Claims
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1. A method for detecting somatic genetic variants of cell-free deoxyribonucleic acid (DNA) in a human subject, the method comprising:
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a) obtaining 10 to 100 nanograms (ng) of double-stranded cell-free DNA from a blood sample from the human subject; b) ligating adapters comprising molecular barcodes to ends of a plurality of molecules of the double-stranded cell-free DNA to produce tagged parent polynucleotides, wherein the molecular barcodes together constitute a set of 5-100 molecular barcodes from 5-20 nucleotides in length; c) amplifying a plurality of the tagged parent polynucleotides to produce progeny polynucleotides with associated molecular barcodes; d) selectively enriching the progeny polynucleotides for target regions associated with cancer, whereby enriched progeny polynucleotides are generated; e) sequencing a portion of the enriched progeny polynucleotides to produce sequencing reads of the progeny polynucleotides with associated molecular barcodes; f) aligning mappable portions of the sequencing reads to a human reference genome; g) grouping a plurality of the sequencing reads into families based on the sequence information of the molecular barcodes and the beginning and end base positions of the mapped portion of the progeny polynucleotides; and h) detecting, from among a plurality of the families, the presence or absence of one or more somatic genetic variants comprising a single nucleotide variant (SNV), a copy number variation (CNV), an insertion or deletion (indel), a gene fusion, or any combination thereof. - 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)
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Specification