Systems and methods to detect rare mutations and copy number variation

  • US 9,902,992 B2
  • Filed: 03/21/2016
  • Issued: 02/27/2018
  • Est. Priority Date: 09/04/2012
  • Status: Active Grant
First Claim
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1. A method for detecting genetic aberrations in cell-free DNA (“

  • cfDNA”

    ) molecules from a subject, comprising;

    a) providing cfDNA molecules obtained from a bodily sample of the subject;

    b) attaching tags comprising barcodes having a plurality of different barcode sequences to the cfDNA molecules to tag at least 20% of the cfDNA molecules, which attaching comprises ligating adaptors comprising the barcodes to both ends of the cfDNA molecules, wherein ligating comprises using more than 10×

    molar excess of the adaptors as compared to the cfDNA molecules, thereby generating tagged parent polynucleotides;

    c) amplifying the tagged parent polynucleotides to produce amplified tagged progeny polynucleotides;

    d) sequencing the amplified tagged progeny polynucleotides to produce a plurality of sequence reads from each of the tagged parent polynucleotides, wherein each sequence read of the plurality of sequence reads comprises a barcode sequence and a sequence derived from a cfDNA molecule of the cfDNA molecules;

    e) mapping sequence reads of the plurality of sequence reads to one or more reference sequences from a human genome;

    f) grouping the sequence reads mapped in e) into families based at least on barcode sequences of the sequence reads, each of the families comprising sequence reads comprising the same barcode sequence, whereby each of the families comprises sequence reads amplified from the same tagged parent polynucleotide;

    g) at each of a plurality of genetic loci in the one or more reference sequences, collapsing sequence reads in each family to yield a base call for each family at the genetic locus; and

    h) detecting, at one or more genetic loci, a plurality of genetic aberrations, wherein the plurality of genetic aberrations comprises two or more different members selected from the group of members consisting of a single base substitution, a copy number variation (CNV), an insertion or deletion (indel), and a gene fusion.

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