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Genotyping by next-generation sequencing

  • US 9,951,384 B2
  • Filed: 05/28/2015
  • Issued: 04/24/2018
  • Est. Priority Date: 01/13/2012
  • Status: Active Grant
First Claim
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1. A method for genotyping-by-sequencing a sample, the method comprising:

  • 1) providing a first fully single-stranded oligonucleotide comprising a first primer binding site and a second fully single-stranded oligonucleotide comprising a second primer binding site;

    2) ligating said first fully single-stranded oligonucleotide to the first ends of nucleic acid fragments produced by digesting nucleic acids from the sample with a restriction enzyme and ligating said second fully single-stranded oligonucleotide to the second ends of the nucleic acid fragments to produce a plurality of templates comprising single-stranded ends;

    3) amplifying said templates using a first amplification primer that hybridizes to the first primer binding site or complement thereof and a second amplification primer that hybridizes to the second primer binding site or complement thereof to produce amplicons;

    4) acquiring sequence reads from the amplicons; and

    5) genotyping-by-sequencing the sample by determining a genotype of the sample using one or more sequence reads, wherein;

    a) said first amplification primer comprises a 5′

    portion and a 3′

    portion, wherein;

    1) said 5′

    portion of said first amplification primer hybridizes to said first primer binding site or complement thereof; and

    2) said 3′

    portion of said first amplification primer hybridizes to one or more bases of nucleic acid fragments, said bases being adjacent to the first primer binding site in a subset of the plurality of templates; and

    /orb) said second amplification primer comprises a 5′

    portion and a 3′

    portion, wherein;

    1) said 5′

    portion of the second amplification primer hybridizes to said second primer binding site or complement thereof; and

    2) said 3′

    portion of said second amplification primer hybridizes to one or more bases of nucleic acid fragments, said bases being adjacent to the second primer binding site in a subset of the plurality of templates.

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