CPC C12N 15/1058 (2013.01) [C12N 15/101 (2013.01); C12N 15/1093 (2013.01); C12N 15/1096 (2013.01); C12Q 1/6844 (2013.01); C12Q 1/6869 (2013.01); C12Q 1/6874 (2013.01); C12Q 1/6886 (2013.01); C40B 40/08 (2013.01); C12Q 2600/106 (2013.01); C12Q 2600/112 (2013.01); C12Q 2600/118 (2013.01); C12Q 2600/16 (2013.01); C40B 20/00 (2013.01)] | 18 Claims |
1. A method for preparing an enriched sequencing library, the method comprising:
(a) obtaining a test sample comprising a plurality of double-stranded deoxyribonucleic acid (dsDNA) fragments;
(b) preparing an enriched sequencing library from the test sample, wherein preparing the sequencing library comprises:
(i) ligating double-stranded DNA adapters to both ends of the dsDNA fragments to generate a plurality of adapter-fragment constructs;
(ii) adding a first set of primers to the adapter-fragment constructs, wherein the first set of primers comprise single-stranded oligonucleotides less than 50 nucleotides in length;
(iii) hybridizing the first set of primers to the adapter-fragment constructs and extending the first set of primers in a first nucleic acid extension reaction using a polymerase to generate a plurality of amplified adapter-fragment constructs;
(iv) enriching the amplified adapter-fragment constructs for adapter-fragment constructs derived from dsDNA fragments less than 150 bp in length to generate an enriched sample comprising enriched adapter-fragment constructs;
(v) adding a second set of primers to the enriched sample, wherein the second set of primers comprise single-stranded oligonucleotides greater than 50 nucleotides in length; and
(vi) hybridizing the second set of primers to the enriched adapter-fragment constructs and extending the second set of primers in a second nucleic acid extension reaction using a polymerase to generate a sequencing library.
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