US 11,851,650 B2
Enrichment of short nucleic acid fragments in sequencing library preparation
Byoungsok Jung, Atherton, CA (US); and Alex Aravanis, San Mateo, CA (US)
Assigned to GRAIL, LLC, Menlo Park, CA (US)
Filed by GRAIL, LLC, Menlo Park, CA (US)
Filed on Sep. 28, 2018, as Appl. No. 16/146,901.
Claims priority of provisional application 62/564,891, filed on Sep. 28, 2017.
Prior Publication US 2019/0093102 A1, Mar. 28, 2019
Int. Cl. C12N 15/10 (2006.01); C12Q 1/68 (2018.01); C40B 20/00 (2006.01); C40B 40/08 (2006.01); C12Q 1/6874 (2018.01); C12Q 1/6844 (2018.01); C12Q 1/6886 (2018.01); C12Q 1/6869 (2018.01)
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
OG exemplary drawing
 
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.