US 11,718,848 B1
Methods for depletion of high-copy sequences in multiplexed whole genome sequencing libraries
David Lee, San Francisco, CA (US); and Justin Lock, San Francisco, CA (US)
Assigned to Color Health, Inc., Burlingame, CA (US)
Filed by Color Health, Inc., Burlingame, CA (US)
Filed on May 27, 2021, as Appl. No. 17/332,610.
Claims priority of provisional application 63/032,006, filed on May 29, 2020.
Int. Cl. C12N 15/10 (2006.01); C12Q 1/6806 (2018.01)
CPC C12N 15/1065 (2013.01) [C12N 15/1093 (2013.01); C12Q 1/6806 (2013.01)] 17 Claims
OG exemplary drawing
 
1. A method for preparing a depleted sequencing library comprising:
a) providing a composition comprising a heterogeneous mixture of linear nucleic acids having a first terminus and a second terminus, wherein the heterogeneous mixture comprises a plurality of target nucleic acids and a plurality of non-target nucleic acids, wherein
a first subset of the plurality of target nucleic acids and a second subset of the plurality of non-target nucleic acids comprise an at least partially double-stranded first adaptor region at the first terminus and an at least partially double-stranded second adaptor region at the second terminus, wherein the first adaptor region comprises a first adaptor sequence and a sequence at least partially complementary to the first adaptor sequence, wherein the second adaptor region comprises a second adaptor sequence and a sequence at least partially complementary to the second adaptor sequence, and
a third subset of the plurality of target nucleic acids and a fourth subset of the plurality of non-target nucleic acids comprise the at least partially double-stranded second adaptor region at the first terminus and the at least partially double-stranded second adaptor region at the second terminus;
b) adding removable blocker oligonucleotides to the composition, wherein at least a portion of the removable blocker oligonucleotides are RNA oligonucleotides, and wherein the removable blocker oligonucleotides comprise:
(i) first removable blocker oligonucleotides that can anneal to the first adaptor sequence; and
(ii) second removable blocker oligonucleotides that can anneal to the second adaptor sequence;
wherein at least one of the removable blocker oligonucleotides selected from the removable blocker oligonucleotides in (i) and (ii) is added in excess over the adaptor sequence to which it hybridizes so that a quantity of said at least one of the removable blocker oligonucleotides is not annealed to an adaptor sequence and is free in solution;
c) removing non-target nucleic acids from the composition by sequence capture to bait oligonucleotides thereby depleting non-target nucleic acids from the composition; and
d) treating the composition by at least an addition of divalent ions to reduce a quantity of the RNA oligonucleotides that are not annealed to an adaptor sequence or to a sequence substantially complementary to an adaptor sequence,
whereby the composition comprises a depleted sequencing library.