US 11,725,305 B2
Rapid library construction for high throughput sequencing
Joseph Dunham, El Segundo, CA (US)
Assigned to SEQONCE BIOSCIENCES, INC., Pasadena, CA (US)
Appl. No. 16/631,321
Filed by SEQONCE BIOSCIENCES, INC., Pasadena, CA (US)
PCT Filed Jul. 17, 2018, PCT No. PCT/US2018/042500
§ 371(c)(1), (2) Date Jan. 15, 2020,
PCT Pub. No. WO2019/018404, PCT Pub. Date Jan. 24, 2019.
Claims priority of provisional application 62/533,483, filed on Jul. 17, 2017.
Prior Publication US 2020/0208299 A1, Jul. 2, 2020
Int. Cl. C40B 50/06 (2006.01); C12N 15/10 (2006.01); C40B 70/00 (2006.01); C40B 80/00 (2006.01)
CPC C40B 50/06 (2013.01) [C12N 15/1068 (2013.01); C40B 70/00 (2013.01); C40B 80/00 (2013.01)] 20 Claims
 
1. A method for preparing a high-throughput sequencing library comprising the steps of:
A. generating double-stranded fragments from a plurality of polynucleotides;
B. fraying the ends of the fragments to expose single-stranded regions;
C. hybridizing oligonucleotide probes to the single-strand regions to form double-stranded duplexes, wherein the probes comprise a cleavable:
(1) 5′ chemically active group;
(2) 3′ chemically active group; or
(3) 5′ and 3′ chemically active groups;
wherein the lengths of the probes are at least equivalent to the exposed single-stranded regions of the frayed fragments;
D. contacting the double-stranded duplexes with at least one cleaving agent to cleave the chemically active group, or groups, to produce a single-stranded 3′ overhang, a single-stranded 5′ overhang, or single-stranded 3′ and 5′ overhangs in the double-stranded duplexes; and
E. ligating at least two sets of double-stranded DNA adaptors to the double-stranded duplexes, wherein
(1) the adaptors in a first set of adaptors comprise at least one of the following:
i. a 3′ overhang complementary to the overhang end of the 3′ single-stranded overhang of the double-stranded duplex, if present; or
ii. a 5′ overhang complementary to the overhang end of the 5′ single-stranded overhang of the double-stranded duplex, if present, and
(2) the adaptors in a second set of adaptors comprise at least one of the following:
i. a 3′ overhang complementary to the overhang end of the 3′ single-stranded overhang of the double-stranded duplex, if present;
ii. a 5′ overhang complementary to the overhang end of the 5′ single-stranded overhang of the double-stranded duplex, if present; or
iii. a blunt end to ligate to the blunt end of the double-stranded duplex, if present.