US 12,270,070 B2
Single stranded circular DNA libraries for circular consensus sequencing
Rui Chen, Fremont, CA (US); Toumy Guettouche, Pleasanton, CA (US); and Aaron Richardson, Palo Alto, CA (US)
Assigned to Roche Sequencing Solutions, Inc., Pleasanton, CA (US)
Filed by Roche Sequencing Solutions, Inc., Pleasanton, CA (US)
Filed on Sep. 21, 2021, as Appl. No. 17/448,248.
Application 17/448,248 is a division of application No. 16/444,092, filed on Jun. 18, 2019, granted, now 11,155,855.
Application 16/444,092 is a continuation of application No. PCT/EP2017/083115, filed on Dec. 15, 2017.
Claims priority of provisional application 62/436,819, filed on Dec. 20, 2016.
Prior Publication US 2022/0002787 A1, Jan. 6, 2022
Int. Cl. C12Q 1/6811 (2018.01); C12Q 1/6806 (2018.01); C12Q 1/6834 (2018.01); C12Q 1/6855 (2018.01); C12Q 1/6876 (2018.01)
CPC C12Q 1/6811 (2013.01) [C12Q 1/6806 (2013.01); C12Q 1/6834 (2013.01); C12Q 1/6855 (2013.01); C12Q 1/6876 (2013.01)] 4 Claims
 
1. A method of making a library of circular single-stranded target nucleic acid molecules from a sample comprising a plurality of double-stranded target nucleic acid molecules, the method comprising:
(a) denaturing the double-stranded molecule, thereby forming two strands of the target molecule;
(b) annealing a capture molecule to each strand of the target molecule, wherein the capture molecule is a circular single-stranded nucleic acid molecule comprising two sequences, wherein the two sequences comprise: (i) one sequence that is complementary to at least a portion of the 5′-end of the strand of the target molecule, and (ii) one sequence that is complementary to at least a portion of the 3′-end of the strand of the target molecule, thereby forming a hybrid molecule comprising the capture molecule hybridized to the sequences at the 5′-end and the 3′-end of the strand of the target molecule;
(c) extending the 3′-end of the strand of the target molecule to reach the 5′-end of the strand of the target molecule;
(d) ligating the 5′-end and the 3′-end of the strand of the target molecule, thereby forming a hybrid molecule comprising the capture molecule and a circularized strand of the target molecule; and
(e) separating the capture molecule from the circularized strand of the target molecule, thereby forming a library of circular single-stranded target nucleic acid molecules.