US 12,077,816 B2
Linked paired strand sequencing
Eli N. Glezer, Del Mar, CA (US); Martin Maria Fabani, Encinitas, CA (US); Sabrina Shore, San Diego, CA (US); and Daan Witters, San Diego, CA (US)
Assigned to Singular Genomics Systems, Inc., San Diego, CA (US)
Filed by SINGULAR GENOMICS SYSTEMS, INC., San Diego, CA (US)
Filed on Oct. 13, 2021, as Appl. No. 17/500,733.
Application 17/500,733 is a division of application No. 17/194,023, filed on Mar. 5, 2021, granted, now 11,359,238.
Claims priority of provisional application 63/087,125, filed on Oct. 2, 2020.
Claims priority of provisional application 63/020,881, filed on May 6, 2020.
Claims priority of provisional application 62/986,527, filed on Mar. 6, 2020.
Prior Publication US 2022/0033895 A1, Feb. 3, 2022
Int. Cl. C12Q 1/68 (2018.01); C12Q 1/6855 (2018.01); C12Q 1/6869 (2018.01)
CPC C12Q 1/6869 (2013.01) [C12Q 1/6855 (2013.01)] 22 Claims
OG exemplary drawing
 
1. A method of selectively sequencing a double-stranded nucleic acid, the method comprising:
(a) ligating a first adapter to a first end of the double-stranded nucleic acid, and ligating a second adapter to a second end of the double-stranded nucleic acid, wherein the second adapter is a hairpin adapter, thereby forming a nucleic acid template;
(b) displacing at least a portion of one strand of the nucleic acid template from step (a);
(c) hybridizing a probe oligonucleotide to the displaced portion of the nucleic acid template, thereby forming a probe-template complex;
(d) separating the probe-template complex from nucleic acids not hybridized to a probe; and
(e) sequencing the probe-template complex, wherein sequencing comprises:
hybridizing a blocking primer to the hairpin adapter and extending the blocking primer with a polymerase to generate a blocking strand, thereby displacing the first strand of the probe-template complex;
annealing a first sequencing primer to the first strand, extending the first sequencing primer with one or more labeled nucleotides to generate a first extension strand, and detecting the one or more labeled nucleotides; and
annealing a second sequencing primer to the hairpin adapter, wherein the blocking strand is removed prior to annealing the second sequencing primer, extending the second sequencing primer with one or more labeled nucleotides to generate a second extension strand, and detecting the one or more labeled nucleotides.
 
22. A method of selectively sequencing a double-stranded nucleic acid, the method comprising:
contacting a plurality of nucleic acid templates with a plurality of probe oligonucleotides and binding a probe oligonucleotide to a nucleic acid template thereby forming a probe-template complex, wherein said nucleic acid template each comprise a first adapter, a double-stranded nucleic acid portion, and a hairpin adapter;
separating the probe-template complex from nucleic acid templates not bound to a probe; and
removing the probe from the probe-template complex;
hybridizing a blocking primer to the hairpin adapter and extending the blocking primer with a polymerase to generate a blocking strand, thereby displacing the first strand of the double-stranded nucleic acid portion;
annealing a first sequencing primer to the first strand, extending the first sequencing primer with one or more labeled nucleotides to generate a first extension strand, and detecting the one or more labeled nucleotides; and
annealing a second sequencing primer to the hairpin adapter, wherein the blocking strand is removed prior to annealing the second sequencing primer, extending the second sequencing primer with one or more labeled nucleotides to generate a second extension strand, and detecting the one or more labeled nucleotides.