	US 12,006,545 B2
	Methods of lowering the error rate of massively parallel DNA sequencing using duplex consensus sequencing
Jesse Salk, Seattle, WA (US); Lawrence A. Loeb, Bellevue, WA (US); and Michael Schmitt, Seattle, WA (US)
Assigned to UNIVERSITY OF WASHINGTON THROUGH ITS CENTER FOR COMMERCIALIZATION, Seattle, WA (US)
Filed by UNIVERSITY OF WASHINGTON THROUGH ITS CENTER FOR COMMERCIALIZATION, Seattle, WA (US)
Filed on Aug. 2, 2021, as Appl. No. 17/392,180.
Application 17/392,180 is a continuation of application No. 17/008,395, filed on Aug. 31, 2020, granted, now 11,118,225.
Application 17/008,395 is a continuation of application No. 16/503,382, filed on Jul. 3, 2019, granted, now 10,760,127, issued on Sep. 1, 2020.
Application 16/503,382 is a continuation of application No. 16/120,072, filed on Aug. 31, 2018, granted, now 10,385,393, issued on Aug. 20, 2019.
Application 16/120,072 is a continuation of application No. 15/660,785, filed on Jul. 26, 2017, granted, now 10,287,631, issued on May 14, 2019.
Application 15/660,785 is a continuation of application No. 14/386,800, granted, now 9,752,188, issued on Sep. 5, 2017, previously published as PCT/US2013/032665, filed on Mar. 15, 2013.
Claims priority of provisional application 61/613,413, filed on Mar. 20, 2012.
Claims priority of provisional application 61/625,623, filed on Apr. 17, 2012.
Claims priority of provisional application 61/625,319, filed on Apr. 17, 2012.
Prior Publication US 2021/0371921 A1, Dec. 2, 2021
This patent is subject to a terminal disclaimer.
Int. Cl. C12Q 1/68 (2018.01); C12Q 1/6806 (2018.01); C12Q 1/6869 (2018.01); C12Q 1/6876 (2018.01)

CPC C12Q 1/6876 (2013.01) [C12Q 1/6806 (2013.01); C12Q 1/6869 (2013.01)]

19 Claims

1. A method for sequencing a double-stranded nucleic acid molecule, comprising:

(a) preparing a sequencing library, wherein preparing a sequencing library comprises:

providing a set of hairpin adapters having a double-stranded region and a linker region;

ligating the hairpin adapters to a plurality of double-stranded nucleic acid molecules to generate a sequencing library comprising a plurality of adapter-nucleic acid molecule complexes; and

transitioning the adapter-nucleic acid molecule complexes from a double-stranded form to a linear single-stranded form, wherein each linear single-stranded adapter-nucleic acid molecule complex comprises at a first strand of a double-stranded nucleic acid molecule and a second strand of the same double-stranded nucleic acid molecule, separated by an adapter sequence;

(b) sequencing at least a portion of the linear single-stranded adapter-nucleic acid molecule complexes to obtain a plurality of sequence reads, wherein sequencing comprises cluster amplifying the portion of the linear single-stranded adapter-nucleic acid molecule complexes on a sequencing substrate;

(c) grouping the plurality of sequence reads into a plurality of families based at least in part by cluster on the sequencing substrate; and

(d) comparing sequence reads within a family to generate a consensus sequence for that family.