US 11,952,628 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. 17/392,175.
Application 17/392,175 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 61/625,623, filed on Apr. 17, 2012.
Claims priority of provisional application 61/625,319, filed on Apr. 17, 2012.
Claims priority of provisional application 61/613,413, filed on Mar. 20, 2012.
Prior Publication US 2021/0371920 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)] 26 Claims
 
1. A method of sequencing DNA comprising:
a) attaching partially single-stranded adapters comprising barcodes selected from a plurality of distinct barcode sequences to double-stranded DNA fragments obtained from a bodily sample, wherein attachment of the adapters to double-stranded DNA fragments generates a library of tagged double-stranded adapter-DNA molecules;
b) amplifying strands from a plurality of the double-stranded adapter-DNA molecules in the library to produce strand copies;
c) sequencing a plurality of the strand copies to obtain strand sequence reads comprising one or more barcode sequences and DNA fragment-specific information; and
d) for at least some of the double-stranded adapter-DNA molecules in the library—
grouping the strand sequence reads into families based on i) the barcode sequence, and ii) DNA fragment-specific information;
collapsing a plurality of strand sequence reads within the families to provide a consensus sequence for each of the at least some of the double-stranded adapter-DNA molecules in the library;
comparing the consensus sequence to a reference sequence;
analyzing one or more correspondences between the consensus sequence and the reference sequence to identify a sequence variation,
wherein the library comprises at least a subset of non-uniquely tagged double-stranded adapter-DNA molecules,
wherein non-uniquely tagged double-stranded adapter-DNA molecules are substantially identifiable with respect to other non-uniquely tagged double-stranded adapter-DNA molecules in the bodily sample using the one or more barcode sequences and DNA fragment-specific information,
wherein following step (d), the method further comprises identifying a genetic mutation conferring drug resistance present in one or more of the consensus sequences derived from the double-stranded DNA fragments obtained from a tumor cell population present in the bodily sample,
wherein the adapter comprises a hairpin loop with a uracil linker, and
wherein the barcode sequences are 6 nucleotides in length.