US 12,234,512 B2
Compositions and methods for detecting rare sequence variants
Shengrong Lin, Fremont, CA (US); Zhaohui Sun, Albany, CA (US); Grace Qizhi Zhao, Palo Alto, CA (US); and Paul Ling-Fung Tang, San Francisco, CA (US)
Assigned to AccuraGen Holdings Limited, George Town (KY)
Filed by ACCURAGEN HOLDINGS LIMITED, Grand Cayman (KY)
Filed on Feb. 3, 2023, as Appl. No. 18/164,439.
Application 18/164,439 is a continuation of application No. 16/945,553, filed on Jul. 31, 2020, granted, now 11,597,973.
Application 16/945,553 is a continuation of application No. 15/102,241, granted, now 10,767,222, issued on Sep. 8, 2020, previously published as PCT/US2014/069848, filed on Dec. 11, 2014.
Claims priority of provisional application 62/010,975, filed on Jun. 11, 2014.
Claims priority of provisional application 61/987,414, filed on May 1, 2014.
Claims priority of provisional application 61/914,907, filed on Dec. 11, 2013.
Prior Publication US 2023/0416819 A1, Dec. 28, 2023
This patent is subject to a terminal disclaimer.
Int. Cl. C12Q 1/6874 (2018.01); C12Q 1/6827 (2018.01); C12Q 1/6869 (2018.01)
CPC C12Q 1/6874 (2013.01) [C12Q 1/6827 (2013.01); C12Q 1/6869 (2013.01)] 15 Claims
 
1. A method of distinguishing a sequence variant from an error in a cell-free deoxyribonucleic acid (DNA) polynucleotide, comprising:
(a) circularizing said cell-free DNA polynucleotide or a derivative thereof to form a circular DNA polynucleotide, wherein said cell-free DNA polynucleotide is not enzymatically phosphorylated prior to circularization, and wherein said cell-free DNA polynucleotide is single-stranded;
(b) amplifying said circular DNA polynucleotide of (a) to produce a plurality of concatemers, wherein a concatemer of said plurality of concatemers comprises at least two copies of a sequence of said circular DNA polynucleotide;
(c) sequencing said plurality of concatemers to produce a plurality of sequencing reads; and
(d) identifying a sequence difference between a sequencing read of said plurality of sequencing reads and a reference sequence as said sequence variant only when said sequence difference occurs in at least two copies of said circular DNA polynucleotide in said concatemer and not when said sequence difference occurs in only one copy of said circular DNA polynucleotide in said concatemer, thereby distinguishing said sequence variant from said error.