US 12,006,532 B2
Methods for targeted nucleic acid sequence enrichment with applications to error corrected nucleic acid sequencing
Scott R. Kennedy, Seattle, WA (US); Jesse J. Salk, Seattle, WA (US); Michael Hipp, Seattle, WA (US); Elizabeth Schmidt, Seattle, WA (US); Rosa Ana Risques, Seattle, WA (US); and Daniela Nachmanson, Seattle, WA (US)
Assigned to UNIVERSITY OF WASHINGTON, Seattle, WA (US)
Filed by UNIVERSITY OF WASHINGTON, Seattle, WA (US)
Filed on Sep. 16, 2022, as Appl. No. 17/933,058.
Application 17/933,058 is a continuation of application No. 16/496,936, granted, now 11,479,807, previously published as PCT/US2018/024194, filed on Mar. 23, 2018.
Claims priority of provisional application 62/475,682, filed on Mar. 23, 2017.
Claims priority of provisional application 62/575,958, filed on Oct. 23, 2017.
Prior Publication US 2023/0295686 A1, Sep. 21, 2023
This patent is subject to a terminal disclaimer.
Int. Cl. C12Q 1/6806 (2018.01); C12Q 1/6855 (2018.01); C12Q 1/686 (2018.01)
CPC C12Q 1/6806 (2013.01) [C12Q 1/6855 (2013.01); C12Q 1/686 (2013.01); C12Q 2521/501 (2013.01); C12Q 2525/191 (2013.01); C12Q 2531/113 (2013.01); C12Q 2535/119 (2013.01); C12Q 2535/122 (2013.01)] 22 Claims
 
1. A method comprising:
(a) providing nucleic acid constructs each comprising:
a double-stranded nucleic acid fragment comprising a first strand and a second strand;
a first adapter polynucleotide attached to one end of the fragment; and
a second adapter polynucleotide attached to the other end of the fragment,
wherein the first and second adapter polynucleotides each comprise:
an end portion comprising a 5′-associated sequence and a 3′-associated sequence that are at least partially non-complementary, and
a portion between the end portion and the nucleic acid fragment comprising a double-stranded single molecule identifier (SMI), and
wherein the nucleic acid fragment of at least one of the constructs comprises a target of interest;
(b) amplifying at least a portion of the constructs to generate first and second strand amplicons, wherein a portion of the first and the second strand amplicons comprise the target of interest;
(c) separating the amplicons of (b) into a first sample and a second sample each comprising a plurality of the first strand amplicons and a plurality of the second strand amplicons;
(d) performing targeted amplification of the first sample and the second sample,
wherein the targeted amplification of the first sample comprises exponentially amplifying only the first strand amplicons to generate a first nucleic acid product using a primer specific to the 5′-associated sequence and a primer specific to the target of interest, such that the SMI of the first adapter polynucleotide or second adapter polynucleotide is maintained in the first nucleic acid product, and
wherein the targeted amplification of the second sample comprises exponentially amplifying only the second strand amplicons to generate a second nucleic acid product using a primer specific to the 3′-associated sequence and a primer specific to the target of interest, such that the SMI that is maintained in the first nucleic acid product is also maintained in the second nucleic acid product;
(e) sequencing the first nucleic acid product and the second nucleic acid product to obtain sequence reads each comprising an SMI sequence and a sequence of the target of interest; and
(f) identifying a set of the sequence reads as deriving from a same original nucleic acid construct based at least in part on the SMI sequence of the sequence reads in the set, wherein at least one of the reads in the set is obtained from the first nucleic acid product and at least one of the reads in the set is obtained from the second nucleic acid product.