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US 12,692,539 B2Granted
Methods and compositions for enrichment of target polynucleotides
AssigneeMyriad Women's Health, Inc.
InventorsClement S. Chu, Noah C. Welker, Henry H. Lai
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Abstract
High-fidelity, high-throughput nucleic acid sequencing enables healthcare practitioners and patients to gain insight into genetic variants and potential health risks. However, previous methods of nucleic acid sequencing often introduce sequencing errors (for example, mutations that arise during the preparation of a nucleic acid library, during amplification, or sequencing). Provided herein are methods and compositions for sequencing nucleic acids. Further provided are methods of identifying an error in a nucleic acid sequence.
Claims27
A method for sequencing a target nucleic acid duplex molecule, comprising:
- (a) ligating an adaptor to each end of a target nucleic acid duplex,
- wherein the nucleic acid duplex comprises first and second nucleic acid strands that are complementary to one another,
- wherein each of said adaptors comprises:
- (i) a double stranded region that comprises a molecular barcode sequence;
- and (ii) first and second single stranded regions, wherein the molecular barcode sequence does not include a homopolymer sequence and is not self-complementary,
- wherein the first single stranded region and a portion of the double stranded region of each of said adaptors comprises a sequence S1 that is 5′ of the molecular barcode sequence and the second single stranded region and a portion of the double stranded region of each adaptor comprises sequence S2′ that is 3′ of the molecular barcode sequence, wherein sequences S1 and S2′ are different;
- (b) amplifying the ligated nucleic acids produced in (a) using primers with sequence S1 and the complement of S2′, thereby producing (i) amplified copies of the first strand that comprise sequence S1 at the 5′ end and a first molecular barcode sequence A between S1 and the target nucleic acid sequence of the first strand, and sequence S2′ at the 3′ end and a second molecular barcode sequence B between S2′ and the target nucleic acid sequence of the first strand;
- (ii) amplified copies of the second strand that comprise sequence S1 at the 5′ end and the complement B′ of the second molecular barcode sequence between S1 and the target nucleic acid sequence of the second strand, and sequence S2′ at the 3′ end and the complement A′ of the first molecular barcode sequence between S2′ and the target nucleic acid sequence of the second strand;
- and amplified complements of (i) and (ii);
- (c) hybridizing and extending a primer that comprises:
- (i) a probe sequence that is complementary to a portion of the target nucleic acid sequence of the first and/or second strand, and (ii) a sequence S3, thereby producing primer extension products complementary to the second strand that comprise S3 at the 5′ end and either S1′ or S2′ at the 3′ end and that comprise molecular barcode sequence B between the target nucleic acid sequence and S1′ or S2′, and/or primer extension products complementary to the first strand that comprise S3 at the 5′ end and either S1′ or S2′ at the 3′ end and that comprise molecular barcode sequence A′ between the target nucleic acid sequence and S1′ or S2′;
- (d) differentially amplifying the primer extension products,
- wherein a first reaction comprises amplification using a first primer that comprises a sequence complementary to S3 and one or more sample index sequence(s), and a second primer that comprises S2 and one or more sample index sequence(s), and
- wherein a second reaction comprises amplification using a first primer that comprises a sequence complementary to S3 and one or more sample index sequence(s), and a second primer that comprises S1 and one or more sample index sequence(s);
- and
- (e) sequencing the amplified primer extension products.
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