Methods of lowering the error rate of massively parallel DNA sequencing using duplex consensus sequencing

Patent metadata

Field	Value
Publication number	US-12241123-B2
Application number	US-202117392203-A
Country	US
Kind code	B2
Filing date	Aug 2, 2021
Priority date	Mar 20, 2012
Publication date	Mar 4, 2025
Grant date	Mar 4, 2025

Abstract

Next Generation DNA sequencing promises to revolutionize clinical medicine and basic research. However, while this technology has the capacity to generate hundreds of billions of nucleotides of DNA sequence in a single experiment, the error rate of approximately 1% results in hundreds of millions of sequencing mistakes. These scattered errors can be tolerated in some applications but become extremely problematic when “deep sequencing” genetically heterogeneous mixtures, such as tumors or mixed microbial populations. To overcome limitations in sequencing accuracy, a method Duplex Consensus Sequencing (DCS) is provided. This approach greatly reduces errors by independently tagging and sequencing each of the two strands of a DNA duplex. As the two strands are complementary, true mutations are found at the same position in both strands. In contrast, PCR or sequencing errors will result in errors in only one strand. This method uniquely capitalizes on the redundant information stored in double-stranded DNA, thus overcoming technical limitations of prior methods utilizing data from only one of the two strands.

First claim

What is claimed is:

1. A method of sequencing a double stranded nucleic acid, the method comprising:
(a) ligating a first adapter to a first end of the double stranded nucleic acid, wherein the first adapter is a Y-adapter, wherein the Y-adapter comprises (i) a first strand haying a 5′-arm and a 3′-portion, and (ii) a second strand having a 5′-portion and a 3′-arm, wherein the 3′-portion of the first strand is substantially complementary to the 5′-portion of the second strand, and the 5′-arm of the first strand is not substantially complementary to the 3′-arm of the second strand, and ligating a second adapter to a second end of the double stranded nucleic acid, wherein the second adapter is a hairpin adapter, thereby forming a nucleic acid template;
(b) annealing a first primer to the nucleic acid template, wherein the first primer comprises a sequence that is complementary to a portion of the first adapter, or a complement thereof;
(c) sequencing a first portion of the nucleic acid template by extending the first primer, thereby generating a first read comprising a first nucleic acid sequence of at least a first portion of the double stranded nucleic acid;
(d) annealing a second primer to the nucleic acid template, wherein the second primer comprises a sequence that is complementary to a sequence within a loop of the hairpin adapter, or a complement thereof; and
(e) sequencing a second portion of the nucleic acid template by extending the second primer, thereby generating a second read comprising a nucleic acid sequence of at least a second portion of the double stranded nucleic acid.

2. The method of claim 1 , wherein the ligating of the first adapter comprises ligating a 3′-end of the first strand of the Y-adapter to a 5′-end of the forward strand of the double stranded nucleic acid, and ligating a 5′-end of the second strand of the Y-adapter to a 3′-end of the reverse strand of the double stranded nucleic acid.

3. The method of claim 1 , wherein the first primer anneals to the second strand of the Y-adapter.

4. The method of claim 1 , wherein the second adapter comprises a nucleic acid having a 5′-end, a 5′-portion, the loop, a 3′-portion and a 3′-end, and the 5′-portion of the second adapter is substantially complementary to the 3′-portion of the second adapter.

5. The method of claim 4 , wherein the ligating of the second adapter comprises ligating the 5′-end of the second adapter to a 3′-end of the forward strand of the double stranded nucleic acid and ligating the 3′-end of the second adapter to a 5′-end of the reverse strand of the double stranded nucleic acid.

6. The method of claim 1 , wherein the method further comprises, after (a) and prior to (b), generating amplicons of the nucleic acid template.

7. The method of claim 6 , wherein the method of generating amplicons of the nucleic acid template comprises a polymerase chain reaction.

8. The method of claim 7 , wherein the polymerase chain reaction comprises a bridge PCR amplification method.

9. The method of claim 1 , wherein the sequencing of (c) and the sequencing of (e) comprise a process comprising sequencing by synthesis.

10. The method of claim 1 , wherein the first adapter comprises one or more of a sample barcode sequence, a molecular identifier sequence, or both a sample barcode sequence and a molecular identifier sequence.

11. The method of claim 1 , wherein the second adapter comprises one or more of a sample barcode sequence, a molecular identifier sequence, or both a sample barcode sequence and a molecular identifier sequence.

12. The method of claim 6 , wherein the generating of amplicons comprises attaching the nucleic acid template to a substrate.

Assignees

• Univ Washington Through Its Center For Commercialization

Inventors

• Salk Jesse
• Loeb Lawrence A
• Schmitt Michael

Classifications

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• C12Q1/6806

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• C12Q1/6876 Primary

  Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes · CPC title

• C12Q2565/514

  characterised by the use of the arrayed oligonucleotides as identifier tags, e.g. universal addressable array, anti-tag or tag complement array · CPC title

• C12Q2563/179

  the label being a nucleic acid · CPC title