Generation of single-stranded circular DNA templates for single molecule sequencing

Patent metadata

Field	Value
Publication number	US-12110534-B2
Application number	US-202016945099-A
Country	US
Kind code	B2
Filing date	Jul 31, 2020
Priority date	Feb 5, 2018
Publication date	Oct 8, 2024
Grant date	Oct 8, 2024

Abstract

The invention is a novel method of sequencing nucleic acids involving making and sequencing a library of single stranded circular target nucleic acids.

First claim

The invention claimed is:

1. A method of separately sequencing each strand of a double-stranded target nucleic acid, wherein the method comprises the following steps:
(a) in a reaction mixture, joining a double-stranded target nucleic acid to an adaptor to form an adapted target nucleic acid, wherein the adaptor comprises primer-binding sites, and wherein the adaptor does not consist of a long strand and a short strand;
(b) amplifying the adapted target nucleic acid with a pair of primers, wherein the pair of primers are complementary to the primer-binding sites, thereby forming an amplicon, wherein one primer in the pair of primers comprises a modified nucleotide affecting a rate of digestion by an exonuclease;
(c) contacting the reaction mixture with an exonuclease, thereby eliminating from the reaction mixture the first of the two complementary strands of the amplicon;
(d) circularizing the second of the two complementary strands of the amplicon to form a single stranded circle;
(e) annealing a sequencing primer to the single-stranded circle; and
(f) extending the primer, thereby sequencing one strand of the target nucleic acid.

2. The method of claim 1 , wherein the joining of the double-stranded target nucleic acid to the adaptor is by ligation.

3. The method of claim 2 , wherein the ligation is by joining of cohesive ends of the target nucleic acid and the adaptor.

4. The method of claim 1 , wherein the adaptor comprises a double-stranded part and a single-stranded part comprising two non-annealed portions.

5. The method of claim 1 , wherein the adaptor comprises at least one barcode.

6. The method of claim 1 , wherein the modified nucleotide is a 5 ′-phosphorylated terminal nucleotide and the exonuclease is Lambda exonuclease.

7. The method of claim 1 , further comprising a second exonuclease digestion step, after the circularization step (d).

8. The method of claim 1 , wherein circularization is by splint ligation.

9. The method of claim 1 , wherein the sequencing primer-binding site is in the adaptor.

10. The method of claim 1 , further comprising, prior to step (c), contacting the reaction mixture with a DNA damage-specific agent selected from glycosylase and endonuclease.

11. The method of claim 1 , wherein the steps (a)-(f) are performed on a plurality of target nucleic acids in a sample thereby forming and sequencing a library of nucleic acids from the sample.

Assignees

• Roche Sequencing Solutions Inc
• Kapa Biosystems Inc

Inventors

• Badenhorst Daleen
• Berry Michael S
• Dannebaum Richard
• Hayes Ashley
• Margeridon Severine
• Ranik Martin
• Slabbert Etienne

Classifications

• C12Q2535/122

  Massive parallel sequencing · CPC title

• C12Q2525/307

  Circular oligonucleotides · CPC title

• C12Q2525/191

  incorporating an adaptor · CPC title

• C12Q2525/155

  incorporating/generating a new priming site · CPC title

• C12Q2521/319

  Exonuclease · CPC title