Methods of nucleic acid sequencing

• US 10,267,804 B2
• Filed: 02/01/2017
• Issued: 04/23/2019
• Est. Priority Date: 03/06/2012
• Status: Active Grant

• Current Assignee: Illumina Cambridge Limited (Illumina Incorporated)
• Original Assignee: Illumina Cambridge Limited (Illumina Incorporated)
• Inventors: Gormley, Niall Anthony, Fraser, Louise, Kokko-Gonzales, Paula
• Primary Examiner(s): Dunston, Jennifer
• Application Number:
  US15/421,979
• Publication Number:
  US 20170212123A1
• Time in Patent Office: 811 Days
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target

Abstract

Provided herein is a method of using transposition to improve methods of sequencing RNA molecules. Provided herein is a method of tagging nucleic acid duplexes, such as DNA:RNA duplexes or DNA:DNA duplexes. The method includes the steps of providing a transposase and a transposon composition, providing one or more nucleic acid duplexes immobilized on a support, and contacting the transposase and transposon composition with the one or more nucleic acid duplexes under conditions wherein the one or more nucleic acid duplexes and transposon composition undergo a transposition reaction to produce one or more tagged nucleic acid duplexes, wherein the transposon composition comprises a double stranded nucleic acid molecule comprising a transferred strand and a non-transferred strand.

Claims

1. A method of detecting one or more target nucleic acids comprising the steps of:

(a) capturing one or more target nucleic acids on a support, wherein the one or more target nucleic acids comprise single stranded DNA (ssDNA);

(b) using the one or more captured target nucleic acids as a template strand to produce one or more nucleic acid duplexes immobilized on the support;

(c) contacting a transposase and transposon with the one or more nucleic acid duplexes under conditions wherein the one or more nucleic acid duplexes and transposon composition undergo a transposition reaction to produce one or more tagged nucleic acid duplexes, wherein the transposon composition comprises a double stranded nucleic acid molecule comprising a transferred strand and a non-transferred strand;

(d) contacting the one or more tagged nucleic acid duplexes with a nucleic acid modifying enzyme under conditions to extend the 3′

end of the immobilized strand to the 5′

end of the template strand to produce one or more end-extended tagged nucleic acid duplexes;

(e) amplifying the one or more end-extended tagged nucleic acid duplexes to produce a plurality of tagged nucleic acid strands; and

(f) detecting the plurality of tagged nucleic acid strands.

2. The method of claim 1, wherein the one or more target nucleic acids are genomic DNA molecules.

3. The method of claim 1, wherein capturing the one or more target nucleic acids comprises hybridization to one or more capture probes immobilized on the support that are complementary to at least a portion of the one or more target nucleic acids.

4. The method of claim 1, wherein the one or more target nucleic acids each comprise a 3′

adaptor, the 3′

adaptor comprising a sequence complementary to one or more capture probes immobilized on the support, and wherein the capturing step comprises hybridizing the one or more target nucleic acids comprising the 3′

-adaptor to the one or more capture probes immobilized on the support.

5. The method of claim 1, wherein the support is a bead or a plurality of beads.

6. The method of claim 1, wherein the support is a planar support.

7. The method of claim 1, wherein the amplification of the one or more end-extended tagged nucleic acid duplexes produces at least one copy of the one or more tagged nucleic acid strands.

8. The method of claim 1, wherein the amplification of the one or more end-extended tagged nucleic acid duplexes produces a plurality of copies of the tagged nucleic acid strands.

9. The method of claim 1, wherein the amplification comprises bridge amplification.

10. The method of claim 1, wherein the detection of the plurality of tagged nucleic acid strands occurs on the solid support.

11. The method of claim 10, wherein the detection comprises sequencing at least a portion of a tagged nucleic acid strand of the plurality of tagged nucleic acid strands.

12. A method of detecting one or more target nucleic acids comprising the steps of:

(a) capturing the one or more target nucleic acids on a support by hybridizing a 3′

adaptor of the one or more target nucleic acids to a plurality of primers or a subset thereof, wherein the primers are immobilized on a support, and wherein the one or more target nucleic acids comprise ssDNA;

(b) using the one or more captured target nucleic acids as a template strand to produce one or more nucleic acid duplexes immobilized on the support;

(c) contacting a transposase and transposon with the one or more nucleic acid duplexes under conditions wherein the one or more nucleic acid duplexes and transposon composition undergo a transposition reaction to produce one or more tagged nucleic acid duplexes, wherein the transposon composition comprises a double stranded nucleic acid molecule comprising a transferred strand and a non-transferred strand;

(d) contacting the one or more tagged nucleic acid duplexes with a nucleic acid modifying enzyme under conditions to extend the 3′

end of the immobilized strand to the 5′

end of the template strand to produce one or more end-extended tagged nucleic acid duplexes;

(e) amplifying the one or more end-extended tagged nucleic acid duplexes to produce a plurality of tagged nucleic acid strands; and

(f) detecting the plurality of tagged nucleic acid strands.

13. The method of claim 12, wherein the one or more target nucleic acids are genomic DNA molecules.

14. The method of claim 12, wherein the tagged nucleic acid duplexes are DNA:

DNA duplexes.

15. A method of detecting one or more target nucleic acids comprising the steps of:

(a) providing one or more target nucleic acids, wherein the one or more target nucleic acids comprise ssDNA;

(b) capturing the one or more target nucleic acids on a support, the support comprising a plurality of immobilized nucleic acids each having a sequence complementary to at least a portion of the one or more target nucleic acids;

(c) using the one or more captured target nucleic acids as a template strand to produce one or more nucleic acid duplexes immobilized on the support;

(d) tagging the one or more nucleic acid duplexes via a transposition reaction to produce tagged nucleic acid duplexes;

(e) amplifying the one or more tagged nucleic acid duplexes to produce a plurality of tagged nucleic acid strands; and

(f) detecting the plurality of tagged nucleic acid strands.