Methods of nucleic acid sequencing

• US 9,574,226 B2
• Filed: 03/06/2013
• Issued: 02/21/2017
• Est. Priority Date: 03/06/2012
• Status: Active Grant

• Current Assignee: Illumina Cambridge Limited (Illumina Incorporated)
• Original Assignee: Illumina Incorporated
• Inventors: Gormley, Niall Anthony, Fraser, Louise, Kokko-Gonzales, Paula
• Primary Examiner(s): DUNSTON, JENNIFER ANN
• Application Number:
  US14/378,613
• Publication Number:
  US 20150087534A1
• Time in Patent Office: 1,448 Days
• US Class Current: 1/1
• 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 tagging nucleic acid duplexes comprising the steps of:

(a) providing a transposase and a transposon composition;

(b) providing one or more nucleic acid duplexes immobilized on a support, wherein the one or more nucleic acid duplexes are DNA;

RNA duplexes; and

(c) contacting the transposase and transposon composition with the one or more nucleic acid duplexes under conditions wherein the one or more DNA;

RNA duplexes and transposon composition undergo a transposition reaction to produce one or more tagged DNA;

RNA duplexes, wherein the DNA;

RNA duplexes are tagged on the 5′

end of the RNA strand, and wherein the transposon composition comprises a double stranded nucleic acid molecule comprising a transferred strand and a non-transferred strand.

2. The method of claim 1, wherein the support comprises a plurality of immobilized primers.

3. The method of claim 2, wherein an RNA strand of the one or more nucleic acid duplexes comprises a sequence complementary to at least a portion of one or more of the immobilized primers.

4. The method of claim 3, wherein the DNA:

RNA duplexes are provided by hybridizing one or more RNA molecules to the immobilized primers on the support and extending the primers hybridized to the RNA molecules using the RNA molecules as templates to produce the one or more DNA;

RNA duplexes.

5. The method of claim 3, wherein the immobilized primers comprise a polyT sequence.

6. The method of claim 2, wherein the plurality of immobilized primers comprises a first subset of primers of a first sequence and a second subset of primers of a second sequence.

7. The method of claim 6, wherein the first subset of primers comprises a polyT sequence.

8. The method of claim 4, wherein the plurality of immobilized primers comprise target specific primers capable of hybridizing to the one or more RNA molecules.

9. The method of claim 4, wherein the one or more RNA molecules each comprise a 3′

adaptor comprising a sequence complementary to the plurality of immobilized primers or a subset thereof; and

wherein the hybridizing comprises hybridizing the one or more RNA molecules comprising the 3′

adaptor to the immobilized primers.

10. The method of claim 1, wherein the transposition reaction results in the 5′

tagged RNA strand comprising the transferred strand of the transposon composition and a gap between the 3′

end of a complementary DNA strand and the non-transferred strand of the transposon composition.

11. The method of claim 10, further comprising contacting the one or more tagged DNA:

RNA duplexes with a nucleic acid modifying enzyme under conditions to extend the 3′

end of DNA strands to copy RNA strands to their 5′

end.

12. The method of claim 11, wherein the nucleic acid modifying enzyme displaces the non-transferred strand of the transposon composition.

13. The method of claim 11, further comprising removing the RNA strands from the DNA:

RNA duplexes to yield remaining DNA strands.

14. The method of claim 13, further comprising sequencing at least a portion of the remaining DNA strands.

15. The method of claim 13, further comprising copying at least a portion of the DNA strands to produce a second DNA strand complementary to the DNA strand of the DNA:

RNA duplexes.

16. The method of claim 15, further comprising sequencing the second complementary DNA strand.

17. The method of claim 16, wherein the DNA strand of the DNA:

RNA duplexes is removed prior to sequencing the second complementary DNA strand.

18. The method of claim 13, further comprising amplifying the remaining DNA strands to produce a plurality of double stranded DNA molecules comprising first and second amplified strands.

19. The method of claim 18, further comprising removing the first amplified strands.

20. The method of claim 19, further comprising sequencing at least a portion of the second amplified strands.

21. The method of claim 20, further comprising copying at least a portion of the second amplified strands to regenerate the first amplified strands.

22. The method of claim 21, further comprising removing the second amplified strands.

23. The method of claim 22, further comprising sequencing at least a portion of the first amplified strands.

24. The method of claim 1, wherein the support is selected from the group consisting of a bead, a plurality of beads, and planar support.

25. The method of claim 24, wherein a plurality of duplexes is provided, each duplex immobilized on a single bead.

26. The method of claim 18, wherein the amplification produces a cluster of amplicons.

27. The method of claim 1, wherein the transferred strand comprises a tag to preserve strand information.

28. A method of tagging nucleic acid duplexes comprising the steps of:

(a) providing a transposase and a transposon composition;

(b) providing one or more nucleic acid duplexes immobilized on a support, wherein the support comprises a plurality of immobilized primers comprising a first subset of primers comprising a polyT sequence and a second subset of primers comprising a second sequence; and

(c) 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.

29. The method of claim 28, wherein the one or more nucleic acid duplexes are DNA:

RNA duplexes and wherein the DNA;

RNA duplexes are tagged on the 5′

end of the RNA strand.

30. The method of claim 29, wherein the transposition reaction results in a 5′

tagged RNA strand comprising the transferred strand of the transposon composition and a gap between the 3′

end of a complementary DNA strand and the non-transferred strand of the transposon composition.

31. The method of claim 28, wherein the nucleic acid duplexes are DNA:

DNA duplexes and wherein one of the strands of the DNA;

DNA duplex is tagged at the 5′

end of the DNA strand.

32. The method of claim 31, wherein the transposition reaction results in a 5′

tagged DNA strand comprising the transferred strand of the transposon composition and a gap between the 3′

end of a complementary DNA strand and the non-transferred strand of the transposon composition.

33. A method of tagging nucleic acid duplexes comprising the steps of:

(a) providing a transposase and a transposon composition;

(b) providing one or more nucleic acid duplexes immobilized on a support, wherein the support comprises a plurality of immobilized primers wherein the one or more nucleic acid duplexes comprises one or more DNA;

RNA duplexes having an RNA strand or one or more DNA;

DNA duplexes having a DNA strand, wherein the RNA strand or the DNA strand comprises a sequence complementary to at least a portion of one or more of the immobilized primers and wherein the immobilized primers comprise a polyT sequence; and

(c) 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.

34. The method of claim 33, wherein the one or more nucleic acid duplexes are DNA:

RNA duplexes and wherein the DNA;

RNA duplexes are tagged on the 5′

end of the RNA strand.

35. The method of claim 33, wherein the nucleic acid duplexes are DNA:

DNA duplexes and wherein one of the strands of the DNA;

DNA duplex is tagged at the 5′

end of the DNA strand.