METHODS OF NUCLEIC ACID SEQUENCING

• US 20150087534A1
• Filed: 03/06/2013
• Published: 03/26/2015
• 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
• Granted Patent: US 9,574,226 B2
• Time in Patent Office: Days
• US Class Current: 506/4
• 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; 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.

2. The method of claim 1, 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.

3. The method of claim 1, 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.

4. The method of claims 1 to 3, wherein the support comprises a plurality of immobilized primers.

5. The method of claim 4, wherein the RNA strand of the one or more DNA:

RNA duplexes or the DNA strand of the DNA;

DNA duplexes comprises a sequence complementary to at least a portion of one or more of the immobilized primers.

6. The method of claim 4 or 5, wherein the DNA:

RNA duplexes or the DNA;

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

RNA duplexes or DNA;

DNA duplexes.

7. The method of claim 4 or 5, wherein a plurality of DNA:

RNA duplexes or DNA;

DNA duplexes are provided by hybridizing a plurality of RNA molecules or single stranded DNA molecules to the immobilized primers on the support and extending the primers hybridized to the RNA molecules or single stranded DNA molecules using the RNA molecules or single stranded DNA molecules as template to produce the plurality of DNA;

RNA duplexes or DNA;

DNA molecules.

8. The method of any one of claims 4-7, wherein the immobilized primers comprise a polyT sequence.

9. The method of claim 4, 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.

10. The method of claim 9, wherein the first subset of primers comprises a polyT sequence.

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

12. The method of claim 4, further comprising adding a 3′

adaptor to the plurality of RNA molecules or single stranded DNA molecules, the 3′

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

hybridizing the 3′

-adaptor ligated RNA molecules or single stranded DNA molecules to the immobilized primers.

13. The method of any one of claims 1-12, wherein the transposition reaction results in a 5′

tagged RNA strand or 5′

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

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

14. The method of claim 13, further comprising contacting the one or more tagged DNA:

RNA duplexes or DNA;

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

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

end or copy the single stranded DNA strands to their 5′

ends.

15. The method of claim 14, wherein the nucleic acid modifying enzyme displaces the non-transferred strand of the transposon composition.

16. The method of claim 14, further comprising removing the RNA strands from the DNA:

RNA duplexes.

17. The method of claim 16, further comprising sequencing at least a portion of the DNA strands.

18. The method of claim 16 or 17, 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.

19. The method of claim 18, further comprising sequencing the second complementary DNA strand.

20. The method of claim 19, wherein the DNA strand of the DNA:

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

21. The method of claim 16, further comprising amplifying the DNA strands to produce a plurality of double stranded DNA molecules comprising first and second amplified strands.

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

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

24. The method of claim 23, further comprising copying at least a portion of the second amplified strands to regenerate the first amplified strands.

25. The method of claim 24, further comprising removing the second amplified strands.

26. The method of claim 25, further comprising sequencing at least a portion of the first amplified strands.

27. The method of any one of the preceding claims, wherein the support is a bead or a plurality of beads.

28. The method of any one of the preceding claims, wherein the support is a planar support.

29. The method of claim 27, wherein a plurality of duplexes is provided, each duplex located on a single bead.

30. The method of claim 21, wherein the amplification produces a cluster of amplicons.

31. The method of any one of the preceding claims, wherein the transferred strand comprises a tag to preserve strand information.