Method for sequencing a polynucleotide template

• US 20090088327A1
• Filed: 10/05/2007
• Published: 04/02/2009
• Est. Priority Date: 10/06/2006
• Status: Active Grant

• Current Assignee: Illumina Cambridge Limited (Illumina Incorporated)
• Original Assignee: Illumina Cambridge Limited (Illumina Incorporated)
• Inventors: Barr Ost, Tobias William, Fashena, Sarah, Rigatti, Roberto
• Granted Patent: US 7,754,429 B2
• Time in Patent Office: Days
• US Class Current: 506/4
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2535/119 Double strand sequencing
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2565/513 characterised by the pattern of the arrayed oligonucleotides
• CPC Class Codes: C12Q 2565/543 characterised by the use of two or more capture oligonucleotide primers in concert, e.g. bridge amplification

Abstract

The invention relates to methods for pairwise sequencing of a double-stranded polynucleotide template, which methods result in the sequential determination of nucleotide sequences in two distinct and separate regions of the polynucleotide template. Using the methods of the invention it is possible to obtain two linked or paired reads of sequence information from each double-stranded template on a clustered array, rather than just a single sequencing read from one strand of the template.

Claims

1. A method for pairwise sequencing of first and second regions of a target double-stranded polynucleotide, wherein said first and second regions are in the same target double-stranded polynucleotide, the method comprising:

(a) providing a solid support having immobilised thereon a plurality of double stranded template polynucleotides each formed from complementary first and second template strands linked to the solid support at their 5′

ends, and multiple copies of one or more 5′

-end immobilised primers capable of hybridising to the 3′

end of the first template strand;

(b) treating the plurality of double stranded template polynucleotides such that the first template strands are hybridised to 5′

-end immobilised primers;

(c) carrying out a first sequencing read to determine the sequence of a first region of the template polynucleotide;

(d) carrying out an extension reaction to extend one or more of the immobilised primers to copy the first template strand to generate a second immobilised template strand;

(e) treating the plurality of first and second immobilised template strands to remove the first template strand from the solid support; and

(f) carrying out a second sequencing read to determine the sequence of a second region of the template polynucleotide, wherein determining the sequences of the first and second regions of the target polynucleotide achieves pairwise sequencing of said first and second regions of said target double-stranded polynucleotide.

3. The method according to claims 1 or 2, wherein the sequencing reads are performed using labelled nucleotides or oligonucleotides.

4. The method according to claims 1 or 2, wherein the templates are immobilised on a single planar solid support or on a plurality of microspheres.

5. The method according to claim 4, wherein the microspheres are immobilised on a single solid support.

6. The method according to claim 1 or 2, wherein the treating in steps 1(b), 1(e) or 2(e) involves nicking the immobilised double stranded template polynucleotides with an endonuclease.

7. The method according to claim 1 or 2, wherein the treating in steps 1(b), 1(e) or 2(e) involves formation and cleavage of an abasic site in the immobilised double stranded template polynucleotides.

8. The method according to claim 7, wherein said abasic site is generated from a uracil base or from an 8-oxo-guanine base.

9. The method according to claim 8, wherein the uracil base is removed by treatment with Uracil DNA glycosylase (UDG) and DNA glycosylase-lyase Endonuclease VIII or FPG glycosylase.

10. The method according to claim 1 (c), wherein the sequencing read is performed on a double stranded template using a polymerase with strand displacing activity.

11. The method according to claim 1, further comprising an additional step prior to step 1(c) of making the immobilised template single stranded.

12. The method according to claim 11, wherein the additional step involves treating with a 5′

-3′

exonuclease to digest strands not immobilised at their 5′

-ends or treating with a chemical denaturant.

13. The method according to claim 1, wherein the first sequencing read is performed using an immobilised primer.

14. The method according to claim 1, further comprising an additional step prior to step 1(c) of hybridising a non immobilised sequencing primer to the immobilised template.

15. The method according to claim 1, further comprising an additional step prior to step 1 (d) of removing the bases added in step 1 (c).

16. The method according to claim 15, wherein the removing is carried out using a nicking enzyme followed by treatment with a 5′

-3′

exonuclease or by denaturing the non immobilised sequencing primer.

17. The method according to claims 1(e) or 2(e), wherein the first template strand is attached via a diol linkage which is cleaved by treatment with a chemical cleavage agent comprising periodate, and the duplex is subsequently denatured.

19. The method according to claim 9, wherein the surface is treated with a phosphatase to remove the 3′

-phosphate group left by the action of Uracil DNA glycosylase.

20. The method according to claim 1, further comprising a step of treating with a restriction enzyme prior to step 1(d) to shorten the immobilised primer and release a free 3′

hydroxyl for extension.

21. The method according to claim 1, wherein the immobilised primer is extended by hybridisation of a non-immobilised complementary sequence with a 5′

-overhang, and the immobilised primer is extended to copy the overhang prior to step 1d.

22. The method according to claim 1, wherein step 1d is repeated through multiple cycles of extension and denaturation.

23. The method according to claim 1, wherein at least one of the immobilised primers is blocked at the 3′

end, and the block is removed prior to step 1(d).

24. The method of claim 23, wherein said block is a phosphate moiety, a 3′

-deoxynucleotide or wherein the immobilised primer comprises a self complementary hairpin with a sequence that binds to a restriction enzyme that can be cleaved using said restriction enzyme.

25. A clustered array prepared according to claim 1(a-d) or claim 2.

2. A method for sequencing distal end regions A and B of a target double-stranded polynucleotide, wherein said distal end regions A and B are in the same target double-stranded polynucleotide, the method comprising:

(a) providing a solid support having immobilised thereon a plurality of double stranded template polynucleotides each formed from complementary first and second template strands linked to the solid support at their 5′

ends;

(b) treating the double stranded template polynucleotides such that each double stranded template polynucleotide is cut in at least two places to generate two shortened double stranded template fragments A and B immobilised at one end, wherein A and B are no longer directly connected;

(c) treating the two shortened double stranded template fragments A and B immobilised at one end to make the two non-immobilised ends a blunt ended duplex;

(d) treating the two blunt ended duplexes such that the two blunt ends A and B are connected to form a double stranded nucleotide sequence containing both ends A and B of the original target fragment in a shortened contiguous sequence, immobilised at both ends;

(e) cleaving one strand of the double stranded nucleotide sequence containing both distal ends A and B of the original target fragment joined in a shortened contiguous sequence immobilised at both ends to generate a single stranded nucleotide target sequence containing both distal ends A and B of the original target fragment in a shortened contiguous sequence, wherein said single stranded nucleotide target sequence is immobilised at a single 5′

or 3′

end;

(f) hybridising a sequencing primer to the single stranded nucleotide target sequence containing both distal ends A and B of the original target fragment in a shortened contiguous sequence; and

(g) carrying out a single sequencing reaction to determine a contiguous sequence of both ends A and B of the original target fragment.

18. The method of claim 2 (b), wherein said treating is performed by a remote cutting restriction enzyme which cuts between 10-50 bases remote from its sequence dependent binding site.