Method for sequencing a polynucleotide template

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 complementary strands of the target polynucleotide, the method comprising:

(a) providing a solid support having immobilized thereon a mixture of first and second template polynucleotide duplexes, each comprising the same double-stranded target polynucleotide, wherein both strands of each template polynucleotide duplex are linked to the solid support at the 5′ end, a first template strand of both the first and second template polynucleotide duplexes includes a cleavage site Y, and a second template strand of the first template polynucleotide duplexes but not the second template polynucleotide duplexes includes a cleavage site X, which is different from the cleavage site Y;

(b) cleaving the second template strand of the first template polynucleotide duplexes at the cleavage site X;

(c) hybridizing a first sequencing primer to first template strand of the first template polynucleotide duplexes;

(d) carrying out a first sequencing reaction by sequential addition of nucleotides to the first sequencing primer to determine the sequence of a first region of the target polynucleotide in the first template strand;

(e) cleaving the first template strand of both the first and second template polynucleotide duplexes at the cleavage site Y;

(f) hybridizing a second sequencing primer to the second template strand of the second template polynucleotide duplexes; and

(g) carrying out a second sequencing reaction by sequential addition of nucleotides to the second sequencing primer to determine the sequence of a second region of the target polynucleotide in the second template strand.

2. The method of claim 1 , wherein the ratio of the first template polynucleotide duplexes to the second template polynucleotide duplexes in the mixture is approximately 1:1.

3. The method of claim 1 , wherein the cleavage site X permits enzymatic, chemical or photochemical cleavage of the second template strand of the first template polynucleotide duplexes.

4. The method of claim 3 , wherein the cleavage site X comprises a disulfide linkage which is cleaved by treatment with a chemical cleavage agent comprising tris-(2-carboxyethyl)-phosphine (TCEP) or a salt thereof.

5. The method of claim 1 , wherein the cleavage site Y permits enzymatic, chemical or photochemical cleavage of the first template strand of both the first and second template polynucleotide duplexes.

6. The method of claim 5 , wherein the cleavage site Y comprises a diol linkage which is cleaved by treatment with a chemical cleavage agent comprising periodate.

7. The method of claim 1 , wherein the cleavage site X and the cleavage site Y permit chemical cleavage, and a chemical agent used to cleave at the cleavage site X does not cleave at the cleavage site Y.

8. The method of claim 1 , wherein cleavage site X is a site for enzymatic cleavage, and cleavage site Y is a site for chemical or photochemical cleavage.

9. The method of claim 8 , wherein cleavage site X is a site for cleavage with a restriction endonuclease.

10. The method of claim 1 , wherein the mixture of first and second template polynucleotide duplexes of step (a) are present on a clustered array.

11. The method of claim 10 , wherein the clustered array is formed by solid-phase nucleic acid amplification.

12. The method of claim 10 , wherein the mixture of first and second template polynucleotide duplexes of step (a) are present within a single cluster on said clustered array.

13. The method of claim 12 , wherein each individual cluster on said clustered array comprises first and second template polynucleotide duplexes derived from amplification of a single target polynucleotide molecule.

14. The method of claim 12 , wherein at least 95% of the clusters on the array comprise template polynucleotide duplexes derived from amplification of different single target polynucleotides.

15. The method of claim 1 , wherein the mixture of first and second template polynucleotide duplexes is formed by an amplification reaction using a combination of amplification primers immobilized on said solid support, wherein said combination includes first and second forward primers and reverse primers having the structures:

First forward primer: A-L-X-S1,

Second forward primer: A-L-S1,

Reverse primer: A-L-Y-S2,

wherein A is a moiety which allows attachment to the solid support, L is an optional linker sequence, X is a cleavage site, Y is a cleavage site different to X, S1 and S2 are sequences which permit amplification of a nucleic acid molecule comprising the target double-stranded polynucleotide.