Method for pairwise sequencing of target polynucleotides
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.
1. A method for pairwise sequencing of first and second regions of target polynucleotides having different sequences, the method comprising:
(a) providing a solid support comprising immobilized first strands of the target polynucleotides;
(b) sequencing the first region of the first strands of the target polynucleotides using a sequencing primer in solution, wherein the sequencing comprises extending the sequencing primer to generate an extended sequencing primer;
(c) copying at least a portion of the first strands of the target polynucleotides by further extending the extended sequencing primer, thereby generating second strands having the second regions of the target polynucleotides;
(d) removing the first strands or a portion of the first strands from the solid support; and
(e) sequencing the second region of the second strands of the target polynucleotides using an immobilized sequencing primer, wherein determining the sequence of the first and second regions of the target polynucleotides achieves pairwise sequencing of the first and second regions of the target polynucleotides.
2. The method of claim 1 , wherein the removal is carried out by nicking the first strands followed by digesting the first strands.
3. The method of claim 1 , wherein the target polynucleotides further comprise a tag.
4. The method of claim 3 , further comprising sequencing the tag.
5. The method of claim 4 , wherein the tag is sequenced after step (b).
6. The method of claim 1 , wherein the first and second regions are located at the ends of the target polynucleotides.
7. The method of claim 1 , wherein the sequencing comprises extension of a sequencing primer by sequential additions of nucleotides.
8. The method of claim 7 , wherein the nucleotides comprise labeled nucleotides.
9. The method of claim 8 , wherein the nature of the nucleotide added is determined after each of the sequential additions.
10. The method of claim 1 , wherein the support is a planar solid support.
11. The method of claim 1 , wherein the first strands comprise a cleavage site that allows for controlled cleavage of the first strands.
12. The method of claim 11 , wherein step (d) is carried out using chemical cleavage.
13. The method of claim 11 , wherein step (d) is carried out using enzymatic cleavage.
14. The method of claim 11 , wherein step (d) is carried out using photochemical cleavage.
15. The method of claim 1 , wherein the sequencing is carried out by pyrosequencing.
16. The method of claim 1 , wherein the sequencing is carried out by sequencing-by-synthesis.
17. The method of claim 1 , wherein the sequencing is carried out by sequencing-by-ligation.
18. The method of claim 1 , wherein the first strand is treated to free a 3′ hydroxyl moiety that is used as the immobilized sequencing primer.
19. The method of claim 1 , wherein step (a) comprises providing a solid support comprising immobilized single-stranded first strands of the target polynucleotides.
20. The method of claim 1 , wherein the solid support comprises beads.
21. The method of claim 20 , wherein the beads are in wells.
22. The method of claim 1 , wherein the different target polynucleotides have the same universal primer recognition regions.
23. The method of claim 22 , wherein the sequencing primer in solution comprises a universal primer that is hybridized to the universal primer recognition regions of the target polynucleotides in step (b).
24. The method of claim 22 , wherein the immobilized sequencing primer comprises a universal primer that is hybridized to the universal primer recognition regions of the target polynucleotides in step (e).