Method for sequencing a polynucleotide template
The invention relates to methods for pairwise sequencing of a double-stranded polynucleotide template, which permit the sequential determination of nucleotide sequences in two distinct and separate regions on complementary strands of the double-stranded polynucleotide template. The two regions for sequence determination may or may not be complementary to each other.
1. A method for pairwise sequencing of first and second strands of target polynucleotides, wherein the first and second strands of each target polynucleotide have complementary sequences, the method comprising:
(a) forming a plurality of nucleic acid colonies by solid phase nucleic acid amplification, wherein each of the nucleic acid colonies comprises multiple copies of a first strand and a complementary second strand formed by amplification of a single originating target polynucleotide;
(b) forming a single-stranded region in a subset of the first strands in each of the colonies and hybridizing first sequencing primers to the single-stranded region; whereby after hybridizing said sequencing primer, the colonies comprise a plurality of intact duplexes on the solid support, the intact duplexes comprising a portion of the multiple colonies of the first strand and a complementary second strand;
(c) carrying out a first sequencing reaction by sequential addition of nucleotides to the first sequencing primers to determine sequences from the first strands;
(d) cleaving the first strands;
(e) removing at least a portion of the first strands from the colonies, whereby a single-stranded region is formed in second template strands of the intact duplexes;
(f) hybridizing second sequencing primers to second strands of the colonies;
(g) carrying out a second sequencing reaction by sequential addition of nucleotides to the second sequencing primers to determine sequences from the second strands, wherein the first sequencing reaction and the second sequencing reaction permit pairwise sequencing of the target polynucleotides.
2. The method of claim 1 , wherein the nucleic acid colonies are attached to beads.
3. The method of claim 1 , wherein the nucleic acid colonies are attached to a planar surface.
4. The method of claim 1 , wherein the nucleotides that are added in the first sequencing reaction and in the second sequencing reaction comprise reversible blocking groups.
5. The method of claim 1 , wherein the nucleotides that are added in the first sequencing reaction and in the second sequencing reaction comprise labels.
6. The method of claim 1 , wherein the first sequencing reaction and the second sequencing reaction comprise pyrosequencing.
7. The method of claim 1 , wherein the first sequencing reaction and the second sequencing reaction comprise sequencing-by-synthesis.
8. The method of claim 1 , wherein the cleaving of the first strands comprises enzymatic cleavage.
9. The method of claim 1 , wherein the cleaving of the first strands comprises chemical cleavage.
10. The method of claim 1 , wherein the cleaving of the first strands comprises photochemical cleavage.
11. The method of claim 1 , wherein the cleaving of the first strands comprises cleaving an abasic site.
12. The method of claim 1 , wherein the cleaving of the first strands comprises nicking the first strands.
13. The method of claim 1 , wherein the sequencing is carried out using a sequencing primer in solution.
14. The method of claim 1 , wherein the target polynucleotides comprise known adaptor sequences on the 5′ and 3′ ends of the first and second strands.
15. The method of claim 1 , wherein the first sequencing reaction and the second sequencing reaction permit pairwise sequencing of separate and distinct regions on the complementary strands of each of the target polynucleotides.
16. The method of claim 1 , wherein the first sequencing reaction determines sequences of regions of the first strands, wherein the second sequencing reaction determines sequences of regions of the second strands, and wherein the regions of the first strands are not complementary to the regions of the second strands.
17. The method of claim 1 , wherein the first sequencing reaction determines sequences of regions of the first strands, wherein the second sequencing reaction determines sequences of regions of the second strands, and wherein the regions of the first strands are complementary to the regions of the second strands.