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 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 immobilized 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 immobilized at one end and non-immobilized at another end, wherein A and B are no longer directly connected;
(c) treating the two shortened double stranded template fragments A and B immobilized at one end to make the non-immobilized ends of each of the two shortened double stranded template fragments A and B a blunt ended duplex, thereby forming a first blunt ended duplex having end A and a second blunt ended duplex having end B;
(d) treating the first and second blunt ended duplexes such that the 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, immobilized 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 immobilized at both ends to generate a polynucleotide having a target sequence containing both distal ends A and B of the original target fragment in a shortened contiguous sequence, wherein said polynucleotide having the target sequence is immobilized at a single 5′ or 3′ end; and
(f) carrying out a single sequencing reaction to determine a contiguous sequence of both ends A and B of the original target fragment.
2. The method according to claim 1 , wherein the sequencing reaction is performed using labelled nucleotides or oligonucleotides.
3. The method according to claim 1 , wherein the polynucleotide having the target sequence is immobilized on a single planar solid support or on a plurality of microspheres.
4. The method according to claim 3 , wherein the microspheres are immobilized on a single solid support.
5. The method according to claim 1 , wherein said treating the double stranded template polynucleotides involves nicking the immobilized double stranded template polynucleotides with an endonuclease.
6. The method according to claim 1 , wherein said treating the double stranded template polynucleotides involves formation and cleavage of an abasic site in the immobilized double stranded template polynucleotides.
7. The method according to claim 6 , wherein said abasic site is generated from a uracil base or from an 8-oxo-guanine base.
8. The method according to claim 7 , wherein the uracil base is removed by treatment with Uracil DNA glycosylase (UDG) and DNA glycosylase-lyase Endonuclease VIII or FPG glycosylase.
9. The method according to claim 1 , wherein the single sequencing reaction is performed on a double stranded template using a polymerase with strand displacing activity.
10. The method according to claim 1 , further comprising making the polynucleotide having the target sequence single stranded prior to carrying out the single sequencing reaction.
11. The method according to claim 10 , wherein making the polynucleotide having the target sequence single stranded comprises treating with a 5′-3′ exonuclease to digest strands not immobilized at their 5′-ends or treating with a chemical denaturant.
12. The method according to claim 1 , wherein the single sequencing reaction is performed using an immobilized primer.
13. The method according to claim 1 , wherein carrying out the single sequencing reaction comprises hybridizing a non-immobilized sequencing primer to the polynucleotide having the target sequence.
14. The method according to claim 1 , wherein the first template strand is attached via a diol linkage which is cleaved by treatment with a chemical cleavage agent comprising periodate.
15. The method of claim 1 , wherein said treating the double stranded template polynucleotides is performed by a remote cutting restriction enzyme which cuts between 10-50 bases remote from its sequence dependent binding site.
16. The method according to claim 8 , wherein a surface to which the immobilized double stranded template polynucleotides were immobilized is treated with a phosphatase to remove a 3′-phosphate group left by the action of Uracil DNA glycosylase.
17. 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 immobilized 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 immobilized at one end, wherein A and B are no longer directly connected;
(c) treating the two shortened double stranded template fragments such that the two fragments 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, immobilized at both ends;
(d) 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 immobilized at both ends to generate a nucleotide target sequence containing both distal ends A and B of the original target fragment in a shortened contiguous sequence, wherein said nucleotide target sequence is immobilized at a single 5′ or 3′ end; and
(e) 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 17 , comprising, between steps (b) and (c), a step of treating the two shortened double stranded template fragments A and B immobilized at one end to make the two non-immobilized ends a blunt ended duplex.