Method for pairwise sequencing of target polynucleotides
First Claim
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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.
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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.
41 Citations
18 Claims
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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:
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(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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16)
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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:
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(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. - View Dependent Claims (18)
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Specification