Method for pairwise sequencing of target polynucleotides
First Claim
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1. A method of pairwise sequencing, the method comprising:
- (a) providing a solid support having immobilised thereon a plurality of first template polynucleotides linked to the solid support at their 5′
ends, the solid support further comprising a plurality of 5′
-end immobilized amplification primers;
(b) obtaining sequence data from a first region of the first template polynucleotides;
(c) hybridizing one or more extension oligonucleotides to the amplification primers, each extension oligonucleotide comprising a first portion complementary to an immobilized amplification primer, and a second portion whose sequence is the same sequence as a selected region of the first template polynucleotide;
(d) performing an extension reaction to extend hybridized immobilized amplification primers to produce a population of extended primers;
(e) de-hybridizing the extension oligonucleotides, hybridizing the selected regions of the templates to the extended primers and further extending the extended primers to copy the first template polynucleotides to generate a plurality of second immobilised template polynucleotides;
(f) obtaining sequence data for a second region of each of the second immobilised template polynucleotides, wherein determining the sequences of the first and second regions of the first and second template polynucleotides achieves pairwise sequencing of said first and second regions of said target polynucleotides.
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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.
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12 Claims
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1. A method of pairwise sequencing, the method comprising:
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(a) providing a solid support having immobilised thereon a plurality of first template polynucleotides linked to the solid support at their 5′
ends, the solid support further comprising a plurality of 5′
-end immobilized amplification primers;(b) obtaining sequence data from a first region of the first template polynucleotides; (c) hybridizing one or more extension oligonucleotides to the amplification primers, each extension oligonucleotide comprising a first portion complementary to an immobilized amplification primer, and a second portion whose sequence is the same sequence as a selected region of the first template polynucleotide; (d) performing an extension reaction to extend hybridized immobilized amplification primers to produce a population of extended primers; (e) de-hybridizing the extension oligonucleotides, hybridizing the selected regions of the templates to the extended primers and further extending the extended primers to copy the first template polynucleotides to generate a plurality of second immobilised template polynucleotides; (f) obtaining sequence data for a second region of each of the second immobilised template polynucleotides, wherein determining the sequences of the first and second regions of the first and second template polynucleotides achieves pairwise sequencing of said first and second regions of said target polynucleotides. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12)
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Specification