Method for pair-wise sequencing a plurity of target polynucleotides
AssigneeIllumina Cambridge Limited
InventorsRoberto Rigatti, Tobias William Barr Ost
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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.
Claims20
A method for pairwise sequencing of first and second regions in each of a plurality of target polynucleotides, 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, wherein a 5′-end immobilised primer is hybridised to the 3′ end of each of the first template polynucleotides;
- (b) obtaining sequence data from a first region of the first template polynucleotides;
- (c) extending the 5′-end immobilised primers to copy the first template polynucleotides to generate a plurality of second immobilised template polynucleotides;
- (d) removing the plurality of first template polynucleotides from the solid support;
- and
- (e) 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.
Dependencies are inferred from the claim text.