Method for pairwise sequencing 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.
Claims24
A method for pairwise sequencing of first and second regions of target polynucleotides having different sequences, the method comprising:
- (a) providing a solid support comprising immobilized first strands of the target polynucleotides;
- (b) sequencing the first region of the first strands of the target polynucleotides using a sequencing primer in solution, wherein the sequencing comprises extending the sequencing primer to generate an extended sequencing primer;
- (c) copying at least a portion of the first strands of the target polynucleotides by further extending the extended sequencing primer, thereby generating second strands having the second regions of the target polynucleotides;
- (d) removing the first strands or a portion of the first strands from the solid support;
- and
- (e) sequencing the second region of the second strands of the target polynucleotides using an immobilized sequencing primer, wherein determining the sequence of the first and second regions of the target polynucleotides achieves pairwise sequencing of the first and second regions of the target polynucleotides.
Dependencies are inferred from the claim text.