Method for pairwise sequencing of target polynucleotides
First Claim
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1. A method for sequencing of first and second regions of target polynucleotides having different sequences, the method comprising:
- (a) providing beads comprising immobilized first strands of the target polynucleotides;
(b) hybridizing a sequencing primer in solution to the immobilized first strands and sequencing the first region of the first strands of the of the target polynucleotides, wherein the sequencing comprises extension of a sequencing primer by sequential additions of nucleotides 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 a portion of the first strands from the beads, thereby generating an immobilized extension primer having a free 3′
-hydroxyl; and
(e) sequencing the second region of the second strands of the target polynucleotides using the immobilized extension primer, wherein determining the sequence of the first and second regions of the target polynucleotides achieves sequencing of the first and second regions of the 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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16 Claims
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1. A method for sequencing of first and second regions of target polynucleotides having different sequences, the method comprising:
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(a) providing beads comprising immobilized first strands of the target polynucleotides; (b) hybridizing a sequencing primer in solution to the immobilized first strands and sequencing the first region of the first strands of the of the target polynucleotides, wherein the sequencing comprises extension of a sequencing primer by sequential additions of nucleotides 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 a portion of the first strands from the beads, thereby generating an immobilized extension primer having a free 3′
-hydroxyl; and(e) sequencing the second region of the second strands of the target polynucleotides using the immobilized extension primer, wherein determining the sequence of the first and second regions of the target polynucleotides achieves sequencing of the first and second regions of the target polynucleotides. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16)
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Specification