CPC C12Q 1/6806 (2013.01) [C12Q 1/6834 (2013.01); C12Q 1/6853 (2013.01); C12Q 1/6874 (2013.01); G01N 21/6428 (2013.01); G01N 21/6458 (2013.01); C12Q 2600/158 (2013.01)] | 46 Claims |
1. A method for pairwise sequencing, comprising:
a) providing a plurality of immobilized single stranded nucleic acid concatemer template molecules, wherein individual concatemer template molecules in the plurality comprise at least one nucleotide having a scissile moiety that can be cleaved to generate an abasic site in the concatemer template molecule, wherein each individual concatemer template molecule in the plurality is immobilized to a first surface primer that is immobilized to a support, and wherein the immobilized first surface primers lack nucleotides having a scissile moiety;
b) sequencing the plurality of immobilized concatemer template molecules, thereby generating a plurality of extended forward sequencing primer strands, wherein individual immobilized concatemer template molecules have two or more extended forward sequencing primer strands hybridized thereon;
c) retaining the plurality of immobilized concatemer template molecules and replacing the plurality of extended forward sequencing primer strands with a plurality of forward extension strands that are hybridized to the retained immobilized concatemer template molecules by conducting a primer extension reaction;
d) removing the retained immobilized concatemer template molecules by generating abasic sites in the immobilized concatemer template molecules at the nucleotide(s) having the scissile moiety and generating gaps at the abasic sites to generate a plurality of gap-containing single stranded nucleic acid concatemer template molecules while retaining the plurality of forward extension strands and retaining the plurality of immobilized surface primers; and
e) sequencing the plurality of retained forward extension strands, thereby generating a plurality of extended reverse sequencing primer strands, wherein individual retained forward extension strands have two or more extended reverse sequencing primer strands hybridized thereon.
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