Methods and compositions for paired end sequencing using a single surface primer

A method for pairwise sequencing of first and second regions of a target nucleic acid, wherein the first and second regions are in complementary strands of the target nucleic acid, the method comprising:

• (a) providing an array comprising a plurality of amplification sites,
• wherein the amplification sites comprise (i) a plurality of capture nucleic acids, and (ii) a plurality of clonal single-stranded amplicons,
• wherein each single-stranded amplicon is attached at its 5′ end to a capture nucleic acid,
• wherein the capture nucleic acids comprise a cleavage site;
• (b) hybridizing a first sequencing primer to a universal sequence present on each single-stranded amplicon;
• (c) carrying out a first sequencing reaction by sequential addition of nucleotides to the first sequencing primer using the single-stranded amplicon as a first template to determine the sequence of a first region and to produce a complementary strand of the first region;
• (d) extending the complementary strand of the first region to form a double-stranded amplicon that comprises the first sequencing primer, nucleotides incorporated during the sequencing reaction, and nucleotides incorporated during the extending;
• (e) cleaving the capture nucleic acids attached to the single-stranded amplicons, wherein the cleavage converts the single-stranded amplicons into (i) shortened capture nucleic acids and (ii) unattached first templates that are not attached at the 5′ end to the capture nucleic acids;
• and
• (f) carrying out a second sequencing reaction by sequential addition of nucleotides to a second sequencing primer hybridized to the complementary strand and using the complementary strand as a second template to determine the sequence of a second region, wherein the second sequencing primer is in solution, wherein the complementary strand is not attached to the surface of the amplification site, and wherein the second sequencing reaction comprises subjecting the cleaved double-stranded amplicons to conditions that remove the unattached first templates, and hybridizing the second sequencing primer to a universal sequence present on the complementary strands.