Primer sets and methods and kits incorporating the primer sets

A reusable flow cell, comprising:

• a substrate;
• a polymeric hydrogel attached to at least a portion of the substrate;
• and
• a primer set attached to the polymeric hydrogel, the primer set including:
• a first nuclease resistant primer including a cleavage site;
• and
• a second nuclease resistant primer without any cleavage site.

A method, comprising:

• introducing a first sample into a flow cell including:
• a substrate;
• a polymeric hydrogel attached to at least a portion of the substrate;
• and
• a plurality of primer sets attached to the polymeric hydrogel, each primer set in the plurality including a first nuclease resistant primer including a cleavage site and a second nuclease resistant primer without any cleavage site;
• amplifying a library fragment of the first sample, thereby generating a first plurality of first sample library fragment amplicons respectively attached to a plurality of the first nuclease resistant primers and a second plurality of first sample library fragment amplicons respectively attached to a plurality of the second nuclease resistant primers;
• introducing a cleaving fluid into the flow cell, thereby cleaving the first nuclease resistant primers at the cleavage sites, whereby the first plurality of first sample library fragment amplicons are removed;
• sequencing the second plurality of first sample library fragment amplicons, thereby generating a plurality of first sample library fragment nascent strands;
• introducing a nuclease enzyme into the flow cell, thereby cleaving the second plurality of first sample library fragment amplicons and the plurality of first sample library fragment nascent strands;
• introducing a plurality of regeneration oligonucleotides into the flow cell, whereby first portions of at least some of the regeneration oligonucleotides respectively hybridize to remaining portions of at least some of the first nuclease resistant primers, wherein each regeneration oligonucleotide further includes a second portion that is complementary to a cleaved portion of the first nuclease resistant primer;
• and
• initiating polymerase extension along respective second portions of the hybridized regeneration oligonucleotides to regenerate the cleaved portion of the at least some of the first nuclease resistant primers.

A flow cell, comprising:

• a substrate;
• a polymeric hydrogel attached to at least a portion of the substrate;
• and
• a primer set attached to the polymeric hydrogel, the primer set including:
• a first primer having a first cleavage site and a second cleavage site that is different from the first cleavage site;
• and
• a second primer having the first cleavage site without the second cleavage site.

A method, comprising:

• introducing a first sample into a flow cell including:
• a substrate;
• a polymeric hydrogel attached to at least a portion of the substrate;
• and
• a plurality of primer sets attached to the polymeric hydrogel, each of the primer sets including a first primer having a first cleavage site and a second cleavage site that is different from the first cleavage site, and a second primer having the first cleavage site without the second cleavage site;
• amplifying a first library fragment of the first sample, thereby generating a first plurality of first sample library fragment amplicons respectively attached to a plurality of the first primers and a second plurality of first sample library fragment amplicons respectively attached to a plurality of the second primers;
• introducing a cleaving fluid into the flow cell, thereby cleaving the first primers at the second cleavage sites, whereby the first plurality of first sample library fragment amplicons are removed;
• sequencing the second plurality of first sample library fragment amplicons, thereby generating a plurality of first sample library fragment nascent strands;
• introducing a second cleaving fluid into the flow cell, thereby cleaving each of the first primers and the second primers at their respective first cleavage sites and leaving remaining portions of the first primers and of the second primers attached to the polymeric hydrogel;
• and
• sequentially or simultaneously regenerating i) a cleaved portion of at least some of the first primers at the remaining portions of the at least some of the first primers, and ii) a cleaved portion of at least some of the second primers at the remaining portions of the at least some of the second primers.