Compositions and methods for pairwise sequencing

A method for pairwise sequencing, comprising steps of:

• a) providing a plurality of immobilized single stranded nucleic acid concatemer template molecules each comprising at least one nucleotide having a scissile moiety that can be cleaved to generate an abasic site, wherein the plurality of immobilized single stranded nucleic acid concatemer template molecules comprises individual immobilized concatemer template molecules that are covalently joined or hybridized to surface primers that are immobilized to a support, wherein the surface primers lack nucleotides having the scissile moiety, and wherein the scissile moiety comprises uridine;
• b) sequencing the plurality of immobilized single stranded nucleic acid concatemer template molecules with a first plurality of sequencing polymerases, a plurality of soluble forward sequencing primers and a first plurality of multivalent 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 single stranded nucleic acid concatemer template molecules thereby generating retained 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 an abasic site at the at least one nucleotide having the scissile moiety in each of the retained immobilized concatemer template molecules and generating a gap at the abasic site to generate a plurality of gap-containing concatemer template molecules while retaining the plurality of forward extension strands and retaining the surface primers;
• and
• e) sequencing the plurality of forward extension strands with a second plurality of sequencing polymerases, a plurality of soluble reverse sequencing primers and a second plurality of multivalent molecules, thereby generating a plurality of extended reverse sequencing primer strands, wherein individual forward extension strands have two or more extended reverse sequencing primer strands hybridized thereon,
• wherein individual multivalent molecules in the first plurality of multivalent molecules of step (b) and in the second plurality of multivalent molecules of step (e) comprise (i) a core;
• and (ii) a plurality of nucleotide arms which comprise a core attachment moiety, a spacer, a linker, and a nucleotide unit, wherein the core is attached to the plurality of nucleotide arms via their core attachment moiety, wherein the spacer is attached to the linker, wherein the linker is attached to the nucleotide unit.