Compositions and methods for pairwise sequencing
The present disclosure provides compositions and methods that employ the compositions for conducting pairwise sequencing and for generating concatemer template molecules for pairwise sequencing. The concatemers can be generated using a rolling circle amplification reaction which is conducted either on-support, or conducted in-solution and then distributed onto a support. The rolling circle amplification reaction generates concatemers containing tandem copies of a sequence of interest and at least one universal adaptor sequence. An increase in the number of tandem copies in a given concatemer increases the number of sites along the concatemer for hybridizing to multiple sequencing primers which serve as multiple initiation sites for polymerase-catalyzed sequencing reactions. When the sequencing reaction employs detectably labeled nucleotides and/or detectably labeled multivalent molecules (e.g., having nucleotide units), the signals emitted by the nucleotides or nucleotide units that participate in the parallel sequencing reactions along the concatemer yields an increased signal intensity for each concatemer.
1. 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.
2. The method of claim 1 , wherein the nucleotide units of individual multivalent molecules of step (b) bind a first polymerase which is bound to a nucleic acid duplex comprising an immobilized concatemer template molecule hybridized to a forward sequencing primer.
3. The method of claim 1 , wherein the nucleotide units of individual multivalent molecules of step (e) bind a second polymerase which is bound to a nucleic acid duplex comprising a retained forward extension strand hybridized to a reverse sequencing primer.
4. The method of claim 1 , wherein the core comprises streptavidin and the core attachment moiety comprises biotin.
5. The method of claim 1 , wherein in the spacer comprises a polyethylene glycol moiety, and wherein the linker comprises an aliphatic chain having 2-6 subunits or an oligo ethylene glycol chain having 2-6 subunits.
6. The method of claim 1 , wherein the plurality of nucleotide arms attached to the core have a same type of nucleotide unit, and wherein the same type of nucleotide unit is selected from the group consisting of dATP, dGTP, dCTP, dTTP and dUTP.
7. The method of claim 1 , wherein all of the individual multivalent molecules in the first plurality of multivalent molecules of step (b) have a same type of nucleotide unit, and all of the individual multivalent molecules in the second plurality of multivalent molecules of step (e) have a same type of nucleotide unit, and wherein the type of nucleotide unit is selected from the group consisting of dATP, dGTP, dCTP, dTTP and dUTP.
8. The method of claim 1 , wherein the first plurality of multivalent molecules of step (b) and the second plurality of multivalent molecules of step (e) comprises a mixture of any combination of two or more types of multivalent molecules each type having nucleotide units selected from the group consisting of dATP, dGTP, dCTP, dTTP and dUTP.
9. The method of claim 1 , wherein at least one multivalent molecule in the first plurality of multivalent molecules of step (b) is labeled with a fluorophore, and wherein at least one multivalent molecule in the second plurality of multivalent molecules of step (e) is labeled with a fluorophore.
10. The method of claim 1 , wherein generating abasic sites at step (d) comprises contacting the immobilized concatemer template molecules with uracil DNA glycosylase (UDG).
11. The method of claim 1 , wherein generating the plurality of gap-containing concatemer template molecules of step (d) comprises contacting the retained immobilized concatemer template molecules containing one or more abasic sites with an endonuclease IV, AP lyase, FPG glycosylase/AP lyase and/or endo VIII glycosylase/AP lyase.
12. The method of claim 11 , wherein the AP lyase comprises DNA-apurinic lyase or DNA-apyrimidinic lyase.