Paired-end reads in sequencing by synthesis

Abstract

The disclosure provides methods of generating paired reads in sequencing-by-synthesis process, particularly, in systems with relatively short read lengths (e.g., 15-35 bases), such as for example, in single molecule sequencing by synthesis. Several implementations of the methods are provided. Of particular advantage are the methods that permit re-sequencing of the template, which yields lower error rates. The invention further provides methods of using paired reads, for example, for positioning them over repeats or for assembly into large sequences, including whole genome assembly.

1 - 26 . (canceled) 
     
     
         27 . A method for generating paired reads from a strand of a nucleic acid duplex, the method comprising:
 a) providing a nucleic acid template;   b) conducting a sequencing-by-synthesis reaction using detectably labeled nucleotides to obtain a first read of the template; and   c) conducting a sequencing-by-synthesis reaction using detectably labeled nucleotides to obtain a second read of the template, wherein the second read is separated from the first read along the length of the template.   
     
     
         28 . The method according to  claim 27 , wherein the template is directly attached to a solid support. 
     
     
         29 . The method according to  claim 27 , wherein the template is indirectly associated with a solid support. 
     
     
         30 . The method according to  claim 27 , wherein the first and second reads are separated by unlabeled nucleotides. 
     
     
         31 . The method according to  claim 27 , wherein the first and second reads are separated by further extending a portion of a copy of the template after the first read. 
     
     
         32 . The method according to  claim 27 , wherein the length between the first and second reads is synthesized by incorporating at least one labeled nucleotide and removing the label from the nucleotide. 
     
     
         33 . The method according to  claim 27 , further comprising:
 dissociating the copy of the template from the template; and   repeating steps (b) and (c).   
     
     
         34 . The method according to  claim 27 , wherein the first and/or the second reads are resequenced by a multiple pass sequencing. 
     
     
         35 . The method according to  claim 27 , wherein the first and the second reads are obtained from the same copy of the template. 
     
     
         36 . The method according to  claim 27 , wherein the first and the second reads are obtained from different copies of the template. 
     
     
         37 . A method for generating paired reads from a strand of a nucleic acid duplex, the method comprising:
 a) providing a nucleic acid template;   b) conducting a sequencing-by-synthesis reaction using detectably labeled nucleotides to obtain a first read of the template;   c) synthesizing a spacer, wherein the synthesized spacer comprises unlabeled nucleotides; and   d) conducting a sequencing-by-synthesis reaction using detectably labeled nucleotides to obtain a second read of the template, wherein the second read is separated from the first read by the spacer.   
     
     
         38 . The method according to  claim 37 , wherein the template is directly attached to a solid support. 
     
     
         39 . The method according to  claim 37 , wherein the template is indirectly associated with a solid support. 
     
     
         40 . The method according to  claim 37 , wherein synthesizing the spacer comprises:
 incorporating at least one labeled nucleotide into a copy of the template; and   removing the label from the nucleotide.   
     
     
         41 . The method according to  claim 37 , synthesizing the spacer comprises: extending a portion of a copy of the template after the first read. 
     
     
         42 . A method for generating paired reads from a strand of a nucleic acid duplex, the method comprising:
 a) providing a nucleic acid template, wherein the template is indirectly associated with a solid support;   b) conducting a sequencing-by-synthesis reaction using detectably labeled nucleotides to obtain a first read of the template; and   c) conducting a sequencing-by-synthesis reaction using detectably labeled nucleotides to obtain a second read of the template, wherein the second read is separated from the first read along the length of the template.   
     
     
         43 . The method according to  claim 42 , wherein the first and second reads are separated by unlabeled nucleotides. 
     
     
         44 . The method according to  claim 42 , wherein the first and second reads are separated by further extending a portion of a copy of the template after the first read. 
     
     
         45 . The method according to  claim 42 , wherein the length between the first and second reads is synthesized by incorporating at least one labeled nucleotide and removing the label from the nucleotide.