Paired-end reads in sequencing by synthesis
View Patent ↗The disclosure provides methods of generating paired reads in sequencing-by-synthesis process, particularly, in systems with relatively short read lengths (e.g., 15-35bases), 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. A method for generating paired reads from a strand of a nucleic acid duplex, the method comprising:
a) providing a nucleic acid template attached, either directly or indirectly, to a solid support;
b) conducting a sequencing-by-synthesis reaction using detectably labeled nucleotides to obtain a first read of the template;
c) synthesizing a spacer of a defined length; and
d) conducting a sequencing-by-synthesis reaction using detectably labeled nucleotides to obtain a second read of the template, said second read being separated from the first read by the spacer.
2. The method of claim 1 , wherein the template is individually optically resolvable.
3. The method of claim 1 , further comprising mapping the first and second reads onto a reference sequence.
4. The method of claim 1 , wherein the first and/or the second reads are 5-100 nucleotides long.
5. The method of claim 1 , wherein the spacer length is greater than the length of the first or the second read.
6. The method of claim 1 , wherein the spacer length is 20-1000 nucleotides.
7. The method of claim 1 , wherein the spacer length is fixed by the length of the template.
8. The method of claim 1 , wherein the spacer length is dynamically controlled.
9. The method of claim 1 , wherein the first and/or the second reads are resequenced by a multiple pass sequencing.
10. The method of claim 1 , wherein a copy of the template comprises a phosphothioate nucleotide or another nucleotide analog resistant to enzymatic digestion.
11. The method of claim 1 , wherein the first and the second reads are obtained from the same copy of the template.
12. The method of claim 1 , wherein the first and the second reads are obtained from different copies of the template.
13. The method of claim 1 , wherein the template is attached to a solid support at the 5′ end that is hybridized to a universal primer covalently attached to the support.
14. The method of claim 13 , wherein the universal primer is covalently attached to the support.
15. The method of claim 1 , wherein the template is attached to a solid support at the 3′ end.
16. The method of claim 15 , wherein the template is attached to the support at the 5 40 end.
17. The method of claim 16 , wherein the template comprises a universal primer site at the 3′ end.
18. The method of claim 1 , wherein the method comprises:
i) hybridizing the template to a first universal primer which is attached to a solid support;
ii) obtaining the first read by copying a portion of the template proximal to the support;
iii) further extending the copy of the template thereby creating the spacer; and
iv) obtaining the second read by copying a distal portion of the template.
19. The method of claim 1 , wherein the method comprises:
i) covalently attaching the template to a solid support, said template comprising a complement of a universal primer;
ii) hybridizing the universal primer to the template;
iii) obtaining the first read by copying a first portion of the template;
iv) further extending the copy of the template thereby creating the spacer; and
v) obtaining the second read by copying a second portion of the template.
20. The method of claim 19 , wherein the method further comprises melting off the copy of the template and, optionally, repeating steps ii) - v).
21. The method of claim 19 , wherein the method further comprises incorporating a phosphothioate nucleotide or another nucleotide analog resistant to enzymatic digestion, performing enzymatic digestion of the copy, and optionally, repeating steps ii) - v).
22. The method of claim 19 , wherein the template is covalently attached to the support at the 3′ end and the universal primer is proximal to the support.
23. The method of claim 19 , wherein the template is covalently attached to the support at the 5′ end and the universal primer is distal to the support.
24. The method of claim 19 , wherein the length of the spacer is dynamically controlled.
25. The method of claim 1 , further comprising resequencing the first and/or the second reads.
26. A method of using paired reads, the method comprising: mapping the paired reads generated by the method of claim 1 onto a reference sequence.
27. The method according to claim 26 , wherein using comprises positioning the paired reads over repeats in a reference sequence.
28. The method according to claim 26 , wherein using comprises assembling a portion of a genome.
29. The method according to claim 26 , wherein using comprises assembling a whole genome.
30. A method for generating paired reads from a strand of a nucleic acid duplex, the method comprising:
a) providing individually optically resolvable nucleic acid templates anchored, directly or indirectly, to a solid support;
b) conducting a sequencing-by-synthesis reaction using detectably labeled nucleotides to obtain a first read of a template, said first read consisting of 20-150 bases;
c) synthesizing a spacer of 20-1000 bases; and
d) conducting a sequencing-by-synthesis reaction using detectably labeled nucleotides to obtain a second read of the template, said second read consisting of 20-150 bases and separated from the first read by the spacer.
31. The method of claim 30 , further comprising resequencing the first and/or the second reads prior to the mapping step.
32. A method of using paired reads, the method comprising: mapping the paired reads generated by the method of claim 30 onto a reference sequence.