METHOD FOR SEQUENCING A POLYNUCLEOTIDE TEMPLATE

• US 20120165205A1
• Filed: 06/30/2010
• Published: 06/28/2012
• Est. Priority Date: 07/24/2009
• Status: Active Grant

• Current Assignee: Illumina Incorporated
• Original Assignee: Illumina Incorporated
• Inventors: West, John Stephen
• Granted Patent: US 10,036,063 B2
• Time in Patent Office: Days
• US Class Current: 506/7
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target

Abstract

A method of determining the sequence of a target nucleic acid is provided. The method can include the steps of (a) performing a defined number of incremental extension cycles to produce a population of nucleic acid fragments having different portions of the target nucleic acid wherein the individual nucleic acid fragments in the population have a defined length that is correlated with the number of incremental extension cycles; (b) determining the sequence of the first end of individual nucleic acid fragments in the population, thereby providing first end sequences; (c) determining the sequence of the second end of individual nucleic acid fragments in the population, thereby providing second end sequences; and (d) determining the sequence of the target nucleic acid based on the first end sequences, the second end sequences and the defined length.

Claims

1. A method of determining the sequence of a target nucleic acid, comprising

(a) performing a defined number of incremental extension cycles to produce a population of nucleic acid fragments comprising different portions of the target nucleic acid wherein the individual nucleic acid fragments in the population comprise a defined length that is correlated with the number of incremental extension cycles;

(b) determining the sequence of the first end of individual nucleic acid fragments in the population, thereby providing first end sequences;

(c) determining the sequence of the second end of individual nucleic acid fragments in the population, thereby providing second end sequences; and

(d) determining the sequence of the target nucleic acid based on the first end sequences, the second end sequences and the defined length.

2. The method of claim 1, wherein individual cycles in the incremental extension cycles comprise a wash step to remove an unreacted reagent.

3. The method of claim 1, wherein the population of nucleic acid fragments is produced on a solid support.

4. The method of claim 1, wherein step (a) further comprises cleaving the target nucleic acid to form a plurality of templates for the nucleic acid fragments.

5. The method of claim 4, wherein the plurality of templates are ligated to a universal adapter.

6. The method of claim 5, further comprising annealing primer nucleic acids to the universal adapter to form nucleic acid hybrids.

7. The method of claim 6, wherein the incremental extension cycles comprise contacting the nucleic acid hybrids with nucleotides comprising reversible blocking moieties.

8. The method of claim 7, wherein the nucleotides are unlabelled.

9. The method of claim 6, wherein the incremental extension cycles comprise contacting the nucleic acid hybrids with oligonucleotides.

10. The method of claim 9, wherein the oligonucleotides carry a 3′

or 5′

hydroxyl group, and the annealed primers comprise a terminal 3′

or 5′

phosphate group.

11. The method of claim 9 wherein the oligonucleotides are unlabelled.

12. The method of claim 6, wherein the primer nucleic acids are attached to a solid support.

13. The method according to claim 12 wherein the primer nucleic acids are attached to a solid support via a cleavable linkage.

14. The method of claim 3, wherein the fragments are removed from the support before determining the sequence of the first end of individual nucleic acid fragments.

15. The method of claim 14, wherein the step of determining the sequence of the first end of individual nucleic acid fragments is performed on a second solid support.

16. The method of claim 15, wherein the second solid support is a flow cell which enables the transfer of reagents to the surface of the support.

17. The method of claim 1, wherein the defined length of the fragments is such that at least 50% of the fragments have a defined length that varies by less than 5 nucleotides.

18. The method of claim 1, wherein the step of determining the sequence of the target nucleic acid comprises determining the relative locations of the first end sequences and the second end sequences in the sequence of the target nucleic acid based on the defined length of the individual nucleic acid fragments.

19. The method of claim 18, wherein the step of determining the sequence of the target nucleic acid comprises carrying out a sequence alignment wherein the relative locations of the first end and second end sequences are fixed based on the defined length of the individual nucleic acid fragments.

20. The method of claim 1 wherein the defined length is between 200-1000 bases.