Method for sequencing a polynucleotide template

• US 10,036,063 B2
• Filed: 06/30/2010
• Issued: 07/31/2018
• Est. Priority Date: 07/24/2009
• Status: Active Grant

• Current Assignee: Illumina Incorporated
• Original Assignee: Illumina Incorporated
• Inventors: West, John Stephen
• Primary Examiner(s): Sisson, Bradley L.
• Application Number:
  US13/384,302
• Publication Number:
  US 20120165205A1
• Time in Patent Office: 2,953 Days
• Field of Search: None
• 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 preparing a library of nucleic acid fragments for sequence analysis, comprising:

(a) cleaving a target nucleic acid to produce a plurality of double-stranded cleavage products, wherein the target nucleic acid comprises human genomic DNA;

(b) ligating a first universal adapter to the double-stranded cleavage products to form a plurality of double-stranded templates having known ends, wherein the first universal adapter comprises a region of double-stranded sequence for ligation to the double-stranded cleavage products, and a first universal adapter sequence;

(c) denaturing the double-stranded templates to produce a plurality of single-stranded templates comprising the first universal adapter sequence;

(d) annealing the single-stranded templates to a population of complementary primer nucleic acids attached to a solid support;

(e) performing a defined number of at least 50 incremental extension cycles, each extension cycle comprising;

(i) extending each of the primer nucleic acids by incorporating an unlabeled nucleotide complementary to the annealed template and comprising a reversible blocking moiety; and

(ii) contacting the incorporated nucleotide with a deblocking agent to remove the blocking moiety;

thereby producing a population of extended primers comprising different portions of the target nucleic acid, wherein the individual extended primers in the population comprise a defined length that is correlated with the number of incremental extension cycles;

(f) subjecting the annealed templates to denaturing conditions to remove the templates from the solid support; and

(g) ligating a second universal adapter sequence to the 3′

ends of the extended primers to produce a population of immobilized nucleic acid fragments having universal adapter sequences at both ends;

thereby preparing a library of nucleic acid fragments for sequence analysis.

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 according to claim 1, wherein the primer nucleic acids attached to the solid support comprise a cleavable linkage.

4. The method of claim 1, further comprising:

(h) removing the nucleic acid fragments having a defined length and universal adapter sequences at both ends from the solid support.

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

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

7. The method of claim 1, wherein the number of incremental extension cycles performed in step (e) is between 100-1000 cycles.

8. The method of claim 1, wherein the first universal adapter comprises a region of single-stranded sequence comprising the first universal adapter sequence.

9. A method of preparing a library of nucleic acid fragments for sequence analysis, comprising:

(a) cleaving a target nucleic acid to produce a plurality of double-stranded cleavage products, wherein the target nucleic acid comprises human genomic DNA;

(b) ligating a single-stranded first universal adapter to the 3′

ends of the double-stranded cleavage products to form a plurality of double-stranded templates having known 3′

ends, wherein the first universal adapter comprises a first universal adapter sequence;

(c) denaturing the double-stranded templates to produce a plurality of single-stranded templates comprising the first universal adapter sequence;

(d) annealing the single-stranded templates to a population of complementary primer nucleic acids attached to a solid support;

(e) performing a defined number of at least 50 incremental extension cycles, each extension cycle comprising;

(i) extending each of the primer nucleic acids by incorporating an unlabeled nucleotide complementary to the annealed template and comprising a reversible blocking moiety; and

(ii) contacting the incorporated nucleotide with a deblocking agent to remove the blocking moiety;

thereby producing a population of extended primers comprising different portions of the target nucleic acid, wherein the individual extended primers in the population comprise a defined length that is correlated with the number of incremental extension cycles;

(f) subjecting the annealed templates to denaturing conditions to remove the templates from the solid support; and

(g) ligating a second universal adapter sequence to the 3′

ends of the extended primers to produce a population of immobilized nucleic acid fragments having universal adapter sequences at both ends;

thereby preparing a library of nucleic acid fragments for sequence analysis.

10. The method of claim 9, wherein individual cycles in the incremental extension cycles comprise a wash step to remove an unreacted reagent.

11. The method of claim 9, wherein the primer nucleic acids attached to the solid support comprise a cleavable linkage.

12. The method of claim 9, further comprising:

(h) removing the nucleic acid fragments having a defined length and universal adapter sequences at both ends from the solid support.

13. The method of claim 9, wherein the defined length of the immobilized nucleic acid fragments is such that at least 50% of the immobilized nucleic acid fragments have a defined length that varies by less than 5 nucleotides.

14. The method of claim 9, wherein the defined length is between 200-1000 bases.

15. The method of claim 9, wherein the number of incremental extension cycles is between 100-1000 cycles.