Method for sequencing a polynucelotide template

• US 8,541,172 B2
• Filed: 04/12/2012
• Issued: 09/24/2013
• Est. Priority Date: 09/15/2009
• Status: Active Grant

• Current Assignee: Illumina Cambridge Ltd. (California) (Illumina Incorporated)
• Original Assignee: Illumina Cambridge Ltd. (California) (Illumina Incorporated)
• Inventors: Boutell, Jonathan Mark, Kersey, Alan D.
• Primary Examiner(s): Horlick, Kenneth R.
• Assistant Examiner(s): THOMAS, DAVID C
• Application Number:
  US13/445,072
• Publication Number:
  US 20120258869A1
• Time in Patent Office: 530 Days
• Field of Search: None
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2565/543 characterised by the use of two or more capture oligonucleotide primers in concert, e.g. bridge amplification

Abstract

Improved compositions, methods, apparatus, and kits for high-throughput nucleic acid amplification, detection and sequencing are disclosed. A nucleic acid cluster having an identifiable center is produced by generating on a solid support an immobilized nucleic acid complement from a template, one of which comprises a detectable label; and amplifying the complement and the template to obtain a nucleic acid cluster on the support, the cluster having a substantially central location marked by the detectable label and a surrounding region comprising immobilized copies. Also disclosed are nucleotide sequence determination in nucleic acid clusters so produced, center position annotation in the clusters, assignment of sequence information to overlapping clusters, and related compositions and methods.

Claims

1. A method for sequencing nucleic acids, comprising

(a) attaching a plurality of different nucleic acids to a surface;

(b) amplifying the different nucleic acids on the surface to form a plurality of clusters on the surface, wherein clusters in the plurality of clusters abut or overlap with each other on the surface, wherein each of the clusters comprises copies of a single nucleic acid from the plurality of different nucleic acids; and

(c) determining the sequences of the amplified copies in the clusters, wherein the sequences are determined by a method comprising;

(i) obtaining images of overlapping signals emanating from the clusters that abut or overlap with each other on the surface, and

(ii) processing the images to assign nucleic acid sequence information to a single cluster, wherein one or more nucleic acids in a cluster are distinguished from surrounding nucleic acids in the same cluster.

2. The method of claim 1, wherein the surface is contained in a flow cell.

3. The method of claim 1, wherein the amplifying in (b) is isothermal amplification.

4. The method of claim 1, wherein the diameter of each of the clusters is between 0.2 μ

m to 6 μ

m.

5. The method of claim 1, wherein the density of the plurality of clusters is in the range from 100,000 clusters per mm² to 10,000,000 clusters per mm².

6. The method of claim 1, wherein the density of the plurality of clusters is in the range from 1,000,000 clusters per mm² to 10,000,000 clusters per mm².

7. The method of claim 1, wherein the clusters comprise a circular shape.

8. The method of claim 1, wherein the clusters comprise a shape other than a circular shape.

9. The method of claim 1, wherein the amplifying uses only one species of oligonucleotide primers immobilized on the surface.

10. The method of claim 1, wherein the amplifying uses first and second different species of oligonucleotide primers immobilized on the surface.

11. The method of claim 1, wherein the image detects 100,000,000 or more clusters simultaneously.

12. The method of claim 1, wherein the one or more nucleic acids in the cluster that are distinguished from the surrounding nucleic acids in the same cluster comprise the identifiable center of the cluster.

13. The method of claim 1, wherein the determining of the sequences comprises determining a reference position in each of the clusters, wherein the reference position distinguishes the signals detected from the clusters that abut or overlap with each other.

14. The method of claim 13, wherein the reference position of each of the clusters is surrounded by a region of the respective cluster.

15. The method of claim 13, wherein the reference position of each of the clusters comprises the identifiable center of the cluster.

16. The method of claim 1, wherein the determining of the sequences comprises sequencing by synthesis.

17. The method of claim 1, wherein the determining of the sequences comprises sequencing by ligation.

18. The method of claim 1, wherein the determining of the sequences comprises repeated cycles of the obtaining of the images and the processing of the images.

19. The method of claim 1, wherein the signals comprise fluorescent signals.

20. The method of claim 1, wherein each of the clusters comprises at least 500 copies of a single nucleic acid from the plurality of different nucleic acids.

21. The method of claim 1, wherein a plurality of oligonucleotide primers is immobilized on the surface.

22. The method of claim 21, wherein the attaching comprises hybridizing the different nucleic acids to the oligonucleotide primers that are immobilized on the surface.

23. The method of claim 22, wherein the plurality of different nucleic acids comprise universal sequences that hybridize to the oligonucleotide primers that are immobilized on the surface.

24. The method of claim 23, wherein the universal sequences are present at the 5′

ends of the different nucleic acids.

25. The method of claim 23, wherein the universal sequences are present at the 3′

ends of the different nucleic acids.

26. An automated system comprising a processor and a non-transitory computer readable medium comprising instructions for

(a) attaching a plurality of different nucleic acids to a surface;

(b) amplifying the different nucleic acids on the surface to form a plurality of clusters on the surface, wherein clusters in the plurality of clusters abut or overlap with each other on the surface, wherein each of the clusters comprises copies of a single nucleic acid from the plurality of different nucleic acids; and

(c) determining the sequences of the amplified copies in the clusters, wherein the sequences are determined by a method comprising

(i) obtaining images of overlapping signals emanating from the clusters that abut or overlap with each other on the surface, and

(ii) processing the image to assign nucleic acid sequence information to a single cluster, wherein one or more nucleic acids in a cluster are distinguished from surrounding nucleic acids in the same cluster.