Methods and compositions for nucleic acid sequencing

• US 10,287,629 B2
• Filed: 07/12/2016
• Issued: 05/14/2019
• Est. Priority Date: 09/23/2011
• Status: Active Grant

• Current Assignee: Illumina Incorporated
• Original Assignee: Illumina Incorporated
• Inventors: Kain, Robert C., Liu, Xiaohai, Feng, Wenyi, Hirschbein, Bernard, Eltoukhy, Helmy A., Wu, Xiaolin, Smith, Geoffrey Paul, Boutell, Jonathan Mark, Joseph, Thomas, Smith, Randall, Shen, Min-Jui Richard, Tregidgo, Carolyn, Klausing, Kay
• Primary Examiner(s): Priest, Aaron A
• Application Number:
  US15/207,892
• Publication Number:
  US 20170022559A1
• Time in Patent Office: 1,036 Days
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2525/113 incorporating modified backbone
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin

Abstract

The present disclosure provides methods and systems for detecting multiple different nucleotides in a sample. In particular, the disclosure provides for detection of multiple different nucleotides in a sample utilizing fewer detection moieties than the number of nucleotides being detected and/or fewer imaging events than the number of nucleotides being detected.

Claims

1. A method for determining sequences of polynucleotides, the method comprising:

(a) performing a sequencing reaction that comprises repeated cycles of;

(i) incorporating nucleotide conjugates into a plurality of polynucleotides to produce extended polynucleotides,

(ii) detecting a first collection of signals from the extended polynucleotides, wherein the first collection of signals comprises signals from a first label attached to a first type of the nucleotide conjugates and a second label attached to a second type of the nucleotide conjugates,

(iii) adding a third label to a third type of the nucleotide conjugates comprising a hapten that are incorporated into the extended polynucleotides, thereby producing modified polynucleotides; and

(iv) detecting a second collection of signals from the modified polynucleotides, wherein the second collection of signals comprises signals from the third label added to the third type of the nucleotide conjugates; and

(b) determining incorporation of a fourth type of the nucleotide conjugates based on a detection pattern of the first, second and third types of the nucleotide conjugates into the extended polynucleotides, wherein the incorporation of the fourth type of the nucleotide conjugates is determined from a dark state in the first and second collection of signals.

2. The method of claim 1, wherein the sequencing reaction further comprises polymerase catalyzed incorporation of the nucleotide conjugates into the plurality of polynucleotides.

3. The method of claim 2, wherein the nucleotide conjugates are simultaneously present and compete for incorporation into the polynucleotides during the polymerase catalyzed incorporation.

4. The method of claim 3, wherein the nucleotide conjugates comprise reversible terminator moieties, and wherein the sequencing reaction further comprises cleaving the reversible terminator moieties from nucleotide conjugates that are incorporated into the polynucleotides.

5. The method of claim 1, wherein the first and second labels are identical.

6. The method of claim 5, wherein the first, second and third labels are identical.

7. The method of claim 6, wherein the first, second and third labels comprise the same fluorescent moieties.

8. The method of claim 1, wherein the first and second labels are different.

9. The method of claim 8, wherein the first, second and third labels are different.

10. The method of claim 9, wherein the first collection of signals and the second collection of signals comprise similar signals produced by the different first, second and third labels.

11. The method of claim 9, wherein the different first, second and third labels comprise different fluorescent moieties.

12. The method of claim 10, wherein the different fluorescent moieties are detected using the same emission filter.

13. The method of claim 10, wherein the different fluorescent moieties are detected using the same excitation source.

14. The method of claim 6, wherein the fourth type of the nucleotide conjugates is not the label that is attached to the first, second and third type of the nucleotide conjugates.

15. The method of claim 1, wherein the plurality of polynucleotides is attached to a substrate.

16. The method of claim 15, wherein the detecting of the first and second collections of signals comprises obtaining images of the substrate.

17. The method of claim 1, wherein step (a)(iii) further comprises removing the second label attached to the second type of the nucleotide conjugates.

18. The method of claim 1, wherein the adding of the third label comprises binding a labeled receptor to the hapten of the third type of the nucleotide conjugates that are incorporated into the extended polynucleotides.

19. The method of claim 1, wherein the sequencing reaction comprises at least 100 of the repeated cycles, and wherein the sequences that are determined comprise at least 100 nucleotides of the polynucleotides.