Methods and compositions for nucleic acid sequencing

• US 9,453,258 B2
• Filed: 09/21/2012
• Issued: 09/27/2016
• Est. Priority Date: 09/23/2011
• Status: Active Grant

• Current Assignee: Illumina Incorporated
• Original Assignee: Illumina Incorporated
• Inventors: Joseph, Thomas, Tregidgo, Carolyn, Kain, Robert C., Liu, Xiaohai, Feng, Wenyi, Hirschbein, Bernard, Eltoukhy, Helmy A., Wu, Xiaolin, Smith, Geoffrey Paul, Boutell, Jonathan Mark, Smith, Randall, Shen, Min-Jui Richard, Klausing, Kay
• Primary Examiner(s): Priest, Aaron
• Application Number:
  US13/624,200
• Publication Number:
  US 20130079232A1
• Time in Patent Office: 1,467 Days
• Field of Search: None
• US Class Current: 1/1
• 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, comprising

(a) performing a sequencing reaction that comprises at least 50 repeated cycles of

(i) polymerase catalyzed incorporation of different types of nucleotide conjugates into a plurality of polynucleotides, wherein the different types of nucleotide conjugates comprise reversible terminator moieties, wherein the different types of nucleotide conjugates are simultaneously present and compete for incorporation into the polynucleotides during the polymerase catalyzed incorporation, and

(ii) cleaving the reversible terminator moieties from nucleotide conjugates that are incorporated into the polynucleotides;

(b) detecting signals in the sequencing reaction for the incorporation of three different types of the nucleotide conjugates into the polynucleotides, wherein signals are not detected for a fourth type of the nucleotide conjugates incorporated into the polynucleotides; and

(c) determining the incorporation of the fourth type of the nucleotide conjugates based on the detection pattern of the three different types of the nucleotide conjugates into the polynucleotides,

thereby determining sequences comprising at least 50 sequential nucleotides of the polynucleotides, wherein the incorporation of the three different types of the nucleotide conjugates is determined from a signal state and wherein the incorporation of the fourth type of the nucleotide conjugates is determined from a dark state.

2. The method of claim 1, wherein the nucleotide conjugates comprise nucleotide types selected from the group consisting of dATP, dTTP, dUTP, dCTP, dGTP or non-natural nucleotide analogs thereof.

3. The method of claim 1, wherein the three different types of the nucleotide conjugates are each attached to a fluorescent moiety that produces the signals that are detected in the sequencing reaction.

4. The method of claim 3, wherein the fluorescent moiety is the same for the three different types of the nucleotide conjugates.

5. The method of claim 3, wherein one or more different fluorescent moieties are detected by one or more emission filters.

6. The method of claim 3, wherein the fourth type of the nucleotide conjugates is not attached to a fluorescent moiety that produces the signals that are detected in the sequencing reaction.

7. The method of claim 6, wherein a fluorescence detection pattern for the three different types of the nucleotide conjugates is determined by a first and a second imaging event.

8. The method of claim 7, wherein the first imaging event detects a pattern of fluorescence that is different from the pattern of fluorescence detected by the second imaging event.

9. The method of claim 8, wherein the incorporation of one or more of the different types of nucleotide conjugates is determined by the difference in the pattern of fluorescence between the first and second imaging events.

10. The method of claim 3, wherein the fluorescent moiety is different for different types of the nucleotide conjugates.

11. The method of claim 1, wherein the signals comprise images from an array of the polynucleotides that is detected in the sequencing reaction.

12. The method of claim 1, wherein a first type of the three different types of the nucleotide conjugates comprises a donor moiety and acceptor moiety of a fluorescent resonance energy transfer system.

13. The method of claim 12, wherein individual cycles of the sequencing reaction comprise removing the donor or the acceptor from the first type of the three different types of the nucleotide conjugates.

14. The method of claim 1, wherein the detecting of the signals comprises:

(i) detecting a first collection of signals from the three different types of the nucleotide conjugates that are incorporated into the plurality of polynucleotides;

(ii) modifying at least one of the different types of nucleotide conjugates that is incorporated into the plurality of polynucleotides, thereby producing modified polynucleotides; and

(iii) detecting a second collection of signals from the modified polynucleotides, wherein at least one of the different types of nucleotide conjugates is in different state in the first collection of signals compared to the second collection of signals.

15. The method of claim 14, wherein the modifying comprises removing a label from the at least one of the different types of nucleotide conjugates.

16. The method of claim 14, wherein the modifying comprises adding a label to the at least one of the different types of nucleotide conjugates.

17. The method of claim 14, wherein the modifying comprises chemically modifying at least one of the different types of nucleotide conjugates.

18. The method of claim 14, wherein the modifying comprises photo-chemically modifying at least one of the different types of nucleotide conjugates.

19. The method of claim 14, wherein the modifying comprises binding a receptor to recruit a label to at least one of the different types of nucleotide conjugates.

20. The method of claim 1, wherein the signals comprise fluorescence from the three different types of the nucleotide conjugates.

21. 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.

22. A method for determining sequences of polynucleotides, comprising

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

(i) polymerase catalyzed incorporation of different types of nucleotide conjugates into a plurality of polynucleotides, wherein the different types of nucleotide conjugates comprise reversible terminator moieties, wherein the different types of nucleotide conjugates are simultaneously present and compete for incorporation into the polynucleotides during the polymerase catalyzed incorporation, and

(ii) cleaving the reversible terminator moieties from nucleotide conjugates that are incorporated into the polynucleotides;

(b) detecting signals in the sequencing reaction for the incorporation of three different types of the nucleotide conjugates into the polynucleotides, wherein the detecting of the signals comprises;

(i) detecting a first type of the three different types of the nucleotide conjugates in a first channel;

(ii) detecting a second type of the three different types of the nucleotide conjugates in a second channel; and

(iii) detecting a third type of the three different types of the nucleotide conjugates in the first channel and in the second channel,

wherein the third type of the three different types of the nucleotide conjugates comprises a mixture of different nucleotide conjugates that differ by being attached to a first fluorophore that is detected in the first channel or a second fluorophore that is detected in the second channel,

wherein signals are not detected for a fourth type of the nucleotide conjugates incorporated into the polynucleotides; and

(c) determining the incorporation of the fourth type of the nucleotide conjugates based on the detection pattern of the three different types of the nucleotide conjugates into the polynucleotides,

thereby determining the sequences of the polynucleotides, wherein the incorporation of the three different types of the nucleotide conjugates is determined from a signal state and wherein the incorporation of the fourth type of the nucleotide conjugates is determined from a dark state.

23. The method of claim 22, wherein the first type of the three different types of the nucleotide conjugates comprises nucleotide conjugates attached to the first fluorophore and the second type of the three different types of the nucleotide conjugates comprises nucleotide conjugates attached to the second fluorophore.

24. The method of claim 23, wherein the fourth type of the nucleotide conjugates is not attached to a fluorescent moiety that produces the signals that are detected in the sequencing reaction.

25. The method of claim 22, wherein the signals comprise images from an array of the polynucleotides that is detected in the sequencing reaction.

26. The method of claim 22, 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.