METHODS AND COMPOSITIONS FOR NUCLEIC ACID SEQUENCING

• US 20130079232A1
• Filed: 09/21/2012
• Published: 03/28/2013
• Est. Priority Date: 09/23/2011
• Status: Active Grant

• Current Assignee: Illumina Incorporated
• Original Assignee: Illumina Incorporated
• Inventors: FENG, WENYI, HIRSCHBEIN, BERNARD, SHEN, MIN-JUI RICHARD, ELTOUKHY, HELMY A., BOUTELL, JONATHAN MARK, JOSEPH, THOMAS, LIU, XIAOHAI, SMITH, GEOFFREY PAUL, TREGIDGO, CAROLYN, KLAUSING, KAY, WU, XIAOLIN, KAIN, ROBERT C., SMITH, RANDALL
• Granted Patent: US 9,453,258 B2
• Time in Patent Office: Days
• US Class Current: 506/2
• 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 the sequence of a polynucleotide comprising detecting in a sequencing reaction the incorporation of three different types of detectable nucleotide conjugates into a polynucleotide and determining the incorporation of a fourth type of nucleotide based on the detection pattern of the three different types of detectable nucleotides into the polynucleotide thereby determining the sequence of a polynucleotide, wherein the incorporation of three different types of detectable nucleotide conjugates is detected from a signal state and wherein the incorporation of the fourth type of nucleotide is determined from a dark state.

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

3. The method of claim 2, where the non-natural nucleotide analog comprises a reversible terminator moiety and is selected from the group consisting of rbATP, rbTTP, rbUTP, rbCTP and rbGTP.

4. The method of claim 1, wherein the three nucleotide conjugates are detected by detecting a fluorescent moiety.

5. The method of claim 4, wherein the fluorescent moiety is the same for the three nucleotide conjugates or wherein the fluorescent moiety is one or more different fluorescent moieties.

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

7. The method of claim 5, wherein the incorporation of the fourth nucleotide is determined by the fluorescent detection pattern of the fluorescent moiety.

8. The method of claim 7, wherein the fluorescence detection pattern is determined by a first and a second imaging event.

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

10. The method of claim 9, wherein the incorporation of one or more nucleotides is determined by the difference in the pattern of fluorescence between the first and second imaging events.

11. The method of claim 1, wherein the one or more nucleotide type conjugates further comprise one or more linker sequences.

12. The method of claim 11, wherein the one or more linker sequences comprises one or more of a cleavable linker and a spacer linker wherein the cleavable linker comprises one or more cleavable linkage groups selected from the group consisting of a disulfide, a diol, a diazo, an ester, a sulfone, an azide, an alyl and a silyl ether.

13. The method of claim 12, wherein the spacer linker is one or more of polyethylene glycol or concatamers thereof and 2-{2-[3-(2-amino-ethylcarbornyl)-phenoxy]-1-azido-ethoxy}-ethoxy-acetic acid.

14. The method of claim 12, wherein the one or more spacer linkers further comprise one or more cleavable linkage groups wherein the cleavable linkage group is selected from the group consisting of a disulfide, a diol, a diazo, an ester, a sulfone, an azide, an alyl and a silyl ether.

15. The method of claim 13, wherein the one or more nucleotide conjugates comprise a polyethylene glycol linker and a 2-{2-[3-(2-amino-ethylcarbornyl)-phenoxy]-1-azido-ethoxy}-ethoxy-acetic acid linker.

16. The method of claim 15, wherein the nucleotide conjugate comprising a polyethylene glycol linker and a 2-{2-[3-(2-amino-ethylcarbornyl)-phenoxy]-1-azido-ethoxy}-ethoxy-acetic acid linker further comprises a hapten and a fluorescent moiety.

17. The method of claim 16, wherein the hapten is selected from the group consisting of biotin, digoxigenin and dinitrophenol.

18. The method of claim 17, wherein the one or more nucleotide conjugates comprises a streptavidin-fluorescent moiety conjugate.

19. A method for determining the sequence of a polynucleotide comprising:

a) applying to a polynucleotide sample for sequencing a solution comprising four modified nucleotide types wherein three modified nucleotide types are conjugated to one or more detection moieties and one or more linkers positioned between the nucleotide and the one or more detection moieties, and wherein a fourth nucleotide type lacks a detection moiety,

b) detecting a pattern of incorporation of said modified nucleotides in a sequencing reaction thereby capturing a first detectable pattern,

c) applying one or more compositions to the sequencing reaction thereby changing the first detectable pattern,

d) detecting a second detectable pattern, and

e) determining the sequence of the polynucleotide sample based on the detectable patterns.

20. The method of claim 19, wherein the modified nucleotide types are selected from the list comprising rbATP, rbTTP, rbUTP, rbCTP and rbGTP.

21. The method of claim 19, wherein the one or more detection moieties is a fluorescent moiety.

22. The method of claim 21, wherein the fluorescent moiety is the same or the fluorescent moiety is one or more different fluorescent moieties.

23. The method of claim 22, wherein the one or more different fluorescent moieties are detected by one or more emission filters.

24. The method of claim 19, wherein said detecting is an imaging event and wherein the detectable patterns are fluorescent patterns.

25. The method of claim 24, wherein incorporation of the fourth nucleotide type is determined by the fluorescence detection patterns of the other three nucleotide types in both the first and second detectable patterns.

26. The method of claim 19, wherein the one or more nucleotide type conjugates further comprise one or more linker sequences.

27. The method of claim 26, wherein the one or more linkers comprise one or more of a cleavable linker and a spacer linker.

28. The method of claim 27, wherein the cleavable linker comprises one or more cleavable linkage groups from the group consisting of a disulfide, a diol, a diazo, an ester, a sulfone, an azide, an alyl and a silyl ether.

29. The method of claim 27, wherein the spacer linker is one or more of polyethylene glycol or concatamers thereof and 2-{2-[3-(2-amino-ethylcarbornyl)-phenoxy]-1-azido-ethoxy}-ethoxy-acetic acid.

30. The method of claim 29, wherein the one or more spacer linkers further comprise one or more cleavable linkage groups.

31. The method of claim 30, wherein the cleavable linkage group is selected from the group consisting of a disulfide, a diol, a diazo, an ester, a sulfone, an azide, an alyl and a silyl ether.

32. The method of claim 29, wherein the one or more nucleotide type conjugates comprise a polyethylene glycol linker and a 2-{2-[3-(2-amino-ethylcarbornyl)-phenoxy]-1-azido-ethoxy}-ethoxy-acetic acid linker.

33. The method of claim 32, wherein the nucleotide type conjugate comprising a polyethylene glycol linker and a 2-{2-[3-(2-amino-ethylcarbornyl)-phenoxy]-1-azido-ethoxy}-ethoxy-acetic acid linker further comprises a hapten and a fluorescent moiety.

34. The method of claim 33, wherein the hapten is selected from the group consisting of biotin, digoxigenin and dinitrophenol.

35. The method of claim 34, wherein said detecting further comprises a streptavidin-fluorescent moiety conjugate.

36. The method of claim 19, wherein the one or more compositions added to a sequencing reaction thereby changing the first detectable pattern comprises adding one or more of a cleavage compound and a binding partner-detection moiety conjugate.

37. The method of claim 36, wherein the cleavage compound is a reducing agent comprising one or more compositions selected from the group consisting of dithioerythritol, dithiothreitol, 2-mercaptoethanol, glutathione, thioglycolic acid, 2,3-dimercaptopropanol and tris(2-carboxyethyl)phosphine.

38. The method of claim 36, wherein the binding partner-detection moiety conjugate is selected from the group consisting of a streptavidin-fluorescent moiety conjugate, an anti-digoxigenin-fluorescent moiety conjugate and an anti-dinitrophenol-fluorescent moiety conjugate.

39. A composition for sequencing a nucleic acid comprising three different modified nucleotide type conjugates comprising a fluorescent moiety and a fourth different modified nucleotide type, wherein said fourth different modified nucleotide type lacks a fluorescent moiety, and wherein the incorporation of the four modified nucleotide types in the composition into a sequencing reaction is determined by the fluorescent detection of the three different modified nucleotide type conjugates comprising a fluorescent moiety in the composition.

40. The composition of claim 39, wherein the fluorescent moiety is the same for the three modified nucleotides or the fluorescent moiety is one or more different fluorescent moieties.

41. A method for determining the sequence of a polynucleotide comprising the composition of claim 39, comprising:

a) detecting by one or more imaging events the incorporation of the three different types of detectable nucleotide type conjugates into a polynucleotide and

b) determining the incorporation of the fourth type of nucleotide based on the detection pattern of the three different types of detectable nucleotide type conjugates into a polynucleotide, wherein detecting comprises fewer imaging events than different types of nucleotides.

42. The method of claim 41, wherein the fluorescent moiety is the same for the three nucleotide conjugates or the fluorescent moiety is one or more different fluorescent moieties.

43. The method of claim 42, wherein the incorporation of the fourth nucleotide is determined by lack of detection.

44. The method of claim 41, wherein said imaging events comprise a first and a second imaging event.

45. The method of claim 44, wherein the first imaging event detects a pattern of fluorescence that is different from the pattern of fluorescence detected by the second imaging event.

46. The method of claim 45, wherein the incorporation of one or more nucleotides is determined by the difference in the pattern of fluorescence between the first and second imaging events.