PHASE-PROTECTING REAGENT FLOW ORDERINGS FOR USE IN SEQUENCING-BY-SYNTHESIS

• US 20120264621A1
• Filed: 04/05/2012
• Published: 10/18/2012
• Est. Priority Date: 04/08/2011
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: HUBBELL, Earl, SCHULTZ, Jonathan
• Granted Patent: US 9,428,807 B2
• Time in Patent Office: Days
• US Class Current: 506/4
• 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 2533/101 Primer extension
• CPC Class Codes: C12Q 2565/301 Pyrophosphate (PPi)
• CPC Class Codes: C12Q 2565/607 being a sensor, e.g. electrode

Abstract

A method for nucleic acid sequencing includes disposing template polynucleotide strands in defined spaces on a sensor array, at least some of the template polynucleotide strands having a sequencing primer and a polymerase operably bound therewith; exposing the template polynucleotide strands to a series of flows of nucleotide species flowed according to a predetermined ordering; and determining, for each of the series of flows of nucleotide species, how many nucleotide incorporations occurred for that particular flow to determine a predicted sequence of nucleotides corresponding to the template polynucleotide strands, wherein the predetermined ordering (a) is not a series of consecutive repetitions of a 4-flow permutation of four different nucleotide species, (b) is not specifically tailored to a particular combination of a particular template polynucleotide strand to be sequenced and a particular sequencing primer to be used, and (c) comprises a phase-protecting flow ordering.

Claims

1. A method for nucleic acid sequencing, comprising:

disposing a plurality of template polynucleotide strands in a plurality of defined spaces disposed on a sensor array, at least some of the template polynucleotide strands having a sequencing primer and a polymerase operably bound therewith;

exposing the template polynucleotide strands with the sequencing primer and a polymerase operably bound therewith to a series of flows of nucleotide species flowed according to a predetermined ordering; and

determining, for each of the series of flows of nucleotide species, how many nucleotide incorporations occurred for that particular flow to determine a predicted sequence of nucleotides corresponding to the template polynucleotide strands,

wherein the predetermined ordering (a) is not a series of consecutive repetitions of a 4-flow permutation of four different nucleotide species, (b) is not specifically tailored to a particular combination of a particular template polynucleotide strand to be sequenced and a particular sequencing primer to be used, and (c) comprises a phase-protecting flow ordering.

2. The method of claim 1, wherein the phase-protecting flow ordering comprises a de Bruijn sequence of four predetermined nucleotide species having a de Bruijn subsequence length parameter of two or three and without any consecutive repeats of the same nucleotide species.

3. The method of claim 2, wherein the de Bruijn subsequence length parameter is two.

4. The method of claim 2, wherein the de Bruijn subsequence length parameter is three.

5. The method of claim 1, wherein the predetermined ordering comprises a portion that is a de Bruijn sequence and a portion that is not a de Bruijn sequence.

6. The method of claim 1, wherein the four nucleotide species are A, C, G, and T and the predetermined ordering consists of only the phase-protecting flow ordering.

7. The method of claim 2, wherein the phase-protecting flow ordering comprising the de Bruijn subsequence ordering comprises a flow of T followed by a flow of A followed by a flow of C followed by a flow of G followed by a flow of T followed by a flow of C followed by a flow of T followed by a flow of G followed by a flow of A followed by a flow of G followed by a flow of C followed by a flow of A.

8. The method of claim 1, wherein the phase-protecting flow ordering comprises a flow ordering that includes all possible distinct dimer pairs of four nucleotide species.

9. The method of claim 1, wherein the phase-protecting flow ordering comprises a flow of N followed immediately by a flow of X, where X and N represent different nucleotide species, and further comprises, immediately thereafter or elsewhere in the ordering, a flow of N followed immediately by a flow of Y, where Y represents a nucleotide species different from both X and N.

10. The method of claim 1, wherein the phase-protecting flow ordering comprises a flow of X followed immediately by a flow of N, where X and N represent different nucleotide species, and further comprises a flow of Y followed immediately by a flow of N, where Y represents a nucleotide species different from both X and N.

11. The method of claim 1, wherein the phase-protecting flow ordering comprises a first flow ordering in which a first nucleotide species, a second nucleotide species, and a third nucleotide species are flowed at least twice before a fourth nucleotide species is flowed.

12. The method of claim 11, wherein the phase-protecting flow ordering further comprises a second flow ordering in which the second nucleotide species, the third nucleotide species, and the fourth nucleotide species are flowed at least twice before the first nucleotide species is flowed.

13. The method of claim 12, wherein the phase-protecting flow ordering further comprises a third flow ordering in which the first nucleotide species, the third nucleotide species, and the fourth nucleotide species are flowed at least twice before the second nucleotide species is flowed.

14. The method of claim 13, wherein the phase-protecting flow ordering further comprises a fourth flow ordering in which the first nucleotide species, the second nucleotide species, and the fourth nucleotide species are flowed at least twice before the third nucleotide species is flowed.

15. The method of claim 1, wherein the phase-protecting flow ordering comprises a first set of flows and a second set of flows, the second set of flows being derived from a remapping of two or more of four nucleotide species flowed in the first set of flows.

16. The method of claim 15, wherein the second set of flows is derived from a remapping of all four of the nucleotide species in the first set of flows.

17. The method of claim 1, wherein the phase-protecting flow ordering comprises a flow ordering in which at least one given nucleotide species is contiguously flowed two or more times.

18. The method of claim 1, wherein the predetermined ordering is selected based on an assessment of dephasing merit and efficiency of extension calculated for a plurality of candidate flow orderings using simulation sequencing data obtained for a plurality of random test sequences.

19. The method of claim 1, wherein the sensor array is configured to detect hydrogen ions released by incorporation of nucleotides.

20. The method of claim 1, wherein the sensor array is configured to detect inorganic pyrophosphate released by incorporation of nucleotides.

21. A system for nucleic acid sequencing, comprising:

a machine-readable memory; and

a processor configured to execute machine-readable instructions, which, when executed by the processor, cause the system to perform steps including;

exposing a plurality of template polynucleotide strands in a plurality of defined spaces disposed on a sensor array, at least some of the template polynucleotide strands having a sequencing primer and a polymerase operably bound therewith, to a series of flows of nucleotide species flowed according to a predetermined ordering; and

determining, for each of the series of flows of nucleotide species, how many nucleotide incorporations occurred for that particular flow to determine a predicted sequence of nucleotides corresponding to the template polynucleotide strands,

wherein the predetermined ordering (a) is not a series of consecutive repetitions of a 4-flow permutation of four different nucleotide species, (b) is not specifically tailored to a particular combination of a particular template polynucleotide strand to be sequenced and a particular sequencing primer to be used, and (c) comprises a phase-protecting flow ordering.

22. The system of claim 21, wherein the phase-protecting flow ordering comprises a de Bruijn sequence of four predetermined nucleotide species having a de Bruijn subsequence length parameter of two or three and without any consecutive repeats of the same nucleotide species.

23. The system of claim 21, wherein the phase-protecting flow ordering comprises a flow ordering that includes all possible distinct dimer pairs of four nucleotide species.

24. The system of claim 21, wherein the phase-protecting flow ordering comprises a flow of N followed immediately by a flow of X, where X and N represent different nucleotide species, and further comprises, immediately thereafter or elsewhere in the ordering, a flow of N followed immediately by a flow of Y, where Y represents a nucleotide species different from both X and N.

25. The system of claim 21, wherein the phase-protecting flow ordering comprises a flow of X followed immediately by a flow of N, where X and N represent different nucleotide species, and further comprises a flow of Y followed immediately by a flow of N, where Y represents a nucleotide species different from both X and N.

26. The system of claim 21, wherein the phase-protecting flow ordering comprises a first flow ordering in which a first nucleotide species, a second nucleotide species, and a third nucleotide species are flowed at least twice before a fourth nucleotide species is flowed.

27. The system of claim 26, wherein the phase-protecting flow ordering further comprises a second flow ordering in which the second nucleotide species, the third nucleotide species, and the fourth nucleotide species are flowed at least twice before the first nucleotide species is flowed.

28. The system of claim 27, wherein the phase-protecting flow ordering further comprises a third flow ordering in which the first nucleotide species, the third nucleotide species, and the fourth nucleotide species are flowed at least twice before the second nucleotide species is flowed.

29. The system of claim 28, wherein the phase-protecting flow ordering further comprises a fourth flow ordering in which the first nucleotide species, the second nucleotide species, and the fourth nucleotide species are flowed at least twice before the third nucleotide species is flowed.

30. The system of claim 21, wherein the phase-protecting flow ordering comprises a first set of flows and a second set of flows, the second set of flows being derived from a remapping of two or more of four nucleotide species flowed in the first set of flows.

31. The system of claim 21, wherein the phase-protecting flow ordering comprises a flow ordering in which at least one given nucleotide species is contiguously flowed two or more times.

32. The system of claim 21, wherein the predetermined ordering is selected based on an assessment of dephasing merit and efficiency of extension calculated for a plurality of candidate flow orderings using simulation sequencing data obtained for a plurality of random test sequences.

33. The system of claim 21, wherein the sensor array is configured to detect hydrogen ions released by incorporation of nucleotides.

34. The system of claim 21, wherein the sensor array is configured to detect inorganic pyrophosphate released by incorporation of nucleotides.

35. A non-transitory machine-readable storage medium comprising instructions which, when executed by a processor, cause the processor to perform a method for nucleic acid sequencing comprising:

exposing a plurality of template polynucleotide strands in a plurality of defined spaces disposed on a sensor array, at least some of the template polynucleotide strands having a sequencing primer and a polymerase operably bound therewith, to a series of flows of nucleotide species flowed according to a predetermined ordering; and

determining, for each of the series of flows of nucleotide species, how many nucleotide incorporations occurred for that particular flow to determine a predicted sequence of nucleotides corresponding to the template polynucleotide strands,

wherein the predetermined ordering (a) is not a series of consecutive repetitions of a 4-flow permutation of four different nucleotide species, (b) is not specifically tailored to a particular combination of a particular template polynucleotide strand to be sequenced and a particular sequencing primer to be used, and (c) comprises a phase-protecting flow ordering.