Phase-protecting reagent flow orderings for use in sequencing-by-synthesis

• US 9,428,807 B2
• Filed: 04/05/2012
• Issued: 08/30/2016
• 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
• Primary Examiner(s): Chunduru, Prabha
• Application Number:
  US13/440,849
• Publication Number:
  US 20120264621A1
• Time in Patent Office: 1,608 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 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 the 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 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 that comprises a de Bruijn sequence of nucleotide species A, C, G, and T such that;

(i) every possible subsequence of two of the nucleotide species A, C, G, and T appears once as a sequence of consecutive nucleotide species in the de Bruijn sequence or

(ii) every possible subsequence of three of the nucleotide species A, C, G, and T appears once as a sequence of consecutive nucleotide species in the de Bruijn sequence.

2. The method of claim 1, wherein the de Bruijn sequence does not include any consecutive repeats of the same nucleotide species.

3. The method of claim 1, wherein the de Bruijn sequence is such that every possible subsequence of two of the nucleotide species A, C, G, and T appears once as a sequence of consecutive nucleotide species in the de Bruijn sequence.

4. The method of claim 1, wherein the de Bruijn sequence is such that every possible subsequence of three of the nucleotide species A, C, G, and T appears once as a sequence of consecutive nucleotide species in the de Bruijn sequence.

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 predetermined ordering consists of only the phase-protecting flow ordering.

7. The method of claim 1, wherein the phase-protecting flow ordering comprising the de Bruijn sequence 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 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.

9. The method of claim 8, wherein the second set of flows is derived from a remapping of all four of the nucleotide species in the first set of flows.

10. 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.

11. 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.

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

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