# US 10,370,708 B2 · Phase-protecting reagent flow ordering for use in sequencing-by-synthesis > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,370,708 B2 - Document ID: US10370708B2 - Status: Granted - Kind: B2 - Application number: 15/236,172 - Filed: 2016-08-12 - Published: 2017-02-16 - Granted: 2019-08-06 - Earliest claimed: 2011-04-08 - Assignees: Life Technologies Corporation - Applicants: Life Technologies Corporation - Inventors: Earl Hubbell; Jonathan Schultz - Web page: https://patentgrep.com/patent/US10370708B2 ## CPC - C12Q 1/6869: Methods for sequencing - C12Q 1/6874: involving nucleic acid arrays, e.g. sequencing by hybridisation - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES ## Abstract A system and machine readable medium for nucleic acid sequencing includes disposing template polynucleotide strands in 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 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 ### Claim 1 A system for nucleic acid sequencing, comprising: a device configured to sequence nucleic acids; a non-transitory 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 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. ### Claim 2 (depends on 1) The system of claim 1, wherein the de Bruijn sequence does not include any consecutive repeats of the same nucleotide species. ### Claim 3 A system for nucleic acid sequencing, comprising: a device configured to sequence nucleic acids; a non-transitory 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 that comprises a flow ordering that includes all possible distinct dimer pairs of four nucleotide species. ### Claim 4 (depends on 1) The system 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. ### Claim 5 (depends on 1) The system 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. ### Claim 6 A system for nucleic acid sequencing, comprising: a device configured to sequence nucleic acids; a non-transitory 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 that 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. ### Claim 7 (depends on 6) The system of claim 6, 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. ### Claim 8 (depends on 7) The system of claim 7, 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. ### Claim 9 (depends on 8) The system of claim 8, 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. ### Claim 10 (depends on 1) The system 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. ### Claim 11 (depends on 1) The system 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. ### Claim 12 (depends on 1) The system 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. ### Claim 13 (depends on 1) The system of claim 1, wherein the sensor array is configured to detect hydrogen ions released by incorporation of nucleotides. ### Claim 14 (depends on 1) The system of claim 1, wherein the sensor array is configured to detect inorganic pyrophosphate released by incorporation of nucleotides. ### Claim 15 A non-transitory machine-readable storage medium comprising instructions which, when executed by a processor, cause a system comprising the processor and a device configured to sequence nucleic acids 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 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. ### Claim 16 (depends on 15) The non-transitory machine-readable storage medium of claim 15, wherein the de Bruijn sequence does not include any consecutive repeats of the same nucleotide species. ### Claim 17 A non-transitory machine-readable storage medium comprising instructions which, when executed by a processor, cause a system comprising the processor and a device configured to sequence nucleic acids 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 that comprises a flow ordering that includes all possible distinct dimer pairs of four nucleotide species. ### Claim 18 (depends on 15) The non-transitory machine-readable storage medium of claim 15, 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. ### Claim 19 (depends on 15) The non-transitory machine-readable storage medium of claim 15, 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. ### Claim 20 A non-transitory machine-readable storage medium comprising instructions which, when executed by a processor, cause a system comprising the processor and a device configured to sequence nucleic acids 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 that 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.