Phase-protecting reagent flow ordering for use in sequencing-by-synthesis
First 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.
1 Assignment
0 Petitions
Accused Products
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.
112 Citations
20 Claims
-
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. - View Dependent Claims (2, 4, 5, 10, 11, 12, 13, 14)
-
-
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.
-
-
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. - View Dependent Claims (7, 8, 9)
-
-
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. - View Dependent Claims (16, 18, 19)
-
-
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.
-
-
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.
-
Specification