Alternative nucleotide flows in sequencing-by-synthesis methods
First Claim
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1. A method for improving homopolymer classification accuracy, comprising:
- providing a template polynucleotide strand, a primer, and a polymerase in a microwell, the template polynucleotide strand being coupled to or associated with a bead;
successively exposing the template polynucleotide strand to a plurality of each of four kinds of dNTP reagent flows, each dNTP reagent flow having a single kind of dNTP, wherein the plurality of reagent flows are selected according to an ordering, the ordering being such that (a) a flow of one kind of dNTP reagent is always followed by a flow of a different kind of dNTP reagent, (b) at least one flow of each kind of dNTP reagent is followed by a flow of the same kind of dNTP reagent after a single intervening flow of a different kind of dNTP reagent, and (c) the number of flows of each of the four kinds of dNTP reagent flows in the ordering is the same, wherein the successively exposing comprises consecutively repeating the plurality of reagent flows according to the ordering one or more times; and
obtaining, using a sensor configured to provide information about reactions taking place in the microwell, an incorporation signal comprising values generally proportional to a number of dNTP incorporation(s) that result when a base in the template polynucleotide strand is complementary to one of the flowed dNTP reagents.
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Abstract
A method for sequencing a polynucleotide strand by using sequencing-by-synthesis techniques. To address the problem of incomplete extension (IE) and/or carry forward (CF) errors that can occur in sequencing-by-synthesis reactions, an alternative flow ordering of dNTPs is used. In contrast to conventional flow orderings, the dNTPs are flowed in an ordering that is not a continuous repeat of an ordering of the four different dNTPs. This alternate flow ordering may reduce the loss of phasic synchrony in the population of template polynucleotide strands that result from IE and/or CF errors.
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Citations
11 Claims
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1. A method for improving homopolymer classification accuracy, comprising:
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providing a template polynucleotide strand, a primer, and a polymerase in a microwell, the template polynucleotide strand being coupled to or associated with a bead; successively exposing the template polynucleotide strand to a plurality of each of four kinds of dNTP reagent flows, each dNTP reagent flow having a single kind of dNTP, wherein the plurality of reagent flows are selected according to an ordering, the ordering being such that (a) a flow of one kind of dNTP reagent is always followed by a flow of a different kind of dNTP reagent, (b) at least one flow of each kind of dNTP reagent is followed by a flow of the same kind of dNTP reagent after a single intervening flow of a different kind of dNTP reagent, and (c) the number of flows of each of the four kinds of dNTP reagent flows in the ordering is the same, wherein the successively exposing comprises consecutively repeating the plurality of reagent flows according to the ordering one or more times; and obtaining, using a sensor configured to provide information about reactions taking place in the microwell, an incorporation signal comprising values generally proportional to a number of dNTP incorporation(s) that result when a base in the template polynucleotide strand is complementary to one of the flowed dNTP reagents. - View Dependent Claims (6, 9)
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2. A method for improving homopolymer classification accuracy, comprising:
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(a) delivering a known nucleoside triphosphate precursor to a template-based primer extension reaction of a template polynucleotide strand, the known nucleoside triphosphate precursor being delivered according to a predetermined ordering of dNTP flows, the template polynucleotide strand being coupled to or associated with a bead loaded in a microwell; (b) detecting incorporation of the known nucleoside triphosphate using a pH-based sensor configured to provide information about reactions taking place in the microwell and generate an incorporation signal comprising values generally proportional to a number of dNTP incorporation(s) occurring when the complement of the known nucleoside triphosphate is present in the template polynucleotide strand adjacent to the primer; and (c) repeating steps (a) and (b) until each dNTP flow in the predetermined ordering of dNTP flows has been flowed;
wherein the predetermined ordering of dNTP flows is such that (i) a flow of one kind of dNTP is always followed by a flow of a different kind of dNTP, (ii) at least one flow of each kind of dNTP is followed by a flow of the same kind of dNTP after a single intervening flow of a different kind of dNTP, and (iii) the ordering includes four consecutive groups of three dNTP flows, the second dNTP flow in each of the four consecutive groups of three dNTP flows being different than the second dNTP flow in the other three consecutive groups of three dNTP flows, and the first and third dNTP flows in each of the four consecutive groups of three dNTP flows being both of the same kind of dNTP and being different than the second dNTP flow, wherein the delivering comprises consecutively repeating a delivery of the predetermined ordering of dNTP flows one or more times. - View Dependent Claims (7)
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3. A method for improving homopolymer classification accuracy, comprising:
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providing a polynucleotide strand, a primer, and a polymerase in a microwell, the polynucleotide strand being coupled to or associated with a bead; successively exposing the polynucleotide strand coupled to or associated with the bead to flows of four different dNTPs, one kind of dNTP per flow, according to a first predetermined ordering, wherein the first predetermined ordering comprises sequential repetitions of the same sequence of four different dNTPs; successively exposing the polynucleotide strand coupled to or associated with the bead to flows of a plurality of dNTPs, one kind of dNTP per flow, according to a second predetermined ordering, the second predetermined ordering being such that a flow of one kind of dNTP reagent is always followed by a flow of a different kind of dNTP reagent, and at least one flow of each kind of dNTP reagent is followed by a flow of the same kind of dNTP reagent after a single intervening flow of a different kind of dNTP reagent, wherein the second predetermined ordering occurs after the first predetermined ordering, and wherein the successive exposing of the polynucleotide strand according to the second predetermined ordering is consecutively repeated one or more times; and obtaining, using a sensor configured to provide information about reactions taking place in the microwell, an incorporation signal comprising values generally proportional to a number of dNTP incorporation(s) that result when a base in the polynucleotide strand is complementary to one of the flowed dNTPs. - View Dependent Claims (4, 5, 8, 10, 11)
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Specification