Alternative nucleotide flows in sequencing-by-synthesis methods
AssigneeLife Technologies Corporation
InventorsJonathan SCHULTZ, John DAVIDSON
Life of this patent
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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.
Claims11
1-35. (canceled)
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 according to an ordering that improves homopolymer classification accuracy, 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, and (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
- 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 abase in the template polynucleotide strand is complementary to one of the flowed dNTP reagents.
A method for improving homopolymer classification accuracy, comprising:
- (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 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 improves homopolymer classification accuracy and is such that (i) a flow of one kind of dNTP is always followed by a flow of a different kind of dNTP, and (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.
A method for improving homopolymer classification accuracy, comprising:
- 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 according to a first predetermined ordering;
- successively exposing the polynucleotide strand coupled to or associated with the bead to flows of for different dNTPs according to a second predetermined ordering, wherein the second predetermined ordering improves homopolymer classification accuracy and is different from the first predetermined ordering;
- 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.
Dependencies are inferred from the claim text.