Alternative nucleotide flows in sequencing-by-synthesis methods
First Claim
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1. An apparatus for sequencing a polynucleotide strand, comprising:
- a flow chamber for receiving flows of different dNTP reagents;
multiple reservoirs that each contain a different dNTP reagent;
flow paths from each of the reservoirs to the flow chamber; and
a fluidics controller that controls the flow from the reservoirs to the flow chamber, wherein the fluidics controller is programmed to successively provide flow from the multiple reservoirs to the flow chamber according to a predetermined ordering, wherein the predetermined ordering comprises an alternate ordering which is not a continuous and ordered repeat of four different dNTP reagent flows, the alternate ordering comprising a same number of flows for each of four different dNTP reagent flows, a flow of a nucleotide N, which is followed directly by a flow of a different nucleotide W, which is followed directly by a flow of the same nucleotide N, wherein the N-W-N flow is further followed directly by a flow of a nucleotide Z that is different from N and W, and the alternate ordering being successively repeated at least once.
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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.
109 Citations
17 Claims
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1. An apparatus for sequencing a polynucleotide strand, comprising:
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a flow chamber for receiving flows of different dNTP reagents; multiple reservoirs that each contain a different dNTP reagent; flow paths from each of the reservoirs to the flow chamber; and a fluidics controller that controls the flow from the reservoirs to the flow chamber, wherein the fluidics controller is programmed to successively provide flow from the multiple reservoirs to the flow chamber according to a predetermined ordering, wherein the predetermined ordering comprises an alternate ordering which is not a continuous and ordered repeat of four different dNTP reagent flows, the alternate ordering comprising a same number of flows for each of four different dNTP reagent flows, a flow of a nucleotide N, which is followed directly by a flow of a different nucleotide W, which is followed directly by a flow of the same nucleotide N, wherein the N-W-N flow is further followed directly by a flow of a nucleotide Z that is different from N and W, and the alternate ordering being successively repeated at least once. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15)
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13. An apparatus for sequencing a polynucleotide strand, comprising:
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a flow chamber for receiving flows of different dNTP reagents; multiple reservoirs that each contain a different dNTP reagent; flow paths from each of the reservoirs to the flow chamber; and a fluidics controller that controls the flow from the reservoirs to the flow chamber, wherein the fluidics controller is programmed to successively provide flow from the multiple reservoirs to the flow chamber according to a predetermined ordering, wherein the predetermined ordering comprises an alternate ordering which is not a continuous and ordered repeat of four different dNTP reagent flows, the alternate ordering comprising a flow of one kind of dNTP reagent always followed by a flow of a different kind of dNTP reagent, at least one flow of each kind of dNTP reagent followed by a flow of the same kind of dNTP reagent after a single intervening flow of a different kind of dNTP reagent, a same number of flows of each of the four kinds of dNTP reagent flows in the alternate ordering, and wherein repetition of the alternate ordering successively occurs at least once to sequence a desired template length. - View Dependent Claims (14, 16)
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17. An apparatus for sequencing a polynucleotide strand, comprising:
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a flow chamber for receiving flows of different dNTP reagents; multiple reservoirs that each contain a different dNTP reagent; flow paths from each of the reservoirs to the flow chamber; and a fluidics controller that controls the flow from the reservoirs to the flow chamber, wherein the fluidics controller is programmed to successively provide flow from the multiple reservoirs to the flow chamber according to a predetermined ordering, wherein the predetermined ordering consists of only an alternate ordering which is not a continuous and ordered repeat of four different dNTP reagent flows, the alternate ordering comprising a same number of flows for each of four different dNTP reagent flows, a flow of a nucleotide N, which is followed directly by a flow of a different nucleotide W, which is followed directly by a flow of the same nucleotide N, wherein the N-W-N flow is further followed directly by a flow of a nucleotide Z that is different from N and W, and the alternate ordering being repeated at least once.
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Specification