Alternative nucleotide flows in sequencing-by-synthesis methods

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.

1 . A method of sequencing a polynucleotide strand, comprising:
 providing the polynucleotide strand with a primer annealed thereto and a polymerase operably bound to the polynucleotide strand; and   successively exposing the polynucleotide strand to the flow of four different dNTPs according to a predetermined ordering, wherein the predetermined ordering comprises an alternate ordering which is not a continuous repeat of an ordering of the four different dNTPs.   
     
     
         2 . The method of  claim 1 , wherein the alternate ordering is not a continuous repeat of the order in which the first four dNTPs in the predetermined ordering are flowed. 
     
     
         3 . The method of  claim 1 , wherein the alternate ordering includes 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. 
     
     
         4 . The method of  claim 3 , wherein the N-W-N flow is further followed directly by a flow of a nucleotide Z that is different from N and W. 
     
     
         5 . The method of  claim 1 , wherein the polynucleotide strand is disposed inside a reaction chamber and the flow of dNTPs is introduced into the reaction chamber. 
     
     
         6 . The method of  claim 1 , wherein the ordering of the four different dNTPs are: dATP, then dCTP, then dGTP, and then dTTP; or a permutation thereof. 
     
     
         7 . The method of  claim 2 , wherein the ordering of the first four dNTPs are: dATP, then dCTP, then dGTP, and then dTTP; or a permutation thereof. 
     
     
         8 . The method of  claim 1 , wherein the predetermined ordering consists of only the alternate ordering. 
     
     
         9 . The method of  claim 1 , wherein the predetermined ordering includes a portion that is an alternate ordering and a portion that is not an alternate ordering. 
     
     
         10 . The method of  claim 1 , wherein the alternate ordering includes a first dNTP flow, a second dNTP flow, a third dNTP flow, and a fourth dNTP flow, with each flow being a different dNTP; wherein the fourth dNTP flow does not occur until at least one of the first, second, or third dNTP flows are repeated at least once. 
     
     
         11 . The method of  claim 1 , wherein the number of flows for each of the four different dNTPs in the alternate ordering is the same. 
     
     
         12 . The method of  claim 1 , wherein in the predetermined ordering, the flow of one of the four dNTPs is always followed by a flow of a different one of the four dNTPs. 
     
     
         13 . The method of  claim 1 , wherein the length of the alternate ordering is a multiple of four. 
     
     
         14 . The method of  claim 13 , wherein the length of the alternate ordering is 8, 12, 16, 20, 24, 28, or 32. 
     
     
         15 . The method of  claim 1 , further comprising detecting hydrogen ions released by dNTP incorporation reactions. 
     
     
         16 . The method of  claim 1 , further comprising detecting inorganic pyrophosphate released by dNTP incorporation reactions. 
     
     
         17 . The method of  claim 16 , wherein the inorganic pyrophosphate is detected by light emitted from an enzyme cascade initiated by the inorganic pyrophosphate. 
     
     
         18 . 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 repeat of an ordering of the four different dNTP reagent flows.   
     
     
         19 . The apparatus of  claim 18 , wherein the alternate ordering is not a continuous repeat of the order in which the first four dNTP reagents in the predetermined ordering are flowed. 
     
     
         20 . The apparatus of  claim 18 , further comprising a flow cell loaded into the flow chamber, wherein the flow cell comprises a microwell array that contains multiple copies of the polynucleotide strand with a primer annealed thereto. 
     
     
         21 . The apparatus of  claim 20 , wherein the flow cell further comprises a chemFET sensor array for detecting the reaction of the dNTP reagent with the contents of the microwell array. 
     
     
         22 . The apparatus of  claim 20 , wherein the polynucleotide strands are attached to beads that are contained in the microwells of the microwell array. 
     
     
         23 . The apparatus of  claim 18 , wherein the alternate ordering includes 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. 
     
     
         24 . The apparatus of  claim 23 , wherein the N-W-N flow is further followed directly by a flow of a nucleotide Z that is different from N and W. 
     
     
         25 . The apparatus of  claim 18 , wherein the predetermined ordering consists of only the alternate ordering. 
     
     
         26 . A method of performing template-based extension of a primer, comprising:
 providing at least one template polynucleotide strand having a primer and polymerase operably bound thereto; and   successively exposing the template polynucleotide strand to a plurality of each kind of flow such that (a) a flow of one kind is always followed by a flow of a different kind; and (b) at least one flow of each kind is followed by a flow of the same kind after a single intervening flow of a different kind.   
     
     
         27 . The method of  claim 26 , wherein the number of flows of each kind in the plurality is the same. 
     
     
         28 . A method of determining the sequence of a template polynucleotide strand by template-based extension of a primer, comprising:
 (a) delivering a known nucleoside triphosphate precursor to a template-based primer extension reaction of a polynucleotide strand, the known nucleoside triphosphate precursor being delivered according to a predetermined ordering of dNTP flows;   (b) detecting incorporation of the known nucleoside triphosphate whenever its complement is present in the template polynucleotide strand adjacent to the primer; and   (c) repeating steps (a) and (b) until the sequence of the template polynucleotide strand is determined; wherein the predetermined ordering of dNTP flows is defined by (i) a flow of one kind is always followed by a flow of a different kind; and (ii) at least one flow of each kind is followed by a flow of the same kind after a single intervening flow of a different kind.   
     
     
         29 . The method of  claim 28 , wherein the number of flows of each kind in the plurality is the same. 
     
     
         30 . A method for sequencing a template polynucleotide strand comprising:
 (a) disposing a plurality of template polynucleotide strands into a plurality of reaction chambers, each reaction chamber comprising a template polynucleotide strand having a sequencing primer hybridized thereto and a polymerase operably bound thereto;   (b) introducing a known nucleoside triphosphates into each reaction chamber according to a predetermined ordering of dNTP flows;   (c) detecting sequential incorporation at the 3′ end of the sequencing primer of one or more nucleoside triphosphates if the known nucleoside triphosphate is complementary to corresponding nucleotides in the template nucleic acid;   (d) washing away unincorporated nucleoside triphosphates from the reaction chamber; and   (e) repeating steps (b) through (d) until the polynucleotide strand is sequenced;   wherein the predetermined ordering of dNTP flows is defined by (i) a flow of one kind is always followed by a flow of a different kind; and (iii) at least one flow of each kind is followed by a flow of the same kind after a single intervening flow of a different kind.   
     
     
         31 . The method of  claim 30 , wherein the number of flows of each kind in the plurality is the same. 
     
     
         32 . A method of sequencing a polynucleotide strand, comprising:
 providing the polynucleotide strand with a primer annealed thereto and a polymerase operably bound to the polynucleotide strand;   successively exposing the polynucleotide strand to the flow of four different dNTPs according to a first predetermined ordering; and   successively exposing the polynucleotide strand to the flow of four different dNTPs according to a second predetermined ordering, wherein the second predetermined ordering is different from the first predetermined ordering.   
     
     
         33 . The method of  claim 32 , wherein the second predetermined ordering comprises an alternate ordering which is not a continuous repeat of the first predetermined ordering of the four different dNTPs. 
     
     
         34 . The method of  claim 32 , further comprising successively exposing the polynucleotide strand to the flow of four different dNTPs according to a third predetermined ordering, wherein the third predetermined ordering comprises an alternate ordering which is not a continuous repeat the of the first predetermined ordering of the four different dNTPs or of the second predetermined ordering of the four different dNTPs. 
     
     
         35 . The method of  claim 32 , further comprising successively exposing the polynucleotide strand to the flow of four different dNTPs according to the first predetermined ordering after successively exposing the polynucleotide strand to the flow of four different dNTPs according to the second predetermined ordering.