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Alternative nucleotide flows in sequencing-by-synthesis methods

  • US 10,392,660 B2
  • Filed: 01/12/2017
  • Issued: 08/27/2019
  • Est. Priority Date: 06/11/2010
  • Status: Active Grant
First Claim
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1. A method for sequencing a template polynucleotide strand comprising:

  • (a) disposing a plurality of template polynucleotide strands into a plurality of reaction chambers, such that each reaction chamber has a template polynucleotide strand of the plurality having a sequencing primer hybridized thereto and a polymerase operably bound thereto;

    (b) introducing a known nucleoside triphosphate into each reaction chamber according to a predetermined ordering of dNTP reagent 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 polynucleotide strands;

    (d) washing away unincorporated nucleoside triphosphates from the reaction chamber; and

    (e) repeating steps (b) through (d) until a desired template length of the plurality of template polynucleotide strands is sequenced;

    wherein the predetermined ordering of dNTP reagent flows 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, and 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, and wherein repetition of the alternate ordering successively occurs at least once to sequence the desired template length.

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