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Alternative nucleotide flows in sequencing-by-synthesis methods

  • US 9,416,413 B2
  • Filed: 04/09/2013
  • Issued: 08/16/2016
  • Est. Priority Date: 06/11/2010
  • Status: Active Grant
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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