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SEQUENCING AND HIGH RESOLUTION IMAGING

  • US 20190323080A1
  • Filed: 07/01/2019
  • Published: 10/24/2019
  • Est. Priority Date: 03/17/2017
  • Status: Active Grant
First Claim
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1. A method for sequencing a plurality of polynucleotides immobilized at high density on a surface of a substrate, comprising:

  • a. providing a substrate comprising a surface, wherein the surface comprises a plurality of polynucleotides immobilized on the surface at discrete locations, and wherein said surface comprises reagents for sequencing by synthesis;

    b. performing a plurality of cycles of single molecule sequencing by synthesis comprising, each cycle comprising;

    i. contacting said polynucleotides with a set of reversible terminator nucleotides comprising a detectable label;

    ii. imaging a field of said surface with an optical system to detect an optical signal from each nucleotide incorporated into said polynucleotides, thereby detecting a plurality of optical signals in said field for said cycle;

    c. determining a peak location from each of said plurality of optical signals from images of said field from at least two of said plurality of cycles;

    d. overlaying said peak locations for each optical signal and applying an optical distribution model at each cluster of optical signals to determine a relative position of each detected incorporated nucleotide on said surface with improved accuracy;

    e. deconvolving said optical signals in each field image from each cycle using said determined relative position and a deconvolution function;

    f. identifying said detectable labels incorporated into said polynucleotide for each field and each cycle from said deconvolved optical signals; and

    g. sequencing said plurality of polynucleotides immobilized on the surface of the substrate from said identified detectable labels across said plurality of cycles at each polynucleotide position.

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