SEQUENCING AND HIGH RESOLUTION IMAGING
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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Abstract
Disclosed herein are methods and systems for detection and discrimination of optical signals from a densely packed substrate. These have broad applications for biomolecule detection near or below the diffraction limit of optical systems, including in improving the efficiency and accuracy of polynucleotide sequencing applications.
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Citations
30 Claims
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1. A method for sequencing a plurality of polynucleotides immobilized at high density on a surface of a substrate, comprising:
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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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12)
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13. A method for accurately determining a relative position of analytes immobilized on the surface of a densely packed substrate, comprising:
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a. providing a substrate comprising a surface, wherein the surface comprises a plurality of analytes immobilized on the surface at discrete locations; b. performing a plurality of cycles of probe binding and signal detection on said surface, each cycle comprising; i. contacting said analytes with a plurality of probes from a probe set, wherein said probes comprise a detectable label, wherein each of said probes binds specifically to a target analyte; and ii. imaging a field of said surface with an optical system to detect a plurality of optical signals from individual probes bound to said analytes at discrete locations on said surface; 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; and 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 analyte on said surface with improved accuracy. - View Dependent Claims (14, 15, 16, 17, 18, 19, 20, 21, 22, 23)
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24. A method for identifying a plurality of densely packed analytes immobilized on a surface of a substrate, comprising:
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a. providing a substrate comprising a surface, wherein the surface comprises a plurality of analytes immobilized on the surface at discrete locations; b. performing a plurality of cycles of probe binding and signal detection on said surface, each cycle comprising; i. contacting said analytes with a plurality of probes from a probe set, wherein said probes comprise a detectable label, wherein each of said probes binds specifically to a target analyte; and ii. imaging a field of said surface with an optical system to detect a plurality of optical signals from individual probes bound to said analytes; 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 analyte 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 bound to said immobilized analytes for each field and each cycle from said deconvolved optical signals; and g. using said detectable label identity for each analyte detected at each cycle to determine the identity of a plurality of said analytes on said substrate.
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25. A system for determining the identity of a plurality of analytes, comprising
a. an optical imaging device configured to image a plurality of optical signals from a field of a substrate over a plurality of cycles of probe binding to analytes immobilized on a surface of the substrate; - and
b. an image processing module, said module configured to; i. determine 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; ii. determine a relative position of each detected analyte on said surface with improved accuracy by applying an optical distribution model to each cluster of optical signals from said plurality of cycles; and iii. deconvolve said optical signals in each field image from each cycle using said determined relative position and a deconvolution function. - View Dependent Claims (26, 27, 28, 29, 30)
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Specification