Methods of analyte detection
AssigneePacific Biosciences of California, Inc.
InventorsBryan P. STAKER, Niandong LIU, Michael David MCLAUGHLIN, Bart Lee STAKER
Life of this patent
Estimating the term…
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.
Claims20
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.
A method for accurately determining a relative position of analytes immobilized on the surface of a densely packed substrate, comprising:
- 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.
Dependencies are inferred from the claim text.