METHODS OF ANALYTE DETECTION
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.
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.
2 . The method of claim 1 , wherein said polynucleotides are DNA concatemers.
3 . The method of claim 1 , wherein said set of reversible terminator nucleotides comprises at least four distinct nucleotides each with a distinct detectable label.
4 . The method of claim 1 , wherein said deconvolution comprises removing interfering optical signals from neighboring polynucleotides using a center-to-center distance between said neighboring polynucleotides from said determined relative positions.
5 . The method of claim 4 , wherein said deconvolution function comprises nearest neighbor variable regression.
6 . The method of claim 1 , wherein said polynucleotides are densely packed on said substrate such that there is overlap between optical signals emitted by said detectable labels from nucleotides incorporated into adjacent polynucleotides, and wherein said adjacent polynucleotides each comprise a distinct sequence.
7 . The method of claim 1 , wherein the polynucleotides are immobilized on said surface at an average density of more than 4 molecules per square micron.
8 . The method of claim 1 , wherein said imaging of said surface is performed at a resolution of one pixel per 300 nm or higher along an axis of the image field.
9 . The method of claim 1 , further comprising generating an oversampled image with a higher pixel density from each of said field images from each cycle.
10 . The method of claim 1 , wherein said overlaying said peak locations comprises aligning positions of said optical signal peaks detected in each field for a plurality of said cycles to generate a cluster of optical peak positions for each polynucleotide from said plurality of cycles.
11 . The method of claim 1 , wherein said optical distribution model comprises a point spread function.
12 . The method of claim 1 , wherein said relative position of said analytes immobilized to the surface of the substrate is determined within 10 nm RMS.
13 . 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.
14 . The method of claim 13 , further comprising
a) deconvolving said optical signals in each field image from each cycle using said determined relative position and a deconvolution function; and
b) identifying said detectable labels bound to said immobilized analytes for each field and each cycle from said deconvolved optical signals.
15 . The method of claim 14 , further comprising using said detectable label identity for each analyte detected at each cycle to identify a plurality of said analytes on said substrate.
16 . The method of claim 14 , wherein said deconvolution comprises removing interfering optical signals from neighboring analytes using a center-to-center distance between said neighboring analytes from said determined relative positions of said neighboring analytes.
17 . The method of claim 16 , wherein said deconvolution function comprises nearest neighbor variable regression.
18 . The method of claim 13 , wherein said analytes are single biomolecules.
19 . The method of claim 13 , wherein said analytes immobilized on said surface are spaced apart on average less than the diffraction limit of the light emitted by the detectable labels and imaged by the optical system.
20 . The method of claim 13 , wherein the immobilized analytes comprises an average center-to-center distance between each analyte and the nearest adjacent analyte of less than 500 nm.