Sequencing and high resolution imaging
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 said surface comprises a plurality of polynucleotides immobilized on said 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, each cycle comprising:
i. contacting said polynucleotides with a set of reversible terminator nucleotides, each reversible terminator nucleotide comprising a detectable label;
ii. detecting, with an optical imaging system, a plurality of optical signals emitted from said reversible terminator nucleotides to generate a field image of a field of said surface;
c. determining a position of a peak intensity within each of said plurality of optical signals based on said field image from at least two cycles of said cycles;
d. overlaying said peak intensity positions to generate clusters of said peak intensity positions for said field image from said at least two cycles of said cycles, and applying an optical distribution model to each cluster to determine a relative positions of said reversible terminator nucleotides emitting an optical signal;
e. deconvolving said optical signals detected for each cycle using said determined relative position and a deconvolution function;
f. identifying said reversible terminator nucleotides from each said field image using said deconvolved optical signals; and
g. sequencing said plurality of polynucleotides immobilized on the surface of the substrate based on said identified said reversible terminator nucleotides.
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 and distinct detectable labels.
4. The method of claim 1 , wherein said deconvolving 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 deconvolving function comprises nearest neighbor variable regression.
6. The method of claim 1 , wherein said polynucleotides are densely packed on said substrate such that optical signals emitted from said reversible terminator nucleotides binding to adjacent polynucleotides of said polynucleotides overlap, wherein each of said adjacent polynucleotides 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 field image is generated with a resolution of at least one pixel per 300 nm along at least one axis of said field.
9. The method of claim 1 , further comprising generating an oversampled image based on said generated field image from each cycle of said cycles.
10. The method of claim 1 , wherein said overlaying said peak intensity positions to generate said clusters of said peak intensity positions for said field image comprises aligning said peak intensities positions for said field image from at least two of said 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 determined relative position is determined within 10 nm RMS.