Systems and methods of sequencing polynucleotides

Abstract

The application relates to DNA sequencing systems and methods. Systems and methods for determining the nucleotide sequence of a polynucleotide include attaching three different fluorescent dyes to three different nucleotides during incorporation. In particular, long Stokes shifted dyes may be used to determine the sequence of polynucleotides in a sequencing by synthesis system.

What is claimed is: 
     
         1 . A method of sequencing clusters of labeled polynucleotides bound to a flowcell, comprising:
 exciting the clusters at a first excitation wavelength and detecting fluorescent emissions at a first detection wavelength from a first labeled nucleotide,   exciting the clusters at a second excitation wavelength and detecting fluorescent emissions at a second detection wavelength from a second labeled nucleotide,   exciting the clusters at the first excitation wavelength and detecting fluorescent emissions at the second detection wavelength from a third labeled nucleotide,   determining clusters that did not emit fluorescent emissions after excitation at the first or second excitation wavelengths to detect the presence of a fourth unlabeled nucleotide, and   determining the nucleotide sequence of the polynucleotides based on the detected fluorescent emissions.   
     
     
         2 . The method of  claim 1 , wherein the first excitation wavelength and the second excitation wavelength are different wavelengths. 
     
     
         3 . The method of  claim 2 , wherein the first detection wavelength and the second detection wavelength are different wavelengths. 
     
     
         4 . The method of  claim 3 , wherein the first labeled nucleotide, the second labeled nucleotide, and the third labeled nucleotide are each labeled with a single fluorescent dye. 
     
     
         5 . The method of  claim 4 , wherein the third labeled nucleotide is labeled with a long Stokes shift dye, wherein the long Stokes shift dye has a Stokes shift ranging from about 300 meV to 850 meV. 
     
     
         6 . The method of  claim 4 , wherein the third labeled nucleotide is labeled with a long Stokes shift dye, wherein the long Stokes shift dye has an emission shifted by about 50 nm to 150 nm. 
     
     
         7 . The method of  claim 6 , wherein the long Stokes shift dye is excited at about 300 nm. 
     
     
         8 . The method of  claim 4 , wherein the third labeled nucleotide is labeled with a chromenoquinoline dye. 
     
     
         9 . The method of  claim 4 , wherein the first labeled nucleotide is labeled with dye NR455BoC. 
     
     
         10 . The method of  claim 4 , wherein the second labeled nucleotide is labeled with dye NR550SO. 
     
     
         11 . The method of  claim 4 , wherein the third labeled nucleotide is labeled with dye MC485CQ-O. 
     
     
         12 . The method of  claim 11 , wherein the first detection wavelength is imaged by a first charge coupled device, and the second detection wavelength is imaged by a second charge coupled device. 
     
     
         13 . The method of  claim 4 , wherein exciting at the first excitation wavelength and detecting fluorescent emissions at the first emission wavelength is performed separately from exciting at the first excitation wavelength and detecting fluorescent emissions at the second emission wavelength. 
     
     
         14 . The method  claim 4 , wherein exciting at the first excitation wavelength and detecting fluorescent emissions at the first emission wavelength is performed at the same time as exciting at the first excitation wavelength and detecting fluorescent emissions at the second emission wavelength. 
     
     
         15 . The method of  claim 4 , wherein the detecting fluorescent emissions from the first labeled nucleotide, the second labeled nucleotide and the third labeled nucleotide are performed using a Time Delay Integration (TDI) imaging method. 
     
     
         16 . A method of sequencing clusters of labeled polynucleotides bound to a flowcell, comprising:
 exciting the clusters of labeled polynucleotides at a first excitation wavelength and detecting fluorescent emissions from the clusters at a first detection wavelength to detect the presence of a first labeled nucleotide,   exciting the clusters of labeled polynucleotides at a second excitation wavelength and detecting fluorescent emissions from the clusters at a second detection wavelength to detect the presence of a second labeled nucleotide,   exciting the clusters of labeled polynucleotides at a third excitation wavelength and detecting fluorescent emissions from the clusters at a third detection wavelength to detect the presence of a third labeled nucleotide,   determining clusters that did not emit fluorescent emissions after excitation at the first, second or third excitation wavelengths to detect the presence of a fourth unlabeled nucleotide, and   determining the nucleotide sequence of the labeled polynucleotides based on the detected fluorescent emissions.   
     
     
         17 . The method of  claim 16 , wherein the first excitation wavelength and the second excitation wavelength are the same wavelengths. 
     
     
         18 . The method of  claim 17  wherein the first detection wavelength and the second detection wavelength are different wavelengths. 
     
     
         19 . The method of  claim 18 , wherein the signal to noise ratio (SNR) between the first labeled nucleotide and the fourth unlabeled nucleotide is at least about 10 dB. 
     
     
         20 . The method of  claim 19 , wherein the signal to noise ratio (SNR) between the first labeled nucleotide and the fourth unlabeled nucleotide is at least about 15 dB. 
     
     
         21 . The method of  claim 19 , wherein the SNR between the second labeled nucleotide and the fourth unlabeled nucleotide is at least about 10 dB. 
     
     
         22 . The method of  claim 19 , wherein the SNR between the second labeled nucleotide and the fourth unlabeled nucleotide is at least about 15 dB. 
     
     
         23 . The method of  claim 21 , wherein the SNR between the third labeled nucleotide and the fourth unlabeled nucleotide is at least about 10 dB. 
     
     
         24 . The method of  claim 21 , wherein the SNR between the third labeled nucleotide and the fourth unlabeled nucleotide is at least about 15 dB. 
     
     
         25 . The method of  claim 24 , wherein the first excitation wavelength is between 300 nm to 540 nm. 
     
     
         26 . The method of  claim 25 , wherein the first detection wavelength is between 310 nm and 600 nm. 
     
     
         27 . The method of  claim 26 , wherein the second excitation wavelength is between 350 nm to 600 nm. 
     
     
         28 . The method of  claim 27 , wherein the first detection wavelength is between 360 nm to 660 nm. 
     
     
         29 . The method of  claim 28 , wherein the third excitation wavelength is between 520 nm to 700 nm. 
     
     
         30 . The method of  claim 29 , wherein the first detection wavelength is between 530 nm to 760 nm. 
     
     
         31 . A system for sequencing polynucleotides bound to a flowcell, comprising:
 a machine-readable memory, and   a processor configured to execute machine-readable instructions, which, when executed by the processor, cause the system to perform steps according to  claim 1 .   
     
     
         32 . A non-transitory computer-readable medium storing a polynucleotide sequencing program including instructions that, when executed by a processor, causes a polynucleotide sequencing apparatus, to perform steps according to  claim 1 .