Methods and compositions for incorporating nucleotides

Abstract

The invention provides methods and compositions, including, without limitation, algorithms, computer readable media, computer programs, apparatus, and systems for determining the identity of nucleic acids in nucleotide sequences using, for example, data obtained from sequencing by synthesis methods. The methods of the invention include correcting one or more phenomena that are encountered during nucleotide sequencing, such as using sequencing by synthesis methods. These phenomena include, without limitation, sequence lead, sequence lag, spectral crosstalk, and noise resulting from variations in illumination and/or filter responses.

We claim: 
     
         1 . A spectral crosstalk identification method for identifying spectral crosstalk associated with at least one detectable fluorescent label upon incorporation of a nucleotide analogue in a nucleic acid molecule, comprising:
 providing:
 a fluorescence measuring device configured for measuring fluorescence in a plurality of detector channels, 
 at least one flow cell including a plurality of spots, each spot comprising a plurality of nucleic acid molecules, 
 a solution comprising a mixture including a plurality of nucleotide analogues comprising a plurality of first nucleotide analogues including at least one detectable fluorescent label, and a plurality of second nucleotide analogues without a detectable label, wherein each of the nucleotide analogues in the mixture includes a reversible terminator chemical moiety at the 3′-OH group; and 
 a polymerase, 
   incorporating at least one first nucleotide analogue from the mixture into at least one nucleic acid molecule of at least one spot of the at least one flow cell,   washing remaining, non-incorporated nucleotide analogues from the at least one flow cell,   emitting light to cause fluorescence of the at least one detectable fluorescent label of the first nucleotide analogue incorporated in the at least one nucleic acid molecule,   detecting a first fluorescence intensity of the at least one detectable fluorescent label from the at least one nucleic acid molecule in a first detector channel,   detecting a second fluorescence intensity of the at least one detectable fluorescent label from the at least one nucleic acid in a second detector channel,   and   determining at least one spectral crosstalk factor associated with the at least one detectable fluorescent label in at least one of the first detector channel and the second detector channel, wherein the spectral crosstalk factor is determined based on the contribution of at least one of the first fluorescence intensity and the second fluorescence intensity.   
     
     
         2 . The method of  claim 1 , wherein that at least one spectral crosstalk factor is calculated for two or more detectable fluorescent labels in at least two detector channels. 
     
     
         3 . The method of  claim 2 , wherein the at least one spectral crosstalk factor for the two or more detectable fluorescent labels are used to determine a spectral crosstalk matrix. 
     
     
         4 . The method of  claim 1 , wherein the at least one spectral crosstalk factor comprises a spectral crosstalk ratio determined by calculating the ratio between the first fluorescence intensity detected in the first detector channel and the second fluorescence intensity in the second detector channel. 
     
     
         5 . The method of  claim 3 , wherein the spectral crosstalk matrix is determined using equation: 
       
         
           
             
               
                 [ 
                 
                   
                     
                       
                         M 
                         A 
                       
                     
                   
                   
                     
                       
                         M 
                         B 
                       
                     
                   
                 
                 ] 
               
               = 
               
                 
                   
                     K 
                     [ 
                     
                       
                         
                           A 
                         
                       
                       
                         
                           B 
                         
                       
                     
                     ] 
                   
                   ⁢ 
                       
                   where 
                   ⁢ 
                       
                   K 
                 
                 = 
                 
                   [ 
                   
                     
                       
                         1 
                       
                       
                         
                           R 
                           AB 
                         
                       
                     
                     
                       
                         
                           R 
                           BA 
                         
                       
                       
                         1 
                       
                     
                   
                   ] 
                 
               
             
           
         
         wherein: 
         M A  is a detected fluorescence intensity of a first fluorescent detectable label, 
         M B  is a detected fluorescence intensity of a second fluorescent detectable label, 
         A is an actual fluorescence intensity of the first fluorescent detectable label, 
         B is an actual fluorescence intensity of the second fluorescent detectable label, 
         R AB  is a ratio between (a) a portion of fluorescence intensity for the first fluorescent detectable label that is contributed by the second fluorescent detectable label, and (b) the actual fluorescent intensity of the second fluorescent detectable label, 
         and 
         R BA  is the ratio between (a) a portion of fluorescence intensity for the second fluorescent detectable label that is contributed by the first fluorescent detectable label, and (b) the actual fluorescent intensity of the first fluorescent detectable label. 
       
     
     
         6 . The method of  claim 5 , wherein the equation is solved to determine spectral crosstalk matrix K −1  using equation 
       
         
           
             
               
                 [ 
                 
                   
                     
                       A 
                     
                   
                   
                     
                       B 
                     
                   
                 
                 ] 
               
               = 
               
                 
                   
                     K 
                     
                       - 
                       1 
                     
                   
                   [ 
                   
                     
                       
                         
                           M 
                           A 
                         
                       
                     
                     
                       
                         
                           M 
                           B 
                         
                       
                     
                   
                   ] 
                 
                 . 
               
             
           
         
       
     
     
         7 . The method of  claim 3 , wherein the spectral crosstalk matrix is determined using equation: 
       
         
           
             
               
                 [ 
                 
                   
                     
                       
                         M 
                         A 
                       
                     
                   
                   
                     
                       
                         M 
                         B 
                       
                     
                   
                   
                     
                       
                         M 
                         C 
                       
                     
                   
                 
                 ] 
               
               = 
               
                 
                   
                     K 
                     [ 
                     
                       
                         
                           A 
                         
                       
                       
                         
                           B 
                         
                       
                       
                         
                           C 
                         
                       
                     
                     ] 
                   
                   ⁢ 
                       
                   where 
                   ⁢ 
                       
                   K 
                 
                 = 
                 
                   [ 
                   
                     
                       
                         1 
                       
                       
                         
                           R 
                           AB 
                         
                       
                       
                         0 
                       
                     
                     
                       
                         
                           R 
                           BA 
                         
                       
                       
                         1 
                       
                       
                         
                           R 
                           BC 
                         
                       
                     
                     
                       
                         0 
                       
                       
                         
                           R 
                           CB 
                         
                       
                       
                         1 
                       
                     
                   
                   ] 
                 
               
             
           
         
         wherein: 
         M A  is a detected fluorescence intensity of a first fluorescent detectable label, 
         M B  is a detected fluorescence intensity of a second fluorescent detectable label, 
         M C  is a detected fluorescence intensity of a third fluorescent detectable label, 
         A is an actual fluorescence intensity of the first fluorescent detectable label, 
         B is an actual fluorescence intensity of the second fluorescent detectable label, 
         C is an actual fluorescence intensity of the third fluorescent detectable label, 
         R AB  is a ratio between (a) a portion of fluorescence intensity for the first fluorescent detectable label that is contributed by the second fluorescent detectable label, and (b) an actual fluorescent intensity of the second fluorescent detectable label, 
         R BA  is the ratio between (a) a portion of fluorescence intensity for the second fluorescent detectable label that is contributed by the first fluorescent detectable label, and (b) an actual fluorescent intensity of the first fluorescent detectable label, 
         R BC  is the ratio between (a) a portion of fluorescence intensity for the second fluorescent detectable label that is contributed by the third fluorescent detectable label, and (b) an actual fluorescent intensity of the third fluorescent detectable label, 
         R CB  is the ratio between (a) a portion of fluorescence intensity for the third fluorescent detectable label that is contributed by the second fluorescent detectable label, and (b) an actual fluorescent intensity of the second fluorescent detectable label. 
       
     
     
         8 . The method of  claim 7 , wherein the equation is solved to determine the spectral crosstalk matrix K −1  comprises: 
       
         
           
             
               
                 [ 
                 
                   
                     
                       A 
                     
                   
                   
                     
                       B 
                     
                   
                   
                     
                       C 
                     
                   
                 
                 ] 
               
               = 
               
                 
                   
                     K 
                     
                       - 
                       1 
                     
                   
                   [ 
                   
                     
                       
                         
                           M 
                           A 
                         
                       
                     
                     
                       
                         
                           M 
                           B 
                         
                       
                     
                     
                       
                         
                           M 
                           C 
                         
                       
                     
                   
                   ] 
                 
                 . 
               
             
           
         
       
     
     
         9 . The method of  claim 3 , further comprising correcting at least one fluorescence intensity in at least one of the detector channels based on the at least one spectral crosstalk factor or the spectral crosstalk matrix associated with the at least one detectable label. 
     
     
         10 . The method of  claim 9 , further comprising identifying the at least one incorporated first nucleotide analogue by calculating a measurement from at least one corrected fluorescence intensity. 
     
     
         11 . The method of  claim 1 , wherein the reversible terminator chemical moiety at the 3′-OH group is an azidomethyl group, an aminoxy group, a disulfide group, or an allyl group. 
     
     
         12 . The method of  claim 1 , wherein the at least one fluorescent label is BODIPY, Rhodamine, Carboxyrhodamine, Cyanine, or a combination thereof. 
     
     
         13 . The method of  claim 1 , wherein detecting of fluorescence intensity is via an imaging system, wherein the imaging system comprises one or more bandpass filters. 
     
     
         14 . The method of  claim 1 , wherein the plurality of detector channels comprise at least two of blue, green, yellow, and red channels. 
     
     
         15 . The method of  claim 1 , wherein the plurality of detector channels comprise two detector channels. 
     
     
         16 . The method of  claim 15 , wherein the two channels are: red and green, red and yellow, red and blue, green and yellow, green and blue, or yellow and blue. 
     
     
         17 . The method of  claim 1 , wherein the plurality of spots within the at least one flow cell are arranged in an array. 
     
     
         18 . The method of  claim 1 , wherein the at least one flow cell is motionless. 
     
     
         19 . The method of  claim 1 , wherein the at least one flow cell is moveable. 
     
     
         20 . The method of  claim 19 , wherein the at least one flow cell is mounted on a rotary or linear stage. 
     
     
         21 . The method of  claim 1 , wherein the flow cell is in fluid communication with a fluidics system. 
     
     
         22 . The method of  claim 21 , wherein the fluidics system comprises at least one reagent reservoir and a valve configured to permit the reagent to flow through the flow valve and into a collection reservoir.