Methods, Systems, and Computer Readable Media for Evaluating Variant Likelihood

Abstract

A method for evaluating variant likelihood includes: providing a plurality of template polynucleotide strands, sequencing primers, and polymerase in a plurality of defined spaces disposed on a sensor array; exposing the plurality of template polynucleotide strands, sequencing primers, and polymerase to a series of flows of nucleotide species according to a predetermined order; obtaining measured values corresponding to an ensemble of sequencing reads for at least some of the template polynucleotide strands in at least one of the defined spaces; and evaluating a likelihood that a variant sequence is present given the measured values corresponding to the ensemble of sequencing reads, the evaluating comprising: determining a measurement confidence value for each read in the ensemble of sequencing reads and modifying at least some model-predicted values using a first bias for forward strands and a second bias for reverse strands.

1 . A method for evaluating variant likelihood in nucleic acid sequencing, comprising:
 (a) providing a plurality of template polynucleotide strands, sequencing primers, and polymerase in a plurality of defined spaces disposed on a sensor array;   (b) exposing the plurality of template polynucleotide strands, sequencing primers, and polymerase to a series of flows of nucleotide species according to a predetermined order;   (c) obtaining measured values corresponding to an ensemble of sequencing reads for at least some of the template polynucleotide strands in at least one of the defined spaces; and   (d) evaluating a likelihood that a variant sequence is present given the measured values corresponding to the ensemble of sequencing reads, the evaluating comprising:
 (i) determining a measurement confidence value for each read in the ensemble of sequencing reads, wherein the determining is based on variations between the measured values and model-predicted values for hypothesized sequences obtained using a predictive model of nucleotide incorporations responsive to flows of nucleotide species; and 
 (ii) modifying at least some model-predicted values using a first bias for forward strands and a second bias for reverse strands, wherein the modifying is based on variations between model-predicted values for different hypothesized sequences obtained using the predictive model of nucleotide incorporations responsive to flows of nucleotide species. 
   
     
     
         2 . The method of  claim 1 , wherein modifying the at least some model-predicted values comprises applying a transformation including a product of (i) one of the first and second biases and (ii) a discriminant vector representing a difference between model-predicted values corresponding to different hypothesized sequences. 
     
     
         3 . The method of  claim 1 , wherein evaluating the likelihood further comprises assigning a first frequency to a variant sequence and a second frequency to a non-variant sequence, and calculating a likelihood of having observed the ensemble of sequencing reads conditioned on the first frequency as a function of a product of the likelihoods of having observed each of the sequencing reads given the first frequency. 
     
     
         4 . The method of  claim 1 , wherein evaluating the likelihood further comprises assigning a first frequency to a variant sequence, a second frequency to a non-variant sequence, and a third frequency to an outlier event. 
     
     
         5 . The method of  claim 4 , wherein the outlier event has a flat density across all sequencing reads in the ensemble. 
     
     
         6 . The method of  claim 4 , wherein evaluating the likelihood further comprises calculating a likelihood of having observed the ensemble of sequencing reads conditioned on the third frequency as a function of a product of the likelihoods of having observed each of the sequencing reads given the third frequency. 
     
     
         7 . The method of  claim 1 , wherein the measurement confidence values are estimated using a function comprising a sum of log-likelihood of values measured for a given flow given a hypothesized sequence. 
     
     
         8 . The method of  claim 7 , wherein the measurement confidence values are estimated using a function comprising differences between the measured values and the model-predicted values. 
     
     
         9 . The method of  claim 8 , wherein the differences between measured and model-predicted values at each nucleotide flow are assumed to follow independent normal distributions each having a mean and a variance. 
     
     
         10 . The method of  claim 9 , wherein the differences between measured and model-predicted values at each nucleotide flow are assumed to follow independent t-distributions. 
     
     
         11 . The method of  claim 1 , wherein the measurement confidence values are estimated using an expression comprising 
       
         
           
             
               
                 ε 
                 i 
               
               = 
               
                 1 
                 
                   1 
                   + 
                   
                     exp 
                      
                     
                       ( 
                       
                         
                           LL 
                           yi 
                         
                         - 
                         
                           LL 
                           xi 
                         
                       
                       ) 
                     
                   
                 
               
             
           
         
       
       where LL yi  and LL xi  are log-likelihoods of values measured for a given sequencing read under hypothesized sequences y and x, respectively. 
     
     
         12 . The method of  claim 11 , wherein the measurement confidence values are estimated using an expression for responsibility comprising 
       
         
           
             
               
                 ρ 
                 i 
               
               = 
               
                 π 
                 
                   π 
                   + 
                   
                     
                       ( 
                       
                         1 
                         - 
                         π 
                       
                       ) 
                     
                     * 
                     
                       exp 
                        
                       
                         ( 
                         
                           
                             LL 
                             yi 
                           
                           - 
                           
                             LL 
                             xi 
                           
                         
                         ) 
                       
                     
                   
                 
               
             
           
         
       
       where π represents a first frequency assigned to a variant sequence, 1−π represents a second frequency assigned to a non-variant sequence, and ρ i  represents a measure of responsibility for each of the sequencing reads in the ensemble. 
     
     
         13 . The method of  claim 11 , where the variance is estimated by decomposition of the variance in a flow and sequencing read into underlying latent components. 
     
     
         14 . The method of  claim 13 , wherein each latent component corresponds to a homopolymer having an integer length. 
     
     
         15 . The method of  claim 13 , wherein the latent components include a null variance component representing contribution to a flow regardless of any nucleotide incorporation, a residual variance component representing contribution for nucleotide incorporations not explicitly modeled, and one or more additional variance components. 
     
     
         16 . The method of  claim 15 , wherein the one or more additional variance components comprise variance components associated with homopolymers having an integer length. 
     
     
         17 . The method of  claim 13 , wherein the latent components are estimated using an EM methodology and a method of moments approximation. 
     
     
         18 . The method of  claim 1 , wherein evaluating the likelihood comprises estimating (i) a first frequency π assigned to a variant sequence, (ii) at least one of a measurement confidence value ε i  for each of the sequencing reads in the ensemble and a measure of responsibility ρ i  for each of the sequencing reads in the ensemble, and (iii) a variance σ ij   2  for each of the flows and sequencing reads in the ensemble. 
     
     
         19 . A non-transitory machine-readable storage medium comprising instructions which, when executed by a processor, cause the processor to perform a method for evaluating variant likelihood in nucleic acid sequencing comprising:
 (a) obtaining measured values corresponding to an ensemble of sequencing reads for at least some template polynucleotide strands in at least one defined space, wherein a plurality of template polynucleotide strands, sequencing primers, and polymerase have been provided in a plurality of defined spaces disposed on a sensor array, and wherein the plurality of template polynucleotide strands, sequencing primers, and polymerase have been exposed to a series of flows of nucleotide species according to a predetermined order; and   (b) evaluating a likelihood that a variant sequence is present given the measured values corresponding to the ensemble of sequencing reads, the evaluating comprising:
 (i) determining a measurement confidence value for each read in the ensemble of sequencing reads, wherein the determining is based on variations between the measured values and model-predicted values for hypothesized sequences obtained using a predictive model of nucleotide incorporations responsive to flows of nucleotide species; and 
 (ii) modifying at least some model-predicted values using a first bias for forward strands and a second bias for reverse strands, wherein the modifying is based on variations between model-predicted values for different hypothesized sequences obtained using the predictive model of nucleotide incorporations responsive to flows of nucleotide species. 
   
     
     
         20 . A system for evaluating variant likelihood in nucleic acid sequencing, including:
 a plurality of template polynucleotide strands, sequencing primers, and polymerase provided in a plurality of defined spaces disposed on a sensor array;   an apparatus configured to expose the plurality of template polynucleotide strands, sequencing primers, and polymerase to a series of flows of nucleotide species according to a predetermined order;   a machine-readable memory; and   a processor configured to execute machine-readable instructions, which, when executed by the processor, cause the system to perform a method for evaluating variant likelihood, comprising:   (a) obtaining measured values corresponding to an ensemble of sequencing reads for at least some of the template polynucleotide strands in at least one of the defined spaces; and   (b) evaluating a likelihood that a variant sequence is present given the measured values corresponding to the ensemble of sequencing reads, the evaluating comprising:
 (i) determining a measurement confidence value for each read in the ensemble of sequencing reads, wherein the determining is based on variations between the measured values and model-predicted values for hypothesized sequences obtained using a predictive model of nucleotide incorporations responsive to flows of nucleotide species; and 
 (ii) modifying at least some model-predicted values using a first bias for forward strands and a second bias for reverse strands, wherein the modifying is based on variations between model-predicted values for different hypothesized sequences obtained using the predictive model of nucleotide incorporations responsive to flows of nucleotide species.