Methods and systems for analyzing image data

Abstract

Methods and systems for analysis of image data generated from various reference points. Particularly, the methods and systems provided are useful for real time analysis of image and sequence data generated during DNA sequencing methodologies.

What is claimed is: 
     
         1 . A system comprising:
 at least one processor; and   a non-transitory computer readable medium comprising instructions that, when executed by the at least one processor, cause the system to:
 determine a base call for a nucleotide base of a polynucleotide; 
 generate an online-overlap metric, a purity metric, a phasing metric, and a start5 metric for the base call; 
 determine a quality score for the base call based on the online-overlap metric, the purity metric, the phasing metric, and the start5 metric; and 
 generate output-base-calling data comprising the base call for the nucleotide base and the quality score. 
   
     
     
         2 . The system of  claim 1 , further comprising instructions that, when executed by the at least one processor, cause the system to determine the online-overlap metric for the base call by determining a measure of separation between foreground intensity values corresponding to the nucleotide base and background intensity values. 
     
     
         3 . The system of  claim 1 , further comprising instructions that, when executed by the at least one processor, cause the system to determine the purity metric for the base call by determining a likelihood that the base call for the nucleotide base is reliable based on data for a current sequencing cycle. 
     
     
         4 . The system of  claim 1 , further comprising instructions that, when executed by the at least one processor, cause the system to determine the phasing metric for the base call by determining, for a signal corresponding to the base call, a measure of noise from a previous sequencing cycle and a next sequencing cycle. 
     
     
         5 . The system of  claim 1 , further comprising instructions that, when executed by the at least one processor, cause the system to determine the start5 metric for the base call by determining whether the nucleotide base for the base call is located within an initial set of nucleotide bases of a read or determined during an initial set of sequencing cycles. 
     
     
         6 . The system of  claim 1 , further comprising instructions that, when executed by the at least one processor, cause the system to:
 determine the quality score is above or below a quality-score threshold;   categorize the quality score for the base call in a group of quality scores based on the quality score being above or below the quality-score threshold; and   generate the output-base-calling data comprising an indicator that the quality score for the base call corresponds to the group of quality scores.   
     
     
         7 . The system of  claim 1 , further comprising instructions that, when executed by the at least one processor, cause the system to determine the quality score for the base call from a quality table generated by a Phred algorithm based on online-overlap metrics, purity metrics, phasing metrics, and start5 metrics. 
     
     
         8 . The system of  claim 1 , further comprising instructions that, when executed by the at least one processor, cause the system to:
 determine that the quality score does not satisfy a reliability threshold; and   discount the quality score as unreliable based on the quality score not satisfying the reliability threshold.   
     
     
         9 . The system of  claim 8 , further comprising instructions that, when executed by the at least one processor, cause the system to determine the reliability threshold by identifying transition points between quality scores for reliable base calls and quality scores for unreliable base calls. 
     
     
         10 . A non-transitory computer-readable medium storing instructions that, when executed by at least one processor, cause a computing device to:
 determine a base call for a nucleotide base of a polynucleotide;   generate an online-overlap metric, a purity metric, a phasing metric, and a start5 metric for the base call;   determine a quality score for the base call based on the online-overlap metric, the purity metric, the phasing metric, and the start5 metric; and   generate output-base-calling data comprising the base call for the nucleotide base and the quality score.   
     
     
         11 . The non-transitory computer-readable medium of  claim 10 , further comprising instructions that, when executed by the at least one processor, cause the computing device to determine the online-overlap metric for the base call by determining a measure of separation between foreground intensity values corresponding to the nucleotide base and background intensity values. 
     
     
         12 . The non-transitory computer-readable medium of  claim 10 , further comprising instructions that, when executed by the at least one processor, cause the computing device to determine the purity metric for the base call by determining a likelihood that the base call for the nucleotide base is reliable relative to other non-called nucleotide bases based on data for a current sequencing cycle. 
     
     
         13 . The non-transitory computer-readable medium of  claim 10 , further comprising instructions that, when executed by the at least one processor, cause the computing device to determine the phasing metric for the base call by determining, for a signal corresponding to the base call, a measure of noise from a previous sequencing cycle and a next sequencing cycle. 
     
     
         14 . The non-transitory computer-readable medium of  claim 10 , further comprising instructions that, when executed by the at least one processor, cause the computing device to determine the start5 metric for the base call by determining whether the nucleotide base for the base call is located within an initial set of nucleotide bases of a read or determined during an initial set of sequencing cycles. 
     
     
         15 . The non-transitory computer-readable medium of  claim 10 , further comprising instructions that, when executed by the at least one processor, cause the computing device to:
 determine the nucleotide base is incorporated at an end of a read for the polynucleotide;   determine that the quality score does not satisfy a reliability threshold; and   discount the quality score as unreliable based on the nucleotide base being incorporated at the end of the read and the quality score not satisfying the reliability threshold.   
     
     
         16 . A method comprising:
 determining a base call for a nucleotide base of a polynucleotide;   generating an online-overlap metric, a purity metric, a phasing metric, and a start5 metric for the base call;   determining a quality score for the base call based on the online-overlap metric, the purity metric, the phasing metric, and the start5 metric; and   generating output-base-calling data comprising the base call for the nucleotide base and the quality score.   
     
     
         17 . The method of  claim 16 , further comprising:
 determining the quality score is above or below a quality-score threshold;   categorizing the quality score for the base call in a group of quality scores based on the quality score being above or below the quality-score threshold; and   generating the output-base-calling data comprising an indicator that the quality score for the base call corresponds to the group of quality scores.   
     
     
         18 . The method of  claim 16 , further comprising determining the quality score for the base call from a quality table generated by a Phred algorithm based on online-overlap metrics, purity metrics, phasing metrics, and start5 metrics. 
     
     
         19 . The method of  claim 16 , further comprising:
 determining that the quality score does not satisfy a reliability threshold; and   discounting the quality score as unreliable based on the quality score not satisfying the reliability threshold.   
     
     
         20 . The method of  claim 19 , further comprising determining the reliability threshold by identifying transition points between quality scores for reliable base calls and quality scores for unreliable base calls.