Systems and methods for off-target sequence detection

Abstract

A computer-implemented method, computer system and computer-readable medium for identifying off-target matches from a set of candidate primer sequences on a genome reference sequence can include: receiving onto a data storage unit a plurality of candidate primer sequences; for each candidate primer sequence, calculating, using a processor, a plurality of candidate matches on the genome reference sequence for the candidate primer sequences; calculating, using the processor, verified matches on the genome reference sequence based on the candidate matching locations satisfying a plurality of matching verification rules; performing matching calculations of the verified matches to determine whether the verified matches form a match condition on the genome reference sequence; and generating a location profile on the genome reference sequence based on the match condition from the verified matches that meet a predetermined threshold.

1 - 20 . (canceled) 
     
     
         21 . A computer-implemented method of identifying off-target matches from a set of candidate primer sequences on a genome reference sequence, the method comprising:
 receiving onto a data storage unit a plurality of candidate primer sequences;   decomposing each candidate primer sequence into k-mers comprising substrings or subsequences of length k;   for each candidate primer sequence, identifying a plurality of candidate matches on the genome reference sequence for the candidate primer sequences, wherein identifying the plurality of candidate matches comprises searching a k-mer index of the genome reference sequence for occurrences of each k-mer;   calculating verified matches on the genome reference sequence based on the candidate matches satisfying a plurality of matching verification rules, wherein calculating the verified matches comprises reusing one or more rule satisfaction calculations for k-mers when present in a cache;   performing matching calculations to determine off-target match conditions for one or more of the verified matches with respect to the genome reference sequence; and   selecting, in response to determining off-target match conditions, candidate primer sequences that do not form an off-target match on the genome reference sequence as acceptable primer sequences for use in a multiplex PCR assay.   
     
     
         22 . The method of  claim 21 , further comprising updating the cache with one or more respective rule satisfaction calculations for respective k-mers based on the respective k-mers satisfying one or more matching verification rules. 
     
     
         23 . The method of  claim 22 , wherein the one or more matching verification rules comprise a rule that there are at least k consecutive matching nucleotides. 
     
     
         24 . The method of  claim 22 , wherein the one or more matching verification rules comprise a rule that a threshold number of nucleotides mismatches is not exceeded. 
     
     
         25 . The method of  claim 24 , wherein the threshold number is a function of the length of the respective candidate primer sequence. 
     
     
         26 . The method of  claim 22 , wherein the one or more matching verification rules comprise a rule that a threshold number of nucleotide mismatches on an end of the respective candidate primer sequence is not exceeded. 
     
     
         27 . The method of  claim 22 , wherein identifying the plurality of candidate matches comprises reusing one or more rule satisfaction calculations for k-mers when present in the cache. 
     
     
         28 . The method of  claim 21 , further comprising generating a location profile on the genome reference sequence based on the off-target match condition from the verified matches that meet a predetermined off-target threshold. 
     
     
         29 . The method of  claim 21 , wherein:
 the cache is organized based on different sequence lengths.   
     
     
         30 . The method of  claim 21 , wherein the candidate matches comprise a candidate matching location of the corresponding candidate primer sequence on the genome reference sequence, and
 wherein the verified matches comprise a verified matching location of the verified matches on the genome reference sequence.   
     
     
         31 . The method of  claim 21 , wherein the plurality of candidate primer sequences comprises a first candidate primer sequence, the method further comprising:
 generating a prediction of a number of matches on the reference genome sequence for the first candidate primer sequence by comparing k-mers derived from the first candidate primer sequence to the k-mer index of the genome reference sequence;   responsive to determining that the predicted number of matches exceeds a threshold, discarding the first candidate primer sequence from further evaluation.   
     
     
         32 . The method of  claim 21  further comprising:
 clustering a plurality of sequences corresponding to the verified matches into sequence proximity groupings; and 
 using the sequence proximity groupings to identify the off-target match condition. 
 
     
     
         33 . The method of  claim 32  wherein candidate primer sequences that form an off-target match cause amplification of non-target sequences or interference with amplification of target locations. 
     
     
         34 . A computing system for identifying off-target matches from a set of candidate primer sequences on a genome reference sequence, the computing system comprising:
 at least one processor; and   a memory storing instructions that, when executed by the at least one processor, causes the computing system to perform:
 receiving onto a data storage unit a plurality of candidate primer sequences; 
 decomposing each candidate primer sequence into k-mers comprising sub strings or subsequences of length k; 
 for each candidate primer sequence, identifying a plurality of candidate matches on the genome reference sequence for the candidate primer sequences, wherein identifying the plurality of candidate matches comprises searching a k-mer index of the genome reference sequence for occurrences of each k-mer; 
 calculating verified matches on the genome reference sequence based on the candidate matches satisfying a plurality of matching verification rules, wherein calculating the verified matches comprises reusing one or more rule satisfaction calculations for k-mers when present in a cache; 
 performing matching calculations to determine off-target match conditions for one or more of the verified matches with respect to the genome reference sequence; and 
 selecting, in response to determining off-target match conditions, candidate primer sequences that do not form an off-target match on the genome reference sequence as acceptable primer sequences for use in a multiplex PCR assay. 
   
     
     
         35 . The computing system of  claim 34 , wherein:
 the cache is coupled to the memory, and   wherein the cache stores rule satisfaction calculations separately for different parameters of a respective rule.   
     
     
         36 . The computing system of  claim 34  wherein the memory further stores instructions that when executed by the at least one processor causes the computer system to perform skipping a rule satisfaction calculation for at least one of the candidate primer sequences based on rule satisfaction calculations stored in the cache. 
     
     
         37 . The computing system of  claim 34  wherein:
 the cache is organized based on different sequence lengths. 
 
     
     
         38 . The computing system of  claim 34  wherein the plurality of matching verification rules comprise a rule that there are at least k consecutive matching nucleotides. 
     
     
         39 . The computing system of  claim 34  wherein the plurality of matching verification rules comprise a rule that a threshold number of nucleotide mismatches is not exceeded. 
     
     
         40 . The computing system of  claim 34 , wherein the plurality of matching verification rules comprise a rule that a threshold number of nucleotide mismatches on an end of the respective candidate primer sequence is not exceeded. 
     
     
         41 . A non-transitory computer-readable storage medium for identifying off-target matches from a set of candidate primer sequences on a genome reference sequence comprising computer-executable instructions that when executed cause a computing system to:
 receive onto a data storage unit a plurality of candidate primer sequences;   decompose each candidate primer sequence into k-mers comprising substrings or sub sequences of length k;   for each candidate primer sequence, identify a plurality of candidate matches on the genome reference sequence for the candidate primer sequences, wherein identifying the plurality of candidate matches comprises searching a k-mer index of the genome reference sequence for occurrences of each k-mer;   calculate verified matches on the genome reference sequence based on the candidate matches satisfying a plurality of matching verification rules, wherein calculating the verified matches comprises reusing one or more rule satisfaction calculations for k-mers when present in a cache;   perform matching calculations to determine off-target match conditions for one or more of the verified matches with respect to the genome reference sequence; and   selecting, in response to determining off-target match conditions, candidate primer sequences that do not form an off-target match on the genome reference sequence as acceptable primer sequences for use in a multiplex PCR assay.