Alignment free filtering for identifying fusions

Abstract

Cell free nucleic acids from a test sample obtained from an individual are analyzed to identify possible fusion events. Cell free nucleic acids are sequenced and processed to generate fragments. Fragments are decomposed into kmers and the kmers are either analyzed de novo or compared to targeted nucleic acid sequences that are known to be associated with fusion gene pairs of interest. Thus, kmers that may have originated from a fusion event can be identified. These kmers are consolidated to generate gene ranges from various genes that match sequences in the fragment. A candidate fusion event can be called given the spanning of one or more gene ranges across the fragment.

What is claimed is: 
     
         1 . A kit comprising:
 reagents for isolating nucleic acids from a test sample and sequencing the isolated nucleic acids to obtain a plurality of sequence read pairs;   instructions for using the reagents; and   a non-transitory computer readable storage medium comprising instructions for the processing the plurality of sequence read pairs that when executed by a computer, cause the computer to:
 query an index structure with one or more kmers to determine which of the one or more matches a gene segment of one or more genes, each kmer having a length of k nucleic acids; 
 determine a maximum gene span for a pair of genes based on a plurality of gene ranges, each gene range comprising one or more kmers on a gene segment of one gene of the pair that map to a kmer in the index structure; and 
 assign a candidate gene fusion event to the pair of genes based on the maximum gene span. 
   
     
     
         2 . The kit of  claim 1 , wherein the test sample comprises cell-free RNA nucleic acid fragments and the plurality of sequence read pairs comprise RNA sequencing reads. 
     
     
         3 . The kit of  claim 1 , wherein the test sample comprises cell-free DNA nucleic acid fragments and the plurality of sequence read pairs comprise DNA sequencing reads. 
     
     
         4 . The kit of  claim 1 , wherein the instructions for using the reagents comprise one or more of:
 instructions for extracting the nucleic acid from the tests sample;   instructions describing the order in which the reagents are to be added; and   centrifugal speeds for isolating nucleic acids from the test sample.   
     
     
         5 . The kit of  claim 1 , wherein the instructions stored on the computer-readable medium further comprise instructions that, when executed by the processor, cause the computer to:
 generate a plurality of kmers from the plurality of sequence read pairs, each kmer having a length of k nucleic acids.   
     
     
         6 . The kit of  claim 5 , wherein the instructions to generate the plurality of kmers from the plurality of sequence read pairs further comprise instructions that, when executed by the processor, cause the computer to:
 process one or more of the plurality of sequence read pairs to generate a plurality of fragments; and   for each fragment of the plurality of fragments, decompose the fragment into the plurality of kmers.   
     
     
         7 . The kit of  claim 6 , wherein the instructions to process one or more of the plurality of sequence read pairs to generate a plurality of fragments further comprise instructions that, when executed by the processor, cause the computer to:
 decompose a sequence read pair comprising a first sequence read and a second sequence read into a plurality of kmers;   identify a common kmer from the plurality of kmers that occurs in the first sequence read and second sequence read; and   concatenate a prefix of the first sequence read, the common kmer, and a suffix of the second sequence read.   
     
     
         8 . The kit of  claim 6 , wherein the instructions to process one or more of the plurality of sequence read pairs further comprise instructions that, when executed by the processor, cause the computer to:
 identify an overlapping region between a first sequence read of a sequence read pair and a second sequence read of the sequence read pair; and   trim an overhang of the first sequence read that is beyond the second sequence read.   
     
     
         9 . The kit of  claim 6 , wherein the instructions to process one or more of the plurality of sequence read pairs further comprise instructions that, when executed by the processor, cause the computer to concatenate a first sequence read of a sequence read pair with a reverse of a second sequence read of the sequence read pair. 
     
     
         10 . The kit of  claim 1 , wherein the instructions stored on the computer-readable medium further comprise instructions that, when executed by the processor, cause the computer to:
 generate a plurality of gene ranges, each gene range comprising one or more kmers that map to a gene segment from a gene and map to a kmer in the index structure   
     
     
         11 . The kit of  claim 1 , wherein the instructions to determine the maximum gene span further comprise instructions that, when executed by the processor, cause the computer to:
 for one or more pairs of gene ranges of the plurality of gene ranges, determine a score for each pair of gene ranges, wherein each of the one or more pairs of gene ranges are derived from two different genes; and   determine a maximally scored pair of gene ranges, the maximally scored pair of gene ranges derived from the first gene and the second gene.   
     
     
         12 . The kit of  claim 11 , wherein the score determined for each pair of gene ranges comprises a summation of a length of a first gene range of the pair of gene ranges, a length of a second gene range of the pair of gene ranges, and one or more lengths of gene ranges that have a lower order in comparison to the first gene range. 
     
     
         13 . The kit of  claim 12 , wherein each of the first gene range and gene ranges corresponding to the one or more lengths of gene ranges are derived from a common gene. 
     
     
         14 . The kit of  claim 1 , wherein the instructions to determine the maximum gene span further comprise instructions that, when executed by the processor, cause the computer to:
 compare a length of the determined maximum gene span to a threshold length; and   identify the first gene and the second gene as corresponding to the candidate gene fusion event based on the comparison.   
     
     
         15 . The kit of  claim 1 , wherein the instructions stored on the computer-readable medium further comprise instructions that, when executed by the processor, cause the computer to:
 remove one or more of the plurality of sequence read pairs that are of low complexity, wherein a low complexity sequence read pair includes one or two nucleotides that occur over a threshold percentage of all nucleotides in the sequence read pair.   
     
     
         16 . The kit of  claim 1 , wherein the instructions stored on the computer-readable medium further comprise instructions that, when executed by the processor, cause the computer to:
 remove one or more of the plurality of sequence read pairs that are of low complexity, wherein a low complexity sequence read pair is less than a threshold read length.   
     
     
         17 . The kit of  claim 1 , wherein the instructions stored on the computer-readable medium further comprise instructions that, when executed by the processor, cause the computer to:
 pre-filter the plurality of sequence read pairs to remove sequence read pairs that meet one or more criteria prior to generating the plurality of kmers from the plurality of sequence read pairs.   
     
     
         18 . The kit of  claim 17 , wherein the one or more criteria comprise whether a sequence read is a singleton, whether a sequence read is a hard clip, a threshold template length, a threshold alignment score, a threshold base quality score, a bit flag, a cigar, an edit distance, a suboptimal alignment score, or a supplementary alignment measure. 
     
     
         19 . The kit of  claim 1 , wherein the instructions stored on the computer-readable medium further comprise instructions that, when executed by the processor, cause the computer to:
 apply an exon boundary filter to determine whether the candidate gene fusion event is a false positive fusion gene event after assigning the candidate gene fusion event to the first gene and the second gene.   
     
     
         20 . The kit of  claim 1 , wherein the instructions to apply the exon boundary filter further comprise instructions that, when executed by the processor, cause the computer to:
 for each of one or more sequence read pairs of the plurality of sequence read pairs that support the candidate gene fusion event,
 identify a breakpoint of the sequence read pair corresponding to the candidate gene fusion event; 
 identify nucleotide bases within a threshold number of nucleotide bases; and 
 compare the identified nucleotide bases to regions in the first gene and the second gene.