Methods and applications of gene fusion detection in cell-free dna analysis

Abstract

Systems and methods are disclosed for determining gene fusion by determining a fused read containing sequencing data of a portion of a fused chromosome DNA molecule; determining a predetermined point on the genome with least one mapped portion of the fused read clipped at the predetermined point (a breakpoint); identifying two mapped read portions from two breakpoints (breakpoint pair) as a potential fusion candidate; creating one or more fusion sets based on breakpoint pairs and clustering the fusion sets into one or more fusion clusters; and identifying each fusion cluster meeting a predetermined criterion as a gene fusion.

What is claimed is: 
     
         1 . A method for processing genetic sequence read data from a sample, the method comprising:
 determining a fused read containing sequencing data of at least a portion of a fused chromosome DNA molecule;   determining a predetermined point on the genome with least one mapped portion of the fused read clipped at the predetermined point (a breakpoint);   identifying two mapped read portions from two breakpoints (breakpoint pair) as a potential fusion candidate;   creating one or more fusion sets based on breakpoint pairs and clustering the fusion sets into one or more fusion clusters; and   identifying each fusion cluster meeting a predetermined criterion as a gene fusion.   
     
     
         2 . The method of  claim 1 , comprising assigning a unique molecule or read identifier (read ID) to each read. 
     
     
         3 . The method of  claim 1 , comprising clipping each mapped portion of the reads from one or both sides. 
     
     
         4 . The method of  claim 1 , wherein the breakpoints are independent of the reads in identity and are identified by a sign, a chromosome and a position. 
     
     
         5 . (canceled) 
     
     
         6 . The method of  claim 2 , comprising selecting every two mapped read portions with common read IDs that belong to two breakpoints with appropriate signs as a potential fusion candidate. 
     
     
         7 . The method of  claim 6 , wherein the potential fusion candidate location in the original read before mapping shows the read portion as originally located next to each other. 
     
     
         8 . The method of  claim 6 , comprising checking if read portions are mapped on one strand for differences in the breakpoints' signs. 
     
     
         9 . (canceled) 
     
     
         10 . (canceled) 
     
     
         11 . The method of  claim 1 , comprising grouping fusion sets with similar breakpoints in a fusion cluster. 
     
     
         12 . The method of  claim 11 , wherein the similar breakpoints are breakpoints no more than 5 nucleotides, no more than 10 nucleotides or no more than 25 nucleotides apart. 
     
     
         13 . (canceled) 
     
     
         14 . The method of  claim 1 , comprising determining for the fusion cluster one or more of a number of fused molecules for each partner, a number of fused reads for each partner, a number of wild-type reads or molecules for each partner, or a fusion percentage for each partner as a ratio of fused molecules to total number of molecules for each partner. 
     
     
         15 . (canceled) 
     
     
         16 . (canceled) 
     
     
         17 . (canceled) 
     
     
         18 . (canceled) 
     
     
         19 . (canceled) 
     
     
         20 . (canceled) 
     
     
         21 . The method of  claim 1 , comprising determining a downstream gene of the fusion cluster. 
     
     
         22 . The method of  claim 1 , wherein the criteria comprises having more than one molecule in the cluster or having at least a molecule with both Watson and Crick strands. 
     
     
         23 . (canceled) 
     
     
         24 . A method comprising:
 (a) sequencing DNA molecules with a DNA sequencer to generate a collection of sequences;   (b) mapping the collection of sequences to a reference genome;   (c) identifying fused reads from the mapped collection, wherein a fused read contains sub-sequences, wherein a first sub-sequence of the sub-sequences maps to a first genetic locus and a second sub-sequence of the sub-sequences maps to a second, distinct genetic locus;   (d) for each fused read, identifying a first breakpoint at the first genetic locus and a second breakpoint at the second genetic locus, wherein a breakpoint is a point on the reference genome where a sequence of a fused read is clipped, and wherein the first and second breakpoints form a breakpoint pair;   (e) generating sets of fused reads, each set comprising fused reads having the same breakpoint pair;   (f) clustering sets of fused reads, wherein each cluster is formed from sets of fused reads having first breakpoints within a first predetermined nucleotide distance and second breakpoints within a second predetermined nucleotide distance; and   (g) determining a gene fusion for one or more clusters, wherein a gene fusion for a cluster has, as a first fusion gene breakpoint, a breakpoint selected from the first breakpoints in the cluster and, as a second fusion gene breakpoint, a breakpoint selected from the second breakpoints in the cluster, and wherein the first and second fusion gene breakpoints are each selected based on selection criteria.   
     
     
         25 . The method of  claim 24 , wherein the distinct genetic loci are located on different chromosomes or on different genes of the same chromosome. 
     
     
         26 . The method of  claim 24 , wherein the first and second predetermined distances are each no more than 5 nucleotides, no more than 10 nucleotides or no more than 25 nucleotides. 
     
     
         27 . The method of  claim 24 , wherein the selection criteria include the breakpoint having the most fused reads in the cluster. 
     
     
         28 . The method of  claim 24 , comprising determining a gene fusion for a plurality of gene clusters. 
     
     
         29 . A method comprising:
 (a) sequencing a plurality of DNA molecules with a DNA sequencer;   (b) tagging each of the plurality of sequenced molecules with an identifier thereby generating tagged sequences;   (c) mapping each of the tagged sequences to a reference genome;   (d) identifying clipped reads from the mapped tagged sequences, wherein a clipped read is a tagged sequence containing a mapped portion and a clipped portion, wherein the mapped portion maps to a genetic locus and the clipped portion does not map to the genetic locus;   (e) determining a breakpoint of each clipped read, wherein a breakpoint is a point on the reference genome where a sequence of a clipped read is clipped;   (f) creating breakpoint sets, each breakpoint set comprising identifiers of clipped reads having the same breakpoint;   (g) creating sets of breakpoint pairs by comparing pairs of breakpoint sets, each set of breakpoint pairs including identifiers present in both members of a compared pair of breakpoint sets;   (h) clustering sets of breakpoint pairs, wherein each cluster includes sets of breakpoint pairs having a first breakpoint of the pair within a first predetermined genetic distance and a second breakpoint of the pair within a second predetermined genetic distance; and   (i) determining a gene fusion for one or more of the clusters, wherein a gene fusion for a cluster has, as a first fusion gene breakpoint, a breakpoint selected from the first breakpoints in the cluster and, as a second fusion gene breakpoint, a breakpoint selected from the second breakpoints in the cluster, and wherein the first and second fusion gene breakpoints are each selected based on a selection criteria.   
     
     
         30 . The method of  claim 29 , wherein the selection criteria include the breakpoint having the most fused reads in the cluster. 
     
     
         31 . (canceled) 
     
     
         32 . (canceled) 
     
     
         33 . (canceled) 
     
     
         34 . (canceled) 
     
     
         35 . (canceled) 
     
     
         36 . The method of  claim 1 , further comprising identifying a breakpoint of the gene fusion as the fusion gene breakpoint.