Gene Fusion Detection
The DRAGEN Gene Fusion module uses the DRAGEN RNA splice-aware aligner to detect gene fusion events. The supplementary (chimeric) alignments are used to find potential breakpoints and read evidence is accumulated for the resulting fusion event candidates. Then, an ML model is applied to score the putative fusion events to filter potential false positives. The ML scoring model is currently available on human samples only, and does not support non-human reference genomes.
Running DRAGEN Gene Fusion
You can run the DRAGEN Gene Fusion module together with a regular RNA-Seq map/align job. To enable the DRAGEN Gene Fusion module, set --enable-rna-gene-fusion to "true". The DRAGEN Gene Fusion module requires a gene annotations file in GTF or GFF format.
The following is an example command line for running an end-to-end RNA-Seq experiment with RNA fusion detection.
At the end of a run, a summary of detected gene fusion events is output, which is like the following example.
Gene Fusion Output
The <output-file-prefix>.fusion_candidates.features.csv file lists the detected gene fusion events. The output CSV file includes the following columns.
#FusionGene: Parent gene names (in 5' to 3' order of transcript) participating in the fusion; hereafter referred to as Gene 1 and Gene 2. If a fusion breakpoint overlaps multiple genes, the genes are listed by default as separate candidates (rows). To show them as a semi-colon separated gene list on the same row, the option--rna-gf-merge-callscan be set to "true" as described in the Gene Fusion Options and Filters section.Score: Fusion call confidence score predicted by the ML model. If the ML model is used, the score can be 0 (low confidence) to 1 (high-confidence call). Currently the ML model only supports human references. In the case an ML model is not available, the number of supporting reads will be reported as the score.LeftBreakpoint: Gene 1 breakpoint formatted as<Chromosome>:<Position>:<Strand>.RightBreakpoint: Gene 2 breakpoint formatted as<Chromosome>:<Position>:<Strand>.Filter: Semicolon separated list of filter flags. TheLOW_SCOREfilter is used to filter low confidence fusion candidates. If--rna-gf-enable-post-filtering=true, other confidence filters will also be applied. Informative filters, on the other hand, do not fail the fusion. In the absence of the ML model scoring (i.e. a non-human reference is used), a more aggressive post-filtering will take place and all confidence and informative filters will be applied.
The following are the available filters.
LOW_SCORE
Confidence (always applied)
The fusion candidate has low score (< 0.5) as determined by the ML model.
--rna-gf-min-score
MIN_SUPPORT
Confidence (optional)
The fusion has < 2 supporting read pairs.
--rna-gf-min-split-support
LOW_UNIQUE_ALIGNMENTS
Confidence (optional)
The minimum number of unique supporting read alignments required at each breakpoint in not met. Unique alignments have unique start and end positions and are not PCR duplicates.
--rna-gf-min-unique-alignments
LOW_MAPQ
Confidence (optional)
All fusion-supporting read alignments at either breakpoint have MAPQ < 20.
--rna-gf-min-breakpoint-mapq
DOUBLE_BROKEN_EXON
Confidence (optional)
If both breakpoints are >50 bp away from annotated exon boundaries, then the number of supporting reads do not satisfy a high threshold requirement (≥10 supporting reads). The distance indicates an intronic fusion.
--rna-gf-exon-snap
--rna-gf-min-support-be
UNENRICHED_GENES
Confidence (optional)
--rna-gf-enriched-only
MITOCHONDRIAL_GENES
Confidence (optional)
The fusion candidate involves mitochondrial genes. Set --rna-gf-filter-chrm=false to disable this filter. Default value is "true".
--rna-gf-filter-chrm
READ_THROUGH
Confidence (optional)
The breakpoints are cis neighbors (< 200,000 bp) on the reference genome.
--rna-gf-min-cis-distance
ANCHOR_SUPPORT
Information only
Read alignments of fusion-supporting reads are not long enough (less than 12 bp) at either breakpoint.
--rna-gf-min-anchor
HOMOLOGOUS
Information only
The candidate is likely to be a false candidate generated because the two genes involved have high gene homology. Default threshold is 1e-100.
--rna-gf-min-blast-pairs-eval
LOW_ALT_TO_REF
Information only
The number of reads supporting the fusion is < 1% of the number of reads supporting the reference transcript at either breakpoint.
--rna-gf-min-alt-to-ref
LOW_GENE_COVERAGE
Information only
Either breakpoint has less than 125 bp with nonzero read coverage.
--rna-gf-min-covered-bases
Note that the specific features and column values are subject to change in future DRAGEN versions as more RNA data is analyzed.
#SplitScore: Combined count of fusion-supporting read pairs reported as split reads and soft-clipped reads#NumSplitReads: Number of fusion-supporting read pairs with at least one split read alignment.#NumSoftClippedReads: Number of fusion-supporting read pairs with no split read alignment, but at least one soft clipped alignment. Included inSplitScoreand includes soft-clipped reads for both Gene1 and Gene2#NumSoftClippedReadsGene1: Number of fusion-supporting read pairs with no split read alignment, but at least one soft clipped alignment to Gene 1#NumSoftClippedReadsGene2: See above (NumSoftClippedReadsGene1) for Gene 2#NumPairedReads: Number of fusion-supporting read pairs such that one of the reads maps to Gene1 and the other maps to Gene2, without any breakpoint overlap#NumRefSplitReadsGene1: Number of read pairs that map fully within Gene 1 such that at least one of the reads aligns across the breakpoint. These reads support the reference transcript and do not support the fusion.#NumRefSplitReadsGene2: See above (NumRefSplitReadsGene1) for Gene 2#NumRefPairedReadsGene1: Number of read pairs such that one of the reads maps on the left side of the Gene 1 breakpoint and the other maps on the right side of the Gene 1 breakpoint, without overlapping the break. These reads support the reference transcript and do not support the fusion.#NumRefPairedReadsGene2: See above (NumRefPairedReadsGene1) for Gene 2#RefToAlt-- Log2 value of the ratio of max(NumRefSplitReadsGene1, NumRefSplitReadsGene2) / (fusion split + soft clipped reads); used for theLOW_ALT_TO_REFfilter#UniqueAlignmentsGene1: Unique (start-end) positions of fusion-supporting read alignments to Gene 1 (after dedup); used for theLOW_UNIQUE_ALIGNMENTSfilter#UniqueAlignmentsGene2: Unique (start-end) positions of fusion-supporting read alignments to Gene 2 (after dedup); used for theLOW_UNIQUE_ALIGNMENTSfilter#MaxMapqGene1: Maximum MAPQ for fusion-supporting reads in Gene 1#AvgMapqGene1: Average MAPQ for fusion-supporting reads in Gene 1#MaxMapqGene2: Maximum MAPQ for fusion-supporting reads in Gene 2#AvgMapqGene2: Average MAPQ for fusion-supporting reads in Gene 2#CoverageBasesGene1: Bases in Gene 1 with read coverage within a certain distance (default 1000 bp) of the breakpoint in the direction of the breakpoint strand which is part of the fusion transcript#CoverageBasesGene2: See above (CoverageBasesGene1) for Gene 2#DeltaExonBoundaryGene1: Distance from the Gene 1 breakpoint for the closest fusion-supporting alignment (higher distance to boundary lowers score)#DeltaExonBoundaryGene2: See above (DeltaExonBoundaryGene1) for Gene 2#IsRestrictedGene1: Indicator variable of whether Gene 1 is tagged as protein coding in the annotation file#IsRestrictedGene2: Indicator variable of whether Gene 2 is tagged as protein coding in the annotation file#IsEnrichedGene1: If enrichment or amplicon assay, then indicates whether Gene 1 is enriched. If whole transcriptome sequencing, then set to 1#IsEnrichedGene2: See above (IsEnrichedGene1) for Gene 2#CisDistance: Distance between breakpoints if they are adjacent to each other and on the same strand. Large value (3.2G) if not a CIS break; used for theREAD_THROUGHfilter.#BreakpointDistance: Distance between breakpoints if they are adjacent. Large value (3.2G) if not within same chromosome#GenePairHomologyEval: E-value of pairwise BLAST alignment of the parent genes#AnchorLength1: Longest alignment of a fusion-supporting read to Gene 1#AnchorLength2: Longest alignment of a fusion-supporting read to Gene 2#NormalizedAnchorLength1: Normalized value ofAnchorLength1by the maximum read length.#NormalizedAnchorLength2: Normalized value ofAnchorLength2by the maximum read length.#FusionLengthGene1: Distance from breakpoint to the end of Gene 1#FusionLengthGene2: Distance from breakpoint to the end of Gene 2#NonFusionLengthGene1: Breakpoint distance to the end of transcript not part of the fusion for Gene 1#NonFusionLengthGene2: Breakpoint distance to the end of transcript not part of the fusion for Gene 2#Gene1Id: Gene ID reported in the annotation file for Gene 1#Gene2Id: Gene ID reported in the annotation file for Gene 2#Gene1Location:IntactExon: Breakpoint matches exon boundary,
BrokenExon: Breakpoint is within an exon but does not match the exon boundary,
Intron: Breakpoint is within an intron,
Intergenic: Breakpoint does not overlap any gene
#Gene2Location: See above (Gene1Location) for Gene 2#Gene1Sense: "TRUE" if the Gene 1 5' to 3' direction matches the breakpoint order, indicating that the gene is the upstream gene in the fusion transcript#Gene2Sense: See above (Gene1Sense) for Gene 2
In addition, if --rna-gf-merge-calls is enabled, DRAGEN will merge the fusion candidates that overlap the same breakpoint into a single row reporting the feature values for the highest scoring passing candidate (or highest scoring failing candidate if no passing candidate is reported). For each breakpoint, in the column #FusionGene, it reports a semi-colon separated list of names of all overlapping genes with a passing candidate. The following two columns are added to the features.csv output file:
#AdditionalGenes1: If a mix of passing and failing candidates are reported for the same breakpoint of Gene 1, genes with only failing candidates are listed. If no passing candidate exists, then all overlapping genes are reported in the#FusionGenecolumn.#AdditionalGenes2: See above (AdditionalGenes1) for Gene 2
The <output-file-prefix>.fusion_candidates.final output file lists each passing fusion along with the read names that support the fusion, including Split Reads, Soft-clipped reads, and Paired (discordant) Reads and the passing scores. These reads can be extracted from the output BAM file and then used to visualize the fusions (i.e. in IGV). The same information for the non-passing fusions is provided in the <output-file-prefix>.filter_info output file.
The <output-file-prefix>.fusion_candidates.vcf.gz output file provides the VCF representation for all of the breakpoints for the candidate fusions using structural variant-style BND notation. The VCF header is annotated with ##source=DRAGEN_RNA_GF to indicate the file is generated by the DRAGEN RNA Gene Fusion pipeline. All fusion candidates (passing and failing) are represented in the VCF output with one entry for each side of the fusion breakpoint (Gene 1 and Gene 2).
The <output-file-prefix>.final.fusion_candidates.vcf.gz output file provides the filtered VCF for all passing breakpoints for the candidate fusions.
The <output-file-prefix>.fusion_metrics.csv output file provides a simple count of the total number of fusion candidates, those passing the scoring filter, and the number of unique left-right gene combinations that are found.
Gene Fusion Options and Filters
The following thresholds and options may be used to configure the fusion caller:
--rna-gf-enriched-regionsAlternative to--rna-gf-enriched-genes, but input is provided as a bed-file with regions' coordinates instead of a gene list. All the genes in the provided annotation file that overlap such regions are included. Genes that are extracted in this way are summarized in output in the*.fusion.enriched_genes.txtfile. This option cannot be provided together with--rna-gf-enriched-genes.--rna-repeat-genesText file that contains the names or IDs (from the annotation file) of targeted repetitive genes for sensitive fusion detection. Exclusive from--rna-repeat-intervals. This option overrides the default BED file. The repeat genes list should only contain genes listed in the input annotation file.--rna-repeat-intervalsBED file that contains a target list of repeat intervals for sensitive fusion detection. Exclusive from--rna-repeat-genes. This option overrides the default files, which contain the genes CIC, DUX4, NPM1, PSPH, and SEPTIN14 for GRCh38 and hg19 reference genomes.--rna-gf-restrict-genesWhen parsing the gene annotations file for use in the DRAGEN Gene Fusion module, you can use this option to restrict the entries of interest to only protein-coding regions. Restricting the annotation to only the protein-coding genes reduces false positive rates in currently studied fusion events. To report non-coding gene fusions such as pseudo genes and lincRNAs, turn off this option. The default value is "true".--rna-gf-merge-callsIf multiple genes overlap a fusion breakpoint, DRAGEN generates and scores a separate fusion candidate for each gene pair overlapping the breakpoint. The default value is "false" so that each reported fusion event only has one left and right gene in the fusion, and overlapping genes are output as separate events.--rna-gf-allow-overlapping-genesAllows for fusion calls between overlapping genes. The default value is "false".--rna-gf-enable-post-filtersEnable post-filtering of RNA gene fusion candidates by confidence flags. The filter flags are listed in the table above. The default value is "false".--rna-gf-output-fusion-sequenceAdd a "FusionSequence" column for all passing fusions in the<output-file-prefix>.fusion_candidates.finalfile based on the contig assembly of all supporting reads. If no assembly was generated, then "NoAssembly" is reported. Setting this option to "true" also updates the fusion breakpoints based on the alignment of the assembled contig to the reference for the passing fusions in the<output-file-prefix>.fusion_candidates.finalfile and the output VCF files. The left and right breakpoint positions are chosen such that the alignment score between the assembled contig and the reference sequences is maximized. If there are multiple maximal alignments, the positions minimizing the distance from the original fusion breakpoints is reported. An additional "BreakpointLeeway" column is also added to the<output-file-prefix>.fusion_candidates.finalfile. This column has the form "-XX|+YY" where XX is the number of bases the reported breakpoint can be shifted left (relative to the left side of the fusion) while maintaining the maximal alignment score and YY is similarly the number of bases it can be shifted to the right. For example, "-2|+1" indicates the breakpoint could be shifted 2 to the left or 1 to the right and still have a maximal alignment score (due to identical bases occurring adjacent to the breakpoint). "NA" is output if no assembly is generated. The default value for this option is "true".--rna-gf-sv-vcfStructural Variant VCF file output from DRAGEN DNA structural variant caller run in somatic mode. See below for more information.
Merging Fusion Caller with the Splice Variant Caller
When the splice variant caller and gene fusion caller are both enabled, the passing and failed intergenic splice variants will be passed to the gene fusion caller to be reported as candidate fusion events. This merging only occurs for genomes supported by the ML model for gene fusions (currently only Human genomes are supported). The passing calls are output to the fusion caller's <output-file-prefix>.fusion_candidates.final file. The tab separated fields are described below.
Field Names
Description
FusionGene
Left and Right gene names (separated by "--")
Score
Value between 0 and 1, from the splice variant caller
LeftBreakpoint, RightBreakpoint
The location for left and right sides of the splice with three colon separated fields: chromosome:coordinate:strand(+/-)
Gene1Location, Gene2Location
Splice Variant caller always outputs "SpliceVar" here instead of Exon/Intron location
Gene1Sense, Gene2Sense
Always TRUE for by design
Gene1Id, Gene2Id
Long form ID (i.e. for Gencode it is usually "ENSG.version")
NumSplitReads
Taken from the split_unique_reads_alt column value of the splice_varian_fusions.tsv file
NumSoftClippedReads, NumPairedReads
These values are not used by RSV caller and are set to "0"
ReadNames
Not provided by this caller and set to "N/A"
The passing splice variant calls will also be output to the VCF outputs: <output-file-prefix>.final.fusion_candidates.vcf and <output-file-prefix>.fusion_candidates.vcf with the "SPLICE_VARIANT" flag in the info field.
Reporting read-through fusions
Read-through gene fusions occur when neighboring genes are spliced together. These fusions are detected by the Splice Variant Caller as intergenic splice variants on adjacent genes and by default are not passed to the gene fusion caller. To detect them, enable the gene fusion and splice caller together with the following options:
Running RNA fusion detection with somatic SV evidence
When the SV VCF input is provided to the RNA fusion caller, the following additional features will be reported in the features.csv output file:
#SvEvent: A semi-colon separated string representation of SV events matching the fusion candidate.#SvType: A semi-colon separated list of type of the matching SV events.#SomaticScore: The highest SomaticScore value of the matching SV events.#SvDistance: The maximum distance between any SV breakpoint to any fusion breakpoints (if multiple matching SV events, then minimum of all maximum distances over all SV events).#LeftSvDistance: The distance between the left fusion breakpoint and the corresponding SV breakpoint (if multiple matching SV events, then minimum over all SV events).#RightSvDistance: The distance between the right fusion breakpoint and the corresponding SV breakpoint (if multiple matching SV events, then minimum over all SV events).#SvPresent: Set to 1 if matching SV event is present, otherwise 0.#SvAbsent: Set to 1 if no matching SV event is present, otherwise 0.
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