# RNA Output
Refer to [RNA Analysis Methods](https://help.connected.illumina.com/tso500/dragen-tso-500-guides/dragen-tso-500-v2.5/analysis-methods/rna-analysis-methods) for more information.
## Splice Variant VCF
The splice variant VCF contains all candidate splice variants targeted by the analysis panel identified by the RNA analysis pipeline. You can apply the following filters for each variant call:
| Filter Name | Description |
| ------------------- | ---------------------------------------------------------------------------------------------------------------- |
| LowQ | Splice variant score < passing quality score threshold value of 1. |
| PASS | Splice variant score ≥ passing quality score threshold value of 1. |
| LowUniqueAlignments | All splice junction supporting reads map to a unique genomic interval near at least one of the two splice sites. |
Refer to the headers in the output for more information about each column.
## Splice Variant Annotated JSON
If available, each splice variant is annotated using the Illumina Annotation Engine. The following information is captured in the JSON:
* HGNC Gene
* Transcript
* Exons
* Introns
* Canonical
* Consequence
## All Fusions CSV
The all fusions CSV file contains all candidate fusions identified by the DRAGEN RNA pipeline. Two output columns in the file describe the candidate fusions: Filter and KeepFusion.
The following table describes the semicolon-separated output found in the Filter columns. The output is either a confidence filter or information only as indicated. If none of the confidence filters are triggered, the Filter column contains the output PASS, else it contains the output FAIL.
Filter Column Output
| Filter | Filter Type | Description |
| -------------------------- | ----------------- | ------------------------------------------------------------------------------------------------------------------------------------------------------------------- |
| DOUBLE\_BROKEN\_EXON | Confidence filter | If both breakpoints are distant from annotated exon boundaries, the number of supporting reads do not satisfy a high threshold requirement (≥ 10 supporting reads). |
| LOW\_MAPQ | Confidence filter | All fusion supporting read alignments at either of the breakpoints have MAPQ < 20. |
| LOW\_UNIQUE\_ALIGNMENTS | Confidence filter | All fusion supporting read alignments map to a unique genomic interval at either of the breakpoints. |
| LOW\_SCORE | Confidence filter | The fusion candidate has probabilistic score as determined by the features of the candidate. |
| MIN\_SUPPORT | Confidence filter | The fusion candidate has very few fusion supporting reads (< 5 supporting read pairs). |
| READ\_THROUGH | Confidence filter | The breakpoints are cis neighbors (< 200 kbp) on the reference genome. |
| ANCHOR\_SUPPORT | Information only | Read alignments of fusion supporting reads are not long enough (12 bp) at either of the two breakpoints. |
| HOMOLOGOUS | Information only | The candidate is likely a false candidate generated because the two genes involved have high gene homology. |
| LOW\_ALT\_TO\_REF | Information only | The number of fusion supporting reads is < 1% of the number of reads supporting the reference transcript at either of the two breakpoints. |
| LOW\_GENE\_COVERAGE | Information only | Each breakpoint in an enriched gene has fewer than 125 bp with nonzero read coverage. |
| NO\_COMPLETE\_SPLIT\_READS | Confidence filter | For every fusion-supporting split read, the total number of aligned bases across two breakpoints is less 60% of the read length. |
| UNENRICHED\_GENE | Confidence filter | Neither of the two parent genes is in the enrichment panel. |
The KeepFusion column of the output has a value of TRUE when none of the confidence filters are triggered.
Refer to the headers in the output for more information about each column.
Fusion Columns
| Fusion Object Field | Source |
| --------------------------- | ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ |
| Gene A | The gene associated with the A side of the fusion. A semicolon delimited list is used for multiple genes. |
| Gene B | The gene associated with the B side of the fusion. A semicolon delimited list is used for multiple genes. |
| Gene A Breakpoint | \[Information only] The chromosome and offset of the Gene A side of the fusion. |
| Gene A Location | Location of the breakpoint within Gene A:
- IntactExon—Matches exon boundary
- BrokenExon—Inside an exon
- Intronic—Within an intron
- Intergenic—No gene overlap (currently excluded)
If multiple genes are in Gene A, then semicolon separated list of locations. This column is used internally to identify genes to report when a breakpoint occurs in a region overlapping multiple genes. Occasionally, additional values are listed for genes that were excluded from the GeneA list.
|
| Gene A Sense | Boolean indicating whether left/right breakpoint order suggests fusion transcript is in the same sense of Gene A. If multiple genes are in Gene A, then semicolon separated list of bools. |
| Gene A Strand | Strand of Gene A, + for forward, - for reverse. |
| Gene B Breakpoint | \[Information only] The chromosome and offset of the Gene B side of the fusion. |
| Gene B Location | Location of the breakpoint within Gene B:
- IntactExon—Matches exon boundary
- BrokenExon—Inside an exon
- Intronic—Within an intron
- Intergenic—No gene overlap (currently excluded)
If multiple genes in Gene B, then semicolon separated list of locations. This column is used internally to identify genes to report when a breakpoint occurs in a region overlapping multiple genes. Occasionally, additional values are listed for genes that were excluded from the GeneB list.
|
| Gene B Sense | Boolean indicating whether left/right breakpoint order suggests fusion transcript is in the same sense of Gene B. If multiple genes are in Gene B, then semicolon separated list of bools. |
| Gene B Strand | Strand of Gene B, + for forward, - for reverse. |
| Score | The quality of fusion as determined by DRAGEN server. |
| Filter | The filter associated with the fusion as determined by the respective caller. Results from different callers are not equivalent. |
| Ref A Dedup | Gene A uniquely mapping reads paired across or split by the junction. Does not support fusion. Duplicate reads are not included. |
| Ref B Dedup | Gene B uniquely mapping reads paired across or split by the junction. Does not support fusion. Duplicate reads are not included. |
| Alt Split Dedup | Uniquely mapping reads split by the junction. Supports fusion. Duplicate reads are not included. |
| Alt Pair Dedup | Uniquely mapping reads paired across junction. Supports fusion. Duplicate reads are not included. |
| KeepFusion | The determination whether the fusion should be kept or dropped from the list of fusions. |
| Fusion Directionality Known | Whether fusion directionality is known and indicated by gene order. |
When using Microsoft Excel to view this report, genes that are convertible to dates (such as MARCH1 automatically convert to dd-mm format (1 Mar) by Excel. The following are fusion allow list genes:
* ABL1
* AKT3
* ALK
* AR
* AXL
* BCL2
* BRAF
* BRCA1
* BRCA2
* CDK4
* CSF1R
* EGFR
* EML4
* ERBB2
* ERG
* ESR1
* ETS1
* ETV1
* ETV4
* ETV5
* EWSR1
* FGFR1
* FGFR2
* FGFR3
* FGFR4
* FLI1
* FLT1
* FLT3
* JAK2
* KDR
* KIF5B
* KIT
* KMT2A
* MET
* MLLT3
* MSH2
* MYC
* NOTCH1
* NOTCH2
* NOTCH3
* NRG1
* NTRK1
* NTRK2
* NTRK3
* PAX3
* PAX7
* PDGFRA
* PDGFRB
* PIK3CA
* PPARG
* RAF1
* RET
* ROS1
* RPS6KB1
* TMPRSS2