# Combined Variant Output

File name: `{Pair_ID}_CombinedVariantOutput.tsv`

The combined variant output file contains the variants and biomarkers in a single file that is based on a single sample. If using pair ID, the file is based on paired DNA and RNA samples from the same individual. The output contains the following variant types and biomarkers:

* Small variants
* Copy number variants (CNV) (with absolute copy number when HRD Assay is run)
* TMB
* MSI
* Fusions
* Splice variants
* GIS (when HRD Assay is run)
* Gene-level Loss of Heterozygosity (when HRD Assay is run)
* Large Rearrangements

The combined variant output file also contains Analysis Details and Sequencing Run Details sections. The details of each are listed in the following table:

| Analysis Details                                                                                                                                                                                                     | Sequencing Run Details                                                                                                                                                                                                                                       |
| -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- | ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ |
| <p>- Pair ID<br>- DNA Sample ID (if DNA is run)<br>- RNA Sample ID (if RNA is run)</p><p>- Library Prep Kit<br>- Output Date<br>- Output Time<br>- Module Version<br>- Pipeline Version (Docker image version #)</p> | <p>- Run Name<br>- Run Date<br>- DNA Sample Index ID (if DNA is run)<br>- RNA Sample Index ID (if RNA is run)<br>- Instrument ID<br>- Instrument Control Software Version<br>- Instrument Type</p><p>- Instrument Platform</p><p>- \[HRD] Sample feature</p> |

Combined variant output produces small variants with blank fields in the following situations:

* The variant has been matched to a canonical RefSeq transcript on an overlapping gene not targeted by TruSight Oncology 500.
* The variant is located in a region designated iSNP, indel, or Flanking in the `TST500_Manifest.bed` file located in the Resources folder.

## Variant Filtering Rules

* **Small Variants -** All variants with the FILTER field marked as PASS in the hard-filtered genome VCF are present in the combined variant output.
  * Gene information is only present for variants belonging to canonical transcripts that are within the Gene Allow List–Small Variants.
  * Transcript information is only present for variants belonging to canonical transcripts that are within the Gene Allow List–Small Variants.
* **Copy Number Variants -** All variants with the FILTER field marked as PASS and ALT field is \<DUP or \<DEL> in cnv.vcf file from DRAGEN DNA call or abcn\_annotated.vcf from GIS step(HRD assay))are present in the combined variant output:
  * Gene Name.
  * Fold Change.
  * Copy Number Variant (\<DUP or \<DEL>) .
  * Absolute Copy Number (HRD Assay) – This value is included only if the HRD assay was run and the sample passed HRD sample QC. If the HRD assay failed QC , the Absolute Copy Number will be reported as NA, and the CNVs will be sourced from the DRAGEN DNA call (cnv.vcf).
* **Fusion Variants -** Fusion variants must meet the following conditions:
  * Passing variant call (KeepFusion field is true).
  * Contains at least one gene on the fusion allow list.
  * Genes separated by a dash (-) indicate that the fusion directionality could be determined. Genes separated by a slash (/) indicate that the fusion directionality could not be determined.
* **Biomarkers TMB/MSI -** Always present when DNA sample is processed.
* **Splice Variants -** Passing splice variants that are contained on genes EGFR, MET, and AR.
* **Biomarker GIS -** Present only if TruSight Oncology 500 HRD analysis is performed
* **Loss of Heterozygosity** - Present only when TruSight Oncology 500 HRD is run. Loss of heterozygosity (LOH) must meet the following condition:
  * MCN field is equal to 0
* **Large Rearrangements CNV -** Large Rearrangements CNVs must meet the following conditions:
  * BRCA1 or BRCA2 contains at least one affected exon.
  * ALT field is \<DUP> or \<LOSS> .