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)

• Exon-level CNVs

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
- Pair ID - DNA sample ID (if DNA is run) - RNA sample ID (if RNA is run) - Output date - Output time - Module version - Pipeline version (Docker image version #)	- Run name - Run date - DNA sample index ID (if DNA is run) - RNA sample index ID (if RNA is run) - [HRD] Sample feature - Instrument ID - Instrument control software version - Instrument type - RTA version - Reagent cartridge lot number

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 - Copy number variants must meet the following conditions:

  • FILTER field marked as PASS.

  • ALT field is <DUP or <DEL> .

• 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

• Exon-level CNVs - Exon-levels CNVs must meet the following conditions:

  • BRCA1 or BRCA2 contains at least one affected exon.

  • ALT field is <DUP> or <LOSS> .