Sample QC

These metrics and guidelines apply to DRAGEN TSO 500 v2.1 and above.

DNA Sample QC

DNA library QC results are available in the MetricsOutput.tsv file.

Metric	Description	Recommended Guideline Quality Threshold	Variant Class
CONTAMINATION_SCORE	The contamination score is based on VAF distribution of SNPs.	≤ 1457	All
MEDIAN_EXON_COVERAGE	Median exon fragment coverage across all exon bases.	≥ 150	Small variant TMB
PCT_CHIMERIC_READS	Proportion of total number of non-supplementary, non-secondary, and passing QC reads after alignment to the whole genome sequence.	≤ 8	Small variant TMB
PCT_EXON_50X	Percent exon bases with 50x fragment coverage.	≥ 90.0	Small variant TMB
MEDIAN_INSERT_SIZE	The median fragment length in the sample.	≥ 70	Small variant TMB
USABLE_MSI_SITES	The number of MSI sites usable for MSI calling.	≥ 40	MSI
MEDIAN_BIN_COUNT_CNV_TARGET	The median raw bin count per CNV target.	≥ 1.0	CNV
PCT_TARGET_HRD_50X (HRD samples)	Percent of HRD probe SNP panel covered by at least 50X coverage	≥ 50	GIS
EXCESSIVE_TF (HRD samples)	EXCESSIVE TF indicates if there is excessive tumor content in sample. Troubleshooting: Samples with pure tumor fraction >90% are outside the design for GIS estimation (this includes pure tumor cell lines)	= 0 (= 1 indicates Excessive TF)	GIS

RNA Sample QC

The input for RNA Library QC is RNA alignment. Metrics and guideline thresholds can be found in the MetricsOutput.tsv file.

Metric	Description	Recommended Guideline Quality Threshold	Variant Classes
MEDIAN_CV_GENE_500X	The median CV for all genes with median coverage > 500x. Genes with median coverage > 500x are likely to be highly expressed. Higher CV median > 500x indicates an issue with library preparation (poor sample input and/or probes pulldown issue).	≤ 0.93	Fusion, Splice
MEDIAN_INSERT_SIZE	The median fragment length in the sample.	≥ 80	Fusion, Splice
TOTAL_ON_TARGET_READS*	The total number of reads that map to the target regions.	≥ 9000000 (v1) ≥ 2,500,000 (v2)	Fusion, Splice
GENE_MEDIAN_COVERAGE**	The median deduped coverage across all genes in the RNA panel (55 genes).	N/A	Fusion, Splice

*TOTAL_ON_TARGET_READS is the only QC metric with guidelines specific to chemistry (v1 vs. v2 assay); all other guidelines are applicable to both

** To avoid failing RNA samples unnecessarily, Illumina does not recommend a universal threshold for GENE_MEDIAN_COVERAGE to determine RNA sample quality. RNA expression varies significantly across tissue types and a small panel size (55 genes), which makes normalization challenging. Tissue-specific thresholds could be considered for normalization.