# Sample QC

{% hint style="success" %}
These metrics and guidelines apply to DRAGEN TSO 500 v2.1 and above.
{% endhint %}

## 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) | <p>= 0<br>(= 1 indicates Excessive TF)</p> | 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.                                                                                                                                                                                           | <p>≥ 9000000 (v1)</p><p>≥ 2,500,000 (v2)</p> | 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.


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