# Quality Control The software calculates several quality control metrics for runs and samples. {% hint style="success" %} These metrics and guidelines apply to DRAGEN TSO 500 v2.1 and above. {% endhint %} ## Run QC The Run Metrics section of the metrics output report provides sequencing run quality metrics along with suggested values to determine if they are within an acceptable range. The overall percentage of reads passing filter is compared to a minimum threshold. For Read 1 and Read 2, the average percentage of bases ≥ Q30, which gives a prediction of the probability of an incorrect base call (Q‑score), are also compared to a minimum threshold. The following tables show run metric and quality threshold information for different systems. The values in the Run Metrics section are listed as NA in the following situations: * If the analysis was started from FASTQ files. * If the analysis was started from BCL files and the InterOp files are missing or corrupt. ### NextSeq 500/550 or NextSeq 550Dx (RUO) | Metric | Description | Recommended Guideline Quality Threshold | Variant Class | | ------------------ | --------------------------------------------------- | --------------------------------------- | ------------- | | PCT\_PF\_READS (%) | Total percentage of reads passing filter. | ≥80.0 | All | | PCT\_Q30\_R1 (%) | Percentage of Read 1 reads with quality score ≥ 30. | ≥80.0 | All | | PCT\_Q30\_R2 (%) | Percentage of Read 2 reads with quality score ≥ 30. | ≥80.0 | All | ### NovaSeq 6000 or NovaSeq 6000Dx (RUO) | Metric | Description | Recommended Guideline Quality Threshold | Variant Class | | ------------------ | --------------------------------------------------- | --------------------------------------- | ------------- | | PCT\_PF\_READS (%) | Total percentage of reads passing filter. | ≥55.0 | All | | PCT\_Q30\_R1 (%) | Percentage of Read 1 reads with quality score ≥ 30. | ≥80.0 | All | | PCT\_Q30\_R2 (%) | Percentage of Read 2 reads with quality score ≥ 30. | ≥80.0 | All | ### NextSeq 1000/2000 | Metric | Description | Recommended Guideline Quality Threshold | Variant Class | | ---------------- | --------------------------------------------------- | --------------------------------------- | ------------- | | PCT\_Q30\_R1 (%) | Percentage of Read 1 reads with quality score ≥ 30. | ≥85.0 | All | | PCT\_Q30\_R2 (%) | Percentage of Read 2 reads with quality score ≥ 30. | ≥85.0 | All | ### NovaSeq X | Metric | Description | Recommended Guideline Quality Threshold | Variant Class | | ---------------- | --------------------------------------------------- | --------------------------------------- | ------------- | | PCT\_Q30\_R1 (%) | Percentage of Read 1 reads with quality score ≥ 30. | ≥85.0 | All | | PCT\_Q30\_R2 (%) | Percentage of Read 2 reads with quality score ≥ 30. | ≥85.0 | All | ## DNA Sample QC DRAGEN TruSight Oncology 500 uses QC metrics to assess the validity of analysis for DNA libraries that pass contamination quality control. If the library fails one or more quality metrics, then the corresponding variant type or biomarker is not reported, and the associated QC category in the report header displays FAIL. Additionally, a companion diagnostic result may not be available if it relies on QC passing for one or more of the following QC categories. 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.