Quality Control

The software calculates several quality control metrics for runs and samples.

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. Refer to Metrics Output for details.

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. Refer to Metrics Output for details.

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.