# Minimal Checklist This section provides a succinct checklist of critical Quality Control (QC) metrics for evaluating DRAGEN run performance. These metrics are located in the standard CSV output files in the run directory. ## 1. Basic Run & Alignment QC **Source File:** `.mapping_metrics.csv`\ **Default Status:** Enabled Note that mapping metrics are computed on the raw input sample (prior to optional UMI collapsing). | Metric Name | Critical Trend / Success Criteria | | --------------------------------------- | ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- | | **Total input reads** | **Volume check:** Confirm read count matches sequencer output and assay expectations. E.g \~**600–900M reads** for 30X human WGS. Low counts indicate flow cell loading issues or demultiplexing failures. | | **Q30 bases** | **Base quality:** Generally **> 85–90%**. A global drop indicates chemistry or flow cell issues (see FastQC metrics for positional detail). | | **Mapped reads** | **Alignment success:** Aim for **> 95%** (human WGS). Low mapping (< 90%) suggests wrong reference genome, severe contamination, or poor library quality. | | **Supplementary (chimeric) alignments** | **Structural integrity:** Reads split across distant loci. For high-quality human germline WGS, expected values are typically **< 2–3%**. Values **> 3–5%** warrant investigation and may indicate library chimera artifacts (e.g. PCR stitching, FFPE-related fragmentation) or, in somatic samples, true structural variation. Sustained levels **> 5%** in germline samples are generally considered abnormal. | | **Soft-clipped bases (R1/R2)** | **Adapter/quality trimming:** High percentages indicate adapter read-through, short insert sizes, or poor-quality read ends (e.g. FFPE artifacts). For high-quality libraries, typical values are **< 2–3%**. Values **> 3–5%** suggest suboptimal trimming or degraded DNA. Levels **> 5%** should be treated as a QC concern and reviewed alongside FastQC metrics. | | **Estimated sample contamination** | **Purity check:** (Requires `--qc-detect-contamination=true`). For human germline samples, **> 1–2%** contamination can materially impact variant calling accuracy, especially for low-frequency somatic variants. | | **Duplicate reads** | **Library complexity:** Elevated duplication rates suggest reduced library complexity or over-amplification. For high-quality germline WGS, typical values are **< 20%**. For WES and other targeted assays, higher duplication rates (e.g. **20–50%**) are common and should be interpreted in the context of on-target coverage and assay design. | | **Insert length (median)** | **Fragment size:** Should match the library prep target (e.g. \~350 bp). Deviations can affect coverage uniformity. | ## 2. FastQC (Sequence Composition) **Source File:** `.fastqc_metrics.csv`\ **Default Status:** Enabled Note that FastQC metrics are computed on the raw input sample (prior to optional UMI collapsing). | Metric Name | Critical Trend / Success Criteria | | --------------------------------- | ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ | | **Positional base mean quality** | **Cycle decay:** Identify quality drop-off at read ends or specific cycles (e.g. fluidics issues). | | **Read GC content** | **Contamination/bias:** Deviation from expected distribution (e.g. human \~40–45% GC) suggests contamination or severe PCR bias. | | **Sequence positions (adapters)** | **Adapter content:** High levels indicate untrimmed adapters or short inserts. This can lead to high levels (e.g. **> 5%**) of reported soft-clipped bases in the mapping metrics. Confirm trimming options are enabled. | ## 3. UMI QC (Applies Only to UMI Designs) **Source File:** `.umi_metrics.csv`\ **Default Status:** Enabled as part of the UMI pipeline (requires `--umi-enable=true`) | Metric Name | Critical Trend / Success Criteria | | -------------------- | ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ | | **Consensus reads** | **Conversion efficiency:** Low ratio relative to total input reads suggests low molecular complexity or insufficient sequencing depth. | | **Mean family size** | **Saturation:** Family sizes near **1.0** indicate under-sequencing; error correction is ineffective without duplicate families. Very high mean family sizes may indicate excessive PCR amplification. | ## 4. Coverage QC **Source File:** `.*_coverage_metrics.csv`\ (e.g. `wgs_coverage_metrics.csv` or `target_bed_coverage_metrics.csv`)\ **Default Status:** Enabled Please note coverage metrics are computed post read deduplication or UMI collapsing. Reads with MAPQ=0 are ignored. | Metric Name | Critical Trend / Success Criteria | | ------------------------------------------------------- | ---------------------------------------------------------------------------------------------------------------------------------------------------------------------- | | **Average alignment coverage over target bed / genome** | **Depth check:** Primary driver of sensitivity. Ensure it meets the assay target (e.g. **30×** germline, **100×+** somatic). | | **Uniformity of coverage (PCT > 0.2× mean)** | **Bias check:** Low uniformity indicates coverage bias (e.g. GC bias), leading to variant calling blind spots. Germline WGS typically expects **≥ 80–90%** uniformity. | | **PCT of genome with coverage \[x: inf)** | **Callability:** (e.g. `PCT ≥ 20×`). Germline WGS typically requires **> 95% at 20×**. | | **Aligned bases in target bed / genome** | **Yield:** Total usable data. Useful for normalizing performance across runs or flow cells. | ## 5. Variant-Level Sanity Check (Optional) **Source File:** `.vc_metrics.csv`\ **Default Status:** Conditional (variant caller enabled) | Metric Name | Critical Trend / Success Criteria | | --------------- | ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- | | **Ti/Tv Ratio** | **Biological plausibility:** For human germline WGS, expect approximately **1.9–2.2**. Significantly lower values (e.g. **< 1.7**) often indicate elevated false-positive rates due to sequencing or alignment errors. | **Note:**\ Ti/Tv is a biological sanity check, not a standalone QC pass/fail metric. Expected values depend on organism, assay type, and genomic region. ### How to Use This Checklist * Treat this as a **minimum QC review**, not an exhaustive list. * Values outside these ranges warrant investigation but are **not automatic failures**. * Some metrics are assay-specific and may require optimization for particular use cases.