Missed Variant Calls

If an expected variant is not reported in the VCF output, check the following in order:

1. Coverage Overall

Check the Average alignment coverage over target region QC metric. Coverage of 150x–200x or higher is typically required for sensitivity down to 5% VAF. If coverage is low, consider:

• Deeper sequencing on the affected sample

• Repeating library preparation with increased input DNA

2. Coverage at Position

Check coverage at the specific genomic position where the variant was expected:

• If the variant appears in the VCF: Check the DP field for the read depth at that position.

• If the variant is missing from the VCF: Visualize the BAM file in a genome browser such as IGV (Integrative Genomics Viewer) to inspect the position directly.

Position-level coverage above 100x is typically required for sensitivity at 5% VAF.

3. Low-Quality FFPE Mode

If you ran the sample in low-quality FFPE mode, try rerunning it as a standard solid sample. The FFPE filters increase specificity but may slightly reduce sensitivity for borderline calls.

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If none of the above resolves the issue, contact Illumina Technical Support at [email protected].