Dry Lab / Computational: low alignment / mapping rate

1. Ranked causes

   1. Wrong or outdated reference genome/index buildMapping rate is uniformly low across every sample, not just one.
   2. Sample contamination (wrong species / rRNA depletion failure)Unmapped reads BLAST to an unexpected organism.
   3. Adapter contamination not trimmed before alignmentFastQC shows adapter content warnings pre-alignment.
   4. Aligner parameters too strict for read length/qualityMapping rate improves substantially when mismatch tolerance is relaxed.

2. Recovery steps

   1. Confirm the reference build matches the organism and annotation version used elsewhere in the pipelineA stale or mismatched index is the most common systemic cause.
   2. Run adapter/quality trimming (e.g. Trim Galore, fastp) before re-aligningUntrimmed adapters directly reduce mapping rate.
   3. BLAST a sample of unmapped reads to check for species/contamination mismatchConfirms whether the sample itself, not the pipeline, is the issue.
   4. Relax aligner mismatch/seed parameters for shorter or noisier readsFixes rate for legitimately lower-quality but on-target reads.

3. Controls first

   • Did a positive-control library (known good sample) map at the expected rate on this reference?If no: Reference build or aligner parameters — not just one bad sample.
   • Is the low mapping rate consistent across every sample in the batch?If yes: Systemic pipeline issue (reference build or trimming), not one bad sample.

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