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Document Qualimap read counting limitation #1625

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33168d3
Add warning about Qualimap read counting bug
pinin4fjords Nov 14, 2025
f2ddd31
Update CHANGELOG.md for Qualimap documentation PR
pinin4fjords Nov 14, 2025
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1 change: 1 addition & 0 deletions CHANGELOG.md
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Expand Up @@ -15,6 +15,7 @@ Special thanks to the following for their contributions to the release:

- [PR #1608](https://github.com/nf-core/rnaseq/pull/1608) - Bump version after release 3.21.0
- [PR #1617](https://github.com/nf-core/rnaseq/pull/1617) - Update bbmap/bbsplit module
- [PR #1625](https://github.com/nf-core/rnaseq/pull/1625) - Add documentation warning about Qualimap read counting bug ([#1273](https://github.com/nf-core/rnaseq/issues/1273))

## [[3.21.0](https://github.com/nf-core/rnaseq/releases/tag/3.21.0)] - 2025-09-18

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12 changes: 12 additions & 0 deletions docs/output.md
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Expand Up @@ -614,6 +614,18 @@ RSeQC documentation: [tin.py](http://rseqc.sourceforge.net/#tin-py)

The [Qualimap RNA-seq QC module](http://qualimap.bioinfo.cipf.es/doc_html/analysis.html#rna-seq-qc) is used within this pipeline to assess the overall mapping and coverage relative to gene features.

:::warning
**Known limitation**: Qualimap has a known bug ([#1273](https://github.com/nf-core/rnaseq/issues/1273), [Qualimap issue #81](https://bitbucket.org/kokonech/qualimap/issues/81)) where it may report more reads assigned to genomic features (exons, introns, intergenic regions) than the total number of reads in the BAM file. This can lead to inflated read counts and incorrect genomic origin statistics.

If accurate read distribution metrics are critical for your analysis, we recommend:

- Cross-referencing Qualimap results with RSeQC output (particularly `read_distribution.txt`)
- Using `--skip_qualimap` to disable Qualimap and rely on RSeQC for genomic feature distribution
- Validating suspicious results with alternative tools or custom scripts

This is an upstream tool issue that cannot be fixed at the pipeline level.
:::

![MultiQC - Qualimap gene coverage plot](images/mqc_qualimap_coverage.png)

![MultiQC - Qualimap genomic origin plot](images/mqc_qualimap_features.png)
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