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Document RSeQC inner_distance limitation for large chromosomes #1624

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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 #1624](https://github.com/nf-core/rnaseq/pull/1624) - Document RSeQC inner_distance limitation for genomes with large chromosomes (>500 Mb), such as plant genomes

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

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17 changes: 17 additions & 0 deletions docs/usage.md
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Expand Up @@ -378,6 +378,23 @@ This pipeline uses featureCounts to generate QC metrics based on [biotype](http:

Please get in touch with us on the #rnaseq channel in the [nf-core Slack workspace](https://nf-co.re/join) if you are having problems or need any advice.

#### Large chromosomes (plant genomes)

Genomes with very large chromosomes (>500 Mb), such as plant genomes, may encounter failures in the RSeQC `inner_distance` module due to a known limitation in the underlying bx-python library. The bx-python BitSet implementation has a maximum capacity of approximately 537 million bases, which can be exceeded by chromosomes in organisms like wheat, barley, and other plants.

If you encounter an error message similar to `IndexError: [coordinate] is larger than the size of this BitSet (536870912)`, you can work around this by excluding the `inner_distance` module from RSeQC analysis:

```bash
--rseqc_modules 'bam_stat,infer_experiment,junction_annotation,junction_saturation,read_distribution,read_duplication'
```

This removes `inner_distance` from the default list of RSeQC modules while retaining all other quality control metrics. Note that the inner_distance metric is only relevant for paired-end data and provides information about fragment size distribution.

For more information, see the upstream issues:

- [nf-core/rnaseq#608](https://github.com/nf-core/rnaseq/issues/608)
- [bxlab/bx-python#67](https://github.com/bxlab/bx-python/issues/67)

### iGenomes (not recommended)

If the `--genome` parameter is provided (e.g. `--genome GRCh37`) then the FASTA and GTF files (and existing indices) will be automatically obtained from AWS-iGenomes unless these have already been downloaded locally in the path specified by `--igenomes_base`.
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