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fix: wire NanoPlot pre/post stats into MultiQC #181

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1bc9048
fix: wire NanoPlot pre/post stats into MultiQC
ljwharbers Aug 13, 2026
f23b9b5
test: update default snapshot for NanoPlot MultiQC entries
ljwharbers Aug 13, 2026
edf169d
style: shorten NANOPLOT_PRE prefix comment
ljwharbers Sep 3, 2026
29e2427
Merge origin/dev into fix_nanoplot_multiqc
ljwharbers Sep 8, 2026
0ca9302
Update extended nf-test snapshots for NanoPlot MultiQC wiring
ljwharbers Sep 9, 2026
2b51e0f
Name samtools and mosdepth QC outputs per BAM so MultiQC keeps tumor …
ljwharbers Sep 9, 2026
5372cd1
Regenerate all five nf-test snapshots for the samtools/mosdepth prefi…
ljwharbers Sep 9, 2026
85d2edb
Raise MultiQC log_filesize_limit so long-read samtools stats are parsed
ljwharbers Sep 9, 2026
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4 changes: 4 additions & 0 deletions CHANGELOG.md
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Expand Up @@ -25,6 +25,10 @@ and this project adheres to [Semantic Versioning](https://semver.org/spec/v2.0.0

### `Fixed`

- [#181](https://github.com/IntGenomicsLab/lrsomatic/pull/181) - NanoPlot pre- and post-alignment statistics now reach MultiQC. `ch_nanoplot_pre_txt` and `ch_nanoplot_post_txt` were declared empty and mixed into the MultiQC inputs, but never assigned from `NANOPLOT_PRE.out.txt` / `NANOPLOT_POST.out.txt`, so the NanoStat section was silently missing from every report (@ljwharbers).
- [#181](https://github.com/IntGenomicsLab/lrsomatic/pull/181) - `NANOPLOT_PRE` now includes the replicate in its output prefix, so samples with more than one replicate no longer collapse into a single MultiQC sample (@ljwharbers).
- [#181](https://github.com/IntGenomicsLab/lrsomatic/pull/181) - samtools stats/flagstat/idxstats and mosdepth outputs are now prefixed `<sample>_<type>` instead of `<sample>`. The tumor and normal files of a matched pair shared a basename, so MultiQC logged `Duplicate sample name found! Overwriting` and reported only one of the two; the general statistics table now has one row per BAM (`<sample>_tumor`, `<sample>_normal`), which the post-alignment NanoStat row joins (@ljwharbers).
- [#181](https://github.com/IntGenomicsLab/lrsomatic/pull/181) - Raised MultiQC's `log_filesize_limit` to 500 MB. `samtools stats` output for long reads is 70 MB and more, above the 50 MB default, so MultiQC silently skipped it and the samtools stats section covered only small files (@ljwharbers).
Comment on lines +28 to +31
- [#186](https://github.com/IntGenomicsLab/lrsomatic/pull/186) - Stopped snapshotting the md5 of sample4's merged tumour BAM and its index in the `clair_only` nf-test: `samtools merge` gives the colliding `@PG` IDs of the two replicates a random hex suffix, so neither digest is reproducible. The alignment records are, and are now asserted with `bam().getReadsMD5()` instead (@ljwharbers).
- [#182](https://github.com/IntGenomicsLab/lrsomatic/pull/182) - Added `--vcf` to the default `vep_args` so VEP writes VCF output rather than its default tab-delimited format (@AmberVerhasselt).
- [#183](https://github.com/IntGenomicsLab/lrsomatic/pull/183) - Corrected the `github` URL for Laurens Lambrechts in `manifest.contributors`, which was a copy of @MariosEft97's, to @laulambr. RO-Crate metadata updated to match (@ljwharbers).
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4 changes: 4 additions & 0 deletions CITATIONS.md
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Expand Up @@ -70,6 +70,10 @@

> Ewels P, Magnusson M, Lundin S, Käller M. MultiQC: summarize analysis results for multiple tools and samples in a single report. Bioinformatics. 2016 Oct 1;32(19):3047-8. doi: 10.1093/bioinformatics/btw354. Epub 2016 Jun 16. PubMed PMID: 27312411; PubMed Central PMCID: PMC5039924.

- [NanoPlot](https://pubmed.ncbi.nlm.nih.gov/37171891/)

> De Coster W, Rademakers R. NanoPack2: population-scale evaluation of long-read sequencing data. Bioinformatics. 2023 May 4;39(5):btad311. doi: 10.1093/bioinformatics/btad311. PubMed PMID: 37171891; PubMed Central PMCID: PMC10196664.

- [SAMtools](https://pubmed.ncbi.nlm.nih.gov/19505943/)

> Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R; 1000 Genome Project Data Processing Subgroup. The Sequence Alignment/Map format and SAMtools. Bioinformatics. 2009 Aug 15;25(16):2078-9. doi: 10.1093/bioinformatics/btp352. Epub 2009 Jun 8. PubMed PMID: 19505943; PubMed Central PMCID: PMC2723002.
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10 changes: 10 additions & 0 deletions assets/multiqc_config.yml
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Expand Up @@ -10,4 +10,14 @@ report_section_order:

export_plots: true

# Post-alignment NanoPlot files are named <sample>_<type>_aln_NanoStats.txt. Dropping "_aln"
# puts their NanoStat row on the same <sample>_<type> row as samtools and mosdepth for that BAM.
extra_fn_clean_exts:
- "_aln"
Comment on lines +13 to +16

# samtools stats on long reads writes 70+ MB files (its read-length and coverage histograms run to
# the longest read and deepest base). MultiQC silently skips any file above log_filesize_limit,
# whose default is 50 MB, so the samtools stats section would only ever cover small files.
log_filesize_limit: 500000000

disable_version_detection: true
8 changes: 6 additions & 2 deletions conf/modules.config
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Expand Up @@ -32,7 +32,8 @@ process {
}

withName: '.*:BAM_STATS_SAMTOOLS:.*' {
ext.prefix = { "${meta.id}" }
// Type in the prefix, otherwise the tumor and normal files of a pair share a basename and MultiQC keeps only one
ext.prefix = { "${meta.id}_${meta.type}" }
publishDir = [
Comment on lines 34 to 37
path: { "${params.outdir}/${meta.id}/qc/${meta.type}/samtools/" },
mode: params.publish_dir_mode,
Expand Down Expand Up @@ -60,7 +61,8 @@ process {
}

withName: '.*:NANOPLOT_PRE' {
ext.prefix = { "${meta.id}_${meta.type}_ubam" }
// Replicate in the prefix, otherwise MultiQC keeps only one identically named NanoStats.txt
ext.prefix = { "${meta.id}_${meta.type}_rep${meta.replicate}_ubam" }
publishDir = [
path: { "${params.outdir}/${meta.id}/qc/${meta.type}/nanoplot_ubam_rep${meta.replicate}" },
mode: params.publish_dir_mode,
Expand Down Expand Up @@ -88,6 +90,8 @@ process {

withName: '.*:MOSDEPTH' {
ext.args = { '-n -x' }
// Type in the prefix, otherwise the tumor and normal files of a pair share a basename and MultiQC keeps only one
ext.prefix = { "${meta.id}_${meta.type}" }
publishDir = [
path: { "${params.outdir}/${meta.id}/qc/${meta.type}/mosdepth" },
mode: params.publish_dir_mode,
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50 changes: 27 additions & 23 deletions docs/output.md
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Expand Up @@ -178,41 +178,41 @@ QC outputs are placed under `tumor/` for all samples, and additionally under `no
│ │ ├── fibertoolsrs
│ │ │ ├── sample_qc.txt
│ │ ├── mosdepth
│ │ │ ├── sample.mosdepth.global.dist.txt
│ │ │ ├── sample.mosdepth.summary.txt
│ │ │ ├── sample_tumor.mosdepth.global.dist.txt
│ │ │ ├── sample_tumor.mosdepth.summary.txt
│ │ ├── nanoplot_aln
│ │ │ ├── sample_tumor_aln_NanoStats.txt
│ │ │ ├── sample_tumor_aln_NanoPlot-report.html
│ │ ├── nanoplot_ubam_rep1
│ │ │ ├── sample_tumor_ubam_NanoStats.txt
│ │ │ ├── sample_tumor_ubam_NanoPlot-report.html
│ │ │ ├── sample_tumor_rep1_ubam_NanoStats.txt
│ │ │ ├── sample_tumor_rep1_ubam_NanoPlot-report.html
│ │ ├── samtools
│ │ │ ├── sample.flagstat
│ │ │ ├── sample.idxstats
│ │ │ ├── sample.stats
│ │ │ ├── sample_tumor.flagstat
│ │ │ ├── sample_tumor.idxstats
│ │ │ ├── sample_tumor.stats
│ ├── normal # paired samples only
│ │ └── [same subdirectories as tumor]
│ ├── whatshap_stats
│ │ ├── sample.stats.tsv
│ │ ├── sample.blocklist.tsv
```

| File | Description |
| ------------------------------------------------------------ | ------------------------------------------------------------------------------------------------------------------------ |
| `cramino_aln/sample_{type}_cramino.txt` | cramino QC summary statistics for the aligned bam file |
| `cramino_ubam_rep1/sample_{type}_cramino.txt` | cramino QC summary statistics for the unaligned bam files |
| `fibertoolsrs/sample_qc.txt` | fibertools QC summary for the bam file |
| `mosdepth/sample.mosdepth.global.dist.txt` | a cumulative distribution indicating the proportion of total bases that were covered for at least a given coverage value |
| `mosdepth/sample.mosdepth.summary.txt` | overall summary file from mosdepth tool |
| `nanoplot_aln/sample_{type}_aln_NanoStats.txt` | NanoPlot summary statistics for the aligned BAM file |
| `nanoplot_aln/sample_{type}_aln_NanoPlot-report.html` | NanoPlot interactive HTML report for the aligned BAM file |
| `nanoplot_ubam_rep1/sample_{type}_ubam_NanoStats.txt` | NanoPlot summary statistics for the unaligned BAM file |
| `nanoplot_ubam_rep1/sample_{type}_ubam_NanoPlot-report.html` | NanoPlot interactive HTML report for the unaligned BAM file |
| `samtools/sample.flagstat` | a summary of the counts of different samtools flags |
| `samtools/sample.idxstats` | a summary of the number of mapped and unmapped reads |
| `samtools/sample.stats` | summary statistics from the bamfile |
| `whatshap_stats/sample.stats.tsv` | WhatsHap phasing statistics per chromosome including phase block N50 and switch error rates |
| `whatshap_stats/sample.blocklist.tsv` | list of all phase blocks with their genomic coordinates |
| File | Description |
| ----------------------------------------------------------------- | ------------------------------------------------------------------------------------------------------------------------ |
| `cramino_aln/sample_{type}_cramino.txt` | cramino QC summary statistics for the aligned bam file |
| `cramino_ubam_rep1/sample_{type}_cramino.txt` | cramino QC summary statistics for the unaligned bam files |
| `fibertoolsrs/sample_qc.txt` | fibertools QC summary for the bam file |
| `mosdepth/sample_{type}.mosdepth.global.dist.txt` | a cumulative distribution indicating the proportion of total bases that were covered for at least a given coverage value |
| `mosdepth/sample_{type}.mosdepth.summary.txt` | overall summary file from mosdepth tool |
| `nanoplot_aln/sample_{type}_aln_NanoStats.txt` | NanoPlot summary statistics for the aligned BAM file |
| `nanoplot_aln/sample_{type}_aln_NanoPlot-report.html` | NanoPlot interactive HTML report for the aligned BAM file |
| `nanoplot_ubam_rep1/sample_{type}_rep1_ubam_NanoStats.txt` | NanoPlot summary statistics for the unaligned BAM file |
| `nanoplot_ubam_rep1/sample_{type}_rep1_ubam_NanoPlot-report.html` | NanoPlot interactive HTML report for the unaligned BAM file |
Comment on lines +209 to +210
| `samtools/sample_{type}.flagstat` | a summary of the counts of different samtools flags |
| `samtools/sample_{type}.idxstats` | a summary of the number of mapped and unmapped reads |
| `samtools/sample_{type}.stats` | summary statistics from the bamfile |
| `whatshap_stats/sample.stats.tsv` | WhatsHap phasing statistics per chromosome including phase block N50 and switch error rates |
| `whatshap_stats/sample.blocklist.tsv` | list of all phase blocks with their genomic coordinates |

</details>

Expand Down Expand Up @@ -552,6 +552,8 @@ The report is one self-contained file — plots and tables are embedded, so it c

### `multiqc`

Sample rows are named per BAM: `{sample}_tumor` and `{sample}_normal` carry the samtools, mosdepth and post-alignment NanoPlot statistics of that BAM, `{sample}_{type}_rep{N}_ubam` rows carry the pre-alignment NanoPlot statistics of each unaligned replicate, and WhatsHap phasing statistics sit on a plain `{sample}` row because phasing is done once per sample.

<details markdown="1">
<summary>Output files</summary>

Expand All @@ -570,8 +572,10 @@ The report is one self-contained file — plots and tables are embedded, so it c
│ │ ├── multiqc_data.json
│ │ ├── multiqc_general_stats.txt
│ │ ├── multiqc_software_versions.txt
│ │ ├── multiqc_nanostat.txt
│ │ ├── multiqc_sources.txt
│ │ ├── multiqc.log
│ │ ├── nanostat_fasta_stats_table.txt
│ ├── multiqc_plots
│ │ ├── pdf
│ │ │ ├── mosdepth-coverage-per-contig-multi-cnt.pdf
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