Document Qualimap read counting limitation #1625

pinin4fjords · 2025-11-14T18:05:43Z

Description

Adds documentation warning about a known Qualimap bug where it reports more reads assigned to genomic features than exist in the BAM file.

Details

Adds a warning box in the Qualimap section of output.md
Links to both the pipeline issue ( Qualimap assigns more reads to exonic regions than total reads in the sample #1273) and the upstream Qualimap issue
Provides workarounds: using RSeQC instead, --skip_qualimap flag, or validation with custom tools
Clarifies this is an upstream tool issue that cannot be fixed at the pipeline level

Addresses

Closes #1273 (via documentation)

References

PR checklist

This comment contains a description of changes (with reason).
CHANGELOG.md is updated (will update after review if needed)
If you've fixed a bug or added code that should be tested, add tests!
Documentation in docs is updated.
If necessary, also make a PR on the nf-core/rnaseq branch on the nf-core/test-datasets repository.

Document known limitation where Qualimap reports more reads than exist in the BAM file when assigning reads to genomic features. Provide workarounds including using RSeQC as alternative and --skip_qualimap flag. Addresses #1273

pinin4fjords · 2025-11-14T18:08:13Z

Note: This PR cannot be merged until #1618 is merged first, as that PR contains CI fixes needed for tests to pass.

github-actions · 2025-11-14T18:08:53Z

`nf-core pipelines lint` overall result: Passed ✅ ⚠️

Posted for pipeline commit a353fa4

+| ✅ 284 tests passed       |+
#| ❔   8 tests were ignored |#
#| ❔   1 tests had warnings |#
!| ❗   9 tests had warnings |!

❗ Test warnings:

files_exist - File not found: assets/multiqc_config.yml
pipeline_todos - TODO string in base.config: Check the defaults for all processes
pipeline_todos - TODO string in awsfulltest.yml: You can customise AWS full pipeline tests as required
pipeline_todos - TODO string in methods_description_template.yml: #Update the HTML below to your preferred methods description, e.g. add publication citation for this pipeline
pipeline_todos - TODO string in main.nf: Optionally add in-text citation tools to this list.
pipeline_todos - TODO string in main.nf: Optionally add bibliographic entries to this list.
pipeline_todos - TODO string in main.nf: Only uncomment below if logic in toolCitationText/toolBibliographyText has been filled!
pipeline_todos - TODO string in nextflow.config: Specify any additional parameters here
pipeline_if_empty_null - ifEmpty(null) found in /home/runner/work/rnaseq/rnaseq/subworkflows/local/prepare_genome/main.nf: _ versions = ch_versions.ifEmpty(null) // channel: [ versions.yml ]
_

❔ Tests ignored:

files_exist - File is ignored: conf/modules.config
nextflow_config - Config default ignored: params.ribo_database_manifest
files_unchanged - File ignored due to lint config: assets/email_template.html
files_unchanged - File ignored due to lint config: assets/email_template.txt
files_unchanged - File ignored due to lint config: .gitignore or .prettierignore
actions_nf_test - actions_nf_test
multiqc_config - multiqc_config
modules_config - modules_config

❔ Tests fixed:

rocrate_readme_sync - Mismatch fixed: RO-Crate description updated from README.md.

✅ Tests passed:

files_exist - File found: .gitattributes
files_exist - File found: .gitignore
files_exist - File found: .nf-core.yml
files_exist - File found: .prettierignore
files_exist - File found: .prettierrc.yml
files_exist - File found: CHANGELOG.md
files_exist - File found: CITATIONS.md
files_exist - File found: CODE_OF_CONDUCT.md
files_exist - File found: LICENSE or LICENSE.md or LICENCE or LICENCE.md
files_exist - File found: nextflow_schema.json
files_exist - File found: nextflow.config
files_exist - File found: README.md
files_exist - File found: .github/.dockstore.yml
files_exist - File found: .github/CONTRIBUTING.md
files_exist - File found: .github/ISSUE_TEMPLATE/bug_report.yml
files_exist - File found: .github/ISSUE_TEMPLATE/config.yml
files_exist - File found: .github/ISSUE_TEMPLATE/feature_request.yml
files_exist - File found: .github/PULL_REQUEST_TEMPLATE.md
files_exist - File found: .github/workflows/branch.yml
files_exist - File found: .github/workflows/nf-test.yml
files_exist - File found: .github/actions/get-shards/action.yml
files_exist - File found: .github/actions/nf-test/action.yml
files_exist - File found: .github/workflows/linting_comment.yml
files_exist - File found: .github/workflows/linting.yml
files_exist - File found: assets/email_template.html
files_exist - File found: assets/email_template.txt
files_exist - File found: assets/sendmail_template.txt
files_exist - File found: assets/nf-core-rnaseq_logo_light.png
files_exist - File found: conf/test.config
files_exist - File found: conf/test_full.config
files_exist - File found: docs/images/nf-core-rnaseq_logo_light.png
files_exist - File found: docs/images/nf-core-rnaseq_logo_dark.png
files_exist - File found: docs/output.md
files_exist - File found: docs/README.md
files_exist - File found: docs/README.md
files_exist - File found: docs/usage.md
files_exist - File found: nf-test.config
files_exist - File found: tests/default.nf.test
files_exist - File found: main.nf
files_exist - File found: conf/base.config
files_exist - File found: conf/igenomes.config
files_exist - File found: conf/igenomes_ignored.config
files_exist - File found: .github/workflows/awstest.yml
files_exist - File found: .github/workflows/awsfulltest.yml
files_exist - File found: modules.json
files_exist - File found: ro-crate-metadata.json
files_exist - File not found check: .github/ISSUE_TEMPLATE/bug_report.md
files_exist - File not found check: .github/ISSUE_TEMPLATE/feature_request.md
files_exist - File not found check: .github/workflows/push_dockerhub.yml
files_exist - File not found check: .markdownlint.yml
files_exist - File not found check: .nf-core.yaml
files_exist - File not found check: .yamllint.yml
files_exist - File not found check: bin/markdown_to_html.r
files_exist - File not found check: conf/aws.config
files_exist - File not found check: docs/images/nf-core-rnaseq_logo.png
files_exist - File not found check: lib/Checks.groovy
files_exist - File not found check: lib/Completion.groovy
files_exist - File not found check: lib/NfcoreTemplate.groovy
files_exist - File not found check: lib/Utils.groovy
files_exist - File not found check: lib/Workflow.groovy
files_exist - File not found check: lib/WorkflowMain.groovy
files_exist - File not found check: lib/WorkflowRnaseq.groovy
files_exist - File not found check: parameters.settings.json
files_exist - File not found check: pipeline_template.yml
files_exist - File not found check: Singularity
files_exist - File not found check: lib/nfcore_external_java_deps.jar
files_exist - File not found check: .travis.yml
nextflow_config - Found nf-schema plugin
nextflow_config - Config variable found: manifest.name
nextflow_config - Config variable found: manifest.nextflowVersion
nextflow_config - Config variable found: manifest.description
nextflow_config - Config variable found: manifest.version
nextflow_config - Config variable found: manifest.homePage
nextflow_config - Config variable found: timeline.enabled
nextflow_config - Config variable found: trace.enabled
nextflow_config - Config variable found: report.enabled
nextflow_config - Config variable found: dag.enabled
nextflow_config - Config variable found: process.cpus
nextflow_config - Config variable found: process.memory
nextflow_config - Config variable found: process.time
nextflow_config - Config variable found: params.outdir
nextflow_config - Config variable found: params.input
nextflow_config - Config variable found: manifest.mainScript
nextflow_config - Config variable found: timeline.file
nextflow_config - Config variable found: trace.file
nextflow_config - Config variable found: report.file
nextflow_config - Config variable found: dag.file
nextflow_config - Config variable (correctly) not found: params.nf_required_version
nextflow_config - Config variable (correctly) not found: params.container
nextflow_config - Config variable (correctly) not found: params.singleEnd
nextflow_config - Config variable (correctly) not found: params.igenomesIgnore
nextflow_config - Config variable (correctly) not found: params.name
nextflow_config - Config variable (correctly) not found: params.enable_conda
nextflow_config - Config variable (correctly) not found: params.max_cpus
nextflow_config - Config variable (correctly) not found: params.max_memory
nextflow_config - Config variable (correctly) not found: params.max_time
nextflow_config - Config variable (correctly) not found: params.validationFailUnrecognisedParams
nextflow_config - Config variable (correctly) not found: params.validationLenientMode
nextflow_config - Config variable (correctly) not found: params.validationSchemaIgnoreParams
nextflow_config - Config variable (correctly) not found: params.validationShowHiddenParams
nextflow_config - Config variable (correctly) not found: validation.failUnrecognisedParams
nextflow_config - Config variable (correctly) not found: validation.failUnrecognisedHeaders
nextflow_config - Config timeline.enabled had correct value: true
nextflow_config - Config report.enabled had correct value: true
nextflow_config - Config trace.enabled had correct value: true
nextflow_config - Config dag.enabled had correct value: true
nextflow_config - Config manifest.name began with nf-core/
nextflow_config - Config variable manifest.homePage began with https://github.com/nf-core/
nextflow_config - Config dag.file ended with .html
nextflow_config - Config variable manifest.nextflowVersion started with >= or !>=
nextflow_config - Config manifest.version ends in dev: 3.22.0dev
nextflow_config - Config params.custom_config_version is set to master
nextflow_config - Config params.custom_config_base is set to https://raw.githubusercontent.com/nf-core/configs/master
nextflow_config - Lines for loading custom profiles found
nextflow_config - nextflow.config contains configuration profile test
nextflow_config - Config default value correct: params.hisat2_build_memory= 200.GB
nextflow_config - Config default value correct: params.gtf_extra_attributes= gene_name
nextflow_config - Config default value correct: params.gtf_group_features= gene_id
nextflow_config - Config default value correct: params.featurecounts_group_type= gene_biotype
nextflow_config - Config default value correct: params.featurecounts_feature_type= exon
nextflow_config - Config default value correct: params.igenomes_base= s3://ngi-igenomes/igenomes/
nextflow_config - Config default value correct: params.trimmer= trimgalore
nextflow_config - Config default value correct: params.min_trimmed_reads= 10000
nextflow_config - Config default value correct: params.umi_dedup_tool= umitools
nextflow_config - Config default value correct: params.umitools_extract_method= string
nextflow_config - Config default value correct: params.umitools_grouping_method= directional
nextflow_config - Config default value correct: params.aligner= star_salmon
nextflow_config - Config default value correct: params.pseudo_aligner_kmer_size= 31
nextflow_config - Config default value correct: params.min_mapped_reads= 5.0
nextflow_config - Config default value correct: params.kallisto_quant_fraglen= 200
nextflow_config - Config default value correct: params.kallisto_quant_fraglen_sd= 200
nextflow_config - Config default value correct: params.stranded_threshold= 0.8
nextflow_config - Config default value correct: params.unstranded_threshold= 0.1
nextflow_config - Config default value correct: params.extra_fqlint_args= --disable-validator P001
nextflow_config - Config default value correct: params.deseq2_vst= true
nextflow_config - Config default value correct: params.rseqc_modules= bam_stat,inner_distance,infer_experiment,junction_annotation,junction_saturation,read_distribution,read_duplication
nextflow_config - Config default value correct: params.bracken_precision= S
nextflow_config - Config default value correct: params.skip_bbsplit= true
nextflow_config - Config default value correct: params.skip_preseq= true
nextflow_config - Config default value correct: params.custom_config_version= master
nextflow_config - Config default value correct: params.custom_config_base= https://raw.githubusercontent.com/nf-core/configs/master
nextflow_config - Config default value correct: params.publish_dir_mode= copy
nextflow_config - Config default value correct: params.max_multiqc_email_size= 25.MB
nextflow_config - Config default value correct: params.validate_params= true
nextflow_config - Config default value correct: params.pipelines_testdata_base_path= https://raw.githubusercontent.com/nf-core/test-datasets/7f1614baeb0ddf66e60be78c3d9fa55440465ac8/
nf_test_content - 'tests/hisat2.nf.test' contains outdir parameter
nf_test_content - 'tests/hisat2.nf.test' snapshots a 'versions.yml' file
nf_test_content - 'tests/hisat2.nf.test' snapshots a 'versions.yml' file
nf_test_content - 'tests/hisat2.nf.test' contains outdir parameter
nf_test_content - 'tests/hisat2.nf.test' snapshots a 'versions.yml' file
nf_test_content - 'tests/hisat2.nf.test' snapshots a 'versions.yml' file
nf_test_content - 'tests/kallisto.nf.test' contains outdir parameter
nf_test_content - 'tests/kallisto.nf.test' snapshots a 'versions.yml' file
nf_test_content - 'tests/kallisto.nf.test' snapshots a 'versions.yml' file
nf_test_content - 'tests/kallisto.nf.test' contains outdir parameter
nf_test_content - 'tests/kallisto.nf.test' snapshots a 'versions.yml' file
nf_test_content - 'tests/kallisto.nf.test' snapshots a 'versions.yml' file
nf_test_content - 'tests/default.nf.test' contains outdir parameter
nf_test_content - 'tests/default.nf.test' snapshots a 'versions.yml' file
nf_test_content - 'tests/default.nf.test' snapshots a 'versions.yml' file
nf_test_content - 'tests/default.nf.test' contains outdir parameter
nf_test_content - 'tests/default.nf.test' snapshots a 'versions.yml' file
nf_test_content - 'tests/default.nf.test' snapshots a 'versions.yml' file
nf_test_content - 'tests/bam_input.nf.test' contains outdir parameter
nf_test_content - 'tests/bam_input.nf.test' snapshots a 'versions.yml' file
nf_test_content - 'tests/bam_input.nf.test' snapshots a 'versions.yml' file
nf_test_content - 'tests/bam_input.nf.test' contains outdir parameter
nf_test_content - 'tests/bam_input.nf.test' snapshots a 'versions.yml' file
nf_test_content - 'tests/bam_input.nf.test' snapshots a 'versions.yml' file
nf_test_content - 'tests/remove_ribo_rna.nf.test' contains outdir parameter
nf_test_content - 'tests/remove_ribo_rna.nf.test' snapshots a 'versions.yml' file
nf_test_content - 'tests/remove_ribo_rna.nf.test' snapshots a 'versions.yml' file
nf_test_content - 'tests/remove_ribo_rna.nf.test' contains outdir parameter
nf_test_content - 'tests/remove_ribo_rna.nf.test' snapshots a 'versions.yml' file
nf_test_content - 'tests/remove_ribo_rna.nf.test' snapshots a 'versions.yml' file
nf_test_content - 'tests/salmon.nf.test' contains outdir parameter
nf_test_content - 'tests/salmon.nf.test' snapshots a 'versions.yml' file
nf_test_content - 'tests/salmon.nf.test' snapshots a 'versions.yml' file
nf_test_content - 'tests/salmon.nf.test' contains outdir parameter
nf_test_content - 'tests/salmon.nf.test' snapshots a 'versions.yml' file
nf_test_content - 'tests/salmon.nf.test' snapshots a 'versions.yml' file
nf_test_content - 'tests/min_mapped_reads.nf.test' contains outdir parameter
nf_test_content - 'tests/min_mapped_reads.nf.test' snapshots a 'versions.yml' file
nf_test_content - 'tests/min_mapped_reads.nf.test' snapshots a 'versions.yml' file
nf_test_content - 'tests/min_mapped_reads.nf.test' contains outdir parameter
nf_test_content - 'tests/min_mapped_reads.nf.test' snapshots a 'versions.yml' file
nf_test_content - 'tests/min_mapped_reads.nf.test' snapshots a 'versions.yml' file
nf_test_content - 'tests/sentieon_default.nf.test' contains outdir parameter
nf_test_content - 'tests/sentieon_default.nf.test' snapshots a 'versions.yml' file
nf_test_content - 'tests/sentieon_default.nf.test' snapshots a 'versions.yml' file
nf_test_content - 'tests/sentieon_default.nf.test' contains outdir parameter
nf_test_content - 'tests/sentieon_default.nf.test' snapshots a 'versions.yml' file
nf_test_content - 'tests/sentieon_default.nf.test' snapshots a 'versions.yml' file
nf_test_content - 'tests/nofasta.nf.test' contains outdir parameter
nf_test_content - 'tests/nofasta.nf.test' snapshots a 'versions.yml' file
nf_test_content - 'tests/nofasta.nf.test' snapshots a 'versions.yml' file
nf_test_content - 'tests/skip_trimming.nf.test' contains outdir parameter
nf_test_content - 'tests/skip_trimming.nf.test' snapshots a 'versions.yml' file
nf_test_content - 'tests/skip_trimming.nf.test' snapshots a 'versions.yml' file
nf_test_content - 'tests/featurecounts_group_type.nf.test' contains outdir parameter
nf_test_content - 'tests/featurecounts_group_type.nf.test' snapshots a 'versions.yml' file
nf_test_content - 'tests/featurecounts_group_type.nf.test' snapshots a 'versions.yml' file
nf_test_content - 'tests/featurecounts_group_type.nf.test' contains outdir parameter
nf_test_content - 'tests/featurecounts_group_type.nf.test' snapshots a 'versions.yml' file
nf_test_content - 'tests/featurecounts_group_type.nf.test' snapshots a 'versions.yml' file
nf_test_content - 'tests/skip_qc.nf.test' contains outdir parameter
nf_test_content - 'tests/skip_qc.nf.test' snapshots a 'versions.yml' file
nf_test_content - 'tests/skip_qc.nf.test' snapshots a 'versions.yml' file
nf_test_content - 'tests/skip_qc.nf.test' contains outdir parameter
nf_test_content - 'tests/skip_qc.nf.test' snapshots a 'versions.yml' file
nf_test_content - 'tests/skip_qc.nf.test' snapshots a 'versions.yml' file
nf_test_content - 'tests/umi.nf.test' contains outdir parameter
nf_test_content - 'tests/umi.nf.test' snapshots a 'versions.yml' file
nf_test_content - 'tests/umi.nf.test' snapshots a 'versions.yml' file
nf_test_content - 'tests/umi.nf.test' contains outdir parameter
nf_test_content - 'tests/umi.nf.test' snapshots a 'versions.yml' file
nf_test_content - 'tests/umi.nf.test' snapshots a 'versions.yml' file
nf_test_content - 'tests/umi.nf.test' contains outdir parameter
nf_test_content - 'tests/umi.nf.test' snapshots a 'versions.yml' file
nf_test_content - 'tests/umi.nf.test' snapshots a 'versions.yml' file
nf_test_content - 'tests/star_rsem.nf.test' contains outdir parameter
nf_test_content - 'tests/star_rsem.nf.test' snapshots a 'versions.yml' file
nf_test_content - 'tests/star_rsem.nf.test' snapshots a 'versions.yml' file
nf_test_content - 'tests/star_rsem.nf.test' contains outdir parameter
nf_test_content - 'tests/star_rsem.nf.test' snapshots a 'versions.yml' file
nf_test_content - 'tests/star_rsem.nf.test' snapshots a 'versions.yml' file
nf_test_content - 'tests/nextflow.config' contains modules_testdata_base_path
nf_test_content - 'tests/nextflow.config' contains pipelines_testdata_base_path
nf_test_content - 'nf-test.config' sets a testsDir
nf_test_content - 'nf-test.config' sets a workDir
nf_test_content - 'nf-test.config' sets a configFile
files_unchanged - .gitattributes matches the template
files_unchanged - .prettierrc.yml matches the template
files_unchanged - CODE_OF_CONDUCT.md matches the template
files_unchanged - LICENSE matches the template
files_unchanged - .github/.dockstore.yml matches the template
files_unchanged - .github/CONTRIBUTING.md matches the template
files_unchanged - .github/ISSUE_TEMPLATE/bug_report.yml matches the template
files_unchanged - .github/ISSUE_TEMPLATE/config.yml matches the template
files_unchanged - .github/ISSUE_TEMPLATE/feature_request.yml matches the template
files_unchanged - .github/PULL_REQUEST_TEMPLATE.md matches the template
files_unchanged - .github/workflows/branch.yml matches the template
files_unchanged - .github/workflows/linting_comment.yml matches the template
files_unchanged - .github/workflows/linting.yml matches the template
files_unchanged - assets/sendmail_template.txt matches the template
files_unchanged - assets/nf-core-rnaseq_logo_light.png matches the template
files_unchanged - docs/images/nf-core-rnaseq_logo_light.png matches the template
files_unchanged - docs/images/nf-core-rnaseq_logo_dark.png matches the template
files_unchanged - docs/README.md matches the template
actions_awstest - '.github/workflows/awstest.yml' is triggered correctly
actions_awsfulltest - .github/workflows/awsfulltest.yml is triggered correctly
actions_awsfulltest - .github/workflows/awsfulltest.yml does not use -profile test
readme - README Nextflow minimum version badge matched config. Badge: 25.04.0, Config: 25.04.0
readme - README nf-core template version badge found.
readme - README Zenodo placeholder was replaced with DOI.
plugin_includes - No wrong validation plugin imports have been found
pipeline_name_conventions - Name adheres to nf-core convention
template_strings - Did not find any Jinja template strings (0 files)
schema_lint - Schema lint passed
schema_lint - Schema title + description lint passed
schema_lint - Input mimetype lint passed: 'text/csv'
schema_params - Schema matched params returned from nextflow config
system_exit - No System.exit calls found
actions_schema_validation - Workflow validation passed: linting.yml
actions_schema_validation - Workflow validation passed: download_pipeline.yml
actions_schema_validation - Workflow validation passed: cloud_tests_small.yml
actions_schema_validation - Workflow validation passed: template-version-comment.yml
actions_schema_validation - Workflow validation passed: linting_comment.yml
actions_schema_validation - Workflow validation passed: release-announcements.yml
actions_schema_validation - Workflow validation passed: fix_linting.yml
actions_schema_validation - Workflow validation passed: nf-test.yml
actions_schema_validation - Workflow validation passed: awsfulltest.yml
actions_schema_validation - Workflow validation passed: cloud_tests_full.yml
actions_schema_validation - Workflow validation passed: branch.yml
actions_schema_validation - Workflow validation passed: awstest.yml
actions_schema_validation - Workflow validation passed: clean-up.yml
merge_markers - No merge markers found in pipeline files
modules_json - Only installed modules found in modules.json
modules_structure - modules directory structure is correct 'modules/nf-core/TOOL/SUBTOOL'
local_component_structure - local subworkflows directory structure is correct 'subworkflows/local/TOOL/SUBTOOL'
base_config - conf/base.config found and not ignored.
nfcore_yml - Repository type in .nf-core.yml is valid: pipeline
nfcore_yml - nf-core version in .nf-core.yml is set to the latest version: 3.5.1
rocrate_readme_sync - RO-Crate description matches the README.md.

Run details

nf-core/tools version 3.5.1
Run at 2025-11-25 17:25:12

nf-core-bot · 2025-11-25T14:30:26Z

Warning

Newer version of the nf-core template is available.

Your pipeline is using an old version of the nf-core template: 3.4.1.
Please update your pipeline to the latest version.

For more documentation on how to update your pipeline, please see the nf-core documentation and Synchronisation documentation.

Add warning about Qualimap read counting bug

33168d3

Document known limitation where Qualimap reports more reads than exist in the BAM file when assigning reads to genomic features. Provide workarounds including using RSeQC as alternative and --skip_qualimap flag. Addresses #1273

pinin4fjords mentioned this pull request Nov 14, 2025

Qualimap assigns more reads to exonic regions than total reads in the sample #1273

Closed

Update CHANGELOG.md for Qualimap documentation PR

f2ddd31

pinin4fjords added this to the 3.22.0 milestone Nov 14, 2025

Merge branch 'dev' into docs-qualimap-limitation

11eb565

pinin4fjords added the Review & Merge label Nov 25, 2025

maxulysse approved these changes Nov 25, 2025

View reviewed changes

Merge branch 'dev' into docs-qualimap-limitation

a353fa4

pinin4fjords merged commit 84c35ee into dev Nov 25, 2025
6 checks passed

pinin4fjords deleted the docs-qualimap-limitation branch November 25, 2025 17:26

pinin4fjords mentioned this pull request Nov 26, 2025

Dev -> master for nf-core/rnaseq 3.22.0 #1639

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Document Qualimap read counting limitation #1625

Document Qualimap read counting limitation #1625

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Document Qualimap read counting limitation #1625

Document Qualimap read counting limitation #1625

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pinin4fjords commented Nov 14, 2025

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nf-core pipelines lint overall result: Passed ✅ ⚠️

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`nf-core pipelines lint` overall result: Passed ✅ ⚠️