Automated qc reports

Version Compatibility

Reference examples tested with: MultiQC 1.21+ (Plotly era), FastQC 0.12+, STAR 2.7.11+, Subread 2.0+, salmon 1.10+, samtools 1.19+, Picard 3.1+, fastp 0.23+

Before using code patterns, verify installed versions match. If versions differ:

• CLI: <tool> --version then <tool> --help to confirm flags

Config keys and defaults move between MultiQC releases (the plotting backend changed from HighCharts to Plotly at 1.20; flat-plot and AI thresholds shifted). When a default matters, confirm it against the installed version: python -c "import multiqc; print(multiqc.__version__)" and check that version's config_defaults.yaml.

If code throws an error, run multiqc --help and adapt flags to the installed version rather than retrying.

Automated QC Reports with MultiQC

"Aggregate QC results into one report" -> Walk a directory of tool outputs, parse the metrics those tools already wrote, and render one interactive HTML report plus a parseable multiqc_data/ directory.

• CLI: multiqc <dir> (scans for recognized tool outputs)

The Load-Bearing Idea: MultiQC Aggregates, It Does Not Measure

MultiQC computes nothing. It SCRAPES the log/metrics files that FastQC, STAR, Picard, salmon, bcftools, etc. already wrote, re-tabulates those numbers, and renders them. Every value in a report traces back to an upstream tool's output file. Four consequences that drive every real decision below:

• The report is a triage SNAPSHOT, not a pass/fail gate. There is no "MultiQC said FAIL -> stop the pipeline." MultiQC has no fail-on-threshold and exits 0 on bad QC. Green/amber/red is either the upstream tool's own status (FastQC writes PASS/WARN/FAIL; MultiQC just displays it) or a threshold a human configured. Gating is a SEPARATE step (see From Report to Gate).
• Garbage upstream becomes a clean-looking report. Run a tool with the wrong strandedness, wrong reference, or dedup on amplicon data, and MultiQC faithfully aggregates the wrong numbers into a polished HTML. Polish is not evidence of correctness.
• An empty report means "nothing matched," not "QC passed." Point MultiQC at the wrong directory or over-filter modules and it emits a near-empty report with a log warning and exit 0. Always check the sample count in the header against the roster expected.
• It is only as current as its parsers. Each module is a hand-written parser keyed to a specific log format. An upstream version bump that changes a header can silently drop a file or mis-map a column.

Basic Usage

multiqc results/ -o qc_report/            # scan results/, write qc_report/multiqc_report.html
multiqc results/ -n project_qc -o qc/     # custom report name
multiqc results/ -m fastqc -m star        # ONLY these modules (see scoping below)
multiqc results/ -c multiqc_config.yaml   # reproducible config-driven report

Supported Tools

MultiQC ships parsers for 100+ tools. Common assay groupings:

Stage	Tools with modules
Read QC	FastQC, fastp, Cutadapt, falco
Alignment	STAR, HISAT2, BWA, Bowtie2, samtools, Qualimap, Picard
Quantification	featureCounts, Salmon, kallisto, RSeQC
Variant calling	bcftools, GATK, Picard, SnpEff, VEP
Single-cell	Cell Ranger, STARsolo

Module Detection Is Regex - Scope It

Detection runs off search_patterns.yaml: each module declares a filename glob/regex (fn/fn_re) and/or a file-content match (contents/contents_re, bounded by num_lines). Loose patterns (*.txt, *.log, *.json) in a messy directory cause FALSE module matches and PHANTOM samples - a file that is not really that tool's output gets parsed as one. A single file can also satisfy two modules.

Scope explicitly rather than trusting auto-detection across thousands of samples:

multiqc results/ --ignore "*_tmp/" --ignore "work/"   # drop paths from the search
multiqc results/ -m fastqc -m star -m salmon          # run ONLY named modules
multiqc results/ -e snippy -e custom_content          # run all EXCEPT named modules

Tighten an over-loose pattern by overriding sp: in the config (sp: {mytool: {fn: 'real_name_*.txt'}}). Production configs pin sp: and module_order instead of relying on detection.

Sample Names Are Derived, Not Declared

Sample names are NOT read from a manifest. MultiQC derives each name from the matched filename (or a sample column inside the file), then "cleans" it by trimming a ~100-entry default list of extensions (fn_clean_exts: .gz, .fastq, .bam, _fastqc, ...). This is how sampleA_R1.fastq.gz, sampleA.sorted.bam, and sampleA.salmon/ all collapse to one sampleA row gathering read, alignment, and quant metrics.

The same mechanism is the #1 large-cohort bug:

• Merge - two genuinely different inputs clean to the same name (two lanes both reduce to sampleA) and silently overwrite each other's metrics.
• Split - one sample appears as several rows because different tools cleaned its name differently (one kept _L001, another stripped it).

multiqc_data/multiqc_sources.txt maps every parsed file to the sample name it produced - read it first when diagnosing duplicate/missing rows. Controls:

Need	Control
Add suffixes to strip (keep defaults)	extra_fn_clean_exts: in config (do NOT override fn_clean_exts, which replaces the defaults)
Use the log filename as the name	--fn_as_s_name (config use_filename_as_sample_name)
Disambiguate by directory	--dirs / -d, --dirs-depth N
Keep full names, no cleaning	--fullnames / -s
Rename at report time	--replace-names map.tsv (pattern -> replacement, two columns)
Offer toggleable name sets	--sample-names headered.tsv (relabel buttons, does not merge rows)

General Statistics and Conditional Formatting Are Configured, Not Authoritative

The General Statistics table is one row per sample with columns each module contributes. Cell colors come from table_cond_formatting_rules (numeric gt/lt/eq/ge/le, string s_eq/s_contains/s_ne). A red ">10% duplication" cell is red because someone wrote that rule (or because a module ships a built-in default rule), not because biology says 10% is bad. Treat formatting as a configured convenience; absence of red is not a pass, and presence of red is not a biological verdict. Column visibility/order/naming are config too (table_columns_visible, table_columns_placement, table_columns_name).

Large Cohorts: MultiQC Downgrades Automatically

To keep the single HTML openable, MultiQC silently changes rendering as series counts grow. The exact thresholds have moved across versions - verify against the installed config_defaults.yaml - but the behaviors are:

Behavior	Config key	Effect
Table -> violin/beeswarm plot	max_table_rows (~500)	above the limit the General Stats "table" becomes a distribution plot; per-cell view is lost
Interactive plot deferred	plots_defer_loading_numseries (~100)	viewer must click to render
Interactive -> flat image	plots_flat_numseries (moved across versions; HighCharts-era 100, current default much higher)	plots render as static PNG/SVG

Force a mode for reproducible visuals across cohort sizes: --flat / --interactive (config plots_force_flat / plots_force_interactive). At tens of thousands of samples, also scope with -m/--ignore or split into per-batch reports - MultiQC holds all parsed data in memory before rendering.

Custom Content (Injecting Custom Metrics)

Two mechanisms; --custom-data-file does NOT exist.

• _mqc suffix - any file named *_mqc.{tsv,csv,txt,yaml,json,png,...} is auto-discovered and rendered with no config. The suffix is what makes it findable.
• custom_data in the config - define a section with plot_type (bargraph, linegraph, table, generalstats, image, ...) and inline data or a search pattern. plot_type: generalstats injects columns straight into General Statistics.

From Report to Gate (the decision MultiQC does not make)

MultiQC is a viewer; QC GATING is separate. The machine-readable truth lives in multiqc_data/: multiqc_data.json (all parsed values), per-module multiqc_*.txt tables, and multiqc_general_stats.txt. Build a gate ON TOP of that file, not by scraping the HTML:

multiqc results/ -o qc/ --data-format json     # write multiqc_data.json
# a downstream script parses qc/multiqc_data/multiqc_data.json,
# applies thresholds, and exits non-zero / quarantines failing samples.

This is the correct division of labor: MultiQC presents; the pipeline (nf-core modules, a purpose-built gater like CheckQC, a Nextflow/Snakemake check, or a parse-and-exit script) decides. Building fail-on-threshold logic inside MultiQC is a category error.

AI Summaries Send Data Off-Network

MultiQC (1.27+) can prepend an LLM-written natural-language summary (--ai / --ai-summary, --ai-summary-full; providers via ai_provider: seqera, openai, anthropic, aws_bedrock, custom; keys via OPENAI_API_KEY / ANTHROPIC_API_KEY / SEQERA_ACCESS_TOKEN). It is OFF by default. When enabled it transmits the aggregated QC metrics - and, unless ai_anonymize_samples is set, the SAMPLE NAMES - to an external API over the internet. For clinical, patient, or embargoed data this can be a data-governance violation; use --no-ai to strip AI controls from a shared report, or the in-browser on-demand mode (summary stays in browser local storage, not baked into the distributed HTML). Confirm the exact key spelling against the installed version.

Reproducibility

• Pin both the MultiQC version and the upstream tool versions. Parsers, default thresholds, and column sets shift between releases; a report from 1.30 is not byte-stable against one from 1.14.
• Pin a config, treat the report as a deliverable. The nf-core pattern: ship multiqc_config.yml locking title, module_order, sample-name cleaning, and sp: patterns; expose --multiqc_config to layer a user config on top (both apply, user wins). nf-core also emits a methods_description_template.yml so the report carries auto-generated methods text and citations for only the tools that ran, plus a consolidated software-versions table.
• Cross-check the roster. If 3 of 100 BAMs failed earlier and produced no metrics, MultiQC reports a clean 97-sample report with no indication 3 are missing. It aggregates what exists and has no notion of an expected sample set.

Common Errors

Symptom	Cause	Fix
Near-empty report, exit 0	No files matched a search pattern (wrong dir, over-filtered)	Check header sample count; read multiqc_sources.txt; relax -m/--ignore
Two samples merged into one row	Names collide after fn_clean_exts cleaning	extra_fn_clean_exts, --dirs, or --replace-names; verify in multiqc_sources.txt
One sample split across rows	Tools cleaned the name differently	--fn_as_s_name or extra_fn_clean_exts to normalize
Phantom sample / wrong module	Loose pattern matched an unrelated file	--ignore the path or tighten sp:; restrict with -m
Metric missing after a tool upgrade	Upstream log-format drift broke the parser	Pin tool + MultiQC versions; check the module changelog
"table" rendered as a violin plot	Rows exceeded max_table_rows	Raise the limit or split the cohort
Sensitive sample names left the network	AI summary enabled	--no-ai, or ai_anonymize_samples; default is off

Related Skills

• read-qc/quality-reports - Generate the FastQC/falco inputs MultiQC aggregates
• read-qc/fastp-workflow - Preprocessing QC that feeds the report
• read-qc/rnaseq-qc - Post-alignment RNA-seq metrics surfaced in MultiQC
• workflows/rnaseq-to-de - Full pipeline that emits a MultiQC report as a deliverable

References

• Ewels P, Magnusson M, Lundin S, Käller M. MultiQC: summarize analysis results for multiple tools and samples in a single report. Bioinformatics. 2016;32(19):3047-3048. doi:10.1093/bioinformatics/btw354
• MultiQC documentation: docs.seqera.io/multiqc (post-2024 features incl. Plotly plots and AI summaries are documented here and in the GitHub CHANGELOG, not a separate paper)