Bulk RNA-seq

Overview

This skill prepares bulk RNA-seq reads and quantification output for a reproducible gene-level comparison. It owns sample validation, reads-to-counts recipes, and the count/metadata handoff; downstream statistical fitting and enrichment remain in their specialist skills.

"Defensible" means three things, applied throughout:

• Reproducible — pinned pipeline/tool versions, containers where possible, recorded parameters, fixed random seeds.
• Quality-gated — QC is inspected and acted on before, during, and after quantification, not skipped.
• Statistically sound — adequate replication, a design that matches the biology, counts handled correctly, and FDR-controlled testing.

The pipeline is: FastQC/trim → align/quant (STAR/Salmon) → counts → DE (pydeseq2) → enrichment (pathway-enrichment) → figures.

When to Use This Skill

Use this skill when the user wants to:

• Go from FASTQ files (or a sequencing run) to differentially expressed genes and pathways.
• Run or configure nf-core/rnaseq, or align/quantify with STAR, Salmon, or featureCounts.
• Turn Salmon/STAR/featureCounts output into a counts matrix ready for DESeq2/PyDESeq2.
• Design or sanity-check a bulk RNA-seq experiment (replicates, batch, strandedness) before committing compute.
• Scope an end-to-end RNA-seq analysis and decide which tools and skills to chain.

This is bulk RNA-seq (samples = biological specimens). For single-cell/nuclei data use scanpy; for the DE statistics alone use pydeseq2; for enrichment alone use pathway-enrichment.

The Pipeline at a Glance

flowchart TD
    fastq["Raw FASTQ + samplesheet"] --> qc["FastQC + MultiQC"]
    qc --> trim["Trim: fastp / Trim Galore"]
    trim --> align["Align + quant: STAR and/or Salmon"]
    align --> counts["Gene-level counts matrix"]
    counts --> de["Differential expression"]
    de --> enrich["Pathway / GSEA enrichment"]
    de --> fig["Figures"]
    enrich --> fig
    nfcore["nf-core/rnaseq via nextflow skill"] -.->|"path A"| align
    manual["Standalone recipes (this skill)"] -.->|"path B"| align
    bridge["build_counts_matrix.py (this skill)"] -.-> counts
    pydeseq2skill["pydeseq2 skill"] -.-> de
    pwskill["pathway-enrichment skill"] -.-> enrich
    vizskill["scientific-visualization skill"] -.-> fig

Two Upstream Paths — Pick One

The reads → counts stage can be run two ways. Both produce gene-level counts, but STAR/featureCounts and Salmon/tximport do not generally give identical or interchangeable values: assignment rules, multimapping, and effective-length corrections differ. Choose one quantification route for the entire comparison.

When unsure, prefer Path A: nf-core/rnaseq already wires together FastQC → trimming → STAR/Salmon → quantification → tximport → MultiQC with sensible, reviewed defaults, which is the most defensible option. Path B exists for transparency and constrained setups.

Both paths converge on a gene-level counts matrix. Preserve whether counts are raw or length-scaled, the transcript-to-gene mapping release, and any offsets. Length-scaled counts must not receive a second transcript-length correction.

Setup

# This skill's glue (bridge + handoffs) — Python
uv pip install "pytximport==0.13.0" pandas numpy

# Downstream skills install their own deps:
#   pydeseq2 skill           -> uv pip install pydeseq2
#   pathway-enrichment skill -> uv pip install gseapy gprofiler-official

# Path A (nf-core): only Nextflow + a container engine are needed — see the `nextflow` skill.

# Path B (standalone tools): install via bioconda. Pin versions for reproducibility.
conda create -n rnaseq -c conda-forge -c bioconda --strict-channel-priority \
  fastqc fastp trim-galore "star=2.7.11b" "salmon=2.8.0" subread multiqc

Reviewed against nf-core/rnaseq 3.27.0, STAR 2.7.11b documentation, Salmon 2.8.0, pytximport 0.13.0 and PyDESeq2 0.5.4. The bundled Python bridge and a tiny Salmon index/quant run were executed; the full Nextflow/STAR/trimming recipes are illustrative, not an end-to-end validation. Salmon 2.x cannot read older C++ indexes: rebuild them. The bridge's length-scaled Salmon route is for full-length bulk RNA-seq; 3′ counting assays need original counts without transcript-length correction.

Record the exact versions you use (pipeline revision, tool versions, reference genome + annotation release) — they belong in the methods section and make the analysis reproducible.

Version 2.0 makes STAR strandedness explicit and rejects ambiguous samples, gene sets, transcript mappings and fractional featureCounts data that older bridge versions accepted.

Quick Start

Path A — nf-core/rnaseq (recommended)

# 0. Validate the samplesheet first (catches the most common failures early)
python scripts/validate_samplesheet.py --samplesheet samplesheet.csv --nfcore

# 1. Smoke-test the environment with tiny bundled data
nextflow run nf-core/rnaseq -r 3.27.0 -profile test,docker --outdir test_results

# 2. Real run: pin the revision, pick an aligner, pass a samplesheet + reference
nextflow run nf-core/rnaseq -r 3.27.0 \
  -profile docker \
  --input samplesheet.csv \
  --genome GRCh38 \
  --aligner star_salmon \
  --outdir results \
  -resume

nf-core/rnaseq runs tximport internally, so gene counts come out already merged — no bridge script needed. Use results/star_salmon/salmon.merged.gene_counts_length_scaled.tsv for DE. Samplesheet format, aligner choice, and outputs: references/upstream-nfcore.md. For engine/HPC/cloud/container detail, use the **nextflow** skill.

Path B — standalone STAR/Salmon (abbreviated)

mkdir -p qc/
fastqc -o qc/ reads/*.fastq.gz                      # 1. QC raw reads
fastp -i s1_R1.fq.gz -I s1_R2.fq.gz \
      -o s1_R1.trim.fq.gz -O s1_R2.trim.fq.gz \
      --thread 4 -j s1.fastp.json                   # 2. Trim adapters/low-quality
salmon quant -i salmon_index -l A \
      -1 s1_R1.trim.fq.gz -2 s1_R2.trim.fq.gz \
      --gcBias --seqBias -p 8 -o quant/s1            # 3. Quantify (per sample)

Full recipes (FastQC, fastp/Trim Galore, STAR index+align+--quantMode GeneCounts, Salmon decoy-aware index, featureCounts, strandedness): references/upstream-manual.md.

Counts → DE → enrichment (both paths)

# Path B only: assemble a gene x sample counts matrix + metadata template for PyDESeq2
python scripts/build_counts_matrix.py --from salmon \
  --quant-dir quant/ --tx2gene tx2gene.tsv --output-dir counts/

# Then hand off (see the dedicated skills):
#   pydeseq2:           counts.csv + metadata.csv -> DE table (log2FC, padj, stat)
#   pathway-enrichment: rank by `stat` (GSEA) or padj+|LFC| hit list (ORA)
#   scientific-visualization / matplotlib: volcano, MA, heatmap, PCA, enrichment dotplot

Stage-by-Stage Workflow

Work top to bottom. Each stage names the skill or file that owns the detail. Don't skip the design/QC stages — they are where bulk RNA-seq studies most often go wrong.

1. Design & sample sheet. Confirm ≥3 biological replicates per group, identify batch/confounders, and choose the comparison(s). Build the samplesheet and validate it with scripts/validate_samplesheet.py. Rationale and rules: references/design-and-qc.md.
2. Raw-read QC. FastQC per file; aggregate with MultiQC. Check per-base quality, adapter content, duplication, and over-representation. Thresholds: references/design-and-qc.md.
3. Trimming. Remove adapters and low-quality tails (via fastp or Trim Galore). Re-run FastQC to confirm. Recipes: references/upstream-manual.md (Path A does this for you).
4. Align / quantify. STAR (genome alignment + --quantMode GeneCounts) and/or Salmon (decoy-aware selective alignment). Determine strandedness — the wrong convention can silently discard most assigned reads. Detail: references/upstream-manual.md; pipeline params: references/upstream-nfcore.md.
5. Build the counts matrix. Turn quant output into a gene × sample integer matrix and a metadata template (scripts/build_counts_matrix.py). The estimated-count and gene-ID-mapping nuances live in references/counts-and-handoff.md.
6. **Differential expression → pydeseq2 skill.** Load counts.csv + metadata.csv, set the design (e.g. ~batch + condition), check full rank and residual degrees of freedom, fit, and test an explicit contrast (e.g. treated versus control) with FDR control. Inspect the PCA and p-value histogram as QC.
7. **Enrichment → pathway-enrichment skill.** For GSEA, rank the full gene list by the DESeq2 stat; for ORA, pass the thresholded hit list (padj < 0.05, optionally |log2FC| > 1). Match identifiers to the selected library; retain an auditable mapping and an assay-specific ORA background.
8. **Figures → scientific-visualization skill.** Volcano, MA, sample-distance heatmap, PCA, and enrichment dotplots, plus the MultiQC report for the QC narrative.

The counts → DE bridge (the key glue)

This is the one stage with no upstream/downstream skill, so this skill owns it. scripts/build_counts_matrix.py converts quant output into exactly what pydeseq2 expects:

• Salmon (--from salmon): aggregates per-sample quant.sf to gene level with pytximport using counts_from_abundance="length_scaled_tpm" (an offset-free choice for full-length gene-level DE), needs a tx2gene map.
• STAR (--from star): reads each ReadsPerGene.out.tab, selecting the column for your --strandedness (unstranded/forward/reverse).
• featureCounts (--from featurecounts): parses the combined featureCounts matrix.

It writes counts.csv (genes × samples, integers), counts_provenance.json (input hashes, import mode and sample order), and metadata_template.csv (one row per sample) for you to fill in. Salmon/RSEM counts are estimates (non-integer); they are rounded to integers because PyDESeq2 requires integer counts — see references/counts-and-handoff.md for why this is acceptable with length_scaled_tpm and how it differs from the offset-based DESeq2+tximport route. That reference also covers identifier mapping (when required by the selected enrichment library) and the exact orientation PyDESeq2 wants.

Common Pitfalls

These cause most wrong or irreproducible bulk RNA-seq results:

1. Too few replicates. Plan biological replication from expected variability and effect size; three per group is a starting point, not a power guarantee. Technical lanes do not increase biological sample size.
2. Confounded batch and condition. If every treated sample was processed on a different day/lane than controls, the effect is unrecoverable. Randomize, and model known batches (~batch + condition). See references/design-and-qc.md.
3. Wrong strandedness. Choosing the wrong STAR column or featureCounts -s/Salmon library type can discard most assigned reads; there is no universal 50% loss. Use Salmon -l A or infer strandedness, and verify the assigned-reads fraction.
4. Feeding TPM/FPKM to DESeq2. DESeq2 needs raw (or length-scaled) counts, never TPM/FPKM/normalized values. The bridge handles this.
5. Non-integer counts. The bridge rounds length-scaled Salmon estimates only; it rejects fractional featureCounts values rather than truncating them. Its Salmon route is for full-length assays, not 3′ tag counts.
6. Gene-ID mismatch into enrichment. Match gene IDs to the selected organism and gene-set release. Many GMT libraries use symbols; g:Profiler can accept Ensembl IDs directly. Do not collapse ambiguous mappings silently.
7. Skipping post-quant QC. Always look at the PCA and sample-distance heatmap before trusting DE — they expose swapped labels, outliers, and hidden batches.
8. Mixing aligners across samples. Quantify every sample with the same tool, version, reference, and parameters.
9. Unpinned versions. "latest" pipelines/genomes make results unreproducible; pin -r, tool versions, and the genome/annotation release.

Integration with Other Skills

• Upstream execution: nextflow (runs nf-core/rnaseq, Path A; HPC/cloud/containers).
• Reference data / gene IDs: gget (gget ref for genome+GTF, gget info/gget search for ID mapping), database-lookup (Ensembl/NCBI), biopython/pysam (FASTA/BAM handling).
• Differential expression: pydeseq2 (the DE engine this skill hands counts to).
• Enrichment: pathway-enrichment (ORA + GSEA; its scripts/run_enrichment.py reads a DESeq2 results CSV directly).
• Figures & reporting: scientific-visualization, matplotlib, seaborn; scientific-writing for the methods/results narrative.
• Related but distinct: scanpy (single-cell), statistical-analysis (multiple-testing depth).

Reference Files

Read the relevant file when you need depth — each is self-contained:

• references/upstream-nfcore.md — Path A: samplesheet format, --aligner/--pseudo_aligner choice, key params, the salmon.merged.gene_counts*.tsv outputs, MultiQC, and what to hand to pydeseq2.
• references/upstream-manual.md — Path B: FastQC, fastp/Trim Galore, STAR genome index + alignment + --quantMode GeneCounts, Salmon decoy-aware index + quant, featureCounts, and how to determine strandedness.
• references/counts-and-handoff.md — turning quant output into PyDESeq2-ready counts.csv/metadata.csv (pytximport, STAR column selection, featureCounts), the integer/estimated-count nuance, Ensembl→symbol mapping, and the DE→enrichment rank/hit-list recipe.
• references/design-and-qc.md — experimental design (replication, batch, confounding, design formulas) and QC-metric interpretation (mapping rate, duplication, rRNA, complexity, PCA/outliers) — the defensible-pipeline backbone.

Resources

• nf-core/rnaseq: https://nf-co.re/rnaseq · STAR: https://github.com/alexdobin/STAR · Salmon: https://combine-lab.github.io/salmon/
• fastp: https://github.com/OpenGene/fastp · Trim Galore: https://github.com/FelixKrueger/TrimGalore · MultiQC: https://multiqc.info
• pytximport: https://pytximport.complextissue.com · featureCounts (Subread): https://subread.sourceforge.net
• Method background: Love et al. 2014 (DESeq2) DOI 10.1186/s13059-014-0550-8 · Soneson et al. 2015 (tximport) DOI 10.12688/f1000research.7563.2