Bulk Rnaseq

Bulk RNA-seq

Overview

This skill orchestrates a complete, defensible bulk RNA-seq differential-expression study, from raw sequencing reads to enriched pathways and figures. It is a router, not a reimplementation: most stages already have dedicated skills in this repo, and this skill connects them in the right order, fills the one real gap (raw reads → a gene-level counts matrix), and enforces the design and QC decisions that determine whether the final result is trustworthy.

"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. They produce equivalent gene counts; choose by context, then stay on that path.

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, after which the workflow is identical.

Setup

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

# 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 bioconda -c conda-forge \
  fastqc fastp trim-galore "star=2.7.11b" "salmon=1.10.3" subread multiqc

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.

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

# 1. Smoke-test the environment with tiny bundled data
nextflow run nf-core/rnaseq -r 3.26.0 -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 3.26.0 \
  -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)

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 (transcript quasi-mapping, decoy-aware). Determine strandedness — it is easy to get wrong and silently halves your counts. 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), fit, and test 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). Map gene IDs to symbols first.
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" (the right choice for 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) 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 Ensembl→symbol mapping (needed before enrichment) and the exact orientation PyDESeq2 wants.

Common Pitfalls

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

1. Too few replicates. <3 biological replicates per group gives almost no power and unstable dispersion estimates. More replicates beat deeper sequencing.
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 silently discards ~half the reads. 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. PyDESeq2 requires integers; round Salmon estimates (the bridge does this).
6. Gene-ID mismatch into enrichment. DESeq2 output is often Ensembl IDs; Enrichr/MSigDB want symbols. Map IDs before pathway-enrichment or "nothing is significant".
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://salmon.readthedocs.io
• 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