Bulk RNA-seq Pathway Enrichment

Pathway enrichment analysis for bulk RNA-seq differential expression results. Primary engine is GSEApy for ORA (Enrichr) and GSEA (pre-ranked); falls back to a built-in hypergeometric test with Benjamini-Hochberg FDR correction when GSEApy is not installed.

Why This Exists

• Without it: Researchers must manually extract significant gene lists, format them for external enrichment tools, and cross-reference multiple pathway databases.
• With it: A single Python command runs ORA or GSEA on DE results, produces publication-ready bar and dot plots, and exports filtered enrichment tables ready for biological interpretation.
• Why OmicsClaw: Wraps the standard hypergeometric and rank-based enrichment approaches into the OmicsClaw reporting framework with automatic fallback when optional dependencies are unavailable.

Workflow

1. Load: Read a DE results table (CSV with gene, log2FoldChange, pvalue, padj columns).
2. Filter: For ORA, extract significant genes at user-specified padj and log2FC cutoffs. For GSEA, rank all genes by a combined score.
3. Enrich: Test gene lists against pathway gene sets using hypergeometric ORA or rank-based GSEA.
4. Correct: Apply Benjamini-Hochberg multiple testing correction across all terms.
5. Visualize: Generate enrichment bar plot and dot plot of top enriched terms.
6. Report: Write markdown report, result.json, full and filtered enrichment tables, and a reproducibility script.

CLI Reference

python bulkrna_enrichment.py --input <de_results.csv> --output <dir> --method ora
python bulkrna_enrichment.py --input <de_results.csv> --output <dir> --method gsea
python bulkrna_enrichment.py --demo --output /tmp/bulkrna_enrichment_demo
python bulkrna_enrichment.py --input <de.csv> --output <dir> --gene-set-file custom_sets.json
python omicsclaw.py run bulkrna-enrichment --demo

Example Queries

• "Run pathway enrichment on my bulk RNA DE results"
• "Perform GSEA on this differential expression table"
• "Which GO terms are enriched in my upregulated genes?"
• "Run ORA with KEGG pathways on these DEGs"

Algorithm / Methodology

1. ORA (Over-Representation Analysis): For each gene set, compute a hypergeometric test p-value measuring the overlap between the user's significant gene list and the gene set, relative to the background gene count.
2. GSEA (Gene Set Enrichment Analysis): Rank all genes by log2FC * -log10(pvalue), then for each gene set compute the mean rank compared to random gene sets via permutation testing.
3. Multiple testing: Benjamini-Hochberg correction across all tested terms.
4. GSEApy integration: When gseapy is installed, use its Enrichr and pre-ranked GSEA implementations. Otherwise, the built-in hypergeometric and rank-based methods provide equivalent core functionality.

Input Formats

.csv

gene

log2FoldChange

pvalue

padj

bulkrna-de

Output Structure

output_directory/
├── report.md
├── result.json
├── figures/
│   ├── enrichment_barplot.png
│   └── enrichment_dotplot.png
├── tables/
│   ├── enrichment_results.csv
│   └── enrichment_significant.csv
└── reproducibility/
    └── commands.sh

Parameters

--method

ora

ora

gsea

--padj-cutoff

0.05

--lfc-cutoff

1.0

--gene-set-file

Dependencies

Required: numpy, pandas, scipy, matplotlib Optional: gseapy (recommended; provides Enrichr and full GSEA functionality)

Safety

• Local-first: All processing runs locally; no data is uploaded to external services.
• Disclaimer: Every report includes the standard OmicsClaw disclaimer.
• Audit trail: Parameters, method used (including fallback events), and input checksums are recorded in result.json.

Integration with Orchestrator

Trigger conditions:

• Automatically invoked when user intent matches bulk RNA pathway enrichment keywords.

Chaining partners:

• bulkrna-de

  -- Upstream: differential expression to produce DE tables

• bulkrna-coexpression

  -- Parallel: co-expression modules for functional interpretation

Citations

• GSEApy -- Python wrapper for GSEA/Enrichr
• MSigDB -- Molecular Signatures Database
• Benjamini-Hochberg -- Benjamini & Hochberg, JRSSB 1995
• Subramanian et al. -- Gene set enrichment analysis, PNAS 2005

Related Skills

• bulkrna-de

  -- Differential expression analysis upstream

• bulkrna-coexpression

  -- Co-expression network analysis

• bulkrna-deconvolution

  -- Cell type deconvolution of bulk samples