beita6969 When to Trigger
Activate this skill when the user mentions:
- Pathway analysis, KEGG, Reactome, WikiPathways
- Gene Ontology (GO) enrichment, biological process, molecular function
- Protein-protein interaction (PPI) networks, STRING, BioGRID
- Multi-omics integration (transcriptomics + proteomics + metabolomics)
- Gene set enrichment analysis (GSEA), over-representation analysis (ORA)
- Sequence databases, UniProt, NCBI, Ensembl queries
- Single-cell RNA-seq analysis, clustering, trajectory inference
Step-by-Step Methodology
- Data preparation - Standardize gene/protein identifiers (convert to Entrez, Ensembl, or UniProt IDs as needed). Remove duplicates and handle ambiguous mappings. Verify organism and genome build.
- Differential analysis - For transcriptomics: DESeq2 or edgeR (count data), limma-voom (normalized). For proteomics: limma with appropriate normalization. Apply multiple testing correction (BH-FDR). Set thresholds (|log2FC| > 1, padj < 0.05 as defaults, adjustable).
- Functional enrichment - Perform GO enrichment (BP, MF, CC) using clusterProfiler, g:Profiler, or DAVID. Run KEGG/Reactome pathway enrichment. Use GSEA for ranked gene lists (no arbitrary cutoff). Report enriched terms with gene ratio, p-value, adjusted p-value, and gene members.
- Network analysis - Build PPI networks from STRING (confidence > 0.7 for high confidence). Identify hub genes (degree centrality), bottleneck nodes (betweenness centrality), and functional modules (MCODE, Louvain clustering). Overlay expression data on network.
- Multi-omics integration - For paired omics: correlation analysis, canonical correlation (CCA), or MOFA/DIABLO. Map features across omics layers using shared identifiers or known biological connections. Identify convergent pathways.
- Single-cell analysis - QC filtering (genes/cell, UMI/cell, mitochondrial %). Normalization (scran, SCTransform). Dimensionality reduction (PCA, UMAP). Clustering (Leiden, Louvain). Cell type annotation (SingleR, scType, marker genes). Trajectory inference (Monocle3, Slingshot).
- Visualization - Generate volcano plots, heatmaps (with hierarchical clustering), dot plots (enrichment), network diagrams, UMAP/tSNE plots (single-cell), and circos plots (multi-omics).
Key Databases and Tools
- Gene Ontology (GO) - Functional annotations
- KEGG / Reactome / WikiPathways - Pathway databases
- STRING / BioGRID / IntAct - PPI databases
- Ensembl / NCBI / UniProt - Sequence and annotation databases
- clusterProfiler / g:Profiler / DAVID - Enrichment tools
- Seurat / Scanpy - Single-cell analysis frameworks
- Cytoscape - Network visualization
Output Format
- Enrichment results as tables: term, description, gene ratio, p-value, padj, gene list.
- Volcano plots with labeled significant genes and fold-change thresholds.
- Network figures with node coloring (expression), size (degree), and module highlighting.
- UMAP/tSNE plots with cluster labels and cell type annotations.
- Heatmaps with dendrograms and annotation bars.
Quality Checklist
- Gene ID mapping verified (conversion losses reported)
- Background gene set appropriate for enrichment analysis
- Multiple testing correction applied (BH-FDR or equivalent)
- Redundant GO terms handled (semantic similarity, REVIGO)
- Network confidence threshold specified and justified
- Single-cell QC thresholds documented
- Batch effects assessed and corrected if present
- Results cross-validated across databases or methods
- Biological interpretation grounded in literature