@bgicli/bgicli 2.1.1 → 2.2.1

package/data/workflows/functional-enrichment-from-degs/references/gsea_ora_comparison.md

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+# GSEA vs ORA: Detailed Method Comparison
+
+This document provides an in-depth comparison of Gene Set Enrichment Analysis
+(GSEA) and Over-Representation Analysis (ORA) to help you understand when to use
+each method.
+
+---
+
+## GSEA (Gene Set Enrichment Analysis)
+
+### Method Overview
+
+**What it does:** Tests whether genes in a pathway tend to be at the TOP or
+BOTTOM of your ranked gene list (by fold change or test statistic).
+
+**Input:** ALL genes ranked by expression change (no cutoff applied)
+
+**Question answered:** "Are genes in this pathway coordinately up- or
+down-regulated?"
+
+**Statistical approach:** Uses a running enrichment score that walks down the
+ranked gene list, increasing when a gene is in the pathway, decreasing when not.
+Computes significance via permutation testing.
+
+### Strengths
+
+- No arbitrary significance cutoffs needed
+- Detects subtle but coordinated changes across many genes
+- Works well even when few genes pass strict thresholds
+- Provides direction (activated vs suppressed pathways)
+- More sensitive to modest changes across entire pathways
+- Uses all available information from the experiment
+
+### Limitations
+
+- Requires ranking metric (fold changes or test statistics)
+- Computationally more expensive (permutation testing)
+- Results can be harder to interpret for non-bioinformaticians
+- Requires careful selection of ranking metric
+
+### Output Interpretation
+
+- **Positive NES (Normalized Enrichment Score):** Pathway is ACTIVATED (genes
+  tend to be upregulated)
+- **Negative NES:** Pathway is SUPPRESSED (genes tend to be downregulated)
+- **Leading edge genes:** Core genes driving the enrichment signal
+- **FDR < 0.05:** Statistically significant after multiple testing correction
+
+### Best Ranking Metrics (in order of preference)
+
+1. **Test statistic (t-statistic, Wald statistic)** - BEST, accounts for both
+   effect size and significance
+2. **Signed -log10(pvalue)** - Good alternative if test statistic unavailable
+3. **Log2 fold change** - Avoid if possible, ignores statistical significance
+
+---
+
+## ORA (Over-Representation Analysis)
+
+### Method Overview
+
+**What it does:** Tests whether your significant genes overlap with a pathway
+more than expected by chance.
+
+**Input:** A discrete list of significant genes (e.g., padj < 0.05,
+|log2FC| > 1)
+
+**Question answered:** "Do my significant genes contain more pathway X members
+than expected?"
+
+**Statistical approach:** Uses Fisher's exact test / hypergeometric test to
+compare observed vs. expected overlap between gene list and pathway.
+
+### Strengths
+
+- Simple and intuitive
+- Fast computation
+- Good for validating GSEA findings
+- Works with any gene list (not just DE results)
+- Easy to explain to collaborators
+
+### Limitations
+
+- Requires arbitrary cutoffs (which genes are "significant"?)
+- Loses information about genes just below threshold
+- Must analyze UP and DOWN genes separately to preserve direction
+- Less sensitive to subtle coordinated changes
+- Can be biased by gene list size
+
+### Output Interpretation
+
+- **GeneRatio:** k/n (genes in pathway / total query genes)
+- **BgRatio:** M/N (genes in pathway in background / total background genes)
+- **p.adjust < 0.05:** Statistically significant enrichment
+- **Higher GeneRatio** = stronger enrichment
+- **Must run separately for up/down genes** to preserve directionality
+
+### Critical ORA Requirements
+
+1. **Specify background genes:** Use all tested genes, not all known genes
+2. **Separate up/down genes:** Don't combine opposite directions
+3. **Appropriate thresholds:** padj < 0.05, |log2FC| ≥ 1 (adjust as needed)
+
+---
+
+## When to Use Which Method
+
+| Scenario                                           | Recommended Method | Rationale                                |
+| -------------------------------------------------- | ------------------ | ---------------------------------------- |
+| **Default / no preference stated**                 | GSEA               | More sensitive, no arbitrary cutoffs     |
+| User has full DE results with fold changes         | GSEA               | Can use all information                  |
+| User wants to detect subtle coordinated changes    | GSEA               | Designed for this                        |
+| User asks "what pathways are activated/suppressed" | GSEA               | Provides directionality via NES          |
+| User has a specific gene list (no fold changes)    | ORA                | GSEA requires ranking                    |
+| User wants quick validation of GSEA results        | ORA                | Complementary approach                   |
+| User explicitly requests ORA                       | ORA                | Honor user preference                    |
+| Few genes pass significance threshold (<50)        | GSEA               | ORA underpowered with few genes          |
+| User wants to compare to Enrichr results           | ORA                | Enrichr uses ORA method                  |
+| Very large gene list (>1000 significant genes)     | Both               | GSEA for sensitivity, ORA for validation |
+
+---
+
+## Decision Flowchart
+
+```
+User requests enrichment analysis
+            │
+            ▼
+    Do they have full DE results
+    (with fold changes)?
+            │
+       ┌────┴────┐
+       │         │
+      YES        NO
+       │         │
+       ▼         ▼
+   Use GSEA    Use ORA
+   (default)   (gene list only)
+       │
+       ▼
+   Did user specifically
+   request ORA?
+       │
+   ┌───┴───┐
+   │       │
+  YES      NO
+   │       │
+   ▼       ▼
+Run both  Run GSEA
+          only
+```
+
+---
+
+## Comparing Results Between Methods
+
+When you run both GSEA and ORA, you may see:
+
+### Concordant Results (expected)
+
+- Pathways significant in both GSEA and ORA
+- This is the strongest evidence for pathway enrichment
+
+### GSEA significant, ORA not significant
+
+- Pathway has modest but coordinated changes across many genes
+- Few genes pass strict ORA thresholds
+- Still biologically meaningful
+
+### ORA significant, GSEA not significant
+
+- Strong signal in a few genes, but not coordinated across pathway
+- May indicate a subset of pathway is affected
+- Less common than scenario above
+
+### Neither significant
+
+- Pathway not affected in your experiment
+- Or: wrong database, wrong species, poor gene identifier mapping
+
+---
+
+## Common Pitfalls
+
+### GSEA Pitfalls
+
+1. ❌ Using fold change alone for ranking (ignores significance)
+2. ❌ Not removing duplicate genes
+3. ❌ Using too few permutations (use ≥10,000)
+4. ❌ Filtering genes before ranking (use ALL genes)
+
+### ORA Pitfalls
+
+1. ❌ Not specifying background genes (biases results)
+2. ❌ Combining up and down genes (loses biological meaning)
+3. ❌ Using all known genes as background (wrong denominator)
+4. ❌ Filtering by keyword instead of statistics
+
+---
+
+## References
+
+1. **GSEA original paper:** Subramanian A, et al. (2005) Gene set enrichment
+   analysis: A knowledge-based approach for interpreting genome-wide expression
+   profiles. PNAS. DOI: 10.1073/pnas.0506580102
+
+2. **Method comparison:** Geistlinger L, et al. (2021) Toward a gold standard
+   for benchmarking gene set enrichment analysis. Brief Bioinform. DOI:
+   10.1093/bib/bbz158
+
+3. **Best practices:** Reimand J, et al. (2019) Pathway enrichment analysis and
+   visualization of omics data using g:Profiler, GSEA, Cytoscape and
+   EnrichmentMap. Nat Protoc. DOI: 10.1038/s41596-018-0103-9

package/data/workflows/functional-enrichment-from-degs/references/gsea_ora_validation_framework.md

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+# GSEA/ORA Workflow Validation Framework v3.0
+
+## Overview
+
+This validation framework tests **unique clusterProfiler capabilities** using
+pre-processed datasets from the vignette. Each test focuses on a distinct
+feature or visualization type to avoid redundancy.
+
+**Primary References:**
+
+- Yu G. _Biomedical Knowledge Mining using GOSemSim and clusterProfiler_ [1]
+- Xu S, Hu E, Cai Y, et al. _Using clusterProfiler to characterize multiomics
+  data._ Nature Protocols 19, 3292–3320 (2024) [2]
+
+---
+
+## Validation Test Summary Table
+
+| Test ID | Unique Capability                | Dataset                      | Function                    | Key Output/Plot               |
+| ------- | -------------------------------- | ---------------------------- | --------------------------- | ----------------------------- |
+| 1       | **KEGG ORA**                     | `geneList` (DOSE)            | `enrichKEGG()`              | `barplot()`                   |
+| 2       | **KEGG GSEA**                    | `geneList` (DOSE)            | `gseKEGG()`                 | `gseaplot2()` + `ridgeplot()` |
+| 3       | **GO ORA with simplify**         | `geneList` (DOSE)            | `enrichGO()` + `simplify()` | `dotplot()`                   |
+| 4       | **GO DAG visualization**         | `geneList` (DOSE)            | `enrichGO()`                | `goplot()`                    |
+| 5       | **MSigDB/Custom gene sets**      | `geneList` (DOSE)            | `enricher()` / `GSEA()`     | `cnetplot()`                  |
+| 6       | **compareCluster (multi-group)** | `gcSample` (clusterProfiler) | `compareCluster()`          | `dotplot()` with facets       |
+| 7       | **Enrichment map**               | `geneList` (DOSE)            | `enrichGO()`                | `emapplot()` + `treeplot()`   |
+| 8       | **Gene-concept heatmap**         | `geneList` (DOSE)            | `enrichKEGG()`              | `heatplot()`                  |
+| 9       | **UpSet plot**                   | `geneList` (DOSE)            | `enrichGO()`                | `upsetplot()`                 |
+| 10      | **KEGG pathway visualization**   | `geneList` (DOSE)            | `enrichKEGG()`              | `browseKEGG()` / `pathview()` |
+
+---
+
+## Test Details
+
+### Test 1: KEGG ORA with Bar Plot
+
+| Field                 | Value                                         |
+| --------------------- | --------------------------------------------- |
+| **Unique Capability** | Basic KEGG over-representation analysis       |
+| **Dataset**           | `data(geneList, package="DOSE")` - pre-loaded |
+| **Function**          | `enrichKEGG()`                                |
+| **Visualization**     | `barplot()`                                   |
+
+**Natural Language Prompt:**
+
+> "Using the DOSE geneList dataset, perform KEGG pathway enrichment on genes
+> with |log2FC| > 2 and show results as a bar plot."
+
+**Expected Results:**
+
+- Cell cycle (hsa04110) as top pathway
+- At least 5 significant pathways (padj < 0.05)
+
+**Validation Criteria:**
+
+- [ ] `enrichKEGG()` returns results
+- [ ] `barplot()` generates visualization
+- [ ] Cell cycle pathway is enriched
+
+**Code:**
+
+```r
+library(clusterProfiler)
+data(geneList, package="DOSE")
+gene <- names(geneList)[abs(geneList) > 2]
+kk <- enrichKEGG(gene = gene, organism = 'hsa')
+barplot(kk, showCategory = 10)
+```
+
+---
+
+### Test 2: KEGG GSEA with Running Score Plot and Ridge Plot
+
+| Field                 | Value                                         |
+| --------------------- | --------------------------------------------- |
+| **Unique Capability** | GSEA with enrichment score visualization      |
+| **Dataset**           | `data(geneList, package="DOSE")` - pre-loaded |
+| **Function**          | `gseKEGG()`                                   |
+| **Visualization**     | `gseaplot2()` + `ridgeplot()`                 |
+
+**Natural Language Prompt:**
+
+> "Using the DOSE geneList dataset, perform KEGG GSEA and show the running
+> enrichment score plot for the top pathway, plus a ridge plot of all
+> significant pathways."
+
+**Expected Results:**
+
+- Mix of positive and negative NES values
+- Running score plot shows gene rank distribution
+
+**Validation Criteria:**
+
+- [ ] `gseKEGG()` returns results with NES values
+- [ ] `gseaplot2()` shows running enrichment score
+- [ ] `ridgeplot()` shows expression distribution across pathways
+
+**Code:**
+
+```r
+library(clusterProfiler)
+library(enrichplot)
+data(geneList, package="DOSE")
+kk2 <- gseKEGG(geneList = geneList, organism = 'hsa', minGSSize = 120)
+gseaplot2(kk2, geneSetID = 1, title = kk2$Description[1])
+ridgeplot(kk2)
+```
+
+---
+
+### Test 3: GO ORA with Redundancy Reduction (simplify)
+
+| Field                 | Value                                         |
+| --------------------- | --------------------------------------------- |
+| **Unique Capability** | GO term simplification to reduce redundancy   |
+| **Dataset**           | `data(geneList, package="DOSE")` - pre-loaded |
+| **Function**          | `enrichGO()` + `simplify()`                   |
+| **Visualization**     | `dotplot()` comparing before/after            |
+
+**Natural Language Prompt:**
+
+> "Using the DOSE geneList dataset, perform GO Biological Process enrichment,
+> then use simplify() to reduce redundant terms. Show dotplots before and after
+> simplification."
+
+**Expected Results:**
+
+- Simplified results have fewer, non-redundant terms
+- Key biological themes preserved
+
+**Validation Criteria:**
+
+- [ ] `enrichGO()` returns many GO terms
+- [ ] `simplify()` reduces term count significantly
+- [ ] `dotplot()` shows cleaner, non-redundant results
+
+**Code:**
+
+```r
+library(clusterProfiler)
+library(org.Hs.eg.db)
+data(geneList, package="DOSE")
+gene <- names(geneList)[abs(geneList) > 2]
+ego <- enrichGO(gene = gene, OrgDb = org.Hs.eg.db, ont = "BP", readable = TRUE)
+ego_simplified <- simplify(ego, cutoff = 0.7, by = "p.adjust")
+dotplot(ego, showCategory = 15, title = "Before simplify")
+dotplot(ego_simplified, showCategory = 15, title = "After simplify")
+```
+
+---
+
+### Test 4: GO DAG Visualization
+
+| Field                 | Value                                         |
+| --------------------- | --------------------------------------------- |
+| **Unique Capability** | Visualize GO terms as directed acyclic graph  |
+| **Dataset**           | `data(geneList, package="DOSE")` - pre-loaded |
+| **Function**          | `enrichGO()`                                  |
+| **Visualization**     | `goplot()`                                    |
+
+**Natural Language Prompt:**
+
+> "Using the DOSE geneList dataset, perform GO Cellular Component enrichment and
+> visualize the enriched terms as a GO DAG showing parent-child relationships."
+
+**Expected Results:**
+
+- DAG shows hierarchical GO term relationships
+- Enriched terms highlighted with color
+
+**Validation Criteria:**
+
+- [ ] `goplot()` generates DAG visualization
+- [ ] Parent-child relationships visible
+- [ ] Color indicates enrichment significance
+
+**Code:**
+
+```r
+library(clusterProfiler)
+library(org.Hs.eg.db)
+data(geneList, package="DOSE")
+gene <- names(geneList)[abs(geneList) > 2]
+ego <- enrichGO(gene = gene, OrgDb = org.Hs.eg.db, ont = "CC")
+goplot(ego)
+```
+
+---
+
+### Test 5: Custom Gene Sets (MSigDB) with Gene-Concept Network
+
+| Field                 | Value                                                              |
+| --------------------- | ------------------------------------------------------------------ |
+| **Unique Capability** | Universal enrichment with custom gene sets + network visualization |
+| **Dataset**           | `data(geneList, package="DOSE")` - pre-loaded                      |
+| **Function**          | `enricher()` or `GSEA()` with MSigDB                               |
+| **Visualization**     | `cnetplot()`                                                       |
+
+**Natural Language Prompt:**
+
+> "Using the DOSE geneList dataset, perform enrichment analysis with MSigDB
+> Hallmark gene sets and visualize the gene-concept network showing which genes
+> belong to which pathways."
+
+**Expected Results:**
+
+- Custom gene sets successfully used
+- Network shows gene-pathway connections
+
+**Validation Criteria:**
+
+- [ ] `enricher()` or `GSEA()` works with custom TERM2GENE
+- [ ] `cnetplot()` shows gene-pathway network
+- [ ] Genes colored by fold change
+
+**Code:**
+
+```r
+library(clusterProfiler)
+library(msigdbr)
+library(enrichplot)
+data(geneList, package="DOSE")
+gene <- names(geneList)[abs(geneList) > 2]
+
+# Get Hallmark gene sets
+h_gene_sets <- msigdbr(species = "Homo sapiens", category = "H") %>%
+  dplyr::select(gs_name, entrez_gene)
+
+em <- enricher(gene, TERM2GENE = h_gene_sets)
+cnetplot(em, categorySize = "pvalue", foldChange = geneList)
+```
+
+---
+
+### Test 6: compareCluster (Multi-Group Comparison)
+
+| Field                 | Value                                            |
+| --------------------- | ------------------------------------------------ |
+| **Unique Capability** | Compare enrichment across multiple gene clusters |
+| **Dataset**           | `data(gcSample)` - pre-loaded in clusterProfiler |
+| **Function**          | `compareCluster()`                               |
+| **Visualization**     | `dotplot()` with multiple columns                |
+
+**Natural Language Prompt:**
+
+> "Using the gcSample dataset from clusterProfiler, compare GO enrichment across
+> all 8 gene clusters and visualize with a dotplot."
+
+**Expected Results:**
+
+- 8 clusters compared simultaneously
+- Shared and unique pathways visible
+
+**Validation Criteria:**
+
+- [ ] `compareCluster()` processes all 8 clusters
+- [ ] `dotplot()` shows multi-column comparison
+- [ ] Different clusters show different enrichment patterns
+
+**Code:**
+
+```r
+library(clusterProfiler)
+library(org.Hs.eg.db)
+data(gcSample)
+ck <- compareCluster(geneCluster = gcSample, fun = enrichGO,
+                     OrgDb = org.Hs.eg.db, ont = "BP")
+dotplot(ck)
+```
+
+---
+
+### Test 7: Enrichment Map and Tree Plot
+
+| Field                 | Value                                         |
+| --------------------- | --------------------------------------------- |
+| **Unique Capability** | Visualize term similarity as network/tree     |
+| **Dataset**           | `data(geneList, package="DOSE")` - pre-loaded |
+| **Function**          | `enrichGO()` + `pairwise_termsim()`           |
+| **Visualization**     | `emapplot()` + `treeplot()`                   |
+
+**Natural Language Prompt:**
+
+> "Using the DOSE geneList dataset, perform GO enrichment and visualize term
+> relationships using an enrichment map (network of similar terms) and a tree
+> plot (hierarchical clustering)."
+
+**Expected Results:**
+
+- Similar GO terms cluster together
+- Tree shows hierarchical grouping of terms
+
+**Validation Criteria:**
+
+- [ ] `pairwise_termsim()` calculates term similarity
+- [ ] `emapplot()` shows term similarity network
+- [ ] `treeplot()` shows hierarchical clustering
+
+**Code:**
+
+```r
+library(clusterProfiler)
+library(org.Hs.eg.db)
+library(enrichplot)
+data(geneList, package="DOSE")
+gene <- names(geneList)[abs(geneList) > 2]
+ego <- enrichGO(gene = gene, OrgDb = org.Hs.eg.db, ont = "BP")
+ego <- pairwise_termsim(ego)
+emapplot(ego)
+treeplot(ego)
+```
+
+---
+
+### Test 8: Gene-Concept Heatmap
+
+| Field                 | Value                                         |
+| --------------------- | --------------------------------------------- |
+| **Unique Capability** | Heatmap of genes vs pathways                  |
+| **Dataset**           | `data(geneList, package="DOSE")` - pre-loaded |
+| **Function**          | `enrichKEGG()`                                |
+| **Visualization**     | `heatplot()`                                  |
+
+**Natural Language Prompt:**
+
+> "Using the DOSE geneList dataset, perform KEGG enrichment and visualize as a
+> heatmap showing which genes are in which pathways, with fold change indicated
+> by color."
+
+**Expected Results:**
+
+- Matrix view of gene-pathway membership
+- Fold change shown as color gradient
+
+**Validation Criteria:**
+
+- [ ] `heatplot()` generates gene-pathway matrix
+- [ ] Fold change mapped to color
+- [ ] Pathways and genes clearly labeled
+
+**Code:**
+
+```r
+library(clusterProfiler)
+library(enrichplot)
+data(geneList, package="DOSE")
+gene <- names(geneList)[abs(geneList) > 2]
+kk <- enrichKEGG(gene = gene, organism = 'hsa')
+heatplot(kk, foldChange = geneList, showCategory = 5)
+```
+
+---
+
+### Test 9: UpSet Plot for Pathway Overlap
+
+| Field                 | Value                                         |
+| --------------------- | --------------------------------------------- |
+| **Unique Capability** | Visualize gene overlap across pathways        |
+| **Dataset**           | `data(geneList, package="DOSE")` - pre-loaded |
+| **Function**          | `enrichGO()`                                  |
+| **Visualization**     | `upsetplot()`                                 |
+
+**Natural Language Prompt:**
+
+> "Using the DOSE geneList dataset, perform GO enrichment and create an UpSet
+> plot showing how genes overlap across the top enriched terms."
+
+**Expected Results:**
+
+- UpSet plot shows intersection sizes
+- Identifies genes shared across multiple terms
+
+**Validation Criteria:**
+
+- [ ] `upsetplot()` generates visualization
+- [ ] Intersection matrix visible
+- [ ] Bar heights show intersection sizes
+
+**Code:**
+
+```r
+library(clusterProfiler)
+library(org.Hs.eg.db)
+library(enrichplot)
+data(geneList, package="DOSE")
+gene <- names(geneList)[abs(geneList) > 2]
+ego <- enrichGO(gene = gene, OrgDb = org.Hs.eg.db, ont = "CC")
+upsetplot(ego)
+```
+
+---
+
+### Test 10: KEGG Pathway Map Visualization
+
+| Field                 | Value                                         |
+| --------------------- | --------------------------------------------- |
+| **Unique Capability** | Overlay expression on KEGG pathway diagrams   |
+| **Dataset**           | `data(geneList, package="DOSE")` - pre-loaded |
+| **Function**          | `enrichKEGG()`                                |
+| **Visualization**     | `browseKEGG()` or `pathview()`                |
+
+**Natural Language Prompt:**
+
+> "Using the DOSE geneList dataset, perform KEGG enrichment and visualize the
+> Cell Cycle pathway with gene expression values overlaid on the pathway
+> diagram."
+
+**Expected Results:**
+
+- KEGG pathway diagram with colored genes
+- Expression values mapped to color scale
+
+**Validation Criteria:**
+
+- [ ] `browseKEGG()` opens pathway in browser OR
+- [ ] `pathview()` generates pathway image file
+- [ ] Genes colored by expression level
+
+**Code:**
+
+```r
+library(clusterProfiler)
+library(pathview)
+data(geneList, package="DOSE")
+gene <- names(geneList)[abs(geneList) > 2]
+kk <- enrichKEGG(gene = gene, organism = 'hsa')
+
+# Option 1: Browser-based
+browseKEGG(kk, 'hsa04110')
+
+# Option 2: Generate image file
+pathview(gene.data = geneList, pathway.id = "hsa04110", species = "hsa")
+```
+
+---
+
+## Quick Reference: Pre-loaded Datasets
+
+| Dataset    | Package         | Description                                  | Load Command                     |
+| ---------- | --------------- | -------------------------------------------- | -------------------------------- |
+| `geneList` | DOSE            | Ranked gene list (log2FC) from breast cancer | `data(geneList, package="DOSE")` |
+| `gcSample` | clusterProfiler | 8 gene clusters for comparison               | `data(gcSample)`                 |
+
+---
+
+## Quick Reference: Visualization Functions
+
+| Function       | Input                  | Output     | Use Case                 |
+| -------------- | ---------------------- | ---------- | ------------------------ |
+| `barplot()`    | enrichResult           | Bar chart  | Simple pathway counts    |
+| `dotplot()`    | enrichResult           | Dot plot   | Gene ratio + p-value     |
+| `cnetplot()`   | enrichResult           | Network    | Gene-pathway connections |
+| `heatplot()`   | enrichResult           | Heatmap    | Gene-pathway matrix      |
+| `emapplot()`   | enrichResult + termsim | Network    | Term similarity          |
+| `treeplot()`   | enrichResult + termsim | Dendrogram | Term clustering          |
+| `upsetplot()`  | enrichResult           | UpSet      | Gene overlap             |
+| `goplot()`     | enrichGO result        | DAG        | GO hierarchy             |
+| `gseaplot2()`  | gseaResult             | Line plot  | Running enrichment score |
+| `ridgeplot()`  | gseaResult             | Ridge plot | Expression distribution  |
+| `browseKEGG()` | enrichKEGG result      | Browser    | Interactive pathway      |
+| `pathview()`   | gene list              | Image file | Static pathway diagram   |
+
+---
+
+## References
+
+1. Yu G. Biomedical Knowledge Mining using GOSemSim and clusterProfiler.
+   https://yulab-smu.top/biomedical-knowledge-mining-book/
+2. Xu S, Hu E, Cai Y, et al. Using clusterProfiler to characterize multiomics
+   data. _Nature Protocols_ 19, 3292–3320 (2024).
+
+---
+
+## Changelog
+
+### v3.0 (Current)
+
+- Streamlined to 10 tests focusing on unique capabilities
+- Each test covers a distinct clusterProfiler feature or visualization
+- All tests use pre-loaded datasets (`geneList`, `gcSample`)
+- Removed redundant tests across different datasets
+- Added comprehensive visualization function reference