Bio Workflows Expression To Pathways

Version Compatibility

Reference examples tested with: clusterProfiler 4.10+, org.Hs.eg.db 3.18+, ReactomePA 1.46+, enrichplot 1.22+.

Before using code patterns, verify installed versions match. If versions differ:

• R: packageVersion('<pkg>') then ?function_name to verify parameters

If code throws ImportError, AttributeError, or TypeError, introspect the installed package and adapt the example to match the actual API rather than retrying.

Expression to Pathways Workflow

"Find enriched pathways from my differential expression results" -> Decide the generation (ORA vs GSEA) FIRST, then convert IDs to the form each method needs, run enrichment against the chosen database, and collapse redundancy before interpreting - because the enrichment result is a claim conditioned on the method, the background universe, and the database version, not a discovery the algorithm hands back.

• R: enrichGO(...) / gseGO(...) / enrichKEGG(...) / enrichPathway(...) (clusterProfiler, ReactomePA)

Scope: the ORCHESTRATION of a DE-to-enrichment pipeline - the generation fork, per-method ID conversion, the universe decision, the live-vs-local database caveat, and the handoff to redundancy-collapsed visualization. This workflow does NOT re-teach each method. The null/universe/reproducibility theory and the master method-selection tree -> the pathway-analysis README and the per-method skills (go-enrichment, gsea). ORA mechanics -> go-enrichment; GSEA mechanics -> gsea; per-database IDs and live-DB behavior -> kegg-pathways, reactome-pathways, wikipathways; the DE list and ranking statistic -> differential-expression/de-results; plotting -> enrichment-visualization.

The Single Most Important Modern Insight -- The First Decision Is the Generation, and It Is Set by the Input, Not by Preference

Pathway analysis has three generations (Khatri 2012 PLoS Comput Biol 8:e1002375): over-representation analysis (ORA), functional class scoring / GSEA (FCS), and pathway topology. A workflow that "runs enrichment" without first deciding which generation applies has already made the choice silently - usually ORA, the worst-calibrated corner of the space. The fork is mechanical:

1. Is there a meaningful per-gene ranking for (nearly) ALL measured genes? A signed test statistic, the DESeq2 Wald stat, or -sign(log2FC)*log10(p) for every tested gene -> GSEA (a NAMED vector sorted in DECREASING order; the ranking metric IS the experiment). No arbitrary cutoff; detects coordinated weak shifts that ORA misses.
2. Only a pre-selected LIST (DE hits past a cutoff, a co-expression module, GWAS loci, screen hits)? -> ORA, and the deliverable hinges on a defensible background universe - the genes that were testable, not the whole genome. ORA's p-value is whatever the denominator says it is.

The dangerous default is running ORA on data that has a full ranking (binarizing away the signal) or running ORA against the genome (measuring expression, not enrichment). Decide the fork out loud, record it, and record the why (see the pathway-analysis README) - this workflow owns the routing, not the derivation.

Pipeline Overview

DE results (differential-expression/de-results)
    |
    v
[0. Decide the generation: ranking for all genes? -> GSEA | pre-selected list? -> ORA]
    |
    +--> ORA branch: define the TESTABLE-gene universe, convert IDs per method
    |        +--> enrichGO     (OrgDb keyType)        -> go-enrichment
    |        +--> enrichKEGG   ('kegg' / 'ncbi-geneid', LIVE DB)  -> kegg-pathways
    |        +--> enrichPathway (ENTREZ, local DB)    -> reactome-pathways
    |        +--> enrichWP      (ENTREZ, LIVE GMT)    -> wikipathways
    |
    +--> GSEA branch: build a NAMED decreasing vector of ALL genes, set.seed
    |        +--> gseGO / gseKEGG / GSEA(+msigdbr)    -> gsea
    |
    v
[Redundancy collapse + visualization: simplify, pairwise_termsim, dotplot/emapplot/gseaplot2]   (enrichment-visualization)
    |
    v
A claim conditioned on universe + method + database version (record provenance)

Stage Map

Decision Tree by Scenario

Stage 1: Prepare the Input (list, ranked vector, and the universe)

Goal: Turn a DE table into the two possible inputs - a gene LIST for ORA and a NAMED decreasing vector for GSEA - and define the background universe as the testable genes.

Approach: Read the DE result, derive the significant list, build the ranked vector from a signed statistic (not a bare log2FC), and set the universe to exactly the genes that entered the DE test. The DE mechanics (the $padj vs $adj.P.Val column, shrinkage) live at differential-expression/de-results - this is only input shaping.

library(clusterProfiler)
library(org.Hs.eg.db)

res <- read.csv('deseq2_results.csv', row.names = 1)

# ORA input: a pre-selected list (DESeq2 padj column; limma/edgeR name it differently)
sig_genes <- rownames(subset(res, padj < 0.05 & abs(log2FoldChange) > 1))

# Background universe = genes that were TESTABLE (entered the DE test), NOT the genome.
# Using the genome measures expression bias, not enrichment (the universe rule; see pathway-analysis/go-enrichment).
universe_genes <- rownames(res[!is.na(res$pvalue), ])

# GSEA input: a NAMED vector of ALL genes, sorted DECREASING by a signed metric.
# Prefer the Wald stat (magnitude + precision); a bare log2FC over-weights noisy low-count genes.
# TRAP: a table from lfcShrink(type='apeglm'/'ashr') has NO `stat` column -- shrinkage drops it.
# Rank from the UNSHRUNK results(dds)$stat; use edgeR `sign(logFC)*-log10(PValue)` or limma `t`.
ranked <- res$stat
names(ranked) <- rownames(res)
ranked <- sort(ranked[!is.na(ranked)], decreasing = TRUE)

Stage 2: Convert Gene IDs to What Each Method Needs

Goal: Map identifiers to the exact ID type each enrichment function expects, because a mismatch returns zero hits silently.

Approach: Use bitr (OrgDb) for SYMBOL/ENSEMBL -> ENTREZ, keep both list and ranked vector in the same ID space, deduplicate, and track the conversion rate. Per-method ID rules are owned by each DB skill; the table below is the routing summary.

# enrichGO accepts ENSEMBL/SYMBOL/ENTREZ via keyType=; ENTREZ is the safe lingua franca downstream
sig_entrez <- bitr(sig_genes, fromType = 'SYMBOL', toType = 'ENTREZID', OrgDb = org.Hs.eg.db)
bg_entrez <- bitr(universe_genes, fromType = 'SYMBOL', toType = 'ENTREZID', OrgDb = org.Hs.eg.db)

# Carry the ranking through conversion: name the kept stat by its ENTREZ id
ranked_map <- bitr(names(ranked), fromType = 'SYMBOL', toType = 'ENTREZID', OrgDb = org.Hs.eg.db)
ranked_list <- ranked[ranked_map$SYMBOL]
names(ranked_list) <- ranked_map$ENTREZID
ranked_list <- ranked_list[!duplicated(names(ranked_list))]   # dedup or GSEA biases the score
ranked_list <- sort(ranked_list, decreasing = TRUE)           # re-sort: bitr remap can reorder rows

conv_rate <- nrow(sig_entrez) / length(sig_genes)   # report it; <0.85 -> wrong ID type/organism

Stage 3a: ORA Branch (pre-selected list + universe)

Goal: Test each gene set for over-representation of the list against the testable-gene background.

Approach: Always pass universe=; run GO ontologies separately; KEGG/Reactome/WikiPathways each need their own ID form. KEGG and WikiPathways query a LIVE database (internet-dependent, not reproducible across releases - pin the run date); GO and Reactome read local annotation (reproducible given the Bioconductor release).

# GO ORA - universe is the decision; simplify() collapses DAG redundancy (BP/MF/CC separately, not 'ALL')
go_bp <- enrichGO(sig_entrez$ENTREZID, universe = bg_entrez$ENTREZID, OrgDb = org.Hs.eg.db,
                  pAdjustMethod = 'BH', pvalueCutoff = 0.05, readable = TRUE)
go_bp <- simplify(go_bp, cutoff = 0.7, by = 'p.adjust')

# KEGG ORA - LIVE KEGG REST API; needs internet; record the access date for reproducibility
kegg <- enrichKEGG(sig_entrez$ENTREZID, universe = bg_entrez$ENTREZID, organism = 'hsa', keyType = 'ncbi-geneid', pvalueCutoff = 0.05)   # Entrez input; keyType='kegg' = Entrez for eukaryotes / locus tags for prokaryotes, 'ncbi-geneid' is explicit
kegg <- setReadable(kegg, OrgDb = org.Hs.eg.db, keyType = 'ENTREZID')

# Reactome ORA - ENTREZ required; LOCAL reactome.db so reproducible given the release
library(ReactomePA)
reactome <- enrichPathway(sig_entrez$ENTREZID, universe = bg_entrez$ENTREZID, organism = 'human', pvalueCutoff = 0.05, readable = TRUE)

Stage 3b: GSEA Branch (named decreasing vector of all genes)

Goal: Find gene sets whose genes shift coordinately across the full ranking, without a significance cutoff.

Approach: Run on the named decreasing ranked_list, fix the permutation seed so p-values are reproducible, then read the leading edge as the interpretable core. clusterProfiler GSEA is preranked / gene-permutation (the inter-gene-correlation-UNcorrected null) - a discovery screen; see pathway-analysis/gsea for the calibration caveat (CAMERA/ROAST).

set.seed(123)   # permutation reproducibility; without it p-values drift across runs

gsea_go <- gseGO(ranked_list, OrgDb = org.Hs.eg.db,
                 minGSSize = 10, maxGSSize = 500, pvalueCutoff = 0.05, verbose = FALSE)

gsea_kegg <- gseKEGG(ranked_list, organism = 'hsa',
                     minGSSize = 10, maxGSSize = 500, pvalueCutoff = 0.05, verbose = FALSE)

Stage 4: Collapse Redundancy, Then Visualize

Goal: Reduce overlapping terms to distinct findings before drawing conclusions, then plot deliberately.

Approach: A list of 40 significant GO terms is often a few biological stories told many times (shared genes via the GO true-path rule). Collapse with simplify/pairwise_termsim, then plot - emapplot/treeplot require pairwise_termsim() first (cnetplot does NOT; it draws the gene-concept network from the geneID column directly), and gseaplot2 is for a gseaResult not an enrichResult. Encoding choice and required pre-steps are owned by pathway-analysis/enrichment-visualization.

library(enrichplot)

go_bp <- pairwise_termsim(go_bp)              # required before emapplot/treeplot
dotplot(go_bp, showCategory = 20)             # GeneRatio vs Count: pick the encoding deliberately
emapplot(go_bp, showCategory = 30)            # redundancy-collapsed term-similarity map
gseaplot2(gsea_go, geneSetID = 1:3)           # gseaResult only, not enrichResult

Multi-Condition Comparison

Goal: Compare enrichment across conditions in one model instead of comparing p-values from separate runs.

Approach: compareCluster fits all gene lists together and facets the dotplot; never compare raw -log10(p) across separate enrichments (it scales with set size and sample size). For GSEA, compare NES, not p.

gene_clusters <- list(A = sig_A, B = sig_B, C = sig_C)
cc <- compareCluster(gene_clusters, fun = 'enrichKEGG', organism = 'hsa',
                     universe = bg_entrez$ENTREZID)   # compareCluster forwards ... to fun; omitting universe silently reverts to the whole-genome background
dotplot(cc, showCategory = 10)

Per-Method Failure Modes

Whole-genome background

Trigger: universe= left at default while only ~12k genes were expressed. Mechanism: the hypergeometric p-value is fully determined by the denominator; the genome inflates any set whose members are expressed in the tissue. Symptom: many tissue-specific terms enrich with tiny p. Fix: set universe to the genes that entered the DE test (the testable set).

ORA on a ranked dataset

Trigger: filtering all-gene DE results to a list and running ORA. Mechanism: binarizing at an arbitrary cutoff discards magnitude and the coordinated-weak signal. Symptom: GSEA finds sets ORA missed. Fix: if a ranking exists for all genes, run GSEA; reserve ORA for genuinely unranked lists.

Wrong ID type for the database

Trigger: ENSEMBL/SYMBOL passed to enrichKEGG/enrichPathway/enrichWP. Mechanism: those expect kegg-id/ENTREZ; unmatched IDs are dropped. Symptom: zero terms, no error. Fix: bitr/bitr_kegg to the required ID; check conv_rate.

GSEA without set.seed or with an unsorted vector

Trigger: no seed, or a list that is not named and decreasing. Mechanism: permutation p-values drift run to run; an unsorted/unnamed vector errors or mis-ranks. Symptom: different leading edges each run, or a names error. Fix: build the named decreasing vector and set.seed.

Ranking a GSEA vector off a shrunken-LFC object

Trigger: building the ranked vector from a lfcShrink(type='apeglm'/'ashr') table, or ranking by bare log2FoldChange. Mechanism: apeglm/ashr DROP the stat column, so the vector silently falls back to shrunken LFC; low-count genes with unstable large FC then hijack the leading edge. Symptom: the leading edge is dominated by low-baseMean genes, or res$stat is NULL. Fix: rank from the unshrunken results(dds)$stat (DESeq2), limma topTable$t, or edgeR sign(logFC)*-log10(PValue); reserve shrunken LFC for visualization.

Live-DB result reported as reproducible

Trigger: KEGG/WikiPathways result with no recorded date. Mechanism: those query the current data release; the same code returns different pathways later. Symptom: a collaborator cannot reproduce the figure. Fix: record the access date and data version; prefer local GO/Reactome when reproducibility is paramount.

Redundancy read as replication

Trigger: interpreting 40 overlapping GO terms as 40 findings. Mechanism: the true-path rule and pathway overlap mean shared genes drive many sets. Symptom: the same 3-5 genes explain the top 20 terms. Fix: simplify/pairwise_termsim, inspect the leading-edge/geneID core, report clusters of terms.

Quantitative Thresholds

Common Errors

References

• Khatri P, Sirota M, Butte AJ. 2012. Ten years of pathway analysis: current approaches and outstanding challenges. PLoS Comput Biol 8:e1002375.
• Subramanian A, Tamayo P, Mootha VK, et al. 2005. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. PNAS 102:15545-15550.
• Goeman JJ, Buhlmann P. 2007. Analyzing gene expression data in terms of gene sets: methodological issues. Bioinformatics 23:980-987.
• Wu T, Hu E, Xu S, et al. 2021. clusterProfiler 4.0: a universal enrichment tool for interpreting omics data. The Innovation 2:100141.
• Yu G, He QY. 2016. ReactomePA: an R/Bioconductor package for reactome pathway analysis and visualization. Mol BioSyst 12:477-479.
• Kanehisa M, Goto S. 2000. KEGG: Kyoto Encyclopedia of Genes and Genomes. Nucleic Acids Res 28:27-30.
• Young MD, Wakefield MJ, Smyth GK, Oshlack A. 2010. Gene ontology analysis for RNA-seq: accounting for selection bias. Genome Biol 11:R14.
• Wijesooriya K, Jadaan SA, Perera KL, Kaur T, Ziemann M. 2022. Urgent need for consistent standards in functional enrichment analysis. PLoS Comput Biol 18:e1009935.

Related Skills

• pathway-analysis/go-enrichment - GO over-representation, background universe, redundancy reduction, length bias
• pathway-analysis/gsea - Ranked-list GSEA, named decreasing vector, ranking metric, leading edge, NES
• pathway-analysis/kegg-pathways - KEGG pathway/module enrichment, live DB, prokaryotes, multi-condition
• pathway-analysis/reactome-pathways - Reactome curated-pathway ORA and GSEA, ENTREZ IDs, reproducible local DB
• pathway-analysis/wikipathways - WikiPathways community-pathway enrichment, versioned GMT, broad species
• pathway-analysis/enrichment-visualization - Dot/bar/cnet/emap/GSEA plots and required pre-steps
• differential-expression/de-results - Source of the gene list and the ranking statistic