Over-representation (or enrichment) analysis is a statistical method that determines whether genes from pre-defined sets (ex: those beloging to a specific GO term or KEGG pathway) are present more than would be expected (over-represented) in a subset of your data. In this case, the subset is your set of under or over expressed genes. For example, the fruit fly transcriptome has about 10,000 genes. If 260 genes are categorized as “axon guidance” (2.6% of all genes have category “axon guidance”), and in an experiment we find 1000 genes are differentially expressed and 200 of those genes are in the category “axon guidance” (20% of DE genes have category “axon guidance”), is that significant?
This R Notebook describes the implementation of over-representation analysis using the clusterProfiler package. For more information please see the full documentation here: https://bioconductor.org/packages/release/bioc/vignettes/clusterProfiler/inst/doc/clusterProfiler.html
Follow along interactively with the R Markdown Notebook:
Install and load packages
#BiocManager::install("clusterProfiler", version = "3.8") #BiocManager::install("pathview") #install.packages("wordcloud") library(clusterProfiler) library(wordcloud)
I’m using D melanogaster data, so I install and load the annotation “org.Dm.eg.db” below. See all annotations available here: http://bioconductor.org/packages/release/BiocViews.html#___OrgDb (there are 19 presently available).
organism = "org.Dm.eg.db" #BiocManager::install(organism, character.only = TRUE) library(organism, character.only = TRUE)
# reading in input from deseq2 df = read.csv("drosphila_example_de.csv", header=TRUE) # we want the log2 fold change original_gene_list <- df$log2FoldChange # name the vector names(original_gene_list) <- df$X # omit any NA values gene_list<-na.omit(original_gene_list) # sort the list in decreasing order (required for clusterProfiler) gene_list = sort(gene_list, decreasing = TRUE) # Exctract significant results (padj < 0.05) sig_genes_df = subset(df, padj < 0.05) # From significant results, we want to filter on log2fold change genes <- sig_genes_df$log2FoldChange # Name the vector names(genes) <- sig_genes_df$X # omit NA values genes <- na.omit(genes) # filter on min log2fold change (log2FoldChange > 2) genes <- names(genes)[abs(genes) > 2]
Create enrichGO object
Ontology Options: [“BP”, “MF”, “CC”]
keyType This is the source of the annotation (gene ids). The options vary for each annotation. In the example of org.Dm.eg.db, the options are:
“ACCNUM” “ALIAS” “ENSEMBL” “ENSEMBLPROT” “ENSEMBLTRANS” “ENTREZID”
“ENZYME” “EVIDENCE” “EVIDENCEALL” “FLYBASE” “FLYBASECG” “FLYBASEPROT”
“GENENAME” “GO” “GOALL” “MAP” “ONTOLOGY” “ONTOLOGYALL”
“PATH” “PMID” “REFSEQ” “SYMBOL” “UNIGENE” “UNIPROT”
Check which options are available with the
keytypes command, for example
Create the object
go_enrich <- enrichGO(gene = genes, universe = names(gene_list), OrgDb = organism, keyType = 'ENSEMBL', readable = T, ont = "BP", pvalueCutoff = 0.05, qvalueCutoff = 0.10)
Emphasizes the genes overlapping among different gene sets.
#BiocManager::install("enrichplot") library(enrichplot) upsetplot(go_enrich)
wcdf<-read.table(text=go_enrich$GeneRatio, sep = "/") wcdf$term<-go_enrich[,2] wordcloud(words = wcdf$term, freq = wcdf$V1, scale=(c(4, .1)), colors=brewer.pal(8, "Dark2"), max.words = 25)
barplot(go_enrich, drop = TRUE, showCategory = 10, title = "GO Biological Pathways", font.size = 8)
Enrichment map organizes enriched terms into a network with edges connecting overlapping gene sets. In this way, mutually overlapping gene sets are tend to cluster together, making it easy to identify functional modules.
Enriched GO induced graph:
goplot(go_enrich, showCategory = 10)
The cnetplot depicts the linkages of genes and biological concepts (e.g. GO terms or KEGG pathways) as a network (helpful to see which genes are involved in enriched pathways and genes that may belong to multiple annotation categories).
# categorySize can be either 'pvalue' or 'geneNum' cnetplot(go_enrich, categorySize="pvalue", foldChange=gene_list)