Hi there! I'm trying to perform gene ontology analysis comparing tumor and normal samples. There are about 250 and 350 differentially enriched peaks in the tumor and normal samples respectively.

However, there are 0 enriched terms found when I performed the enrichGO() function on the tumor and normal gene lists containing the respective entrez ID of the differentially enriched genes.

May I ask if anyone encountered a similar issue and if there are any ways to resolve this please?

Thank you!

samplefiles <- list.files("/path/diffbind_enriched_bed", 
                          pattern= ".bed",
                          full.names=T) 

samplefiles <- as.list(samplefiles) 

names(samplefiles) <- c("Normal", "Tumor") 

txdb <- TxDb.Hsapiens.UCSC.hg38.knownGene

peakAnnoList <- lapply(samplefiles, 
                       annotatePeak, 
                       TxDb=txdb,
                       tssRegion=c(-1000, 1000), 
                       verbose=FALSE) 

# ====================== Retrieve annotations ======================

normal_annot <- data.frame(peakAnnoList[["Normal"]]@anno)


tumor_annot <- data.frame(peakAnnoList[["Tumor"]]@anno)

# ====== Obtain the gene symbols from the annotations ======

entrez_normal <- normal_annot$geneId
annotations_edb_normal <- AnnotationDbi::select(EnsDb.Hsapiens.v86, 
                                                keys = entrez_normal, 
                                                columns = c("GENENAME"), 
                                                keytype = "ENTREZID") 

normal_annot %>% 
  left_join(annotations_edb_normal, by=c("geneId"="ENTREZID")) %>% 
  write.table(file="results/chipseeker/Normal_peak_annotation.txt", sep="\t", quote=F, row.names=F)

entrez_tumor <- tumor_annot$geneId
annotations_edb_tumor <- AnnotationDbi::select(EnsDb.Hsapiens.v86, 
                                                keys = entrez_tumor, 
                                                columns = c("GENENAME"), 
                                                keytype = "ENTREZID")

annotations_edb_tumor$ENTREZID <- as.character(annotations_edb_tumor$ENTREZID)

tumor_annot %>% 
  left_join(annotations_edb_tumor, by=c("geneId"="ENTREZID")) %>% 
  write.table(file="results/chipseeker/Tumor_peak_annotation.txt", sep="\t", quote=F, row.names=F)


# ================================= GO enrichment analysis ==============================================

# Over-representation analysis
ego_normal <- enrichGO(gene = entrez_normal, 
                keyType = "ENTREZID", 
                OrgDb = org.Hs.eg.db, 
                ont = "BP", 
                pAdjustMethod = "BH", 
                qvalueCutoff = 0.05, 
                readable = TRUE)

> ego_normal
# over-representation test
#
#...@organism    Homo sapiens 
#...@ontology    BP 
#...@keytype     ENTREZID 
#...@gene    chr [1:350] "54541" "2027" "79633" "5097" "81565" "54508" "7580" "5048" "494470" "143425" "124900546" "2274" ...
#...pvalues adjusted by 'BH' with cutoff <0.05 
#...0 enriched terms found
#...Citation
 T Wu, E Hu, S Xu, M Chen, P Guo, Z Dai, T Feng, L Zhou, W Tang, L Zhan, X Fu, S Liu, X Bo, and G Yu.
 clusterProfiler 4.0: A universal enrichment tool for interpreting omics data.
 The Innovation. 2021, 2(3):100141 

ego_tumor <- enrichGO(gene = entrez_tumor,
                       keyType = "ENTREZID", 
                       OrgDb = org.Hs.eg.db, 
                       ont = "BP", 
                       pAdjustMethod = "BH", 
                       qvalueCutoff = 0.05, 
                       readable = TRUE)
> ego_tumor
#
# over-representation test
#
#...@organism    Homo sapiens 
#...@ontology    BP 
#...@keytype     ENTREZID 
#...@gene    chr [1:240] "9684" "587" "341" "340371" "439965" "2743" "387893" "29894" "57142" "8733" "4796" "10485" "8644" ...
#...pvalues adjusted by 'BH' with cutoff <0.05 
#...0 enriched terms found
#...Citation
 T Wu, E Hu, S Xu, M Chen, P Guo, Z Dai, T Feng, L Zhou, W Tang, L Zhan, X Fu, S Liu, X Bo, and G Yu.
 clusterProfiler 4.0: A universal enrichment tool for interpreting omics data.
 The Innovation. 2021, 2(3):100141 

sessionInfo( )
R version 4.3.3 (2024-02-29)
Platform: aarch64-apple-darwin20 (64-bit)
Running under: macOS Sonoma 14.1.2

Matrix products: default
BLAS:   /System/Library/Frameworks/Accelerate.framework/Versions/A/Frameworks/vecLib.framework/Versions/A/libBLAS.dylib 
LAPACK: /Library/Frameworks/R.framework/Versions/4.3-arm64/Resources/lib/libRlapack.dylib;  LAPACK version 3.11.0

locale:
[1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8

tzcode source: internal

attached base packages:
[1] stats4    stats     graphics  grDevices utils     datasets  methods   base     

other attached packages:
 [1] org.Hs.eg.db_3.18.0                      dplyr_1.1.4                             
 [3] ChIPseeker_1.38.0                        clusterProfiler_4.10.1                  
 [5] EnsDb.Hsapiens.v86_2.99.0                ensembldb_2.26.0                        
 [7] AnnotationFilter_1.26.0                  TxDb.Hsapiens.UCSC.hg38.knownGene_3.18.0
 [9] GenomicFeatures_1.54.4                   AnnotationDbi_1.64.1                    
[11] Biobase_2.62.0                           GenomicRanges_1.54.1                    
[13] GenomeInfoDb_1.38.8                      IRanges_2.36.0                          
[15] S4Vectors_0.40.2                         BiocGenerics_0.48.1                     

loaded via a namespace (and not attached):
  [1] splines_4.3.3                           BiocIO_1.12.0                          
  [3] ggplotify_0.1.2                         bitops_1.0-7                           
  [5] filelock_1.0.3                          tibble_3.2.1                           
  [7] polyclip_1.10-6                         XML_3.99-0.16.1                        
  [9] lifecycle_1.0.4                         mixsqp_0.3-54                          
 [11] lattice_0.22-6                          MASS_7.3-60.0.1                        
 [13] systemPipeR_2.8.0                       magrittr_2.0.3                         
 [15] limma_3.58.1                            plotrix_3.8-4                          
 [17] yaml_2.3.8                              cowplot_1.1.3                          
 [19] DBI_1.2.2                               RColorBrewer_1.1-3                     
 [21] abind_1.4-5                             ShortRead_1.60.0                       
 [23] zlibbioc_1.48.2                         purrr_1.0.2                            
 [25] ggraph_2.2.1                            RCurl_1.98-1.14                        
 [27] yulab.utils_0.1.4                       tweenr_2.0.3                           
 [29] rappdirs_0.3.3                          GenomeInfoDbData_1.2.11                
 [31] enrichplot_1.22.0                       ggrepel_0.9.5                          
 [33] irlba_2.3.5.1                           tidytree_0.4.6                         
 [35] codetools_0.2-20                        DelayedArray_0.28.0                    
 [37] DOSE_3.28.2                             xml2_1.3.6                             
 [39] ggforce_0.4.2                           tidyselect_1.2.1                       
 [41] aplot_0.2.2                             farver_2.1.1                           
 [43] viridis_0.6.5                           matrixStats_1.3.0                      
 [45] BiocFileCache_2.10.2                    jsonlite_1.8.8                         
 [47] GenomicAlignments_1.38.2                tidygraph_1.3.1                        
 [49] bbmle_1.0.25.1                          tools_4.3.3                            
 [51] progress_1.2.3                          TxDb.Hsapiens.UCSC.hg19.knownGene_3.2.2
 [53] treeio_1.26.0                           Rcpp_1.0.12                            
 [55] glue_1.7.0                              gridExtra_2.3                          
 [57] SparseArray_1.2.4                       DESeq2_1.42.1                          
 [59] qvalue_2.34.0                           MatrixGenerics_1.14.0                  
 [61] amap_0.8-19                             withr_3.0.0                            
 [63] numDeriv_2016.8-1.1                     BiocManager_1.30.22                    
 [65] fastmap_1.1.1                           boot_1.3-30                            
 [67] latticeExtra_0.6-30                     fansi_1.0.6                            
 [69] caTools_1.18.2                          digest_0.6.35                          
 [71] truncnorm_1.0-9                         gridGraphics_0.5-1                     
 [73] R6_2.5.1                                colorspace_2.1-0                       
 [75] GO.db_3.18.0                            GreyListChIP_1.34.0                    
 [77] gtools_3.9.5                            jpeg_0.1-10                            
 [79] biomaRt_2.58.2                          RSQLite_2.3.6                          
 [81] utf8_1.2.4                              tidyr_1.3.1                            
 [83] generics_0.1.3                          data.table_1.15.4                      
 [85] rtracklayer_1.62.0                      graphlayouts_1.1.1                     
 [87] prettyunits_1.2.0                       httr_1.4.7                             
 [89] htmlwidgets_1.6.4                       S4Arrays_1.2.1                         
 [91] scatterpie_0.2.2                        pkgconfig_2.0.3                        
 [93] gtable_0.3.4                            blob_1.2.4                             
 [95] hwriter_1.3.2.1                         XVector_0.42.0                         
 [97] shadowtext_0.1.3                        htmltools_0.5.8.1                      
 [99] fgsea_1.28.0                            ProtGenerics_1.34.0                    
[101] scales_1.3.0                            png_0.1-8                              
[103] ggfun_0.1.4                             ashr_2.2-63                            
[105] rstudioapi_0.16.0                       reshape2_1.4.4                         
[107] rjson_0.2.21                            nlme_3.1-164                           
[109] coda_0.19-4.1                           curl_5.2.1                             
[111] bdsmatrix_1.3-7                         cachem_1.0.8                           
[113] stringr_1.5.1                           KernSmooth_2.23-22                     
[115] parallel_4.3.3                          HDO.db_0.99.1                          
[117] restfulr_0.0.15                         apeglm_1.24.0                          
[119] pillar_1.9.0                            grid_4.3.3                             
[121] vctrs_0.6.5                             gplots_3.1.3.1                         
[123] dbplyr_2.5.0                            invgamma_1.1                           
[125] mvtnorm_1.2-4                           cli_3.6.2                              
[127] locfit_1.5-9.9                          compiler_4.3.3                         
[129] Rsamtools_2.18.0                        rlang_1.1.3                            
[131] crayon_1.5.2                            SQUAREM_2021.1                         
[133] labeling_0.4.3                          interp_1.1-6                           
[135] emdbook_1.3.13                          plyr_1.8.9                             
[137] fs_1.6.3                                stringi_1.8.3                          
[139] viridisLite_0.4.2                       deldir_2.0-4                           
[141] BiocParallel_1.36.0                     munsell_0.5.1                          
[143] Biostrings_2.70.3                       lazyeval_0.2.2                         
[145] GOSemSim_2.28.1                         Matrix_1.6-5                           
[147] BSgenome_1.70.2                         patchwork_1.2.0                        
[149] hms_1.1.3                               bit64_4.0.5                            
[151] ggplot2_3.5.0                           KEGGREST_1.42.0                        
[153] statmod_1.5.0                           SummarizedExperiment_1.32.0            
[155] igraph_2.0.3                            memoise_2.0.1                          
[157] ggtree_3.10.1                           fastmatch_1.1-4                        
[159] DiffBind_3.12.0                         bit_4.0.5                              
[161] gson_0.1.0                              ape_5.8