Last updated: 2021-01-27

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Rmd 687a6ca davetang 2021-01-27 Gene Ontology Enrichment Analysis

The Gene Ontology Enrichment Analysis (GOEA) is a typical analysis carried out on transcriptome data. Online tools for performing a GOEA include DAVID, Enrichr, and PANTHER just to name a few. While web-based tools are easy to use, it becomes tedious when you have to analyse (or re-analyse) lots of datasets. Therefore, it is preferable to use a programmatic approach and in this post we will check out some Bioconductor packages that allow to perform a GOEA.

First install the following packages, if necessary, and then load them.

if (!requireNamespace("BiocManager", quietly = TRUE))
    install.packages("BiocManager")

my_packages <- c("clusterProfiler",
                 "GOstats",
                 "GO.db",
                 "org.Hs.eg.db")

to_install <- my_packages[!my_packages %in% installed.packages()]
BiocManager::install(pkgs = to_install)

# load all packages and suppress output of sapply
invisible(sapply(my_packages, library, character.only = TRUE))

Create a positive control where the gene set are composed of genes that are all associated with GO:0007411 (axon guidance); we will use the org.Hs.eg.db package to achieve this based on the vignette.

Methods that can be applied to AnnotationDbi objects such as org.Hs.eg.db include: columns, keytypes, keys, and select.

Use columns to find out what data can be retrived using select.

columns(org.Hs.eg.db)
 [1] "ACCNUM"       "ALIAS"        "ENSEMBL"      "ENSEMBLPROT"  "ENSEMBLTRANS"
 [6] "ENTREZID"     "ENZYME"       "EVIDENCE"     "EVIDENCEALL"  "GENENAME"    
[11] "GO"           "GOALL"        "IPI"          "MAP"          "OMIM"        
[16] "ONTOLOGY"     "ONTOLOGYALL"  "PATH"         "PFAM"         "PMID"        
[21] "PROSITE"      "REFSEQ"       "SYMBOL"       "UCSCKG"       "UNIGENE"     
[26] "UNIPROT"     

Use keytypes to find out what fields we can use as keys to query the database.

keytypes(org.Hs.eg.db)
 [1] "ACCNUM"       "ALIAS"        "ENSEMBL"      "ENSEMBLPROT"  "ENSEMBLTRANS"
 [6] "ENTREZID"     "ENZYME"       "EVIDENCE"     "EVIDENCEALL"  "GENENAME"    
[11] "GO"           "GOALL"        "IPI"          "MAP"          "OMIM"        
[16] "ONTOLOGY"     "ONTOLOGYALL"  "PATH"         "PFAM"         "PMID"        
[21] "PROSITE"      "REFSEQ"       "SYMBOL"       "UCSCKG"       "UNIGENE"     
[26] "UNIPROT"     

Select all genes with GO:0007411.

go_to_entrez <- select(org.Hs.eg.db,
                       keys = "GO:0007411",
                       columns = "ENTREZID",
                       keytype = "GO")
'select()' returned 1:many mapping between keys and columns
axon_gene <- unique(go_to_entrez$ENTREZID)
length(axon_gene)
[1] 205

To perform the GOEA we need to create a gene background called the universe and we will use all genes with a GO term. Normally the universe should be the list of genes that were actually assayed in your transcriptome analysis.

all_go_terms <- keys(org.Hs.eg.db, keytype = "GO")
all_go <- select(org.Hs.eg.db, keys = all_go_terms, columns = c("ENTREZID", "GO"), keytype = "GO")
'select()' returned 1:many mapping between keys and columns
universe <- unique(all_go$ENTREZID)
length(universe)
[1] 20488

The function hyperGTest will perform the GOEA based on a set of parameters; in this example, we are testing for the over-representation of biological process (BP) terms and using a p-value cutoff of 0.001 or less.

params <- new('GOHyperGParams',
              geneIds = axon_gene,
              universeGeneIds = universe,
              ontology = 'BP',
              pvalueCutoff = 0.001,
              conditional = FALSE,
              testDirection = 'over',
              annotation = "org.Hs.eg.db"
             )
 
my_test <- hyperGTest(params)
my_test
Gene to GO BP  test for over-representation 
4723 GO BP ids tested (1069 have p < 0.001)
Selected gene set size: 205 
    Gene universe size: 18670 
    Annotation package: org.Hs.eg 

Use summary to get a summary of the results. The summary contains the GOID, Pvalue, OddsRatio, ExpCount, Count, and Size.

  • ExpCount is the expected count
  • Count is how many instances of that term were actually observed in your gene list
  • Size is the number that could have been found in your gene list if every instance had turned up
head(summary(my_test))
      GOBPID       Pvalue OddsRatio ExpCount Count Size
1 GO:0007409  0.00000e+00       Inf 5.138725   205  468
2 GO:0007411  0.00000e+00       Inf 3.030530   205  276
3 GO:0048667  0.00000e+00       Inf 6.401446   205  583
4 GO:0061564  0.00000e+00       Inf 5.643814   205  514
5 GO:0097485  0.00000e+00       Inf 3.041510   205  277
6 GO:0006935 6.87054e-316       Inf 7.071237   205  644
                                                   Term
1                                          axonogenesis
2                                         axon guidance
3 cell morphogenesis involved in neuron differentiation
4                                      axon development
5                            neuron projection guidance
6                                            chemotaxis

GO terms associated to axons are enriched as expected. Note that the Count and Size for GO:0007411 is not identical even though we had selected all genes associated with GO:0007411.

If we manually select Entrez gene IDs using org.Hs.egGO, we still get the same list of genes, so I’m not sure how the size is calculated by hyperGTest.

my_df <- as.data.frame(org.Hs.egGO)
my_idx <- my_df$go_id == "GO:0007411"
length(unique(my_df[my_idx, "gene_id"])) == length(axon_gene)
[1] TRUE

What if my gene list IDs are not Entrez gene IDs?

We can use the biomaRt package for converting between different gene identifiers and in this example, we will convert Ensembl gene IDs to Entrez gene IDs.

if (!"biomaRt" %in% installed.packages()){
  BiocManager::install("biomaRt")
}

library("biomaRt")

We will fetch every Ensembl gene ID and randomly select 10 IDs to convert into Entrez gene IDs.

ensembl <- useMart("ensembl", dataset="hsapiens_gene_ensembl")
 
my_chr <- c(1:22, 'M', 'X', 'Y')
my_ensembl_gene <- getBM(attributes = 'ensembl_gene_id',
                         filters = 'chromosome_name',
                         values = my_chr,
                         mart = ensembl)
 
head(my_ensembl_gene)
  ensembl_gene_id
1 ENSG00000223972
2 ENSG00000227232
3 ENSG00000278267
4 ENSG00000243485
5 ENSG00000284332
6 ENSG00000237613

Select 10 Ensembl gene IDs.

set.seed(1984)
to_convert <- sample(x = my_ensembl_gene$ensembl_gene_id, size = 10, replace = FALSE)

Now to convert the IDs.

to_entrez <- getBM(attributes = c('ensembl_gene_id', 'entrezgene_id'),
                   filters = 'ensembl_gene_id',
                   values = to_convert,
                   mart = ensembl)

to_entrez
   ensembl_gene_id entrezgene_id
1  ENSG00000124568          6568
2  ENSG00000131400          9476
3  ENSG00000212191            NA
4  ENSG00000225315            NA
5  ENSG00000228658            NA
6  ENSG00000256659     101927694
7  ENSG00000257890            NA
8  ENSG00000267552            NA
9  ENSG00000280344            NA
10 ENSG00000281133            NA

Note that not all Ensembl IDs have Entrez IDs. We can find out how many Ensembl IDs do not have Entrez IDs.

my_entrez_gene <- getBM(attributes = c('ensembl_gene_id', 'entrezgene_id'),
                        filters = 'ensembl_gene_id',
                        values = my_ensembl_gene,
                        mart = ensembl)

table(is.na(my_entrez_gene$entrezgene_id))

FALSE  TRUE 
25628 35099 

35099 out of 60727 Ensembl gene IDs do not have corresponding Entrez gene IDs. To learn more about the missing Entrez ID values from the Ensembl conversion see this useful post on BioStars.


sessionInfo()
R version 4.0.3 (2020-10-10)
Platform: x86_64-apple-darwin17.0 (64-bit)
Running under: macOS Big Sur 10.16

Matrix products: default
BLAS:   /Library/Frameworks/R.framework/Versions/4.0/Resources/lib/libRblas.dylib
LAPACK: /Library/Frameworks/R.framework/Versions/4.0/Resources/lib/libRlapack.dylib

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

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

other attached packages:
 [1] biomaRt_2.44.4         org.Hs.eg.db_3.11.4    GO.db_3.11.4          
 [4] GOstats_2.54.0         graph_1.66.0           Category_2.54.0       
 [7] Matrix_1.3-2           AnnotationDbi_1.50.3   IRanges_2.22.2        
[10] S4Vectors_0.26.1       Biobase_2.48.0         BiocGenerics_0.34.0   
[13] clusterProfiler_3.16.1 workflowr_1.6.2       

loaded via a namespace (and not attached):
  [1] fgsea_1.14.0           colorspace_2.0-0       ellipsis_0.3.1        
  [4] ggridges_0.5.3         rprojroot_2.0.2        qvalue_2.20.0         
  [7] fs_1.5.0               rstudioapi_0.13        farver_2.0.3          
 [10] urltools_1.7.3         graphlayouts_0.7.1     ggrepel_0.9.1         
 [13] bit64_4.0.5            scatterpie_0.1.5       xml2_1.3.2            
 [16] splines_4.0.3          cachem_1.0.1           GOSemSim_2.14.2       
 [19] knitr_1.30             polyclip_1.10-0        jsonlite_1.7.2        
 [22] annotate_1.66.0        dbplyr_2.0.0           ggforce_0.3.2         
 [25] BiocManager_1.30.10    compiler_4.0.3         httr_1.4.2            
 [28] rvcheck_0.1.8          assertthat_0.2.1       fastmap_1.1.0         
 [31] later_1.1.0.1          tweenr_1.0.1           htmltools_0.5.1.1     
 [34] prettyunits_1.1.1      tools_4.0.3            igraph_1.2.6          
 [37] gtable_0.3.0           glue_1.4.2             reshape2_1.4.4        
 [40] DO.db_2.9              dplyr_1.0.3            rappdirs_0.3.1        
 [43] fastmatch_1.1-0        Rcpp_1.0.6             enrichplot_1.8.1      
 [46] vctrs_0.3.6            ggraph_2.0.4           xfun_0.20             
 [49] stringr_1.4.0          lifecycle_0.2.0        XML_3.99-0.5          
 [52] DOSE_3.14.0            europepmc_0.4          MASS_7.3-53           
 [55] scales_1.1.1           tidygraph_1.2.0        hms_1.0.0             
 [58] promises_1.1.1         RBGL_1.64.0            RColorBrewer_1.1-2    
 [61] curl_4.3               yaml_2.2.1             memoise_2.0.0         
 [64] gridExtra_2.3          ggplot2_3.3.3          downloader_0.4        
 [67] triebeard_0.3.0        stringi_1.5.3          RSQLite_2.2.3         
 [70] genefilter_1.70.0      BiocParallel_1.22.0    rlang_0.4.10          
 [73] pkgconfig_2.0.3        bitops_1.0-6           evaluate_0.14         
 [76] lattice_0.20-41        purrr_0.3.4            cowplot_1.1.1         
 [79] bit_4.0.4              tidyselect_1.1.0       AnnotationForge_1.30.1
 [82] GSEABase_1.50.1        plyr_1.8.6             magrittr_2.0.1        
 [85] R6_2.5.0               generics_0.1.0         DBI_1.1.1             
 [88] withr_2.4.1            pillar_1.4.7           whisker_0.4           
 [91] survival_3.2-7         RCurl_1.98-1.2         tibble_3.0.5          
 [94] crayon_1.3.4           BiocFileCache_1.12.1   rmarkdown_2.6         
 [97] viridis_0.5.1          progress_1.2.2         grid_4.0.3            
[100] data.table_1.13.6      Rgraphviz_2.32.0       blob_1.2.1            
[103] git2r_0.28.0           digest_0.6.27          xtable_1.8-4          
[106] tidyr_1.1.2            httpuv_1.5.5           gridGraphics_0.5-1    
[109] openssl_1.4.3          munsell_0.5.0          viridisLite_0.3.0     
[112] ggplotify_0.0.5        askpass_1.1