Hi all!

I am trying to do KEGG enrichment with enrichKEGG, I was wondering if I need to specify universe argument. Could you please help me with it?

1. In the documentation, it says "universe: background genes. If missing, the all genes listed in the database (eg TERM2GENE table) will be used as background." However, enrichKEGG doesn't take TERM2GENE argument. If the universe is not specified, what will be used as the universe?

It returns unused argument (TERM2GENE = Transcriptome) If I specify TERM2GENE.

1. Should I use the KEGG column from the "KEGG Orthology to Genes mapping" as the universe?

enr_results <- enrichKEGG(DEG$KEGG, organism='ko', universe = Transcriptome$KEGG, pvalueCutoff = 0.05, pAdjustMethod = "BH", qvalueCutoff = 0.05, minGSSize = 5)

Here are what my files look like:

1. KEGG to DEG mappings

   > head(DEG)
    KEGG Gene
   1 K17277  FS_gene_3
   2 K14700 FS_gene_11
   3 K14701 FS_gene_11
   

2. KEGG to whole transcriptome mappings

   head(Transcriptome)
    KEGG Gene
   1 K02727  FS_gene_1
   2 K17277  FS_gene_3
   3 K17307 FS_gene_10
   

Thanks a lot!

This very insightful paper was released last year on the good practice to conduct when running GO : https://journals.plos.org/ploscompbiol/article?id=10.1371/journal.pcbi.1009935

According to this paper, "In the case of ORA for differential expression (eg: RNA-seq), a whole genome background is inappropriate because in any tissue, most genes are not expressed and therefore have no chance of being classified as DEGs. A good rule of thumb is to use a background gene list consisting of genes detected in the assay at a level where they have a chance of being classified as DEG [5–7]. Using the whole genome background gene list may be suitable in cases where all genes have the capacity of being detected, for example in studies of genetic variation (eg: [8]). However the problem becomes more acute when the proportion of measured genes/proteins is small, for example in proteomics and single-cell RNA-sequencing where only a few thousand analytes are detected."

Then I think you should select all the genes expressed in your experiment as the background gene list.

There is no need to specify universe argument. When you use enrichKEGG to do enrichment analysis, itwill automatically use all genes that can be annotated to the pathways as the background.