Hi,

I am new to transcriptomic data analysis and I am currently working on pathway analysis. To do so, I am using the clusterProfiler package on R. I'm going to use both enrichGO and enrichKEGG but they both have a "universe" parameter and I am struggling understanding it right.

So, from what I have understood is that, I can either use every genes that I have in my assay (around 56k) and it corresponds to the defaults parameter of the function, ie "all the genes listed in the database will be used as background", or I can use every genes that I have kept to do my differential gene expression analysis, since I have filtered them to get rid of the low counts. I have filtered them as follow, where dds is a DESeq2 object that I got using DESeqDataSetFromMatrix function with the data tables that I have :

keep <- rowSums(counts(dds) >= 10) > 3
dds <- dds[keep,]

I get around 18k genes left. Then, I used the enrichGO function in two differents ways, as follows :

enrich18k <- enrichGO(gene=rownames(subset(resyoungAC, log2FoldChange>=1 & padj<0.05)),
                         universe=rownames(resyoungAC),
                         OrgDb = "org.Mm.eg.db",
                         keyType = "ENSEMBL",
                         ont="BP")

enrich56k <- enrichGO(gene=rownames(subset(resyoungAC, log2FoldChange>=1 & padj<0.05)),
                         OrgDb = "org.Mm.eg.db",
                         keyType = "ENSEMBL",
                         ont="BP")

Here, resyoungAC is the object that I got using results() function from DESeq. I am filtering to keep only the up-regulated genes. The only difference between both functions is the universe parameter, where I set it as rownames(resyoungAC) in the first function, corresponding to the 18k genes that were left after filtering. The second function is using the default parameter ie the 56k genes (I verified that it was indeed using these genes, and it is).

My problem is that I have few of the most expressed pathways in the enrich18k object that don't make it in the second object (enrich56k) and I can't understand why.

In the enrich18k object, the most expressed pathway is "response to bacterium", with 27/127 as GeneRatio and 497/15489 as BgRatio (padj~7.2e-12). In the enrich56k object, the most expressed pathway is "leukocyte chemotaxis" with 14/127 as GeneRatio and 235/23012 as BgRatio (padj~1.05e-07).

I don't understand why the "response to bacterium" is not in the enrich56k results since my 127 genes of interest remain the same and I apparently have 27 of these genes that are expressed in that pathway in enrich18k.

Thanks a lot for you help.

The reason you get different results is because you are changing the universe. The GO hypergeometric test is basically asking if the proportion of genes in a given term are enriched in your set of genes as compared to the universe from which they were selected.

You can think of this in percentage terms. As an example, say that a given term contains 10% of the genes in the universe, but that term is enriched in your set of significant genes, containing 25% of the genes in that set. The term will likely be significant, because that's a big enrichment.

But the comparison is to the proportions in your universe, so let's use another example. Say the term of interest is 10% of the genes in the universe, and it's also 10% of the significant genes. In that case it won't be significant. But if you filter the genes, and after having done so, the proportion of that term that remain in your filtered set has been reduced to only 5% of the genes, you are very likely to have a significant result, because now that 10% in your significant gene set is twice the 5% in your universe.

As a general rule I mostly use the unfiltered set of genes as my universe, and my rationale for doing so is that a priori all of those genes could have been detected, and it's only a posteriori that I find out which genes have been detected. You can of course make the opposite argument, using the conventional balls in an urn analogy. If you think of a hypergeometric test in that heuristic, then when you reach into the urn to select the balls, the only balls you can actually grab are the ones in the urn, not all the balls that could have been in the urn if you were able to measure them.

You as the analyst have to make the decision as to which analogy makes more sense to you, and that you feel more comfortable defending if anybody asks why you made that choice.