interpreting R code function - r

Question

I would like to perform pathway enrichment analyses.
I have 21 list of significant genes, and mutiple types of pathways I would like to check (ie. check for enrichment in KEGG pathways, GOterms, complexes etc.).
I found this example of code, on an old BioC post. However, I am having trouble adapting it for myself.
Firstly,
1- what does this mean? I don't know this multiple colon syntax.
hyperg <- Category:::.doHyperGInternal
2 - I don't understand how this line works. hyperg.test is a function that needs 3 variables passed to it, correct? Is this line somehow passing "genes.by.pathways, significant.genes, and all.geneIDs to thr hyperg.test?
pVals.by.pathway<-t(sapply(genes.by.pathway, hyperg.test, significant.genes, all.geneIDs))
Code that I would like to adapt
library(KEGGREST)
library(org.Hs.eg.db)
# created named list, length 449, eg:
# path:hsa00010: "Glycolysis / Gluconeogenesis"
pathways <- keggList("pathway", "hsa")
# make them into KEGG-style human pathway identifiers
human.pathways <- sub("path:", "", names(pathways))
# for demonstration, just use the first ten pathways
demo.pathway.ids <- head(human.pathways, 10)
demo.pathways <- setNames(keggGet(demo.pathway.ids), demo.pathway.ids)
genes.by.pathway <- lapply(demo.pathways, function(demo.pathway) {
demo.pathway$GENE[c(TRUE, FALSE)]
})
all.geneIDs <- keys(org.Hs.eg.db)
# chose one of these for demonstration. the first (a whole genome random
# set of 100 genes) has very little enrichment, the second, a random set
# from the pathways themselves, has very good enrichment in some pathways
set.seed(123)
significant.genes <- sample(all.geneIDs, size=100)
#significant.genes <- sample(unique(unlist(genes.by.pathway)), size=10)
# the hypergeometric distribution is traditionally explained in terms of
# drawing a sample of balls from an urn containing black and white balls.
# to keep the arguments straight (in my mind at least), I use these terms
# here also
hyperg <- Category:::.doHyperGInternal
hyperg.test <-
function(pathway.genes, significant.genes, all.genes, over=TRUE)
{
white.balls.drawn <- length(intersect(significant.genes, pathway.genes))
white.balls.in.urn <- length(pathway.genes)
total.balls.in.urn <- length(all.genes)
black.balls.in.urn <- total.balls.in.urn - white.balls.in.urn
balls.pulled.from.urn <- length(significant.genes)
hyperg(white.balls.in.urn, black.balls.in.urn,
balls.pulled.from.urn, white.balls.drawn, over)
}
pVals.by.pathway <-
t(sapply(genes.by.pathway, hyperg.test, significant.genes, all.geneIDs))
print(pVals.by.pathway)

Answer 1

The reason you are getting your error is because it appears you don't have the Category package installed from bioconductor. I suspect this because of the triple colon operator :::. This operator is very similar to the double colon operator ::. Whereas with :: you can access exported objects from a package without loading it, the ::: allows access to non-exported objects (in this case the hyperg function from Category). If you install the Category package the code runs without error.
With regard to the sapply statement:
pVals.by.pathway<-t(sapply(genes.by.pathway, hyperg.test, significant.genes, all.geneIDs))
You can break this down into the separate parts to understand it. Firstly, the sapply is iterating over the elements of gene.by.pathway and passing them to the first argument of hyperg.test. The following arguments are the two addition parameters. It is a little unclear and I personally recommend that people explicitly identify the parameters to avoid unexpected surprises and avoids the need for the exact same order. This is a little repetitive in this case but a good way to avoid a silly bug (e.g. putting significant.genes after all.geneIds)
Rewritten:
pVals.by.pathway <-
t(sapply(genes.by.pathway, hyperg.test, significant.genes=significant.genes, all.genes=all.geneIDs))
Once this loop completes, the sapply function simplifies the output in to a matrix. However, the output is much more user-friendly by taking the transpose t.
Generally speaking, when trying to understand complex apply statements I find it best to break them apart in to smaller parts and see what the objects themselves look like.