Gene ontology testing for Ilumina methylation array data

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Description

Tests gene ontology enrichment for significant CpGs from Illumina's Infinium HumanMethylation450 or MethylationEPIC array, taking into account the differing number of probes per gene present on the array.

Usage

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gometh(sig.cpg, all.cpg = NULL, collection = c("GO","KEGG"), array.type = c("450K","EPIC"), plot.bias = FALSE, prior.prob = TRUE)

Arguments

sig.cpg

character vector of significant CpG sites to test for GO term enrichment

all.cpg

character vector of all CpG sites tested. Defaults to all CpG sites on the array.

collection

the collection of pathways to test. Options are "GO" and "KEGG". Defaults to "GO".

array.type

the Illumina methylation array used. Options are "450K" or "EPIC". Defaults to "450K".

plot.bias

logical, if true a plot showing the bias due to the differing numbers of probes per gene will be displayed

prior.prob

logical, if true will take into account the probability of significant differentially methylation due to numbers of probes per gene. If false, a hypergeometric test is performed ignoring any bias in the data.

Details

This function takes a character vector of significant CpG sites, maps the CpG sites to Entrez Gene IDs, and tests for GO term or KEGG pathway enrichment using a hypergeometric test, taking into account the number of CpG sites per gene on the 450K/EPIC array.

Geeleher et al. (2013) showed that a severe bias exists when performing gene set analysis for genome-wide methylation data that occurs due to the differing numbers of CpG sites profiled for each gene. gometh is based on the goseq method (Young et al., 2010) and calls the goana function for GO testing, or the kegga function for KEGG testing, both of which are from the limma package (Ritchie et al. 2015). If prior.prob is set to FALSE, then prior probabilities are not used and it is assumed that each gene is equally likely to have a significant CpG site associated with it.

Genes associated with each CpG site are obtained from the annotation package IlluminaHumanMethylation450kanno.ilmn12.hg19 if the array type is "450K". For the EPIC array, the annotation package IlluminaHumanMethylationEPICanno.ilm10b2.hg19 is used. In order to get a list which contains the mapped Entrez gene IDS, please use the getMappedEntrezIDs function.

gometh tests all GO or KEGG terms, and false discovery rates are calculated using the method of Benjamini and Hochberg (1995).

The limma functions topGO and topKEGG can be used to display the top 20 most enriched pathways.

For more generalised gene set testing where the user can specify the gene set/s of interest to be tested, please use the gsameth function.

Value

A data frame with a row for each GO or KEGG term and the following columns:

Term

GO term if testing GO pathways

Ont

ontology that the GO term belongs to if testing GO pathways. "BP" - biological process, "CC" - cellular component, "MF" - molecular function.

Pathway

the KEGG pathway being tested if testing KEGG terms.

N

number of genes in the GO or KEGG term

DE

number of genes that are differentially methylated

P.DE

p-value for over-representation of the GO or KEGG term term

FDR

False discovery rate

Author(s)

Belinda Phipson

References

Phipson, B., Maksimovic, J., and Oshlack, A. (2016). missMethyl: an R package for analysing methylation data from Illuminas HumanMethylation450 platform. Bioinformatics, 15;32(2), 286–8.

Geeleher, P., Hartnett, L., Egan, L. J., Golden, A., Ali, R. A. R., and Seoighe, C. (2013). Gene-set analysis is severely biased when applied to genome-wide methylation data. Bioinformatics, 29(15), 1851–1857.

Young, M. D., Wakefield, M. J., Smyth, G. K., and Oshlack, A. (2010). Gene ontology analysis for RNA-seq: accounting for selection bias. Genome Biology, 11, R14.

Ritchie, M. E., Phipson, B., Wu, D., Hu, Y., Law, C. W., Shi, W., and Smyth, G. K. (2015). limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Research, gkv007.

Benjamini, Y., and Hochberg, Y. (1995). Controlling the false discovery rate: a practical and powerful approach to multiple testing. Journal of the Royal Statistical Society Series, B, 57, 289-300.

See Also

goana,kegga,gsameth

Examples

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library(IlluminaHumanMethylation450kanno.ilmn12.hg19)
library(limma)
ann <- getAnnotation(IlluminaHumanMethylation450kanno.ilmn12.hg19)

# Randomly select 1000 CpGs to be significantly differentially methylated
sigcpgs <- sample(rownames(ann),1000,replace=FALSE)

# All CpG sites tested
allcpgs <- rownames(ann)

# GO testing with prior probabilities taken into account
# Plot of bias due to differing numbers of CpG sites per gene
gst <- gometh(sig.cpg = sigcpgs, all.cpg = allcpgs, collection = "GO", plot.bias = TRUE, prior.prob = TRUE)

# Total number of GO categories significant at 5% FDR
table(gst$FDR<0.05)

# Table of top GO results
topGO(gst)

# GO testing ignoring bias
gst.bias <- gometh(sig.cpg = sigcpgs, all.cpg = allcpgs, collection = "GO", prior.prob=FALSE)

# Total number of GO categories significant at 5% FDR ignoring bias
table(gst.bias$FDR<0.05)

# Table of top GO results ignoring bias
topGO(gst.bias)

# KEGG testing
kegg <- gometh(sig.cpg = sigcpgs, all.cpg = allcpgs, collection = "KEGG", prior.prob=TRUE)

# Table of top KEGG results
topKEGG(kegg)