gometh {missMethyl} | R Documentation |
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.
gometh( sig.cpg, all.cpg = NULL, collection = c("GO", "KEGG"), array.type = c("450K", "EPIC"), plot.bias = FALSE, prior.prob = TRUE, anno = NULL, equiv.cpg = TRUE, fract.counts = TRUE )
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. |
anno |
Optional. A |
equiv.cpg |
Logical, if true then equivalent numbers of cpgs are used
for odds calculation rather than total number cpgs. Only used if
|
fract.counts |
Logical, if true then fractional counting of cpgs is used
to account for cgps that map to multiple genes. Only used if
|
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.
The testing now also takes into account that some CpGs map to multiple genes.
For a small number of gene families, this previously caused their associated
GO categories/gene sets to be erroneously overrepresented and thus highly
significant. If fract.counts=FALSE
then CpGs are allowed to map to
multiple genes (this is NOT recommended).
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.ilm10b4.hg19
is used. To use a
different annotation package, please supply it using the anno
argument.
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.
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 |
Belinda Phipson
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.
## Not run: # to avoid timeout on Bioconductor build 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, anno = ann) # Total number of GO categories significant at 5% FDR table(gst$FDR<0.05) # Table of top GO results topGSA(gst) # GO testing ignoring bias gst.bias <- gometh(sig.cpg = sigcpgs, all.cpg = allcpgs, collection = "GO", prior.prob=FALSE, anno = ann) # Total number of GO categories significant at 5% FDR ignoring bias table(gst.bias$FDR<0.05) # Table of top GO results ignoring bias topGSA(gst.bias) # KEGG testing kegg <- gometh(sig.cpg = sigcpgs, all.cpg = allcpgs, collection = "KEGG", prior.prob=TRUE, anno = ann) # Table of top KEGG results topGSA(kegg) ## End(Not run)