gsaregion: Generalised gene set testing for Illumina's methylation array...

In Oshlack/missMethyl: Analysing Illumina HumanMethylation BeadChip Data

Generalised gene set testing for Illumina's methylation array data

Description

Given a user specified list of gene sets to test, gsaregion tests whether differentially methylated regions (DMRs) identified from Illumina's Infinium HumanMethylation450 or MethylationEPIC array are enriched, taking into account the differing number of probes per gene present on the array.

Usage

gsaregion(
  regions,
  all.cpg = NULL,
  collection,
  array.type = c("450K", "EPIC"),
  plot.bias = FALSE,
  prior.prob = TRUE,
  anno = NULL,
  equiv.cpg = TRUE,
  fract.counts = TRUE,
  genomic.features = c("ALL", "TSS200", "TSS1500", "Body", "1stExon", "3'UTR", "5'UTR",
    "ExonBnd"),
  sig.genes = FALSE
)

Arguments

regions	GRanges Object of DMR coordinates to test for GO term enrichment.
all.cpg	Character vector of all CpG sites tested. Defaults to all CpG sites on the array.
collection	A list of user specified gene sets to test. Can also be a single character vector gene set. Gene identifiers must be Entrez Gene IDs.
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 DataFrame object containing the complete array annotation as generated by the minfi getAnnotation function. Speeds up execution, if provided.
equiv.cpg	Logical, if true then equivalent numbers of cpgs are used for odds calculation rather than total number cpgs. Only used if prior.prob=TRUE.
fract.counts	Logical, if true then fractional counting of cpgs is used to account for cgps that map to multiple genes. Only used if prior.prob=TRUE.
genomic.features	Character vector or scalar indicating whether the gene set enrichment analysis should be restricted to CpGs from specific genomic locations. Options are "ALL", "TSS200","TSS1500","Body","1stExon", "3'UTR","5'UTR","ExonBnd"; and the user can select any combination. Defaults to "ALL".
sig.genes	Logical, if true then the significant differentially methylated genes that overlap with the gene set of interest is outputted as the final column in the results table. Default is FALSE.

Details

This function extends goregion, which only tests GO and KEGG pathways. gsaregion can take a list of user specified gene sets and test whether the significant DMRs are enriched in these pathways. This function takes a GRanges object of DMR coordinates, maps them to CpG sites on the array and then to Entrez Gene IDs, and tests for enrichment using Wallenius' noncentral hypergeometric test, taking into account the number of CpG sites per gene on the 450K/EPIC array. 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. The topGSA function can be used to display the top 20 most enriched pathways.

If you are interested in which genes overlap with the genes in the gene set, setting sig.genes to TRUE will output an additional column in the results data frame that contains all the significant differentially methylated gene symbols, comma separated. The default is FALSE.

Value

A data frame with a row for each gene set and the following columns:

N	number of genes in the gene set
DE	number of genes that are differentially methylated
P.DE	p-value for over-representation of the gene set
FDR	False discovery rate, calculated using the method of Benjamini and Hochberg (1995).
SigGenesInSet	Significant differentially methylated genes overlapping with the gene set of interest.

Author(s)

Jovana Maksimovic

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

gometh,goregion,gsameth, getMappedEntrezIDs

Examples

## Not run:  # to avoid timeout on Bioconductor build
library(IlluminaHumanMethylationEPICanno.ilm10b4.hg19)
library(limma)
library(DMRcate)
library(ExperimentHub)
library(org.Hs.eg.db)

# Follow the example for the dmrcate function to get some EPIC data from
# ExperimentHub
eh <- ExperimentHub()
FlowSorted.Blood.EPIC <- eh[["EH1136"]]
tcell <- FlowSorted.Blood.EPIC[,colData(FlowSorted.Blood.EPIC)$CD4T==100 |
                                colData(FlowSorted.Blood.EPIC)$CD8T==100]
detP <- detectionP(tcell)
remove <- apply(detP, 1, function (x) any(x > 0.01))
tcell <- tcell[!remove,]
tcell <- preprocessFunnorm(tcell)
#Subset to chr2 only
tcell <- tcell[seqnames(tcell) == "chr2",]
tcellms <- getM(tcell)
tcellms.noSNPs <- rmSNPandCH(tcellms, dist=2, mafcut=0.05)
tcell$Replicate[tcell$Replicate==""] <- tcell$Sample_Name[tcell$Replicate==""]
tcellms.noSNPs <- avearrays(tcellms.noSNPs, tcell$Replicate)
tcell <- tcell[,!duplicated(tcell$Replicate)]
tcell <- tcell[rownames(tcellms.noSNPs),]
colnames(tcellms.noSNPs) <- colnames(tcell)
assays(tcell)[["M"]] <- tcellms.noSNPs
assays(tcell)[["Beta"]] <- ilogit2(tcellms.noSNPs)

# Perform region analysis
type <- factor(tcell$CellType)
design <- model.matrix(~type) 
myannotation <- cpg.annotate("array", tcell, arraytype = "EPIC",
                             analysis.type="differential", design=design, 
                             coef=2)
# Run DMRCate with beta value cut-off filter of 0.1                              
dmrcoutput <- dmrcate(myannotation, lambda=1000, C=2, betacutoff = 0.1)
regions <- extractRanges(dmrcoutput)
length(regions)

ann <- getAnnotation(IlluminaHumanMethylationEPICanno.ilm10b4.hg19)
# All CpG sites tested (limited to chr 2)
allcpgs <- rownames(tcell)
# Use org.Hs.eg.db to extract a GO term
GOtoID <- suppressMessages(select(org.Hs.eg.db, keys=keys(org.Hs.eg.db), 
                                  columns=c("ENTREZID","GO"), 
                                  keytype="ENTREZID"))
keep.set1 <- GOtoID$GO %in% "GO:0010951"
set1 <- GOtoID$ENTREZID[keep.set1]
keep.set2 <- GOtoID$GO %in% "GO:0042742"
set2 <- GOtoID$ENTREZID[keep.set2]
keep.set3 <- GOtoID$GO %in% "GO:0031295"
set3 <- GOtoID$ENTREZID[keep.set3]
# Make the gene sets into a list
sets <- list(set1, set2, set3)
names(sets) <- c("GO:0010951","GO:0042742","GO:0031295")

# Testing with prior probabilities taken into account
# Plot of bias due to differing numbers of CpG sites per gene
gst <- gsaregion(regions = regions, all.cpg = allcpgs, collection = sets, 
                 array.type = "EPIC", plot.bias = TRUE, prior.prob = TRUE,
                 anno = ann)
topGSA(gst)

# Add significant genes in gene set to output
gst <- gsaregion(regions = regions, all.cpg = allcpgs, collection = sets, 
                 array.type = "EPIC", plot.bias = TRUE, prior.prob = TRUE,
                 anno = ann, sig.genes = TRUE)
topGSA(gst)

## End(Not run)

Oshlack/missMethyl documentation built on March 26, 2023, 1:50 p.m.