methylGSA: Gene Set Analysis for DNA Methylation Datasets

In methylGSA: Gene Set Analysis Using the Outcome of Differential Methylation

knitr::opts_chunk$set(comment = "", message=FALSE, warning = FALSE)

Installation

To install and load methylGSA

if (!requireNamespace("BiocManager", quietly = TRUE))
    install.packages("BiocManager")

BiocManager::install("methylGSA")

library(methylGSA)

Depending on the DNA methylation array type, other packages may be needed before running the analysis.

If analyzing 450K array, IlluminaHumanMethylation450kanno.ilmn12.hg19 needs to be loaded.

library(IlluminaHumanMethylation450kanno.ilmn12.hg19)

If analyzing EPIC array, IlluminaHumanMethylationEPICanno.ilm10b4.hg19 needs to be loaded.

library(IlluminaHumanMethylationEPICanno.ilm10b4.hg19)

If analyzing user-supplied mapping between CpG ID and gene name, neither IlluminaHumanMethylation450kanno.ilmn12.hg19 nor IlluminaHumanMethylationEPICanno.ilm10b4.hg19 needs to be loaded.

Introduction

The methylGSA package contains functions to carry out gene set analysis adjusting for the number of CpGs of each gene. It has been shown by Geeleher et al [1] that gene set analysis is extremely biased for DNA methylation data. This package contains three main functions to adjust for the bias in gene set analysis.

• methylglm: Incorporating number of CpGs as a covariate in logistic regression.

• methylRRA: Adjusting for multiple p-values of each gene by Robust Rank Aggregation [2], and then apply either over-representation analysis (ORA) or Preranked version of Gene Set Enrichment Analysis (GSEAPreranked) [3] in gene set testing.

• methylgometh: Adjusting the number of CpGs for each gene by weighted resampling and Wallenius non-central hypergeometric approximation. (via missMethyl [4])

Supported gene sets and gene ID types

• Gene Ontology (via org.Hs.eg.db [5])
• KEGG (via org.Hs.eg.db [5])
• Reactome (via reactome.db [6])
• User-supplied gene sets. Supported input gene ID types:
  • "SYMBOL"
  • "ENSEMBL"
  • "ENTREZID"
  • "REFSEQ"

Supported array types

• Infinium Human Methylation 450K BeadChip (via IlluminaHumanMethylation450kanno.ilmn12.hg19 [7])
• Infinium Methylation EPIC BeadChip (via IlluminaHumanMethylationEPICanno.ilm10b4.hg19 [8])
• User supplied mapping between CpG ID and gene name

Description of methylglm

methylglm is an extention of GOglm [9]. GOglm adjusts length bias for RNA-Seq data by incorporating gene length as a covariate in logistic regression model. methylglm adjusts length bias in DNA methylation by the number of CpGs instead of gene length. For each gene set, we fit a logistic regression model:

$$logit (\pi_{i}) = \beta_{0} + \beta_{1}x_{i} + \beta_{2}c_{i}$$ For each gene $i$, $\pi_{i}$ = Pr(gene $i$ is in gene set), $x_{i}$ represents negative logarithmic transform of its minimum p-value in differential methylation analysis, and $c_{i}$ is logarithmic transform of its number of CpGs.

methylglm requires only a simple named vector. This vector contains p-values of each CpG. Names should be their corresponding CpG IDs.

Example

Here is what the input vector looks like:

data(cpgtoy)
head(cpg.pval, 20)

Please note that the p-values here in cpg.pval is just for illustration purposes. They are used to illustrate how to use the functions in methylGSA. The actual p-values in differential methylation analysis may be quite different from the p-values in cpg.pval in terms of the magnitude.

Then call methylglm:

res1 = methylglm(cpg.pval = cpg.pval, minsize = 200, 
                 maxsize = 500, GS.type = "KEGG")
head(res1, 15)

Result is a data frame ranked by p-values of gene sets.

Description of methylRRA

Robust rank aggregation [2] is a parameter free model that aggregates several ranked gene lists into a single gene list. The aggregation assumes random order of input lists and assign each gene a p-value based on order statistics. We apply this order statistics idea to adjust for number of CpGs.

For gene $i$, let $P_{1}, P_{2}, ... P_{n}$ be the p-values of individual CpGs in differential methylation analysis. Under the null hypothesis, $P_{1}, P_{2}, ... P_{n} ~ \overset{i.i.d}{\sim} Unif[0, 1]$. Let $P_{(1)}, P_{(2)}, ... P_{(n)}$ be the order statistics. Define: $$\rho = \text{min}{\text{Pr}(P_{(1)}<P_{(1)\text{obs}}), \text{Pr}(P_{(2)}<P_{(2)\text{obs}})..., \text{Pr}(P_{(n)}<P_{(n)\text{obs}}) } $$

methylRRA supports two approaches to adjust for number of CpGs, ORA and GSEAPreranked [3]. In ORA approach, for gene $i$, conversion from $\rho$ score into p-value is done by Bonferroni correction [2]. We get a p-value for each gene and these p-values are then corrected for multiple testing use Benjamini & Hochberg procedure [10]. By default, genes satisfy FDR<0.05 are considered DE genes. If there are no DE genes under FDR 0.05, users are able to use sig.cut option to specify a higher FDR cut-off or topDE option to declare top genes to be differentially expressed. We then apply ORA based on these DE genes.

In GSEAPreranked approach, for gene $i$, we also convert $\rho$ score into p-value by Bonferroni correction. p-values are converted into z-scores. We then apply Preranked version of Gene Set Enrichment Analysis (GSEAPreranked) on the gene list ranked by the z-scores.

Example

To apply ORA approach, we use argument method = "ORA" in methylRRA

res2 = methylRRA(cpg.pval = cpg.pval, method = "ORA", 
                    minsize = 200, maxsize = 210)
head(res2, 15)

To apply GSEAPreranked approach, we use argument method = "GSEA" in methylRRA

res3 = methylRRA(cpg.pval = cpg.pval, method = "GSEA", 
                    minsize = 200, maxsize = 210)
head(res3, 10)

Description of methylgometh

methylgometh calls gometh or gsameth function in missMethyl package [4] to adjust number of CpGs in gene set testing. gometh modifies goseq method [11] by fitting a probability weighting function and resampling from Wallenius non-central hypergeometric distribution.

methylgometh requires two inputs, cpg.pval and sig.cut. sig.cut specifies the cut-off point to declare a CpG as differentially methylated. By default, sig.cut is 0.001. Similar to methylRRA, if no CpG is significant, users are able to specify a higher cut-off or use topDE option to declare top CpGs to be differentially methylated.

Example

res4 = methylgometh(cpg.pval = cpg.pval, sig.cut = 0.001, 
                        minsize = 200, maxsize = 210)
head(res4, 15)

Other options

methylGSA provides many other options for users to customize the analysis.

• array.type is to specify which array type to use. It is either "450K" or "EPIC". Default is "450K". This argument will be ignored if FullAnnot is provided.
• FullAnnot is preprocessed mapping between CpG ID and gene name provided by prepareAnnot function. Default is NULL. Check example below for details.
• group is the type of CpG to be considered in methylRRA or methylglm. By default, group is "all", which means all CpGs are considered regardless of their gene group. If group is "body", only CpGs on gene body will be considered. If group is "promoter1" or "promoter2", only CpGs on promoters will be considered. Based on the annotation in IlluminaHumanMethylation450kanno.ilmn12.hg19 and IlluminaHumanMethylationEPICanno.ilm10b4.hg19, "body", "promoter1" and "promoter2" are defined as:
  • body: CpGs whose gene group correspond to "Body" or "1stExon"
  • promoter1: CpGs whose gene group correspond to "TSS1500" or "TSS200"
  • promoter2: CpGs whose gene group correspond to "TSS1500", "TSS200", "1stExon", or "5'UTR"
• GS.list is user supplied gene sets to be tested. It should be a list with entry names gene sets IDs and elements correpond to genes that gene sets contain. If there is no input list, Gene Ontology is used.
• GS.idtype is the type of gene ID in user supplied gene sets. If GS.list is not empty, then the user is expected to provide gene ID type. Supported ID types are "SYMBOL", "ENSEMBL", "ENTREZID", "REFSEQ". Default is "SYMBOL".
• GS.type is the published gene sets/pathways to be tested if GS.list is empty. Supported pathways are "GO", "KEGG", and "Reactome". Default is "GO".
• minsize is an integer. If the number of genes in a gene set is less than this integer, this gene set is not tested. Default is 100.
• maxsize is also an integer. If the number of genes in a gene set is greater than this integer, this gene set is not tested. Default is 500.
• In methylRRA, method is to specify gene set test method. It is either "ORA" or "GSEA" as described above.
• In methylglm, parallel is either TRUE or FALSE indicating whether parallel should be used.

Examples

Here an example of user supplied gene sets. The gene ID type is gene symbol

data(GSlisttoy)
## to make the display compact, only a proportion of each gene set is shown
head(lapply(GS.list, function(x) x[1:30]), 3)   

This is an example of running methylglm with parallel

library(BiocParallel)
res_p = methylglm(cpg.pval = cpg.pval, minsize = 200, 
                  maxsize = 500, GS.type = "KEGG", parallel = TRUE)

methylglm and methylRRA support user supplied CpG ID to gene mapping. The mapping is expected to be a matrix, or a data frame or a list. For a matrix or data frame, 1st column should be CpG ID and 2nd column should be gene name. For a list, entry names should be gene names and elements correpond to CpG IDs. This is an example of user supplied CpG to gene mapping:

data(CpG2Genetoy)
head(CpG2Gene)   

To use user supplied mapping in methylglm or methylRRA, first preprocess the mapping by prepareAnnot function

FullAnnot = prepareAnnot(CpG2Gene) 

Test the gene sets using "ORA" in methylRRA, use FullAnnot argument to provide the preprocessed CpG ID to gene mapping

GS.list = GS.list[1:10]
res5 = methylRRA(cpg.pval = cpg.pval, FullAnnot = FullAnnot, method = "ORA", 
                    GS.list = GS.list, GS.idtype = "SYMBOL", 
                    minsize = 100, maxsize = 300)
head(res5, 10)

Here is another example. Test Reactome pathways using methylglm

res6 = methylglm(cpg.pval = cpg.pval, array.type = "450K", 
                    GS.type = "Reactome", minsize = 100, maxsize = 110)
head(res6, 10)

Visualization

Following bar plot implemented in enrichplot [12], we also provide bar plot to visualize the gene set analysis results. The input of barplot function can be any result returned by methylglm, methylRRA, or methylgometh. Various options are provided for users to customize the plot.

• xaxis is to specify the label in x-axis. It is either "Count" (number of significant genes in gene set) or "Size" (total number of genes in gene set). "Count" option is not available for methylglm and methylRRA(GSEA). Default is "Size".
• num is to specify the number of genes sets to display on the bar plot. Default is 5.
• colorby is a string. Either "pvalue" or "padj". Default is "padj".
• title is a string. The title to display on the bar plot. Default is NULL.

Example

Here is an example of using barplot to visualize the result of methylglm

barplot(res1, num = 8, colorby = "pvalue")

Session info

sessionInfo()

References

[1] Geeleher, Paul, Lori Hartnett, Laurance J Egan, Aaron Golden, Raja Affendi Raja Ali, and Cathal Seoighe. 2013. Gene-Set Analysis Is Severely Biased When Applied to Genome-Wide Methylation Data. Bioinformatics 29 (15). Oxford University Press: 1851–7.

[2] Kolde, Raivo, Sven Laur, Priit Adler, and Jaak Vilo. 2012. Robust Rank Aggregation for Gene List Integration and Meta-Analysis. Bioinformatics 28 (4). Oxford University Press: 573–80.

[3] Subramanian, Aravind, Pablo Tamayo, Vamsi K Mootha, Sayan Mukherjee, Benjamin L Ebert, Michael A Gillette, Amanda Paulovich, et al. 2005. Gene Set Enrichment Analysis: A Knowledge-Based Approach for Interpreting Genome-Wide Expression Profiles. Proceedings of the National Academy of Sciences 102 (43). National Acad Sciences: 15545–50.

[4] Phipson, Belinda, Jovana Maksimovic, and Alicia Oshlack. 2015. MissMethyl: An R Package for Analyzing Data from Illumina's Humanmethylation450 Platform. Bioinformatics 32 (2). Oxford University Press: 286–88.

[5] Carlson M (2018). org.Hs.eg.db: Genome wide annotation for Human. R package version 3.6.0.

[6] Ligtenberg W (2018). reactome.db: A set of annotation maps for reactome. R package version 1.64.0.

[7] Hansen, KD. (2016). IlluminaHumanMethylation450kanno.ilmn12.hg19: Annotation for Illumina’s 450k Methylation Arrays. R Package, Version 0.6.0 1.

[8] Hansen, KD. (2017). IlluminaHumanMethylationEPICanno.ilm10b4.hg19: Annotation for Illumina’s Epic Methylation Arrays. R Package, Version 0.6.0 1.

[9] Mi, Gu, Yanming Di, Sarah Emerson, Jason S Cumbie, and Jeff H Chang. 2012. Length Bias Correction in Gene Ontology Enrichment Analysis Using Logistic Regression. PloS One 7 (10). Public Library of Science: e46128.

[10] Benjamini, Yoav, and Yosef Hochberg. 1995. Controlling the False Discovery Rate: A Practical and Powerful Approach to Multiple Testing. Journal of the Royal Statistical Society. Series B (Methodological). JSTOR, 289–300.

[11] Young, Matthew D, Matthew J Wakefield, Gordon K Smyth, and Alicia Oshlack. 2012. Goseq: Gene Ontology Testing for Rna-Seq Datasets. R Bioconductor.

[12] Yu G (2018). enrichplot: Visualization of Functional Enrichment Result. R package version 1.0.2, https://github.com/GuangchuangYu/enrichplot.

methylGSA documentation built on Nov. 8, 2020, 8:29 p.m.