Comprehensive analysis of DNA methylation data with RnBeads

Abstract

RnBeads is a software tool for large-scale analysis and interpretation of DNA methylation data, providing a user-friendly analysis workflow that yields detailed hypertext reports (http://rnbeads.mpi-inf.mpg.de/). Supported assays include whole-genome bisulfite sequencing, reduced representation bisulfite sequencing, Infinium microarrays and any other protocol that produces high-resolution DNA methylation data. Notable applications of RnBeads include the analysis of epigenome-wide association studies and epigenetic biomarker discovery in cancer cohorts.

Figure 1. RnBeads workflow for analyzing large-scale DNA methylation data

The RnBeads workflow consists of seven modules and is essentially self-configuring based on a sample annotation table provided by the user. Each module generates part of the RnBeads hypertext report, which includes method descriptions, diagrams, and links to data tables. Furthermore, all data and annotations are stored in RnBSet objects to facilitate custom analysis workflows in R, and they are exported for visualization using genome browsers and follow-up analyses using other software tools. IDAT, signal intensity data; GS, Illumina GenomeStudio data; TAB, TAB-delimited data; GEO, Gene Expression Omnibus data.

Figure 2. Analysis of DNA methylation during adult stem cell differentiation

RnBeads was used to re-analyze an RRBS dataset comprising 19 cell types of the blood and skin lineages. All diagrams shown were calculated by RnBeads but have been reformatted according to journal standards. (a) Global distribution of DNA methylation levels among retained and removed CpGs after the preprocessing step. (b) Relative similarity and differences of DNA methylation profiles between cell types. Two maximally informative dimensions were calculated using multi-dimensional scaling (MDS) based on the matrix of average methylation levels in 5kb tiling regions. Samples are color-coded according to cell type. (c) Composite plot of DNA methylation levels in blood (green) and skin (orange) cell types averaged across all genes. Each gene was covered by six equally sized bins and by two flanking regions of the same size. Smoothing was done using cubic splines. (d) Heatmap with hierarchical clustering of DNA methylation levels among lineage marker genes that are specifically expressed in the blood lineage. Clustering used average linkage and Manhattan distance. (e) Scatterplot of groupwise mean DNA methylation levels across genes, with the 1,000 highest-ranking differentially methylated genes highlighted in red. Point density is shown as blue shading. (f) Volcano plot illustrating effect size and statistical significance across genes, with the 1,000 highest-ranking differentially methylated genes highlighted in red. Point density is shown as blue shading. (g) DNA methylation profile of the Hoxb3 gene locus on Chromosome 11 (brown triangle). Heatmaps show DNA methylation levels of single CpGs according to the color scheme in panel d. Smoothing of DNA methylation levels (bottom) was done using cubic splines.