Reference-free deconvolution, visualization and interpretation of complex DNA methylation data using DecompPipeline, MeDeCom and FactorViz

DNA methylation assays are typically performed on bulk tissue samples, which show substantial cellular heterogeneity. To dissect these heterogeneous DNA methylomes, several deconvolution approaches have been proposed (for a list of tools see this GitHub page). Reference-free deconvolution tools do not rely on purified cell type profiles, and address heterogeneity in an unbiased way. These methods rely on high-quality data and pose challenges for the interpretation of deconvolution results. We propse a three-stage protocol to perform reference-free deconvolution of complex DNA methylation data:

Data preparation

Input DNA methylation data either obtained from bisulfite sequencing technologies (BED files) or the Illumina Infinium BeadArrays (IDAT files) are processed using the RnBeads sofware package. The data set is checked for quality using RnBeads' reporting functionality, and then filtered according to user-specified options, which removes intensity/coverage outliers, or annotated single nucleotide polymorphisms. These steps are compiled in the comprehensive software suite DecompPipeline. Confounding factors are addressed for using Independent Component Analysis (ICA).

Deconvolution

The second stage comprises reference-free deconvolution of the processed DNA methylation data matrix. The protocol supports MeDeCom, as well as RefFreeCellMix and EDec. All of these methods are based on non-negative matrix factoriation (NMF).

Interpretation

To visualize deconvolution results and to get from the results to biological interpretation, we implemented the R/shiny graphical user interface FactorViz. FactorViz provides functions to link the obtained components (latent methylation components=LMCs) to phenotypic traits and to perform enrichment analysis on the sites that are particularly hypo-methylated in an LMC.

Publication

The protocol has been published in Nature Protocols, 2020