Recent studies indicate that DNA methylation can be used to identify changes at transcriptional enhancers and other cis-regulatory elements in primary human samples. A systematic approach to inferring gene regulatory networks has been provided by the R/Bioconductor package ELMER (Enhancer Linking by Methylation/Expression Relationships), which first identifies DNA methylation changes in distal regulatory elements and correlates these with the expression of nearby genes to identify direct transcriptional targets. Next, ELMER performs a transcription factor binding motif analysis and integrates with expression profiling of all human transcription factors, to identify master regulatory TFs and place each differentially methylated regulatory element into the context of an altered gene regulatory network (GRN).Here we present a completely updated version of the package (ELMER v. 2.0), which uses the latest Bioconductor data structures including the popular MultiAsssayExperiment, supports multiple reference genome assemblies as well as the DNA methylation platforms Infinium MethylationEPIC and Infinium HumanMethylation450, and provides a "Supervised" analysis mode for paired sample study designs (such as treated vs. untreated replicate samples). It also supports data import from the new NCI Genomic Data Commons (GDC) database. The new version is substantially re-written, improving stability, performance, and extensibility. It also uses improved databases for transcription factor binding domain families and binding motif specificities, and has newly designed output plots for publication-quality figures.Below, we describe the methods and new features of ELMER v. 2.0 and present two use case demonstrating how the tool can be used to analyze TCGA data in either Unsupervised or Supervised mode. ELMER (v2.0.0) is available as an R/Bioconductor package at