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An R package for DNA methylation analysis and annotation from high-throughput bisulfite sequencing. methylKit is designed to deal with sequencing data from RRBS and its variants, but also target-capture methods such as Agilent SureSelect methyl-seq. In addition, methylKit can deal with base-pair resolution data for 5hmC obtained from Tab-seq or oxBS-seq. It can also handle whole-genome bisulfite sequencing data if proper input format is provided.

GBSA / Genome Bisulfite Sequencing Analyser

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An open-source software tool capable of analysing whole-genome bisulfite sequencing data with either a gene-centric or gene-independent focus. GBSA’s output can be easily integrated with other high-throughput sequencing data, such as RNA-Seq or ChIP-seq, to elucidate the role of methylated intergenic regions in gene regulation. In essence, GBSA allows an investigator to explore not only known loci but also all the genomic regions, for which methylation studies could lead to the discovery of new regulatory mechanisms.


Infers epiallelic haplotypes, annotated with CpG methylation marks and polymorphisms, from whole genome bisulphite sequencing data, and nucleosome occupancy from NOMe-seq data. The performance of epiG depends on a number of user-adjustable parameters. These parameters control how noise is treated and how reads are clustered together to form haplotype chains. epiG starts by assigning each read to its own haplotype chain and then, iteratively, it improves the haplotype chains by re-assigning reads to chains one at a time, using an optimization procedure and prior genetic information.

bicycle / BIsulfite-based methylCYtosine CalLEr

Analyzes whole genome bisulfite sequencing (WGBS) data. bicycle is a next-generation sequencing (NGS) bioinformatic pipeline that can process data from directional (Lister) and non-directional (Cokus) bisulfite sequencing protocols and from single-end and paired-end sequencing. It also performs methylation calls for cytosines in CG and non-CG contexts (CHG and CHH). It provides statistical methylcytosine calling and offers several filters to screen reads.


A comprehensive tool for identification and analysis of the methylation patterns of genomic regions from bisulfite sequencing data. CpG_MPs first normalizes bisulfite sequencing reads into methylation level of CpGs. Then it identifies unmethylated and methylated regions using the methylation status of neighboring CpGs by hotspot extension algorithm without knowledge of pre-defined regions. Furthermore, the conservatively and differentially methylated regions across paired or multiple samples (cells or tissues) are identified by combining a combinatorial algorithm with Shannon entropy.

SMAP / Streamlined Methylation Analysis Pipeline

Allows to extract multiple types of information (such as DMCs, DMRs, SNPs and ASM) from various types of RRBS and Bis-seq data. SMAP is designed to be an easy-to-use, one-stop and sophisticated package for methylation analyses. The pipeline consists of seven operational stages: (i) reference preparation, (ii) read preparation, (iii) alignment, (iv) calculation of methylation rate, (v) differentially methylated regions (DMR) detection, (vi) single nucleotide polymorphism (SNP) and allele-specific DNA methylation (ASM) calling and (vii) summarization.


A software tool that not only fulfills the core data analysis requirements (e.g. sequence alignment, differential methylation analysis, etc.) but also provides useful tools for methylation data annotation and visualization. Specifically, Methy-Pipe uses Burrow-Wheeler Transform (BWT) algorithm to directly align bisulfite sequencing reads to a reference genome and implements a novel sliding window based approach with statistical methods for the identification of differentially methylated regions (DMRs). The capability of processing data parallelly allows it to outperform a number of other bisulfite alignment software packages.

WBSA / Web Service for Bisulfite Sequencing Data Analysis

A free web application for analysis of whole-genome bisulfite-sequencing (WGBS) and genome-wide reduced representation bisulfite sequencing (RRBS) data. WBSA not only focuses on CpG methylation, but also allows CHG and CHH analysis. BWA is incorporated as its mapping software. WBSA can be applied to DNA methylation researches for animals and plants and it provides advanced analysis for both WGBS and RRBS. It can also identify differently methylated regions (DMRs) in different strings. WBSA includes six modules: Home, WGBS, RRBS, DMR, Documents and Downloads, and provides the executable package for downloads and local installation. WGBS and RRBS modules include four main steps: pre-processing of reads and reference, alignment to the reference, identification of methylcytosines and annotation. DMR module includes DMRs identification and annotation of the correlative genes.


A comprehensive genome-scale DNA methylation analysis server based on RRBS data. RRBS-Analyser can assess sequencing quality, generate detailed statistical information, align the bisulfite-treated short reads to reference genome, identify and annotate the methylcytosines (5mCs) and associate them with different genomic features in CG, CHG, and CHH content. RRBS-Analyser supports detection, annotation, and visualization of differentially methylated regions (DMRs) for multiple samples from nine reference organisms. Moreover, RRBS-Analyser provides researchers with detailed annotation of DMR-containing genes, which will greatly aid subsequent studies. The input of RRBS-Analyser can be raw FASTQ reads, generic SAM format, or self-defined format containing individual 5mC sites. RRBS-Analyser can be widely used by researchers wanting to unravel the complexities of DNA methylome in the epigenetic community.