TECHNICAL NOTEOpen Access MethVisual - visualization and exploratory statistical analysis of DNA methylation profiles from bisulfite sequencing Arie Zackay1,2, Christine Steinhoff1* Abstract Background: Exploration of DNA methylation and its impact on various regulatory mechanisms has become a very active field of research. Simultaneously there is an arising need for tools to process and analyse the data together with statistical investigation and visualisation. Findings: MethVisual is a new application that enables exploratory analysis and intuitive visualization of DNA methylation data as is typically generated by bisulfite sequencing. The package allows the import of DNA methylation sequences, aligns them and performs quality control comparison. It comprises basic analysis steps as lollipop visualization, co-occurrence display of methylation of neighbouring and distant CpG sites, summary statistics on methylation status, clustering and correspondence analysis. The package has been developed for methylation data but can be also used for other data types for which binary coding can be inferred. The application of the package, as well as a comparison to existing DNA methylation analysis tools and its workflow based on two datasets is presented in this paper. Conclusions: The R package MethVisual offers various analysis procedures for data that can be binarized, in particular for bisulfite sequenced methylation data. R/Bioconductor has become one of the most important environments for statistical analysis of various types of biological and medical data. Therefore, any data analysis within R that allows the integration of various data types as provided from different technological platforms is convenient. It is the first and so far the only specific package for DNA methylation analysis, in particular for bisulfite sequenced data available in R/Bioconductor enviroment. The package is available for free at http:/methvisual. molgen.mpg.de/ and from the Bioconductor Consortium http:/www.bioconductor.org. Findings Motivation DNA Methylation is a biochemical modification of DNA which occurs in vertebrates almost exclusively at CpG sites, e.g. a methyl group is added at the 5 C position of cytosines. Recently, exploration of DNA methylation and its impact on various regulatory mechanisms has become a very active field of research. Specific DNA methylation patterns and/or their impact on a number of regulatory processes have been described for cancer 1,2, silencing of repetitive elements 3-5, ageing, development and embryonic stem cell profiling 6-8, correlation with chromatin remodeling, X chromosome inactivation 9, RNA interference 10; imprinting 11,12, tissue specific expression profiles 13 and evo- lutionary mutation processes 14. The variety of regula- tory mechanisms for which involvement of methylation has been reported demonstrates its high impact. Furthermore, recent results suggest a very close func- tional relationship between DNA methylation and his- tone modification 15. Thus, in combination with other epigenetic events, DNA methylation might have an important regulatory impact. While a number of technologies for quantitative and qualitative analysis of CpG methylation have been pub- lished over the last years, the most accurate experimen- tal procedures are still based on bisulfite treatment followed by conversion of non methylated cytosines to * Correspondence: christine.steinhoffmolgen.mpg.de 1Department of Computational Biology, Max Planck Institute for Molecular Genetics, Ihnestr 73, 14195 Berlin, Germany Full list of author information is available at the end of the article Zackay and Steinhoff BMC Research Notes 2010, 3:337 2010 Steinhoff et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http:/creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. uracil and sequencing. Analyzing this kind of data is complicated. Several steps, like alignment of bisulfite treated sequence to reference sequence, detection of low conversion rates of C to T in the bisulfite treatment or the conversion process and quality control, are necessary before actually extracting methylation profiles for further statistical analysis. However, this procedure is a prerequi- site for the investigation of functionality of DNA methy- lation. Tools that allow appropriate processing of this kind of data are needed and currently being developed with different focuses 16-21. The exploration of combi- natorially acting factors for the determination of epige- netic features is a field of current research 22. Bisulfite based DNA methylation data can be pro- duced in a high throughput fashion and on the basis of single gene investigation. While high throughput DNA methylation profiling is a very new technological ap