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methylKit is an R package for DNA methylation analysis and annotation from high-throughput bisulfite sequencing. The package 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.
You can subscribe to our googlegroups page to get the latest information about new releases and features (low-frequency, only updates are posted)
To ask questions please use methylKit_discussion forum
You can also check out the blogposts we make on using methylKit
in R console,
library(devtools)
install_github("al2na/methylKit", build_vignettes=FALSE,
repos=BiocManager::repositories(),
dependencies=TRUE)
if this doesn't work, you might need to add type="source"
argument.
library(devtools)
install_github("al2na/methylKit", build_vignettes=FALSE,
repos=BiocManager::repositories(),ref="development",
dependencies=TRUE)
if this doesn't work, you might need to add type="source"
argument.
Typically, bisulfite converted reads are aligned to the genome and % methylation value per base is calculated by processing alignments. methylKit
takes that % methylation value per base information as input. Such input file may be obtained from AMP pipeline for aligning RRBS reads. A typical input file looks like this:
chrBase chr base strand coverage freqC freqT
chr21.9764539 chr21 9764539 R 12 25.00 75.00
chr21.9764513 chr21 9764513 R 12 0.00 100.00
chr21.9820622 chr21 9820622 F 13 0.00 100.00
chr21.9837545 chr21 9837545 F 11 0.00 100.00
chr21.9849022 chr21 9849022 F 124 72.58 27.42
chr21.9853326 chr21 9853326 F 17 70.59 29.41
methylKit
reads in those files and performs basic statistical analysis and annotation for differentially methylated regions/bases. Also a tab separated text file with a generic format can be read in, such as methylation ratio files from BSMAP, see here for an example. Alternatively, read.bismark
function can read SAM file(s) output by Bismark(using bowtie/bowtie2) aligner (the SAM file must be sorted based on chromosome and read start). The sorting must be done by unix sort or samtools, sorting using other tools may change the column order of the SAM file and that will cause an error.
Below, there are several options showing how to do basic analysis with methylKit
.
Annotation files in BED format are needed for annotating your differentially methylated regions. You can download annotation files from UCSC table browser for your genome of interest. Go to [http://genome.ucsc.edu/cgi-bin/hgGateway]. On the top menu click on "tools" then "table browser". Select your "genome" of interest and "assembly" of interest from the drop down menus. Make sure you select the correct genome and assembly. Selecting wrong genome and/or assembly will return unintelligible results in downstream analysis.
From here on you can either download gene annotation or CpG island annotation.
In addition, you can check this tutorial to learn how to download any track from UCSC in BED format (http://www.openhelix.com/cgi/tutorialInfo.cgi?id=28)
The most recent version of the R script in the Genome Biology manuscript is here.
If you used methylKit please cite:
If you used flat-file objects or over-dispersion corrected tests please consider citing:
and also consider citing the following publication as a use-case with specific cutoffs:
e-mail to methylkit_discussion@googlegroups.com or post a question using the web interface.
if you are going to submit bug reports or ask questions, please send sessionInfo() output from R console as well.
Questions are very welcome, although we suggest you read the paper, documentation(function help pages and the vignette) and blog entries first. The answer to your question might be there already.
See the trello board for methylKit development. You can contribute to the methylKit development via github ([http://github.com/al2na/methylKit/]) by opening an issue and discussing what you want to contribute, we will guide you from there. In addition, you should:
Bump up the version in the DESCRIPTION file on the 3rd number. For example, the master branch has the version numbering as in "X.Y.1". If you make a change to master branch you should bump up the version in the DESCRIPTION file to "X.Y.2".
Add your changes to the NEWS file as well under the correct version and appropriate section. Attribute the changes to yourself, such as "Contributed by X"
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