README.md

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methylKit

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GitHub R package version codecov

Introduction

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.

Current Features

Staying up-to-date

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

Installation

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.

Install the development version

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.

How to Use

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.

Documentation

Downloading Annotation Files

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.

  1. For gene annotation, select "Genes and Gene prediction tracks" from the "group" drop-down menu. Following that, select "Refseq Genes" from the "track" drop-down menu. Select "BED- browser extensible data" for the "output format". Click "get output" and on the following page click "get BED" without changing any options. save the output as a text file.
  2. For CpG island annotation, select "Regulation" from the "group" drop-down menu. Following that, select "CpG islands" from the "track" drop-down menu. Select "BED- browser extensible data" for the "output format". Click "get output" and on the following page click "get BED" without changing any options. save the output as a text file.

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)

R script for Genome Biology publication

The most recent version of the R script in the Genome Biology manuscript is here.

Citing methylKit

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:

Contact & Questions

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.

Contribute to the development

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:

License

Artistic License/GPL



al2na/methylKit documentation built on Feb. 1, 2024, 4:42 p.m.