Methylation analysis using MethTools 


Methylation analysis is carried out using a modified version of MethTools, a collection of open source Perl scripts that makes graphical representation of bisulphite sequencing results and calculates methylation densities (http://genome.imb-jena.de/methtools/). Instead of using the web interface, we downloaded and modified the scripts to suit our specific needs. For our purpose, only CpG methylation is analysed and clones are ordered by methylation level in the graphical representation. A shell script was also created to automate the analysis. The .exp.seq is included in the output graph to demonstrate the expected positions of CpGs.

MethTools has strict requirements for input file format: all sequences have to be aligned, in fasta format, and the first sequence has to be the genomic sequence and contains no illegal characters (only A, T, C, G, N are allowed) or gaps.

1.	Align sequences in MUSCLE (http://www.ebi.ac.uk/Tools/muscle/index.html, better for local alignment than CLUSTALW).
2.	Check the alignments in Genedoc (http://www.nrbsc.org/gfx/genedoc/index.html) to ensure that all CpGs are correctly aligned. 
3.	Put the MethTools script files and the aligned input file in the same directory, run autometh.sh on commandline to carry out the analysis. Output is a zipped file containing three folders and all results (methylation graph etc.) are in the ‘stats’ folder.