e.g. [http://seqanswers.com/forums/showthread.php?t=5502|Bismark]?
A colleague produced an alignment (SAM format) of bisulfite-treated sequences using Novoalign. About 85% of the sequences seem correctly aligned (which may need to be resolved first). Is there a tool or set of tools available for finding and quantifying the C->T conversions? The read length for each aligned sequence is roughly 40bp.
I'm most familiar with Perl, but I'm open to tools in any language that would be helpful.
8 answers
I had that same problem with existing pipelines for BS-treated reads--it's hard to get per-base methylation scores after the alignment.
So, (shamelss plug) I wrote MethylCoder, which takes FASTQ or Fasta reads files runs the alignment, and outputs (in addition to a SAM file) a text file like:
#seqid mt bp c t
chr1 3 1354 0 1
chr1 1 1358 6 1
chr1 2 1393 0 1
chr1 6 1394 4 9
indicating the methylation status at every base-- the methylation is c / (c + t).
where mt is the methylation type or context (1/4 are CG +/- strand, 2/5 are CHG +/- strand and 3/6 are CHH +/- ).
The pipeline is documented at the link above and also includes scripts for visualization and analysis for doing things like finding regions that are differentially methylated between 2 runs of BS-Seq. Also the author is very responsive to questions and feature requests ;)
I understand that this question was answered ages ago, but since it is the most relevant question tagged "Bisulfite" and thus a natural first step for those new to the technology and seeking general guidance on how to interpret it (as I did a month ago), I think the following update is relevant:
Felix Krueger wrote the following review in early 2012 detailing the steps for analysis of bisulfite-treated libraries, common sources of errors and a list of tools. Among these tools are Bismark(ref) (written by Krueger), MethylCoder (ref) (written by brentp), BRAT (ref), BS-Seeker (ref) and BSMAP (ref).
I'd also recommend going to the SeqAnswers software list and searching for "bisulfite"/"bisulphite".
I've been very pleased with the GNUmap software and the response of its developers. They have a flag for doing bisulfite analysis. However, I'm not sure if you can do the bisulfite analysis on an existing alignment, you may have to re-map the reads if you use this tool. But definitely for the future I would recommend this tool.
There were new tools advertised in the ISMB this year. I have not tried them myself but search the proceedings Especially the poster sessions.
e.g. Bismark(http://seqanswers.com/forums/showthread.php?t=5502)?
GNUMAP (mentioned by Daniel Standage) was also presented in a poster.
You may want to look at methVisual, a Bioconductor package and BSMAP, a tool to map bisulfite reads to genome sequence. Disclaimer: I have not used either, so can't be sure of their suitability for your problem.
As another answer, GSNAP (part of GMAP) maps BS-Seq reads. In my experience, it is able to map more reads, at the expense of running time.
There is a comparison of what I consider the full-featured BS-Seq mapping programs here: https://github.com/brentp/methylcode/tree/master/bench
Hope ViewBS can help you. https://github.com/readbio/ViewBS
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@Pierre Lindenbaum. Where did your answer go?