WGBS data analysis in Galaxy?

Question

[Gema Sanz]

Hi,

Is there any way to follow a pipeline to analyze bisulfite sequencing data?

I know the alignment is tricky, but I've read some people do it with Bowtie without bisulfite genome conversion but I can't guess how to set the parameters to allow the penalty, as expected, I got 95% unmapped. I've also read about using a peak caller to call methylation "peaks".

Of course this is not the best way to do, but after trying a lot, I'm not able to compile any other software in my computer (windows), I'm bench person. I tried to install bismark, bsmooth... but errors everywhere :(

/G

[Gema Sanz]

Thank you for your answer. I agree with your points. I have a mac available, is it installed from the galaxy tool shed?

[Devon Ryan]

I don't know of any BSseq tools available in galaxy (I don't know if bismark was ever fully added, but it's the most likely to be in the toolshed). In general, you might have better luck getting that installed on a Mac than windows. Having said that, if you just don't bother with Galaxy at all then installation would be even easier.

[Biomonika (Noolean)]

Maybe you may get some advice here: https://biostar.usegalaxy.org/

Answer 1

[Devon Ryan]

You won't have any luck with bowtie2 by itself. Anyone you saw using that alone simply had no idea what they were doing and got completely crap results. You might have luck with integrating bwa-meth with galaxy, since it's pretty minimal on its requirements (just bwa I think). The better question is, "why bother with Galaxy"? It's easier (and vastly faster) to not use it than to integrate a new tool into it. If all you have is windows, then try to steal a labmate's Mac.

Regarding calling methylation peaks, you don't want to do that. Perhaps you've read papers about using within-sample methylation changes to find accessibility changes, such as promoter sites. In general, what you do after you have the resulting methylation metrics will be dependent on the biological question.

Whatever you do, make sure to make an M-bias plot before extracting methylation metrics. There's no sense in starting a downstream analysis with noisier data than needed.

[wanziyi89]

Hi Ryan,

Thanks for the primer regarding M-bias plot. Can you elaborate more on the application of M-bias plot and how do we interpret and applied it for downstream analysis, e.g. methylation calls or DMR testing.

Answer 2

[Evgeniia Golovina]

If you are interested in building pipeline for bisulfite sequencing data analysis, you can do it easily on Genestack platform. What you need is just to sign up for free, upload your data and build your pipeline. It can include preprocessing of you raw reads, mapping step (Genestack Bisulfite Sequencing Mapping app is based on bsmap) and Methylation Ratio Analysis (to determine methylation percent).

Answer 3

[Czh3]

Here is WGBS pipeline on Github, you may try it.

https://github.com/Czh3/NGSTools/blob/master/BS_seq.py