MethylDackel, GTN Tutorial Galaxies, and DNA Methylation

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I was following the tutorial “DNA Methylation data analysis” and the MethylDackel tool that it says to use for Methylation bias and metric extraction can not be found. I tried to download the tutorial’s workflow and it says it is missing tools from the toolshed. Is there anyway to still use that tool? I have all my files in BAM format from using the bwameth tool but without the next tool I am stuck

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Hi @ntillquist

The tool may not be installed at the public Galaxy server you are working at currently. Or, you own Galaxy if that is where you are working.

Try working at one of the servers (or the Docker instance, if available) listed for the tutorial under the pull-down menu “Available on these Galaxies”.

GTN Tutorial DNA Methylation data analysis

GTN Topic: Epigenetics Galaxy Training!

Screenshot with that menu expanded. UseGalaxy.eu is one good choice as GTN tutorials are routinely tested there. This tutorial happens to also be associated with a pre-configured Docker image – navigate back up to the topic level to find the details if that interests you.

screenshot-methylation-seq-gtn-galaxies-menu
screenshot-methylation-seq-gtn-galaxies-menu1798×1364 373 KB

Hope that helps!

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Hi @jennaj . I am trying to run this tutorial on use galaxy.eu.
In the “Hands-on: Methylation extraction with MethylDackel section”
I cannot find the option to " * Set the parameter Extract fractional methylation (only) at each position. This is mutually exclusive with --counts, --logit, and --methylKit to Yes." , as mentioned in the tutorial.
Please suggest

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Hi @akanksha_bafna

Try clicking to open the tool directly. That points to the EU server, and opens the version of the tool supported in the tutorial. Be sure to look under advanced options too for options.

Let us know if that is not enough and can look closer. These tutorials were just updated so are current.

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Hi,
@jennaj . I opened the tool from usegalaxy.eu server and executed a job.

I looked in advanced options as well but could not find the specified function to set the parameter.

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Hi @akanksha_bafna

The tool can be opened directly from the tutorial. That points to the specific version of the tool that matches the tutorial.

Or, you can navigate using the version’s menu Changing the tool version

I’m explaining since you might need to do this across tutorials and tools, so is good to learn how :slight_smile: Give that a try and if still stuck, I’ll explain the steps with screenshots.

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@jennaj . Indeed an older version of the tool , had the missing parameter as advanced settings that helped me .

Another question based on the course tutorial : for compute matrix it uses imported file CpGislands.bed. As per my understanding, this is for human genome . I am using mm10 methylation datasets. Please could you help me where can I find the CpGislands. bed to carry out the plotProfile and differential methylation.

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UCSC https://genome.ucsc.edu/ has a track for this data associated with mm10. See their Table Browser. You can go there first and check the box for output to Galaxy, or open it from within Galaxy with the tool UCSC main.

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Hi @jennaj

Thanks . I will do that . I reckon in the tutorial at no point it is mentioned to import CpGislands.bed as input and can be confusing . Perhaps adding it as data to upload prior might be useful.

On a separate note I checked the workflow and would just like to clarify few parameters.
In the tutorial :
MethylDackel tool with the following parameters:

  • Choose at the first option Load reference genome from Local cache and for Using reference genomethe value hg38.
  • Select for the option sorted_alignments.bam the computed bam file of step 4 of the bwameth alignment.
  • Use for What do you want to do? the value Determine the position-dependent methylation bias in the dataset, producing diagnostic SVG images.
  • Set the parameters By default, if only one read in a pair aligns (a singleton) then it's ignored. and By default, paired-end alignments with the properly-paired bit unset in the FLAG field are ignored. Note that the definition of concordant and discordant is based on your aligner settings. to Yes.

Should we change the default settings of the above mentioned parameters and set it to “yes”.

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Hello @akanksha_bafna

If your reads are paired-end, then choose the pair-end end option with this tool, too. The concordant alignments flag in the BAM will be important to avoid over counting. SAMtools stats can count those up, and Filter BAM can remove the excess reads if you want to reduce the BAM size for some reason (example: to reduce the load on downstream tools).