RSEM prepare reference

[mathew_harvey]

Hi, I am currently trying to run RNA-SEQ on an mm39 sample. But when i use this tool with steps provided in a previous forum post linked here. Updating the mouse genome to GRCm39/mm39 on Galaxy. All that the tool produces is an ascii text file. I can not figure out what is wrong with the issue is.

here is a page with my settings, any help would be greatly appreciated.

[jennaj]

Welcome, @mathew_harvey

I can reproduce what I think you are describing in this test history.

• https://usegalaxy.eu/u/jenj/h/test-rsem-prepare-reference

With a screenshot here showing that the downstream tool is not recognizing the index from the history.

And this one showing the file contents and creation details (i-icon).

I was working at UseGalaxy.eu. Is that also where you were working?

Let’s report the issue here. → Issues with RSEM reference index · Issue #1385 · usegalaxy-eu/infrastructure-playbook · GitHub

Thanks for reporting the problem!

[mathew_harvey]

Hi, Thanks for your reply. I am working in UseGalaxy.eu and thats the same problem i am having. Is there anything else you need me to do or that would help solve the issue?

[jennaj]

Hi @mathew_harvey Thanks for confirming the server! There isn’t anything to be done on your or my part besides reporting the issue. This tool is only hosted at the EU server (not the other UseGalaxy servers) and is a bit older. It might have larger technical issues – not sure – but the EU admins will know.

Meanwhile, I can let you know that there are alternative methods you could explore. Fully assembling the transcripts is not needed for expression analysis. Maybe consider following the analysis guides here to see if these will work for you?

• Transcriptomics / Tutorial List

This is the version you might be most interested in.

• Hands-on: Reference-based RNA-Seq data analysis / Reference-based RNA-Seq data analysis / Transcriptomics

But you could also look at this one, since it includes discovery?

• Hands-on: De novo transcriptome reconstruction with RNA-Seq / De novo transcriptome reconstruction with RNA-Seq / Transcriptomics

Then down in the advanced sections, you’ll find tutorial examples like this one. It was just updated and is another way to explore the kind of data you have. All of the reference data needed should be available for mm39.

• Hands-on: Genome-wide alternative splicing analysis / Genome-wide alternative splicing analysis / Transcriptomics

All of these include a workflow that you could use as a template to see how the results are reported. I have run those workflows on the example data recently, and everything worked as expected but we can work through any new troubles here at this forum. Maybe also review the other end-to-end tutorials in that group to learn about other ways to calculate and visualize DE results from RNA-seq data?

Hope this helps!