Cuffdiff error while running RNA seq

Question: Cuffdiff error while running RNA seq

9 months ago by

gozrom • 0

gozrom • 0 wrote:

Hi, there,

I've aligned my reads with HISAT2, then did cufflinks on the aligned read and cuffmerge, the next step of cuffdiff however failed, I tried running it twice and got the same error:

R is free software and comes with ABSOLUTELY NO WARRANTY. You are welcome to redistribute it under

Not sure whether I am doing something wrong or it is an actual error.

Any help is appreciated,

thank you.

error biomart rna-seq cuffdiff • 648 views

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modified 9 months ago by Jennifer Hillman Jackson ♦ 25k • written 9 months ago by gozrom • 0

Side question: If I want to use TPM metrics instead of FPKM what should I use for that in Galaxy.

ADD REPLY • link written 9 months ago by gozrom • 0

9 months ago by

Jennifer Hillman Jackson ♦ 25k

United States

Jennifer Hillman Jackson ♦ 25k wrote:

Hello,

Did you set the alignment reporting option on the HISAT2 tool form for Cufflinks? This is how: https://galaxyproject.org/tutorials/nt_rnaseq/#spliced-mapping-with-hisat

If yes, and there is still an error:

If working at https://usegalaxy.org and this error occurred last week - rerun the job - many server updates were in progress and tools corrected.
Review common troubleshooting FAQs to make sure the usage is correct: https://galaxyproject.org/support/#getting-inputs-right
Post the full error back here as a comment for community review (click on the bug icon to see the full message). Please note which version of the tool you are using and where you are working.
- The tool version is included at the top of the tool form (end of the name) or "View details" report (click on the "i" icon for the error dataset)
- A public Galaxy server? What is the URL?
- Or your own local/docker/cloud Galaxy? Which version of Galaxy is being used?
After reviewing common solutions in the FAQs, send in a bug report if working at https://usegalaxy.org or can reproduce the issue at this server: https://galaxyproject.org/support/#unexpected-results

TPM metrics are generated by both Salmon and Sailfish. This tutorial covers usage: https://galaxyproject.org/tutorials/rb_rnaseq/

Galaxy tutorials: https://galaxyproject.org/learn/

Support FAQs: https://galaxyproject.org/support/

Thanks! Jen, Galaxy team

ADD COMMENT • link written 9 months ago by Jennifer Hillman Jackson ♦ 25k

Thanks, about the HISAT2 specific for Cufflinks - no, I used default settings, I'll rerun with specific to cufflinks. I'm new to Galaxy, so thank you for the tutorials, so far I used some of it and in some cases just got youtube videos. I used web GUI public server usegalaxy.org Since I never used Salmon I would like to review the tutorial first and then come back to you If I have any trouble using it.

Additionally, I tried to import GRCm38.p5 from Biomart ensemble mouse gene database and got an error, too: The uploaded file contains inappropriate HTML content. The process seems simple, just send the DB to Galaxy button, but maybe I'm doing something wrong? Any suggestion? Thanks.

ADD REPLY • link written 9 months ago by gozrom • 0

Trying to use Salmon, there is no tutorial in the link that you have sent, just an explanation of the principle. Any tutorial on salmon?

ADD REPLY • link written 9 months ago by gozrom • 0

Sorry, some of these tutorials overlap and are edited for content. This one from the GTN (also linked from the primary tutorials hub page) covers usage: https://galaxyproject.github.io/training-material/topics/transcriptomics/tutorials/srna/tutorial.html

ADD REPLY • link written 9 months ago by Jennifer Hillman Jackson ♦ 25k

When Biomart returns this error it indicates that the data selected for output is formatted for HTML. This is usually a description field of some type. Omitting these resolves the problem.

The entire genome would be too large to send back to Galaxy (if that is what you are doing) - and the output from Biomart is probably returning a warning that the data is too large to transfer (in HTML -- or what appears to be HTML). Without reproducing your query I cannot be 100% certain. But if you are trying to extract a large fasta file, I would suggest downloading it locally first, review to make sure it is intact, then upload using FTP to Galaxy.

If that doesn't work, you can post back the Biomart query for review. The server at https://usegalaxy.org is undergoing updates and some functions are still problematic. If this is one of them, and the file is actually valid but failing the transfer, it would be new issue and we'll want to clarify and fix it. Screenshot of the steps (there are usually a few) would be one way to share your query exactly. Graphics must be placed in a public location then linked to this site. I use https://imgur.com/ but Dropbox, Google drive, etc are also alternatives. Just make sure the permissions are set so that others can view publically.

ADD REPLY • link modified 9 months ago • written 9 months ago by Jennifer Hillman Jackson ♦ 25k

I was trying to download mouse transcriptome file in the FASTA format from Ensemble,

If you have a different solution that works for that I would use it instead, thanks.

ADD REPLY • link modified 9 months ago • written 9 months ago by gozrom • 0

I ran some tests and did a search to see if this was recently reported by the development community.

The Get Data > Biomart tool itself is problematic. I was able to download some data locally and upload it to Galaxy (a tabular dataset). Local file download for fasta failed (directly from Biomart, did not involve Galaxy). I am testing the compressed URL method. More details and the test history I am using: https://github.com/galaxyproject/galaxy/issues/5302

Please follow the ticket for updates. Thanks for following up!

ADD REPLY • link written 9 months ago by Jennifer Hillman Jackson ♦ 25k

Thank you I'll follow the ticket.

Rerun the cuffdiff again after cufflinks on hisat2 specific for cufflinks same error as before -

R is free software and comes with ABSOLUTELY NO WARRANTY. You are welcome to redistribute it under

ADD REPLY • link written 9 months ago by gozrom • 0

Hello, Some of the HISAT2 runs did not include the Cufflinks alignment reporting. Example: data collection 129. Please review all HISAT2 inputs and rerun any jobs that did not have this parameter set, then rerun the downstream tools that use the output. If you get this error again, the same problem exists in at least one of the input BAMs from HISAT2.

Tip: To review the error for a dataset, click on the bug icon. You do not have to submit this if you can figure out the problem. However, if you do need help, the error can be sent in that way for feedback. https://galaxyproject.org/issues/#usage-problem-reporting

ADD REPLY • link modified 9 months ago • written 9 months ago by Jennifer Hillman Jackson ♦ 25k

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