Unexpected failure when I perform cufflinks: Deprecated tool. Solution: Use current Differential Expression Tools, Workflows, Tutorials, FAQs

I was running Cufflinks with my BAM files. I have done it with success for the first time. However, when I try to do the second file with the same setting it comes out a wronging alert:
“Remote job server indicated a problem running or monitoring this job.”
I have searched the similar error in Galaxy Help. If I understood it right, the file formats and size or the server problems may cause this error. The second time I apply a 6Gb BAM file, which is slightly larger than my first time BAM file. Is there anything I should notice but I missed or it could be the server error?

Thanks for your reading! It would be nice to have your response!

Szu Shuo

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Hi @userlee7655

All of the older Tuxedo tools are in a deprecated state but some still work. This includes Tophat/Tophat2, all Cuff* tools, and cummeRbund. Tophat for Illumia, in particular, should definitely be avoided. Cuffdiff does have a known issue with the SQLlite output option (the input for cummeRbund) and that will not be fixed. There are probably other issues with that tool and/or other deprecated tools.

We strongly recommend updating to use current/supported (eg: maintained) differential expression tools/methods. Tutorial links below, plus an FAQ that covers the various usage issues that can come up and be addressed – many will apply to the tools/methods you are using now – as well as other tools/workflows. It is a list of summarized help for the most common usage errors.

That said, you could try at least one more rerun. Sometimes the particular error you got comes up due to transient cluster issues. Even if you reran already, try one more time unless you discover that there was an input problem first.

If it fails again, then check your inputs more carefully. If those are correct, you probably will need to move to use the current tools/methods for scientifically correct results, even if a deprecated tool does not actually error. We will not be addressing fixes for any deprecated tools and can offer only limited support if you decide to use them anyway for some reason.

The FAQ that includes both DE troubleshooting tips plus the current DE tutorial links: https://galaxyproject.org/support >> FAQ >> Extended Help for Differential Expression Analysis Tools

Note: If you are using HISAT2 for mapping or decide to use it going forward, a specific setting to format the output for Cufflinks is required. The same BAM result cannot be used with both Cufflinks/Cuffdiff and Stringtie. Find the setting on the tool form under Advanced Options > Spliced alignment options > Transcriptome assembly reporting. Again, it is strongly recommended to NOT use Tophat or Tophat2 – both can produce unreliable scientific results or metadata problems that can be difficult to detect, even if the mapping job did not actually error for technical reasons. For metadata problems, sometimes you’ll need to expand the mapping dataset to see the warning in a green, putatively “successful” job. A link is given to “redetect” the metadata. Sometimes it works, sometimes not. And even if it works, there could still be content issues.

More troubleshooting help, summarized with more tips/links for common usage issues and solutions:

Hope this advice helps!

Hi Jennaj,
thanks so much for your reply.
I am a total rooky in sequencing result analysis.
I will follow your suggestion to use other tools to perform my analysis.
Thank you again, I am pretty grateful.

Szu Shuo

1 Like