Hello everyone,
I am analyzing RNA-seq data from a non-model halophyte plant. Because no reference genome is available, I aligned the reads to an available genome from a closely related species in the same genus.
Workflow:
Aligned raw RNA-seq reads to the available reference genome using HISAT2
Mapping completed successfully and produced a BAM file
BAM was sorted and indexed using samtools
Ran StringTie using the provided GFF3 annotation
Issue: StringTie fails with: Fatal error: Exit code 1 (), then I tried using AGAT’s “fixing” option on the GFF3, but it did not resolve the issue.
What could be causing this error? Is there a reliable way to fix it within Galaxy (e.g., using seqID renaming tools like AGAT or gffread), or is re-aligning to a genome that exactly matches the annotation the better approach? For non-model plants, is it generally advisable to run StringTie without a reference annotation?
Thank you for any advice.
•
written
by
Chamara Lakshitha •
Hi Juke, thank you! I followed your advice and used the dos2unix tool directly on the Galaxy platform. It fixed the formatting issues I was having, and now StringTie is running perfectly without errors. Really appreciate the help!"
Log in to answer this question.
Check/validate the GFF3 file with https://github.com/gpertea/gffread or a similar tool.
Hi Arup, thank you for the link! I actually used the built-in GFF-to-GTF conversion tools available in Galaxy for my current files. I don't have a native Linux machine, but I will try your suggestion using WSL on Windows, as this looks very helpful for fixing the underlying formatting issues I've been hitting. Thanks for the help!
This appears to be related to running the analysis on
galaxy. It may be best to post this on their help forum since they can access your data on backend : https://help.galaxyproject.org/Hi GenoMax, thank you! I actually submitted a bug report as well, and I've now managed to get it fixed. Cleaning the file formatting resolved the issue, and it is all working well now. Thanks for the guidance!