featureCounts to analyse miR RNA-seq data



Hi there:

I am writing to ask your help in regard to use featureCounts to analyse miR RNA-seq data in https://usegalaxy.eu/. I have tried to use a gene annotation file mmu.gff3 that I previously downloaded from miRBase. However, I always get the same error:

Fatal error: Exit code 255 ()

I guess I must select some option from Advance options panel, but I can not find which one is. Would you be so kind to help me with this issue?

Thanks in advance


Hello, Featurecounts expects GTF as the input format and will fail with GFF3.

The tool Convert Formats >> gffread might be an option. The tool is deprecated at https://usegalaxy.org but still active at https://usegalaxy.eu. You’ll need to carefully inspect the output and possibly make other changes to get the data into a standardized GTF format that meets the other criteria (many advanced options are specifying what features to count on, gene/transcript attribute content/format – and your data needs to match up).

GTF formatting and related data manipulation help:

You also might want to consider using a public Galaxy server that is domain-specific for miRNA analysis. Please see this archived Galaxy Biostars post for details and links: https://biostar.usegalaxy.org/p/28003/index.html

Contacting the MiRBase authors is also an option – they could have alternative advice for obtaining the annotation data in GTF format. Please find the contact information at the bottom of this page: http://www.mirbase.org/ftp.shtml

All (known!) Public Galaxy servers: https://galaxyproject.org/use/

The EU team may post back with additional suggestions. Thanks!