Feature count on exon level instead gene level

Hello dear Galaxy community,

I need your help!

I have analyzed RNA-seq data by mapping with RNA STAR and then performing featureCounts, followed by DESeq2 and other downstream analyses. Initially, I conducted the analysis at the gene level. However, my colleagues have now decided that they want to see the analysis at the exon level.

I adjusted the featureCounts settings by enabling the “on feature level” option. However, when I use gene_id as an identifier, I get repeated occurrences of the gene name corresponding to the number of exons, for example:

Gene A 0
Gene A 0
Gene A 1

When I use exon_id as an identifier, the results are even less useful. Unfortunately, I cannot use both the gene and exon IDs simultaneously in the “GFF Gene Identifier” field, as this results in an error.

How can I resolve this issue so that I obtain counts with both the gene ID and exon number?

I would be very grateful for any advice.

Welcome, @Sabsida

I wonder if DEXseq instead of DESeq2 is what you want?

Search the tool panel at the UseGalaxy.eu server to find it. Notice there are two modes with the upstream tool: one is a special way to generate a GTF with DEXSeq-Count that has all of the exon-level information encoded in a way these tools can understand. Run that first. Then use that GTF to actually generate the counts in the other mode. Then the main tool can generate the DE.

This tutorial might also interest you. → Hands-on: Genome-wide alternative splicing analysis / Genome-wide alternative splicing analysis / Transcriptomics

It doesn’t sound like your GTF has content issues but double checking can’t hurt. All of these tools are picky and in slightly different ways. Using the most basic standard formats for reference data is usually a good idea. This guide here has some tips. → https://training.galaxyproject.org/training-material/faqs/galaxy/analysis_differential_expression_help.html

Hope this helps and you can ask follow up questions. :slight_smile:

Ohh, thank you very much for the advice. It worked! :grinning:

1 Like

Great! Very glad it worked out :rocket: