Hi @Dongqi_Xie
It sounds like everything is set up well so far!
- Known: Gencode annotation will have CDS regions annotated.
- Discovery: Stringtie annotation will NOT have CDS regions annotated (does not predict coding region footprints)
These two are then compared to put all of the annotations together into a reconciled “master annotation” file. Then this is what is used for the differential exon expression analysis.
The exact step is what was discussed in the original topic.
Did you run your data through this way? Or am I misunderstanding where the problem is coming up?
If this was the step, and the annotation didn’t actually merge together for some reason, there is likely a mismatch somewhere between reference data assembly versions (the actual chromosome names and strings of bases). We can help to troubleshoot here if you would like to share back your history for review. (you can unshare after!). Screenshots may also work but you’ll need to capture a lot of details for the jobs, parameters, and the input data content.
Another example for getting Gencode data synched up was in this topic. The user decided to use the Gencode human assembly – both the fasta and the gtf – since this avoided issues that can come up between patch assembly alt/haplotype fragments that they cared about. UCSC and Gencode have slightly different versions – we have the UCSC version indexed on the server but you can use a custom genome from Gencode if you want to. The first files in this shared history are the data they selected and each have the original file names as hosted at Gencode → https://www.gencodegenes.org/human/.
These concerns about reference data would happen anywhere a large analysis like this would be done, especially when the comparisons are so detailed – differential exons or variants in particular. The tools are reviewing the reference very closely and will complain over even minor unexpected file format/content variations.
Ok, so that’s a lot of information! But the correction is probably small, we just need to review to learn where to make the adjustment. I’ll watch for your reply. 