Merged vs. Ensembl GTF for htseq-count

Question

[kousi31]

I have downloaded genome and gtf files from Ensembl. I have aligned my reads using hisat2. I am planning to get raw read counts using htseq count to use in deseq2. Many pipelines have used Ref GTF files for htseq count. I have a doubt as whether I should create a merged gtf file using stringtie or use ensembl gtf? Your suggestions will be helpful.

Answer 1

[i.sudbery]

If you are working in a well annotated species (which seems likely if you are getting the GTF from Ensembl), then using the Ensembl annotation is almost certainly sufficient. This is particularly true if you are going to use gene level counts via htseq count - at best StringTie will detect some new isoforms of existing genes (which may or may not be correct), but likely that will be new combinations of existing exons, and since htseq doesn't distinguish between isoforms anyway, the gene level count will be the same.

Answer 2

[Kristoffer Vitting-Seerup]

I've written a bit about such considerations here - hope this is helpfull.