How can I differentiate exogenous from endogenous transcripts in RNAseq data?

Question

[dsytan]

We have RNA-seq data from different time points in a reprogramming experiment. The reprogramming was done by retroviral overexpression of the TFs. How can we distinguish the endogenous and exogenous expression of this TF?

[ATpoint]

I guess your best chance is to perform differential exon usage analysis focusing on the UTR regions which should be present in the endogenous but not exogenous transcripts.

I would go with: https://f1000research.com/articles/7-952 and probably also worth reading (but covered in this workflow) is https://bioconductor.org/packages/devel/bioc/vignettes/DEXSeq/inst/doc/DEXSeq.html

Just to add: What Ian suggests below with "creating a transcript with and without UTR" refers to the transcriptome that you use when quantifying reads with salmon. You would need to create an additional transcript (the one you overexpress) without the UTRs and add this to the reference transcriptome, and then use salmon to quantify your reads against that reference.

[i.sudbery]

We've also found that creating a transcript with and without the UTR and quantifying with Salmon works pretty well.

[ksong]

Dear friends dsytan, ATpoint, and i.sudbery,

We want to do the same thing but found a problem. Please see this conversation. https://support.bioconductor.org/p/9143106/

Basically the exogenous expression of this gene is not zero when cells have high endogenous expression. Do you have time to review the method we use? Is it possible to share with us some successful examples? we can also share our own data if think that can work better? Michael has already replied us on the bioconductor forum. We are trying his suggestion. If you have other suggestions please let us know. I really appreciate your help. Kai