how can i quantify expression using own my gff ?

Question

[dlyedms1019]

Hi,

I want to quantify the leads in my defined region.

This is not a gene, but an enhancer region. (from ENCODE)

The data is a bam file that maps total RNA-seq using tophat.

I want to measure the expression of a region that is thought to be an enhancer RNA that is not defined in human gtf.

So I wondered if I could use the gffs for the regions I want to use for quantification.

But, I am not sure which tool to use and how.

Thanks you.

Answer 1

[i.sudbery]

Both featureCounts and htseq count will take any GTF file to get regions to quantify over. I recommend featureCounts - its faster and has more features. You just need to make sure you tell featureCounts which feautre type to quantify over (depends what feature type your enhancers are enocded as) and tell it to aggregate at the feature level, not the gene or transcript level.

[dlyedms1019]

Thanks you for your comments! I made a SAF(simple annotation format) for featureCounts and quantified eRNA and mRNA all.

Answer 2

[wuyc]

Just check this pipeline for enhancer RNA quantification from RNA-seq,

http://fun-science.club/PET/

You can also check the paper: Wu, Y, Yang, Y, Gu, H, et al. Multi‐omics analysis reveals the functional transcription and potential translation of enhancers. Int. J. Cancer. 2020; 1– 15. https://doi.org/10.1002/ijc.33132