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Old 02-18-2017, 08:07 AM   #1
achamess
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Location: North Carolina, USA

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Default How to use HTSeq to count RNA-Seq overlap w/ whole genes (intron+exon) vs. exon only

So I recently performed RNA-seq using the SmartSeq2 on RNA from neuronal nuclei.


My sample was a pool of sorted neuronal nuclei from mouse. Uing STAR to align, I found that about 20% is exonic, and 60-70% is intronic. Even though I polyA selected, there is still a ton of retained intron, which is consistent with a recent paper that did single nucleus RNASeq in the human brain https://www.ncbi.nlm.nih.gov/pubmed/27339989

But the question is, what to do with the intronic reads?

One of my goals is differential gene expression. I want to take my RNASeq reads and count the overlap with the whole gene (intron + exon), rather than just exons.

How would I do this? What annotation file should I use for HTSeq? I have a GTF from UCSC, but I see exons, CDS but not the whole gene. How would I go about doing this? Do I need to make a custom GTF using the transcription start and stop sites?
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Old 02-20-2017, 11:36 PM   #2
Michael.Ante
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Default

I don't know if it's a good idea to do it this way, since you'll get a higher amount of overlapping features. I hope you have a stranded library.

Nevertheless, in order to get what you described you'll need a GTF which has also "transcript" in the third field ( head -n 1000 my.gtf | cut -f 3 | sort -u ; should also report transcript). ENSEMBL's gtf has it for instance.
With such a gtf, you can use this feature type instead of exons for counting:

Code:
htseq-count -t transcript my_alignment.sam my.gtf
Cheers,

Michael

Last edited by Michael.Ante; 02-20-2017 at 11:41 PM.
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