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  • 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?

  • #2
    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; 02-21-2017, 12:41 AM.

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