which feature type and id attribute to use in ht-seq count

Question

[kousi31]

Hi all I want to use ht-seq count for counting reads. I am planning to do both read counts for exons and gene, and compare them later. If I have understood right, we calculate exon counts to find differential expression between alternatively spliced transcripts which may not be visible in gene counts. If so, which feature to use ?

Mygff file is like this

• gene 3503760 3523716 . - . ID=gene7;
• mRNA 3503760 3523716 . - . ID=rna12;Parent=gene7;
• exon 3515344 3515454 . - . ID=id60;Parent=rna12;
• CDS ID=cds9;Parent=rna12
• CDS ID=cds9;Parent=rna12
• CDS ID=cds9;Parent=rna12
• mRNA 3503760 3523659 . - . ID=rna13;Parent=gene7;
• exon 3523589 3523659 . - . ID=id63;Parent=rna13;
• exon

If the 'mRNAs' represents alternatively spliced transcripts of gene7, then in ht-seq count,

1. is it ok use --type mRNA and --i ID or should I use --type exon and -i ID ? and why?

2. to calculate the raw counts for genes, is the option --type gene and -i ID are correct?

Thanks in advance for your suggestions.

[Buffo]

mRNA will count annotated isoforms, to look for new isoforms you can use StringTie, and by default htseq-count count genes, so yes, option 2 is fine.

[kousi31]

According to the manual - https://htseq.readthedocs.io/en/release_0.11.1/count.html, by default it counts exon when aGTF file is provided.

(-t <feature type="">, --type=<feature type="">

feature type (3rd column in GFF file) to be used, all features of other type are ignored (default, suitable for RNA-Seq analysis using an Ensembl GTF file: exon))

However, I want to understand the significance of using --type exon over --type mRNA.

[Buffo]

To count genes (complete, all annotated exons):

--type exon
  

To count mRNAs (if a gen has to isoforms it will count two mRNAs):

--type mRNA