Ht-seq count stranded=Yes question

Question

[Krish Srinivasan]

Hello Everyone,

I just had a questions about the stranded option of HT-seq Count. I have a first stranded library synthesized using dUTP (therefore stranded=reverse) what would happen if I had put stranded = yes? I still get counts, however they are very few in number? Is using stranded=yes in this case just throwing away data evenly, or is it introducing some sort of bias?

Thanks

Answer 1

[WouterDeCoster]

You will throw away all reads which are in the incorrect direction, so only reads which are antisense to your actual annotation will be counted if you select the wrong strandedness option. I'm not sure why you would want to do that.

[Devon Ryan]

I've had cases of people interested in anti-sense transcription, in which case using the opposite setting made sense. Aside from that though...

[Krish Srinivasan]

Thank you for the response! I just wanted to clarify how HT-seq count is counting aligned reads. I am just not understanding how the reads are being counted and the directional aspect of the reads. I'm asking because I made the mistake of using the default HT-seq count option which is stranded = yes