Hi,
I'm fairly new to RNA-Seq data analysis and I'm a bit confused about something and I'd like to get your views on it. I have run an RNA-Seq analysis for a set of samples that i have been told they were prepared as second strand. So I've run tophat2 (didn't include strandness there, left if as default), then I counted reads with HTSeq (using -s reverse), and done DESeq and DEXSeq. I was happy with my data, since I could detect our positive control splicing events, until I had to run another data set, and something went wrong.
I've contacted the people that had prepared the libraries and run the sequencing for us and it turns out that the first data set had to be run using fr-firststrand.
Now I'm very confused, why does my analysis show differential splicing in positive control splicing events, if it seems that i have run the wrong commands? is the fact that I run the mapping as default the explanation? but then how about counting with HTSeq? surely that should be counting the wrong strand and throwing away a lot of useful data?
I would appreciate if anyone could help, i'm extremely confused here.
Thank you very much for your time and help,
Miriam
I'm fairly new to RNA-Seq data analysis and I'm a bit confused about something and I'd like to get your views on it. I have run an RNA-Seq analysis for a set of samples that i have been told they were prepared as second strand. So I've run tophat2 (didn't include strandness there, left if as default), then I counted reads with HTSeq (using -s reverse), and done DESeq and DEXSeq. I was happy with my data, since I could detect our positive control splicing events, until I had to run another data set, and something went wrong.
I've contacted the people that had prepared the libraries and run the sequencing for us and it turns out that the first data set had to be run using fr-firststrand.
Now I'm very confused, why does my analysis show differential splicing in positive control splicing events, if it seems that i have run the wrong commands? is the fact that I run the mapping as default the explanation? but then how about counting with HTSeq? surely that should be counting the wrong strand and throwing away a lot of useful data?
I would appreciate if anyone could help, i'm extremely confused here.
Thank you very much for your time and help,
Miriam