I am ready to order mRNA sequencing on case and control rats for an experiment I am conducting measuring transcription levels of genes in rats. I'd like to identify differentially expressed (DE) transcripts and correlate mRNA transcript expression level with micro-RNA expression levels obtained in a previous experiment. I need to be able to detect differences in expression of about ~1.5x reliably from brain tissue (amygdala, etc.) I also need to be able to detect splice variants, which I think means I need longer reads and possibly paired end reads.
Like many researchers, I am limited by cost. In my preliminary studies, I think I have identified three key variables that I think will increase cost efficacy (and therefore statistical power) of my study, they are:
1) I can increase sample size at the cost of trading-off read depth
2) I can increase read length to get better mapping, but it will cost more
3) I can opt for paired end reads (PE), which will cost more than SE but will map better and might help more with detecting splice variants.
Reading through the literature, I think what I have figured out is that it makes a lot more sense to do a higher number of cases and controls at lower depth for 1).
However, for 2) and 3), I cannot assess the degree of the trade off between single vs paired end read, or optimal read length that gives me combination of (mean mapping efficiency)*(cost efficiency per read) = highest total mapped reads per unit price. A complicating factor I don't understand well is how good the quality of the rat reference genome is, and how that will influence my mapping efficiency with respect to optimizing read length and SE vs PE.
Summary - If in the end I am interested in Differential Expression analysis primarily, but also want to be able to learn about splice variants, then what is the best combination of read depth, read length, and single vs paired ends? Therefore, ultimately my goal is to optimize statistical power to detect association of DE genes - what is the optimal experimental design?
I recognize there may be no "right" answer to this, but answers from your experience would be helpful to me. Thank you very much in advance.