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  • Experimental Design Violation?

    Hello,
    I am hoping to get some help with the experimental design of my RNAseq project. We have 4 populations and 2 treatments, which in the end results in 24 individuals per treatment per population - this is set in stone and cannot be changed. We would like to sample over a time course, but with the limited number of individuals and minimum number of replicates required to detected differential expression (8 individuals will be sampled of the 24 in order to get at least 6 individuals sequenced) this results in only 3 time points before we run into the issue of repeat sampling of individuals.

    My supervisors would like at least 5 time points sampled, but this would result in the duplicate sampling of 16 individuals (as all 24 individuals were sampled in the first 3 timepoints). My question/concern is whether repeating the sampling of only some of the individuals and not all will skew the results as its no longer balanced. But perhaps I'm thinking of this more on an individual basis and not a population basis (new to RNA, previously worked with DNA). Has anyone seen this design before? I do not want to lower the number of individuals sampled per time point in order to get more time points sampled, so trying to see if this duplicate sampling is too flawed to proceed.
    Thank you.

  • #2
    Hello,
    I don't understand why you don't sequence all of your individuals at every time point?
    This would give you the best time course experimental design, because you'll have all your replicates at all time points. I would not go for a down-sampling at each time point and especially not by choosing different individuals, because you won't be able to follow the inter-individual variability. If for cost / experimental reasons you need to lower your number of sequenced individuals, just choose like 8 per experimental condition and sample only those for all desired time points. This way, you can have reliable data and use as many time points as you (or your supervisor like!
    Good luck with your design and experiments!

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