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  • Ref column for 2 factor RNA-Seq time course

    Dear all,

    I seek some help with normalization strategy for my experiment. I have described the experimental design below.

    Tissue gathered one wild type and 8 mutants - so genotype may be considered 'factor #1'.
    For each of these genotypes, 4 time points have been used for library generation - so time may be considered 'factor #2'

    The reason why I do not clump libraries across ALL genotypes is because I think it is like comparing apples to oranges to bananas to peaches....
    Since the statistical validity of RNA-Seq comparisons allows for only a small fraction of DE genes in a background of largely unchanged gene expression, I think that comparing different mutants and wild type will violate this assumption. Do you agree? Or do you think I'd have to empirically prove this theoretical prediction before I conclude its a 2-factor experiment?

    Now, IF you agree that the experiment is indeed a 2-factor one, then how do I go about

    1. choosing the reference column / library for TMM normalization - should this TMM normalization be performed for one genotype at a time? I am leaning towards TMM normalization for each genotype separately.

    2. since each library is quadruplicated, does edgeR allow independently replicated reference libraries to be ALL used for normalization against, or can I use just one of the 4 reference libraries at any one time (which would defeat the purpose of replication)?

    For the purpose of creating a ref library, I a am thinking of making an RLE-based (geometric mean) pseudo-library from the 4 lib reps for the reference 'conditions'.
    However, before calculating the pseudo-ref library, for the 4 ref libs I am first considering removing genes / rows where any expression falls beyond +/- 3 SDs. Your opinions on this?

    To provide a context for my questions, the final goals of my research are to:
    a. cluster and identify co-expressed genes within a genotype, and
    b. identify genes with variant expression patterns across genotypes
    Last edited by anandksrao; 03-25-2012, 10:10 PM. Reason: clarity

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