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 06-26-2013, 05:11 PM #1 alittleboy Member   Location: USA Join Date: Apr 2011 Posts: 60 DEXSeq for multi-factor design I am using DEXSeq for testing differential exon usage between two conditions: control and treatment. For each condition, I have 8 biological replicates (C1-C8, and T1-T8). The design is listed below. condition subject C1 control 1 C2 control 2 C3 control 3 C4 control 4 C5 control 5 C6 control 6 C7 control 7 C8 control 8 T1 treatment 1 T2 treatment 2 T3 treatment 3 T4 treatment 4 T5 treatment 5 T6 treatment 6 T7 treatment 7 T8 treatment 8 As you can see from the last column, we have 8 subjects involved in the experiment. Subject 1 has both the control and the treatment, and so on for all the other subjects. This is different from the situation discussed in the DEXSeq vignette here, for example: design(pasillaExons) gives: condition type treated1fb treated single-read treated2fb treated paired-end treated3fb treated paired-end untreated1fb untreated single-read untreated2fb untreated single-read untreated3fb untreated paired-end untreated4fb untreated paired-end I think in the pasilla example, the biological replicates are all different. Thus in my situation, in order to see if there is differential exon usage between the treatment and control, can I do: (1) ignore the fact that each subject had both control and treatment? In this case, in my implementation, shall I write: f_dispersion = count ~ sample + condition * exon pExons = estimateDispersions(pExons, formula=f_dispersion) pExons = fitDispersionFunction(pExons) Null model: f_0 = count ~ sample + condition Alternative model: f_1 = count ~ sample + condition * I(exon == exonID) pExons = testForDEU(pExons, formula0 = f_0, formula1 = f_1) (2) incorporate the subject as a corvariate (coded that column as a factor), and then analyze in the GLM framework? In this case, in my implementation, shall I write: f_dispersion = count ~ sample + (condition + subject) * exon Null model: f_0 = count ~ sample + subject * exon + condition Alternative model: f_1 = count ~ sample + subject * exon + condition * I(exon == exonID) (3) I am not sure if including subject as a corvariate is the best approach in my situation. Are there any other options that I can consider? (4) I write the formula for null and alternative models exactly according to the vignette, but I am not sure if they are what I should put in R implementation. Thank you so much ;-) Last edited by alittleboy; 06-26-2013 at 05:16 PM.