Dear Bioconductors, I would like to ask for some advice/suggestions on the set-up of DEXSeq with multiple condictions. At the moment, I am using DEXSeq in a "vanilla" fashion: - 2 conditions, knockdown and control - 2 biological replicates per condition - output exons that change upon knockdown. So far this is working fine. But I also have another experimental variable: sub-cellular fractions (total vs fraction). The goal is obtain exons whose expression is changed in the knockdown but only in the fraction, that is a combined effect of knockdown and sub-cellular localization. Following the vignette, I was thinking of an experimental design like this: condition type sample1_a control total sample1_b control total sample2_a knockdown total sample2_b knockdown total sample3_a control fraction sample3_b control fraction sample4_a knockdown fraction sample4_b knockdown fraction and the code would be: formuladispersion <- count ~ sample + ( condition + type ) * exon ecs <- estimateDispersions( ecs, formula = formuladispersion ) ecs <- fitDispersionFunction(ecs) formula0 <- count ~ sample + type * exon + condition formula1 <- count ~ sample + type * exon + condition * I(exon == exonID) ecs <- testForDEU( ecs, formula0=formula0, formula1=formula1 ) res2 <- DEUresultTable( ecs ) would this work and is this design correct? Thank you, António -- --------------------------------------------------------------------- António Miguel de Jesus Domingues, PhD Neugebauer group Max Planck Institute of Molecular Cell Biology and Genetics Pfotenhauerstrasse 108 01307 Dresden Germany http://people.mpi-cbg.de/domingue/home.html e-mail: domingue@mpi-cbg.de tel. +49 351 210 2481 The Unbearable Lightness of Molecular Biology [[alternative HTML version deleted]]