Hi, I've time course RNA-seq data that i would like to analyze using edgeR : Q1) shall i normalize using calcNormFactors() or glmFit() and glmLRT() do this normalization ,therefore no need to use calcNormFactors()?? Q2) when created design matrix using model.matrix(~mydata$samples$group) i got a matrix in which the control has been removed ,i then calculates dispersion using: estimateGLMCommonDips(mydata,design matrix) followed by testing using glmFit(),glmLRT() Does the matrix created in a correct way? Q3) In glmLRT() how can i determine the coefficient to be used if it is my first time to analyze RNA-seq data?? Q4) After using glmFit(),glmLRT() followed by topTags() ,i got FDR =1 for all genes,Any idea why FDR=1?? Thank you in advance. Best Regards, Rabe [[alternative HTML version deleted]]

---------- Forwarded message ---------- From: Asma rabe <asma.rabe@gmail.com> Date: Wed, Oct 19, 2011 at 3:55 PM Subject: edgeR questions To: Bioconductor@r-project.org Hi, I've time course RNA-seq data that i would like to analyze using edgeR : Q1) shall i normalize using calcNormFactors() or glmFit() and glmLRT() do this normalization ,therefore no need to use calcNormFactors()?? Q2) when created design matrix using model.matrix(~mydata$samples$group) i got a matrix in which the control has been removed ,i then calculates dispersion using: estimateGLMCommonDips(mydata,design matrix) followed by testing using glmFit(),glmLRT() Does the matrix created in a correct way? Q3) In glmLRT() how can i determine the coefficient to be used if it is my first time to analyze RNA-seq data?? Q4) After using glmFit(),glmLRT() followed by topTags() ,i got FDR =1 for all genes,Any idea why FDR=1?? Thank you in advance. Best Regards, Rabe [[alternative HTML version deleted]]