am trying to run a regression analysis on RNA-seq expected counts generated using RSEM. To perform the analysis I performed TMM normalization and extracted CPM values. I then used voomWithQualityWeights, lmFit and eBayes to perform association analysis. Since RNA has a different data distribution than the ELISA values, I would like to know if the steps that I followed are correct. I appreciate all your help and suggestions.
Following is my code:
d <- DGEList(counts=data, group=groups) TMM <- calcNormFactors(d, method="TMM") logCPM <- cpm(TMM, log=TRUE) design <- model.matrix(~LN_AB, data=Pheno) voom <- voomWithQualityWeights(data, design=design, lib.size=NULL, normalize.method="none") fit1 <- lmFit(normalizeddata,design) fit2 <- eBayes(fit1) newFrame <- topTableF(fit2)