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A flexible statistical framework is developed for the analysis of read counts from RNA-Seq gene expression studies. It provides the ability to analyse complex experiments involving multiple treatment conditions and blocking variables while still taking full account of biological variation. Biological variation between RNA samples is estimated separately from the technical variation associated with sequencing technologies. Novel empirical Bayes methods allow each gene to have its own specific variability, even when there are relatively few biological replicates from which to estimate such variability. The pipeline is implemented in the edgeR package of the Bioconductor project. A case study analysis of carcinoma data demonstrates the ability of generalized linear model methods (GLMs) to detect differential expression in a paired design, and even to detect tumour-specific expression changes. The case study demonstrates the need to allow for gene-specific variability, rather than assuming a common dispersion across genes or a fixed relationship between abundance and variability. Genewise dispersions de-prioritize genes with inconsistent results and allow the main analysis to focus on changes that are consistent between biological replicates. Parallel computational approaches are developed to make non-linear model fitting faster and more reliable, making the application of GLMs to genomic data more convenient and practical. Simulations demonstrate the ability of adjusted profile likelihood estimators to return accurate estimators of biological variability in complex situations. When variation is gene-specific, empirical Bayes estimators provide an advantageous compromise between the extremes of assuming common dispersion or separate genewise dispersion. The methods developed here can also be applied to count data arising from DNA-Seq applications, including ChIP-Seq for epigenetic marks and DNA methylation analyses.

Motivation: Statistical methods are used to test for the differential expression of genes in microarray experiments. The most widely used methods successfully test whether the true differential expression is different from zero, but give no assurance that the differences found are large enough to be biologically meaningful. Results: We present a method, t-tests relative to a threshold (TREAT), that allows researchers to test formally the hypothesis (with associated p-values) that the differential expression in a microarray experiment is greater than a given (biologically meaningful) threshold. We have evaluated the method using simulated data, a dataset from a quality control experiment for microarrays and data from a biological experiment investigating histone deacetylase inhibitors. When the magnitude of differential expression is taken into account, TREAT improves upon the false discovery rate of existing methods and identifies more biologically relevant genes. Availability: R code implementing our methods is contributed to the software package limma available at http://www.bioconductor.org. Contact: smyth@wehi.edu.au