The Empirical analysis of DGE tool implements the 'Exact Test' for two-group comparisons developed by Robinson and Smyth [Robinson and Smyth, 2008] and incorporated in the EdgeR Bioconductor package [Robinson et al., 2010]. The test is applicable to count data only, and is designed specifically to deal with situations in which many features are studied simultaneously (e.g. genes in a genome) but where only a few biological replicates are available for each of the experimental groups studied. This is typically the case for RNA-seq expression analysis.
Empirical analysis of DGE
The test uses the raw counts, and implicitly carries out normalization and transformation of these counts (see below for details). It is based on the assumption that the count data follows a Negative Binomial distribution, which in contrast to the Poisson distribution has the characteristic that it allows for a non-constant mean-variance relationship. The test is also appropriate for larger numbers of samples.
The 'Exact Test' of Robinson and Smyth is similar to Fisher's Exact Test, but also accounts for overdispersion caused by biological variability. Whereas Fisher's Exact Test compares the counts in one sample against those of another, the 'Exact Test' compares the counts in one set of count samples against those in another set of count samples. This is achieved by replacing the Hypergeometric distributions of Fisher's Exact Test by Negative binomial distributions, whereby the variability within each of the two groups of samples compared is taken into account. This only works if the dispersions in the two groups compared are identical. As this cannot generally be assumed to be the case for the original (nor for the normalized) data, pseudodata for which the dispersion is identical is generated from the original data, and the test is carried out on this pseudodata. The generation of the pseudodata is performed simultaneously with the estimation of the dispersion, in an iterative procedure called quantile-adjusted conditional maximum likelihood. Either a single common dispersion for all features may be assumed (as in [Robinson and Smyth, 2008]), or it may be assumed that the dispersion for each feature (e.g. gene) is a 'weighted average' of the common dispersion and feature (e.g. gene) specific dispersions (as suggested in [Robinson and Smyth, 2007]). The weight given to each of the components depends on the number of samples in the groups: the more samples there are in the groups, the higher the weight will be given to the gene-specific component.
The Exact Test in the EdgeR Bioconductor package provides the user with the option to set a large number of parameters. The implementation of the 'Empirical analysis of DGE' algorithm in the Genomics Workbench uses for the most parts the default settings in the edgeR package, version 3.4.0. A detailed outline of the parameter settings is given in Empirical analysis of DGE parameters).