We model read counts Kij as following a negative binomial distribution (sometimes also called a gamma-Poisson distribution) with mean μij and dispersion αi. The mean is taken as a quantity qij, proportional to the concentration of cDNA fragments from the gene in the sample, scaled by a normalization factor sij, i.e., μij=sijqij. For many applications, the same constant sj can be used for all genes in a sample, which then accounts for differences in sequencing depth between samples. To estimate these size factors, the DESeq2 package offers the median-of-ratios method already used in DESeq [4]. However, it can be advantageous to calculate gene-specific normalization factors sij to account for further sources of technical biases such as differing dependence on GC content, gene length or the like, using published methods [13,14], and these can be supplied instead.
