Applying the Classic Test dN/dS to Detect Selection in Archaea

Compared with the long history of research on Bacteria and Eukaryotes, Archaea are a young domain of study for life sciences. Before sequencing technologies, the scientific community primarily understood Archaea to be limited to extreme environments. However, it is now recognized that Archaea occupy a broad diversity of environments, including soil and marine habitats, where they play essential geochemical roles, as well as being animal and plant symbionts. Due to their late recognition as a new domain, the study of archaeal evolution has been delayed relative to Bacteria. Recent progress in high-throughput sequencing technologies has improved the accessibility of genomic data to broader phylogenetic breadths, including uncultivated clades. Here, we provide a step-by-step computational pipeline to estimate the ratio of nonsynonymous to synonymous substitution rates (dN/dS), a measure of the strength and direction of selection on protein-coding genes. We focus our chapter on the estimation of dN/dS to disentangle the evolutionary forces acting on the core genome of archaeal genomes, but this workflow is also applicable to Bacteria. Importantly, we present an overview of the biases to consider when estimating dN/dS, as well as potential strategies to circumvent technical limitations. We describe a case example of the use of dN/dS to explore differences in the evolutionary constraints acting on different archaeal species belonging to the genus Methanosarcina. We provide custom Python scripts, which are freely available and applicable to a broad diversity of questions on the evolution of Archaea.