Fig. 1: Design of genomic analyses to detect potential signals of adaptation.

A S. aureus colonies cultured from swabs taken from typical carriage sites of the same individual. (the human body silhouette icon was released into the public domain and obtained from https://commons.wikimedia.org/wiki/File:Human_body_silhouette.svg; the culture plate was created manually; and the inoculation loop obtained from https://bioicons.com/ under license CC0 1.0). B Multiple isolates are whole genome sequenced from the same individual (the sequencing machine icon was created manually). C A core-genome phylogeny is used to ensure isolates from the same host are clonal and to identify an appropriate outgroup. Isolate short reads are mapped to the outgroup assembly to call genetic variants. The sequence of the most recent common ancestor (MRCA) of all isolates from the same host is reconstructed. D The short reads of each isolate are mapped to the MRCA reconstructed sequence to call variants wherein the reference allele represents the ancestral allele and the alternative allele the evolved one. The coordinates of variants in a complete and well-annotated reference genome (Reference lift-over) are determined. Variants on repetitive, low-complexity and phage regions are removed as well as those attributable to recombination (Variant filtering). In the last step, the effect of variants on genes is annotated (Variant annotation). E The number of protein-altering mutations are counted on protein-coding genes (CDS), transcriptional units (operons) and high-level functional units across all individuals. F Each functional unit is tested for an enrichment of protein-altering mutations compared to the rest of the genome. G The mutant isolate (with a putative adaptive mutation) and a closely related wildtype isolate obtained from the same individual are tested in vitro for antibiotic susceptibility (AST), delta-haemolytic activity, and growth under a variety of nitrogen sources to validate the phenotypic effect of putative adaptive mutations (the petri dish and multi-well plate icons by Servier https://smart.servier.com/ were obtained from https://bioicons.com/ under license CC-BY 3.0; the δ-haemolysis plate and the growth curves were obtained from this work).