Average Nucleotide Identity based Staphylococcus aureus strain grouping allows identification of strain-specific genes in the pangenome

Staphylococcus aureus causes both hospital and community acquired infections in humans worldwide. Due to the high incidence of infection S. aureus is also one of the most sampled and sequenced pathogens today, providing an outstanding resource to understand variation at the bacterial subspecies level. We processed and downsampled 83,383 public S. aureus Illumina whole genome shotgun sequences and 1,263 complete genomes to produce 7,954 representative substrains. Pairwise comparison of core gene Average Nucleotide Identity (ANI) revealed a natural boundary of 99.5% that could be used to define 145 distinct strains within the species. We found that intermediate frequency genes in the pangenome (present in 10-95% of genomes) could be divided into those closely linked to strain background ("strain-concentrated") and those highly variable within strains ("strain-diffuse"). Non-core genes had different patterns of chromosome location; notably, strain-diffuse associated with prophages, strain-concentrated with the vSa{beta} genome island and rare genes (84,000 genomes) that was re-processed and subsampled to remove redundancy. We found that individual genomes could be grouped into strains by sharing > 99.5% identical nucleotide sequence of the core part of their genome. We also showed that a portion of genes that are present in intermediate frequency in the species are strongly associated with some strains but completely absent from others, suggesting a role in strain-specificity. This work lays the foundation for understanding individual gene histories of the S. aureus species and also outlines strategies for processing large bacterial genomic datasets.