FastANI, Mash and Dashing equally differentiate between species.

Bacteria of the genus are among the most important multi-drug resistant human pathogens, though they have been isolated from a variety of environments. The importance and ubiquity of these organisms call for quick and accurate methods for their classification. Average Nucleotide Identity (ANI) is becoming a standard for species delimitation based on whole genome sequence comparison. However, much faster genome comparison tools have been appearing in the literature. In this study we tested the quality of different approaches for genome-based species delineation against ANI. To this end, we compared 1,189 genomes using measures calculated with Mash, Dashing, and DNA compositional signatures, all of which run in a fraction of the time required to obtain ANI. Receiver Operating Characteristic (ROC) curve analyses showed equal quality in species discrimination for ANI, Mash and Dashing, with Area Under the Curve (AUC) values above 0.99, followed by DNA signatures (AUC: 0.96). Accordingly, groups obtained at optimized cutoffs largely agree with species designation, with ANI, Mash and Dashing producing 15 species-level groups. DNA signatures broke the dataset into more than 30 groups. Testing Mash to map species after adding draft genomes to the dataset also showed excellent results (AUC above 0.99), producing a total of 26 species-level groups. The ecological niches of strains were found to neither be related to species delimitation, nor to protein functional content, suggesting that a single species can have a wide repertoire of ecological functions.