EvANI benchmarking workflow for evolutionary distance estimation

1Advances in long-read sequencing technology has led to a rapid increase in high-quality genome assemblies. These make it possible to compare genome sequences across the Tree of Life, deepening our understanding of evolutionary relationships. Average nucleotide identity (ANI) is a distance measure that has been applied to species delineation, building of guide trees, and searching large sequence databases. Since computing ANI is computationally expensive, the field has increasingly turned to sketch-based approaches that use assumptions and heuristics to speed this up. We propose a suite of simulated and real benchmark datasets, together with a rank-correlation-based metric, to study how these assumptions and heuristics impact distance estimates. We call this evaluation framework EvANI. With EvANI, we show that ANIb is the ANI estimation algorithm that best captures tree distance, though it is also the least efficient. We show that k-mer based approaches are extremely efficient and have consistently strong accuracy. We also show that some clades have inter-sequence distances that are best computed using multiple values of k, e.g. k = 10 and k = 19 for Chlamydiales. Finally, we highlight that approaches based on maximal exact matches may represent an advantageous compromise, achieving an intermediate level of computational efficiency while avoiding over-reliance on a single fixed k-mer length.