Benchmarking the topological accuracy of bacterial phylogenomic workflows using in silico evolution

Phylogenetic analyses are widely used in microbiological research, for example to trace the progression of bacterial outbreaks based on whole-genome sequencing data. In practice, multiple analysis steps such as de novo assembly, alignment and phylogenetic inference are combined to form phylogenetic workflows. Comprehensive benchmarking of the accuracy of complete phylogenetic workflows is lacking. To benchmark different phylogenetic workflows, we simulated bacterial evolution under a wide range of evolutionary models, varying the relative rates of substitution, insertion, deletion, gene duplication, gene loss and lateral gene transfer events. The generated datasets corresponded to a genetic diversity usually observed within bacterial species (≥95 % average nucleotide identity). We replicated each simulation three times to assess replicability. In total, we benchmarked 19 distinct phylogenetic workflows using 8 different simulated datasets. We found that recently developed k -mer alignment methods such as kSNP and ska achieve similar accuracy as reference mapping. The high accuracy of k -mer alignment methods can be explained by the large fractions of genomes these methods can align, relative to other approaches. We also found that the choice of de novo assembly algorithm influences the accuracy of phylogenetic reconstruction, with workflows employing SPAdes or skesa outperforming those employing Velvet. Finally, we found that the results of phylogenetic benchmarking are highly variable between replicates. We conclude that for phylogenomic reconstruction, k -mer alignment methods are relevant alternatives to reference mapping at the species level, especially in the absence of suitable reference genomes. We show de novo genome assembly accuracy to be an underappreciated parameter required for accurate phylogenomic reconstruction.