Using Core Genome Alignments To Assign Bacterial Species

ANI, dDDH, and CGASI correlation analysis. CGASI, ANI, and dDDH values were calculated for 7,264 intragenus pairwise comparisons of genomes for Rickettsia, Orientia, Ehrlichia, Anaplasma, Neorickettsia, Wolbachia, Caulobacter, Erwinia, Neisseria, Polaribacter, Ralstonia, and Thermus. (A) The CGASI and ANI values for the intragenus comparisons follow a second-degree polynomial model (r² = 0.977), with the ANI species cutoff of ≥95% being equivalent to a CGASI of 96.8%, indicated by the blue dashed box. (B) The CGASI and dDDH values for all pairwise comparisons follow a third-degree polynomial model (r² = 0.978), with the dDDH species cutoff of ≥70% being equivalent to a CGASI of 97.6%, indicated by the red dashed box. (C) To identify the optimal CGASI cutoff to use when classifying species, for each increment of the CGASI species cutoff plotted on the x axis, the percentage of intraspecies and interspecies comparisons correctly assigned was determined based on classically defined species designations. The ideal cutoff should maximize the prediction of classically defined species for both interspecies and intraspecies comparisons. The ANI-equivalent CGASI species cutoff is represented by the blue dashed line, while the dDDH-equivalent CGASI species cutoff is represented by the red dashed line.

Analysis of the ANI, dDDH, and CGASI values of 69 Rickettsia genomes. Across 69 Rickettsia genomes, the ANI and dDDH values (A) and CGASI values (B) were calculated for each pairwise genome comparison. The shape next to each Rickettsia genome represents whether the genome originates from an ancestral (⚫), transitional (), typhus group (▲), or spotted fever group (■) Rickettsia species, while the colors of the shapes on the axes represent species designations as determined by a CGASI cutoff of ≥96.8%. (C) An ML phylogenomic tree with 1,000 bootstraps was generated using the core genome alignment. (D) The relationships in the green box in panel C cannot be adequately visualized at the necessary scale, so they are illustrated separately with a different scale. For both trees, red branches represent branches with <100 bootstrap support.

Analysis of the ANI, dDDH, and CGASI values of 16 Ehrlichia genomes. For 16 Ehrlichia genomes, the ANI and dDDH values (A) and CGASI values (B) were calculated for each pairwise genome comparison. The colors of the shapes next to each Ehrlichia genome represent species designations as determined by a CGASI cutoff of ≥96.8%. (C) An ML phylogenomic tree with 1,000 bootstraps was generated using the core genome, with red branches representing branches with <100 bootstrap support.

Analysis of the ANI, dDDH, and CGASI values of 30 Anaplasma genomes. (A) For 30 Anaplasma genomes, the ANI and dDDH values were calculated for each genome comparison and color-coded to illustrate the results with respect to ANI cutoffs of ≥95% and dDDH cutoffs of ≥70%. (B) When we attempted to construct a core genome alignment using all 30 Anaplasma genomes, only a 20-kbp alignment was generated, accounting for <1% of the average Anaplasma genome size. Therefore, CGASI values were calculated after the Anaplasma genomes were split into two subsets containing 20 A. phagocytophilum genomes and the remaining 10 Anaplasma genomes. In all panels, the shape next to each genome denotes genus designations as determined by the size of their core genome alignments, while the color of the shape denotes the species as defined by a CGASI cutoff of ≥96.8%.

Analysis of the ANI, dDDH, and CGASI values of 23 Wolbachia genomes. For 23 Wolbachia genomes, the ANI and dDDH values (A) and CGASI values (B) were calculated for each pairwise genome comparison. The shape next to each Wolbachia genome represents supergroup A (⚫), B (▲), C (■), D (), E (), F (), and L (◆) designations, while the color of the shape indicates species as defined by a CGASI cutoff of ≥96.8%. (C) An ML phylogenomic tree was generated using the Wolbachia core genome alignment constructed using 23 Wolbachia genomes, with red branches representing branches with <100 bootstrap support. The species designations in supergroup B show the CGASI-designated species clusters of wPip_Pel, wPip_JHB, wAus, and wStri to be polyphyletic.