<i>Bacillus pseudomycoides</i> CHAES I 2_2 digital DNA-DNA hybridization results

The genome sequence data were uploaded to the Type (Strain) Genome Server (TYGS), a free bioinformatics platform available under https://tygs.dsmz.de, for a whole genome-based taxonomic analysis [1]. The analysis also made use of recently introduced methodological updates and features [2,3]. Information on nomenclature, synonymy and associated taxonomic literature was provided by TYGS's sister database, the List of Prokaryotic names with Standing in Nomenclature (LPSN, available at https://lpsn.dsmz.de) [2,3]. The results were provided by the TYGS on 2026-01-30. The TYGS analysis was subdivided into the following steps: Determination of closely related type strainsDetermination of closest type strain genomes was done in two complementary ways: First, all user genomes were compared against all type strain genomes available in the TYGS database via the MASH algorithm, a fast approximation of intergenomic relatedness [4], and, the ten type strains with the smallest MASH distances chosen per user genome. Second, an additional set of ten closely related type strains was determined via the 16S rDNA gene sequences. These were extracted from the user genomes using RNAmmer [5] and each sequence was subsequently BLASTed [6] against the 16S rDNA gene sequence of each of the currently 24050 type strains available in the TYGS database. This was used as a proxy to find the best 50 matching type strains (according to the bitscore) for each user genome and to subsequently calculate precise distances using the Genome BLAST Distance Phylogeny approach (GBDP) under the algorithm 'coverage' and distance formula d5 [7]. These distances were finally used to determine the 10 closest type strain genomes for each of the user genomes. Pairwise comparison of genome sequencesFor the phylogenomic inference, all pairwise comparisons among the set of genomes were conducted using GBDP and accurate intergenomic distances inferred under the algorithm 'trimming' and distance formula d5 [7]. 100 distance replicates were calculated each. Digital DDH values and confidence intervals were calculated using the recommended settings of the GGDC 4.0 [2,7]. Phylogenetic inferenceThe resulting intergenomic distances were used to infer a balanced minimum evolution tree with branch support via FASTME 2.1.6.1 including SPR postprocessing [8]. Branch support was inferred from 100 pseudo-bootstrap replicates each. The trees were rooted at the midpoint [9] and visualized with PhyD3 [10]. Type-based species and subspecies clusteringThe type-based species clustering using a 70% dDDH radius around each of the 17 type strains was done as previously described [1]. The resulting groups are shown in Table 1 and 4. Subspecies clustering was done using a 79% dDDH threshold as previously introduced [11]. [1] Meier-Kolthoff JP, Göker M. TYGS is an automated high-throughput platform for state-of-the-art genome-based taxonomy. Nat. Commun. 2019;10: 2182. DOI: 10.1038/s41467-019-10210-3 [2] Meier-Kolthoff JP, Sardà Carbasse J, Peinado-Olarte RL, Göker M. TYGS and LPSN: a database tandem for fast and reliable genome-based classification and nomenclature of prokaryotes. Nucleic Acid Res. 2022;50: D801–D807. DOI: 10.1093/nar/gkab902 [3] Freese HM, Meier-Kolthoff JP, Sardà Carbasse J, Afolayan AO, Göker M. TYGS and LPSN in 2025: a Global Core Biodata Resource for genome-based classification and nomenclature of prokaryotes within DSMZ Digital Diversity. Nucleic Acid Res. 2025, gkaf1110. DOI: 10.1093/nar/gkaf1110 [4] Ondov BD, Treangen TJ, Melsted P, et al. Mash: Fast genome and metagenome distance estimation using MinHash. Genome Biol 2016;17: 1–14. DOI: 10.1186/s13059-016-0997-x [5] Lagesen K, Hallin P. RNAmmer: consistent and rapid annotation of ribosomal RNA genes. Nucleic Acids Res. Oxford Univ Press; 2007;35: 3100–3108. DOI: 10.1093/nar/gkm160 [6] Camacho C, Coulouris G, Avagyan V, Ma N, Papadopoulos J, Bealer K, et al. BLAST+: architecture and applications. BMC Bioinformatics. 2009;10: 421. DOI: 10.1186/1471-2105-10-421 [7] Meier-Kolthoff JP, Auch AF, Klenk H-P, Göker M. Genome sequence-based species delimitation with confidence intervals and improved distance functions. BMC Bioinformatics. 2013;14: 60. DOI: 10.1186/1471-2105-14-60 [8] Lefort V, Desper R, Gascuel O. FastME 2.0: A comprehensive, accurate, and fast distance-based phylogeny inference program. Mol Biol Evol. 2015;32: 2798–2800. DOI: 10.1093/molbev/msv150 [9] Farris JS. Estimating phylogenetic trees from distance matrices. Am Nat. 1972;106: 645–667. [10] Kreft L, Botzki A, Coppens F, Vandepoele K, Van Bel M. PhyD3: A phylogenetic tree viewer with extended phyloXML support for functional genomics data visualization. Bioinformatics. 2017;33: 2946–2947. DOI: 10.1093/bioinformatics/btx324 [11] Meier-Kolthoff JP, Hahnke RL, Petersen J, Scheuner C, Michael V, Fiebig A, et al. Complete genome sequence of DSM 30083T, the type strain (U5/41T) of Escherichia coli, and a proposal for delineating subspecies in microbial taxonomy. Stand Genomic Sci. 2014;9: 2. DOI: 10.1186/1944-3277-9-2