Comprehensive 16S rRNA gene sequence curation

The up-to-date reference 16S rRNA gene sequences were maintained as described earlier [3 (link)]. We attempted to select a sequence with the best quality for each species by using the following strategy. For cases in which multiple sequences were available for a type strain, the sequence extracted from its whole-genome assembly (WGA) was selected. As for PCR-derived sequences, the quality of sequencing was checked manually by secondary-structure-aware alignment using the EzEditor program [13 (link)]. Maximum-likelihood phylogenetic trees of each taxonomic group, such as phyla, classes, orders or families, were generated from manually aligned 16S rRNA gene sequences using RAxML software [14 (link)]. All 16S rRNA gene sequences were assigned taxonomically to the species level as a part of the complete taxonomic hierarchy which consisted of phylum, class, order, family, genus and species (subspecies if applicable).