OTUs clustering should be avoided for defining oral microbiome

This in silico investigation aimed to: 1) evaluate a set of primer pairs with high coverage, including those most commonly used in the literature, to find the different oral species with 16S rRNA gene amplicon similarity/identity (ASI) values [≥]97%; and 2) identify oral species that may be erroneously clustered in the same operational taxonomic unit (OTU) and ascertain whether they belong to distinct genera or other higher taxonomic ranks. Thirty-nine primer pairs were employed to obtain amplicon sequence variants (ASVs) from the complete genomes of 186 bacterial and 135 archaeal species. For each primer, ASVs without mismatches were aligned using BLASTN and their similarity values were obtained. Finally, we selected ASVs from different species with an ASI value [≥]97% that were covered 100% by the query sequences. For each primer, the percentage of species-level coverage with no ASI[≥]97% (SC-NASI[≥]97%) was calculated. Based on the SC-NASI[≥]97% values, the best primer pairs were OP_F053-KP_R020 for bacteria (65.05%), KP_F018-KP_R002 for archaea (51.11%), and OP_F114-KP_R031 for bacteria and archaea together (52.02%). Eighty percent of the oral-bacteria and oralarchaea species shared an ASI[≥]97% with at least one other taxa, including Campylobacter, Rothia, Streptococcus, and Tannerella, which played conflicting roles in the oral microbiota. Moreover, around a quarter and a third of these two-by-two similarity relationships were between species from different bacteria and archaea genera, respectively. Furthermore, even taxa from distinct families, orders, and classes could be grouped in the same cluster. Consequently, irrespective of the primer pair used, OTUs constructed with a 97% similarity provide an inaccurate description of oral-bacterial and oral-archaeal species, greatly affecting microbial diversity parameters. As a result, clustering by OTUs impacts the credibility of the associations between some oral species and certain health and disease conditions. This limits significantly the comparability of the microbial diversity findings reported in oral microbiome literature.