Optimal 16S rRNA gene amplicon sequencing analysis for oral microbiota to avoid the potential bias introduced by trimming length, primer, and database

16S rRNA gene amplicon sequencing analysis is used to investigate bacterial communities; however, the estimated bacterial composition can differ from the original due to experimental and analytical biases. Therefore, this study determines the optimal conditions to minimize the potential biases in 16S rRNA gene amplicon sequencing analysis using different DNA samples, trimming lengths, primers, and databases. The results of the mock1 community with 250 and 300 bp paired-end (PE), comprising 15 bacteria from various environments, showed the highest similarity between the theoretical value and the data using the Greengenes2 and the V3 region at the genus level and V5-V6 at the species level. In the 300 bp PE sequencing analysis of the mock2 community, comprising six major oral bacteria, the data using the V3-V4, V4, and V5-V6 regions with the SILVA, Greengenes2, and the Human Oral Microbiome Database (HOMD) showed the highest similarity to the theoretical values at the genus level. At the species level, the data using the V3-V4 and V4 regions with Greengenes2 and the data using the V1-V2 with HOMD exhibited the highest alignment with the theoretical values. In the species analysis of the dental calculus samples with 300 bp PE, the Shannon index value was higher with the V1-V2 region and HOMD than with others. Our results suggest that the optimal conditions for oral microbiome analysis are the combinations of 300 bp PE and the V3-V4 or V4 region with the Greengenes2 and the V1-V2 with HOMD.IMPORTANCEThe 16S rRNA gene amplicon sequencing analysis is frequently used in oral microbiome research. However, this method can have biases that distort the experimental data, which depend on the methodological steps, including sequencing length, trimming length, selected amplification regions, and referenced databases. In this study, a combination of 300 bp PE and the primer targeting the V3-V4 or V4 regions with the Greengenes2 and the V1-V2 region with the HOMD was the most bias-minimizing condition for oral microbiota analysis. In addition, this is the first report of such analyses in modern Japanese dental calculus. The methods used in this study will aid in setting appropriate conditions for sequence analysis of microbiota obtained not only from the oral cavity but also from any environment. The 16S rRNA gene amplicon sequencing analysis is frequently used in oral microbiome research. However, this method can have biases that distort the experimental data, which depend on the methodological steps, including sequencing length, trimming length, selected amplification regions, and referenced databases. In this study, a combination of 300 bp PE and the primer targeting the V3-V4 or V4 regions with the Greengenes2 and the V1-V2 region with the HOMD was the most bias-minimizing condition for oral microbiota analysis. In addition, this is the first report of such analyses in modern Japanese dental calculus. The methods used in this study will aid in setting appropriate conditions for sequence analysis of microbiota obtained not only from the oral cavity but also from any environment.