Background and objective: 16S rRNA gene amplicon sequencing analysis is widely used to investigate the diversity and complexity of microbial communities in oral diseases. However, the selected amplification regions may distort the accurate data assessment. This study aimed to evaluate the bias of 16S rRNA gene amplicon analysis using primers targeting different hypervariable regions. Materials and
Methods: Bacterial DNA samples were prepared from mock1 community comprising of 15 bacteria from various environments, mock2 community comprising of 6 major oral bacteria, and samples from the dental calculus of 5 patients. DNA samples were amplified with 9 primers and sequenced with 300 bp paired ends on Illumina Miseq platform, and the data was analyzed using QIIME2 with databases; SILVA, Greengenes, and Human Oral Microbiome Database databases (HOMD).
Results: In the genus-level analysis of mock1 community, the similarity of relative bacterial abundance ratio to the theoretical value was highest in V3-V4 region with SILVA. In the analysis of mock2 community, the highest levels were evident in V3-V4 region with SILVA at the genus-level and V1-V2 region with HOMD at the species-level. In the species-level analysis of the dental calculus samples, the ratios between V1-V2 and V3-V4 regions with HOMD were dissimilar, and the number of detected bacteria was higher in V1-V2 than that of V3-V4 region with HOMD.
Conclusion: The use of primers targeted V1-V2 region, 300 bp paired ends sequencing, and HOMD database might show the most reliable result for 16S rRNA gene amplification sequencing analysis of the oral microbiome.
