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

The sequenced DNA of two mock communities was made by mixing the various bacterial species. Mock1 is composed of 15 bacterial species which habit various environments. Mock2 is composed of 6 bacterial species which habit an oral cavity. The DNA of mock1 was sequenced with 250 and 300 bp paired ends and the DNA of mock2 was sequenced with 300 bp paired ends. In this study on oral microbiota, two mock communities and dental calculus samples, stepwise trimming of the paired-end sequenced reads, nine types of primers targeting different hypervariable regions, and three reference databases were used to comprehensively investigate each methodological step in order to determine the optimal condition for oral microbiota in 16S rRNA gene amplicon sequencing analysis.