Abstract
The profiling of bacterial communities by the sequencing of housekeeping genes such as that encoding the small subunit ribosomal RNA has revealed the extensive diversity of bacterial life on earth. Standard protocols have been developed and are widely used for this application, but individual habitats may require modification of methods. This review discusses the sequencing and analysis methods most appropriate for the study of the bacterial component of the human oral microbiota. If possible, DNA should be extracted from samples soon after collection. If samples have to be stored for practical reasons, precautions to avoid DNA degradation on freezing should be taken. A critical aspect of profiling oral bacterial communities is the choice of region of the 16S rRNA gene for sequencing. The V1-V2 region provides the best discrimination between species of the genus Streptococcus, the most common genus in the mouth and important in health and disease. The MiSeq platform is most commonly used for sequencing, but long-read technologies are now becoming available that should improve the resolution of analyses. There are a variety of well-established data analysis pipelines available, including mothur and QIIME, which identify sequence reads as phylotypes by comparing them to reference data sets or grouping them into operational taxonomic units. DADA2 has improved sequence error correction capabilities and resolves reads to unique variants. Two curated oral 16S rRNA databases are available: HOMD and CORE. Expert interpretation of community profiles is required, both to detect the presence of contaminating DNA, which is commonly present in the reagents used in analysis, and to differentiate oral and nonoral bacteria and determine the significance of findings. Despite advances in shotgun whole-genome metagenomic methods, oral bacterial community profiling via 16S rRNA sequence analysis remains a valuable technique for the characterization of oral bacterial populations.