Long journey of 16S rRNA-amplicon sequencing toward cell-based functional bacterial microbiota characterization

Bacteria often exist and function as a community, known as the bacterial microbiota, which consists of vast numbers of bacteria belonging to many bacterial species (taxa). Characterizing the bacterial microbiota needs high-throughput approaches that enable the identification and quantification of many bacterial cells, and such approaches have been under development for more than 30 years. In this review, we describe the history of high-throughput technologies based on 16S ribosomal RNA (rRNA) gene-amplicon sequencing for the characterization of bacterial microbiotas. Then, we summarize the features and applications of current 16S rRNA gene-amplicon sequencing approaches, including a recent achievement that enables the identification of individual cells with single-base accuracy for 16S rRNA genes and the quantification of many identified cells. Furthermore, we present the prospects for further technical development, including the combined use of high-throughput methods and other informative analyses, such as whole-genome sequencing in the common unit of the cell, which enables bacterial microbiota characterization based on both the number of cells and their functions.