Over the last decade, biologists have come to appreciate that the human body is inhabited by thousands of bacterial species in diverse communities unique to each body site. Moreover, due to high-throughput sequencing methods for microbial characterization in a culture-independent manner, it is becoming evident that the microbiome plays an important role in human health and disease. This chapter focuses on the most common form of bacterial microbiome profiling, targeted amplicon sequencing of the 16S ribosomal RNA (rRNA) subunit encoded by 16S rDNA. We discuss important features for designing and performing microbiome experiments on human specimens, including experimental design, sample collection, DNA preparation, and selection of the 16S rDNA sequencing target. We also provide details for designing fusion primers required for targeted amplicon sequencing and selecting the most appropriate high-throughput sequencing platform. We conclude with a review of the fundamental concepts of data analysis and interpretation for these kinds of experiments. Our goal is to provide the reader with the essential knowledge needed to undertake microbiome experiments for application to human disease research questions.
Keywords: 16S rDNA; 16S rRNA; Bacteria; Microbiome; Targeted amplicon sequencing.