Characterization of Sinus Microbiota by 16S Sequencing from Swabs
New culture-independent microbiology methods are leading to a paradigm shift in our understanding of how the microbial community at the mucosal surface impacts sinonasal health and disease. Whereas traditional culture-based protocols were designed to identify specific pathogens in order to direct antibiotic therapies and eradicate bacteria, newer molecular techniques allow for the identification of both culturable and nonculturable bacteria in diverse communities. As a result of the recent explosion in the use of molecular techniques, we are gaining an understanding of how commensal bacteria may help modulate the host immune response and promote homeostasis. Here, we describe the general workflow of microbiome sequencing including the detailed methods for extracting mixed-community genomic DNA from sinonasal swabs, amplifying bacterial 16S rRNA genes using quantitative PCR, and preparing the samples for next-generation sequencing on the most commonly used sequencing platforms.
Key wordsSinus swabs DNA extraction Bacterial 16S rRNA Sinusitis Culture-independent microbiology Next generation sequencing Microbiome
The authors would like to acknowledge Diana Ir for generously sharing her knowledge of 16S microbiome characterization, reviewing early drafts and providing valuable feedback that improved the manuscript significantly. In addition, we would like to thank Dr. Daniel N. Frank, Ph.D., for his generous support and collaboration. Research reported in this publication was supported by the National Institute On Deafness And Other Communication Disorders of the National Institutes of Health under award numbers K23DC014747 (V.R. Ramakrishnan) and T32DC01228003 (T.W. Vickery), as well as the Flight Attendants Medical Research Institute grant CIA13006 (V.R. Ramakrishnan and D.N. Frank). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health or the Flight Attendants Medical Research Institute.