Protocol

Diagnostic Bacteriology

Volume 1616 of the series Methods in Molecular Biology pp 23-38

Date:

Characterization of Sinus Microbiota by 16S Sequencing from Swabs

  • Thad W. VickeryAffiliated withUniversity of Colorado School of Medicine
  • , Jennifer M. KofonowAffiliated withDivision of Infectious Disease, Department of Medicine, University of Colorado
  • , Vijay R. RamakrishnanAffiliated withDepartment of Otolaryngology–Head and Neck Surgery, University of Colorado Email author 

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Abstract

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 words

Sinus swabs DNA extraction Bacterial 16S rRNA Sinusitis Culture-independent microbiology Next generation sequencing Microbiome