Efficient Nucleic Acid Extraction and 16S rRNA Gene Sequencing for Bacterial Community Characterization

• Main Authors: Anahtar, Melis N. (Contributor), Bowman, Brittany A. (Contributor), Kwon, Douglas (Contributor)
• Other Authors: Ragon Institute of MGH, MIT and Harvard (Contributor), Koch Institute for Integrative Cancer Research at MIT (Contributor)
• Format: Article
• Language: English
• Published: MyJoVE Corporation, 2016-07-07T18:46:22Z.
• Subjects: Article
• Online Access: Get fulltext

There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing technologies have allowed for the rapid and efficient characterization of bacterial communities using 16S rRNA gene sequencing from a variety of sources. Although readily available tools for 16S rRNA sequence analysis have standardized computational workflows, sample processing for DNA extraction remains a continued source of variability across studies. Here we describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs. We also delineate downstream methods for 16S rRNA gene sequencing, including generation of sequencing libraries, data quality control, and sequence analysis. The workflow can accommodate multiple samples types, including stool and swabs collected from a variety of anatomical locations and host species. Additionally, recovered DNA and RNA can be separated and used for other applications, including whole genome sequencing or RNA-seq. The method described allows for a common processing approach for multiple sample types and accommodates downstream analysis of genomic, metagenomic and transcriptional information.
Bill & Melinda Gates Foundation
National Institute of Allergy and Infectious Diseases (U.S.) (NIAID (1R01AI111918))
Burroughs Wellcome Fund
National Institute of General Medical Sciences (U.S.) (NIGMS T32GM007753)
Paul & Daisy Soros Fellowships for New Americans (New York, N.Y.)