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http://dx.doi.org/10.4014/jmb.1403.03032

Development of a Novel Long-Range 16S rRNA Universal Primer Set for Metagenomic Analysis of Gastrointestinal Microbiota in Newborn Infants  

Ku, Hye-Jin (Department of Food Science and Biotechnology, Institute of Life Sciences and Resources, Kyung Hee University)
Lee, Ju-Hoon (Department of Food Science and Biotechnology, Institute of Life Sciences and Resources, Kyung Hee University)
Publication Information
Journal of Microbiology and Biotechnology / v.24, no.6, 2014 , pp. 812-822 More about this Journal
Abstract
Metagenomic analysis of the human intestinal microbiota has extended our understanding of the role of these bacteria in improving human intestinal health; however, a number of reports have shown that current total fecal DNA extraction methods and 16S rRNA universal primer sets could affect the species coverage and resolution of these analyses. Here, we improved the extraction method for total DNA from human fecal samples by optimization of the lysis buffer, boiling time (10 min), and bead-beating time (0 min). In addition, we developed a new long-range 16S rRNA universal PCR primer set targeting the V6 to V9 regions with a 580 bp DNA product length. This new 16S rRNA primer set was evaluated by comparison with two previously developed 16S rRNA universal primer sets and showed high species coverage and resolution. The optimized total fecal DNA extraction method and newly designed long-range 16S rRNA universal primer set will be useful for the highly accurate metagenomic analysis of adult and infant intestinal microbiota with minimization of any bias.
Keywords
16S rRNA universal primer; fecal DNA extraction; gastrointestinal microbiota; metagenomics; next-generation sequencing;
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