title: Design and experimental application of a novel non-degenerate universal primer set that amplifies prokaryotic 16S rRNA genes with a low possibility to amplify eukaryotic rRNA genes.
abstract: The deep sequencing of 16S rRNA genes amplified by universal primers has revolutionized our understanding of microbial communities by allowing the characterization of the diversity of the uncultured majority. However, some universal primers also amplify eukaryotic rRNA genes, leading to a decrease in the efficiency of sequencing of prokaryotic 16S rRNA genes with possible mischaracterization of the diversity in the microbial community. In this study, we compared 16S rRNA gene sequences from genome-sequenced strains and identified candidates for non-degenerate universal primers that could be used for the amplification of prokaryotic 16S rRNA genes. The 50 identified candidates were investigated to calculate their coverage for prokaryotic and eukaryotic rRNA genes, including those from uncultured taxa and eukaryotic organelles, and a novel universal primer set, 342F-806R, covering many prokaryotic, but not eukaryotic, rRNA genes was identified. This primer set was validated by the amplification of 16S rRNA genes from a soil metagenomic sample and subsequent pyrosequencing using the Roche 454 platform. The same sample was also used for pyrosequencing of the amplicons by employing a commonly used primer set, 338F-533R, and for shotgun metagenomic sequencing using the Illumina platform. Our comparison of the taxonomic compositions inferred by the three sequencing experiments indicated that the non-degenerate 342F-806R primer set can characterize the taxonomic composition of the microbial community without substantial bias, and is highly expected to be applicable to the analysis of a wide variety of microbial communities.
authors.affiliation: 1Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 B-36, Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Mori
authors.forename: Fumito
authors.initials: F
authors.lastname: Maruyama
authors.forename: Hiromi
authors.initials: H
authors.lastname: Kato
authors.forename: Atsushi
authors.initials: A
authors.lastname: Toyoda
authors.forename: Ayumi
authors.initials: A
authors.lastname: Dozono
authors.forename: Yoshiyuki
authors.initials: Y
authors.lastname: Ohtsubo
authors.forename: Yuji
authors.initials: Y
authors.lastname: Nagata
authors.forename: Asao
authors.initials: A
authors.lastname: Fujiyama
authors.forename: Masataka
authors.initials: M
authors.lastname: Tsuda
authors.forename: Ken
authors.initials: K
authors.lastname: Kurokawa
mesh_terms: D001105:Archaea / Q000145:classification / Q000235:genetics*; D001419:Bacteria / Q000145:classification / Q000235:genetics*; D017931:DNA Primers / Q000737:chemistry* / Q000235:genetics; D056890:Eukaryota / Q000235:genetics; D020459:Genes, rRNA; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D012988:Soil Microbiology
chemical_list: D017931:DNA Primers; D012336:RNA, Ribosomal, 16S
keywords: 16S rRNA; microbial community; non-degenerate primer; primer design