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.

Responses of bacterial community structure and denitrifying bacteria in biofilm to submerged macrophytes and nitrate

“…The bacterial 16S rRNA gene was amplified using the primers 342F (5′-CTACGGGGGGCAGCAG-3′) and 806R (5′-GGACTACCGGGGTATCT-3′)48. The PCR reactions were performed in a 20 μL reaction mixture containing 4 μL of 5X FastPfu Buffer, 2 μL of 2.5 mM dNTPs, 0.8 μL of each primer (5 μM), 0.4 μL of FastPfu Polymerase, and 10 ng of template DNA.…”

Microbial regulation of the soil carbon cycle: evidence from gene–enzyme relationships

“…Barcoded pyrosequencing and analysis Fusion primers 341F-806R (Mori et al, 2013) and LR3-LR0R (Liu et al, 2012) were used to amplify multiplexed bar-coded 16S rRNA and large subunit rRNA gene sequences to profile bacterial and fungal communities, respectively. PCR products were purified, pooled and sequenced on a 454 GS FLX Titanium sequencer (Roche 454 Life Sciences, Branford, CT, USA).…”

Assay for estimating total bacterial load: relative qPCR normalisation of bacterial load with associated clinical implications

“…Primer candidates, listed in Supplementary Table 2, are tested to satisfy 2 criteria: a) no presence of signal in the negative control and b) preservation of same limits of detection (LoDs), as previously reported by equivalent assays (Clifford et al, 2012;Liu et al, 2012;Nadkarni et al, 2002). As a result, a new reverse primer was discovered, completely eliminating "previously reported contamination-like priming" in the absence of unintended targets (Clifford et al, 2012;Huys et al, 2008;Liu et al, 2012;Mori et al, 2014;Nadkarni et al, 2002). The novel hybridization probe was successfully added for the first time, thus allowing assay compatibility with hybridization-based diagnostic qPCR chemistry (TaqMan like).…”