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Development of a prokaryotic universal primer for simultaneous analysis of Bacteria and Archaea using next-generation sequencing.

ABSTRACT: For the analysis of microbial community structure based on 16S rDNA sequence diversity, sensitive and robust PCR amplification of 16S rDNA is a critical step. To obtain accurate microbial composition data, PCR amplification must be free of bias; however, amplifying all 16S rDNA species with equal efficiency from a sample containing a large variety of microorganisms remains challenging. Here, we designed a universal primer based on the V3-V4 hypervariable region of prokaryotic 16S rDNA for the simultaneous detection of Bacteria and Archaea in fecal samples from crossbred pigs (Landrace × Large white × Duroc) using an Illumina MiSeq next-generation sequencer. In-silico analysis showed that the newly designed universal prokaryotic primers matched approximately 98.0% of Bacteria and 94.6% of Archaea rRNA gene sequences in the Ribosomal Database Project database. For each sequencing reaction performed with the prokaryotic universal primer, an average of 69,330 (± 20,482) reads were obtained, of which archaeal rRNA genes comprised approximately 1.2% to 3.2% of all prokaryotic reads. In addition, the detection frequency of Bacteria belonging to the phylum Verrucomicrobia, including members of the classes Verrucomicrobiae and Opitutae, was higher in the NGS analysis using the prokaryotic universal primer than that performed with the bacterial universal primer. Importantly, this new prokaryotic universal primer set had markedly lower bias than that of most previously designed universal primers. Our findings demonstrate that the prokaryotic universal primer set designed in the present study will permit the simultaneous detection of Bacteria and Archaea, and will therefore allow for a more comprehensive understanding of microbial community structures in environmental samples.

SUBMITTER: Takahashi S

PROVIDER: S-EPMC4140814 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Development of a prokaryotic universal primer for simultaneous analysis of Bacteria and Archaea using next-generation sequencing.

Takahashi Shunsuke S Tomita Junko J Nishioka Kaori K Hisada Takayoshi T Nishijima Miyuki M

PloS one 20140821 8

For the analysis of microbial community structure based on 16S rDNA sequence diversity, sensitive and robust PCR amplification of 16S rDNA is a critical step. To obtain accurate microbial composition data, PCR amplification must be free of bias; however, amplifying all 16S rDNA species with equal efficiency from a sample containing a large variety of microorganisms remains challenging. Here, we designed a universal primer based on the V3-V4 hypervariable region of prokaryotic 16S rDNA for the si ...[more]

PMID: 25144201

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Project description:Pollen, fungi, and bacteria are the main microscopic biological entities present in outdoor air, causing allergy symptoms and disease transmission and having a significant role in atmosphere dynamics. Despite their relevance, a method for monitoring simultaneously these biological particles in metropolitan environments has not yet been developed. Here, we assessed the use of the Hirst-type spore trap to characterize the global airborne biota by high-throughput DNA sequencing, selecting regions of the 16S rRNA gene and internal transcribed spacer for the taxonomic assignment. We showed that aerobiological communities are well represented by this approach. The operational taxonomic units (OTUs) of two traps working synchronically compiled >87% of the total relative abundance for bacterial diversity collected in each sampler, >89% for fungi, and >97% for pollen. We found a good correspondence between traditional characterization by microscopy and genetic identification, obtaining more-accurate taxonomic assignments and detecting a greater diversity using the latter. We also demonstrated that DNA sequencing accurately detects differences in biodiversity between samples. We concluded that high-throughput DNA sequencing applied to aerobiological samples obtained with Hirst spore traps provides reliable results and can be easily implemented for monitoring prokaryotic and eukaryotic entities present in the air of urban areas.IMPORTANCE Detection, monitoring, and characterization of the wide diversity of biological entities present in the air are difficult tasks that require time and expertise in different disciplines. We have evaluated the use of the Hirst spore trap (an instrument broadly employed in aerobiological studies) to detect and identify these organisms by DNA-based analyses. Our results showed a consistent collection of DNA and a good concordance with traditional methods for identification, suggesting that these devices can be used as a tool for continuous monitoring of the airborne biodiversity, improving taxonomic resolution and characterization together. They are also suitable for acquiring novel DNA amplicon-based information in order to gain a better understanding of the biological particles present in a scarcely known environment such as the air.

Dataset Information

Development of a prokaryotic universal primer for simultaneous analysis of Bacteria and Archaea using next-generation sequencing.

Publications

Development of a prokaryotic universal primer for simultaneous analysis of Bacteria and Archaea using next-generation sequencing.

Similar Datasets

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