Dataset Information

Comparison of Boiling and Robotics Automation Method in DNA Extraction for Metagenomic Sequencing of Human Oral Microbes.

ABSTRACT: The rapid improvement of next-generation sequencing performance now enables us to analyze huge sample sets with more than ten thousand specimens. However, DNA extraction can still be a limiting step in such metagenomic approaches. In this study, we analyzed human oral microbes to compare the performance of three DNA extraction methods: PowerSoil (a method widely used in this field), QIAsymphony (a robotics method), and a simple boiling method. Dental plaque was initially collected from three volunteers in the pilot study and then expanded to 12 volunteers in the follow-up study. Bacterial flora was estimated by sequencing the V4 region of 16S rRNA following species-level profiling. Our results indicate that the efficiency of PowerSoil and QIAsymphony was comparable to the boiling method. Therefore, the boiling method may be a promising alternative because of its simplicity, cost effectiveness, and short handling time. Moreover, this method was reliable for estimating bacterial species and could be used in the future to examine the correlation between oral flora and health status. Despite this, differences in the efficiency of DNA extraction for various bacterial species were observed among the three methods. Based on these findings, there is no "gold standard" for DNA extraction. In future, we suggest that the DNA extraction method should be selected on a case-by-case basis considering the aims and specimens of the study.

SUBMITTER: Yamagishi J

PROVIDER: S-EPMC4841512 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Comparison of Boiling and Robotics Automation Method in DNA Extraction for Metagenomic Sequencing of Human Oral Microbes.

Yamagishi Junya J Sato Yukuto Y Shinozaki Natsuko N Ye Bin B Tsuboi Akito A Nagasaki Masao M Yamashita Riu R

PloS one 20160422 4

The rapid improvement of next-generation sequencing performance now enables us to analyze huge sample sets with more than ten thousand specimens. However, DNA extraction can still be a limiting step in such metagenomic approaches. In this study, we analyzed human oral microbes to compare the performance of three DNA extraction methods: PowerSoil (a method widely used in this field), QIAsymphony (a robotics method), and a simple boiling method. Dental plaque was initially collected from three vol ...[more]

PMID: 27104353

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Project description:Traditionally slurry is used as source of nitrogen, phosphorous, and potassium in bio fertilizers to improve crop production. However, poorly managed slurry causes a hazardous effect to the environment by producing greenhouse gases, causing the eutrophication of water bodies, and polluting the groundwater. It has been largely reported that the microbial presence in slurry causing a diverse effect on its storage and disposal system. However, the diversity of bacterial populations in pig slurries remains largely unexplored. Here we report the bacterial diversity present in the slurry from slurry pits, and the effect of storage time on bacterial population. We collected 42 samples from three different pig slurry pits, as three replicates from each one until the 14th week. We used the 16S rRNA, Quantitative Insights Into Microbial Ecology (QIIME) and Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) protocols for the metagenomic downstream analysis. Taxonomic annotation using the Greengenes metagenomic database indicated that on an average 76.2% Firmicutes, 14.4% Bacteroidetes, 4.9% Proteobacteria, etc. microbial populations were present. Comparative microbial analysis showed that the population of Firmicutes decreased from the first to the 14th week, whereas the population of Bacteroidetes increased from the first to the 14th week. Through principal coordinate analysis (PCoA), (linear discriminant analysis effect size (LEfSe), and Pearson's correlation analysis, we found microbial biomarkers according to the storage time point. All bacterial populations were well clustered according to the early, middle, and last weeks of storage. LEfSe showed that Actinobacteria, Lachnospiraceae, Ruminococcaceae, and Bacteroidia are dominantly present in first, seventh, ninth, and 14th week, respectively. Lachnospiraceae and Ruminococcaceae are ubiquitous gastrointestinal non-pathogenic bacteria. KEGG pathways, such as membrane transport, carbohydrate and amino acid metabolism, genetic replication and repair, were significant among all samples. Such a KEGG pathway may indicate the association between the host organism's metabolic activity and the microbes present in the gastro intestinal tract (GIT).

Project description:IntroductionAdvancements in DNA extraction and sequencing technologies have been fundamental in deciphering the significance of the microbiome related to human health and pathology. Whole metagenome shotgun sequencing (WMS) is gaining popularity in use compared to its predecessor (i.e., amplicon-based approaches). However, like amplicon-based approaches, WMS is subject to bias from DNA extraction methods that can compromise the integrity of sequencing and subsequent findings. The purpose of this study was to evaluate systematic differences among four commercially available DNA extraction kits frequently used for WMS analysis of the microbiome.MethodsOral, vaginal, and rectal swabs were collected in replicates of four by a healthcare provider from five participants and randomized to one of four DNA extraction kits. Two extraction blanks and three replicate mock community samples were also extracted using each extraction kit. WMS was completed with NovaSeq 6000 for all samples. Sequencing and microbial communities were analyzed using nonmetric multidimensional scaling and compositional bias analysis.ResultsExtraction kits differentially biased the percentage of reads attributed to microbial taxa across samples and body sites. The PowerSoil Pro kit performed best in approximating expected proportions of mock communities. While HostZERO was biased against gram-negative bacteria, the kit outperformed other kits in extracting fungal DNA. In clinical samples, HostZERO yielded a smaller fraction of reads assigned to Homo sapiens across sites and had a higher fraction of reads assigned to bacterial taxa compared to other kits. However, HostZERO appears to bias representation of microbial communities and demonstrated the most dispersion by site, particularly for vaginal and rectal samples.ConclusionsSystematic differences exist among four frequently referenced DNA extraction kits when used for WMS analysis of the human microbiome. Consideration of such differences in study design and data interpretation is imperative to safeguard the integrity of microbiome research and reproducibility of results.

Dataset Information

Comparison of Boiling and Robotics Automation Method in DNA Extraction for Metagenomic Sequencing of Human Oral Microbes.

Publications

Comparison of Boiling and Robotics Automation Method in DNA Extraction for Metagenomic Sequencing of Human Oral Microbes.

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