Comparison of Boiling and Robotics Automation Method in DNA Extraction for Metagenomic Sequencing of Human Oral Microbes.
Comparison of Boiling and Robotics Automation Method in DNA Extraction for Metagenomic Sequencing of Human Oral Microbes.
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DOI：
10.1371/journal.pone.0154389
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Yamashita R
中科院分区：
文献类型：
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作者：
Yamagishi J;Sato Y;Shinozaki N;Ye B;Tsuboi A;Nagasaki M;Yamashita R
下一代测序性能的快速改进使我们能够分析超过一万个样本的庞大样本集。然而，DNA提取仍然是这种宏基因组方法中的限制性步骤。在这项研究中，我们分析了人类口腔微生物，比较了三种DNA提取方法的性能：PowerSoil（该领域广泛使用的方法），QIAsymphony（机器人方法）和简单的煮沸方法。在初步研究中，最初从三名志愿者中收集牙菌斑，然后在后续研究中扩大到12名志愿者。细菌植物群通过在物种水平分析后对16S rRNA的V4区进行测序来估计。我们的研究结果表明，PowerSoil和QIAsymphony的效率与煮沸法相当。因此，煮沸法可能是一种有前途的替代方法，因为它简单，成本效益高，处理时间短。此外，该方法用于估计细菌种类是可靠的，并且可以在将来用于检查口腔植物群与健康状况之间的相关性。尽管如此，在三种方法中观察到不同细菌物种的DNA提取效率的差异。基于这些发现，DNA提取没有“金标准”。在未来，我们建议，DNA提取方法应选择在个案的基础上，考虑到研究的目的和标本。
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.