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16S rRNA gene sequencing of mock microbial populations-impact of DNA extraction method, primer choice and sequencing platform

机译:模拟微生物种群的16S rRNA基因测序-DNA提取方法,引物选择和测序平台的影响

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摘要

Background: Next-generation sequencing platforms have revolutionised our ability to investigate the microbiota composition of complex environments, frequently through 16S rRNA gene sequencing of the bacterial component of the community. Numerous factors, including DNA extraction method, primer sequences and sequencing platform employed, can affect the accuracy of the results achieved. The aim of this study was to determine the impact of these three factors on 16S rRNA gene sequencing results, using mock communities and mock community DNA. Results: The use of different primer sequences (V4-V5, V1-V2 and V1-V2 degenerate primers) resulted in differences in the genera and species detected. The V4-V5 primers gave the most comparable results across platforms. The three Ion PGM primer sets detected more of the 20 mock community species than the equivalent MiSeq primer sets. Data generated from DNA extracted using the 2 extraction methods were very similar. Conclusions: Microbiota compositional data differed depending on the primers and sequencing platform that were used. The results demonstrate the risks in comparing data generated using different sequencing approaches and highlight the merits of choosing a standardised approach for sequencing in situations where a comparison across multiple sequencing runs is required.
机译:背景:新一代测序平台已经彻底改变了我们研究复杂环境中微生物群落组成的能力,通常通过对该社区细菌组成部分进行16S rRNA基因测序。包括DNA提取方法,引物序列和测序平台在内的许多因素都会影响所获得结果的准确性。这项研究的目的是使用模拟社区和模拟社区DNA确定这三个因素对16S rRNA基因测序结果的影响。结果:使用不同的引物序列(V4-V5,V1-V2和V1-V2简并引物)会导致检测到的属和种不同。 V4-V5引物在各个平台上给出的结果最为可比。与等效的MiSeq引物组相比,三个离子PGM引物组检测到20个模拟群落物种更多。使用两种提取方法提取的DNA产生的数据非常相似。结论:微生物群组成数据取决于所使用的引物和测序平台。结果证明了比较使用不同测序方法生成的数据的风​​险,并强调了在需要跨多个测序运行进行比较的情况下选择标准化测序方法的优点。

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