Impact of DNA polymerase choice on assessment of bacterial communities by a Legionella genus-specific next-generation sequencing approach
Impact of DNA polymerase choice on assessment of bacterial communities by a Legionella genus-specific next-generation sequencing approach
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DOI：
10.1101/247445
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发表时间：
2018-01
期刊：
影响因子：
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通讯作者：
R. P. A. Pereira;J. Peplies;I. Brettar;M. Höfle
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文献类型：
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作者：
R. P. A. Pereira;J. Peplies;I. Brettar;M. Höfle
文库制备步骤是基于 NGS 的研究中偏差的主要来源。一些与 PCR 相关的因素可能会对 NGS 工具在环境研究和诊断中的应用产生负面影响。 DNA 聚合酶是研究最多的因素之一。在我们的研究中，我们使用属特异性 NGS 方法评估了 DNA 聚合酶类型对细菌群落（更准确地说是军团菌）特征的影响。使用校对高保真 KAPA HiFi 进行的检测比使用广泛使用的非校对 HotStarTaq 进行的检测显示出更好的扩增产量。 HotStarTaq 显着高估了军团菌群落丰富度指标。然而，DNA 聚合酶的选择并没有显着改变群落特征和组成。这些结果证实了在 NGS 检测中校对高保真度 DNA 聚合酶的使用，并强调需要在比较研究和基于 NGS 的诊断工具的未来指南中考虑不同 DNA 聚合酶的影响。
The library preparation step is a major source of bias in NGS-based studies. Several PCR-related factors might negatively influence the application of NGS tools in environmental studies and diagnostics. Among the most understudied factors are DNA polymerases. In our study, we evaluated the effect of DNA polymerase type on the characterisation of bacterial communities, more precisely Legionella, using a genus-specific NGS approach. The assay with proof-reading high fidelity KAPA HiFi showed better amplification yield than the one with widely used non-proofreading HotStarTaq. Legionella community richness metrics were significantly overestimated with HotStarTaq. However, the choice of DNA polymerase did not significantly change the community profiling and composition. These results substantiate the use of proof-reading high fidelity DNA polymerases in NGS assays and highlight the need of considering the impact of different DNA polymerases in comparative studies and future guidelines for NGS-based diagnostic tools.