Understanding PCR Processes to Draw Meaningful Conclusions from Environmental DNA Studies
Understanding PCR Processes to Draw Meaningful Conclusions from Environmental DNA Studies
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DOI：
10.1038/s41598-019-48546-x
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发表时间：
2019-08-20
影响因子：
4.6
通讯作者：
Gallego, Ramon
中科院分区：
文献类型：
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作者：
Kelly, Ryan P.;Shelton, Andrew Olaf;Gallego, Ramon
随着环境DNA（eDNA）研究在生态学应用中的普及，很明显，它们的结果与传统的非PCR调查有很大的不同。一般来说，依赖于扩增子测序的eDNA研究可能会检测到采样环境中存在的数百个物种，但所产生的物种组成可能是特异性的，反映物种的真实生物量丰度很差或根本没有。在这里，我们使用一组模拟来开发一个机制的理解的过程，导致常见的混合模板PCR为基础的（metabarcoding）研究的结果。特别是，我们专注于PCR循环数和引物扩增效率对测序研究中多样性度量结果的影响。然后，我们表明，比例指数的扩增子读取捕获的趋势，在分类单位的生物量与高精度，特别是在扩增效率高（中位数相关性高达0.97）。我们的研究结果解释了基于PCR的研究中观察到的大部分行为，并为该领域的最佳实践提供了建议。
As environmental DNA (eDNA) studies have grown in popularity for use in ecological applications, it has become clear that their results differ in significant ways from those of traditional, non-PCR-based surveys. In general, eDNA studies that rely on amplicon sequencing may detect hundreds of species present in a sampled environment, but the resulting species composition can be idiosyncratic, reflecting species' true biomass abundances poorly or not at all. Here, we use a set of simulations to develop a mechanistic understanding of the processes leading to the kinds of results common in mixed-template PCR-based (metabarcoding) studies. In particular, we focus on the effects of PCR cycle number and primer amplification efficiency on the results of diversity metrics in sequencing studies. We then show that proportional indices of amplicon reads capture trends in taxon biomass with high accuracy, particularly where amplification efficiency is high (median correlation up to 0.97). Our results explain much of the observed behavior of PCR-based studies, and lead to recommendations for best practices in the field.