Geisen S, Laros I, Vizcaíno A, Bonkowski M, de Groot G A
Department of Terrestrial Ecology, Netherlands Institute of Ecology (NIOO-KNAW), PO Box 50, 6700 AB, Wageningen, the Netherlands.
Department of Terrestrial Ecology, Institute of Zoology, University of Cologne, Zülpicher Str 47b, 50674, Cologne, Germany.
Mol Ecol. 2015 Sep;24(17):4556-69. doi: 10.1111/mec.13238. Epub 2015 Jun 5.
Protists, the most diverse eukaryotes, are largely considered to be free-living bacterivores, but vast numbers of taxa are known to parasitize plants or animals. High-throughput sequencing (HTS) approaches now commonly replace cultivation-based approaches in studying soil protists, but insights into common biases associated with this method are limited to aquatic taxa and samples. We created a mock community of common free-living soil protists (amoebae, flagellates, ciliates), extracted DNA and amplified it in the presence of metazoan DNA using 454 HTS. We aimed at evaluating whether HTS quantitatively reveals true relative abundances of soil protists and at investigating whether the expected protist community structure is altered by the co-amplification of metazoan-associated protist taxa. Indeed, HTS revealed fundamentally different protist communities from those expected. Ciliate sequences were highly over-represented, while those of most amoebae and flagellates were under-represented or totally absent. These results underpin the biases introduced by HTS that prevent reliable quantitative estimations of free-living protist communities. Furthermore, we detected a wide range of nonadded protist taxa probably introduced along with metazoan DNA, which altered the protist community structure. Among those, 20 taxa most closely resembled parasitic, often pathogenic taxa. Therewith, we provide the first HTS data in support of classical observational studies that showed that potential protist parasites are hosted by soil metazoa. Taken together, profound differences in amplification success between protist taxa and an inevitable co-extraction of protist taxa parasitizing soil metazoa obscure the true diversity of free-living soil protist communities.
原生生物是最多样化的真核生物，在很大程度上被认为是自由生活的食细菌者，但已知有大量的分类群寄生于植物或动物。高通量测序（HTS）方法现在在研究土壤原生生物时普遍取代了基于培养的方法，但对与该方法相关的常见偏差的认识仅限于水生分类群和样本。我们创建了一个由常见的自由生活土壤原生生物（变形虫、鞭毛虫、纤毛虫）组成的模拟群落，提取DNA并在存在后生动物DNA的情况下使用454高通量测序进行扩增。我们旨在评估高通量测序是否能定量揭示土壤原生生物的真实相对丰度，并调查后生动物相关原生生物分类群的共扩增是否会改变预期的原生生物群落结构。事实上，高通量测序揭示的原生生物群落与预期的群落有根本不同。纤毛虫序列高度过度代表，而大多数变形虫和鞭毛虫的序列则代表性不足或完全缺失。这些结果强调了高通量测序引入的偏差，这些偏差阻碍了对自由生活原生生物群落进行可靠的定量估计。此外，我们检测到了一系列可能与后生动物DNA一起引入的未添加的原生生物分类群，这改变了原生生物群落结构。其中，20个分类群与寄生性、通常是致病性的分类群最为相似。因此，我们提供了第一批高通量测序数据，以支持经典的观察性研究，这些研究表明土壤后生动物携带潜在的原生生物寄生虫。综上所述，原生生物分类群之间扩增成功率的巨大差异以及寄生在土壤后生动物上的原生生物分类群不可避免的共提取掩盖了自由生活土壤原生生物群落的真实多样性。
