Song Zewei, Schlatter Dan, Kennedy Peter, Kinkel Linda L, Kistler H Corby, Nguyen Nhu, Bates Scott T
Department of Plant Pathology, University of Minnesota, Saint Paul, MN 55108, United States of America.
Department of Plant Biology, University of Minnesota, Saint Paul, MN 55108, United States of America.
PLoS One. 2015 May 14;10(5):e0127234. doi: 10.1371/journal.pone.0127234. eCollection 2015.
Next generation fungal amplicon sequencing is being used with increasing frequency to study fungal diversity in various ecosystems; however, the influence of sample preparation on the characterization of fungal community is poorly understood. We investigated the effects of four procedural modifications to library preparation for high-throughput sequencing (HTS). The following treatments were considered: 1) the amount of soil used in DNA extraction, 2) the inclusion of additional steps (freeze/thaw cycles, sonication, or hot water bath incubation) in the extraction procedure, 3) the amount of DNA template used in PCR, and 4) the effect of sample pooling, either physically or computationally. Soils from two different ecosystems in Minnesota, USA, one prairie and one forest site, were used to assess the generality of our results. The first three treatments did not significantly influence observed fungal OTU richness or community structure at either site. Physical pooling captured more OTU richness compared to individual samples, but total OTU richness at each site was highest when individual samples were computationally combined. We conclude that standard extraction kit protocols are well optimized for fungal HTS surveys, but because sample pooling can significantly influence OTU richness estimates, it is important to carefully consider the study aims when planning sampling procedures.
下一代真菌扩增子测序正越来越频繁地用于研究各种生态系统中的真菌多样性；然而，样本制备对真菌群落特征的影响却鲜为人知。我们研究了高通量测序（HTS）文库制备过程中四个程序修改的影响。考虑了以下处理方法：1）DNA提取中使用的土壤量，2）提取过程中增加额外步骤（冻融循环、超声处理或热水浴孵育），3）PCR中使用的DNA模板量，4）物理或计算样本合并的影响。来自美国明尼苏达州两个不同生态系统（一个草原和一个森林地点）的土壤用于评估我们结果的普遍性。前三种处理对两个地点观察到的真菌OTU丰富度或群落结构均无显著影响。与单个样本相比，物理合并捕获了更多的OTU丰富度，但当单个样本进行计算合并时，每个地点的总OTU丰富度最高。我们得出结论，标准提取试剂盒方案已针对真菌HTS调查进行了良好优化，但由于样本合并会显著影响OTU丰富度估计，因此在规划采样程序时仔细考虑研究目的很重要。
