Tissue storage and primer selection influence pyrosequencing-based inferences of diversity and community composition of endolichenic and endophytic fungi

Next-generation sequencing technologies have provided unprecedented insights into fungal diversity and ecology. However, intrinsic biases and insufficient quality control in next-generation methods can lead to difficult-to-detect errors in estimating fungal community richness, distributions, and composition. The aim of this study was to examine how tissue storage prior to DNA extraction, primer design, and various quality-control approaches commonly used in 454 amplicon pyrosequencing might influence ecological inferences in studies of endophytic and endolichenic fungi. We first contrast 454 data sets generated contemporaneously from subsets of the same plant and lichen tissues that were stored in CTAB buffer, dried in silica gel, or freshly frozen prior to DNA extraction. We show that storage in silica gel markedly limits the recovery of sequence data and yields a small fraction of the diversity observed by the other two methods. Using lichen mycobiont sequences as internal positive co...下一代测序（Next-generation sequencing）技术为真菌多样性与生态学研究提供了前所未有的见解。然而，下一代测序方法本身存在的内在偏倚与质控不充分问题，可能在估算真菌群落丰度、分布及组成时引发难以检出的误差。本研究旨在探究DNA提取前的组织储存方式、引物设计，以及454扩增子焦磷酸测序（454 amplicon pyrosequencing）中常用的各类质控方法，会如何影响内生真菌（endophytic fungi）与地衣内生真菌（endolichenic fungi）相关研究中的生态学推断。本研究首先对取自同一植物与地衣组织的亚组同期生成的454数据集进行对比，这些亚组在DNA提取前分别采用CTAB缓冲液（CTAB buffer）储存、硅胶干燥或新鲜冷冻三种方式处理。研究结果表明，硅胶干燥储存方式会显著限制序列数据的回收效率，仅能获取另外两种储存方式所观测到的多样性中的极小一部分。我们以地衣真菌共生体序列作为内部阳性对照（原文后续内容未完整呈现）