Data from: 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 controls, we next show that despite careful filtering of raw reads and utilization of current best-practice OTU clustering methods, homopolymer errors in sequences representing rare taxa artificially increased estimates of richness ca. 15-fold in a model data set. Third, we show that inferences regarding endolichenic diversity can be improved by using a novel primer that reduces amplification of the mycobiont. Together, our results provide a rationale for selecting tissue treatment regimes prior to DNA extraction, demonstrate the efficacy of reducing mycobiont amplification in studies of the fungal microbiomes of lichen thalli, and highlight the difficulties in differentiating true information about fungal biodiversity from methodological artifacts.

下一代测序技术（next-generation sequencing）为真菌多样性与生态学研究带来了前所未有的认知突破。然而，下一代测序方法固有的偏差与不完善的质量控制，可能会在估算真菌群落丰富度、分布与组成时产生难以察觉的误差。本研究旨在探讨DNA提取前的组织保存方式、引物设计，以及454扩增子焦磷酸测序（454 amplicon pyrosequencing）中常用的各类质量控制方法，会如何影响内生真菌与地衣内生真菌相关研究中的生态学推断。我们首先对同期生成的454数据集进行对比，这些数据集取自同一批植物与地衣组织的亚样本，这些亚样本在DNA提取前分别以CTAB缓冲液（CTAB buffer）保存、硅胶干燥或新鲜冷冻处理。研究结果表明，硅胶干燥保存会显著限制序列数据的回收率，仅能获取另外两种处理方式所观测到的多样性的极小一部分。以地衣真菌共生体序列作为内部阳性对照，我们进一步证实：即便已严格过滤原始读段（reads）并采用当前主流的最佳实践操作分类单元（OTU）聚类方法，代表稀有类别的序列中的均聚物错误，仍会在模型数据集中使丰富度估算值被人为抬高约15倍。第三，我们证实，使用可降低真菌共生体扩增的新型引物，能够优化关于地衣内生真菌多样性的生态学推断。综上，本研究结果为DNA提取前的组织处理方案选择提供了理论依据，证实了在地衣体真菌微生物组研究中降低真菌共生体扩增的有效性，并凸显了从方法学伪影中甄别真菌生物多样性真实信息的难点。