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（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，下一代测序方法固有的偏倚与质量控制不足，可能导致真菌群落丰富度、分布及组成估算中难以被检出的误差。本研究旨在探讨DNA提取前的组织保存方式、引物设计（primer design），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，对内生真菌与地衣内生真菌（endophytic and endolichenic fungi）研究中的生态学推断所产生的影响。
我们首先对比了同期制备的同一批植物与地衣组织亚样本所生成的454数据集：这些亚样本分别保存于CTAB缓冲液（CTAB buffer）、经硅胶（silica gel）干燥，或在DNA提取前进行新鲜冷冻。结果显示，硅胶干燥保存会显著降低序列数据的回收率，仅能检出另外两种保存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参阳性对照（internal positive controls），我们进一步发现，尽管已对原始reads（raw reads）开展严格过滤，并采用当前通用的最佳实践OTU聚类方法，代表稀有类群的序列中的均聚物错误（homopolymer errors）仍会使模型数据集的丰富度估计值虚高约15倍。
第三，我们证实，使用可抑制真菌共生体扩增的新型引物，能够优化地衣内生真菌多样性的相关研究推断。综上，我们的研究为DNA提取前的组织处理方案选择提供了理论依据，证明了在地衣体（lichen thalli）真菌微生物组（fungal microbiomes）研究中降低真菌共生体扩增的有效性，并凸显了区分真菌生物多样性的真实信息与方法学伪影（methodological artifacts）的难度。