Data from: Ion Torrent PGM as tool for fungal community analysis: a case study of endophytes in Eucalyptus grandis reveals high taxonomic diversity

The Kingdom Fungi adds substantially to the diversity of life, but due to their cryptic morphology and lifestyle, tremendous diversity, paucity of formally described specimens, and the difficulty in isolating environmental strains into culture, fungal communities are difficult to characterize. This is especially true for endophytic communities of fungi living in healthy plant tissue. The developments in next generation sequencing technologies are, however, starting to reveal the true extent of fungal diversity. One of the promising new technologies, namely semiconductor sequencing, has thus far not been used in fungal diversity assessments. In this study we sequenced the internal transcribed spacer 1 (ITS1) nuclear encoded ribosomal RNA of the endophytic community of the economically important tree, Eucalyptus grandis, from South Africa using the Ion Torrent Personal Genome Machine (PGM). We determined the impact of various analysis parameters on the interpretation of the results, namely different sequence quality parameter settings, different sequence similarity cutoffs for clustering and filtering of databases for removal of sequences with insufficient taxonomy. Sequence similarity cutoff values only had a marginal effect on the identified family numbers, whereas different sequence quality filters had a large effect (89 vs. 48 families between least and most stringent filters). Database filtering had a small, but statistically significant, effect on the assignment of sequences to reference sequences. The community was dominated by Ascomycota, and particularly by families in the Dothidiomycetes that harbor well-known plant pathogens. The study demonstrates that semiconductor sequencing is an ideal strategy for environmental sequencing of fungal communities. It also highlights some potential pitfalls in subsequent data analyses when using a technology with relatively short read lengths.真菌界极大地丰富了生命多样性，但由于其形态与生活史隐蔽、多样性极高、正式描述的标本稀缺，且难以将环境菌株分离培养，真菌群落的特征分析始终颇具挑战。针对健康植物组织内定殖的内生真菌群落而言，这一难题尤为突出。不过，下一代测序技术的发展正逐步揭示真菌多样性的真实面貌。然而，作为极具前景的新兴技术之一，半导体测序此前尚未被应用于真菌多样性评估。本研究依托南非经济树种巨桉的内生真菌群落，利用离子激流个人基因组测序仪（Ion Torrent Personal Genome Machine，PGM）对其核编码核糖体RNA的内部转录间隔区1（internal transcribed spacer 1，ITS1）进行了测序。我们探究了多种分析参数对结果解读的影响，具体包括不同的序列质量参数设置、用于聚类的序列相似性阈值，以及用于剔除分类学信息不足序列的数据库过滤策略。研究发现，序列相似性阈值对所鉴定的科级分类单元数量仅存在微弱影响，而不同的序列质量过滤策略则会产生显著差异（最宽松与最严格的过滤条件下，鉴定得到的科数分别为89与48）。数据库过滤对序列匹配参考序列的分配结果存在小幅但具有统计学意义的影响。该群落以子囊菌门为主，尤其包含多个隶属于座囊菌纲的类群，其中不乏知名植物病原菌。本研究证实，半导体测序是开展真菌群落环境测序的理想策略，同时也点明了针对该技术较短读长特性开展后续数据分析时可能存在的潜在陷阱。