With the continual improvement in high-throughput sequencing technology and constant updates to fungal reference databases, the use of amplicon-based DNA markers as a tool to reveal fungal diversity and composition in various ecosystems has become feasible. However, both primer selection and the experimental procedure require meticulous verification. Here, we computationally and experimentally evaluated the accuracy and specificity of three widely used or newly designed internal transcribed spacer (ITS) primer sets (ITS1F/ITS2, gITS7/ITS4 and 5.8S-Fun/ITS4-Fun). In silico evaluation revealed that primer coverage varied at different taxonomic levels due to differences in degeneracy and the location of primer sets. Using even and staggered mock community standards, we identified different proportions of chimeric and mismatch reads generated by different primer sets, as well as great variation in species abundances, suggesting that primer selection would affect the results of amplicon-based metabarcoding studies. Choosing proofreading and high-fidelity polymerase (KAPA HiFi) could significantly reduce the percentage of chimeric and mismatch sequences, further reducing inflation of operational taxonomic units. Moreover, for two types of environmental fungal communities, plant endophytic and soil fungi, it was demonstrated that the three primer sets could not reach a consensus on fungal community composition or diversity, and that primer selection, not experimental treatment, determines observed soil fungal community diversity and composition. Future DNA marker surveys should pay greater attention to potential primer effects and improve the experimental scheme to increase credibility and accuracy.

中文翻译：

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探索用于真菌群落的基于扩增子的内部转录间隔子标记物的准确性。

随着高通量测序技术的不断改进和真菌参考数据库的不断更新，使用基于扩增子的DNA标记作为揭示各种生态系统中真菌多样性和组成的工具已变得可行。但是，引物的选择和实验程序都需要进行仔细的验证。在这里，我们通过计算和实验评估了三种广泛使用或新设计的内部转录间隔子（ITS）引物组（ITS1F / ITS2，gITS7 / ITS4和5.8S-Fun / ITS4-Fun）的准确性和特异性。在计算机分析中，由于简并性和引物组位置的差异，引物覆盖率在不同的分类学水平上有所不同。使用偶数和交错的模拟社区标准，我们确定了不同比例的引物集产生的嵌合和错配读数的不同比例，以及物种丰度的巨大差异，这表明引物的选择将影响基于扩增子的元条形码研究的结果。选择校对和高保真聚合酶（KAPA HiFi）可以显着降低嵌合和错配序列的百分比，从而进一步减少操作分类单元的膨胀。此外，对于两种类型的环境真菌群落，植物内生真菌和土壤真菌，已证明这三种引物对真菌群落的组成或多样性没有达成共识，并且引物的选择而非实验处理决定了观察到的土壤真菌群落。多样性和组成。