Key Laboratory of Industrial Ecology and Environmental Engineering (Ministry of Education), School of Environmental Science and Technology, Dalian University of Technology, Dalian, China.
CAS Key Laboratory of Environmental Biotechnology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing, China.
Mol Ecol Resour. 2020 Jan;20(1):170-184. doi: 10.1111/1755-0998.13097. Epub 2019 Nov 6.
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.
随着高通量测序技术的不断提高和真菌参考数据库的不断更新，基于扩增子的 DNA 标记物作为揭示各种生态系统中真菌多样性和组成的工具已成为可能。然而，无论是引物的选择还是实验程序都需要进行仔细的验证。在这里，我们从计算和实验两个方面评估了三种广泛使用或新设计的内部转录间隔区（ITS）引物（ITS1F/ITS2、gITS7/ITS4 和 5.8S-Fun/ITS4-Fun）的准确性和特异性。计算机模拟评估结果表明，由于引物的简并性和位置不同，不同分类水平的引物覆盖度也不同。使用均匀和交错的模拟群落标准品，我们确定了不同引物产生的嵌合体和错配reads 的比例不同，以及物种丰度的巨大差异，这表明引物选择会影响基于扩增子的宏条形码研究的结果。选择具有校对和高保真聚合酶（KAPA HiFi）的引物可以显著降低嵌合体和错配序列的比例，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，结果表明，这三种引物不能在真菌群落组成或多样性上达成共识，而且是引物选择而不是实验处理决定了观察到的土壤真菌群落的多样性和组成。未来的 DNA 标记物调查应更加关注潜在的引物效应，并改进实验方案以提高可信度和准确性。
