期刊
MOLECULAR ECOLOGY RESOURCES
卷 20, 期 1, 页码 170-184出版社
WILEY
DOI: 10.1111/1755-0998.13097
关键词
fungal diversity; high-throughput sequencing; metabarcoding; mock communities; polymerase; primer
资金
- Key Research Program of the Chinese Academy of Sciences [KFZD-SW-219-3]
- Key Research Program of Frontier Sciences, CAS [QYZDB-SSW-DQC026]
- Strategic Priority Research Program of the Chinese Academy of Sciences [XDB15010302]
- CAS 135 program [2017XTBG-F01]
- National Natural Science Foundation of China (NSFC) [31870467, U1602234]
- National Key Research and Development Program [2016YFC0500702]
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.
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