Dierickx Glen, Tondeleir Lowie, Asselman Pieter, Vandekerkhove Kris, Verbeken Annemieke
Research Group Mycology, Ghent University, 9000 Gent, Belgium.
Research Institute for Nature and Forest, 9500 Geraardsbergen, Belgium.
J Fungi (Basel). 2024 Oct 10;10(10):708. doi: 10.3390/jof10100708.
Nanopore raw read accuracy has improved to over 99%, making it a potential tool for metabarcoding. For broad adoption, guidelines on quality filtering are needed to ensure reliable taxonomic unit recovery. This study aims to provide those guidelines for a fungal metabarcoding context and to apply them to a case study of ectomycorrhizae in the decaying bark of . We introduce the eNano pipeline to test two standard metabarcoding approaches: (1) Reference-based mapping leveraging UNITE's species hypothesis system (SH approach); (2) Constructing 98% OTUs (OTU approach). Our results demonstrate that both approaches are effective with Nanopore data. When using a reference database, we recommend strict mapping criteria rather than Phred-based filtering. Leveraging the SH-system further enhances reproducibility and facilitates cross-study communication. For the 98% OTUs, filtering reads at ≥Q25 is recommended. Our case study reveals that the decay gradient is a primary determinant of community composition and that specific mycorrhizal fungi colonize decaying bark. Complementing our metabarcoding results with root tip morphotypification, we identify and as key ectomycorrhizae of saplings on decaying logs. These findings demonstrate that Nanopore sequencing can provide valuable ecological insights and support its broader use in fungal metabarcoding as read quality continues to improve.
纳米孔原始读数的准确率已提高到99%以上，使其成为元条形码分析的潜在工具。为了广泛应用，需要质量过滤指南以确保可靠的分类单元恢复。本研究旨在为真菌元条形码分析提供此类指南，并将其应用于腐烂树皮中外生菌根的案例研究。我们引入了eNano管道来测试两种标准的元条形码分析方法：（1）基于参考的映射，利用UNITE的物种假设系统（SH方法）；（2）构建98%的操作分类单元（OTU方法）。我们的结果表明，这两种方法对纳米孔数据均有效。使用参考数据库时，我们建议采用严格的映射标准而非基于Phred的过滤。利用SH系统可进一步提高可重复性并促进跨研究交流。对于98%的OTU，建议在≥Q25时过滤读数。我们的案例研究表明，腐烂梯度是群落组成的主要决定因素，特定的菌根真菌定殖于腐烂树皮中。通过根尖形态分型对我们的元条形码分析结果进行补充，我们确定了 和 为腐烂原木上幼树的关键外生菌根。这些发现表明，随着读数质量不断提高，纳米孔测序可为生态学提供有价值的见解，并支持其在真菌元条形码分析中的更广泛应用。
