What Quality Suffices for Nanopore Metabarcoding? Reconsidering Methodology and Ectomycorrhizae in Decaying Fagus sylvatica Bark as Case Study.

IF 4.2 2区 生物学 Q2 MICROBIOLOGY Journal of Fungi Pub Date : 2024-10-10 DOI:10.3390/jof10100708
Glen Dierickx, Lowie Tondeleir, Pieter Asselman, Kris Vandekerkhove, Annemieke Verbeken
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Abstract

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 Fagus sylvatica. 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 Laccaria amethystina and Tomentella sublilacina 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.

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纳米孔元标码的质量如何?重新考虑方法并以腐烂的欧洲椑树皮中的外生菌根为案例研究。
纳米孔原始读数的准确率已提高到 99% 以上,使其成为一种潜在的元标码工具。为了广泛应用,需要制定质量过滤指南,以确保可靠的分类单元恢复。本研究旨在为真菌元标码提供这些指导原则,并将其应用于Fagus sylvatica腐烂树皮中外生菌根的案例研究。我们引入 eNano 管道来测试两种标准的元标码方法:(1) 利用 UNITE 的物种假设系统(SH 方法)进行基于参考的绘图;(2) 构建 98% 的 OTU(OTU 方法)。我们的研究结果表明,这两种方法对 Nanopore 数据都很有效。在使用参考数据库时,我们建议使用严格的映射标准,而不是基于 Phred 的筛选。利用 SH 系统可进一步提高可重复性,促进跨研究交流。对于 98% 的 OTUs,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,特定的菌根真菌在腐烂的树皮上定植。通过根尖形态分型对代谢编码结果进行补充,我们发现 Laccaria amethystina 和 Tomentella sublilacina 是腐烂原木上树苗的主要外生菌根。这些研究结果表明,纳米孔测序可以提供有价值的生态学见解,并支持随着读数质量的不断提高,在真菌代谢编码中更广泛地使用纳米孔测序。
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来源期刊
Journal of Fungi
Journal of Fungi Medicine-Microbiology (medical)
CiteScore
6.70
自引率
14.90%
发文量
1151
审稿时长
11 weeks
期刊介绍: Journal of Fungi (ISSN 2309-608X) is an international, peer-reviewed scientific open access journal that provides an advanced forum for studies related to pathogenic fungi, fungal biology, and all other aspects of fungal research. The journal publishes reviews, regular research papers, and communications in quarterly issues. Our aim is to encourage scientists to publish their experimental and theoretical results in as much detail as possible. Therefore, there is no restriction on paper length. Full experimental details must be provided so that the results can be reproduced.
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