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Third-generation sequencing platforms, such as Oxford Nanopore Technology (ONT), have made it possible to characterize communities through the sequencing of long amplicons. While this theoretically allows for an increased taxonomic resolution compared to short-read sequencing platforms such as Illumina, the high error rate remains problematic for accurately identifying the community members present within a sample. Here, we present and validate CONCOMPRA, a tool that allows the detection of closely related strains within a community by drafting and mapping to consensus sequences. We show that CONCOMPRA outperforms several other tools for profiling bacterial communities using full-length 16S rRNA gene sequencing. Since CONCOMPRA does not rely on a sequence database for profiling communities, it is applicable to systems and amplicons for which little to no reference data exists. Our validation test shows that the amplification of long PCR products is likely to produce chimeric byproducts that inflate alpha diversity and skew community structure, stressing the importance of chimera detection. CONCOMPRA is available on GitHub (https://github.com/willem-stock/CONCOMPRA). [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Understanding and describing the diversity of living organisms is a great challenge. Fungi have for a long time been, and unfortunately still are, underestimated when it comes to taxonomic research. The foundations were laid by the first mycologists through field observations. These important fundamental works have been and remain vital reference works. Nevertheless, a non-negligible part of the studied funga escaped their attention. Thanks to modern developments in molecular techniques, the study of fungal diversity has been revolutionized in terms of tools and knowledge. Despite a number of disadvantages inherent to these techniques, traditional field-based inventory work has been increasingly superseded and neglected. This perspective aims to demonstrate the central importance of field-based research in fungal diversity studies, and encourages researchers not to be blinded by the sole use of molecular methods., Competing Interests: TEM and DH were employed by Operation Wallacea Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Cazabonne, Bartrop, Dierickx, Gafforov, Hofmann, Martin, Piepenbring, Rivas-Ferreiro and Haelewaters.)