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Mol Ecol Resour. 2018 Sep 21. doi: 10.1111/1755-0998.12944. [Epub ahead of print]

Introducing ribosomal tandem repeat barcoding for fungi.

Author information

1
Department of Biological and Environmental Sciences, University of Gothenburg, Göteborg, Sweden.
2
Chair of Urban Water Systems Engineering, Technical University of Munich, Garching, Germany.
3
Gothenburg Global Biodiversity Centre, Göteborg, Sweden.
4
Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, Wisconsin.
5
Department of Infectious Diseases, Institute of Biomedicine, The Sahlgrenska Academy, University of Gothenburg, Göteborg, Sweden.
6
Leibniz-Institute of Freshwater Ecology and Inland Fisheries Berlin, Stechlin, Germany.
7
Department of Mathematical Sciences, Chalmers University of Technology and University of Gothenburg, Göteborg, Sweden.
8
Department of Environmental Science, Faculty of Science, Toho University, Funabashi, Japan.
9
Graduate School of Environment and Information Sciences, Yokohama National University, Hodogayaku, Yokohama, Japan.

Abstract

Sequence comparison and analysis of the various ribosomal genetic markers are the dominant molecular methods for identification and description of fungi. However, new environmental fungal lineages known only from DNA data reveal significant gaps in our sampling of the fungal kingdom in terms of both taxonomy and marker coverage in the reference sequence databases. To facilitate the integration of reference data from all of the ribosomal markers, we present three sets of general primers that allow for amplification of the complete ribosomal operon from the ribosomal tandem repeats. The primers cover all ribosomal markers: ETS, SSU, ITS1, 5.8S, ITS2, LSU and IGS. We coupled these primers successfully with third-generation sequencing (PacBio and Nanopore sequencing) to showcase our approach on authentic fungal herbarium specimens (Basidiomycota), aquatic chytrids (Chytridiomycota) and a poorly understood lineage of early diverging fungi (Nephridiophagidae). In particular, we were able to generate high-quality reference data with Nanopore sequencing in a high-throughput manner, showing that the generation of reference data can be achieved on a regular desktop computer without the involvement of any large-scale sequencing facility. The quality of the Nanopore generated sequences was 99.85%, which is comparable with the 99.78% accuracy described for Sanger sequencing. With this work, we hope to stimulate the generation of a new comprehensive standard of ribosomal reference data with the ultimate aim to close the huge gaps in our reference datasets.

KEYWORDS:

IGS; Nanopore; PacBio; Sanger; ribosomal operon; third-generation sequencing

PMID:
30240145
DOI:
10.1111/1755-0998.12944

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