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Introducing ribosomal tandem repeat barcoding for fungi

Journal article
Authors Christian Wurzbacher
Ellen Larsson
Johan Bengtsson-Palme
Silke Van den Wyngaert
Sten Svantesson
Erik Kristiansson
Maiko Kagami
R. Henrik Nilsson
Published in Molecular Ecology Resources
Volume 19
Issue 2
Pages 118-127
ISSN 1755-098X
Publication year 2019
Published at Department of Mathematical Sciences
Department of Biological and Environmental Sciences
Institute of Biomedicine, Department of Infectious Medicine
Pages 118-127
Language en
Links https://onlinelibrary.wiley.com/doi...
Keywords IGS, Nanopore, PacBio, ribosomal operon, Sanger, third-generation sequencing
Subject categories Biological Systematics

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.

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