Introducing ribosomal tandem repeat barcoding for fungi

Abstract Sequence analysis of the various ribosomal genetic markers is the dominant molecular method for identification and description of fungi. However, there is little agreement on what ribosomal markers should be used, and research groups utilize different markers depending on what fungal groups are targeted. New environmental fungal lineages known only from DNA data reveal significant gaps in the coverage of the fungal kingdom both in terms of taxonomy and marker coverage in the reference sequence databases. In order to integrate references covering all of the ribosomal markers, we present three sets of general primers that allow the 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) from the 5’ end of the ribosomal operon all the way to the 3’ end. We coupled these primers successfully with third generation sequencing (PacBio and Nanopore sequencing) to showcase our approach on authentic fungal herbarium specimens. 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 need for a 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.