Introducing ribosomal tandem repeat barcoding for fungi

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. K E Y W O R D S discussions on the implementation of long-read sequencing. The authors would like to acknowledge support from Science for Life Laboratory, the National Genomics Infrastructure, NGI and Uppmax for providing assistance in massive parallel sequencing and computational infrastructure. CW and RHN gratefully acknowledge financial support from Stiftelsen Olle Engkvist Byggmästare, Stiftelsen Lars Hiertas Minne, Kapten Carl Stenholms Donationsfond and Birgit och Birger Wålhströms Minnesfond.

Fungi in aquatic ecosystems

“…Sanger and high throughput sequencing) have played a central role in expanding our understanding of the dimensions of aquatic fungal diversity by accessing DNA sequences from environmental samples. However, since the early diverging fungal lineages do not share a consistent barcode and suffer from severe reference database gaps, a unifying ribosomal barcode is currently being established and refined 30,47 for third generation long-read sequencers (e.g. PacBio, Oxford Nanopore Technologies).…”

“…32,33 ). Furthermore, when coupled to single cell microscope inspections, third generation long-read sequencing may help to close the huge reference data gaps for aquatic fungi 47,86 . The increased read length allows to sequencing whole genes (e.g., the complete ribosomal operon) or gene clusters, which facilitate a potential subspecies level of taxonomic resolution.…”

“…Chytridiomycota and Rozellomycota 13,[43][44][45] . In particular, the latter is hyperdiverse and poorly known, which requires new explorative approaches such as single cell genome sequencing 47 . This will allow for a better understanding of the evolution of the entire fungal kingdom.…”

“…This 'annotation gap' does not only affect functional genes, but is even clearly visible in curated fungal barcode reference data. Currently, attempts have been made towards unifying all ribosomal regions into one single ribosomal fungal marker that covers all of the eukaryotic ribosomal regions at once 30,47 , which greatly improves taxonomic resolution of non-cultured and unknown fungi. However, we want to highlight that there is a great need for increased cultivation of fungal strains both for phylogenetic and physiological characterization which allows for linking phylogenetic, morphological as well as physiological features in a reliable manner.…”

Identification of Mycoses in Developing Countries

“…Ashikawa et al [157] used nanopore sequencing to identify five species of Candida in positive blood culture bottles and compared its performance with Sanger sequencing. Wurzbacher et al [158] used nanopore sequencing to screen fungal herbarium specimens for a rarely studied fungal ribosomal repeat (intergenic spacer, IGS), which is longer than the internal transcribed spacer (ITS), but it is not a good choice for Sanger sequencing due to the numerous reactions that are required to cover the length, time it takes to complete the multiple reactions, and costs. The MinION sequencer easily processes these regions, as this platform can sequence templates hundreds of kilobases long, and showed similar accuracy to Sanger sequencing [159].…”