Improvements in DNA sequencing has impacted research in many ways. Known as “3rd generation” sequencing platforms, PacBio and Oxford Nanopore technologies allow sequencing of very long reads (>100kb) compared to classical Sanger Sequencing (<800bp). Common DNA regions that are sequenced for identifying fungi to a species level include the external transcribed spacer (ETS), small ribosomal subunit (SSU), internal transcribed spacer 1 (ITS1), 5.8S ribosomal RNA sequence (5.8S), internal transcribed spacer 2 (ITS2), large ribosomal subunit (LSU), and intergenic spacer (IGS). These sequences are very close together, and are often found clustered in tandem repeats in fungal genomes known as a ribosomal operon (Fig. 1).
A recent report in Molecular Ecology Resources by Wurzbacher et al. shows the power of 3rd generation sequencing technologies over Sanger Sequencing. By performing PCR and sequencing the entire ribosomal operon (~10kb) instead of sequencing each region individually through Sanger Sequencing, scientists can gain high quality data in a high throughput manner, providing clarity and resolution of fungal species relatedness. They test this new method on herbarium samples of Basidiomycetes, aquatic Chytridiomycetes, and cryptic single-celled fungi in the Nephridiophagidae family