Correct and fast identification of the agents of mycoses is of great importance to enable early diagnosis and targeted antifungal therapy. DNA barcoding offers an accurate, fast, cost-effective, culture independent approach for species identification. The current primary fungal barcode is the internal transcribed spacer (ITS) region and recently a secondary barcode, the translation elongation factor 1α (TEF1α) has been introduced. The ISHAM-ITS database was extended with the addition of secondary barcode sequences to form the new “ISHAM BARCODE DATABASE”, which currently contains 4200 ITS and 908 TEF1α sequences. The application of the dual DNA barcoding system increases the ability to accurate identification of all clinically important fungal pathogens.

Recently, there has been a fundamental shift away from Sanger sequencing to next generation sequencing (NGS) allowing sequence based identification of complex samples (metabarcoding). One of those new technologies is the nanopore based long reads sequencing. To assess the advantages and pitfalls of the technology in clinical diagnosis we used the MinION^TM, a palm-sized sequencer to sequence DNA directly from human sputum. Our results indicate that false-positive and error-prone reads currently represent a real challenge for metabarcoding studies. To overcome these issues, more accurate taxonomy assignment algorithms and reference databases are needed.