Rapid and real-time identification of fungi up to species level with long amplicon nanopore sequencing from clinical samples

Abstract

The availability of long-read technologies, like Oxford Nanopore Technologies, provides the opportunity to sequence longer fragments of the fungal ribosomal operon, up to 6 Kb (18S-ITS1-5.8S-ITS2-28S) and to improve the taxonomy assignment of the communities up to species level and in real-time. We assess the applicability for taxonomic assignment of amplicons targeting a 3.5 Kb region (V3 18S-ITS1-5.8S-ITS2-28S D2) and a 6 Kb region (V1 18S-ITS1-5.8S-ITS2-28S D12) with the What's in my pot (WIMP) classifier. We used the ZymoBIOMICS^TM mock community and different microbiological fungal cultures as positive controls. Long amplicon sequencing correctly identified <i>Saccharomyces cerevisiae</i> and <i>Cryptococcus neoformans</i> from the mock community and <i>Malassezia pachydermatis</i>, <i>Microsporum canis</i> and <i>Aspergillus fumigatus</i> from the microbiological cultures. Besides, we identified <i>Rhodotorula graminis</i> in a culture mislabelled as <i>Candida</i> spp. We applied the same approach to external otitis in dogs. <i>Malassezia</i> was the dominant fungal genus in dogs' ear skin, whereas <i>Ma. pachydermatis</i> was the main species in the healthy sample. Conversely, we identified a higher representation of <i>Ma. globosa</i> and <i>Ma. sympodialis</i> in otitis affected samples. We demonstrate the suitability of long ribosomal amplicons to characterize the fungal community of complex samples, either healthy or with clinical signs of infection.