MinION application: performing long fragment analysis on pure fungal cultures (3.5 kb and 6 Kb) and genome analysis of Malassezia pachydermatis
Sara described how taxonomic resolution of fungi at the species level using short-read amplicon sequencing is challenging; marker genes amplified for fungal species identification typically include ITS1, ITS2, 18S rRNA gene, or 28S rRNA gene.Sara explained how she has used nanopore sequencing of long amplicons for the detection of fungal communities from the skin of dogs.PCR amplicons covering both the complete ITS1-5.8S-ITS2 locus (3.5 kb), and the entire fungal ribosomal operon (18S-ITS1-5.8S-ITS2-28S) (6 kb) were sequenced. Her sequencing and analysis workflow involved: library preparation using the PCR Barcoding Kit I (EXP-PBC001) and Ligation Sequencing Kit 1D (SQK-LSK109); MinION sequencing (FLO-MIN106 flow cells); basecalling with Albacore or Guppy v2.3.5; Porechop v0.2.3 QC; and EPI2ME's 'What’s in my pot (WIMP)' for fungal species identification.
From the microbial cultures, the predominant species identified were: Malassezia pachydermatis, Cryptococcus neoformans, yeast Saccharomyces cerevisiae, and Microsporum canis. Except for M. canis, 100% coverage of both the ITS locus and entire operon was obtained. Taxonomic identification at the species level was improved with sequencing of the entire ribosomal operon. Aspergillus species were also detected, including Aspergillus fumigatus.
Sara next explained the application of her workflow for species resolution in complex Malassezia fungal cultures from dog skin samples. Malassezia is a fungus commonly found on the skin of dogs and is thought to be associated with diseases such as atopic dermatitis and otitis externa. Sara successfully detected the etiological agent, M. pachydermatis, in complex otitis culture samples using her nanopore sequencing workflow and Canu read alignment. Her future work will be to perform whole-genome sequencing and de novo assembly of M. pachydermatis. She stated that there is also a significant need to update fungal databases. To end her talk Sara suggested that you need to be patient when studying fungal diseases as often "you are not finding what you are expecting".