Application of Long-Read Sequencing Technologies to the Diagnosis of Pathogenic Fungal Species

Abstract

The advent of long-read sequencing technologies changed the landscape of next generation sequencing technologies which had previously been dominated by short-read sequencers. These can be applied using a metabarcoding approach, which involves the sequencing of amplified genomic regions, or metagenomic approach, in which all genomic material is sequenced. These new technologies, capable of ultra-long read lengths have a high potential for their application to fungal diagnostics, however, the application to the identification of pathogenic fungal species is currently limited. This thesis aims to contribute to the improvement of metabarcoding and metagenomic long-read sequencing of pathogenic fungal species.

Chapter 1 outlines the current routine diagnostic methods, the long-read sequencing technologies, and their application to fungal identification. Chapter 2 describes the generation of barcode sequences to improve the database of fungal reference barcodes. The ISHAM Barcoding database was extended in Chapter 3, to contain both primary and secondary fungal barcode reference sequences. Chapter 4 compares the barcodes and shows that the secondary fungal barcode improves identification in all analysed taxa. Chapter 5 establishes a new bioinformatic tool for the identification of fungal species. Chapter 6 investigates a metagenomic sequencing approach using Oxford Nanopore Technology MinION flow cells to identify fungal pathogens from clinical samples and Chapter 7 reveals the inability of this new flow cell to identify fungal pathogens in the clinical samples sequenced. Chapter 8 compares DNA extraction and human DNA depletion methods to develop an optimised workflow that improves fungal identification from spiked blood samples. In Chapter 9 bioinformatic tools and databases are compared to determine a workflow for successful fungal identification. Chapter 10 summarises the findings within this thesis and provides future directions to progress this research field.