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Gauthier, Nicholas

University of British Columbia

The application of next-generation sequencing technologies to answer more routine diagnostic and applied microbiological questions is becoming increasingly feasible. Current reference-standard molecular diagnostic strategies are limited in that they require a priori knowledge of a pathogen’s genome. Metagenomic next generation sequencing (mNGS) is a pathogen-agnostic diagnostic approach that can be used for detection of viral pathogens and characterization of viral infections. Chapter 1 of this thesis describes technological advancements in mNGS technology for viral pathogen detection and diagnosis. This narrative review highlights technical, logistical, and translational barriers to the widespread use of this technology in clinical settings. In chapter 2, I describe the use of full-length 16S rRNA nanopore sequencing for characterization of the upper respiratory tract microbiome in hospitalized and community-dwelling individuals with and without active SARS-CoV-2 infection. We found significant differences in beta-diversity, but not alpha-diversity in our study groups and identified several differentially abundant taxa associated with SARS-CoV-2 infection status and disease severity. Chapter 3 outlines the feasibility and performance of long-read mNGS as a diagnostic tool for detection and characterization of SARS-CoV-2. We reported that mNGS is a highly specific method for SARS-CoV-2 detection and is sensitive for specimens with higher viral loads. We showed that this technology can also be used to characterize viral genomes. Finally, chapters 4 and 5 outline the development, optimization, and validation of a novel mNGS assay for detection of viral pathogens. This assay was developed to address translational barriers to mNGS adoption. We developed and clinically validated an assay that is sensitive, specific, rapid, cost-effective, and inclusive. In addition to successfully detecting four known viral pathogens, the assay detected a total of nine respiratory pathogens that were missed by conventional molecular diagnostic testing when originally tested. In summary, the work presented in this thesis highlights the feasibility of using next-generation sequencing workflows in routine clinical service for diagnosis of viral pathogens and characterization of viral infections. This thesis represents novel, applied work that contributes to the field of microbiology and can have immediate impact on clinical microbiology and diagnostic services for human and animal health and public health surveillance.

Text

2024-04-04

Attribution-NoDerivatives 4.0 International

http://hdl.handle.net/2429/87692

Microbiology and Immunology

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nd/4.0/