title: Rapid and precise detection of gastrointestinal infections using metagenome sequencing
Creators: name: Trivett, Hannah
Publisher: name: University of Liverpool Repository
Year: 2024

description: Metagenomic sequencing is the study of all nucleotide sequences of all organisms contained within a sample. Typically, metagenomic studies look at microorganisms in a specific environment; the specific environment of interest for this thesis is the gastrointestinal tract. Since its introduction in the early 2000s, this ‘omics method has begun to transition from research into clinical laboratories as a diagnostic and surveillance tool. However, medical microbiology laboratories still favour traditional culture-dependent molecular diagnostic methods over culture-independent sequencing; therefore, this project evaluates long-read metagenomic sequencing as an alternative approach for culture-independent rapid and accurate pathogen detection. 
Semi-structured interviews were conducted using an interpretive phenomenological approach to understand the utility of integrating new diagnostic technologies into clinical settings. Stakeholders reflected on the barriers to incorporating new diagnostics, which included standardisation, cost, and communication. These discussions provided potential guidance for the user requirements of metagenomic sequencing to integrate into the current diagnostic landscape.
Applying the guidance from the qualitative research, 15 stool samples underwent PacBio long-read metagenomic HiFi sequencing to develop a clinically useful pathogen identification workflow. Of these samples, 12 possessed gastrointestinal pathogens, characterised using traditional molecular diagnostics. The study revealed that high-quality draft genomes could be resolved for the pathogens of interest from shotgun metagenomic data and had the potential to provide intraspecies diversity, where more than one metagenome-assembled genome of the same species was resolved from the same faecal sample. 
The main goal of the workflow was to identify clinically relevant pathogens directly from faecal samples, with additional virulence gene identification and antimicrobial susceptibility profiles, to assist in treatment management and clinical decision-making. Shotgun metagenomics was validated using a selection of extraintestinal E. coli isolates as a negative control for virulence gene profiling and Shigella isolates as a positive control. This investigation highlighted the challenge of profiling enteric pathogens when virulence genes are encoded on extrachromosomal elements. Virulence genes could only be identified within the metagenomic assembly, providing a virulence profile for the metagenome, which could be used to infer the presence of E. coli pathotypes in the metagenome.
To supplement the shotgun metagenomics data, comprehensive profiles of five faecal samples were achieved through Hi-C. The study highlighted the success of Hi-C in resolving strain-level metagenome-assembled genomes, extrachromosomal linkage, and virulence and antimicrobial resistance gene profiling. The addition of Hi-C enabled the identification of co-occurring E. coli pathotypes in the gastrointestinal tract, along with novel EAEC/ETEC strains. 
The ability to successfully extract the gastrointestinal pathogens of interest highlights the potential for using long-read metagenomic sequencing to provide comprehensive metagenomic profiles, which can be used to identify pathogens and antimicrobial resistance genotypes from a single sequencing run. This body of evidence is crucial as proof of principle for the capabilities of long-read metagenomic sequencing for infection treatment and public health management. Whilst this project focuses on gastrointestinal infections, this agnostic approach to pathogen identification could be applied to various infectious diseases, adding to the armoury of current diagnostic tests available in clinical laboratories to provide a comprehensive yet efficient diagnostic in its approach to pathogen identification and treatment.