Improving pathogen analysis by pushing the boundaries of long read sequencing

Rudder, Steven John (2025) Improving pathogen analysis by pushing the boundaries of long read sequencing. Doctoral thesis, University of East Anglia.

Preview

PDF Download (8MB) | Preview

Abstract

Salmonella and Campylobacter are leading foodborne pathogens responsible for gastroenteritis globally, yet their detection and characterisation remain limited by culturing challenges, DNA extraction constraints, and preservation-related biases. Advances in long read sequencing platforms and metagenomic approaches offer exciting opportunities to overcome these barriers by enabling culture-free recovery of complete genomes directly from stool.

This work combined laboratory automation for high molecular weight (HMW) DNA extraction with short- and long- read sequencing to address four key challenges: (i) development of semi-automated Fire Monkey protocols for HMW DNA extraction on a Tecan A200 robotic platform for clinical bacterial isolates and stool, (ii) investigation of within-host diversity of Salmonella enterica from gastroenteritis patients, (iii) evaluation of stool preservation conditions for metagenomic recovery of Campylobacter genomes, and (iv) implementation of HMW-DNA extraction and long-read sequencing from stool for metagenomic recovery of Campylobacter genomes.

Developed Fire Monkey protocols produced DNA of sufficient length and purity for long-read sequencing and hybrid assembly. This enabled single contig bacterial genome to be assembled with DNA extracted from both isolates and stool. Sequencing of up to 20 Salmonella colonies per patient revealed within-host diversity was limited to single nucleotide polymorphisms (SNPs) and antimicrobial resistance gene profiles. In the Campylobacter storage study, stool frozen untreated and stool frozen with glycerol outperformed Zymo DNA/RNA Shield for preserving genome coverage and typing accuracy over nine months at –80 °C. The Fire Monkey stool HMW DNA protocol developed as part of the project enabled recovery of a single-contig Campylobacter genome, which facilitated typing at SNP resolution. Comparative evaluation of Fire Monkey and Maxwell extractions further demonstrated that DNA quality strongly influenced the completeness and reliability of metagenome-derived genomes.

Together, these findings help to inform best practices for public health surveillance, outbreak investigations, and the future integration of metagenomics.

Item Type:	Thesis (Doctoral)
Faculty \ School:	Faculty of Science > School of Biological Sciences
Depositing User:	Chris White
Date Deposited:	02 Feb 2026 14:47
Last Modified:	02 Feb 2026 14:47
URI:	https://ueaeprints.uea.ac.uk/id/eprint/101812
DOI:

Downloads

Actions (login required)

View Item