Application of rapid sequencing for the detection and epidemiology of respiratory pathogens

Aydin, Alp (2022) Application of rapid sequencing for the detection and epidemiology of respiratory pathogens. Doctoral thesis, University of East Anglia.

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Abstract

Lower respiratory tract infections (LRTI) are a leading cause of morbidity and mortality globally, and the rise of Antimicrobial Resistance (AMR) complicates their treatment. To achieve the best patient outcomes and avoid contributing to the rise of AMR, timely and appropriate antimicrobial treatment needs to be prescribed. However, the current gold standard for aetiological investigation of LRTIs (microbiological culture) is too slow to guide initial therapy.

Clinical metagenomics (CMg) has emerged as a potential solution to this problem; however, existing methods are too laborious. In this study, we optimise our previously published CMg pipeline to achieve a sensitive workflow with a 3.5 hour turnaround time. Evaluating the workflow, we show efficient depletion (>99.8%) of host DNA with our new 15 minute host depletion method. Sensitivity and specificity are 90.5% and 62.5%, respectively, rising to 96.6% and 100% when qPCR is used to investigate discordance. We also show that 30 minutes of sequencing is sufficient to make an accurate pathogen call.

For pathogen surveillance, targeted sequencing approaches are more appropriate. Sequencing of SARS-CoV-2 for genomic epidemiology became a valuable tool during the ongoing COVID-19 pandemic. However, early on, methods were low-throughput and inflexible. We responded to this by developing a high-throughput library preparation method, CoronaHiT, which can be used for sequencing SARS-CoV-2 on Illumina or Oxford Nanopore Technologies platforms. The method was shown to be cheap and accurate, while also being more robust for samples with lower viral loads. CoronaHiT has subsequently been used to sequence hundreds of thousands of SARS-CoV-2 genomes in the UK.

In conclusion, we have developed and optimised two different approaches for investigating respiratory infections (CMg and targeted) for two different applications, demonstrating the potential of rapid sequencing. Methods like these will continue to reshape diagnostics and public health in the future.

Item Type:	Thesis (Doctoral)
Faculty \ School:	Faculty of Medicine and Health Sciences > Norwich Medical School
Depositing User:	Chris White
Date Deposited:	16 Feb 2023 15:09
Last Modified:	16 Feb 2023 15:09
URI:	https://ueaeprints.uea.ac.uk/id/eprint/91157
DOI:

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