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Evaluating the use of a nanopore metagenomic sequencing approach for the detection and characterisation of infectious causes of disease
Tattersall, M. M. 2025. Evaluating the use of a nanopore metagenomic sequencing approach for the detection and characterisation of infectious causes of disease. Unpublished masters thesis. Stellenbosch: Stellenbosch University [online]. Available: https://scholar.sun.ac.za/items/62249438-b7ed-4001-9...
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Stellenbosch : Stellenbosch University
2025
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| access_status_str | Open Access |
| author | Tattersall, Megan May |
| author2 | Moleleki, Malefu |
| author_browse | Moleleki, Malefu Tattersall, Megan May |
| author_facet | Moleleki, Malefu Tattersall, Megan May |
| author_sort | Tattersall, Megan May |
| collection | Thesis |
| dc_rights_str_mv | Stellenbosch University |
| description | Tattersall, M. M. 2025. Evaluating the use of a nanopore metagenomic sequencing approach for the detection and characterisation of infectious causes of disease. Unpublished masters thesis. Stellenbosch: Stellenbosch University [online]. Available: https://scholar.sun.ac.za/items/62249438-b7ed-4001-995a-0bf1b7d5daae |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/132410 |
| institution | Stellenbosch University (South Africa) |
| language | English |
| last_indexed | 2026-06-10T12:43:30.254Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from SUNScholar — Stellenbosch University Repository |
| publishDate | 2025 |
| publishDateRange | 2025 |
| publishDateSort | 2025 |
| publisher | Stellenbosch : Stellenbosch University |
| publisherStr | Stellenbosch : Stellenbosch University |
| record_format | dspace |
| source_str | SUNScholar — Stellenbosch University Repository |
| spelling | oai:scholar.sun.ac.za:10019.1/132410 Evaluating the use of a nanopore metagenomic sequencing approach for the detection and characterisation of infectious causes of disease Tattersall, Megan May Moleleki, Malefu Van Zyl, Kristien Nel Nel, Pieter Stellenbosch University. Faculty of Science. Dept. Department of Pathology. Medical Microbiology. Pathogenic microorganisms -- Identification Metagenomics High-throughput nucleotide sequencing UCTD Tattersall, M. M. 2025. Evaluating the use of a nanopore metagenomic sequencing approach for the detection and characterisation of infectious causes of disease. Unpublished masters thesis. Stellenbosch: Stellenbosch University [online]. Available: https://scholar.sun.ac.za/items/62249438-b7ed-4001-995a-0bf1b7d5daae Thesis (MSc)--Stellenbosch University, 2025. ENGLISH ABSTRACT: Introduction: Target-specificity and microorganism viability are limitations of traditional methods for pathogen detection, which need to be addressed to improve public health and the overall impact of infectious diseases. Metagenomic next generation sequencing (mNGS) could circumvent the challenges associated with traditional methods, improving pathogen detection and reducing the number of false negative results. However, the lack of protocol standardisation, presence of host DNA, and the difficulty in interpreting results need to be addressed. This study aimed to evaluate an Oxford Nanopore Technologies (ONT) mNGS approach for the detection and characterisation of pathogens in a range of clinical samples at a tertiary hospital in South Africa. Methods: Protocol verification involved comparing the mNGS relative abundance (RA) results of 13 clinical specimens to routine culture results, which were supplemented with ONT whole genome sequencing (WGS) of the corresponding bacterial isolates. Adaptive sampling was implemented during mNGS, to reduce the proportion of human reads generated, followed by further removal using Bowtie2. Antimicrobial resistance (AMR) associated genes and virulence factors were identified and compared to phenotypic and WGS-based results. The mNGS protocol was then evaluated on 48 clinical specimens (20 culture positive and 28 culture negative). The mNGS results were compared to routine culture results for pathogen identification and AMR. Results: Of the 13 specimens tested for protocol verification, mNGS identified multiple organisms in most specimens, with species matching culture being the most dominant in terms of abundance in 11/13 (85%) specimens. mNGS and culture AMR profiles matched in 31% (4/13) of samples. No virulence genes were detected in 4/13 (31%) mNGS samples with low read counts, that had WGS gene identification. Of the 48 specimens tested, following protocol verification, culture identified a pathogen in 42% (20/48) of specimens while mNGS identified a potential pathogen in 54% (26/48). Some of the organisms identified could be clinically significant; however, aetiology could not be confirmed in this study. mNGS detected microorganisms in 38% (11/28) of culture negative specimens. Culture and mNGS results were congruent in 45% (9/20) of culture positive specimens; however, the cultured organism was rarely dominant in terms of abundance on mNGS (15% [3/20]). Of the 11/20 (55%) culture positive samples with phenotypic resistance, 5/11 (45%) had corresponding AMR-associated genes detected using mNGS. Antimicrobial resistance genes were also detected among three of the culture negative samples (3/10, 30%) in which a microorganism was identified using mNGS. Conclusion: The mNGS protocol verification indicated comparable performance to culture and WGS for pathogen identification. The mNGS protocol evaluation indicated a potential pathogen in 48% of the specimens, including 29% of culture negative samples. Minimal correlation was observed between mNGS and phenotypic AMR profiles, indicating that mNGS, alone, may not be a suitable approach for describing AMR results. The overall congruency of pathogen identification using mNGS and culture during protocol evaluation was moderate, with CSF being the most congruent specimen type. Discrepancies may be attributed to low DNA input and quality or contamination. While the study indicates that RA could be useful as an indicator of the pathogens of clinical relevance, potential pathogens detected using mNGS must be assessed in conjunction with additional laboratory and clinical information on a case-by-case basis. AFRIKAANSE OPSOMMING: Inleiding: Teikenspesifisiteit en lewensvatbaarheid van mikro-organismes is beperkings van tradisionele metodes vir patogeenopsporing, wat aangespreek moet word om openbare gesondheid en die algehele impak van aansteeklike siektes te verbeter. Metagenomiese volgende-generasie-volgordebepaling (mNGS) kan die uitdagings wat met tradisionele metodes geassosieer word vermy, patogeenopsporing verbeter, en die aantal vals negatiewe resultate verminder. Die gebrek aan protokolstandaardisering, teenwoordigheid van gasheer-DNA en die moeilike interpretasie van resultate moet egter aangespreek word. Die doel van hierdie studie was om 'n Oxford Nanopore Technologies (ONT) mNGS-benadering vir die opsporing en karakterisering van patogene in 'n reeks kliniese monsters by 'n tersie re hospitaal in Suid-Afrika te evalueer. Metodes: Protokolverifikasie het die vergelyking van die mNGS relatiewe hoeveelheid (“Relative abundance” [RA]) resultate van 13 kliniese monsters met roetinekultuurresultate behels, en is met ONT-heelgenoomvolgordebepaling (WGS) van die ooreenstemmende bakterie le isolate aangevul. “Adaptive sampling” is tydens mNGS geï mplementeer, om die persentasie menslike volgordebepalingsdata wat gegenereer word te verminder; dit is gevolg deur verdere verwydering van menslike volgordebepalingsdata met behulp van Bowtie2. Gene geassosieer met antimikrobiese weerstand (AMR) en virulensiefaktore is geï dentifiseer en vergelyk met fenotipiese- en WGS-gebaseerde resultate. Die mNGS-protokol is daarna op 48 kliniese monsters (20 kultuurpositief en 28 kultuurnegatief) gee valueer. Die mNGS-resultate met betrekking tot patogeenidentifikasie en AMR, is met roetine-kultuurresultate vergelyk. Resultate: Van die 13 monsters wat tydens protokolverifikasie getoets is, het mNGS veelvuldige organismes in die meeste monsters geï dentifiseer. Spesies wat ooreengestem het met kultuur was die mees dominante organisme, in terme van RA, in 11/13 (85%) monsters. mNGS en kultuur AMR-profiele het in 31% (4/13) van monsters ooreengestem. Geen virulensiegene is in 4/13 (31%) mNGS-monsters met lae leestellings wat wel WGSgeenidentifikasie gehad het, opgespoor nie. Van die 48 monsters wat getoets is tydens protokolverifikasie, het kultuur in 42% (20/48) van monsters ’n patogeen geï dentifiseer, terwyl mNGS 'n potensie le patogeen in 54% geï dentifiseer het (26/48). mNGS het mikroo rganismes in 38% (11/28) van kultuurnegatiewe monsters opgespoor. Kultuuren mNGS-resultate het in 45% (9/20) van kultuurpositiewe monsters ooreengestem; die gekweekte organisme was egter selde dominant in terme van RA met mNGS (15% [3/20]). Van die 11/20 (55%) kultuurpositiewe monsters met fenotipiese weerstand, het mNGS in 5/11 (45%) ooreenstemmende AMR-geassosieerde gene opgespoor. Antimikrobiese weerstandsgene is ook in drie van die kultuurnegatiewe monsters (3/10, 30%) waarin 'n mikro-organisme met behulp van mNGS geï dentifiseer is, opgespoor. Gevolgtrekking: Die mNGS-protokolverifikasie dui dat kultuur en WGS vir patogeenidentifikasie vergelykbaar is. Die mNGS-protokolevaluering het 'n potensie le patogeen in 48% van die monsters opgespoor, insluitend 29% van kultuurnegatiewe monsters. Minimale korrelasie is tussen mNGS en fenotipiese AMR-profiele waargeneem, wat aandui dat mNGS alleen dalk nie 'n geskikte benadering is om AMR-resultate te beskryf nie. Die algehele ooreenstemming van patogeenidentifikasie met behulp van mNGS en kultuur tydens protokolevaluering was matig; die monstertipe met die beste ooreenstemming was serebrospinale vloeistof. Teenstrydige resultate kan toegeskryf word aan lae DNAinsette en kwaliteit, of kontaminasie. Terwyl die studie aandui dat RA nuttig kan wees as 'n aanduiding van kliniese relevante patogene, moet potensie le patogene wat met behulp van mNGS opgespoor word, van geval tot geval met behulp van addisionele laboratoriumen kliniese inligting beoordeel word. Masters 2025-06-06T08:03:29Z 2025-06-06T08:03:29Z 2025-03 Thesis https://scholar.sun.ac.za/handle/10019.1/132410 en Stellenbosch University 107 pages : illustrations application/pdf Stellenbosch : Stellenbosch University |
| spellingShingle | Pathogenic microorganisms -- Identification Metagenomics High-throughput nucleotide sequencing UCTD Tattersall, Megan May Evaluating the use of a nanopore metagenomic sequencing approach for the detection and characterisation of infectious causes of disease |
| title | Evaluating the use of a nanopore metagenomic sequencing approach for the detection and characterisation of infectious causes of disease |
| title_full | Evaluating the use of a nanopore metagenomic sequencing approach for the detection and characterisation of infectious causes of disease |
| title_fullStr | Evaluating the use of a nanopore metagenomic sequencing approach for the detection and characterisation of infectious causes of disease |
| title_full_unstemmed | Evaluating the use of a nanopore metagenomic sequencing approach for the detection and characterisation of infectious causes of disease |
| title_short | Evaluating the use of a nanopore metagenomic sequencing approach for the detection and characterisation of infectious causes of disease |
| title_sort | evaluating the use of a nanopore metagenomic sequencing approach for the detection and characterisation of infectious causes of disease |
| topic | Pathogenic microorganisms -- Identification Metagenomics High-throughput nucleotide sequencing UCTD |
| url | https://scholar.sun.ac.za/handle/10019.1/132410 |
| work_keys_str_mv | AT tattersallmeganmay evaluatingtheuseofananoporemetagenomicsequencingapproachforthedetectionandcharacterisationofinfectiouscausesofdisease |