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Detection of microorganisms and genes conferring antibiotic resistance in human plasma using nanopore sequencing with MinION for early sepsis diagnosis
University of Skövde, School of Bioscience.
2025 (English)Independent thesis Basic level (degree of Bachelor), 20 credits / 30 HE creditsStudent thesis
Abstract [en]

Sepsis is a serious condition caused by a dysregulated immune reaction. It is a common cause of death and can have many long-lasting health consequences for the survivors. The lack of early and appropriate treatment options decreases the chances of survival. The current sepsis diagnostic is based on time-consuming blood culturing (BC), with low sensitivity. Therefore, the ongoing research on sepsis focuses largely on finding new biomarkers and methods for early sepsis diagnosis. One of the promising biomarkers is cell-free DNA (cfDNA), which can provide information about the current condition as well as the prognosis of the patient. This project aimed to analyse plasma samples, non-spiked and spiked with microbial DNA, through sequencing with the MinION device to evaluate the effectiveness of nanopore sequencing for detecting different species and genes conferring antibiotic resistance. To achieve this, the plasma isolated from healthy human blood was spiked with different amounts of microbial DNA. The total DNA was extracted from the spiked and non-spiked plasma samples. The extracted DNA was sequenced using the MinION device and a Flongle flow cell. The obtained data was analysed with EPI2ME wf-metagenomics, and the Bacterial and Viral Bioinformatics Resource Center (BV-BRC) Taxonomic Classification (TC) and Metagenomic Read Mapping (MRM) tools. The microorganisms were identified at the species and genus levels. The identification of genes conferring antibiotic resistance was not as efficient as species identification, allowing for the detection of a fraction of the expected genes. The reason could be the low quality of reads arising from high fragmentation of DNA. It might be due to a low concentration of extracted DNA, resulting in highly fragmented DNA during library preparation. An optimisation of the procedure or the use of a different sequencing kit might improve the results. More research, however, is needed to assess that.

Place, publisher, year, edition, pages
2025. , p. 54
National Category
Microbiology in the Medical Area
Identifiers
URN: urn:nbn:se:his:diva-25556OAI: oai:DiVA.org:his-25556DiVA, id: diva2:1985314
Subject / course
Bioscience
Educational program
Molekylär biodesign 180 hp
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Available from: 2025-07-23 Created: 2025-07-23 Last updated: 2025-09-29Bibliographically approved

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