Evaluation of nanopore-based sequencing technology for gene marker based analysis of complex microbial communities. Method development for accurate 16S rRNA gene amplicon sequencing

Calus, Szymon Tomasz (2018) Evaluation of nanopore-based sequencing technology for gene marker based analysis of complex microbial communities. Method development for accurate 16S rRNA gene amplicon sequencing. PhD thesis, University of Glasgow.

Abstract

Nucleic acid sequencing can provide a detailed overview of microbial communities in comparison with standard plate-culture methods. Expansion of high-throughput sequencing (HTS) technologies and reduction in analysis costs has allowed for detailed exploration of various habitats with use of amplicon, metagenomics, and metatranscriptomics approaches. However, due to a capital cost of HTS platforms and requirements for batch analysis, genomics-based studies are still not being used as a standard method for the comprehensive examination of environmental or clinical samples for microbial characterization.

This research project investigated the potential of a novel nanopore-based sequencing platform from Oxford Nanopore Technologies (ONT) for rapid and accurate analysis of various environmentally complex samples. ONT is an emerging company that developed the first-ever portable nanopore-based sequencing platform called MinIONTM. Portability and miniaturised size of the device gives an immense opportunity for de-centralised, in-field, and real-time analysis of environmental and clinical samples. Nonetheless, benchmarking of this new technology against the current gold-standard platform (i.e., Illumina sequencers) is necessary to evaluate nanopore data and understand its benefits and limitations.

The focus of this study is on the evaluation of nanopore sequencing data: read quality, sequencing errors, alignment quality but also bacterial community structure. For this reason, mock bacterial community samples were generated, sequenced and analysed with use of multiple bioinformatics approaches. Furthermore, this study developed sophisticated library preparation and data analyses methods to enable high-accuracy analysis of amplicon libraries from complex microbial communities for sequencing on the nanopore platform. Besides, the best performing library preparation and data analyses methods were used for analysis of environmental samples and compared to high-quality Illumina metagenomics data. This work opens a new possibility for accurate, in-field amplicon analysis of complex samples with the use of MinIONTM and for the development of autonomous biosensing technology for culture-free detection of pathogenic and non-pathogenic microorganisms in water, soil, food, drinks or blood.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Keywords: Nanopore, sequencing, 16S rRNA, bacteria, protocol, development.
• Subjects: T Technology > T Technology (General)
• Colleges/Schools: College of Science and Engineering > School of Engineering
• Funder's Name: Engineering and Physical Sciences Research Council (EPSRC), Engineering and Physical Sciences Research Council (EPSRC)
• Supervisor's Name: Ijaz, Dr. Umer Z. and Pinto, Dr. Ameet J.
• Date of Award: 2018
• Depositing User: PhD Szymon T Calus
• Unique ID: glathesis:2018-41086
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 26 Mar 2019 11:34
• Last Modified: 08 Jan 2024 16:30
• Thesis DOI: 10.5525/gla.thesis.41086
• URI: https://theses.gla.ac.uk/id/eprint/41086

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