Nanopore sequencing technology has the potential to revolutionise metagenomics by providing long reads, which can improve taxonomic classification and assembly contiguity, near real-time analysis, enabling rapid results and improved sequencing efficiency, and portability, allowing sequencing in the field. However, the full potential of these features is largely unrealised due to the lack of available tools and methods. In this thesis, we report on tools and analysis methods that facilitate the use of nanopore sequencing technology for metagenomics and real-time analysis.
Applying metagenomics to samples containing a mix of eukaryote species, such as bee-collected pollen, is challenging due to lack of available reference genomes. This thesis presents a new method, RevMet (Reverse Metagenomics), for semi-quantitative characterisation of mixed eukaryote samples without the need for complete reference genomes. Instead, each reference species is represented by a low-cost genome skim. The short-read reference skims are mapped to the long nanopore query reads to individually classify them, which is the reverse of the standard metagenomic approach of mapping reads to (assembled) references.
Recognising the need for an open-source software tool for real-time analysis and visualisation of metagenomic sequencing data, we developed MARTi (Metagenomic Analysis in Real-Time). MARTi provides a rapid, lightweight web interface that allows users to view community composition and identify antimicrobial resistance genes in real time. MARTi consists of two main parts, the Engine and the GUI, and can be configured in multiple ways to suit the needs of the user. We demonstrate MARTi on live nanopore sequencing runs - firstly, using a mock gut community and, secondly, using clinical faecal gut microbiome samples taken from patients suffering from liver disease
