• Research

Nanopore sequencing for interactive, real-time metagenomics

Unleashing the potential of real-time, clinical and environmental nanopore sequencing applications.

Project summary.

Led by: Leggett Group

Start date: Oct 2017

End date: Sept 2022

Duration: 4 years

Grants: UKRI / BBSRC DTP studentship

The Oxford Nanopore Technologies MinION represents the first truly portable sequencing platform capable of being deployed in field alongside sample collection, making it ideally suited to metagenomic, clinical and environmental sequencing applications. Additionally, its streamed mode of operation offers the potential to analyse sequencing data as it is generated. Despite the excitement associated with the MinION, analysis tools and pipelines have lagged behind, making it difficult to realise the potential of the device; typically users analyse data offline, rather than exploiting the real-time functionality of the platform, and there are often many manual steps involved.

In this project, we are researching a number of novel techniques for exploiting the real-time nature of the MinION. This has included developing the NanoOK RT tool and its successor, MARTi (Metagenomic Analysis in Real Time), which perform real-time analysis and visualisation of taxonomy and antimicrobial resistance analysis. We are also developing approaches for analysis of metagenomic sequence data using Oxford Nanopore’s ‘Read Until’ programming interface to selectively enrichfrom  mixed samples by analysis of raw signal data during sequencing.

Details.

The MinION is a single molecule sequencer that arrived in pre-release form in 2014. Offering multi-kilobase reads, it attracted interest due to its compact size, low cost, and a streamed mode of operation enabling analysis of data as it is generated. The technology involves detection of current changes across biological nanopores through which DNA molecules move. EI were one of the first organisations enrolled in the MinION Access Programme (MAP), leading to our development of NanoOK, a tool for comprehensive alignment-based analysis and sample comparison of Nanopore data.

A significant amount of EI’s work with the MinION has focused on metagenomic applications and real-time surveillance - e.g. airborne pathogens (EI project), pre-term baby microbiomes (with Lindsay Hall, Quadram Bioscience Institute), bee pollen metagenomics (with Douglas Yu, UEA). Such applications play to the unique strengths of the MinION, as it represents the first truly portable sequencing platform capable of being deployed in the field alongside sample collection.

Above: A screenshot from MARTi, a real-time metagenomic analysis tool developed by the Leggett Group.

Despite the excitement associated with the MinION, analysis tools and pipelines currently lag behind and make it difficult to realise the potential of the device; typically users analyse data offline, rather than exploiting the real-time functionality of the platform, and there are often many manual steps involved. Oxford Nanopore provide a service called What’s In My Pot (WIMP), which provides taxonomic assignment of bacteria, virus and fungi reads through a web interface, but it is a closed system that does not allow user-defined databases (e.g. resistance genes, eukaryotes) and does not facilitate deeper analysis beyond initial classification. There remains a need for an open and comprehensive platform for real-time metagenomic analysis of nanopore data.

Impact statement.

The idea of applying nanopore sequencing to in-field environmental and clinical metagenomics has generated a great deal of excitement. The main impact of this project will be to help to realise the potential of the platform by freeing up researchers to concentrate on scientific questions rather than on developing complex (often single-use) pipelines.

The outputs of the project will be particularly relevant to environmental applications (e.g. pathogen identification, in-field species identification) and to clinical applications (e.g. pathogen identification, antimicrobial resistance identification). A drive to ensure an open platform will enable its adaptation and extension beyond the original vision.