New software for nanopore based diagnostics and surveillance

The Oxford Nanopore Technologies MinION is a new DNA sequencer developed in the UK from UK technology, it is only a few inches long and plugs into the USB port of a laptop. Its low cost and portability make it extremely attractive for "in field" and clinical sequencing applications. These kinds of samples are typically metagenomic (mixed samples of many different organisms) and often the key questions researchers want to answer are:
1) what species are in the sample?
2) how much of each is there?
3) What kinds of antibiotic resistance bacteria are there?

Unlike previous sequencing technologies, the MinION is capable of working in real-time, which means that data can be analysed progressively as it is generated. In a clinical setting, this could mean getting test results back far quicker than would otherwise be possible. However there is a lack of software capable of performing real-time analysis and many users fail to take advantage of this unique feature of the technology.

The aim of this project is to develop a software tool that can provide real-time analysis of clinical and environmental samples in an easy-to-install and easy-to-use way, but which enables tailoring of the analysis to the specific needs of individual applications. We are also very keen to take advantage of the fact that the MinION can sequence much longer stretches of DNA than older technologies in order to more accurately identify the bacteria which harbour antibiotic resistance. Ultimately, this will lead to improved treatment regimes for illnesses linked to bacteria.

The Oxford Nanopore Technologies MinION sequencer has become an attractive platform for analysis of metagenomic datasets, particularly in clinical and in-field settings. Its low cost of ownership, compact size, simple sample preparations and the fact that it requires only a laptop and a USB port to function have meant that it is relatively easy to deploy outside of a traditional lab environment. It also has the unique ability of being able to sequence in real-time, thus providing data progressively as sequencing continues. However, real-time analysis approaches have lagged behind.

In this project, we will develop a general-purpose user-friendly tool for real-time analysis of nanopore metagenomic data. The software will be designed to have wide applicability to any sample, but will include some features that will be particularly relevant to antimicrobial diagnostic applications. We will provide a simple interface for configuring analyses and a browser-based GUI for straightforward visualisation of results. Support will be provided for a range of third party tools, but an API will allow integration of new tools and custom analysis steps. We will take advantage of the long reads provided by nanopore sequencing to implement an approach we call "walking out" to locate the species containing detected antimicrobial resistance genes. Recognising that there are many exciting applications of real-time metagenomics that may take place in remote locations, or without access to an HPC environment, we will delivery software capable of running on an analysis laptop or compute stick connected to the sequencing laptop. As well as providing open source code, we will use the latest containerisation technologies to deliver an easy-to-install application that does not require specialist computing knowledge.

Academic impact

This project will result in an enabling tool and the direct impact on academic researchers will be to give them the necessary tools to ask questions that were previously impossible (e.g. in remote locations or in real time).

The software will be valuable to researchers working in applying nanopore sequencing to almost any area of metagenomics. Our focus is on real-time diagnostics and surveillance, but there is nothing about the design of the tool that will make it inappropriate to use in applications where speed is less critical. Instead, analysing data while it is still be sequenced will save time and free researchers to ask more questions of their hypothesis and data than they otherwise may have been able to.

Making the tool extensible and providing an API for developers will enable its impact to be felt more widely. Making it open source enables others to develop it further and to reuse code in other projects, enabling its impact to be felt further afield.

Economic and societal impact

The application of real-time diagnostics in healthcare has the potential to dramatically improve clinical outcomes and to save money. An example is our own work with necrotising enterocolitis (NEC) [Leggett et al. bioRxiv 2017] in which we used MinION diagnostics to obtain clinically relevant pathogen and antibiotic resistance information in under 6 hours, a vast improvement on typical clinical microbiology tests which take around 48hrs. This has the potential to improve survival rates and cut the costs of treatment for NEC. In the US, the cost of caring for NEC infants is estimated to be between $500m and $1bn per year (PMID: 21247316). Similar figures are not available for the UK, but each day in neonatal intensive care is estimated to cost around £4,000. As well as NEC, there are a wide range of health problems in children and adults which are linked to the microbiome and for which research and treatment would benefit enormously from nanopore sequencing combined with the analysis tool we propose.

Beyond health, metagenomic analysis touches almost every part of biology. Rapid analysis of crop pathogens could lead to better yields, reduction in crop damage and a reduction in pesticide use. In other domains, the use of in-field metagenomics will enable experiments to be carried out without the experimental difficulties and societal concern associated with removing biological specimens from one country to another.

Sequencing technology is a collection of great British success stories, e.g., Fred Sanger won his second Nobel Prize in Chemistry in 1980 for developing methods to determine DNA sequences and the Sanger Institute (named in his honour) determined much of the sequence of the human genome using the very dideoxy chemistry he invented. Fellow Cambridge scientists Shankar Balasubramanian, and David Klenerman, developed the sequencing-by-synthesis (SBS) technology (funded by a BBSRC grant) that underpins the Illumina sequencers which currently dominate the market. Similarly Oxford Nanopore Technologies are now pioneering a new type of sequencing which could democratise genetic and genomic analysis to any life science laboratory. Provision of easy to use analysis will contribute towards the uptake of this exciting (British) technology.