New software for nanopore based diagnostics and surveillance

Lead Research Organisation: Earlham Institute
Department Name: Research Faculty

Abstract

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.

Technical Summary

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.

Planned Impact

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.
 
Description We developed software for real-time analysis of metagenomic samples (mixed, microbial communities) sequenced with nanopore sequencers. We have applied it to a number of sample types, but perhaps most significantly, the software was used in a proof-of concept project to diagnose pathogen and antibiotic resistance in preterm babies suffering from necrotising enterocolitis and sepsis. Using the approach, we were able to diagnose pathogen and antibiotic resistance in around 5 hours, which compares very well to the ~ 48hrs taken with conventional tests. This work was published in the journal Nature Microbiology (Leggett et al. 2019, Nature Microbiology).

Additionally, we worked to implement software enrichment of metagenomic samples using Oxford Nanopore's ReadUntil software API. We are currently preparing a manuscript for publication.
Exploitation Route Other scientists can use our software to perform real-time analysis of their metagenomic samples. The use cases are very wide.
Sectors Agriculture, Food and Drink,Environment,Healthcare,Other

 
Description DNA sequencing for biological threat monitoring
Amount $5,270,000 (USD)
Funding ID HR001119C0031 
Organisation Defense Advanced Research Projects Agency (DARPA) 
Sector Public
Country United States
Start 12/2018 
End 12/2023
 
Description DNA sequencing for biological threat monitoring
Amount $5,270,000 (USD)
Funding ID HR001119C0031 
Organisation Defense Advanced Research Projects Agency (DARPA) 
Sector Public
Country United States
Start 12/2018 
End 12/2023
 
Description Beth Okamura (Natural History Museum) collaboration 
Organisation Natural History Museum
Country United Kingdom 
Sector Public 
PI Contribution Richard Leggett is co-I on Beth Okamura's Leverhulme Trust funded project aiming to characterise parasites in frog populations using metagenomic techniques.
Collaborator Contribution Obtained grant funding from Leverhulme Trust
Impact Successful application to Leverhulme Trust.
Start Year 2020
 
Description Johnson Biosignatures Lab 
Organisation Georgetown University
Country United States 
Sector Academic/University 
PI Contribution We updated our NanoOK RT software to enable in-field use and accompanied Sarah Johnson and her team on a field trip to Iceland in August 2018.
Collaborator Contribution Sarah Johnson invited me to join her group on field work in Iceland, covering all costs. We are collaborating on data analysis and publications.
Impact Talks and posters at conferences. Updates to our software. Sequencing datasets.
Start Year 2018
 
Description Livestock gut microbiome 'fingerprinting' with Entomics 
Organisation Entomics Biosystems
Country United Kingdom 
Sector Private 
PI Contribution We have been working with Entomics to apply nanopore sequencing and metagenomic analysis approaches to understanding salmon fish microbiomes and the effect of different feeding regimes. We have brought nanopore sequencing and bioinformatics knowledge to the partnership.
Collaborator Contribution Entomics have expertise in developing livestock feeds
Impact Sequencing datasets and preliminary analysis. An application for further funding from the FTM to expand the research.
Start Year 2018
 
Description Plymouth Dental School collaboration 
Organisation University of Plymouth
Country United Kingdom 
Sector Academic/University 
PI Contribution Richard Leggett has been asked to be on a PhD supervisory team for a project looking at using the MinION for oral screening in dentistry. Richard has also been named as a collaborator on a grant looking at metagenomics of the oral microbiome. This work came as a result of our work on MinION sequencing of preterm babies.
Collaborator Contribution Applied for PhD studentship and grant.
Impact Grant application.
Start Year 2019
 
Title Legopore 
Description Software to control the Lego DNA sequencer model built for public engagement. 
Type Of Technology Software 
Year Produced 2018 
Open Source License? Yes  
Impact Hundreds of members of the public, adults and children, have used the software at one of our engagement events. 
 
Title NanoOK RT software tool 
Description A real-time analysis tool for metagenomic classification and identification of antimicrobial resistances form nanopore sequence data. 
Type Of Technology Software 
Year Produced 2017 
Open Source License? Yes  
Impact The software was developed for and initially used in our work with pre-term babies suffering from Necrotizing Enterocolitis. We are now working to apply it to a wide range of other application areas and have had discussions with a number of interested parties at national and international institutes. 
 
Description BAMBI Diagnosing infections earlier in preterm babies with real time genomic analysis - 16/12/2019 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact Website article highlighting a new method for profiling the microbiome of preterm babies that can significantly speed-up the identification of infections and indicate more effective treatments developed in collaboration with Quadram Bioscience and NNUH. News stories highlight important updates that also have broad relevance and interest to the national and/or specialised media. This story generated 293K estimated coverage reads and 298K social shares.
Year(s) Of Engagement Activity 2019
URL https://www.earlham.ac.uk/newsroom/diagnosing-infections-earlier-preterm-babies-real-time-genomic-an...
 
Description Inside EI - Open Day 2019 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact One day in May 2019 EI opened its doors to the public and schools from the region. Offering an overview of the Tree of Life with the branches of our tree culminating in posters displaying colourful imagery of our research, visitors were able to talk to researchers about their projects and why the research is important. There were hands on activities providing insight into de novo genome assembly, nanopore sequencing technology, synthetic biology and high performance/cloud computing.

In addition to the activities, there were talks and tours of the laboratories, which were fully subscribed.

We were joined by schools from across the region in the morning, and opened our doors to the general public for the afternoon, with a total of over 400 visitors, media coverage (TV and radio) reaching an estimated 500+. The feedback was incredibly positive and we achieved an unbelievably successful set of outcomes. Comparison of pre- and post Open Day attitudes demonstrated that: Participants reported very high increases in understanding of a range of topics, while overwhelmingly agreed that research at EI should be funded by the government. The majority of people left with an increased appreciation of the importance of bioinformatics, genomics, etc. for their every day life, the environment, and more.
Year(s) Of Engagement Activity 2019
URL https://www.earlham.ac.uk/articles/inside-ei-public-engagement-science-impact
 
Description Invited talk - Real-time sequencing and analysis of microbial communities with nanopores 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Professional Practitioners
Results and Impact Research day on microbe research attended by approx. 200.
Year(s) Of Engagement Activity 2019
URL http://microbesinnorwich.org/
 
Description Invited talk - Semiquantitative characterisation of mixed pollen samples using nanopore sequencing and Reverse Metagenomics (RevMet) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Invited speaker at Natural History Museum Next Generation Sequencing and Molecular Workshop, 13th February 2020.
Year(s) Of Engagement Activity 2020
 
Description NanoOK of the North and South 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact EI website article about our work developing the nanopore analysis software NanoOK
Year(s) Of Engagement Activity 2019
URL http://www.earlham.ac.uk/articles/nanook-north-south-life-in-antarctic
 
Description New rapid test diagnoses pneumonia and lower respiratory tract infections - 24/06/2019 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact Website article highlighting research carried out by Quadram Institute, University of East Anglia (UEA) and Earlham Institute (EI) to develop a new, rapid way of diagnosing bacterial lower respiratory tract infections in hours rather than days that could improve patient care and slow the spread of antimicrobial resistance. The MinION technology was applied using software developed at EI as part of the CSP. Article showcased collaborative work between the research institutes and NHS. News stories highlight important updates that also have broad relevance and interest to the national and/or specialised media.
Year(s) Of Engagement Activity 2019
URL https://www.earlham.ac.uk/newsroom/new-rapid-test-diagnoses-pneumonia-and-lower-respiratory-infectio...
 
Description Norwich Science Festival - 22/10/2019 to 24/10/2019 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Norwich Science Festival is a wonderful showcase of science from Norwich and beyond for learning and excellent public engagement. This year, EI presented 11 talks and poems covering a fascinating range of science topics, from Salmonella and guts through to sequencing algae in the Antarctic, covering the whole tree of life in the process, bringing an innovative Bee Trail, LEGO sequencer, robots and more. Researchers from the CSP participated, presented their work and engaged with the public. Coverage on TV, radio and media with estimated coverage views of 698K
Year(s) Of Engagement Activity 2019
URL https://earlhaminstitute.coveragebook.com/b/02819684cc7da5de
 
Description Norwich Science Festival - Lego DNA sequencer 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact We created a working Lego Mindstorms model of a DNA sequencer, along with software to run it. We arranged activities around it during the Norwich Science Festival and engaged lots of adults and children.
Year(s) Of Engagement Activity 2018
URL https://twitter.com/brickopore/status/1055043270999465985
 
Description Norwich Science Festival - The Nedome 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact During Norwich Science Festival, we ran live demonstrations of nanopore sequencing in which we sequenced the "Ned-ome" (DNA kindly provided by Ned, a PhD student).
Year(s) Of Engagement Activity 2018
URL https://twitter.com/hashtag/nedome?lang=en
 
Description Poster presentation - Experiments with ReadUntil 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Poster presentation at London Calling conference, May 2018
Year(s) Of Engagement Activity 2018
 
Description Poster presentation - The long and the short of eukaryotic metagenomics: Identification and quantification of plant species in bee pollen 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Poster presentation at the Advances in Genome Biology and Technology (AGBT) conference in Marco Island, Florida.
Year(s) Of Engagement Activity 2019
URL https://www.agbt.org
 
Description Presentation "DNA Sequencing for Biological Threat Monitoring" 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Presentation at DARPA headquarters in Arlington Virginia, USA, at the "SIGMA+ Sensor Kickoff Meeting" I had an audience of ~100 US, UK and Israeli stakeholders from government agencies (including military and homeland security) and companies that develop and supply technology to US military. This talk raised a lot of interest and questions about the capabilities of the new (potentially disruptive) technology.
Year(s) Of Engagement Activity 2018
 
Description Presentation - Lego sequencer at the Women of the Fututre event 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Activity at the Women of the Fututre event
Year(s) Of Engagement Activity 2019
 
Description Talk - Characterisation of bee-collected pollen using MinION and RevMet 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Talk at London Calling 2019 given by Ned Peel.
Year(s) Of Engagement Activity 2019
 
Description Talk - Developing a Streamlined Field Protocol for In Situ Sequencing and Analysis of Extremophiles 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Talk at American Geophysical Union Fall Meeting, Washington DC, 10-14 December 2018
Year(s) Of Engagement Activity 2018
 
Description Talk and poster presentation - Taking the lab to the sample: enabling new ways of working with nanopore sequencing 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Poster and flash talk at the Advances in Genome Biology and Technology (AGBT) conference in Marco Island, Florida.
Year(s) Of Engagement Activity 2019
URL https://www.agbt.org
 
Description The Earlham Institute LEGO sequencer 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Article on EI website about our engagement project in which we built a LEGO DNA sequencer
Year(s) Of Engagement Activity 2018
URL http://www.earlham.ac.uk/articles/earlham-institute-lego-sequencer
 
Description Visit from Poulton group UEA to Richard Leggett 01/08/2019 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Professional Practitioners
Results and Impact To observe and receive training using nanopore technologies
Year(s) Of Engagement Activity 2019
 
Description Web article - Women in computing: Yuxuan Lan - 11.02.2019 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Articles such as this pertain to our publically funded research such that the information gleaned can be disseminated to the general public. As of March 2019, our articles have reached people in all but 6 countries worldwide, with over 50000 pageviews on our website in the year 2018. On social media, this reach has exceeded a million people, monthly. As part of an expanding portfolio covering the range of science that we do, each article forms a vital component of how we engage the wider international community with important scientific breakthroughs and knowledge.

This is part of our women in computing to promote this as a career addressing the skill shortage and lack of gender balance in the UK
Year(s) Of Engagement Activity 2019
URL http://www.earlham.ac.uk/articles/women-computing-yuxuan-lan
 
Description Where are the bees? Tracking down which flowers they pollinate - 07/08/2019 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Media article highlighting work carried out at EI and as part of CSP to develop a new method to rapidly identify the sources of bee pollen to understand which flowers are important for bees. News stories highlight important updates that also have broad relevance and interest to the national and/or specialised media, this story has estimated coverage views of 398K.
Year(s) Of Engagement Activity 2019
URL http://www.earlham.ac.uk/newsroom/where-are-the-bees
 
Description Where bees roam: pollinate this! - 07/08/2019 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Website article highlighting reverse metagenomics project to track what plants bees have visited via the pollen grains they pick up, using data gleaned from next-, and third-generation genome sequencing technologies and software developed at EI supported through the CSP.
Year(s) Of Engagement Activity 2019
URL http://www.earlham.ac.uk/articles/where-bees-roam-pollinate-this
 
Description brickopore.co.uk 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Created a website to provide information on our Lego DNA sequencer, details of events and instructions to build. Designed to promote the institute's engagement and to encourage it elsewhere.
Year(s) Of Engagement Activity 2019
URL http://brickopore.co.uk