Rapid in-field Nanopore-based identification of plant and animal pathogens

Lead Research Organisation: Earlham Institute
Department Name: Research Faculty

Abstract

Airborne crop diseases pose a serious threat to food security and are responsible for devastating loss of yield and over-reliance on pesticides. Early detection enables farmers to take preventative action, drastically reducing damage and cost. Current detection regimes often rely on expert identification of the pathogen from plant damage. More recently, other molecular techniques have emerged. However, these methods suffer the same problems - being specific for a single species and a need for relatively large quantities of pathogenic material.

Recently, TGAC has been working on an approach dubbed Air-seq that seeks to identify pathogens through sequencing of biological particles present in air. This overcomes both problems associated with current techniques as it is unbiased (not limited by species) and requires very small quantities of material. Our ultimate aim is to put sample collection, sequencing and analysis in a single box that can be deployed in the field. Key to success is a compact sequencing technology and this has recently emerged in the form of Oxford Nanopore Technologies' (ONT) MinION.

The MinION is a new compact, low-cost sequencing technology that offers long reads (thousands of bases of DNA) and a streamed mode of operation enabling analysis of data as it is generated. These attributes make it ideally suited to in-field use. However, part of the process of generating sequencing data involves converting an electrical signal from the DNA sensing pore into a sequence of bases (letters) and this is performed via an internet 'basecalling' service. For in-field deployment, this is unsatisfactory, as we cannot rely on high speed, reliable data connections. We believe a completely new approach is required in which we utilise the raw signal data in order to identify species, instead of searching against basecalled sequence.

In this project, we will develop a tool that searches Nanopore signal data looking for the characteristic signal traces of pathogens of interest, building up a report on abundance levels in the process.

Technical Summary

Airborne crop pathogens pose a serious threat to food security and are responsible for devastating loss of yield and over-reliance on pesticides. Early detection enables farmers to take prophylactic action, drastically reducing damage and cost. Current detection regimes often rely on expert identification of the pathogen from plant damage. More recently, techniques have emerged utilising PCR or antibody-based assays. However, these methods suffer the same problems - being specific for a single species and a need for relatively large quantities of pathogenic material.

Recently, TGAC has been working on an approach dubbed Air-seq that seeks to identify pathogens through sequencing of biological particles present in air. This overcomes both problems associated with current techniques as it is unbiased and requires very small quantities of material. Our ultimate aim is to put sample collection, sequencing and analysis in a single box that can be deployed in the field. Key to success is a compact sequencing technology and this has recently emerged in the form of Oxford Nanopore Technologies' (ONT) MinION.

The MinION is a compact, low cost single molecule sequencing technology that offers multi-kilobase reads and a streamed mode of operation enabling analysis of data as it is generated. These attributes make it ideally suited to in-field use. However, ONT's basecalling utilises a cloud-based system in which pore electrical signal data is uploaded and basecalled sequence downloaded. For in-field deployment, this is unsatisfactory, as we cannot rely on high bandwidth data connections. We believe a completely new approach is required in which we utilise the raw signal data in order to identify species, instead of searching against basecalled sequence.

In this project, we will develop a tool that searches Nanopore signal data looking for the characteristic signal traces of pathogens of interest, building up a report on abundance levels in the process.

Planned Impact

Academic impact

This work will result in the production of a valuable tool for scientists working on in-field uses of Nanopore sequencing. Our initial target is pathogen detection applications, but target sequences could be anything and this therefore widens the tools' usefulness to a wide range of clinical, ecological and conservation diagnostic and surveillance applications.

The availability of an API widens use further, enabling others to build new tools tailored to specific applications that sit on top of the API.

The development of the tool will generate new opportunities for collaborative work with R&D groups in industry and with academic institutions. We anticipate that it will also form part of an ongoing relationship with Dstl.

The postdoctoral researcher employed for the project will gain important knowledge of bioinformatics, signal processing and Nanopore sequencing. They will develop extremely valuable skills in the use of high performance computing environments and will gain further opportunities to develop their written and verbal communication skills.

Economic and societal impacts

Early detection and quantification of crop pathogens has the potential to dramatically change agriculture in significant ways:
1. Reduction in the use of pesticides: by early harvesting or by spraying only when a) pathogen levels reach dangerous levels b) the pathogen race is a known to be able to overcome the cultivars resistance c) targeted use of pesticides the pathogen is not resistant to. The knock-on effect will be to reduce the economic impact of pesticides in non-target species, estimated to be around $8 billion annually (Aktar et al. 2009 PMID: 21217838).
2. Reduction in crop damage by ensuring spraying occurs before infection can take hold.
3. Improved results from crop spraying due to better selection of fungicides based on exact knowledge of pathogen levels and strain.
4. Detection of novel pathogens and new strains of known pathogens
5. Better modelling of disease epidemiology.

Reducing the damage caused by crop disease and reducing the level of fungicide use will have positive economic impacts on farmers and will help to ensure UK food security. It will also allow (with wind direction) the early detection of pathogens that are carried from the continent or have just established a "bridgehead" in the UK.

Within Dstl's remit, this project has the potential to revolutionise the detection of biological warfare agents in the field. This would have immediate implications for defending the UK against the use of biological weapons and against bioterrorism.
 
Description We have developed a tool for real-time analysis of environmental and clinical samples, in-field. This enables researchers to perform DNA sequencing with nanopore sequencers in a remote location and to obtain live analysis of the species composition of the sample.

We have also developed tools for software enrichment of samples, enabling targeted sequencing of particular regions/organism. This is via Oxford Nanopore's ReadUntil programming interface.
Exploitation Route The software we produce is available to others to use. A number of groups have developed or begun to develop collaborations with us.
Sectors Aerospace, Defence and Marine,Agriculture, Food and Drink,Environment,Healthcare

 
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 Interactive real-time metagenomics algorithms for Nanopore sequencing (LEGGETT_E17DTP1)
Amount £90,000 (GBP)
Funding ID 1937486 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 09/2017 
End 09/2021
 
Description New software for nanopore based diagnostics and surveillance
Amount £151,571 (GBP)
Funding ID BB/R022445/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 08/2018 
End 01/2020
 
Description Ship-seq: Nanopore sequencing of polar microbes on board icebreakers
Amount £90,000 (GBP)
Funding ID 1942119 
Organisation Natural Environment Research Council 
Sector Public
Country United Kingdom
Start 09/2017 
End 05/2021
 
Description ARM DSP technology 
Organisation ARM Holdings
Country United Kingdom 
Sector Private 
PI Contribution We engaged ARM to discuss the potential for using their DSP technology on this project. This resulted in a meeting, a follow-up call and an exchange of emails. We have sent code and papers to ARM.
Collaborator Contribution ARM are offering advice on hardware solutions to some of the problems we are trying to solve and on the most appropriate development kits etc.
Impact Outputs are so far better understanding for ARM of the needs of genomics and medical sequencing. For us, it is better understanding of low-level processor technology. Collaboration is multi-disciplinary - we are a team of biologists and computer scientists and ARM are mainly engineers.
Start Year 2017
 
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 Kromek 
Organisation Kromek Group plc
Country United Kingdom 
Sector Private 
PI Contribution Kromek approached us about applying for DARPA funding for a project to sequence airborne DNA for threat monitoring (see separate grant). We supplied expertise in sequencing, bioinformatics and molecular biology and helped to write a grant proposal.
Collaborator Contribution Kromek have engineering expertise and have previously delivered a DARPA radiation detector contract.
Impact Successful application of funding to DARPA. Continued discussions on future work.
Start Year 2018
 
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 Conference talk: Data analysis tools - NanoOK 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Presentation of NanoOK tool at international nanopore conference, London Calling 2017.
Year(s) Of Engagement Activity 2017
 
Description Conference talk: Real-time microbial disease diagnostics with MinION and NanoOK 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Presentation of research at international nanopore conference, London Calling 2017.
Year(s) Of Engagement Activity 2017
 
Description Conference talk: Tackling microbial diseases using MinION diagnostics and NanoOK 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Invited plenary speaker at the Bern Nanopore Technology Day.
Year(s) Of Engagement Activity 2017
 
Description Engineering day at the Norwich Science Festival 
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 Through a series of talks and activities, we highlighted Earlham Institute's important work in aquaculture and synthetic biology, among other things.

Disseminating this sort of research is of vital importance, as is collecting feedback from both participants and volunteers alike, in order that we can better stimulate conversations around complex science and increase understanding of the taxpayer funded research that we do.

In depth feedback showed that people were more aware of the important research that we do, and how it influences how we understand and conserve important ecosystems and increase sustainable food production.
Year(s) Of Engagement Activity 2018
 
Description Extreme environments: genome sequencing & space 
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 on our fieldwork in Iceland
Year(s) Of Engagement Activity 2018
URL http://www.earlham.ac.uk/articles/extreme-environments-genome-sequencing-space
 
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 Member of MinION Analysis and Reference Consortium (MARC) 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact MARC is a consortium of scientists who seek to work together to understand how to get the best out of nanopore technology and to collaborate on international experiments and publications. So far, we have published two papers together which seek to describe and evaluate MinION technology. We are now working on a third publication based on an experiment to sequence river water around the world using nanopore sequencing.
Year(s) Of Engagement Activity 2015,2016,2017,2018
 
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 Nature Day at Norwich Science Festival 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Through a series of engaging, innovative activities, we showcased the valuable research we are doing into understanding genomes and bottlenecks.

We had a hugely popular "Pink Pigeon Trail", which allowed us to engage with children and adults simultaneously in an interactive, exploratory activity that allowed participants to understand the concepts around genetic bottlenecks and how they affect species.

Feedback from the event suggested that the public came away with a better understanding of the issues surrounding conservation and genetic diversity, while thinking more about the role of Institutes such as EI in tackling global issues from protecting biodiversity through to improving food security.

Over the whole week of Norwich Science Festival, the activities and talks we put on in relation to genetic bottlenecks and genetic diversity reached well over 20 000 people.

Importantly, feedback from the organisers suggested that our public engagement activities have "set the standard" and "raised the bar" for future Science Festivals, which was a fantastic achievement for the Institute and the communications team.
Year(s) Of Engagement Activity 2018
URL http://www.earlham.ac.uk/articles/celebration-science-norwich
 
Description Norwich Research Park Day at the Norwich Science Festival 
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 This, a celebration of science from across the NRP, was a fantastic chance for us to showcase the work across our collective CSPs and NCs.

All in all, we had dozens of staff involved from across the institute who helped us to deliver a set of multiple activities, workshops and talks on the day. Activities included a live LEGO sequencer with BLAST analysis of genomes and a live DNA sequencing experiment. Additionally, there were talks on the Institute and how it applies big data and computational approaches to understanding life on earth, the interactions underpinning ecosystems and communities, as well as how we can better understand evolution to drive trait improvement.

The event was a spectacular success, with desired outcomes being a greater awareness of the important research undertaken by EI, as well as the value of applying computational approaches to tackling a swathe of biological questions. The specific feedback showed that this event had definitely increased understanding among participants, who numbered more than 8000, of the role that EI and its core programmes play at the cutting edge of science.

Social media coverage ensured that the activities we put on reached many thousands more people than just those who attended the event on the day.
Year(s) Of Engagement Activity 2018
URL https://www.youtube.com/watch?v=Uf0h8Q8PVxI
 
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 - Eukaryotic metagenomics: Identification and quantification of bee collected 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 London Calling conference, May 2018
Year(s) Of Engagement Activity 2018
 
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 - Real-time surveillance and diagnostics with NanoOK RT 
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 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 Poster presentation: Tackling microbial diseases of the preterm infant gut using MinION diagnostics and NanoOK 
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 AGBT 2018 conference, Florida, USA, February 2018.
Year(s) Of Engagement Activity 2018
 
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 Real time sequencing solutions: taking the lab to the sample and beyond 
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 the EI website about our work with real-time nanopore sequencing
Year(s) Of Engagement Activity 2018
URL http://www.earlham.ac.uk/articles/real-time-sequencing-solutions-taking-lab-sample-and-beyond
 
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 Techology Day at Norwich Science Festival 
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 7500 members of the general public attended Nature Day at Norwich Science Festival. A number of activities were available to engage the general public with the work of the institute. A number of talks/presentations were scheduled also.
Year(s) Of Engagement Activity 2018
 
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 Women in Computing: Yuxuan Lan 
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 An article on the EI website featuring an interview with Yuxuan Lan, a postdoc employed on these grants.
Year(s) Of Engagement Activity 2019
URL http://www.earlham.ac.uk/articles/women-computing-yuxuan-lan