Overview

Project Highlights:

  • Develop rapid and affordable methods for vertebrate species identification by nanopore DNA sequencing
  • Collaborate with Twycross Zoo & Zoological Society of London (ZSL) in testing method on a wide variety of species and substrates (blood, hair, muscle)
  • In collaboration with ZSL explore its application in the field to combatting illegal trade in animal products both within and passing through Mongolia a key location in trafficking protected species

Overview

Illegal trading in animal products is commonplace, widespread and diverse, ranging from trafficking high-value, CITES-protected, endangered species between countries, fraudulent misidentification of by-catch fish filleted at sea (estimated at ~25%), through to substitution of low-value meat from one species into foodstuffs advertised as containing another (e.g. the 2013 horse meat scandal).

Once stripped of species-specific morphological characters many products can only be reliably identified by DNA testing, which has until now been costly and time-consuming, requiring both bulky specialist equipment and highly trained lab personnel. However, a recent development in third-generation DNA sequencing, the Oxford Nanopore Technologies (ONT) MinION, offers the possibility of affordable and portable DNA sequencing capacity at a customs post, quayside or food processing plant, providing rapid confirmation of species of origin and the option to seize illegal products and detain those trading in them on the spot.

We intend to develop and test a universal species ID test applicable to all these scenarios in partnership with the East Midlands Zoological Society (Twycross Zoo) and our collaborators at the Zoological Society of London (ZSL). ZSL has been working with the Mongolian Government to tackle the Illegal Wildlife Trade (IWT) since 2013, building law enforcement capacity and raising awareness within local communities. Mongolia is a key source and transit country for IWT, driving catastrophic declines across a broad spectrum of Mongolia’s wildlife populations, including most of their iconic medium and large-sized mammals. Unfortunately many of its frontline law enforcement officers are unable to identify seized wildlife products. This project will seek to trial the use of nanopore sequencing at key IWT border and market hotspots as a proof of concept to aid the rapid and accurate identification of (previously unidentifiable) wildlife products such as meat, fur and traditional medicine ingredients and in the long-term, establishing a chain-of-evidence to aid ultimate prosecution of illicit wildlife product smugglers. We will also explore the global issue of bushmeat, which threatens not only wild species but also human consumers exposed to pathogens borne by them (which can also be detected by nanopore sequencing).

DNA extracted from samples such as blood passes through one of 512 protein nanopores within the laptop-powered MinION DNA sequencer.  Each nanopore is embedded in a membrane separating two chambers, a current flowing between the chambers is differentially blocked by the transit of an A, G, C or T causing base specific fluctuations which are converted back to bases and compared with species databases to highlight a match.

Methodology

The test will build upon methods we are currently developing with ONT to preferentially sequence mitochondrial DNA and compare results with the BOLD & GenBank databases of species-specific mtDNA sequences that already cover many species affected by illegal trade. The student will exploit a number of products planned for release next year to meet ONT’s declared aim of allowing “anyone to sequence anything, anywhere”. Twycross & ZSL will supply samples for DNA extraction and processing optimization, and source reference samples from both the target and closely related species to evaluate accuracy in blind tests.  

Training and Skills

At UoL

- bioinformatics; screening publically available resources for sequencing targets, developing appropriate pipelines for NGS interpretation, in the context of in-field analysis and case reporting on bushmeat;

- DNA extraction: from variety of substrates (blood, hair, bushmeat etc.);

- multiplex PCR; design and validation;

- DNA sequencing; traditional Sanger; Illumina MiSeq & ThermoFisher Ion Torrent NGS; ONT MinION nanopore approaches

- training in forensic validation methods

At Twycross

- work alongside zoo professionals, including studbook keepers, vets and research staff, engage with other university students conducting a range of research activities.

Timeline

Year 1: Sample collection, DNA preparation; Checking of reference materials using conventional Sanger and Illumina sequencing; Development of nanopore sequencing methods.

Year 2: Nanopore analysis and development of in-house bioinformatics; protocol development and optimization of field toolkit

Year 3: Testing of nanopore sequencing in the field; demonstration in enforcement setting; bushmeat testing.

Partners and collaboration (including CASE)

CAM, JHW & MAJ have expertise in the analysis of mitochondrial DNA, emerging sequencing technologies (including nanopore) and the application of fgenetics in forensic investigation. Twycross and ZSL have both contributed to scoping this proposal and their knowledge will be key to formulating the project plan, guiding the research and contributing to reports and publications arising from this project. ZSL will work with the student to trial the test in an enforcement setting in Mongolia (separate funding will be sought) and to develop awareness and training materials for their customs officials. We will publicize the research through outreach activities.

Further Details

https://www2.le.ac.uk/centres/forensic-science/ajfgu/wildlife_nanopore

https://www.zsl.org/conservation/threats/illegal-wildlife-trade-crisis/illegal-wildlife-trade-in-mongolia