Whole Genome Sequencing

Source tracking for the food industry

Date: July 2017, Version 1

NGS platforms have been available for over a decade, and are nowbecoming sufficiently inexpensive that their application by suitablyskilled people to real world problems in the food industry, such asbacterial genome sequencing, is increasingly feasible. There are anumber of different sequencing technologies available, each one havingtheir own advantages and disadvantages in terms of the cost of theinstrument, cost per run, run time and the length and number of readsproduced (a read being an individual DNA sequence). The technologychosen will depend on the specific task to be accomplished. Outlinedbelow are some of the instances where NGS has the potential to make areal difference for the food industry.

Food safetyFood safety is perhaps the most obvious and advanced area where NGS can play a role. High throughput technologies are capable of rapidly

producing draft-quality genome sequences of the bacteria responsiblefor important foodborne diseases (including Salmonella enterica, Listeria monocytogenes and Campylobacter jejuni). The US FDA’sGenomeTrakr programme has been uploading DNA sequence data toNational Center for Biotechnology Information (NCBI) since 2013, andhas so far sequenced more than 35,000 isolates1. When these sequencedata are uploaded and compared to similar data from clinical patients itbecomes possible to rapidly detect outbreaks of foodborne disease at anearly stage, when they are small and not geographically clustered.Outbreaks can also be traced back to their source, from the scale of ageneral foodstuff and geographical origin, such as tuna from westernIndia2 to individual processing plants, as in the case of Roos FoodsHispanic-style cheeses contaminated with L. monocytogenes3.

Other organisations, such as the Global Microbial Identifier (GMI), are now trying to apply this whole genome sequencing (WGS) approach

Source Tracking for the food industry Recent advances in rapid, high throughput DNA sequencing have revolutionised the study of biology, and have the capacity to do the same for food safety and security. So called ‘Next Generation Sequencing’ (NGS) technologies have a range of applications, including the sequencing of microbial genomes and culture-free identification of microbial communities. The implications of this for the food industry range from source tracking of bacterial contaminants, identification of spoilage communities, and authentication of the origins of foodstuffs.

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on a global scale, by working to harmonise qualitystandards in wet lab and computational approaches,and to overcome potential legislative issues. Fera hascontributed to this effort in part by hosting the 7th GMIMeeting in late 2014, and contributing open accessWGS data. There is a great potential benefit to industryin adopting WGS approaches. By providing the mostdiscriminative method possible for distinguishingbetween bacterial strains, it may be possible to tracecontamination within a food business back to anindividual faulty process or piece of equipment. Forexample, a study of single DNA base pair differenceswithin and between subtypes of L. monocytogenesisolated from a number of delicatessens was able todistinguish between long-term persistence within adeli and repeated introduction from external sources4. This is very usefulinformation for a food business trying to deal with a long termcontamination problem. If sequences from processing plant isolates areuploaded to the public database (anonymously, if desired) they can berapidly compared to other isolates from around the globe. Not onlywould this contribute to global public health, it could allow closematches to isolates from other foodstuffs to be detected, identifyingpotentially contaminated ingredients within the supply chain.

Food spoilageSpoilage of food is another area where NGS has a role to play. Food wasteis a hot topic at the moment attracting publicity and interest from UK andEU Governments. It is estimated that a total of over 14 million tonnes offood was wasted in the UK in 20065 with each tonne having a value of at least £9506, with a proportion of this waste due to spoilage. NGS applications which do not rely on culturing individual microbes canbe used identify the communities present on food items as they undergospoilage, and look for critical changes that indicate which microbes areresponsible for spoilage.

The benefits to industry of a clearer understanding of whatinfluences microbial communities associated with spoilage would be ameans to increase shelf-life and reduce waste. Identifying microbialcommunity changes or physiological shifts associated with the onset of

spoilage would allow rapid assays or biosensors to be designed. Thesecould detect the growth of spoilage organisms before they become aproblem, thereby facilitating stock control. Alternatively, microbialcommunities which competitively exclude the growth of spoilageorganisms may be identified, which would suggest the potential forinoculation with beneficial microbes to extend product shelf life. Further down the line, there is the potential to identify safe biocontrolagents (such as bacteriophages) to suppress problematic organisms.This approach is starting to be used for both spoilage organisms such asLactobacillus brevis in beer7, and pathogenic organisms like Cronobactersakazakii in powdered infant formula8.

Details of different sequencing approaches that may be applied to understanding spoilage organismsDifferent metagenomic approaches can be taken for communityprofiling, with most analyses being either metabarcoding approaches orshotgun metagenomics. Metabarcoding involves amplification of a partof a conserved gene (normally part of the 16S rRNA gene for bacteria, forexample) from a mixed sample and then sequencing the products on anNGS platform. This will give you all the different sequences of that gene present in the community, and allow inference of which microbesare present. Shotgun metagenomics, on the other hand, involvessequencing all (or a subset of all) the DNA present in a sample in an un -

targeted fashion. The sequences produced are thencompared to existing genome databases to try andidentify which organisms they came from.

Metabarcoding has the benefit of being anestablished, repeatable and cost effective techniquethat is becoming standardised. However, being basedon the sequence of a single gene it does not alwaysprovide sufficient resolution to identify individualmicrobial species or genera. Additionally, the specificsof the gene fragment under analysis as well as down -stream computational approaches can influence the apparent abundance of different microbes. Theshotgun metagenomics approach of sequencing allDNA present requires the generation of large amountsof data in order to detect rare organisms. So muchdata requires greater computing power and analysistime, and methods for identifying microbial species

The Oxford NanoporeMinion, a small instrumentcapable of rapidly generatinglong DNA sequences

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species-specific genetic tests can detect a panel of specified species frommixed samples. The development of an NGS approach would allow thedetection of multiple meat species from a mixed sample, includingunexpected species for which an individual assay has not been designed.By adapting the amplification target this technique could be expandedto look at other products, such as herbs and spices, where adulterationcan be a problem9.

In terms of identifying food origin, NGS may also have a role to play.Many foodstuffs have their own microbiota, either from processesinherent to their production, such as fermentation, or simply picked up from the foodstuff’s environment or constituent ingredients. An understanding of the microbiota inherent to different products, andhow they change based on storage conditions, geographical origins etc,may eventually allow confirmation or refutation of origin. Many types offood appear to be amenable to these analyses. For food products wherea premium is placed on traceability and authenticity, tools like this will bean asset to food businesses when auditing their supply chains.

SummaryNext generation sequencing approaches therefore have a range ofpotential applications in the food industry in terms of keeping

consumers safe and avoiding costly recalls, reducing waste and money through combatting spoilage, and confirming the authenticity of products. As the price of sequencing per base continues to go down, and new rapid, portable sequencing platforms are being developed, NGS will become an increasingly attractive option for the food industry to replace or augment their current testing methods. At the moment NGS requires specialised equipment and skilled staff are needed to interpret the data, but we are now at the point where the industry can use those conversant in the technology to provide authoritative solutions.

from the data are also still being developed. Beyond metagenomics, the field of metatranscriptomics (the sequencing of RNA to identify which genes are being expressed under which conditions) takes us into the realm of functional genomics, where we will begin to understand which specific microbial pro -cesses are responsible for or markers of different stages of spoilage.

Food authenticityFood authenticity is an area of great strategic importance to Fera, as we manage the EU Food Integrity Project (https://secure.fera.defra.gov.uk/foodintegrity/) and played a pivotal role in the detection of beef substitution with horse meat during the horse meat scandal. Conventional DNA sequences (such as the cytochrome b gene) can be used to identify the species of single origin meat products, and

1. FDA. GenomeTrakr Fast Facts 2015 [cited 2015 12/11/2015]. Available from:http://www.fda.gov/Food/FoodScienceResearch/WholeGenomeSequencingProgramWGS/ucm403550.htm.

2. Hoffmann M, Luo Y, Monday SR, Gonzalez-Escalona N, Ottesen AR, Muruvanda T, et al. TracingOrigins of the Salmonella Bareilly Strain Causing a Food-borne Outbreak in the United States.Journal of Infectious Diseases. 2015:jiv297.

3. FDA. FDA Investigates presence of Listeria in some Hispanic-style Cheeses 2014 [cited 201520th March]. Available from: http://www.fda.gov/Food/RecallsOutbreaksEmergencies/Outbreaks/ucm386726.htm.

4. Stasiewicz MJ, Oliver HF, Wiedmann M, den Bakker HC, Elkins CA. Whole-Genome SequencingAllows for Improved Identification of Persistent Listeria monocytogenes in Food-AssociatedEnvironments. Applied and Environmental Microbiology. 2015;81(17):6024-37.

5. Monier V, Mudgal S, Escalon V, O'Connor C, Gibon T, Anderson G, et al. Preparatory Study onFood Waste Across EU 27. 2010, Technical Report – 2010 – 054.

6. House of Lords European Union Committee. Counting the Cost of Food Waste: EU Food WastePrevention. The House of Lords, 2013.

7. Deasy T, Mahony J, Neve H, Heller KJ, van Sinderen D. Isolation of a Virulent Lactobacillusbrevis Phage and Its Application in the Control of Beer Spoilage. Journal of Food Protection.2011;12(5):2157-61.

8. Lee J-H, Bai J, Shin H, Kim Y, Park B, Heu S, et al. A Novel Bacteriophage Targeting Cronobactersakazakii is a Potential Biocontrol Agent in Foods. Applied and Environmental Microbiology.2015:AEM.01827-15.

9. Does your spice rack contain fake oregano? Which? News2015 [08/01/2016].Available from: http://www.which.co.uk/news/2015/07/does-your-spice-rack-contain-fake-oregano-408737/.

References

High-throughput technologies are capable of producing sequencesof bacteria responsible for foodborne diseases

The illumina MiSeq, a benchtop sequencer used for metagenomic approaches and sequencingmicrobial genomes

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Want to know more?

To find out more about how Fera can be your partner with regards to improving understanding of emerging risks and issues, ultimately aiming to reduce your business risk and likelihood of product recall, please visit for more information;

www.fera.co.uk/food-safety-quality/riskidentification/

Want to know more?

To find out more about how Fera can be your partner with regards to improving understanding of emerging risks and issues, ultimately aiming to reduce your business risk and likelihood of product recall, please contact;

Fera Science Ltd (Fera), National Agri-Food Innovation Campus, Sand Hutton, York, YO41 1LZ, United Kingdom

T: +44 (0) 300 100 0321 E: info@fera.co.uk W: www.fera.co.uk/food-safety-quality/riskidentification/

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