Today’s food industry is truly global, involving producers and manufacturers from around the world. Consumers are increasingly demanding transparency about food composition. However, ensuring traceability along the entire supply chain, from primary production to the end-consumer product, is challenging. The number of intermediaries and geographical locations involved in manufacturing processes creates a network that requires the most advanced traceability systems.
On the analytical side, food traceability remains a challenging topic. The aim of the traceability system is to guarantee the integrity of food from a raw material to a final product for the end consumer. Many methods have been proposed to track ingredient composition and identification along the supply chain. However, until now, very few methods have been identified that can really tackle this complex problem. These include DNA-based methods more focused on species identification and chemical methods, like stable isotopic analysis, which is a very powerful tool for origin and wild/farmed ingredient tracking.
Within food traceability, one of the hot topics is food authenticity to guarantee the correct composition of a product according to the description of that product and what is expected to be included in it.
Today we are seeing food authenticity being introduced to the routine testing and regulatory arena. Recent food fraud scandals mean it is imperative that the industry be able to identify the food ingredients that compose each food product, whether meat-, fish-, or plant-based. However, ready-to-eat products that are generally composed of several ingredients are more complex, particularly if those ingredients are sourced from different geographical origins, each with its own requirements. This means that the global food industry needs to adapt to the challenges presented by a dynamic and rapidly growing food market.
Popularity of NGS
The introduction of DNA-based tracing methods brings new and very powerful tools for identification of many ingredients in processed food products. One of the most recent DNA-based methods introduced for food analysis is next-generation sequencing (NGS). This method is dramatically changing the analytic approach, moving from the detection of one or a set of species to determining all species in a sample.
Currently, NGS is the only method that ensures the correct identification of species in complex foods. Its use by all major laboratories for food authenticity analysis is increasing.
The NGS method is based on DNA analysis through DNA sequencing and produces millions of individual DNA sequences all grouped in a single file. With NGS, different sequences can be produced from the various DNAs composing the food product. This means that the method is appropriate to use in products containing many ingredients visually not identifiable and mixed. Basically, since each different ingredient contains a unique DNA sequence (its own fingerprint), NGS will virtually sequence each one of the DNA molecules present in a sample to produce individual DNA sequences for each. Therefore, unlike the Sanger DNA sequencing method that originates only one DNA sequence from a food sample, NGS is the method of choice for DNA sequencing identification of products containing multiple ingredients.
Using appropriate software, the scope of NGS is virtually unlimited and it can be used on any kind of sample DNA, whether it contains different DNA sequences or not. This means that any kind of species can be detected, as the analytical method is no longer focused on detection of a limited number of species. Despite different NGS platforms available in the market (Illumina, Thermo Fisher Scientific, Pacific Biosciences, Oxford Nanopore Technologies), all of them are used to obtain sequences of defined regions in the DNA molecules and produce huge text files containing millions of individual sequences.
Specific genes are well known for species identification and include nuclear (e.g., ribosomal RNA genes), mitochondrial (e.g., COI), and chloroplast (e.g., rbcl). When a sample is analyzed the question is no longer: “Are species X, Y, or Z present in the sample?” Using NGS the question is: “Which species are present in the sample?”
Since all sequences obtained can be compared with a specific DNA database, each match between the obtained NGS sequences and the database originate a species ID result, producing a list of species instead of a presence/absence result for targeted species. Additionally, using appropriate software, a ratio of DNA sequences obtained for each species can be created. Due to the untargeted nature of this method even exotic species can be identified.
The Challenge of Fragmented DNA
DNA-based methods are limited by the need to obtain DNA fragments with the necessary integrity to perform the analysis. In some products, specifically those that have been highly processed, ingredient DNA can be highly fragmented or even absent. When DNA is highly fragmented, it is essential to guarantee that the DNA-based method used will allow the detection of DNA fragments as small as 100 base pairs, or even lower.
The smaller the DNA fragment to be analyzed, the more difficult it is to differentiate between closely related species. The best strategy is to use a DNA sequencing method that obtains the full nucleotide (A, T, G, C) sequence of the target region to be analyzed. Real-time polymerase chain reaction’s (PCR’s) fluorescent signal is a limitation for the detection of cross species reactivity, and may produce false positive results, especially in complex food products containing multiple ingredients.
DNA Barcoding Strategy
Probably the most well-known use of DNA sequencing for food authenticity is the DNA barcoding strategy that is already in use by many regulatory entities in the sector. Perhaps one of the most widely used barcoding methods is the one for fish-based products, enabling fish species identification by regulatory bodies in the U.S. and Europe. However, this method is not suitable for processed samples that contain multiple ingredients (species) as it only enables the identification of a unique species. Food products containing multiple species cannot be analyzed with this approach.
With NGS a similar barcoding approach can be used by sequencing defined DNA regions and comparing the results with the same DNA/species databases used for the classic Sanger DNA sequencing approach.
The DNA Sequence Database
One of the key points when using a DNA-sequence producing method like NGS is the reliability of the databases that are used for species identification. Many efforts have been made in recent years to try to ensure the reliability of the DNA sequences contained in the databases, including using reference material that is sequenced and included on the database. Using bioinformatic tools to analyze public data is also valuable work so long as the DNA sequence analysis tools are used correctly. The use of multiple DNA alignments and phylogenetic analyses is crucial for ensuring the reliability of the sequence included on the databases. Because NGS is highly customizable, it makes it possible for any lab to produce its own DNA database to ensure its quality.
Wider Availability of NGS
Given the recognition of NGS as a powerful tool, the first workflow for using NGS for species identification on food was announced for the market in November 2018, making the method available to any laboratory working in food production. Additionally, NGS has been introduced into standardization, namely at the ISO level, to start to define the minimum requirements related with all pre- and post-bioinformatic analyses required during NGS analysis. This includes not only the DNA sequence itself that depends on the NGS platform used, but also the definition of the DNA regions to be analyzed and the DNA databases used for species identification.
The availability and use of an untargeted approach is of great importance. Experience tells us that when authenticity issues are involved, a targeted approach is not suitable, as it will only deliver a result for the species targeted. If a product contains any additional species besides those targeted by PCR analysis, no information will be available.
A Changing Regulatory Landscape
Along with issues of authenticity, local regulators respond to increased concern about anything that can impact human health. This adds more layers of regulation to food markets.
Furthermore, today’s consumers are much more concerned about a product’s ingredients. There is often a financial concern that they are paying for something that is not as labeled, or is not what they paid for. Additional consumer concerns relate to allergens, food intolerances, species protection, and species sustainability, amongst others. Nutritional content is highly dependent on a product’s ingredients, and the full or partial substitution of any specific ingredient can impact this. Any of these concerns can be highly damaging to a food brand as consumers can rapidly lose confidence.
One of the biggest advantages of NGS testing is its untargeted nature that enables full knowledge of the DNA content of a food sample. In addition, virtually any kind of DNA sequence can be identified using the appropriate bioinformatic tools available. The use of NGS can have a huge impact on all matters related to food integrity including authenticity, safety, and traceability.
Dr. Gadanho is the global food molecular business development manager for SGS Molecular. Reach him at email@example.com. Dr. Pandiani is the global food molecular business manager for SGS Molecular. Reach him at firstname.lastname@example.org.