A proposal for standardization of transgenic reference sequences used in food forensics

The need for standardized procedures is of paramount importance in the highly regulated and often controversial field of genetically modified organisms (GMOs) and food forensics [ , , ]. A genetically modified (GM) or transgenic plant contains a gene or genes that have been introduced artificially into their genome in order to improve agronomic traits [ , ]. Despite the benefits, public concern regarding the potential impacts of GMO on human health and environment has increased over the years [ , ]. These concerns have fuelled the implementation of strict legislation on the planting, marketing, labelling and trade of GMOs. The collaboration between research and enforcement laboratories (e.g., the European Network of GMO Laboratories) has resulted in the harmonisation and standardisation of means and methods for GMOs analysis. The use of certified reference materials (CRMs) is mandatory for the certification and accreditation of laboratories [ ]. The CRMs are available in the form of powder, extracted DNA or plasmids with transgenic DNA of a known GMO and are used for the validation of analytical procedures regarding the detection and quantification of GMOs [ ]. In contrast with the accessibility of CRMs, professionals working with GMOs face great difficulties when looking for reference DNA sequences of transgenic elements. Several countries have established a legal framework for risk assessment and authorization of GMOs that includes freely accessible databases [ , , , ], however, there is no indication of which reference sequences should be used for indexing purposes. In fact, the transgenic sequences used in many GMOs are usually kept confidential by biotech companies in order to protect their technology against competitors. It is also difficult to find the most common transgenic sequences used in many commercialized GMOs by searching public databases (e.g., NCBI Entrez Nucleotide database). The problem often resides in the different names used for the same element (e.g., P35S, p35S, P-35s, CMV 35S, CaMV 35S), making it difficult to find the correct sequence. Moreover, the sequences used in GMOs (e.g., inserts or plasmids) are retrieved simultaneously with hundreds of sequences from population studies of the donor species, which makes it very difficult to choose an adequate sequence for reference purposes. In many cases, a search for a transgenic element only retrieves complete chromosome sequences or large sequence contigs, forcing the researcher to do additional sequence analyses with specialized software. Furthermore, there is no sequence information available for some elements in public databases, particularly for junction regions between elements, which are often the target of DNA-based detection methods. In this case, the researcher has to reconstruct the junction regions by the tedious process of downloading, aligning and editing sequences from different elements. The absence of reference sequences also hinders the description of methods and communication of results among laboratories. The simple process of describing polymorphisms on a transgenic element or sharing the location of PCR primers is problematic and highly prone to mistakes without a proper reference. By all these reasons, it is becoming evident that the existence of an organized list of reference sequences for the most common transgenic elements would facilitate the development, study and detection of GMOs. The use of reference sequences is a common practice on forensic investigations [ , ]. Here we propose the standardization of transgenic reference sequences and provide a catalogue of curated sequences for the most common transgenic elements used in GM plants. The list is freely accessible via the web at http://portugene.com/GMOrefseq.html and a subset of the database is described in Table 1 as an example. The reference sequences are organized in a dynamic table with hyperlinks to the NCBI Sequence Viewer, Taxonomy and PubMed. The sequences can be visualized and downloaded with full annotations. Our reference dataset was also compared by blast with the GMO-related sequences available in the JRC GMO-Amplicons database [ ].