Model-Based Classification for Digital PCR: Your Umbrella for Rain.

Standard data analysis pipelines for digital PCR estimate the concentration of a target nucleic acid by digitizing the end-point fluorescence of the parallel micro-PCR reactions, using an automated hard threshold. While it is known that misclassification has a major impact on the concentration estimate and substantially reduces accuracy, the uncertainty of this classification is typically ignored. We introduce a model-based clustering method to estimate the probability that the target is present (absent) in a partition conditional on its observed fluorescence and the distributional shape in no-template control samples. This methodology acknowledges the inherent uncertainty of the classification and provides a natural measure of precision, both at individual partition level and at the level of the global concentration. We illustrate our method on genetically modified organism, inhibition, dynamic range, and mutation detection experiments. We show that our method provides concentration estimates of similar accuracy or better than the current standard, along with a more realistic measure of precision. The individual partition probabilities and diagnostic density plots further allow for some quality control. An R implementation of our method, called Umbrella, is available, providing a more objective and automated data analysis procedure for absolute dPCR quantification.

Pilot market surveillance of GMM contaminations in alpha-amylase food enzyme products: A detection strategy strengthened by a newly developed qPCR method targeting a GM Bacillus licheniformis producing alpha-amylase.

Using high-throughput metagenomics on commercial microbial fermentation products, DNA from a new unauthorized genetically modified microorganism (GMM), namely the GM B. licheniformis strain producing alpha-amylase (GMM alpha-amylase2), was recently discovered and characterized. On this basis, a new qPCR method targeting an unnatural association of sequences specific to the GMM alpha-amylase2 strain was designed and developed in this study, allowing to strengthen the current GMM detection strategy. The performance of the newly developed qPCR method was assessed for its specificity and sensitivity to comply with the minimum performance requirements established by the European Network of GMO Laboratories for GMO analysis. Moreover, the transferability of the in house validated qPCR method was demonstrated. Finally, its applicability was confirmed by a pilot market surveillance of GMM contaminations conducted for the first time on 40 alpha-amylase food enzyme products labelled as containing alpha-amylase. This pilot market surveillance allowed also to highlight numerous contaminations with GMM alpha-amylase2, including frequent cross-contaminations with other GMM strains previously characterized. In addition, the presence of full-length AMR genes, raising health concerns, was also reported.

Development and validation of a ddPCR assay to detect and quantify tobacco DNA in smoke and smokeless tobacco and tobacco-free products.

The last decade, smoke and smokeless products claiming to be tobacco-free, including herbal cigarettes and herbal shisha, became available on the European market and gained popularity. This study proposes a new digital droplet PCR (ddPCR) method, designed based on a previously developed real-time PCR (qPCR) method being currently used by the U.S. Food and Drug Administration (FDA) to specifically detect the presence of tobacco DNA in targeting a sequence from the Nicotiana tabacum nia-1 gene. To ensure a harmonized and reliable control by enforcement laboratories, both of these qPCR and ddPCR methods were then evaluated and validated for their compliance to an international standard. First, the performance of these PCR-based methods was successfully assessed as specific and sensitive, and in line with minimum performance requirements from international standard. Secondly, the transferability to external laboratory was confirmed for these PCR-based methods. Finally, the applicability of these PCR-based methods was demonstrated using 7 ground tobacco reference materials from the Tobacco Research Center (TRC) Toronto University as well as 6 commercial smokeless and tobacco-free smoke and smokeless products. Based on this study, the previously developed qPCR method was confirmed as complying with international standard, ensuring a efficient and harmonize use by enforcement laboratories for tobacco control on the European market. Moreover, this study proposed to enforcement laboratories the possibility to use a ddPCR method, enabling the simultaneous detection and absolute quantification of tobacco DNA as well as a limited impact of PCR inhibitors.

Detection and identification of authorized and unauthorized GMOs using high-throughput sequencing with the support of a sequence-based GMO database.

The increasing number and diversity of genetically modified organisms (GMOs) for the food and feed market calls for the development of advanced methods for their detection and identification. This issue can be addressed by next generation sequencing (NGS). However, the efficiency of NGS-based strategies depends on the availability of bioinformatic methods to find sequences of the transgenic insert and junction regions, which is a challenging topic. To facilitate this task, we have developed Nexplorer, a sequence-based database in which annotated sequences of GM events are stored in a structured, searchable and extractable format. As a proof of concept, we have developed a methodology for the analysis of sequencing data of DNA walking libraries of samples containing GMOs using the database. The efficiency of the method has been tested on datasets representing various scenarios that can be encountered in routine GMO analysis. Database-guided analysis allowed obtaining detailed and reliable information with limited hands-on time. As the database allows for efficient analysis of NGS data, it paves the way for the use of NGS sequencing technology to aid routine detection and identification of GMO.

DNA walking strategy to identify unauthorized genetically modified bacteria in microbial fermentation products.

Recently, unexpected contaminations of unauthorized genetically modified microorganisms (GMM) carrying antimicrobial resistance (AMR) genes were reported in microbial fermentation products commercialized on the food and feed chain. To guarantee the traceability and safety of the food and feed chain, whole-genome sequencing (WGS) has played a key role to prove GMM contaminations via the characterization of unnatural associations of sequences. However, WGS requires a prior microbial isolation of the GMM strain, which can be difficult to successfully achieve. Therefore, in order to avoid such bottleneck, a culture-independent approach was proposed in this study. First, the screening for the aadD gene, an AMR gene conferring a resistance to kanamycin, and for the pUB110 shuttle vector, carrying the aadD gene and commonly used to produce GMM, is performed. In case of a positive signal, DNA walking methods anchored on the two borders of the detected pUB110 shuttle vector are applied to characterize unknown flanking regions. Following to the sequencing of the generated amplicons, unnatural associations of sequences can be identified, allowing to demonstrate the presence of unauthorized GMM. The developed culture-independent strategy was successfully applied on commercialized microbial fermentation products, allowing to prove the presence of GMM contaminations in the food and feed chain.

Development of a real-time PCR marker targeting a new unauthorized genetically modified microorganism producing protease identified by DNA walking.

A PCR-based DNA walking analysis was performed on a protease product suspected to contain a new unauthorized genetically modified microorganism (GMM). Though the characterization of unnatural associations of sequences between the pUB110 shuttle vector and a Bacillus amyloliquefaciens gene coding for a protease, the presence of the GMM was shown. Based on these sequences of interest, a real-time PCR marker was developed to target specifically the newly discovered GMM, namely GMM protease2. The performance of the real-time PCR marker was assessed in terms of specificity and sensitivity. The applicability of the real-time PCR GMM protease2 marker was also demonstrated on microbial fermentation products. To confirm its use by other GMO enforcement laboratories, the transferability of the in-house validated real-time PCR marker was demonstrated by assays performed by an external laboratory.

A shotgun metagenomics approach to detect and characterize unauthorized genetically modified microorganisms in microbial fermentation products.

The presence of a genetically modified microorganism (GMM) or its DNA, often harboring antimicrobial resistance (AMR) genes, in microbial fermentation products on the market is prohibited by European regulations. GMMs are currently screened for through qPCR assays targeting AMR genes and vectors, and then confirmed by targeting known specific GM constructs/events. However, when the GMM was not previously characterized and an isolate cannot be obtained, its presence cannot be proven. We present a metagenomics approach capable of delivering the proof of presence of a GMM in a microbial fermentation product, with characterization based on the detection of AMR genes and vectors, species and unnatural associations in the GMM genome. In our proof-of-concept study, this approach was performed on a case with a previously isolated and sequenced GMM, an unresolved case for which no isolate was obtained, and a non-GMM-contaminated sample, all representative for the possible scenarios to occur in routine setting. Both short and long read sequencing were used. This workflow paves the way for a strategy to detect and characterize unknown GMMs by enforcement laboratories.

Stability of the MON 810 transgene in maize.

We analysed the DNA variability of the transgene insert and its flanking regions in maize MON 810 commercial varieties. Southern analysis demonstrates that breeding, since the initial transformation event more than 10 years ago, has not resulted in any rearrangements. A detailed analysis on the DNA variability at the nucleotide level, using DNA mismatch endonuclease assays, showed the lack of polymorphisms in the transgene insert. We conclude that the mutation rate of the transgene is not significantly different from that observed in the maize endogenous genes. Six SNPs were observed in the 5'flanking region, corresponding to a Zeon1 retrotransposon long terminal repeat. All six SNPs are more than 500 bp upstream of the point of insertion of the transgene and do not affect the reliability of the established PCR-based transgene detection and quantification methods. The mutation rate of the flanking region is similar to that expected for a maize repetitive sequence. We detected low levels of cytosine methylation in leaves of different transgenic varieties, with no significant differences on comparing different transgenic varieties, and minor differences in cytosine methylation when comparing leaves at different developmental stages. There was also a reduction in cryIAb mRNA accumulation during leaf development.

Evaluation of the reliability of maize reference assays for GMO quantification.

A reliable PCR reference assay for relative genetically modified organism (GMO) quantification must be specific for the target taxon and amplify uniformly along the commercialised varieties within the considered taxon. Different reference assays for maize (Zea mays L.) are used in official methods for GMO quantification. In this study, we evaluated the reliability of eight existing maize reference assays, four of which are used in combination with an event-specific polymerase chain reaction (PCR) assay validated and published by the Community Reference Laboratory (CRL). We analysed the nucleotide sequence variation in the target genomic regions in a broad range of transgenic and conventional varieties and lines: MON 810 varieties cultivated in Spain and conventional varieties from various geographical origins and breeding history. In addition, the reliability of the assays was evaluated based on their PCR amplification performance. A single base pair substitution, corresponding to a single nucleotide polymorphism (SNP) reported in an earlier study, was observed in the forward primer of one of the studied alcohol dehydrogenase 1 (Adh1) (70) assays in a large number of varieties. The SNP presence is consistent with a poor PCR performance observed for this assay along the tested varieties. The obtained data show that the Adh1 (70) assay used in the official CRL NK603 assay is unreliable. Based on our results from both the nucleotide stability study and the PCR performance test, we can conclude that the Adh1 (136) reference assay (T25 and Bt11 assays) as well as the tested high mobility group protein gene assay, which also form parts of CRL methods for quantification, are highly reliable. Despite the observed uniformity in the nucleotide sequence of the invertase gene assay, the PCR performance test reveals that this target sequence might occur in more than one copy. Finally, although currently not forming a part of official quantification methods, zein and SSIIb assays are found to be highly reliable in terms of nucleotide stability and PCR performance and are proposed as good alternative targets for a reference assay for maize.

Are antimicrobial resistance genes key targets to detect genetically modified microorganisms in fermentation products?

As genetically modified microorganisms (GMM), commonly used by the food and feed industry to produce additives, enzymes and flavourings, are frequently harbouring antimicrobial resistance (AMR) genes as selection markers, health and environmental concerns were consequently raised. For this reason, the interest of the competent authorities to control such microbial fermentation products has strongly increased, especially since several recent accidental contaminations of unauthorized GMM, or associated recombinant DNA, in bacterial fermentation products intended for the European food and feed chain. However, no global screening strategy is currently available in enforcement laboratories to assess the presence of GMM harbouring AMR genes and/or the presence of full-length AMR genes. Moreover, the confidentiality of the related GMM dossiers strongly hampers the development of methods to perform such control. To overcome this issue, an analysis of related publicly available patents was performed in this study to identify all reported AMR genes. On this basis, the aminoglycoside adenyltransferase (aadD) gene, conferring a resistance to both kanamycin and neomycin, was identified as a key target to cover a large spectrum of GM bacteria. A real-time PCR method to screen for its potential presence as well as a nested-PCR method associated with a sequencing analysis to assess its full-length were developed to target this aadD gene. The performance of these new methods were successfully evaluated in terms of specificity, sensitivity and applicability, allowing their easy implementation in enforcement laboratories. Moreover, the integration of these newly developed methods to our very recently proposed strategy, initially targeting GMM carrying a chloramphenicol resistance gene, allows to drastically increase the detection spectrum of GM bacteria producing fermentation food and feed products. The data generated by the proposed strategy represents therefore a crucial support for the competent authorities, especially to evaluate potential risks for the food and feed safety.

Identification of an unauthorized genetically modified bacteria in food enzyme through whole-genome sequencing.

Recently, the unexpected presence of a viable unauthorized genetically modified bacterium in a commercialized food enzyme (protease) product originating from a microbial fermentation process has been notified at the European level (RASFF 2019.3332). This finding was made possible thanks to the use of the next-generation sequencing technology, as reported in this study. Whole-genome sequencing was used to characterize the genetic modification comprising a sequence from the pUB110 shuttle vector (GenBank: M19465.1), harbouring antimicrobial resistance genes conferring a resistance to kanamycine, neomycin and bleomycin, flanked on each side by a sequence coding for a protease (GenBank: WP_032874795.1). In addition, based on these data, two real-time PCR methods, that can be used by enforcement laboratories, specific to this unauthorized genetically modified bacterium were developed and validated. The present study emphasizes the key role that whole-genome sequencing can take for detection of unknown and unauthorized genetically modified microorganisms in commercialized microbial fermentation products intended for the food and feed chain. Moreover, current issues encountered by the Competent Authorities and enforcement laboratories with such unexpected contaminations and the importance of performing official controls were highlighted.

MinION sequencing technology to characterize unauthorized GM petunia plants circulating on the European Union market.

In order to characterize unauthorized genetically modified petunia, an integrated strategy has been applied here on several suspected petunia samples from the European market. More precisely, DNA fragments of interest were produced by DNA walking anchored on key targets, earlier detected by real-time PCR screening analysis, to be subsequently sequenced using the MinION platform from Oxford Nanopore Technologies. This way, the presence of genetically modified petunia was demonstrated via the characterization of their transgene flanking regions as well as unnatural associations of elements from their transgenic cassette.

An integrated strategy combining DNA walking and NGS to detect GMOs.

Recently, we developed a DNA walking system for the detection and characterization of a broad spectrum of GMOs in routine analysis of food/feed matrices. Here, we present a new version with improved throughput and sensitivity by coupling the DNA walking system to Pacific Bioscience® Next-generation sequencing technology. The performance of the new strategy was thoroughly assessed through several assays. First, we tested its detection and identification capability on grains with high or low GMO content. Second, the potential impacts of food processing were investigated using rice noodle samples. Finally, GMO mixtures and a real-life sample were analyzed to illustrate the applicability of the proposed strategy in routine GMO analysis. In all tested samples, the presence of multiple GMOs was unambiguously proven by the characterization of transgene flanking regions and the combinations of elements that are typical for transgene constructs.

Assessment of primer/template mismatch effects on real-time PCR amplification of target taxa for GMO quantification.

GMO quantification, based on real-time PCR, relies on the amplification of an event-specific transgene assay and a species-specific reference assay. The uniformity of the nucleotide sequences targeted by both assays across various transgenic varieties is an important prerequisite for correct quantification. Single nucleotide polymorphisms (SNPs) frequently occur in the maize genome and might lead to nucleotide variation in regions used to design primers and probes for reference assays. Further, they may affect the annealing of the primer to the template and reduce the efficiency of DNA amplification. We assessed the effect of a minor DNA template modification, such as a single base pair mismatch in the primer attachment site, on real-time PCR quantification. A model system was used based on the introduction of artificial mismatches between the forward primer and the DNA template in the reference assay targeting the maize starch synthase (SSIIb) gene. The results show that the presence of a mismatch between the primer and the DNA template causes partial to complete failure of the amplification of the initial DNA template depending on the type and location of the nucleotide mismatch. With this study, we show that the presence of a primer/template mismatch affects the estimated total DNA quantity to a varying degree.

Gene stacking in transgenic plants: towards compliance between definitions, terminology, and detection within the EU regulatory framework.

The combination or stacking of different traits or genes in plants is rapidly gaining popularity in biotech crop production. Here we review the existing terminology regarding gene stacking in plants, and its implications in relation to genetics, biosafety, detectability and European regulations. Different methods of production of stacked gene traits, as well as the status of their cultivation and approval, are reviewed. Related to the different techniques of transformation and production, including classical breeding, and to differences in global authorization and commercialization practices, there are many types, definitions, and perceptions of stacking. These include: (1) stacking of traits and (2) stacking of events, which are the most widely accepted perceptions of stacking, and (3) stacking of genes, which from the analytical and traceability point of view may be a more appropriate perception. These differences in perceptions and definitions are discussed, as are their implications for analytical detection and regulatory compliance according to (in particular) European Union (EU) regulations. A comprehensive terminology regarding gene stacking with regulatory relevance is proposed. The haploid genome equivalent is proposed as the prevailing unit of measurement at all stages throughout the chain, in order to ensure that terminology and definitions of gene stacks are adapted to analytical detection, traceability, and compliance with EU regulations.

Stability of the T-DNA flanking regions in transgenic Arabidopsis thaliana plants under influence of abiotic stress and cultivation practices.

Genetic transformation is often associated with different rearrangements of the plant genome at the site of insertion. Therefore the question remains weather these T-DNA insertion sites are more prone to genotoxic stresses. Here, we studied the impact of propagation through generations, the influence of gene stacking and of photo oxidative stress caused by high light intensity on the stability of the transgene flanking regions in the model plant Arabidopsis thaliana. Conformational Sensitive Capillary Electrophoresis (CSCE), RFLP and sequencing were deployed in this analysis in order to study the proximal 100 bp and the long-range T-DNA flanking sequences. By screening seven transgenic lines no evidence for occurrence of mutation events were found, implying that the nucleotide sequence of the T-DNA flanking regions of the studied events is unlikely to be unstable.

Sequence stability of the T-DNA - plant junctions in tissue culture in Arabidopsis transgenic lines.

The stability of the inserted transgenes and particularly the junction regions of transgenic events is a concern of food labeling, traceability and post release monitoring, as these regions are used for development of event-specific DNA-based detection methods. During the standard agricultural breeding practices, the transgenic lines can be exposed to completely different conditions than those in the laboratory environment. Some of these conditions have the potential to affect the stability of the transgenic locus and the surrounding DNA. As tissue culture is recognized as a stressful and mutagenic factor, we have analyzed the effect of this process on the stability of the junction regions at nucleotide level in five Arabidopsis thaliana transgenic lines in comparison with the respective integration loci in ColO and C24 ecotypes. No indication of any kind of alteration at nucleotide level of the junctions was found. The relevance of the stability of the plant-T-DNA junction regions for application of the DNA-based methods in commercial transgenic plants is discussed.