Similar to genetically modified organisms (GMOs) produced by classical genetic engineering, gene-edited (GE) organisms and their derived food/feed products commercialized on the European Union market fall within the scope of European Union Directive 2001/18/EC. Consequently, their control in the food/feed chain by GMO enforcement laboratories is required by the competent authorities to guarantee food/feed safety and traceability (2003/1829/EC; 2003/1830/EC). However, their detection is potentially challenging at both the analytical and interpretation levels since this requires methodological approaches that can target and detect a specific single nucleotide variation (SNV) introduced into a GE organism. In this study, we propose a targeted high-throughput sequencing approach, including (i) a prior PCR-based enrichment step to amplify regions of interest, (ii) a sequencing step, and (iii) a data analysis methodology to identify SNVs of interest. To investigate if the performance of this targeted high-throughput sequencing approach is compatible with the performance criteria used in the GMO detection field, several samples containing different percentages of a GE rice line carrying a single adenosine insertion in OsMADS26 were prepared and analyzed. The SNV of interest in samples containing the GE rice line could successfully be detected, both at high and low percentages. No impact related to food processing or to the presence of other crop species was observed. The present proof-of-concept study has allowed us to deliver the first experimental-based evidence indicating that the proposed targeted high-throughput sequencing approach may constitute, in the future, a specific and sensitive tool to support the safety and traceability of the food/feed chain regarding GE plants carrying SNVs.

Next-generation sequencing (NGS) is being integrated into routine clinical practice in the field of (hemato-) oncology to search for variants with diagnostic, prognostic, or therapeutic value at potentially low allelic frequencies. The complex sequencing workflows used require careful validation and continuous quality control. Participation in external quality assessments (EQA) helps laboratories evaluate their performance and guarantee the validity of tests results with the ultimate goal of ensuring high-quality patient care. Here, we describe three benchmarking trials performed during the period 2017–2018 aiming firstly at establishing the state-of-the-art and secondly setting up a NGS-specific EQA program at the national level in the field of clinical (hemato-) oncology in Belgium. DNA samples derived from cell line mixes and artificially mutated cell lines, designed to carry variants of clinical relevance occurring in solid tumors, hematological malignancies, and BRCA1/BRCA2 genes, were sent to Belgian human genetics, anatomic pathology, and clinical biology laboratories, to be processed following routine practices, together with surveys covering technical aspects of the NGS workflows. Despite the wide variety of platforms and workflows currently applied in routine clinical practice, performance was satisfactory, since participating laboratories identified the targeted variants with success rates ranging between 93.06% and 97.63% depending on the benchmark, and few false negative or repeatability issues were identified. However, variant reporting and interpretation varied, underlining the need for further standardization. Our approach showcases the feasibility of developing and implementing EQA for routine clinical practice in the field of (hemato-) oncology, while highlighting the challenges faced.