Foreign DNA detection by high-throughput sequencing to regulate genome-edited agricultural products

Abstract

Although the advent of several new breeding techniques (NBTs) is revolutionizing agricultural production processes, technical information necessary for their regulation is yet to be provided. Here, we show that high-throughput DNA sequencing is effective for the detection of unintended remaining foreign DNA segments in genome-edited rice. A simple k-mer detection method is presented and validated through a series of computer simulations and real data analyses. The data show that a short foreign DNA segment of 20 nucleotides can be detected and the probability that the segment is overlooked is 10⁻³ or less if the average sequencing depth is 30 or more, while the number of false hits is less than 1 on average. This method was applied to real sequencing data, and the presence and absence of an external DNA segment were successfully proven. Additionally, our in-depth analyses also identified some weaknesses in current DNA sequencing technologies. Hence, for a rigorous safety assessment, the combination of k-mer detection and another method, such as Southern blot assay, is recommended. The results presented in this study will lay the foundation for the regulation of NBT products, where foreign DNA is utilized during their generation.