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

Takeshi Itoh,Ryo Ohsawa,Ritsuko Onuki,Hiroaki Sakai,Masao Oshima,Yutaka Tabei,Mai Tsuda,Masaki Endo,Tsuyoshi Tanaka

doi:10.1038/s41598-020-61949-5

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.

Highlights

The advent of several new breeding techniques (NBTs) is revolutionizing agricultural production processes, technical information necessary for their regulation is yet to be provided
Because current high-throughput sequencing technologies are promising for the detection of foreign DNA remnants in host genomes[11,12,13,14,15,16], guidelines for large-scale sequence data submission have been developed for the safety assessment of genetically-modified organisms (GMOs)[17,18]
The importance of next-generation sequencing (NGS) for the genome-edited agricultural products (GEAPs)/GMO safety assessment is recognized, the results of NGS data analyses vary depending on the bioinformatics analytical programs used[27], and this could hamper the standardization of regulation process for GEAPs and related materials

Summary

Introduction

The advent of several new breeding techniques (NBTs) is revolutionizing agricultural production processes, technical information necessary for their regulation is yet to be provided. Because current high-throughput sequencing technologies are promising for the detection of foreign DNA remnants in host genomes[11,12,13,14,15,16], guidelines for large-scale sequence data submission have been developed for the safety assessment of genetically-modified organisms (GMOs)[17,18]. We present a simple method to unambiguously detect unintended remaining foreign DNA segments that compares k-mers between the genome and vector sequences without a reference genome sequence. This method was validated through computer simulation and using real data analysis of the rice genome, which currently has the best quality among the genomes of agricultural organisms[28]. The limitations and weaknesses of the k-mer analysis and the amount of DNA sequence data needed for this analysis were investigated

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Conclusion