Rapid and Detailed Characterization of Transgene Insertion Sites in Genetically Modified Plants via Nanopore Sequencing

Abstract

Molecular characterization of genetically modified plants can provide crucial information for the development of detection and identification methods, to comply with traceability, and labeling requirements prior to commercialization. Detailed description of the genetic modification was previously a challenging step in the safety assessment, since it required the use of laborious and time-consuming techniques. In this study an accurate, simple, and fast method was developed for molecular characterization of genetically modified (GM) plants, following a user-friendly workflow for researchers with limited bioinformatic capabilities. Three GM events from a diverse array of crop species—perennial ryegrass, white clover, and canola—were used to test the approach that exploits long-read sequencing by the MinION device, from Oxford Nanopore Technologies. The method delivered a higher degree of resolution of the transgenic events within the host genome than has previously been possible with the standard Illumina short-range sequencing strategies. The flanking sequences, copy number, and presence of backbone sequences, and overall transgene insertion structure were determined for each of the plant genomes, with the additional identification of moderate-sized secondary insertions that would have previously been missed. The proposed workflow takes only about 1 week from DNA extraction to analyzed result, and the method will complement the existing approaches for molecular characterization of GM plants, since it makes the process faster, simpler, and more cost-effective.

LIFE‐Seq: a universal Large Integrated DNA Fragment Enrichment Sequencing strategy for deciphering the transgene integration of genetically modified organisms

“…Osamu et al identified a single transgene insertion and confirmed a large genomic deletion during transgene insertion in a transgenic mouse strain (Suzuki et al, 2020). Paula et al reported the molecular characterization of three GM plant species using MinION at the WGS level (Giraldo et al, 2021). Anne-Laure et al identified genomic insertion and flanking sequences in transgenic drought-tolerant maize line 'SbSNAC1-382' using single-molecule real-time (SMRT) sequencing coupled with plasmid rescue (Boutigny et al, 2020).…”

“…Paula et al . reported the molecular characterization of three GM plant species using MinION at the WGS level (Giraldo et al ., 2021 ). Anne‐Laure et al .…”

Rapid and precise genotyping of transgene zygosity in mice using an allele-specific method

“…To date, progeny testing ( McHugh et al, 2012 ), quantitative Southern blotting analysis ( Stumpel et al, 2018 ), fluorescent in situ hybridization ( McHugh et al, 2012 ), direct fluorescence imaging ( Lin et al, 2015 ), and quantitative PCR (qPCR) assay ( Tesson et al, 2010 ) are commonly used methods to discriminate homozygous from heterozygous transgenic mice. Computation biology-based methods such as Illumina whole genome sequencing ( Yong et al, 2015 ; Irie et al, 2017) , nanopore sequencing ( Giraldo et al, 2020 ), Xdrop indirect sequencing (Samplix) ( Blondal et al, 2021 ), and nanopore adaptive sampling ( Ulrich et al, 2022 ) have recently demonstrated their abilities in determination of transgene zygosity. However, these methods are either time-consuming, or require knowledge and capability of biological computation.…”

Using Combined Methods of Genetic Mapping and Nanopore-Based Sequencing Technology to Analyze the Insertion Positions of G10evo-EPSPS and Cry1Ab/Cry2Aj Transgenes in Maize

“…Despite conducting a bioinformatics analysis of that flanking sequence, it was still difficult to determine the insert position of SK12-5 by using the conventional methods. To overcome this problem, in this study, the combined methods of genetic mapping (Zhang et al, 2015 , and Michelmore et al, 1991 ) and nanopore-based sequencing technology (Giraldo et al, 2020 ) were applied to further analyze the insertion position of SK12-5. The findings of this study are therefore very helpful for understanding the molecular characterization of SK12-5.…”