A bioinformatics approach for identifying transgene insertion sites using whole genome sequencing data

Abstract

BackgroundGenetically modified crops (GM crops) have been developed to improve the agricultural traits of modern crop cultivars. Safety assessments of GM crops are of paramount importance in research at developmental stages and before releasing transgenic plants into the marketplace. Sequencing technology is developing rapidly, with higher output and labor efficiencies, and will eventually replace existing methods for the molecular characterization of genetically modified organisms.MethodsTo detect the transgenic insertion locations in the three GM rice gnomes, Illumina sequencing reads are mapped and classified to the rice genome and plasmid sequence. The both mapped reads are classified to characterize the junction site between plant and transgene sequence by sequence alignment.ResultsHerein, we present a next generation sequencing (NGS)-based molecular characterization method, using transgenic rice plants SNU-Bt9–5, SNU-Bt9–30, and SNU-Bt9–109. Specifically, using bioinformatics tools, we detected the precise insertion locations and copy numbers of transfer DNA, genetic rearrangements, and the absence of backbone sequences, which were equivalent to results obtained from Southern blot analyses.ConclusionNGS methods have been suggested as an effective means of characterizing and detecting transgenic insertion locations in genomes. Our results demonstrate the use of a combination of NGS technology and bioinformatics approaches that offers cost- and time-effective methods for assessing the safety of transgenic plants.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-017-0386-x) contains supplementary material, which is available to authorized users.

Screening of oleaginous algal strains from Chlamydomonas reinhardtii mutant libraries via density gradient centrifugation

“…The discordant reads were collected and remapped to detect read coverage pattern and find the clipping regions which can be the insertion site using Integrative Genomics Viewer (IGV; Robinson et al, ). From IGV results, reads aligned to both reference sequence and target sequence were obtained to identify the insert junction location on the C. reinhardtii chromosome based on NCBI (Park et al, ).…”

Safety Assessment of Genetically Modified Feed: Is There Any Difference From Food?

“…Following identification of low-copy number transformants, the precise location(s) of the transgene(s) in the crop genome is required to be identified (Park et al, 2017). DNA sequencing approaches have been used for this purpose, and this process may also identify backbone sequence(s), which were not intended to be introduced from the transformation vector into the host genome (Kononov et al, 1997).…”

“…SGS technologies have also been proposed to comply with the requirements for GM traceability due to the ability to detect all target sequences in multiple samples without the development and validation of target-specific methods and reference material (Arulandhu et al, 2018). However, the requirement of bioinformatics knowledge for data analysis and more sophisticated devices limits its use in routine GM event detection (Park et al, 2017). Figure S4 compares qPCR, ddPCR, and SGS in terms of GM identification and quantification, multiplexing capacity and ability to detect known and unknown sequences.…”

TransgeneR: a one-stop tool for transgene integration and rearrangement discovery using sequencing data

“…Next generation sequencing has revolutionized genomic/gene expression studies and its utility in GM animals and crops is anticipated to increase. Attempts have been made towards this goal, such as Target Loci Amplification (TLA) [8], VISPA2 [9,10], CONTRAILS [11] and analysis pipelines [12]. Among them, TLA is an integrated platform by combining experimental targeted locus amplification and sequencing technologies to study the transgene events.…”