Park et al. BMC Biotechnology (2017) 17:67 DOI 10.1186/s12896-017-0386-x METHODOLOGY ARTICLE Open Access A bioinformatics approach for identifying transgene insertion sites using whole genome sequencing data Doori Park1,3, Su-Hyun Park2,6, Yong Wook Ban1,4, Youn Shic Kim2, Kyoung-Cheul Park1,5, Nam-Soo Kim3, Ju-Kon Kim2* and Ik-Young Choi1,5* Abstract Background: Genetically 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. Methods: To 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. Results: Herein, 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. Conclusion: NGS 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. Keywords: Genetically modified organism (GMO), GM rice, Next-generation sequencing (NGS), Molecular characterization, GM safety, Bioinformatics Background domesticated animals [2]. One such method of creating Domesticated plants and animals have been modified genetically modified (GM) crops involves introducing (using artificial selection and crossbreeding) to meet random insertions of recombinant DNA into genomes human needs for at least 10,000 years. However, these of crops or animals through transformation techniques, domestication processes have resulted in severe genetic such as Agrobacterium-mediated transformation and erosion in modern crops and animals, which has left particle bombardment [3, 4]. However, several issues re- them vulnerable to biotic and abiotic stresses [1]. In lated to the production of genetically modified (GM) response, many genetic modification techniques have crops are still under debate. These issues include poten- been invented to improve modern crop cultivars and tial ecological impacts, concerns for food safety, and the genetic stability of crops [5, 6]. * Correspondence: [email protected]; [email protected] Before releasing new GM crop varieties into the 2Graduate School of International Agricultural Technology and Crop Biotech marketplace, molecular characterization of the modified Institute/GreenBio Science and Technology, Seoul National University, 1447, plants is a critical step for assessing their safety and Pyeongchang, Gangwon 25354, Republic of Korea 1Department of Agriculture and Life Industry, Kangwon National University, 1 obtaining regulatory approvals [7]. Weber et al. [8] dis- Kangwondaehak-gil, Chuncheon, Gangwon 24341, Republic of Korea cussed the potential risks of genomic instability on the Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Park et al. BMC Biotechnology (2017) 17:67 Page 2 of 8 genomic plasticity of GM plants and its effects on the Methods safety of food and animal feed [8]. They concluded that Rice transformation and DNA extraction there is no evidence that GM plants are less stable than The conventional rice variety (Oryza sativa japonica cv. non-GM plants and that the risks of introducing new Illmi) was used as the background cultivar to generate food hazards into the food supply are no different than transgenic rice lines. T-DNA was composed of the rice introducing food derived from conventional breeding ribulose-1,5-bisphosphate carboxylase/oxygenase small process. Even so, practical concerns remain regarding subunit 3 (RbcS3) promoter linked to transit peptide the genomic locations of transgenic integrations [9, 10]. (TP3), insect-resistance gene Cry1Ac (Bt9), terminator If the transgene integrates into a heterochromatin re- PinII and herbicide resistant gene expression cassette gion, then it will not be expressed as desired; alterna- (35S::bar::3’nos), which was used as a selection marker tively, if the transgene is inserted by using a strong (Additional file 1: FigureS1). Transgenic rice lines were promoter, then its expression will be suppressed by host obtained by Agrobacterium-mediated co-cultivation gene silencing mechanisms. (detailed information available in [21]). Three T3 homo- Molecular characterization is necessary for the zygous transgenic rice plants, SNU-Bt9–5, SNU-Bt9–30, commercialization of transgenic crops. Using a and SNU-Bt9–109, were used as plant materials, and sequence-specific probe that is homologous to the trans- Illmi rice was used as a control. Genomic DNA from gene, Southern blot (SB) analysis has been widely used 0.5 g of leaf tissue was isolated from each of the four in molecular characterization of transgenic events to de- plants using a modified DNAzol®ES method (Molecular termine the presence, and copy numbers of, transgenes Research Center, Cincinnati, OH, USA). [11, 12]. Transgenes can also be detected through poly- merase chain reaction (PCR) and Sanger sequencing [13, Sequencing library preparation and whole genome 14] or using microarray methods [15]. Although SB and sequencing other methods are typically carried out for the selection Genomic DNA quality was evaluated by 0.5% agarose of GM events, these methods are laborious and time gel electrophoresis. Following a quality check, genomic consuming and suffer from limitations such as detection DNA for shotgun sequencing was sheared to a 500 bp of single base substitutions or small insertions/deletions, average fragment size (Covaris, Woburn, MA, USA). A which can occur within the transfer DNA (T-DNA) and Truseq DNA PCR-free Library Preparation Kit (Illumina its insertion site [16]. Recently, several publications Inc. San Diego, CA, USA) was used to construct DNA showed sufficient evidence that next generation se- libraries following the manufacturer’s protocol. The quencing (NGS) could be used to replace or comple- quality of constructed DNA libraries was confirmed by ment traditional methods [17–20]. NGS technologies using the LabChip GX system (PerkinElmer, Waltham, will likely offer rapid and cost-effective protocols for de- MA, USA). DNA libraries were sequenced with 150-bp tecting the exact copy numbers and genomic locations of paired-end sequencing on an Illumina Hiseq 2500. All transgenes, the presence of vector backbones, and the sta- reads are available in the NCBI Sequence Read Archive bility of T-DNA across generations. NGS is also sensitive repository [SRX2762614, SRX2762615, SRX2762616, enough to identify nucleotide substitutions other than SRX2762617, SRX2762618, and SRX2762619]. SNPs, including small insertions and deletions, which en- able comparative studies across events and reference ge- T-DNA insert site analysis nomes [16]. Initially, Illumina paired-end sequencing reads with In this paper, three T3 GM rice plants, (Oryza sativa average Phred scores ≥20 were retained, and duplicate japonica cv. Illmi) SNU-Bt9–5, SNU-Bt9–30, and SNU- sequences were removed using FastQC. These qualified Bt9–109, were characterized by NGS technique before reads were classified into three groups: 1) reads derived commercialization. Transfer DNA carried an insect- from rice endogenous genomic regions; 2) reads derived resistance gene, Cry1Ac, and plants were transformed from a plasmid sequence containing transfer DNA; and using Agrobacterium-mediated transformation. From high 3) reads derived from the location of transgene integra- throughput re-sequencing and bioinformatics analysis, we tion sites that spanned the junction between plant and identified the precise genomic locations of the transgene transgene sequences. To obtain the third group of reads, insertions and determined the presence or absence of reads were first mapped back to transformation plasmid plasmid backbone sequences in GM plants. We intended vector sequences (pPZP200 including T-DNA) using the to provide insights into the distinct advantages of applying Burrows-Wheeler Aligner with maximum exact matches various bioinformatics approaches, particularly regarding (BWA-MEM) with a minimum seed length