Application Prospect Analysis Of NGS Technology In Molecular Characterization Of Genetically Modified Crops

Molecular characteristics refer to the integration,expression and genetic stability of exogenous DNA fragments in the receptor genome of transgenic plants.The main contents include the copy number of inserted exogenous genes,the flanking sequence of insertion site,the integrity of insertion sequence,and whether there is insertion of carrier bone frame sequence,etc.Molecular characteristics are the basis for researchers to screen and identify transformed weight,and also the main content of safety evaluation of transgenic plants.At present,the copy number of inserted exogenous gene can be analyzed by Southern hybridization,RT-qPCR or digital PCR,and the insertion site and flanking sequence can be obtained by PCR based genome walking.These techniques are applicable to the analysis of the molecular characteristics of genetically modified crops with simple integration of exogenous inserted fragments,but the analysis of the molecular characteristics of the complex integration of multiple copies inserted at the same site,the complex genetically modified crops with multiple exogenous genes,and the new genetically modified crops developed by gene editing and synthetic biology are often not accurate enough.With the development of DNA sequencing technology and the arrival of the era of big data,NGS(next generation sequencing)technology with its characteristics of high throughput,high sensitivity,strong operability,and repeatability,provides a new technical scheme for accurate analysis of the molecular characteristics of transgenic plants.Based on the background investigation of the development of NGS technology,the application of NGS in the analysis of molecular characteristics of transgenic plants,and the requirements of high-throughput sequencing data in the safety evaluation of transgenic plants in foreign countries,this study summarizes the technical methods of NGS technology in the analysis of molecular characteristics of genetically modified crops,and the technical origin of NGS and traditional methods in the analysis of molecular characteristics of genetically modified crops.The sequencing results and data processing methods of different sequencing platforms of NGS were compared systematically.The main research contents and results are as follows:1.The technical method of analyzing the molecular characteristics of genetically modified crops with NGS technology was established.Taking transgenic glyphosate resistant rice G2-7 transformant and its parent ZH11 as samples,the whole genome was sequenced using Illumina NovaSeq 6000 platform,and a total of 129.72 GB was obtained.Compared with the reference genome and transgenic vector sequence of rice,we determined that the exogenous gene of G2-7 was single copy insertion,without plasmid backbone insertion,and the insertion site was Oryza Sativa Japonica Group chromosome 1,the integration process resulted in 16 bp deletion of rice genome and the 49 bp deletion at both ends of the inserted exogenous DNA reference sequence,375 bp in the left and 353 bp in the right flanking sequences were found by sequence assemble methord.These results proved that the combination of NGS technology and bioinformatics method can be used to safety assessment of genetically modified crops.NGS provides an effective way to save time and cost.2.Analysis of the molecular characteristics of the transformants with complex integration.Using Illumina HiSeq 4000 platform to sequence the whole genome of transgenic glyphosate resistant rice G2-6 transformant and its parent ZH11,with the depth of 20×,30×,40× and 70×,respectively.A total of 149.35 GB sequencing data were obtained.Using PacBio Sequal platform to sequence the whole genome of the transgenic glyphosate resistant rice G2-6 transformant and its parent ZH11,the data volume was 20 GB.By combining the sequencing results of Illumina platform and PacBio platform,the molecular characteristics of G2-6 transformant were analyzed.We determined that the exogenous gene of G2-6 transformation event was single copy insertion,without plasmid backbone insertion,and the insertion site was Oryza Sativa Japonica Group chromosome 8,resulting in 31 bp deletion of rice genome and 69 insertion of exogenous DNA reference sequence at both ends.Based on the analysis of the sequencing data of Illumina and PacBio,278 bp and 773 bp flanking sequences were obtained at the left and right ends of the insertion sites which could not be obtained by traditional methods,and 482 bp unexpected insertion sequences at the 3' end of G2-6 were found.These results show that PacBio sequencing platform has unique advantages in solving the complex exogenous DNA insertion.3.The application of NGS technology in transgenic safety evaluation was compared,including different sequencing platforms,different data analysis software and different sequencing depth.In this study,using the results of Illumina and PacBio sequencing to analyze the molecular characteristics can get the same conclusion in terms of copy number,insertion site and plasmid backbone insertion.For the flanking sequence of complex transformants,PacBio can be used combining Sanger to analyze the sequencing results.Two alignment and visualization software can get a consistent conclusion,and the selection of software can be based on the amount of sequencing data and the size of reference sequence.Under the condition of building a library and sequencing in the same batch,the amount of data obtained by Illumina sequencing is directly proportional to the depth of sequencing.When assembling flanking sequence,it is recommended to use more than 30× sequencing data to ensure that the amount of data obtained by the junction area is sufficient.The above results provided a reference for the application of NGS in genetically modified crops safety assessment.In conclusion,the research results of this paper show that NGS technology can get the same results as traditional methods in analyzing the molecular characteristics of genetically modified crops.NGS technology can overcome shortcomings of traditional methods in analyzing the molecular characteristics of complex integrands,with a more efficient and rapid advantage.This paper provides theoretical support for the application of NGS technology in the safety assessment of genetically modified organisms in China.