Molecular Characterization Of Transgenic Maize GM11048 Based On PacBio Sequencing Technology

With the rapid development of biotechnology and genomics,high-throughput sequencing technology has become the mainstream technology in the field of nucleic acid sequence analysis due to its high throughput,wide coverage and large amount of information,and is widely used in basic research,such as agriculture,medicine and microbiology.Highthroughput sequencing technology can be used as a new technology to identify the molecular characteristics of transgenic crops.Analysis of the molecular characteristics of genetically modified crops is a prerequisite for the commercial approval of genetically modified crops.This mainly includes insertion site and flanking sequence analysis,copy number analysis,exogenous fragment insertion integrity analysis,and whether it is a carrier skeleton insertion,etc.In this study,GM11048 was selected as the research object.Based on Illumina secondgeneration sequencing data and Pac Bio third-generation sequencing data,a personalized bioinformatics data analysis process was used to identify the complete molecular characteristics of the GM11048 event,and the accuracy of the above analysis results was verified by experiments.The main research contents and results are as follows:1.Illumina sequencing technology and Pac Bio sequencing technology were used to analyze the molecular characteristics of transgenic crops.Due to the increasing complexity of information on exogenous insertion of transgenic crops,conventional methods for molecular trait analysis can no longer meet the requirements.In this study,the analysis of molecular characteristics of GM11048 was taken as an example.A data analysis process based on Illumina second-generation sequencing technology and Pac Bio third-generation sequencing technology was established on the bioinformatics platform,and the information on the molecular characteristics of GM11048 was quickly and accurately obtained.The feasibility and reliability of the system was confirmed by subsequent experiments.It is also suitable for complex transformants and can technically support the study of molecular characteristics of transgenic crops.2.The molecular characteristic information of transgenic maize GM11048 was analyzed.Based on the Illumina second-generation sequencing platform and Pac Bio third-generation sequencing platform,the whole genome of GM11048 was resequenced and the molecular characteristics of GM11048 were identified based on the self-built bioinformatics data analysis platform in combination with the on-board data,recipient genome sequence information and vector sequence information.For the transformation event of transgenic maize GM11048,the analysis results showed that the exogenous fragment was inserted into the recipient genome at 252,281,07 bp-252,281,66 bp in the form of a single copy,and the length of the exogenous fragment was 133 bp-10,486 bp without the insertion of the vector skeleton sequence.The integration process resulted in a 58 bp deletion of the receptor genome,a 132 bp deletion of the LB terminal,and a 123 bp deletion of the RB terminal.At the same time,the conventional PCR assay combined with Sanger sequencing technology confirmed the accuracy of the above method in the analysis of molecular characteristics.3.The application of Pac Bio sequencing technology in molecular characterization was compared and analyzed.In this study,the whole-genome resequencing data of GM11048 were obtained based on Illumina sequencing platform and Pac Bio sequencing platform.In the molecular characteristic analysis,Illumina sequencing technology showed that the reads length of Illumina sequencing technology was short,the insertion information was often affected by the sequencing depth,but the base accuracy was high.Pac Bio third-generation sequencing technology provides longer reads and uniform coverage,which offers greater advantages in identifying complex insertion events,such as unexpected fragment insertions.The high single base error rate in Pac Bio third generation sequencing data can be improved by combining it with Illumina sequencing technology or by improving the sequencing depth for specific transformation events.Due to the application characteristics of Pac Bio third generation sequencing technology,it is a more efficient choice to analyze the molecular characteristics of transgenic plants together with second generation sequencing technology.In summary,the research results of this paper show that the technical system integrating Illumina sequencing technology and Pac Bio sequencing technology to analyze the molecular characteristics of transgenic crops has high feasibility and reliability,and has a wide range of applications,which is of great significance for the safety assessment of transgenic crops.In addition,the system will also serve as a technical reference for detecting the composition of biotech plants.