Molecular Characterization of Transgenic Events Using Next Generation Sequencing Approach

Abstract

Demand for the commercial use of genetically modified (GM) crops has been increasing in light of the projected growth of world population to nine billion by 2050. A prerequisite of paramount importance for regulatory submissions is the rigorous safety assessment of GM crops. One of the components of safety assessment is molecular characterization at DNA level which helps to determine the copy number, integrity and stability of a transgene; characterize the integration site within a host genome; and confirm the absence of vector DNA. Historically, molecular characterization has been carried out using Southern blot analysis coupled with Sanger sequencing. While this is a robust approach to characterize the transgenic crops, it is both time- and resource-consuming. The emergence of next-generation sequencing (NGS) technologies has provided highly sensitive and cost- and labor-effective alternative for molecular characterization compared to traditional Southern blot analysis. Herein, we have demonstrated the successful application of both whole genome sequencing and target capture sequencing approaches for the characterization of single and stacked transgenic events and compared the results and inferences with traditional method with respect to key criteria required for regulatory submissions.

Fig 1. Molecular characterization of transgenic events by traditional and advanced approaches.

Fig 2. Molecular characterization of transgenic soybean Transgenic Event 1 (TE1) and Transgenic Event 2 (TE2) using whole genome sequencing.

Genomic DNA of TE1 and TE2 was randomly sheared and sequenced using Illumina's HiSeq2000 instrument. The genome coverage was ~ 10X, i.e. 10 copies of soybean haploid genome. Short HiSeq2000 reads (A) spanning entire T-DNA within TE1 and TE2 (B) were mapped back to transformation plasmid that contained intended T-DNA (C1) and backbone (C2). Uninterrupted blue bars aligned to the intended T-DNA (C1) of the transformation plasmid confirms the integrity of T-DNA within events. No blue bars over plasmid backbone (C2) confirms the absence of those sequences within the genome of TE1 and TE2 events. Twenty chromosomes (Gm1-20) of soybean reference genome (Williams 82 version X) are represented in circular fashion (D). Reads spanning junction regions were mapped back to soybean reference genome, which showed single insertion site on chromosome 6 (E) in TE1 and chromosome 2 (E) in TE2.

Fig 3. Molecular characterization of soybean breeding stack Transgenic Event 1 x Transgenic Event 2 (TE1 x TE2) using whole genome sequencing.

Genomic DNA of TE1 x TE2 was randomly sheared and sequenced using Illumina's HiSeq2000 instrument. The genome coverage was ~ 14X, i.e. 14 copies of soybean haploid genome. Short HiSeq2000 reads (A) spanning entire T-DNA within TE1 and TE2 (B) were mapped back to transformation plasmid that contained intended T-DNA (C1) and backbone (C2). Uninterrupted blue bars aligned to the intended T-DNA (C1) of the transformation plasmid confirms the integrity of T-DNA within TE1 and TE2. No blue bars over plasmid backbone (C2) confirms the absence of those sequences within the genome of TE1 and TE2. Twenty chromosomes (Gm1-20) of soybean reference genome (Williams 82 version X) are represented in circular fashion (D). Reads spanning junction regions were mapped back to soybean reference genome, which showed single insertion site on chromosome 6 (E) in TE1 and on chromosome 2 (E) in TE2. T-DNA insert in both TE1 and TE2 share the same fragment at the 3'border region (F).

Fig 4. Sensitivity of paired end chemistry of NGS in detecting small DNA aberrations (insertion, deletion, and inversion) within T-DNA.