Fig 1.
Molecular characterization of transgenic events by traditional and advanced approaches.
Table 1.
Insertion location, integration site in soybean genome and 5’ and 3’ Flanking Junction reads by whole genome sequencing of TE1 and TE2 single and TE1 x TE2 breeding stack events.
Table 2.
Insertion location, integration site in soybean genome and 5’ and 3’ Flanking Junction reads by Target genome sequencing of TE1 and TE2 single and TE1 x TE2 breeding stack events.
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).
Table 3.
Molecular characterization of soybean single events, TE 1 and TE2, and their breeding stack TE1× TE2 using whole genome sequencing sequencing approaches.
Each element of the T-DNA is represented by the “X” amount of coverage depth. Both T-DNA inserts within TE1 and TE2 events share several identical elements, such as T1_E5 = T2_E1 (promoters), T1_E7 = T2_E3 = T2_E6 (terminators), T1_E8 = T2_E4 = T2_E7 (promoters), T1_E9 = T2_E8 (gene of interest, GOI), and T1_E10 = T2_E9 (terminators).
Table 4.
Molecular characterization of soybean single events, TE 1 and TE2, and their breeding stack TE1× TE2 using target capture sequencing approaches.
Each element of the T-DNA is represented by the “X” amount of coverage depth. Both T-DNA inserts within TE1 and TE2 events share several identical elements, such as T1_E5 = T2_E1 (promoters), T1_E7 = T2_E3 = T2_E6 (terminators), T1_E8 = T2_E4 = T2_E7 (promoters), T1_E9 = T2_E8 (gene of interest, GOI), and T1_E10 = T2_E9 (terminators).
Fig 4.
Sensitivity of paired end chemistry of NGS in detecting small DNA aberrations (insertion, deletion, and inversion) within T-DNA.
Table 5.
Comparison of concepts between Southern blot analysis, Target capture sequencing and Whole Genome Sequencing.