Figure 1.

Molecular methods and their resultant characterization endpoints. Panel A details the current methodology (reliant on Southern blot analysis): the junction sequence analysis (JSA) method described in this paper and the resultant characterization endpoints. Panel B shows locus-specific polymerase chain reaction (PCR) and sequencing methods and endpoints, which are common to both the Southern-based and the JSA characterization method.

 


Figure 2.

Illustration of a simple transformation event leading to detectable novel junction sequences. Schematic detail of a simple example transformation event, wherein single copy of a transfer DNA (T-DNA) cassette has been inserted at a single locus within the genome. The relationship of the control and test loci are shown along with the inserted T-DNA cassette of the test material and two novel chimeric sequences detectable around the two T-DNA to genomic flanking junctions. These novel sequences are characteristic of the transformed locus and are detectable by next generation (NexGen) sequencing and bioinformatics. Throughout this paper we term these sequences “junction sequences.”

 


Figure 3.

Panel A: circular map of the transformation plasmid PV-GMGOX20 containing the transfer DNA (T-DNA) used for Agrobacterium-mediated transformation to create MON17903. Elements of the plasmid are shown and the locations of Southern blot probes 1–6 are indicated. Panel B: circular map of the transformation plasmid PV-GMPQ/HT4404 containing the two T-DNAs used for Agrobacterium-mediated transformation to create MON87704.

 


Figure 4.

A linear map of the insert and adjacent DNA flanking the insert in MON17903. Identified on the map are the locations of the transfer DNA (T-DNA) border regions as well as restriction sites with positions relative to the size of the linear map for enzymes used in the Southern analysis. The T-DNA insert is depicted by the thick black bar shown between the 5′ and 3′ genomic flanks. The position of T-DNA probes used for Southern blot analysis is shown (labeled probe 1–3). The positions of the polymerase chain reaction (PCR) amplicons produced for directed sequencing of the locus are also shown (PCR amplicon 1–3). The resultant consensus sequence covers the entire T-DNA insert, 989 bp of 5′ flanking sequence, and 930 bp of 3′ flanking sequence.

 


Figure 5.

Genomic DNA from the test and control material were sequenced using Illumina HiSeq/TruSeq technology (Illumina, Inc.) that produces large numbers of short sequence reads approximately 100 bp in length. Sufficient numbers of these sequence fragments were obtained to comprehensively cover the genomes of each sample at >75x average coverage. Using these genome sequence reads, bioinformatics search tools were used to select all sequence reads that are significantly similar (as defined in the text) to the transformation plasmid. Only the selected sequence reads were used in further bioinformatics analysis to determine the insert number by detecting and characterizing all junction sequences and the presence or absence of the plasmid backbone sequences by lack of detectable sequences, including the use of suitable controls for experimental comprehensiveness and sensitivity.

 


Figure 6.

Southern blot analysis of MON17903 using transfer DNA (T-DNA) probes. Panel A (probe 1 and probe 3): The blot was hybridized with two 32P-labeled probes that spanned a portion of the T-DNA sequence (probe 1 and probe 3 in Fig. 4). Each lane contains approximately 10 μg of digested genomic DNA isolated from leaf tissue. Lane designations are as follows: Lane 1: parental conventional control (AseI). Lane 2: MON17903 (AseI). Lane 3: parental conventional control (StyI). Lane 4: MON17903 (StyI). Lane 5: parental conventional control (StyI) spiked with probe 1 and probe 3 (∼1.0 genome equivalent). Lane 6: parental conventional control (StyI) spiked with probe 1 and probe 3 (∼0.1 genome equivalent). Lane 7: parental conventional control (StyI) spiked with PV-GMGOX20 (BamHI and ScaI) (∼1.0 genome equivalent). Lane 8: parental conventional control (AseI). Lane 9: MON17903 (AseI). Lane 10: parental conventional control (StyI). Lane 11: MON17903 (StyI). Arrows denote the size of the DNA, in kilobase pairs, obtained from the 1 kb DNA Extension Ladder (Invitrogen) on the ethidium bromide stained gel. Long run (32 V for 14.5 h followed by 35 V for 8 h) or short run (35 V for approximately 8 h) are indicated above the lanes. Panel B (probe 2): The blot was hybridized with one 32P-labeled probe that spanned a portion of the T-DNA sequence (probe 2 in Fig. 4). Each lane contains approximately 10 μg of digested genomic DNA isolated from leaf tissue. Lane designations are as follows: Lane 1: parental conventional control (AseI). Lane 2: MON17903 (AseI). Lane 3: parental conventional control (StyI). Lane 4: MON17903 (StyI). Lane 5: parental conventional control (StyI) spiked with PV-GMGOX20 (BamHI and ScaI) (∼1.0 genome equivalent). Lane 6: parental conventional control (StyI) spiked with PV-GMGOX20 (BamHI and ScaI) (∼0.1 genome equivalent). Lane 7: parental conventional control (AseI). Lane 8: MON17903 (AseI). Lane 9: parental conventional control (StyI). Lane 10: MON17903 (StyI). Arrows denote the size of the DNA, in kilobase pairs, obtained from the 1 kb DNA Extension Ladder (Invitrogen) on the ethidium bromide stained gel. Long run (32 V for 14.5 h followed by 35 V for 8 h) or short run (35 V for approximately 8 h) are indicated above the lanes.

 


Figure 7.

Southern blot analysis of MON17903 genomic DNA using backbone probes. Each blot was hybridized with one 32P-labeled probe that spanned a portion of the backbone sequence and each lane contains approximately 10 μg of digested genomic DNA isolated from leaf tissue. Lane designations are as follows: Lane 1: parental conventional control (AseI). Lane 2: MON17903 (AseI). Lane 3: parental conventional control (StyI). Lane 4: MON17903 (StyI). Lane 5: parental conventional control (StyI) spiked with PV-GMGOX20 (BamHI and ScaI) (∼1.0 genome equivalent). Lane 6: parental conventional control (StyI) spiked with PV-GMGOX20 (BamHI and ScaI) (∼0.1 genome equivalent). Lane 7: parental conventional control (AseI). Lane 8: MON17903 (AseI). Lane 9: parental conventional control (StyI). Lane 10: MON17903 (StyI). Panel A: backbone probe 4. Panel B: backbone probe 5. Panel C: backbone probe 6 (see Fig. 3, panel A). Arrows denote the size of the DNA, in kilobase pairs, obtained from the 1 kb DNA Extension Ladder (Invitrogen) on the ethidium bromide stained gel. Long run (30 V for 14.5 h followed by 40 V for 5.5 h) or short run (40 V for 5.5 h) are indicated above the lanes.

 


Figure 8.

Schematic representation of the insert DNA in soybean MON87704. Panel A represents a linear map of the transfer DNA (T-DNA) I and T-DNA II arrangement in the transformation plasmid, PV-GMPQ/HT4404. The numbers at the bottom are the corresponding coordinates on PV-GMPQ/HT4404. Panel A also represents a linear map of the arrangement of the two T-DNAs as they exist in MON87704. The numbers at the bottom are the corresponding coordinates on MON87704. In MON87704, the T-DNA II region between two vertical dotted lines is inverted and linked in tandem to the T-DNA I region at its partial right border. Panel B: The numbers correspond to the basepair locations of the T-DNA I and T-DNA II regions in the transformation plasmid PV-GMPQ/HT4404 and those of in MON87704 insert sequence. Also shown is the correspondence of the junction sequences determined from high throughput sequencing and junction sequence analysis (JSA) bioinformatics with the full insert sequence characterized by in planta locus-specific sequence. Junction points are indicated by the “^” character in the alignment sequence text.

 


Figure 9.

Bar graph of sequence read selection results. Three different selection query sequences (PV-GMGOX20 backbone, the endogenous single copy Le1 gene, or PV-GMGOX20 transfer DNA [T-DNA]) were used to select sequences from either the MON17903 test or A3244 control Illumina sequence datasets (labeled Sequencing Sample) as described in the text. The number of selected bases (normalized per kilobase pair of Selection Query Sequence length and by sample effective coverage to a 1x depth) in each dataset is shown in this graph.

 


Figure 10.

Junction sequences detected by junction sequence analysis (JSA). Panel A: linear map of the event illustrating the relationship of the detected junction sequences to the event locus (note that the junction sequences, schematically represented by red lines, are for illustration purposes and their lengths are not drawn to scale to aid viewing). Panel B: trimmed nucleotide alignment of the detected junction sequences. The detected junction sequences are ordered by junction sequence class (defined by break point as discussed in the text) and then by alignment start site. The junction point between the transfer DNA (T-DNA) border and genomic flanking sequence is indicated by the “^” character. Both alignments are trimmed to include only the 30 plasmid bases proximal to the break point as well as all sequence of the genomic flank. Both alignments are shown 5′→3′ beginning with the detected plasmid sequence. Panel C: full consensus sequence for JSC-A and JSC-B showing perfect alignment to the independently determined in planta locus-specific sequence.