Fig 1.
Probe design and workflow of the T-DNA capture.
A. Schematic illustration of the location of the hybridization probes designed to capture T-DNA-genome junctions. The black lines represent genomic DNA. Grey rectangles represent T-DNA sequences and light green rectangles represent the left and right border repeats (LB and RB). Black triangles indicate the nicking sites. 70 mer probes represented by magenta lines are designed to match 90 bp to 20 bp (5’ to 3’) inside of the nicking sites and are 5’ biotinylated (purple circles). B. Workflow of T-DNA capture and sequencing processes.
Fig 2.
A, B. Genome browser view of the T-DNA insertion sites found in sample pPLV26-Cas9_C4 (A) and pPLV26-Cas9_C3 (B). Red reads map to the Watson strand and blue reads map to the Crick strand. Coverage distribution tracks are positioned above (grey histograms). Schematic representations of the insert-T-DNA junctions are represented below (T-DNA in grey, border sequences in light green and genomic sequences in black). For pPLV26-Cas9_C3, the peak shown was the only one detected and only one end of the insertion was recovered.
Fig 3.
A. Schematic view of read pairs that span the T-DNA—genome junctions. One read maps to the end of the T-DNA and the paired read maps to the genomic sequence around the T-DNA insertion site. B. Genome browser view showing junction reads mapping to the genomic DNA flanking the T-DNA insertion site in sample pPLV26-MeGI_2–3. C. Genome browser view of aligned reads from the same junction read pairs showing the end of the reads that are mapping to the T-DNA plasmid sequences. Elements in the T-DNA plasmid and the locations of probes for capture are shown below. The extra peak in tNOS downstream of the MeGI gene (black arrow) is due to the presence of another tNOS adjacent to the LB and the fact that Bowtie2 randomly selects among the best alignments when more than one is present. D. Schematic illustration of the inferred structure of the T-DNA insertion in sample pPLV26-MeGI_2–3 and primers that were used to confirm the insertion site. E. PCR confirmation of the T-DNA insertion structure in pPLV26-MeGI_2–3.