Fig. 1

Schematic representation of the materials and overview of the method. a The transgene border and adjacent plant genomic sequence are amplified in a single PCR consisting of two phases: a high-temperature linear phase which favors transgene-specific primer binding and extension, and a low-temperature exponential phase to favor binding of genome walking primers to the linearly amplified transgene-containing template. b PCR products from step one are used in a second PCR to attach 5’ overhang and enrich for transgene-containing targets using a separate, nested transgene-specific reverse primer. with a 5’ overhang sequence for adapter attachment. c PCR is performed to incorporate unique barcodes and a P5 and P7 Illumina adapter. d Indexed PCR amplicons are pooled together into a single multiplex sequencing library and sequenced on an Illumina platform. e Sequences are processed as single-end reads starting with alignment of the transgene-containing read (read 2) to the transgene border / vector backbone sequence. Remaining unaligned regions are submitted as BLAST database query against the reference genome of the transformed species. Left- and right-border derived libraries are processed independently. Final BLAST results are then combined and compared to identify “hotspots” or regions of high read support across multiple libraries to obtain a list of putative insertion sites for further investigation. f Primers are designed to span the predicted insertion site and amplify the full genomic region in the “T-DNA flanking PCR”. A second PCR is performed with a forward or reverse insertion site-specific primer along with a transgene-specific primer in the “T-DNA anchored PCR” to confirm the presence of a T-DNA/plant DNA junction. PCR products are then sanger sequenced to verify the sequence of the plant genomic region associated with the insertion using longer read lengths