TTLOC: A Tn5 transposase-based approach to localize T-DNA integration sites

Thermal asymmetric interlaced-polymerase chain reaction-based and whole-genome sequencing-based T-DNA localization approaches have been developed for the recovery of T-DNA integration sites (TISs). Nevertheless, a low-cost and high-throughput technique for the detection of TISs, which would facilitate the identification of genetically engineered plants, is in high demand for rapid crop breeding and plant synthetic biology. Here, we present Tn5 transposase-based T-DNA integration site localization (TTLOC), a Tn5-based approach for TIS localization. TTLOC employs specialized adaptor-assembled Tn5 transposases for genomic DNA tagmentation. TTLOC library construction is straightforward, involving only six steps that requires two and a half hours to complete. The resulting pooled library is compatible with next-generation sequencing, which enables high-throughput determination. We demonstrate the ability of TTLOC to recover 95 non-redundant TISs from 65 transgenic Arabidopsis (Arabidopsis thaliana) lines, and 37 non-redundant TISs from the genomes of transgenic rice (Oryza sativa), soybean (Glycine max), tomato (Solanum lycopersicum), potato (Solanum tuberosum), and from the large hexaploid wheat (Triticum aestivum) genome. TTLOC is a cost-effective method, as 1 to 2 Gb of raw data for each multiplexing library are sufficient for efficient TIS calling, independent of the genome size. Our results establish TTLOC as a promising strategy for evaluation of genome engineered plants and for selecting genome safe harbors for trait stacking in crop breeding and plant synthetic biology.