biostudies-literatureTao XYKey Research and Development Program of ZhejiangCentral Government guided Local Science and Technology Development FundsProject of Xianghu LaboratoryAustralian Research Councilkiaf102https://www.ebi.ac.uk/biostudies/studies/S-EPMC11961865biostudies-literatureUnknown197(4)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 comPlant physiologyTTLOC: A Tn5 transposase-based approach to localize T-DNA integration sites.PMC119618652023ZY10162023C1S02001FT2101003662024SSYS0099Gilliham MFeng SLLi XJWang WLi YJChen ZHXu SCTao XYfalseTTLOC: 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 com2025-01-01T00:00:00Z2025 Mar2026-04-08T19:46:13.564Z2026-04-08T14:28:31.948ZS-EPMC119618654013178010.1093/plphys/kiaf102