LIFE‐Seq: a universal Large Integrated DNA Fragment Enrichment Sequencing strategy for deciphering the transgene integration of genetically modified organisms

Abstract

Summary Molecular characterization of genetically modified organisms (GMOs) yields basic information on exogenous DNA integration, including integration sites, entire inserted sequences and structures, flanking sequences and copy number, providing key data for biosafety assessment. However, there are few effective methods for deciphering transgene integration, especially for large DNA fragment integration with complex rearrangement, inversion and tandem repeats. Herein, we developed a universal L arge I ntegrated DNA F ragments E nrichment strategy combined with PacBio Seq uencing (LIFE‐Seq) for deciphering transgene integration in GMOs. Universal tilling DNA probes targeting transgenic elements and exogenous genes facilitate specific enrichment of large inserted DNA fragments associated with transgenes from plant genomes, followed by PacBio sequencing. LIFE‐Seq were evaluated using six GM events and four crop species. Target DNA fragments averaging ~6275 bp were enriched and sequenced, generating ~26 352 high fidelity reads for each sample. Transgene integration structures were determined with high repeatability and sensitivity. Compared with next‐generation whole‐genome sequencing, LIFE‐Seq achieved better data integrity and accuracy, greater universality and lower cost, especially for transgenic crops with complex inserted DNA structures. LIFE‐Seq could be applied in molecular characterization of transgenic crops and animals, and complex DNA structure analysis in genetics research.

T-DNA characterization of genetically modified 3-R-gene late blight-resistant potato events with a novel procedure utilizing the Samplix Xdrop® enrichment technology

“…Access to the Xdrop ® instrument in a local laboratory or the availability of an Xdrop ® targeted enrichment service, within a technical research facility, would be cost-effective and efficient for researchers that only require occasional use of the instrument. As described in the introduction, the techniques, target capture sequencing (TCS) coupled with Illumina sequencing ( Magembe et al., 2023 ) and LIFE‐Seq (a universal Large Integrated DNA Fragment Enrichment Sequencing) coupled with PacBio sequencing ( Zhang et al., 2022 ), also offer improved and cost-effective approaches to characterizing T-DNA in genetically modified crops.…”

“…Coverage across the entire T-DNA was not the focus of the paper and was not fully analyzed with the TCS method. Another strategy for T-DNA characterizing involves targeted fragment enrichment called LIFE‐Seq (a universal Large Integrated DNA Fragment Enrichment Sequencing) which has been reported for use in four crops—maize, rice, canola, and soybean ( Zhang et al., 2022 ). This approach utilizes long target DNA enrichment using several applications, including genome DNA random fragmentation, end repair, A-tailing ligation, barcode adapter ligation, size selection, tiling probe hybridization and capture, and long-range PCR.…”