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 Large Integrated DNA Fragments Enrichment strategy combined with PacBio Sequencing (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.

中文翻译：

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LIFE-Seq：一种通用的大整合 DNA 片段富集测序策略，用于破译转基因生物的转基因整合

转基因生物 (GMOs) 的分子表征可提供有关外源 DNA 整合的基本信息，包括整合位点、整个插入序列和结构、侧翼序列和拷贝数，为生物安全评估提供关键数据。然而，破译转基因整合的有效方法很少，特别是对于具有复杂重排、倒位和串联重复的大 DNA 片段整合。在此，我们结合 PacBio Seq开发了一种通用的L arge I ntegrated DNA Fragments Enrichment策略uencing (LIFE-Seq) 用于破译转基因生物中的转基因整合。针对转基因元件和外源基因的通用耕作 DNA 探针有助于特异性富集与植物基因组中的转基因相关的大插入 DNA 片段，然后进行 PacBio 测序。LIFE-Seq 使用六个 GM 事件和四个作物物种进行评估。对平均约 6275 bp 的目标 DNA 片段进行富集和测序，为每个样本生成约 26 352 个高保真读数。转基因整合结构的确定具有高重复性和灵敏度。与下一代全基因组测序相比，LIFE-Seq 实现了更好的数据完整性和准确性、更大的通用性和更低的成本，特别是对于插入 DNA 结构复杂的转基因作物。