Elucidation of the genomic organizations of transgene insertion sites is essential for the genetic studies of transgenic plants. Herein, we establish an analysis pipeline that identifies the transgene insertion sites as well as the presence of vector backbones, through de novo genome assembly with high-throughput sequencing data in two transgenic soybean lines, AtYUCCA6-#5 and 35S-UGT72E3/2-#7. Sequencing data of approximately 28× and 29× genome coverages for each line generated by high-throughput sequencing were de novo assembled. The databases generated from the de novo assembled sequences were used to search contigs that contained putative insertion sites and their flanking sequences (integration sites) of transgene fragments using transgenic vector sequences as queries. The predicted integration site sequences, which are located at three annotated genes that might regulate plant development or confer disease resistance, were then confirmed by local alignment against the soybean reference genome and PCR amplification. As results, we revealed the precise transgene-flanking sequences and sequence rearrangements at insertion sites in both the transgenic lines, as well as the aberrant insertion of a transgene fragment. Consequently, relative to experimental or enrichment technologies, our approach is straightforward and time-effective, providing an alternative method for the identification of insertion sites in transgenic plants.

阐明转基因插入位点的基因组组织对于转基因植物的遗传研究至关重要。在此，我们建立了一个分析流程，通过从头基因组组装和两个转基因大豆品系 AtYUCCA6-#5 和 35S-UGT72E3/2- 中的高通量测序数据来识别转基因插入位点以及载体主链的存在。 #7. 通过高通量测序生成的每个品系的大约 28× 和 29× 基因组覆盖度的测序数据被从头组装。使用转基因载体序列作为查询，从头组装序列生成的数据库用于搜索包含转基因片段的推定插入位点及其侧翼序列（整合位点）的重叠群。预测的整合位点序列位于可能调节植物发育或赋予抗病性的三个注释基因处，然后通过与大豆参考基因组的局部比对和 PCR 扩增来确认。结果，我们揭示了两个转基因系中插入位点的精确转基因侧翼序列和序列重排，以及转基因片段的异常插入。因此，相对于实验或富集技术，我们的方法简单且省时，为鉴定转基因植物中的插入位点提供了替代方法。