Abstract The rapid development of genetically modified (GM) technologies, increasing GM plant varieties and differentiated policies towards GM organisms (GMOs), make it more challenging for GMO testing nowadays. PCR-based approaches, as current routine methods, require the detailed information of transgenic events before assay. Recently reported probe-based capture strategies coupled with next-generation sequencing (NGS) offer a practical but costly way for detecting unauthorized or unknown GMOs (UGMOs) that still depend on the known GM elements as targets or start-points. Herein, we present a novel method, ‘SSH-seq’, for GMO testing based on the combination of suppressive subtractive hybridization (SSH) and NGS strategies, which has potentials for detecting UGMOs without the prior knowledge of GM cassettes. As a result, single SSH-seq experiment yielded approximately 33.8 times of reads derived from the MON87769 transgene compared to endogenous gene Lectin. For multiple SSH-seq started with eight GM soybean reference materials, part of targets were successfully enriched. Although our method might be influenced by several factors, such as the choice of degrading enzymes, GMO insertion size, sequence specificity of targets, dosage of driver DNA, NGS production, and experimental parameters, we have reasons to believe that SSH-seq will act as a powerful tool in the field of GMO testing after further optimizations, especially for UGMOs containing novel elements.

中文翻译：

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通过基于PCR的抑制消减杂交和下一代测序的组合策略检测未知转基因生物的可行方法

摘要 转基因（GM）技术的快速发展、转基因植物品种的增加以及对转基因生物（GMO）的差异化政策，使得当今转基因检测更具挑战性。作为当前的常规方法，基于 PCR 的方法需要在检测前获得转基因事件的详细信息。最近报道的基于探针的捕获策略与下一代测序 (NGS) 相结合，提供了一种实用但成本高昂的方法来检测仍然依赖于已知 GM 元素作为目标或起点的未经授权或未知的转基因生物 (UGMO)。在此，我们提出了一种新方法“SSH-seq”，用于基于抑制性消减杂交 (SSH) 和 NGS 策略相结合的 GMO 测试，该方法有可能在没有 GM 盒的先验知识的情况下检测 UGMO。因此，与内源基因凝集素相比，单个 SSH-seq 实验产生了大约 33.8 倍的来自 MON87769 转基因的读数。对于从八种转基因大豆参考材料开始的多个 SSH-seq，成功富集了部分目标。尽管我们的方法可能受到多种因素的影响，例如降解酶的选择、GMO 插入大小、目标序列特异性、驱动 DNA 的剂量、NGS 生产和实验参数，但我们有理由相信 SSH-seq 会起作用作为经过进一步优化的转基因测试领域的强大工具，特别是对于包含新元素的UGMO。部分目标被成功富集。尽管我们的方法可能受到多种因素的影响，例如降解酶的选择、GMO 插入大小、目标序列特异性、驱动 DNA 的剂量、NGS 生产和实验参数，但我们有理由相信 SSH-seq 会起作用作为经过进一步优化的转基因测试领域的强大工具，特别是对于包含新元素的UGMO。部分目标被成功富集。尽管我们的方法可能受到多种因素的影响，例如降解酶的选择、GMO 插入大小、目标序列特异性、驱动 DNA 的剂量、NGS 生产和实验参数，但我们有理由相信 SSH-seq 会起作用作为经过进一步优化的转基因测试领域的强大工具，特别是对于包含新元素的UGMO。