The main challenge associated with genotyping based on conventional length polymorphisms is the cross-laboratory standardization of allele sizes. This step requires the inclusion of standards and manual sizing to avoid false results. Capillary electrophoresis (CE) approaches limit the information to the length polymorphism and do not allow the determination of a complete marker sequence. As an alternative, high-throughput sequencing (HTS) offers complete information regarding marker sequences and their flanking regions. In this work, we investigated the suitability of a semi-quantitative sequencing approach for microsatellite genotyping using Illumina paired-end technology. Twelve microsatellite loci that are well established for grapevine CE typing were analysed on 96 grapevine samples from six different countries. We redesigned primers to the length of the amplicon for short sequencing (~100 bp). The primer pair was flanked with a 10 bp overhang for the introduction of barcodes on both sides of the amplicon to enable high multiplexing. The highest data peaks were determined as simple sequence repeat (SSR) alleles and compared with the CE dataset based on 12 reference samples. The comparison showed that HTS SSR genotyping can successfully replace the CE system in further experiments. We believe that, with next-generation sequencing, genotyping can be improved in terms of its speed, accuracy, and price.

中文翻译：

---

HTS 方法在葡萄 (Vitis vinifera L.) 中准确基因分型的潜力

与基于常规长度多态性的基因分型相关的主要挑战是等位基因大小的跨实验室标准化。此步骤需要包含标准和手动调整大小以避免错误结果。毛细管电泳 (CE) 方法将信息限制为长度多态性，并且不允许确定完整的标记序列。作为替代方案，高通量测序 (HTS) 可提供有关标记序列及其侧翼区域的完整信息。在这项工作中，我们研究了使用 Illumina 双端技术进行微卫星基因分型的半定量测序方法的适用性。对来自六个不同国家的 96 个葡萄树样本分析了 12 个为葡萄树 CE 分型而建立的微卫星位点。我们根据扩增子的长度重新设计引物，以进行短测序 (~100 bp)。引物对两侧有一个 10 bp 的突出端，用于在扩增子的两侧引入条形码，以实现高多重化。最高数据峰被确定为简单序列重复 (SSR) 等位基因，并与基于 12 个参考样本的 CE 数据集进行比较。比较表明，HTS SSR 基因分型可以在进一步的实验中成功取代 CE 系统。我们相信，通过新一代测序，基因分型可以在速度、准确性和价格方面得到改善。最高数据峰被确定为简单序列重复 (SSR) 等位基因，并与基于 12 个参考样本的 CE 数据集进行比较。比较表明，HTS SSR 基因分型可以在进一步的实验中成功取代 CE 系统。我们相信，通过新一代测序，基因分型可以在速度、准确性和价格方面得到改善。最高数据峰被确定为简单序列重复 (SSR) 等位基因，并与基于 12 个参考样本的 CE 数据集进行比较。比较表明，HTS SSR 基因分型可以在进一步的实验中成功取代 CE 系统。我们相信，通过新一代测序，基因分型可以在速度、准确性和价格方面得到改善。