Magbanua Zenaida V, Hsu Chuan-Yu, Pechanova Olga, Arick Mark, Peterson Daniel G
Institute for Genomics, Biocomputing, and Biotechnology, Mississippi State University, Mississippi State, MS, USA.
Methods Mol Biol. 2025;2943:189-204. doi: 10.1007/978-1-0716-4642-7_16.
ddRAD-Seq is a reduced representation sequencing technique that results in sequence datasets that can be compared and used to identify SNPs. We present an improved ddRAD-Seq protocol that increases efficiency and decreases the time to complete a ddRAD-Seq experiment. It utilizes selected restriction enzyme digestion fragments, quick acting ligases that are neutral with the restriction enzyme buffer eliminating buffer exchange steps, and adapters designed to be compatible with Illumina index primers. Enzyme deactivation steps are eliminated, library amplification and barcoding are completed in one PCR step, highly-efficient and precise size selection with BluePippin system and cleanup steps using magnetic beads are consolidated at the end of the library generation step. The SNPs that we identified using this streamlined protocol were validated in population and evolutionary studies of cotton (plant) (Magbanua et al., Anal Biochem 662:115001, https://doi.org/10.1016/j.ab.2022.115001 , 2023) and rohu carp (animal) (Arick et al., G3 (Bethesda) 13, https://doi.org/10.1093/g3journal/jkad009 , 2023).
ddRAD-Seq是一种简化代表性测序技术，可生成可用于比较和识别单核苷酸多态性（SNP）的序列数据集。我们提出了一种改进的ddRAD-Seq方案，该方案提高了效率并缩短了完成ddRAD-Seq实验的时间。它利用选定的限制性内切酶消化片段、对限制性内切酶缓冲液呈中性的快速作用连接酶以消除缓冲液交换步骤，以及设计为与Illumina索引引物兼容的接头。省去了酶失活步骤，在一个PCR步骤中完成文库扩增和条形码标记，使用BluePippin系统进行高效精确的大小选择，并在文库生成步骤结束时合并使用磁珠的纯化步骤。我们使用这种简化方案鉴定出的SNP在棉花（植物）（Magbanua等人，《分析生物化学》662:115001，https://doi.org/10.1016/j.ab.2022.115001，2023）和印度鲤（动物）（Arick等人，《G3（贝塞斯达）》13，https://doi.org/10.1093/g3journal/jkad009，2023）的群体和进化研究中得到了验证。
