Snyder-Mackler Noah, Voyles Tawni, Tung Jenny
Department of Evolutionary Anthropology, Duke University, Durham, NC, USA.
Department of Psychology, University of Washington, Seattle, WA, USA.
Methods Mol Biol. 2019;1963:107-120. doi: 10.1007/978-1-4939-9176-1_12.
Capture-based enrichment techniques have revolutionized genomic analysis of species and populations for which only low-quality or contaminated DNA samples (e.g., ancient DNA, noninvasively collected DNA, environmental DNA) are available. This chapter outlines an optimized laboratory protocol for generating RNA "baits" for genome-wide capture of target DNA from a larger pool of DNA. This method relies on the in vitro transcription of biotinylated RNA baits, which has the dual benefit of eliminating the high cost of synthesizing custom baits and producing a bait set that targets the majority of regions genome-wide. We provide a detailed protocol for the three main steps involved in bait library construction: (1) making a DNA library from a high-quality DNA sample for the organism of interest or a closely related species; (2) using duplex-specific nuclease digestion to reduce the representation of repetitive regions in the DNA library; and (3) performing in vitro transcription of the repetitive region-depleted DNA library to generate biotinylated RNA baits. Where applicable, we include notes and recommendations based on our own experiences.
基于捕获的富集技术彻底改变了对那些只能获得低质量或受污染DNA样本（如古代DNA、非侵入性收集的DNA、环境DNA）的物种和种群的基因组分析。本章概述了一种优化的实验室方案，用于生成RNA“诱饵”，以便从更大的DNA池中对目标DNA进行全基因组捕获。该方法依赖于生物素化RNA诱饵的体外转录，这具有双重好处，既消除了合成定制诱饵的高成本，又产生了一套针对全基因组大部分区域的诱饵。我们提供了诱饵文库构建所涉及的三个主要步骤的详细方案：（1）从感兴趣的生物体或密切相关物种的高质量DNA样本制作DNA文库；（2）使用双链特异性核酸酶消化以减少DNA文库中重复区域的占比；（3）对去除重复区域的DNA文库进行体外转录以生成生物素化RNA诱饵。在适用的地方，我们根据自己的经验给出了注释和建议。
