Detecting DNA double-stranded breaks in mammalian genomes by linear amplification-mediated high-throughput genome-wide translocation sequencing.
Detecting DNA double-stranded breaks in mammalian genomes by linear amplification-mediated high-throughput genome-wide translocation sequencing.
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DOI：
10.1038/nprot.2016.043
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发表时间：
2016-05
期刊：
影响因子：
14.8
通讯作者：
Frock RL
中科院分区：
文献类型：
--
作者：
Hu J;Meyers RM;Dong J;Panchakshari RA;Alt FW;Frock RL
无偏，高通量检测和定量DNA双链断裂（DSB）在哺乳动物细胞的全基因组将促进产生和修复内源性DSB的机制的基础研究。它们还将使更多的应用研究成为可能，例如评估工程化核酸酶的靶向和脱靶活性。在这里，我们描述了一种线性扩增介导的高通量全基因组测序（LAM-HTGTS）方法检测全基因组的“猎物”DSB通过其易位在培养的哺乳动物细胞中的固定的“诱饵”DSB。使用LAM-PCR从分离的基因组DNA直接克隆诱饵-猎物连接，并单向连接到桥接头;随后的PCR步骤扩增单链DNA连接文库，为Illumina配对末端Miseq测序做准备。一个定制的生物信息学管道识别有助于连接的猎物序列，并将它们映射到整个基因组。LAM-HTGTS与相关方法不同，因为它以核苷酸水平分辨率检测广泛的断裂末端结构。熟悉核酸方法和下一代测序分析对于文库生成和数据解释是必要的。LAM-HTGTS检测灵敏、可重复、相对便宜、可扩展且易于实施，周转时间不到一周。
Unbiased, high-throughput assays to detect and quantify DNA double-stranded breaks (DSBs) genome-wide in mammalian cells will facilitate basic studies of mechanisms that generate and repair endogenous DSBs. They will also enable more applied studies, such as evaluating on- and off-target activities of engineered nucleases. Here we describe a linear amplification-mediated high-throughput genome-wide sequencing (LAM-HTGTS) method for detecting genome-wide “prey” DSBs via their translocation in cultured mammalian cells to a fixed “bait” DSB. Bait-prey junctions are cloned directly from isolated genomic DNA using LAM-PCR and unidirectionally ligated to bridge adapters; subsequent PCR steps amplify the single-stranded DNA junction library in preparation for Illumina paired-end Miseq sequencing. A custom bioinformatic pipeline identifies prey sequences that contribute to junctions and maps them across the genome. LAM-HTGTS differs from related approaches because it detects a wide range of broken end structures with nucleotide level resolution. Familiarity with nucleic acid methods and next-generation sequencing analysis are necessary for library generation and data interpretation. LAM-HTGTS assays are sensitive, reproducible, relatively inexpensive, scalable, and straightforward to implement with a turnaround time of less than one week.