Background Ligation-mediated PCR protocols have diverse uses including the identification of integration sites of insertional mutagens, integrating vectors and naturally occurring mobile genetic elements. For approaches that employ NGS sequencing, the relative abundance of integrations within a complex mixture is typically determined through the use of read counts or unique fragment lengths from a ligation of sheared DNA; however, these estimates may be skewed by PCR amplification biases and saturation of sequencing coverage. Results Here we describe a modification of our previous splinkerette based ligation-mediated PCR using a novel Illumina-compatible adapter design that prevents amplification of non-target DNA and incorporates unique molecular identifiers. This design reduces the number of PCR cycles required and improves relative quantitation of integration abundance for saturating sequencing coverage. By inverting the forked adapter strands from a standard orientation, the integration-genome junction can be sequenced without affecting the sequence diversity required for cluster generation on the flow cell. Replicate libraries of murine leukemia virus-infected spleen samples yielded highly reproducible quantitation of clonal integrations as well as a deep coverage of subclonal integrations. A dilution series of DNAs bearing integrations of MuLV or piggyBac transposon shows linearity of the quantitation over a range of concentrations. Conclusions Merging ligation and library generation steps can reduce total PCR amplification cycles without sacrificing coverage or fidelity. The protocol is robust enough for use in a 96 well format using an automated liquid handler and we include programs for use of a Beckman Biomek liquid handling workstation. We also include an informatics pipeline that maps reads, builds integration contigs and quantitates integration abundance using both fragment lengths and unique molecular identifiers. Suggestions for optimizing the protocol to other target DNA sequences are included. The reproducible distinction of clonal and subclonal integration sites from each other allows for analysis of populations of cells undergoing selection, such as those found in insertional mutagenesis screens.

中文翻译：

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LUMI-PCR：一种用于整合位点克隆的 Illumina 平台连接介导的 PCR 协​​议，提供整合位点的分子定量。

背景 连接介导的 PCR 方案具有多种用途，包括识别插入诱变剂的整合位点、整合载体和天然存在的可移动遗传元件。对于采用 NGS 测序的方法，复杂混合物中整合的相对丰度通常通过使用读取计数或剪切 DNA 连接的独特片段长度来确定；然而，这些估计可能会受到 PCR 扩增偏差和测序覆盖率饱和的影响。结果 在这里，我们描述了对我们之前基于 splinkerette 的连接介导 PCR 的修改，该修改使用了一种新的 Illumina 兼容适配器设计，可防止非目标 DNA 的扩增并结合独特的分子标识符。这种设计减少了所需的 PCR 循环次数，并提高了整合丰度的相对定量，以达到饱和测序覆盖率。通过从标准方向反转分叉的衔接子链，可以对整合基因组连接进行测序，而不会影响流动槽上簇生成所需的序列多样性。小鼠白血病病毒感染的脾脏样本的复制文库产生了高度可重复的克隆整合定量以及亚克隆整合的深度覆盖。一系列稀释的带有 MuLV 或 piggyBac 转座子整合的 DNA 显示出在一定浓度范围内的定量线性。结论 合并连接和文库生成步骤可以在不牺牲覆盖率或保真度的情况下减少总 PCR 扩增循环。该协议足够强大，可以使用自动液体处理器在 96 孔格式中使用，我们包括用于使用 Beckman Biomek 液体处理工作站的程序。我们还包括一个信息学管道，它使用片段长度和唯一的分子标识符来映射读取、构建整合重叠群和量化整合丰度。包括针对其他目标 DNA 序列优化协议的建议。克隆和亚克隆整合位点彼此之间的可重复区分允许分析正在选择的细胞群，例如在插入诱变筛选中发现的细胞群。使用片段长度和唯一的分子标识符构建整合重叠群并量化整合丰度。包括针对其他目标 DNA 序列优化协议的建议。克隆和亚克隆整合位点彼此之间的可重复区分允许分析正在选择的细胞群，例如在插入诱变筛选中发现的细胞群。使用片段长度和唯一的分子标识符构建整合重叠群并量化整合丰度。包括针对其他目标 DNA 序列优化协议的建议。克隆和亚克隆整合位点彼此之间的可重复区分允许分析正在选择的细胞群，例如在插入诱变筛选中发现的细胞群。