Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites
Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites
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DOI：
10.3791/53840
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发表时间：
2016-03-01
影响因子：
1.2
通讯作者：
Engelman, Alan N.
中科院分区：
文献类型：
--
作者：
Serrao, Erik;Cherepanov, Peter;Engelman, Alan N.
逆转录病毒在局部和全局尺度上都表现出特征整合偏好。在这里，我们提出了一个详细的协议（1）使用连接介导的PCR（LM-PCR）扩增和下一代测序（NGS）的逆转录病毒整合位点的不同文库的生成，（2）使用BEDTools绘制每个病毒-宿主连接的基因组位置，（3）分析数据的统计相关性。从感染细胞中提取的基因组DNA通过限制酶消化或超声处理进行片段化。在合适的DNA末端修复后，将双链接头连接到DNA末端上，并使用与病毒的长末端重复（LTR）末端和连接的接头DNA互补的引物进行半巢式PCR。PCR引物携带NGS期间DNA聚类所需的序列，从而不需要单独的衔接子连接。在NGS之前进行质量控制（QC）以评估DNA片段大小分布和衔接子DNA掺入。过滤序列输出文件以获得含LTR的读段，并裁剪掉定义LTR和接头的序列。使用BLAT将修剪的宿主细胞序列映射到参考基因组，并过滤与参考基因组中的独特点的最低97%同一性。针对相邻核苷酸（nt）序列和相对于各种基因组特征的分布来仔细检查独特的整合位点。使用该方案，可以在三天内从基因组DNA构建高复杂性的整合位点文库。因此，包括易感组织培养细胞的外源性病毒感染到整合位点分析的整个方案可以在大约1至2周内进行。这项技术的最新应用涉及从HIV感染患者的整合位点的纵向分析。
Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients.