NGS-based protocol for rapid identification of virus integration sites (VIS)

The protocol selectively amplifies VIS-genome junctions using the retroviral vector neomycin (NEO) gene, Genomic Walker Adapter approach, linker-mediated NEO-PCR (LM-NEO-PCR) or biotinylated NEO-capture, followed by long terminal repeats PCR (LTR-PCR). LTR-genome junctions were sequenced (NGS), reads mapped, quantified, and linked to genes. The protocol was tested on DNA from single clones holding twenty reported VIS loci and on multiplex and diluted clone DNA samples.本实验方案可借助逆转录病毒载体新霉素（NEO）基因、基因组步移接头法（Genomic Walker Adapter）、接头介导的新霉素PCR（LM-NEO-PCR）或生物素标记的新霉素捕获技术，对VIS-基因组连接区（VIS-genome junctions）进行选择性扩增，随后通过长末端重复序列PCR（LTR-PCR）完成后续扩增步骤。对所得LTR-基因组连接区进行下一代测序（NGS），对测序读段进行比对、定量，并将其与相关基因进行关联分析。本方案已在携带20个已报道VIS位点的单克隆DNA样本，以及多重混合与稀释处理的克隆DNA样本中完成性能验证。