Modified Genomic Sequencing PCR Using the MiSeq Platform to Identify Retroviral Integration Sites

使用 MiSeq 平台改进基因组测序 PCR 来识别逆转录病毒整合位点

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作者:Dustin T Rae, Casey P Collins, Jonah D Hocum, Diana L Browning, Grant D Trobridge

Abstract

High-throughput mapping of retroviral vector integration sites (RIS) has become an invaluable tool to evaluate novel gene therapy vectors and to track clonal contribution in preclinical and clinical studies. Beard et al. (Methods Mol Biol 2014;1185:321-344) described an improved protocol developed for efficient capture, sequencing, and analysis of RIS that preserves gene-modified clonal contribution information. Here we describe adaptations to the previously published modified genomic sequencing PCR (MGS-PCR) protocol using the Illumina MiSeq paired-end sequencing platform. Lentiviral, gammaretroviral, and foamy virus vector integrations were analyzed. MGS-PCR using the MiSeq platform allows for the use of merged paired-end reads, which allows for efficient localization of RIS to published genomes.

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