Novel protocol for mapping virus integration sites in genes involved in therapy resistance

Abstract

Abstract Retroviral transduction of cancer cell lines has been used to find genes related to therapy resistance. Characterization of virus integration sites (VIS) pinpointing these genes has been cumbersome. This study defines a sequencing-based protocol to rapidly characterize genomic loci near 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. Our VIS-NGS protocol enriched significantly (p < 0.02) more loci at high reads coverage in samples with VIS compared to negative controls. The protocol found seventeen reported VIS loci (85%) in single clone DNAs, of which fifteen loci (88%) were also detected in multiplex samples. Six loci were evaluated for all dilutions, with three loci detected at lowest 1% clone proportion. The protocol can be conducted in two weeks and successfully found almost all VIS loci in single VIS clones and detected half of the evaluated loci at low clone proportion.