Nanopore sequencing as a scalable, cost-effective platform for analyzing polyclonal vector integration sites following clinical T cell therapy

Abstract

Background Analysis of vector integration sites in gene-modified cells can provide critical information on clonality and potential biological impact on nearby genes. Current short-read next-generation sequencing methods require specialized instruments and large batch runs. Methods We used nanopore sequencing to analyze the vector integration sites of T cells transduced by the gammaretroviral vector, SFG.iCasp9.2A.I "CD19. DNA from oligoclonal cell lines and polyclonal clinical samples were restriction enzyme digested with two 6-cutters, NcoI and BspHI; and the flanking genomic DNA amplified by inverse PCR or cassette ligation PCR. Following nested PCR and barcoding, the amplicons were sequen..