Abstract
High-throughput integration site profiling has become a feasible tool to assess vector biosafety and to monitor the cell fate of the gene-corrected cell population in clinical gene therapy studies. Here we report a step-by-step protocol for universal genome-wide and comprehensive integrome analysis that can be performed on >102–103 samples of interest in parallel. This assay is composed of fast and cost-efficient non-restrictive linear amplification–mediated PCR; optimized sample preparation for pyrosequencing; and automated bioinformatic data mining, including sequence trimming, alignment to the cellular genome and further annotation. Moreover, the workflow of this large-scale assay can be adapted to any PCR-based method aiming to characterize unknown flanking DNA adjacent to a known DNA region. Thus, in combination with next-generation sequencing technologies, large-scale integrome analysis of >4 × 105–1 × 106 integration site sequences can be accomplished within a single week.
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Acknowledgements
Funding was provided by the Deutsche Forschungsgemeinschaft DFG (SPP1230, grant of the Tumor Center Heidelberg/Mannheim), by the Bundesministerium für Bildung und Forschung BMBF (iGene), by the European Commission (VIth + VIIth Framework Program, CONSERT, CLINIGENE and PERSIST) and by the Initiative and Networking Fund of the Helmholtz Association within the Helmholtz Alliance on Immunotherapy of Cancer.
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A.P., R.G., H.G., M.S. and C.v.K. developed the concept of nrLAM-PCR. A.P., A.A., R.G., C.C.B. and S.S. established integration site analysis with nrLAM-PCR. S.W. established preparation of nrLAM-PCR and LAM-PCR products for pyrosequencing. A.A. established the bioinformatic analysis of nrLAM-PCR and LAM-PCR products. W.W. provided vectors and performed experiments. A.P., A.A., R.G., C.C.B., S.W., M.S. and C.v.K. prepared and wrote the manuscript.
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Supplementary Figs. 1–4 & Supplementary Table 1 (PDF 241 kb)
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Paruzynski, A., Arens, A., Gabriel, R. et al. Genome-wide high-throughput integrome analyses by nrLAM-PCR and next-generation sequencing. Nat Protoc 5, 1379–1395 (2010). https://doi.org/10.1038/nprot.2010.87
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DOI: https://doi.org/10.1038/nprot.2010.87
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