Genome-wide high-throughput integrome analyses by nrLAM-PCR and next-generation sequencing
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...
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| Hauptverfasser: | , , , , , , , , , |
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| Dokumenttyp: | Article (Journal) |
| Sprache: | Englisch |
| Veröffentlicht: |
08 July 2010
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| In: |
Nature protocols
Year: 2010, Jahrgang: 5, Heft: 8, Pages: 1379-1395 |
| ISSN: | 1750-2799 |
| DOI: | 10.1038/nprot.2010.87 |
| Online-Zugang: | Verlag, lizenzpflichtig, Volltext: https://doi.org/10.1038/nprot.2010.87 Verlag, lizenzpflichtig, Volltext: https://www.nature.com/articles/nprot.2010.87 
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| Verfasserangaben: | Anna Paruzynski, Anne Arens, Richard Gabriel, Cynthia C. Bartholomae, Simone Scholz, Wei Wang, Stephan Wolf, Hanno Glimm, Manfred Schmidt & Christof von Kalle |
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| 520 | |a 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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