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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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Main Authors: Paruzynski, Anna (Author) , Arens, Anne (Author) , Gabriel, Richard (Author) , Bartholomä, Cynthia C. (Author) , Scholz, Simone (Author) , Wang, Wei (Author) , Wolf, Stephan (Author) , Glimm, Hanno (Author) , Schmidt, Manfred (Author) , Kalle, Christof von (Author)
Format: Article (Journal)
Language:English
Published: 08 July 2010
In: Nature protocols
Year: 2010, Volume: 5, Issue: 8, Pages: 1379-1395
ISSN:1750-2799
DOI:10.1038/nprot.2010.87
Online Access: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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Author Notes: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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Summary: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.
Item Description:Gesehen am 26.06.2023
Physical Description:Online Resource
ISSN:1750-2799
DOI:10.1038/nprot.2010.87

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