Nat Protoc. 2010 Aug;5(8):1379-95. Epub 2010 Jul 8.

Genome-wide high-throughput integrome analyses by nrLAM-PCR and next-generation sequencing.

Paruzynski A, Arens A, Gabriel R, Bartholomae CC, Scholz S, Wang W, Wolf S, Glimm H, Schmidt M, von Kalle C.

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Department of Translational Oncology, National Center for Tumor Diseases (NCT), Heidelberg, Germany.

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 >10(2)-10(3) 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 x 10(5)-1 x 10(6) integration site sequences can be accomplished within a single week.

PMID:: 20671722; [PubMed - indexed for MEDLINE]

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Research Support, Non-U.S. Gov't

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DNA Primers

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