title: Genome-wide high-throughput integrome analyses by nrLAM-PCR and next-generation sequencing.
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.
authors.affiliation: Department of Translational Oncology, National Center for Tumor Diseases (NCT), Heidelberg, Germany.
authors.forename: Anna
authors.initials: A
authors.lastname: Paruzynski
authors.forename: Anne
authors.initials: A
authors.lastname: Arens
authors.forename: Richard
authors.initials: R
authors.lastname: Gabriel
authors.forename: Cynthia C
authors.initials: CC
authors.lastname: Bartholomae
authors.forename: Simone
authors.initials: S
authors.lastname: Scholz
authors.forename: Wei
authors.initials: W
authors.lastname: Wang
authors.forename: Stephan
authors.initials: S
authors.lastname: Wolf
authors.forename: Hanno
authors.initials: H
authors.lastname: Glimm
authors.forename: Manfred
authors.initials: M
authors.lastname: Schmidt
authors.forename: Christof
authors.initials: C
authors.lastname: von Kalle
mesh_terms: D019295:Computational Biology / Q000379:methods; D017931:DNA Primers; D015316:Genetic Therapy; D023281:Genomics; D008863:Microspheres; D016133:Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D017931:DNA Primers