Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 5 (8), 1379-95

Genome-wide High-Throughput Integrome Analyses by nrLAM-PCR and Next-Generation Sequencing

Affiliations

Genome-wide High-Throughput Integrome Analyses by nrLAM-PCR and Next-Generation Sequencing

Anna Paruzynski et al. Nat Protoc.

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.

Similar articles

See all similar articles

Cited by 78 PubMed Central articles

See all "Cited by" articles

References

    1. Nat Biotechnol. 2006 Jun;24(6):687-96 - PubMed
    1. Blood. 2006 Oct 15;108(8):2545-53 - PubMed
    1. Nat Med. 2006 Apr;12(4):401-9 - PubMed
    1. Nucleic Acids Res. 2007;35(19):e130 - PubMed
    1. Nucleic Acids Res. 1991 Apr 11;19(7):1713 - PubMed

Publication types

LinkOut - more resources

Feedback