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

Nat Protoc. 2010 Aug;5(8):1379-95. doi: 10.1038/nprot.2010.87. Epub 2010 Jul 8.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Computational Biology / methods
  • DNA Primers
  • Genetic Therapy
  • Genomics
  • Microspheres
  • Polymerase Chain Reaction / methods*
  • Sequence Analysis, DNA / methods*


  • DNA Primers