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 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.
Paruzynski
Anna
A
Department of Translational Oncology, National Center for Tumor Diseases (NCT), Heidelberg, Germany.
Arens
Anne
A
Gabriel
Richard
R
Bartholomae
Cynthia C
CC
Scholz
Simone
S
Wang
Wei
W
Wolf
Stephan
S
Glimm
Hanno
H
Schmidt
Manfred
M
von Kalle
Christof
C
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*
D017931:DNA Primers