High performance analysis of genome integrating gene transfer agents

Viral clinical gene therapy trails successfully restored the function of defect genes but also introduced adverse side effects due to insertional mutagenesis. Therefore, integration site (IS) profiling of genome integrating gene transfer agents is inevitable. Viral and non-viral IS can be accessed via LAM-or nrLAM-PCR, which amplify e.g. virus or transposon flanking genomic regions. By incorporating pyrosequencing primers and probe specific tags, hundreds of samples can be pooled and directly sequenced with the 454/Roche Titanium technology. These sequences are then automatically analyzed by Perl scripting together with standardized bioinformatics programs. Pooled samples are sorted by tag and gene transfer vector-specific sequences are removed. The sequences are mapped against the host genome via BLAT and genomic IS are annotated with the next RefSeq gene, CpG island and repeat. Further (semi)automated analysis gives an overview of typical characteristics, such as IS distribution in genes and on chromosomes, IS present at different time points or fractions within one study as well as IS hot spots of certain vector types which are summarized to identify potential harmful clones. With an optimal infrastructure the bioinformatical analysis of more than 400.000 raw sequence reads can be archived in a couple of days. Combination of LAM- or nrLAM-PCR with high-throughput sequencing and an adequate high performance bioinformatics infrastructure gives a time efficient and broad insight into gene transfer studies.