INSPIIRED: a pipeline for quantitative analysis of sites of new DNA integration in cellular genomes. Homo sapiens

Integration of new DNA into host genomes is a required step in the replication of retroviruses or transposons, and these reactions have been adapted for use in therapeutic gene transfer. Intense interest focuses on tracking the distributions of integration sites to characterize populations of transduced cells and to monitor potential outgrow of cell clones. In the associated manuscript, we describe a pipeline for analyzing integration site distributions named INSPIIRED (integration site pipeline for paired-end reads). We describe optimized biochemical steps for site-isolation using Illumina paired-end sequencing, then software for alignment, quality control, and management of integration sites in unique and repeated sequences. In the method, DNA sequences are broken by sonication following isolation, so that the number of break sites can be used to infer abundance (the SonicAbundance method). To test the pipeline, we generated integration sites of known positions in silico with simulated sequence error, and describe optimization of sample processing parameters based on comparison to truth. We demonstrate the use of this pipeline to characterize the repeated structure in sample data sets from experimental infections in cell culture and human gene therapy. Software to implement this pipeline is available at https://github.com/BushmanLab/INSPIIRED.