94. AAV Integration Site Determination Using Illumina Mate Pair Sequencing

Abstract

Integration of AAV into genomic loci has been well studied using low throughput techniques, which has revealed clear integration hotspots. Recent studies using high throughput sequencing have uncovered new genomic loci where AAV may integrate. However, the low integration frequency combined with the AAV inverted terminal repeats (ITR) makes amplification technologically challenging and prone to artifacts that may mis-identify or miss some integration sites. Our lab has utilized a modified Illumina Mate Pair sequencing technique to uncover genomic integration sites and bypass the need to amplify through the AAV ITR. We believe that this technique can accurately establish bona-fide integration sites, uncover novel integration sites, and provide further evidence for existing known hotspots. At its core, mate pair sequencing involves self-circularization of fractured genomic fragments followed by recovery and sequencing of the junction. In essence, it bridges the low throughput shuttle vector systems used in the past with high throughput sequencing. Here we have utilized biotinylated loxP linkers added to the ends of genomic fragments and at unique sites in AAV vectors followed by Cre circularization (Van Nieuwerburgh, 2012). The circularized fragments are enriched by exonuclease treatment to remove linear fragments then fractured to ~400 bp. The fragments containing the loxP junction are purified using magnetic strepavadin coated beads and Illumina sequencing adapters are added before sequencing. Using this technique, we have successfully recovered control sequences of plasmids mixed with genomic DNA. Importantly, the Cre recombination reaction allows us to accurately distinguish self-circularized fragments from cross-recombined fragments and eliminate apparent false positive integration sites. We are working on applying this technique to DNA samples from mice injected with AAV. Continued study of AAV integration will be extremely important for establishing the safety profile of AAV used in clinical applications.