The SSV‐Seq 2.0 PCR‐Free Method Improves the Sequencing of Adeno‐Associated Viral Vector Genomes Containing GC‐Rich Regions and Homopolymers

Abstract Adeno‐associated viral vectors (AAV) are efficient engineered tools for delivering genetic material into host cells. The commercialization of AAV‐based drugs must be accompanied by the development of appropriate quality control (QC) assays. Given the potential risk of co‐transfer of oncogenic or immunogenic sequences with therapeutic vectors, accurate methods to assess the level of residual DNA in AAV vector stocks are particularly important. An assay based on high‐throughput sequencing (HTS) to identify and quantify DNA species in recombinant AAV batches is developed. Here, it is shown that PCR amplification of regions that have a local GC content >90% and include successive mononucleotide stretches, such as the CAG promoter, can introduce bias during DNA library preparation, leading to drops in sequencing coverage. To circumvent this problem, SSV‐Seq 2.0, a PCR‐free protocol for sequencing AAV vector genomes containing such sequences, is developed. The PCR‐free protocol improves the evenness of the rAAV genome coverage and consequently leads to a more accurate relative quantification of residual DNA. HTS‐based assays provide a more comprehensive assessment of DNA impurities and AAV vector genome integrity than conventional QC tests based on real‐time PCR and are useful methods to improve the safety and efficacy of these viral vectors.