Fast-Seq, a simple method for rapid and inexpensive validation of packaged ssAAV genomes in academic settings.

Adeno-associated viral (AAV) vectors have shown great promise in gene delivery as evidenced by recent FDA approvals. Despite efforts to optimize manufacturing for GMP productions, few academic labs have the resources to assess vector composition. One critical component of vector quality is packaged genome fidelity. Errors in viral genome replication and packaging can result in the incorporation of faulty genomes with mutations, truncations or rearrangements, compromising vector potency. Thus, sequence validation of packaged genome composition is an important quality control, even in academic settings. We developed Fast-Seq, an end-to-end method for extraction, purification, sequencing and data analysis of packaged ssAAV genomes intended for non-GMP preclinical environments. We validated Fast-Seq on ssAAV vectors with 3 different genome compositions (CAG-tdTomato, EF1⍺-FLuc, CAG-GFP), 3 different genome sizes (2.9kb, 3.6kb, 4.3kb), packaged in 4 different capsid serotypes (AAV1, AAV2, AAV5, AAV8), and produced using the two most common production methods (Baculovirus-Sf9, human HEK293), from both common commercial vendors and academic core facilities supplying academic laboratories. We achieved an average genome coverage of >1,400X and an average ITR coverage of >280X, despite the many differences in composition of each ssAAV sample. When compared to other ssAAV NGS methods for GMP settings, Fast-Seq has several unique advantages: Tn5 transposase-based fragmentation rather than sonication, 125x less input DNA, simpler adapter ligation, compatibility with commonly available inexpensive sequencing instruments, and free open-source data analysis code in a pre-assembled customizable Docker container designed for novices. Fast-Seq can be completed in 18 hours, is more cost-effective than other NGS methods, and is more accurate than Sanger sequencing which is generally only applied at 1-2x sequencing depth. Fast-Seq is a rapid, simple, and inexpensive methodology to validate packaged ssAAV genomes in academic settings.