SSV-Seq 2.0, a more accurate PCR-free method for high-throughput
sequencing of adeno-associated viral vector genomes
Abstract
Adeno-associated viral vectors (AAV) are one of the most efficient
engineered tools for delivering genetic material into host cells. The
commercialization of AAV-based drugs goes hand in hand with the need to
increase manufacturing capacities and to develop appropriate quality
controls. In particular, accurate methods to assess the level of
residual DNA in AAV vector stocks are needed, considering the potential
risk of co-transferring oncogenic or immunogenic sequences with the
therapeutic vectors. Our laboratory has developed an assay based on
high-throughput sequencing (HTS) to exhaustively identify and quantify
DNA species in recombinant AAV batches. Compiled with a computational
analysis of the single nucleotide variants (SNV), Single-Stranded Virus
Sequencing (SSV-Seq) also provides information regarding rAAV genome
identity. In this study, we show that the PCR amplification of regions
with high GC content and including mononucleotide stretches can be
biased during the DNA library preparation, leading to drops in the
sequencing coverage along the AAV vector genome. To circumvent this
problem, we have developed a PCR-free protocol, named SSV-Seq 2.0, that
is optimized for the sequencing of rAAV genomes that contain sequences
with a high percentage of GC and homopolymers, such as the CAG promoter.
HTS-based assays are indispensable to provide accurate data to the
regulatory agencies regarding nucleic acids content in AAV vector
batches and to improve the safety and efficacy of these viral vectors.