Quantifying DNA point mutations in commercially available AAV reporter vectors and plasmids

To compare the accuracy of three sequencing methods for quantifying point mutations in AAV products, we sequenced the green fluorescent protein (GFP) transgene from two AAV9 reporter vectors, three lots each, and their respective plasmids, using highly accurate single template amplification (STA) followed by Sanger sequencing (STA-Sanger), and two next generation sequencing (NGS) platforms (Illumina and Oxford Nanopore). Overall, our results showed evidence for single nucleotide mutations in AAV products and a lack of consistency among sequencing platforms, which underscores the need for the AAV industry to develop sequencing methodologies with improved accuracy as a standard QC protocol.