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 three sequencing approaches: single-template amplification (STA) followed by Sanger sequencing (STA-Sanger) and two next-generation sequencing (NGS) platforms (Illumina and Oxford Nanopore Technologies [ONT]). Similar vector per-base mutation rates were found for both STA-Sanger (0.016%) and Illumina (0.013%), with slightly lower rates in plasmid sequences (STA-Sanger: 0.0019%; Illumina: 0.0074%), whereas ONT per-base mutation rates were much higher (vector: 2.2%; plasmid: 1.3%). The non-reference majority variant frequency (MVF) was more strongly correlated among vector lots for ONT (R² = 0.91) compared with Illumina (R² = 0.46), although correlation between plasmid and vectors was similar for both platforms (R² = 0.23) and virtually non-existent between platforms for the same sample (R² < 0.05). Overall, our results showed evidence for single-nucleotide mutations in AAV products, although there was 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.