Recombinant AAV batch profiling by nanopore sequencing elucidates product-related DNA impurities and vector genome length distribution

Recombinant adeno-associated virus (rAAV) vectors can contain unintended DNA impurities that could pose safety risks. Current methods to assess purity either overlook unknown contaminants, require large input amounts, or cannot accurately resolve the highly structured inverted terminal repeats in rAAV genomes. Dunker-Seidler and Breunig et al. employed Oxford Nanopore sequencing for full-length vector genome analysis, achieving real-time comprehensive profiling that outperformed other methods.

Key points:

• Oxford Nanopore sequencing successfully identified impurities in rAAV vector genomes, including plasmid-derived DNA, helper plasmid DNA, and host-cell DNA impurities

• The authors detected a considerably improved read quality with Oxford Nanopore V14 compared with V9 chemistry, reducing false indels from 1.42% to 0.55%

• Compared with PacBio long-read sequencing, Oxford Nanopore sequencing provided comparable or superior results, especially for detecting vector genome truncations and impurities within the viral capsid

• Oxford Nanopore technology requires lower sample input compared with other sequencing platforms, making it feasible for early-stage gene therapy research

Sample type: viral vectors

Kit: Native Barcoding Kit