Nanopore sequencing of native adeno-associated virus single-stranded DNA using a transposase-based rapid protocol

Monitoring DNA integrity and DNA contaminants in adeno-associated virus (AAV) gene therapy vectors is of major interest, because of clinical applications with increasing therapeutic doses.

We here report direct, amplification-free nanopore sequencing of single-stranded AAV DNA using a rapid transposase-based protocol.

Direct sequencing of bacteriophage M13 single-stranded DNA supports the finding that single-stranded DNA in general is amenable to direct transposase-based library generation, albeit with increased insertion bias. Sequencing AAV DNA from purified viral particles readily covered the otherwise notoriously difficult to sequence inverted terminal repeats and revealed single-nucleotide variants across the transgene cassette. Significant methylation of the packaged DNA was not identified.

Furthermore, nanopore sequencing provided long reads up to full genome coverage and enabled detection of a priori unknown packaged DNA, which sets it apart from short read techniques or qPCR. Long reads directly revealed packaging of two fused genomes and fusions of a genome to the plasmid backbone. Preferred packaging of distinct forms of backbone DNA from producer plasmids, caused by a so far unknown mechanism, were uncovered.

The findings promote direct nanopore sequencing as a fast and versatile platform for AAV vector characterization in research and clinical settings even on single-stranded DNA viruses.