AAV Nanopore Seq

Monitoring of DNA contaminants in Adeno-associated virus (AAV) gene therapy vectors is of major interest, due to its clinical application with increasing therapeutic doses. We report here direct, amplification-free Nanopore sequencing of single-stranded AAV DNA using a rapid transposase-based protocol. Our findings are supported by direct sequencing of bacteriophage M13 single-stranded DNA, which leads us to deduce that single-stranded DNA in general is amenable to direct transposase-based library generation. Sequencing of packaged AAV DNA revealed single-nucleotide variants within the inverted terminal repeats and across the transgene cassette. Unexpectedly, Nanopore sequencing also revealed that both recombinant genome strands harbour CpG methylations, possibly explaining part of the reduced transduction efficiency seen for recombinant AAV compared to the wild type genome. The share of contaminating DNA found by sequencing was comparative to qPCR measurements, however, we find that the obtained long reads hold another intriguing layer of information. Long reads directly revealed packaging of genome homodimers and genome-backbone heterodimers and furthermore uncovered preferred packaging of distinct forms of backbone DNA from producer plasmids by a so far unknown mechanism. The findings promote direct Nanopore sequencing as a fast and versatile platform for AAV quality control and basic research.