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

Abstract

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 transposasebased 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 singlestranded DNA viruses.

SSV-Seq 2.0, a more accurate PCR-free method for high-throughput sequencing of adeno-associated viral vector genomes

“…On the other hand, sequencing technologies such as single molecule real-time (SMRT) sequencing (Pacific Biosciences) is described as giving a less biased coverage across GC-rich regions (Ross et al, 2013). Offering long read lengths, single molecule sequencing technologies also allow to study rAAV vector genome integrity (Radukic et al, 2019;Tai et al, 2018;Xie et al, 2017). Interestingly, rAAV genome truncations have been detected at hairpin-like structures using the AAV-GPseq SMRT-based assay, creating self-complementary viral genomes (Xie et al, 2017).…”

“…In addition to the relative percentage of each DNA species, SSV-Seq can provide information regarding vector genome identity with a computational analysis of the single nucleotide variants (SNV) and the sequencing coverage over the recombinant AAV genome. Since then, and as sign of interest for HTS-based methods applied to rAAV quality control, other protocols have been developed to analyze the identity (Guerin et al, 2020;Maynard et al, 2019) or integrity (Radukic et al, 2019;Tai et al, 2018;Xie et al, 2017) of AAV vector genomes by sequencing.…”

Exploring the SSBreakome: genome‐wide mapping of DNA single‐strand breaks by next‐generation sequencing

“…Oxford Nanopore) [97], promises new opportunities for simplified SSB mapping. Following heat denaturation of genomic DNA and one-step ligation of relevant adaptor-motor enzyme complexes for nanopore sequencing, protocols for the sequencing of ssDNA fragments [98] could potentially reveal the positions of 5 0 and 3 0 extremities resulting from SSBs directly and without PCR amplification, and even provide information about their absolute frequency.…”

Herpes Simplex Virus Co-Infection Facilitates Rolling Circle Replication of the Adeno-Associated Virus Genome

“…In addition, we present nanopore sequencing as a novel, high-throughput approach to investigate the mechanism of viral genome replication. Nanopore sequencing has previously been applied to analyze the integrity of genomes of recombinant AAV vector stocks (27). However, we are unaware of any studies using nanopore sequencing to assess the mechanism of viral genome replication.…”