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

ABSTRACT: Next-generation sequencing of single-stranded DNA (ssDNA) enables transgene characterization of gene therapy vectors such as adeno-associated virus (AAV), but current library generation uses complicated and potentially biased second-strand synthesis. We report that libraries for nanopore sequencing of ssDNA can be conveniently created without second-strand synthesis using a transposase-based protocol. We show for bacteriophage M13 ssDNA that the MuA transposase has unexpected residual activity on ssDNA, explained in part by transposase action on transient double-stranded hairpins. In case of AAV, library creation is additionally aided by genome hybridization. We demonstrate the power of direct sequencing combined with nanopore long reads by characterizing AAV vector transgenes. Sequencing yielded reads up to full genome length, including GC-rich inverted terminal repeats. Unlike short-read techniques, single reads covered genome-genome and genome-contaminant fusions and other recombination events, whilst additionally providing information on epigenetic methylation. Single-nucleotide variants across the transgene cassette were revealed and secondary genome packaging signals were readily identified. Moreover, comparison of sequence abundance with quantitative polymerase chain reaction results demonstrated the technique's future potential for quantification of DNA impurities in AAV vector stocks. The findings promote direct nanopore sequencing as a fast and versatile platform for ssDNA characterization, such as AAV ssDNA in research and clinical settings.

SUBMITTER: Radukic MT

PROVIDER: S-EPMC7671332 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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

Radukic Marco T MT Brandt David D Haak Markus M Müller Kristian M KM Kalinowski Jörn J

NAR genomics and bioinformatics 20200928 4

Next-generation sequencing of single-stranded DNA (ssDNA) enables transgene characterization of gene therapy vectors such as adeno-associated virus (AAV), but current library generation uses complicated and potentially biased second-strand synthesis. We report that libraries for nanopore sequencing of ssDNA can be conveniently created without second-strand synthesis using a transposase-based protocol. We show for bacteriophage M13 ssDNA that the MuA transposase has unexpected residual activity o ...[more]

PMID: 33575623

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Project description:Recombinant adeno-associated viruses (rAAVs) are currently the most prominently investigated vector platform for human gene therapy. The rAAV capsid serves as a potent and efficient vehicle for delivering genetic payloads into the host cell, while the vector genome determines the function and effectiveness of these biotherapies. However, current production schemes yield vectors that may consist of heterogeneous populations, compromising their potencies. The development of next-generation sequencing methods within the past few years have helped investigators profile the diversity and relative abundances of heterogenous species in vector preparations. Specifically, long-read sequencing methods, like single molecule real-time (SMRT) sequencing, have been used to uncover truncations, chimeric genomes, and inverted terminal repeat (ITR) mutations in vectors. Unfortunately, these sequencing platforms may be inaccessible to investigators with limited resources, require large amounts of input material, or may require long wait times for sequencing and analyses. Recent advances with nanopore sequencing have helped to bridge the gap for quick and relatively inexpensive long-read sequencing needs. However, their limitations and sample biases are not well-defined for sequencing rAAV. In this study, we explored the capacity for nanopore sequencing to directly interrogate rAAV content to obtain full-length resolution of encapsidated genomes. We found that the nanopore platform can cover the entirety of rAAV genomes from ITR to ITR without the need for pre-fragmentation. However, the accuracy for base calling was low, resulting in a high degree of miscalled bases and false indels. These false indels led to read-length compression; thus, assessing heterogeneity based on read length is not advisable with current nanopore technologies. Nonetheless, nanopore sequencing was able to correctly identify truncation hotspots in single-strand and self-complementary vectors similar to SMRT sequencing. In summary, nanopore sequencing can serve as a rapid and low-cost alternative for proofing AAV vectors.

Project description:Adeno-associated viral (AAV) vectors have shown great promise in gene delivery as evidenced by recent FDA approvals. Despite efforts to optimize manufacturing for good manufacturing practice (GMP) productions, few academic laboratories have the resources to assess vector composition. One critical component of vector quality is packaged genome fidelity. Errors in viral genome replication and packaging can result in the incorporation of faulty genomes with mutations, truncations, or rearrangements, compromising vector potency. Thus, sequence validation of packaged genome composition is an important quality control (QC), even in academic settings. We developed Fast-Seq, an end-to-end method for extraction, purification, sequencing, and data analysis of packaged single-stranded AAV (ssAAV) genomes intended for non-GMP preclinical environments. We validated Fast-Seq on ssAAV vectors with three different genome compositions (CAG-GFP, CAG-tdTomato, EF1α-FLuc), three different genome sizes (2.9, 3.6, 4.4 kb), packaged in four different capsid serotypes (AAV1, AAV2, AAV5, and AAV8), and produced using the two most common production methods (Baculovirus-Sf9 and human HEK293), from both common commercial vendors and academic core facilities supplying academic laboratories. We achieved an average genome coverage of >1,400 × and an average inverted terminal repeat coverage of >280 × , despite the many differences in composition of each ssAAV sample. When compared with other ssAAV next-generation sequencing (NGS) methods for GMP settings, Fast-Seq has several unique advantages: Tn5 transposase-based fragmentation rather than sonication, 125 × less input DNA, simpler adapter ligation, compatibility with commonly available inexpensive sequencing instruments, and free open-source data analysis code in a preassembled customizable Docker container designed for novices. Fast-Seq can be completed in 18 h, is more cost-effective than other NGS methods, and is more accurate than Sanger sequencing, which is generally only applied at 1-2 × sequencing depth. Fast-Seq is a rapid, simple, and inexpensive methodology to validate packaged ssAAV genomes in academic settings.

Dataset Information

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

Publications

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

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