Oxford Nanopore Sequencing of Native Adeno-Associated Virus Vectors for Quality Control

Introduction

Adeno-associated virus (AAV) is a non-enveloped single-stranded DNA virus used in gene therapy. Accurate validation, contamination detection, and quality control (QC) of recombinant AAV (rAAV) vectors are crucial to ensure the correct rAAV genomes are packaged into capsids before therapeutic use, confirming the safety and efficacy of the therapy. Legacy short-read sequencing technology can present limitations, particularly with features like inverted terminal repeats (ITRs) in AAV genomes, which are difficult to map due to their high GC content, palindromic nature, and complex secondary structure. Short-read sequencing also struggles with accurately characterizing genome truncations and heterogeneous vector populations, impacting functionality and efficacy.

In contrast, long Oxford Nanopore reads can sequence full-length, native rAAV genomes, including both single-stranded and self-complementary AAV vectors, end to end. This enables full characterization of ITRs, identification of any truncated rAAV genomes, contamination, or mutations. Transgenes and promoters of interest can also be identified to support validation of rAAV vectors.

Prior to rAAV sequencing, whole-plasmid sequencing should be performed for complete QC across the entire AAV production process. Further details can be found in the whole-plasmid sequencing workflow: nanoporetech.com/resource-centre/plasmid-sequencing-workflow.

This document presents a workflow to sequence and characterize full-length, native rAAV vectors using MinION™ or GridION™ sequencing devices and the EPI2ME™ analysis platform.

Extraction: Obtaining High Molecular-Weight DNA

For full-length sequencing of rAAV genomes in single reads, it is important to select an extraction method that preserves the ~4.7 kb DNA fragments. The PureLink Viral RNA/DNA Mini Kit is recommended as it has been observed to produce more full-length rAAV genomes and ITR sequences compared to other methods, such as proteinase K and heat-based extractions. Prior to extraction, samples should be treated with DNase I to remove any unencapsidated DNA contamination.

Self-annealing of any remaining single strands of rAAV vector before extraction is optional; omitting this step is recommended to maximize the number of full-length ITR sequences. Quantifying samples using the Qubit ssDNA and dsDNA HS Assay Kits and a Qubit fluorometer is recommended before proceeding to library preparation.

Find extraction protocols and guidance on DNA storage and handling: nanoporetech.com/documentation/prepare.

Process:

~160 min
rAAV lysate
DNase I treatment and rAAV extraction with PureLink RNA/DNA Mini Kit
DNA from extracted rAAV

Library Preparation: Multiplexing Samples

To prepare samples for sequencing in multiplex, use the Oxford Nanopore Native Barcoding Kit 24. This PCR-free library preparation method is optimized for high sequencing output of native DNA.

Multiplexed sequencing of samples on a single MinION Flow Cell can reduce the cost per sample. Flow cells can also be washed and reused with the Flow Cell Wash Kit.

Learn more about Oxford Nanopore library preparation: nanoporetech.com/prepare.

Process:

~135 min
DNA from extracted rAAV
End-prep
Ligation of barcodes
Ligation of sequencing adapters
Loading

Sequencing

We recommend sequencing up to six barcoded rAAV libraries on a MinION Flow Cell. This can be run on a portable MinION device for easily accessible, routine sequencing. For higher throughput needs, the GridION device can be used, enabling on-demand sequencing on up to five independent flow cells at a time.

For full-length rAAV sequencing, basecalling in high accuracy (HAC) mode using the MinKNOW™ Software is recommended. Sequencing should continue until the necessary coverage for experimental goals is achieved. To detect shorter reads that may indicate contamination, such as ITR-tetramers, the minimum read length can be reduced in MinKNOW.

Find out more about Oxford Nanopore sequencing devices: nanoporetech.com/sequence.

Device: GridION

Analysis: Using the EPI2ME wf-aav-qc Workflow

QC of rAAV vectors is achieved using the wf-aav-qc workflow. This workflow, an EPI2ME solution, integrates several tools for the quick assessment of rAAV vectors. These include minimap2¹ to map reads to a combined reference sequence (containing host cell reference genome, the transgene plasmid, and other AAV plasmids) and Medaka² to call transgene plasmid variants and generate a consensus sequence.

EPI2ME is the user-friendly Oxford Nanopore data analysis platform, suitable for all levels of bioinformatics expertise. The report generated by this EPI2ME workflow includes contamination plots (showing read sources distribution), a truncations plot, an ITR-ITR read coverage plot, and a genome type frequency plot.

View the dedicated EPI2ME workflow: nanoporetech.com/epi2me-wf-aav-qc.

Analysis Components:

AAV-QC
Outputs
Pipeline
FAQ's

Further Information

Find out more at: nanoporetech.com/biopharma.

View the end-to-end protocol: nanoporetech.com/aav-sequencing-protocol.

References

Li, H. Minimap2: pairwise alignment for nucleotide sequences. Bioinformatics 34(18):3094-3100 (2018). DOI: https://doi.org/10.1093/bioinformatics/bty191
GitHub. Medaka. Available at: https://github.com/nanoporetech/medaka [Accessed 05 June 2025]

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