Workflow: AAV sequencing
Nanopore sequencing of native adeno-associated virus vectors for quality control
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 is crucial to ensure the correct rAAV genomes are packaged into cells before therapeutic use, to confirm the safety and efficacy of the therapy. However, using traditional short-read sequencing technology for QC can present limitations: features such as inverted terminal repeats (ITRs) in AAV genomes are especially hard to map due to their high GC content, palindromic nature, and complex secondary structure.
In contrast, long nanopore sequencing reads can be used to sequence full-length, native rAAV genomes — both single-stranded and self-complementary AAV vectors — end to end, to aid their QC. This allows for ITRs to be fully characterised, enabling identification of any truncated rAAV genomes, contamination, or mutations. Transgenes and promoters of interest can also be identified to support validation of rAAV vectors.
Here we present a workflow to sequence and characterise full-length native rAAV vectors using MinION Flow Cells on MinION or GridION sequencing devices and the EPI2ME analysis platform.