Optimizing single molecule, real-time sequencing for enhanced characterization of adeno-associated viral vector genomes

Monitoring the payload of recombinant adeno-associated viral vectors (rAAVs) used for gene therapy applications is essential. Long-read sequencing is a promising method to confirm not only the identity of the packaged DNA but also to assess its integrity with regard to truncations, deletions, insertions, and chimeras. Given its high accuracy, even in regions that are notoriously difficult to sequence, such as the rAAV's inverted-terminal repeats, single molecule, real-time (SMRT) sequencing by Pacific Biosciences might be well suited for quality control purposes. However, the library preparation workflow involves multiple steps that could potentially introduce biases and thereby misrepresent the sample composition. Here, we present a systematic study investigating the influence of several steps on the quality of the output data. Based on these insights, we introduce an adapted protocol to enhance the SMRT technology for accurate and reproducible in-depth characterization of the ssDNA payload in rAAVs.