Adapting A Hybrid Analysis For AAV Characterization By Combining Long And Short Read Sequencing Techniques

By Brianna McManus, Rebecca Bova, Ph.D., Christina Kalinyak, Maggie Luce, Servane Orozco, and Bradley Hasson

This poster delves into the intricate characterization of adeno-associated viruses (AAV) by employing a hybrid sequencing approach that integrates both long-read and short-read techniques. This innovative methodology demonstrates remarkable efficacy, achieving over 96% identity mapping to target genes with long-read samples averaging 3,000 base pairs, while short-read samples exceeded 99% identity. The importance of pre-treatment with Benzonase® endonuclease, which effectively eliminates non-encapsidated sequences before sequencing, thereby enhancing the accuracy of the results is highlighted. Utilizing the Illumina® platform for short-read sequencing and Oxford Nanopore technologies for long-read sequencing, the challenges posed by sequencing complex regions such as inverted terminal repeats (ITRs) are addressed. The developed analysis pipeline not only improves the understanding of AAV capsid contents but also aids in identifying residual sequences from the manufacturing process, which is crucial for biosafety assessments in gene therapy applications.

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