Nanopore sequencing enables scalable, end-to-end quality control and capsid library profiling of recombinant AAV vectors | LC26

Abstract
Recombinant adeno-associated viruses (rAAVs) are among the most widely used vectors for gene therapy, requiring stringent quality control to ensure vector integrity, safety, and therapeutic performance. Oxford Nanopore Technologies (ONT) sequencing enables unbiased, real-time analysis of native DNA and RNA, and offers unique advantages for comprehensive rAAV characterization through long-read sequencing. Here, we established ONT sequencing as a scalable, end-to-end platform for rAAV quality management and capsid library assessment. Genomic DNA from purified individual rAAV vectors and large, technically non-equimolar rAAV capsid libraries was directly sequenced using ONT native barcoding (SQK-NBD114.96) on MinION and PromethION 2 Solo devices. Following transduction of multiple cell lines, rAAV-derived transcripts were analyzed by ONT direct RNA sequencing (SQK-RNA004) as well as by cDNA sequencing using the cDNA-PCR Barcoding Kit V14 (SQK-PCB114.24), enabling a direct comparison of RNA- and cDNA-based approaches. All genomic DNA datasets were analyzed using the dedicated wf-aav-qc bioinformatic pipeline. Long-read sequencing revealed no detectable backbone contamination from production plasmids and only minimal genome truncations, with a remarkably high proportion of full-length (ITR–ITR) rAAV genomes. Both direct RNA and cDNA sequencing confirmed accurate and reproducible transcription of the gene of interest across multiple cell lines. Importantly, ONT sequencing enabled precise quantification of inherently non-equimolar rAAV libraries, allowing high-resolution determination of capsid variant distributions and reliable identification of the desired rAAV capsid candidate. In summary, ONT sequencing provides a robust, scalable, and comprehensive solution for rAAV quality control, transcript validation, and library screening, supporting accelerated vector development from discovery to production.

Biography
Stefan Hardy Lung is a PhD candidate at the Institute of Virology in Innsbruck, working in the fields of gene therapy, viral vector research, and virus diagnostics. He has been responsible for the nanopore sequencing core facility for over four years, with a focus on long-read sequencing, rAAV vector quality control, capsid library profiling, transcript analysis, and diagnostic applications. Stefan’s work integrates Oxford Nanopore sequencing technologies with bioinformatic workflows for scalable and comprehensive viral characterization.