Residual DNA in AAV9 sample

This study investigates the presence of residual plasmid DNA and host-cell derived nucleic acids in recombinant AAV9 vector preparations obtained from CsCl ultracentrifugation gradients. Capsid fractions representing heavy, full, intermediate, and empty particles were collected and processed either with or without DNase I pre-treatment in order to differentiate encapsidated DNA from externally associated contaminants. DNase I was heat-inactivated using 0.5 M EDTA at 75 °C for 10 minutes prior to downstream steps. Total DNA was extracted from each fraction using the QIAamp MinElute Virus Spin Kit following the manufacturer's protocol for viral nucleic acid purification, with minor modifications to accommodate the low DNA yields. Eluates were quantified using the Qubit dsDNA High Sensitivity assay, and DNA amounts below 10 ng/µL were concentrated with 1.8× AMPure XP beads. Because DNA recovery from gradient fractions did not meet the recommended input for ligation-based library preparation, a trace amount of BstEII-HF–digested lambda DNA (provided in the ONT LSK109 kit) was added to all samples as carrier DNA prior to library construction. Nanopore sequencing libraries were prepared using the Ligation Sequencing Kit SQK-LSK109 combined with EXP-NBD104 native barcodes, following Oxford Nanopore Technologies' official protocol. Barcoded libraries were pooled and loaded onto an R9.4.1 flow cell and sequenced for up to 72 hours on a MinION device. Long-read sequencing data generated in this study enable the characterization of AAV9 genome packaging, the identification of encapsidated versus surface-associated nucleic acids, and the assessment of residual plasmid backbones or host-cell DNA. These datasets provide a high-resolution resource for evaluating vector purity and understanding packaging heterogeneity across CsCl-separated AAV9 fractions.