AviNP-Seq: A Blindspot-Free Single-Molecule Framework for Unmasking AAV Genome Heterogeneity and Determining Packaging Limits.

Comprehensive recombinant adeno-associated virus characterization is essential for establishing the knowledge base required to ensure clinical safety and efficacy, yet current long-read methods suffer from library preparation biases that obscure genome integrity. We present AviNP-seq, a blindspot-free nanopore sequencing framework utilizing one-end-sufficient ligation and Cas9-ribonucleoprotein (RNP) linearization to minimize terminal selection. Applied to a 1.5-6.5 kb panel, AviNP-seq delineates a sharp packaging cliff at 5.0-5.2 kb and reveals that sequence structure modulates integrity by 2-5× at fixed lengths. It unmasks covalent head-to-tail tandems in sub-3 kb vectors, detecting them with significantly higher sensitivity than PacBio HiFi. The Cas9-RNP step boosts ligation yield ∼7-fold, providing an unbiased assessment of genome integrity (≥95% inverted terminal repeat [ITR]-to-ITR). In addition, the assay quantifies plasmid impurities down to 0.05% with linear response. By integrating integrity mapping, tandem detection, and impurity profiling into a rapid (<36 h), low-input workflow, AviNP-seq provides a robust analytical tool to guide vector design and de-risk early-stage process development.