Nanopore sequencing of native adeno-associated virus (AAV) single-stranded DNA using a transposase-based rapid protocol.

ABSTRACT: Next-generation sequencing of single-stranded DNA (ssDNA) enables transgene characterization of gene therapy vectors such as adeno-associated virus (AAV), but current library generation uses complicated and potentially biased second-strand synthesis. We report that libraries for nanopore sequencing of ssDNA can be conveniently created without second-strand synthesis using a transposase-based protocol. We show for bacteriophage M13 ssDNA that the MuA transposase has unexpected residual activity on ssDNA, explained in part by transposase action on transient double-stranded hairpins. In case of AAV, library creation is additionally aided by genome hybridization. We demonstrate the power of direct sequencing combined with nanopore long reads by characterizing AAV vector transgenes. Sequencing

SUBMITTER: Radukic MT 

PROVIDER: S-EPMC7671332 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature