Accurate and Rapid Sequence Analysis of Adeno-Associated Virus Plasmids by Illumina Next-Generation Sequencing.

Sequence validation of plasmid DNA is a crucial quality control step that must occur prior to adeno-associated virus (AAV) vector packaging through plasmid transfection. AAV cis-plasmids present unique challenges to sequence analysis, as they contain inverted terminal repeats and are prone to sequence rearrangements. An accurate and rapid next-generation sequencing approach has been established to analyze full-length sequences of AAV cis-plasmids within 3.5 days. Here, a step-by-step protocol is described that can reliably detect and identify the location and frequency of sequence variants commonly observed in AAV cis-plasmids.