PacBio sequencing of recombinant AAV9
PacBio long-read sequencing and data analysis was performed by SeqCenter (Pittsburgh, PA). Briefly, AAV DNA was extracted following the ThermoFisher Scientific PureLink Viral RNA/DNA mini kit (Thermo Fisher, 12280050) per PacBio recommendations. Samples were eluted into a final volume of 50 ul. Sequencing libraries were prepared following the Preparing multiplexed AAV SMRTbell libraries protocol, without thermal annealing, and using the SMRTbell barcoded adapter plate 3.0 (Pacific Biosciences). Pooled libraries underwent the binding and cleanup steps using the Revio Polymerase kit (Pacific Biosciences). Prepared libraries were loaded onto the PacBio Revio instrument. Demultiplexing, quality control, and adapter trimming were performed on instrument via Lima (PacBio barcode demultiplexer and primer remover). BAM files generated through HiFi sequencing were converted to FastQ files using the SamTools Fastq algorithm. Generated reads from the PacBio instrument (Revio) were used as input for AAV Analysis using the Magdoll pipeline (https://github.com/Magdoll/AAV).