We are proud to be a PacBio-sponsored adeno-associated virus (AAV) sequencing facility and to take part in this growing field where accuracy is key to the development of new therapeutics. PacBio’s SMRT platform with CCS is ideal for identifying mutations, truncations, and host integration events in AAV genome populations. We use PacBio’s protocol for preparing multiplexed AAV SMRTbell libraries using the SMRTbell prep kit 3.0, which accommodates both self-complementing (scAAV) and single-stranded (ssAAV) constructs within a sample.
The AAV library preparation first converts ssAAV DNA to dsDNA by thermal annealing, if ssAAV is present. Once in double-stranded form, barcoded adaptors are added onto all fragments for multiplexed sequencing.
Because AAV nucleic acids may be in multiple forms in a sample and dsDNA is required for successful sequencing, it is critical that the sample form is disclosed. This allows us to address any non-complementary and self-complementary regions with interior inserted repeats before final library preparation.