Figure 1.

Flowchart of the library preparation and subsequent sequencing by TMCA-AAVseq

The process commences with the extraction of DNA from both normal and noncanonical vectors. Subsequently, second-strand synthesis is carried out from the 3′ ITR, followed by end repair and modification. Next, adapters designed for SMRT sequencing are ligated with the library DNA molecules. In the accompanying chart, original AAV DNA strands are represented by solid red lines, and newly synthesized strands are depicted with dashed black lines. Normal ITRs are illustrated in blue, and mutant ITRs are shown in pink. The ultimate output of this process comprises high-quality CCSs, ensuring a minimum quality threshold of 0.99 and a minimum of 3 passes. Finally, AAV genome sequence and configuration were retrieved. scAAV genomes were not tested here.