Figure 1.

Workflow for shuffling and selection, sequencing, and analysis

After library creation by shuffling and the selection process, successful capsids are isolated by PCR. ONT R2C2 sequencing is conducted by generating concatemers by circularization with a splint and rolling circle amplification, followed by ONT sequencing. Sequencing data are processed by AAVolve on a per-read basis to generate consensus sequences from concatemer reads, then alignment to a reference, parental region identification, error correction, and counting.