Fig. 2: Observed rAAV chimerism is robust to the long-read library preparation procedure.
From: Pool-packaged AAV libraries exhibit extensive length-dependent and homology-dependent chimerism
a, Comparison of PCR versus direct long-read library preparation for ONT sequencing of paired barcode libraries. Left, schematic of the workflow. For plasmid DNA, we compare digested BC1–BC2 inserts versus PCR products. For AAV-packaged DNA, we either extracted DNA and performed PCR before long-read sample preparation or submitted the AAV particles directly (library preparation using the annealing strategy19). All ONT sequencing was performed with Plasmidsaurus. The graph shows the fraction of discordant barcode pairs from PCR-derived libraries (x axis) versus direct (same data as Fig. 1c; y axis), as also shown in Supplementary Fig. 4b. AAV samples only include the PHP.eB serotype with standard packaging conditions. Error bars correspond to the 20th–80th percentiles from bootstrap resampling to document read counting noise (smaller than symbol size for PCR libraries along x axis because of high sequencing coverage for these libraries; center: quantification from all reads). The AAV data (circles) are from one packaging replicate per library, with ONT preparation either through direct (annealing-based) or PCR-based approaches. The plasmid-derived data (squares) are from a single replicate (one preparation per library). b, Comparison of the fraction of discordant BC1–BC2 pairs from AAV-packaged DNA from the same libraries (p151:mid-hom in blue and p152:mid-nonhom in orange) across ONT (both direct and PCR-based) and PacBio libraries. Quantification on the PacBio reads was stratified by class of molecules (produced from intramolecular extension or through annealing), as also shown in Extended Data Fig. 5.
