Fig. 1: Chimera formation during rAAV packaging revealed by barcode swapping experiments.
From: Pool-packaged AAV libraries exhibit extensive length-dependent and homology-dependent chimerism
a, A complex doubly barcoded cloning dock with associated dictionary of valid BC1–BC2 pairs was constructed and served as the starting point to clone libraries of inserts of varying lengths and homology class within AAV2 ITRs (six separate libraries: [short ~0.2 kb, mid ~0.8 kb, long ~2.1 kb] × [homologous, nonhomologous]). The seven different libraries considered are schematized (Extended Data Figs. 1 and 2). b, Each cloned barcoded library was separately: (1) digested to liberate the barcoded insert and (2) AAV-packaged. Both plasmid-derived insert and AAV DNA were submitted for direct long-read sequencing. Resulting long reads were scanned for barcodes and the fraction of discordant BC1–BC2 pairs, as compared to the bona fide parental dictionary, was determined. c, Quantification of the fraction of discordant barcode pairs as a function of the full-length BC-to-BC average size. Left, plasmid DNA; middle, AAV-packaged DNA; right, zoomed-in view of y axis range 0–0.1. Each point corresponds to swap quantification for a library for both plasmid-derived (square) and AAV-derived (circle) material (n = 1 replicate per library). For each data point, we analyzed full-length BC-to-BC reads passing quality control filters and having separately valid BC1 and BC2 (Supplementary Figs. 3a and 4a). Quantifications derived from library p153× corresponding to the pool of multiple-sized inserts in a single AAV-packaged sample are marked by an ×. Swaps are significantly higher (one-sided bootstrap FDR < 10−5) for the homologous versus their respective size-matched nonhomologous libraries. d, The impact of starting plasmid dose on BC1–BC2 chimerism was assessed by packaging library p151:mid-hom with different starting amount per 15-cm plate of producing cells, ranging from 15 μg (100% dose) to 15 ng (0.1% dose). The total input of transfected DNA was kept fixed by adding ITR-free plasmid (Supplementary Fig. 6). Vertical error bars correspond to the 20th–80th percentiles from bootstrap resampling to document read counting noise (center: quantification from all reads). Horizontal error bars to the 10th–90th percentile of the BC-to-BC length distribution from plasmid digest inserts. Data from each library or condition is from one packaging replicate, with separate packaging for distinct libraries (Supplementary Data 2). A second biological packaging replicate is presented in Fig. 2b, with quantitative reproducibility, for libraries p151 and p152.
