Extended Data Fig. 1: Barcode pairs AAV constructs and cloning strategy.
From: Pool-packaged AAV libraries exhibit extensive length-dependent and homology-dependent chimerism
(a) Schematic of components of plasmid AiP11839 (Addgene#163509) between the AAV2 ITRs. Segments under the map highlight constant regions (cloned by PCR) used both as homologous library inserts (blue) and filler sequences to fix the ITR-to-ITR length (white). These segments are shown panel c below. Positions of PacI and BbsI restriction sites are marked by green carets, NotI and MluI sites by pale blue carets. (b) Molecular cloning scheme used. First complex BC1-BC2 parental dock with GFP stuffer p146 was cloned, and served as template for cloning secondary docks p147 and p148 with filler sequences. Complex library p146 was used as template for barcode dictionary generation. All parental docks were digested with SapI to liberate the GFP, which was replaced by respective internally indexed inserts to generate the final series p149-p154 (which were all bottlenecked to a target of 20k transformants). (c) At scale schematics of ITR-to-ITR components of cloned parental and insert-containing libraries. Position of SapI restriction sites are marked by pale red carets, BglII and PmlI sites by grey carets. (d) Sequences surrounding the two barcodes highlighting alignment signposts (yellow) used to create local position reference frames around barcodes in the long-read data. The ‘Insert index’ is shown as Ns, but is fixed and different for each type of insert (not degenerate). Constant Nextera handles between the barcodes (which constitute short homologous regions even in the non-homologous libraries) are shown.
