Fig. 3

Generation and analysis of paired end reads from integrated AAV vector cassettes. a Diagrammatic representation of PBR_I and PBR_II DNA junction fragment library preparation. The integrated vector cassette and flanking genomic DNA was subjected to restriction enzyme digestion using MluC1, and PCR amplicons generated from both the resultant 5′ and 3′ ends of the vector cassette, using PBR_I and PBR_II primer sets (respectively) pooled prior to sequencing. Ub-ISAP was run separately to extract ISs using both primer sets from the same raw sequencing read file. b Comparison of the top 20 IS-containing genes (X axis) extracted from unique ISs output derived from sequential Ub-ISAP analysis of a pooled DNA fragment library prepared with alternative primer sets to derive the PBR_I and PBR_II datasets. Red columns show the number of ISs identified for each gene from the PBR_I data compared with the number identified from the PBR_II data (green columns)