Figure 2. Impact of GC and homopolymers content on the sequencing coverage of the recombinant AAV genome. (a)Sequencing coverage and percentage of GC along the AAV vector genome. The sequencing coverages obtained from the 3.2-kb AAV8-CAG-GFP vector (red) and the internal normalizer (vector plasmid) (blue) were normalized by dividing the read coverage at each base by the sum of the coverage for all bases mapped along the rAAV genome. The grey boxes indicate two 300 bp-regions showing a drastic sequencing coverage drop. Region 1 and 2 were centered around the minimal number of reads at position 666 and 1421 of the rAAV genome, respectively. The percentage of GC (black) was determined using the program NTContent with the following parameters: window size, 200 bases and step, 20 bases. The rAAV genome map is represented above the graph. (b, c)Nucleotide content along regions 1 (b) and 2 (c). Each base was represented at each position by a colored dot: G (green), C (brown), T (blue) and A (purple). Colored boxes represent homopolymers of ≥ 6 nucleotides. Magnified sequencing coverages are represented as black lines.