title: Target-Enrichment Sequencing of AAV-FVIII from Canis Lupus Familiaris Adult Liver
design: TES (target enrichment sequencing)was performed by double-capture using two different RNA 120 bp-long bait sets, both designed based on 8x tiling. The first bait set was homologous to the whole vector sequence and the second covered only vector regions diverging between the vector and the dog genome: ITRs, promoter-transgene junction, transgene exon-exon junctions and the polyA. TES was performed first on control samples consisting of vector plasmid spiked into untransduced canine genomic DNA simulating a vector load of 1 vector genome/cell and non-spiked control. Samples were analyzed in duplicate using 1000 ng DNA per replicate. DNA was sheared to ~500bp length using ultrasonicator (Covaris) and fragment size was verified by TapeStation (Agilent). Libraries were prepared using the Agilent SureSelectXT2 kit in line with manufacturers instructions. Additional hybridization and PCR enrichment steps were performed on the final library. After library concentration and size distribution evaluation, libraries were sequenced by 2x250bp symmetric paired-end on the Illumina MiSeq platform.