title: LAM_PCR and gDNA-seq of AAV-FVIII from Canis Lupus Familiaris Adult Liver
design: Mediated PCR (LAM-PCR) primers were tested on limiting dilutions of the vector plasmid ranging from 1pg to 0.001pg and samples were analyzed in triplicates using an input of 500ng per replicate. LAM-PCR was performed as previously described 2. A biotinylated primer hybridizing next to the 3'- inverted terminal repeats (ITR) of the vector was used to amplify vector-flanking sequences in two 50-cycles linear PCR amplification steps. Biotinylated PCR-products were purified by magnetic capture followed by double strand synthesis and restriction digest using MluCI and MseI. After digestion, a cohesive double-stranded linker cassette including a sample barcode was ligated to the restricted PCR products. Subsequently, two nested exponential PCR steps were performed on the purified biotinylated products using up to 2/5 and 2/20 of the eluate, respectively, using biotinylated vector- and adaptor-specific primers. Preparation for sequencing was performed in a PCR with fusion-primers carrying MiSeq-specific sequencing adaptors, as well as an additional sample barcode 3,4. Libraries were sequenced on the Illumina MiSeq platform by 450+50bp asymmetric paired-end sequencing.