Targetted FLG resequencing in homo sapiens: Fluidigm Access Array 454 design
FLG primers for the Roche 454 GS FLX+ were designed based on the FLG primers used by the McLean group and gave overlapping PCR products with an average length of 538 bp (Sandilands et al. 2007; Chen et al. 2011). Samples and primers were cross-reacted with the Fluidigm Access Array 48.48 IFC microfluidic chip. PCR product pool for each DNA sample was harvested from the IFC and individually quantified and checked for quality using Agilent DNA 1000 chip run on Bioanalyzer. When correct size range of products was obtained, 1?L of PCR products from each sample (up to a maximum of 48 samples) were pooled and purified using Agencourt� AMPure� XP Reagent Beads (Beckman Coulter Genomics) according to the instructions provided in the Access Array Manual. Two pools of purified products were then further combined into a single sequencing pool containing 96 samples.