Targetted FLG resequencing in homo sapiens: Fluidigm Access Array 2x250bp design
FLG primers were designed specifically for Illumina Reagent Kit V2 MiSeq 2 x 250 bp sequencing so that the 12-repeat version of FLG (Sandilands et al. 2007) would be covered with partial overlap of amplicons no more than 500bp in length. 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 AgencourtŽ AMPureŽ XP Reagent Beads (Beckman Coulter Genomics) according to the instructions provided in the Access Array Manual. Two or four pools of purified products were then further combined into a single sequencing pool containing 96 samples or 192 samples respectively.