Targetted FLG resequencing in homo sapiens: Fluidigm Access Array 2x300bp design
Partially redesigned primers of FLG coding sequence PCR from (Sandilands et al. 2007) were used to produce amplicons of no more than 600 bp for use in Illumina MiSeq 2 x 300 bp sequencing. 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 pools of purified products were then further combined into a single sequencing pool containing 96 samples.