Amplicons of CHRNA5 exons (1–6) were verified by agarose gel electrophoresis prior to Sanger sequencing. Sequencher 4.9 (GeneCodes Corporation, Ann Arbor, MI, USA) auto-called common polymorphisms and rare polymorphisms were called using the ‘call secondary peaks’ command with secondary peak height set to 45%. Initial quality control was by visual inspection of chromatogram peaks with additional verification done by sequencing the opposite strands of amplicons, by sequencing a second amplicon from the same individual or by TaqMan® allelic discrimination assays (Life Technologies, Grand Island, NY, USA). The frame-shift deletion in CHRNA5 exon 2 was verified by TA-cloning of amplicons into pCRII-TOPO vector (Life Technologies), transforming E. coli, purifying plasmid DNA and fully sequencing both alleles. TaqMan® assays used were: C_1502678_10 (rs2036527:G>A), C_26000428_20 (rs16969968:G>A) and custom assays for the exon 2 frame-shift deletion, rs80087508:A>G and rs79109919:T>A. TaqMan® assays were cycled in a 9700 PCR machine under standard parameters in 384 well plates with 4 ng gDNA per 5 μl reaction, post-read into SDS 2.2.2 on a 7900HT and final genotypes called automatically using TaqMan® Genotyper (Life Technologies) prior to export into Excel for recoding and analysis using Haploview 4.2 (Barrett et al., 2005).
