Beadstudio genomestudio software

DCCT/EDIC and WESDR subjects were genotyped using the Illumina (Illumina^® Inc., San Diego, CA, USA) Human 1M and HumanOmni1-Quad assays, respectively. Genotypes were called per study using Illumina’s proprietary GenCall algorithm implemented in BEADSTUDIO/GENOMESTUDIO software (Illumina^®). Genotype calls were exported to PLINK v1.07 (http://pngu.mgh.harvard.edu/purcell/plink/) for further analysis. Extensive QC measures were undertaken to remove samples with low genotyping quality and individuals with cryptic relatedness or ethnic admixture (Paterson et al. 2009 (link)). All analyses were limited to individuals from a white European ancestry who clustered with CEU and TSI samples of HapMap 3 (The International HapMap 3 Consortium 2010 (link)) in principal component analysis. As another measure against population stratification, the association between the first three principal components (PC) and the outcome was assessed using logistic regression which was not significant in either univariate or multivariate models.

In order to assess genetic variation across all study sites, we selected a subset of purified DNA samples (N = 72) that included 48 samples randomly selected from the two South Australia sites along with all 24 samples from the other sites. These samples were submitted to Neogen, Inc. (Lincoln, NE, USA) for genotyping on the GigaMUGA [26 (link)] Illumina Infinium II array (Illumina Inc.). Genotypes were initially called using Illumina BeadStudio/GenomeStudio software. The argyle R package [30 (link)] was subsequently used for genotype quality control following threshold quantile normalization of raw intensity values (function tQN). Genotypes were then filtered for minimum minor allele frequency (MAF > 0.05) and subjected to linkage disequilibrium pruning (window size 1kb, step size 1, R² = 0.5) in PLINK v1.9 [31 (link)]. To evaluate population genetic structure, principal components analysis (PCA) was carried out using the adegenet R package [32 (link)] and visualized on a biplot.

Total RNA was isolated from mouse liver with Trizol® (Invitrogen) and then hybridized to BD-202-0202 Illumina BeadChips. Total RNA was quality-controlled using the Agilent Bioanalyzer RNA 6000 Chip (Agilent) and labeled according to the manufacturer’s instructions using the Illumina® TotalPrepTM RNA amplification kit. A total of 750 ng biotinylated aRNA was hybridized to mouse Ref-8v 2BeadChips (Illumina). Following posthybridization rinses, arrays were incubated with streptavidin conjugated Cy3 and scanned using an Illumina BeadStation 500X Genetic Analysis Systems scanner. Hybridization intensity data were extracted from the scanned images using Illumina BeadStudio GenomeStudio software, V2011.1. Raw data were subjected to Z-normalization, as described elsewhere^{51 (link),52 (link)}. PCA was performed on the normalized Z-scores of all of the detectable probes in the samples. Significant genes were selected by the z-test < 0.05, false discovery rate < 0.30, as well as z-ratio > 1.5 in both directions and ANOVA P-value < 0.05. PAGE was analyzed as previously described⁵³ ; n = 5 per group; age = 57 weeks; diet = 41 weeks.