Computational protocol: Integrated Genetic and Epigenetic Analysis Identifies Haplotype-Specific Methylation in the FTO Type 2 Diabetes and Obesity Susceptibility Locus

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Protocol publication

[…] Linkage Disequilibrium blocks around genotyped susceptibility genes were defined as per Gabriel et al. , as implemented in HAPLOVIEW v.4.1 . At each block, subjects were grouped by genotype without reference to case or control status, for each T2D susceptibility SNP. Average methylation score was calculated per block by summating scores for all BATMAN windows within in it and dividing by the number of windows. Non-parametric (Kruskal-Wallis) and parametric (Linear Regression) tests generated p value statistics for the mean methylation score with respect to genotype status. Permutation empirical p-values were calculated by retaining observed methylation scores and shuffling genotype assignment 10,000 times.A sliding windows analysis was performed across these LD blocks using 100 bp BATMAN methylation output windows, similar to that used in the context of contiguous SNP haplotypes . Starting with a window size of one and moving one window along per calculation across the entire block, Kruskal–Wallis and Linear Regression analyses were performed for the genotype groups with respect to methylation scores. Window size was increased by one on each pass and the analysis repeated, until the window size equalled the entire LD block. The resulting p-values were outputted and plotted at the midway point for each window. Detection of both non-linear and a biologically plausible linear association, between genotype and methylation status provides robust support to the significant relationship at the FTO LD block (Supplementary and ). Sliding windows analysis, statistical calculation, and permutation scripts were written with the R package . [...] RNA-Seq data generated by an Illumina Genome Analyser from RNA derived from <24 hr post-mortem Cerebellum tissue samples of six anonymous unrelated donor males was available from the paper of Wang et al. . These data were aligned using TopHat 1.0, which incorporates the Bowtie aligner and additionally generates splice junction reads . SAM output files were visualised with the Integrative Genomics Viewer (version 1.4.01, http://www.broadinstitute.org/igv). […]

Pipeline specifications

Software tools Haploview, TopHat, Bowtie, IGV
Applications RNA-seq analysis, GWAS
Diseases Diabetes Mellitus, Diabetes Mellitus, Type 2
Chemicals Nitroprusside