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To focus on biologically relevant CpG sites we have selected those with differences in average beta value greater than 0.15 (more than 15% difference in methylation level).
Expression levels were expressed as an average Beta value.
We found the EMT gene set to be negatively enriched when genes were ranked according to the rho value between gene expression and the average beta value in our integrative analysis (enrichment score -0.571, P < 0.001).
Initially, we have selected CpG sites with differences in average beta value greater than 0.15 (more than 15% difference in mean methylation level) as this method is relatively resistant to potential confounding BeadChip effects.
A total of 1,738 probes (mapping to 818 unique genes) were differentially methylated after setting the FDR at 5%% and constraining the absolute difference of the average beta value within the preM and postM groups to be >0.1 (Fig. 2a).
The average degree of DNA methylation, i.e., the average beta value for the 319,596 analyzed probes, was higher in fat cells from obese cases (0.425 ± 0.366, mean ± SD) as compared to never-obese controls (0.420 ± 0.375) (P = 1.3 × 10−7).
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Random Forests [47](http://www.math.usu.edu/~adele/forests/),was used to build classifiers of invasive stage (versus noninvasive) by CpG average beta values.
Under the assumption that the average beta values follow a beta distribution [46], a quasi-binomial model (logit link, binomial variance, and non-unit scale parameter) was used; note that the logit link function imposes an appropriate constraint on the mean average beta.
Additional genes were removed if the difference in average beta values between groups was less than 0.34.
The resulting data were inspected for complete bisulfite conversion and average beta values for each targeted CpG residue determined using the Illumina Genome Studio Methylation Module, Version 3.2.
Validation of genome-wide DNA methylation results is crucial, considering that the average beta values between smokers and non-smokers at numerous loci differed merely by about 0.1.
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