To confirm the results obtained from the Illumina HumanMethylation450 arrays, we performed pyrosequencing analysis of bisulphite-converted DNA with appropriate oligos using the PyroMark Q24 System and advanced CpG Reagents (Qiagen ®).
The p-value for methylation differences between case and normal groups at each locus was calculated as previously described [32 (link)]. Filtering criteria for p-values were set at <0.05 and also <0.01 in order to identify the most differentiating cytosines. P-values were calculated with and without Benjamini and Hochberg False Discovery Rate (FDR) correction for multiple testing [33 ]. The Benjamini and Hochberg correction tolerates more false positive genes than the Bonferroni correction.
Further analysis of the differentially methylated genes was conducted for potential biological significance. ROC curves and ROC AUC were calculated to determine the diagnostic accuracy of specific cytosine loci to differentiate AVS from control groups. Data were normalized using the Controls Normalization Method.
The p-value for methylation differences between case and normal groups at each locus was calculated as previously described [32 (link)]. Filtering criteria for p-values were set at <0.05 and also <0.01 in order to identify the most differentiating cytosines. P-values were calculated with and without Benjamini and Hochberg False Discovery Rate (FDR) correction for multiple testing [33 ]. The Benjamini and Hochberg correction tolerates more false positive genes than the Bonferroni correction.
Further analysis of the differentially methylated genes was conducted for potential biological significance. ROC curves and ROC AUC were calculated to determine the diagnostic accuracy of specific cytosine loci to differentiate AVS from control groups. Data were normalized using the Controls Normalization Method.
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