Repeatability of methylation measures using a QIAseq targeted methyl panel and comparison with the Illumina HumanMethylation450 assay

Abstract

Objective In previous studies using Illumina Infinium methylation arrays, we have identified DNA methylation marks associated with cancer predisposition and progression. In the present study, we have sought to find appropriate technology to both technically validate our data and expand our understanding of DNA methylation in these genomic regions. Here, we aimed to assess the repeatability of methylation measures made using QIAseq targeted methyl panel and to compare them with those obtained from the Illumina HumanMethylation450 (HM450K) assay. We included in the analysis high molecular weight DNA extracted from whole blood (WB) and DNA extracted from formalin-fixed paraffin-embedded tissues (FFPE). Results The repeatability of QIAseq-methylation measures was assessed at 40 CpGs, using the Intraclass Correlation Coefficient (ICC). The mean ICCs and 95% confidence intervals (CI) were 0.72 (0.62–0.81), 0.59 (0.47–0.71) and 0.80 (0.73–0.88) for WB, FFPE and both sample types combined, respectively. For technical replicates measured using QIAseq and HM450K, the mean ICCs (95% CI) were 0.53 (0.39–0.68), 0.43 (0.31–0.56) and 0.70 (0.59–0.80), respectively. Bland–Altman plots indicated good agreement between QIAseq and HM450K measurements. These results demonstrate that the QIAseq targeted methyl panel produces reliable and reproducible methylation measurements across the 40 CpGs that were examined.

The Role of the IGF2 Methylation Score in Diagnosing Adrenocortical Tumors with Unclear Malignant Potential—Feasibility of Formalin-Fixed Paraffin-Embedded Tissue

“…Some studies show a promising correlation [17][18][19][20][21][22][23][24]. However, upon closer examination of the results, many studies experience high failure rates when using FFPE tissue [23][24][25][26][27], and both random and non-random deviations from the methylation percentage were found in frozen tissue [19][20][21][22][25][26][27][28][29]. Similar articles using other methods for methylation determination, such as Infinium Beadchip, Methylight, or methylation-specific PCR, show the same problems and limitations.…”

“…Similar articles using other methods for methylation determination, such as Infinium Beadchip, Methylight, or methylation-specific PCR, show the same problems and limitations. Some studies find a good correlation between FFPE and frozen tissues [28][29][30][31][32][33], while others experience problems with failure rates [27,28,[34][35][36] and poor correlation [28,29,36,37]. A possible explanation for the higher failure rate in FFPE tissue and the lack of replicability and correlation can be found in the degradation of DNA during storage [38].…”