Abstract
BackgroundHigh-throughput profiling of DNA methylation status of CpG islands is crucial to understand the epigenetic regulation of genes. The microarray-based Infinium methylation assay by Illumina is one platform for low-cost high-throughput methylation profiling. Both Beta-value and M-value statistics have been used as metrics to measure methylation levels. However, there are no detailed studies of their relations and their strengths and limitations.ResultsWe demonstrate that the relationship between the Beta-value and M-value methods is a Logit transformation, and show that the Beta-value method has severe heteroscedasticity for highly methylated or unmethylated CpG sites. In order to evaluate the performance of the Beta-value and M-value methods for identifying differentially methylated CpG sites, we designed a methylation titration experiment. The evaluation results show that the M-value method provides much better performance in terms of Detection Rate (DR) and True Positive Rate (TPR) for both highly methylated and unmethylated CpG sites. Imposing a minimum threshold of difference can improve the performance of the M-value method but not the Beta-value method. We also provide guidance for how to select the threshold of methylation differences.ConclusionsThe Beta-value has a more intuitive biological interpretation, but the M-value is more statistically valid for the differential analysis of methylation levels. Therefore, we recommend using the M-value method for conducting differential methylation analysis and including the Beta-value statistics when reporting the results to investigators.
Highlights
High-throughput profiling of DNA methylation status of CpG islands is crucial to understand the epigenetic regulation of genes
High-throughput profiling of DNA methylation status of CpG islands is crucial for forwarding our understanding of the influence of epigenomics [4,5,6]
To estimate the methylation status, the Illumina Infinium assay utilizes a pair of probes to measure the intensities of the methylated and unmethylated alleles at the interrogated CpG site [10]
Summary
High-throughput profiling of DNA methylation status of CpG islands is crucial to understand the epigenetic regulation of genes. The microarray-based Infinium methylation assay by Illumina is one platform for low-cost high-throughput methylation profiling Both Beta-value and M-value statistics have been used as metrics to measure methylation levels. The first one is called Beta-value, ranging from 0 to 1, which has been widely used to measure the percentage of methylation This is the method currently recommended by Illumina [11,12]. We have referred to it as the M-value method because it has been widely used in the mRNA expression microarray analysis Since both methods have their own strengths and limitations, understanding the performance characteristics of both measures is very important in providing the best methylation analysis. We will evaluate the performance of these two methods in detecting differentially methylated CpG sites
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