Comparison of Beta-value and M-value methods for quantifying methylation levels by microarray analysis

Pan Du, Xiao Zhang, Chiang-Ching Huang, Nadereh Jafari, Warren A Kibbe, Lifang Hou, Simon M Lin, Pan Du, Xiao Zhang, Chiang-Ching Huang, Nadereh Jafari, Warren A Kibbe, Lifang Hou, Simon M Lin

Abstract

Background: 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. However, there are no detailed studies of their relations and their strengths and limitations.

Results: We 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.

Conclusions: The 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.

Figures

Figure 1
Figure 1
The relationship curve between M-value and Beta-value.
Figure 2
Figure 2
The histograms of Beta-value (left) and M-value (right) (27578 interrogated CpG sites in total).
Figure 3
Figure 3
The mean and standard deviation relations of technical replicates. Beta-value (left) and M-value (right).
Figure 4
Figure 4
Performance comparisons of Beta- and M-value in the range of low, middle and high methylation levels based on the relationship of 1 - Detection Rate versus True Positive Rate.
Figure 5
Figure 5
Performance comparisons of Beta and M-value based on the True Positive Rate (TPR) and Detection Rate (DR) at different thresholds of methylation difference. (A) TPR versus threshold of difference of Beta-value; (B) TPR versus threshold of difference of M-value; (C) DR versus threshold of difference of Beta-value; (D) DR versus threshold of difference of M-value.

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