CpG site- and methylation level-dependent measurement variability across three bisulfite-based DNA methylation assays

Accurate quantification of DNA methylation at specific CpG sites is essential for epigenetic studies, yet measurements can vary depending on assay design and experimental context. Here, we examined locus-specific DNA methylation quantification across three widely used bisulfite-based assays, Illumina Infinium MethylationEPIC array, QIAseq targeted methyl sequencing (TMS), and pyrosequencing, using identical DNA samples. This controlled framework enabled evaluation of method-dependent measurement behavior across CpG sites and a range of methylation levels while minimizing biological variability. Across shared CpGs, all three platforms consistently captured relative methylation trends; however, absolute methylation values differed systematically in a CpG- and methylation-dependent manner. Pyrosequencing generally reported higher methylation levels compared with TMS, whereas EPIC array measurements showed increased variability at low methylation levels (<10%), resulting in reduced inter-method agreement in this range. In contrast, concordance between methods improved at moderate-to-high methylation levels. Error estimates and agreement analyses further indicated that measurement variability was not uniform across CpGs, highlighting site-specific and methylation-level-dependent effects. Together, these results demonstrate that locus-specific DNA methylation quantification using bisulfite-based assays is influenced by local CpG context and methylation level. While relative methylation patterns are preserved across platforms, absolute values are not directly interchangeable, particularly at low methylation levels. These findings underscore the importance of context-aware interpretation and orthogonal validation when precise, locus-specific DNA methylation quantification is required.