Single-cell DNA methylation profiling: Technologies, computation, and applications in precision oncology

Cancer is an intrinsically heterogeneous disease characterized by distinct malignant subclones defined by specific genetic and epigenetic alterations, such as aberrant DNA methylation. Traditional bulk sequencing methods analyze large populations of cells in aggregate, yielding an averaged methylome signal that masks the rare but clinically significant epigenetic patterns driving tumor initiation, metastasis, and therapeutic resistance. The emergence of single-cell DNA methylation (scDNAme) sequencing has driven a paradigm shift in oncology by providing the resolution required to dissect this intratumoral heterogeneity. By profiling the epigenome of individual cells, scDNAme analysis enables the discovery of novel aberrant patterns, the precise reconstruction of cellular lineages, and the characterization of specific populations-such as cancer stem cells (CSCs) or drug-resistant clones-that possess distinct methylome signatures. This technological advance is not merely an incremental improvement; it is a prerequisite for understanding core cancer hallmarks, such as the evasion of growth control and resistance to apoptosis, which are frequently governed by these specific subclones. This review specifically provides a comprehensive comparative overview evaluating the technical and chemical capabilities of emerging single-cell modalities to distinguish DNA methylation profiles. By highlighting the strategic workflows of these emergent technologies alongside advanced computational tools, we emphasize how resolving these discrete cytosine variants empowers precise cell lineage tracing, the identification of refractory clones, and the implementation of locus-specific epigenetic editing therapies against causal tumor subclones.