Evolution of genome-wide methylation profiling technologies

Abstract

In this mini-review, we explore the advancements in genome-wide DNA methylation profiling, tracing the evolution from traditional methods such as methylation arrays and whole-genome bisulfite sequencing to the cutting-edge single-molecule profiling enabled by long-read sequencing (LRS) technologies. We highlight how LRS is transforming clinical and translational research, particularly by its ability to simultaneously measure genetic and epigenetic information, providing a more comprehensive understanding of complex disease mechanisms. We discuss current challenges and future directions in the field, emphasizing the need for innovative computational tools and robust, reproducible approaches to fully harness the capabilities of LRS in molecular diagnostics.

Figure 1.

Methods to detect genome-wide 5mC. DNAm can be detected through enrichment, bisulfite conversion, enzymatic modification, or single-molecule approaches. (A) Enrichment methods use restriction enzymes or immunoprecipitation to separate methylated and unmethylated DNA. (B) Bisulfite treatment converts unmethylated cytosine to uracil, allowing differentiation between methylated and unmethylated DNA that can be assayed using microarrays or short-read sequencing. (C) Newer enzymatic methods use a series of proteins that oxidize and glycosylate modified cytosines, followed by deamination that is achieved either through specific enzymes or borane reduction. (D) Single-molecule methods directly measure DNAm as DNA passes through the nanopore (Oxford Nanopore, top panel) or as fluorescent nucleotides are incorporated into the growing strand by a polymerase (PacBio, middle panel). Created in BioRender. Montano, C. (2024). https://BioRender.com/i42h077.

Figure 2.

Diagnostic applications of haplotype-aware methylation analysis. Haplotype assignment after phasing significantly enhances the diagnostic utility of LRS. By enabling the simultaneous detection of SNVs, SVs, CNVs, and DNA modifications, it allows disease mechanisms that previously required separate specialized tests to be studied under a single, unified approach, such as imprinting (A), epimutations (B), and repeat expansions (C). It also facilitates variant prioritization (D), enables the investigation of skewed XCI where, instead of mixed inactivation, one haplotype is inactivated consistently (E), and can even be applied to study cell-free DNA (F). Created in BioRender. Montano, C. (2024). https://BioRender.com/h78t893.