5mC and 5hmC methylation sequencing: the power of 6-base sequencing in a multiomic era

ABSTRACT The integration of genetic and epigenetic information is essential for a comprehensive understanding of genome function and regulation. Traditional sequencing methods often fall short in capturing both genetic variants and epigenetic modifications such as 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) simultaneously. Recent advances in 6-base sequencing have enabled the simultaneous, base-resolution detection of canonical bases and key cytosine modifications in a single workflow. This review explores the biological significance of 5mC and 5hmC, discusses current methods to achieve 6-base sequencing, and highlights recent applications in academic and clinical settings.

• Publication year

  2025

• Venue

  Epigenomics

• Publication date

  2025-11-12

• Fields of study

  Biology, Medicine

• Identifiers
  DOI 10.1080/17501911.2025.2586452PMID 41222620PMCID 12826727
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar, PubMed

• No claims are published for this paper.

• No concepts are published for this paper.

• The technology landscape for detection of DNA methylation in cancer liquid biopsies

  2025cited by this paper
• SCoTCH-seq reveals that 5-hydroxymethylcytosine encodes regulatory information across DNA strands

  2025cited by this paper
• Cell identity and 5-hydroxymethylcytosine

  2025cited by this paper
• Temporally discordant chromatin accessibility and DNA demethylation define short- and long-term enhancer regulation during cell fate specification

  2025influential reference
• Detection of early Parkinson's disease using 5-hydroxymethylcytosine episignatures in blood DNA.

  2025cited by this paper
• Epigenome-wide DNA methylation association study of CHIP provides insight into perturbed gene regulation

  2025cited by this paper
• Whole-genome sequencing of 490,640 UK Biobank participants

  2025cited by this paper
• OGT prevents DNA demethylation and suppresses the expression of transposable elements in heterochromatin by restraining TET activity genome-wide

  2025influential reference
• Brain 5-hydroxymethylcytosine alterations are associated with Alzheimer’s disease neuropathology

  2025cited by this paper
• Colorectal cancer progression to metastasis is associated with dynamic genome-wide biphasic 5-hydroxymethylcytosine accumulation

  2025cited by this paper
• Double and single stranded detection of 5-methylcytosine and 5-hydroxymethylcytosine with nanopore sequencing

  2025influential reference
• 5-methylcytosine and 5-hydroxymethylcytosine are synergistic biomarkers for early detection of colorectal cancer

  2024influential reference
• Base-excision repair pathway shapes 5-methylcytosine deamination signatures in pan-cancer genomes

  2024cited by this paper
• An Overview of Global, Local, and Base-Resolution Methods for the Detection of 5-Hydroxymethylcytosine in Genomic DNA.

  2024cited by this paper
• A cfDNA methylation-based tissue-of-origin classifier for cancers of unknown primary

  2024cited by this paper
• Advances in the joint profiling technologies of 5mC and 5hmC

  2024cited by this paper
• A comparison of methods for detecting DNA methylation from long-read sequencing of human genomes

  2024cited by this paper
• Plasma cell-free DNA hydroxymethylation profiling reveals anti-PD-1 treatment response and resistance biology in non-small cell lung cancer

  2024cited by this paper
• Genome-wide characterization of dynamic DNA 5-hydroxymethylcytosine and TET2-related DNA demethylation during breast tumorigenesis

  2024cited by this paper
• CDCA7 is an evolutionarily conserved hemimethylated DNA sensor in eukaryotes

  2024cited by this paper
• The C-terminal 4CXXC-type zinc finger domain of CDCA7 recognizes hemimethylated DNA and modulates activities of chromatin remodeling enzyme HELLS

  2024cited by this paper
• Direct haplotype-resolved 5-base HiFi sequencing for genome-wide profiling of hypermethylation outliers in a rare disease cohort

  2023cited by this paper
• Pan-precancer and cancer DNA methylation profiles revealed significant tissue specificity of interrupted biological processes in tumorigenesis

  2023cited by this paper
• Direct enzymatic sequencing of 5-methylcytosine at single-base resolution

  2023cited by this paper
• Simultaneous sequencing of genetic and epigenetic bases in DNA

  2023influential reference
• The methylome and cell-free DNA: current applications in medicine and pediatric disease

  2023cited by this paper
• Role of Transposable Elements in Genome Stability: Implications for Health and Disease

  2022cited by this paper
• Simultaneous analysis of mutations and methylations in circulating cell-free DNA for hepatocellular carcinoma detection

  2022cited by this paper
• Methylation biomarkers for early cancer detection and diagnosis: Current and future perspectives.

  2022cited by this paper
• Out of LINE: Transposons, genome integrity, and neurodegeneration.

  2022cited by this paper
• The 5-Hydroxymethylcytosine Landscape of Prostate Cancer

  2022cited by this paper
• Discovering DNA Methylation, the History and Future of the Writing on DNA

  2022cited by this paper
• The rs1001179 SNP and CpG methylation regulate catalase expression in chronic lymphocytic leukemia

  2022cited by this paper
• DNA methylation: Precise modulation of chromatin structure and dynamics.

  2022cited by this paper
• Large-scale manipulation of promoter DNA methylation reveals context-specific transcriptional responses and stability

  2022cited by this paper
• Genomic and epigenomic BRCA alterations predict adaptive resistance and response to platinum-based therapy in patients with triple negative breast and ovarian carcinomas

  2022cited by this paper
• DNA methylation signatures of Alzheimer’s disease neuropathology in the cortex are primarily driven by variation in non-neuronal cell-types

  2022cited by this paper
• CRISPRi screens reveal a DNA methylation-mediated 3D genome dependent causal mechanism in prostate cancer

  2021cited by this paper
• Subtraction-free and bisulfite-free specific sequencing of 5-methylcytosine and its oxidized derivatives at base resolution

  2021cited by this paper
• Nanopore sequencing technology, bioinformatics and applications

  2021cited by this paper
• Distinct cytosine modification profiles define epithelial-to-mesenchymal cell-state transitions

  2021cited by this paper
• Comparison of EM-seq and PBAT methylome library methods for low-input DNA

  2021cited by this paper
• Bona Fide Tumor Suppressor Genes Hypermethylated in Melanoma: A Narrative Review

  2021cited by this paper
• Applications and potentials of nanopore sequencing in the (epi)genome and (epi)transcriptome era

  2021cited by this paper
• Hydroxymethylation profile of cell-free DNA is a biomarker for early colorectal cancer

  2021cited by this paper
• Somatic mutation rates scale with lifespan across mammals

  2021influential reference
• Non-coding driver mutations in human cancer

  2021cited by this paper
• Clinical validation of a targeted methylation-based multi-cancer early detection test using an independent validation set.

  2021cited by this paper
• Navigating the DNA methylation landscape of cancer.

  2021cited by this paper
• Losing DNA methylation at repetitive elements and breaking bad

  2021cited by this paper
• TAB-seq and ACE-seq Data Processing for Genome-Wide DNA hydroxymethylation Profiling.

  2021cited by this paper
• DNA methylation-calling tools for Oxford Nanopore sequencing: a survey and human epigenome-wide evaluation

  2021cited by this paper
• Evaluation of whole-genome DNA methylation sequencing library preparation protocols

  2021cited by this paper
• Endonuclease enrichment TAPS for cost-effective genome-wide base-resolution DNA methylation detection

  2021cited by this paper
• Liquid Chromatography-Mass Spectrometry Analysis of Cytosine Modifications.

  2021cited by this paper
• Evaluation of whole-genome DNA methylation sequencing library preparation protocols

  2021cited by this paper
• Analyses of non-coding somatic drivers in 2,658 cancer whole genomes

  2020cited by this paper
• Towards a mechanistic understanding of DNA methylation readout by transcription factors.

  2020cited by this paper
• A brief history of human disease genetics

  2020cited by this paper
• ATAC-Me Captures Prolonged DNA Methylation of Dynamic Chromatin Accessibility Loci during Cell Fate Transitions.

  2020cited by this paper
• DNA methylation of the TERT promoter and its impact on human cancer.

  2020cited by this paper
• A meta-analysis of epigenome-wide association studies in Alzheimer’s disease highlights novel differentially methylated loci across cortex

  2020cited by this paper
• Loss of 5-Hydroxymethylcytosine is an Epigenetic Hallmark of Thyroid Carcinomas with TERT Promoter Mutations

  2020cited by this paper
• Methylation: An Ineluctable Biochemical and Physiological Process Essential to the Transmission of Life

  2020cited by this paper
• Staying true to yourself: mechanisms of DNA methylation maintenance in mammals

  2020cited by this paper
• Tissue-specific 5-hydroxymethylcytosine landscape of the human genome

  2020cited by this paper
• 5-Methylcytosine

  2020cited by this paper
• Structural insights into the specific recognition of 5-methylcytosine and 5-hydroxymethylcytosine by TAL effectors.

  2019cited by this paper
• Clonal hematopoiesis in human aging and disease

  2019cited by this paper
• The diverse roles of DNA methylation in mammalian development and disease

  2019cited by this paper
• Transposable elements drive widespread expression of oncogenes in human cancers

  2019cited by this paper
• Bisulfite-free direct detection of 5-methylcytosine and 5-hydroxymethylcytosine at base resolution

  2019cited by this paper
• Parallel profiling of DNA methylation and hydroxymethylation highlights neuropathology-associated epigenetic variation in Alzheimer’s disease

  2019cited by this paper
• Longitudinal Epigenome-Wide Methylation Study of Cognitive Decline and Motor Progression in Parkinson’s Disease

  2019cited by this paper
• Epigenetic signatures of methylated DNA cytosine in Alzheimer’s disease

  2019cited by this paper
• Analysis of DNA methylation in cancer: location revisited

  2018cited by this paper
• Universal Patterns of Selection in Cancer and Somatic Tissues

  2018cited by this paper
• DNA methylation 101: what is important to know about DNA methylation and its role in SLE risk and disease heterogeneity

  2018cited by this paper
• The repertoire of mutational signatures in human cancer

  2018cited by this paper
• Two are better than one: HPoxBS - hairpin oxidative bisulfite sequencing

  2018cited by this paper
• Association analyses of more than 140,000 men identify 63 new prostate cancer susceptibility loci

  2018cited by this paper
• Fine-mapping of prostate cancer susceptibility loci in a large meta-analysis identifies candidate causal variants

  2018cited by this paper
• New guidelines for DNA methylome studies regarding 5-hydroxymethylcytosine for understanding transcriptional regulation

  2018cited by this paper
• Utility of circulating tumor DNA in cancer diagnostics with emphasis on early detection

  2018cited by this paper
• Subtelomeric methylation distinguishes between subtypes of Immunodeficiency, Centromeric instability and Facial anomalies syndrome

  2018cited by this paper
• Comparison of whole-genome bisulfite sequencing library preparation strategies identifies sources of biases affecting DNA methylation data

  2017cited by this paper
• CpG and Non-CpG Methylation in Epigenetic Gene Regulation and Brain Function

  2017cited by this paper
• Universal Patterns of Selection in Cancer and Somatic Tissues

  2017cited by this paper
• Modeling methyl-sensitive transcription factor motifs with an expanded epigenetic alphabet

  2016cited by this paper
• An interplay of the base excision repair and mismatch repair pathways in active DNA demethylation

  2016cited by this paper
• The Conundrum of Genetic “Drivers” in Benign Conditions

  2016cited by this paper
• Understanding TERT Promoter Mutations: A Common Path to Immortality

  2016cited by this paper
• 5-Hydroxymethylcytosine in E-box motifs ACAT|GTG and ACAC|GTG increases DNA-binding of the B-HLH transcription factor TCF4.

  2016cited by this paper
• 5-Methylcytosine (5mC) and 5-Hydroxymethylcytosine (5hmC) Enhance the DNA Binding of CREB1 to the C/EBP Half-Site Tetranucleotide GCAA.

  2016cited by this paper
• Epigenetic Determinants of Cancer.

  2016cited by this paper
• DNA Methylation Analysis: Choosing the Right Method

  2016cited by this paper
• Causal role of histone acetylations in enhancer function

  2016cited by this paper
• 5-Hydroxymethylcytosine, the “Sixth Base”, during brain development and ageing

  2015cited by this paper
• C/EBPβ (CEBPB) protein binding to the C/EBP|CRE DNA 8-mer TTGC|GTCA is inhibited by 5hmC and enhanced by 5mC, 5fC, and 5caC in the CG dinucleotide.

  2015cited by this paper
• 5-Hydroxymethylcytosine and disease.

  2014cited by this paper

• Integrative dual ctDNA 5mC/5hmC methylomics and clonal reconstruction infer tumor transcription and resistance phenotypes in metastatic prostate cancer

  2026influential citation
• Methylation profiling in neuropathological tumors diagnosis: a comprehensive review

  2025cites this paper