Direct, bisulfite-free 5mC and 5hmC sequencing at single-cell resolution
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ABSTRACT:
PROVIDER: PRJNA1076505 | ENA |
REPOSITORIES: ENA
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Project description:We report the first direct sequencing methodologies, termed scTAPS and scCAPS+ (exclusive to 5hmC), which enable quantitative detection of 5mC and 5hmC at single-base resolution and single-cell level. With ~90% mapping efficacy, our methods accurately benchmark 5mC and 5hmC profiles in CD8+ T and mES cells, respectively. Notably, scCAPS+ revealed a global increase in 5hmC within the hippocampus of aging mice, particularly evident in non-neurons.
2025-07-18 | GSE255771 | GEO
Project description:Although various methods have been developed for sequencing cytosine epigenetic modifications, specific and quantitative sequencing of the two major epigenetic modifications of cytosines, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) at base-resolution is still challenging. Most times it requires subtraction of two methods to obtain both the true 5mC and 5hmC information, which increases noise and requires high sequencing depth. Recently we developed TET assisted pyridine borane sequencing (TAPS) for bisulfite-free direct sequencing of DNA methylation, which provides the sum of 5mC and 5hmC. Here we extend it to two sister methods, TAPSβ and CAPS (Chemical-Assisted Pyridine borane Sequencing), for whole-genome subtraction-free and specific sequencing of 5mC and 5hmC, respectively. We also demonstrated Pyridine borane Sequencing (PS) of whole-genome 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC), the further oxidized derivatives of 5mC and 5hmC. This completes the versatile borane reduction chemistry-based methods as a comprehensive suite for direct and quantitative sequencing of all four individual cytosine epigenetic modifications.
2020-12-14 | GSE155613 | GEO
Project description:We report a new bisulfite-free 5mC and 5hmC base-resolution sequencing method: TET Assisted Pyridine borane Sequencing (TAPS). TAPS relies on mild reactions and detects DNA modifications directly, without affecting unmodified cytosines. We applied this method for the first time to whole genome sequencing in E14 mESC cell line. For comparsion we prepared whole-genome bisulfite sequencing in the same cell line. Compared with bisulfite sequencing, TAPS results in higher mapping rates, more even coverage and lower sequencing cost, enabling more informative and cheaper methylome analyses. We expect TAPS to become the new standard in epigenetic DNA sequencing.
2018-12-24 | GSE112520 | GEO
Project description:5-hydroxymethylcytosine (5hmC), an oxidized derivative of 5-methylcytosine (5mC), has been implicated as an important epigenetic regulator of mammalian development. Current procedures use cost-prohibitive DNA sequencing methods to discriminate 5hmC from 5mC, limiting their accessibility to the scientific community. Here we report a method that combines TET-assisted bisulfite conversion with Illumina 450K DNA methylation arrays for a low-cost high-throughput approach that distinguishes 5hmC and 5mC signals. Implementing this approach, termed TAB-array, we assessed DNA methylation dynamics in the differentiation of human pluripotent stem cells into cardiovascular and neural progenitors. With the ability to discriminate 5mC and 5hmC, we found a much larger number of dynamically methylated genomic regions implicated in the development of these lineages than we could detect by 5mC analysis alone. The increased resolution and accuracy afforded by this approach provides a powerful means to investigate the distinct contributions of 5mC and 5hmC in human development and disease. We generated illumina 450k DNA methylation data for a total of 9 sample groups with two biological replicates for each group. Data for 4/9 groups were generated from glucosylated and bisulfite converted DNA, from human induced plurupotent stem cells (hIPSCs), differentiated cardiovascular progenitors (CVPs), differentiated neural progenitors (NPCs), and fibroblasts. Data for the next 4/9 groups were generated from glucosylated, TET-oxidized and bisulfite converted DNA, from and included replicates of hIPSCs, CVPs, NPCs, and fibroblasts. Data for the last group was generated from standard bisulfite converted DNA (not glucosylated) from fibroblasts.
2014-12-12 | E-GEOD-60225 | biostudies-arrayexpress
Project description:DNA methylation of C5-cytosine (5mC) in the mammalian genome is a key epigenetic event that is critical for various cellular processes. However, how the genome-wide 5mC pattern is dynamically regulated remains a fundamental question in epigenetic biology. The TET family of 5mC hydroxylases, which convert 5mC to 5-hydroxymethylcytosine (5hmC), have provided a new potential mechanism for the dynamic regulation of DNA methylation. The extent to which individual Tet family members contribute to the genome-wide 5mC and 5hmC patterns and associated gene network remains largely unknown. Here we report genome-wide mapping of Tet1 and 5hmC in mESCs and reveal a mechanism of action by which Tet1 controls 5hmC and 5mC levels in mESCs. In combination with microarray and mRNA-seq expression profiling, we identify a comprehensive yet intricate gene network influenced by Tet1. We propose a model whereby Tet1 controls DNA methylation both by binding to CpG-rich regions to prevent unwanted DNA methyltransferase activity, and by converting the existing 5mC to 5hmC through its enzymatic activity. This Tet1-mediated antagonism of CpG methylation imparts differential maintenance of DNA methylation status at Tet1 target loci, thereby providing a new regulatory mechanism for establishing the epigenetic landscape of mESCs, which ultimately contributes to mESC differentiation and the onset of embryonic development. To determine the genome-wide DNA methylation changes caused by Tet1 depletion in mouse ES cells. Tet1 protein was depleted by specific siRNA treatment. The DNA methylation levels in control and Tet1 siRNA-transfected ES cells were determined by targeted bisulfite sequencing.
2011-04-21 | E-GEOD-28533 | biostudies-arrayexpress
Project description:5-hydroxymethylcytosine (5hmC) constitutes up to 20% of 5-methylcytosine (5mC) in mammalian cells. As conventional bisulfite sequencing is unable to distinguish 5hmC from 5mC, several methods have been proposed to detect 5hmC and infer 5mC levels by subtracting 5hmC from whole-genome bisulfite sequencing data. This subtraction approach, however, can lead to the underestimation of 5mC levels at sites with high 5hmC abundance. Here, we present two orthogonal methods: CMD1-Deaminase sequencing and CMD1-TET bisulfite sequencing, which enable the direct and independent detection of 5mC. In combination with ACE-seq or TAB-seq, these techniques facilitate an accurate distinction between 5mC and 5hmC, eliminating the need for subtraction. Using these approaches, we generated a comprehensive landscape of 5mC and 5hmC in the hippocampus of the Alzheimer's disease (AD) model mice. Notably, while 5hmC levels were substantially reduced in the AD mice, 5mC levels remained largely unchanged, suggesting a critical role for 5hmC as an independent epigenetic mark in the pathogenesis of AD.
2025-07-17 | GSE290950 | GEO
Project description:5-hydroxymethylcytosine (5hmC) constitutes up to 20% of 5-methylcytosine (5mC) in mammalian cells. As conventional bisulfite sequencing is unable to distinguish 5hmC from 5mC, several methods have been proposed to detect 5hmC and infer 5mC levels by subtracting 5hmC from whole-genome bisulfite sequencing data. This subtraction approach, however, can lead to the underestimation of 5mC levels at sites with high 5hmC abundance. Here, we present two orthogonal methods: CMD1-Deaminase sequencing and CMD1-TET bisulfite sequencing, which enable the direct and independent detection of 5mC. In combination with ACE-seq or TAB-seq, these techniques facilitate an accurate distinction between 5mC and 5hmC, eliminating the need for subtraction. Using these approaches, we generated a comprehensive landscape of 5mC and 5hmC in the hippocampus of the Alzheimer's disease (AD) model mice. Notably, while 5hmC levels were substantially reduced in the AD mice, 5mC levels remained largely unchanged, suggesting a critical role for 5hmC as an independent epigenetic mark in the pathogenesis of AD.
2025-07-17 | GSE290949 | GEO
Project description:5-hydroxymethylcytosine (5hmC) constitutes up to 20% of 5-methylcytosine (5mC) in mammalian cells. As conventional bisulfite sequencing is unable to distinguish 5hmC from 5mC, several methods have been proposed to detect 5hmC and infer 5mC levels by subtracting 5hmC from whole-genome bisulfite sequencing data. This subtraction approach, however, can lead to the underestimation of 5mC levels at sites with high 5hmC abundance. Here, we present two orthogonal methods: CMD1-Deaminase sequencing and CMD1-TET bisulfite sequencing, which enable the direct and independent detection of 5mC. In combination with ACE-seq or TAB-seq, these techniques facilitate an accurate distinction between 5mC and 5hmC, eliminating the need for subtraction. Using these approaches, we generated a comprehensive landscape of 5mC and 5hmC in the hippocampus of the Alzheimer's disease (AD) model mice. Notably, while 5hmC levels were substantially reduced in the AD mice, 5mC levels remained largely unchanged, suggesting a critical role for 5hmC as an independent epigenetic mark in the pathogenesis of AD.
2025-07-17 | GSE290951 | GEO
Project description:5-hydroxymethylcytosine (5hmC), an oxidized derivative of 5-methylcytosine (5mC), has been implicated as an important epigenetic regulator of mammalian development. Current procedures use cost-prohibitive DNA sequencing methods to discriminate 5hmC from 5mC, limiting their accessibility to the scientific community. Here we report a method that combines TET-assisted bisulfite conversion with Illumina 450K DNA methylation arrays for a low-cost high-throughput approach that distinguishes 5hmC and 5mC signals. Implementing this approach, termed TAB-array, we assessed DNA methylation dynamics in the differentiation of human pluripotent stem cells into cardiovascular and neural progenitors. With the ability to discriminate 5mC and 5hmC, we found a much larger number of dynamically methylated genomic regions implicated in the development of these lineages than we could detect by 5mC analysis alone. The increased resolution and accuracy afforded by this approach provides a powerful means to investigate the distinct contributions of 5mC and 5hmC in human development and disease.
2014-12-12 | GSE60225 | GEO
Project description:Oxidative modification of 5-methylcytosine (5mC) by TET DNA dioxygenases generates 5-hydroxymethylcytosine (5hmC), the most abundant form of oxidized 5mC. Existing single-cell bisulfite sequencing methods cannot resolve 5mC and 5hmC, leaving the cell-type-specific regulatory mechanisms of TET and 5hmC largely unknown. Here we present Joint single-nucleus (hydroxy)methylcytosine sequencing (Joint-snhmC-seq), a scalable and quantitative approach that simultaneously profiles 5hmC and true 5mC in single cells by harnessing differential deaminase activity of APOBEC3A towards 5mC and chemically protected 5hmC. Joint-snhmC-seq profiling of single nuclei from the mouse brains reveals an unprecedented level of epigenetic heterogeneity of both 5hmC and true 5mC at single-cell resolution. We show that cell-type-specific profiles of 5hmC or true 5mC improve multi-modal single-cell data integration, enable accurate identification of neuronal subtypes, and uncover context-specific regulatory effects of cell-type-specific genes by TET enzymes.
2023-07-14 | GSE236769 | GEO