Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Unnatural cytosine analogs potentiate a customizable, enzymatic method for integrated epigenetic and four-base genetic sequencing

ABSTRACT: The interplay of genetic and epigenetic information shapes cell identity, development, and disease. However, standard methods for profiling DNA modifications (e.g., bisulfite-sequencing) rely on selective C-to-T conversions, hindering the simultaneous examination of both genetic and epigenetic information. Here, we introduce Integrated-Sequencing, which provides high-fidelity mapping of DNA modifications while preserving the native four-base genetic code in single molecules. Integrated-Seq leverages the synthesis of a tethered copy strand with unnatural cytosine analogs that resist enzymatic conversion, combined with a novel DNA deaminase-helicase fusion that drives selective C-to-T conversion of natural cytosines in the original template strand. We demonstrate that Integrated-Seq is compatible with customizable enzymatic readouts to parse 5-methylcytosine and 5-hydroxymethylcytosine, and that preservation of the original four-base genetic code markedly improved enrichment, facilitating analysis of targeted genomic regions. Integrated-Seq thus provides a platform for simultaneous genetic and epigenetic analyses, paving the way for deep insights into fundamental biology and next-generation diagnostics.

ORGANISM(S): Mus musculus

PROVIDER: GSE317731 | GEO | 2026/03/17

REPOSITORIES: GEO

ACCESS DATA

Json Xml

Dataset's files

Source:

			Action	DRS
		Other

Items per page:

1 - 1 of 1

Similar Datasets

A Ternary-code DNA Methylome Atlas of Mouse Tissues

Project description:DNA cytosine modifications, including 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC), are key epigenetic regulators with distinct functions. Dissecting the ternary code (C, 5mC, 5hmC) across tissues and cell types remains a critical priority due to the limitations of traditional profiling methods based on bisulfite conversion. Here, we leverage the combined bisulfite and enzymatic (bACE) conversion with the Mouse Methylation BeadChip to generate 265 base-resolution ternary-code modification maps of 5mC and 5hmC across 29 mouse tissue types spanning 8-76 weeks of age and both sexes. Our atlas reveals a complex grammar of 5hmC distribution, jointly shaped by cell mitotic activity, chromatin states, and interplay with 5mC at the same and neighboring CpG sites. Of note, we demonstrate that 5hmC significantly complements 5mC-based biomarkers in delineating cell identity in both brain and non-brain tissues. Each modification state, including 5hmC alone, accurately discriminates tissue types, enabling high-precision machine learning classification of epigenetic identity. Furthermore, the ternary methylome variations extensively implicate gene transcriptional variation, with age-related changes correlated with gene expression in a tissue-dependent manner. Our work examining the interplay of tissue type, sex, and age on methylome dynamics deepens our understanding of cytosine modification biology, augments the scope of DNA modification biomarker discovery, and provides a reference atlas to explore epigenetic dynamics in development and disease.

2025-09-17 | GSE308297 | GEO

RRHP : a tag-based approach for 5-hydroxymethylcytosine mapping at single-site resolution

Project description:We present here a novel approach called Reduced Representation 5-Hydroxymethylcytosine Profiling (RRHP), which exploits ?-glucosyltransferase (?-GT) to inhibit restriction digestion at adapters ligated to a genomic library, such that only fragments presenting glucosylated 5hmC residues at adapter junctions will be amplified and sequenced. This assay profiles 5hmC sites with single-base resolution in a strand-specific manner. The absence of harsh chemical conversion steps allows for sequencing of native DNA with less inputs, enhancing both sequencing quality and mapping efficiency. Most importantly, the method proves highly reproducible and is a positive display method, sensitive enough to interrogate 5hmC sites with low abundance. When combined with existing RRBS data, it allows simultaneous comparison of 5mC and 5hmC at specific site. developing a new assay for genomic profiling of 5hmC

2014-08-26 | E-GEOD-49546 | biostudies-arrayexpress

Efficient and accurate determination of genome-wide DNA methylation patterns in Arabidopsis thaliana with enzymatic methyl sequencing

Project description:5′ methylation of cytosines in DNA molecules is an important epigenetic mark in eukaryotes. Bisulfite sequencing is the gold standard of DNA methylation detection, and whole-genome bisulfite sequencing (WGBS) has been widely used to detect methylation at single-nucleotide resolution on a genome-wide scale. However, sodium bisulfite is known to severely degrade DNA, which, in combination with biases introduced during PCR amplification, leads to unbalanced base representation in the final sequencing libraries. Enzymatic conversion of unmethylated cytosines to uracils can achieve the same end product for sequencing as does bisulfite treatment and does not affect the integrity of the DNA; enzymatic methylation sequencing may, thus, provide advantages over bisulfite sequencing.

2020-10-07 | GSE151616 | GEO

RRHP : a tag-based approach for 5-hydroxymethylcytosine mapping at single-site resolution

Project description:We present here a novel approach called Reduced Representation 5-Hydroxymethylcytosine Profiling (RRHP), which exploits β-glucosyltransferase (β-GT) to inhibit restriction digestion at adapters ligated to a genomic library, such that only fragments presenting glucosylated 5hmC residues at adapter junctions will be amplified and sequenced. This assay profiles 5hmC sites with single-base resolution in a strand-specific manner. The absence of harsh chemical conversion steps allows for sequencing of native DNA with less inputs, enhancing both sequencing quality and mapping efficiency. Most importantly, the method proves highly reproducible and is a positive display method, sensitive enough to interrogate 5hmC sites with low abundance. When combined with existing RRBS data, it allows simultaneous comparison of 5mC and 5hmC at specific site.

2014-08-26 | GSE49546 | GEO

Nanoluc synthesized by the PURE system with the tRNA array method

Project description:Transfer RNA (tRNA) plays a central role in translation. Simultaneously synthesizing the minimal yet sufficient number of tRNA species (at least 21) in vitro poses a challenge to constructing a self-reproducible artificial cell. In this study, we developed a simplified processing method that enables the simultaneous expression of all 21 tRNAs in a reconstituted transcription/translation system (PURE system). By combining direct tRNA linkage and HDVR attachment methods, termed the tRNA array method, we achieved simultaneous expression of all 21 tRNAs from a single polycistronic DNA template in the PURE system. This method allows for the easy customization of the genetic code by introducing additional tRNAs. To demonstrate genetic code reassignment, we replaced five Leu residues (CUU) in Nanoluc with ACG, which is assigned to Thr in the native codon table. To confirm amino acid reassignment, we performed LC-MS/MS analysis of translated Nanoluc proteins. The results confirmed the incorporation of threonine at the reassigned ACG codon in the presence of tRNA^Thr_CGU, and leucine incorporation when using tRNA^Leu_CGU.

2025-07-25 | PXD066302 | JPOST Repository

Methyl-Micro-C

Project description:Simultaneous measurement of epigenetic signatures from the same cell and tissue provides significant benefits for research, especially when resources are limited, and precise analysis is essential. Here, we report a technique called Methyl-Micro-C to simultaneously map chromatin accessibility, interaction, and DNA methylation in the same sample. Methyl-Micro-C enhances the resolution of chromatin interaction and the coverage of CpGs by combining MNase-mediated fragmentation with enzymatic conversion. This technique is relatively straightforward, making it easy for researchers to apply. It also allows for the profiling of three-dimensional epigenomes with consistent chromatin accessibility, interaction, and DNA methylation signals in a cost-efficient manner.

2025-06-04 | GSE276687 | GEO

A Ternary-code DNA Methylome Atlas of Mouse Tissues

Project description:DNA cytosine modifications, including 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC), are key epigenetic regulators with distinct functions. To better understand the ternary code (C, 5mC, 5hmC) across different tissues and cell types, we used bACE conversion with scalable assays to map these modifications at base resolution in 29 mouse tissues across various ages and sexes. Our atlas revealed complex patterns of 5hmC distribution, shaped by mitotic activity, chromatin states, and interactions with 5mC. Notably, 5hmC complements 5mC in identifying cell types, extending beyond the brain. Each modification, including 5hmC alone, distinguishes tissue types, enabling high-precision classification of epigenetic identity. Additionally, changes in the methylome correlate with gene transcription, with age-related effects varying by tissue type, offering new insights into cytosine modification biology and potential biomarkers.

2025-09-26 | GSE290585 | GEO

Epigenetic analyses of methylation and nucleosome occupancy in cell-free DNA (cfNOMe)

Project description:Cell-free DNA (cfDNA) has become essential in cancer diagnostics and prenatal testing. We investigate enzymatic cytosine conversion for epigenetic analyses of cfDNA and find that it reliably measures cytosine methylation and nucleosome occupancy. Whereas previously, separate experiments had to be performed to study either epigenetic marking, this approach delivers reliable results for both, enabling more comprehensive and inexpensive epigenetic profiling of cfDNA.

| EGAS00001004370 | EGA

Bisulfite-Free Whole-Genome Mapping of 5-Methylcytosine at Single-Base Resolution by NTD-seq

Project description:5-Methylcytosine (5mC) is a crucial epigenetic modification plays a significant role in the regulation of gene expression. Accurate and quantitative detection of 5mC at single-base resolution is essential for understanding its epigenetic functions within genomes. In this study, we develop a novel NaegleriaTET-assisted deaminase sequencing (NTD-seq) method for the base-resolution and quantitative detection of 5mC in genomic DNA. The TAD-seq method utilizes a Naegleria TET-like dioxygenase (nTET) to oxidize 5mC, generating 5-methylcytosine oxidation products (5moC). We also engineered a variant of the human apolipoprotein B mRNA-editing catalytic polypeptide-like 3A (A3A), creating an A3A mutant (A3Am). Treatment with A3Am results in the conversion of cytosine to uracil, while 5moC remains unchanged. Consequently, TAD-seq enables the direct deamination of cytosine to uracil by A3Am, which is sequenced as thymine, whereas 5mC, once oxidized to 5moC by nTET, resists deamination and is sequenced as cytosine. Therefore, the cytosines that persist in the sequencing data represent the original 5mC sites. We applied NTD-seq to HEK293T cells, generating a base-resolution map of 5mC that exhibits strong concordance with maps generated by conventional BS-seq. NTD-seq emerges as a powerful, bisulfite-free approach for the single-base resolution mapping of 5mC stoichiometry in genomic DNA.

2025-04-02 | GSE263426 | GEO

Bisulfite-Free Whole-Genome Mapping of 5-Methylcytosine at Single-Base Resolution by TAD-seq

Project description:5-Methylcytosine (5mC) is a crucial epigenetic modification plays a significant role in the regulation of gene expression. Accurate and quantitative detection of 5mC at single-base resolution is essential for understanding its epigenetic functions within genomes. In this study, we develop a novel nTET-assisted deaminase sequencing (TAD-seq) method for the base-resolution and quantitative detection of 5mC in genomic DNA. The TAD-seq method utilizes a Naegleria TET-like dioxygenase (nTET) to oxidize 5mC, generating 5-methylcytosine oxidation products (5moC). We also engineered a variant of the human apolipoprotein B mRNA-editing catalytic polypeptide-like 3A (A3A), creating an A3A mutant (A3Am). Treatment with A3Am results in the conversion of cytosine to uracil, while 5moC remains unchanged. Consequently, TAD-seq enables the direct deamination of cytosine to uracil by A3Am, which is sequenced as thymine, whereas 5mC, once oxidized to 5moC by nTET, resists deamination and is sequenced as cytosine. Therefore, the cytosines that persist in the sequencing data represent the original 5mC sites. We applied TAD-seq to HEK293T cells, generating a base-resolution map of 5mC that exhibits strong concordance with maps generated by conventional BS-seq. TAD-seq emerges as a powerful, bisulfite-free approach for the single-base resolution mapping of 5mC stoichiometry in genomic DNA.

2024-06-30 | GSE254203 | GEO

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data