Quantitative sequencing of 5-methylcytosine and 5-hydroxymethylcytosine at single nucleotide resolution
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Project description:To investigate the genomic levels of 5-hydroxymethylcytosine at single-base resolution. The current study developed a method which allows one to study hydroxymethylation of cytosines in the genome via a subtractive method of RRBS and oxidative RRBS.
2012-04-24 | E-MTAB-1042 | biostudies-arrayexpress
Project description:Bisulfite-free direct detection of 5-methylcytosine and 5-hydroxymethylcytosine at base resolution
| PRJNA448064 | ENA
Project description:Cytosine base modifications 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5fC) are present in mammalian DNA. Here, reduced bisulfite sequencing is developed for quantitatively sequencing 5fC at single-base resolution. This method is then applied with oxidative bisulfite sequencing to gain a map of 5mC, 5hmC and 5fC in mouse embryonic stem cells.
2014-04-08 | GSE56572 | GEO
Project description:Cytosine base modifications 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5fC) are present in mammalian DNA. Here, reduced bisulfite sequencing is developed for quantitatively sequencing 5fC at single-base resolution. This method is then applied with oxidative bisulfite sequencing to gain a map of 5mC, 5hmC and 5fC in mouse embryonic stem cells. 12 samples, reduced representation bisulphite treatment: 4 replicates each for bisulphite (BS), oxidative BS (oxBS) and reduced BS (redBS) for the detection of 5mC, 5hmC and 5fC. Mouse (strain B6C) embryonic stem cells.
2014-04-08 | E-GEOD-56572 | biostudies-arrayexpress
Project description:Glioblastoma multiforme is the most common and aggressive type of brain cancer. Little is known about the complex relationship between genomic and epigenomic as tumour progresses. We present the following base resolution whole genome maps of matched tumour/margin and blood samples from a glioblastoma multiforme patient:<br>* Single nucleotide variations (SNVs), copy number variations (CNVs) and structural variations (SVs) as revealed by DNA sequencing. </br> <br>* 5-methylcytosine and 5-hydroxymethylcytosine levels obtained using (oxidative)bisulfite sequencing. </br> <br>* Transcript levels produced using RNA sequencing.</br> <br>For the three samples with very large bam raw data files ('Blood DNA-seq', 'Margin DNA-seq' and 'Tumour DNA-seq'), bai index files are available from https://www.ebi.ac.uk/arrayexpress/files/E-MTAB-5171/E-MTAB-5171.additional.1.zip
2017-02-24 | E-MTAB-5171 | biostudies-arrayexpress
Project description:5-Hydroxymethylcytosine (5hmC) is an important epigenetic mark that can regulate gene expression. While some methods were developed to detect 5hmC, direct genome-wide mapping of 5hmC at base resolution are still highly desirable. Herein, we proposed a single-step deamination sequencing (SSD-seq) method for the genome-wide mapping of 5hmC at single-base resolution. This method capitalizes on a screened engineered human apolipoprotein B mRNA-editing catalytic polypeptide-like 3A (A3A) protein to produce differential deamination activity toward cytosine (C), 5-methylcytosine (5mC), and 5hmC. In SSD-seq, an engineered A3A protein (eA3A-v10) can adequately deaminate C and 5mC, but not 5hmC. The original C and 5mC in DNA are deaminated by eA3A-v10 to form uracil (U) and thymine (T), both of which are read as T during sequencing. However, 5hmC is resistant to the deamination by eA3A-v10 and is still read as C during sequencing. Therefore, the remaining C in the sequence reads manifests the original 5hmC. Applying SSD-seq to generate a base-resolution map of 5hmC in human lung tissue, we found that 5hmC was almost entirely confined to CpG dinucleotides. The base-resolution map of 5hmC from human lung tissue generated by SSD-seq correlated strongly with that generated by prior ACE-seq. Taken together, the SSD-seq method is single-step, bisulfite-free and does not require DNA glycosylation or chemical treatment, which offers a valuable tool for the direct and quantitative detection of 5hmC in genomes at single-base resolution.
2023-12-04 | GSE236353 | GEO
Project description:Active DNA demethylation in mammals involves TET-mediated iterative oxidation of 5-methylcytosine (5mC)/5-hydroxymethylcytosine (5hmC) and subsequent excision repair of highly oxidized cytosine bases 5-formylcytosine (5fC)/5-carboxylcytosine (5caC) by Thymine DNA glycosylase (TDG). However, quantitative and high-resolution analysis of active DNA demethylation activity remains challenging. Here we describe M.SssI methylase-assisted bisulfite sequencing (MAB-seq), a method that directly maps 5fC/5caC at single-base resolution. Genome-wide MAB-seq allows systematic identification of 5fC/5caC in Tdg-depleted embryonic stem cells, thereby generating a base-resolution map of active DNA demethylome. A comparison of 5fC/5caC and 5hmC distribution maps indicates that catalytic processivity of TET enzymes correlates with local chromatin accessibility. MAB-seq also reveals strong strand asymmetry of active demethylation within palindromic CpGs. Integrating MAB-seq with other base-resolution mapping methods enables quantitative measurement of cytosine modification states at key transitioning steps of active demethylation pathway, and reveals a regulatory role of 5fC/5caC excision repair in active DNA demethylation cascade. Analysis of 5fC/5caC excision repair-dependent active DNA demethylome by MAB-seq in mouse embryonic stem cells.
2014-11-02 | E-GEOD-62631 | biostudies-arrayexpress
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:Mapping genome-wide 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5fC) at single-base resolution is important to understand their biological functions. We present a cost-efficient mapping method that combines 5hmC-specific restriction enzyme PvuRts1I with a 5hmC enrichment method. The sensitive method enables detection of low abundant 5hmC sites, providing a more complete 5hmC landscape than available bisulfite-based methods. This method generated the first genome-wide 5fC map at single-base resolution. Parallel analyses revealed that 5hmC and 5fC existed with lower abundance and more dynamically in non-CpG context than in CpG context. In the genic region, distribution of 5hmCpG and 5fCpG differed from 5hmCH and 5fCH (H=A, T, C). 5hmC and 5fC were distributed distinctly at regulatory protein-DNA binding sites, depleted in permissive transcription factor binding sites, and enriched at active and poised enhancers. This sensitive bisulfite-conversion free method can be applied to biological samples with limited starting material or low abundance of cytosine modifications. Sensitive mapping of genome-wide 5-hydroxymethylcytosine and 5-formylcytosine in mouse embryonic stem cell at single-base resolution by combining 5-hydroxymethylcytosine specific restriction enzyme PvuRts1I and 5-hydroxymethylcytosine enrichment method (selective chemical labeling or SEAL)
2014-12-22 | E-GEOD-64186 | biostudies-arrayexpress
Project description:Here we present APOBEC-coupled epigenetic sequencing (ACE-seq), a bisulfite-free method for localizing 5-hydroxymethylcytosine (5hmC) at single-base resolution with low DNA input. The method builds on the observation that AID/APOBEC family DNA deaminase enzymes can potently discriminate between cytosine modification states and exploits the non-destructive nature of enzymatic, rather than chemical, deamination. ACE-seq yielded high-confidence 5hmC profiles with at least 1,000-fold less DNA input than conventional methods. Applying ACE-seq to generate a base-resolution map of 5hmC in tissue-derived cortical excitatory neurons, we found that 5hmC was almost entirely confined to CG dinucleotides. The whole-genome map permitted cytosine, 5-methylcytosine (5mC) and 5hmC to be parsed and revealed genomic features that diverged from global patterns, including enhancers and imprinting control regions with high and low 5hmC/5mC ratios, respectively. Enzymatic deamination overcomes many challenges posed by bisulfite-based methods, thus expanding the scope of epigenome profiling to include scarce samples and opening new lines of inquiry regarding the role of cytosine modifications in genome biology.
2018-10-08 | GSE116016 | GEO