RRHP : a tag-based approach for 5-hydroxymethylcytosine mapping at single-site resolution
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE49546 | GEO | 2014/08/26
SECONDARY ACCESSION(S): PRJNA214228
REPOSITORIES: GEO
Similar Datasets
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
Project description:We report the setup of a new method to map 5-hydroxymethylcytosine (5hmC) genome-wide at CpG resolution. The method combines selective chemical labeling by 5hmC b-glucosyltransferase and exonuclease digestion of the DNA molecules bound to streptavidin beads after biotinylation of the 5-glucosylmethylcytosines. Associated with a straightforward bioinformatic analysis, this new procedure provides a cost-effective and fast method for mapping 5hmC at high resolution.
2016-03-04 | GSE70635 | GEO
Project description:While the role of 5-methylcytosine has been well studied, the biological role of 5-hydroxymethylcytosine still remains unclear due to the limited methods available for single-base detection of 5-hydroxymethylcytosine (5hmC). Here we present Mirror bisulfite sequencing for the detection of 5hmC at a single CpG site by synthesizing a DNA strand to mirror the parental strand. This semi-conservative duplex is sequentially treated with β-glucosyltransferase and M.SssI methylase. A glucosyl-5hmCpG in the parental strand inhibits methylation of the mirroring CpG site, and after bisulfite conversion, a thymine in the mirroring strand indicates a 5hmCpG site in the parental strand whereas a cytosine indicates a non-5hmC site. Using this method, the 5hmC levels of various human tissues and paired tumor liver tissues were mapped genome-wide.
2018-05-22 | GSE69272 | 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:5-methylcytosine (5-mC) can be oxidized to 5-hydroxymethylcytosine (5-hmC). Genome-wide profiling of 5-hmC thus far indicated 5-hmC may not only be an intermediate form of DNA demethylation but could also constitute an epigenetic mark per se. We describe a cost-effective and selective method to detect both the hydroxymethylation and methylation status of cytosines in more than 1.8 million MspI sites in the human genome. This method involves the selective glucosylation of 5-hmC residues, short-sequence tag generation and high-throughput sequencing. We tested this method by screening H9 human embryonic stem cells and their differentiated embroid body cells, and found that differential hydroxymethylation preferentially occur in bivalent genes during cellular differentiation. Especially, our results support hydroxymethylation can regulate key transcription regulators with bivalent marks through demethylation and affect cellular decision on choosing active or inactive state of these genes upon cellular differentiation. We developed a cost-effective and selective method to detect both the hydroxymethylation and methylation status of cytosines in more than 1.8 million MspI sites in the human genome. In this method, we took advantage of the differential enzymatic sensitivities of the isoschizomers MspI and HpaII. HpaII cleaves only a completely unmodified site, any modification at either cytosine blocks the cleavage, while MspI recognizes and cleaves both 5-mC and 5-hmC, but not the newly discovered 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC). Furthermore, beta-glucosyltransferase (beta-GT) can transfer a glucose to the hydroxyl group of 5-hmC and generate beta-glucosyl-5-hydroxymethylcytosine (5-ghmC) that blocks MspI digestion. Thus, either by combining beta-GT treatment with MspI digestion or simply applying MspI/HpaII digestion, short sequence tags generated can be used for inferring hydroxymethylation or methylation status in around 1.8 million cytosine sites in the human genome. We tested this method by screening H9 human embryonic stem cells and their differentiated embroid body cells.
2012-12-20 | E-GEOD-41069 | 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
Project description:Background: 5-hydroxymethylcytosine (5-hmC) is a recently discovered epigenetic modification that is altered in cancers. Genome wide assays for 5-hmC determination are needed as many of the techniques commonly used to assay 5-methylcytosine (5-mC), including conventional methyl-sensitive restriction digest and bisulfite sequencing, are incapable of distinguishing between 5-mC and 5-hmC. Results: Glycosylation of 5-hmC residues by beta-Glucosyl Transferase (beta-GT) can make CCGG residues insensitive to digestion by MspI. We used this premise to modify the HELP-tagging assay to identify both 5-mC and 5-hmC loci in the genome. Comparison of sequencing libraries after HpaII, MspI and MspI+ beta-GT conversion resulted in locus specific 5-mC and 5-hmC determination. A custom bioinformatics pipeline was created to identify 5-hmC sites that were validated at global level by LS-MS and the locus specific level by qRT-PCR of 5-hmC pulldown DNA. Hydroxymethylation at both promoter and intragenic locations correlated positively with gene expression. Analysis of pancreatic cancer samples revealed striking redistribution of 5-hmC sites in cancer cells and demonstrated enrichment of this modification at many oncogenic promoters such as GATA6. Conclusions: The HELP-GT assay allows a high resolution, simultaneous determination of 5-hmC and 5-mC loci from small amounts of DNA with the utilisation of modest sequencing resources. Redistribution of 5-hmC seen in cancer highlights the importance of examining this modification in conjugation with conventional methylome analysis. We did methylation and hydroxymethylation tests for one control and two pancreatic cancer cases
2013-07-30 | E-GEOD-42723 | biostudies-arrayexpress
Project description:We developed a novel approach, J-binding protein 1 sequencing (JBP1-seq), that combines the benefits of an improved recombinant JBP1 protein, Nextera-based library construction, and nextgeneration sequencing (NGS) for genome-wide profiling of 5-hydroxymethylcytosine (5hmC). Compared with the original JBP1, this new recombinant JBP1 was biotinylatedin vivo and conjugated to magnetic beads via biotin-streptavidin interactions. These modifications allowed a more efficient and consistent pull-down of β-glucosyl-5-hydroxymethylcytosine (β-glu-5hmC), and sequence-ready libraries can be generated within 4.5 hours from DNA inputs as low as 50 ng. 5hmC enrichment of human brain DNA using the new JBP1 resulted in over 25,000 peaks called, which is significantly higher than the 4,003 peaks enriched using the old JBP1. Comparison of the technical duplicates and validations with other platforms indicated the results are reproducible and reliable. Thus, JBP1-seq provides a fast, efficient, cost-effective method for accurate 5hmC genome-wide profiling. An improvement of JBP1-Seq
2014-11-26 | E-GEOD-61193 | biostudies-arrayexpress
Project description:5-Methylcytosine (5-mC) is an important DNA modification found in eukaryotes that impacts gene regulation and disease pathogenesis. Recently, 5-hydroxymethylcytosine (5-hmC), another form of DNA modification, has been identified in substantial amounts in certain mammalian cell types; however, its roles as well as its distribution in mammalian genomes are unknown. Here we present a selective chemical labeling method for 5-hmC by utilizing T4 bacteriophage BGT-glucosyltransferase to transfer an engineered glucose moiety containing an azide group onto the hydroxyl group of 5-hmC, which in turn can chemically incorporate a biotin group for detection, affinity enrichment, and sequencing of 5-hmC in mammalian genomes. Using this highly effective method, we demonstrate that 5-hmC is present in human cell lines beyond those previously recognized. We also find a gene expression level-dependent enrichment of intragenic 5-hmC in mouse cerebellum and an age-dependent acquisition of this modification in specific gene bodies linked to neurodegenerative disorders Identification of 5hmC enriched genmoic regions in mouse cerebellum
2010-12-12 | E-GEOD-25398 | biostudies-arrayexpress
Project description:Background: 5-hydroxymethylcytosine (5-hmC) is a recently discovered epigenetic modification that is altered in cancers. Genome wide assays for 5-hmC determination are needed as many of the techniques commonly used to assay 5-methylcytosine (5-mC), including conventional methyl-sensitive restriction digest and bisulfite sequencing, are incapable of distinguishing between 5-mC and 5-hmC. Results: Glycosylation of 5-hmC residues by beta-Glucosyl Transferase (beta-GT) can make CCGG residues insensitive to digestion by MspI. We used this premise to modify the HELP-tagging assay to identify both 5-mC and 5-hmC loci in the genome. Comparison of sequencing libraries after HpaII, MspI and MspI+ beta-GT conversion resulted in locus specific 5-mC and 5-hmC determination. A custom bioinformatics pipeline was created to identify 5-hmC sites that were validated at global level by LS-MS and the locus specific level by qRT-PCR of 5-hmC pulldown DNA. Hydroxymethylation at both promoter and intragenic locations correlated positively with gene expression. Analysis of pancreatic cancer samples revealed striking redistribution of 5-hmC sites in cancer cells and demonstrated enrichment of this modification at many oncogenic promoters such as GATA6. Conclusions: The HELP-GT assay allows a high resolution, simultaneous determination of 5-hmC and 5-mC loci from small amounts of DNA with the utilisation of modest sequencing resources. Redistribution of 5-hmC seen in cancer highlights the importance of examining this modification in conjugation with conventional methylome analysis.
2013-07-30 | GSE42723 | GEO