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

ABSTRACT: Current methods for genomic mapping of 5-hydroxymethylcytosine (5hmC) have been limited by either costly sequencing depth, high DNA input, or lack of single-base resolution. We present an approach called Reduced Representation 5-Hydroxymethylcytosine Profiling (RRHP) to map 5hmC sites at single-base resolution by exploiting the use of beta-glucosyltransferase to inhibit enzymatic digestion at the junction where adapters are ligated to a genomic library. Therefore, only library fragments presenting glucosylated 5hmC residues at the junction are sequenced. RRHP can detect sites with low 5hmC abundance, and when combined with RRBS data, 5-methylcytosine and 5-hydroxymethylcytosine can be compared at a specific site.

SUBMITTER: Petterson A

PROVIDER: S-EPMC4212096 | biostudies-literature |

REPOSITORIES: biostudies-literature

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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

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

Single-Base Resolution Mapping of 5-Hydroxymethylcytosine Modifications in Hippocampus of Alzheimer's Disease Subjects.

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EBS-seq: enrichment-based method for accurate analysis of 5-hydroxymethylcytosine at single-base resolution.

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Single-CpG resolution mapping of 5-hydroxymethylcytosine by chemical labeling and exonuclease digestion identifies evolutionarily unconserved CpGs as TET targets.

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| S-EPMC3743234 | biostudies-literature

Bisulfite-Free, Nanoscale Analysis of 5-Hydroxymethylcytosine at Single Base Resolution.

Project description:High-resolution detection of genome-wide 5-hydroxymethylcytosine (5hmC) sites of small-scale samples remains challenging. Here, we present hmC-CATCH, a bisulfite-free, base-resolution method for the genome-wide detection of 5hmC. hmC-CATCH is based on selective 5hmC oxidation, chemical labeling and subsequent C-to-T transition during PCR. Requiring only nanoscale input genomic DNA samples, hmC-CATCH enabled us to detect genome-wide hydroxymethylome of human embryonic stem cells in a cost-effective manner. Further application of hmC-CATCH to cell-free DNA (cfDNA) of healthy donors and cancer patients revealed base-resolution hydroxymethylome in the human cfDNA for the first time. We anticipate that our chemical biology approach will find broad applications in hydroxymethylome analysis of limited biological and clinical samples.

| S-EPMC6423965 | biostudies-literature

Characterizing 5-hydroxymethylcytosine in human prefrontal cortex at single base resolution.

Project description:BackgroundThe recent discovery that methylated cytosines are converted to 5-hydroxymethylated cytosines (5hmC) by the family of ten-eleven translocation enzymes has sparked significant interest on the genomic location, the abundance in different tissues, the putative functions, and the stability of this epigenetic mark. 5hmC plays a key role in the brain, where it is particularly abundant and dynamic during development.ResultsHere, we comprehensively characterize 5hmC in the prefrontal cortices of 24 subjects. We show that, although there is inter-individual variability in 5hmC content among unrelated individuals, approximately 8 % of all CpGs on autosomal chromosomes contain 5hmC, while sex chromosomes contain far less. Our data also provide evidence suggesting that 5hmC has transcriptional regulatory properties, as the density of 5hmC was highest in enhancer regions and within exons. Furthermore, we link increased 5hmC density to histone modification binding sites, to the gene bodies of actively transcribed genes, and to exon-intron boundaries. Finally, we provide several genomic regions of interest that contain gender-specific 5hmC.ConclusionsCollectively, these results present an important reference for the growing number of studies that are interested in the investigation of the role of 5hmC in brain and mental disorders.

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High-resolution mapping of injury-site dependent functional recovery in a single axon in zebrafish.

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A Sensitive Approach to Map Genome-wide 5-Hydroxymethylcytosine and 5-Formylcytosine at Single-base Resolution

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2014-12-22 | E-GEOD-64186 | biostudies-arrayexpress

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