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Direct sequencing of 5-methylcytosine and 5-hydroxymethylcytosine at single-base resolution unravels their distinct roles in Alzheimer’s disease [BS-Seq mESC]

ABSTRACT: Direct sequencing of 5-methylcytosine and 5-hydroxymethylcytosine at single-base resolution unravels their distinct roles in Alzheimer’s disease [BS-Seq mESC]

PROVIDER: PRJNA1231068 | ENA |

REPOSITORIES: ENA

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Comparison of ox-BS-450k 5-hydroxymethylcytosine and BS-450k 5-methylcytosine profiles in the human placenta

Project description:Bisulphite (BS) and oxidative bisulphite (oxBS) converted DNA from 4 normal human placentas were hybridized to the Illumina HumanMethylation450 Beadchip v1.2, obtaining the BS and oxBS DNA methylation profiles across approximately 450,000 CpGs. By using the oxBS treatment, the selective chemical oxidation of 5-hydroxymethylcytosine (5hmC) to 5-formylcytosine (5fC) and the deamination of the latter to uracil during the BS conversion allowed the quantification of independent 5-methylcytosine (5mC) and 5hmC methylation levels at every single CpG. In consequence, this data set characterizes the genome-wide distribution of 5hmC in the human placenta offerring a high-confidence list of intervals enriched for 5hmC in this tissue.

2017-09-12 | GSE93429 | GEO

Direct sequencing of 5-methylcytosine and 5-hydroxymethylcytosine at single-base resolution unravels their distinct roles in Alzheimer’s disease [BS-Seq mESC]

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.

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Direct sequencing of 5-methylcytosine and 5-hydroxymethylcytosine at single-base resolution unravels their distinct roles in Alzheimer’s disease [BS-Seq]

Project description:Direct sequencing of 5-methylcytosine and 5-hydroxymethylcytosine at single-base resolution unravels their distinct roles in Alzheimer’s disease [BS-Seq]

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Direct sequencing of 5-methylcytosine and 5-hydroxymethylcytosine at single-base resolution unravels their distinct roles in Alzheimer’s disease [BS-Seq]

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Accurate measurement of 5-methylcytosine and 5-hydroxymethylcytosine in human cerebellum DNA

Project description:The Infinium 450K Methylation array is an established tool for measuring methylation. However, the bisulfite (BS) reaction commonly used with the 450K array cannot distinguish between 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). The oxidative-bisulfite assay disambiguates 5mC and 5hmC. We describe the use of oxBS in conjunction with the 450K array (oxBS-array) to analyse 5hmC/5mC in cerebellum DNA. The methylation level derived by the BS reaction is the combined level of 5mC and 5hmC at a given base, while the oxBS reaction gives the level of 5mC alone. The level of 5hmC is derived by subtracting the oxBS level from the BS level. Here we present an analysis method that distinguishes genuine positive levels of 5hmC at levels as low as 3 %. We preformed four replicates of the same sample of cerebellum and found a high level of reproducibility (average r for BS = 98.3, and average r for oxBS = 96.8). In total, 114,734 probes showed a significant positive measurement for 5hmC. The range at which we were able to distinguish 5hmC occupancy was between 3 % and 42 %. In order to investigate the effects of multiple replicates on 5hmC detection we also simulated fewer replicates and found that decreasing the number of replicates to two reduced the number of positive probes identified by > 50%. We validated our results using qPCR in conjunction with glucosylation of 5hmC sites followed by MspI digestion and we found good concordance with the array estimates (r = 0.94). This experiment provides a map of 5hmC in the cerebellum and a robust dataset for use as a standard in future 5hmC analyses. We also provide a novel method for validating the presence of 5hmC at low levels, and highlight some of the pitfalls associated with measuring 5hmC and 5mC.

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Direct sequencing of 5-methylcytosine and 5-hydroxymethylcytosine at single-base resolution unravels their distinct roles in Alzheimer’s disease [RNA-Seq]

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Differential 5-methylcytosine and 5-hydroxymethylcytosine in Parkinson’s and Alzheimer’s Disease.

Project description:Background: 5-methylcytosine (5-mC) and its oxidized form, 5-hydroxymethylcytosine (5-hmC), are distinct epigenetic marks that help regulate gene expression in the mammalian brain. Existing studies have identified associations between 5-mC and neurodegenerative disease, but little work has examined the potential role of 5-hmC in Parkinson’s disease (PD) or Alzheimer’s disease (AD). Here, we utilized PD postmortem brain tissue and a public dataset from postmortem AD brains to analyze the effect of neurodegenerative disease on paired 5-mC and 5-hmC levels. Results: In PD samples, we measured genome-wide 5-mC and 5-hmC from control (n=3) and PD (n=6) brains using the Illumina EPIC array combined with bisulfite and oxidative bisulfite treatments (BS/oxBS-EPIC). In publicly sourced data, genome-wide 5-mC and 5-hmC were measured from control (n=25) and AD (n=62) human entorhinal cortex tissue using the Illumina 450K array combined with bisulfite and oxidative bisulfite treatment (BS/oxBS-450K). Paired 5-mC and 5-hmC beta values were generated using a custom pipeline of bioinformatics tools, and we modeled the effect of disease on paired 5-mC and 5-hmC data using a mixed effects beta regression model with a random effect for ID and an interaction term between disease category and DNA modification category (“5-mC” or “5-hmC”). We identified a number of CpG probes (AD: n=699, PD: total n = 80) that showed a significant interaction between disease status and DNA modification category (p-value < 2.4x10-7). Conclusions: While our PD data requires additional validation, our analyses suggest that there are widespread shifts in the balance between 5-mC and 5-hmC in Parkinson’s and Alzheimer’s disease.

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