Base-resolution detection of N4-methylcytosine in genomic DNA using 4mC-TAB-seq
Ontology highlight
ABSTRACT:
ORGANISM(S): Caldicellulosiruptor kristjanssonii
SUBMITTER: Robert Schmitz
PROVIDER: E-GEOD-63371 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:N4-methylcytosine is a major DNA modification integral to restriction-modification (R-M) systems in bacterial genomes. Here we describe 4mC-Tet-Assisted Bisulfite-sequencing (4mC-TAB-seq), a method that accurately and rapidly reveals the genome-wide locations of N4-methylcytosines at single-base resolution. By coupling Tet-mediated oxidation with a modified sodium bisulfite conversion reaction, unmodified cytosines and 5-methylcytosines are read out as thymines, whereas N4-methylcytosines are read out as cytosines revealing their positions throughout the genome.
2015-07-11 | GSE63371 | GEO
Project description:Stochastic changes in cytosine methylation are a source of heritable epigenetic and phenotypic diversity in plants. Using the model plant Arabidopsis thaliana, we derive robust estimates of the rate at which methylation is spontaneously gained (forward epimutation) or lost (backward epimutation) at individual cytosines and construct a comprehensive picture of the epimutation landscape in this species. We demonstrate that the dynamic interplay between forward and backward epimutations is modulated by genomic context and show that subtle contextual differences have profoundly shaped patterns of methylation diversity in A. thaliana natural populations over evolutionary timescales. Theoretical arguments indicate that the epimutation rates reported here are high enough to rapidly uncouple genetic from epigenetic variation, but low enough for new epialleles to sustain long-term selection responses. Our results provide new insights into methylome evolution and its population-level consequences. MethylC-seq of Arabidopsis thaliana
2015-05-13 | E-GEOD-64463 | biostudies-arrayexpress
Project description:The reprogramming of parental methylomes is essential for embryonic development. In mammals, paternal 5-methylcytosines (5mCs) have been proposed to be actively converted to oxidized bases. These paternal oxidized bases and maternal 5mCs are believed to be passively diluted by cell divisions. By generating single-base resolution, allele-specific DNA methylomes from mouse gametes, early embryos, and primordial germ cell (PGC), as well as single-base-resolution maps of oxidized cytosine bases for early embryos, we report the existence of 5hmC and 5fC in both maternal and paternal genomes and find that 5mC or its oxidized derivatives, at the majority of demethylated CpGs, are converted to unmodified cytosines independent of passive dilution from gametes to four-cell embryos. Therefore, we conclude that paternal methylome and at least a significant proportion of maternal methylome go through active demethylation during embryonic development. Additionally, all the known imprinting control regions (ICRs) were classified into germ-line or somatic ICRs. The cross of two mouse strains was performed using DBA/2J as the paternal strain and C57BL/6J as the maternal strain. The hybrid embryos were collected at 2-cell, 4-cell, ICM, E6.5, E7.5 stages. Female and male E13.5 PGC samples (B6; 129S4-Pou5f1tm2Jae/J) were collected from timed mating of C57BL/6J female mice. MethylC-Seq: oocytes (C57BL/6J), sperm (DBA/2J), 2-cell embryos, 4-cell embryos, ICM, E6.5 embryos, E7.5 embryos, E13.5 female PGCs and E13.5 male PGCs. TAB-Seq: 2-cell embryos. fCAB-Seq: 2-cell embryos. RNA-Seq: oocytes (C57BL/6J).
2014-05-12 | E-GEOD-56697 | biostudies-arrayexpress
Project description:5-hydroxymethylcytosines (5hmC) is particularly abundant in mammalian brain with little-known functions. Here we present the first genome-wide and single-base-resolution maps of 5hmC and 5mC in human brain by combined application of TAB-Seq and MethylC-Seq. We report that the majority of modified cytosines are hydroxymethylated in adult human brain, a significant proportion of which are highly-hydroxymethylated with enrichment in active genic regions and distal-regulatory elements. 5hmC is more enriched in poised than active enhancers, and CpG island shores and enhancers show comparable 5hmC profiles. Notably, 5hmC spikes were identified at the 5’ splicing sites, suggesting a link between 5hmC and splicing. Additionally, we identified a transcription-correlated 5hmC bias towards to sense strand and a 5mC bias towards antisense strand of gene bodies, and a bias towards C-rich sequences surrounding the 5hmC sites. Our data imply multiple roles for 5hmC in alternative splicing and gene regulation in addition to be an intermediate of DNA demethylation in human brain. Examination of hydroxymethylomes of 1 adult and 1 fetal brain tissue of frontal lobe, as well as 1 methylome of the same adult.
2014-02-14 | E-GEOD-46710 | biostudies-arrayexpress
Project description:The development of whole-genome bisulfite sequencing (WGBS) has led to a number of exciting discoveries about how genomes utilize DNA methylation and has led to a plethora of novel testable hypotheses. Methods for constructing sodium bisulfite-converted and amplified libraries have recently excelled to the point that the bottleneck for experiments that use WGBS has shifted to data analysis and interpretation. Here we present empirical evidence for an over-representation of methylated DNA from WGBS. This enrichment for methylated DNA is exacerbated by higher cycles of PCR and is influenced by the type of uracil-insensitive DNA polymerase used for amplifying the sequencing library. Future efforts to computationally correct for this enrichment bias will be essential to increasing the accuracy of determining methylation levels for individual cytosines. MethylC-Seq of Arabidopsis thaliana
2014-09-11 | E-GEOD-58217 | biostudies-arrayexpress
Project description:We report a new bisulfite-free 5mC and 5hmC base-resolution sequencing method: TET Assisted Pyridine borane Sequencing (TAPS). TAPS relies on mild reactions and detects DNA modifications directly, without affecting unmodified cytosines. We applied this method for the first time to whole genome sequencing in E14 mESC cell line. For comparsion we prepared whole-genome bisulfite sequencing in the same cell line. Compared with bisulfite sequencing, TAPS results in higher mapping rates, more even coverage and lower sequencing cost, enabling more informative and cheaper methylome analyses. We expect TAPS to become the new standard in epigenetic DNA sequencing.
2018-12-24 | GSE112520 | GEO
Project description:Mouse KrasG12D/P53-/-(KP) lung tumor cells were transfected with pLKO.1-Tet on-shNpm1-3(N3), pLKO.1-Tet on-shNpm1-4(N4) or pLKO.1-Tet on-shNpm1-5 (N5) and then selected by using 2ug/ml puromycin, to establish the 3 stable cell lines. These 3 stable cell lines (N3, N4 or N5) were treated with 100ng/ml Doxycline and then the total RNA of the treated and untreated cells were harvested for RNA seq.
2020-06-22 | GSE133555 | 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:The non-methylable cytosine analogs, 5-azacytidine and zebularine, are widely used to disrupt DNA methyltransferase activity and reduce genomic DNA methylation. In this study, whole-genome bisulfite sequencing is used to construct maps of DNA methylation with single base pair resolution in Arabidopsis thaliana seedlings treated with each demethylating agent. We find that 5-azacytidine and zebularine-treated seedlings have nearly indistinguishable patterns of DNA methylation genome-wide and that 5-azacytidine, despite being more unstable in aqueous solution, has a slightly greater demethylating effect at higher concentrations across the genome. Transcriptome analyses revealed a substantial number of up-regulated genes and transposable element genes, particularly CACTA-like elements, demonstrating that chemical demethylating agents have a disproportionately large effect on loci that are silenced by DNA methylation. Bisulfite-Seq and RNA-Seq
2016-06-24 | E-GEOD-80300 | biostudies-arrayexpress
Project description:DNA methylation is a complex epigenetic marker that can be analysed using a wide variety of methods. Interpretation and visualisation of DNA methylation data can mask complexity in terms of methylation status at each CpG site, cellular heterogeneity of samples and allelic DNA methylation patterns within a given DNA strand. Bisulfite sequencing is considered the gold standard, however visualisation of massively parallel sequencing results remains a significant challenge. We created a program called Methpat that facilitates visualisation and interpretation of bisulfite sequencing data generated by massively parallel sequencing. To demonstrate this, we performed multiplex PCR that targeted 48 regions of interest across 95 human samples. The regions selected included known gene promoters associated with cancer, repetitive elements, known imprinted regions and mitochondrial genomic sequences. We interrogated a range of samples including human cell lines, primary tumours and primary tissue samples. Methpat generates two forms of output: a tab delimited text file for each sample that summarises DNA methylation patterns and their read counts for each amplicon and a HTML file that summarises this data visually. Methpat can be used with publicly available whole genome bisulfite sequencing (WGBS) and reduced representation bisulfite sequencing (RRBS) datasets with sufficient read depths. Using Methpat, complex DNA methylation data derived from massively parallel sequencing can be summarised and visualised for biological interpretation. By accounting for allelic DNA methylation states and their abundance in a sample, Methpat can unmask the complexity of DNA methylation and reveal further biological insight in existing datasets. Multiplex bisulfite PCR and Next Generation sequencing of primary human samples and breast cancer cell lines.
2015-11-23 | E-GEOD-71804 | biostudies-arrayexpress