DNA methylation estimation using methylation-sensitive restriction enzyme bisulfite sequencing (MREBS)
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE123566 | GEO | 2018/12/11
REPOSITORIES: GEO
Similar Datasets
Project description:Whole-genome bisulfite sequencing (WGBS) and reduced representation bisulfite sequencing (RRBS) are widely used for measuring DNA methylation levels on a genome-wide scale. Both methods have limitations: WGBS is expensive and prohibitive for most large-scale projects; RRBS only interrogates 6-12% of the CpGs in the human genome. Here, we introduce methylation-sensitive restriction enzyme bisulfite sequencing (MREBS) which has the reduced sequencing requirements of RRBS, but significantly expands the coverage of CpG sites in the genome. We built a multiple regression model that combines the two features of MREBS: the bisulfite conversion ratios of single cytosines (as in WGBS and RRBS) as well as the number of reads that cover each locus (as in MRE-seq). This combined approach allowed us to estimate differential methylation across 60% of the genome using read count data alone, and where counts were sufficiently high in both samples (about 1.5% of the genome), our estimates were significantly improved by the single CpG conversion information. We show that differential DNA methylation values based on MREBS data correlate well with those based on WGBS and RRBS. This newly developed technique combines the sequencing cost of RRBS and DNA methylation estimates on a portion of the genome similar to WGBS, making it ideal for large-scale projects of mammalian genomes.
| S-EPMC6448829 | biostudies-literature
Project description:DNA methylation estimation using methylation-sensitive restriction enzyme bisulfite sequencing (MREBS)
| PRJNA509235 | ENA
Project description:Single-cell multi-omics are powerful means to study cell-to-cell heterogeneity. Here, we present a single-tube, bisulfite-free method for the simultaneous, genome-wide analysis of DNA methylation and genetic variants in single cells: epigenomics and genomics of single cells analyzed by restriction (epi-gSCAR). By applying this method, we obtained DNA methylation measurements of up to 506,063 CpGs and up to 1,244,188 single-nucleotide variants from single acute myeloid leukemia-derived cells. We demonstrate that epi-gSCAR generates accurate and reproducible measurements of DNA methylation and allows to differentiate between cell lines based on the DNA methylation and genetic profiles.
| S-EPMC7851132 | biostudies-literature
Project description:BACKGROUND: Genomic DNA methylation affects approximately 1% of DNA bases in humans, with the most common event being the addition of a methyl group to the cytosine residue present in the CpG (cytosine-guanine) dinucleotide. Methylation is of particular interest because of its role in gene silencing in many pathological conditions. CpG methylation can be measured using a wide range of techniques, including methylation-specific (MS) PCR, pyrosequencing (PSQ), bisulfite sequencing (BS) and methylation-sensitive restriction enzyme (MSRE) PCR. However, although it is possible to utilise these methods to measure CpG methylation, optimisation of the assays can be complicated due to the absence of suitable control DNA samples. RESULTS: To address this problem, we have developed an approach that employs multiple displacement based whole genome amplification (WGA) with or without SssI-methylase treatment to generate CpG methylated and CpG unmethylated DNA, respectively, that come from the same source DNA. CONCLUSION: Using these alternately methylated DNA samples, we have been able to develop and optimise reliable MS-PCR, PSQ, BS and MRSE-PCR assays for CpG methylation detection, which would otherwise not have been possible, or at least have been significantly more difficult.
| S-EPMC2175516 | biostudies-literature
Project description:Epigenetic regulation is important in human health and disease, but the exact mechanisms remain largely enigmatic. DNA methylation represents one epigenetic aspect but is challenging to quantify. In this study, we introduce a digital approach for the quantification of the amount and density of DNA methylation. We designed an experimental setup combining efficient methylation-sensitive restriction enzymes with digital polymerase chain reaction (PCR) to quantify a targeted density of DNA methylation independent of bisulfite conversion. By using a stable reference and comparing experiments treated and untreated with these enzymes, copy number instability could be properly normalized. In silico simulations demonstrated the mathematical validity of the setup and showed that the measurement precision depends on the amount of input DNA and the fraction methylated alleles. This uncertainty could be successfully estimated by the confidence intervals. Quantification of RASSF1 promoter methylation in a variety of healthy and malignant samples and in a calibration curve confirmed the high accuracy of our approach, even in minute amounts of DNA. Overall, our results indicate the possibility of quantifying DNA methylation with digital PCR, independent of bisulfite conversion. Moreover, as the context-density of methylation can also be determined, biological mechanisms can now be quantitatively assessed.
| S-EPMC7756443 | biostudies-literature
Project description:Aberrant CpG dinucleotide methylation in a specific region of the telomerase reverse transcriptase (TERT) promoter is associated with increased TERT mRNA levels and malignancy in several cancer types. However, routine screening of this region to aid cancer diagnosis can be challenging because i) several established methylation assays may inaccurately report on hypermethylation of this particular region, ii) interpreting the results of methylation assays can sometimes be difficult for clinical laboratories, and iii) use of high-throughput methylation assays for a few patient samples can be cost prohibitive. Herein, we describe the use of combined bisulfite restriction enzyme analysis (COBRA) as a diagnostic tool for detecting the hypermethylated TERT promoter using in vitro methylated and unmethylated genomic DNA as well as genomic DNA from four melanomas and two benign melanocytic lesions. We compare COBRA with MassARRAY, a more commonly used high-throughput approach, in screening for promoter hypermethylation in 28 formalin-fixed, paraffin-embedded neuroblastoma samples. COBRA sensitively and specifically detected samples with hypermethylated TERT promoter and was as effective as MassARRAY at differentiating high-risk from benign or low-risk tumors. This study demonstrates the utility of this low-cost, technically straightforward, and easily interpretable assay for cancer diagnosis in tumors of an ambiguous nature.
| S-EPMC5417004 | biostudies-literature
Project description:Genotyping-by-sequencing (GBS) or restriction-site associated DNA marker sequencing (RAD-seq) is a practical and cost-effective method for analysing large genomes from high diversity species. This method of sequencing, coupled with methylation-sensitive enzymes (often referred to as methylation-sensitive restriction enzyme sequencing or MRE-seq), is an effective tool to study DNA methylation in parts of the genome that are inaccessible in other sequencing techniques or are not annotated in microarray technologies. Current software tools do not fulfil all methylation-sensitive restriction sequencing assays for determining differences in DNA methylation between samples. To fill this computational need, we present msgbsR, an R package that contains tools for the analysis of methylation-sensitive restriction enzyme sequencing experiments. msgbsR can be used to identify and quantify read counts at methylated sites directly from alignment files (BAM files) and enables verification of restriction enzyme cut sites with the correct recognition sequence of the individual enzyme. In addition, msgbsR assesses DNA methylation based on read coverage, similar to RNA sequencing experiments, rather than methylation proportion and is a useful tool in analysing differential methylation on large populations. The package is fully documented and available freely online as a Bioconductor package ( https://bioconductor.org/packages/release/bioc/html/msgbsR.html ).
| S-EPMC5794748 | biostudies-literature
Project description:DNA methylation is an epigenetic modification that plays a key role in several cellular processes mediating the fine regulation of gene expression. Aberrant DNA methylation is observed in a wide range of pathologies, including cancer. Since these DNA modifications are transferred to the cell progenies and are stable over the time, the analysis of DNA methylation status has been proposed for diagnostic and prognostic purposes in cancer. Currently, DNA bisulfite conversion is the gold standard method for the high‑throughput analysis of DNA methylation alterations. However, bisulfite treatment induces DNA fragmentation affecting its quality for the downstream analyses. In this field, it is mandatory to identify novel methods to overcome the limits of conventional approaches. In the present study, the Methylation‑Sensitive Restriction Enzyme‑droplet digital PCR (MSRE‑ddPCR) assay was developed as a novel sensitive method for the analysis of DNA methylation of short genomic regions, combining the MSRE assay with the high‑sensitivity ddPCR and using an exogenous methylation sequence as control. Setup and validation experiments were performed analyzing a methylation hotspot of the Solute Carrier Family 22 Member 17 in DNA samples derived from melanoma cell lines as well as from tissues and serum samples obtained from patients with melanoma and healthy controls. Compared with the standard MSRE approaches, the MSRE‑ddPCR assay is more appropriate for the analysis of DNA methylation (methDNA) in samples with low amounts of DNA (up to 0.651 ng) showing a greater sensitivity. These findings suggested the potential clinical application of MSRE‑ddPCR paving the way to the analysis of other methDNA hotspots in different tumors.
| S-EPMC10998716 | biostudies-literature
Project description:We performed RRBS and WGBS on primary human chronic lymphocytic leukemia and normal healthy donor B cell samples Due to patient privacy concerns, the raw data is being made available via controlled access in dbGaP (http://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/study.cgi?study_id=phs000435.v1.p1). cross-sectional/longitudinal
2015-01-01 | E-GEOD-58889 | biostudies-arrayexpress
Project description:BackgroundReduced representation bisulfite sequencing (RRBS) was developed to measure DNA methylation of high-CG regions at single base-pair resolution, and has been widely used because of its minimal DNA requirements and cost efficacy; however, the CpG coverage of genomic regions is restricted and important regions with low-CG will be ignored in DNA methylation profiling. This method could be improved to generate a more comprehensive representation.ResultsBased on in silico simulation of enzyme digestion of human and mouse genomes, we have optimized the current single-enzyme RRBS by applying double enzyme digestion in the library construction to interrogate more representative regions. CpG coverage of genomic regions was considerably increased in both high-CG and low-CG regions using the double-enzyme RRBS method, leading to more accurate detection of their average methylation levels and identification of differential methylation regions between samples. We also applied this double-enzyme RRBS method to comprehensively analyze the CpG methylation profiles of two colorectal cancer cell lines.ConclusionThe double-enzyme RRBS increases the CpG coverage of genomic regions considerably over the previous single-enzyme RRBS method, leading to more accurate detection of their average methylation levels. It will facilitate genome-wide DNA methylation studies in multiple and complex clinical samples.
| S-EPMC3570491 | biostudies-literature