Dataset Information

Quantitative sequencing of 5-formylcytosine in DNA at single-base resolution.

ABSTRACT: Recently, the cytosine modifications 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5fC) were found to exist in the genomic deoxyribonucleic acid (DNA) of a wide range of mammalian cell types. It is now important to understand their role in normal biological function and disease. Here we introduce reduced bisulfite sequencing (redBS-Seq), a quantitative method to decode 5fC in DNA at single-base resolution, based on a selective chemical reduction of 5fC to 5hmC followed by bisulfite treatment. After extensive validation on synthetic and genomic DNA, we combined redBS-Seq and oxidative bisulfite sequencing (oxBS-Seq) to generate the first combined genomic map of 5-methylcytosine, 5hmC and 5fC in mouse embryonic stem cells. Our experiments revealed that in certain genomic locations 5fC is present at comparable levels to 5hmC and 5mC. The combination of these chemical methods can quantify and precisely map these three cytosine derivatives in the genome and will help provide insights into their function.

SUBMITTER: Booth MJ

PROVIDER: S-EPMC4188980 | biostudies-literature | 2014 May

REPOSITORIES: biostudies-literature

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Quantitative sequencing of 5-formylcytosine in DNA at single-base resolution.

Booth Michael J MJ Marsico Giovanni G Bachman Martin M Beraldi Dario D Balasubramanian Shankar S

Nature chemistry 20140323 5

Recently, the cytosine modifications 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5fC) were found to exist in the genomic deoxyribonucleic acid (DNA) of a wide range of mammalian cell types. It is now important to understand their role in normal biological function and disease. Here we introduce reduced bisulfite sequencing (redBS-Seq), a quantitative method to decode 5fC in DNA at single-base resolution, based on a selective chemical reduction of 5fC to 5hmC followed by bisulfite treatm ...[more]

PMID: 24755596

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Project description:BackgroundDetection of DNA methylome at single-base resolution is a significant challenge but promises to shed considerable light on human disease etiology. Current technologies could not detect DNA methylation genome-wide at single-base resolution with small amount of sequencing data and could not avoid detecting the methylation of repetitive elements which are considered as "junk DNA".MethodsIn this study, we have developed a novel DNA methylome profiling technology named MB-seq with its ability to identify genome-wide 5mC and quantify DNA methylation levels by introduced an assistant adapter AluI-linker This linker can be ligated to sonicated DNA and then be digested after the bisulfite treatment and amplification, which has no effect of MeDIP enrichment. Because many researchers are interested in investigating the methylation of functional regions such as promoters and gene bodies, we have also developed a novel alternative method named MRB-seq, which can be used to investigate the DNA methylation of functional regions by removing the repeats with Cot-1 DNA.ResultsIn this study, we have developed MB-seq, a novel DNA methylome profiling technology combining MeDIP-seq with bisulfite conversion, which can precisely detect the 5mC sites and determine their DNA methylation level at single-base resolution in a cost-effective way. In addition, we have developed a new alternative method, MRB-seq (MeDIP-repetitive elements removal-bisulfite sequencing), which interrogates 5mCs in functional regions by depleting nearly half of repeat fragments enriched by MeDIP. Comparing MB-seq and MRB-seq to whole-genome BS-seq using the same batch of DNA from YH peripheral blood mononuclear cells. We found that the sequencing data of MB-seq and MRB-seq almost reaches saturation after generating 7-8 Gbp data, whereas BS-seq requires about 100 Gbp data to achieve the same effect. In comparison to MeDIP-seq and BS-seq, MB-seq offers several key advantages, including single-base resolution, discriminating the methylated sites within a CpG and non-CpG pattern and overcoming the false positive of MeDIP-seq due to the non-specific binding of 5-methylcytidine antibody to genomic fragments.ConclusionOur novel developed method MB-seq can accelerate the decoding process of DNA methylation mechanism in human diseases because it requires 7-8 Gbp data to measure human methylome with enough coverage and sequencing depth, affording it a direct and practical application in the study of multiple samples. In addition, we have also provided a novel alternative MRB-seq method, which removes most repetitive sequences and allows researchers to genome-wide characterize DNA methylation of functional regions.

Dataset Information

Quantitative sequencing of 5-formylcytosine in DNA at single-base resolution.

Publications

Quantitative sequencing of 5-formylcytosine in DNA at single-base resolution.

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