Dataset Information

Sequencing 5-Hydroxymethyluracil at Single-Base Resolution.

ABSTRACT: 5-hydroxymethyluracil (5hmU) is formed through oxidation of thymine both enzymatically and non-enzymatically in various biological systems. Although 5hmU has been reported to affect biological processes such as protein-DNA interactions, the consequences of 5hmU formation in genomes have not been yet fully explored. Herein, we report a method to sequence 5hmU at single-base resolution. We employ chemical oxidation to transform 5hmU to 5-formyluracil (5fU), followed by the polymerase extension to induce T-to-C base changes owing to the inherent ability of 5fU to form 5fU:G base pairing. In combination with the Illumina next generation sequencing technology, we developed polymerase chain reaction (PCR) conditions to amplify the T-to-C base changes and demonstrate the method in three different synthetic oligonucleotide models as well as part of the genome of a 5hmU-rich eukaryotic pathogen. Our method has the potential capability to map 5hmU in genomic DNA and thus will contribute to promote the understanding of this modified base.

SUBMITTER: Kawasaki F

PROVIDER: S-EPMC6100112 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

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Publications

Sequencing 5-Hydroxymethyluracil at Single-Base Resolution.

Kawasaki Fumiko F Martínez Cuesta Sergio S Beraldi Dario D Mahtey Areeb A Hardisty Robyn E RE Carrington Mark M Balasubramanian Shankar S

Angewandte Chemie (International ed. in English) 20180704 31

5-hydroxymethyluracil (5hmU) is formed through oxidation of thymine both enzymatically and non-enzymatically in various biological systems. Although 5hmU has been reported to affect biological processes such as protein-DNA interactions, the consequences of 5hmU formation in genomes have not been yet fully explored. Herein, we report a method to sequence 5hmU at single-base resolution. We employ chemical oxidation to transform 5hmU to 5-formyluracil (5fU), followed by the polymerase extension to ...[more]

PMID: 29882366

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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

Sequencing 5-Hydroxymethyluracil at Single-Base Resolution.

Publications

Sequencing 5-Hydroxymethyluracil at Single-Base Resolution.

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