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Bisulfite-free direct detection of 5-methylcytosine and 5-hydroxymethylcytosine at base resolution

ABSTRACT: Bisulfite-free direct detection of 5-methylcytosine and 5-hydroxymethylcytosine at base resolution

PROVIDER: PRJNA448064 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR6918160_1.fastq.gz	Fastqsanger.gz
	SRR6918160_2.fastq.gz	Fastqsanger.gz
	SRR6918157_1.fastq.gz	Fastqsanger.gz
	SRR6918157_2.fastq.gz	Fastqsanger.gz
	SRR6918158_1.fastq.gz	Fastqsanger.gz

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Bisulfite-free direct detection of 5-methylcytosine and 5-hydroxymethylcytosine at base resolution

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

Bisulfite-free, nano-scale analysis of 5-hydroxymethylcytosine at single base resolution

Project description:High-resolution detection of genome-wide 5-hydroxymethylcytosine (5hmC) sites of small-scale samples represents a continuous challenge. Here, we present CATCH-seq, a bisulfite-free, base-resolution method for the genome-wide detection of 5hmC. CATCH-seq is based on selective 5hmC oxidation, labeling and subsequent C-to-T transition during PCR. Applications of CATCH-seq to nano-scale DNA samples reveal previously underappreciated non-CG 5hmCs in the hESC genome and base-resolution hydroxymethylome in human cell-free DNA.

2018-10-09 | GSE112048 | GEO

Subtraction-free and bisulfite-free specific sequencing of 5-methylcytosine and its oxidized derivatives at base resolution

Project description:Although various methods have been developed for sequencing cytosine epigenetic modifications, specific and quantitative sequencing of the two major epigenetic modifications of cytosines, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) at base-resolution is still challenging. Most times it requires subtraction of two methods to obtain both the true 5mC and 5hmC information, which increases noise and requires high sequencing depth. Recently we developed TET assisted pyridine borane sequencing (TAPS) for bisulfite-free direct sequencing of DNA methylation, which provides the sum of 5mC and 5hmC. Here we extend it to two sister methods, TAPSβ and CAPS (Chemical-Assisted Pyridine borane Sequencing), for whole-genome subtraction-free and specific sequencing of 5mC and 5hmC, respectively. We also demonstrated Pyridine borane Sequencing (PS) of whole-genome 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC), the further oxidized derivatives of 5mC and 5hmC. This completes the versatile borane reduction chemistry-based methods as a comprehensive suite for direct and quantitative sequencing of all four individual cytosine epigenetic modifications.

2020-12-14 | GSE155613 | GEO

Mirror bisulfite sequencing – a method for single-base resolution of hydroxymethylcytosine

Project description:While the role of 5-methylcytosine has been well studied, the biological role of 5-hydroxymethylcytosine still remains unclear due to the limited methods available for single-base detection of 5-hydroxymethylcytosine (5hmC). Here we present Mirror bisulfite sequencing for the detection of 5hmC at a single CpG site by synthesizing a DNA strand to mirror the parental strand. This semi-conservative duplex is sequentially treated with β-glucosyltransferase and M.SssI methylase. A glucosyl-5hmCpG in the parental strand inhibits methylation of the mirroring CpG site, and after bisulfite conversion, a thymine in the mirroring strand indicates a 5hmCpG site in the parental strand whereas a cytosine indicates a non-5hmC site. Using this method, the 5hmC levels of various human tissues and paired tumor liver tissues were mapped genome-wide.

2018-05-22 | GSE69272 | GEO

Bisulfite-free and Base-resolution Analysis of 5-formylcytosine at Whole-genome Scale

Project description:Active DNA demethylation in mammals involves TET-mediated oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxycytosine (5caC). However, genome-wide detection of 5fC at single-base resolution remains challenging. Here we present a bisulfite-free method for the whole-genome analysis of 5fC, based on a selective chemical labeling of 5fC and subsequent C-to-T transition during PCR. Base-resolution 5fC maps reveal limited overlap with 5hmC, with 5fC-marked regions more active than 5hmC-marked ones. Utilization of cyclization-enabled C-to-T transition of 5fC (hence “fC-CET”) to obtain genome-wide map of 5fC at single-base resolution WT and TdgKO mES cell lines. Two non-enriched input DNAs (Input: preAI), two AI labeled samples (Input: AI), two pull-down output samples.

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Bisulfite-Free Whole-Genome Mapping of 5-Methylcytosine at Single-Base Resolution by TAD-seq

Project description:5-Methylcytosine (5mC) is a crucial epigenetic modification plays a significant role in the regulation of gene expression. Accurate and quantitative detection of 5mC at single-base resolution is essential for understanding its epigenetic functions within genomes. In this study, we develop a novel nTET-assisted deaminase sequencing (TAD-seq) method for the base-resolution and quantitative detection of 5mC in genomic DNA. The TAD-seq method utilizes a Naegleria TET-like dioxygenase (nTET) to oxidize 5mC, generating 5-methylcytosine oxidation products (5moC). We also engineered a variant of the human apolipoprotein B mRNA-editing catalytic polypeptide-like 3A (A3A), creating an A3A mutant (A3Am). Treatment with A3Am results in the conversion of cytosine to uracil, while 5moC remains unchanged. Consequently, TAD-seq enables the direct deamination of cytosine to uracil by A3Am, which is sequenced as thymine, whereas 5mC, once oxidized to 5moC by nTET, resists deamination and is sequenced as cytosine. Therefore, the cytosines that persist in the sequencing data represent the original 5mC sites. We applied TAD-seq to HEK293T cells, generating a base-resolution map of 5mC that exhibits strong concordance with maps generated by conventional BS-seq. TAD-seq emerges as a powerful, bisulfite-free approach for the single-base resolution mapping of 5mC stoichiometry in genomic DNA.

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Quantitative sequencing of 5-methylcytosine and 5-hydroxymethylcytosine at single base resolution

Project description:To investigate the genomic levels of 5-hydroxymethylcytosine at single-base resolution. The current study developed a method which allows one to study hydroxymethylation of cytosines in the genome via a subtractive method of RRBS and oxidative RRBS.

2012-04-24 | E-MTAB-1042 | biostudies-arrayexpress

Double and single stranded detection of 5-methylcytosine and 5-hydroxymethylcytosine with nanopore sequencing

Project description:5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are modified versions of cytosine in DNA with roles in regulating gene expression. Using whole genomic DNA from mouse cerebellum, we have benchmarked 5mC and 5hmC detection by Oxford Nanopore Technologies sequencing against other standard techniques. In addition, we assessed the ability of duplex base-calling to study strand asymmetric modification. Nanopore detection of 5mC and 5hmC is accurate relative to compared techniques and opens new means of studying these modifications. Strand asymmetric modification is widespread across the genome but reduced at imprinting control regions and CTCF binding sites in mouse cerebellum. This study demonstrates the unique ability of nanopore sequencing to improve the resolution and detail of cytosine modification mapping.

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Bisulfite-free, nano-scale analysis of 5-hydroxymethylcytosine at single base resolution

Project description:Bisulfite-free, nano-scale analysis of 5-hydroxymethylcytosine at single base resolution

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