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Base-resolution detection of N4-methylcytosine in genomic DNA using 4mC-TAB-seq

ABSTRACT: Base-resolution detection of N4-methylcytosine in genomic DNA using 4mC-TAB-seq

PROVIDER: PRJNA267586 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR1748403.fastq.gz	Fastqsanger.gz
	SRR1748404.fastq.gz	Fastqsanger.gz
	SRR1748405.fastq.gz	Fastqsanger.gz
	SRR1748406.fastq.gz	Fastqsanger.gz
	SRR1748407.fastq.gz	Fastqsanger.gz

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Base-resolution detection of N4-methylcytosine in genomic DNA using 4mC-TAB-seq

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. 4mC-TAB-seq

2015-07-11 | E-GEOD-63371 | biostudies-arrayexpress

Base-resolution detection of N4-methylcytosine in genomic DNA using 4mC-TAB-seq

2015-07-11 | GSE63371 | GEO

Genome-wide mapping of N4-methylcytosine at single-base resolution by APOBEC3A-mediated deamination sequencing

Project description:Genome-wide mapping of N4-methylcytosine at single-base resolution by APOBEC3A-mediated deamination sequencing

| PRJNA785432 | ENA

Genome-wide mapping of N4-methylcytosine at single-base resolution by APOBEC3A-mediated deamination sequencing

Project description:N 4-methylcytosine (4mC) is a natural DNA modification occurring in thermophiles and plays important roles in restriction-modification (R-M) systems in bacterial genomes. However, the precise location and sequence context of 4mC in the whole genome are limited. In this study, we developed an APOBEC3A-mediated deamination sequencing (4mC-AMD-seq) method for genome-wide mapping of 4mC at single-base resolution. In the 4mC-AMD-seq method, cytosine and 5-methylcytosine (5mC) are deaminated by APOBEC3A (A3A) protein to generate uracil and thymine, both of which are read as thymine in sequencing, while 4mC is resistant to deamination and therefore read as cytosine. Thus, the readouts of cytosines from sequencing could manifest the original 4mC sites in genomes. With the 4mC-AMD-seq method, we achieved the genome-wide mapping of 4mC in Deinococcus radiodurans (D. radiodurans). In addition, we confirmed that 4mC, but not 5mC, was the major modification in the D. radiodurans genome. We identified 1586 4mC sites in the genome of D. radiodurans, among which 564 sites were located in the CCGCGG motif. The average methylation levels in the CCGCGG motif and non-CCGCGG sequence were 70.0% and 22.8%, respectively. We envision that the 4mC-AMD-seq method will facilitate the investigation of 4mC functions, including the 4mC-involved R-M systems, in uncharacterized but potentially useful strains.

2021-12-15 | GSE189965 | GEO

Base-resolution detection of N4-methylcytosine in genomic DNA using 4mC-Tet-assisted-bisulfite- sequencing.

Project description:Restriction-modification (R-M) systems pose a major barrier to DNA transformation and genetic engineering of bacterial species. Systematic identification of DNA methylation in R-M systems, including N(6)-methyladenine (6mA), 5-methylcytosine (5mC) and N(4)-methylcytosine (4mC), will enable strategies to make these species genetically tractable. Although single-molecule, real time (SMRT) sequencing technology is capable of detecting 4mC directly for any bacterial species regardless of whether an assembled genome exists or not, it is not as scalable to profiling hundreds to thousands of samples compared with the commonly used next-generation sequencing technologies. Here, we present 4mC-Tet-assisted bisulfite-sequencing (4mC-TAB-seq), a next-generation sequencing method that rapidly and cost efficiently reveals the genome-wide locations of 4mC for bacterial species with an available assembled reference genome. In 4mC-TAB-seq, both cytosines and 5mCs are read out as thymines, whereas only 4mCs are read out as cytosines, revealing their specific positions throughout the genome. We applied 4mC-TAB-seq to study the methylation of a member of the hyperthermophilc genus, Caldicellulosiruptor, in which 4mC-related restriction is a major barrier to DNA transformation from other species. In combination with MethylC-seq, both 4mC- and 5mC-containing motifs are identified which can assist in rapid and efficient genetic engineering of these bacteria in the future.

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

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

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Direct enzymatic sequencing of 5-methylcytosine at single-base resolution

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Single-base resolution methylome of 5-methylcytosine in mammalian transcriptome

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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.

2024-06-30 | GSE254203 | GEO

Direct enzymatic sequencing of 5-methylcytosine at single-base resolution [1]

Project description:Direct enzymatic sequencing of 5-methylcytosine at single-base resolution [1]

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