Ontology highlight
ABSTRACT:
PROVIDER: PRJNA1144670 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| Other | ||||
| SRR30150148_1.fastq.gz | Fastqsanger.gz | |||
| SRR30150152_1.fastq.gz | Fastqsanger.gz | |||
| SRR30150153_1.fastq.gz | Fastqsanger.gz | |||
| SRR30150154_1.fastq.gz | Fastqsanger.gz |
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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 benchmark 5mC and 5hmC detection by Oxford Nanopore Technologies sequencing against other standard techniques. In addition, we assess 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. Here we demonstrate the unique ability of nanopore sequencing to improve the resolution and detail of cytosine modification mapping.
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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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Project description:Cytosine base modifications 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5fC) are present in mammalian DNA. Here, reduced bisulfite sequencing is developed for quantitatively sequencing 5fC at single-base resolution. This method is then applied with oxidative bisulfite sequencing to gain a map of 5mC, 5hmC and 5fC in mouse embryonic stem cells. 12 samples, reduced representation bisulphite treatment: 4 replicates each for bisulphite (BS), oxidative BS (oxBS) and reduced BS (redBS) for the detection of 5mC, 5hmC and 5fC. Mouse (strain B6C) embryonic stem cells.
2014-04-08 | E-GEOD-56572 | biostudies-arrayexpress
Project description:Cytosine deaminases have important uses in the detection of epigenetic modifications and in genome editing. However, the range of applications of deaminases is limited by their substrate preference. To expand the toolkit of deaminases, we developed an in-vitro approach that bypasses a major hurdle with their severe toxicity in expression hosts. We screened 175 putative cytosine deaminases, primarily from bacteria, and found enzymes with strong activity on double- and single-stranded DNA in various sequence contexts, including some without any sequence constraints. We also found enzymes that do not deaminate modified cytosines. The remarkable diversity of cytosine deaminases opens new avenues for biotechnological and medical applications. As a demonstration, we developed a single-enzyme methylation sequencing (SEM-seq) method for 5-methylcytosine detection using a novel non-specific, modification-sensitive double-stranded DNA deaminase, MsddA. SEM-seq generated accurate base-resolution maps of 5-methylcytosine in human genome samples including cell free DNA and samples of 10 pg DNA, equivalent to single cell input. This simple and efficient protocol has the potential to allow high-throughput epigenome profiling of scarce biological material.
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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.
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