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Ultrafast Bisulfite Sequencing for Efficient and Accurate 5-Methylcytosine Detection in DNA and RNA

ABSTRACT: Ultrafast Bisulfite Sequencing for Efficient and Accurate 5-Methylcytosine Detection in DNA and RNA

PROVIDER: PRJNA936787 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR23538300_1.fastq.gz	Fastqsanger.gz
	SRR23538300_2.fastq.gz	Fastqsanger.gz
	SRR23538301_1.fastq.gz	Fastqsanger.gz
	SRR23538301_2.fastq.gz	Fastqsanger.gz
	SRR23538302_1.fastq.gz	Fastqsanger.gz

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Ultrafast Bisulfite Sequencing for Efficient and Accurate 5-Methylcytosine Detection in DNA and RNA

Project description:We present an ultrafast bisulfite sequencing (UBS-seq) method, which accelerates the bisulfite reaction by about 13-fold, resulting in minimal DNA damage and much lower background in all DNA sequences. This ultrafast BS protocol is also ideal for mapping of RNA 5-methylcytosine (m5C) because of much shorter reaction time and dramatically reduced RNA background. And allowed detection of m5C stoichiometry in highly structured RNA species.

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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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Project description:The Infinium 450K Methylation array is an established tool for measuring methylation. However, the bisulfite (BS) reaction commonly used with the 450K array cannot distinguish between 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). The oxidative-bisulfite assay disambiguates 5mC and 5hmC. We describe the use of oxBS in conjunction with the 450K array (oxBS-array) to analyse 5hmC/5mC in cerebellum DNA. The methylation level derived by the BS reaction is the combined level of 5mC and 5hmC at a given base, while the oxBS reaction gives the level of 5mC alone. The level of 5hmC is derived by subtracting the oxBS level from the BS level. Here we present an analysis method that distinguishes genuine positive levels of 5hmC at levels as low as 3 %. We preformed four replicates of the same sample of cerebellum and found a high level of reproducibility (average r for BS = 98.3, and average r for oxBS = 96.8). In total, 114,734 probes showed a significant positive measurement for 5hmC. The range at which we were able to distinguish 5hmC occupancy was between 3 % and 42 %. In order to investigate the effects of multiple replicates on 5hmC detection we also simulated fewer replicates and found that decreasing the number of replicates to two reduced the number of positive probes identified by > 50%. We validated our results using qPCR in conjunction with glucosylation of 5hmC sites followed by MspI digestion and we found good concordance with the array estimates (r = 0.94). This experiment provides a map of 5hmC in the cerebellum and a robust dataset for use as a standard in future 5hmC analyses. We also provide a novel method for validating the presence of 5hmC at low levels, and highlight some of the pitfalls associated with measuring 5hmC and 5mC.

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