Non-destructive enzymatic deamination enables single molecule long read sequencing for the determination of 5-methylcytosine and 5- hydroxymethylcytosine at single base resolution
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA595150 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR10676800_1.fastq.gz | Fastqsanger.gz | |||
| SRR10676800_2.fastq.gz | Fastqsanger.gz | |||
| SRR10676801_1.fastq.gz | Fastqsanger.gz | |||
| SRR10676801_2.fastq.gz | Fastqsanger.gz | |||
| SRR10676802_1.fastq.gz | Fastqsanger.gz |
Items per page: 5 1 - 5 of 78 |
Similar Datasets
Project description:Bisulfite treatment damages the DNA leading to fragmentation and loss of long-range methylation information. To overcome this limitation of bisulfite treated DNA we applied a new enzymatic deamination approach, termed EM-seq (Enzymatic Methyl-seq) to long-range sequencing technologies. Our methodology, named LR-EM-seq (Long Range Enzymatic Methyl-seq) preserves the integrity of DNA allowing long-range methylation profiling of 5-mC and 5-hmC) over several kilobases of genomic DNA. Applied to known differentially methylated regions (DMR), LR-EM-seq achieves phasing of over 5 kb resulting in much broader and better defined DMRs than previously reported.
2020-09-01 | GSE141908 | GEO
Project description:5-Hydroxymethylcytosine (5hmC) is an important epigenetic mark that can regulate gene expression. While some methods were developed to detect 5hmC, direct genome-wide mapping of 5hmC at base resolution are still highly desirable. Herein, we proposed a single-step deamination sequencing (SSD-seq) method for the genome-wide mapping of 5hmC at single-base resolution. This method capitalizes on a screened engineered human apolipoprotein B mRNA-editing catalytic polypeptide-like 3A (A3A) protein to produce differential deamination activity toward cytosine (C), 5-methylcytosine (5mC), and 5hmC. In SSD-seq, an engineered A3A protein (eA3A-v10) can adequately deaminate C and 5mC, but not 5hmC. The original C and 5mC in DNA are deaminated by eA3A-v10 to form uracil (U) and thymine (T), both of which are read as T during sequencing. However, 5hmC is resistant to the deamination by eA3A-v10 and is still read as C during sequencing. Therefore, the remaining C in the sequence reads manifests the original 5hmC. Applying SSD-seq to generate a base-resolution map of 5hmC in human lung tissue, we found that 5hmC was almost entirely confined to CpG dinucleotides. The base-resolution map of 5hmC from human lung tissue generated by SSD-seq correlated strongly with that generated by prior ACE-seq. Taken together, the SSD-seq method is single-step, bisulfite-free and does not require DNA glycosylation or chemical treatment, which offers a valuable tool for the direct and quantitative detection of 5hmC in genomes at single-base resolution.
2023-12-04 | GSE236353 | GEO
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
Project description:The predominant methodology for DNA methylation analysis relies on the chemical deamination by sodium bisulfite of unmodified cytosine to uracil to permit the differential readout of methylated cytosines. Bisulfite treatment damages the DNA, leading to fragmentation and loss of long-range methylation information. To overcome this limitation of bisulfite-treated DNA, we applied a new enzymatic deamination approach, termed enzymatic methyl-seq (EM-seq), to long-range sequencing technologies. Our methodology, named long-read enzymatic modification sequencing (LR-EM-seq), preserves the integrity of DNA, allowing long-range methylation profiling of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) over multikilobase length of genomic DNA. When applied to known differentially methylated regions (DMRs), LR-EM-seq achieves phasing of >5 kb, resulting in broader and better defined DMRs compared with that previously reported. This result showed the importance of phasing methylation for biologically relevant questions and the applicability of LR-EM-seq for long-range epigenetic analysis at single-molecule and single-nucleotide resolution.
| S-EPMC7849414 | biostudies-literature
Project description:Transition of cytosine to thymine in CpG dinucleotides is the most frequent type of mutation in cancer. This increased mutability is commonly attributed to the spontaneous deamination of 5-methylcytosine (5mC), which is normally repaired by the base-excision repair (BER) pathway. However, the contribution of 5mC deamination in the increasing diversity of cancer mutational signatures remains poorly explored. Here, we integrate mutational signatures analysis in a large series of tumor whole genomes with lineage-specific epigenomic data to draw a detailed view on 5mC deamination in cancer. We uncover tumor type-specific patterns of 5mC deamination signatures in CpG and non-CpG contexts. We demonstrate that the BER glycosylase MBD4 preferentially binds to active chromatin and early replicating DNA, which correlates with lower mutational burden in these domains. We validate our findings by modeling BER deficiencies in isogenic cell models. Overall, we establish MBD4 as the main actor responsible for 5mC deamination repair in humans.
2024-09-12 | GSE275181 | GEO
Project description:Genome-wide mapping of N4-methylcytosine at single-base resolution by APOBEC3A-mediated deamination sequencing
| PRJNA785432 | ENA
Project description:5-Hydroxymethylcytosine (5hmC) is a crucial epigenetic mark that plays a key role in regulating various physiological and pathological processes. Although several methods have been developed to detect 5hmC, direct genome-wide mapping at single-base resolution remains highly desirable. Herein, we developed a double-stranded deamination sequencing (DDD-seq) method for genome-wide mapping of 5hmC at single-base resolution. This method utilizes the double-stranded DNA deaminase SsdAcat from Pseudomonas syringae, which efficiently deaminates cytosine (C), 5-methylcytosine (5mC), 5hmC, 5-formylcytosine (5fC), and 5-carboxycytosine (5caC) in duplex DNA, but not glycosylated 5hmC (5gmC). In DDD-seq, C, 5mC, 5fC, and 5caC in dsDNA are deaminated by SsdAcat and read as T during sequencing, while 5gmC is resistant to deamination, allowing its identification by detecting the remaining C in sequence reads. The map of 5hmC generated by DDD-seq in mouse cerebellum tissue closely aligns with results obtained from the ACE-seq method. Additionally, applying DDD-seq to cerebellum tissue of mouse subjected to chronic sleep deprivation revealed significant global changes in 5hmC distribution on genomic DNA. In contrast to previous single-stranded deaminase APOBEC3A-based mapping methods that require denaturation of dsDNA into ssDNA, DDD-seq eliminates this step, reducing risks associated with incomplete denaturation and simplifying sequencing library construction. Additionally, SsdAcat demonstrates superior thermostability and activity across a wide range of pH values and temperatures, making DDD-seq applicable in broader scenarios with more accessible conditions. Collectively, the DDD-seq method is straightforward, bisulfite-free, and eliminates the need for DNA denaturation step, making it a valuable tool for direct and quantitative detection of 5hmC in genomes at single-base resolution.
2025-07-24 | GSE275113 | GEO
Project description:5-methylcytosine (5mC) is the most important DNA modification in mammalian genomes as a lineage-defining mark dynamically altered in development and disease. The ideal method for 5mC localization would be both non-destructive of DNA and direct, without requiring inference based on detection of unmodified cytosines. Here, we present Direct Methylation Sequencing (DM-Seq), a bisulfite-free method for profiling 5mC at single-base resolution, using nanogram quantities of input DNA. DM-Seq employs two key DNA modifying enzymes: a neomorphic DNA methyltransferase engineered to generate the unnatural base 5-carboxymethylcytosine, and a DNA deaminase capable of precise discrimination between cytosine modification states. Coupling these activities requires a novel adapter strategy employing 5-propynylcytosine, ultimately resulting in the accurate and direct detection of only 5mC via a C-to-T transition in sequencing. In performing comparisons to DM-Seq, we uncover a systematic bias in 5mC detection seen with the hybrid enzymatic-chemical TAPS sequencing approach. Furthermore, by applying DM-Seq to a human glioblastoma tumor, we demonstrate that DM-Seq, unlike bisulfite-sequencing, detects 5mC at prognostically-important CpGs, without confounding by 5-hydroxymethylcytosine. DM-Seq thus leverages unnatural DNA modifications to create the first method for direct 5mC profiling entirely using enzymes rather than chemical reagents.
2023-04-24 | GSE225964 | GEO
Project description:5-methylcytosine (5mC) is the most important DNA modification in mammalian genomes as a lineage-defining mark dynamically altered in development and disease. The ideal method for 5mC localization would be both non-destructive of DNA and direct, without requiring inference based on detection of unmodified cytosines. Here, we present Direct Methylation Sequencing (DM-Seq), a bisulfite-free method for profiling 5mC at single-base resolution, using nanogram quantities of input DNA. DM-Seq employs two key DNA modifying enzymes: a neomorphic DNA methyltransferase engineered to generate the unnatural base 5-carboxymethylcytosine, and a DNA deaminase capable of precise discrimination between cytosine modification states. Coupling these activities requires a novel adapter strategy employing 5-propynylcytosine, ultimately resulting in the accurate and direct detection of only 5mC via a C-to-T transition in sequencing. In performing comparisons to DM-Seq, we uncover a systematic bias in 5mC detection seen with the hybrid enzymatic-chemical TAPS sequencing approach. Furthermore, by applying DM-Seq to a human glioblastoma tumor, we demonstrate that DM-Seq, unlike bisulfite-sequencing, detects 5mC at prognostically-important CpGs, without confounding by 5-hydroxymethylcytosine. DM-Seq thus leverages unnatural DNA modifications to create the first method for direct 5mC profiling entirely using enzymes rather than chemical reagents.
2023-04-24 | GSE225973 | GEO
Project description:5-methylcytosine (5mC) is the most important DNA modification in mammalian genomes as a lineage-defining mark dynamically altered in development and disease. The ideal method for 5mC localization would be both non-destructive of DNA and direct, without requiring inference based on detection of unmodified cytosines. Here, we present Direct Methylation Sequencing (DM-Seq), a bisulfite-free method for profiling 5mC at single-base resolution, using nanogram quantities of input DNA. DM-Seq employs two key DNA modifying enzymes: a neomorphic DNA methyltransferase engineered to generate the unnatural base 5-carboxymethylcytosine, and a DNA deaminase capable of precise discrimination between cytosine modification states. Coupling these activities requires a novel adapter strategy employing 5-propynylcytosine, ultimately resulting in the accurate and direct detection of only 5mC via a C-to-T transition in sequencing. In performing comparisons to DM-Seq, we uncover a systematic bias in 5mC detection seen with the hybrid enzymatic-chemical TAPS sequencing approach. Furthermore, by applying DM-Seq to a human glioblastoma tumor, we demonstrate that DM-Seq, unlike bisulfite-sequencing, detects 5mC at prognostically-important CpGs, without confounding by 5-hydroxymethylcytosine. DM-Seq thus leverages unnatural DNA modifications to create the first method for direct 5mC profiling entirely using enzymes rather than chemical reagents.
2023-04-24 | GSE225971 | GEO