Direct enzymatic sequencing of 5-methylcytosine at single-base resolution
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ABSTRACT:
ORGANISM(S): Cloning vector pUC19 Escherichia phage Lambda Homo sapiens Escherichia phage T4
PROVIDER: GSE225975 | GEO | 2023/04/24
REPOSITORIES: GEO
Similar Datasets
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
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 | GSE225967 | 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: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
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
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:Bisulfite sequencing detects 5mC and 5hmC at single-base resolution. However, bisulfite treatment damages DNA, which results in fragmentation, DNA loss, and biased sequencing data. To overcome these problems, enzymatic methyl-seq (EM-seq) was developed. This method detects 5mC and 5hmC using two sets of enzymatic reactions. In the first reaction, TET2 and T4-BGT convert 5mC and 5hmC into products that cannot be deaminated by APOBEC3A. In the second reaction, APOBEC3A deaminates unmodified cytosines by converting them to uracils. Therefore, these three enzymes enable the identification of 5mC and 5hmC. EM-seq libraries were compared with bisulfite-converted DNA, and each library type was ligated to Illumina adaptors before conversion. Libraries were made using NA12878 genomic DNA, cell-free DNA, and FFPE DNA over a range of DNA inputs. The 5mC and 5hmC detected in EM-seq libraries were similar to those of bisulfite libraries. However, libraries made using EM-seq outperformed bisulfite-converted libraries in all specific measures examined (coverage, duplication, sensitivity, etc.). EM-seq libraries displayed even GC distribution, better correlations across DNA inputs, increased numbers of CpGs within genomic features, and accuracy of cytosine methylation calls. EM-seq was effective using as little as 100 pg of DNA, and these libraries maintained the described advantages over bisulfite sequencing. EM-seq library construction, using challenging samples and lower DNA inputs, opens new avenues for research and clinical applications.
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Project description:mRNAs of siCTRL, siNSUN2 and ALYREF-RIP HeLa cells, and multiple mouse tissues were purified using oligo (dT)-conjugated magnetic beads followed by RiboMinus treatement. mRNAs were fragmented to ~200 nt and treated by Sodium bisulfite solution (pH 5.1) containing Hydroquinone. Bisulfite treated mRNAs were reverse transcribed to cDNA using ACT random hexamer primers. The cDNAs were subjected to libraries construction using KAPA Stranded mRNA-Seq Kit (KAPA) and performed sequencing on HiSeq2500 (Illumina) in pair-end mode, creating reads with a length of 125 bp. Sequencing chemistry v4 (Illumina) was used and every sample was sequenced at one lane.
2017-04-19 | GSE93749 | GEO
Project description:The underlying biological mechanisms through which epidemiologically defined breast cancer risk factors contribute to disease risk remain poorly understood. Here we investigated the impact cancer risk factors have on the normal breast epigenome by analyzing DNA methylation genome-wide (Infinium 450K array) in cancer-free women. We tested the relation of established breast cancer risk factors with DNA methylation adjusting for potential variation in cell-type proportions.
2017-06-29 | GSE74214 | GEO