Ontology highlight
ABSTRACT:
PROVIDER: PRJNA980280 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR24829800_1.fastq.gz | Fastqsanger.gz | |||
| SRR24829800_2.fastq.gz | Fastqsanger.gz | |||
| SRR24829801_1.fastq.gz | Fastqsanger.gz | |||
| SRR24829801_2.fastq.gz | Fastqsanger.gz | |||
| SRR24829802_1.fastq.gz | Fastqsanger.gz |
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Project description:Whole-genome bisulfite sequencing (BS-Seq) measures cytosine methylation changes at single-base resolution and can be used to profile cell-free DNA (cfDNA). In plasma, ultrashort single-stranded cfDNA (uscfDNA, ~50 nt) has been identified together with 167 bp double-stranded mononucleosomal cell-free DNA (mncfDNA). However, the methylation profile of uscfDNA has not been described. Conventional BS-Seq workflows may not be helpful because bisulfite conversion degrades larger DNA into smaller fragments, leading to erroneous categorization as uscfDNA. We describe the “5mCAdpBS-Seq” workflow in which pre-methylated 5mC (5-methylcytosine) single-stranded adapters are ligated to heat-denatured cfDNA before bisulfite conversion. This method retains only DNA fragments that are unaltered by bisulfite treatment, resulting in less biased uscfDNA methylation analysis. Using 5mCAdpBS-Seq, uscfDNA had lower levels of DNA methylation (~15%) and was enriched in promoters and CpG islands. Hypomethylated uscfDNA fragments were enriched in upstream transcription start sites (TSSs), and the intensity of enrichment positively affected gene expression of hemopoietic cells. Using tissue-of-origin deconvolution, we inferred that uscfDNA is derived primarily from eosinophils, neutrophils, and monocytes. As proof-of-principle, we show that characteristics of the methylation profile of uscfDNA can distinguish non-small cell lung carcinoma from non-cancer samples. The 5mCAdpBS-Seq workflow is recommended for any cfDNA methylation-based investigations.
2024-03-28 | GSE252088 | GEO
Project description:NFATc2 is a sequence specific DNA binding protein in the Rel family that binds a 5-mer motif CGGAA better when both cytosines in the CG dinucleotide are methylated. Using protein binding microarrays (PBMs) we examined the DNA binding of NFATc2 to three additional types of DNA: (1) single-stranded DNA (ssDNA), (2) double stranded DNA (dsDNA) with 5-methylcytosine (5mC, M) in one strand and cytosine in the second strand (dsDNA(5mC|C)) and (3) DNA with 5-hydroxymethylcytosine (5hmC, H) in one strand and cytosine in the second strand (dsDNA(5hmC|C)). NFATc2 binding to ssDNA is strongest for the 7-mer ATTTCCA. While NFAT can bind ssDNA, the average binding to the 40,000 DNA features of the PBM was twice as high for ssDNA compared to dsDNA, with maximal binding observed to dsDNA, suggesting that NFAT binding to dsDNA is more specific. dsDNA sequences containing the 5-mer MGGAA with 5mC in one DNA strand are better bound than CGGAA, with methylation of a single cytosine being sufficient for the stronger binding seen when both cytosines in the CG dinucleotide are methylated. Several DNA sequences containing HGGAA are also better bound than those containing cytosine, however the effect of 5hmC is not as dramatic as that of 5mC. Analysis of available NFATc2:DNA complexes rationalizes our PBM data.
2018-12-31 | GSE104631 | GEO
Project description:Genome-wide maps and functional peak analysis of ultrashort single-stranded cell-free DNA (uscfDNA) and mononucleosomal cell-free DNA (mncfDNA) in human plasma
| PRJNA836112 | ENA
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:Whole-genome bisulfite sequencing (WGBS) is currently the gold standard for DNA methylation (5-methylcytosine, 5mC) profiling, however the destructive nature of sodium bisulfite results in DNA fragmentation and subsequent biases in sequencing data. Such issues have led to the development of bisulfite-free methods for 5mC detection. Nanopore sequencing is a long read non-destructive approach that directly analyzes DNA and RNA fragments in real time. Recently, computational tools have been developed that enable base-resolution detection of 5mC from Oxford Nanopore sequencing data. In this chapter we provide a detailed protocol for preparation, sequencing, read assembly and analysis of genome-wide 5mC using Nanopore sequencing technologies.
2021-12-03 | GSE179673 | GEO