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.
Project description:We used the nanopore Cas9 targeted sequencing (nCATS) strategy to specifically sequence 125 L1HS-containing loci in parallel and measure their DNA methylation levels using nanopore long-read sequencing. Each targeted locus is sequenced at high coverage (~45X) with unambiguously mapped reads spanning the entire L1 element, as well as its flanking sequences over several kilobases. The genome-wide profile of L1 methylation was also assessed by bs-ATLAS-seq in the same cell lines (E-MTAB-10895).
Project description:Probing epigenetic features on long molecules of DNA has tremendous potential to advance our understanding of the phased epigenome. In this study, we evaluate CpG methylation and chromatin accessibility simultaneously on long strands of DNA using GpC methyltransferase to exogenously label open chromatin, coupled with nanopore sequencing technology. We performed nanopore sequencing of Nucleosome Occupancy and Methylome (nanoNOMe) on four human cell lines (GM12878, MCF-10A, MCF-7, MDA-MB-231), and demonstrate the ability to directly measure methylation and chromatin accessibility in genomic features such as structural variations and repetitive elements. The long single-molecule resolution allows footprinting of protein and nucleosome binding and determining the combinatorial promoter epigenetic state on individual molecules. Long-read sequencing makes it possible to robustly assign reads to haplotypes, enabling allele-specific epigenetic analysis across the genome. We use existing SNV data on GM12878 to present the first fully phased human Probing epigenetic features on long molecules of DNA has tremendous potential to advance our understanding of the phased epigenome. We evaluate CpG methylation and chromatin accessibility simultaneously on long strands of DNA using GpC methyltransferase to exogenously label open chromatin, coupled with nanopore sequencing technology. We performed nanopore sequencing of Nucleosome Occupancy and Methylome (nanoNOMe) on four human cell lines (GM12878, MCF-10A, MCF-7, MDA-MB-231), and demonstrate the ability to directly measure methylation and chromatin accessibility in genomic features such as structural variations and repetitive elements. The long single-molecule resolution allows footprinting of protein and nucleosome binding and determining the combinatorial promoter epigenetic state on individual molecules. Long-read sequencing makes it possible to robustly assign reads to haplotypes, enabling allele-specific epigenetic analysis across the genome. We use existing SNV data on GM12878 to present the first fully phased human epigenome, consisting of chromosome-level allele-specific profiles of CpG methylation and chromatin accessibility.mosome-level allele-specific profiles of CpG methylation and chromatin accessibility.
Project description:State-of-the-art algorithms for m6A detection and quantification via nanopore direct RNA sequencing have been continuously developed, little is known about their capacities and limitations, which makes a comprehensive assessment in urgent need. Therefore, we performed comprehensive benchmarking of 10 computational tools relying on current-based and base-calling “errors” strategies for m6A detection by nanopore sequencing.
Project description:This SuperSeries is composed of the following subset Series: GSE15746: Methylation detection Oligonucleotide Microarray Analysis: high resolution method for CpG island methylation detection 1 GSE15747: Methylation detection Oligonucleotide Microarray Analysis: high resolution method for CpG island methylation detection 2 Refer to individual Series
Project description:Nanopore Sequencing and assembly of Col-0 carrying seed coat expressed GFP and RFP transgenes flanking the centromere of chromosome 3 (CTL 3.9) - additionally, DNA methylation was derived using deepsignal-plant using these reads.
Project description:We present scNanoATAC-seq (Single-cell Assay for Transposase Accessible Chromatin by Oxford Nanopore Technologies Sequencing), an effective method for simultaneous detection of chromatin accessibility and genetic variation. Long fragments (about 4-5Kb) of single-cell ATAC-seq library were enriched and sequenced by Oxford Nanopore Technologies platform. Ends of long ATAC-seq fragments are regarded as chromatin accessibility signal in downstream analysis.
Project description:We present scNanoATAC-seq (Single-cell Assay for Transposase Accessible Chromatin by Oxford Nanopore Technologies Sequencing), an effective method for simultaneous detection of chromatin accessibility and genetic variation. Long fragments (about 4-5Kb) of single-cell ATAC-seq library were enriched and sequenced by Oxford Nanopore Technologies platform. Ends of long ATAC-seq fragments are regarded as chromatin accessibility signal in downstream analysis.
Project description:Aberrant DNA-methylation at CpG dinucleotides is a hallmark of cancer and is associated with the emergence of resistance to anti-cancer treatment, though molecular mechanisms and biological signifi- cance remain elusive. Genome-scale methylation maps by currently used methods are based on chemical modification of DNA and are best suited for analyses of methylation at CpG-rich regions (CpG islands). We report the first high-coverage whole-genome map in cancer using the long-read nanopore technol- ogy, which allows simultaneous DNA-sequence and -methylation analyses on native DNA. We analyzed clonal epigenomic/genomic evolution in Acute Myeloid Leukemias (AMLs) at diagnosis and relapse, af- ter chemotherapy. Long-read sequencing coupled to a novel computational method allowed definition of differential methylation at unprecedented resolution (> 99% CpGs), extending analyses of CpG is- lands to sparse CpGs, which represent half of all differentially-methylated regions. We showed that the
Project description:Aberrant DNA-methylation at CpG dinucleotides is a hallmark of cancer and is associated with the emergence of resistance to anti-cancer treatment, though molecular mechanisms and biological signifi- cance remain elusive. Genome-scale methylation maps by currently used methods are based on chemical modification of DNA and are best suited for analyses of methylation at CpG-rich regions (CpG islands). We report the first high-coverage whole-genome map in cancer using the long-read nanopore technol- ogy, which allows simultaneous DNA-sequence and -methylation analyses on native DNA. We analyzed clonal epigenomic/genomic evolution in Acute Myeloid Leukemias (AMLs) at diagnosis and relapse, af- ter chemotherapy. Long-read sequencing coupled to a novel computational method allowed definition of differential methylation at unprecedented resolution (> 99% CpGs), extending analyses of CpG is- lands to sparse CpGs, which represent half of all differentially-methylated regions. We showed that the