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:Replicon-seq is a method to study the progression of sister replisomes during DNA replication. This method relies excision of the full-length of replicons by the fusion of MNase to MCM4 and sequencing via Nanopore technology.
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:Human cancer cell lines were pulsed with thymidine analogues EdU and BrdU, sequenced on the Oxford Nanopore platform, and analysed with our DNAscent software to measure DNA replication stress.
Project description:Over the last 30 years the soil bacterium Agrobacterium tumefaciens has been the workhorse tool for plant genome engineering. Replacement of native tumor-inducing (Ti) plasmid elements with customizable cassettes enabled insertion of a sequence of interest as “Transfer DNA” (T-DNA) into the plant genome of interest. Although these T-DNA transfer mechanisms are well understood, detailed understanding of the structure and epigenomic status of insertion events was limited by current technologies. To fill this gap, we analyzed transgenic Arabidopsis thaliana lines from three widely used collections (SALK, SAIL and WISC) with two single molecule technologies, optical genome mapping and nanopore sequencing. Optical maps for four randomly selected T-DNA lines revealed between one and seven insertions/rearrangements with unexpectedly large sizes ranging from 27 to 236 kilobases. De novo nanopore-based genome assemblies for two heterozygous lines resolved T-DNA structures up to 36 kb and revealed large-scale T-DNA associated translocations and exchange of chromosome arm ends. The multiple internally rearranged nature of T-DNA arrays, consisting of identical T-DNA/backbone concatemers made full assembly even for long nanopore reads impossible. For the current TAIR10 reference genome, nanopore contigs corrected 83% of non-centromeric misassemblies. This unprecedented nucleotide-level definition of T-DNA insertions enabled the mapping of epigenome data. The SALK_059379 T-DNA insertions were enriched for 24nt siRNAs and contained dense cytosine DNA methylation. Transgene silencing via the RNA directed DNA methylation pathway was confirmed by in planta assays. In contrast, SAIL_232 T-DNA sequence was predominantly targeted by 21/22nt siRNAs, and DNA methylation and silencing was limited to the GUS gene, but not the resistance gene. With the emergence of genome editing technologies that rely on Agrobacterium for gene delivery, this study provides new insights into the structural impact of engineering plant genomes and demonstrates the utility of state-of-the-art long-range sequencing technologies to rapidly identify unanticipated genomic changes.
Project description:Transposon insertion site sequencing (TIS) is a powerful method for associating genotype to phenotype. However, all TIS methods described to date use short nucleotide sequence reads which cannot uniquely determine the locations of transposon insertions within repeating genomic sequences where the repeat units are longer than the sequence read length. To overcome this limitation, we have developed a TIS method using Oxford Nanopore sequencing technology that generates and uses long nucleotide sequence reads; we have called this method LoRTIS (Long Read Transposon Insertion-site Sequencing). This experiment data contains sequence files generated using Nanopore and Illumina platforms. Biotin1308.fastq.gz and Biotin2508.fastq.gz are fastq files generated from nanopore technology. Rep1-Tn.fastq.gz and Rep1-Tn.fastq.gz are fastq files generated using Illumina platform. In this study, we have compared the efficiency of two methods in identification of transposon insertion sites.
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.