Project description:S. meliloti strains with a bi- and monopartite genome configuration were constructed by consecutive Cre/lox-mediated site-specific fusions of the secondary replicons. Beside the correct genomic arrangements, these strains and precursors were tested for variations in the nucleotide sequence. Futher, a marker fequency analysis was performed to test if replication is initiated at all origins and to determine the replication termination regions of the triple replicon fusion molecule. To gain the sequence data for these analyses, respective strains were applied to whole genome sequencing using an Illumina MiSeq-System and Oxford Nanopore (MinION) sequencing technology.
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: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:We performed genomic sequencing of whole-genome amplified DNA and native DNA isolated during growth in one of five conditions. We sequenced the DNA using Oxford Nanopore and compared the signals from the whole genome amplified DNA to the native DNA to infer sites at which the native DNA was methylated. The file names here are denoted via the strain name (SC419, SC452, or SC469), the growth condition (37C M9, 42C M9, 25C M9, rich media LB, 96 hours of growth), and in two cases, the replicate culture (M9_rep1 and M9_rep2)
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:BmN4 cells are cultured cells derived from Bombyx mori ovaries and widely used to study transposon silencing by PIWI-interacting RNAs (piRNAs). A high-accurate genome sequence of BmN4 cells is required to analyze the piRNA pathway using RNA-seq. The genome sequence of BmN4 cells was assembled using Pacific Biosciences (PacBio) HiFi and Oxford Nanopore technology Ultralong (ONT-UL) reads. Microscopic observation and image analysis showed that BmN4 cells were octoploid on average, and the number of chromosomes per cell was highly variable. We concluded the haplotype-resolved assembly of such a complex genome would be difficult; therefore, we assembled a consensus genome sequence. RNA-seq analysis of Siwi knockdown cells also revealed that Siwi-piRISC may target Countdown (Cd), an LTR retrotransposon. By comparing the consensus genome sequence with the reads, we identified differences between haplotypes, particulary structural variants, suggesting that some transposons, including Countdown, increased their copy number in BmN4 cells.
Project description:Higher-order chromatin structure arises from the combinatorial physical interactions of many genomic loci. To investigate this aspect of genome architecture we developed Pore-C, which couples chromatin conformation capture with Oxford Nanopore Technologies (ONT) long reads to directly sequence multi-way chromatin contacts without amplification.
Project description:This is the whole genome and epigenome nasopharyngeal cancer sequencing from clinical nasopharyngeal biopsy. The methylation information is derived from nanopore sequencing in its native form. The 5mC information per position is extracted and presented here as a bedgraph file.
Project description:Recent completion of the telomere-to-telomere (T2T) genome assembly has enabled a comprehensive characterization of pericentromeric SatⅠ, SatII, SatⅢ and centromeric α-satellite repeats. SatⅢ DNA constitutes ~1.56% of the genome with a reported localization across 16 chromosomes. The transcription activity of SatⅢ DNA across genome and the sequence of SatⅢ transcripts remained largely unclear. We performed nanopore long-read RNA sequencing (RNA-seq) in untreated (UN), sodium arsenite (SA: 0.1mM, 5 h) and heat shock (HS: 42°C, 2 h; 37°C, 1 h) stressed HeLa cells to characterize SatⅢ transcripts . Since a portion of SatⅢ transcripts is non-polyadenylated, We performed polyadenylated (poly(A)+) and rRNA-depleted (ribo-) nanopore cDNA long-read RNA-seq.
Project description:5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are modified versions of cytosine in DNA with roles in regulating gene expression. Using whole genomic DNA from mouse cerebellum, we have benchmarked 5mC and 5hmC detection by Oxford Nanopore Technologies sequencing against other standard techniques. In addition, we assessed the ability of duplex base-calling to study strand asymmetric modification. Nanopore detection of 5mC and 5hmC is accurate relative to compared techniques and opens new means of studying these modifications. Strand asymmetric modification is widespread across the genome but reduced at imprinting control regions and CTCF binding sites in mouse cerebellum. This study demonstrates the unique ability of nanopore sequencing to improve the resolution and detail of cytosine modification mapping.