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 identified hankyphage prophages within B. thetaiotaomicron isolates gathered from French hospitals. We extracted genomic DNA from an overnight culture from a single colony of each strain and sequenced them using Nanopore sequencing using the Plasmidsaurus platform. This long-read approach helped the assembly of the phages and determination of the hankyphage ends. We also improved the annotation of the reference hankyphage (hankyphage p00 from P. dorei HM719) using a structural prediction approach and annotated our B. thetaiotaomicron hankyphages using this new annotation. In this project we upload the genomic raw reads of nanopore sequencing of our hankyphage-bearing B. thetaiotaomicron collection (jmh strains) and the processed assembled hankyphages.
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:Primary objectives: The primary objective is to investigate circulating tumor DNA (ctDNA) via deep sequencing for mutation detection and by whole genome sequencing for copy number analyses before start (baseline) with regorafenib and at defined time points during administration of regorafenib for treatment efficacy in colorectal cancer patients in terms of overall survival (OS).
Primary endpoints: circulating tumor DNA (ctDNA) via deep sequencing for mutation detection and by whole genome sequencing for copy number analyses before start (baseline) with regorafenib and at defined time points during administration of regorafenib for treatment efficacy in colorectal cancer patients in terms of overall survival (OS).
Project description:We identified exosomal miRNA biomarkers for pancreatic cancer diagnostics by isolating exosomes using a recently developed magnetic nanopore isolation technology and small RNA sequencing.
2019-02-11 | GSE111750 | GEO
Project description:Real time, field-deployable whole genome sequencing of malaria parasites using nanopore technology
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:Antimicrobial resistance (AMR) arises from complex genetic and regulatory changes, including single mutations, gene acquisitions or cumulative effects. Advancements in genomics and proteomics facilitate more comprehensive understanding of the mechanisms behind antimicrobial resistance. In this study, 74 clinically obtained Klebsiella pneumoniae isolates with increased meropenem and/or imipenem MICs were characterized by broth microdilution and PCR to check for the presence of carbapenemase genes. Subsequently, a representative subset of 15 isolates was selected for whole genome sequencing (WGS) by Illumina and Nanopore sequencing, and proteomic analysis by liquid chromatography-mass spectrometry (LC-MS/MS) to investigate the mechanisms underlying the differences in carbapenem susceptibility of Klebsiella pneumoniae isolates. Identical techniques were applied to characterize 4 mutants obtained after sequential meropenem exposure. We demonstrated that in clinically obtained isolates, increased copy numbers of blaOXA-48 containing plasmids, combined with OmpK36 loss, contributed to high carbapenem MICs without involvement of OmpK35 or other porins or efflux systems. In the meropenem exposed mutants, increased copy numbers of blaCTX-M-15 or blaOXA-48 containing plasmids, combined with OmpK36 loss was demonstrated. The OmpK36 loss resulted from the insertion of IS1 transposable elements or partial deletion of the ompK36 gene. Additionally, we identified two mutations, C59A and C58A, in the DNA coding the copA antisense RNA of IncFII plasmids and multiple mutations of an IncR plasmid, associated with increased plasmid copy numbers. This study demonstrates that by combining WGS and LC-MS/MS, the effect of genomic changes on protein expression related to antibiotic resistance and the mechanisms behind antibiotic resistance can be elucidated.
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)