Project description:To evaluate targeted MinION next generation sequencing as a diagnostic method for detection of pathogens in human blood and plasma, human blood or plasma samples were spiked with measured amounts of viruses, bacteria, protozoan parasites or tested pathogen-free as negative controls. Nucleic acid was extracted from samples and PCR amplification performed in multiplex primer pools with a procedure described in ArrayExpress experiment submission ID 18379. The PCR products were used for library preparation. The libraries sequenced on an Oxford Nanopore MinION. The passed reads aligned with a custom reference file to determine the identity of the pathogen in the sample.
Project description:The LRGASP challenge encompasses different human, mouse, and manatee samples sequenced using multiple combinations of protocols and platforms. Different challenges will use distinct subsets of the samples for evaluation. The long-read sequencing platforms used in these challenges are the Pacific Biosciences (PacBio) Sequel II, Oxford Nanopore (ONT) MinION and PromethION. Samples will also be sequenced on the Illumina HiSeq 2500. The primary LRGASP library prep protocols are “standard” cDNA sequencing, direct RNA sequencing, R2C2, and CapTrap. Each sample will also include Lexogen SIRV-Set 4 spike-ins. We will also provide simulated PacBio and ONT data as part of the evaluations. This particular study focuses on single strand CAGE sequencing of human iPSCs, defining CAGE peaks from Illumina HiSeq 2500 (SR: 150 cycles) of two biological replicates for use in the LRGASP challenge.
Project description:In this experiment we wanted to see how the binding behavior of the S. Cerevisiae transcription factor Leu3, on of the main regulators of leucine biosynthesis, is affected by different availability of the branched chain amino acids. For this we grow the cells in shake flask under glucose limitation and treated them 2 hours before sampling. The cells were then cross-linked with formaldehyde and ChIP-seq was performed using the Oxford Nanopore MinIon.
Project description:DNA microarrays and specific RT-PCR assays were used to reveal transcriptional patterns in the blood of children presenting with dengue shock syndrome (DSS) and well-matched patients with uncomplicated dengue. The transcriptome of patients with acute uncomplicated dengue was characterized by a metabolically demanding "host defense" profile; transcripts related to oxidative metabolism, interferon signaling, protein ubiquination, apoptosis and cytokines were prominent. In contrast, the transcriptome of DSS patients was surprisingly benign, particularly with regard to transcripts derived from apoptotic and type I interferon pathways. These data highlight significant heterogeneity in the type or timing of host transcriptional immune responses precipitated by DENV infection independent of the duration of illness. In particular, they suggest that if transcriptional events in the blood compartment contribute to capillary leakage leading to hypovolemic shock, they occur before cardiovascular decompensation occurs, a finding that has implications for rational adjuvant therapy in this syndrome. Whole blood transcriptional profiles of children infected with dengue virus with different clinical outcomes were compared. The subjects including 9 acute dengue shock samples, 9 acute uncomplicated dengue samples, 6 autologous follow up dengue samples and 6 autologous follow up uncomplicated dengue patients. Microarray data was normalised using Genespring GX7 software, statistical analysis was performed in Multiexperiment viewer software. Pathway analysis was performed using Ingenuity Pathway analysis online software.
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:DNA microarrays and specific RT-PCR assays were used to reveal transcriptional patterns in the blood of children presenting with dengue shock syndrome (DSS) and well-matched patients with uncomplicated dengue. The transcriptome of patients with acute uncomplicated dengue was characterized by a metabolically demanding "host defense" profile; transcripts related to oxidative metabolism, interferon signaling, protein ubiquination, apoptosis and cytokines were prominent. In contrast, the transcriptome of DSS patients was surprisingly benign, particularly with regard to transcripts derived from apoptotic and type I interferon pathways. These data highlight significant heterogeneity in the type or timing of host transcriptional immune responses precipitated by DENV infection independent of the duration of illness. In particular, they suggest that if transcriptional events in the blood compartment contribute to capillary leakage leading to hypovolemic shock, they occur before cardiovascular decompensation occurs, a finding that has implications for rational adjuvant therapy in this syndrome.
Project description:Whole blood from patients with acute dengue infection (as determined with PCR) were assessed for global transcriptional changes during different stages of the disease with reference to dengue virus IgG status at study inclusion
Project description:Whole blood from patients with acute dengue infection (as determined with PCR) were assessed for global transcriptional changes during different stages of the disease with reference to dengue virus IgG status at study inclusion Whole blood collected in PAX-gene tubes and extracted for total RNA