Ontology highlight
ABSTRACT:
PROVIDER: PRJNA1211591 | ENA |
REPOSITORIES: ENA
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Project description:Transposon-directed insertion-site sequencing (TraDIS) of a 1 million mutant library of Salmonella Typhi
| PRJEB2004 | ENA
Project description:Generation of a Tn5 transposon library in Haemophilus parasuis and analysis by Transposon-Directed Insertion-Site Sequencing (TraDIS)
| PRJEB4161 | ENA
Project description:A whole genome screen was used to assay every gene of Escherichia coli strain BW25113 to identify genes involved in susceptibility to the monobactam (beta-lactam) antibiotic aztreonam. The methodology has been called TraDIS-Xpress, and is a version of TraDIS or Tn-seq. A transposon mutant library consisting of several hundred thousand mutants was constructed using a Tn5-derived transposon incorporating an inducible outward transcribing promoter. All the mutants were grown in LB broth cultures supplemented with aztreonam at 2 x, 1 x, 0.5 x and 0.25 x MIC with induction of the transposon promoter using 0.2 mM IPTG or 1 mM IPTG or without induction. Following growth, mutants with increased susceptibility show reduced numbers and those with reduced susceptibility show increased numbers. Each condition was performed in duplicate. The methodology enable genes to be assayed by insertional inactivation or by changes in expression. Expression changes result from altered transcription from upstream transposon insertions transcribing into the gene, or downstream insertions transcribing into the gene in the reverse direction leading to RNA interference through the generation of reverse and complementary RNA. Thus, essential genes into which transposon insertions are not tolerated may be assayed also by changes in numbers of upstream or downstream insertion mutants. Changes to high throughput sequencing protocols permit the generation of nucleotide sequence reads from the known transposon sequences into the surrounding insertion site for all the mutants in the mixture simultaneously. Matching the sequence of the reads to the genome nucleotide sequence of E. coli BW25113 then allows the precise locations of all the transposon insertion sites of all the mutants to be mapped simultaneously. The relative changes in mutants between control (without) and selective condition (with aztreonam) then indicates which genes are involved in susceptibility. The numbers of sequence reads that match is reflected by the number of mutants, and so the degree of susceptibility can also be estimated.
2024-07-06 | E-MTAB-11576 | biostudies-arrayexpress
Project description:Whole genome sequence and transposon-directed insertion site sequencing (TraDIS) reads of a uropathogenic Escherichia coli strain
| PRJNA1162300 | ENA
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.
2022-05-30 | E-MTAB-11351 | biostudies-arrayexpress
Project description:Pasteurella multocida is a Gram-negative capsulated bacterium responsible for a range of diseases that cause severe morbidity and mortality in livestock animals. The hyaluronic acid (HA) capsule produced by P. multocida serogroup A strains is a critical virulence factor. In this study, we utilised transposon-directed insertion site sequencing (TraDIS) to identify genes essential for in vitro growth of P. multocida, and combined TraDIS with discontinuous density gradients (TraDISort) to identify genes required for HA capsule production and regulation in this pathogen. Analysis of mutants with a high cell density phenotype, indicative of the loss of extracellular capsule, led to the identification of 69 genes important for capsule production. These genes included all previously characterized genes in the capsule biosynthesis locus, and fis and hfq that encode known positive regulators of P. multocida capsule. Many of the other capsule-associated genes identified in this study were involved in regulation or activation of the stringent response, including spoT and relA that encode proteins that regulate the concentration of guanosine alarmones. Disruption of the autoregulatory domains in the C-terminal half of SpoT using insertional mutagenesis resulted in reduced expression of capsule biosynthesis genes and an acapsular phenotype. Overall, these findings have greatly increased the understanding of hyaluronic acid capsule production and regulation in P. multocida.
2022-03-14 | GSE198087 | GEO
Project description:Enterotoxigeneic Escherichia coli (ETEC) is a leading cause of diarrhoeal infections in young children living in endemic regions in low and middle-income countries and adults travelling to these destinations. CFA/I fimbriae have been identified as the predominant colonisation factor associated with human ETEC infections. Here we used used Transposon-directed insertion-site sequencing (TraDIS) and transcriptomic analysis to identify the essential genome of the prototypical CFA/I expressing ETEC strain H10407 and uncover the survival mechanisms that enhance persistence of ETEC isolates in water and within mammalian hosts. RNA transcription profiles of H10407 were identified under different in vitro growth conditions including aerobic growth in neutral LB media (pH7); aerobic growth in acidic media (pH5); aerobic growth in alkaline media (pH9); anaerobic growth in neutral LB media (pH7); and survival in fresh water. Research work including RNA preparation and bioinformatics and statistical analyses were conducted at the Wellcome Trust Sanger Institute (WTSI) and the Westmead Institute for Medical Research (WIMR), the University of Sydney. The sequencing data was generated in the Bioscience Core Laboratory at King Abdullah University of Science and Technology (KAUST).
2021-04-21 | E-MTAB-9364 | biostudies-arrayexpress