Unknown,Transcriptomics,Genomics,Proteomics

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Whole genome TraDIS-Xpress assay to determine gene roles during growth of Escherichia coli in competition with Pseudomonas aeruginosa, in the presence or absence of glucose

ABSTRACT: The aim of the experiment was to assay every gene in the Escherichia coli genome to identify those that contribute to competition with Pseudomonas aeruginosa. A library of transposon-insertion mutants was grown overnight either alone or together with an equal starting number of P. aeruginosa cells. The experiment was also conducted in the presence or absence of glucose to interrogate the effect of a plentiful supply of carbon source. DNA sequencing was then used to reveal the locations of the transposons in every mutant. By comparing the numbers of each mutant between conditions, information can be gained about the relative fitness of that mutant under the conditions tested.

INSTRUMENT(S): NextSeq 500

ORGANISM(S): Escherichia coli K-12

SUBMITTER: Nicholas Thomson

PROVIDER: E-MTAB-13391 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Somatic transposon mutagenesis in mice is an efficient strategy to investigate the genetic mechanisms of tumorigenesis. The identification of tumor driving transposon insertions traditionally requires the generation of large tumor cohorts to obtain information about common insertion sites. Tumor driving insertions are also characterized by their clonal expansion in tumor tissue, a phenomenon that is facilitated by the slow and evolving transformation process of transposon mutagenesis. We describe here an improved approach for the detection of tumor driving insertions that assesses the clonal expansion of insertions by quantifying the relative proportion of sequence reads obtained in individual tumors. To this end, we have developed a protocol for insertion site sequencing that utilizes acoustic shearing of tumor DNA and Illumina sequencing. We analyzed various solid tumors generated by PiggyBac mutagenesis and for each tumor >10^6 reads corresponding to >10^4 insertion sites were obtained. In each tumor, 9 to 25 insertions stood out by their enriched sequence read frequencies when compared to frequencies obtained from tail DNA controls. These enriched insertions are potential clonally expanded tumor driving insertions, and thus identify candidate cancer genes. The candidate cancer genes of our study comprised many established cancer genes, but also novel candidate genes such as Mastermind-like1 (Mamld1) and Diacylglycerolkinase delta (Dgkd). We show that clonal expansion analysis by high-throughput sequencing is a robust approach for the identification of candidate cancer genes in insertional mutagenesis screens on the level of individual tumors. Solid tumors in mice were generated by somatic transposon mutagenesis with a PiggyBac transposon system. Insertion sites of transposons in 11 tumors and 6 non-cancerous tail controls were determined by Illumina high-throughput sequencing. Insertions were determined both on 5' and 3' sides of the transposon (PB5 and PB3, respectively). Quantitative analysis of read numbers revealed enrichment of certain insertions in tumors, but not in controls, and these enriched insertions identify candidate cancer genes.

Dataset Information

Whole genome TraDIS-Xpress assay to determine gene roles during growth of Escherichia coli in competition with Pseudomonas aeruginosa, in the presence or absence of glucose

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