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ABSTRACT:
PROVIDER: PRJEB28011 | ENA |
REPOSITORIES: ENA
Similar Datasets
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.
2013-08-28 | E-GEOD-46210 | biostudies-arrayexpress
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.
2013-08-28 | GSE46210 | GEO
Project description:This dataset was used to assess the random insertion by tranposases of lox sites in Mycoplasma pneumoniae. This is part of the protocol LoxTnSeq, a new methodology to generate and catalogue libraries of genome reduction mutants. LoxTnSeq combines random integration of Lox sites by transposon mutagenesis, and the generation of mutants via cre recombinase, catalogued via deep-sequencing. When LoxTnSeq was applied to the naturally genome reduced bacterium Mycoplasma pneumoniae, we obtained a mutant pool containing 285 unique deletions. These deletions spanned from >50 bp to 28 Kb, which represent 21% of the total genome. LoxTnSeq also highlighted large regions of non-essential genes that could be removed simultaneously, and other similar regions that could not, providing a guide for future genome reductions.
2021-08-18 | E-MTAB-9590 | biostudies-arrayexpress
Project description:In this work, a plasmid-based system is applied to inhibit the transposition of bacterial insertion sequences (IS). Using multiple guide RNAs, inactivated Cas9 was directed to simultaneously bind to the left end of IS1, IS5, IS3 and IS150 in Escherichia coli, in vivo. As a result, the transcription of IS1, and IS5 was successfully silenced, in certain cases by two orders of magnitude. The transposition rate of all four targeted elements nevertheless dropped to negligible levels, as verified at the cycA and bgl chromosomal loci. A GFP-expressing plasmid, known to be predominantly inactivated by insertion mutations also displayed a significant increase in stability. The transposition-silencing effect was easily transferable between various E. coli strains by plasmid transformation. Our portable system, or other plasmids constructed likewise can serve as useful tools to eliminate insertion mutagenesis or selectively study distinct transposable elements in numerous prokaryotic species.
2019-02-21 | GSE110946 | GEO
Project description:We present LoxTnSeq, a new methodology to generate and catalogue libraries of genome reduction mutants. LoxTnSeq combines random integration of Lox sites by transposon mutagenesis, and the generation of mutants via cre recombinase, catalogued via deep-sequencing. When LoxTnSeq was applied to the naturally genome reduced bacterium Mycoplasma pneumoniae, we obtained a mutant pool containing 285 unique deletions. These deletions spanned from >50 bp to 28 Kb, which represent 21% of the total genome. LoxTnSeq also highlighted large regions of non-essential genes that could be removed simultaneously, and other similar regions that could not, providing a guide for future genome reductions.
2021-08-18 | E-MTAB-9582 | biostudies-arrayexpress
Project description:Kir2.1 has been implicated in a number of channelopathies including Andersen-Tawil Syndrome, Short QT Syndrome and catecholaminergic polymorphic ventricular tachycardia. We developed a mass spectrometry-based assay to identify phosphorylation sites and test the functional consequence of removing those sites by site-directed mutagenesis. Our study identified novel sites of phosphorylation and suggests that the site of phosphorylation can influence channel function. We envision our approach can be readily adapted to study additional mutations and other ion channels.
2021-11-03 | PXD024662 | Pride
Project description:Transposable elements (TEs) are often the primary determinant of genome size differences among eukaryotes. In plants, the proliferation of TEs is countered through epigenetic silencing mechanisms that prevent transposition. Recent studies using the model plant Arabidopsis thaliana have revealed that methylated TE insertions are often associated with reduced expression of nearby genes, and these insertions may be subject to purifying selection due to their effect on nearby genes. Less is known about the genome-wide patterns of epigenetic silencing of TEs in other plant species. Here, we compare the 24-nt siRNA complement from Arabidopsis thaliana and a closely related congener with a two- to three-fold higher TE copy number, A. lyrata. We show that TEs, and particularly siRNA-targeted TEs, are associated with reduced gene expression within both species and also with gene expression differences between orthologs. In addition, A. lyrata TEs are targeted by a lower fraction of uniquely matching siRNAs, which are associated with more effective silencing of TE expression. Overall, our results suggest that the efficacy of RNA-directed DNA methylation silencing is lower in A. lyrata, a finding that may shed light on the causes of differential TE proliferation among species. 4 A. lyrata mRNA-seq samples
2011-02-11 | E-GEOD-24571 | biostudies-arrayexpress
Project description:Site-directed mutagenesis of soybean
| PRJDB10633 | ENA
Project description:Amino acid insertions and deletions (indels) are an abundant class of genetic variants. However, compared to substitutions, the effects of indels are not well understood and poorly predicted. Here we address this shortcoming by performing deep indel mutagenesis (DIM) of structurally diverse proteins. Indel tolerance is strikingly different to substitution tolerance and varies extensively both between different proteins and within different regions of the same protein. Although state of the art variant effect predictors perform poorly on indels, we show that both experimentally-measured and computationally-predicted substitution scores can be repurposed as good indel variant effect predictors by incorporating information on protein secondary structures. Quantifying the effects of indels on protein-protein interactions reveals that insertions can be an important class of gain-of-function variants. Our results provide an overview of the impact of indels on proteins and a method to predict their effects genome-wide.
2023-09-28 | GSE244096 | GEO
Project description:Using an integrated systems approach, the expressed proteome of B. diazoefficiens strain 110scp4 was measured under i) normal, oxic growth, and ii) microoxic growth condtions. This included, as a first step, the sequencing and de novo assembly of the genome of this widely used rhizobial model strain, which turned out to harbor several deletions and insertions compared to the B. diazoefficiens USDA 110 NCBI reference genome. With this optimal basis in hand, a shotgun proteomics approach relying on a slightly adapated FASP protocol was carried out, allowing to identify 2900 (oxia) and 2826 (microoxia) proteins, respectively, thereby largely expanding the proteome known to be expressed under microoxic conditions.
2019-05-30 | PXD012491 | Pride