Dataset Information

High-throughput, quantitative analyses of genetic interactions in E. coli.

ABSTRACT: Large-scale genetic interaction studies provide the basis for defining gene function and pathway architecture. Recent advances in the ability to generate double mutants en masse in Saccharomyces cerevisiae have dramatically accelerated the acquisition of genetic interaction information and the biological inferences that follow. Here we describe a method based on F factor-driven conjugation, which allows for high-throughput generation of double mutants in Escherichia coli. This method, termed genetic interaction analysis technology for E. coli (GIANT-coli), permits us to systematically generate and array double-mutant cells on solid media in high-density arrays. We show that colony size provides a robust and quantitative output of cellular fitness and that GIANT-coli can recapitulate known synthetic interactions and identify previously unidentified negative (synthetic sickness or lethality) and positive (suppressive or epistatic) relationships. Finally, we describe a complementary strategy for genome-wide suppressor-mutant identification. Together, these methods permit rapid, large-scale genetic interaction studies in E. coli.

SUBMITTER: Typas A

PROVIDER: S-EPMC2700713 | biostudies-literature | 2008 Sep

REPOSITORIES: biostudies-literature

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High-throughput, quantitative analyses of genetic interactions in E. coli.

Typas Athanasios A Nichols Robert J RJ Siegele Deborah A DA Shales Michael M Collins Sean R SR Lim Bentley B Braberg Hannes H Yamamoto Natsuko N Takeuchi Rikiya R Wanner Barry L BL Mori Hirotada H Weissman Jonathan S JS Krogan Nevan J NJ Gross Carol A CA

Nature methods 20080901 9

Large-scale genetic interaction studies provide the basis for defining gene function and pathway architecture. Recent advances in the ability to generate double mutants en masse in Saccharomyces cerevisiae have dramatically accelerated the acquisition of genetic interaction information and the biological inferences that follow. Here we describe a method based on F factor-driven conjugation, which allows for high-throughput generation of double mutants in Escherichia coli. This method, termed gen ...[more]

PMID: 19160513

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Project description:DNA from buccal brush samples is being used for high-throughput analyses in a variety of applications, but the impact of sample type on genotyping success and downstream statistical analysis remains unclear. The objective of the current study was to determine laboratory predictors of genotyping failure among buccal DNA samples, and to evaluate the successfully genotyped results with respect to analytic quality control metrics. Sample and genotyping characteristics were compared between buccal and blood samples collected in the population-based Genetic and Environmental Risk Factors for Hemorrhagic Stroke (GERFHS) study (https://gerfhs.phs.wfubmc.edu/public/index.cfm).Seven-hundred eight (708) buccal and 142 blood DNA samples were analyzed for laboratory-based and analysis metrics. Overall genotyping failure rates were not statistically different between buccal (11.3%) and blood (7.0%, p?=?0.18) samples; however, both the Contrast Quality Control (cQC) rate and the dynamic model (DM) call rates were lower among buccal DNA samples (p?<?0.0001). The ratio of double-stranded to total DNA (ds/total ratio) in the buccal samples was the only laboratory characteristic predicting sample success (p?<?0.0001). A threshold of at least 34% ds/total DNA provided specificity of 98.7% with a 90.5% negative predictive value for eliminating probable failures. After genotyping, median sample call rates (99.1% vs. 99.4%, p?<?0.0001) and heterozygosity rates (25.6% vs. 25.7%, p?=?0.006) were lower for buccal versus blood DNA samples, respectively, but absolute differences were small. Minor allele frequency differences from HapMap were smaller for buccal than blood samples, and both sample types demonstrated tight genotyping clusters, even for rare alleles.We identified a buccal sample characteristic, a ratio of ds/total DNA <34%, which distinguished buccal DNA samples likely to fail high-throughput genotyping. Applying this threshold, the quality of final genotyping resulting from buccal samples is somewhat lower, but compares favorably to blood. Caution is warranted if cases and controls have different sample types, but buccal samples provide comparable results to blood samples in large-scale genotyping analyses.

Dataset Information

High-throughput, quantitative analyses of genetic interactions in E. coli.

Publications

High-throughput, quantitative analyses of genetic interactions in E. coli.

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