A defined zebrafish line for high-throughput genetics and genomics: NHGRI-1.
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ABSTRACT:
SUBMITTER: LaFave MC
PROVIDER: S-EPMC4174928 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:PurposeThe National Human Genome Research Institute (NHGRI) at the National Institutes of Health (NIH) recognizes an urgent need for educator resources on cutting edge scientific topics due to increased public interest in genetics and genomics. We developed a Short Course in Genomics ("Short Course") to inspire new teaching materials through collaborative course development sessions and lectures, to expand access to cutting edge scientific information, and to provide a framework to consider when crafting new coursework related to scientific education.MethodsWe compared publicly available participant data from 2015 to 2019 with data from the National Center for Education Statistics to assess our progress in serving diverse educator and student populations. We also evaluated course agendas and interviewed participants and instructors.ResultsMiddle school, high school, community college, and tribal college course attendees from the last five years were more likely to teach students from diverse communities underrepresented in science, technology, engineering, and mathematics (STEM). Both attendees and Short Course instructors emphasized the importance of bidirectional learning through interactive curriculum development.ConclusionThis course has the potential to facilitate the engagement of educators and students at all levels, recruit and maintain a diverse STEM workforce, and improve genomic literacy and future health decision-making.
| S-EPMC7796976 | biostudies-literature
Project description:We show that targeted single-cell RNA-sequencing (TAP-seq) permits reliable mapping of cell (sub)types with as little as 100 reads per cell and reduces the sequencing costs for differential expression testing by a factor of 10-30, thereby enabling a cost-effective profiling of a large number of genotypes at the single cell level. We demonstrate the use of TAP-seq by generating comprehensive perturbation-based enhancer-target gene maps for 1.5% of the human genome.
2020-03-23 | GSE135497 | GEO
Project description:The zebrafish is a popular model organism for studying development and disease, and genetically modified zebrafish provide an essential tool for functional genomic studies. Numerous publications have demonstrated the efficacy of gene targeting in zebrafish using CRISPR/Cas9, and they have included descriptions of a variety of tools and methods for guide RNA synthesis and mutant identification. However, most of the published techniques are not readily scalable to increase throughput. We recently described a CRISPR/Cas9-based high-throughput mutagenesis and phenotyping pipeline in zebrafish. Here, we present a complete workflow for this pipeline, including target selection; cloning-free single-guide RNA (sgRNA) synthesis; microinjection; validation of the target-specific activity of the sgRNAs; founder screening to identify germline-transmitting mutations by fluorescence PCR; determination of the exact lesion by Sanger or next-generation sequencing (including software for analysis); and genotyping in the F1 or subsequent generations. Using these methods, sgRNAs can be evaluated in 3 d, zebrafish germline-transmitting mutations can be identified within 3 months and stable lines can be established within 6 months. Realistically, two researchers can target tens to hundreds of genes per year using this protocol.
| S-EPMC5630457 | biostudies-literature
Project description:Nitroreductase enzymes are responsible for the reduction of nitro functional groups to amino functional groups, and are found in a range of animal models, zebrafish (Danio rerio) excluded. Transgenic zebrafish models have been developed for tissue-specific cell ablation, which use nitroreductase to ablate specific tissues or cell types following exposure to the non-toxic pro-drug metronidazole (MTZ). When metabolized by nitroreductase, MTZ produces a potent cytotoxin, which specifically ablates the tissue in which metabolism occurs. Uses, beyond tissue-specific cell ablation, are possible for the hepatocyte-specific Tg(l-fabp:CFP-NTR)s891 zebrafish line, including investigations of the role of nitroreductase in the toxicity of nitrated compounds. The hepatic ablation characteristics of this transgenic line were explored, in order to expand its potential uses. Embryos were exposed at 48, 72, or 96 hours post fertilization (hpf) to a range of MTZ concentrations, and the ablation profiles were compared. Ablation occurred at a 10-fold lower concentration than previously reported. Embryos were exposed to a selection of other compounds, with and without MTZ, in order to investigate alternative uses for this transgenic line. Test compounds were selected based on: their ability to undergo nitroreduction, known importance of hepatic metabolism to toxicity, and known pharmaceutical hepatotoxins. Selected compounds included nitrated polycyclic aromatic hydrocarbons (nitro-PAHs), the PAHs retene and benzo[a]pyrene, and the pharmaceuticals acetaminophen and flutamide. The results suggest a range of potential roles of the liver in the toxicity of these compounds, and highlight the additional uses of this transgenic model in toxicity testing.
| S-EPMC5527975 | biostudies-literature
Project description:Protein X-ray crystallography recently celebrated its 50th anniversary. The structures of myoglobin and hemoglobin determined by Kendrew and Perutz provided the first glimpses into the complex protein architecture and chemistry. Since then, the field of structural molecular biology has experienced extraordinary progress and now more than 55000 protein structures have been deposited into the Protein Data Bank. In the past decade many advances in macromolecular crystallography have been driven by world-wide structural genomics efforts. This was made possible because of third-generation synchrotron sources, structure phasing approaches using anomalous signal, and cryo-crystallography. Complementary progress in molecular biology, proteomics, hardware and software for crystallographic data collection, structure determination and refinement, computer science, databases, robotics and automation improved and accelerated many processes. These advancements provide the robust foundation for structural molecular biology and assure strong contribution to science in the future. In this report we focus mainly on reviewing structural genomics high-throughput X-ray crystallography technologies and their impact.
| S-EPMC2764548 | biostudies-literature
Project description:Excess lipid storage is an epidemic problem in human populations. Thus, the identification of small molecules to treat or prevent lipid storage-related metabolic complications is of great interest. Here we screened >320.000 compounds for their ability to prevent a cellular lipid accumulation phenotype. We used fly cells because the multifarious tools available for this organism should facilitate unraveling the mechanism-of-action of active small molecules. Of the several hundred lipid storage inhibitors identified in the primary screen we concentrated on three structurally diverse and potent compound classes active in cells of multiple species (including human) and negligible cytotoxicity. Together with Drosophila in vivo epistasis experiments, RNA-Seq expression profiles suggested that the target of one of the small molecules was diacylglycerol acyltransferase 1 (DGAT1), a key enzyme in the production of triacylglycerols and prominent human drug target. We confirmed this prediction by biochemical and enzymatic activity tests.
| S-EPMC4919474 | biostudies-literature
Project description:BACKGROUND:Visualization plays an essential role in genomics research by making it possible to observe correlations and trends in large datasets as well as communicate findings to others. Visual analysis, which combines visualization with analysis tools to enable seamless use of both approaches for scientific investigation, offers a powerful method for performing complex genomic analyses. However, there are numerous challenges that arise when creating rich, interactive Web-based visualizations/visual analysis applications for high-throughput genomics. These challenges include managing data flow from Web server to Web browser, integrating analysis tools and visualizations, and sharing visualizations with colleagues. RESULTS:We have created a platform simplifies the creation of Web-based visualization/visual analysis applications for high-throughput genomics. This platform provides components that make it simple to efficiently query very large datasets, draw common representations of genomic data, integrate with analysis tools, and share or publish fully interactive visualizations. Using this platform, we have created a Circos-style genome-wide viewer, a generic scatter plot for correlation analysis, an interactive phylogenetic tree, a scalable genome browser for next-generation sequencing data, and an application for systematically exploring tool parameter spaces to find good parameter values. All visualizations are interactive and fully customizable. The platform is integrated with the Galaxy (http://galaxyproject.org) genomics workbench, making it easy to integrate new visual applications into Galaxy. CONCLUSIONS:Visualization and visual analysis play an important role in high-throughput genomics experiments, and approaches are needed to make it easier to create applications for these activities. Our framework provides a foundation for creating Web-based visualizations and integrating them into Galaxy. Finally, the visualizations we have created using the framework are useful tools for high-throughput genomics experiments.
| S-EPMC3691752 | biostudies-literature
Project description:Despite tremendous progress in understanding the nature of the immune system, the full diversity of an organism's antibody repertoire is unknown. We used high-throughput sequencing of the variable domain of the antibody heavy chain from 14 zebrafish to analyze VDJ usage and antibody sequence. Zebrafish were found to use between 50 and 86% of all possible VDJ combinations and shared a similar frequency distribution, with some correlation of VDJ patterns between individuals. Zebrafish antibodies retained a few thousand unique heavy chains that also exhibited a shared frequency distribution. We found evidence of convergence, in which different individuals made the same antibody. This approach provides insight into the breadth of the expressed antibody repertoire and immunological diversity at the level of an individual organism.
| S-EPMC3086368 | biostudies-literature
Project description:During the past three decades, we have witnessed remarkable advances in our understanding of the molecular etiologies of hereditary neurodegenerative diseases, which have been accomplished by 'positional cloning' strategies. The discoveries of the causative genes for hereditary neurodegenerative diseases accelerated not only the studies on the pathophysiologic mechanisms of diseases, but also the studies for the development of disease-modifying therapies. Genome-wide association studies (GWAS) based on the 'common disease-common variants hypothesis' are currently undertaken to elucidate disease-relevant alleles. Although GWAS have successfully revealed numerous susceptibility genes for neurodegenerative diseases, odds ratios associated with risk alleles are generally low and account for only a small proportion of estimated heritability. Recent studies have revealed that the effect sizes of the disease-relevant alleles that are identified based on comprehensive resequencing of large data sets of Parkinson disease are substantially larger than those identified by GWAS. These findings strongly argue for the role of the 'common disease-multiple rare variants hypothesis' in sporadic neurodegenerative diseases. Given the rapidly improving technologies of next-generation sequencing next-generation sequencing (NGS), we expect that NGS will eventually enable us to identify all the variants in an individual's personal genome, in particular, clinically relevant alleles. Beyond this, whole genome resequencing is expected to bring a paradigm shift in clinical practice, where clinical practice including diagnosis and decision-making for appropriate therapeutic procedures is based on the 'personal genome'. The personal genome era is expected to be realized in the near future, and society needs to prepare for this new era.
| S-EPMC2875051 | biostudies-literature
Project description:The major histocompatibility complex (MHC) region on the short arm of chromosome 6 harbors the largest number of replicated associations across the human genome for a wide range of diseases, but the functional basis for these associations is still poorly understood. One fundamental challenge in fine-mapping associations to functional alleles is the enormous sequence diversity and broad linkage disequilibrium of the MHC, both of which hamper the cost-effective interrogation in large patient samples and the identification of causal variants. In this review, we argue that there is now a valuable opportunity to leverage existing genome-wide association study (GWAS) datasets for in-depth investigation to identify independent effects in the MHC. Application of imputation to GWAS data facilitates comprehensive interrogation of the classical human leukocyte antigen (HLA) loci. These datasets are, in many cases, sufficiently large to give investigators the ability to disentangle effects at different loci. We also explain how querying variation at individual amino acid positions for association can be powerful and expand traditional analyses that focus only on the classical HLA types.
| S-EPMC3459647 | biostudies-literature