Project description:The ability to perform complex bioassays in parallel enables experiments otherwise impossible due to throughput and cost constraints. By way of example, highly parallel chemical-genetic screens using pooled collections of thousands of defined Saccharomyces cerevisiae gene deletion strains are feasible because each strain is barcoded with unique DNA sequences. It is, however, time consuming and expensive to individually barcode individual strains. To provide a simple and general method of barcoding yeast collections, we built a set of donor strains, called Barcoders, with unique barcodes that can be systematically transferred to any S. cerevisiae collection. We applied this technology by generating a collection of barcoded DAmP (Decreased Abundance by mRNA Perturbation) loss-of-function strains comprising 87.1% of all essential yeast genes. This test collection validates both the Barcoders and the DAmP collection as useful tools for genome-wide chemical genetic assays.
Project description:Systematic analyses of essential gene function using mutant collections in Saccharomyces cerevisiae have been conducted using collections of heterozygous diploids, promoter shut-off alleles, through alleles with destabilized mRNA, destabilized protein, or bearing mutations that lead to a temperature-sensitive (ts) phenotype. We previously described a method for construction of barcoded ts alleles in a systematic fashion. Here we report the completion of this collection of alleles covering 600 essential yeast genes. This resource covers a larger gene repertoire than previous collections and provides a complementary set of strains suitable for single gene and genomic analyses. We use deep sequencing to characterize the amino acid changes leading to the ts phenotype in half of the alleles. We also use high-throughput approaches to describe the relative ts behavior of the alleles. Finally, we demonstrate the experimental usefulness of the collection in a high-content, functional genomic screen for ts alleles that increase spontaneous P-body formation. By increasing the number of alleles and improving the annotation, this ts collection will serve as a community resource for probing new aspects of biology for essential yeast genes.
Project description:To profile off-target effects of psychoactive drugs, 87 genome-wide drug fitness assays (of which 5 were performed in duplicate) and 18 genome-wide control experiments were performed in Saccharomyces cerevisiae. Heterozygous and homozygous yeast single gene deletion strains from the BY collection of the Saccharomyces cerevisiae genome deletion consortium were used.
Project description:Short-read DNA sequencing technologies provide new tools to answer biological questions. However, high cost and low throughput limit their widespread use, particularly in organisms with smaller genomes such as S. cerevisiae. Although ChIP-Seq in mammalian cell lines is replacing array-based ChIP-chip as the standard for transcription factor binding studies, ChIP-Seq in yeast is still underutilized compared to ChIP-chip. We developed a multiplex barcoding system that allows simultaneous sequencing and analysis of multiple samples using Illumina’s platform. We applied this method to analyze the chromosomal distributions of three yeast DNA binding proteins (Ste12, Cse4 and RNA PolII) and a reference sample (input DNA) in a single experiment and demonstrate its utility for rapid and accurate results at reduced costs. We developed a barcoding ChIP-Seq method for the concurrent analysis of transcription factor binding sites for yeast. Our multiplex strategy generated high quality data that was indistinguishable from data obtained with non-barcoded libraries. None of the barcoded adapters induced differences relative to a non-barcoded adapter when applied to the same DNA sample. We used this method to map the binding sites for Cse4, Ste12 and Pol II throughout the yeast genome and we found 148 binding targets for Cse4, 823 targets for Ste12 and 2508 targets for PolII. Cse4 was strongly bound to all yeast centromeres as expected and the remaining non-centromeric targets correspond to highly expressed genes in rich media, the latter constituting a novel finding. We designed a multiplex short-read DNA sequencing method to perform efficient ChIP-Seq in yeast and other small genome model organisms. This method produces accurate results with higher throughput and reduced cost. Given constant improvements in high-throughput sequencing technologies, increasing multiplexing will be possible to further decrease costs per sample and to accelerate the completion of large consortium projects such as modENCODE.
Project description:Yeast knockout collection TAG microarrays are an emergent platform for rapid, genome-wide functional characterization of yeast genes. We describe a method for analyzing two-color array data to efficiently represent differential knockout strain representation across two experimental conditions. Using a fully defined spike-in pool, we show that the sensitivity and specificity of this method exceed typical current approaches. Keywords: Saccharomyces cerevisiae, yeast, self_vs_self, spike-in pools
Project description:The MMRC reference collection is a dataset of gene expression profiling, array comparative genomic hybridization, and re-sequencing created as a resource for the Multiple Myeloma research community.
Project description:Cancer is a heterogeneous disease, where multiple, phenotypically distinct subpopulations co-exist. Tumour evolution is a result of a complex interplay of genetic and epigenetic factors. To predict the molecular drivers of distinct cancer responses, we apply single-cell lineage tracing (scRNA-Seq of barcoded cells) on a triple-negative breast cancer model. We propose GALILEO, a framework providing lineage tracing, transcriptomic, and chromatin accessibility information simultaneously at single-cell resolution (Multiome ATAC + gene expression on barcoded cells). The combination of single-cell lineage tracing with phenotypic assays allows to link a cell state with its fate.