A comprehensive platform for highly multiplexed mammalian functional genetic screens

<p>Abstract</p> <p>Background</p> <p>Genome-wide screening in human and mouse cells using RNA interference and open reading frame over-expression libraries is rapidly becoming a viable experimental approach for many research labs. There are a variety of gene expression modulation libraries commercially available, however, detailed and validated protocols as well as the reagents necessary for deconvolving genome-scale gene screens using these libraries are lacking. As a solution, we designed a comprehensive platform for highly multiplexed functional genetic screens in human, mouse and yeast cells using popular, commercially available gene modulation libraries. The Gene Modulation Array Platform (GMAP) is a single microarray-based detection solution for deconvolution of loss and gain-of-function pooled screens.</p> <p>Results</p> <p>Experiments with specially constructed lentiviral-based plasmid pools containing ~78,000 shRNAs demonstrated that the GMAP is capable of deconvolving genome-wide shRNA "dropout" screens. Further experiments with a larger, ~90,000 shRNA pool demonstrate that equivalent results are obtained from plasmid pools and from genomic DNA derived from lentivirus infected cells. Parallel testing of large shRNA pools using GMAP and next-generation sequencing methods revealed that the two methods provide valid and complementary approaches to deconvolution of genome-wide shRNA screens. Additional experiments demonstrated that GMAP is equivalent to similar microarray-based products when used for deconvolution of open reading frame over-expression screens.</p> <p>Conclusion</p> <p>Herein, we demonstrate four major applications for the GMAP resource, including deconvolution of pooled RNAi screens in cells with at least 90,000 distinct shRNAs. We also provide detailed methodologies for pooled shRNA screen readout using GMAP and compare next-generation sequencing to GMAP (i.e. microarray) based deconvolution methods.</p

Genome annotation for A. gallica (JGI Armillaria gallica 21-2 v1)

Provisional genome annotation was done with GeneMarkES under the default settings and SNPs were annotated with snpEff

Data from: Clonal evolution and genome stability in a 2,500-year-old fungal individual

Individuals of the basidiomycete fungus Armillaria are well-known for their ability to spread from woody substrate to substrate on the forest floor through the growth of rhizomoprhs. Here we made 248 collections of A. gallica in one locality in Michigan’s Upper Peninsula. To identify individuals, we genotyped collections with molecular markers and somatic compatibility testing. We found several different individuals in proximity to one another, but one genetic individual stood out as exceptionally large, covering hundreds of tree root systems over approximate 75 hectares of forest floor. Based on observed growth rates of the fungus, we estimate the minimum age of the large individual as 2,500 years. With whole-genome sequencing and variant discovery, we also found that mutation had occurred within the somatic cells of the individual, reflecting its historical pattern of growth from a single point. The overall rate of mutation over the 90 mb genome, however, was extremely low. This same individual was first discovered in the late 1980s, but its full spatial extent and internal mutation dynamic was unkown at that time. The large individual of A. gallica has been remarkably resistant to genomic change as it has persisted in place

Menin links the stress response to genome stability in Drosophila melanogaster.

BACKGROUND: The multiple endocrine neoplasia type I gene functions as a tumor suppressor gene in humans and mouse models. In Drosophila melanogaster, mutants of the menin gene (Mnn1) are hypersensitive to mutagens or gamma irradiation and have profound defects in the response to several stresses including heat shock, hypoxia, hyperosmolarity and oxidative stress. However, it is not known if the function of menin in the stress response contributes to genome stability. The objective of this study was to examine the role of menin in the control of the stress response and genome stability. METHODOLOGY/PRINCIPAL FINDINGS: Using a test of loss-of-heterozygosity, we show that Drosophila strains lacking a functional Mnn1 gene or expressing a Mnn1 dsRNA display increased genome instability in response to non-lethal heat shock or hypoxia treatments. This is also true for strains lacking all Hsp70 genes, implying that a precise control of the stress response is required for genome stability. While menin is required for Hsp70 expression, the results of epistatic studies indicate that the increase in genome instability observed in Mnn1 lack-of-function mutants cannot be accounted for by mis-expression of Hsp70. Therefore, menin may promote genome stability by controlling the expression of other stress-responsive genes. In agreement with this notion, gene profiling reveals that Mnn1 is required for sustained expression of all heat shock protein genes but is dispensable for early induction of the heat shock response. CONCLUSIONS/SIGNIFICANCE: Mutants of the Mnn1 gene are hypersensitive to several stresses and display increased genome instability when subjected to conditions, such as heat shock, generally regarded as non-genotoxic. In this report, we describe a role for menin as a global regulator of heat shock gene expression and critical factor in the maintenance of genome integrity. Therefore, menin links the stress response to the control of genome stability in Drosophila melanogaster

Data from: Population genomics reveals structure at the individual, host-tree scale and persistence of genotypic variants of the undomesticated yeast Saccharomyces paradoxus in a natural woodland

Genetic diversity in experimental, domesticated and wild populations of the related yeasts, Saccharomyces cerevisiae and S. paradoxus has been well described at the global scale. We investigated the population genomics of a local population on a small spatial scale to address two main questions. First, is there genomic variation in a S. paradoxus population at a spatial scale spanning centimeters (microsites) to tens of meters? Second, does the distribution of genomic variants persist over time? Our sample consisted of 42 S. paradoxus strains from 2014 and 43 strains from 2015 collected from the same 72 microsites around four host trees (Quercus rubra and Q. alba) within 1km2 in a mixed hardwood forest in southern Ontario. Six additional S. paradoxus strains recovered from adjacent maple and beech trees in 2015 are also included in the sample. Whole-genome sequencing and genomic SNP analysis revealed five differentiated groups (clades) within the sampled area. The signal of persistence of genotypes in their microsites from 2014 to 2015 was highly significant. Isolates from the same tree tended to be more related than strains from different trees, with limited evidence of dispersal between trees. In growth assays, one genotype had a significantly longer lag phase than the other strains. Our results indicate that different clades co-exist at fine spatial scale, and that population structure persists over at least a one year interval in these wild yeasts, suggesting the efficacy of yearly sampling to follow longer term genetic dynamics in future studies

Data from: Persistence of resident and transplanted genotypes of the undomesticated yeast, Saccharomyces paradoxus in forest soil

One might expect yeasts in soil to be highly dispersed via water or insects, forming ephemeral, genetically heterogeneous populations subject to competition and environmental stochasticity. Here, we report persistence of genotypes of the yeast Saccharomyces paradoxus in space and time. Within 1 km2 in a mixed hardwood forest on scales from centimeters to tens of meters, we detect persistence over 3 years of native genotypes, identified by SNPs genome-wide, of the wild yeast, Saccharomyces paradoxus around Quercus rubra and Q. alba. Yeasts were recovered by enrichment in ethanol-containing medium, which measures only presence or absence, not abundance. Additional transplantation experiments employed strains marked with spontaneous defects in the URA3 gene, which also confer resistance to 5-Fluoroorotic acid (5FOA). Plating soil suspensions from transplant sites on 5FOA medium permitted one-step quantification of yeast colony-forming units, with no interference from other unmarked yeasts or microorganisms. After an initial steep decrease in abundance, the yeast densities fluctuated over time, increasing in association with rainfall and decreasing in association with drought. After 18 months, the transplanted yeasts remain in place on the nine sites. In vitro transplantation experiments into non-sterile soil in petri dishes showed similar patterns of persistence and response to moisture and drought. To determine whether S. cerevisiae, not previously recovered from soils regionally, can persist in our cold-climate sites, we transplanted marked S. cerevisiae alone and in mixture with S. paradoxus in fall, 2017. Five months on, S. cerevisiae persist to the same extent as S. paradoxus

Persistence of Resident and Transplanted Genotypes of the Undomesticated Yeast Saccharomyces paradoxus in Forest Soil

Saccharomyces yeasts are intensively studied in biological research and in their domesticated roles in brewing and baking, and yet, remarkably little is known about their mode of life in forest soils. We report here that resident genotypes of the yeast S. paradoxus are persistent on a time scale of years in their microhabitats in forest soils. We also show that resident genotypes can be replaced by transplanted yeast genotypes. The high inoculum levels in experimental transplantations rapidly decreased over time, but the transplanted genotypes persisted at low abundance. We conclude that, in forest soils, Saccharomyces yeasts exist at very low abundance and that dispersal events are rare.One might expect yeasts in soil to be highly dispersed via water or insects, forming ephemeral, genetically heterogeneous populations subject to competition and environmental stochasticity. Here, we report persistence of genotypes of the yeast Saccharomyces paradoxus in space and time. Within 1 km2 in a mixed hardwood forest on scales from centimeters to tens of meters, we detected persistence over 3 years of native genotypes, identified by single nucleotide polymorphisms (SNPs) genome-wide, of the wild yeast Saccharomyces paradoxus growing around Quercus rubra and Quercus alba. Yeasts were recovered by enrichment in ethanol-containing medium, which measures only presence or absence, not abundance. Additional transplantation experiments employed strains marked with spontaneous defects in the URA3 gene, which also confer resistance to 5-fluoroorotic acid (5FOA). Plating soil suspensions from transplant sites on 5FOA-containing medium permitted one-step quantification of yeast CFU, with no interference from other unmarked yeasts or microorganisms. After an initial steep decrease in abundance, the yeast densities fluctuated over time, increasing in association with rainfall and decreasing in association with drought. After 18 months, the transplanted yeasts remained in place on the nine sites. In vitro transplantation experiments into nonsterile soil in petri dishes showed similar patterns of persistence and response to moisture and drought. To determine whether Saccharomyces cerevisiae, not previously recovered from soils regionally, can persist in our cold climate sites, we transplanted marked S. cerevisiae alone and in mixture with S. paradoxus in the fall of 2017. Five months later, S. cerevisiae persisted to the same extent as S. paradoxus