scholarly journals Measuring error rates in genomic perturbation screens: gold standards for human functional genomics

2014 ◽  
Author(s):  
Traver Hart ◽  
Kevin R. Brown ◽  
Fabrice Sircoulomb ◽  
Robert Rottapel ◽  
Jason Moffat
Keyword(s):  
Mammalian Cells ◽  
Large Scale ◽  
Cellular Proliferation ◽  
Gene Knockout ◽  
Error Rates ◽  
Measuring Error ◽  
Technological Advancement ◽  
Loss Of Function ◽  
False Discovery Rates ◽  
Gold Standard Reference

AbstractTechnological advancement has opened the door to systematic genetics in mammalian cells. Genome-scale loss-of-function screens can assay fitness defects induced by partial gene knockdown, using RNA interference, or complete gene knockout, using new CRISPR techniques. These screens can reveal the basic blueprint required for cellular proliferation. Moreover, comparing healthy to cancerous tissue can uncover genes that are essential only in the tumor; these genes are targets for the development of specific anticancer therapies. Unfortunately, progress in this field has been hampered by offtarget effects of perturbation reagents and poorly quantified error rates in large-scale screens. To improve the quality of information derived from these screens, and to provide a framework for understanding the capabilities and limitations of CRISPR technology, we derive gold-standard reference sets of essential and nonessential genes, and provide a Bayesian classifier of gene essentiality that outperforms current methods on both RNAi and CRISPR screens. Our results indicate that CRISPR technology is more sensitive than RNAi, and that both techniques have nontrivial false discovery rates that can be mitigated by rigorous analytical methods.

scholarly journals BAGEL: A computational framework for identifying essential genes from pooled library screens.

2015 ◽  
Author(s):  
Traver Hart ◽  
Jason Moffat
Keyword(s):  
Mammalian Cells ◽  
State Of The Art ◽  
Gene Knockout ◽  
High Sensitivity ◽  
Major Advance ◽  
Computational Framework ◽  
New Methods ◽  
Gold Standard Reference ◽  
Order Of Magnitude ◽  
Prior State

Background: The adaptation of the CRISPR-Cas9 system to pooled library gene knockout screens in mammalian cells represents a major technological leap over RNA interference, the prior state of the art. New methods for analyzing the data and evaluating results are needed. Results: We offer BAGEL (Bayesian Analysis of Gene EssentiaLity), a supervised learning method for analyzing gene knockout screens. Coupled with gold-standard reference sets of essential and nonessential genes, BAGEL offers significantly greater sensitivity than current methods, while computational optimizations reduce runtime by an order of magnitude. Conclusions: Using BAGEL, we identify ~2,000 fitness genes in pooled library knockout screens in human cell lines at 5% FDR, a major advance over competing platforms. BAGEL shows high sensitivity and specificity even across screens with highly variable reagent quality.

scholarly journals Evaluation of RNAi and CRISPR technologies by large-scale gene expression profiling in the Connectivity Map

2017 ◽  
Author(s):  
Ian Smith ◽  
Peyton G Greenside ◽  
David Wadden ◽  
Itay Tirosh ◽  
Ted Natoli ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mammalian Cells ◽  
Large Scale ◽  
Gene Signature ◽  
Loss Of Function ◽  
Connectivity Map ◽  
Rnai Technology ◽  
Short Palindromic Repeat ◽  
Short Hairpin Rnas ◽  
Target Effects

AbstractThe application of RNA interference (RNAi) to mammalian cells has provided the means to perform phenotypic screens to determine the functions of genes. Although RNAi has revolutionized loss of function genetic experiments, it has been difficult to systematically assess the prevalence and consequences of off-target effects. The Connectivity Map (CMAP) represents an unprecedented resource to study the gene expression consequences of expressing short hairpin RNAs (shRNAs). Analysis of signatures for over 13,000 shRNAs applied in 9 cell lines revealed that miRNA-like off-target effects of RNAi are far stronger and more pervasive than generally appreciated. We show that mitigating off-target effects is feasible in these datasets via computational methodologies to produce a Consensus Gene Signature (CGS). In addition, we compared RNAi technology to clustered regularly interspaced short palindromic repeat (CRISPR)-based knockout by analysis of 373 sgRNAs in 6 cells lines, and show that the on-target efficacies are comparable, but CRISPR technology is far less susceptible to systematic off-target effects. These results will help guide the proper use and analysis of loss-of-function reagents for the determination of gene function.

Haploid Genetic Screens in Human Cells Identify Host Factors Used by Pathogens

Science ◽  
2009 ◽  
Vol 326 (5957) ◽  
pp. 1231-1235 ◽  
Author(s):  
Jan E. Carette ◽  
Carla P. Guimaraes ◽  
Malini Varadarajan ◽  
Annie S. Park ◽  
Irene Wuethrich ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Large Scale ◽  
Insertional Mutagenesis ◽  
Screening Method ◽  
Chromosome 8 ◽  
Host Factors ◽  
Null Alleles ◽  
Model Organisms ◽  
Loss Of Function ◽  
Genetic Screens

Loss-of-function genetic screens in model organisms have elucidated numerous biological processes, but the diploid genome of mammalian cells has precluded large-scale gene disruption. We used insertional mutagenesis to develop a screening method to generate null alleles in a human cell line haploid for all chromosomes except chromosome 8. Using this approach, we identified host factors essential for infection with influenza and genes encoding important elements of the biosynthetic pathway of diphthamide, which are required for the cytotoxic effects of diphtheria toxin and exotoxin A. We also identified genes needed for the action of cytolethal distending toxin, including a cell-surface protein that interacts with the toxin. This approach has both conceptual and practical parallels with genetic approaches in haploid yeast.

Optical Scanning for Large-Scale Studies

1975 ◽  
Vol 14 (01) ◽  
pp. 32-34
Author(s):  
Elisabeth Schach
Keyword(s):  
Large Scale ◽  
Error Rates ◽  
Data Reporting ◽  
Reading Error ◽  
Optical Scanning ◽  
Rejection Rates

Data reporting the experience with an optical mark page reader is presented (IBM 1231Ν1). Information from 52,000 persons was gathered in seven countries, decentrally coded and centrally processed. Reader performance rates (i.e. sheets read per hour, sheet rejection rates, reading error rates) and costs (coding, verification, reading, etc.) are given.

Suppressors of a Cold-Sensitive Mutation in Yeast U4 RNA Define Five Domains in the Splicing Factor Prp8 That Influence Spliceosome Activation

Genetics ◽  
2000 ◽  
Vol 155 (4) ◽  
pp. 1667-1682 ◽  
Author(s):  
Andreas N Kuhn ◽  
David A Brow
Keyword(s):  
Large Scale ◽  
Splicing Factor ◽  
Cold Sensitivity ◽  
Splicing Factors ◽  
Loss Of Function ◽  
U1 Snrnp ◽  
Snrnp Protein ◽  
Cold Sensitive ◽  
Spliceosome Activation ◽  
U4 Rna

AbstractThe highly conserved splicing factor Prp8 has been implicated in multiple stages of the splicing reaction. However, assignment of a specific function to any part of the 280-kD U5 snRNP protein has been difficult, in part because Prp8 lacks recognizable functional or structural motifs. We have used a large-scale screen for Saccharomyces cerevisiae PRP8 alleles that suppress the cold sensitivity caused by U4-cs1, a mutant U4 RNA that blocks U4/U6 unwinding, to identify with high resolution five distinct regions of PRP8 involved in the control of spliceosome activation. Genetic interactions between two of these regions reveal a potential long-range intramolecular fold. Identification of a yeast two-hybrid interaction, together with previously reported results, implicates two other regions in direct and indirect contacts to the U1 snRNP. In contrast to the suppressor mutations in PRP8, loss-of-function mutations in the genes for two other splicing factors implicated in U4/U6 unwinding, Prp44 (Brr2/Rss1/Slt22/Snu246) and Prp24, show synthetic enhancement with U4-cs1. On the basis of these results we propose a model in which allosteric changes in Prp8 initiate spliceosome activation by (1) disrupting contacts between the U1 snRNP and the U4/U6-U5 tri-snRNP and (2) orchestrating the activities of Prp44 and Prp24.

scholarly journals Analysis of local habitat selection and large-scale attraction/avoidance based on animal tracking data: is there a single best method?

2021 ◽  
Vol 9 (1) ◽  
Author(s):  
Moritz Mercker ◽  
Philipp Schwemmer ◽  
Verena Peschko ◽  
Leonie Enners ◽  
Stefan Garthe
Keyword(s):  
Habitat Selection ◽  
Large Scale ◽  
Error Rates ◽  
Process Models ◽  
Statistical Hypothesis ◽  
Type I ◽  
Tracking Data ◽  
Selection Models ◽  
Animal Tracking ◽  
Step Selection

Abstract Background New wildlife telemetry and tracking technologies have become available in the last decade, leading to a large increase in the volume and resolution of animal tracking data. These technical developments have been accompanied by various statistical tools aimed at analysing the data obtained by these methods. Methods We used simulated habitat and tracking data to compare some of the different statistical methods frequently used to infer local resource selection and large-scale attraction/avoidance from tracking data. Notably, we compared spatial logistic regression models (SLRMs), spatio-temporal point process models (ST-PPMs), step selection models (SSMs), and integrated step selection models (iSSMs) and their interplay with habitat and animal movement properties in terms of statistical hypothesis testing. Results We demonstrated that only iSSMs and ST-PPMs showed nominal type I error rates in all studied cases, whereas SSMs may slightly and SLRMs may frequently and strongly exceed these levels. iSSMs appeared to have on average a more robust and higher statistical power than ST-PPMs. Conclusions Based on our results, we recommend the use of iSSMs to infer habitat selection or large-scale attraction/avoidance from animal tracking data. Further advantages over other approaches include short computation times, predictive capacity, and the possibility of deriving mechanistic movement models.

scholarly journals Variable Expressivity of the Beckwith-Wiedemann Syndrome in Four Pedigrees Segregating Loss-of-Function Variants of CDKN1C

Genes ◽  
2021 ◽  
Vol 12 (5) ◽  
pp. 706
Author(s):  
Angela Sparago ◽  
Flavia Cerrato ◽  
Laura Pignata ◽  
Francisco Cammarata-Scalisi ◽  
Livia Garavelli ◽  
...  
Keyword(s):  
Cellular Proliferation ◽  
Missense Variant ◽  
Abdominal Wall Defects ◽  
Loss Of Function ◽  
Nonsense Mutations ◽  
Molecular Defects ◽  
Variable Expressivity ◽  
Imprinting Disorder ◽  
Perinatal Lethality ◽  
Intrafamilial Variability

Beckwith-Wiedemann syndrome (BWS) is an imprinting disorder characterized by prenatal and/or postnatal overgrowth, organomegaly, abdominal wall defects and tumor predisposition. CDKN1C is a maternally expressed gene of the 11p15.5 chromosomal region and is regulated by the imprinting control region IC2. It negatively controls cellular proliferation, and its expression or activity are frequently reduced in BWS. In particular, loss of IC2 methylation is associated with CDKN1C silencing in the majority of sporadic BWS cases, and maternally inherited loss-of-function variants of CDKN1C are the most frequent molecular defects of familial BWS. We have identified, using Sanger sequencing, novel CDKN1C variants in three families with recurrent cases of BWS, and a previously reported variant in a woman with recurrent miscarriages with exomphalos. Clinical evaluation of the patients showed variable manifestation of the disease. The frameshift and nonsense variants were consistently associated with exomphalos, while the missense variant caused a less severe phenotype. Pregnancy loss and perinatal lethality were found in the families segregating nonsense mutations. Intrafamilial variability of the clinical BWS features was observed, even between siblings. Our data are indicative of severe BWS phenotypes that, with variable expressivity, may be associated with both frameshift and nonsense variants of CDKN1C.

scholarly journals Cross-Species RNAi Rescue Platform in Drosophila melanogaster

Genetics ◽  
2009 ◽  
Vol 183 (3) ◽  
pp. 1165-1173 ◽  
Author(s):  
Shu Kondo ◽  
Matthew Booker ◽  
Norbert Perrimon
Keyword(s):  
Cell Culture ◽  
Drosophila Melanogaster ◽  
Large Scale ◽  
Transgenic Animals ◽  
Selection Marker ◽  
Bacterial Artificial Chromosomes ◽  
Loss Of Function ◽  
Artificial Chromosomes ◽  
Target Effects

RNAi-mediated gene knockdown in Drosophila melanogaster is a powerful method to analyze loss-of-function phenotypes both in cell culture and in vivo. However, it has also become clear that false positives caused by off-target effects are prevalent, requiring careful validation of RNAi-induced phenotypes. The most rigorous proof that an RNAi-induced phenotype is due to loss of its intended target is to rescue the phenotype by a transgene impervious to RNAi. For large-scale validations in the mouse and Caenorhabditis elegans, this has been accomplished by using bacterial artificial chromosomes (BACs) of related species. However, in Drosophila, this approach is not feasible because transformation of large BACs is inefficient. We have therefore developed a general RNAi rescue approach for Drosophila that employs Cre/loxP-mediated recombination to rapidly retrofit existing fosmid clones into rescue constructs. Retrofitted fosmid clones carry a selection marker and a phiC31 attB site, which facilitates the production of transgenic animals. Here, we describe our approach and demonstrate proof-of-principle experiments showing that D. pseudoobscura fosmids can successfully rescue RNAi-induced phenotypes in D. melanogaster, both in cell culture and in vivo. Altogether, the tools and method that we have developed provide a gold standard for validation of Drosophila RNAi experiments.

scholarly journals Automation of large scale transient protein expression in mammalian cells

2011 ◽  
Vol 175 (2) ◽  
pp. 209-215 ◽  
Author(s):  
Yuguang Zhao ◽  
Benjamin Bishop ◽  
Jordan E. Clay ◽  
Weixian Lu ◽  
Margaret Jones ◽  
...  
Keyword(s):  
Protein Expression ◽  
Mammalian Cells ◽  
Large Scale ◽  
Transient Protein Expression ◽  
Expression In Mammalian Cells
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