Unlocking the Bottleneck in Forward Genetics Using Whole-Genome Sequencing and Identity by Descent to Isolate Causative Mutations

Forward genetics screens with N-ethyl-N-nitrosourea (ENU) provide a powerful way to illuminate gene function and generate mouse models of human disease; however, the identification of causative mutations remains a limiting step. Current strategies depend on conventional mapping, so the propagation of affected mice requires non-lethal screens; accurate tracking of phenotypes through pedigrees is complex and uncertain; out-crossing can introduce unexpected modifiers; and Sanger sequencing of candidate genes is inefficient. Here we show how these problems can be efficiently overcome using whole-genome sequencing (WGS) to detect the ENU mutations and then identify regions that are identical by descent (IBD) in multiple affected mice. In this strategy, we use a modification of the Lander-Green algorithm to isolate causative recessive and dominant mutations, even at low coverage, on a pure strain background. Analysis of the IBD regions also allows us to calculate the ENU mutation rate (1.54 mutations per Mb) and to model future strategies for genetic screens in mice. The introduction of this approach will accelerate the discovery of causal variants, permit broader and more informative lethal screens to be used, reduce animal costs, and herald a new era for ENU mutagenesis.     

Genomewide two-generation screens for recessive mutations by ES cell mutagenesis

Forward genetic mutation screens in mice are typically begun by mutagenizing the germline of male mice with N-ethyl-N-nitrosourea (ENU). Genomewide recessive mutations transmitted by these males can be rendered homozygous after three generations of breeding, at which time phenotype screens can be performed. An alternative strategy for randomly mutagenizing the mouse genome is by chemical treatment of embryonic stem (ES) cells. Here we demonstrate the feasibility of performing genomewide mutation screens with only two generations of breeding. Mice potentially homozygous for mutations were obtained by crossing chimeras derived from ethylmethane sulfonate (EMS)–mutagenized ES cells to their daughters, or by intercrossing offspring of chimeras. This strategy was possible because chimeras transmit variations of the same mutagenized diploid genome, whereas ENU-treated males transmit numerous unrelated genomes. This also results in a doubling of screenable mutations in a pedigree compared to germline ENU mutagenesis. Coupled with the flexibility to treat ES cells with a variety of potent mutagens and the ease of producing distributable, quality-controlled, long-term supplies of cells in a single experiment, this strategy offers a number of advantages for conducting forward genetic screens in mice.     

A primer on using pooled shRNA libraries for functional genomic screens

The discovery of RNA interference (RNAi) has revolutionized genetic analysis in mammalian cells. Loss-of-function RNAi screens enable rapid, functional annotation of the genome. Of the various RNAi approaches, pooled shRNA libraries have received considerable attention because of their versatility. A number of genome-wide shRNA libraries have been constructed against the human and mouse genomes, and these libraries can be readily applied to a variety of screens to interrogate the function of human and mouse genes in an unbiased fashion. We provide an introduction to the technical aspects of using pooled shRNA libraries for genetic screens.     

Analysis of Multiple Ethyl Methanesulfonate-Mutagenized Caenorhabditis elegans Strains by Whole-Genome Sequencing

Whole-genome sequencing (WGS) of organisms displaying a specific mutant phenotype is a powerful approach to identify the genetic determinants of a plethora of biological processes. We have previously validated the feasibility of this approach by identifying a point-mutated locus responsible for a specific phenotype, observed in an ethyl methanesulfonate (EMS)-mutagenized Caenorhabditis elegans strain. Here we describe the genome-wide mutational profile of 17 EMS-mutagenized genomes as assessed with a bioinformatic pipeline, called MAQGene. Surprisingly, we find that while outcrossing mutagenized strains does reduce the total number of mutations, a striking mutational load is still observed even in outcrossed strains. Such genetic complexity has to be taken into account when establishing a causative relationship between genotype and phenotype. Even though unintentional, the 17 sequenced strains described here provide a resource of allelic variants in almost 1000 genes, including 62 premature stop codons, which represent candidate knockout alleles that will be of further use for the C. elegans community to study gene function.INDUCING molecular lesions in a genome is an effective approach to interrogate the genome for its functional elements. Molecular lesions can be induced using a variety of methods. Because of their efficiency and their ability to generate alleles with various different alterations in gene activity (e.g., amorphic, antimorphic, hypomorphic, and hypermorphic), chemical mutagens, such as ethyl methanesulfonate (EMS), are frequently used in genetic mutant screens (Anderson 1995). However, due to mutagen efficiency, a mutant animal selected for a single-locus phenotype invariably contains EMS-induced “background mutations” in its genome. Experimenters try to minimize the potential impact of background mutations through outcrossing to animals with a wild-type genome. Yet no full snapshots of genome sequences right after EMS mutagenesis and after outcrossing have so far been provided to illustrate the extent of background mutations and the extent to which they can indeed be eliminated.Another caveat of using base-changing chemical mutagens is the relative difficulty associated with identifying the phenotype-causing molecular lesion. In multicellular genetic model organisms, mutant identification involves time-consuming positional cloning approaches, usually involving breeding with genetically marked strains that allow pinpointing of the location of a molecular lesion. Even with rapid, SNP-based mapping approaches in animals with short generation times, such as Caenorhabditis elegans, substantial time hurdles, particularly in the final, fine-mapping stages, still exist. Conceptually similar problems in defining the location of a molecular lesion are encountered by human geneticists who attempt to identify disease-causing genetic lesions.Whole-genome sequencing (WGS) is beginning to emerge as an efficient and cost-effective tool to shortcut time-consuming mapping and positional cloning efforts (Hobert 2010). The sequencing of an entire genome and its ensuing comparison to a wild-type reference genome can potentially directly pinpoint the molecular lesion that results in the mutant phenotype the animal has been selected for. Proof-of-concept studies in bacteria, yeast, plants, worms, and flies have validated the applicability of this approach (Sarin et al. 2008; Smith et al. 2008; Srivatsan et al. 2008; Blumenstiel et al. 2009; Irvine et al. 2009; Flowers et al. 2010).Present-day deep sequencing platforms used for WGS generate relatively short sequence reads, thereby posing the bioinformatic challenge to align those reads to a reference genome. We previously described a software pipeline, MAQGene, which is based on the standard alignment program MAQ (Li et al. 2008) and facilitates this bioinformatic step by providing the end user with an extensively curated list of sequence variants from a WGS run of a mutated genome compared to a reference genome (Bigelow et al. 2009). This pipeline can be used for well-annotated, assembled genomes, such as C. elegans or Drosophila. In this article, we describe that this pipeline can identify not only point mutations but also deletions. We then use this pipeline to analyze a total of 17 EMS-mutagenized genomes. We find that EMS-mutagenized genomes carry a significant mutational load including presumptive loss-of-function alleles in several protein-coding genes that can lead to synthetic genetic interactions, one of which we describe here in more detail. We show that outcrossing to wild-type animals can lighten the mutational load; however, a substantial number of sequence variants are also introduced during outcrossing. Even though background mutations uncovered by WGS may complicate the interpretation of mutant phenotypes, they do provide a potentially useful source for functional studies of the affected genes.     

Seamless site-directed mutagenesis of the <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> genome using CRISPR-Cas9

CRISPR assisted homology directed repair enables the introduction of virtually any modification to the Saccharomyces cerevisiae genome. Of obvious interest is the marker-free and seamless introduction of point mutations. To fulfill this promise, a strategy that effects single nucleotide changes while preventing repeated recognition and cutting by the gRNA/Cas9 complex is needed. We demonstrate a two-step method to introduce point mutations at 17 positions in the S. cerevisiae genome. We show the general applicability of the method, enabling the seamless introduction of single nucleotide changes at any location, including essential genes and non-coding regions. We also show a quantifiable phenotype for a point mutation introduced in gene GSH1. The ease and wide applicability of this general method, combined with the demonstration of its feasibility will enable genome editing at an unprecedented level of detail in yeast and other organisms.     

Dissemination of Cephalosporin Resistance Genes between Escherichia coli Strains from Farm Animals and Humans by Specific Plasmid Lineages

Third-generation cephalosporins are a class of β-lactam antibiotics that are often used for the treatment of human infections caused by Gram-negative bacteria, especially Escherichia coli. Worryingly, the incidence of human infections caused by third-generation cephalosporin-resistant E. coli is increasing worldwide. Recent studies have suggested that these E. coli strains, and their antibiotic resistance genes, can spread from food-producing animals, via the food-chain, to humans. However, these studies used traditional typing methods, which may not have provided sufficient resolution to reliably assess the relatedness of these strains. We therefore used whole-genome sequencing (WGS) to study the relatedness of cephalosporin-resistant E. coli from humans, chicken meat, poultry and pigs. One strain collection included pairs of human and poultry-associated strains that had previously been considered to be identical based on Multi-Locus Sequence Typing, plasmid typing and antibiotic resistance gene sequencing. The second collection included isolates from farmers and their pigs. WGS analysis revealed considerable heterogeneity between human and poultry-associated isolates. The most closely related pairs of strains from both sources carried 1263 Single-Nucleotide Polymorphisms (SNPs) per Mbp core genome. In contrast, epidemiologically linked strains from humans and pigs differed by only 1.8 SNPs per Mbp core genome. WGS-based plasmid reconstructions revealed three distinct plasmid lineages (IncI1- and IncK-type) that carried cephalosporin resistance genes of the Extended-Spectrum Beta-Lactamase (ESBL)- and AmpC-types. The plasmid backbones within each lineage were virtually identical and were shared by genetically unrelated human and animal isolates. Plasmid reconstructions from short-read sequencing data were validated by long-read DNA sequencing for two strains. Our findings failed to demonstrate evidence for recent clonal transmission of cephalosporin-resistant E. coli strains from poultry to humans, as has been suggested based on traditional, low-resolution typing methods. Instead, our data suggest that cephalosporin resistance genes are mainly disseminated in animals and humans via distinct plasmids.     

Clonal Architectures and Driver Mutations in Metastatic Melanomas

To reveal the clonal architecture of melanoma and associated driver mutations, whole genome sequencing (WGS) and targeted extension sequencing were used to characterize 124 melanoma cases. Significantly mutated gene analysis using 13 WGS cases and 15 additional paired extension cases identified known melanoma genes such as BRAF, NRAS, and CDKN2A, as well as a novel gene EPHA3, previously implicated in other cancer types. Extension studies using tumors from another 96 patients discovered a large number of truncation mutations in tumor suppressors (TP53 and RB1), protein phosphatases (e.g., PTEN, PTPRB, PTPRD, and PTPRT), as well as chromatin remodeling genes (e.g., ASXL3, MLL2, and ARID2). Deep sequencing of mutations revealed subclones in the majority of metastatic tumors from 13 WGS cases. Validated mutations from 12 out of 13 WGS patients exhibited a predominant UV signature characterized by a high frequency of C->T transitions occurring at the 3′ base of dipyrimidine sequences while one patient (MEL9) with a hypermutator phenotype lacked this signature. Strikingly, a subclonal mutation signature analysis revealed that the founding clone in MEL9 exhibited UV signature but the secondary clone did not, suggesting different mutational mechanisms for two clonal populations from the same tumor. Further analysis of four metastases from different geographic locations in 2 melanoma cases revealed phylogenetic relationships and highlighted the genetic alterations responsible for differential drug resistance among metastatic tumors. Our study suggests that clonal evaluation is crucial for understanding tumor etiology and drug resistance in melanoma.     

Anecdotal,Historical and Critical Commentaries on Genetics: The Impact of Whole Genome Sequencing on Model System Genetics: Get Ready for the Ride

Much of our understanding of how organisms develop and function is derived from the extraordinarily powerful, classic approach of screening for mutant organisms in which a specific biological process is disrupted. Reaping the fruits of such forward genetic screens in metazoan model systems like Drosophila, Caenorhabditis elegans, or zebrafish traditionally involves time-consuming positional cloning strategies that result in the identification of the mutant locus. Whole genome sequencing (WGS) has begun to provide an effective alternative to this approach through direct pinpointing of the molecular lesion in a mutated strain isolated from a genetic screen. Apart from significantly altering the pace and costs of genetic analysis, WGS also provides new perspectives on solving genetic problems that are difficult to tackle with conventional approaches, such as identifying the molecular basis of multigenic and complex traits.GENETIC model systems, from bacteria, yeast, plants, worms, flies, and fish to mice allow the dissection of the genetic basis of virtually any biological process by isolating mutants obtained through random mutagenesis, in which the biological process under investigation is defective. Such forward genetic analysis is unbiased and free of assumptions. The rigor and conceptual simplicity of forward genetic analysis is striking, some may say, beautiful; and the unpredictability of what one finds—be that an unexpected phenotype popping out of a screen or the eventual molecular nature of the gene (take the discovery of miRNAs as an example; Lee et al. 1993)—appeals to the adventurous. Even though mutant phenotypic analysis alone can reveal the logic of underlying biological processes (take Ed Lewis'' analysis of homeotic mutants as an example; Lewis 1978)—it is the identification of the molecular lesions in mutant animals that provides the key mechanistic and molecular details that propel our understanding of biological processes.The identification of the molecular lesion in mutant organisms depends on how the mutation was introduced. Classically, two types of mutagens have been used in most model systems: biological agents such as plasmids, viruses, or transposons whose insertions disrupt functional DNA elements (either coding or regulatory elements) or chemical mutagens, such as ethyl methane sulfonate (EMS) or N-ethyl N-nitroso urea (ENU), that introduce point mutations or deletions. Point mutation-inducing chemical mutagens are in many ways a superior mutagenic agent because their mutational frequency is high and because the spectrum of their effects on a given locus—producing hypomorphs, hypermorphs, amorphs, neomorphs, etc.—is hard to match by biological mutagens. Moreover, chemical mutagens do not display the positional bias of many biological agents. In addition, point mutations in a gene are often crucial in dissecting the functionally relevant domains of the gene product. In spite of the advantages of chemical mutagens, model system geneticists often prefer biological mutagens simply because the molecular lesions induced by those agents are characterized by the easily locatable DNA footprint that these agents generate. In contrast, the location of a point mutation (or deletion) has to be identified through conventional mapping strategies, which tend to be tedious and time consuming. Even in model systems in which positional cloning is quite fast and straightforward (e.g., Caenorhabditis elegans, which has a short generation time and a multitude of mapping tools available), it nevertheless is a significant effort that can occasionally present hurdles that are difficult to surmount (e.g., if the gene maps into a region with few genetic markers that allow for mapping). These difficulties explain why RNAi-based “genetic screens” have gained significant popularity in C. elegans; they circumvent mapping and reveal molecular identities of genes involved in a given process straight away (Kamath and Ahringer 2003). However, genes and cells show differential susceptibility to RNAi; off-target effects and lack of reproducibility can be a problem, and the range of effects that RNAi has on gene activity is generally more limited compared to chemically induced gene mutations.The recent application of next generation, deep sequencing technology (see Bentley 2006; Morozova and Marra 2008 for technology reviews) is beginning to significantly alter the landscape of genetic analysis as it allows the use of chemical mutagens without having to deal with its disadvantages. Deep sequencing technology incorporated into platforms such as Illumina''s Genome Analyzer or ABI''s SOLiD, allows one-shot sequencing of the entire model system''s genome, resulting in the detection of mutagen-induced sequence alterations compared to a nonmutagenized reference genome. Proof-of-concept studies have so far been conducted in bacteria, yeast, plant, worms, and flies, all published within the last year (Sarin et al. 2008; Smith et al. 2008; Srivatsan et al. 2008; Blumenstiel et al. 2009; Irvine et al. 2009; Rigola et al. 2009). Many more studies are under way; for example, since our first proof-of-principle study (Sarin et al. 2008), my laboratory has identified the molecular basis of >10 C. elegans strains defective in neuronal development and homeostasis (V. Bertrand, unpublished data; M. Doitsidou, unpublished data; E. Flowers, unpublished data; S. Sarin, unpublished data).The advantages of whole genome sequencing (WGS) are obvious. The process is extraordinarily fast with the sequencing taking only ∼5 days and the subsequent sequence data analysis only a few hours, particularly if the end user employs bioinformatic tools customized for mutant detection (Bigelow et al. 2009). The process is also remarkably cost effective. For example, a C. elegans genome can be sequenced with a required sequence coverage of ∼10 times for <$2,000 in reagent and machine operating costs. The capacity of deep sequencing machines—and hence the costs associated with sequencing a genome—apparently follow Moore''s law of doubling its capacity about every 2 years, like many technological innovations do (Pettersson et al. 2009). That is, the <$1,000 genome for C. elegans (∼100-Mb genome) and Drosophila (∼123-Mb genome) is just around the corner and other models will sooner or later follow suit. The cost effectiveness becomes particularly apparent if one compares the cost of WGS to the personnel and reagent costs associated with multiple-month to multiple-year mapping-based cloning efforts.WGS identifies sequence variants between a mutated genome and a premutagenesis reference genome. Chemical mutagens randomly introduce many mutations in the genome and, therefore, the phenotype-causing sequence variant needs to be identified as such out of a large pool of sequence variants. Sequence variants that have no impact on the phenotype can be outcrossed before sequencing or eliminated through some rough mapping of the mutation, which allows the experimenter to focus only on those variants contained in a specific sequence interval. Ensuing functional tests such as transformation rescue or phenocopy by RNAi and the availability of other alleles of the same locus are critical means to validate a phenotype-causing sequence variant (Sarin et al. 2008). The latter approach—the availability of multiple alleles of the same locus—is in many ways the most powerful one to sift through a number of candidate variants revealed by WGS. In this approach, candidate loci revealed by WGS are resequenced by conventional Sanger sequencing in allelic strains and only those that are indeed phenotype causing will show up mutated in all allelic variants of the locus (Sarin et al. 2008). The availability of multiple alleles of a locus is highly desirable for many aspects of genetic analysis anyway and therefore does not represent an additional and specific burden for undertaking a WGS project.The importance of WGS on model system genetics will be substantial and wide ranging. Speed and cost effectiveness means that the wastelands of genetic mapping can be trespassed fast enough to allow an experimenter to multitask a whole mutant collection in parallel, thereby closing in on the “holy grail” of genetic analysis—the as-complete-as-possible mutational saturation of a biological process and the resulting deciphering of complete genetic pathways and networks. What will become limiting steps are not any more the tediousness of mapping, but rather the effectiveness with which mutant collections can be built. Novel technologies that involve machine-based, semiautomated selection of mutant animals have been developed over the past few years to study a variety of distinct biological processes in several metazoan model systems, e.g., gfp-based morphology or cell fate screens in worms (Crane et al. 2009; Doitsidou et al. 2008) or behavioral screens in flies (Dankert et al. 2009) and are important steps in this direction. Such an “industrial revolution” of genetic screening (i.e., the mutant selection part, followed by WGS) moves us geneticists away from, not into the trenches of factory life and frees us up to do what we should like to enjoy most—thinking of designing interesting screens, seeing how genes interact, and interpreting it all.Another important impact of WGS is that it will allow tackling problems that were previously hard to deal with. For example, the tediousness of following subtle phenotypes, low penetrance phenotypes, or phenotypes that are cumbersome to score often hampers positional cloning approaches that rely on identifying rare recombinants in a large sibling pool. Moreover, many genetic traits such as behavioral genetic traits are very sensitive to genetic background and are therefore also often hard to map in the conventional way. WGS hones in on candidate genes straight away. Taking this notion a step further, WGS will also be able to get at the molecular basis of multigenic traits and quantitative trait loci, which again are hard to molecularly identify through conventional mapping strategies; a proof-of-principle study has made this point already in bacteria (Srivatsan et al. 2008). In principle, such multigenic traits may have been mutationally induced or could be present in natural variants of a species, which provides intriguing perspective for the population geneticist.Model organisms of biological interest that were previously relatively intractable for classic genetic mutant analysis due to the absence of genetic markers or other practical problems such as prohibitive generation times, may also now be movable into the arena of genetic model systems, through the WGS-mediated molecular analysis of mutagen-induced variants or through the study of natural variants.The sequencing of human cancer genomes has already begun to illustrate the impact of WGS on human genetics (Campbell et al. 2008; Ley et al. 2008). However, those human WGS studies illustrate why model systems will continue to be extremely important—their experimental accessibility allows us to address which of the many variants detected by WGS is indeed the phenotype-causing one.The message to model system geneticists is clear: Get access to a deep sequencer, buckle up, and get ready for the ride.     

Conditional DNA repair mutants enable highly precise genome engineering

Oligonucleotide-mediated multiplex genome engineering is an important tool for bacterial genome editing. The efficient application of this technique requires the inactivation of the endogenous methyl-directed mismatch repair system that in turn leads to a drastically elevated genomic mutation rate and the consequent accumulation of undesired off-target mutations. Here, we present a novel strategy for mismatch repair evasion using temperature-sensitive DNA repair mutants and temporal inactivation of the mismatch repair protein complex in Escherichia coli. Our method relies on the transient suppression of DNA repair during mismatch carrying oligonucleotide integration. Using temperature-sensitive control of methyl-directed mismatch repair protein activity during multiplex genome engineering, we reduced the number of off-target mutations by 85%, concurrently maintaining highly efficient and unbiased allelic replacement.     

N-Ethyl-N-Nitrosourea Mutagenesis: Boarding the Mouse Mutant Express

In the mouse, random mutagenesis with N-ethyl-N-nitrosourea (ENU) has been used since the 1970s in forward mutagenesis screens. However, only in the last decade has ENU mutagenesis been harnessed to generate a myriad of new mouse mutations in large-scale genetic screens and focused, smaller efforts. The development of additional genetic tools, such as balancer chromosomes, refinements in genetic mapping strategies, and evolution of specialized assays, has allowed these screens to achieve new levels of sophistication. The impressive productivity of these screens has led to a deluge of mouse mutants that wait to be harnessed. Here the basic large- and small-scale strategies are described, as are the basics of screen design. Finally, and importantly, this review describes the mechanisms by which such mutants may be accessed now and in the future. Thus, this review should serve both as an overview of the power of forward mutagenesis in the mouse and as a resource for those interested in developing their own screens, adding onto existing efforts, or obtaining specific mouse mutants that have already been generated.     

Large Scale Identification of Genes Involved in Cell Surface Biosynthesis and Architecture in Saccharomyces Cerevisiae

The sequenced yeast genome offers a unique resource for the analysis of eukaryotic cell function and enables genome-wide screens for genes involved in cellular processes. We have identified genes involved in cell surface assembly by screening transposon-mutagenized cells for altered sensitivity to calcofluor white, followed by supplementary screens to further characterize mutant phenotypes. The mutated genes were directly retrieved from genomic DNA and then matched uniquely to a gene in the yeast genome database. Eighty-two genes with apparent perturbation of the cell surface were identified, with mutations in 65 of them displaying at least one further cell surface phenotype in addition to their modified sensitivity to calcofluor. Fifty of these genes were previously known, 17 encoded proteins whose function could be anticipated through sequence homology or previously recognized phenotypes and 15 genes had no previously known phenotype.     

Plasmid Flux in Escherichia coli ST131 Sublineages,Analyzed by Plasmid Constellation Network (PLACNET), a New Method for Plasmid Reconstruction from Whole Genome Sequences

Bacterial whole genome sequence (WGS) methods are rapidly overtaking classical sequence analysis. Many bacterial sequencing projects focus on mobilome changes, since macroevolutionary events, such as the acquisition or loss of mobile genetic elements, mainly plasmids, play essential roles in adaptive evolution. Existing WGS analysis protocols do not assort contigs between plasmids and the main chromosome, thus hampering full analysis of plasmid sequences. We developed a method (called plasmid constellation networks or PLACNET) that identifies, visualizes and analyzes plasmids in WGS projects by creating a network of contig interactions, thus allowing comprehensive plasmid analysis within WGS datasets. The workflow of the method is based on three types of data: assembly information (including scaffold links and coverage), comparison to reference sequences and plasmid-diagnostic sequence features. The resulting network is pruned by expert analysis, to eliminate confounding data, and implemented in a Cytoscape-based graphic representation. To demonstrate PLACNET sensitivity and efficacy, the plasmidome of the Escherichia coli lineage ST131 was analyzed. ST131 is a globally spread clonal group of extraintestinal pathogenic E. coli (ExPEC), comprising different sublineages with ability to acquire and spread antibiotic resistance and virulence genes via plasmids. Results show that plasmids flux in the evolution of this lineage, which is wide open for plasmid exchange. MOB_F12/IncF plasmids were pervasive, adding just by themselves more than 350 protein families to the ST131 pangenome. Nearly 50% of the most frequent γ–proteobacterial plasmid groups were found to be present in our limited sample of ten analyzed ST131 genomes, which represent the main ST131 sublineages.     

Systematic Screening of Drosophila Deficiency Mutations for Embryonic Phenotypes and Orphan Receptor Ligands

This paper defines a collection of Drosophila deletion mutations (deficiencies) that can be systematically screened for embryonic phenotypes, orphan receptor ligands, and genes affecting protein localization. It reports the results of deficiency screens we have conducted that have revealed new axon guidance phenotypes in the central nervous system and neuromuscular system and permitted a quantitative assessment of the number of potential genes involved in regulating guidance of specific motor axon branches. Deficiency “kits” that cover the genome with a minimum number of lines have been established to facilitate gene mapping. These kits cannot be systematically analyzed for phenotypes, however, since embryos homozygous for many deficiencies in these kits fail to develop due to the loss of key gene products encoded within the deficiency. To create new kits that can be screened for phenotype, we have examined the development of the nervous system in embryos homozygous for more than 700 distinct deficiency mutations. A kit of ∼400 deficiency lines for which homozygotes have a recognizable nervous system and intact body walls encompasses >80% of the genome. Here we show examples of screens of this kit for orphan receptor ligands and neuronal antigen expression. It can also be used to find genes involved in expression, patterning, and subcellular localization of any protein that can be visualized by antibody staining. A subset kit of 233 deficiency lines, for which homozygotes develop relatively normally to late stage 16, covers ∼50% of the genome. We have screened it for axon guidance phenotypes, and we present examples of new phenotypes we have identified. The subset kit can be used to screen for phenotypes affecting all embryonic organs. In the future, these deficiency kits will allow Drosophila researchers to rapidly and efficiently execute genome-wide anatomical screens that require examination of individual embryos at high magnification.     

Alignment-free design of highly discriminatory diagnostic primer sets for Escherichia coli O104:H4 outbreak strains

Background

An Escherichia coli O104:H4 outbreak in Germany in summer 2011 caused 53 deaths, over 4000 individual infections across Europe, and considerable economic, social and political impact. This outbreak was the first in a position to exploit rapid, benchtop high-throughput sequencing (HTS) technologies and crowdsourced data analysis early in its investigation, establishing a new paradigm for rapid response to disease threats. We describe a novel strategy for design of diagnostic PCR primers that exploited this rapid draft bacterial genome sequencing to distinguish between E. coli O104:H4 outbreak isolates and other pathogenic E. coli isolates, including the historical hæmolytic uræmic syndrome (HUSEC) E. coli HUSEC041 O104:H4 strain, which possesses the same serotype as the outbreak isolates.

Methodology/Principal Findings

Primers were designed using a novel alignment-free strategy against eleven draft whole genome assemblies of E. coli O104:H4 German outbreak isolates from the E. coli O104:H4 Genome Analysis Crowd-Sourcing Consortium website, and a negative sequence set containing 69 E. coli chromosome and plasmid sequences from public databases. Validation in vitro against 21 ‘positive’ E. coli O104:H4 outbreak and 32 ‘negative’ non-outbreak EHEC isolates indicated that individual primer sets exhibited 100% sensitivity for outbreak isolates, with false positive rates of between 9% and 22%. A minimal combination of two primers discriminated between outbreak and non-outbreak E. coli isolates with 100% sensitivity and 100% specificity.

Conclusions/Significance

Draft genomes of isolates of disease outbreak bacteria enable high throughput primer design and enhanced diagnostic performance in comparison to traditional molecular assays. Future outbreak investigations will be able to harness HTS rapidly to generate draft genome sequences and diagnostic primer sets, greatly facilitating epidemiology and clinical diagnostics. We expect that high throughput primer design strategies will enable faster, more precise responses to future disease outbreaks of bacterial origin, and help to mitigate their societal impact.     

FRUIT,a Scar-Free System for Targeted Chromosomal Mutagenesis,Epitope Tagging,and Promoter Replacement in Escherichia coli and Salmonella enterica

Recombineering is a widely-used approach to delete genes, introduce insertions and point mutations, and introduce epitope tags into bacterial chromosomes. Many recombineering methods have been described, for a wide range of bacterial species. These methods are often limited by (i) low efficiency, and/or (ii) introduction of “scar” DNA into the chromosome. Here, we describe a rapid, efficient, PCR-based recombineering method, FRUIT, that can be used to introduce scar-free point mutations, deletions, epitope tags, and promoters into the genomes of enteric bacteria. The efficiency of FRUIT is far higher than that of the most widely-used recombineering method for Escherichia coli. We have used FRUIT to introduce point mutations and epitope tags into the chromosomes of E. coli K-12, Enterotoxigenic E. coli, and Salmonella enterica. We have also used FRUIT to introduce constitutive and inducible promoters into the chromosome of E. coli K-12. Thus, FRUIT is a versatile, efficient recombineering approach that can be applied in multiple species of enteric bacteria.     

Development of a genome-targeting mutator for the adaptive evolution of microbial cells

Methods that can randomly introduce mutations in the microbial genome have been used for classical genetic screening and, more recently, the evolutionary engineering of microbial cells. However, most methods rely on either cell-damaging agents or disruptive mutations of genes that are involved in accurate DNA replication, of which the latter requires prior knowledge of gene functions, and thus, is not easily transferable to other species. In this study, we developed a new mutator for in vivo mutagenesis that can directly modify the genomic DNA. Mutator protein, MutaEco, in which a DNA-modifying enzyme is fused to the α-subunit of Escherichia coli RNA polymerase, increases the mutation rate without compromising the cell viability and accelerates the adaptive evolution of E. coli for stress tolerance and utilization of unconventional carbon sources. This fusion strategy is expected to accommodate diverse DNA-modifying enzymes and may be easily adapted to various bacterial species.     

Whole Genome Sequencing versus Traditional Genotyping for Investigation of a Mycobacterium tuberculosis Outbreak: A Longitudinal Molecular Epidemiological Study

Background

Understanding Mycobacterium tuberculosis (Mtb) transmission is essential to guide efficient tuberculosis control strategies. Traditional strain typing lacks sufficient discriminatory power to resolve large outbreaks. Here, we tested the potential of using next generation genome sequencing for identification of outbreak-related transmission chains.

Methods and Findings

During long-term (1997 to 2010) prospective population-based molecular epidemiological surveillance comprising a total of 2,301 patients, we identified a large outbreak caused by an Mtb strain of the Haarlem lineage. The main performance outcome measure of whole genome sequencing (WGS) analyses was the degree of correlation of the WGS analyses with contact tracing data and the spatio-temporal distribution of the outbreak cases. WGS analyses of the 86 isolates revealed 85 single nucleotide polymorphisms (SNPs), subdividing the outbreak into seven genome clusters (two to 24 isolates each), plus 36 unique SNP profiles. WGS results showed that the first outbreak isolates detected in 1997 were falsely clustered by classical genotyping. In 1998, one clone (termed “Hamburg clone”) started expanding, apparently independently from differences in the social environment of early cases. Genome-based clustering patterns were in better accordance with contact tracing data and the geographical distribution of the cases than clustering patterns based on classical genotyping. A maximum of three SNPs were identified in eight confirmed human-to-human transmission chains, involving 31 patients. We estimated the Mtb genome evolutionary rate at 0.4 mutations per genome per year. This rate suggests that Mtb grows in its natural host with a doubling time of approximately 22 h (400 generations per year). Based on the genome variation discovered, emergence of the Hamburg clone was dated back to a period between 1993 and 1997, hence shortly before the discovery of the outbreak through epidemiological surveillance.

Conclusions

Our findings suggest that WGS is superior to conventional genotyping for Mtb pathogen tracing and investigating micro-epidemics. WGS provides a measure of Mtb genome evolution over time in its natural host context. Please see later in the article for the Editors'' Summary