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Random transgene integration is a powerful tool for developing new genome-wide screening approaches. These techniques have already been used for functional gene annotation by transposon-insertion sequencing, for identif ication of transcription factor binding sites and regulatory sequences, and for dissecting chromatin position effects. Precise localization of transgenes and accurate artifact f iltration are essential for this type of method. To date, many mapping assays have been developed, including Inverse-PCR, TLA, LAM-PCR, and splinkerette PCR. However, none of them is able to ensure localization of both transgene's f lanking regions simultaneously, which would be necessary for some applications. Here we proposed a cheap and simple NGS-based approach that overcomes this limitation. The developed assay requires using intentionally designed vectors that lack recognition sites of one or a set of restriction enzymes used for DNA fragmentation. By looping and sequencing these DNA fragments, we obtain special data that allows us to link the two f lanking regions of the transposon. This can be useful for precise insertion mapping and for screening approaches in the f ield of chromosome engineering, where chromosomal recombination events between transgenes occur in a cell population. To demonstrate the method's feasibility, we applied it for mapping SB transposon integration in the human HAP1 cell line. Our technique allowed us to eff iciently localize genomic transposon integrations, which was conf irmed via PCR analysis. For practical application of this approach, we proposed a set of recommendations and a normalization strategy. The developed method can be used for multiplex transgene localization and detection of rearrangements between them.Полногеномные скрининговые методы, основанные на случайной интеграции экзогенных генетических конструкций, – новейший класс инструментов, открывающий возможности для изучения широкого спектра геномных процессов. Данный подход уже был применен к функциональному аннотированию генов млекопитающих, скринингу приспособленности бактерий, определению сайтов связывания факторов транскрипции, идентификации регуляторных генетических элементов и исследованию хромосомного эффекта положения. Все эти эксперименты требуют точной локализации трансгенов в геноме. Существующие на сегодняшний день методы картирования, такие как Inverse-PCR, TLA, splinkerette PCR и LAM-PCR, не позволяют одновременно определять оба участка генома, фланкирующие одну интеграцию трансгена, что ограничивает применимость подходов, в том числе связанных с хромосомной инженерией. В настоящей работе мы предлагаем метод, с помощью которого можно преодолеть это ограничение. Разработанная технология основана на фрагментации геномной ДНК, не затрагивающей интеграции трансгена. Это достигается путем исключения из последовательности вектора сайтов узнавания одного или нескольких ферментов рестрикции. Затем, как и в Inverse-PCR, были закольцованы молекулы лигированием в разбавленной смеси и секвенированы. Полученные данные дают возможность с высокой точностью идентифицировать перестройки и отделить их от артефактов лигирования, и, кроме того, отследить события транслокаций между интеграциями трансгенов. Это может быть использовано в экспериментах по изучению индуцируемых хромосомных перестроек. Для доказательства применимости метода мы с его помощью картировали интеграции транспозона Sleeping Beauty в клетки человека линии Hap1. Картированные интеграции были валидированы с помощью ПЦР-анализа. В статье приведен ряд рекомендаций для практического использования этого метода в экспериментах по множественной локализации интеграций трансгенных конструкций.

As promising biomarkers and therapy targets, microRNAs (miRNAs) are involved in various physiological and tumorigenic processes. Genetic variants in miRNA‐binding sites can lead to dysfunction of miRNAs and contribute to disease. However, systematic investigation of the miRNA‐related single nucleotide polymorphisms (SNPs) for pancreatic cancer (PC) risk remains elusive. We performed integrative bioinformatics analyses to select 31 SNPs located in miRNA‐target binding sites using the miRNASNP v2.0, a solid database providing miRNA‐related SNPs for genetic research, and investigated their associations with risk of PC in two large case‐control studies totally including 1847 cases and 5713 controls. We observed that the SNP rs3802266 is significantly associated with increased risk of PC (odds ratio (OR) = 1.21, 95% confidence intervals (CI) = 1.11‐1.31, P = 1.29E‐05). Following luciferase reporter gene assays show that rs3802266‐G creates a stronger binding site for miR‐181a‐2‐3p in 3′ untranslated region (3′UTR) of the gene ZHX2. Expression quantitative trait loci (eQTL) analysis suggests that ZHX2 expression is lower in individuals carrying rs3802266‐G with increased PC risk. In conclusion, our findings highlight the involvement of miRNA‐binding SNPs in PC susceptibility and provide new clues for PC carcinogenesis.

Background Macrophages, besides resting latently infected CD4+ T cells, constitute the predominant stable, major non-T cell HIV reservoirs. Therefore, it is essential to eliminate both latently infected CD4+ T cells and tissue macrophages to completely eradicate HIV in patients. Until now, most of the research focus is directed towards eliminating latently infected CD4+ T cells. However, few approaches have been directed at killing of HIV-infected macrophages either in vitro or in vivo. HIV infection dysregulates the expression of many host genes essential for the survival of infected cells. We postulated that exploiting this alteration may yield novel targets for the selective killing of infected macrophages. Methods We applied a pooled shRNA-based genome-wide approach by employing a lentivirus-based library of shRNAs to screen novel gene targets whose inhibition should selectively induce apoptosis in HIV-infected macrophages. Primary human MDMs were infected with HIV-eGFP and HIV-HSA viruses. Infected MDMs were transfected with siRNAs specific for the promising genes followed by analysis of apoptosis by flow cytometry using labelled Annexin-V in HIV-infected, HIV-exposed but uninfected bystander MDMs and uninfected MDMs. The results were analyzed using student’s t-test from at least four independent experiments. Results We validated 28 top hits in two independent HIV infection models. This culminated in the identification of four target genes, Cox7a2, Znf484, Cstf2t, and Cdk2, whose loss-of-function induced apoptosis preferentially in HIV-infected macrophages. Silencing these single genes killed significantly higher number of HIV-HSA-infected MDMs compared to the HIV-HSA-exposed, uninfected bystander macrophages, indicating the specificity in the killing of HIV-infected macrophages. The mechanism governing Cox7a2-mediated apoptosis of HIV-infected macrophages revealed that targeting respiratory chain complex II and IV genes also selectively induced apoptosis of HIV-infected macrophages possibly through enhanced ROS production. Conclusions We have identified above-mentioned novel genes and specifically the respiratory chain complex II and IV genes whose silencing may cause selective elimination of HIV-infected macrophages and eventually the HIV-macrophage reservoirs. The results highlight the potential of the identified genes as targets for eliminating HIV-infected macrophages in physiological environment as part of an HIV cure strategy.

Conjugal transfer is a major driving force of genetic exchange in eubacteria, and the system in IncP1-type broad-host-range plasmids transfers DNA even to eukaryotes and archaea in a process known as trans-kingdom conjugation (TKC). Although conjugation factors encoded on plasmids have been extensively analyzed, those on the donor chromosome have not. To identify the potential conjugation factor(s), a genome-wide survey on a comprehensive collection of Escherichia coli gene knockout mutants (Keio collection) as donors to Saccharomyces cerevisiae recipients was performed using a conjugal transfer system mediated by the type IV secretion system (T4SS) of the IncP1α plasmid. Out of 3,884 mutants, three mutants (ΔfrmR, ΔsufA, and ΔiscA) were isolated, which showed an increase by one order of magnitude in both E. coli–E. coli and E. coli–yeast conjugations without an increase in the mRNA accumulation level for the conjugation related genes examined. The double-knockout mutants for these genes (ΔfrmRΔsufA and ΔiscAΔfrmR) did not show synergistic effects on the conjugation efficiency, suggesting that these factors affect a common step in the conjugation machinery. The three mutants demonstrated increased conjugation efficiency in IncP1β-type but not in IncN- and IncW-type broad-host-range plasmid transfers, and the homologous gene knockout mutants against the three genes in Agrobacterium tumefaciens also showed increased TKC efficiency. These results suggest the existence of a specific regulatory system in IncP1 plasmids that enables the control of conjugation efficiency in different hosts, which could be utilized for the development of donor strains as gene introduction tools into bacteria, eukaryotes, and archaea.

Shiga toxin (STx) or Vero toxin is a virulence factor produced by enterohemorrhagic Escherichia coli. The toxin binds to the glycosphingolipid globotriaosylceramide (Gb3) for its entry, and causes cell death by inhibiting ribosome function. Previously, we performed a loss-of-function screen in HeLa cells using a human CRISPR knockout (KO) library and identified various host genes required for STx-induced cell death. To determine whether this library targeted to the human genome is applicable to non-human primate cells and to identify previously unrecognized factors crucial for STx-induced cell death, we herein performed a similar screen in the African green monkey kidney-derived Vero C1008 subline. Many genes relevant to metabolic enzymes and membrane trafficking were enriched, although the number of enriched genes was less than that obtained in the screening for HeLa cells. Of note, several genes that had not been enriched in the previous screening were enriched: one of these genes was SYS1, which encodes a multi-spanning membrane protein in the Golgi apparatus. In SYS1 KO Vero cells, expression of Gb3 and sphingomyelin was decreased, while that of glucosylceramide and lactosylceramide was increased. In addition, loss of SYS1 inhibited the biosynthesis of protein glycans, deformed the Golgi apparatus, and perturbed the localization of trans-Golgi network protein (TGN) 46. These results indicate that the human CRISPR KO library is applicable to Vero cell lines, and SYS1 has a widespread effect on glycan biosynthesis via regulation of intra-Golgi and endosome–TGN retrograde transports.

The use of oxidizing agents is one of the most favorable approaches to kill bacteria in daily life. However, bacteria have been evolving to survive in the presence of different oxidizing agents. In this study, we aimed to obtain a comprehensive list of genes whose expression can make Escherichiacoli cells resistant to different oxidizing agents. For this purpose, we utilized the ASKA library and performed a genome-wide screening of ~4200 E. coli genes. Hydrogen peroxide (H2O2) and hypochlorite (HOCl) were tested as representative oxidizing agents in this study. To further validate our screening results, we used different E. coli strains as host cells to express or inactivate selected resistance genes individually. More than 100 genes obtained in this screening were not known to associate with oxidative stress responses before. Thus, this study is expected to facilitate both basic studies on oxidative stress and the development of antibacterial agents.

The emergence of widespread antimicrobial resistance has led to the need for development of novel therapeutic interventions. Antivirulence strategies are an attractive alternative to classic antimicrobial therapy; however, they require identification of new specific targets which can be exploited in drug discovery programs., The opportunistic pathogen Pseudomonas aeruginosa produces an arsenal of virulence factors causing a wide range of diseases in multiple hosts and is difficult to eradicate due to its intrinsic resistance to antibiotics. With the antibacterial pipeline drying up, antivirulence therapy has become an attractive alternative strategy to the traditional use of antibiotics to treat P. aeruginosa infections. To identify P. aeruginosa genes required for virulence in multiple hosts, a random library of Tn5 mutants in strain PAO1-L was previously screened in vitro for those showing pleiotropic effects in the production of virulence phenotypes. Using this strategy, we identified a Tn5 mutant with an insertion in PA4130 showing reduced levels of a number of virulence traits in vitro. Construction of an isogenic mutant in this gene presented results similar to those for the Tn5 mutant. Furthermore, the PA4130 isogenic mutant showed substantial attenuation in disease models of Drosophila melanogaster and Caenorhabditis elegans as well as reduced toxicity in human cell lines. Mice infected with this mutant demonstrated an 80% increased survival rate in acute and agar bead lung infection models. PA4130 codes for a protein with homology to nitrite and sulfite reductases. Overexpression of PA4130 in the presence of the siroheme synthase CysG enabled its purification as a soluble protein. Methyl viologen oxidation assays with purified PA4130 showed that this enzyme is a nitrite reductase operating in a ferredoxin-dependent manner. The preference for nitrite and production of ammonium revealed that PA4130 is an ammonia:ferredoxin nitrite reductase and hence was named NirA.

Significance Wnt activation is critically important during both early development and throughout adulthood for the maintenance and regeneration of tissue-resident stem cells. However, the underlying mechanism regulating Wnt-dependent epithelial renewal and differentiation in the stomach remains poorly understood. Here we apply genome-scale CRISPR-Cas9 knockout screening to three-dimensional ex vivo stomach organoids to reveal important mechanistic insight into Wnt-dependent epithelial renewal in mouse gastric epithelia. Our organoid-based screening strategy can be readily applied to a wide variety of organoids derived from different sources, potentially providing important insights on multiple biological phenomena., An ability to safely harness the powerful regenerative potential of adult stem cells for clinical applications is critically dependent on a comprehensive understanding of the underlying mechanisms regulating their activity. Epithelial organoid cultures accurately recapitulate many features of in vivo stem cell-driven epithelial renewal, providing an excellent ex vivo platform for interrogation of key regulatory mechanisms. Here, we employed a genome-scale clustered, regularly interspaced, short palindromic repeats (CRISPR) knockout (KO) screening assay using mouse gastric epithelial organoids to identify modulators of Wnt-driven stem cell-dependent epithelial renewal in the gastric mucosa. In addition to known Wnt pathway regulators, such as Apc, we found that KO of Alk, Bclaf3, or Prkra supports the Wnt independent self-renewal of gastric epithelial cells ex vivo. In adult mice, expression of these factors is predominantly restricted to non-Lgr5–expressing stem cell zones above the gland base, implicating a critical role for these factors in suppressing self-renewal or promoting differentiation of gastric epithelia. Notably, we found that Alk inhibits Wnt signaling by phosphorylating the tyrosine of Gsk3β, while Bclaf3 and Prkra suppress regenerating islet-derived (Reg) genes by regulating the expression of epithelial interleukins. Therefore, Alk, Bclaf3, and Prkra may suppress stemness/proliferation and function as novel regulators of gastric epithelial differentiation.

Extracellular interactions between cell surface receptors are necessary for signaling and adhesion but identifying them remains technically challenging. We describe a cell-based genome-wide approach employing CRISPR activation to identify receptors for a defined ligand. We show receptors for high-affinity antibodies and low-affinity ligands can be unambiguously identified when used in pools or as individual binding probes. We apply this technique to identify ligands for the adhesion G-protein-coupled receptors and show that the Nogo myelin-associated inhibitory proteins are ligands for ADGRB1. This method will enable extracellular receptor-ligand identification on a genome-wide scale. Electronic supplementary material The online version of this article (10.1186/s13059-018-1581-3) contains supplementary material, which is available to authorized users.

Small GTPases function as molecular switches to active or inactive signaling cascades via binding or hydrolyzing GTP. A type of plant specific small GTPases, the ROPs are known to be involved in plant growth, development and immunity. We determined whether ROPs are conserved in Solanaceous species and whether they are involved in plant growth, development and resistance against Phytophthora capsisi. In genome-wide screening, a total of 66 ROPs in six Solanaceous species (SolROPs) were identified, including 16 ROPs in Solanum tuberosum L. (potato), 9 in Solanum lycopersicum L. (tomato), 5 in Solanum melongena L. (eggplant), 9 in Capsicum annuum L. (pepper), 13 in Nicotiana benthamiana Domin and 14 in Nicotiana tabacum L. (tobacco). Phylogenetic analysis revealed that 11 AtROPs and 66 SolROPs fall into five distinct clades (I-V) and hence a novel and systematic gene nomenclature was proposed. In addition, a comprehensive expression analysis was performed by making use of an online database. This revealed that ROP genes are differentially expressed during plant growth and development. Moreover, gene expression of SlROP-II.1 in S. lycopersicum could be significantly induced by P. capsici. Subsequently, SlROP-II.1 and its homologues in N. benthamiana and C. annuum (NbROP-II.1 and CaROP-II.1) were selected for functional analysis using virus-induced gene silencing. Infection assays with P. capsici on silenced plants revealed that SlROP-II.1, NbROP-II.1 and CaROP-II.1 play a role in P. capsici resistance, suggesting conserved function of ROP-II clade across different Solanaceous species. In addition, NbROP-II.1 is also involved in regulating plant growth and development. This study signified the diversity of Solanaceous ROPs and their potential roles in plant growth, development and immunity.

The conjugal transfer is a major driving force in the spread of antibiotic resistance genes. Nevertheless, an effective approach has not yet been developed to target conjugal transfer to prevent the acquisition of antibiotic resistance by this mechanism. This study aimed to identify potential targets for plasmid transfer blockade by isolating mutants defective in the completion of the acquisition of antibiotic resistance via conjugal transfer. We performed genome-wide screening by combining an IncP1α-type broad host range plasmid conjugation system with a comprehensive collection of Escherichia coli gene knockout mutants (Keio collection; 3884 mutants). We followed a six-step screening procedure to identify the mutants showing conjugation deficiency precisely. No mutants defective in the conjugal transfer were isolated, strongly suggesting that E. coli cannot escape from being a recipient organism for P1α plasmid transfer. However, several mutants with low viability were identified, as well as mutants defective in establishing resistance to chloramphenicol, which was used for transconjugant selection. These results suggest that developing drugs capable of inhibiting the establishment of antibiotic resistance is a better approach than attempting to prevent the conjugal transfer to block the spread of antibiotic resistance genes. Our screening system based on the IncP1α-type plasmid transfer can be extended to isolation of target genes for other drugs. This study could be the foundation for further research to understand its underlying molecular mechanism through functional analysis of the identified genes.

Klebsiella pneumoniae is a common cause of bloodstream infections in immunocompromised and hospitalized patients, and over the last 2 decades, some strains have acquired resistance to nearly all available antibiotics, including broad-spectrum carbapenems. The U.S. Centers for Disease Control and Prevention has listed carbapenem-resistant K. pneumoniae (CR-Kp) as an urgent public health threat. Dense colonization of the intestine by CR-Kp and other antibiotic-resistant bacteria is associated with an increased risk of bacteremia. Reducing the density of gut colonization by CR-Kp is likely to reduce their transmission from patient to patient in health care facilities as well as systemic infections. How CR-Kp expands and persists in the gut lumen, however, is poorly understood. Herein, we generated a highly saturated mutant library in a multidrug-resistant K. pneumoniae strain and identified genetic factors that are associated with dense gut colonization by K. pneumoniae. This study sheds light on host colonization by K. pneumoniae and identifies potential colonization factors that contribute to high-density persistence of K. pneumoniae in the intestine., A diverse, antibiotic-naive microbiota prevents highly antibiotic-resistant microbes, including carbapenem-resistant Klebsiella pneumoniae (CR-Kp), from achieving dense colonization of the intestinal lumen. Antibiotic-mediated destruction of the microbiota leads to expansion of CR-Kp in the gut, markedly increasing the risk of bacteremia in vulnerable patients. While preventing dense colonization represents a rational approach to reduce intra- and interpatient dissemination of CR-Kp, little is known about pathogen-associated factors that enable dense growth and persistence in the intestinal lumen. To identify genetic factors essential for dense colonization of the gut by CR-Kp, we constructed a highly saturated transposon mutant library with >150,000 unique mutations in an ST258 strain of CR-Kp and screened for in vitro growth and in vivo intestinal colonization in antibiotic-treated mice. Stochastic and partially reversible fluctuations in the representation of different mutations during dense colonization revealed the dynamic nature of intestinal microbial populations. We identified genes that are crucial for early and late stages of dense gut colonization and confirmed their role by testing isogenic mutants in in vivo competition assays with wild-type CR-Kp. Screening of the transposon library also identified mutations that enhanced in vivo CR-Kp growth. These newly identified colonization factors may provide novel therapeutic opportunities to reduce intestinal colonization by CR-Kp.

During screening of protein-protein interactions, using human protein arrays carrying 19,676 recombinant glutathione s-transferase (GST)-fused human proteins, we identified the high-mobility protein group 20A (HMG20A) as a novel S100A6 binding partner. We confirmed the Ca2+-dependent interaction of HMG20A with S100A6 by the protein array method, biotinylated S100A6 overlay, and GST-pulldown assay in vitro and in transfected COS-7 cells. Co-immunoprecipitation of S100A6 with HMG20A from HeLa cells in a Ca2+-dependent manner revealed the physiological relevance of the S100A6/HMG20A interaction. In addition, HMG20A has the ability to interact with S100A1, S100A2, and S100B in a Ca2+-dependent manner, but not with S100A4, A11, A12, and calmodulin. S100A6 binding experiments using various HMG20A mutants revealed that Ca2+/S100A6 interacts with the C-terminal region (residues 311–342) of HMG20A with stoichiometric binding (HMG20A:S100A6 dimer = 1:1). This was confirmed by the fact that a GST-HMG20A mutant lacking the S100A6 binding region (residues 311–347, HMG20A-ΔC) failed to interact with endogenous S100A6 in transfected COS-7 cells, unlike wild-type HMG20A. Taken together, these results identify, for the first time, HMG20A as a target of Ca2+/S100 proteins, and may suggest a novel linkage between Ca2+/S100 protein signaling and HMG20A function, including in the regulation of neural differentiation.

We describe the development of an optimized glycolytic flux biosensor and its application in detecting altered flux in a production strain and in a mutant library. The glycolytic flux biosensor is based on the Cra-regulated ppsA promoter of E. coli controlling fluorescent protein synthesis. We validated the glycolytic flux dependency of the biosensor in a range of different carbon sources in six different E. coli strains and during mevalonate production. Furthermore, we studied the flux-altering effects of genome-wide single gene knock-outs in E. coli in a multiplex FlowSeq experiment. From a library consisting of 2126 knock-out mutants, we identified 3 mutants with high-flux and 95 mutants with low-flux phenotypes that did not have severe growth defects. This approach can improve our understanding of glycolytic flux regulation improving metabolic models and engineering efforts., Graphical abstract fx1

Fermentation at low temperatures is one of the most popular current winemaking practices because of its reported positive impact on the aromatic profile of wines. However, low temperature is an additional hurdle to develop Saccharomyces cerevisiae wine yeasts, which are already stressed by high osmotic pressure, low pH and poor availability of nitrogen sources in grape must. Understanding the mechanisms of adaptation of S. cerevisiae to fermentation at low temperature would help to design strategies for process management, and to select and improve wine yeast strains specifically adapted to this winemaking practice. The problem has been addressed by several approaches in recent years, including transcriptomic and other high-throughput strategies. In this work we used a genome-wide screening of S. cerevisiae diploid mutant strain collections to identify genes that potentially contribute to adaptation to low temperature fermentation conditions. Candidate genes, impaired for growth at low temperatures (12 °C and 18 °C), but not at a permissive temperature (28 °C), were deleted in an industrial homozygous genetic background, wine yeast strain FX10, in both heterozygosis and homozygosis. Some candidate genes were required for growth at low temperatures only in the laboratory yeast genetic background, but not in FX10 (namely the genes involved in aromatic amino acid biosynthesis). Other genes related to ribosome biosynthesis (SNU66 and PAP2) were required for low-temperature fermentation of synthetic must (SM) in the industrial genetic background. This result coincides with our previous findings about translation efficiency with the fitness of different wine yeast strains at low temperature., Funding from the Spanish Government trough MINECO and FEDER funds: MINECO AGL2012-32064 and AGL2015-63629-R grants, INIA RM2012-00007-00-00 grant, MINECO RTC-2014-2186-2 and MINECO PCIN-2015-143 grants is acknowledged.

Since the onset of antiviral therapy, viral resistance has compromised the clinical value of small-molecule drugs targeting pathogen components. As intracellular parasites, viruses complete their life cycle by hijacking a multitude of host-factors. Aiming at the latter rather than the pathogen directly, host-directed antiviral therapy has emerged as a concept to counteract evolution of viral resistance and develop broad-spectrum drug classes. This approach is propelled by bioinformatics analysis of genome-wide screens that greatly enhance insights into the complex network of host-pathogen interactions and generate a shortlist of potential gene targets from a multitude of candidates, thus setting the stage for a new era of rational identification of drug targets for host-directed antiviral therapies. With particular emphasis on human immunodeficiency virus and influenza virus, two major human pathogens, we review screens employed to elucidate host-pathogen interactions and discuss the state of database ontology approaches applicable to defining a therapeutic endpoint. The value of this strategy for drug discovery is evaluated, and perspectives for bioinformatics-driven hit identification are outlined.

Background The parasite Leishmania panamensis is the main cause of leishmaniasis in Panama. The disease is largely uncontrolled, with a rising incidence and no appropriate control measures. While microsatellites are considered some of the best genetic markers to study population genetics and molecular epidemiology in these and other parasites, none has been developed for L. panamensis. Findings Here we have developed and tested a new panel of microsatellites for this species, based on high-throughput genome-wide screening. The new set of microsatellites is composed of seventeen loci, mainly spanning trinucleotide or longer motifs. We have evaluated the sensitivity and specificity of the panel based on a sample of 27 isolates obtained from cutaneous leishmaniasis patients from central Panama and also several reference species from both L. (Leishmania) and L. (Viannia) subgenera. The genetic equilibrium was assessed both intra- and inter-loci, while the reproductive mode was evaluated using several tests. The new SSR panel shows high polymorphism and sensitivity, as well as good specificity. The preliminary data described here for L. panamensis suggest extensive departure from Hardy-Weinberg proportions, significant linkage disequilibrium and strong deficit of heterozygotes. Several recombination tests involving multilocus linkage disequilibrium and a phylogenetic approach allowed rejection of frequent recombination in our dataset. Conclusions The genome-wide strategy described here proved to be useful to identify and test new polymorphic SSR loci in Leishmania. The new panel of polymorphic microsatellites is a valuable contribution to the existing molecular markers for the study of genetic structure and other aspects of this important species. Electronic supplementary material The online version of this article (doi:10.1186/s13071-015-1153-2) contains supplementary material, which is available to authorized users.

Background RNA interference (RNAi) leads to sequence specific knock-down of gene expression and has emerged as an important tool to analyse gene functions, pathway analysis and gene therapy. Although RNAi is a conserved cellular process involving common elements and factors, species-specific differences have been observed among different eukaryotes. Identification of components for RNAi pathway is pursued intensively and successful genome-wide screens have been performed for components of RNAi pathways in various organisms. Functional comparative genomics analysis offers evolutionary insight that forms basis of discoveries of novel RNAi-factors within related organisms. Keeping in view the academic and commercial utility of insect derived cell-line from Spodoptera frugiperda, we pursued the identification and functional analysis of components of RNAi-machinery of Sf21 cell-line using genome-wide application. Results The genome and transcriptome of Sf21 was assembled and annotated. In silico application of comparative genome analysis among insects allowed us to identify several RNAi factors in Sf21 line. The candidate RNAi factors from assembled genome were validated by knockdown analysis of candidate factors using the siRNA screens on the Sf21-gfp reporter cell-line. Forty two (42) potential factors were identified using the cell based assay. These include core RNAi elements including Dicer-2, Argonaute-1, Drosha, Aubergine and auxiliary modules like chromatin factors, RNA helicases, RNA processing module, signalling allied proteins and others. Phylogenetic analyses and domain architecture revealed that Spodoptera frugiperda homologs retained identity with Lepidoptera (Bombyx mori) or Coleoptera (Tribolium castaneum) sustaining an evolutionary conserved scaffold in post-transcriptional gene silencing paradigm within insects. Conclusion The database of RNAi-factors generated by whole genome association survey offers comprehensive outlook about conservation as well as specific differences of the proteins of RNAi machinery. Understanding the interior involved in different phases of gene silencing also offers impending tool for RNAi-based applications. Electronic supplementary material The online version of this article (doi:10.1186/1471-2164-15-775) contains supplementary material, which is available to authorized users.

The PBAT software package (v2.5) provides a unique set of tools for complex family-based association analysis at a genome-wide level. PBAT can handle nuclear families with missing parental genotypes, extended pedigrees with missing genotypic information, analysis of single nucleotide polymorphisms (SNPs), haplotype analysis, quantitative traits, multivariate/longitudinal data and time to onset phenotypes. The data analysis can be adjusted for covariates and gene/environment interactions. Haplotype-based features include sliding windows and the reconstruction of the haplotypes of the probands. PBAT's screening tools allow the user successfully to handle the multiple comparisons problem at a genome-wide level, even for 100,000 SNPs and more. Moreover, PBAT is computationally fast. A genome scan of 300,000 SNPs in 2,000 trios takes 4 central processing unit (CPU)-days. PBAT is available for Linux, Sun Solaris and Windows XP. ispartof: Human Genomics vol:2 issue:1 pages:67-69 ispartof: location:England status: published

The chromosomal translocation t(12;21) resulting in the ETV6/RUNX1 fusion gene is the most frequent structural cytogenetic abnormality among patients with childhood acute lymphoblastic leukaemia (ALL). We investigated 62 ETV6/RUNX1‐positive childhood ALL patients by single nucleotide polymorphism array to explore acquired copy number alterations (CNAs) at diagnosis. The mean number of CNAs was 2·82 (range 0–14). Concordance with available G‐band karyotyping and comparative genomic hybridization was 93%. Based on three major protein‐protein complexes disrupted by these CNAs, patients could be categorized into four distinct subgroups, defined by different underlying biological mechanisms relevant to the aetiology of childhood ALL. When recurrent CNAs were evaluated by an oncogenetic tree analysis classifying their sequential order, the most common genetic aberrations (deletions of 6q, 9p, 13q and X, and gains of 10 and 21) seemed independent of each other. Finally, we identified the most common regions with recurrent gains and losses, which comprise microRNA clusters with known oncogenic or tumour‐suppressive roles. The present study sheds further light on the genetic diversity of ETV6/RUNX1‐positive childhood ALL, which may be important for understanding poor responses among this otherwise highly curable subset of ALL and lead to novel targeted treatment strategies.

Item does not contain fulltext Array-based whole genome investigation or molecular karyotyping enables the genome-wide detection of submicroscopic imbalances. Proof-of-principle experiments have demonstrated that molecular karyotyping outperforms conventional karyotyping with regard to detection of chromosomal imbalances. This article identifies areas for which the technology seems matured and areas that require more investigations. Molecular karyotyping should be part of the genetic diagnostic work-up of patients with developmental disorders. For the implementation of the technique for other constitutional indications and in prenatal diagnosis, more research is appropriate. Also, the article aims to provide best practice guidelines for the application of array comparative genomic hybridisation to ensure both technical and clinical quality criteria that will optimise and standardise results and reports in diagnostic laboratories. In short, both the specificity and the sensitivity of the arrays should be evaluated in every laboratory offering the diagnostic test. Internal and external quality control programmes are urgently needed to evaluate and standardise the test results between laboratories.