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Sample records for amplicon resequencing experiments

  1. Analysing 454 amplicon resequencing experiments using the modular and database oriented Variant Identification Pipeline

    Directory of Open Access Journals (Sweden)

    Deforce Dieter

    2010-05-01

    Full Text Available Abstract Background Next-generation amplicon sequencing enables high-throughput genetic diagnostics, sequencing multiple genes in several patients together in one sequencing run. Currently, no open-source out-of-the-box software solution exists that reliably reports detected genetic variations and that can be used to improve future sequencing effectiveness by analyzing the PCR reactions. Results We developed an integrated database oriented software pipeline for analysis of 454/Roche GS-FLX amplicon resequencing experiments using Perl and a relational database. The pipeline enables variation detection, variation detection validation, and advanced data analysis, which provides information that can be used to optimize PCR efficiency using traditional means. The modular approach enables customization of the pipeline where needed and allows researchers to adopt their analysis pipeline to their experiments. Clear documentation and training data is available to test and validate the pipeline prior to using it on real sequencing data. Conclusions We designed an open-source database oriented pipeline that enables advanced analysis of 454/Roche GS-FLX amplicon resequencing experiments using SQL-statements. This modular database approach allows easy coupling with other pipeline modules such as variant interpretation or a LIMS system. There is also a set of standard reporting scripts available.

  2. Genomic resequencing combined with quantitative proteomic analyses elucidate the survival mechanisms of Lactobacillus plantarum P-8 in a long-term glucose-limited experiment.

    Science.gov (United States)

    He, Qiuwen; Cao, Chenxia; Hui, Wenyan; Yu, Jie; Zhang, Heping; Zhang, Wenyi

    2018-03-30

    Lactobacillus plantarum, commonly isolated from plant material, is widely used to produce various types of fermented foods. However, nutrient-limiting conditions are often encountered during industrial applications. The present study aimed to investigate the response of L. plantarum P-8 to glucose-limited conditions in a long-term experiment. Genotypic and proteomic changes in L. plantarum P-8 were monitored over 3 years in glucose-limited and glucose-normal media using whole-genome resequencing and tandem mass tag-based quantitative proteomic analysis. Results showed that L. plantarum employed numerous survival mechanisms, including alteration of the cell envelope, activation of the PTS system, accumulation and consumption of amino acids, increase in the metabolism of carbohydrates (via glycolysis, citric acid cycle, and pyruvate metabolism), and increase in the production of ATP in response to glucose starvation. This study demonstrates the feasibility of experimental evolution of L. plantarum P-8, while whole-genome resequencing of adapted isolates provided clues toward bacterial functions involved and a deeper mechanistic understanding of the adaptive response of L. plantarum to glucose-limited conditions. We have conducted a 3-year experiment monitoring genotypic and proteomic changes in Lactobacillus plantarum P-8 in glucose-limited and glucose-normal media. Whole-genome resequencing and tandem mass tag-based quantitative proteomics were performed for analyzing genomic evolution of L. plantarum P-8 in glucose-limited and glucose-normal conditions. In addition, differential expressed proteins in all generations between these two conditions were identified and functions of these proteins specific to L group were predicted. L. plantarum employed numerous survival mechanisms, including alteration of the cell envelope, activation of the PTS system, accumulation and consumption of amino acids, increase in the metabolism of carbohydrates (glycolysis, citric acid

  3. Allele Re-sequencing Technologies

    DEFF Research Database (Denmark)

    Byrne, Stephen; Farrell, Jacqueline Danielle; Asp, Torben

    2013-01-01

    The development of next-generation sequencing technologies has made sequencing an affordable approach for detection of genetic variations associated with various traits. However, the cost of whole genome re-sequencing still remains too high to be feasible for many plant species with large and com...... alternative to whole genome re-sequencing to identify causative genetic variations in plants. One challenge, however, will be efficient bioinformatics strategies for data handling and analysis from the increasing amount of sequence information....... and complex genomes. Recent developments in strategies for target-enrichment, transcriptome re-sequencing, and partial genome re-sequencing allows for enrichment for regions of interest at a scale that is matched to the throughput of next-generation sequencing platforms, and has emerged as a promising...

  4. (SELAX strain) B2 amplicon

    African Journals Online (AJOL)

    USER

    2010-07-12

    Jul 12, 2010 ... Restriction enzyme digests of the clones suggested that the cloned portion of the B2 amplicon was 16 kb. Key words: B2 gene, A2B2 esterase, insecticide resistance. INTRODUCTION. Organophosphate resistance in Culex pipiens complex mosquitoes has been shown to be correlated with the presence of ...

  5. Removing Noise From Pyrosequenced Amplicons

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    Davenport Russell J

    2011-01-01

    Full Text Available Abstract Background In many environmental genomics applications a homologous region of DNA from a diverse sample is first amplified by PCR and then sequenced. The next generation sequencing technology, 454 pyrosequencing, has allowed much larger read numbers from PCR amplicons than ever before. This has revolutionised the study of microbial diversity as it is now possible to sequence a substantial fraction of the 16S rRNA genes in a community. However, there is a growing realisation that because of the large read numbers and the lack of consensus sequences it is vital to distinguish noise from true sequence diversity in this data. Otherwise this leads to inflated estimates of the number of types or operational taxonomic units (OTUs present. Three sources of error are important: sequencing error, PCR single base substitutions and PCR chimeras. We present AmpliconNoise, a development of the PyroNoise algorithm that is capable of separately removing 454 sequencing errors and PCR single base errors. We also introduce a novel chimera removal program, Perseus, that exploits the sequence abundances associated with pyrosequencing data. We use data sets where samples of known diversity have been amplified and sequenced to quantify the effect of each of the sources of error on OTU inflation and to validate these algorithms. Results AmpliconNoise outperforms alternative algorithms substantially reducing per base error rates for both the GS FLX and latest Titanium protocol. All three sources of error lead to inflation of diversity estimates. In particular, chimera formation has a hitherto unrealised importance which varies according to amplification protocol. We show that AmpliconNoise allows accurate estimates of OTU number. Just as importantly AmpliconNoise generates the right OTUs even at low sequence differences. We demonstrate that Perseus has very high sensitivity, able to find 99% of chimeras, which is critical when these are present at high

  6. Annotation and Re-Sequencing of Genes from De Novo Transcriptome Assembly of Abies alba (Pinaceae

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    Anna M. Roschanski

    2013-01-01

    Full Text Available Premise of the study: We present a protocol for the annotation of transcriptome sequence data and the identification of candidate genes therein using the example of the nonmodel conifer Abies alba. Methods and Results: A normalized cDNA library was built from an A. alba seedling. The sequencing on a 454 platform yielded more than 1.5 million reads that were de novo assembled into 25 149 contigs. Two complementary approaches were applied to annotate gene fragments that code for (1 well-known proteins and (2 proteins that are potentially adaptively relevant. Primer development and testing yielded 88 amplicons that could successfully be resequenced from genomic DNA. Conclusions: The annotation workflow offers an efficient way to identify potential adaptively relevant genes from the large quantity of transcriptome sequence data. The primer set presented should be prioritized for single-nucleotide polymorphism detection in adaptively relevant genes in A. alba.

  7. Annotation and re-sequencing of genes from de novo transcriptome assembly of Abies alba (Pinaceae).

    Science.gov (United States)

    Roschanski, Anna M; Fady, Bruno; Ziegenhagen, Birgit; Liepelt, Sascha

    2013-01-01

    We present a protocol for the annotation of transcriptome sequence data and the identification of candidate genes therein using the example of the nonmodel conifer Abies alba. • A normalized cDNA library was built from an A. alba seedling. The sequencing on a 454 platform yielded more than 1.5 million reads that were de novo assembled into 25149 contigs. Two complementary approaches were applied to annotate gene fragments that code for (1) well-known proteins and (2) proteins that are potentially adaptively relevant. Primer development and testing yielded 88 amplicons that could successfully be resequenced from genomic DNA. • The annotation workflow offers an efficient way to identify potential adaptively relevant genes from the large quantity of transcriptome sequence data. The primer set presented should be prioritized for single-nucleotide polymorphism detection in adaptively relevant genes in A. alba.

  8. Cooperative Epigenetic Modulation by Cancer Amplicon Genes

    Science.gov (United States)

    Rui, Lixin; Tolga Emre, N. C.; Kruhlak, Michael J.; Chung, Hye-Jung; Steidl, Christian; Slack, Graham; Wright, George W.; Lenz, Georg; Ngo, Vu N.; Shaffer, Arthur L.; Xu, Weihong; Zhao, Hong; Yang, Yandan; Lamy, Laurence; Davis, R. Eric; Xiao, Wenming; Powell, John; Maloney, David; Thomas, Craig J.; Möller, Peter; Rosenwald, Andreas; Ott, German; Muller-Hermelink, Hans Konrad; Savage, Kerry; Connors, Joseph M.; Rimsza, Lisa M.; Campo, Elias; Jaffe, Elaine S.; Delabie, Jan; Smeland, Erlend B.; Weisenburger, Dennis D.; Chan, Wing C.; Gascoyne, Randy D.; Levens, David; Staudt, Louis M.

    2010-01-01

    Chromosome band 9p24 is frequently amplified in primary mediastinal B-cell lymphoma (PMBL) and Hodgkin lymphoma (HL). To identify oncogenes in this amplicon, we screened an RNA interference library targeting amplicon genes and thereby identified JAK2 and the histone demethylase JMJD2C as essential genes in these lymphomas. Inhibition of JAK2 and JMJD2C cooperated in killing these lymphomas by decreasing tyrosine 41 phosphorylation and increasing lysine 9 trimethylation of histone H3, promoting heterochromatin formation. MYC, a major target of JAK2-mediated histone phosphorylation, was silenced following JAK2 and JMJD2C inhibition, with a corresponding increase in repressive chromatin. Hence, JAK2 and JMJD2C cooperatively remodel the PMBL and HL epigenome, offering a mechanistic rationale for the development of JAK2 and JMJD2C inhibitors in these diseases. PMID:21156283

  9. Annotation and re-sequencing of genes from de novo transcriptome assembly of Abies alba (Pinaceae)1

    Science.gov (United States)

    Roschanski, Anna M.; Fady, Bruno; Ziegenhagen, Birgit; Liepelt, Sascha

    2013-01-01

    • Premise of the study: We present a protocol for the annotation of transcriptome sequence data and the identification of candidate genes therein using the example of the nonmodel conifer Abies alba. • Methods and Results: A normalized cDNA library was built from an A. alba seedling. The sequencing on a 454 platform yielded more than 1.5 million reads that were de novo assembled into 25149 contigs. Two complementary approaches were applied to annotate gene fragments that code for (1) well-known proteins and (2) proteins that are potentially adaptively relevant. Primer development and testing yielded 88 amplicons that could successfully be resequenced from genomic DNA. • Conclusions: The annotation workflow offers an efficient way to identify potential adaptively relevant genes from the large quantity of transcriptome sequence data. The primer set presented should be prioritized for single-nucleotide polymorphism detection in adaptively relevant genes in A. alba. PMID:25202477

  10. Fast and accurate read alignment for resequencing.

    Science.gov (United States)

    Mu, John C; Jiang, Hui; Kiani, Amirhossein; Mohiyuddin, Marghoob; Bani Asadi, Narges; Wong, Wing H

    2012-09-15

    Next-generation sequence analysis has become an important task both in laboratory and clinical settings. A key stage in the majority sequence analysis workflows, such as resequencing, is the alignment of genomic reads to a reference genome. The accurate alignment of reads with large indels is a computationally challenging task for researchers. We introduce SeqAlto as a new algorithm for read alignment. For reads longer than or equal to 100 bp, SeqAlto is up to 10 × faster than existing algorithms, while retaining high accuracy and the ability to align reads with large (up to 50 bp) indels. This improvement in efficiency is particularly important in the analysis of future sequencing data where the number of reads approaches many billions. Furthermore, SeqAlto uses less than 8 GB of memory to align against the human genome. SeqAlto is benchmarked against several existing tools with both real and simulated data. Linux and Mac OS X binaries free for academic use are available at http://www.stanford.edu/group/wonglab/seqalto whwong@stanford.edu.

  11. Ultra-high resolution HLA genotyping and allele discovery by highly multiplexed cDNA amplicon pyrosequencing

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    Lank Simon M

    2012-08-01

    Full Text Available Abstract Background High-resolution HLA genotyping is a critical diagnostic and research assay. Current methods rarely achieve unambiguous high-resolution typing without making population-specific frequency inferences due to a lack of locus coverage and difficulty in exon-phase matching. Achieving high-resolution typing is also becoming more challenging with traditional methods as the database of known HLA alleles increases. Results We designed a cDNA amplicon-based pyrosequencing method to capture 94% of the HLA class I open-reading-frame with only two amplicons per sample, and an analogous method for class II HLA genes, with a primary focus on sequencing the DRB loci. We present a novel Galaxy server-based analysis workflow for determining genotype. During assay validation, we performed two GS Junior sequencing runs to determine the accuracy of the HLA class I amplicons and DRB amplicon at different levels of multiplexing. When 116 amplicons were multiplexed, we unambiguously resolved 99%of class I alleles to four- or six-digit resolution, as well as 100% unambiguous DRB calls. The second experiment, with 271 multiplexed amplicons, missed some alleles, but generated high-resolution, concordant typing for 93% of class I alleles, and 96% for DRB1 alleles. In a third, preliminary experiment we attempted to sequence novel amplicons for other class II loci with mixed success. Conclusions The presented assay is higher-throughput and higher-resolution than existing HLA genotyping methods, and suitable for allele discovery or large cohort sampling. The validated class I and DRB primers successfully generated unambiguously high-resolution genotypes, while further work is needed to validate additional class II genotyping amplicons.

  12. Use of AmpliWax to optimize amplicon sterilization by isopsoralen.

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    De la Viuda, M; Fille, M; Ruiz, J; Aslanzadeh, J

    1996-12-01

    The photochemical inactivation of amplicons by isopsoralen (IP-10) has been suggested as a possible means to prevent PCR carryover contamination. To evaluate the technique, serial dilutions of amplicons (10(11) to 10(3)) from the Borrelia burgdorferi OSP A gene were amplified in the presence of 0, 25, 50, and 100 micrograms of IP-10 per ml for 45 cycles. The PCR products were exposed to UV light for 15 min to activate IP-10 and sterilize the amplicons. One microliter of each sterilized sample was reamplified for an additional 45 cycles. The PCR products were then resolved in an agarose gel, blotted onto a nylon membrane, and probed with an alkaline phosphatase-conjugated chemiluminescent probe. Although IP-10 at concentrations of 50 and 100 micrograms/ml effectively sterilized up to 10(11) amplicons, the compound was inhibitory to PCR. IP-10 at a concentration of 25 micrograms/ml had slight inhibitory effect on PCR and did not completely sterilized all of the amplicons. Therefore, in subsequent experiments AmpliWax was substituted for mineral oil, and PCR was performed on 10(9) to 10(3) amplicons as described above. Following the amplification, the PCR tubes were cooled to solidify the AmpliWax and inoculated with various concentrations of IP-10. With this technique, PCR products produced from as many as 10(9) target amplicons were effectively sterilized with 200 micrograms of IP-10 per ml. Similarly, the addition of IP-10 (50 micrograms/ml) before and after PCR was evaluated for the detection of B. burgdorferi in 62 ticks from a region of Southern Connecticut where the organism is highly endemic. PCR performed in the presence of 50 micrograms of IP-10 per ml detected B. burgdorferi-specific DNA in 17 of 62 ticks (27%) following gel electrophoresis and in 34 of 62 ticks (55%) following Southern blot hybridization of the PCR products. In contrast, post-PCR addition of IP-10 detected borrelia-specific DNA in 31 of 62 ticks (50%) following gel electrophoresis and in

  13. The influence of amplicon length on real-time PCR results

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    Debode, F.

    2017-01-01

    Full Text Available Description of the subject. This paper discusses the influence of amplicon length on real-time PCR results. Objectives. The aim of the experiments was to show that amplicon size has an influence on detection. Method. Tests were performed on genomic and plasmid DNA. Double-dye probes and SYBR® Green were used for detection by real-time PCR. Primers were selected in order to produce fragments with increasing sizes. Experiments dealt with two targets: an endogenous target for soybean (part of the lectin gene and a transgenic target (junction P35S-CTP of the MON40-3-2 soybean. Results. The results show that the kinetics of amplification curves evolve as a function of amplicon length, and smaller amplicons yield a higher level of fluorescence for the plateau phase. DNA degradation within the sample as well as the principles of fluorescence acquisition as a function of the chemistry used can also be factors. Conclusions. It was experimentally shown that the observed effect is linked to the suboptimal elongation temperature used in real-time PCR. Detection using SYBR® Green is less impacted as the loss of efficiency is partially compensated by the greater integration of SYBR® Green molecules in the larger fragments.

  14. Comprehensive Molecular Profiling of Archival Bone Marrow Trephines Using a Commercially Available Leukemia Panel and Semiconductor-Based Targeted Resequencing.

    Science.gov (United States)

    Bartels, Stephan; Schipper, Elisa; Kreipe, Hans Heinrich; Lehmann, Ulrich

    2015-01-01

    Comprehensive mutation profiling becomes more and more important in hematopathology complementing morphological and immunohistochemical evaluation of fixed, decalcified and embedded bone marrow biopsies for diagnostic, prognostic and also predictive purposes. However, the number and the size of relevant genes leave conventional Sanger sequencing impracticable in terms of costs, required input DNA, and turnaround time. Since most published protocols and commercially available reagents for targeted resequencing of gene panels are established and validated for the analysis of fresh bone marrow aspirate or peripheral blood it remains to be proven whether the available technology can be transferred to the analysis of archival trephines. Therefore, the performance of the recently available Ion AmpliSeq AML Research panel (LifeTechnologies) was evaluated for the analysis of fragmented DNA extracted from archival bone marrow trephines. Taking fresh aspirate as gold standard all clinically relevant mutations (n = 17) as well as 25 well-annotated SNPs could be identified reliably with high quality in the corresponding archival trephines of the training set (n = 10). Pre-treatment of the extracted DNA with Uracil-DNA-Glycosylase reduced the number of low level artificial sequence variants by more than 60%, vastly reducing time required for proper evaluation of the sequencing results. Subsequently, randomly picked FFPE samples (n = 41) were analyzed to evaluate sequencing performance under routine conditions. Thereby all known mutations (n = 43) could be verified and 36 additional mutations in genes not yet covered by the routine work-up (e.g., TET2, ASXL1, DNMT3A), demonstrating the feasibility of this approach and the gain of diagnostically relevant information. The dramatically reduced amount of input DNA, the increase in sensitivity as well as calculated cost-effectiveness, low hands on , and turn-around-time, necessary for the analysis of 237 amplicons strongly argue for

  15. Comprehensive Molecular Profiling of Archival Bone Marrow Trephines Using a Commercially Available Leukemia Panel and Semiconductor-Based Targeted Resequencing.

    Directory of Open Access Journals (Sweden)

    Stephan Bartels

    Full Text Available Comprehensive mutation profiling becomes more and more important in hematopathology complementing morphological and immunohistochemical evaluation of fixed, decalcified and embedded bone marrow biopsies for diagnostic, prognostic and also predictive purposes. However, the number and the size of relevant genes leave conventional Sanger sequencing impracticable in terms of costs, required input DNA, and turnaround time. Since most published protocols and commercially available reagents for targeted resequencing of gene panels are established and validated for the analysis of fresh bone marrow aspirate or peripheral blood it remains to be proven whether the available technology can be transferred to the analysis of archival trephines. Therefore, the performance of the recently available Ion AmpliSeq AML Research panel (LifeTechnologies was evaluated for the analysis of fragmented DNA extracted from archival bone marrow trephines. Taking fresh aspirate as gold standard all clinically relevant mutations (n = 17 as well as 25 well-annotated SNPs could be identified reliably with high quality in the corresponding archival trephines of the training set (n = 10. Pre-treatment of the extracted DNA with Uracil-DNA-Glycosylase reduced the number of low level artificial sequence variants by more than 60%, vastly reducing time required for proper evaluation of the sequencing results. Subsequently, randomly picked FFPE samples (n = 41 were analyzed to evaluate sequencing performance under routine conditions. Thereby all known mutations (n = 43 could be verified and 36 additional mutations in genes not yet covered by the routine work-up (e.g., TET2, ASXL1, DNMT3A, demonstrating the feasibility of this approach and the gain of diagnostically relevant information. The dramatically reduced amount of input DNA, the increase in sensitivity as well as calculated cost-effectiveness, low hands on , and turn-around-time, necessary for the analysis of 237 amplicons

  16. Resequencing three candidate genes discovers seven potentially deleterious variants susceptibility to major depressive disorder and suicide attempts in Chinese.

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    Rao, Shitao; Leung, Cherry She Ting; Lam, Macro Hb; Wing, Yun Kwok; Waye, Mary Miu Yee; Tsui, Stephen Kwok Wing

    2017-03-01

    To date almost 200 genes were found to be associated with major depressive disorder (MDD) or suicide attempts (SA), but very few genes were reported for their molecular mechanisms. This study aimed to find out whether there were common or rare variants in three candidate genes altering the risk for MDD and SA in Chinese. Three candidate genes (HOMER1, SLC6A4 and TEF) were chosen for resequencing analysis and association studies as they were reported to be involved in the etiology of MDD and SA. Following that, bioinformatics analyses were applied on those variants of interest. After resequencing analysis and alignment for the amplicons, a total of 34 common or rare variants were found in the randomly selected 36 Hong Kong Chinese patients with both MDD and SA. Among those, seven variants show potentially deleterious features. Rs60029191 and a rare variant located in regulatory region of the HOMER1 gene may affect the promoter activities through interacting with predicted transcription factors. Two missense mutations existed in the SLC6A4 coding regions were firstly reported in Hong Kong Chinese MDD and SA patients, and both of them could affect the transport efficiency of SLC6A4 to serotonin. Moreover, a common variant rs6354 located in the untranslated region of this gene may affect the expression level or exonic splicing of serotonin transporter. In addition, both of a most studied polymorphism rs738499 and a low-frequency variant in the promoter region of the TEF gene were found to be located in potential transcription factor binding sites, which may let the two variants be able to influence the promoter activities of the gene. This study elucidated the potentially molecular mechanisms of the three candidate genes altering the risk for MDD and SA. These findings implied that not only common variants but rare variants could make contributions to the genetic susceptibility to MDD and SA in Chinese. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. Optimal Activation of Isopsoralen To Prevent Amplicon Carryover

    OpenAIRE

    Fahle, Gary A.; Gill, Vee J.; Fischer, Steven H.

    1999-01-01

    We compared the efficiencies of activation of the photochemical isopsoralen compound 10 and its resulting amplicon neutralizations under conditions with a UV transilluminator box at room temperature (RT) and a HRI-300 UV photothermal reaction chamber at RT and at 5°C. Our data suggest that use of the HRI-300 reaction chamber at 5°C results in a statistically significantly higher degree of amplicon neutralization.

  18. High throughput 16S rRNA gene amplicon sequencing

    DEFF Research Database (Denmark)

    Nierychlo, Marta; Larsen, Poul; Jørgensen, Mads Koustrup

    S rRNA gene amplicon sequencing has been developed over the past few years and is now ready to use for more comprehensive studies related to plant operation and optimization thanks to short analysis time, low cost, high throughput, and high taxonomic resolution. In this study we show how 16S r......RNA gene amplicon sequencing can be used to reveal factors of importance for the operation of full-scale nutrient removal plants related to settling problems and floc properties. Using optimized DNA extraction protocols, indexed primers and our in-house Illumina platform, we prepared multiple samples...... be correlated to the presence of the species that are regarded as “strong” and “weak” floc formers. In conclusion, 16S rRNA gene amplicon sequencing provides a high throughput approach for a rapid and cheap community profiling of activated sludge that in combination with multivariate statistics can be used...

  19. MAGERI: Computational pipeline for molecular-barcoded targeted resequencing.

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    Mikhail Shugay

    2017-05-01

    Full Text Available Unique molecular identifiers (UMIs show outstanding performance in targeted high-throughput resequencing, being the most promising approach for the accurate identification of rare variants in complex DNA samples. This approach has application in multiple areas, including cancer diagnostics, thus demanding dedicated software and algorithms. Here we introduce MAGERI, a computational pipeline that efficiently handles all caveats of UMI-based analysis to obtain high-fidelity mutation profiles and call ultra-rare variants. Using an extensive set of benchmark datasets including gold-standard biological samples with known variant frequencies, cell-free DNA from tumor patient blood samples and publicly available UMI-encoded datasets we demonstrate that our method is both robust and efficient in calling rare variants. The versatility of our software is supported by accurate results obtained for both tumor DNA and viral RNA samples in datasets prepared using three different UMI-based protocols.

  20. Whole-genome resequencing and transcriptomic analysis to identify genes involved in leaf-color diversity in ornamental rice plants.

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    Chang-Kug Kim

    Full Text Available Rice field art is a large-scale art form in which people design rice fields using various kinds of ornamental rice plants with different leaf colors. Leaf color-related genes play an important role in the study of chlorophyll biosynthesis, chloroplast structure and function, and anthocyanin biosynthesis. Despite the role of different metabolites in the traditional relationship between leaf and color, comprehensive color-specific metabolite studies of ornamental rice have been limited. We performed whole-genome resequencing and transcriptomic analysis of regulatory patterns and genetic diversity among different rice cultivars to discover new genetic mechanisms that promote enhanced levels of various leaf colors. We resequenced the genomes of 10 rice leaf-color accessions to an average of 40× reads depth and >95% coverage and performed 30 RNA-seq experiments using the 10 rice accessions sampled at three developmental stages. The sequencing results yielded a total of 1,814 × 106 reads and identified an average of 713,114 SNPs per rice accession. Based on our analysis of the DNA variation and gene expression, we selected 47 candidate genes. We used an integrated analysis of the whole-genome resequencing data and the RNA-seq data to divide the candidate genes into two groups: genes related to macronutrient (i.e., magnesium and sulfur transport and genes related to flavonoid pathways, including anthocyanidin biosynthesis. We verified the candidate genes with quantitative real-time PCR using transgenic T-DNA insertion mutants. Our study demonstrates the potential of integrated screening methods combined with genetic-variation and transcriptomic data to isolate genes involved in complex biosynthetic networks and pathways.

  1. Comparison of variations detection between whole-genome amplification methods used in single-cell resequencing

    DEFF Research Database (Denmark)

    Hou, Yong; Wu, Kui; Shi, Xulian

    2015-01-01

    BACKGROUND: Single-cell resequencing (SCRS) provides many biomedical advances in variations detection at the single-cell level, but it currently relies on whole genome amplification (WGA). Three methods are commonly used for WGA: multiple displacement amplification (MDA), degenerate-oligonucleoti......BACKGROUND: Single-cell resequencing (SCRS) provides many biomedical advances in variations detection at the single-cell level, but it currently relies on whole genome amplification (WGA). Three methods are commonly used for WGA: multiple displacement amplification (MDA), degenerate...

  2. An evolutionary framework for association testing in resequencing studies.

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    C Ryan King

    2010-11-01

    Full Text Available Sequencing technologies are becoming cheap enough to apply to large numbers of study participants and promise to provide new insights into human phenotypes by bringing to light rare and previously unknown genetic variants. We develop a new framework for the analysis of sequence data that incorporates all of the major features of previously proposed approaches, including those focused on allele counts and allele burden, but is both more general and more powerful. We harness population genetic theory to provide prior information on effect sizes and to create a pooling strategy for information from rare variants. Our method, EMMPAT (Evolutionary Mixed Model for Pooled Association Testing, generates a single test per gene (substantially reducing multiple testing concerns, facilitates graphical summaries, and improves the interpretation of results by allowing calculation of attributable variance. Simulations show that, relative to previously used approaches, our method increases the power to detect genes that affect phenotype when natural selection has kept alleles with large effect sizes rare. We demonstrate our approach on a population-based re-sequencing study of association between serum triglycerides and variation in ANGPTL4.

  3. A priori Considerations When Conducting High-Throughput Amplicon-Based Sequence Analysis

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    Aditi Sengupta

    2016-03-01

    Full Text Available Amplicon-based sequencing strategies that include 16S rRNA and functional genes, alongside “meta-omics” analyses of communities of microorganisms, have allowed researchers to pose questions and find answers to “who” is present in the environment and “what” they are doing. Next-generation sequencing approaches that aid microbial ecology studies of agricultural systems are fast gaining popularity among agronomy, crop, soil, and environmental science researchers. Given the rapid development of these high-throughput sequencing techniques, researchers with no prior experience will desire information about the best practices that can be used before actually starting high-throughput amplicon-based sequence analyses. We have outlined items that need to be carefully considered in experimental design, sampling, basic bioinformatics, sequencing of mock communities and negative controls, acquisition of metadata, and in standardization of reaction conditions as per experimental requirements. Not all considerations mentioned here may pertain to a particular study. The overall goal is to inform researchers about considerations that must be taken into account when conducting high-throughput microbial DNA sequencing and sequences analysis.

  4. Molecular cloning, sequencing, and distribution of feline GnRH receptor (GnRHR) and resequencing of canine GnRHR.

    Science.gov (United States)

    Samoylov, Alexandre M; Napier, India D; Morrison, Nancy E; Martin, Douglas R; Cox, Nancy R; Samoylova, Tatiana I

    2015-01-15

    GnRH receptors play vital roles in mammalian reproduction via regulation of gonadotropin secretion, which is essential for gametogenesis and production of gonadal steroids. GnRH receptors for more than 20 mammalian species have been sequenced, including human, mouse, and dog. This study reports the molecular cloning and sequencing of GnRH receptor (GnRHR) cDNA from the pituitary gland of the domestic cat, an important species in biomedical research. Feline GnRHR cDNA is composed of 981 nucleotides and encodes a 327 amino acid protein. Unlike the majority of mammalian species sequenced so far, but similar to canine GnRHR, feline GnRHR protein lacks asparagine in position three of the extracellular domain of the protein. At the amino acid level, feline GnRHR exhibits 95.1% identity with canine, 93.8% with human, and 88.9% with mouse GnRHR. Comparative sequence analysis of GnRHRs for multiple mammalian species led to resequencing of canine GnRHR, which differed from that previously published by a single base change that translates to a different amino acid in position 193. This single base change was confirmed in dogs of multiple breeds. Reverse transcriptase PCR analysis of GnRHR messenger RNA in different tissues from four normal cats indicated the presence of amplicons of varying lengths, including full-length as well as shortened GnRHR amplicons, pointing to the existence of truncated GnRHR transcripts in the domestic cat. This study is the first insight into molecular composition and expression of feline GnRHR and promotes better understanding of receptor organization, and distribution in various tissues of this species. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. GWASeq: targeted re-sequencing follow up to GWAS.

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    Salomon, Matthew P; Li, Wai Lok Sibon; Edlund, Christopher K; Morrison, John; Fortini, Barbara K; Win, Aung Ko; Conti, David V; Thomas, Duncan C; Duggan, David; Buchanan, Daniel D; Jenkins, Mark A; Hopper, John L; Gallinger, Steven; Le Marchand, Loïc; Newcomb, Polly A; Casey, Graham; Marjoram, Paul

    2016-03-03

    For the last decade the conceptual framework of the Genome-Wide Association Study (GWAS) has dominated the investigation of human disease and other complex traits. While GWAS have been successful in identifying a large number of variants associated with various phenotypes, the overall amount of heritability explained by these variants remains small. This raises the question of how best to follow up on a GWAS, localize causal variants accounting for GWAS hits, and as a consequence explain more of the so-called "missing" heritability. Advances in high throughput sequencing technologies now allow for the efficient and cost-effective collection of vast amounts of fine-scale genomic data to complement GWAS. We investigate these issues using a colon cancer dataset. After QC, our data consisted of 1993 cases, 899 controls. Using marginal tests of associations, we identify 10 variants distributed among six targeted regions that are significantly associated with colorectal cancer, with eight of the variants being novel to this study. Additionally, we perform so-called 'SNP-set' tests of association and identify two sets of variants that implicate both common and rare variants in the etiology of colorectal cancer. Here we present a large-scale targeted re-sequencing resource focusing on genomic regions implicated in colorectal cancer susceptibility previously identified in several GWAS, which aims to 1) provide fine-scale targeted sequencing data for fine-mapping and 2) provide data resources to address methodological questions regarding the design of sequencing-based follow-up studies to GWAS. Additionally, we show that this strategy successfully identifies novel variants associated with colorectal cancer susceptibility and can implicate both common and rare variants.

  6. BFAST: an alignment tool for large scale genome resequencing.

    Directory of Open Access Journals (Sweden)

    Nils Homer

    2009-11-01

    Full Text Available The new generation of massively parallel DNA sequencers, combined with the challenge of whole human genome resequencing, result in the need for rapid and accurate alignment of billions of short DNA sequence reads to a large reference genome. Speed is obviously of great importance, but equally important is maintaining alignment accuracy of short reads, in the 25-100 base range, in the presence of errors and true biological variation.We introduce a new algorithm specifically optimized for this task, as well as a freely available implementation, BFAST, which can align data produced by any of current sequencing platforms, allows for user-customizable levels of speed and accuracy, supports paired end data, and provides for efficient parallel and multi-threaded computation on a computer cluster. The new method is based on creating flexible, efficient whole genome indexes to rapidly map reads to candidate alignment locations, with arbitrary multiple independent indexes allowed to achieve robustness against read errors and sequence variants. The final local alignment uses a Smith-Waterman method, with gaps to support the detection of small indels.We compare BFAST to a selection of large-scale alignment tools -- BLAT, MAQ, SHRiMP, and SOAP -- in terms of both speed and accuracy, using simulated and real-world datasets. We show BFAST can achieve substantially greater sensitivity of alignment in the context of errors and true variants, especially insertions and deletions, and minimize false mappings, while maintaining adequate speed compared to other current methods. We show BFAST can align the amount of data needed to fully resequence a human genome, one billion reads, with high sensitivity and accuracy, on a modest computer cluster in less than 24 hours. BFAST is available at (http://bfast.sourceforge.net.

  7. Deep resequencing reveals allelic variation in Sesamum indicum.

    Science.gov (United States)

    Wang, Linhai; Han, Xuelian; Zhang, Yanxin; Li, Donghua; Wei, Xin; Ding, Xia; Zhang, Xiurong

    2014-08-20

    Characterization of genome-wide patterns of allelic variation and linkage disequilibrium can be used to detect reliable phenotype-genotype associations and signatures of molecular selection. However, the use of Sesamum indicum germplasm for breeding is limited by the lack of polymorphism data. Here we describe the massively parallel resequencing of 29 sesame strains from 12 countries at a depth of ≥ 13-fold coverage for each of the samples tested. We detected an average of 127,347 SNPs, 17,961 small InDels, and 9,266 structural variants per sample. The population SNP rate, population diversity (π) and Watterson's estimator of segregating sites (θw) were estimated at 8.6 × 10⁻³, 2.5 × 10⁻³ and 3.0 × 10⁻³ bp⁻¹, respectively. Of these SNPs, 23.2% were located within coding regions. Polymorphism patterns were nonrandom among gene families, with genes mediating interactions with the biotic or abiotic environment exhibiting high levels of polymorphism. The linkage disequilibrium (LD) decay distance was estimated at 150 kb, with no distinct structure observed in the population. Phylogenetic relationships between each of the 29 sesame strains were consistent with the hypothesis of sesame originating on the Indian subcontinent. In addition, we proposed novel roles for adenylate isopentenyltransferase (ITP) genes in determining the number of flowers per leaf axil of sesame by mediating zeatin biosynthesis. This study represents the first report of genome-wide patterns of genetic variation in sesame. The high LD distance and abundant polymorphisms described here increase our understanding of the forces shaping population-wide sequence variation in sesame and will be a valuable resource for future gene-phenotype and genome-wide association studies (GWAS).

  8. Swarm: robust and fast clustering method for amplicon-based studies

    Science.gov (United States)

    Rognes, Torbjørn; Quince, Christopher; de Vargas, Colomban; Dunthorn, Micah

    2014-01-01

    Popular de novo amplicon clustering methods suffer from two fundamental flaws: arbitrary global clustering thresholds, and input-order dependency induced by centroid selection. Swarm was developed to address these issues by first clustering nearly identical amplicons iteratively using a local threshold, and then by using clusters’ internal structure and amplicon abundances to refine its results. This fast, scalable, and input-order independent approach reduces the influence of clustering parameters and produces robust operational taxonomic units. PMID:25276506

  9. FlowClus: efficiently filtering and denoising pyrosequenced amplicons.

    Science.gov (United States)

    Gaspar, John M; Thomas, W Kelley

    2015-03-27

    Reducing the effects of sequencing errors and PCR artifacts has emerged as an essential component in amplicon-based metagenomic studies. Denoising algorithms have been designed that can reduce error rates in mock community data, but they change the sequence data in a manner that can be inconsistent with the process of removing errors in studies of real communities. In addition, they are limited by the size of the dataset and the sequencing technology used. FlowClus uses a systematic approach to filter and denoise reads efficiently. When denoising real datasets, FlowClus provides feedback about the process that can be used as the basis to adjust the parameters of the algorithm to suit the particular dataset. When used to analyze a mock community dataset, FlowClus produced a lower error rate compared to other denoising algorithms, while retaining significantly more sequence information. Among its other attributes, FlowClus can analyze longer reads being generated from all stages of 454 sequencing technology, as well as from Ion Torrent. It has processed a large dataset of 2.2 million GS-FLX Titanium reads in twelve hours; using its more efficient (but less precise) trie analysis option, this time was further reduced, to seven minutes. Many of the amplicon-based metagenomics datasets generated over the last several years have been processed through a denoising pipeline that likely caused deleterious effects on the raw data. By using FlowClus, one can avoid such negative outcomes while maintaining control over the filtering and denoising processes. Because of its efficiency, FlowClus can be used to re-analyze multiple large datasets together, thereby leading to more standardized conclusions. FlowClus is freely available on GitHub (jsh58/FlowClus); it is written in C and supported on Linux.

  10. Deep amplicon sequencing reveals mixed phytoplasma infection within single grapevine plants

    DEFF Research Database (Denmark)

    Nicolaisen, Mogens; Contaldo, Nicoletta; Makarova, Olga

    2011-01-01

    The diversity of phytoplasmas within single plants has not yet been fully investigated. In this project, deep amplicon sequencing was used to generate 50,926 phytoplasma sequences from 11 phytoplasma-infected grapevine samples from a PCR amplicon in the 5' end of the 16S region. After clustering ...

  11. Multiplex PCR, amplicon size and hybridization efficiency on the NanoChip electronic microarray

    DEFF Research Database (Denmark)

    Børsting, Claus; Sanchez, Juan J; Morling, Niels

    2004-01-01

    . Hybridization to individual amplicons in multiplexes was less efficient suggesting that intramolecular and intermolecular interactions may block access to the target sequence on the NanoChip array. We observed a high risk of contamination with amplicons shorter than 60 bp and therefore, we recommend the use...

  12. Isolation and analysis of high quality nuclear DNA with reduced organellar DNA for plant genome sequencing and resequencing

    Directory of Open Access Journals (Sweden)

    Zdepski Anna

    2011-05-01

    Full Text Available Abstract Background High throughput sequencing (HTS technologies have revolutionized the field of genomics by drastically reducing the cost of sequencing, making it feasible for individual labs to sequence or resequence plant genomes. Obtaining high quality, high molecular weight DNA from plants poses significant challenges due to the high copy number of chloroplast and mitochondrial DNA, as well as high levels of phenolic compounds and polysaccharides. Multiple methods have been used to isolate DNA from plants; the CTAB method is commonly used to isolate total cellular DNA from plants that contain nuclear DNA, as well as chloroplast and mitochondrial DNA. Alternatively, DNA can be isolated from nuclei to minimize chloroplast and mitochondrial DNA contamination. Results We describe optimized protocols for isolation of nuclear DNA from eight different plant species encompassing both monocot and eudicot species. These protocols use nuclei isolation to minimize chloroplast and mitochondrial DNA contamination. We also developed a protocol to determine the number of chloroplast and mitochondrial DNA copies relative to the nuclear DNA using quantitative real time PCR (qPCR. We compared DNA isolated from nuclei to total cellular DNA isolated with the CTAB method. As expected, DNA isolated from nuclei consistently yielded nuclear DNA with fewer chloroplast and mitochondrial DNA copies, as compared to the total cellular DNA prepared with the CTAB method. This protocol will allow for analysis of the quality and quantity of nuclear DNA before starting a plant whole genome sequencing or resequencing experiment. Conclusions Extracting high quality, high molecular weight nuclear DNA in plants has the potential to be a bottleneck in the era of whole genome sequencing and resequencing. The methods that are described here provide a framework for researchers to extract and quantify nuclear DNA in multiple types of plants.

  13. Genome resequencing and comparative variome analysis in a Brassica rapa and Brassica oleracea collection

    NARCIS (Netherlands)

    Cheng, Feng; Wu, Jian; Cai, Chengcheng; Fu, Lixia; Liang, Jianli; Borm, Theo; Zhuang, Mu; Zhang, Yangyong; Zhang, Fenglan; Bonnema, Guusje; Wang, Xiaowu

    2016-01-01

    The closely related species Brassica rapa and B. oleracea encompass a wide range of vegetable, fodder and oil crops. The release of their reference genomes has facilitated resequencing collections of B. rapa and B. oleracea aiming to build their variome datasets. These data can be used to

  14. Targeted resequencing in epileptic encephalopathies identifies de novo mutations in CHD2 and SYNGAP1

    DEFF Research Database (Denmark)

    Carvill, Gemma L; Heavin, Sinéad B; Yendle, Simone C

    2013-01-01

    Epileptic encephalopathies are a devastating group of epilepsies with poor prognosis, of which the majority are of unknown etiology. We perform targeted massively parallel resequencing of 19 known and 46 candidate genes for epileptic encephalopathy in 500 affected individuals (cases) to identify ...

  15. A systematic, large-scale resequencing screen of X-chromosome coding exons in mental retardation.

    NARCIS (Netherlands)

    Tarpey, P.S.; Smith, R.; Pleasance, E.; Whibley, A.; Edkins, S.; Hardy, C.; O'Meara, S.; Latimer, C.; Dicks, E.; Menzies, A.; Stephens, P.; Blow, M.; Greenman, C.; Xue, Y.; Tyler-Smith, C.; Thompson, D.; Gray, K.; Andrews, J.; Barthorpe, S.; Buck, G.; Cole, J.; Dunmore, R.; Jones, D.; Maddison, M.; Mironenko, T.; Turner, R.; Turrell, K.; Varian, J.; West, S.; Widaa, S.; Wray, P.; Teague, J.; Butler, A.; Jenkinson, A.; Jia, M.; Richardson, D.; Shepherd, R.; Wooster, R.; Tejada, M.I.; Martinez, F.; Carvill, G.; Goliath, R.; Brouwer, A.P.M. de; Bokhoven, H. van; Esch, H. van; Chelly, J.; Raynaud, M.; Ropers, H.H.; Abidi, F.E.; Srivastava, A.K.; Cox, J.; Luo, Y.; Mallya, U.; Moon, J.; Parnau, J.; Mohammed, S.; Tolmie, J.L.; Shoubridge, C.; Corbett, M.; Gardner, A.; Haan, E.; Rujirabanjerd, S.; Shaw, M.A.; Vandeleur, L.; Fullston, T.; Easton, D.F.; Boyle, J.; Partington, M.; Hackett, A.; Field, M.; Skinner, C.; Stevenson, R.E.; Bobrow, M.; Turner, G.; Schwartz, C.E.; Gecz, J.; Raymond, F.L.; Futreal, P.A.; Stratton, M.R.

    2009-01-01

    Large-scale systematic resequencing has been proposed as the key future strategy for the discovery of rare, disease-causing sequence variants across the spectrum of human complex disease. We have sequenced the coding exons of the X chromosome in 208 families with X-linked mental retardation (XLMR),

  16. Metatranscriptomics and Amplicon Sequencing Reveal Mutualisms in Seagrass Microbiomes

    Directory of Open Access Journals (Sweden)

    Byron C. Crump

    2018-03-01

    Full Text Available Terrestrial plants benefit from many well-understood mutualistic relationships with root- and leaf-associated microbiomes, but relatively little is known about these relationships for seagrass and other aquatic plants. We used 16S rRNA gene amplicon sequencing and metatranscriptomics to assess potential mutualisms between microorganisms and the seagrasses Zostera marina and Zostera japonica collected from mixed beds in Netarts Bay, OR, United States. The phylogenetic composition of leaf-, root-, and water column-associated bacterial communities were strikingly different, but these communities were not significantly different between plant species. Many taxa present on leaves were related to organisms capable of consuming the common plant metabolic waste product methanol, and of producing agarases, which can limit the growth of epiphytic algae. Taxa present on roots were related to organisms capable of oxidizing toxic sulfur compounds and of fixing nitrogen. Metatranscriptomic sequencing identified expression of genes involved in all of these microbial metabolic processes at levels greater than typical water column bacterioplankton, and also identified expression of genes involved in denitrification and in bacterial synthesis of the plant growth hormone indole-3-acetate. These results provide the first evidence using metatranscriptomics that seagrass microbiomes carry out a broad range of functions that may benefit their hosts, and imply that microbe–plant mutualisms support the health and growth of aquatic plants.

  17. Use of AmpliWax to optimize amplicon sterilization by isopsoralen.

    OpenAIRE

    De la Viuda, M; Fille, M; Ruiz, J; Aslanzadeh, J

    1996-01-01

    The photochemical inactivation of amplicons by isopsoralen (IP-10) has been suggested as a possible means to prevent PCR carryover contamination. To evaluate the technique, serial dilutions of amplicons (10(11) to 10(3)) from the Borrelia burgdorferi OSP A gene were amplified in the presence of 0, 25, 50, and 100 micrograms of IP-10 per ml for 45 cycles. The PCR products were exposed to UV light for 15 min to activate IP-10 and sterilize the amplicons. One microliter of each sterilized sample...

  18. Characterization of Novel Genes With 8p11-12 Amplicon in Breast Cancer

    National Research Council Canada - National Science Library

    Yang, Zeng-Quan

    2004-01-01

    ...% of human breast cancer (HBC). Using chromosome comparative genomic hybridization (CGH) and array CGH, overlapping amplicons were found to be centered around chromosome 8p11-p12 in 3 breast cancer cell lines developed in Dr...

  19. Genome resequencing and comparative variome analysis in a Brassica rapa and Brassica oleracea collection.

    Science.gov (United States)

    Cheng, Feng; Wu, Jian; Cai, Chengcheng; Fu, Lixia; Liang, Jianli; Borm, Theo; Zhuang, Mu; Zhang, Yangyong; Zhang, Fenglan; Bonnema, Guusje; Wang, Xiaowu

    2016-12-20

    The closely related species Brassica rapa and B. oleracea encompass a wide range of vegetable, fodder and oil crops. The release of their reference genomes has facilitated resequencing collections of B. rapa and B. oleracea aiming to build their variome datasets. These data can be used to investigate the evolutionary relationships between and within the different species and the domestication of the crops, hereafter named morphotypes. These data can also be used in genetic studies aiming at the identification of genes that influence agronomic traits. We selected and resequenced 199 B. rapa and 119 B. oleracea accessions representing 12 and nine morphotypes, respectively. Based on these resequencing data, we obtained 2,249,473 and 3,852,169 high quality SNPs (single-nucleotide polymorphisms), as well as 303,617 and 417,004 InDels for the B. rapa and B. oleracea populations, respectively. The variome datasets of B. rapa and B. oleracea represent valuable resources to researchers working on evolution, domestication or breeding of Brassica vegetable crops.

  20. A modified method for whole exome resequencing from minimal amounts of starting DNA.

    Directory of Open Access Journals (Sweden)

    Iwanka Kozarewa

    Full Text Available Next generation DNA sequencing (NGS technologies have revolutionized the pace at which whole genome and exome sequences can be generated. However, despite these advances, many of the methods for targeted resequencing, such as the generation of high-depth exome sequences, are somewhat limited by the relatively large amounts of starting DNA that are normally required. In the case of tumour analysis this is particularly pertinent as many tumour biopsies often return submicrogram quantities of DNA, especially when tumours are microdissected prior to analysis. Here, we present a method for exome capture and resequencing using as little as 50 ng of starting DNA. The sequencing libraries generated by this minimal starting amount (MSA-Cap method generate datasets that are comparable to standard amount (SA whole exome libraries that use three micrograms of starting DNA. This method, which can be performed in most laboratories using commonly available reagents, has the potential to enhance large scale profiling efforts such as the resequencing of tumour exomes.

  1. A modified method for whole exome resequencing from minimal amounts of starting DNA.

    Science.gov (United States)

    Kozarewa, Iwanka; Rosa-Rosa, Juan Manuel; Wardell, Christopher P; Walker, Brian A; Fenwick, Kerry; Assiotis, Ioannis; Mitsopoulos, Costas; Zvelebil, Marketa; Morgan, Gareth J; Ashworth, Alan; Lord, Christopher J; J, Christopher

    2012-01-01

    Next generation DNA sequencing (NGS) technologies have revolutionized the pace at which whole genome and exome sequences can be generated. However, despite these advances, many of the methods for targeted resequencing, such as the generation of high-depth exome sequences, are somewhat limited by the relatively large amounts of starting DNA that are normally required. In the case of tumour analysis this is particularly pertinent as many tumour biopsies often return submicrogram quantities of DNA, especially when tumours are microdissected prior to analysis. Here, we present a method for exome capture and resequencing using as little as 50 ng of starting DNA. The sequencing libraries generated by this minimal starting amount (MSA-Cap) method generate datasets that are comparable to standard amount (SA) whole exome libraries that use three micrograms of starting DNA. This method, which can be performed in most laboratories using commonly available reagents, has the potential to enhance large scale profiling efforts such as the resequencing of tumour exomes.

  2. JRC GMO-Amplicons: a collection of nucleic acid sequences related to genetically modified organisms.

    Science.gov (United States)

    Petrillo, Mauro; Angers-Loustau, Alexandre; Henriksson, Peter; Bonfini, Laura; Patak, Alex; Kreysa, Joachim

    2015-01-01

    The DNA target sequence is the key element in designing detection methods for genetically modified organisms (GMOs). Unfortunately this information is frequently lacking, especially for unauthorized GMOs. In addition, patent sequences are generally poorly annotated, buried in complex and extensive documentation and hard to link to the corresponding GM event. Here, we present the JRC GMO-Amplicons, a database of amplicons collected by screening public nucleotide sequence databanks by in silico determination of PCR amplification with reference methods for GMO analysis. The European Union Reference Laboratory for Genetically Modified Food and Feed (EU-RL GMFF) provides these methods in the GMOMETHODS database to support enforcement of EU legislation and GM food/feed control. The JRC GMO-Amplicons database is composed of more than 240 000 amplicons, which can be easily accessed and screened through a web interface. To our knowledge, this is the first attempt at pooling and collecting publicly available sequences related to GMOs in food and feed. The JRC GMO-Amplicons supports control laboratories in the design and assessment of GMO methods, providing inter-alia in silico prediction of primers specificity and GM targets coverage. The new tool can assist the laboratories in the analysis of complex issues, such as the detection and identification of unauthorized GMOs. Notably, the JRC GMO-Amplicons database allows the retrieval and characterization of GMO-related sequences included in patents documentation. Finally, it can help annotating poorly described GM sequences and identifying new relevant GMO-related sequences in public databases. The JRC GMO-Amplicons is freely accessible through a web-based portal that is hosted on the EU-RL GMFF website. Database URL: http://gmo-crl.jrc.ec.europa.eu/jrcgmoamplicons/. © The Author(s) 2015. Published by Oxford University Press.

  3. Efficient error correction for next-generation sequencing of viral amplicons

    Directory of Open Access Journals (Sweden)

    Skums Pavel

    2012-06-01

    Full Text Available Abstract Background Next-generation sequencing allows the analysis of an unprecedented number of viral sequence variants from infected patients, presenting a novel opportunity for understanding virus evolution, drug resistance and immune escape. However, sequencing in bulk is error prone. Thus, the generated data require error identification and correction. Most error-correction methods to date are not optimized for amplicon analysis and assume that the error rate is randomly distributed. Recent quality assessment of amplicon sequences obtained using 454-sequencing showed that the error rate is strongly linked to the presence and size of homopolymers, position in the sequence and length of the amplicon. All these parameters are strongly sequence specific and should be incorporated into the calibration of error-correction algorithms designed for amplicon sequencing. Results In this paper, we present two new efficient error correction algorithms optimized for viral amplicons: (i k-mer-based error correction (KEC and (ii empirical frequency threshold (ET. Both were compared to a previously published clustering algorithm (SHORAH, in order to evaluate their relative performance on 24 experimental datasets obtained by 454-sequencing of amplicons with known sequences. All three algorithms show similar accuracy in finding true haplotypes. However, KEC and ET were significantly more efficient than SHORAH in removing false haplotypes and estimating the frequency of true ones. Conclusions Both algorithms, KEC and ET, are highly suitable for rapid recovery of error-free haplotypes obtained by 454-sequencing of amplicons from heterogeneous viruses. The implementations of the algorithms and data sets used for their testing are available at: http://alan.cs.gsu.edu/NGS/?q=content/pyrosequencing-error-correction-algorithm

  4. Resequencing studies of nonmodel organisms using closely related reference genomes: optimal experimental designs and bioinformatics approaches for population genomics.

    Science.gov (United States)

    Nevado, B; Ramos-Onsins, S E; Perez-Enciso, M

    2014-04-01

    Decreasing costs of next-generation sequencing (NGS) experiments have made a wide range of genomic questions open for study with nonmodel organisms. However, experimental designs and analysis of NGS data from less well-known species are challenging because of the lack of genomic resources. In this work, we investigate the performance of alternative experimental designs and bioinformatics approaches in estimating variability and neutrality tests based on the site-frequency-spectrum (SFS) from individual resequencing data. We pay particular attention to challenges faced in the study of nonmodel organisms, in particular the absence of a species-specific reference genome, although phylogenetically close genomes are assumed to be available. We compare the performance of three alternative bioinformatics approaches – genotype calling, genotype–haplotype calling and direct estimation without calling genotypes. We find that relying on genotype calls provides biased estimates of population genetic statistics at low to moderate read depth (2–8X). Genotype–haplotype calling returns more accurate estimates irrespective of the divergence to the reference genome, but requires moderate depth (8–20X). Direct estimation without calling genotypes returns the most accurate estimates of variability and of most SFS tests investigated, including at low read depth (2–4X). Studies without species-specific reference genome should thus aim for low read depth and avoid genotype calling whenever individual genotypes are not essential. Otherwise, aiming for moderate to high depth at the expense of number of individuals, and using genotype–haplotype calling, is recommended.

  5. Evaluation of Quality Assessment Protocols for High Throughput Genome Resequencing Data.

    Science.gov (United States)

    Chiara, Matteo; Pavesi, Giulio

    2017-01-01

    Large-scale initiatives aiming to recover the complete sequence of thousands of human genomes are currently being undertaken worldwide, concurring to the generation of a comprehensive catalog of human genetic variation. The ultimate and most ambitious goal of human population scale genomics is the characterization of the so-called human "variome," through the identification of causal mutations or haplotypes. Several research institutions worldwide currently use genotyping assays based on Next-Generation Sequencing (NGS) for diagnostics and clinical screenings, and the widespread application of such technologies promises major revolutions in medical science. Bioinformatic analysis of human resequencing data is one of the main factors limiting the effectiveness and general applicability of NGS for clinical studies. The requirement for multiple tools, to be combined in dedicated protocols in order to accommodate different types of data (gene panels, exomes, or whole genomes) and the high variability of the data makes difficult the establishment of a ultimate strategy of general use. While there already exist several studies comparing sensitivity and accuracy of bioinformatic pipelines for the identification of single nucleotide variants from resequencing data, little is known about the impact of quality assessment and reads pre-processing strategies. In this work we discuss major strengths and limitations of the various genome resequencing protocols are currently used in molecular diagnostics and for the discovery of novel disease-causing mutations. By taking advantage of publicly available data we devise and suggest a series of best practices for the pre-processing of the data that consistently improve the outcome of genotyping with minimal impacts on computational costs.

  6. Genome editing using FACS enrichment of nuclease-expressing cells and indel detection by amplicon analysis

    DEFF Research Database (Denmark)

    Lonowski, Lindsey A; Narimatsu, Yoshiki; Riaz, Anjum

    2017-01-01

    ). First, Indel Detection by Amplicon Analysis (IDAA) determines the size and frequency of insertions and deletions elicited by nucleases in cells, tissues or embryos through analysis of fluorophore-labeled PCR amplicons covering the nuclease target site by capillary electrophoresis in a sequenator. Second...... the testing of new nuclease reagents and the generation of edited cell pools or clonal cell lines, reducing the number of clones that need to be generated and increasing the ease with which they are screened. The pipeline shortens the time line, but it most prominently reduces the workload of cell...

  7. Resuscitation Resequenced: A Rational Approach to Patients with Trauma in Shock.

    Science.gov (United States)

    Petrosoniak, Andrew; Hicks, Christopher

    2018-02-01

    Trauma resuscitation is a complex and dynamic process that requires a high-performing team to optimize patient outcomes. More than 30 years ago, Advanced Trauma Life Support was developed to formalize and standardize trauma care; however, the sequential nature of the algorithm that is used can lead to ineffective prioritization. An improved understanding of shock mandates an updated approach to trauma resuscitation. This article proposes a resequenced approach that (1) addresses immediate threats to life and (2) targets strategies for the diagnosis and management of shock causes. This updated approach emphasizes evidence-based resuscitation principles that align with physiologic priorities. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. Resequencing 50 accessions of cultivated and wild rice yields markers for identifying agronomically important genes

    DEFF Research Database (Denmark)

    Xu, Xun; Liu, Xin; Ge, Song

    2012-01-01

    Rice is a staple crop that has undergone substantial phenotypic and physiological changes during domestication. Here we resequenced the genomes of 40 cultivated accessions selected from the major groups of rice and 10 accessions of their wild progenitors (Oryza rufipogon and Oryza nivara) to >15 x...... diversity in cultivated but not wild rice, which represent candidate regions selected during domestication. Some of these variants are associated with important biological features, whereas others have yet to be functionally characterized. The molecular markers we have identified should be valuable...

  9. Using surface-enhanced Raman spectroscopy and electrochemically driven melting to discriminate Yersinia pestis from Y. pseudotuberculosis based on single nucleotide polymorphisms within unpurified polymerase chain reaction amplicons.

    Science.gov (United States)

    Papadopoulou, Evanthia; Goodchild, Sarah A; Cleary, David W; Weller, Simon A; Gale, Nittaya; Stubberfield, Michael R; Brown, Tom; Bartlett, Philip N

    2015-02-03

    The development of sensors for the detection of pathogen-specific DNA, including relevant species/strain level discrimination, is critical in molecular diagnostics with major impacts in areas such as bioterrorism and food safety. Herein, we use electrochemically driven denaturation assays monitored by surface-enhanced Raman spectroscopy (SERS) to target single nucleotide polymorphisms (SNPs) that distinguish DNA amplicons generated from Yersinia pestis, the causative agent of plague, from the closely related species Y. pseudotuberculosis. Two assays targeting SNPs within the groEL and metH genes of these two species have been successfully designed. Polymerase chain reaction (PCR) was used to produce Texas Red labeled single-stranded DNA (ssDNA) amplicons of 262 and 251 bases for the groEL and metH targets, respectively. These amplicons were used in an unpurified form to hybridize to immobilized probes then subjected to electrochemically driven melting. In all cases electrochemically driven melting was able to discriminate between fully homologous DNA and that containing SNPs. The metH assay was particularly challenging due to the presence of only a single base mismatch in the middle of the 251 base long PCR amplicon. However, manipulation of assay conditions (conducting the electrochemical experiments at 10 °C) resulted in greater discrimination between the complementary and mismatched DNA. Replicate data were collected and analyzed for each duplex on different days, using different batches of PCR product and different sphere segment void (SSV) substrates. Despite the variability introduced by these differences, the assays are shown to be reliable and robust providing a new platform for strain discrimination using unpurified PCR samples.

  10. Amplicon sequencing for the quantification of spoilage microbiota in complex foods including bacterial spores

    NARCIS (Netherlands)

    Boer, de P.; Caspers, M.; Sanders, J.W.; Kemperman, R.; Wijman, J.; Lommerse, G.; Roeselers, G.; Montijn, R.; Abee, T.; Kort, R.

    2015-01-01

    Background
    Spoilage of food products is frequently caused by bacterial spores and lactic acid bacteria. Identification of these organisms by classic cultivation methods is limited by their ability to form colonies on nutrient agar plates. In this study, we adapted and optimized 16S rRNA amplicon

  11. Overexpressed Genes/ESTs and Characterization of Distinct Amplicons on 17823 in Breast Cancer Cells

    Directory of Open Access Journals (Sweden)

    Ayse E. Erson

    2001-01-01

    Full Text Available 17823 is a frequent site of gene amplification in breast cancer. Several lines of evidence suggest the presence of multiple amplicons on 17823. To characterize distinct amplicons on 17823 and localize putative oncogenes, we screened genes and expressed sequence tags (ESTs in existing physical and radiation hybrid maps for amplification and overexpression in breast cancer cell lines by semiquantitative duplex PCR, semiquantitative duplex RT-PCR, Southern blot, Northern blot analyses. We identified two distinct amplicons on 17823, one including TBX2 and another proximal region including RPS6KB1 (PS6K and MUL. In addition to these previously reported overexpressed genes, we also identified amplification and overexpression of additional uncharacterized genes and ESTs, some of which suggest potential oncogenic activity. In conclusion, we have further defined two distinct regions of gene amplification and overexpression on 17823 with identification of new potential oncogene candidates. Based on the amplification and overexpression patterns of known and as of yet unrecognized genes on 17823, it is likely that some of these genes mapping to the discrete amplicons function as oncogenes and contribute to tumor progression in breast cancer cells.

  12. Global Perspectives on Activated Sludge Community Composition analyzed using 16S rRNA amplicon sequencing

    DEFF Research Database (Denmark)

    Nierychlo, Marta; Saunders, Aaron Marc; Albertsen, Mads

    Activated sludge is the most commonly applied bioprocess throughout the world for wastewater treatment. Microorganisms are key to the process, yet our knowledge of their identity and function is still limited. High-througput16S rRNA amplicon sequencing can reliably characterize microbial...

  13. Improved sensitivity of circulating tumor DNA measurement using short PCR amplicons

    DEFF Research Database (Denmark)

    Andersen, Rikke Fredslund; Spindler, Karen-Lise Garm; Brandslund, Ivan

    2015-01-01

    , however, presents a number of challenges that require attention. The amount of DNA is low and highly fragmented and analyses need to be optimized accordingly. KRAS ARMS-qPCR assays with amplicon lengths of 120 and 85 base pairs, respectively, were compared using positive control material (PCR fragments...

  14. Characterization of the 17p amplicon in human sarcomas: microsatellite marker analysis

    NARCIS (Netherlands)

    Wolf, M.; Tarkkanen, M.; Hulsebos, T.; Larramendy, M. L.; Forus, A.; Myklebost, O.; Aaltonen, L. A.; Elomaa, I.; Knuutila, S.

    1999-01-01

    The structure of the 17p amplicon from 9 human sarcoma specimens evaluated by comparative genomic hybridization (CGH) has been studied by analyzing 28 microsatellite markers by PCR. Eleven sarcoma specimens showing no DNA copy number increases at 17p by CGH were analyzed as control samples. Five

  15. Polyadenylated Sequencing Primers Enable Complete Readability of PCR Amplicons Analyzed by Dideoxynucleotide Sequencing

    Directory of Open Access Journals (Sweden)

    Martin Beránek

    2012-01-01

    Full Text Available Dideoxynucleotide DNA sequencing is one of the principal procedures in molecular biology. Loss of an initial part of nucleotides behind the 3' end of the sequencing primer limits the readability of sequenced amplicons. We present a method which extends the readability by using sequencing primers modified by polyadenylated tails attached to their 5' ends. Performing a polymerase chain reaction, we amplified eight amplicons of six human genes (AMELX, APOE, HFE, MBL2, SERPINA1 and TGFB1 ranging from 106 bp to 680 bp. Polyadenylation of the sequencing primers minimized the loss of bases in all amplicons. Complete sequences of shorter products (AMELX 106 bp, SERPINA1 121 bp, HFE 208 bp, APOE 244 bp, MBL2 317 bp were obtained. In addition, in the case of TGFB1 products (366 bp, 432 bp, and 680 bp, respectively, the lengths of sequencing readings were significantly longer if adenylated primers were used. Thus, single strand dideoxynucleotide sequencing with adenylated primers enables complete or near complete readability of short PCR amplicons.

  16. A database and API for variation, dense genotyping and resequencing data

    Directory of Open Access Journals (Sweden)

    Flicek Paul

    2010-05-01

    Full Text Available Abstract Background Advances in sequencing and genotyping technologies are leading to the widespread availability of multi-species variation data, dense genotype data and large-scale resequencing projects. The 1000 Genomes Project and similar efforts in other species are challenging the methods previously used for storage and manipulation of such data necessitating the redesign of existing genome-wide bioinformatics resources. Results Ensembl has created a database and software library to support data storage, analysis and access to the existing and emerging variation data from large mammalian and vertebrate genomes. These tools scale to thousands of individual genome sequences and are integrated into the Ensembl infrastructure for genome annotation and visualisation. The database and software system is easily expanded to integrate both public and non-public data sources in the context of an Ensembl software installation and is already being used outside of the Ensembl project in a number of database and application environments. Conclusions Ensembl's powerful, flexible and open source infrastructure for the management of variation, genotyping and resequencing data is freely available at http://www.ensembl.org.

  17. Integration of transcriptome and whole genomic resequencing data to identify key genes affecting swine fat deposition.

    Directory of Open Access Journals (Sweden)

    Kai Xing

    Full Text Available Fat deposition is highly correlated with the growth, meat quality, reproductive performance and immunity of pigs. Fatty acid synthesis takes place mainly in the adipose tissue of pigs; therefore, in this study, a high-throughput massively parallel sequencing approach was used to generate adipose tissue transcriptomes from two groups of Songliao black pigs that had opposite backfat thickness phenotypes. The total number of paired-end reads produced for each sample was in the range of 39.29-49.36 millions. Approximately 188 genes were differentially expressed in adipose tissue and were enriched for metabolic processes, such as fatty acid biosynthesis, lipid synthesis, metabolism of fatty acids, etinol, caffeine and arachidonic acid and immunity. Additionally, many genetic variations were detected between the two groups through pooled whole-genome resequencing. Integration of transcriptome and whole-genome resequencing data revealed important genomic variations among the differentially expressed genes for fat deposition, for example, the lipogenic genes. Further studies are required to investigate the roles of candidate genes in fat deposition to improve pig breeding programs.

  18. [Identification of novel pathogenic gene mutations in pediatric acute myeloid leukemia by whole-exome resequencing].

    Science.gov (United States)

    Shiba, Norio

    2015-12-01

    A new class of gene mutations, identified in the pathogenesis of adult acute myeloid leukemia (AML), includes DNMT3A, IDH1/2, TET2 and EZH2. However, these mutations are rare in pediatric AML cases, indicating that pathogeneses differ between adult and pediatric forms of AML. Meanwhile, the recent development of massively parallel sequencing technologies has provided a new opportunity to discover genetic changes across entire genomes or proteincoding sequences. In order to reveal a complete registry of gene mutations, we performed whole exome resequencing of paired tumor-normal specimens from 19 pediatric AML cases using Illumina HiSeq 2000. In total, 80 somatic mutations or 4.2 mutations per sample were identified. Many of the recurrent mutations identified in this study involved previously reported targets in AML, such as FLT3, CEBPA, KIT, CBL, NRAS, WT1 and EZH2. On the other hand, several genes were newly identified in the current study, including BCORL1 and major cohesin components such as SMC3 and RAD21. Whole exome resequencing revealed a complex array of gene mutations in pediatric AML genomes. Our results indicate that a subset of pediatric AML represents a discrete entity that could be discriminated from its adult counterpart, in terms of the spectrum of gene mutations.

  19. Global assessment of genomic variation in cattle by genome resequencing and high-throughput genotyping

    DEFF Research Database (Denmark)

    Zhan, Bujie; Fadista, João; Thomsen, Bo

    2011-01-01

    sequence of a single Holstein Friesian bull with data from single nucleotide polymorphism (SNP) and comparative genomic hybridization (CGH) array technologies to determine a comprehensive spectrum of genomic variation. The performance of resequencing SNP detection was assessed by combining SNPs that were...... of split-read and read-pair approaches proved to be complementary in finding different signatures. CNVs were identified on the basis of the depth of sequenced reads, and by using SNP and CGH arrays. Conclusions Our results provide high resolution mapping of diverse classes of genomic variation...... in an individual bovine genome and demonstrate that structural variation surpasses sequence variation as the main component of genomic variability. Better accuracy of SNP detection was achieved with little loss of sensitivity when algorithms that implemented mapping quality were used. IBD regions were found...

  20. Molecular footprints of domestication and improvement in soybean revealed by whole genome re-sequencing

    DEFF Research Database (Denmark)

    Li, Ying-hui; Zhao, Shan-cen; Ma, Jian-xin

    2013-01-01

    BACKGROUND:Artificial selection played an important role in the origin of modern Glycine max cultivars from the wild soybean Glycine soja. To elucidate the consequences of artificial selection accompanying the domestication and modern improvement of soybean, 25 new and 30 published whole-genome re......-sequencing accessions, which represent wild, domesticated landrace, and Chinese elite soybean populations were analyzed.RESULTS:A total of 5,102,244 single nucleotide polymorphisms (SNPs) and 707,969 insertion/deletions were identified. Among the SNPs detected, 25.5% were not described previously. We found...... that artificial selection during domestication led to more pronounced reduction in the genetic diversity of soybean than the switch from landraces to elite cultivars. Only a small proportion (2.99%) of the whole genomic regions appear to be affected by artificial selection for preferred agricultural traits...

  1. A systematic, large-scale resequencing screen of X-chromosome coding exons in mental retardation

    Science.gov (United States)

    Tarpey, Patrick S; Smith, Raffaella; Pleasance, Erin; Whibley, Annabel; Edkins, Sarah; Hardy, Claire; O'Meara, Sarah; Latimer, Calli; Dicks, Ed; Menzies, Andrew; Stephens, Phil; Blow, Matt; Greenman, Chris; Xue, Yali; Tyler-Smith, Chris; Thompson, Deborah; Gray, Kristian; Andrews, Jenny; Barthorpe, Syd; Buck, Gemma; Cole, Jennifer; Dunmore, Rebecca; Jones, David; Maddison, Mark; Mironenko, Tatiana; Turner, Rachel; Turrell, Kelly; Varian, Jennifer; West, Sofie; Widaa, Sara; Wray, Paul; Teague, Jon; Butler, Adam; Jenkinson, Andrew; Jia, Mingming; Richardson, David; Shepherd, Rebecca; Wooster, Richard; Tejada, M Isabel; Martinez, Francisco; Carvill, Gemma; Goliath, Rene; de Brouwer, Arjan P M; van Bokhoven, Hans; Van Esch, Hilde; Chelly, Jamel; Raynaud, Martine; Ropers, Hans-Hilger; Abidi, Fatima E; Srivastava, Anand K; Cox, James; Luo, Ying; Mallya, Uma; Moon, Jenny; Parnau, Josef; Mohammed, Shehla; Tolmie, John L; Shoubridge, Cheryl; Corbett, Mark; Gardner, Alison; Haan, Eric; Rujirabanjerd, Sinitdhorn; Shaw, Marie; Vandeleur, Lucianne; Fullston, Tod; Easton, Douglas F; Boyle, Jackie; Partington, Michael; Hackett, Anna; Field, Michael; Skinner, Cindy; Stevenson, Roger E; Bobrow, Martin; Turner, Gillian; Schwartz, Charles E; Gecz, Jozef; Raymond, F Lucy; Futreal, P Andrew; Stratton, Michael R

    2010-01-01

    Large-scale systematic resequencing has been proposed as the key future strategy for the discovery of rare, disease-causing sequence variants across the spectrum of human complex disease. We have sequenced the coding exons of the X chromosome in 208 families with X-linked mental retardation (XLMR), the largest direct screen for constitutional disease-causing mutations thus far reported. The screen has discovered nine genes implicated in XLMR, including SYP, ZNF711 and CASK reported here, confirming the power of this strategy. The study has, however, also highlighted issues confronting whole-genome sequencing screens, including the observation that loss of function of 1% or more of X-chromosome genes is compatible with apparently normal existence. PMID:19377476

  2. The draft genome of watermelon (Citrullus lanatus) and resequencing of 20 diverse accessions

    DEFF Research Database (Denmark)

    Guo, Shaogui; Zhang, Jianguo; Sun, Honghe

    2013-01-01

    Watermelon, Citrullus lanatus, is an important cucurbit crop grown throughout the world. Here we report a high-quality draft genome sequence of the east Asia watermelon cultivar 97103 (2n = 2× = 22) containing 23,440 predicted protein-coding genes. Comparative genomics analysis provided...... an evolutionary scenario for the origin of the 11 watermelon chromosomes derived from a 7-chromosome paleohexaploid eudicot ancestor. Resequencing of 20 watermelon accessions representing three different C. lanatus subspecies produced numerous haplotypes and identified the extent of genetic diversity...... and population structure of watermelon germplasm. Genomic regions that were preferentially selected during domestication were identified. Many disease-resistance genes were also found to be lost during domestication. In addition, integrative genomic and transcriptomic analyses yielded important insights...

  3. The draft genome of watermelon (Citrullus lanatus) and resequencing of 20 diverse accessions.

    Science.gov (United States)

    Guo, Shaogui; Zhang, Jianguo; Sun, Honghe; Salse, Jerome; Lucas, William J; Zhang, Haiying; Zheng, Yi; Mao, Linyong; Ren, Yi; Wang, Zhiwen; Min, Jiumeng; Guo, Xiaosen; Murat, Florent; Ham, Byung-Kook; Zhang, Zhaoliang; Gao, Shan; Huang, Mingyun; Xu, Yimin; Zhong, Silin; Bombarely, Aureliano; Mueller, Lukas A; Zhao, Hong; He, Hongju; Zhang, Yan; Zhang, Zhonghua; Huang, Sanwen; Tan, Tao; Pang, Erli; Lin, Kui; Hu, Qun; Kuang, Hanhui; Ni, Peixiang; Wang, Bo; Liu, Jingan; Kou, Qinghe; Hou, Wenju; Zou, Xiaohua; Jiang, Jiao; Gong, Guoyi; Klee, Kathrin; Schoof, Heiko; Huang, Ying; Hu, Xuesong; Dong, Shanshan; Liang, Dequan; Wang, Juan; Wu, Kui; Xia, Yang; Zhao, Xiang; Zheng, Zequn; Xing, Miao; Liang, Xinming; Huang, Bangqing; Lv, Tian; Wang, Junyi; Yin, Ye; Yi, Hongping; Li, Ruiqiang; Wu, Mingzhu; Levi, Amnon; Zhang, Xingping; Giovannoni, James J; Wang, Jun; Li, Yunfu; Fei, Zhangjun; Xu, Yong

    2013-01-01

    Watermelon, Citrullus lanatus, is an important cucurbit crop grown throughout the world. Here we report a high-quality draft genome sequence of the east Asia watermelon cultivar 97103 (2n = 2× = 22) containing 23,440 predicted protein-coding genes. Comparative genomics analysis provided an evolutionary scenario for the origin of the 11 watermelon chromosomes derived from a 7-chromosome paleohexaploid eudicot ancestor. Resequencing of 20 watermelon accessions representing three different C. lanatus subspecies produced numerous haplotypes and identified the extent of genetic diversity and population structure of watermelon germplasm. Genomic regions that were preferentially selected during domestication were identified. Many disease-resistance genes were also found to be lost during domestication. In addition, integrative genomic and transcriptomic analyses yielded important insights into aspects of phloem-based vascular signaling in common between watermelon and cucumber and identified genes crucial to valuable fruit-quality traits, including sugar accumulation and citrulline metabolism.

  4. Evaluation of genetic variation among Brazilian soybean cultivars through genome resequencing.

    Science.gov (United States)

    Maldonado dos Santos, João Vitor; Valliyodan, Babu; Joshi, Trupti; Khan, Saad M; Liu, Yang; Wang, Juexin; Vuong, Tri D; de Oliveira, Marcelo Fernandes; Marcelino-Guimarães, Francismar Corrêa; Xu, Dong; Nguyen, Henry T; Abdelnoor, Ricardo Vilela

    2016-02-13

    Soybean [Glycine max (L.) Merrill] is one of the most important legumes cultivated worldwide, and Brazil is one of the main producers of this crop. Since the sequencing of its reference genome, interest in structural and allelic variations of cultivated and wild soybean germplasm has grown. To investigate the genetics of the Brazilian soybean germplasm, we selected soybean cultivars based on the year of commercialization, geographical region and maturity group and resequenced their genomes. We resequenced the genomes of 28 Brazilian soybean cultivars with an average genome coverage of 14.8X. A total of 5,835,185 single nucleotide polymorphisms (SNPs) and 1,329,844 InDels were identified across the 20 soybean chromosomes, with 541,762 SNPs, 98,922 InDels and 1,093 CNVs that were exclusive to the 28 Brazilian cultivars. In addition, 668 allelic variations of 327 genes were shared among all of the Brazilian cultivars, including genes related to DNA-dependent transcription-elongation, photosynthesis, ATP synthesis-coupled electron transport, cellular respiration, and precursors of metabolite generation and energy. A very homogeneous structure was also observed for the Brazilian soybean germplasm, and we observed 41 regions putatively influenced by positive selection. Finally, we detected 3,880 regions with copy-number variations (CNVs) that could help to explain the divergence among the accessions evaluated. The large number of allelic and structural variations identified in this study can be used in marker-assisted selection programs to detect unique SNPs for cultivar fingerprinting. The results presented here suggest that despite the diversification of modern Brazilian cultivars, the soybean germplasm remains very narrow because of the large number of genome regions that exhibit low diversity. These results emphasize the need to introduce new alleles to increase the genetic diversity of the Brazilian germplasm.

  5. Bacterial and viral identification and differentiation by amplicon sequencing on the MinION nanopore sequencer.

    Science.gov (United States)

    Kilianski, Andy; Haas, Jamie L; Corriveau, Elizabeth J; Liem, Alvin T; Willis, Kristen L; Kadavy, Dana R; Rosenzweig, C Nicole; Minot, Samuel S

    2015-01-01

    The MinION™ nanopore sequencer was recently released to a community of alpha-testers for evaluation using a variety of sequencing applications. Recent reports have tested the ability of the MinION™ to act as a whole genome sequencer and have demonstrated that nanopore sequencing has tremendous potential utility. However, the current nanopore technology still has limitations with respect to error-rate, and this is problematic when attempting to assemble whole genomes without secondary rounds of sequencing to correct errors. In this study, we tested the ability of the MinION™ nanopore sequencer to accurately identify and differentiate bacterial and viral samples via directed sequencing of characteristic genes shared broadly across a target clade. Using a 6 hour sequencing run time, sufficient data were generated to identify an E. coli sample down to the species level from 16S rDNA amplicons. Three poxviruses (cowpox, vaccinia-MVA, and vaccinia-Lister) were identified and differentiated down to the strain level, despite over 98% identity between the vaccinia strains. The ability to differentiate strains by amplicon sequencing on the MinION™ was accomplished despite an observed per-base error rate of approximately 30%. While nanopore sequencing, using the MinION™ platform from Oxford Nanopore in particular, continues to mature into a commercially available technology, practical uses are sought for the current versions of the technology. This study offers evidence of the utility of amplicon sequencing by demonstrating that the current versions of MinION™ technology can accurately identify and differentiate both viral and bacterial species present within biological samples via amplicon sequencing.

  6. Pregnancy-associated glycoprotein (PAG) family: transcripts and gene amplicons in camelids.

    Science.gov (United States)

    Majewska, Marta; Panasiewicz, Grzegorz; Klisch, Karl; Olivera, Louis V M; Mamani, Javier M; Abd-Elnaeim, Mahmoud M; Szafranska, Bozena

    2009-07-01

    In this study, the placental localization of PAG-like transcripts and genomic existence of PAG-like amplicons in new-world (Lp, Lama pacos, alpaca) and old-world camelids (Cb, Camelus bactrianus, bactrian; Cd, Camelus dromedarius; dromedary) are reported for the first time. Sections of Lp (150-347 days post coitum), Cd (43-90 cm crown-rump length) and Cb (term) placentas were used for heterologous (ht; cross-species) autoradiographic in situ hybridization (aISH) with single-stranded diagnostic (antisense) or control (sense) [alpha-(35)S]dATP-labeled 323 nt porcine PAG8 (pPAG8) cDNA probes produced by asymmetric PCRs. The aISH with antisense (35)S-pPAG8 probe identified camelid PAG-like (LpPAG, CbPAG and CdPAG) mRNA expression restricted to chorionic epithelium cells within placentas of camelids. In addition, genomic DNA (gDNA), isolated from placental sections were used as templates for camelid PAG-like gene amplicon production by PCR. Specificity of the obtained multiple camelid gDNA PAG-like amplicons was confirmed by double ht-Southern hybridizations with [alpha-(32)P]dATP-labeled 611 bp pPAG5 and pPAG10 double-stranded cDNA probes. The double ht-Southern hybridizations of camelid gDNA amplicons (with pPAG5 and -10 probes) allowed the identification of length-polymorphism of LpPAG, CbPAG and CdPAG genes, coding catalytically active and potentially inactive forms. Such an application of porcine PAG probes may be advantageous for future identification of still undiscovered PAG-like families in other eutherian species.

  7. Simultaneous analysis of multiple PCR amplicons enhances capillary SSCP discrimination of MHC alleles.

    Science.gov (United States)

    Alcaide, Miguel; López, Lidia; Tanferna, Alessandro; Blas, Julio; Sergio, Fabrizio; Hiraldo, Fernando

    2010-04-01

    Major histocompatibility complex (MHC) genotyping still remains one of the most challenging issues for evolutionary ecologists. To date, none of the proposed methods have proven to be perfect, and all provide both important pros and cons. Although denaturing capillary electrophoresis has become a popular alternative, allele identification commonly relies upon conformational polymorphisms of two single-stranded DNA molecules at the most. Using the MHC class II (beta chain, exon 2) of the black kite (Aves: Accipitridae) as our model system, we show that the simultaneous analysis of overlapping PCR amplicons from the same target region substantially enhances allele discrimination. To cover this aim, we designed a multiplex PCR capable to generate four differentially sized and labeled amplicons from the same allele. Informative peaks to assist allele calling then fourfold those generated by the analysis of single PCR amplicons. Our approach proved successful to differentiate all the alleles (N=13) isolated from eight unrelated birds at a single optimal run temperature and electrophoretic conditions. In particular, we emphasize that this approach may constitute a straightforward and cost-effective alternative for the genotyping of single or duplicated MHC genes displaying low to moderate sets of divergent alleles.

  8. Comparison of somatic mutation calling methods in amplicon and whole exome sequence data.

    Science.gov (United States)

    Xu, Huilei; DiCarlo, John; Satya, Ravi Vijaya; Peng, Quan; Wang, Yexun

    2014-03-28

    High-throughput sequencing is rapidly becoming common practice in clinical diagnosis and cancer research. Many algorithms have been developed for somatic single nucleotide variant (SNV) detection in matched tumor-normal DNA sequencing. Although numerous studies have compared the performance of various algorithms on exome data, there has not yet been a systematic evaluation using PCR-enriched amplicon data with a range of variant allele fractions. The recently developed gold standard variant set for the reference individual NA12878 by the NIST-led "Genome in a Bottle" Consortium (NIST-GIAB) provides a good resource to evaluate admixtures with various SNV fractions. Using the NIST-GIAB gold standard, we compared the performance of five popular somatic SNV calling algorithms (GATK UnifiedGenotyper followed by simple subtraction, MuTect, Strelka, SomaticSniper and VarScan2) for matched tumor-normal amplicon and exome sequencing data. We demonstrated that the five commonly used somatic SNV calling methods are applicable to both targeted amplicon and exome sequencing data. However, the sensitivities of these methods vary based on the allelic fraction of the mutation in the tumor sample. Our analysis can assist researchers in choosing a somatic SNV calling method suitable for their specific needs.

  9. Amplicon sequencing for the quantification of spoilage microbiota in complex foods including bacterial spores.

    Science.gov (United States)

    de Boer, Paulo; Caspers, Martien; Sanders, Jan-Willem; Kemperman, Robèr; Wijman, Janneke; Lommerse, Gijs; Roeselers, Guus; Montijn, Roy; Abee, Tjakko; Kort, Remco

    2015-01-01

    Spoilage of food products is frequently caused by bacterial spores and lactic acid bacteria. Identification of these organisms by classic cultivation methods is limited by their ability to form colonies on nutrient agar plates. In this study, we adapted and optimized 16S rRNA amplicon sequencing for quantification of bacterial spores in a canned food matrix and for monitoring the outgrowth of spoilage microbiota in a ready-to-eat food matrix. The detection limit of bar-coded 16S rRNA amplicon sequencing was determined for the number of bacterial spores in a canned food matrix. Analysis of samples from a canned food matrix spiked with a mixture of equinumerous spores from the thermophiles, Geobacillus stearothermophilus and Geobacillus thermoglucosidans, and the mesophiles, Bacillus sporothermodurans, Bacillus cereus, and Bacillus subtilis, led to the detection of these spores with an average limit of 2 × 10(2) spores ml(-1). The data were normalized by setting the number of sequences resulting from DNA of an inactivated bacterial species, present in the matrix at the same concentration in all samples, to a fixed value for quantitative sample-to-sample comparisons. The 16S rRNA amplicon sequencing method was also employed to monitor population dynamics in a ready-to-eat rice meal, incubated over a period of 12 days at 7 °C. The most predominant outgrowth was observed by the genera Leuconostoc, Bacillus, and Paenibacillus. Analysis of meals pre-treated with weak acids showed inhibition of outgrowth of these three genera. The specificity of the amplicon synthesis was improved by the design of oligonucleotides that minimize the amplification of 16S rRNA genes from chloroplasts originating from plant-based material present in the food. This study shows that the composition of complex spoilage populations, including bacterial spores, can be monitored in complex food matrices by bar-coded amplicon sequencing in a quantitative manner. In order to allow sample

  10. Sequence-specific validation of LAMP amplicons in real-time optomagnetic detection of Dengue serotype 2 synthetic DNA

    DEFF Research Database (Denmark)

    Minero, Gabriel Khose Antonio; Nogueira, Catarina; Rizzi, Giovanni

    2017-01-01

    magnetic nanoparticles attached to biotinylated LAMP amplicons. We demonstrate detection of sub-femtomolar Dengue DNA target concentrations in the ideal contamination-free lab environment within 20 min. The second detection approach is based on sequence-specific binding of functionalised magnetic...... nanoparticles to loops of LAMP amplicons. Melting studies reveal that true positive and spurious amplicons have different melting points and this allows us to discriminate between them. This is found to be in a good agreement with subsequent studies on real-time sequence-specific discrimination of LAMP...... amplicons. The specific binding causes clustering of magnetic nanoparticles via binding to multiple sites (loops) emerging in the elongation phase of LAMP. Formation of nanoclusters is monitored via the depletion of the optomagnetic signal due to free nanoparticles. After sequence-specific validation, we...

  11. Mining environmental high-throughput sequence data sets to identify divergent amplicon clusters for phylogenetic reconstruction and morphotype visualization.

    Science.gov (United States)

    Gimmler, Anna; Stoeck, Thorsten

    2015-08-01

    Environmental high-throughput sequencing (envHTS) is a very powerful tool, which in protistan ecology is predominantly used for the exploration of diversity and its geographic and local patterns. We here used a pyrosequenced V4-SSU rDNA data set from a solar saltern pond as test case to exploit such massive protistan amplicon data sets beyond this descriptive purpose. Therefore, we combined a Swarm-based blastn network including 11 579 ciliate V4 amplicons to identify divergent amplicon clusters with targeted polymerase chain reaction (PCR) primer design for full-length small subunit of the ribosomal DNA retrieval and probe design for fluorescence in situ hybridization (FISH). This powerful strategy allows to benefit from envHTS data sets to (i) reveal the phylogenetic position of the taxon behind divergent amplicons; (ii) improve phylogenetic resolution and evolutionary history of specific taxon groups; (iii) solidly assess an amplicons (species') degree of similarity to its closest described relative; (iv) visualize the morphotype behind a divergent amplicons cluster; (v) rapidly FISH screen many environmental samples for geographic/habitat distribution and abundances of the respective organism and (vi) to monitor the success of enrichment strategies in live samples for cultivation and isolation of the respective organisms. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.

  12. Whole genome resequencing of black Angus and Holstein cattle for SNP and CNV discovery

    Directory of Open Access Journals (Sweden)

    Stothard Paul

    2011-11-01

    Full Text Available Abstract Background One of the goals of livestock genomics research is to identify the genetic differences responsible for variation in phenotypic traits, particularly those of economic importance. Characterizing the genetic variation in livestock species is an important step towards linking genes or genomic regions with phenotypes. The completion of the bovine genome sequence and recent advances in DNA sequencing technology allow for in-depth characterization of the genetic variations present in cattle. Here we describe the whole-genome resequencing of two Bos taurus bulls from distinct breeds for the purpose of identifying and annotating novel forms of genetic variation in cattle. Results The genomes of a Black Angus bull and a Holstein bull were sequenced to 22-fold and 19-fold coverage, respectively, using the ABI SOLiD system. Comparisons of the sequences with the Btau4.0 reference assembly yielded 7 million single nucleotide polymorphisms (SNPs, 24% of which were identified in both animals. Of the total SNPs found in Holstein, Black Angus, and in both animals, 81%, 81%, and 75% respectively are novel. In-depth annotations of the data identified more than 16 thousand distinct non-synonymous SNPs (85% novel between the two datasets. Alignments between the SNP-altered proteins and orthologues from numerous species indicate that many of the SNPs alter well-conserved amino acids. Several SNPs predicted to create or remove stop codons were also found. A comparison between the sequencing SNPs and genotyping results from the BovineHD high-density genotyping chip indicates a detection rate of 91% for homozygous SNPs and 81% for heterozygous SNPs. The false positive rate is estimated to be about 2% for both the Black Angus and Holstein SNP sets, based on follow-up genotyping of 422 and 427 SNPs, respectively. Comparisons of read depth between the two bulls along the reference assembly identified 790 putative copy-number variations (CNVs. Ten

  13. An Efficient Approach for the Development of Locus Specific Primers in Bread Wheat (Triticum aestivum L. and Its Application to Re-Sequencing of Genes Involved in Frost Tolerance.

    Directory of Open Access Journals (Sweden)

    Steve Babben

    Full Text Available Recent declines in costs accelerated sequencing of many species with large genomes, including hexaploid wheat (Triticum aestivum L.. Although the draft sequence of bread wheat is known, it is still one of the major challenges to developlocus specific primers suitable to be used in marker assisted selection procedures, due to the high homology of the three genomes. In this study we describe an efficient approach for the development of locus specific primers comprising four steps, i.e. (i identification of genomic and coding sequences (CDS of candidate genes, (ii intron- and exon-structure reconstruction, (iii identification of wheat A, B and D sub-genome sequences and primer development based on sequence differences between the three sub-genomes, and (iv; testing of primers for functionality, correct size and localisation. This approach was applied to single, low and high copy genes involved in frost tolerance in wheat. In summary for 27 of these genes for which sequences were derived from Triticum aestivum, Triticum monococcum and Hordeum vulgare, a set of 119 primer pairs was developed and after testing on Nulli-tetrasomic (NT lines, a set of 65 primer pairs (54.6%, corresponding to 19 candidate genes, turned out to be specific. Out of these a set of 35 fragments was selected for validation via Sanger's amplicon re-sequencing. All fragments, with the exception of one, could be assigned to the original reference sequence. The approach presented here showed a much higher specificity in primer development in comparison to techniques used so far in bread wheat and can be applied to other polyploid species with a known draft sequence.

  14. Amplicon-based metagenomics identified candidate organisms in soils that caused yield decline in strawberry

    OpenAIRE

    Xiangming Xu; Thomas Passey; Feng Wei; Robert Saville; Richard J. Harrison

    2015-01-01

    A phenomenon of yield decline due to weak plant growth in strawberry was recently observed in non-chemo-fumigated soils, which was not associated with the soil fungal pathogen Verticillium dahliae, the main target of fumigation. Amplicon-based metagenomics was used to profile soil microbiota in order to identify microbial organisms that may have caused the yield decline. A total of 36 soil samples were obtained in 2013 and 2014 from four sites for metagenomic studies; two of the four sites ha...

  15. Analysis and Visualization Tool for Targeted Amplicon Bisulfite Sequencing on Ion Torrent Sequencers.

    Directory of Open Access Journals (Sweden)

    Stephan Pabinger

    Full Text Available Targeted sequencing of PCR amplicons generated from bisulfite deaminated DNA is a flexible, cost-effective way to study methylation of a sample at single CpG resolution and perform subsequent multi-target, multi-sample comparisons. Currently, no platform specific protocol, support, or analysis solution is provided to perform targeted bisulfite sequencing on a Personal Genome Machine (PGM. Here, we present a novel tool, called TABSAT, for analyzing targeted bisulfite sequencing data generated on Ion Torrent sequencers. The workflow starts with raw sequencing data, performs quality assessment, and uses a tailored version of Bismark to map the reads to a reference genome. The pipeline visualizes results as lollipop plots and is able to deduce specific methylation-patterns present in a sample. The obtained profiles are then summarized and compared between samples. In order to assess the performance of the targeted bisulfite sequencing workflow, 48 samples were used to generate 53 different Bisulfite-Sequencing PCR amplicons from each sample, resulting in 2,544 amplicon targets. We obtained a mean coverage of 282X using 1,196,822 aligned reads. Next, we compared the sequencing results of these targets to the methylation level of the corresponding sites on an Illumina 450k methylation chip. The calculated average Pearson correlation coefficient of 0.91 confirms the sequencing results with one of the industry-leading CpG methylation platforms and shows that targeted amplicon bisulfite sequencing provides an accurate and cost-efficient method for DNA methylation studies, e.g., to provide platform-independent confirmation of Illumina Infinium 450k methylation data. TABSAT offers a novel way to analyze data generated by Ion Torrent instruments and can also be used with data from the Illumina MiSeq platform. It can be easily accessed via the Platomics platform, which offers a web-based graphical user interface along with sample and parameter storage

  16. Synthetic spike-in standards for high-throughput 16S rRNA gene amplicon sequencing.

    Science.gov (United States)

    Tourlousse, Dieter M; Yoshiike, Satowa; Ohashi, Akiko; Matsukura, Satoko; Noda, Naohiro; Sekiguchi, Yuji

    2017-02-28

    High-throughput sequencing of 16S rRNA gene amplicons (16S-seq) has become a widely deployed method for profiling complex microbial communities but technical pitfalls related to data reliability and quantification remain to be fully addressed. In this work, we have developed and implemented a set of synthetic 16S rRNA genes to serve as universal spike-in standards for 16S-seq experiments. The spike-ins represent full-length 16S rRNA genes containing artificial variable regions with negligible identity to known nucleotide sequences, permitting unambiguous identification of spike-in sequences in 16S-seq read data from any microbiome sample. Using defined mock communities and environmental microbiota, we characterized the performance of the spike-in standards and demonstrated their utility for evaluating data quality on a per-sample basis. Further, we showed that staggered spike-in mixtures added at the point of DNA extraction enable concurrent estimation of absolute microbial abundances suitable for comparative analysis. Results also underscored that template-specific Illumina sequencing artifacts may lead to biases in the perceived abundance of certain taxa. Taken together, the spike-in standards represent a novel bioanalytical tool that can substantially improve 16S-seq-based microbiome studies by enabling comprehensive quality control along with absolute quantification. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  17. LipidSeq: a next-generation clinical resequencing panel for monogenic dyslipidemias.

    Science.gov (United States)

    Johansen, Christopher T; Dubé, Joseph B; Loyzer, Melissa N; MacDonald, Austin; Carter, David E; McIntyre, Adam D; Cao, Henian; Wang, Jian; Robinson, John F; Hegele, Robert A

    2014-04-01

    We report the design of a targeted resequencing panel for monogenic dyslipidemias, LipidSeq, for the purpose of replacing Sanger sequencing in the clinical detection of dyslipidemia-causing variants. We also evaluate the performance of the LipidSeq approach versus Sanger sequencing in 84 patients with a range of phenotypes including extreme blood lipid concentrations as well as additional dyslipidemias and related metabolic disorders. The panel performs well, with high concordance (95.2%) in samples with known mutations based on Sanger sequencing and a high detection rate (57.9%) of mutations likely to be causative for disease in samples not previously sequenced. Clinical implementation of LipidSeq has the potential to aid in the molecular diagnosis of patients with monogenic dyslipidemias with a high degree of speed and accuracy and at lower cost than either Sanger sequencing or whole exome sequencing. Furthermore, LipidSeq will help to provide a more focused picture of monogenic and polygenic contributors that underlie dyslipidemia while excluding the discovery of incidental pathogenic clinically actionable variants in nonmetabolism-related genes, such as oncogenes, that would otherwise be identified by a whole exome approach, thus minimizing potential ethical issues.

  18. Molecular footprints of domestication and improvement in soybean revealed by whole genome re-sequencing

    Science.gov (United States)

    2013-01-01

    Background Artificial selection played an important role in the origin of modern Glycine max cultivars from the wild soybean Glycine soja. To elucidate the consequences of artificial selection accompanying the domestication and modern improvement of soybean, 25 new and 30 published whole-genome re-sequencing accessions, which represent wild, domesticated landrace, and Chinese elite soybean populations were analyzed. Results A total of 5,102,244 single nucleotide polymorphisms (SNPs) and 707,969 insertion/deletions were identified. Among the SNPs detected, 25.5% were not described previously. We found that artificial selection during domestication led to more pronounced reduction in the genetic diversity of soybean than the switch from landraces to elite cultivars. Only a small proportion (2.99%) of the whole genomic regions appear to be affected by artificial selection for preferred agricultural traits. The selection regions were not distributed randomly or uniformly throughout the genome. Instead, clusters of selection hotspots in certain genomic regions were observed. Moreover, a set of candidate genes (4.38% of the total annotated genes) significantly affected by selection underlying soybean domestication and genetic improvement were identified. Conclusions Given the uniqueness of the soybean germplasm sequenced, this study drew a clear picture of human-mediated evolution of the soybean genomes. The genomic resources and information provided by this study would also facilitate the discovery of genes/loci underlying agronomically important traits. PMID:23984715

  19. Microevolution in cyanobacteria: re-sequencing a motile substrain of Synechocystis sp. PCC 6803.

    Science.gov (United States)

    Trautmann, Danika; Voss, Björn; Wilde, Annegret; Al-Babili, Salim; Hess, Wolfgang R

    2012-12-01

    Synechocystis sp. PCC 6803 is a widely used model cyanobacterium for studying photosynthesis, phototaxis, the production of biofuels and many other aspects. Here we present a re-sequencing study of the genome and seven plasmids of one of the most widely used Synechocystis sp. PCC 6803 substrains, the glucose tolerant and motile Moscow or 'PCC-M' strain, revealing considerable evidence for recent microevolution. Seven single nucleotide polymorphisms (SNPs) specifically shared between 'PCC-M' and the 'PCC-N and PCC-P' substrains indicate that 'PCC-M' belongs to the 'PCC' group of motile strains. The identified indels and SNPs in 'PCC-M' are likely to affect glucose tolerance, motility, phage resistance, certain stress responses as well as functions in the primary metabolism, potentially relevant for the synthesis of alkanes. Three SNPs in intergenic regions could affect the promoter activities of two protein-coding genes and one cis-antisense RNA. Two deletions in 'PCC-M' affect parts of clustered regularly interspaced short palindrome repeats-associated spacer-repeat regions on plasmid pSYSA, in one case by an unusual recombination between spacer sequences.

  20. Three-stage quality control strategies for DNA re-sequencing data.

    Science.gov (United States)

    Guo, Yan; Ye, Fei; Sheng, Quanghu; Clark, Travis; Samuels, David C

    2014-11-01

    Advances in next-generation sequencing (NGS) technologies have greatly improved our ability to detect genomic variants for biomedical research. In particular, NGS technologies have been recently applied with great success to the discovery of mutations associated with the growth of various tumours and in rare Mendelian diseases. The advance in NGS technologies has also created significant challenges in bioinformatics. One of the major challenges is quality control of the sequencing data. In this review, we discuss the proper quality control procedures and parameters for Illumina technology-based human DNA re-sequencing at three different stages of sequencing: raw data, alignment and variant calling. Monitoring quality control metrics at each of the three stages of NGS data provides unique and independent evaluations of data quality from differing perspectives. Properly conducting quality control protocols at all three stages and correctly interpreting the quality control results are crucial to ensure a successful and meaningful study. © The Author 2013. Published by Oxford University Press. For Permissions, please email: journals.permissions@oup.com.

  1. Resequencing Pathogen Microarray (RPM) for prospective detection and identification of emergent pathogen strains and variants

    Science.gov (United States)

    Tibbetts, Clark; Lichanska, Agnieszka M.; Borsuk, Lisa A.; Weslowski, Brian; Morris, Leah M.; Lorence, Matthew C.; Schafer, Klaus O.; Campos, Joseph; Sene, Mohamadou; Myers, Christopher A.; Faix, Dennis; Blair, Patrick J.; Brown, Jason; Metzgar, David

    2010-04-01

    High-density resequencing microarrays support simultaneous detection and identification of multiple viral and bacterial pathogens. Because detection and identification using RPM is based upon multiple specimen-specific target pathogen gene sequences generated in the individual test, the test results enable both a differential diagnostic analysis and epidemiological tracking of detected pathogen strains and variants from one specimen to the next. The RPM assay enables detection and identification of pathogen sequences that share as little as 80% sequence similarity to prototype target gene sequences represented as detector tiles on the array. This capability enables the RPM to detect and identify previously unknown strains and variants of a detected pathogen, as in sentinel cases associated with an infectious disease outbreak. We illustrate this capability using assay results from testing influenza A virus vaccines configured with strains that were first defined years after the design of the RPM microarray. Results are also presented from RPM-Flu testing of three specimens independently confirmed to the positive for the 2009 Novel H1N1 outbreak strain of influenza virus.

  2. Whole genome resequencing reveals natural target site preferences of transposable elements in Drosophila melanogaster.

    Directory of Open Access Journals (Sweden)

    Raquel S Linheiro

    Full Text Available Transposable elements are mobile DNA sequences that integrate into host genomes using diverse mechanisms with varying degrees of target site specificity. While the target site preferences of some engineered transposable elements are well studied, the natural target preferences of most transposable elements are poorly characterized. Using population genomic resequencing data from 166 strains of Drosophila melanogaster, we identified over 8,000 new insertion sites not present in the reference genome sequence that we used to decode the natural target preferences of 22 families of transposable element in this species. We found that terminal inverted repeat transposon and long terminal repeat retrotransposon families present clade-specific target site duplications and target site sequence motifs. Additionally, we found that the sequence motifs at transposable element target sites are always palindromes that extend beyond the target site duplication. Our results demonstrate the utility of population genomics data for high-throughput inference of transposable element targeting preferences in the wild and establish general rules for terminal inverted repeat transposon and long terminal repeat retrotransposon target site selection in eukaryotic genomes.

  3. Amplicon-based metagenomics identified candidate organisms in soils that caused yield decline in strawberry.

    Science.gov (United States)

    Xu, Xiangming; Passey, Thomas; Wei, Feng; Saville, Robert; Harrison, Richard J

    2015-01-01

    A phenomenon of yield decline due to weak plant growth in strawberry was recently observed in non-chemo-fumigated soils, which was not associated with the soil fungal pathogen Verticillium dahliae, the main target of fumigation. Amplicon-based metagenomics was used to profile soil microbiota in order to identify microbial organisms that may have caused the yield decline. A total of 36 soil samples were obtained in 2013 and 2014 from four sites for metagenomic studies; two of the four sites had a yield-decline problem, the other two did not. More than 2000 fungal or bacterial operational taxonomy units (OTUs) were found in these samples. Relative abundance of individual OTUs was statistically compared for differences between samples from sites with or without yield decline. A total of 721 individual comparisons were statistically significant - involving 366 unique bacterial and 44 unique fungal OTUs. Based on further selection criteria, we focused on 34 bacterial and 17 fungal OTUs and found that yield decline resulted probably from one or more of the following four factors: (1) low abundance of Bacillus and Pseudomonas populations, which are well known for their ability of supressing pathogen development and/or promoting plant growth; (2) lack of the nematophagous fungus (Paecilomyces species); (3) a high level of two non-specific fungal root rot pathogens; and (4) wet soil conditions. This study demonstrated the usefulness of an amplicon-based metagenomics approach to profile soil microbiota and to detect differential abundance in microbes.

  4. Multiplex amplicon sequencing for microbe identification in community-based culture collections.

    Science.gov (United States)

    Armanhi, Jaderson Silveira Leite; de Souza, Rafael Soares Correa; de Araújo, Laura Migliorini; Okura, Vagner Katsumi; Mieczkowski, Piotr; Imperial, Juan; Arruda, Paulo

    2016-07-12

    Microbiome analysis using metagenomic sequencing has revealed a vast microbial diversity associated with plants. Identifying the molecular functions associated with microbiome-plant interaction is a significant challenge concerning the development of microbiome-derived technologies applied to agriculture. An alternative to accelerate the discovery of the microbiome benefits to plants is to construct microbial culture collections concomitant with accessing microbial community structure and abundance. However, traditional methods of isolation, cultivation, and identification of microbes are time-consuming and expensive. Here we describe a method for identification of microbes in culture collections constructed by picking colonies from primary platings that may contain single or multiple microorganisms, which we named community-based culture collections (CBC). A multiplexing 16S rRNA gene amplicon sequencing based on two-step PCR amplifications with tagged primers for plates, rows, and columns allowed the identification of the microbial composition regardless if the well contains single or multiple microorganisms. The multiplexing system enables pooling amplicons into a single tube. The sequencing performed on the PacBio platform led to recovery near-full-length 16S rRNA gene sequences allowing accurate identification of microorganism composition in each plate well. Cross-referencing with plant microbiome structure and abundance allowed the estimation of diversity and abundance representation of microorganism in the CBC.

  5. Insight into biases and sequencing errors for amplicon sequencing with the Illumina MiSeq platform.

    Science.gov (United States)

    Schirmer, Melanie; Ijaz, Umer Z; D'Amore, Rosalinda; Hall, Neil; Sloan, William T; Quince, Christopher

    2015-03-31

    With read lengths of currently up to 2 × 300 bp, high throughput and low sequencing costs Illumina's MiSeq is becoming one of the most utilized sequencing platforms worldwide. The platform is manageable and affordable even for smaller labs. This enables quick turnaround on a broad range of applications such as targeted gene sequencing, metagenomics, small genome sequencing and clinical molecular diagnostics. However, Illumina error profiles are still poorly understood and programs are therefore not designed for the idiosyncrasies of Illumina data. A better knowledge of the error patterns is essential for sequence analysis and vital if we are to draw valid conclusions. Studying true genetic variation in a population sample is fundamental for understanding diseases, evolution and origin. We conducted a large study on the error patterns for the MiSeq based on 16S rRNA amplicon sequencing data. We tested state-of-the-art library preparation methods for amplicon sequencing and showed that the library preparation method and the choice of primers are the most significant sources of bias and cause distinct error patterns. Furthermore we tested the efficiency of various error correction strategies and identified quality trimming (Sickle) combined with error correction (BayesHammer) followed by read overlapping (PANDAseq) as the most successful approach, reducing substitution error rates on average by 93%. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  6. CASPER: context-aware scheme for paired-end reads from high-throughput amplicon sequencing.

    Science.gov (United States)

    Kwon, Sunyoung; Lee, Byunghan; Yoon, Sungroh

    2014-01-01

    Merging the forward and reverse reads from paired-end sequencing is a critical task that can significantly improve the performance of downstream tasks, such as genome assembly and mapping, by providing them with virtually elongated reads. However, due to the inherent limitations of most paired-end sequencers, the chance of observing erroneous bases grows rapidly as the end of a read is approached, which becomes a critical hurdle for accurately merging paired-end reads. Although there exist several sophisticated approaches to this problem, their performance in terms of quality of merging often remains unsatisfactory. To address this issue, here we present a context-aware scheme for paired-end reads (CASPER): a computational method to rapidly and robustly merge overlapping paired-end reads. Being particularly well suited to amplicon sequencing applications, CASPER is thoroughly tested with both simulated and real high-throughput amplicon sequencing data. According to our experimental results, CASPER significantly outperforms existing state-of-the art paired-end merging tools in terms of accuracy and robustness. CASPER also exploits the parallelism in the task of paired-end merging and effectively speeds up by multithreading. CASPER is freely available for academic use at http://best.snu.ac.kr/casper.

  7. Direct recovery of infectious Pestivirus from a full-length RT-PCR amplicon

    DEFF Research Database (Denmark)

    Rasmussen, Thomas Bruun; Reimann, Ilona; Hoffmann, Bernd

    2008-01-01

    This study describes the use of a novel and rapid long reverse transcription (RT)-PCR for the generation of infectious full-length cDNA of pestiviruses. To produce rescued viruses, full-length RT-PCR amplicons of 12.3 kb, including a T7-promotor, were transcribed directly in vitro, and the result......This study describes the use of a novel and rapid long reverse transcription (RT)-PCR for the generation of infectious full-length cDNA of pestiviruses. To produce rescued viruses, full-length RT-PCR amplicons of 12.3 kb, including a T7-promotor, were transcribed directly in vitro......, and the resulting RNA transcripts were electroporated into ovine cells. Infectious virus was obtained after one cell culture passage. The rescued viruses had a phenotype similar to the parental Border Disease virus strain. Therefore, direct generation of infectious pestiviruses from full-length RT-PCR cDNA products...

  8. Massively multiplexed microbial identification using resequencing DNA microarrays for outbreak investigation

    Science.gov (United States)

    Leski, T. A.; Ansumana, R.; Jimmy, D. H.; Bangura, U.; Malanoski, A. P.; Lin, B.; Stenger, D. A.

    2011-06-01

    Multiplexed microbial diagnostic assays are a promising method for detection and identification of pathogens causing syndromes characterized by nonspecific symptoms in which traditional differential diagnosis is difficult. Also such assays can play an important role in outbreak investigations and environmental screening for intentional or accidental release of biothreat agents, which requires simultaneous testing for hundreds of potential pathogens. The resequencing pathogen microarray (RPM) is an emerging technological platform, relying on a combination of massively multiplex PCR and high-density DNA microarrays for rapid detection and high-resolution identification of hundreds of infectious agents simultaneously. The RPM diagnostic system was deployed in Sierra Leone, West Africa in collaboration with Njala University and Mercy Hospital Research Laboratory located in Bo. We used the RPM-Flu microarray designed for broad-range detection of human respiratory pathogens, to investigate a suspected outbreak of avian influenza in a number of poultry farms in which significant mortality of chickens was observed. The microarray results were additionally confirmed by influenza specific real-time PCR. The results of the study excluded the possibility that the outbreak was caused by influenza, but implicated Klebsiella pneumoniae as a possible pathogen. The outcome of this feasibility study confirms that application of broad-spectrum detection platforms for outbreak investigation in low-resource locations is possible and allows for rapid discovery of the responsible agents, even in cases when different agents are suspected. This strategy enables quick and cost effective detection of low probability events such as outbreak of a rare disease or intentional release of a biothreat agent.

  9. Whole-genome resequencing uncovers molecular signatures of natural and sexual selection in wild bighorn sheep.

    Science.gov (United States)

    Kardos, Marty; Luikart, Gordon; Bunch, Rowan; Dewey, Sarah; Edwards, William; McWilliam, Sean; Stephenson, John; Allendorf, Fred W; Hogg, John T; Kijas, James

    2015-11-01

    The identification of genes influencing fitness is central to our understanding of the genetic basis of adaptation and how it shapes phenotypic variation in wild populations. Here, we used whole-genome resequencing of wild Rocky Mountain bighorn sheep (Ovis canadensis) to >50-fold coverage to identify 2.8 million single nucleotide polymorphisms (SNPs) and genomic regions bearing signatures of directional selection (i.e. selective sweeps). A comparison of SNP diversity between the X chromosome and the autosomes indicated that bighorn males had a dramatically reduced long-term effective population size compared to females. This probably reflects a long history of intense sexual selection mediated by male-male competition for mates. Selective sweep scans based on heterozygosity and nucleotide diversity revealed evidence for a selective sweep shared across multiple populations at RXFP2, a gene that strongly affects horn size in domestic ungulates. The massive horns carried by bighorn rams appear to have evolved in part via strong positive selection at RXFP2. We identified evidence for selection within individual populations at genes affecting early body growth and cellular response to hypoxia; however, these must be interpreted more cautiously as genetic drift is strong within local populations and may have caused false positives. These results represent a rare example of strong genomic signatures of selection identified at genes with known function in wild populations of a nonmodel species. Our results also showcase the value of reference genome assemblies from agricultural or model species for studies of the genomic basis of adaptation in closely related wild taxa. © 2015 John Wiley & Sons Ltd.

  10. Discovery of Gene Sources for Economic Traits in Hanwoo by Whole-genome Resequencing

    Directory of Open Access Journals (Sweden)

    Younhee Shin

    2016-09-01

    Full Text Available Hanwoo, a Korean native cattle (Bos taurus coreana, has great economic value due to high meat quality. Also, the breed has genetic variations that are associated with production traits such as health, disease resistance, reproduction, growth as well as carcass quality. In this study, next generation sequencing technologies and the availability of an appropriate reference genome were applied to discover a large amount of single nucleotide polymorphisms (SNPs in ten Hanwoo bulls. Analysis of whole-genome resequencing generated a total of 26.5 Gb data, of which 594,716,859 and 592,990,750 reads covered 98.73% and 93.79% of the bovine reference genomes of UMD 3.1 and Btau 4.6.1, respectively. In total, 2,473,884 and 2,402,997 putative SNPs were discovered, of which 1,095,922 (44.3% and 982,674 (40.9% novel SNPs were discovered against UMD3.1 and Btau 4.6.1, respectively. Among the SNPs, the 46,301 (UMD 3.1 and 28,613 SNPs (Btau 4.6.1 that were identified as Hanwoo-specific SNPs were included in the functional genes that may be involved in the mechanisms of milk production, tenderness, juiciness, marbling of Hanwoo beef and yellow hair. Most of the Hanwoo-specific SNPs were identified in the promoter region, suggesting that the SNPs influence differential expression of the regulated genes relative to the relevant traits. In particular, the non-synonymous (ns SNPs found in CORIN, which is a negative regulator of Agouti, might be a causal variant to determine yellow hair of Hanwoo. Our results will provide abundant genetic sources of variation to characterize Hanwoo genetics and for subsequent breeding.

  11. Resequencing array for gene variant detection in malignant hyperthermia and butyrylcholinestherase deficiency.

    Science.gov (United States)

    Levano, Soledad; Gonzalez, Asensio; Singer, Martine; Demougin, Philippe; Rüffert, Henrik; Urwyler, Albert; Girard, Thierry

    2017-05-01

    Malignant hyperthermia (MH) and butyrylcholinestherase (BCHE) deficiency are two relevant pharmacogenetic disorders in anesthetic practice linked with sequence variants, the former in the RyR1 and CACNA1S genes, the latter in the BCHE gene. Genotyping for known pathogenic variants in these genes is useful to help identify susceptible individuals, and others may exist but remain unknown, because full-length sequence of these genes is, in general, not investigated. To facilitate this task, we developed a resequencing DNA array, the perioperative patient safety (POPS) array, to be able to screen the entire coding sequences of the RyR1, CACNA1S and BCHE genes. MH-susceptible individuals (n = 121) identified with the in vitro contracture test, the standard diagnostic tool for MH susceptibility, were genotyped with the arrays. Compared with capillary sequencing, call rates with the arrays could achieve 100% at maximal sensitivity, although to reduce false positive rates, sensitivity was adjusted to 0.85, 0.87 and 0.66 for RyR1, CACNA1S and BCHE respectively, with overall base call specificity exceeding 99%. Detection of 29 predetermined RyR1 variants in 44 individuals was successful in 97% of the cases, among them all 16 variants of established diagnostic value. In a trial application of the arrays, 21 MH-susceptible subjects with no known RyR1 or CACNA1S variants were screened, resulting in the discovery of new variants, all confirmed by capillary sequencing. In conclusion, arrays offer an efficient high-throughput alternative for diagnostic genotyping of candidate genes affecting MH susceptibility, BCHE deficiency and other neuromuscular disorders, simultaneously enabling a comprehensive search for rare variants in these genes. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Simultaneous analysis of all SNPs in genome-wide and re-sequencing association studies.

    Directory of Open Access Journals (Sweden)

    Clive J Hoggart

    2008-07-01

    Full Text Available Testing one SNP at a time does not fully realise the potential of genome-wide association studies to identify multiple causal variants, which is a plausible scenario for many complex diseases. We show that simultaneous analysis of the entire set of SNPs from a genome-wide study to identify the subset that best predicts disease outcome is now feasible, thanks to developments in stochastic search methods. We used a Bayesian-inspired penalised maximum likelihood approach in which every SNP can be considered for additive, dominant, and recessive contributions to disease risk. Posterior mode estimates were obtained for regression coefficients that were each assigned a prior with a sharp mode at zero. A non-zero coefficient estimate was interpreted as corresponding to a significant SNP. We investigated two prior distributions and show that the normal-exponential-gamma prior leads to improved SNP selection in comparison with single-SNP tests. We also derived an explicit approximation for type-I error that avoids the need to use permutation procedures. As well as genome-wide analyses, our method is well-suited to fine mapping with very dense SNP sets obtained from re-sequencing and/or imputation. It can accommodate quantitative as well as case-control phenotypes, covariate adjustment, and can be extended to search for interactions. Here, we demonstrate the power and empirical type-I error of our approach using simulated case-control data sets of up to 500 K SNPs, a real genome-wide data set of 300 K SNPs, and a sequence-based dataset, each of which can be analysed in a few hours on a desktop workstation.

  13. Whole-genome resequencing of 100 healthy individuals using DNA pooling

    Science.gov (United States)

    Wang, Xiaobin; Sui, Weiguo; Wu, Weiqing; Hou, Xianliang; Ou, Minglin; Xiang, Yueying; Dai, Yong

    2016-01-01

    With the advent of next-generation sequencing technology, the cost of sequencing has significantly decreased. However, sequencing costs remain high for large-scale studies. In the present study, DNA pooling was applied as a cost-effective strategy for sequencing. The sequencing results for 100 healthy individuals obtained via whole-genome resequencing and using DNA pooling are presented in the present study. In order to minimise the likelihood of systematic bias in sampling, paired-end libraries with an insert size of 500 bp were prepared for all samples and then subjected to whole-genome sequencing using four lanes for each library and resulting in at least a 30-fold haploid coverage for each sample. The NCBI human genome build37 (hg19) was used as a reference genome for the present study and the short reads were aligned to the reference genome achieving 99.84% coverage. In addition, the average sequencing depth was 32.76. In total, ~3 million single-nucleotide polymorphisms were identified, of which 99.88% were in the NCBI dbSNP database. Furthermore, ~600,000 small insertion/deletions, 500,000 structure variants, 5,000 copy number variations and 13,000 single nucleotide variants were identified. According to the present study, the whole genome has been sequenced for a small sample subjects from southern China for the first time. Furthermore, new variation sites were identified by comparing with the reference sequence, and new knowledge of the human genome variation was added to the human genomic databases. Furthermore, the particular distribution regions of variation were illustrated by analyzing various sites of variation, such as single-nucleotide polymorphisms. PMID:27882129

  14. Targeted resequencing and analysis of the Diamond-Blackfan anemia disease locus RPS19.

    Directory of Open Access Journals (Sweden)

    Alvaro Martinez Barrio

    2009-07-01

    Full Text Available The Ribosomal protein S19 gene locus (RPS19 has been linked to two kinds of red cell aplasia, Diamond-Blackfan Anemia (DBA and Transient Erythroblastopenia in Childhood (TEC. Mutations in RPS19 coding sequences have been found in 25% of DBA patients, but not in TEC patients. It has been suggested that non-coding RPS19 sequence variants contribute to the considerable clinical variability in red cell aplasia. We therefore aimed at identifying non-coding variations associated with DBA or TEC phenotypes.We targeted a region of 19'980 bp encompassing the RPS19 gene in a cohort of 89 DBA and TEC patients for resequencing. We provide here a catalog of the considerable, previously unrecognized degree of variation in this region. We identified 73 variations (65 SNPs, 8 indels that all are located outside of the RPS19 open reading frame, and of which 67.1% are classified as novel. We hypothesize that specific alleles in non-coding regions of RPS19 could alter the binding of regulatory proteins or transcription factors. Therefore, we carried out an extensive analysis to identify transcription factor binding sites (TFBS. A series of putative interaction sites coincide with detected variants. Sixteen of the corresponding transcription factors are of particular interest, as they are housekeeping genes or show a direct link to hematopoiesis, tumorigenesis or leukemia (e.g. GATA-1/2, PU.1, MZF-1.Specific alleles at predicted TFBSs may alter the expression of RPS19, modify an important interaction between transcription factors with overlapping TFBS or remove an important stimulus for hematopoiesis. We suggest that the detected interactions are of importance for hematopoiesis and could provide new insights into individual response to treatment.

  15. Human apolipoprotein e resequencing by proteomic analysis and its application to serotyping.

    Directory of Open Access Journals (Sweden)

    Motoi Nishimura

    Full Text Available BACKGROUND: Apolipoprotein E (ApoE typing is considered important because of the association between ApoE and Alzheimer's disease and familial dyslipidemia and is currently performed by genetic testing (APOE genotyping. ApoE levels in plasma and serum are clinically determined by immunoassay. METHODS: Combining an ApoE immunoassay reagent with proteomic analysis using an Orbitrap mass spectrometer, we attempted to resequence ApoE from trace amounts of serum for typing (serotyping. Most (24 of 33 ApoE mutant proteins registered to date with Online Mendelian Inheritance in Man, such as ApoE2 and ApoE4, involve lysine and arginine mutations. Digestion of mutant ApoE with trypsin will thus result in fragments that differ substantially from wild-type ApoE3 in terms of mass, making serotyping ideally suited to mass spectrometry analysis. RESULTS: The mean coverage of the amino acid sequence of full-length ApoE was 91.6% in the protein resequence. Residues 112 and 158 (which are mutated in ApoE2 and ApoE4 were covered in all samples, and the protein sequences were used for serotyping. Serotypes including all heterozygous combinations (ApoE2/E3, E2/E4, E3/E4 corresponded exactly to the APOE genotyping results in each of the subjects. CONCLUSION: Our novel ApoE serotyping method with protein resequencing requires no synthesis of stable isotope-labeled peptides or genome analysis. The method can use residual blood from samples collected for routine clinical tests, thus enabling retrospective studies with preserved body fluids. The test could be applied to samples from subjects whose DNA is unavailable. In future studies, we hope to demonstrate the capability of our method to detect rare ApoE mutations.

  16. Photochemical immobilization of anthraquinone conjugated oligonucleotides and PCR amplicons on solid surfaces

    DEFF Research Database (Denmark)

    Koch, T.; Jacobsen, N.; Fensholdt, J.

    2000-01-01

    Ligand immobilization on solid surfaces is an essential step in fields such as diagnostics, bio sensor manufacturing, and new material sciences in general. In this paper a photochemical approach based on anthraquinone as the chromophore is presented. Photochemical procedures offer special...... advantages as they are able to generate highly reactive species in an orientation specific manner. As presented here, anthraquinone (AQ) mediated covalent DNA immobilization appears to be superior to currently known procedures. A synthetic procedure providing AQ-phosphoramidites is presented. These reagents...... facilitate AQ conjugation during routine DNA synthesis, thus enabling the AQ-oligonucleotides to be immobilized in a very convenient and efficient manner. AQ-conjugated PCR primers can be used directly in PCR. When the PCR is performed in solution, the amplicons can be immobilized after the PCR. Moreover...

  17. Swarm v2: highly-scalable and high-resolution amplicon clustering.

    Science.gov (United States)

    Mahé, Frédéric; Rognes, Torbjørn; Quince, Christopher; de Vargas, Colomban; Dunthorn, Micah

    2015-01-01

    Previously we presented Swarm v1, a novel and open source amplicon clustering program that produced fine-scale molecular operational taxonomic units (OTUs), free of arbitrary global clustering thresholds and input-order dependency. Swarm v1 worked with an initial phase that used iterative single-linkage with a local clustering threshold (d), followed by a phase that used the internal abundance structures of clusters to break chained OTUs. Here we present Swarm v2, which has two important novel features: (1) a new algorithm for d = 1 that allows the computation time of the program to scale linearly with increasing amounts of data; and (2) the new fastidious option that reduces under-grouping by grafting low abundant OTUs (e.g., singletons and doubletons) onto larger ones. Swarm v2 also directly integrates the clustering and breaking phases, dereplicates sequencing reads with d = 0, outputs OTU representatives in fasta format, and plots individual OTUs as two-dimensional networks.

  18. Nitrogenase gene amplicons from global marine surface waters are dominated by genes of non-cyanobacteria

    DEFF Research Database (Denmark)

    Farnelid, Hanna; Andersson, Anders F.; Bertilsson, Stefan

    2011-01-01

    by unicellular cyanobacteria, 42% of the identified non-cyanobacterial nifH clusters from the corresponding DNA samples were also detected in cDNA. The study indicates that non-cyanobacteria account for a substantial part of the nifH gene pool in marine surface waters and that these genes are at least......Cyanobacteria are thought to be the main N2-fixing organisms (diazotrophs) in marine pelagic waters, but recent molecular analyses indicate that non-cyanobacterial diazotrophs are also present and active. Existing data are, however, restricted geographically and by limited sequencing depths. Our...... of the nifH gene pool in marine waters. Great divergence in nifH composition was observed between sites. Cyanobacteria-like genes were most frequent among amplicons from the warmest waters, but overall the data set was dominated by nifH sequences most closely related to non-cyanobacteria. Clusters related...

  19. Performance of amplicon-based next generation DNA sequencing for diagnostic gene mutation profiling in oncopathology.

    Science.gov (United States)

    Sie, Daoud; Snijders, Peter J F; Meijer, Gerrit A; Doeleman, Marije W; van Moorsel, Marinda I H; van Essen, Hendrik F; Eijk, Paul P; Grünberg, Katrien; van Grieken, Nicole C T; Thunnissen, Erik; Verheul, Henk M; Smit, Egbert F; Ylstra, Bauke; Heideman, Daniëlle A M

    2014-10-01

    Next generation DNA sequencing (NGS) holds promise for diagnostic applications, yet implementation in routine molecular pathology practice requires performance evaluation on DNA derived from routine formalin-fixed paraffin-embedded (FFPE) tissue specimens. The current study presents a comprehensive analysis of TruSeq Amplicon Cancer Panel-based NGS using a MiSeq Personal sequencer (TSACP-MiSeq-NGS) for somatic mutation profiling. TSACP-MiSeq-NGS (testing 212 hotspot mutation amplicons of 48 genes) and a data analysis pipeline were evaluated in a retrospective learning/test set approach (n = 58/n = 45 FFPE-tumor DNA samples) against 'gold standard' high-resolution-melting (HRM)-sequencing for the genes KRAS, EGFR, BRAF and PIK3CA. Next, the performance of the validated test algorithm was assessed in an independent, prospective cohort of FFPE-tumor DNA samples (n = 75). In the learning set, a number of minimum parameter settings was defined to decide whether a FFPE-DNA sample is qualified for TSACP-MiSeq-NGS and for calling mutations. The resulting test algorithm revealed 82% (37/45) compliance to the quality criteria and 95% (35/37) concordant assay findings for KRAS, EGFR, BRAF and PIK3CA with HRM-sequencing (kappa = 0.92; 95% CI = 0.81-1.03) in the test set. Subsequent application of the validated test algorithm to the prospective cohort yielded a success rate of 84% (63/75), and a high concordance with HRM-sequencing (95% (60/63); kappa = 0.92; 95% CI = 0.84-1.01). TSACP-MiSeq-NGS detected 77 mutations in 29 additional genes. TSACP-MiSeq-NGS is suitable for diagnostic gene mutation profiling in oncopathology.

  20. Amplicon Sequencing Reveals Microbiological Signatures in Spent Nuclear Fuel Storage Basins

    Directory of Open Access Journals (Sweden)

    Christopher E. Bagwell

    2018-03-01

    Full Text Available Water quality is an important determinant for the structural integrity of alloy cladded fuels and assemblies during long-term wet storage. Detailed characterization of a water filled storage basin for spent nuclear reactor fuel was performed following the formation and proliferation of an amorphous white flocculent. White precipitant was sampled throughout the storage basin for chemical and spectroscopic characterization, and environmental DNA was extracted for 454 pyrosequencing of bacterial 16S rRNA gene diversity. Accordingly, spectroscopic analyses indicated the precipitant to be primarily amorphous to crystalline aluminum (oxy hydroxides with minor associated elemental components including Fe, Si, Ti, and U. High levels of organic carbon were co-localized with the precipitant relative to bulk dissolved organic concentrations. Bacterial densities were highly variable between sampling locations and with depth within the water filled storage basin; cell numbers ranged from 4 × 103to 4 × 104 cells/mL. Bacterial diversity that was physically associated with the aluminum (oxy hydroxide complexes exceeded an estimated 4,000 OTUs/amplicon library (3% cutoff and the majority of sequences were aligned to the families Burkholderiaceae (23%, Nitrospiraceae (23%, Hyphomicrobiaceae (17%, and Comamonadaceae (6%. We surmise that episodic changes in the physical and chemical properties of the basin contribute to the polymerization of aluminum (oxy hydroxides, which in turn can chemisorb nutrients, carbon ligands and bacterial cells from the surrounding bulk aqueous phase. As such, these precipitants should establish favorable microhabitats for bacterial colonization and growth. Comparative analyses of 16S rRNA gene amplicon libraries across a selection of natural and engineered aquatic ecosystems were performed and microbial community and taxonomic signatures unique to the spent nuclear fuel (SNF storage basin environment were revealed. These insights

  1. Similar diversity of Alphaproteobacteria and nitrogenase gene amplicons on two related Sphagnum mosses

    Directory of Open Access Journals (Sweden)

    Anastasia eBragina

    2012-01-01

    Full Text Available Sphagnum mosses represent a main component in ombrotrophic wetlands. They harbor a specific and diverse microbial community with essential functions for the host. To understand extend and degree of host specificity, Sphagnum fallax and S. angustifolium, two phylogenetically closely related species, which show distinct habitat preference with respect to the nutrient level, were analyzed by a multifaceted approach. Microbial fingerprints obtained by PCR-SSCP (single-strand conformation polymorphism using universal, group-specific and functional primers were highly similar. Similarity was confirmed for colonization patterns obtained by fluorescence in situ hybridization (FISH coupled with confocal laser scanning microscopy (CLSM: Alphaproteobacteria were the main colonizers inside the hyaline cells of Sphagnum leaves. A deeper survey of Alphaproteobacteria by 16S rRNA gene amplicon sequencing reveals a high diversity with Acidocella, Acidisphaera, Rhodopila and Phenylobacterium as major genera for both mosses. Pathogen defense and nitrogen fixation are important functions of Sphagnum-associated bacteria, which are fulfilled by microbial communities of both Sphagna in a similar way. NifH libraries of Sphagnum-associated microbial communities were characterized by high diversity and abundance of Alphaproteobacteria but contained also diverse amplicons of other taxa, e.g. Cyanobacteria, Geobacter and Spirochaeta. Statistically significant differences between the microbial communities of both Sphagnum species could not be discovered in any of the experimental approach. Our results show that the same close relationship, which exists between the physical, morphological and chemical characteristics of Sphagnum mosses and the ecology and function of bog ecosystems, also connects moss plantlets with their associated bacterial communities.

  2. A need for standardization in drinking water analysis – an investigation of DNA extraction procedure, primer choice and detection limit of 16S rRNA amplicon sequencing

    DEFF Research Database (Denmark)

    Brandt, Jakob; Nielsen, Per Halkjær; Albertsen, Mads

    have been made to illuminate the effects specifically related to bacterial communities in drinking water. In this study, we investigated the impact of the DNA extraction and primer choice on the observed community structure, and we also estimated the detection limit of the 16S rRNA amplicon sequencing....... The PowerWater DNA Isolation Kit resulted in significantly higher amounts of isolated DNA compared to the FastDNA SPIN Kit for Soil. Furthermore, extraction with the PowerWater kit lead to detection of a significantly higher number of OTUs. Likewise, the primer experiment revealed great discrepancies in OTU....... coli could be detected in all samples. However, samples with 101 cells/mL had several contaminating OTUs, constituting approximately 8% of the read abundances. For 16S rRNA gene analysis in drinking water samples, we recommend using the PowerWater DNA Isolation Kit for DNA extraction in combination...

  3. Sequence-specific validation of LAMP amplicons in real-time optomagnetic detection of Dengue serotype 2 synthetic DNA.

    Science.gov (United States)

    Minero, Gabriel Antonio S; Nogueira, Catarina; Rizzi, Giovanni; Tian, Bo; Fock, Jeppe; Donolato, Marco; Strömberg, Mattias; Hansen, Mikkel F

    2017-09-08

    We report on an optomagnetic technique optimised for real-time molecular detection of Dengue fever virus under ideal as well as non-ideal laboratory conditions using two different detection approaches. The first approach is based on the detection of the hydrodynamic volume of streptavidin coated magnetic nanoparticles attached to biotinylated LAMP amplicons. We demonstrate detection of sub-femtomolar Dengue DNA target concentrations in the ideal contamination-free lab environment within 20 min. The second detection approach is based on sequence-specific binding of functionalised magnetic nanoparticles to loops of LAMP amplicons. Melting studies reveal that true positive and spurious amplicons have different melting points and this allows us to discriminate between them. This is found to be in a good agreement with subsequent studies on real-time sequence-specific discrimination of LAMP amplicons. The specific binding causes clustering of magnetic nanoparticles via binding to multiple sites (loops) emerging in the elongation phase of LAMP. Formation of nanoclusters is monitored via the depletion of the optomagnetic signal due to free nanoparticles. After sequence-specific validation, we claim detection of down to 100 fM of Dengue target after 20 min of LAMP with a contamination background.

  4. Resequencing three candidate genes for major depressive disorder in a Dutch cohort.

    Directory of Open Access Journals (Sweden)

    Eva C Verbeek

    Full Text Available Major depressive disorder (MDD is a psychiatric disorder, characterized by periods of low mood of more than two weeks, loss of interest in normally enjoyable activities and behavioral changes. MDD is a complex disorder and does not have a single genetic cause. In 2009 a genome wide association study (GWAS was performed on the Dutch GAIN-MDD cohort. Many of the top signals of this GWAS mapped to a region spanning the gene PCLO, and the non-synonymous coding single nucleotide polymorphism (SNP rs2522833 in the PCLO gene became genome wide significant after post-hoc analysis. We performed resequencing of PCLO, GRM7, and SLC6A4 in 50 control samples from the GAIN-MDD cohort, to detect new genomic variants. Subsequently, we genotyped these variants in the entire GAIN-MDD cohort and performed association analysis to investigate if rs2522833 is the causal variant or simply in linkage disequilibrium with a more associated variant. GRM7 and SLC6A4 are both candidate genes for MDD from literature. We aimed to gather more evidence that rs2522833 is indeed the causal variant in the GAIN-MDD cohort or to find a previously undetected common variant in either PCLO, GRM7, or SLC6A4 with a higher association in this cohort. After next generation sequencing and association analysis we excluded the possibility of an undetected common variant to be more associated. For neither PCLO nor GRM7 we found a more associated variant. For SLC6A4, we found a new SNP that showed a lower P-value (P = 0.07 than in the GAIN-MDD GWAS (P = 0.09. However, no evidence for genome-wide significance was found. Although we did not take into account rare variants, we conclude that our results provide further support for the hypothesis that the non-synonymous coding SNP rs2522833 in the PCLO gene is indeed likely to be the causal variant in the GAIN-MDD cohort.

  5. Resequencing Three Candidate Genes for Major Depressive Disorder in a Dutch Cohort

    Science.gov (United States)

    Verbeek, Eva C.; Bevova, Marianna R.; Bochdanovits, Zoltán; Rizzu, Patrizia; Bakker, Ingrid M. C.; Uithuisje, Tiny; De Geus, Eco J.; Smit, Johannes H.; Penninx, Brenda W.; Boomsma, Dorret I.; Hoogendijk, Witte J. G.; Heutink, Peter

    2013-01-01

    Major depressive disorder (MDD) is a psychiatric disorder, characterized by periods of low mood of more than two weeks, loss of interest in normally enjoyable activities and behavioral changes. MDD is a complex disorder and does not have a single genetic cause. In 2009 a genome wide association study (GWAS) was performed on the Dutch GAIN-MDD cohort. Many of the top signals of this GWAS mapped to a region spanning the gene PCLO, and the non-synonymous coding single nucleotide polymorphism (SNP) rs2522833 in the PCLO gene became genome wide significant after post-hoc analysis. We performed resequencing of PCLO, GRM7, and SLC6A4 in 50 control samples from the GAIN-MDD cohort, to detect new genomic variants. Subsequently, we genotyped these variants in the entire GAIN-MDD cohort and performed association analysis to investigate if rs2522833 is the causal variant or simply in linkage disequilibrium with a more associated variant. GRM7 and SLC6A4 are both candidate genes for MDD from literature. We aimed to gather more evidence that rs2522833 is indeed the causal variant in the GAIN-MDD cohort or to find a previously undetected common variant in either PCLO, GRM7, or SLC6A4 with a higher association in this cohort. After next generation sequencing and association analysis we excluded the possibility of an undetected common variant to be more associated. For neither PCLO nor GRM7 we found a more associated variant. For SLC6A4, we found a new SNP that showed a lower P-value (P = 0.07) than in the GAIN-MDD GWAS (P = 0.09). However, no evidence for genome-wide significance was found. Although we did not take into account rare variants, we conclude that our results provide further support for the hypothesis that the non-synonymous coding SNP rs2522833 in the PCLO gene is indeed likely to be the causal variant in the GAIN-MDD cohort. PMID:24278217

  6. Function of the EGR-1/TIS8 radiation inducible promoter in a minimal HSV-1 amplicon system

    International Nuclear Information System (INIS)

    Spear, M.A.; Sakamoto, K.M.; Herrlinger, U.; Pechan, P.; Breakefield, X.O.

    1997-01-01

    Purpose: To evaluate function of the EGR-1/TIS8 promoter region in minimal HSV-1 amplicon system in order to determine the feasibility of using the system to regulate vector replication with radiation. Materials and Methods: A 600-base pair 5' upstream region of the EGR-1 promoter linked to chloramphenicol acetyltransferase (CAT) was recombined into a minimal HSV-1 amplicon vector system (pONEC). pONEC or a control plasmid was transfected into U87 glioma cells using the Lipofectamine method. Thirty-six hours later one aliquot of cells from each transfection was irradiated to a dose of 20 Gy and another identical aliquot served as a control. CAT activity was assayed 8 hours after irradiation. Results: pONEC transfected cells irradiated with 20 Gy demonstrated 2.0 fold increase in CAT activity compared to non-irradiated cells. Cells transfected with control plasmid showed no change in CAT activity. Unirradiated pONEC cells had CAT activity 1.3 times those transfected with control plasmid. Conclusion: We have previously created HSV-1 gene therapy amplicon vector systems which allow virus-amplicon interdependent replication, with the intent of regulating replication. The above data demonstrates that a minimal amplicon system will allow radiation dependent regulation by the EGR-1 promoter, thus indicating the possibility of using this system to regulate onsite, spatially and temporally regulated vector production. Baseline CAT activity was higher and relative induction lower than other reported expression constructs, which raises concern for the ability of the system to produce a differential in transcription levels sufficient for this purpose. This is possibly the result of residual promoter/enhancer elements remaining in the HSV-1 sequences. We are attempting to create constructs lacking these elements. Addition of secondary promoter sequences may also be of use. We are also currently evaluating the efficacy of the putative IEX-1 radiation inducible promoter region in

  7. Herpes simplex virus type 1-based amplicon vectors for fundamental research in neurosciences and gene therapy of neurological diseases.

    Science.gov (United States)

    Jerusalinsky, Diana; Baez, María Verónica; Epstein, Alberto Luis

    2012-01-01

    Somatic manipulation of the nervous system without the involvement of the germinal line appears as a powerful counterpart of the transgenic strategy. The use of viral vectors to produce specific, transient and localized knockout, knockdown, ectopic expression or overexpression of a gene, leads to the possibility of analyzing both in vitro and in vivo molecular basis of neural function. In this approach, viral particles engineered to carry transgenic sequences are delivered into discrete brain regions, to transduce cells that will express the transgenic products. Amplicons are replication-incompetent helper-dependent vectors derived from herpes simplex virus type 1 (HSV-1), with several advantages that potentiate their use in neurosciences: (1) minimal toxicity: amplicons do not encode any virus proteins, are neither toxic for the infected cells nor pathogenic for the inoculated animals and elicit low levels of adaptive immune responses; (2) extensive transgene capacity to carry up to 150-kb of foreign DNA; i.e., entire genes with regulatory sequences could be delivered; (3) widespread cellular tropism: amplicons can experimentally infect several cell types including glial cells, though naturally the virus infects mainly neurons and epithelial cells; (4) since the viral genome does not integrate into cellular chromosomes there is low probability to induce insertional mutagenesis. Recent investigations on gene transfer into the brain using these vectors, have focused on gene therapy of inherited genetic diseases affecting the nervous system, such as ataxias, or on neurodegenerative disorders using experimental models of Parkinson's or Alzheimer's disease. Another group of studies used amplicons to investigate complex neural functions such as neuroplasticity, anxiety, learning and memory. In this short review, we summarize recent data supporting the potential of HSV-1 based amplicon vector model for gene delivery and modulation of gene expression in primary cultures

  8. 16S rRNA Amplicon Sequencing for Epidemiological Surveys of Bacteria in Wildlife.

    Science.gov (United States)

    Galan, Maxime; Razzauti, Maria; Bard, Emilie; Bernard, Maria; Brouat, Carine; Charbonnel, Nathalie; Dehne-Garcia, Alexandre; Loiseau, Anne; Tatard, Caroline; Tamisier, Lucie; Vayssier-Taussat, Muriel; Vignes, Helene; Cosson, Jean-François

    2016-01-01

    The human impact on natural habitats is increasing the complexity of human-wildlife interactions and leading to the emergence of infectious diseases worldwide. Highly successful synanthropic wildlife species, such as rodents, will undoubtedly play an increasingly important role in transmitting zoonotic diseases. We investigated the potential for recent developments in 16S rRNA amplicon sequencing to facilitate the multiplexing of the large numbers of samples needed to improve our understanding of the risk of zoonotic disease transmission posed by urban rodents in West Africa. In addition to listing pathogenic bacteria in wild populations, as in other high-throughput sequencing (HTS) studies, our approach can estimate essential parameters for studies of zoonotic risk, such as prevalence and patterns of coinfection within individual hosts. However, the estimation of these parameters requires cleaning of the raw data to mitigate the biases generated by HTS methods. We present here an extensive review of these biases and of their consequences, and we propose a comprehensive trimming strategy for managing these biases. We demonstrated the application of this strategy using 711 commensal rodents, including 208 Mus musculus domesticus , 189 Rattus rattus , 93 Mastomys natalensis , and 221 Mastomys erythroleucus , collected from 24 villages in Senegal. Seven major genera of pathogenic bacteria were detected in their spleens: Borrelia , Bartonella , Mycoplasma , Ehrlichia , Rickettsia , Streptobacillus , and Orientia . Mycoplasma , Ehrlichia , Rickettsia , Streptobacillus , and Orientia have never before been detected in West African rodents. Bacterial prevalence ranged from 0% to 90% of individuals per site, depending on the bacterial taxon, rodent species, and site considered, and 26% of rodents displayed coinfection. The 16S rRNA amplicon sequencing strategy presented here has the advantage over other molecular surveillance tools of dealing with a large spectrum of

  9. Differential amplicons (ΔAmp)-a new molecular method to assess RNA integrity.

    Science.gov (United States)

    Björkman, J; Švec, D; Lott, E; Kubista, M; Sjöback, R

    2016-01-01

    Integrity of the mRNA in clinical samples has major impact on the quality of measured expression levels. This is independent of the measurement technique being next generation sequencing (NGS), Quantitative real-time PCR (qPCR) or microarray profiling. If mRNA is highly degraded or damaged, measured data will be very unreliable and the whole study is likely a waste of time and money. It is therefore common strategy to test the quality of RNA in samples before conducting large and costly studies. Most methods today to assess the quality of RNA are ignorant to the nature of the RNA and, therefore, reflect the integrity of ribosomal RNA, which is the dominant species, rather than of mRNAs, microRNAs and long non-coding RNAs, which usually are the species of interest. Here, we present a novel molecular approach to assess the quality of the targeted RNA species by measuring the differential amplification (ΔAmp) of an Endogenous RNase Resistant (ERR) marker relative to a reference gene, optionally combined with the measurement of two amplicons of different lengths. The combination reveals any mRNA degradation caused by ribonucleases as well as physical, chemical or UV damage. ΔAmp has superior sensitivity to common microfluidic electrophoretic methods, senses the integrity of the actual targeted RNA species, and allows for a smoother and more cost efficient workflow.

  10. Differential amplicons (ΔAmp—a new molecular method to assess RNA integrity

    Directory of Open Access Journals (Sweden)

    J. Björkman

    2016-01-01

    Full Text Available Integrity of the mRNA in clinical samples has major impact on the quality of measured expression levels. This is independent of the measurement technique being next generation sequencing (NGS, Quantitative real-time PCR (qPCR or microarray profiling. If mRNA is highly degraded or damaged, measured data will be very unreliable and the whole study is likely a waste of time and money. It is therefore common strategy to test the quality of RNA in samples before conducting large and costly studies. Most methods today to assess the quality of RNA are ignorant to the nature of the RNA and, therefore, reflect the integrity of ribosomal RNA, which is the dominant species, rather than of mRNAs, microRNAs and long non-coding RNAs, which usually are the species of interest. Here, we present a novel molecular approach to assess the quality of the targeted RNA species by measuring the differential amplification (ΔAmp of an Endogenous RNase Resistant (ERR marker relative to a reference gene, optionally combined with the measurement of two amplicons of different lengths. The combination reveals any mRNA degradation caused by ribonucleases as well as physical, chemical or UV damage. ΔAmp has superior sensitivity to common microfluidic electrophoretic methods, senses the integrity of the actual targeted RNA species, and allows for a smoother and more cost efficient workflow.

  11. Hi-Plex for Simple, Accurate, and Cost-Effective Amplicon-based Targeted DNA Sequencing.

    Science.gov (United States)

    Pope, Bernard J; Hammet, Fleur; Nguyen-Dumont, Tu; Park, Daniel J

    2018-01-01

    Hi-Plex is a suite of methods to enable simple, accurate, and cost-effective highly multiplex PCR-based targeted sequencing (Nguyen-Dumont et al., Biotechniques 58:33-36, 2015). At its core is the principle of using gene-specific primers (GSPs) to "seed" (or target) the reaction and universal primers to "drive" the majority of the reaction. In this manner, effects on amplification efficiencies across the target amplicons can, to a large extent, be restricted to early seeding cycles. Product sizes are defined within a relatively narrow range to enable high-specificity size selection, replication uniformity across target sites (including in the context of fragmented input DNA such as that derived from fixed tumor specimens (Nguyen-Dumont et al., Biotechniques 55:69-74, 2013; Nguyen-Dumont et al., Anal Biochem 470:48-51, 2015), and application of high-specificity genetic variant calling algorithms (Pope et al., Source Code Biol Med 9:3, 2014; Park et al., BMC Bioinformatics 17:165, 2016). Hi-Plex offers a streamlined workflow that is suitable for testing large numbers of specimens without the need for automation.

  12. Extracellular DNA amplicon sequencing reveals high levels of benthic eukaryotic diversity in the central Red Sea

    KAUST Repository

    Pearman, John K.

    2015-11-01

    The present study aims to characterize the benthic eukaryotic biodiversity patterns at a coarse taxonomic level in three areas of the central Red Sea (a lagoon, an offshore area in Thuwal and a shallow coastal area near Jeddah) based on extracellular DNA. High-throughput amplicon sequencing targeting the V9 region of the 18S rRNA gene was undertaken for 32 sediment samples. High levels of alpha-diversity were detected with 16,089 operational taxonomic units (OTUs) being identified. The majority of the OTUs were assigned to Metazoa (29.2%), Alveolata (22.4%) and Stramenopiles (17.8%). Stramenopiles (Diatomea) and Alveolata (Ciliophora) were frequent in a lagoon and in shallower coastal stations, whereas metazoans (Arthropoda: Maxillopoda) were dominant in deeper offshore stations. Only 24.6% of total OTUs were shared among all areas. Beta-diversity was generally lower between the lagoon and Jeddah (nearshore) than between either of those and the offshore area, suggesting a nearshore–offshore biodiversity gradient. The current approach allowed for a broad-range of benthic eukaryotic biodiversity to be analysed with significantly less labour than would be required by other traditional taxonomic approaches. Our findings suggest that next generation sequencing techniques have the potential to provide a fast and standardised screening of benthic biodiversity at large spatial and temporal scales.

  13. Functional Overexpression of Vomeronasal Receptors Using a Herpes Simplex Virus Type 1 (HSV-1-Derived Amplicon.

    Directory of Open Access Journals (Sweden)

    Benjamin Stein

    Full Text Available In mice, social behaviors such as mating and aggression are mediated by pheromones and related chemosignals. The vomeronasal organ (VNO detects olfactory information from other individuals by sensory neurons tuned to respond to specific chemical cues. Receptors expressed by vomeronasal neurons are implicated in selective detection of these cues. Nearly 400 receptor genes have been identified in the mouse VNO, but the tuning properties of individual receptors remain poorly understood, in part due to the lack of a robust heterologous expression system. Here we develop a herpes virus-based amplicon delivery system to overexpress three types of vomeronasal receptor genes and to characterize cell responses to their proposed ligands. Through Ca2+ imaging in native VNO cells we show that virus-induced overexpression of V1rj2, V2r1b or Fpr3 caused a pronounced increase of responsivity to sulfated steroids, MHC-binding peptide or the synthetic hexapeptide W-peptide, respectively. Other related ligands were not recognized by infected individual neurons, indicating a high degree of selectivity by the overexpressed receptor. Removal of G-protein signaling eliminates Ca2+ responses, indicating that the endogenous second messenger system is essential for observing receptor activation. Our results provide a novel expression system for vomeronasal receptors that should be useful for understanding the molecular logic of VNO ligand detection. Functional expression of vomeronasal receptors and their deorphanization provides an essential requirement for deciphering the neural mechanisms controlling behavior.

  14. Swarm v2: highly-scalable and high-resolution amplicon clustering

    Directory of Open Access Journals (Sweden)

    Frédéric Mahé

    2015-12-01

    Full Text Available Previously we presented Swarm v1, a novel and open source amplicon clustering program that produced fine-scale molecular operational taxonomic units (OTUs, free of arbitrary global clustering thresholds and input-order dependency. Swarm v1 worked with an initial phase that used iterative single-linkage with a local clustering threshold (d, followed by a phase that used the internal abundance structures of clusters to break chained OTUs. Here we present Swarm v2, which has two important novel features: (1 a new algorithm for d = 1 that allows the computation time of the program to scale linearly with increasing amounts of data; and (2 the new fastidious option that reduces under-grouping by grafting low abundant OTUs (e.g., singletons and doubletons onto larger ones. Swarm v2 also directly integrates the clustering and breaking phases, dereplicates sequencing reads with d = 0, outputs OTU representatives in fasta format, and plots individual OTUs as two-dimensional networks.

  15. Shedding light on the microbial community of the macropod foregut using 454-amplicon pyrosequencing.

    Directory of Open Access Journals (Sweden)

    Lisa-Maree Gulino

    Full Text Available Twenty macropods from five locations in Queensland, Australia, grazing on a variety of native pastures were surveyed and the bacterial community of the foregut was examined using 454-amplicon pyrosequencing. Specifically, the V3/V4 region of 16S rRNA gene was examined. A total of 5040 OTUs were identified in the data set (post filtering. Thirty-two OTUs were identified as 'shared' OTUS (i.e. present in all samples belonging to either Firmicutes or Bacteroidetes (Clostridiales/Bacteroidales. These phyla predominated the general microbial community in all macropods. Genera represented within the shared OTUs included: unclassified Ruminococcaceae, unclassified Lachnospiraceae, unclassified Clostridiales, Peptococcus sp. Coprococcus spp., Streptococcus spp., Blautia sp., Ruminoccocus sp., Eubacterium sp., Dorea sp., Oscillospira sp. and Butyrivibrio sp. The composition of the bacterial community of the foregut samples of each the host species (Macropus rufus, Macropus giganteus and Macropus robustus was significantly different allowing differentiation between the host species based on alpha and beta diversity measures. Specifically, eleven dominant OTUs that separated the three host species were identified and classified as: unclassified Ruminococcaceae, unclassified Bacteroidales, Prevotella spp. and a Syntrophococcus sucromutans. Putative reductive acetogens and fibrolytic bacteria were also identified in samples. Future work will investigate the presence and role of fibrolytics and acetogens in these ecosystems. Ideally, the isolation and characterization of these organisms will be used for enhanced feed efficiency in cattle, methane mitigation and potentially for other industries such as the biofuel industry.

  16. High-Throughput Analysis of DNA Break-Induced Chromosome Rearrangements by Amplicon Sequencing.

    Science.gov (United States)

    Brown, Alexander J; Al-Soodani, Aneesa T; Saul, Miles; Her, Stephanie; Garcia, Juan C; Ramsden, Dale A; Her, Chengtao; Roberts, Steven A

    2018-01-01

    The mechanistic understanding of how DNA double-strand breaks (DSB) are repaired is rapidly advancing in part due to the advent of inducible site-specific break model systems as well as the employment of next-generation sequencing (NGS) technologies to sequence repair junctions at high depth. Unfortunately, the sheer volume of data produced by these methods makes it difficult to analyze the structure of repair junctions manually or with other general-purpose software. Here, we describe methods to produce amplicon libraries of DSB repair junctions for sequencing, to map the sequencing reads, and then to use a robust, custom python script, Hi-FiBR, to analyze the sequence structure of mapped reads. The Hi-FiBR analysis processes large data sets quickly and provides information such as number and type of repair events, size of deletion, size of insertion and inserted sequence, microhomology usage, and whether mismatches are due to sequencing error or biological effect. The analysis also corrects for common alignment errors generated by sequencing read mapping tools, allowing high-throughput analysis of DSB break repair fidelity to be accurately conducted regardless of which suite of NGS analysis software is available. © 2018 Elsevier Inc. All rights reserved.

  17. Species tree estimation of North American chorus frogs (Hylidae: Pseudacris) with parallel tagged amplicon sequencing.

    Science.gov (United States)

    Barrow, Lisa N; Ralicki, Hannah F; Emme, Sandra A; Lemmon, Emily Moriarty

    2014-06-01

    The field of phylogenetics is changing rapidly with the application of high-throughput sequencing to non-model organisms. Cost-effective use of this technology for phylogenetic studies, which often include a relatively small portion of the genome but several taxa, requires strategies for genome partitioning and sequencing multiple individuals in parallel. In this study we estimated a multilocus phylogeny for the North American chorus frog genus Pseudacris using anonymous nuclear loci that were recently developed using a reduced representation library approach. We sequenced 27 nuclear loci and three mitochondrial loci for 44 individuals on 1/3 of an Illumina MiSeq run, obtaining 96.5% of the targeted amplicons at less than 20% of the cost of traditional Sanger sequencing. We found heterogeneity among gene trees, although four major clades (Trilling Frog, Fat Frog, crucifer, and West Coast) were consistently supported, and we resolved the relationships among these clades for the first time with strong support. We also found discordance between the mitochondrial and nuclear datasets that we attribute to mitochondrial introgression and a possible selective sweep. Bayesian concordance analysis in BUCKy and species tree analysis in (*)BEAST produced largely similar topologies, although we identify taxa that require additional investigation in order to clarify taxonomic and geographic range boundaries. Overall, we demonstrate the utility of a reduced representation library approach for marker development and parallel tagged sequencing on an Illumina MiSeq for phylogenetic studies of non-model organisms. Copyright © 2014 Elsevier Inc. All rights reserved.

  18. Comparative analyses of amplicon migration behavior in differing denaturing gradient gel electrophoresis (DGGE) systems

    Science.gov (United States)

    Thornhill, D. J.; Kemp, D. W.; Sampayo, E. M.; Schmidt, G. W.

    2010-03-01

    Denaturing gradient gel electrophoresis (DGGE) is commonly utilized to identify and quantify microbial diversity, but the conditions required for different electrophoretic systems to yield equivalent results and optimal resolution have not been assessed. Herein, the influence of different DGGE system configuration parameters on microbial diversity estimates was tested using Symbiodinium, a group of marine eukaryotic microbes that are important constituents of coral reef ecosystems. To accomplish this, bacterial clone libraries were constructed and sequenced from cultured isolates of Symbiodinium for the ribosomal DNA internal transcribed spacer 2 (ITS2) region. From these, 15 clones were subjected to PCR with a GC clamped primer set for DGGE analyses. Migration behaviors of the resulting amplicons were analyzed using a range of conditions, including variation in the composition of the denaturing gradient, electrophoresis time, and applied voltage. All tests were conducted in parallel on two commercial DGGE systems, a C.B.S. Scientific DGGE-2001, and the Bio-Rad DCode system. In this context, identical nucleotide fragments exhibited differing migration behaviors depending on the model of apparatus utilized, with fragments denaturing at a lower gradient concentration and applied voltage on the Bio-Rad DCode system than on the C.B.S. Scientific DGGE-2001 system. Although equivalent PCR-DGGE profiles could be achieved with both brands of DGGE system, the composition of the denaturing gradient and application of electrophoresis time × voltage must be appropriately optimized to achieve congruent results across platforms.

  19. Pooled Resequencing of 122 Ulcerative Colitis Genes in a Large Dutch Cohort Suggests Population-Specific Associations of Rare Variants in MUC2

    NARCIS (Netherlands)

    Visschedijk, Marijn C.; Alberts, Rudi; Mucha, Soren; Deelen, Patrick; de Jong, Dirk J.; Pierik, Marieke; Spekhorst, Lieke M.; Imhann, Floris; van der Meulen-de Jong, Andrea E.; van der Woude, C. Janneke; van Bodegraven, Adriaan A.; Oldenburg, Bas; Löwenberg, Mark; Dijkstra, Gerard; Ellinghaus, David; Schreiber, Stefan; Wijmenga, Cisca; Rivas, Manuel A.; Franke, Andre; van Diemen, Cleo C.; Weersma, Rinse K.

    2016-01-01

    Genome-wide association studies have revealed several common genetic risk variants for ulcerative colitis (UC). However, little is known about the contribution of rare, large effect genetic variants to UC susceptibility. In this study, we performed a deep targeted re-sequencing of 122 genes in Dutch

  20. Genome Re-sequencing of Diverse Sweet Cherry (Prunus avium) Individuals Reveals a Modifier Gene Mutation Conferring Pollen-part Self-compatibility.

    Science.gov (United States)

    Ono, Kentaro; Akagi, Takashi; Morimoto, Takuya; Wünsch, Ana; Tao, Ryutaro

    2018-04-04

    The S-RNase-based gametophytic self-incompatibility (GSI) reproduction barrier is important for maintaining genetic diversity in species of the families Solanaceae, Plantaginaceae, and Rosaceae. Among the plant taxa with S-RNase-based GSI, Prunus species in the family Rosaceae exhibit Prunus-specific self-incompatibility (SI). Although pistil S and pollen S determinants have been identified, the mechanism underlying SI remains uncharacterized in Prunus species. A putative pollen-part modifier was identified in this study. Disruption of this modifier supposedly confers self-compatibility (SC) to sweet cherry (Prunus avium) 'Cristobalina'. To identify the modifier, genome re-sequencing experiments were completed involving sweet cherry individuals from 18 cultivars and 43 individuals in two segregating populations. Cataloging of subsequences (35-bp kmers) from the obtained genomic reads, while referring to the mRNA-sequencing data, enabled the identification of a candidate gene [M locus-encoded GST (MGST)]. Additionally, the insertion of a transposon-like sequence in the putative MGST promoter region in 'Cristobalina' down-regulated MGST expression levels, likely leading to the SC of this cultivar. Phylogenetic, evolutionary, and gene expression analyses revealed that MGST may have undergone lineage-specific evolution, and the encoded protein may function differently from the corresponding proteins encoded by GST orthologs in other species, including members of the subfamily Maloideae (Rosaceae). Thus, MGST may be important for Prunus-specific SI. The identification of this novel modifier will expand our understanding of the Prunus-specific GSI system. We herein discuss the possible functions of MGST in the Prunus-specific GSI system.

  1. Whole-genome re-sequencing for the identification of high contribution susceptibility gene variants in patients with type 2 diabetes

    Science.gov (United States)

    SUN, XIAOJUAN; SUI, WEIGUO; WANG, XIAOBING; HOU, XIANLIANG; OU, MINGLIN; DAI, YONG; XIANG, YUEYING

    2016-01-01

    There is increasing evidence that several genes are associated with an increased risk of type 2 diabetes (T2D); genome-wide association investigations and whole-genome re-sequencing investigations offer a useful approach for the identification of genes involved in common human diseases. To further investigate which polymorphisms confer susceptibility to T2D, the present study screened for high-contribution susceptibility gene variants Chinese patients with T2D using whole-genome re-sequencing with DNA pooling. In total, 100 Chinese individuals with T2D and 100 healthy Chinese individuals were analyzed using whole-genome re-sequencing using DNA pooling. To minimize the likelihood of systematic bias in sampling, paired-end libraries with an insert size of 500 bp were prepared for in T2D in all samples, which were then subjected to whole-genome sequencing. Each library contained four lanes. The average sequencing depth was 35.70. In the present study, 1.36 GB of clean sequence data were generated, and the resulting calculated T2D genome consensus sequence covered 99.88% of the hg19 sequence. A total of 3,974,307 single nucleotide polymorphisms were identified, of which 99.88% were in the dbSNP database. The present study also found 642,189 insertions and deletions, 5,590 structure variants (SVs), 4,713 copy number variants (CNVs) and 13,049 single nucleotide variants. A total of 1,884 somatic CNVs and 74 somatic SVs were significantly different between the cases and controls. Therefore, the present study provided validation of whole-genome re-sequencing using the DNA pooling approach. It also generated a whole-genome re-sequencing genotype database for future investigations of T2D. PMID:27035118

  2. DNA Methylation Analysis by Bisulfite Conversion Coupled to Double Multiplexed Amplicon-Based Next-Generation Sequencing (NGS).

    Science.gov (United States)

    Bashtrykov, Pavel; Jeltsch, Albert

    2018-01-01

    Methylation of cytosine bases in DNA is one of the main epigenetic signals regulating gene expression and chromatin structure. The distribution of DNA methylation in the genome has a cell-type-specific pattern and can be modulated by internal or external stimuli. One of the most powerful approaches to investigate DNA methylation patterns is bisulfite conversion of the DNA followed by DNA sequencing, which allows to determine methylation patterns at a single-cytosine resolution. Here, we present a protocol for bisulfite DNA methylation analysis of targeted genomic regions using amplicon-based next-generation sequencing (NGS) on an Illumina sequencing system. We use a PCR-free library generation approach and implement a nested strategy for double molecular barcoding of samples (combining indexing of adapters and in-line barcoding of individual amplicons) which allows highly multiplexed sequencing. Also, we discuss the main limitations of this technology in particular in relation to clonal DNA amplification and other PCR artifacts.

  3. Network analysis of the microorganism in 25 Danish wastewater treatment plants over 7 years using high-throughput amplicon sequencing

    DEFF Research Database (Denmark)

    Albertsen, Mads; Larsen, Poul; Saunders, Aaron Marc

    Wastewater treatment is the world’s largest biotechnological processes and a perfect model system for microbial ecology as the habitat is well defined and replicated all over the world. Extensive investigations on Danish wastewater treatment plants using fluorescent in situ hybridization have...... the existing knowledge on core genera with high-throughput amplicon sequencing, plant design and process data in order to identify interactions and community shaping factors. We investigated 25 Danish full-scale wastewater treatment plants with nutrient removal over a period of 7 years with two to four samples...... a year, totaling over 400 samples. All samples were subjected to 16S rDNA amplicon sequencing using V13 primers on the Illumina MiSeq platform (2x300bp) to a depth of at least 20.000 quality filtered reads per sample. The OTUs were assigned taxonomy based on a manually curated version of the greengenes...

  4. Prediction of Genes Related to Positive Selection Using Whole-Genome Resequencing in Three Commercial Pig Breeds

    Directory of Open Access Journals (Sweden)

    HyoYoung Kim

    2015-12-01

    Full Text Available Selective sweep can cause genetic differentiation across populations, which allows for the identification of possible causative regions/genes underlying important traits. The pig has experienced a long history of allele frequency changes through artificial selection in the domestication process. We obtained an average of 329,482,871 sequence reads for 24 pigs from three pig breeds: Yorkshire (n = 5, Landrace (n = 13, and Duroc (n = 6. An average read depth of 11.7 was obtained using whole-genome resequencing on an Illumina HiSeq2000 platform. In this study, cross-population extended haplotype homozygosity and cross-population composite likelihood ratio tests were implemented to detect genes experiencing positive selection for the genome-wide resequencing data generated from three commercial pig breeds. In our results, 26, 7, and 14 genes from Yorkshire, Landrace, and Duroc, respectively were detected by two kinds of statistical tests. Significant evidence for positive selection was identified on genes ST6GALNAC2 and EPHX1 in Yorkshire, PARK2 in Landrace, and BMP6, SLA-DQA1, and PRKG1 in Duroc.These genes are reportedly relevant to lactation, reproduction, meat quality, and growth traits. To understand how these single nucleotide polymorphisms (SNPs related positive selection affect protein function, we analyzed the effect of non-synonymous SNPs. Three SNPs (rs324509622, rs80931851, and rs80937718 in the SLA-DQA1 gene were significant in the enrichment tests, indicating strong evidence for positive selection in Duroc. Our analyses identified genes under positive selection for lactation, reproduction, and meat-quality and growth traits in Yorkshire, Landrace, and Duroc, respectively.

  5. Identification of candidate domestication regions in the radish genome based on high-depth resequencing analysis of 17 genotypes.

    Science.gov (United States)

    Kim, Namshin; Jeong, Young-Min; Jeong, Seongmun; Kim, Goon-Bo; Baek, Seunghoon; Kwon, Young-Eun; Cho, Ara; Choi, Sang-Bong; Kim, Jiwoong; Lim, Won-Jun; Kim, Kyoung Hyoun; Park, Won; Kim, Jae-Yoon; Kim, Jin-Hyun; Yim, Bomi; Lee, Young Joon; Chun, Byung-Moon; Lee, Young-Pyo; Park, Beom-Seok; Yu, Hee-Ju; Mun, Jeong-Hwan

    2016-09-01

    This study provides high-quality variation data of diverse radish genotypes. Genome-wide SNP comparison along with RNA-seq analysis identified candidate genes related to domestication that have potential as trait-related markers for genetics and breeding of radish. Radish (Raphanus sativus L.) is an annual root vegetable crop that also encompasses diverse wild species. Radish has a long history of domestication, but the origins and selective sweep of cultivated radishes remain controversial. Here, we present comprehensive whole-genome resequencing analysis of radish to explore genomic variation between the radish genotypes and to identify genetic bottlenecks due to domestication in Asian cultivars. High-depth resequencing and multi-sample genotyping analysis of ten cultivated and seven wild accessions obtained 4.0 million high-quality homozygous single-nucleotide polymorphisms (SNPs)/insertions or deletions. Variation analysis revealed that Asian cultivated radish types are closely related to wild Asian accessions, but are distinct from European/American cultivated radishes, supporting the notion that Asian cultivars were domesticated from wild Asian genotypes. SNP comparison between Asian genotypes identified 153 candidate domestication regions (CDRs) containing 512 genes. Network analysis of the genes in CDRs functioning in plant signaling pathways and biochemical processes identified group of genes related to root architecture, cell wall, sugar metabolism, and glucosinolate biosynthesis. Expression profiling of the genes during root development suggested that domestication-related selective advantages included a main taproot with few branched lateral roots, reduced cell wall rigidity and favorable taste. Overall, this study provides evolutionary insights into domestication-related genetic selection in radish as well as identification of gene candidates with the potential to act as trait-related markers for background selection of elite lines in molecular

  6. Whole Genome Re-Sequencing and Characterization of Powdery Mildew Disease-Associated Allelic Variation in Melon.

    Directory of Open Access Journals (Sweden)

    Sathishkumar Natarajan

    Full Text Available Powdery mildew is one of the most common fungal diseases in the world. This disease frequently affects melon (Cucumis melo L. and other Cucurbitaceous family crops in both open field and greenhouse cultivation. One of the goals of genomics is to identify the polymorphic loci responsible for variation in phenotypic traits. In this study, powdery mildew disease assessment scores were calculated for four melon accessions, 'SCNU1154', 'Edisto47', 'MR-1', and 'PMR5'. To investigate the genetic variation of these accessions, whole genome re-sequencing using the Illumina HiSeq 2000 platform was performed. A total of 754,759,704 quality-filtered reads were generated, with an average of 82.64% coverage relative to the reference genome. Comparisons of the sequences for the melon accessions revealed around 7.4 million single nucleotide polymorphisms (SNPs, 1.9 million InDels, and 182,398 putative structural variations (SVs. Functional enrichment analysis of detected variations classified them into biological process, cellular component and molecular function categories. Further, a disease-associated QTL map was constructed for 390 SNPs and 45 InDels identified as related to defense-response genes. Among them 112 SNPs and 12 InDels were observed in powdery mildew responsive chromosomes. Accordingly, this whole genome re-sequencing study identified SNPs and InDels associated with defense genes that will serve as candidate polymorphisms in the search for sources of resistance against powdery mildew disease and could accelerate marker-assisted breeding in melon.

  7. Allelic association, DNA resequencing and copy number variation at the metabotropic glutamate receptor GRM7 gene locus in bipolar disorder.

    Science.gov (United States)

    Kandaswamy, Radhika; McQuillin, Andrew; Curtis, David; Gurling, Hugh

    2014-06-01

    Genetic markers at the GRM7 gene have shown allelic association with bipolar disorder (BP) in several case-control samples including our own sample. In this report, we present results of resequencing the GRM7 gene in 32 bipolar samples and 32 random controls selected from 553 bipolar cases and 547 control samples (UCL1). Novel and potential etiological base pair changes discovered by resequencing were genotyped in the entire UCL case-control sample. We also report on the association between GRM7 and BP in a second sample of 593 patients and 642 controls (UCL2). The three most significantly associated SNPs in the original UCL1 BP GWAS sample were genotyped in the UCL2 sample, of which none were associated. After combining the genotype data for the two samples only two (rs1508724 and rs6769814) of the original three SNP markers remained significantly associated with BP. DNA sequencing revealed mutations in three cases which were absent in control subjects. A 3'-UTR SNP rs56173829 was found to be significantly associated with BP in the whole UCL sample (P = 0.035; OR = 0.482), the rare allele being less common in cases compared to controls. Bioinformatic analyses predicted a change in the centroid secondary structure of RNA and alterations in the miRNA binding sites for the mutated base of rs56173829. We also validated two deletions and a duplication within GRM7 using quantitative-PCR which provides further support for the pre-existing evidence that copy number variants at GRM7 may have a role in the etiology of BP. © 2014 The Authors. American Journal of Medical Genetics Part B: Neuropsychiatric Published by Wiley Periodicals, Inc.

  8. Competitive amplification of differentially melting amplicons (CADMA improves KRAS hotspot mutation testing in colorectal cancer

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    Kristensen Lasse

    2012-11-01

    Full Text Available Abstract Background Cancer is an extremely heterogeneous group of diseases traditionally categorized according to tissue of origin. However, even among patients with the same cancer subtype the cellular alterations at the molecular level are often very different. Several new therapies targeting specific molecular changes found in individual patients have initiated the era of personalized therapy and significantly improved patient care. In metastatic colorectal cancer (mCRC a selected group of patients with wild-type KRAS respond to antibodies against the epidermal growth factor receptor (EGFR. Testing for KRAS mutations is now required prior to anti-EGFR treatment, however, less sensitive methods based on conventional PCR regularly fail to detect KRAS mutations in clinical samples. Methods We have developed sensitive and specific assays for detection of the seven most common KRAS mutations based on a novel methodology named Competitive Amplification of Differentially Melting Amplicons (CADMA. The clinical applicability of these assays was assessed by analyzing 100 colorectal cancer samples, for which KRAS mutation status has been evaluated by the commercially available TheraScreen® KRAS mutation kit. Results The CADMA assays were sensitive to at least 0.5% mutant alleles in a wild-type background when using 50 nanograms of DNA in the reactions. Consensus between CADMA and the TheraScreen kit was observed in 96% of the colorectal cancer samples. In cases where disagreement was observed the CADMA result could be confirmed by a previously published assay based on TaqMan probes and by fast COLD-PCR followed by Sanger sequencing. Conclusions The high analytical sensitivity and specificity of CADMA may increase diagnostic sensitivity and specificity of KRAS mutation testing in mCRC patients.

  9. Evaluating the accuracy of amplicon-based microbiome computational pipelines on simulated human gut microbial communities.

    Science.gov (United States)

    Golob, Jonathan L; Margolis, Elisa; Hoffman, Noah G; Fredricks, David N

    2017-05-30

    Microbiome studies commonly use 16S rRNA gene amplicon sequencing to characterize microbial communities. Errors introduced at multiple steps in this process can affect the interpretation of the data. Here we evaluate the accuracy of operational taxonomic unit (OTU) generation, taxonomic classification, alpha- and beta-diversity measures for different settings in QIIME, MOTHUR and a pplacer-based classification pipeline, using a novel software package: DECARD. In-silico we generated 100 synthetic bacterial communities approximating human stool microbiomes to be used as a gold-standard for evaluating the colligative performance of microbiome analysis software. Our synthetic data closely matched the composition and complexity of actual healthy human stool microbiomes. Genus-level taxonomic classification was correctly done for only 50.4-74.8% of the source organisms. Miscall rates varied from 11.9 to 23.5%. Species-level classification was less successful, (6.9-18.9% correct); miscall rates were comparable to those of genus-level targets (12.5-26.2%). The degree of miscall varied by clade of organism, pipeline and specific settings used. OTU generation accuracy varied by strategy (closed, de novo or subsampling), reference database, algorithm and software implementation. Shannon diversity estimation accuracy correlated generally with OTU-generation accuracy. Beta-diversity estimates with Double Principle Coordinate Analysis (DPCoA) were more robust against errors introduced in processing than Weighted UniFrac. The settings suggested in the tutorials were among the worst performing in all outcomes tested. Even when using the same classification pipeline, the specific OTU-generation strategy, reference database and downstream analysis methods selection can have a dramatic effect on the accuracy of taxonomic classification, and alpha- and beta-diversity estimation. Even minor changes in settings adversely affected the accuracy of the results, bringing them far from the

  10. De novo origin of VCY2 from autosome to Y-transposed amplicon.

    Directory of Open Access Journals (Sweden)

    Peng-Rong Cao

    Full Text Available The formation of new genes is a primary driving force of evolution in all organisms. The de novo evolution of new genes from non-protein-coding genomic regions is emerging as an important additional mechanism for novel gene creation. Y chromosomes underlie sex determination in mammals and contain genes that are required for male-specific functions. In this study, a search was undertaken for Y chromosome de novo genes derived from non-protein-coding sequences. The Y chromosome orphan gene variable charge, Y-linked (VCY2, is an autosome-derived gene that has sequence similarity to large autosomal fragments but lacks an autosomal protein-coding homolog. VCY2 locates in the amplicon containing long DNA fragments that were transposed from autosomes to the Y chromosome before the ape-monkey split. We confirmed that VCY2 cannot be encoded by autosomes due to the presence of multiple disablers that disrupt the open reading frame, such as the absence of start or stop codons and the presence of premature stop codons. Similar observations have been made for homologs in the autosomes of the chimpanzee, gorilla, rhesus macaque, baboon and out-group marmoset, which suggests that there was a non-protein-coding ancestral VCY2 that was common to apes and monkeys that predated the transposition event. Furthermore, while protein-coding orthologs are absent, a putative non-protein-coding VCY2 with conserved disablers was identified in the rhesus macaque Y chromosome male-specific region. This finding implies that VCY2 might have not acquired its protein-coding ability before the ape-monkey split. VCY2 encodes a testis-specific expressed protein and is involved in the pathologic process of male infertility, and the acquisition of this gene might improve male fertility. This is the first evidence that de novo genes can be generated from transposed autosomal non-protein-coding segments, and this evidence provides novel insights into the evolutionary history of the Y

  11. Development of novel InDel markers and genetic diversity in Chenopodium quinoa through whole-genome re-sequencing.

    Science.gov (United States)

    Zhang, Tifu; Gu, Minfeng; Liu, Yuhe; Lv, Yuanda; Zhou, Ling; Lu, Haiyan; Liang, Shuaiqiang; Bao, Huabin; Zhao, Han

    2017-09-05

    Quinoa (Chenopodium quinoa Willd.) is a balanced nutritional crop, but its breeding improvement has been limited by the lack of information on its genetics and genomics. Therefore, it is necessary to obtain knowledge on genomic variation, population structure, and genetic diversity and to develop novel Insertion/Deletion (InDel) markers for quinoa by whole-genome re-sequencing. We re-sequenced 11 quinoa accessions and obtained a coverage depth between approximately 7× to 23× the quinoa genome. Based on the 1453-megabase (Mb) assembly from the reference accession Riobamba, 8,441,022 filtered bi-allelic single nucleotide polymorphisms (SNPs) and 842,783 filtered InDels were identified, with an estimated SNP and InDel density of 5.81 and 0.58 per kilobase (kb). From the genomic InDel variations, 85 dimorphic InDel markers were newly developed and validated. Together with the 62 simple sequence repeat (SSR) markers reported, a total of 147 markers were used for genotyping the 129 quinoa accessions. Molecular grouping analysis showed classification into two major groups, the Andean highland (composed of the northern and southern highland subgroups) and Chilean coastal, based on combined STRUCTURE, phylogenetic tree and PCA (Principle Component Analysis) analyses. Further analysis of the genetic diversity exhibited a decreasing tendency from the Chilean coast group to the Andean highland group, and the gene flow between subgroups was more frequent than that between the two subgroups and the Chilean coastal group. The majority of the variations (approximately 70%) were found through an analysis of molecular variation (AMOVA) due to the diversity between the groups. This was congruent with the observation of a highly significant F ST value (0.705) between the groups, demonstrating significant genetic differentiation between the Andean highland type of quinoa and the Chilean coastal type. Moreover, a core set of 16 quinoa germplasms that capture all 362 alleles was

  12. Unraveling Core Functional Microbiota in Traditional Solid-State Fermentation by High-Throughput Amplicons and Metatranscriptomics Sequencing

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    Zhewei Song

    2017-07-01

    Full Text Available Fermentation microbiota is specific microorganisms that generate different types of metabolites in many productions. In traditional solid-state fermentation, the structural composition and functional capacity of the core microbiota determine the quality and quantity of products. As a typical example of food fermentation, Chinese Maotai-flavor liquor production involves a complex of various microorganisms and a wide variety of metabolites. However, the microbial succession and functional shift of the core microbiota in this traditional food fermentation remain unclear. Here, high-throughput amplicons (16S rRNA gene amplicon sequencing and internal transcribed space amplicon sequencing and metatranscriptomics sequencing technologies were combined to reveal the structure and function of the core microbiota in Chinese soy sauce aroma type liquor production. In addition, ultra-performance liquid chromatography and headspace-solid phase microextraction-gas chromatography-mass spectrometry were employed to provide qualitative and quantitative analysis of the major flavor metabolites. A total of 10 fungal and 11 bacterial genera were identified as the core microbiota. In addition, metatranscriptomic analysis revealed pyruvate metabolism in yeasts (genera Pichia, Schizosaccharomyces, Saccharomyces, and Zygosaccharomyces and lactic acid bacteria (genus Lactobacillus classified into two stages in the production of flavor components. Stage I involved high-level alcohol (ethanol production, with the genus Schizosaccharomyces serving as the core functional microorganism. Stage II involved high-level acid (lactic acid and acetic acid production, with the genus Lactobacillus serving as the core functional microorganism. The functional shift from the genus Schizosaccharomyces to the genus Lactobacillus drives flavor component conversion from alcohol (ethanol to acid (lactic acid and acetic acid in Chinese Maotai-flavor liquor production. Our findings provide

  13. Obtaining representative community profiles of anaerobic digesters through optimisation of 16S rRNA amplicon sequencing protocols

    DEFF Research Database (Denmark)

    Kirkegaard, Rasmus Hansen; McIlroy, Simon Jon; Karst, Søren Michael

    RNA gene amplicon sequencing is rapid, cheap, high throughput, and has high taxonomic resolution. However, biases are introduced in multiple steps of this approach, including non-representative DNA extraction and uneven taxonomic coverage of selected PCR primers, potentially giving a skewed view...... parameters involving harsher lysis conditions than recommended for the commercial kit, and appeared to be similar for both the mesophilic and thermophilic reactor biomass samples. The community profiling was found to be greatly influenced by the selected PCR primers, and showed that in silico designed...

  14. Next-generation sequencing of multiple individuals per barcoded library by deconvolution of sequenced amplicons using endonuclease fragment analysis.

    Science.gov (United States)

    Andersen, Jeppe D; Pereira, Vania; Pietroni, Carlotta; Mikkelsen, Martin; Johansen, Peter; Børsting, Claus; Morling, Niels

    2014-08-01

    The simultaneous sequencing of samples from multiple individuals increases the efficiency of next-generation sequencing (NGS) while also reducing costs. Here we describe a novel and simple approach for sequencing DNA from multiple individuals per barcode. Our strategy relies on the endonuclease digestion of PCR amplicons prior to library preparation, creating a specific fragment pattern for each individual that can be resolved after sequencing. By using both barcodes and restriction fragment patterns, we demonstrate the ability to sequence the human melanocortin 1 receptor (MC1R) genes from 72 individuals using only 24 barcoded libraries.

  15. Analysis of intra-host genetic diversity of Prunus necrotic ringspot virus (PNRSV using amplicon next generation sequencing.

    Directory of Open Access Journals (Sweden)

    Wycliff M Kinoti

    Full Text Available PCR amplicon next generation sequencing (NGS analysis offers a broadly applicable and targeted approach to detect populations of both high- or low-frequency virus variants in one or more plant samples. In this study, amplicon NGS was used to explore the diversity of the tripartite genome virus, Prunus necrotic ringspot virus (PNRSV from 53 PNRSV-infected trees using amplicons from conserved gene regions of each of PNRSV RNA1, RNA2 and RNA3. Sequencing of the amplicons from 53 PNRSV-infected trees revealed differing levels of polymorphism across the three different components of the PNRSV genome with a total number of 5040, 2083 and 5486 sequence variants observed for RNA1, RNA2 and RNA3 respectively. The RNA2 had the lowest diversity of sequences compared to RNA1 and RNA3, reflecting the lack of flexibility tolerated by the replicase gene that is encoded by this RNA component. Distinct PNRSV phylo-groups, consisting of closely related clusters of sequence variants, were observed in each of PNRSV RNA1, RNA2 and RNA3. Most plant samples had a single phylo-group for each RNA component. Haplotype network analysis showed that smaller clusters of PNRSV sequence variants were genetically connected to the largest sequence variant cluster within a phylo-group of each RNA component. Some plant samples had sequence variants occurring in multiple PNRSV phylo-groups in at least one of each RNA and these phylo-groups formed distinct clades that represent PNRSV genetic strains. Variants within the same phylo-group of each Prunus plant sample had ≥97% similarity and phylo-groups within a Prunus plant sample and between samples had less ≤97% similarity. Based on the analysis of diversity, a definition of a PNRSV genetic strain was proposed. The proposed definition was applied to determine the number of PNRSV genetic strains in each of the plant samples and the complexity in defining genetic strains in multipartite genome viruses was explored.

  16. Development of Insertion and Deletion Markers based on Biparental Resequencing for Fine Mapping Seed Weight in Soybean

    Directory of Open Access Journals (Sweden)

    Ying-hui Li

    2014-11-01

    Full Text Available As a complement to single nucleotide polymorphisms (SNPs and simple sequence repeats (SSRs, biallelic insertions and deletions (InDels represent powerful molecular markers with desirable features for filling the gap in current genetic linkage maps. In this study, 28,908 small InDel polymorphisms (1–5 base pair, bp distributed genome-wide were identified and annotated by comparison of a whole-genome resequencing data set from two soybean [ (L. Merr.] genotypes, cultivar Zhonghunag13 (ZH and line Zhongpin03-5373 (ZP. The physical distribution of InDel polymorphisms in soybean genome was uneven, and matched closely with the distribution of previously annotated genes. The average density of InDel in the arm region was significantly higher than that in the pericentromeric region. The genomic regions that were fixed between the two elites were elucidated. With this information, five InDel markers within a putative quantitative trait locus (QTL for seed weight (SW, , were developed and used to genotype 254 recombinant inbred lines (RILs derived from the cross of ZP × ZH. Adding these five InDel markers to previously used SNP and SSR markers facilitated the discovery of further recombination events allowing fine-mapping the QTL to a 0.5 Mbp region. Our study clearly underlines the high value of InDel markers for map-based cloning and marker-assisted selection in soybean.

  17. Whole Genome Re-Sequencing Identifies a Quantitative Trait Locus Repressing Carbon Reserve Accumulation during Optimal Growth in Chlamydomonas reinhardtii

    Science.gov (United States)

    Goold, Hugh Douglas; Nguyen, Hoa Mai; Kong, Fantao; Beyly-Adriano, Audrey; Légeret, Bertrand; Billon, Emmanuelle; Cuiné, Stéphan; Beisson, Fred; Peltier, Gilles; Li-Beisson, Yonghua

    2016-01-01

    Microalgae have emerged as a promising source for biofuel production. Massive oil and starch accumulation in microalgae is possible, but occurs mostly when biomass growth is impaired. The molecular networks underlying the negative correlation between growth and reserve formation are not known. Thus isolation of strains capable of accumulating carbon reserves during optimal growth would be highly desirable. To this end, we screened an insertional mutant library of Chlamydomonas reinhardtii for alterations in oil content. A mutant accumulating five times more oil and twice more starch than wild-type during optimal growth was isolated and named constitutive oil accumulator 1 (coa1). Growth in photobioreactors under highly controlled conditions revealed that the increase in oil and starch content in coa1 was dependent on light intensity. Genetic analysis and DNA hybridization pointed to a single insertional event responsible for the phenotype. Whole genome re-sequencing identified in coa1 a >200 kb deletion on chromosome 14 containing 41 genes. This study demonstrates that, 1), the generation of algal strains accumulating higher reserve amount without compromising biomass accumulation is feasible; 2), light is an important parameter in phenotypic analysis; and 3), a chromosomal region (Quantitative Trait Locus) acts as suppressor of carbon reserve accumulation during optimal growth. PMID:27141848

  18. Identification of Substrain-Specific Mutations by Massively Parallel Whole-Genome Resequencing of Synechocystis sp. PCC 6803

    Science.gov (United States)

    Kanesaki, Yu; Shiwa, Yuh; Tajima, Naoyuki; Suzuki, Marie; Watanabe, Satoru; Sato, Naoki; Ikeuchi, Masahiko; Yoshikawa, Hirofumi

    2012-01-01

    The cyanobacterium, Synechocystis sp. PCC 6803, was the first photosynthetic organism whose genome sequence was determined in 1996 (Kazusa strain). It thus plays an important role in basic research on the mechanism, evolution, and molecular genetics of the photosynthetic machinery. There are many substrains or laboratory strains derived from the original Berkeley strain including glucose-tolerant (GT) strains. To establish reliable genomic sequence data of this cyanobacterium, we performed resequencing of the genomes of three substrains (GT-I, PCC-P, and PCC-N) and compared the data obtained with those of the original Kazusa strain stored in the public database. We found that each substrain has sequence differences some of which are likely to reflect specific mutations that may contribute to its altered phenotype. Our resequence data of the PCC substrains along with the proposed corrections/refinements of the sequence data for the Kazusa strain and its derivatives are expected to contribute to investigations of the evolutionary events in the photosynthetic and related systems that have occurred in Synechocystis as well as in other cyanobacteria. PMID:22193367

  19. Gene expression profiling and candidate gene resequencing identifies pathways and mutations important for malignant transformation caused by leukemogenic fusion genes.

    Science.gov (United States)

    Novak, Rachel L; Harper, David P; Caudell, David; Slape, Christopher; Beachy, Sarah H; Aplan, Peter D

    2012-12-01

    NUP98-HOXD13 (NHD13) and CALM-AF10 (CA10) are oncogenic fusion proteins produced by recurrent chromosomal translocations in patients with acute myeloid leukemia (AML). Transgenic mice that express these fusions develop AML with a long latency and incomplete penetrance, suggesting that collaborating genetic events are required for leukemic transformation. We employed genetic techniques to identify both preleukemic abnormalities in healthy transgenic mice as well as collaborating events leading to leukemic transformation. Candidate gene resequencing revealed that 6 of 27 (22%) CA10 AMLs spontaneously acquired a Ras pathway mutation and 8 of 27 (30%) acquired an Flt3 mutation. Two CA10 AMLs acquired an Flt3 internal-tandem duplication, demonstrating that these mutations can be acquired in murine as well as human AML. Gene expression profiles revealed a marked upregulation of Hox genes, particularly Hoxa5, Hoxa9, and Hoxa10 in both NHD13 and CA10 mice. Furthermore, mir196b, which is embedded within the Hoxa locus, was overexpressed in both CA10 and NHD13 samples. In contrast, the Hox cofactors Meis1 and Pbx3 were differentially expressed; Meis1 was increased in CA10 AMLs but not NHD13 AMLs, whereas Pbx3 was consistently increased in NHD13 but not CA10 AMLs. Silencing of Pbx3 in NHD13 cells led to decreased proliferation, increased apoptosis, and decreased colony formation in vitro, suggesting a previously unexpected role for Pbx3 in leukemic transformation. Published by Elsevier Inc.

  20. Whole-Genome Resequencing of Experimental Populations Reveals Polygenic Basis of Egg-Size Variation in Drosophila melanogaster.

    Science.gov (United States)

    Jha, Aashish R; Miles, Cecelia M; Lippert, Nodia R; Brown, Christopher D; White, Kevin P; Kreitman, Martin

    2015-10-01

    Complete genome resequencing of populations holds great promise in deconstructing complex polygenic traits to elucidate molecular and developmental mechanisms of adaptation. Egg size is a classic adaptive trait in insects, birds, and other taxa, but its highly polygenic architecture has prevented high-resolution genetic analysis. We used replicated experimental evolution in Drosophila melanogaster and whole-genome sequencing to identify consistent signatures of polygenic egg-size adaptation. A generalized linear-mixed model revealed reproducible allele frequency differences between replicated experimental populations selected for large and small egg volumes at approximately 4,000 single nucleotide polymorphisms (SNPs). Several hundred distinct genomic regions contain clusters of these SNPs and have lower heterozygosity than the genomic background, consistent with selection acting on polymorphisms in these regions. These SNPs are also enriched among genes expressed in Drosophila ovaries and many of these genes have well-defined functions in Drosophila oogenesis. Additional genes regulating egg development, growth, and cell size show evidence of directional selection as genes regulating these biological processes are enriched for highly differentiated SNPs. Genetic crosses performed with a subset of candidate genes demonstrated that these genes influence egg size, at least in the large genetic background. These findings confirm the highly polygenic architecture of this adaptive trait, and suggest the involvement of many novel candidate genes in regulating egg size. © The Author 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  1. Population Structure and Domestication Revealed by High-Depth Resequencing of Korean Cultivated and Wild Soybean Genomes†

    Science.gov (United States)

    Chung, Won-Hyong; Jeong, Namhee; Kim, Jiwoong; Lee, Woo Kyu; Lee, Yun-Gyeong; Lee, Sang-Heon; Yoon, Woongchang; Kim, Jin-Hyun; Choi, Ik-Young; Choi, Hong-Kyu; Moon, Jung-Kyung; Kim, Namshin; Jeong, Soon-Chun

    2014-01-01

    Despite the importance of soybean as a major crop, genome-wide variation and evolution of cultivated soybeans are largely unknown. Here, we catalogued genome variation in an annual soybean population by high-depth resequencing of 10 cultivated and 6 wild accessions and obtained 3.87 million high-quality single-nucleotide polymorphisms (SNPs) after excluding the sites with missing data in any accession. Nuclear genome phylogeny supported a single origin for the cultivated soybeans. We identified 10-fold longer linkage disequilibrium (LD) in the wild soybean relative to wild maize and rice. Despite the small population size, the long LD and large SNP data allowed us to identify 206 candidate domestication regions with significantly lower diversity in the cultivated, but not in the wild, soybeans. Some of the genes in these candidate regions were associated with soybean homologues of canonical domestication genes. However, several examples, which are likely specific to soybean or eudicot crop plants, were also observed. Consequently, the variation data identified in this study should be valuable for breeding and for identifying agronomically important genes in soybeans. However, the long LD of wild soybeans may hinder pinpointing causal gene(s) in the candidate regions. PMID:24271940

  2. Population-based resequencing of experimentally evolved populations reveals the genetic basis of body size variation in Drosophila melanogaster.

    Directory of Open Access Journals (Sweden)

    Thomas L Turner

    2011-03-01

    Full Text Available Body size is a classic quantitative trait with evolutionarily significant variation within many species. Locating the alleles responsible for this variation would help understand the maintenance of variation in body size in particular, as well as quantitative traits in general. However, successful genome-wide association of genotype and phenotype may require very large sample sizes if alleles have low population frequencies or modest effects. As a complementary approach, we propose that population-based resequencing of experimentally evolved populations allows for considerable power to map functional variation. Here, we use this technique to investigate the genetic basis of natural variation in body size in Drosophila melanogaster. Significant differentiation of hundreds of loci in replicate selection populations supports the hypothesis that the genetic basis of body size variation is very polygenic in D. melanogaster. Significantly differentiated variants are limited to single genes at some loci, allowing precise hypotheses to be formed regarding causal polymorphisms, while other significant regions are large and contain many genes. By using significantly associated polymorphisms as a priori candidates in follow-up studies, these data are expected to provide considerable power to determine the genetic basis of natural variation in body size.

  3. |SE|S|AM|E| Barcode: NGS-oriented software for amplicon characterization--application to species and environmental barcoding.

    Science.gov (United States)

    Piry, S; Guivier, E; Realini, A; Martin, J-F

    2012-11-01

    Progress in NGS technologies has opened up new opportunities for characterizing biodiversity, both for individual specimen identification and for environmental barcoding. Although the amount of data available to biologist is increasing, user-friendly tools to facilitate data analysis have yet to be developed. Our aim, with |SE|S|AM|E| Barcode, is to provide such support through a unified platform. The sequences are analysed through a pipeline that (i) processes NGS amplicon runs, filtering markers and samples, (ii) builds reference libraries and finally (iii) identifies (barcodes) the sequences in each amplicon from the reference library. We use a simulated data set for specimen identification and a recently published data set for environmental barcoding to validate the method. The results obtained are consistent with the expected characterizations (in silico and previously published, respectively). |SE|S|AM|E| Barcode and its documentation are freely available under the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported Licence for Windows and Linux from http://www1.montpellier.inra.fr/CBGP/NGS/. © 2012 Blackwell Publishing Ltd.

  4. Improved Efficiency and Reliability of NGS Amplicon Sequencing Data Analysis for Genetic Diagnostic Procedures Using AGSA Software

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    Axel Poulet

    2016-01-01

    Full Text Available Screening for BRCA mutations in women with familial risk of breast or ovarian cancer is an ideal situation for high-throughput sequencing, providing large amounts of low cost data. However, 454, Roche, and Ion Torrent, Thermo Fisher, technologies produce homopolymer-associated indel errors, complicating their use in routine diagnostics. We developed software, named AGSA, which helps to detect false positive mutations in homopolymeric sequences. Seventy-two familial breast cancer cases were analysed in parallel by amplicon 454 pyrosequencing and Sanger dideoxy sequencing for genetic variations of the BRCA genes. All 565 variants detected by dideoxy sequencing were also detected by pyrosequencing. Furthermore, pyrosequencing detected 42 variants that were missed with Sanger technique. Six amplicons contained homopolymer tracts in the coding sequence that were systematically misread by the software supplied by Roche. Read data plotted as histograms by AGSA software aided the analysis considerably and allowed validation of the majority of homopolymers. As an optimisation, additional 250 patients were analysed using microfluidic amplification of regions of interest (Access Array Fluidigm of the BRCA genes, followed by 454 sequencing and AGSA analysis. AGSA complements a complete line of high-throughput diagnostic sequence analysis, reducing time and costs while increasing reliability, notably for homopolymer tracts.

  5. A Systematic Assessment of Accuracy in Detecting Somatic Mosaic Variants by Deep Amplicon Sequencing: Application to NF2 Gene.

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    Elisa Contini

    Full Text Available The accurate detection of low-allelic variants is still challenging, particularly for the identification of somatic mosaicism, where matched control sample is not available. High throughput sequencing, by the simultaneous and independent analysis of thousands of different DNA fragments, might overcome many of the limits of traditional methods, greatly increasing the sensitivity. However, it is necessary to take into account the high number of false positives that may arise due to the lack of matched control samples. Here, we applied deep amplicon sequencing to the analysis of samples with known genotype and variant allele fraction (VAF followed by a tailored statistical analysis. This method allowed to define a minimum value of VAF for detecting mosaic variants with high accuracy. Then, we exploited the estimated VAF to select candidate alterations in NF2 gene in 34 samples with unknown genotype (30 blood and 4 tumor DNAs, demonstrating the suitability of our method. The strategy we propose optimizes the use of deep amplicon sequencing for the identification of low abundance variants. Moreover, our method can be applied to different high throughput sequencing approaches to estimate the background noise and define the accuracy of the experimental design.

  6. A Portable Automatic Endpoint Detection System for Amplicons of Loop Mediated Isothermal Amplification on Microfluidic Compact Disk Platform

    Directory of Open Access Journals (Sweden)

    Shah Mukim Uddin

    2015-03-01

    Full Text Available In recent years, many improvements have been made in foodborne pathogen detection methods to reduce the impact of food contamination. Several rapid methods have been developed with biosensor devices to improve the way of performing pathogen detection. This paper presents an automated endpoint detection system for amplicons generated by loop mediated isothermal amplification (LAMP on a microfluidic compact disk platform. The developed detection system utilizes a monochromatic ultraviolet (UV emitter for excitation of fluorescent labeled LAMP amplicons and a color sensor to detect the emitted florescence from target. Then it processes the sensor output and displays the detection results on liquid crystal display (LCD. The sensitivity test has been performed with detection limit up to 2.5 × 10−3 ng/µL with different DNA concentrations of Salmonella bacteria. This system allows a rapid and automatic endpoint detection which could lead to the development of a point-of-care diagnosis device for foodborne pathogens detection in a resource-limited environment.

  7. Multitracer Positron Emission Tomographic Imaging of Exogenous Gene Expression Mediated by a Universal Herpes Simplex Virus 1 Amplicon Vector

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    Christiane Kummer

    2007-05-01

    Full Text Available To develop efficient and safe gene therapy approaches, the herpes simplex virus type 1 thymidine kinase gene (HSV-1-tk has been shown to function as a marker gene for the direct noninvasive in vivo localization of thymidine kinase (TK expression by positron emission tomography (PET using radiolabeled nucleoside analogues as specific TK substrates. Moreover, the gene encoding dopamine type 2 receptor (d2r could be used as a PET marker gene using specific radiolabeled receptor binding compounds. Here we describe the quantitative colocalization of d2r and HSV-1-tk gene expression mediated from a universal HSV-1 amplicon vector in a subcutaneous human Gli36dEGFR glioma model by PET. The HSV-1 amplicon vector was constructed using a bicistronic gene cassette to contain (1 the d2r80A mutant, which is able to bind its ligand racloprid but unable to activate downstream signal transduction pathways, and (2 the tk39 mutant with enhanced enzymatic activity toward guanosine analogues fused to the green fluorescent protein gene (tk39gfp serving as a marker gene in cell culture. After infection of human Gli36dEGFR glioma cells with the HSV-d2r80AIREStk39gfp (HSV-DITG amplicon vector in cell culture, D2 receptor expression and its targeting to the cell surface were determined by Western blotting and immunolabeling. Vector application in vivo served for quantitative colocalization of d2r80A- and tk39gfp-derived PET signals employing the specific D2 receptor binding compound [11C]racloprid and the specific TK39 substrate 9-(4-[18F]fluoro-3-hydroxymethylbutylguanine. Our results demonstrate that for the range of gene expression studied in vivo, both enzymatic and receptor binding assays give comparable quantitative information on the level of vector-mediated gene expression in vivo. The d2r80A in combination with a specific binding compound passing the intact blood-brain barrier might be an alternative marker gene for the noninvasive assessment of vector

  8. Identification of novel candidate target genes in amplicons of Glioblastoma multiforme tumors detected by expression and CGH microarray profiling

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    Hernández-Moneo Jose-Luis

    2006-09-01

    Full Text Available Abstract Background Conventional cytogenetic and comparative genomic hybridization (CGH studies in brain malignancies have shown that glioblastoma multiforme (GBM is characterized by complex structural and numerical alterations. However, the limited resolution of these techniques has precluded the precise identification of detailed specific gene copy number alterations. Results We performed a genome-wide survey of gene copy number changes in 20 primary GBMs by CGH on cDNA microarrays. A novel amplicon at 4p15, and previously uncharacterized amplicons at 13q32-34 and 1q32 were detected and are analyzed here. These amplicons contained amplified genes not previously reported. Other amplified regions containg well-known oncogenes in GBMs were also detected at 7p12 (EGFR, 7q21 (CDK6, 4q12 (PDGFRA, and 12q13-15 (MDM2 and CDK4. In order to identify the putative target genes of the amplifications, and to determine the changes in gene expression levels associated with copy number change events, we carried out parallel gene expression profiling analyses using the same cDNA microarrays. We detected overexpression of the novel amplified genes SLA/LP and STIM2 (4p15, and TNFSF13B and COL4A2 (13q32-34. Some of the candidate target genes of amplification (EGFR, CDK6, MDM2, CDK4, and TNFSF13B were tested in an independent set of 111 primary GBMs by using FISH and immunohistological assays. The novel candidate 13q-amplification target TNFSF13B was amplified in 8% of the tumors, and showed protein expression in 20% of the GBMs. Conclusion This high-resolution analysis allowed us to propose novel candidate target genes such as STIM2 at 4p15, and TNFSF13B or COL4A2 at 13q32-34 that could potentially contribute to the pathogenesis of these tumors and which would require futher investigations. We showed that overexpression of the amplified genes could be attributable to gene dosage and speculate that deregulation of those genes could be important in the development

  9. Community analysis of picocyanobacteria in an oligotrophic lake by cloning 16S rRNA gene and 16S rRNA gene amplicon sequencing.

    Science.gov (United States)

    Fujimoto, Naoshi; Mizuno, Keigo; Yokoyama, Tomoki; Ohnishi, Akihiro; Suzuki, Masaharu; Watanabe, Satoru; Komatsu, Kenji; Sakata, Yoichi; Kishida, Naohiro; Akiba, Michihiro; Matsukura, Satoko

    2015-01-01

    In this study, the picocyanobacterial species composition of Lake Miyagase was examined by analyzing the 16S rRNA gene in a clone library and by amplicon sequencing using a benchtop next-generation sequencer. Five separate samples were analyzed from different days over a ten-month period. In the picocyanobacterial lineage, 9 and 12 OTUs were identified from a clone library and by amplicon sequencing, respectively. Both analyses suggested that a picocyanobacterium related to Synechococcus sp. MW6B4 was dominant in Lake Miyagase. Our findings suggest that 16S rRNA gene amplicon sequencing enables detailed evaluation of picocyanobacteria composition. One OTU identified was found to be a novel cluster that does not group with any of the known freshwater picocyanobacteria.

  10. Optimized Method of Extracting Rice Chloroplast DNA for High-Quality Plastome Resequencing and de Novo Assembly

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    Takeshi Takamatsu

    2018-02-01

    Full Text Available Chloroplasts, which perform photosynthesis, are one of the most important organelles in green plants and algae. Chloroplasts maintain an independent genome that includes important genes encoding their photosynthetic machinery and various housekeeping functions. Owing to its non-recombinant nature, low mutation rates, and uniparental inheritance, the chloroplast genome (plastome can give insights into plant evolution and ecology and in the development of biotechnological and breeding applications. However, efficient methods to obtain high-quality chloroplast DNA (cpDNA are currently not available, impeding powerful sequencing and further functional genomics research. To investigate effects on rice chloroplast genome quality, we compared cpDNA extraction by three extraction protocols: liquid nitrogen coupled with sucrose density gradient centrifugation, high-salt buffer, and Percoll gradient centrifugation. The liquid nitrogen–sucrose gradient method gave a high yield of high-quality cpDNA with reliable purity. The cpDNA isolated by this technique was evaluated, resequenced, and assembled de novo to build a robust framework for genomic and genetic studies. Comparison of this high-purity cpDNA with total DNAs revealed the read coverage of the sequenced regions; next-generation sequencing data showed that the high-quality cpDNA eliminated noise derived from contamination by nuclear and mitochondrial DNA, which frequently occurs in total DNA. The assembly process produced highly accurate, long contigs. We summarize the extent to which this improved method of isolating cpDNA from rice can provide practical progress in overcoming challenges related to chloroplast genomes and in further exploring the development of new sequencing technologies.

  11. Inferring the demographic history of African farmers and pygmy hunter-gatherers using a multilocus resequencing data set.

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    Etienne Patin

    2009-04-01

    Full Text Available The transition from hunting and gathering to farming involved a major cultural innovation that has spread rapidly over most of the globe in the last ten millennia. In sub-Saharan Africa, hunter-gatherers have begun to shift toward an agriculture-based lifestyle over the last 5,000 years. Only a few populations still base their mode of subsistence on hunting and gathering. The Pygmies are considered to be the largest group of mobile hunter-gatherers of Africa. They dwell in equatorial rainforests and are characterized by their short mean stature. However, little is known about the chronology of the demographic events-size changes, population splits, and gene flow--ultimately giving rise to contemporary Pygmy (Western and Eastern groups and neighboring agricultural populations. We studied the branching history of Pygmy hunter-gatherers and agricultural populations from Africa and estimated separation times and gene flow between these populations. We resequenced 24 independent noncoding regions across the genome, corresponding to a total of approximately 33 kb per individual, in 236 samples from seven Pygmy and five agricultural populations dispersed over the African continent. We used simulation-based inference to identify the historical model best fitting our data. The model identified included the early divergence of the ancestors of Pygmy hunter-gatherers and farming populations approximately 60,000 years ago, followed by a split of the Pygmies' ancestors into the Western and Eastern Pygmy groups approximately 20,000 years ago. Our findings increase knowledge of the history of the peopling of the African continent in a region lacking archaeological data. An appreciation of the demographic and adaptive history of African populations with different modes of subsistence should improve our understanding of the influence of human lifestyles on genome diversity.

  12. QTL Mapping by Whole Genome Re-sequencing and Analysis of Candidate Genes for Nitrogen Use Efficiency in Rice

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    Xinghai Yang

    2017-09-01

    Full Text Available Nitrogen is a major nutritional element in rice production. However, excessive application of nitrogen fertilizer has caused severe environmental pollution. Therefore, development of rice varieties with improved nitrogen use efficiency (NUE is urgent for sustainable agriculture. In this study, bulked segregant analysis (BSA combined with whole genome re-sequencing (WGS technology was applied to finely map quantitative trait loci (QTL for NUE. A key QTL, designated as qNUE6 was identified on chromosome 6 and further validated by Insertion/Deletion (InDel marker-based substitutional mapping in recombinants from F2 population (NIL-13B4 × GH998. Forty-four genes were identified in this 266.5-kb region. According to detection and annotation analysis of variation sites, 39 genes with large-effect single-nucleotide polymorphisms (SNPs and large-effect InDels were selected as candidates and their expression levels were analyzed by qRT-PCR. Significant differences in the expression levels of LOC_Os06g15370 (peptide transporter PTR2 and LOC_Os06g15420 (asparagine synthetase were observed between two parents (Y11 and GH998. Phylogenetic analysis in Arabidopsis thaliana identified two closely related homologs, AT1G68570 (AtNPF3.1 and AT5G65010 (ASN2, which share 72.3 and 87.5% amino acid similarity with LOC_Os06g15370 and LOC_Os06g15420, respectively. Taken together, our results suggested that qNUE6 is a possible candidate gene for NUE in rice. The fine mapping and candidate gene analysis of qNUE6 provide the basis of molecular breeding for genetic improvement of rice varieties with high NUE, and lay the foundation for further cloning and functional analysis.

  13. Next-generation re-sequencing of genes involved in increased platelet reactivity in diabetic patients on acetylsalicylic acid.

    Science.gov (United States)

    Postula, Marek; Janicki, Piotr K; Eyileten, Ceren; Rosiak, Marek; Kaplon-Cieslicka, Agnieszka; Sugino, Shigekazu; Wilimski, Radosław; Kosior, Dariusz A; Opolski, Grzegorz; Filipiak, Krzysztof J; Mirowska-Guzel, Dagmara

    2016-06-01

    The objective of this study was to investigate whether rare missense genetic variants in several genes related to platelet functions and acetylsalicylic acid (ASA) response are associated with the platelet reactivity in patients with diabetes type 2 (T2D) on ASA therapy. Fifty eight exons and corresponding introns of eight selected genes, including PTGS1, PTGS2, TXBAS1, PTGIS, ADRA2A, ADRA2B, TXBA2R, and P2RY1 were re-sequenced in 230 DNA samples from T2D patients by using a pooled PCR amplification and next-generation sequencing by Illumina HiSeq2000. The observed non-synonymous variants were confirmed by individual genotyping of 384 DNA samples comprising of the individuals from the original discovery pools and additional verification cohort of 154 ASA-treated T2DM patients. The association between investigated phenotypes (ASA induced changes in platelets reactivity by PFA-100, VerifyNow and serum thromboxane B2 level [sTxB2]), and accumulation of rare missense variants (genetic burden) in investigated genes was tested using statistical collapsing tests. We identified a total of 35 exonic variants, including 3 common missense variants, 15 rare missense variants, and 17 synonymous variants in 8 investigated genes. The rare missense variants exhibited statistically significant difference in the accumulation pattern between a group of patients with increased and normal platelet reactivity based on PFA-100 assay. Our study suggests that genetic burden of the rare functional variants in eight genes may contribute to differences in the platelet reactivity measured with the PFA-100 assay in the T2DM patients treated with ASA.

  14. Human estrogen sulfotransferase (SULT1E1) pharmacogenomics: gene resequencing and functional genomics

    OpenAIRE

    Adjei, Araba A; Thomae, Bianca A; Prondzinski, Janel L; Eckloff, Bruce W; Wieben, Eric D; Weinshilboum, Richard M

    2003-01-01

    Estrogens are used as drugs and estrogen exposure is a risk factor for hormone-dependent diseases such as breast cancer. Sulfate conjugation is an important pathway for estrogen metabolism. The sulfotransferase (SULT) enzyme SULT1E1 has the lowest Km values for estrogens and catecholestrogens of the 10 known human SULT isoforms.We previously cloned and characterized the human SULT1E1 cDNA and gene as steps toward pharmacogenetic studies. In the present experiments, we set out to determine whe...

  15. HPV Genotyping of Modified General Primer-Amplicons Is More Analytically Sensitive and Specific by Sequencing than by Hybridization.

    Science.gov (United States)

    Meisal, Roger; Rounge, Trine Ballestad; Christiansen, Irene Kraus; Eieland, Alexander Kirkeby; Worren, Merete Molton; Molden, Tor Faksvaag; Kommedal, Øyvind; Hovig, Eivind; Leegaard, Truls Michael; Ambur, Ole Herman

    2017-01-01

    Sensitive and specific genotyping of human papillomaviruses (HPVs) is important for population-based surveillance of carcinogenic HPV types and for monitoring vaccine effectiveness. Here we compare HPV genotyping by Next Generation Sequencing (NGS) to an established DNA hybridization method. In DNA isolated from urine, the overall analytical sensitivity of NGS was found to be 22% higher than that of hybridization. NGS was also found to be the most specific method and expanded the detection repertoire beyond the 37 types of the DNA hybridization assay. Furthermore, NGS provided an increased resolution by identifying genetic variants of individual HPV types. The same Modified General Primers (MGP)-amplicon was used in both methods. The NGS method is described in detail to facilitate implementation in the clinical microbiology laboratory and includes suggestions for new standards for detection and calling of types and variants with improved resolution.

  16. HPV Genotyping of Modified General Primer-Amplicons Is More Analytically Sensitive and Specific by Sequencing than by Hybridization.

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    Roger Meisal

    Full Text Available Sensitive and specific genotyping of human papillomaviruses (HPVs is important for population-based surveillance of carcinogenic HPV types and for monitoring vaccine effectiveness. Here we compare HPV genotyping by Next Generation Sequencing (NGS to an established DNA hybridization method. In DNA isolated from urine, the overall analytical sensitivity of NGS was found to be 22% higher than that of hybridization. NGS was also found to be the most specific method and expanded the detection repertoire beyond the 37 types of the DNA hybridization assay. Furthermore, NGS provided an increased resolution by identifying genetic variants of individual HPV types. The same Modified General Primers (MGP-amplicon was used in both methods. The NGS method is described in detail to facilitate implementation in the clinical microbiology laboratory and includes suggestions for new standards for detection and calling of types and variants with improved resolution.

  17. Shotgun metagenomes and multiple primer pair-barcode combinations of amplicons reveal biases in metabarcoding analyses of fungi

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    Leho Tedersoo

    2015-05-01

    Full Text Available Rapid development of high-throughput (HTS molecular identification methods has revolutionized our knowledge about taxonomic diversity and ecology of fungi. However, PCR-based methods exhibit multiple technical shortcomings that may bias our understanding of the fungal kingdom. This study was initiated to quantify potential biases in fungal community ecology by comparing the relative performance of amplicon-free shotgun metagenomics and amplicons of nine primer pairs over seven nuclear ribosomal DNA (rDNA regions often used in metabarcoding analyses. The internal transcribed spacer (ITS barcodes ITS1 and ITS2 provided greater taxonomic and functional resolution and richness of operational taxonomic units (OTUs at the 97% similarity threshold compared to barcodes located within the ribosomal small subunit (SSU and large subunit (LSU genes. All barcode-primer pair combinations provided consistent results in ranking taxonomic richness and recovering the importance of floristic variables in driving fungal community composition in soils of Papua New Guinea. The choice of forward primer explained up to 2.0% of the variation in OTU-level analysis of the ITS1 and ITS2 barcode data sets. Across the whole data set, barcode-primer pair combination explained 37.6–38.1% of the variation, which surpassed any environmental signal. Overall, the metagenomics data set recovered a similar taxonomic overview, but resulted in much lower fungal rDNA sequencing depth, inability to infer OTUs, and high uncertainty in identification. We recommend the use of ITS2 or the whole ITS region for metabarcoding and we advocate careful choice of primer pairs in consideration of the relative proportion of fungal DNA and expected dominant groups.

  18. Bacterial diversity in Solenopsis invicta and Solenopsis geminata ant colonies characterized by 16S amplicon 454 pyrosequencing.

    Science.gov (United States)

    Ishak, Heather D; Plowes, Rob; Sen, Ruchira; Kellner, Katrin; Meyer, Eli; Estrada, Dora A; Dowd, Scot E; Mueller, Ulrich G

    2011-05-01

    Social insects harbor diverse assemblages of bacterial microbes, which may play a crucial role in the success or failure of biological invasions. The invasive fire ant Solenopsis invicta (Formicidae, Hymenoptera) is a model system for understanding the dynamics of invasive social insects and their biological control. However, little is known about microbes as biotic factors influencing the success or failure of ant invasions. This pilot study is the first attempt to characterize and compare microbial communities associated with the introduced S. invicta and the native Solenopsis geminata in the USA. Using 16S amplicon 454 pyrosequencing, bacterial communities of workers, brood, and soil from nest walls were compared between neighboring S. invicta and S. geminata colonies at Brackenridge Field Laboratory, Austin, Texas, with the aim of identifying potential pathogenic, commensal, or mutualistic microbial associates. Two samples of S. geminata workers showed high counts of Spiroplasma bacteria, a known pathogen or mutualist of other insects. A subsequent analysis using PCR and sequencing confirmed the presence of Spiroplasma in additional colonies of both Solenopsis species. Wolbachia was found in one alate sample of S. geminata, while one brood sample of S. invicta had a high count of Lactococcus. As expected, ant samples from both species showed much lower microbial diversity than the surrounding soil. Both ant species had similar overall bacterial diversities, although little overlap in specific microbes. To properly characterize a single bacterial community associated with a Solenopsis ant sample, rarefaction analyses indicate that it is necessary to obtain 5,000-10,000 sequences. Overall, 16S amplicon 454 pyrosequencing appears to be a cost-effective approach to screen whole microbial diversity associated with invasive ant species.

  19. Analysis of 16S rRNA amplicon sequencing options on the Roche/454 next-generation titanium sequencing platform.

    Directory of Open Access Journals (Sweden)

    Hideyuki Tamaki

    Full Text Available BACKGROUND: 16S rRNA gene pyrosequencing approach has revolutionized studies in microbial ecology. While primer selection and short read length can affect the resulting microbial community profile, little is known about the influence of pyrosequencing methods on the sequencing throughput and the outcome of microbial community analyses. The aim of this study is to compare differences in output, ease, and cost among three different amplicon pyrosequencing methods for the Roche/454 Titanium platform METHODOLOGY/PRINCIPAL FINDINGS: The following three pyrosequencing methods for 16S rRNA genes were selected in this study: Method-1 (standard method is the recommended method for bi-directional sequencing using the LIB-A kit; Method-2 is a new option designed in this study for unidirectional sequencing with the LIB-A kit; and Method-3 uses the LIB-L kit for unidirectional sequencing. In our comparison among these three methods using 10 different environmental samples, Method-2 and Method-3 produced 1.5-1.6 times more useable reads than the standard method (Method-1, after quality-based trimming, and did not compromise the outcome of microbial community analyses. Specifically, Method-3 is the most cost-effective unidirectional amplicon sequencing method as it provided the most reads and required the least effort in consumables management. CONCLUSIONS: Our findings clearly demonstrated that alternative pyrosequencing methods for 16S rRNA genes could drastically affect sequencing output (e.g. number of reads before and after trimming but have little effect on the outcomes of microbial community analysis. This finding is important for both researchers and sequencing facilities utilizing 16S rRNA gene pyrosequencing for microbial ecological studies.

  20. Single nucleotide variants and InDels identified from whole-genome re-sequencing of Guzerat, Gyr, Girolando and Holstein cattle breeds

    OpenAIRE

    Stafuzza, Nedenia Bonvino; Zerlotini, Adhemar; Lobo, Francisco Pereira; Yamagishi, Michel Eduardo Beleza; Chud, Tatiane Cristina Seleguim; Caetano, Alexandre Rodrigues; Munari, Dan?sio Prado; Garrick, Dorian J.; Machado, Marco Antonio; Martins, Marta Fonseca; Carvalho, Maria Raquel; Cole, John Bruce; Barbosa da Silva, Marcos Vinicius Gualberto

    2017-01-01

    Whole-genome re-sequencing, alignment and annotation analyses were undertaken for 12 sires representing four important cattle breeds in Brazil: Guzerat (multi-purpose), Gyr, Girolando and Holstein (dairy production). A total of approximately 4.3 billion reads from an Illumina HiSeq 2000 sequencer generated for each animal 10.7 to 16.4-fold genome coverage. A total of 27,441,279 single nucleotide variations (SNVs) and 3,828,041 insertions/deletions (InDels) were detected in the samples, of whi...

  1. Lactococcus lactis Diversity in Undefined Mixed Dairy Starter Cultures as Revealed by Comparative Genome Analyses and Targeted Amplicon Sequencing of epsD.

    Science.gov (United States)

    Frantzen, Cyril A; Kleppen, Hans Petter; Holo, Helge

    2018-02-01

    Undefined mesophilic mixed (DL) starter cultures are used in the production of continental cheeses and contain unknown strain mixtures of Lactococcus lactis and leuconostocs. The choice of starter culture affects the taste, aroma, and quality of the final product. To gain insight into the diversity of Lactococcus lactis strains in starter cultures, we whole-genome sequenced 95 isolates from three different starter cultures. Pan-genomic analyses, which included 30 publically available complete genomes, grouped the strains into 21 L. lactis subsp . lactis and 28 L. lactis subsp. cremoris lineages. Only one of the 95 isolates grouped with previously sequenced strains, and the three starter cultures showed no overlap in lineage distributions. The culture diversity was assessed by targeted amplicon sequencing using purR , a core gene, and epsD , present in 93 of the 95 starter culture isolates but absent in most of the reference strains. This enabled an unprecedented discrimination of starter culture Lactococcus lactis and revealed substantial differences between the three starter cultures and compositional shifts during the cultivation of cultures in milk. IMPORTANCE In contemporary cheese production, standardized frozen seed stock starter cultures are used to ensure production stability, reproducibility, and quality control of the product. The dairy industry experiences significant disruptions of cheese production due to phage attacks, and one commonly used countermeasure to phage attack is to employ a starter rotation strategy, in which two or more starters with minimal overlap in phage sensitivity are used alternately. A culture-independent analysis of the lactococcal diversity in complex undefined starter cultures revealed large differences between the three starter cultures and temporal shifts in lactococcal composition during the production of bulk starters. A better understanding of the lactococcal diversity in starter cultures will enable the development of

  2. Targeted resequencing of the pericentromere of chromosome 2 linked to constitutional delay of growth and puberty.

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    Diana L Cousminer

    Full Text Available Constitutional delay of growth and puberty (CDGP is the most common cause of pubertal delay. CDGP is defined as the proportion of the normal population who experience pubertal onset at least 2 SD later than the population mean, representing 2.3% of all adolescents. While adolescents with CDGP spontaneously enter puberty, they are at risk for short stature, decreased bone mineral density, and psychosocial problems. Genetic factors contribute heavily to the timing of puberty, but the vast majority of CDGP cases remain biologically unexplained, and there is no definitive test to distinguish CDGP from pathological absence of puberty during adolescence. Recently, we published a study identifying significant linkage between a locus at the pericentromeric region of chromosome 2 (chr 2 and CDGP in Finnish families. To investigate this region for causal variation, we sequenced chr 2 between the genomic coordinates of 79-124 Mb (genome build GRCh37 in the proband and affected parent of the 13 families contributing most to this linkage signal. One gene, DNAH6, harbored 6 protein-altering low-frequency variants (< 6% in the Finnish population in 10 of the CDGP probands. We sequenced an additional 135 unrelated Finnish CDGP subjects and utilized the unique Sequencing Initiative Suomi (SISu population reference exome set to show that while 5 of these variants were present in the CDGP set, they were also present in the Finnish population at similar frequencies. Additional variants in the targeted region could not be prioritized for follow-up, possibly due to gaps in sequencing coverage or lack of functional knowledge of non-genic genomic regions. Thus, despite having a well-characterized sample collection from a genetically homogeneous population with a large population-based reference sequence dataset, we were unable to pinpoint variation in the linked region predisposing delayed puberty. This study highlights the difficulties of detecting genetic variants

  3. Investigation of Microbial Diversity in Geothermal Hot Springs in Unkeshwar, India, Based on 16S rRNA Amplicon Metagenome Sequencing

    OpenAIRE

    Mehetre, Gajanan T.; Paranjpe, Aditi; Dastager, Syed G.; Dharne, Mahesh S.

    2016-01-01

    Microbial diversity in geothermal waters of the Unkeshwar hot springs in Maharashtra, India, was studied using 16S rRNA amplicon metagenomic sequencing. Taxonomic analysis revealed the presence of Bacteroidetes, Proteobacteria, Cyanobacteria, Actinobacteria, Archeae, and OD1 phyla. Metabolic function prediction analysis indicated a battery of biological information systems indicating rich and novel microbial diversity, with potential biotechnological applications in this niche.

  4. Resequencing PNMT in European hypertensive and normotensive individuals: no common susceptibilily variants for hypertension and purifying selection on intron 1

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    Viigimaa Margus

    2007-07-01

    Full Text Available Abstract Background Human linkage and animal QTL studies have indicated the contribution of genes on Chr17 into blood pressure regulation. One candidate gene is PNMT, coding for phenylethanolamine-N-methyltransferase, catalyzing the synthesis of epinephrine from norepinephrine. Methods Fine-scale variation of PNMT was screened by resequencing hypertensive (n = 50 and normotensive (n = 50 individuals from two European populations (Estonians and Czechs. The resulting polymorphism data were analyzed by statistical genetics methods using Genepop 3.4, PHASE 2.1 and DnaSP 4.0 software programs. In silico prediction of transcription factor binding sites for intron 1 was performed with MatInspector 2.2 software. Results PNMT was characterized by minimum variation and excess of rare SNPs in both normo- and hypertensive individuals. None of the SNPs showed significant differences in allelic frequencies among population samples, as well as between screened hypertensives and normotensives. In the joint case-control analysis of the Estonian and the Czech samples, hypertension patients had a significant excess of heterozygotes for two promoter region polymorphisms (SNP-184; SNP-390. The identified variation pattern of PNMT reflects the effect of purifying selection consistent with an important role of PNMT-synthesized epinephrine in the regulation of cardiovascular and metabolic functions, and as a CNS neurotransmitter. A striking feature is the lack of intronic variation. In silico analysis of PNMT intron 1 confirmed the presence of a human-specific putative Glucocorticoid Responsive Element (GRE, inserted by Alu-mediated transfer. Further analysis of intron 1 supported the possible existence of a full Glucocorticoid Responsive Unit (GRU predicted to consist of multiple gene regulatory elements known to cooperate with GRE in driving transcription. The role of these elements in regulating PNMT expression patterns and thus determining the dynamics of the

  5. Resequencing DCDC5 in the Flanking Region of an LD-SNP Derived from a Kidney-Yang Deficiency Syndrome Family

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    Li Ping Zhou

    2011-01-01

    Full Text Available Objective. To explore the genetic traits of Kidney-yang deficiency syndrome (KDS. Design. Twelve KDS subjects and three spouses from a typical KDS family were recruited. Their genomic DNA samples were genotyped by Affymetrix 100K single-nucleotide polymorphism (SNP arrays. The linkage disequilibrium (LD SNPs were generated using GeneChip DNA analysis software (GDAS, Affymetrix. Genes located within 100 bp of the flanks of LD SNPs were mined via GeneView. 29 exons of the doublecortin domain containing 5 (DCDC5, a representative gene within the flank of an LD SNP, were resequenced. Results. Five LD SNPs display midrange linkage with KDS. Two genes with established functions, DCDC5 and Leucyl-tRNA synthetase, were mined in the flanks of LD SNPs. Resequencing of DCDC5 revealed a nonsynonymous variation, in which 3764T/A was replaced by C/G. Accordingly, the Ser1172 was substituted by Pro1172. The S1172P substitution effect was evaluated as “possibly damaging” by PolyPhen. Conclusion. We have identified a genomic variation of DCDC5 based on the LD SNPs derived from a KDS family. DCDC5 and other genes surrounding these SNPs display some relationships with key symptoms of KDS.

  6. Rapid Genome-wide Single Nucleotide Polymorphism Discovery in Soybean and Rice via Deep Resequencing of Reduced Representation Libraries with the Illumina Genome Analyzer

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    Stéphane Deschamps

    2010-07-01

    Full Text Available Massively parallel sequencing platforms have allowed for the rapid discovery of single nucleotide polymorphisms (SNPs among related genotypes within a species. We describe the creation of reduced representation libraries (RRLs using an initial digestion of nuclear genomic DNA with a methylation-sensitive restriction endonuclease followed by a secondary digestion with the 4bp-restriction endonuclease This strategy allows for the enrichment of hypomethylated genomic DNA, which has been shown to be rich in genic sequences, and the digestion with serves to increase the number of common loci resequenced between individuals. Deep resequencing of these RRLs performed with the Illumina Genome Analyzer led to the identification of 2618 SNPs in rice and 1682 SNPs in soybean for two representative genotypes in each of the species. A subset of these SNPs was validated via Sanger sequencing, exhibiting validation rates of 96.4 and 97.0%, in rice ( and soybean (, respectively. Comparative analysis of the read distribution relative to annotated genes in the reference genome assemblies indicated that the RRL strategy was primarily sampling within genic regions for both species. The massively parallel sequencing of methylation-sensitive RRLs for genome-wide SNP discovery can be applied across a wide range of plant species having sufficient reference genomic sequence.

  7. Pooled Resequencing of 122 Ulcerative Colitis Genes in a Large Dutch Cohort Suggests Population-Specific Associations of Rare Variants in MUC2.

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    Marijn C Visschedijk

    Full Text Available Genome-wide association studies have revealed several common genetic risk variants for ulcerative colitis (UC. However, little is known about the contribution of rare, large effect genetic variants to UC susceptibility. In this study, we performed a deep targeted re-sequencing of 122 genes in Dutch UC patients in order to investigate the contribution of rare variants to the genetic susceptibility to UC. The selection of genes consists of 111 established human UC susceptibility genes and 11 genes that lead to spontaneous colitis when knocked-out in mice. In addition, we sequenced the promoter regions of 45 genes where known variants exert cis-eQTL-effects. Targeted pooled re-sequencing was performed on DNA of 790 Dutch UC cases. The Genome of the Netherlands project provided sequence data of 500 healthy controls. After quality control and prioritization based on allele frequency and pathogenicity probability, follow-up genotyping of 171 rare variants was performed on 1021 Dutch UC cases and 1166 Dutch controls. Single-variant association and gene-based analyses identified an association of rare variants in the MUC2 gene with UC. The associated variants in the Dutch population could not be replicated in a German replication cohort (1026 UC cases, 3532 controls. In conclusion, this study has identified a putative role for MUC2 on UC susceptibility in the Dutch population and suggests a population-specific contribution of rare variants to UC.

  8. Pooled Resequencing of 122 Ulcerative Colitis Genes in a Large Dutch Cohort Suggests Population-Specific Associations of Rare Variants in MUC2.

    Science.gov (United States)

    Visschedijk, Marijn C; Alberts, Rudi; Mucha, Soren; Deelen, Patrick; de Jong, Dirk J; Pierik, Marieke; Spekhorst, Lieke M; Imhann, Floris; van der Meulen-de Jong, Andrea E; van der Woude, C Janneke; van Bodegraven, Adriaan A; Oldenburg, Bas; Löwenberg, Mark; Dijkstra, Gerard; Ellinghaus, David; Schreiber, Stefan; Wijmenga, Cisca; Rivas, Manuel A; Franke, Andre; van Diemen, Cleo C; Weersma, Rinse K

    2016-01-01

    Genome-wide association studies have revealed several common genetic risk variants for ulcerative colitis (UC). However, little is known about the contribution of rare, large effect genetic variants to UC susceptibility. In this study, we performed a deep targeted re-sequencing of 122 genes in Dutch UC patients in order to investigate the contribution of rare variants to the genetic susceptibility to UC. The selection of genes consists of 111 established human UC susceptibility genes and 11 genes that lead to spontaneous colitis when knocked-out in mice. In addition, we sequenced the promoter regions of 45 genes where known variants exert cis-eQTL-effects. Targeted pooled re-sequencing was performed on DNA of 790 Dutch UC cases. The Genome of the Netherlands project provided sequence data of 500 healthy controls. After quality control and prioritization based on allele frequency and pathogenicity probability, follow-up genotyping of 171 rare variants was performed on 1021 Dutch UC cases and 1166 Dutch controls. Single-variant association and gene-based analyses identified an association of rare variants in the MUC2 gene with UC. The associated variants in the Dutch population could not be replicated in a German replication cohort (1026 UC cases, 3532 controls). In conclusion, this study has identified a putative role for MUC2 on UC susceptibility in the Dutch population and suggests a population-specific contribution of rare variants to UC.

  9. KAT6A, a Chromatin Modifier from the 8p11-p12 Amplicon is a Candidate Oncogene in Luminal Breast Cancer

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    Brittany Turner-Ivey

    2014-08-01

    Full Text Available The chromosome 8p11-p12 amplicon is present in 12% to 15% of breast cancers, resulting in an increase in copy number and expression of several chromatin modifiers in these tumors, including KAT6A. Previous analyses in SUM-52 breast cancer cells showed amplification and overexpression of KAT6A, and subsequent RNAi screening identified KAT6A as a potential driving oncogene. KAT6A is a histone acetyltransferase previously identified as a fusion partner with CREB binding protein in acute myeloid leukemia. Knockdown of KAT6A in SUM-52 cells, a luminal breast cancer cell line harboring the amplicon, resulted in reduced growth rate compared to non-silencing controls and profound loss of clonogenic capacity both in mono-layer and in soft agar. The normal cell line MCF10A, however, did not exhibit slower growth with knockdown of KAT6A. SUM-52 cells with KAT6A knockdown formed fewer mammospheres in culture compared to controls, suggesting a possible role for KAT6A in self-renewal. Previous data from our laboratory identified FGFR2 as a driving oncogene in SUM-52 cells. The colony forming efficiency of SUM-52 KAT6A knockdown cells in the presence of FGFR inhibition was significantly reduced compared to cells with KAT6A knockdown only. These data suggest that KAT6A may be a novel oncogene in breast cancers bearing the 8p11-p12 amplicon. While there are other putative oncogenes in the amplicon, the identification of KAT6A as a driving oncogene suggests that chromatin-modifying enzymes are a key class of oncogenes in these cancers, and play an important role in the selection of this amplicon in luminal B breast cancers.

  10. Comprehensive evaluation and optimization of amplicon library preparation methods for high-throughput antibody sequencing.

    Science.gov (United States)

    Menzel, Ulrike; Greiff, Victor; Khan, Tarik A; Haessler, Ulrike; Hellmann, Ina; Friedensohn, Simon; Cook, Skylar C; Pogson, Mark; Reddy, Sai T

    2014-01-01

    High-throughput sequencing (HTS) of antibody repertoire libraries has become a powerful tool in the field of systems immunology. However, numerous sources of bias in HTS workflows may affect the obtained antibody repertoire data. A crucial step in antibody library preparation is the addition of short platform-specific nucleotide adapter sequences. As of yet, the impact of the method of adapter addition on experimental library preparation and the resulting antibody repertoire HTS datasets has not been thoroughly investigated. Therefore, we compared three standard library preparation methods by performing Illumina HTS on antibody variable heavy genes from murine antibody-secreting cells. Clonal overlap and rank statistics demonstrated that the investigated methods produced equivalent HTS datasets. PCR-based methods were experimentally superior to ligation with respect to speed, efficiency, and practicality. Finally, using a two-step PCR based method we established a protocol for antibody repertoire library generation, beginning from inputs as low as 1 ng of total RNA. In summary, this study represents a major advance towards a standardized experimental framework for antibody HTS, thus opening up the potential for systems-based, cross-experiment meta-analyses of antibody repertoires.

  11. A one-step real-time multiplex PCR for screening Y-chromosomal microdeletions without downstream amplicon size analysis.

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    Viviana Kozina

    Full Text Available BACKGROUND: Y-chromosomal microdeletions (YCMD are one of the major genetic causes for non-obstructive azoospermia. Genetic testing for YCMD by multiplex polymerase chain reaction (PCR is an established method for quick and robust screening of deletions in the AZF regions of the Y-chromosome. Multiplex PCRs have the advantage of including a control gene in every reaction and significantly reducing the number of reactions needed to screen the relevant genomic markers. PRINCIPAL FINDINGS: The widely established "EAA/EMQN best practice guidelines for molecular diagnosis of Y-chromosomal microdeletions (2004" were used as a basis for designing a real-time multiplex PCR system, in which the YCMD can simply be identified by their melting points. For this reason, some AZF primers were substituted by primers for regions in their genomic proximity, and the ZFX/ZFY control primer was exchanged by the AMELX/AMELY control primer. Furthermore, we substituted the classical SybrGreen I dye by the novel and high-performing DNA-binding dye EvaGreen™ and put substantial effort in titrating the primer combinations in respect to optimal melting peak separation and peak size. SIGNIFICANCE: With these changes, we were able to develop a platform-independent and robust real-time based multiplex PCR, which makes the need for amplicon identification by electrophoretic sizing expendable. By using an open-source system for real-time PCR analysis, we further demonstrate the applicability of automated melting point and YCMD detection.

  12. Simultaneous Whole Mitochondrial Genome Sequencing with Short Overlapping Amplicons Suitable for Degraded DNA Using the Ion Torrent Personal Genome Machine.

    Science.gov (United States)

    Chaitanya, Lakshmi; Ralf, Arwin; van Oven, Mannis; Kupiec, Tomasz; Chang, Joseph; Lagacé, Robert; Kayser, Manfred

    2015-12-01

    Whole mitochondrial (mt) genome analysis enables a considerable increase in analysis throughput, and improves the discriminatory power to the maximum possible phylogenetic resolution. Most established protocols on the different massively parallel sequencing (MPS) platforms, however, invariably involve the PCR amplification of large fragments, typically several kilobases in size, which may fail due to mtDNA fragmentation in the available degraded materials. We introduce a MPS tiling approach for simultaneous whole human mt genome sequencing using 161 short overlapping amplicons (average 200 bp) with the Ion Torrent Personal Genome Machine. We illustrate the performance of this new method by sequencing 20 DNA samples belonging to different worldwide mtDNA haplogroups. Additional quality control, particularly regarding the potential detection of nuclear insertions of mtDNA (NUMTs), was performed by comparative MPS analysis using the conventional long-range amplification method. Preliminary sensitivity testing revealed that detailed haplogroup inference was feasible with 100 pg genomic input DNA. Complete mt genome coverage was achieved from DNA samples experimentally degraded down to genomic fragment sizes of about 220 bp, and up to 90% coverage from naturally degraded samples. Overall, we introduce a new approach for whole mt genome MPS analysis from degraded and nondegraded materials relevant to resolve and infer maternal genetic ancestry at complete resolution in anthropological, evolutionary, medical, and forensic applications. © 2015 The Authors. **Human Mutation published by Wiley Periodicals, Inc.

  13. Factors that affect large subunit ribosomal DNA amplicon sequencing studies of fungal communities: classification method, primer choice, and error.

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    Teresita M Porter

    Full Text Available Nuclear large subunit ribosomal DNA is widely used in fungal phylogenetics and to an increasing extent also amplicon-based environmental sequencing. The relatively short reads produced by next-generation sequencing, however, makes primer choice and sequence error important variables for obtaining accurate taxonomic classifications. In this simulation study we tested the performance of three classification methods: 1 a similarity-based method (BLAST + Metagenomic Analyzer, MEGAN; 2 a composition-based method (Ribosomal Database Project naïve bayesian classifier, NBC; and, 3 a phylogeny-based method (Statistical Assignment Package, SAP. We also tested the effects of sequence length, primer choice, and sequence error on classification accuracy and perceived community composition. Using a leave-one-out cross validation approach, results for classifications to the genus rank were as follows: BLAST + MEGAN had the lowest error rate and was particularly robust to sequence error; SAP accuracy was highest when long LSU query sequences were classified; and, NBC runs significantly faster than the other tested methods. All methods performed poorly with the shortest 50-100 bp sequences. Increasing simulated sequence error reduced classification accuracy. Community shifts were detected due to sequence error and primer selection even though there was no change in the underlying community composition. Short read datasets from individual primers, as well as pooled datasets, appear to only approximate the true community composition. We hope this work informs investigators of some of the factors that affect the quality and interpretation of their environmental gene surveys.

  14. Multilocus amplicon sequencing of Pseudomonas aeruginosa cystic fibrosis airways isolates collected prior to and after early antipseudomonal chemotherapy.

    Science.gov (United States)

    Fischer, Sebastian; Greipel, Leonie; Klockgether, Jens; Dorda, Marie; Wiehlmann, Lutz; Cramer, Nina; Tümmler, Burkhard

    2017-05-01

    Early antimicrobial chemotherapy can prevent or at least delay chronic cystic fibrosis (CF) airways infections with Pseudomonas aeruginosa. During a 10-year study period P. aeruginosa was detected for the first time in 54 CF patients regularly seen at the CF centre Hannover. Amplicon sequencing of 34 loci of the P. aeruginosa core genome was performed in baseline and post-treatment isolates of the 15 CF patients who had remained P. aeruginosa - positive after the first round of antipseudomonal chemotherapy. Deep sequencing uncovered coexisting alternative nucleotides at in total 33 of 55,284 examined genome positions including six non-synonymous polymorphisms in the lasR gene, a key regulator of quorum sensing. After early treatment 42 of 50 novel nucleotide substitutions had emerged in exopolysaccharide biosynthesis, efflux pump and porin genes. Early treatment selects pathoadaptive mutations in P. aeruginosa that are typical for chronic infections of CF lungs. Copyright © 2016 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

  15. Metagenomic Analysis of Slovak Bryndza Cheese Using Next-Generation 16S rDNA Amplicon Sequencing

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    Planý Matej

    2016-06-01

    Full Text Available Knowledge about diversity and taxonomic structure of the microbial population present in traditional fermented foods plays a key role in starter culture selection, safety improvement and quality enhancement of the end product. Aim of this study was to investigate microbial consortia composition in Slovak bryndza cheese. For this purpose, we used culture-independent approach based on 16S rDNA amplicon sequencing using next generation sequencing platform. Results obtained by the analysis of three commercial (produced on industrial scale in winter season and one traditional (artisanal, most valued, produced in May Slovak bryndza cheese sample were compared. A diverse prokaryotic microflora composed mostly of the genera Lactococcus, Streptococcus, Lactobacillus, and Enterococcus was identified. Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris were the dominant taxons in all tested samples. Second most abundant species, detected in all bryndza cheeses, were Lactococcus fujiensis and Lactococcus taiwanensis, independently by two different approaches, using different reference 16S rRNA genes databases (Greengenes and NCBI respectively. They have been detected in bryndza cheese samples in substantial amount for the first time. The narrowest microbial diversity was observed in a sample made with a starter culture from pasteurised milk. Metagenomic analysis by high-throughput sequencing using 16S rRNA genes seems to be a powerful tool for studying the structure of the microbial population in cheeses.

  16. Fungi Sailing the Arctic Ocean: Speciose Communities in North Atlantic Driftwood as Revealed by High-Throughput Amplicon Sequencing.

    Science.gov (United States)

    Rämä, Teppo; Davey, Marie L; Nordén, Jenni; Halvorsen, Rune; Blaalid, Rakel; Mathiassen, Geir H; Alsos, Inger G; Kauserud, Håvard

    2016-08-01

    High amounts of driftwood sail across the oceans and provide habitat for organisms tolerating the rough and saline environment. Fungi have adapted to the extremely cold and saline conditions which driftwood faces in the high north. For the first time, we applied high-throughput sequencing to fungi residing in driftwood to reveal their taxonomic richness, community composition, and ecology in the North Atlantic. Using pyrosequencing of ITS2 amplicons obtained from 49 marine logs, we found 807 fungal operational taxonomic units (OTUs) based on clustering at 97 % sequence similarity cut-off level. The phylum Ascomycota comprised 74 % of the OTUs and 20 % belonged to Basidiomycota. The richness of basidiomycetes decreased with prolonged submersion in the sea, supporting the general view of ascomycetes being more extremotolerant. However, more than one fourth of the fungal OTUs remained unassigned to any fungal class, emphasising the need for better DNA reference data from the marine habitat. Different fungal communities were detected in coniferous and deciduous logs. Our results highlight that driftwood hosts a considerably higher fungal diversity than currently known. The driftwood fungal community is not a terrestrial relic but a speciose assemblage of fungi adapted to the stressful marine environment and different kinds of wooden substrates found in it.

  17. Mutation detection in cholestatic patients using microarray resequencing of ATP8B1 and ABCB11 [v2; ref status: indexed, http://f1000r.es/yv

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    Kirsten E McKay

    2013-03-01

    Full Text Available Background: Neonatal cholestasis is a common presentation of childhood liver diseases and can be a feature of various conditions including disorders of bile acid biogenesis and transport, various inborn errors of metabolism and perinatal infections. Some inherited metabolic diseases can be easily screened using biochemical assays, however many can only be accurately diagnosed by DNA sequencing. Fluorescent capillary Sanger sequencing (FS is the gold standard method used by clinical laboratories for genetic diagnosis of many inherited conditions; however, it does have limitations. Recently microarray resequencing (MR has been introduced into research and clinical practice as an alternative method for genetic diagnosis of heterogeneous conditions. In this report we compared the accuracy of mutation detection for MR with FS in a group of patients with ‘low-normal’ gamma glutamyl transpeptidase (gGT cholestasis without known molecular diagnoses. Methods: 29 patient DNA samples were tested for mutations in the ATP8B1 and ABCB11 genes using both FS and MR. Other known causes of “low gGT cholestasis” such as ARC syndrome and bile acid biosynthesis disorders were excluded. Results: Mutations were identified in 13/29 samples. In 3/29 samples FS and MR gave discordant results: MR had a false positive rate of 3.4% and a false negative rate of 7%. Conclusions: The major advantage of MR over FS is that multiple genes can be screened in one experiment, allowing rapid and cost-effective diagnoses.  However, we have demonstrated that MR technology is limited in sensitivity. We therefore recommend that MR be used as an initial evaluation, with FS deployed when genetic and clinical or histopathological findings are discordant.

  18. Mapping and Identifying a Candidate Gene (Bnmfs for Female-Male Sterility through Whole-Genome Resequencing and RNA-Seq in Rapeseed (Brassica napus L.

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    Changcai Teng

    2017-12-01

    Full Text Available In oilseed crops, carpel and stamen development play vital roles in pollination and rapeseed yield, but the genetic mechanisms underlying carpel and stamen development remain unclear. Herein, a male- and female-sterile mutant was obtained in offspring of a (Brassica napus cv. Qingyou 14 × (Qingyou 14 × B. rapa landrace Dahuang cross. Subsequently, F2–F9 populations were generated through selfing of the heterozygote plants among the progeny of each generation. The male- and female-sterility exhibited stable inheritance in successive generations and was controlled by a recessive gene. The mutant kept the same chromosome number (2n = 38 as B. napus parent but showed abnormal meiosis for male and female. One candidate gene for the sterility was identified by simple sequence repeat (SSR and insertion deletion length polymorphism (InDel markers in F7–F9 plants, and whole-genome resequencing with F8 pools and RNA sequencing with F9 pools. Whole-genome resequencing found three candidate intervals (35.40–35.68, 35.74–35.75, and 45.34–46.45 Mb on chromosome C3 in B. napus and candidate region for Bnmfs was narrowed to approximately 1.11-Mb (45.34–46.45 M by combining SSR and InDel marker analyses with whole-genome resequencing. From transcriptome profiling in 0–2 mm buds, all of the genes in the candidate interval were detected, and only two genes with significant differences (BnaC03g56670D and BnaC03g56870D were revealed. BnaC03g56870D was a candidate gene that shared homology with the CYP86C4 gene of Arabidopsis thaliana. Quantitative reverse transcription (qRT-PCR analysis showed that Bnmfs primarily functioned in flower buds. Thus, sequencing and expression analyses provided evidence that BnaC03g56870D was the candidate gene for male and female sterility in the B. napus mutant.

  19. Genome wide re-sequencing of newly developed Rice Lines from common wild rice (Oryza rufipogon Griff. for the identification of NBS-LRR genes.

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    Wen Liu

    Full Text Available Common wild rice (Oryza rufipogon Griff. is an important germplasm for rice breeding, which contains many resistance genes. Re-sequencing provides an unprecedented opportunity to explore the abundant useful genes at whole genome level. Here, we identified the nucleotide-binding site leucine-rich repeat (NBS-LRR encoding genes by re-sequencing of two wild rice lines (i.e. Huaye 1 and Huaye 2 that were developed from common wild rice. We obtained 128 to 147 million reads with approximately 32.5-fold coverage depth, and uniquely covered more than 89.6% (> = 1 fold of reference genomes. Two wild rice lines showed high SNP (single-nucleotide polymorphisms variation rate in 12 chromosomes against the reference genomes of Nipponbare (japonica cultivar and 93-11 (indica cultivar. InDels (insertion/deletion polymorphisms count-length distribution exhibited normal distribution in the two lines, and most of the InDels were ranged from -5 to 5 bp. With reference to the Nipponbare genome sequence, we detected a total of 1,209,308 SNPs, 161,117 InDels and 4,192 SVs (structural variations in Huaye 1, and 1,387,959 SNPs, 180,226 InDels and 5,305 SVs in Huaye 2. A total of 44.9% and 46.9% genes exhibited sequence variations in two wild rice lines compared to the Nipponbare and 93-11 reference genomes, respectively. Analysis of NBS-LRR mutant candidate genes showed that they were mainly distributed on chromosome 11, and NBS domain was more conserved than LRR domain in both wild rice lines. NBS genes depicted higher levels of genetic diversity in Huaye 1 than that found in Huaye 2. Furthermore, protein-protein interaction analysis showed that NBS genes mostly interacted with the cytochrome C protein (Os05g0420600, Os01g0885000 and BGIOSGA038922, while some NBS genes interacted with heat shock protein, DNA-binding activity, Phosphoinositide 3-kinase and a coiled coil region. We explored abundant NBS-LRR encoding genes in two common wild rice lines through genome

  20. Combined amplicon pyrosequencing assays reveal presence of the apicomplexan "type-N" (cf. Gemmocystis cylindrus and Chromera velia on the Great Barrier Reef, Australia.

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    Jan Slapeta

    Full Text Available BACKGROUND: The coral is predominantly composed of the metabolically dependent coral host and the photosynthetic dinoflagellate Symbiodinium sp. The system as a whole interacts with symbiotic eukaryotes, bacteria and viruses. Gemmocystiscylindrus (cf. "type-N" symbiont belonging to the obligatory parasitic phylum Apicomplexa (Alveolata is ubiquitous in the Caribbean coral, but its presence in the Great Barrier Reef coral has yet to be documented. Approaches allowing identification of the healthy community from the pathogenic or saprobic organisms are needed for sustainable coral reef monitoring. METHODS & PRINCIPAL FINDINGS: We investigated the diversity of eukaryotes associated with a common reef-building corals from the southern Great Barrier Reef. We used three tag encoded 454 amplicon pyrosequencing assays targeting eukaryote small-subunit rRNA gene to demonstrate the presence of the apicomplexan type-N and a photosynthetic sister species to Apicomplexa-Chromeravelia. Amplicon pyrosequencing revealed presence of the small-subunit rRNA genes of known eukaryotic pathogens (Cryptosporidium and Cryptococcus. We therefore conducted bacterial tag encoded amplicon pyrosequencing assay for small-subunit rRNA gene to support effluent exposure of the coral. Bacteria of faecal origin (Enterobacteriales formed 41% of total sequences in contrast to 0-2% of the coral-associated bacterial communities with and without C. velia, respectively. SIGNIFICANCE: This is the first time apicomplexan type-N has been detected in the Great Barrier Reef. Eukaryote tag encoded amplicon pyrosequencing assays demonstrate presence of apicomplexan type-N and C. Velia in total coral DNA. The data highlight the need for combined approaches for eukaryotic diversity studies coupled with bacterial community assessment to achieve a more realistic goals of defining the holobiont community and assessing coral disease. With increasing evidence of Apicomplexa in coral reef

  1. Bacterial community in naturally fermented milk products of Arunachal Pradesh and Sikkim of India analysed by high-throughput amplicon sequencing

    OpenAIRE

    Shangpliang, H. Nakibapher Jones; Rai, Ranjita; Keisam, Santosh; Jeyaram, Kumaraswamy; Tamang, Jyoti Prakash

    2018-01-01

    Naturally fermented milk (NFM) products are popular ethnic fermented foods in Arunachal Pradesh and Sikkim states of India. The present study is the first to have documented the bacterial community in 54 samples of NFM products viz. chhurpi, churkam, dahi and gheu/mar by high-throughput Illumina amplicon sequencing. Metagenomic investigation showed that Firmicutes (Streptococcaceae, Lactobacillaceae) and Proteobacteria (Acetobacteraceae) were the two predominant members of the bacterial commu...

  2. FasL and FADD delivery by a glioma-specific and cell cycle-dependent HSV-1 amplicon virus enhanced apoptosis in primary human brain tumors

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    Lam Paula Y

    2010-10-01

    Full Text Available Abstract Background Glioblastoma multiforme is the most malignant cancer of the brain and is notoriously difficult to treat due to the highly proliferative and infiltrative nature of the cells. Herein, we explored the combination treatment of pre-established human glioma xenograft using multiple therapeutic genes whereby the gene expression is regulated by both cell-type and cell cycle-dependent transcriptional regulatory mechanism conferred by recombinant HSV-1 amplicon vectors. Results We demonstrated for the first time that Ki67-positive proliferating primary human glioma cells cultured from biopsy samples were effectively induced into cell death by the dual-specific function of the pG8-FasL amplicon vectors. These vectors were relatively stable and exhibited minimal cytotoxicity in vivo. Intracranial implantation of pre-transduced glioma cells resulted in better survival outcome when compared with viral vectors inoculated one week post-implantation of tumor cells, indicating that therapeutic efficacy is dependent on the viral spread and mode of viral vectors administration. We further showed that pG8-FasL amplicon vectors are functional in the presence of commonly used treatment regimens for human brain cancer. In fact, the combined therapies of pG8-FasL and pG8-FADD in the presence of temozolomide significantly improved the survival of mice bearing intracranial high-grade gliomas. Conclusion Taken together, our results showed that the glioma-specific and cell cycle-dependent HSV-1 amplicon vector is potentially useful as an adjuvant therapy to complement the current gene therapy strategy for gliomas.

  3. Investigation of Microbial Diversity in Geothermal Hot Springs in Unkeshwar, India, Based on 16S rRNA Amplicon Metagenome Sequencing.

    Science.gov (United States)

    Mehetre, Gajanan T; Paranjpe, Aditi; Dastager, Syed G; Dharne, Mahesh S

    2016-02-25

    Microbial diversity in geothermal waters of the Unkeshwar hot springs in Maharashtra, India, was studied using 16S rRNA amplicon metagenomic sequencing. Taxonomic analysis revealed the presence of Bacteroidetes, Proteobacteria, Cyanobacteria, Actinobacteria, Archeae, and OD1 phyla. Metabolic function prediction analysis indicated a battery of biological information systems indicating rich and novel microbial diversity, with potential biotechnological applications in this niche. Copyright © 2016 Mehetre et al.

  4. Combined amplicon pyrosequencing assays reveal presence of the apicomplexan "type-N" (cf. Gemmocystis cylindrus) and Chromera velia on the Great Barrier Reef, Australia.

    Science.gov (United States)

    Slapeta, Jan; Linares, Marjorie C

    2013-01-01

    The coral is predominantly composed of the metabolically dependent coral host and the photosynthetic dinoflagellate Symbiodinium sp. The system as a whole interacts with symbiotic eukaryotes, bacteria and viruses. Gemmocystiscylindrus (cf. "type-N" symbiont) belonging to the obligatory parasitic phylum Apicomplexa (Alveolata) is ubiquitous in the Caribbean coral, but its presence in the Great Barrier Reef coral has yet to be documented. Approaches allowing identification of the healthy community from the pathogenic or saprobic organisms are needed for sustainable coral reef monitoring. We investigated the diversity of eukaryotes associated with a common reef-building corals from the southern Great Barrier Reef. We used three tag encoded 454 amplicon pyrosequencing assays targeting eukaryote small-subunit rRNA gene to demonstrate the presence of the apicomplexan type-N and a photosynthetic sister species to Apicomplexa-Chromeravelia. Amplicon pyrosequencing revealed presence of the small-subunit rRNA genes of known eukaryotic pathogens (Cryptosporidium and Cryptococcus). We therefore conducted bacterial tag encoded amplicon pyrosequencing assay for small-subunit rRNA gene to support effluent exposure of the coral. Bacteria of faecal origin (Enterobacteriales) formed 41% of total sequences in contrast to 0-2% of the coral-associated bacterial communities with and without C. velia, respectively. This is the first time apicomplexan type-N has been detected in the Great Barrier Reef. Eukaryote tag encoded amplicon pyrosequencing assays demonstrate presence of apicomplexan type-N and C. Velia in total coral DNA. The data highlight the need for combined approaches for eukaryotic diversity studies coupled with bacterial community assessment to achieve a more realistic goals of defining the holobiont community and assessing coral disease. With increasing evidence of Apicomplexa in coral reef environments, it is important not only to understand the evolution of these

  5. A One-Step Real-Time Multiplex PCR for Screening Y-Chromosomal Microdeletions without Downstream Amplicon Size Analysis

    Science.gov (United States)

    Kozina, Viviana; Cappallo-Obermann, Heike; Gromoll, Jörg; Spiess, Andrej-Nikolai

    2011-01-01

    Backgound Y-chromosomal microdeletions (YCMD) are one of the major genetic causes for non-obstructive azoospermia. Genetic testing for YCMD by multiplex polymerase chain reaction (PCR) is an established method for quick and robust screening of deletions in the AZF regions of the Y-chromosome. Multiplex PCRs have the advantage of including a control gene in every reaction and significantly reducing the number of reactions needed to screen the relevant genomic markers. Principal Findings The widely established “EAA/EMQN best practice guidelines for molecular diagnosis of Y-chromosomal microdeletions (2004)” were used as a basis for designing a real-time multiplex PCR system, in which the YCMD can simply be identified by their melting points. For this reason, some AZF primers were substituted by primers for regions in their genomic proximity, and the ZFX/ZFY control primer was exchanged by the AMELX/AMELY control primer. Furthermore, we substituted the classical SybrGreen I dye by the novel and high-performing DNA-binding dye EvaGreen™ and put substantial effort in titrating the primer combinations in respect to optimal melting peak separation and peak size. Significance With these changes, we were able to develop a platform-independent and robust real-time based multiplex PCR, which makes the need for amplicon identification by electrophoretic sizing expendable. By using an open-source system for real-time PCR analysis, we further demonstrate the applicability of automated melting point and YCMD detection. PMID:21887237

  6. Redefining the Human Oral Mycobiome with Improved Practices in Amplicon-based Taxonomy: Discovery of Malassezia as a Prominent Commensal

    Science.gov (United States)

    Dupuy, Amanda K.; David, Marika S.; Li, Lu; Heider, Thomas N.; Peterson, Jason D.; Montano, Elizabeth A.; Dongari-Bagtzoglou, Anna; Diaz, Patricia I.; Strausbaugh, Linda D.

    2014-01-01

    Fungi are a large, complex group, increasingly recognized as emerging threats. Their roles as modifiers of health mandate accurate portrayals of fungal communities in humans. As an entry point into the airways and gastrointestinal tract, fungi in the mouth are relevant to several biocompartments. We have revised current practices in sequence-based taxonomy assignments and employed the improvements to address the question of the fungal genera present in the healthy human mouth. The human oral mycobiome was surveyed using massively parallel, high throughput sequencing of internal transcribed spacer 1 (ITS1) amplicons from saliva following robust extraction methods. Taxonomy was assigned by comparison to a curated reference dataset, followed by filtering with an empirically determined BLAST E-value match statistic (10−42). Nomenclature corrections further refined results by conjoining redundant names for a single fungal genus. Following these curation steps, about two-thirds of the initially identified genera were eliminated. In comparison with the one similar metagenomic study and several earlier culture-based ones, our findings change the current conception of the oral mycobiome, especially with the discovery of the high prevalence and abundance of the genus Malassezia. Previously identified as an important pathogen of the skin, and recently reported as the predominant fungal genus at the nostril and backs of the head and ear, this is the first account of Malassezia in the human mouth. Findings from this study were in good agreement with others on the existence of many consensus members of the core mycobiome, and on unique patterns for individual subjects. This research offered a cautionary note about unconditional acceptance of lengthy lists of community members produced by automated assignments, provided a roadmap for enhancing the likely biological relevance of sequence-based fungal surveys, and built the foundation for understanding the role of fungi in health

  7. Nuclear species-diagnostic SNP markers mined from 454 amplicon sequencing reveal admixture genomic structure of modern citrus varieties.

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    Franck Curk

    Full Text Available Most cultivated Citrus species originated from interspecific hybridisation between four ancestral taxa (C. reticulata, C. maxima, C. medica, and C. micrantha with limited further interspecific recombination due to vegetative propagation. This evolution resulted in admixture genomes with frequent interspecific heterozygosity. Moreover, a major part of the phenotypic diversity of edible citrus results from the initial differentiation between these taxa. Deciphering the phylogenomic structure of citrus germplasm is therefore essential for an efficient utilization of citrus biodiversity in breeding schemes. The objective of this work was to develop a set of species-diagnostic single nucleotide polymorphism (SNP markers for the four Citrus ancestral taxa covering the nine chromosomes, and to use these markers to infer the phylogenomic structure of secondary species and modern cultivars. Species-diagnostic SNPs were mined from 454 amplicon sequencing of 57 gene fragments from 26 genotypes of the four basic taxa. Of the 1,053 SNPs mined from 28,507 kb sequence, 273 were found to be highly diagnostic for a single basic taxon. Species-diagnostic SNP markers (105 were used to analyse the admixture structure of varieties and rootstocks. This revealed C. maxima introgressions in most of the old and in all recent selections of mandarins, and suggested that C. reticulata × C. maxima reticulation and introgression processes were important in edible mandarin domestication. The large range of phylogenomic constitutions between C. reticulata and C. maxima revealed in mandarins, tangelos, tangors, sweet oranges, sour oranges, grapefruits, and orangelos is favourable for genetic association studies based on phylogenomic structures of the germplasm. Inferred admixture structures were in agreement with previous hypotheses regarding the origin of several secondary species and also revealed the probable origin of several acid citrus varieties. The developed species

  8. Microbial community composition and diversity via 16S rRNA gene amplicons: evaluating the illumina platform.

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    Lucas Sinclair

    Full Text Available As new sequencing technologies become cheaper and older ones disappear, laboratories switch vendors and platforms. Validating the new setups is a crucial part of conducting rigorous scientific research. Here we report on the reliability and biases of performing bacterial 16S rRNA gene amplicon paired-end sequencing on the MiSeq Illumina platform. We designed a protocol using 50 barcode pairs to run samples in parallel and coded a pipeline to process the data. Sequencing the same sediment sample in 248 replicates as well as 70 samples from alkaline soda lakes, we evaluated the performance of the method with regards to estimates of alpha and beta diversity. Using different purification and DNA quantification procedures we always found up to 5-fold differences in the yield of sequences between individually barcodes samples. Using either a one-step or a two-step PCR preparation resulted in significantly different estimates in both alpha and beta diversity. Comparing with a previous method based on 454 pyrosequencing, we found that our Illumina protocol performed in a similar manner - with the exception for evenness estimates where correspondence between the methods was low. We further quantified the data loss at every processing step eventually accumulating to 50% of the raw reads. When evaluating different OTU clustering methods, we observed a stark contrast between the results of QIIME with default settings and the more recent UPARSE algorithm when it comes to the number of OTUs generated. Still, overall trends in alpha and beta diversity corresponded highly using both clustering methods. Our procedure performed well considering the precisions of alpha and beta diversity estimates, with insignificant effects of individual barcodes. Comparative analyses suggest that 454 and Illumina sequence data can be combined if the same PCR protocol and bioinformatic workflows are used for describing patterns in richness, beta-diversity and taxonomic

  9. HeurAA: accurate and fast detection of genetic variations with a novel heuristic amplicon aligner program for next generation sequencing.

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    Lőrinc S Pongor

    Full Text Available Next generation sequencing (NGS of PCR amplicons is a standard approach to detect genetic variations in personalized medicine such as cancer diagnostics. Computer programs used in the NGS community often miss insertions and deletions (indels that constitute a large part of known human mutations. We have developed HeurAA, an open source, heuristic amplicon aligner program. We tested the program on simulated datasets as well as experimental data from multiplex sequencing of 40 amplicons in 12 oncogenes collected on a 454 Genome Sequencer from lung cancer cell lines. We found that HeurAA can accurately detect all indels, and is more than an order of magnitude faster than previous programs. HeurAA can compare reads and reference sequences up to several thousand base pairs in length, and it can evaluate data from complex mixtures containing reads of different gene-segments from different samples. HeurAA is written in C and Perl for Linux operating systems, the code and the documentation are available for research applications at http://sourceforge.net/projects/heuraa/

  10. High-throughput multiplex cpDNA resequencing clarifies the genetic diversity and genetic relationships among Brassica napus, Brassica rapa and Brassica oleracea.

    Science.gov (United States)

    Qiao, Jiangwei; Cai, Mengxian; Yan, Guixin; Wang, Nian; Li, Feng; Chen, Binyun; Gao, Guizhen; Xu, Kun; Li, Jun; Wu, Xiaoming

    2016-01-01

    Brassica napus (rapeseed) is a recent allotetraploid plant and the second most important oilseed crop worldwide. The origin of B. napus and the genetic relationships with its diploid ancestor species remain largely unresolved. Here, chloroplast DNA (cpDNA) from 488 B. napus accessions of global origin, 139 B. rapa accessions and 49 B. oleracea accessions were populationally resequenced using Illumina Solexa sequencing technologies. The intraspecific cpDNA variants and their allelic frequencies were called genomewide and further validated via EcoTILLING analyses of the rpo region. The cpDNA of the current global B. napus population comprises more than 400 variants (SNPs and short InDels) and maintains one predominant haplotype (Bncp1). Whole-genome resequencing of the cpDNA of Bncp1 haplotype eliminated its direct inheritance from any accession of the B. rapa or B. oleracea species. The distribution of the polymorphism information content (PIC) values for each variant demonstrated that B. napus has much lower cpDNA diversity than B. rapa; however, a vast majority of the wild and cultivated B. oleracea specimens appeared to share one same distinct cpDNA haplotype, in contrast to its wild C-genome relatives. This finding suggests that the cpDNA of the three Brassica species is well differentiated. The predominant B. napus cpDNA haplotype may have originated from uninvestigated relatives or from interactions between cpDNA mutations and natural/artificial selection during speciation and evolution. These exhaustive data on variation in cpDNA would provide fundamental data for research on cpDNA and chloroplasts. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  11. A Genome Resequencing-Based Genetic Map Reveals the Recombination Landscape of an Outbred Parasitic Nematode in the Presence of Polyploidy and Polyandry.

    Science.gov (United States)

    Doyle, Stephen R; Laing, Roz; Bartley, David J; Britton, Collette; Chaudhry, Umer; Gilleard, John S; Holroyd, Nancy; Mable, Barbara K; Maitland, Kirsty; Morrison, Alison A; Tait, Andy; Tracey, Alan; Berriman, Matthew; Devaney, Eileen; Cotton, James A; Sargison, Neil D

    2018-02-01

    The parasitic nematode Haemonchus contortus is an economically and clinically important pathogen of small ruminants, and a model system for understanding the mechanisms and evolution of traits such as anthelmintic resistance. Anthelmintic resistance is widespread and is a major threat to the sustainability of livestock agriculture globally; however, little is known about the genome architecture and parameters such as recombination that will ultimately influence the rate at which resistance may evolve and spread. Here, we performed a genetic cross between two divergent strains of H. contortus, and subsequently used whole-genome resequencing of a female worm and her brood to identify the distribution of genome-wide variation that characterizes these strains. Using a novel bioinformatic approach to identify variants that segregate as expected in a pseudotestcross, we characterized linkage groups and estimated genetic distances between markers to generate a chromosome-scale F1 genetic map. We exploited this map to reveal the recombination landscape, the first for any helminth species, demonstrating extensive variation in recombination rate within and between chromosomes. Analyses of these data also revealed the extent of polyandry, whereby at least eight males were found to have contributed to the genetic variation of the progeny analyzed. Triploid offspring were also identified, which we hypothesize are the result of nondisjunction during female meiosis or polyspermy. These results expand our knowledge of the genetics of parasitic helminths and the unusual life-history of H. contortus, and enhance ongoing efforts to understand the genetic basis of resistance to the drugs used to control these worms and for related species that infect livestock and humans throughout the world. This study also demonstrates the feasibility of using whole-genome resequencing data to directly construct a genetic map in a single generation cross from a noninbred nonmodel organism with a

  12. Outlier-based identification of copy number variations using targeted resequencing in a small cohort of patients with Tetralogy of Fallot.

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    Vikas Bansal

    Full Text Available Copy number variations (CNVs are one of the main sources of variability in the human genome. Many CNVs are associated with various diseases including cardiovascular disease. In addition to hybridization-based methods, next-generation sequencing (NGS technologies are increasingly used for CNV discovery. However, respective computational methods applicable to NGS data are still limited. We developed a novel CNV calling method based on outlier detection applicable to small cohorts, which is of particular interest for the discovery of individual CNVs within families, de novo CNVs in trios and/or small cohorts of specific phenotypes like rare diseases. Approximately 7,000 rare diseases are currently known, which collectively affect ∼6% of the population. For our method, we applied the Dixon's Q test to detect outliers and used a Hidden Markov Model for their assessment. The method can be used for data obtained by exome and targeted resequencing. We evaluated our outlier-based method in comparison to the CNV calling tool CoNIFER using eight HapMap exome samples and subsequently applied both methods to targeted resequencing data of patients with Tetralogy of Fallot (TOF, the most common cyanotic congenital heart disease. In both the HapMap samples and the TOF cases, our method is superior to CoNIFER, such that it identifies more true positive CNVs. Called CNVs in TOF cases were validated by qPCR and HapMap CNVs were confirmed with available array-CGH data. In the TOF patients, we found four copy number gains affecting three genes, of which two are important regulators of heart development (NOTCH1, ISL1 and one is located in a region associated with cardiac malformations (PRODH at 22q11. In summary, we present a novel CNV calling method based on outlier detection, which will be of particular interest for the analysis of de novo or individual CNVs in trios or cohorts up to 30 individuals, respectively.

  13. Whole genome re-sequencing reveals genome-wide variations among parental lines of 16 mapping populations in chickpea (Cicer arietinum L.).

    Science.gov (United States)

    Thudi, Mahendar; Khan, Aamir W; Kumar, Vinay; Gaur, Pooran M; Katta, Krishnamohan; Garg, Vanika; Roorkiwal, Manish; Samineni, Srinivasan; Varshney, Rajeev K

    2016-01-27

    Chickpea (Cicer arietinum L.) is the second most important grain legume cultivated by resource poor farmers in South Asia and Sub-Saharan Africa. In order to harness the untapped genetic potential available for chickpea improvement, we re-sequenced 35 chickpea genotypes representing parental lines of 16 mapping populations segregating for abiotic (drought, heat, salinity), biotic stresses (Fusarium wilt, Ascochyta blight, Botrytis grey mould, Helicoverpa armigera) and nutritionally important (protein content) traits using whole genome re-sequencing approach. A total of 192.19 Gb data, generated on 35 genotypes of chickpea, comprising 973.13 million reads, with an average sequencing depth of ~10 X for each line. On an average 92.18 % reads from each genotype were aligned to the chickpea reference genome with 82.17 % coverage. A total of 2,058,566 unique single nucleotide polymorphisms (SNPs) and 292,588 Indels were detected while comparing with the reference chickpea genome. Highest number of SNPs were identified on the Ca4 pseudomolecule. In addition, copy number variations (CNVs) such as gene deletions and duplications were identified across the chickpea parental genotypes, which were minimum in PI 489777 (1 gene deletion) and maximum in JG 74 (1,497). A total of 164,856 line specific variations (144,888 SNPs and 19,968 Indels) with the highest percentage were identified in coding regions in ICC 1496 (21 %) followed by ICCV 97105 (12 %). Of 539 miscellaneous variations, 339, 138 and 62 were inter-chromosomal variations (CTX), intra-chromosomal variations (ITX) and inversions (INV) respectively. Genome-wide SNPs, Indels, CNVs, PAVs, and miscellaneous variations identified in different mapping populations are a valuable resource in genetic research and helpful in locating genes/genomic segments responsible for economically important traits. Further, the genome-wide variations identified in the present study can be used for developing high density SNP arrays for

  14. Experimental evolution, genetic analysis and genome re-sequencing reveal the mutation conferring artemisinin resistance in an isogenic lineage of malaria parasites

    KAUST Repository

    Hunt, Paul

    2010-09-16

    Background: Classical and quantitative linkage analyses of genetic crosses have traditionally been used to map genes of interest, such as those conferring chloroquine or quinine resistance in malaria parasites. Next-generation sequencing technologies now present the possibility of determining genome-wide genetic variation at single base-pair resolution. Here, we combine in vivo experimental evolution, a rapid genetic strategy and whole genome re-sequencing to identify the precise genetic basis of artemisinin resistance in a lineage of the rodent malaria parasite, Plasmodium chabaudi. Such genetic markers will further the investigation of resistance and its control in natural infections of the human malaria, P. falciparum.Results: A lineage of isogenic in vivo drug-selected mutant P. chabaudi parasites was investigated. By measuring the artemisinin responses of these clones, the appearance of an in vivo artemisinin resistance phenotype within the lineage was defined. The underlying genetic locus was mapped to a region of chromosome 2 by Linkage Group Selection in two different genetic crosses. Whole-genome deep coverage short-read re-sequencing (IlluminaSolexa) defined the point mutations, insertions, deletions and copy-number variations arising in the lineage. Eight point mutations arise within the mutant lineage, only one of which appears on chromosome 2. This missense mutation arises contemporaneously with artemisinin resistance and maps to a gene encoding a de-ubiquitinating enzyme.Conclusions: This integrated approach facilitates the rapid identification of mutations conferring selectable phenotypes, without prior knowledge of biological and molecular mechanisms. For malaria, this model can identify candidate genes before resistant parasites are commonly observed in natural human malaria populations. 2010 Hunt et al; licensee BioMed Central Ltd.

  15. Experimental evolution, genetic analysis and genome re-sequencing reveal the mutation conferring artemisinin resistance in an isogenic lineage of malaria parasites

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    Hunt Paul

    2010-09-01

    Full Text Available Abstract Background Classical and quantitative linkage analyses of genetic crosses have traditionally been used to map genes of interest, such as those conferring chloroquine or quinine resistance in malaria parasites. Next-generation sequencing technologies now present the possibility of determining genome-wide genetic variation at single base-pair resolution. Here, we combine in vivo experimental evolution, a rapid genetic strategy and whole genome re-sequencing to identify the precise genetic basis of artemisinin resistance in a lineage of the rodent malaria parasite, Plasmodium chabaudi. Such genetic markers will further the investigation of resistance and its control in natural infections of the human malaria, P. falciparum. Results A lineage of isogenic in vivo drug-selected mutant P. chabaudi parasites was investigated. By measuring the artemisinin responses of these clones, the appearance of an in vivo artemisinin resistance phenotype within the lineage was defined. The underlying genetic locus was mapped to a region of chromosome 2 by Linkage Group Selection in two different genetic crosses. Whole-genome deep coverage short-read re-sequencing (Illumina® Solexa defined the point mutations, insertions, deletions and copy-number variations arising in the lineage. Eight point mutations arise within the mutant lineage, only one of which appears on chromosome 2. This missense mutation arises contemporaneously with artemisinin resistance and maps to a gene encoding a de-ubiquitinating enzyme. Conclusions This integrated approach facilitates the rapid identification of mutations conferring selectable phenotypes, without prior knowledge of biological and molecular mechanisms. For malaria, this model can identify candidate genes before resistant parasites are commonly observed in natural human malaria populations.

  16. Recombinant adeno-associated virus type 2 replication and packaging is entirely supported by a herpes simplex virus type 1 amplicon expressing Rep and Cap.

    Science.gov (United States)

    Conway, J E; Zolotukhin, S; Muzyczka, N; Hayward, G S; Byrne, B J

    1997-11-01

    Recombinant adeno-associated virus (AAV) type 2 (rAAV) vectors have recently been shown to have great utility as gene transfer agents both in vitro and in vivo. One of the problems associated with the use of rAAV vectors has been the difficulty of large-scale vector production. Low-efficiency plasmid transfection of the rAAV vector and complementing AAV type 2 (AAV-2) functions (rep and cap) followed by superinfection with adenovirus has been the standard approach to rAAV production. The objectives of this study were to demonstrate the ability of a recombinant herpes simplex virus type 1 (HSV-1) amplicon expressing AAV-2 Rep and Cap to support replication and packaging of rAAV vectors. HSV-1 amplicon vectors were constructed which contain the AAV-2 rep and cap genes under control of their native promoters (p5, p19, and p40). An HSV-1 amplicon vector, HSV-RC/KOS or HSV-RC/d27, was generated by supplying helper functions with either wild-type HSV-1 (KOS strain) or the ICP27-deleted mutant of HSV-1, d27-1, respectively. Replication of the amplicon stocks is not inhibited by the presence of AAV-2 Rep proteins, which highlights important differences between HSV-1 and adenovirus replication and the mechanism of providing helper function for productive AAV infection. Coinfection of rAAV and HSV-RC/KOS resulted in the replication and amplification of rAAV genomes. Similarly, rescue and replication of rAAV genomes occurred when rAAV vector plasmids were transfected into cells followed by HSV-RC/KOS infection and when two rAAV proviral cell lines were infected with HSV-RC/KOS or HSV-RC/d27. Production of infectious rAAV by rescue from two rAAV proviral cell lines has also been achieved with HSV-RC/KOS and HSV-RC/d27. The particle titer of rAAV produced with HSV-RC/d27 is equal to that achieved by supplying rep and cap by transfection followed by adenovirus superinfection. Importantly, no detectable wild-type AAV-2 is generated with this approach. These results demonstrate

  17. Polymicrobial nature of chronic diabetic foot ulcer biofilm infections determined using bacterial tag encoded FLX amplicon pyrosequencing (bTEFAP.

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    Scot E Dowd

    Full Text Available BACKGROUND: Diabetic extremity ulcers are associated with chronic infections. Such ulcer infections are too often followed by amputation because there is little or no understanding of the ecology of such infections or how to control or eliminate this type of chronic infection. A primary impediment to the healing of chronic wounds is biofilm phenotype infections. Diabetic foot ulcers are the most common, disabling, and costly complications of diabetes. Here we seek to derive a better understanding of the polymicrobial nature of chronic diabetic extremity ulcer infections. METHODS AND FINDINGS: Using a new bacterial tag encoded FLX amplicon pyrosequencing (bTEFAP approach we have evaluated the bacterial diversity of 40 chronic diabetic foot ulcers from different patients. The most prevalent bacterial genus associated with diabetic chronic wounds was Corynebacterium spp. Findings also show that obligate anaerobes including Bacteroides, Peptoniphilus, Fingoldia, Anaerococcus, and Peptostreptococcus spp. are ubiquitous in diabetic ulcers, comprising a significant portion of the wound biofilm communities. Other major components of the bacterial communities included commonly cultured genera such as Streptococcus, Serratia, Staphylococcus and Enterococcus spp. CONCLUSIONS: In this article, we highlight the patterns of population diversity observed in the samples and introduce preliminary evidence to support the concept of functional equivalent pathogroups (FEP. Here we introduce FEP as consortia of genotypically distinct bacteria that symbiotically produce a pathogenic community. According to this hypothesis, individual members of these communities when they occur alone may not cause disease but when they coaggregate or consort together into a FEP the synergistic effect provides the functional equivalence of well-known pathogens, such as Staphylococcus aureus, giving the biofilm community the factors necessary to maintain chronic biofilm infections

  18. RNA-Based Amplicon Sequencing Reveals Microbiota Development during Ripening of Artisanal versus Industrial Lard d'Arnad.

    Science.gov (United States)

    Ferrocino, Ilario; Bellio, Alberto; Romano, Angelo; Macori, Guerrino; Rantsiou, Kalliopi; Decastelli, Lucia; Cocolin, Luca

    2017-08-15

    Valle d'Aosta Lard d'Arnad is a protected designation of origin (PDO) product produced from fat of the shoulder and back of heavy pigs. Its manufacturing process can be very diverse, especially regarding the maturation temperature and the NaCl concentration used for the brine; thereby, the main goal of this study was to investigate the impact of those parameters on the microbiota developed during curing and ripening. Three farms producing Lard d'Arnad were selected. Two plants, reflecting the industrial process characterized either by low maturation temperature (plant A [10% NaCl, 2°C]) or by using a low NaCl concentration (plant B [2.5% NaCl, 4°C]), were selected, while the third was characterized by an artisanal process (plant C [30% NaCl, 8°C]). Lard samples were obtained at time 0 and after 7, 15, 30, 60, and 90 days of maturation. From each plant, 3 independent lots were analyzed. The diversity of live microbiota was evaluated by using classical plate counts and amplicon target sequencing of small subunit (SSU) rRNA. The main taxa identified by sequencing were Acinetobacter johnsonii , Psychrobacter , Staphylococcus equorum , Staphylococcus sciuri , Pseudomonas fragi , Brochothrix , Halomonas , and Vibrio , and differences in their relative abundances distinguished samples from the individual plants. The composition of the microbiota was more similar among plants A and B, and it was characterized by the higher presence of taxa recognized as undesired bacteria in food-processing environments. Oligotype analysis of Halomonas and Acinetobacter revealed the presence of several characteristic oligotypes associated with A and B samples. IMPORTANCE Changes in the food production process can drastically affect the microbial community structure, with a possible impact on the final characteristics of the products. The industrial processes of Lard d'Arnad production are characterized by a reduction in the salt concentration in the brines to address a consumer demand

  19. Genome-wide SNPs and re-sequencing of growth habit and inflorescence genes in barley: implications for association mapping in germplasm arrays varying in size and structure

    Directory of Open Access Journals (Sweden)

    Muehlbauer Gary J

    2010-12-01

    Full Text Available Abstract Background Considerations in applying association mapping (AM to plant breeding are population structure and size: not accounting for structure and/or using small populations can lead to elevated false-positive rates. The principal determinants of population structure in cultivated barley are growth habit and inflorescence type. Both are under complex genetic control: growth habit is controlled by the epistatic interactions of several genes. For inflorescence type, multiple loss-of-function alleles in one gene lead to the same phenotype. We used these two traits as models for assessing the effectiveness of AM. This research was initiated using the CAP Core germplasm array (n = 102 assembled at the start of the Barley Coordinated Agricultural Project (CAP. This array was genotyped with 4,608 SNPs and we re-sequenced genes involved in morphology, growth and development. Larger arrays of breeding germplasm were subsequently genotyped and phenotyped under the auspices of the CAP project. This provided sets of 247 accessions phenotyped for growth habit and 2,473 accessions phenotyped for inflorescence type. Each of the larger populations was genotyped with 3,072 SNPs derived from the original set of 4,608. Results Significant associations with SNPs located in the vicinity of the loci involved in growth habit and inflorescence type were found in the CAP Core. Differentiation of true and spurious associations was not possible without a priori knowledge of the candidate genes, based on re-sequencing. The re-sequencing data were used to define allele types of the determinant genes based on functional polymorphisms. In a second round of association mapping, these synthetic markers based on allele types gave the most significant associations. When the synthetic markers were used as anchor points for analysis of interactions, we detected other known-function genes and candidate loci involved in the control of growth habit and inflorescence type. We

  20. Detection of double-stranded PCR amplicons at the attomole level electrosprayed from low nanomolar solutions using FT-ICR mass spectrometry.

    Science.gov (United States)

    Hannis, J C; Muddiman, D C

    2001-02-01

    An 82-base-pair polymerase chain reaction (PCR) product was amplified from the tetranucleotide short tandem repeat locus within the human tyrosine hydroxylase gene. PCR amplification was carried out using 100 ng of human nuclear DNA obtained from an individual who is homozygotic for the 9.3 allele resulting in a 50.5 kDa amplicon. To generate sufficient material for these investigations, several reactions were pooled and subsequently purified and quantified using UV-vis spectrophotometry. A serial dilution was carried out from a 2 microM stock solution providing solution concentrations down to 5 nM. Measurements were made using hexapole accumulation and gated trapping strategies in a 4.7 Telsa Fourier transform ion cyclotron resonance mass spectrometer (FTICR-MS) which facilitated detection of the amplicon at the attomole level when electrosprayed from a 5 nM solution with a single acquisition! The signal-to-noise ratio was determined to be 8.3 for the spectrum derived from the 5 nM solution using the magnitude-mode mass spectral peak height for the most abundant charge-state. This remarkable sensitivity for large PCR amplicons will dramatically improve the ability of electrospray ionization mass spectrometry to address important genetic questions for low copy number genes or when the amount of initial template is limited; the latter issue is commonly encountered in DNA forensics. Furthermore, these data represents over 2 orders of magnitude decrease in detection limits over other existing ESI-MS reports concerning PCR products, including those conducted using FTICR-MS.

  1. Microbial diversity of a full-scale UASB reactor applied to poultry slaughterhouse wastewater treatment: integration of 16S rRNA gene amplicon and shotgun metagenomic sequencing.

    Science.gov (United States)

    Delforno, Tiago Palladino; Lacerda Júnior, Gileno Vieira; Noronha, Melline F; Sakamoto, Isabel K; Varesche, Maria Bernadete A; Oliveira, Valéria M

    2017-06-01

    The 16S rRNA gene amplicon and whole-genome shotgun metagenomic (WGSM) sequencing approaches were used to investigate wide-spectrum profiles of microbial composition and metabolic diversity from a full-scale UASB reactor applied to poultry slaughterhouse wastewater treatment. The data were generated by using MiSeq 2 × 250 bp and HiSeq 2 × 150 bp Illumina sequencing platforms for 16S amplicon and WGSM sequencing, respectively. Each approach revealed a distinct microbial community profile, with Pseudomonas and Psychrobacter as predominant genus for the WGSM dataset and Clostridium and Methanosaeta for the 16S rRNA gene amplicon dataset. The virome characterization revealed the presence of two viral families with Bacteria and Archaea as host, Myoviridae, and Siphoviridae. A wide functional diversity was found with predominance of genes involved in the metabolism of acetone, butanol, and ethanol synthesis; and one-carbon metabolism (e.g., methanogenesis). Genes related to the acetotrophic methanogenesis pathways were more abundant than methylotrophic and hydrogenotrophic, corroborating the taxonomic results that showed the prevalence of the acetotrophic genus Methanosaeta. Moreover, the dataset indicated a variety of metabolic genes involved in sulfur, nitrogen, iron, and phosphorus cycles, with many genera able to act in all cycles. BLAST analysis against Antibiotic Resistance Genes Database (ARDB) revealed that microbial community contained 43 different types of antibiotic resistance genes, some of them were associated with growth chicken promotion (e.g., bacitracin, tetracycline, and polymyxin). © 2017 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

  2. Assessment of Capture and Amplicon-Based Approaches for the Development of a Targeted Next-Generation Sequencing Pipeline to Personalize Lymphoma Management.

    Science.gov (United States)

    Hung, Stacy S; Meissner, Barbara; Chavez, Elizabeth A; Ben-Neriah, Susana; Ennishi, Daisuke; Jones, Martin R; Shulha, Hennady P; Chan, Fong Chun; Boyle, Merrill; Kridel, Robert; Gascoyne, Randy D; Mungall, Andrew J; Marra, Marco A; Scott, David W; Connors, Joseph M; Steidl, Christian

    2018-03-01

    Targeted next-generation sequencing panels are increasingly used to assess the value of gene mutations for clinical diagnostic purposes. For assay development, amplicon-based methods have been preferentially used on the basis of short preparation time and small DNA input amounts. However, capture sequencing has emerged as an alternative approach because of high testing accuracy. We compared capture hybridization and amplicon sequencing approaches using fresh-frozen and formalin-fixed, paraffin-embedded tumor samples from eight lymphoma patients. Next, we developed a targeted sequencing pipeline using a 32-gene panel for accurate detection of actionable mutations in formalin-fixed, paraffin-embedded tumor samples of the most common lymphocytic malignancies: chronic lymphocytic leukemia, diffuse large B-cell lymphoma, and follicular lymphoma. We show that hybrid capture is superior to amplicon sequencing by providing deep more uniform coverage and yielding higher sensitivity for variant calling. Sanger sequencing of 588 variants identified specificity limits of thresholds for mutation calling, and orthogonal validation on 66 cases indicated 93% concordance with whole-genome sequencing. The developed pipeline and assay identified at least one actionable mutation in 91% of tumors from 219 lymphoma patients and revealed subtype-specific mutation patterns and frequencies consistent with the literature. This pipeline is an accurate and sensitive method for identifying actionable gene mutations in routinely acquired biopsy materials, suggesting further assessment of capture-based assays in the context of personalized lymphoma management. Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

  3. Illumina MiSeq 16S amplicon sequence analysis of bovine respiratory disease associated bacteria in lung and mediastinal lymph node tissue.

    Science.gov (United States)

    Johnston, Dayle; Earley, Bernadette; Cormican, Paul; Murray, Gerard; Kenny, David Anthony; Waters, Sinead Mary; McGee, Mark; Kelly, Alan Kieran; McCabe, Matthew Sean

    2017-05-02

    Bovine respiratory disease (BRD) is caused by growth of single or multiple species of pathogenic bacteria in lung tissue following stress and/or viral infection. Next generation sequencing of 16S ribosomal RNA gene PCR amplicons (NGS 16S amplicon analysis) is a powerful culture-independent open reference method that has recently been used to increase understanding of BRD-associated bacteria in the upper respiratory tract of BRD cattle. However, it has not yet been used to examine the microbiome of the bovine lower respiratory tract. The objective of this study was to use NGS 16S amplicon analysis to identify bacteria in post-mortem lung and lymph node tissue samples harvested from fatal BRD cases and clinically healthy animals. Cranial lobe and corresponding mediastinal lymph node post-mortem tissue samples were collected from calves diagnosed as BRD cases by veterinary laboratory pathologists and from clinically healthy calves. NGS 16S amplicon libraries, targeting the V3-V4 region of the bacterial 16S rRNA gene were prepared and sequenced on an Illumina MiSeq. Quantitative insights into microbial ecology (QIIME) was used to determine operational taxonomic units (OTUs) which corresponded to the 16S rRNA gene sequences. Leptotrichiaceae, Mycoplasma, Pasteurellaceae, and Fusobacterium were the most abundant OTUs identified in the lungs and lymph nodes of the calves which died from BRD. Leptotrichiaceae, Fusobacterium, Mycoplasma, Trueperella and Bacteroides had greater relative abundances in post-mortem lung samples collected from fatal cases of BRD in dairy calves, compared with clinically healthy calves without lung lesions. Leptotrichiaceae, Mycoplasma and Pasteurellaceae showed higher relative abundances in post-mortem lymph node samples collected from fatal cases of BRD in dairy calves, compared with clinically healthy calves without lung lesions. Two Leptotrichiaceae sequence contigs were subsequently assembled from bacterial DNA-enriched shotgun sequences

  4. Population-based resequencing analysis of wild and cultivated barley revealed weak domestication signal of selection and bottleneck in the Rrs2 scald resistance gene region.

    Science.gov (United States)

    Fu, Yong-Bi

    2012-02-01

    Many plant disease resistance (R) genes have been cloned, but the potential of utilizing these plant R-gene genomic resources for genetic inferences of plant domestication history remains unexplored. A population-based resequencing analysis of the genomic region near the Rrs2 scald resistance gene was made in 51 accessions of wild and cultivated barley from 41 countries. Fifteen primer pairs were designed to sample the genomic region with a total length of 10 406 bp. More nucleotide diversity was found in wild (π = 0.01846) than cultivated (π = 0.01507) barley samples. Three distinct groups of 29 haplotypes were detected for all 51 samples, and they were well mixed with wild and cultivated barley samples from different countries and regions. The neutrality tests by Tajima's D were not significant, but a significant (P events was 16 in wild barley and 19 in cultivated barley. A coalescence simulation revealed a bottleneck intensity of 1.5 to 2 since barley domestication. Together, the domestication signal in the genomic region was weak both in human selection and domestication bottleneck.

  5. Development of a new resequencing pathogen microarray based assay for detection of broad-spectrum respiratory tract viruses in patients with community-acquired pneumonia.

    Directory of Open Access Journals (Sweden)

    Hongwei Shen

    Full Text Available A Resequencing Pathogen Microarray (RPM is a single, highly multiplexed assay for detecting and differentiating similarly related pathogens by using closely overlapping probe sets to determine a target organism's nucleotide sequence. In this study, a new RPM (RPM-IVDC1 that consisted of 224-bp detector tiles corresponding to 9 influenza A subtypes, 11 rhinoviruses, 28 enteroviruses and 38 other respiratory viruses was developed and optimized to provide individual and simultaneous detection sensitivities ranging from 15 to 750 genomic copies for 16 common respiratory pathogens. A total of 110 consecutive patients with community-acquired pneumonia (CAP admitted to 5 district general hospitals in Beijing during a 1-year period were assessed using the new assay. Among the children (under age 5 and adult patients (above age 18, respiratory syncytial virus (RSV and rhinovirus (RV were the most common etiological agents, respectively, which is consistent with reference assays. Atypical pathogens that may cause CAP-like illness, including rubella virus, measles virus, influenza type C virus, human herpesvirus (HHV were also detected. The results show the capability of RPM-IVDC1 for the accurate detection and identification of multiple virus types, which may be of significant use in epidemic surveillance and outbreak investigations of atypical pathogens.

  6. Forward genetics screen coupled with whole-genome resequencing identifies novel gene targets for improving heterologous enzyme production in Aspergillus niger

    Energy Technology Data Exchange (ETDEWEB)

    Reilly, Morgann C. [Joint BioEnergy Institute, Emeryville, CA (United States); Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Kim, Joonhoon [Joint BioEnergy Institute, Emeryville, CA (United States); Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Lynn, Jed [Joint BioEnergy Institute, Emeryville, CA (United States); Wright-Patterson Air Force Base, Dayton, OH (United States); Simmons, Blake A. [Joint BioEnergy Institute, Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Gladden, John M. [Joint BioEnergy Institute, Emeryville, CA (United States); Sandia National Lab. (SNL-CA), Livermore, CA (United States); Magnuson, Jon K. [Joint BioEnergy Institute, Emeryville, CA (United States); Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Baker, Scott E. [Joint BioEnergy Institute, Emeryville, CA (United States); Pacific Northwest National Lab. (PNNL), Richland, WA (United States)

    2018-01-06

    Plant biomass, once reduced to its composite sugars, can be converted to fuel substitutes. One means of overcoming the recalcitrance of lignocellulose is pretreatment followed by enzymatic hydrolysis. However, currently available commercial enzyme cocktails are inhibited in the presence of residual pretreatment chemicals. Recent studies have identified a number of cellulolytic enzymes from bacteria that are tolerant to pretreatment chemicals such as ionic liquids. The challenge now is generation of these enzymes in copious amounts, an arena where fungal organisms such as Aspergillus niger have proven efficient. Fungal host strains still need to be engineered to increase production titers of heterologous protein over native enzymes, which has been a difficult task. Here, we developed a forward genetics screen coupled with whole-genome resequencing to identify specific lesions responsible for a protein hyper-production phenotype in A. niger. This strategy successfully identified novel targets, including a low-affinity glucose transporter, MstC, whose deletion significantly improved secretion of recombinant proteins driven by a glucoamylase promoter.

  7. Footprints of domestication revealed by RAD-tag resequencing in loquat: SNP data reveals a non-significant domestication bottleneck and a single domestication event.

    Science.gov (United States)

    Wang, Yunsheng; Shahid, Muhammad Qasim; Lin, Shunquan; Chen, Chengjie; Hu, Chen

    2017-05-06

    The process of crop domestication has long been a major area of research to gain insights into the history of human civilization and to understand the process of evolution. Loquat (Eriobotrya japonica Lindl.) is one of the typical subtropical fruit trees, which was domesticated in China at least 2000 years ago. In the present study, we re-sequenced the genome of nine wild loquat accessions collected from wide geographical range and 10 representative cultivated loquat cultivars by using RAD-tag tacit to exploit the molecular footprints of domestication. We obtained 26.4 Gb clean sequencing data from 19 loquat accessions, with an average of 32.64 M reads per genotype. We identified more than 80,000 SNPs distributed throughout the loquat genome. The SNP density and numbers were slightly higher in the wild loquat populations than that in the cultivated populations. All cultivars were clustered together by structure, phylogenetic and PCA analyses. The modern loquat cultivars have experienced a non-significant genetic bottleneck during domestication, and originated from a single domesticated event. Moreover, our study revealed that Hubei province of China is probably the origin center of cultivated loquat.

  8. Forward genetics screen coupled with whole-genome resequencing identifies novel gene targets for improving heterologous enzyme production in Aspergillus niger.

    Science.gov (United States)

    Reilly, Morgann C; Kim, Joonhoon; Lynn, Jed; Simmons, Blake A; Gladden, John M; Magnuson, Jon K; Baker, Scott E

    2018-02-01

    Plant biomass, once reduced to its composite sugars, can be converted to fuel substitutes. One means of overcoming the recalcitrance of lignocellulose is pretreatment followed by enzymatic hydrolysis. However, currently available commercial enzyme cocktails are inhibited in the presence of residual pretreatment chemicals. Recent studies have identified a number of cellulolytic enzymes from bacteria that are tolerant to pretreatment chemicals such as ionic liquids. The challenge now is generation of these enzymes in copious amounts, an arena where fungal organisms such as Aspergillus niger have proven efficient. Fungal host strains still need to be engineered to increase production titers of heterologous protein over native enzymes, which has been a difficult task. Here, we developed a forward genetics screen coupled with whole-genome resequencing to identify specific lesions responsible for a protein hyper-production phenotype in A. niger. This strategy successfully identified novel targets, including a low-affinity glucose transporter, MstC, whose deletion significantly improved secretion of recombinant proteins driven by a glucoamylase promoter.

  9. Conservative fragments in bacterial 16S rRNA genes and primer design for 16S ribosomal DNA amplicons in metagenomic studies.

    Directory of Open Access Journals (Sweden)

    Yong Wang

    Full Text Available Bacterial 16S ribosomal DNA (rDNA amplicons have been widely used in the classification of uncultured bacteria inhabiting environmental niches. Primers targeting conservative regions of the rDNAs are used to generate amplicons of variant regions that are informative in taxonomic assignment. One problem is that the percentage coverage and application scope of the primers used in previous studies are largely unknown. In this study, conservative fragments of available rDNA sequences were first mined and then used to search for candidate primers within the fragments by measuring the coverage rate defined as the percentage of bacterial sequences containing the target. Thirty predicted primers with a high coverage rate (>90% were identified, which were basically located in the same conservative regions as known primers in previous reports, whereas 30% of the known primers were associated with a coverage rate of <90%. The application scope of the primers was also examined by calculating the percentages of failed detections in bacterial phyla. Primers A519-539, E969-983, E1063-1081, U515 and E517, are highly recommended because of their high coverage in almost all phyla. As expected, the three predominant phyla, Firmicutes, Gemmatimonadetes and Proteobacteria, are best covered by the predicted primers. The primers recommended in this report shall facilitate a comprehensive and reliable survey of bacterial diversity in metagenomic studies.

  10. Cowpea mosaic virus RNA-1 acts as an amplicon whose effects can be counteracted by a RNA-2-encoded suppressor of silencing

    International Nuclear Information System (INIS)

    Liu Li; Grainger, Jef; Canizares, M. Carmen; Angell, Susan M.; Lomonossoff, George P.

    2004-01-01

    Lines of Nicotiana benthamiana transgenic for full-length copies of both Cowpea mosaic virus (CPMV) genomic RNAs, either singly or together, have been produced. Plants transgenic for both RNAs developed symptoms characteristic of a CPMV infection. When plants transgenic for RNA-1 were agro-inoculated with RNA-2, no infection developed and the plants were also resistant to challenge with CPMV. By contrast, plants transgenic for RNA-2 became infected when agro-inoculated with RNA-1 and were fully susceptible to CPMV infection. The resistance of RNA-1 transgenic plants was shown to be related to the ability of RNA-1 to self-replicate and act as an amplicon. The ability of transgenically expressed RNA-2 to counteract the amplicon effect suggested that it encodes a suppressor of posttranscriptional gene silencing (PTGS). By examining the ability of portions of RNA-2 to reverse PTGS in N. benthamiana, we have identified the small (S) coat protein as the CPMV RNA-2-encoded suppressor of PTGS

  11. Conservative fragments in bacterial 16S rRNA genes and primer design for 16S ribosomal DNA amplicons in metagenomic studies

    KAUST Repository

    Wang, Yong

    2009-10-09

    Bacterial 16S ribosomal DNA (rDNA) amplicons have been widely used in the classification of uncultured bacteria inhabiting environmental niches. Primers targeting conservative regions of the rDNAs are used to generate amplicons of variant regions that are informative in taxonomic assignment. One problem is that the percentage coverage and application scope of the primers used in previous studies are largely unknown. In this study, conservative fragments of available rDNA sequences were first mined and then used to search for candidate primers within the fragments by measuring the coverage rate defined as the percentage of bacterial sequences containing the target. Thirty predicted primers with a high coverage rate (>90%) were identified, which were basically located in the same conservative regions as known primers in previous reports, whereas 30% of the known primers were associated with a coverage rate of <90%. The application scope of the primers was also examined by calculating the percentages of failed detections in bacterial phyla. Primers A519-539, E969- 983, E1063-1081, U515 and E517, are highly recommended because of their high coverage in almost all phyla. As expected, the three predominant phyla, Firmicutes, Gemmatimonadetes and Proteobacteria, are best covered by the predicted primers. The primers recommended in this report shall facilitate a comprehensive and reliable survey of bacterial diversity in metagenomic studies. © 2009 Wang, Qian.

  12. Single nucleotide variants and InDels identified from whole-genome re-sequencing of Guzerat, Gyr, Girolando and Holstein cattle breeds.

    Science.gov (United States)

    Stafuzza, Nedenia Bonvino; Zerlotini, Adhemar; Lobo, Francisco Pereira; Yamagishi, Michel Eduardo Beleza; Chud, Tatiane Cristina Seleguim; Caetano, Alexandre Rodrigues; Munari, Danísio Prado; Garrick, Dorian J; Machado, Marco Antonio; Martins, Marta Fonseca; Carvalho, Maria Raquel; Cole, John Bruce; Barbosa da Silva, Marcos Vinicius Gualberto

    2017-01-01

    Whole-genome re-sequencing, alignment and annotation analyses were undertaken for 12 sires representing four important cattle breeds in Brazil: Guzerat (multi-purpose), Gyr, Girolando and Holstein (dairy production). A total of approximately 4.3 billion reads from an Illumina HiSeq 2000 sequencer generated for each animal 10.7 to 16.4-fold genome coverage. A total of 27,441,279 single nucleotide variations (SNVs) and 3,828,041 insertions/deletions (InDels) were detected in the samples, of which 2,557,670 SNVs and 883,219 InDels were novel. The submission of these genetic variants to the dbSNP database significantly increased the number of known variants, particularly for the indicine genome. The concordance rate between genotypes obtained using the Bovine HD BeadChip array and the same variants identified by sequencing was about 99.05%. The annotation of variants identified numerous non-synonymous SNVs and frameshift InDels which could affect phenotypic variation. Functional enrichment analysis was performed and revealed that variants in the olfactory transduction pathway was over represented in all four cattle breeds, while the ECM-receptor interaction pathway was over represented in Girolando and Guzerat breeds, the ABC transporters pathway was over represented only in Holstein breed, and the metabolic pathways was over represented only in Gyr breed. The genetic variants discovered here provide a rich resource to help identify potential genomic markers and their associated molecular mechanisms that impact economically important traits for Gyr, Girolando, Guzerat and Holstein breeding programs.

  13. Single nucleotide variants and InDels identified from whole-genome re-sequencing of Guzerat, Gyr, Girolando and Holstein cattle breeds.

    Directory of Open Access Journals (Sweden)

    Nedenia Bonvino Stafuzza

    Full Text Available Whole-genome re-sequencing, alignment and annotation analyses were undertaken for 12 sires representing four important cattle breeds in Brazil: Guzerat (multi-purpose, Gyr, Girolando and Holstein (dairy production. A total of approximately 4.3 billion reads from an Illumina HiSeq 2000 sequencer generated for each animal 10.7 to 16.4-fold genome coverage. A total of 27,441,279 single nucleotide variations (SNVs and 3,828,041 insertions/deletions (InDels were detected in the samples, of which 2,557,670 SNVs and 883,219 InDels were novel. The submission of these genetic variants to the dbSNP database significantly increased the number of known variants, particularly for the indicine genome. The concordance rate between genotypes obtained using the Bovine HD BeadChip array and the same variants identified by sequencing was about 99.05%. The annotation of variants identified numerous non-synonymous SNVs and frameshift InDels which could affect phenotypic variation. Functional enrichment analysis was performed and revealed that variants in the olfactory transduction pathway was over represented in all four cattle breeds, while the ECM-receptor interaction pathway was over represented in Girolando and Guzerat breeds, the ABC transporters pathway was over represented only in Holstein breed, and the metabolic pathways was over represented only in Gyr breed. The genetic variants discovered here provide a rich resource to help identify potential genomic markers and their associated molecular mechanisms that impact economically important traits for Gyr, Girolando, Guzerat and Holstein breeding programs.

  14. Initial association of NR2E1 with bipolar disorder and identification of candidate mutations in bipolar disorder, schizophrenia, and aggression through resequencing.

    Science.gov (United States)

    Kumar, Ravinesh A; McGhee, Kevin A; Leach, Stephen; Bonaguro, Russell; Maclean, Alan; Aguirre-Hernandez, Rosalia; Abrahams, Brett S; Coccaro, Emil F; Hodgins, Sheilagh; Turecki, Gustavo; Condon, Anne; Muir, Walter J; Brooks-Wilson, Angela R; Blackwood, Douglas H; Simpson, Elizabeth M

    2008-09-05

    Nuclear receptor 2E1 gene (NR2E1) resides within a 6q21-22 locus for bipolar disorder and schizophrenia. Mice deleted for Nr2e1 show altered neurogenesis, cortical and limbic abnormalities, aggression, hyperexcitability, and cognitive impairment. NR2E1 is therefore a positional and functional candidate for involvement in mental illness. We performed association analyses in 394 patients with bipolar disorder, 396 with schizophrenia, and 479 controls using six common markers and haplotypes. We also performed a comprehensive mutation screen of NR2E1, resequencing its entire coding region, complete 5' and 3' untranslated regions, consensus splice-sites, and evolutionarily conserved regions in 126 humans with bipolar disorder, schizophrenia, or aggressive disorders. NR2E1 was associated with bipolar disorder I and II [odds ratio (OR = 0.77, P = 0.013), bipolar disorder I (OR = 0.77, P = 0.015), bipolar disorder in females (OR = 0.72, P = 0.009), and with age at onset < or = 25 years (OR = 0.67, P = 0.006)], all of which remained significant after correcting for multiple comparisons. We identified eight novel candidate mutations that were absent in 325 controls; four of these were predicted to alter known neural transcription factor binding sites. Analyses of NR2E1 mRNA in human brain revealed forebrain-specific transcription. The data presented support the hypothesis that genetic variation at NR2E1 may be associated with susceptibility to brain-behavior disorders. 2008 Wiley-Liss, Inc.

  15. Diversity and Genome Analysis of Australian and Global Oilseed Brassica napus L. Germplasm Using Transcriptomics and Whole Genome Re-sequencing

    Directory of Open Access Journals (Sweden)

    M. Michelle Malmberg

    2018-04-01

    Full Text Available Intensive breeding of Brassica napus has resulted in relatively low diversity, such that B. napus would benefit from germplasm improvement schemes that sustain diversity. As such, samples representative of global germplasm pools need to be assessed for existing population structure, diversity and linkage disequilibrium (LD. Complexity reduction genotyping-by-sequencing (GBS methods, including GBS-transcriptomics (GBS-t, enable cost-effective screening of a large number of samples, while whole genome re-sequencing (WGR delivers the ability to generate large numbers of unbiased genomic single nucleotide polymorphisms (SNPs, and identify structural variants (SVs. Furthermore, the development of genomic tools based on whole genomes representative of global oilseed diversity and orientated by the reference genome has substantial industry relevance and will be highly beneficial for canola breeding. As recent studies have focused on European and Chinese varieties, a global diversity panel as well as a substantial number of Australian spring types were included in this study. Focusing on industry relevance, 633 varieties were initially genotyped using GBS-t to examine population structure using 61,037 SNPs. Subsequently, 149 samples representative of global diversity were selected for WGR and both data sets used for a side-by-side evaluation of diversity and LD. The WGR data was further used to develop genomic resources consisting of a list of 4,029,750 high-confidence SNPs annotated using SnpEff, and SVs in the form of 10,976 deletions and 2,556 insertions. These resources form the basis of a reliable and repeatable system allowing greater integration between canola genomics studies, with a strong focus on breeding germplasm and industry applicability.

  16. A Pharmacogenetics Study in Mozambican Patients Treated with Nevirapine: Full Resequencing of TRAF3IP2 Gene Shows a Novel Association with SJS/TEN Susceptibility

    Directory of Open Access Journals (Sweden)

    Cinzia Ciccacci

    2015-03-01

    Full Text Available Steven–Johnson Syndrome (SJS and Toxic Epidermal Necrolysis (TEN are severe adverse drug reactions, characterized by extensive epidermal detachment and erosions of mucous membrane. SJS/TEN is one of the most serious adverse reactions to Nevirapine (NVP treatment, commonly used in developing countries as first-line treatment of human immunodeficiency virus infection. In the last years TRAF3IP2 gene variants had been described as associated with susceptibility to several diseases such as psoriasis and psoriatic arthritis. We hypothesized that this gene, involved in immune response and in NF-κB activation, could also be implicated in the SJS/TEN susceptibility. We performed a full resequencing of TRAF3IP2 gene in a population of patients treated with NVP. Twenty-seven patients with NVP-induced SJS/TEN and 78 controls, all from Mozambique, were enrolled. We identified eight exonic and three intronic already described variants. The case/control association analysis highlighted an association between the rs76228616 SNP in exon 2 and the SJS/TEN susceptibility. In particular, the variant allele (C resulted significantly associated with a higher risk to develop SJS/TEN (p = 0.012 and OR = 3.65 (95% CI 1.33–10.01. A multivariate analysis by logistic regression confirmed its significant contribution (p = 0.027, OR = 4.39 (95% CI 1.19–16.23. In conclusion, our study suggests that a variant in TRAF3IP2 gene could be involved in susceptibility to SJS/TEN.

  17. A pharmacogenetics study in Mozambican patients treated with nevirapine: full resequencing of TRAF3IP2 gene shows a novel association with SJS/TEN susceptibility.

    Science.gov (United States)

    Ciccacci, Cinzia; Rufini, Sara; Mancinelli, Sandro; Buonomo, Ersilia; Giardina, Emiliano; Scarcella, Paola; Marazzi, Maria C; Novelli, Giuseppe; Palombi, Leonardo; Borgiani, Paola

    2015-03-12

    Steven-Johnson Syndrome (SJS) and Toxic Epidermal Necrolysis (TEN) are severe adverse drug reactions, characterized by extensive epidermal detachment and erosions of mucous membrane. SJS/TEN is one of the most serious adverse reactions to Nevirapine (NVP) treatment, commonly used in developing countries as first-line treatment of human immunodeficiency virus infection. In the last years TRAF3IP2 gene variants had been described as associated with susceptibility to several diseases such as psoriasis and psoriatic arthritis. We hypothesized that this gene, involved in immune response and in NF-κB activation, could also be implicated in the SJS/TEN susceptibility. We performed a full resequencing of TRAF3IP2 gene in a population of patients treated with NVP. Twenty-seven patients with NVP-induced SJS/TEN and 78 controls, all from Mozambique, were enrolled. We identified eight exonic and three intronic already described variants. The case/control association analysis highlighted an association between the rs76228616 SNP in exon 2 and the SJS/TEN susceptibility. In particular, the variant allele (C) resulted significantly associated with a higher risk to develop SJS/TEN (p = 0.012 and OR = 3.65 (95% CI 1.33-10.01)). A multivariate analysis by logistic regression confirmed its significant contribution (p = 0.027, OR = 4.39 (95% CI 1.19-16.23)). In conclusion, our study suggests that a variant in TRAF3IP2 gene could be involved in susceptibility to SJS/TEN.

  18. Investigating Drought Tolerance in Chickpea Using Genome-Wide Association Mapping and Genomic Selection Based on Whole-Genome Resequencing Data

    Directory of Open Access Journals (Sweden)

    Yongle Li

    2018-02-01

    Full Text Available Drought tolerance is a complex trait that involves numerous genes. Identifying key causal genes or linked molecular markers can facilitate the fast development of drought tolerant varieties. Using a whole-genome resequencing approach, we sequenced 132 chickpea varieties and advanced breeding lines and found more than 144,000 single nucleotide polymorphisms (SNPs. We measured 13 yield and yield-related traits in three drought-prone environments of Western Australia. The genotypic effects were significant for all traits, and many traits showed highly significant correlations, ranging from 0.83 between grain yield and biomass to -0.67 between seed weight and seed emergence rate. To identify candidate genes, the SNP and trait data were incorporated into the SUPER genome-wide association study (GWAS model, a modified version of the linear mixed model. We found that several SNPs from auxin-related genes, including auxin efflux carrier protein (PIN3, p-glycoprotein, and nodulin MtN21/EamA-like transporter, were significantly associated with yield and yield-related traits under drought-prone environments. We identified four genetic regions containing SNPs significantly associated with several different traits, which was an indication of pleiotropic effects. We also investigated the possibility of incorporating the GWAS results into a genomic selection (GS model, which is another approach to deal with complex traits. Compared to using all SNPs, application of the GS model using subsets of SNPs significantly associated with the traits under investigation increased the prediction accuracies of three yield and yield-related traits by more than twofold. This has important implication for implementing GS in plant breeding programs.

  19. Candidate Gene Resequencing in a Large Bicuspid Aortic Valve-Associated Thoracic Aortic Aneurysm Cohort: SMAD6 as an Important Contributor.

    Science.gov (United States)

    Gillis, Elisabeth; Kumar, Ajay A; Luyckx, Ilse; Preuss, Christoph; Cannaerts, Elyssa; van de Beek, Gerarda; Wieschendorf, Björn; Alaerts, Maaike; Bolar, Nikhita; Vandeweyer, Geert; Meester, Josephina; Wünnemann, Florian; Gould, Russell A; Zhurayev, Rustam; Zerbino, Dmytro; Mohamed, Salah A; Mital, Seema; Mertens, Luc; Björck, Hanna M; Franco-Cereceda, Anders; McCallion, Andrew S; Van Laer, Lut; Verhagen, Judith M A; van de Laar, Ingrid M B H; Wessels, Marja W; Messas, Emmanuel; Goudot, Guillaume; Nemcikova, Michaela; Krebsova, Alice; Kempers, Marlies; Salemink, Simone; Duijnhouwer, Toon; Jeunemaitre, Xavier; Albuisson, Juliette; Eriksson, Per; Andelfinger, Gregor; Dietz, Harry C; Verstraeten, Aline; Loeys, Bart L

    2017-01-01

    Bicuspid aortic valve (BAV) is the most common congenital heart defect. Although many BAV patients remain asymptomatic, at least 20% develop thoracic aortic aneurysm (TAA). Historically, BAV-related TAA was considered as a hemodynamic consequence of the valve defect. Multiple lines of evidence currently suggest that genetic determinants contribute to the pathogenesis of both BAV and TAA in affected individuals. Despite high heritability, only very few genes have been linked to BAV or BAV/TAA, such as NOTCH1, SMAD6 , and MAT2A . Moreover, they only explain a minority of patients. Other candidate genes have been suggested based on the presence of BAV in knockout mouse models (e.g., GATA5, NOS3 ) or in syndromic (e.g., TGFBR1/2, TGFB2/3 ) or non-syndromic (e.g., ACTA2 ) TAA forms. We hypothesized that rare genetic variants in these genes may be enriched in patients presenting with both BAV and TAA. We performed targeted resequencing of 22 candidate genes using Haloplex target enrichment in a strictly defined BAV/TAA cohort ( n = 441; BAV in addition to an aortic root or ascendens diameter ≥ 4.0 cm in adults, or a Z-score ≥ 3 in children) and in a collection of healthy controls with normal echocardiographic evaluation ( n = 183). After additional burden analysis against the Exome Aggregation Consortium database, the strongest candidate susceptibility gene was SMAD6 ( p = 0.002), with 2.5% ( n = 11) of BAV/TAA patients harboring causal variants, including two nonsense, one in-frame deletion and two frameshift mutations. All six missense mutations were located in the functionally important MH1 and MH2 domains. In conclusion, we report a significant contribution of SMAD6 mutations to the etiology of the BAV/TAA phenotype.

  20. Investigating Drought Tolerance in Chickpea Using Genome-Wide Association Mapping and Genomic Selection Based on Whole-Genome Resequencing Data.

    Science.gov (United States)

    Li, Yongle; Ruperao, Pradeep; Batley, Jacqueline; Edwards, David; Khan, Tanveer; Colmer, Timothy D; Pang, Jiayin; Siddique, Kadambot H M; Sutton, Tim

    2018-01-01

    Drought tolerance is a complex trait that involves numerous genes. Identifying key causal genes or linked molecular markers can facilitate the fast development of drought tolerant varieties. Using a whole-genome resequencing approach, we sequenced 132 chickpea varieties and advanced breeding lines and found more than 144,000 single nucleotide polymorphisms (SNPs). We measured 13 yield and yield-related traits in three drought-prone environments of Western Australia. The genotypic effects were significant for all traits, and many traits showed highly significant correlations, ranging from 0.83 between grain yield and biomass to -0.67 between seed weight and seed emergence rate. To identify candidate genes, the SNP and trait data were incorporated into the SUPER genome-wide association study (GWAS) model, a modified version of the linear mixed model. We found that several SNPs from auxin-related genes, including auxin efflux carrier protein (PIN3), p-glycoprotein, and nodulin MtN21/EamA-like transporter, were significantly associated with yield and yield-related traits under drought-prone environments. We identified four genetic regions containing SNPs significantly associated with several different traits, which was an indication of pleiotropic effects. We also investigated the possibility of incorporating the GWAS results into a genomic selection (GS) model, which is another approach to deal with complex traits. Compared to using all SNPs, application of the GS model using subsets of SNPs significantly associated with the traits under investigation increased the prediction accuracies of three yield and yield-related traits by more than twofold. This has important implication for implementing GS in plant breeding programs.

  1. Bacterial tag encoded FLX titanium amplicon pyrosequencing (bTEFAP based assessment of prokaryotic diversity in metagenome of Lonar soda lake, India

    Directory of Open Access Journals (Sweden)

    Pravin Dudhagara

    2015-06-01

    Full Text Available Bacterial diversity and archaeal diversity in metagenome of the Lonar soda lake sediment were assessed by bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP. Metagenome comprised 5093 sequences with 2,531,282 bp and 53 ± 2% G + C content. Metagenome sequence data are available at NCBI under the Bioproject database with accession no. PRJNA218849. Metagenome sequence represented the presence of 83.1% bacterial and 10.5% archaeal origin. A total of 14 different bacteria demonstrating 57 species were recorded with dominating species like Coxiella burnetii (17%, Fibrobacter intestinalis (12% and Candidatus Cloacamonas acidaminovorans (11%. Occurrence of two archaeal phyla representing 24 species, among them Methanosaeta harundinacea (35%, Methanoculleus chikugoensis (12% and Methanolinea tarda (11% were dominating species. Significant presence of 11% sequences as an unclassified indicated the possibilities for unknown novel prokaryotes from the metagenome.

  2. Large-scale benchmarking reveals false discoveries and count transformation sensitivity in 16S rRNA gene amplicon data analysis methods used in microbiome studies

    DEFF Research Database (Denmark)

    Thorsen, Jonathan; Brejnrod, Asker Daniel; Mortensen, Martin Steen

    2016-01-01

    detection power. For beta-diversity-based sample separation, we show that library size normalization has very little effect and that the distance metric is the most important factor in terms of separation power. CONCLUSIONS: Our results, generalizable to datasets from different sequencing platforms......, demonstrate how the choice of method considerably affects analysis outcome. Here, we give recommendations for tools that exhibit low false positive rates, have good retrieval power across effect sizes and case/control proportions, and have low sparsity bias. Result output from some commonly used methods......BACKGROUND: There is an immense scientific interest in the human microbiome and its effects on human physiology, health, and disease. A common approach for examining bacterial communities is high-throughput sequencing of 16S rRNA gene hypervariable regions, aggregating sequence-similar amplicons...

  3. Simplified strategy for rapid first-line screening of fragile X syndrome: closed-tube triplet-primed PCR and amplicon melt peak analysis.

    Science.gov (United States)

    Rajan-Babu, Indhu-Shree; Law, Hai-Yang; Yoon, Chui-Sheun; Lee, Caroline G; Chong, Samuel S

    2015-05-04

    Premutation and full-mutation hyperexpansion of CGG-triplets in the X-linked Fragile X Mental Retardation 1 (FMR1) gene have been implicated in fragile X-associated tremor/ataxia syndrome, fragile X-associated primary ovarian insufficiency, and fragile X syndrome (FXS), respectively. The currently available molecular diagnostic tests are either costly or labour-intensive, which prohibits their application as a first-line FMR1 test in large-scale population-based screening programs. In this study, we demonstrate the utility of a simplified closed-tube strategy for rapid first-line screening of FXS based on melt peak temperature (Tm) analysis of direct triplet-primed polymerase chain reaction amplicons (dTP-PCR MCA). In addition, we also evaluated the correlation between Tm and CGG-repeat size based on capillary electrophoresis (CE) of dTP-PCR amplicons. The assays were initially tested on 29 FMR1 reference DNA samples, followed by a blinded validation on 107 previously characterised patient DNA samples. The dTP-PCR MCA produced distinct melt profiles of higher Tm for samples carrying an expanded allele. Among the samples tested, we also observed a good correlation between Tm and CGG-repeat size. In the blinded validation study, dTP-PCR MCA accurately classified all normal and expansion carriers, and the FMR1 genotypic classification of all samples was completely concordant with the previously determined genotypes as well as the dTP-PCR CE results. This simple and cost-effective MCA-based assay may be useful as a first-line FXS screening tool that could rapidly screen out the large majority of unaffected individuals, thus minimising the number of samples that need to be analysed by Southern blot analysis.

  4. Identification of novel SNPs of ABCD1, ABCD2, ABCD3, and ABCD4 genes in patients with X-linked adrenoleukodystrophy (ALD) based on comprehensive resequencing and association studies with ALD phenotypes.

    Science.gov (United States)

    Matsukawa, Takashi; Asheuer, Muriel; Takahashi, Yuji; Goto, Jun; Suzuki, Yasuyuki; Shimozawa, Nobuyuki; Takano, Hiroki; Onodera, Osamu; Nishizawa, Masatoyo; Aubourg, Patrick; Tsuji, Shoji

    2011-02-01

    Adrenoleukodystrophy (ALD) is an X-linked disorder affecting primarily the white matter of the central nervous system occasionally accompanied by adrenal insufficiency. Despite the discovery of the causative gene, ABCD1, no clear genotype-phenotype correlations have been established. Association studies based on single nucleotide polymorphisms (SNPs) identified by comprehensive resequencing of genes related to ABCD1 may reveal genes modifying ALD phenotypes. We analyzed 40 Japanese patients with ALD. ABCD1 and ABCD2 were analyzed using a newly developed microarray-based resequencing system. ABCD3 and ABCD4 were analyzed by direct nucleotide sequence analysis. Replication studies were conducted on an independent French ALD cohort with extreme phenotypes. All the mutations of ABCD1 were identified, and there was no correlation between the genotypes and phenotypes of ALD. SNPs identified by the comprehensive resequencing of ABCD2, ABCD3, and ABCD4 were used for association studies. There were no significant associations between these SNPs and ALD phenotypes, except for the five SNPs of ABCD4, which are in complete disequilibrium in the Japanese population. These five SNPs were significantly less frequently represented in patients with adrenomyeloneuropathy (AMN) than in controls in the Japanese population (p=0.0468), whereas there were no significant differences in patients with childhood cerebral ALD (CCALD). The replication study employing these five SNPs on an independent French ALD cohort, however, showed no significant associations with CCALD or pure AMN. This study showed that ABCD2, ABCD3, and ABCD4 are less likely the disease-modifying genes, necessitating further studies to identify genes modifying ALD phenotypes.

  5. Identification of novel SNPs of ABCD1, ABCD2, ABCD3, and ABCD4 genes in patients with X-linked adrenoleukodystrophy (ALD) based on comprehensive resequencing and association studies with ALD phenotypes

    Science.gov (United States)

    Matsukawa, Takashi; Asheuer, Muriel; Takahashi, Yuji; Goto, Jun; Suzuki, Yasuyuki; Shimozawa, Nobuyuki; Takano, Hiroki; Onodera, Osamu; Nishizawa, Masatoyo; Aubourg, Patrick

    2010-01-01

    Adrenoleukodystrophy (ALD) is an X-linked disorder affecting primarily the white matter of the central nervous system occasionally accompanied by adrenal insufficiency. Despite the discovery of the causative gene, ABCD1, no clear genotype–phenotype correlations have been established. Association studies based on single nucleotide polymorphisms (SNPs) identified by comprehensive resequencing of genes related to ABCD1 may reveal genes modifying ALD phenotypes. We analyzed 40 Japanese patients with ALD. ABCD1 and ABCD2 were analyzed using a newly developed microarray-based resequencing system. ABCD3 and ABCD4 were analyzed by direct nucleotide sequence analysis. Replication studies were conducted on an independent French ALD cohort with extreme phenotypes. All the mutations of ABCD1 were identified, and there was no correlation between the genotypes and phenotypes of ALD. SNPs identified by the comprehensive resequencing of ABCD2, ABCD3, and ABCD4 were used for association studies. There were no significant associations between these SNPs and ALD phenotypes, except for the five SNPs of ABCD4, which are in complete disequilibrium in the Japanese population. These five SNPs were significantly less frequently represented in patients with adrenomyeloneuropathy (AMN) than in controls in the Japanese population (p = 0.0468), whereas there were no significant differences in patients with childhood cerebral ALD (CCALD). The replication study employing these five SNPs on an independent French ALD cohort, however, showed no significant associations with CCALD or pure AMN. This study showed that ABCD2, ABCD3, and ABCD4 are less likely the disease-modifying genes, necessitating further studies to identify genes modifying ALD phenotypes. Electronic supplementary material The online version of this article (doi:10.1007/s10048-010-0253-6) contains supplementary material, which is available to authorized users. PMID:20661612

  6. Developing high throughput genotyped chromosome segment substitution lines based on population whole-genome re-sequencing in rice (Oryza sativa L.

    Directory of Open Access Journals (Sweden)

    Gu Minghong

    2010-11-01

    Full Text Available Abstract Background Genetic populations provide the basis for a wide range of genetic and genomic studies and have been widely used in genetic mapping, gene discovery and genomics-assisted breeding. Chromosome segment substitution lines (CSSLs are the most powerful tools for the detection and precise mapping of quantitative trait loci (QTLs, for the analysis of complex traits in plant molecular genetics. Results In this study, a wide population consisting of 128 CSSLs was developed, derived from the crossing and back-crossing of two sequenced rice cultivars: 9311, an elite indica cultivar as the recipient and Nipponbare, a japonica cultivar as the donor. First, a physical map of the 128 CSSLs was constructed on the basis of estimates of the lengths and locations of the substituted chromosome segments using 254 PCR-based molecular markers. From this map, the total size of the 142 substituted segments in the population was 882.2 Mb, was 2.37 times that of the rice genome. Second, every CSSL underwent high-throughput genotyping by whole-genome re-sequencing with a 0.13× genome sequence, and an ultrahigh-quality physical map was constructed. This sequencing-based physical map indicated that 117 new segments were detected; almost all were shorter than 3 Mb and were not apparent in the molecular marker map. Furthermore, relative to the molecular marker-based map, the sequencing-based map yielded more precise recombination breakpoint determination and greater accuracy of the lengths of the substituted segments, and provided more accurate background information. Third, using the 128 CSSLs combined with the bin-map converted from the sequencing-based physical map, a multiple linear regression QTL analysis mapped nine QTLs, which explained 89.50% of the phenotypic variance for culm length. A large-effect QTL was located in a 791,655 bp region that contained the rice 'green revolution' gene. Conclusions The present results demonstrated that high

  7. Development of cleaved amplified polymorphic sequence markers and a CAPS-based genetic linkage map in watermelon (Citrullus lanatus [Thunb.] Matsum. and Nakai) constructed using whole-genome re-sequencing data.

    Science.gov (United States)

    Liu, Shi; Gao, Peng; Zhu, Qianglong; Luan, Feishi; Davis, Angela R; Wang, Xiaolu

    2016-03-01

    Cleaved amplified polymorphic sequence (CAPS) markers are useful tools for detecting single nucleotide polymorphisms (SNPs). This study detected and converted SNP sites into CAPS markers based on high-throughput re-sequencing data in watermelon, for linkage map construction and quantitative trait locus (QTL) analysis. Two inbred lines, Cream of Saskatchewan (COS) and LSW-177 had been re-sequenced and analyzed by Perl self-compiled script for CAPS marker development. 88.7% and 78.5% of the assembled sequences of the two parental materials could map to the reference watermelon genome, respectively. Comparative assembled genome data analysis provided 225,693 and 19,268 SNPs and indels between the two materials. 532 pairs of CAPS markers were designed with 16 restriction enzymes, among which 271 pairs of primers gave distinct bands of the expected length and polymorphic bands, via PCR and enzyme digestion, with a polymorphic rate of 50.94%. Using the new CAPS markers, an initial CAPS-based genetic linkage map was constructed with the F2 population, spanning 1836.51 cM with 11 linkage groups and 301 markers. 12 QTLs were detected related to fruit flesh color, length, width, shape index, and brix content. These newly CAPS markers will be a valuable resource for breeding programs and genetic studies of watermelon.

  8. A performance evaluation of Nextera XT and KAPA HyperPlus for rapid Illumina library preparation of long-range mitogenome amplicons.

    Science.gov (United States)

    Ring, Joseph D; Sturk-Andreaggi, Kimberly; Peck, Michelle A; Marshall, Charla

    2017-07-01

    Next-generation sequencing (NGS) facilitates the rapid and high-throughput generation of human mitochondrial genome (mitogenome) data to build population and reference databases for forensic comparisons. To this end, long-range amplification provides an effective method of target enrichment that is amenable to library preparation assays employing DNA fragmentation. This study compared the Nextera XT DNA Library Preparation Kit (Illumina, San Diego, CA) and the KAPA HyperPlus Library Preparation Kit (Kapa Biosystems, Wilmington, MA) for enzymatic fragmentation and indexing of ∼8500bp mitogenome amplicons for Illumina sequencing. The Nextera XT libraries produced low-coverage regions that were consistent across all samples, while the HyperPlus libraries resulted in uniformly high coverage across the mitogenome, even with reduced-volume reaction conditions. The balanced coverage observed from KAPA HyperPlus libraries enables not only low-level variant calling across the mitogenome but also increased sample multiplexing for greater processing efficiency. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Tandem multiplication of the IS26-flanked amplicon with the bla(SHV-5) gene within plasmid p1658/97.

    Science.gov (United States)

    Zienkiewicz, Maksymilian; Kern-Zdanowicz, Izabela; Carattoli, Alessandra; Gniadkowski, Marek; Cegłowski, Piotr

    2013-04-01

    The IncF plasmid p1658/97 (c. 125 kb) from Escherichia coli isolates recovered during a clonal outbreak in a hospital in Warsaw, Poland, in 1997 contains the extended-spectrum β-lactamase (ESBL) gene bla(SHV-5), originated from the Klebsiella pneumoniae chromosome. A region containing the bla(SHV-5) gene is flanked by two IS26 copies and its copy number multiplies spontaneously within p1658/97 and RecA-deficient E. coli strains. Here, we demonstrate that the amplified IS26-bla(SHV-5) units were arranged in tandems, containing up to more than 10 units, which could raise ceftazidime MICs for host strains from 4 μg mL(-1) to more than 128 μg mL(-1). Successive deletions within p1658/97, located outside the amplifiable module and encompassing even as little as c. 15% of the plasmid, blocked the amplification. Moreover, the complementing re-introduction of the deleted fragments in trans did not restore the process. Similarly, insertions of a 1-kb DNA fragment into the amplicon inhibited its self-multiplication ability. The module was able to transmit into another IS26-containing plasmid by recombination. The results prompted us to speculate that local DNA structure, especially favorable in p1658/97, might have been responsible for the IS26-bla(SHV-5) multiplication ability. © 2013 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  10. Application of long amplicon propidium monoazide-PCR to assess the effects of temperature and background microbiota on pathogens in river water.

    Science.gov (United States)

    Banihashemi, Avid; Van Dyke, Michele I; Huck, Peter M

    2017-06-01

    The decay rates of enteric waterborne pathogens were evaluated following the introduction of Yersinia enterocolitica, Salmonella enterica, Campylobacter jejuni and Arcobacter butzleri into river water at different temperatures (5, 15 and 25°C) for a period of 28 days. To improve the accuracy of the results a molecular viability assay, long amplicon propidium monoazide-polymerase chain reaction (PMA-PCR), was used to quantify the viable cell concentration and results from PCR with and without PMA were compared. As well, the effect of background microbiota was assessed for Y. enterocolitica and S. enterica by inoculating cells into sterile and non-sterile river water. Cell persistence was improved by up to 4 log for Y. enterocolitica and 4.5 log for S. enterica in sterile river water compared to natural river water, showing that the autochthonous biological activity in river water can accelerate the die-off of introduced bacteria. Results also showed that low temperature significantly improved the persistence of all four target bacteria in non-sterile river water. There was a more rapid decline in cell concentration in samples with PMA pretreatment; therefore using PMA-PCR analysis can provide more reliable data on viable/active enteric bacteria in aquatic microcosms and allows for improved assessment of pathogens in the environment.

  11. Identification of active denitrifiers by DNA-stable isotope probing and amplicon sequencing reveals Betaproteobacteria as responsible for attenuation of nitrate contamination in a low impacted aquifer.

    Science.gov (United States)

    Bellini, M Inés; Kumaresan, Deepak; Tarlera, Silvana; Murrell, J Colin; Fernández-Scavino, Ana

    2018-02-01

    Groundwater reservoirs constitute important freshwater resources. However, these ecosystems are highly vulnerable to contamination and have to rely on the resident microbiota to attenuate the impact of this contamination. Nitrate is one of the main contaminants found in groundwater, and denitrification is the main process that removes the compound. In this study, the response to nutrient load on indigenous microbial communities in groundwater from a low impacted aquifer in Uruguay was evaluated. Denitrification rates were measured in groundwater samples from three different sites with nitrate, acetate and pyrite amendments. Results showed that denitrification is feasible under in situ nitrate and electron donor concentrations, although the lack of readily available organic energy source would limit the attenuation of higher nitrate concentrations. DNA-stable isotope probing, combined with amplicon sequencing of 16S rRNA, nirS and nirK genes, was used to identify the active denitrifiers. Members of the phylum Betaproteobacteria were the dominant denitrifiers in two of three sites, with different families being observed; members of the genus Vogesella (Neisseriaceae) were key denitrifiers at one site, while the genera Dechloromonas (Rhodocyclaceae) and Comamonas (Comamonadaceae) were the main denitrifiers detected at the other sites. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  12. The use of genus-specific amplicon pyrosequencing to assess phytophthora species diversity using eDNA from soil and water in Northern Spain.

    Science.gov (United States)

    Català, Santiago; Pérez-Sierra, Ana; Abad-Campos, Paloma

    2015-01-01

    Phytophthora is one of the most important and aggressive plant pathogenic genera in agriculture and forestry. Early detection and identification of its pathways of infection and spread are of high importance to minimize the threat they pose to natural ecosystems. eDNA was extracted from soil and water from forests and plantations in the north of Spain. Phytophthora-specific primers were adapted for use in high-throughput Sequencing (HTS). Primers were tested in a control reaction containing eight Phytophthora species and applied to water and soil eDNA samples from northern Spain. Different score coverage threshold values were tested for optimal Phytophthora species separation in a custom-curated database and in the control reaction. Clustering at 99% was the optimal criteria to separate most of the Phytophthora species. Multiple Molecular Operational Taxonomic Units (MOTUs) corresponding to 36 distinct Phytophthora species were amplified in the environmental samples. Pyrosequencing of amplicons from soil samples revealed low Phytophthora diversity (13 species) in comparison with the 35 species detected in water samples. Thirteen of the MOTUs detected in rivers and streams showed no close match to sequences in international sequence databases, revealing that eDNA pyrosequencing is a useful strategy to assess Phytophthora species diversity in natural ecosystems.

  13. Gastrointestinal Bacterial and Methanogenic Archaea Diversity Dynamics Associated with Condensed Tannin-Containing Pine Bark Diet in Goats Using 16S rDNA Amplicon Pyrosequencing.

    Science.gov (United States)

    Min, Byeng R; Solaiman, Sandra; Shange, Raymon; Eun, Jong-Su

    2014-01-01

    Eighteen Kiko-cross meat goats (n = 6) were used to collect gastrointestinal (GI) bacteria and methanogenic archaea for diversity measures when fed condensed tannin-containing pine bark (PB). Three dietary treatments were tested: control diet (0% PB and 30% wheat straw (WS); 0.17% condensed tannins (CT) dry matter (DM)); 15% PB and 15% WS (1.6% CT DM), and 30% PB and 0% WS (3.2% CT DM). A 16S rDNA bacterial tag-encoded FLX amplicon pyrosequencing technique was used to characterize and elucidate changes in GI bacteria and methanogenic archaea diversity among the diets. Proteobacteria was the most dominant phylum in goats with mean relative abundance values ranging from 39.7 (30% PB) to 46.5% (control) and 47.1% (15% PB). Other phyla individually accounted for fewer than 25% of the relative abundance observed. Predominant methanogens were Methanobrevibacter (75, 72, and 49%), Methanosphaera (3.3, 2.3, and 3.4%), and Methanobacteriaceae (1.2, 0.6, and 0.7%) population in control, 15, and 30% PB, respectively. Among methanogens, Methanobrevibacter was linearly decreased (P = 0.05) with increasing PB supplementation. These results indicate that feeding PB selectively altered bacteria and methanogenic archaeal populations in the GI tract of goats.

  14. Gastrointestinal Bacterial and Methanogenic Archaea Diversity Dynamics Associated with Condensed Tannin-Containing Pine Bark Diet in Goats Using 16S rDNA Amplicon Pyrosequencing

    Directory of Open Access Journals (Sweden)

    Byeng R. Min

    2014-01-01

    Full Text Available Eighteen Kiko-cross meat goats (n=6 were used to collect gastrointestinal (GI bacteria and methanogenic archaea for diversity measures when fed condensed tannin-containing pine bark (PB. Three dietary treatments were tested: control diet (0% PB and 30% wheat straw (WS; 0.17% condensed tannins (CT dry matter (DM; 15% PB and 15% WS (1.6% CT DM, and 30% PB and 0% WS (3.2% CT DM. A 16S rDNA bacterial tag-encoded FLX amplicon pyrosequencing technique was used to characterize and elucidate changes in GI bacteria and methanogenic archaea diversity among the diets. Proteobacteria was the most dominant phylum in goats with mean relative abundance values ranging from 39.7 (30% PB to 46.5% (control and 47.1% (15% PB. Other phyla individually accounted for fewer than 25% of the relative abundance observed. Predominant methanogens were Methanobrevibacter (75, 72, and 49%, Methanosphaera (3.3, 2.3, and 3.4%, and Methanobacteriaceae (1.2, 0.6, and 0.7% population in control, 15, and 30% PB, respectively. Among methanogens, Methanobrevibacter was linearly decreased (P=0.05 with increasing PB supplementation. These results indicate that feeding PB selectively altered bacteria and methanogenic archaeal populations in the GI tract of goats.

  15. Size Matters: Assessing Optimum Soil Sample Size for Fungal and Bacterial Community Structure Analyses Using High Throughput Sequencing of rRNA Gene Amplicons

    Directory of Open Access Journals (Sweden)

    Christopher Ryan Penton

    2016-06-01

    Full Text Available We examined the effect of different soil sample sizes obtained from an agricultural field, under a single cropping system uniform in soil properties and aboveground crop responses, on bacterial and fungal community structure and microbial diversity indices. DNA extracted from soil sample sizes of 0.25, 1, 5 and 10 g using MoBIO kits and from 10 and 100 g sizes using a bead-beating method (SARDI were used as templates for high-throughput sequencing of 16S and 28S rRNA gene amplicons for bacteria and fungi, respectively, on the Illumina MiSeq and Roche 454 platforms. Sample size significantly affected overall bacterial and fungal community structure, replicate dispersion and the number of operational taxonomic units (OTUs retrieved. Richness, evenness and diversity were also significantly affected. The largest diversity estimates were always associated with the 10 g MoBIO extractions with a corresponding reduction in replicate dispersion. For the fungal data, smaller MoBIO extractions identified more unclassified Eukaryota incertae sedis and unclassified glomeromycota while the SARDI method retrieved more abundant OTUs containing unclassified Pleosporales and the fungal genera Alternaria and Cercophora. Overall, these findings indicate that a 10 g soil DNA extraction is most suitable for both soil bacterial and fungal communities for retrieving optimal diversity while still capturing rarer taxa in concert with decreasing replicate variation.

  16. Improving the genotyping resolution of Cryptosporidium hominis subtype IbA10G2 using one step PCR-based amplicon sequencing.

    Science.gov (United States)

    Beser, Jessica; Hallström, Björn M; Advani, Abdolreza; Andersson, Sofia; Östlund, Gabriel; Winiecka-Krusnell, Jadwiga; Lebbad, Marianne; Alm, Erik; Troell, Karin; Arrighi, Romanico B G

    2017-11-01

    Cryptosporidium hominis gp60 subtype IbA10G2 is a common cause of cryptosporidiosis. This subtype is responsible for many waterborne outbreaks as well as sporadic cases and is considered virulent and highly important in the epidemiology of cryptosporidiosis. Due to low heterogeneity within the genome of C. hominis it has been difficult to identify epidemiological markers with higher resolution than gp60. However, new markers are required in order to improve outbreak investigations and studies of the transmission dynamics of this clinically important subtype. Based on the whole genome sequences of 17 C. hominis isolates, we have identified several differential loci and developed a new sequence based typing panel with higher resolution than gp60. An amplicon sequencing method was also developed which is based on a one-step PCR which can be sequenced using a Next Generation Sequencing (NGS) platform. Such a system provides a rapid and high-throughput workflow. A panel of nine loci with 10 single nucleotide variants (SNV) was selected and evaluated using clinical IbA10G2 isolates from sporadic, cluster and outbreak associated cases. The specimens were separated into 10 different genetic profiles named sequence types (STs). All isolates within an outbreak or cluster belonged to the same ST, including several samples from the two large waterborne outbreaks which occurred in Sweden between 2010 and 2011 indicating that these outbreaks might be linked. The results demonstrate the methods suitability for improved genotyping of C. hominis IbA10G2. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Fluorescent duplex allele-specific PCR and amplicon melting for rapid homogeneous mtDNA haplogroup H screening and sensitive mixture detection.

    Directory of Open Access Journals (Sweden)

    Harald Niederstätter

    Full Text Available BACKGROUND: For large scale studies aiming at a better understanding of mitochondrial DNA (mtDNA, sequence variation in particular mt haplogroups (hgs and population structure, reliable low-cost high-throughput genotyping assays are needed. Furthermore, methods facilitating sensitive mixture detection and relative quantification of allele proportions are indispensable for the study of heteroplasmy, mitochondrial sequence evolution, and mitochondrial disorders. Here the properties of a homogeneous competitive duplex allele specific PCR (ARMS assay were scrutinized in the light of these requirements. METHODOLOGY/PRINCIPAL FINDINGS: A duplex ARMS assay amplifying either the ancestral mtDNA 2706G allele (non-hg H samples or the derived 7028C allele (hg H samples in the presence of SYBR Green fluorescent reporter dye was developed and characterized. Product detection, allele calling, and hg inference were based on the amplicon-characteristic melting-point temperatures obtained with on-line post-PCR fluorescent dissociation curve analysis (DCA. The analytical window of the assay covered at least 5 orders of magnitude of template DNA input with a detection limit in the low picogram range of genomic DNA. A set of forensically relevant test specimens was analyzed successfully. The presence of mtDNA mixtures was detected over a broad range of input DNA amounts and mixture ratios, and the estimation of allele proportions in samples with known total mtDNA content was feasible with limitations. A qualified DNA analyst successfully analyzed approximately 2,200 DNA extracts within three regular working days, without using robotic lab-equipment. By performing the amplification on-line, the assay also facilitated absolute mtDNA quantification. CONCLUSIONS: Although this assay was developed just for a particular purpose, the approach is general in that it is potentially suitable in a broad variety of assay-layouts for many other applications, including the

  18. Illumina MiSeq Phylogenetic Amplicon Sequencing Shows a Large Reduction of an Uncharacterised Succinivibrionaceae and an Increase of the Methanobrevibacter gottschalkii Clade in Feed Restricted Cattle.

    Directory of Open Access Journals (Sweden)

    Matthew Sean McCabe

    Full Text Available Periodic feed restriction is used in cattle production to reduce feed costs. When normal feed levels are resumed, cattle catch up to a normal weight by an acceleration of normal growth rate, known as compensatory growth, which is not yet fully understood. Illumina Miseq Phylogenetic marker amplicon sequencing of DNA extracted from rumen contents of 55 bulls showed that restriction of feed (70% concentrate, 30% grass silage for 125 days, to levels that caused a 60% reduction of growth rate, resulted in a large increase of relative abundance of Methanobrevibacter gottschalkii clade (designated as OTU-M7, and a large reduction of an uncharacterised Succinivibrionaceae species (designated as OTU-S3004. There was a strong negative Spearman correlation (ρ = -0.72, P = <1x10(-20 between relative abundances of OTU-3004 and OTU-M7 in the liquid rumen fraction. There was also a significant increase in acetate:propionate ratio (A:P in feed restricted animals that showed a negative Spearman correlation (ρ = -0.69, P = <1x10(-20 with the relative abundance of OTU-S3004 in the rumen liquid fraction but not the solid fraction, and a strong positive Spearman correlation with OTU-M7 in the rumen liquid (ρ = 0.74, P = <1x10(-20 and solid (ρ = 0.69, P = <1x10(-20 fractions. Reduced A:P ratios in the rumen are associated with increased feed efficiency and reduced production of methane which has a global warming potential (GWP 100 years of 28. Succinivibrionaceae growth in the rumen was previously suggested to reduce methane emissions as some members of this family utilise hydrogen, which is also utilised by methanogens for methanogenesis, to generate succinate which is converted to propionate. Relative abundance of OTU-S3004 showed a positive Spearman correlation with propionate (ρ = 0.41, P = <0.01 but not acetate in the liquid rumen fraction.

  19. Interspecies hybridization on DNA resequencing microarrays: efficiency of sequence recovery and accuracy of SNP detection in human, ape, and codfish mitochondrial DNA genomes sequenced on a human-specific MitoChip

    Directory of Open Access Journals (Sweden)

    Carr Steven M

    2007-09-01

    Full Text Available Abstract Background Iterative DNA "resequencing" on oligonucleotide microarrays offers a high-throughput method to measure intraspecific biodiversity, one that is especially suited to SNP-dense gene regions such as vertebrate mitochondrial (mtDNA genomes. However, costs of single-species design and microarray fabrication are prohibitive. A cost-effective, multi-species strategy is to hybridize experimental DNAs from diverse species to a common microarray that is tiled with oligonucleotide sets from multiple, homologous reference genomes. Such a strategy requires that cross-hybridization between the experimental DNAs and reference oligos from the different species not interfere with the accurate recovery of species-specific data. To determine the pattern and limits of such interspecific hybridization, we compared the efficiency of sequence recovery and accuracy of SNP identification by a 15,452-base human-specific microarray challenged with human, chimpanzee, gorilla, and codfish mtDNA genomes. Results In the human genome, 99.67% of the sequence was recovered with 100.0% accuracy. Accuracy of SNP identification declines log-linearly with sequence divergence from the reference, from 0.067 to 0.247 errors per SNP in the chimpanzee and gorilla genomes, respectively. Efficiency of sequence recovery declines with the increase of the number of interspecific SNPs in the 25b interval tiled by the reference oligonucleotides. In the gorilla genome, which differs from the human reference by 10%, and in which 46% of these 25b regions contain 3 or more SNP differences from the reference, only 88% of the sequence is recoverable. In the codfish genome, which differs from the reference by > 30%, less than 4% of the sequence is recoverable, in short islands ≥ 12b that are conserved between primates and fish. Conclusion Experimental DNAs bind inefficiently to homologous reference oligonucleotide sets on a re-sequencing microarray when their sequences differ by

  20. An Undergraduate Laboratory Experiment for Upper-Level Forensic Science, Biochemistry, or Molecular Biology Courses: Human DNA Amplification Using STR Single Locus Primers by Real-Time PCR with SYBR Green Detection

    Science.gov (United States)

    Elkins, Kelly M.; Kadunc, Raelynn E.

    2012-01-01

    In this laboratory experiment, real-time polymerase chain reaction (real-time PCR) was conducted using published human TPOX single-locus DNA primers for validation and various student-designed short tandem repeat (STR) primers for Combined DNA Index System (CODIS) loci. SYBR Green was used to detect the amplification of the expected amplicons. The…

  1. Targeted Amplicon Sequencing for Single-Nucleotide-Polymorphism Genotyping of Attaching and Effacing Escherichia coli O26:H11 Cattle Strains via a High-Throughput Library Preparation Technique.

    Science.gov (United States)

    Ison, Sarah A; Delannoy, Sabine; Bugarel, Marie; Nagaraja, Tiruvoor G; Renter, David G; den Bakker, Henk C; Nightingale, Kendra K; Fach, Patrick; Loneragan, Guy H

    2016-01-15

    Enterohemorrhagic Escherichia coli (EHEC) O26:H11, a serotype within Shiga toxin-producing E. coli (STEC) that causes severe human disease, has been considered to have evolved from attaching and effacing E. coli (AEEC) O26:H11 through the acquisition of a Shiga toxin-encoding gene. Targeted amplicon sequencing using next-generation sequencing technology of 48 phylogenetically informative single-nucleotide polymorphisms (SNPs) and three SNPs differentiating Shiga toxin-positive (stx-positive) strains from Shiga toxin-negative (stx-negative) strains were used to infer the phylogenetic relationships of 178 E. coli O26:H11 strains (6 stx-positive strains and 172 stx-negative AEEC strains) from cattle feces to 7 publically available genomes of human clinical strains. The AEEC cattle strains displayed synonymous SNP genotypes with stx2-positive sequence type 29 (ST29) human O26:H11 strains, while stx1 ST21 human and cattle strains clustered separately, demonstrating the close phylogenetic relatedness of these Shiga toxin-negative AEEC cattle strains and human clinical strains. With the exception of seven stx-negative strains, five of which contained espK, three stx-related SNPs differentiated the STEC strains from non-STEC strains, supporting the hypothesis that these AEEC cattle strains could serve as a potential reservoir for new or existing pathogenic human strains. Our results support the idea that targeted amplicon sequencing for SNP genotyping expedites strain identification and genetic characterization of E. coli O26:H11, which is important for food safety and public health. Copyright © 2016 Ison et al.

  2. Genome-Wide Sequence Variation Identification and Floral-Associated Trait Comparisons Based on the Re-sequencing of the 'Nagafu No. 2' and 'Qinguan' Varieties of Apple (Malus domestica Borkh.).

    Science.gov (United States)

    Xing, Libo; Zhang, Dong; Song, Xiaomin; Weng, Kai; Shen, Yawen; Li, Youmei; Zhao, Caiping; Ma, Juanjuan; An, Na; Han, Mingyu

    2016-01-01

    Apple (Malus domestica Borkh.) is a commercially important fruit worldwide. Detailed information on genomic DNA polymorphisms, which are important for understanding phenotypic traits, is lacking for the apple. We re-sequenced two elite apple varieties, 'Nagafu No. 2' and 'Qinguan,' which have different characteristics. We identified many genomic variations, including 2,771,129 single nucleotide polymorphisms (SNPs), 82,663 structural variations (SVs), and 1,572,803 insertion/deletions (INDELs) in 'Nagafu No. 2' and 2,262,888 SNPs, 63,764 SVs, and 1,294,060 INDELs in 'Qinguan.' The 'SNP,' 'INDEL,' and 'SV' distributions were non-random, with variation-rich or -poor regions throughout the genomes. In 'Nagafu No. 2' and 'Qinguan' there were 171,520 and 147,090 non-synonymous SNPs spanning 23,111 and 21,400 genes, respectively; 3,963 and 3,196 SVs in 3,431 and 2,815 genes, respectively; and 1,834 and 1,451 INDELs in 1,681 and 1,345 genes, respectively. Genetic linkage maps of 190 flowering genes associated with multiple flowering pathways in 'Nagafu No. 2,' 'Qinguan,' and 'Golden Delicious,' identified complex regulatory mechanisms involved in floral induction, flower bud formation, and flowering characteristics, which might reflect the genetic variation of the flowering genes. Expression profiling of key flowering genes in buds and leaves suggested that the photoperiod and autonomous flowering pathways are major contributors to the different floral-associated traits between 'Nagafu No. 2' and 'Qinguan.' The genome variation data provided a foundation for the further exploration of apple diversity and gene-phenotype relationships, and for future research on molecular breeding to improve apple and related species.

  3. Genome-Wide Analysis of Simple Sequence Repeats and Efficient Development of Polymorphic SSR Markers Based on Whole Genome Re-Sequencing of Multiple Isolates of the Wheat Stripe Rust Fungus.

    Directory of Open Access Journals (Sweden)

    Huaiyong Luo

    Full Text Available The biotrophic parasitic fungus Puccinia striiformis f. sp. tritici (Pst causes stripe rust, a devastating disease of wheat, endangering global food security. Because the Pst population is highly dynamic, it is difficult to develop wheat cultivars with durable and highly effective resistance. Simple sequence repeats (SSRs are widely used as molecular markers in genetic studies to determine population structure in many organisms. However, only a small number of SSR markers have been developed for Pst. In this study, a total of 4,792 SSR loci were identified using the whole genome sequences of six isolates from different regions of the world, with a marker density of one SSR per 22.95 kb. The majority of the SSRs were di- and tri-nucleotide repeats. A database containing 1,113 SSR markers were established. Through in silico comparison, the previously reported SSR markers were found mainly in exons, whereas the SSR markers in the database were mostly in intergenic regions. Furthermore, 105 polymorphic SSR markers were confirmed in silico by their identical positions and nucleotide variations with INDELs identified among the six isolates. When 104 in silico polymorphic SSR markers were used to genotype 21 Pst isolates, 84 produced the target bands, and 82 of them were polymorphic and revealed the genetic relationships among the isolates. The results show that whole genome re-sequencing of multiple isolates provides an ideal resource for developing SSR markers, and the newly developed SSR markers are useful for genetic and population studies of the wheat stripe rust fungus.

  4. Definitive differentiation between single and mixed mycobacterial infections in red deer (Cervus elaphus) by a combination of duplex amplification of p34 and f57 sequences and Hpy188I enzymatic restriction of duplex amplicons.

    Science.gov (United States)

    Godfroid, Jacques; Delcorps, Cathy; Irenge, Leonid M; Walravens, Karl; Marché, Sylvie; Gala, Jean-Luc

    2005-09-01

    Severe emaciation and mortalities suggestive of mycobacterial infections were recently reported for both adult and young wild red deer (Cervus elaphus) in the southeastern part of Belgium. In deer, tuberculous lesions are not pathognomonic of Mycobacterium bovis infection due to gross and microscopic similarities with lesions caused by Mycobacterium avium subsp. paratuberculosis or M. avium subsp. avium. The aim of this study was to improve molecular methods for the species-specific identification of M. bovis, M. avium subsp. avium, and M. avium subsp. paratuberculosis in mycobacterial infections of deer. DNA banding patterns were assessed prior to and after Hpy188I restriction of f57-upstream (us)-p34 duplex amplicons. The duplex f57-us-p34 PCR differentiated M. bovis from M. avium subsp. paratuberculosis and M. avium subsp. avium infections, whereas the restriction step differentiated single M. avium subsp. paratuberculosis or M. avium subsp. avium infections from mixed M. avium subsp. paratuberculosis/M. avium subsp. avium infections. The endonuclease Hpy188I cleaves DNA between nucleotides N and G in the unique TCNGA sequence. This restriction site was found at position 168 upstream of the us-p34 initiation codon in all M. avium subsp. avium strains tested, regardless of their origin and the results of IS901 PCR. In contrast, the restriction site was abrogated in all M. avium subsp. paratuberculosis strains tested, independent of their origin, Mycobactin J dependency, and IS900 PCR results. Consequently, a two-step strategy, i.e., duplex us-p34-f57 PCR and Hpy188I restriction, allowed us to exclude M. bovis infection and to identify single (M. avium subsp. paratuberculosis or M. avium subsp. avium) or mixed (M. avium subsp. paratuberculosis/M. avium subsp. avium) infections in wild red deer in Belgium. Accordingly, we propose to integrate, in a functional molecular definition of M. avium subsp. paratuberculosis, the absence of the Hpy188I restriction site from

  5. Customer experience

    OpenAIRE

    Koperdáková, Zuzana

    2016-01-01

    Bachelor thesis deals with the theme of customer experience and terms related to this topic. The thesis consists of three parts. The first part explains the terms generally, as the experience or customer loyalty. The second part is dedicated to medotology used for Customer Experience Management. In the third part is described application of Customer Experience Management in practice, particularly in the context Touch Point Analyses in GE Money Bank.

  6. OPERA experiment

    International Nuclear Information System (INIS)

    Komatsu, M.

    2003-01-01

    OPERA is an accelerator experiment designed to explore Super-Kamiokande suggested ν μ ↔ν τ oscillation region in CNGS beam from CERN to LNGS. The key technique in OPERA is modern emulsion read out which applied to CHORUS and DONUT experiments. ECC technique which used in DONUT and OPERA has good modularity to enlarge apparatus for future Neutrino Factory experiments

  7. Guidelines to use tomato in experiments with a controlled environment.

    Directory of Open Access Journals (Sweden)

    Dietmar eSchwarz

    2014-11-01

    Full Text Available Domesticated tomato (Solanum lycopersicum is the most important horticultural crop worldwide. Low polymorphism at the DNA level conflicts with the wealth of morphological variation. Fruits vary widely in size, shape and colour. In contrast, genetic variation between the 16 wild relatives is tremendous. Several large seed banks provide tomato germplasm for both domesticated and wild accessions of tomato. Recently, the genomes of the inbred cultivar Heinz 1706 (≈900 Mb and S. pimpinellifolium (739 Mb were sequenced. Genomic markers and genome re-sequencing data are available for >150 cultivars and accessions. Transformation of tomato is relatively easy and T-DNA insertion line collections are available. Tomato is widely used as a model crop for fruit development but also for diverse physiological, cellular, biochemical, molecular and genetic studies. It can be easily grown in greenhouses or growth chambers. Plants grow, flower, and develop fruits well at daily light lengths between 8-16 hours. The required daily light integral of an experiment depends on growth stage and temperature investigated. Temperature must be 10-35°C, relative humidity 30-90 % and CO2 concentration 200-1500 µmol mol-1. Temperature determines the speed of the phenological development while daily light integral and CO2 concentration affect photosynthesis and biomass production. Seed to seed cultivation takes 100 days at 20°C and can be shortened or delayed by temperature. Tomato may be cultivated in soil, substrates, or aeroponically without any substrate. Root volume and water uptake requirements are primarily determined by transpiration demands of the plants. Many nutrient supply recipes and strategies are available to ensure sufficient supply as well as specific nutrient deficits/surplus. Using appropriate cultivation techniques makes tomato a convenient model plant for researchers, even for beginners.

  8. OPERA experiment

    CERN Document Server

    Komatsu, M

    2003-01-01

    OPERA is an accelerator experiment designed to explore Super- Kamiokande suggested nu//mu arrow left right nu//tau oscillation region in CNGS beam from CERN to LNGS. The key technique in OPERA is modern emulsion read out which applied to CHORUS and DONUT experiments. ECC technique which used in DONUT and OPERA has good modularity to enlarge apparatus for future Neutrino Factory experiments.

  9. TRIO experiment

    Energy Technology Data Exchange (ETDEWEB)

    Clemmer, R.G.; Finn, P.A.; Malecha, R.F.; Misra, B.; Billone, M.C.; Bowers, D.L.; Fischer, A.K.; Greenwood, L.R.; Mattas, R.F.; Tam, S.W.

    1984-09-01

    The TRIO experiment is a test of in-situ tritium recovery and heat transfer performance of a miniaturized solid breeder blanket assembly. The assembly (capsule) was monitored for temperature and neutron flux profiles during irradiation and a sweep gas flowed through the capsule to an anaytical train wherein the amounts of tritium in its various chemical forms were determined. The capsule was designed to operate at different temperatures and sweep gas conditions. At the end of the experiment the amount of tritium retained in the solid was at a concentration of less than 0.1 wppM. More than 99.9% of tritium generated during the experiment was successfully recovered. The results of the experiment showed that the tritium inventories at the beginning and at the end of the experiment follow a relationship which appears to be characteristic of intragranular diffusion.

  10. Pixel Experiments

    DEFF Research Database (Denmark)

    Petersen, Kjell Yngve; Søndergaard, Karin; Augustesen, Christina

    2015-01-01

    Pixel Experiments The term pixel is traditionally defined as any of the minute elements that together constitute a larger context or image. A pixel has its own form and is the smallest unit seen within a larger structure. In working with the potentials of LED technology in architectural lighting...... for using LED lighting in lighting design practice. The speculative experiments that have been set-up have aimed to clarify the variables that can be used as parameters in the design of lighting applications; including, for example, the structuring and software control of light. The experiments also...... elucidate and exemplify already well-known problems in relation to the experience of vertical and horizontal lighting. Pixel Experiments exist as a synergy between speculative test setups and lighting design in practice. This book is one of four books that is published in connection with the research...

  11. Collaborative experience

    DEFF Research Database (Denmark)

    Mortensen, Thomas Bøtker

    Literature review: Collaborative experience has been shown to have a positive effect on the collaborative outcome in general (Anand & Khanna, 2000; Kale, Dyer & Singh, 2002). Furthermore, it has been linked to the ability to exploit the network of the firm for learning (Powell, Koput and Smith...... experience was largest the higher the hypothesized ambiguity. Theoretically contribution: This research project aims at contributing to existing literature by arguing, that collaborative experience is a moderating variable which moderates the effects on collaborative outcome from the level of complexity......, that the largest effects from collaborative experience is from recent collaborative experience, since knowledge depreciates when it is not used. Methodologically contribution: The research project studies the dyad and aims at introducing, to this field of research, an established way of collecting data, a new...

  12. Mixture Experiments

    Energy Technology Data Exchange (ETDEWEB)

    Piepel, Gregory F.

    2007-12-01

    A mixture experiment involves combining two or more components in various proportions or amounts and then measuring one or more responses for the resulting end products. Other factors that affect the response(s), such as process variables and/or the total amount of the mixture, may also be studied in the experiment. A mixture experiment design specifies the combinations of mixture components and other experimental factors (if any) to be studied and the response variable(s) to be measured. Mixture experiment data analyses are then used to achieve the desired goals, which may include (i) understanding the effects of components and other factors on the response(s), (ii) identifying components and other factors with significant and nonsignificant effects on the response(s), (iii) developing models for predicting the response(s) as functions of the mixture components and any other factors, and (iv) developing end-products with desired values and uncertainties of the response(s). Given a mixture experiment problem, a practitioner must consider the possible approaches for designing the experiment and analyzing the data, and then select the approach best suited to the problem. Eight possible approaches include 1) component proportions, 2) mathematically independent variables, 3) slack variable, 4) mixture amount, 5) component amounts, 6) mixture process variable, 7) mixture of mixtures, and 8) multi-factor mixture. The article provides an overview of the mixture experiment designs, models, and data analyses for these approaches.

  13. Simulated experiments

    International Nuclear Information System (INIS)

    Bjerknes, R.

    1977-01-01

    A cybernetic model has been developed to elucidate some of the main principles of the growth regulation system in the epidermis of the hairless mouse. A number of actual and theoretical biological experiments have been simulated on the model. These included simulating the cell kinetics as measured by pulse labelling with tritiated thymidine and by continuous labelling with tritiated thymidine. Other simulated experiments included steady state, wear and tear, painting with a carcinogen, heredity and heredity and tumour. Numerous diagrams illustrate the results of these simulated experiments. (JIW)

  14. Researching experiences

    DEFF Research Database (Denmark)

    Gjedde, Lisa; Ingemann, Bruno

    , dialogue, moods, values and narratives have been investigated qualitatively with more than sixty informants in a range of projects. The processual methodological insights are put into a theoretical perspective and also presented as pragmatic dilemmas. Researching Experiences is relevant not only...... for students and researchers in media and communication studies but also for practitioners within the fields of media, communication and experience design....... for researching experiences in a variety of settings ranging from the museum, to news photography, and interactive media. The research led to the development of a set of methodological tools and approaches we term the reflexivity lab. The interaction in the experimental situation between the media and body...

  15. Researching experiences

    DEFF Research Database (Denmark)

    Gjedde, Lisa; Ingemann, Bruno

    In the beginning was - not the word - but the experience. This phenomenological approach provides the basis for this book, which focuses on how a person-in-situation experiences and constructs meaning from a variety of cultural visual events. This book presents video-based processual methods...... for researching experiences in a variety of settings ranging from the museum, to news photography, and interactive media. The research led to the development of a set of methodological tools and approaches we term the reflexivity lab. The interaction in the experimental situation between the media and body......, dialogue, moods, values and narratives have been investigated qualitatively with more than sixty informants in a range of projects. The processual methodological insights are put into a theoretical perspective and also presented as pragmatic dilemmas. Researching Experiences is relevant not only...

  16. Interpretive Experiments

    Science.gov (United States)

    DeHaan, Frank, Ed.

    1977-01-01

    Describes an interpretative experiment involving the application of symmetry and temperature-dependent proton and fluorine nmr spectroscopy to the solution of structural and kinetic problems in coordination chemistry. (MLH)

  17. Antimatter Experiments

    CERN Multimedia

    2004-01-01

    Antimatter should behave in identical fashion to matter if a form of spacetime symmetry called CPT invariance holds. Two experiments at CERN near Geneva are testing this hypothesis using antihydrogen atoms

  18. Media experiences

    DEFF Research Database (Denmark)

    Buhl, Mie

    2010-01-01

    on their particular iconic systems of meaning perceived through form, lines, contrasts, colours, structures textures, rhythm and composite qualities with embedded interdependent codes and culturally conditioned potential for construction of knowledge and meaning. Mediated experience adds new perspectives...

  19. Experiment Databases

    Science.gov (United States)

    Vanschoren, Joaquin; Blockeel, Hendrik

    Next to running machine learning algorithms based on inductive queries, much can be learned by immediately querying the combined results of many prior studies. Indeed, all around the globe, thousands of machine learning experiments are being executed on a daily basis, generating a constant stream of empirical information on machine learning techniques. While the information contained in these experiments might have many uses beyond their original intent, results are typically described very concisely in papers and discarded afterwards. If we properly store and organize these results in central databases, they can be immediately reused for further analysis, thus boosting future research. In this chapter, we propose the use of experiment databases: databases designed to collect all the necessary details of these experiments, and to intelligently organize them in online repositories to enable fast and thorough analysis of a myriad of collected results. They constitute an additional, queriable source of empirical meta-data based on principled descriptions of algorithm executions, without reimplementing the algorithms in an inductive database. As such, they engender a very dynamic, collaborative approach to experimentation, in which experiments can be freely shared, linked together, and immediately reused by researchers all over the world. They can be set up for personal use, to share results within a lab or to create open, community-wide repositories. Here, we provide a high-level overview of their design, and use an existing experiment database to answer various interesting research questions about machine learning algorithms and to verify a number of recent studies.

  20. Researching Experiences

    DEFF Research Database (Denmark)

    Gjedde, Lisa; Ingemann, Bruno

    In the beginning was - not the word - but the experience. This phenomenological approach provides the basis for this book, which focuses on how a person-in-situation experiences and constructs meaning from a variety of cultural visual events. This book presents video-based processual methods for ...... for students and researchers in media and communication studies but also for practitioners within the fields of media, communication and experience design.......In the beginning was - not the word - but the experience. This phenomenological approach provides the basis for this book, which focuses on how a person-in-situation experiences and constructs meaning from a variety of cultural visual events. This book presents video-based processual methods...... for researching experiences in a variety of settings ranging from the museum, to news photography, and interactive media. The research led to the development of a set of methodological tools and approaches we term the ReflexivityLab. The interaction in the experimental situation between the media and body...

  1. The experience of an experience

    DEFF Research Database (Denmark)

    Rasmussen, Alexandra Marie

    2016-01-01

    This paper examines a popular phenomenon that allows us to see ourselves from various perspectives, and allows us to perpetuate moments enabling us to relive experiences in a different state of mind or at a different time. To examine the phenomenon of experiencing an experience hermeneutic...... is a tool to create a particular mindset, in the developer, to generate a new way of thinking. This model is dynamic by nature and although it is not completed in every branch, it is complete in relation to experiencing your own experience. A company can apply the PoC to gain insight into primary...

  2. The Experiment

    Science.gov (United States)

    Mariana Nicoara, Floare

    2016-04-01

    My name is Nicoara Floarea and I am teacher at Secondary School Calatele and I teach students from preparatory class and the second grade . They are six-eight years old. In my activity, for introducing scientific concepts to my students, I use various and active methods or traditional methods including experiments. The experiment stimulates students' curiosity, their creativity, the understanding and knowledge taught accessibility. I propose you two such experiments: The life cycle of the plants (long-term experiment, with rigorous observation time):We use beans, wheat or other; They are grown in pots and on the cotton soaked with water,keeping under students' observation protecting them ( just soak them regularly) and we waiting the plants rise. For discussions and comments of plant embryo development we use the plants which rose on the cotton soaked with water plants at the end of the first week. Last school year we had in the pot climbing beans which in May made pods. They were not too great but our experiment was a success. The students could deduce that there will develop those big beans which after drying will be planted again. The influence of light on plants (average duration experiment with the necessary observation time): We use two pots in which plants are of the same type (two geraniums), one of them is situated so as to get direct sunlight and other plant we put in a closed box. Although we wet both plants after a week we see that the plant that benefited from sunlight has turned strain in direct sunlight, developing normally in return the plant out of the box I have yellowed leaves, photosynthesis does not She has occurred . Students will understand the vital role of the Sun in plants' life, both in the classroom and in nature. The experiment is a method of teaching students extremely pleasant, with a remarkable percentage of acquiring more knowledge.

  3. Pixel Experiments

    DEFF Research Database (Denmark)

    Søndergaard, Karin; Petersen, Kjell Yngve; Augustesen, Christina

    2015-01-01

    Pixel Experiments The term pixel is traditionally defined as any of the minute elements that together constitute a larger context or image. A pixel has its own form and is the smallest unit seen within a larger structure. In working with the potentials of LED technology in architectural lighting...... design it became relevant to investigate the use of LEDs as the physical equivalent of a pixel as a design approach. In this book our interest has been in identifying how the qualities of LEDs can be used in lighting applications. With experiences in the planning and implementation of architectural...... lighting design in practice, one quickly experiences and realises that there are untapped potentials in the attributes of LED technology. In this research, speculative studies have been made working with the attributes of LEDs in architectural contexts, with the ambition to ascertain new strategies...

  4. Researching Experiences

    DEFF Research Database (Denmark)

    Gjedde, Lisa; Ingemann, Bruno

    , dialogue, moods, values and narratives have been investigated qualitatively with more than sixty informants in a range of projects. The processual methodological insights are put into a theoretical perspective and also presented as pragmatic dilemmas.      Researching Experiences is relevant not only...... for students and researchers in media and communication studies but also for practitioners within the fields of media, communication and experience design....... for researching experiences in a variety of settings ranging from the museum, to news photography, and interactive media. The research led to the development of a set of methodological tools and approaches we term the ReflexivityLab. The interaction in the experimental situation between the media and body...

  5. Fitness is strongly influenced by rare mutations of large effect in a microbial mutation accumulation experiment.

    Science.gov (United States)

    Heilbron, Karl; Toll-Riera, Macarena; Kojadinovic, Mila; MacLean, R Craig

    2014-07-01

    Our understanding of the evolutionary consequences of mutation relies heavily on estimates of the rate and fitness effect of spontaneous mutations generated by mutation accumulation (MA) experiments. We performed a classic MA experiment in which frequent sampling of MA lines was combined with whole genome resequencing to develop a high-resolution picture of the effect of spontaneous mutations in a hypermutator (ΔmutS) strain of the bacterium Pseudomonas aeruginosa. After ∼644 generations of mutation accumulation, MA lines had accumulated an average of 118 mutations, and we found that average fitness across all lines decayed linearly over time. Detailed analyses of the dynamics of fitness change in individual lines revealed that a large fraction of the total decay in fitness (42.3%) was attributable to the fixation of rare, highly deleterious mutations (comprising only 0.5% of fixed mutations). Furthermore, we found that at least 0.64% of mutations were beneficial and probably fixed due to positive selection. The majority of mutations that fixed (82.4%) were base substitutions and we failed to find any signatures of selection on nonsynonymous or intergenic mutations. Short indels made up a much smaller fraction of the mutations that were fixed (17.4%), but we found evidence of strong selection against indels that caused frameshift mutations in coding regions. These results help to quantify the amount of natural selection present in microbial MA experiments and demonstrate that changes in fitness are strongly influenced by rare mutations of large effect. Copyright © 2014 by the Genetics Society of America.

  6. EXPERIENCE WITH

    African Journals Online (AJOL)

    SURGERY DEPT

    Abstract. Background:Island flap techniques currently used in urethroplasty utilize the prepuce and the dorsal penile skin. Our experience with a one-stage island flap urethroplasty for urethral strictures utilizing the ventral penile skin is described. Patients and Method: This is a longitudinal study of seventy six consecutive ...

  7. Targeted resequencing and variant validation using pxlence PCR assays

    Directory of Open Access Journals (Sweden)

    Frauke Coppieters

    2016-01-01

    Full Text Available The advent of next-generation sequencing technologies had a profound impact on molecular diagnostics. PCR is a popular method for target enrichment of disease gene panels. Using our proprietary primer-design pipeline, primerXL, we have created almost one million assays covering over 98% of the human exome. Here we describe the assay specification and both in silico and wet-lab validation of a selected set of 2294 assays using both next-generation sequencing and Sanger sequencing. Using a universal PCR protocol without optimization, these assays result in high coverage uniformity and limited non-specific coverage. In addition, data indicates a positive correlation between the predictive in silico specificity score and the amount of assay non-specific coverage.

  8. Signatures of selection in tilapia revealed by whole genome resequencing.

    Science.gov (United States)

    Xia, Jun Hong; Bai, Zhiyi; Meng, Zining; Zhang, Yong; Wang, Le; Liu, Feng; Jing, Wu; Wan, Zi Yi; Li, Jiale; Lin, Haoran; Yue, Gen Hua

    2015-09-16

    Natural selection and selective breeding for genetic improvement have left detectable signatures within the genome of a species. Identification of selection signatures is important in evolutionary biology and for detecting genes that facilitate to accelerate genetic improvement. However, selection signatures, including artificial selection and natural selection, have only been identified at the whole genome level in several genetically improved fish species. Tilapia is one of the most important genetically improved fish species in the world. Using next-generation sequencing, we sequenced the genomes of 47 tilapia individuals. We identified a total of 1.43 million high-quality SNPs and found that the LD block sizes ranged from 10-100 kb in tilapia. We detected over a hundred putative selective sweep regions in each line of tilapia. Most selection signatures were located in non-coding regions of the tilapia genome. The Wnt signaling, gonadotropin-releasing hormone receptor and integrin signaling pathways were under positive selection in all improved tilapia lines. Our study provides a genome-wide map of genetic variation and selection footprints in tilapia, which could be important for genetic studies and accelerating genetic improvement of tilapia.

  9. SNP detection for massively parallel whole-genome resequencing

    DEFF Research Database (Denmark)

    Li, Ruiqiang; Li, Yingrui; Fang, Xiaodong

    2009-01-01

    Next-generation massively parallel sequencing technologies provide ultrahigh throughput at two orders of magnitude lower unit cost than capillary Sanger sequencing technology. One of the key applications of next-generation sequencing is studying genetic variation between individuals using whole...

  10. A calibrated human Y-chromosomal phylogeny based on resequencing

    Science.gov (United States)

    Wei, Wei; Ayub, Qasim; Chen, Yuan; McCarthy, Shane; Hou, Yiping; Carbone, Ignazio; Xue, Yali; Tyler-Smith, Chris

    2013-01-01

    We have identified variants present in high-coverage complete sequences of 36 diverse human Y chromosomes from Africa, Europe, South Asia, East Asia, and the Americas, representing eight major haplogroups. After restricting our analysis to 8.97 Mb of the unique male-specific Y sequence, we identified 6662 high-confidence variants, including single-nucleotide polymorphisms (SNPs), multi-nucleotide polymorphisms (MNPs), and indels. We constructed phylogenetic trees using these variants, or subsets of them, and recapitulated the known structure of the tree. Assuming a male mutation rate of 1 × 10−9 per base pair per year, the time depth of the tree (haplogroups A3-R) was ∼101,000–115,000 yr, and the lineages found outside Africa dated to 57,000–74,000 yr, both as expected. In addition, we dated a striking Paleolithic male lineage expansion to 41,000–52,000 yr ago and the node representing the major European Y lineage, R1b, to 4000–13,000 yr ago, supporting a Neolithic origin for these modern European Y chromosomes. In all, we provide a nearly 10-fold increase in the number of Y markers with phylogenetic information, and novel historical insights derived from placing them on a calibrated phylogenetic tree. PMID:23038768

  11. Extending Experiences

    DEFF Research Database (Denmark)

    A computer game's player is experiencing not only the game as a designer-made artefact, but also a multitude of social and cultural practices and contexts of both computer game play and everyday life. As a truly multidisciplinary anthology, Extending Experiences sheds new light on the mesh...... of possibilities and influences the player engages with. Part one, Experiential Structures of Play, considers some of the key concepts commonly used to address the experience of a computer game player. The second part, Bordering Play, discusses conceptual and practical overlaps of games and everyday life...... and the impacts of setting up, crossing and breaking the boundaries of game and non-game. Part three, Interfaces of Play, looks at games as technological and historical artefacts and commodities. The fourth part, Beyond Design, introduces new models for the practical and theoretical dimensions of game design....

  12. Dirac experiment

    Energy Technology Data Exchange (ETDEWEB)

    Gomez, F.; Adeva, B.; Afanasev, L.; Benayoun, M.; Brekhovskikh, V.; Caragheorgheopol, G.; Cechak, T.; Chiba, M.; Constantinescu, S.; Doudarev, A.; Dreossi, D.; Drijard, D.; Ferro-Luzzi, M.; Gallas, M.V.; Gerndt, J.; Giacomich, R.; Gianotti, P.; Goldin, D.; Gorin, A.; Gortchakov, O.; Guaraldo, C.; Hansroul, M.; Hosek, R.; Iliescu, M.; Jabitski, M.; Kalinina, N.; Karpoukhine, V.; Kluson, J.; Kobayashi, M.; Kokkas, P.; Komarov, V.; Koulikov, A.; Kouptsov, A.; Krouglov, V.; Krouglova, L.; Kuroda, K.-I.; Lanaro, A.; Lapshine, V.; Lednicky, R.; Leruste, P.; Levisandri, P.; Lopez Aguera, A.; Lucherini, V.; Maki, T.; Manuilov, I.; Montanet, L.; Narjoux, J.-L.; Nemenov, L.; Nikitin, M.; Nunez Pardo, T.; Okada, K.; Olchevskii, V.; Pazos, A.; Pentia, M.; Penzo, A.; Perreau, J.-M.; Petrascu, C.; Plo, M.; Ponta, T.; Pop, D.; Riazantsev, A.; Rodriguez, J.M.; Rodriguez Fernandez, A.; Rykaline, V.; Santamarina, C.; Saborido, J.; Schacher, J.; Sidorov, A.; Smolik, J.; Takeutchi, F.; Tarasov, A.; Tauscher, L.; Tobar, M.J.; Trusov, S.; Vazquez, P.; Vlachos, S.; Yazkov, V.; Yoshimura, Y.; Zrelov, P

    2001-04-01

    The main objective of DIRAC experiment is the measurement of the lifetime {tau} of the exotic hadronic atom consisting of {pi}{sup +} and {pi}{sup -} mesons. The lifetime of this atom is determined by the decay mode {pi}{sup +} {pi}{sup -} {yields} {pi}{sup 0} {pi}{sup 0} due to the strong interaction. Through the precise relationship between the lifetime and the S-wave pion-pion scattering length difference |a{sub 0} - a{sub 2}| for isospin 0 and 2 (respectively), a measurement of {tau} with an accuracy of 10% will allow a determination of |a{sub 0} - a{sub 2}| at a 5% precision level. Pion-pion scattering lengths have been calculated in the framework of chiral perturbation theory with an accuracy below 5%. In this way DIRAC experiment will provide a crucial test of the chiral symmetry breaking scheme in QCD effective theories at low energies.

  13. Dirac experiment

    CERN Document Server

    Gómez, F; Afanasev, L; Benayoun, M; Brekhovskikh, V; Caragheorgheopol, G; Cechák, T; Chiba, M; Constantinescu, S; Doudarev, A; Dreossi, D; Drijard, Daniel; Ferro-Luzzi, M; Gallas, M V; Gerndt, J; Giacomich, R; Gianotti, P; Goldin, D; Gorin, A; Gortchakov, O; Guaraldo, C; Hansroul, M; Hosek, R; Iliescu, M; Jabitski, M; Kalinina, N; Karpoukhine, V; Kluson, J; Kobayshi, M; Kokkas, P; Komarov, V; Koulikov, A; Kouptsov, A; Krouglov, V; Krouglova, L; Kuroda, K I; Lanaro, A; Lapshine, B; Lednicky, R; Leruste, P; Levisandri, P; López-Aguera, A; Lucherini, V; Mäki, T; Manuilov, I; Montanet, L; Narjoux, J L; Nemenov, L; Nikitin, M; Nunez Pardo, T; Okada, K; Olchevskii, V; Pazos, A; Pentia, M; Penzo, Aldo L; Perreau, J M; Petrascu, C; Pló, M; Ponta, T; Pop, D; Riazantsev, A; Rodríguez, J M; Rodriguez Fernandez, A; Rykaline, V; Santamarina, C; Saborido, J; Schacher, J; Sidorov, A; Smolik, J; Takeutchi, F; Tarasov, A; Tauscher, L; Tobar, M J; Trusov, S; Vasquez, P; Vlachos, S; Yazkov, V; Yoshimura, Y; Zrelov, P

    2001-01-01

    The main objective of DIRAC experiment is the measurement of the lifetime tau of the exotic hadronic atom consisting of pi /sup +/ and pi /sup -/ mesons. The lifetime of this atom is determined by the decay mode pi /sup +/ pi /sup -/ to pi /sup 0/ pi /sup 0/ due to the strong interaction. Through the precise relationship between the lifetime and the S-wave pion-pion scattering length difference a/sub 0/-a/sub 2/ for isospin 0 and 2 (respectively), a measurement of tau with an accuracy of 10% will allow a determination of a/sub 0/-a/sub 2/at a 5% precision level. Pion-pion scattering lengths have been calculated in the framework of chiral perturbation theory with an accuracy below 5%. In this way DIRAC experiment will provide a crucial test of the chiral symmetry breaking scheme in QCD effective theories at low energies. (19 refs).

  14. Experiment 7

    CERN Multimedia

    CERN PhotoLab

    1968-01-01

    Over-all view of the arrangement of counters around the polarized target in the kaon-polarized proton experiment. The beam enters through the quadrupole magnet on the left, and strikes the target placed in a 18.5 kG field (one pole of the magnet is just visible above the ring of counters). The scatterred kaon and recoil proton are detected by the counters on the ring and recorded onto magnetic tape.

  15. QUBIC Experiment

    OpenAIRE

    Stolpovskiy, Mikhail

    2016-01-01

    QUBIC is a ground-based experiment, currently under construction, that uses the novel bolometric interferometry technology. It is dedicated to measure the primordial B-modes of CMB. As a bolometric interferometer, QUBIC has high sensitivity and good systematics control. Dust contamination is controlled by operating with two bands -- 150 and 220 GHz. There are two possible sites for QUBIC: either Concordia station in Antarctic or in the Argentinian Puna desert. It is planned to see the first l...

  16. Experiments expectations

    OpenAIRE

    Gorini, B; Meschi, E

    2014-01-01

    This paper presents the expectations and the constraints of the experiments relatively to the commissioning procedure and the running conditions for the 2015 data taking period. The views about the various beam parameters for the p-p period, like beam energy, maximum pileup, bunch spacing and luminosity limitation in IP2 and IP8, are discussed. The goals and the constraints of the 2015 physics program are also presented, including the heavy ions period as well as the special...

  17. Amplicon restriction patterns associated with nitrogenase activity of ...

    Indian Academy of Sciences (India)

    2013-10-01

    Oct 1, 2013 ... Pl. Mol. Biol. Reporter 1 19–21. Jackson ML 1967 Soil chemical analysis (New Delhi, India: Pren- tice Hall). Navarro E, Bousquet J, Moiroud A, Munive A, Piou D and. Normand P 2003 Molecular phylogeny of Alnus (Betulaceae), inferred from nuclear ribosomal DNA ITS sequences. Plant Soil. 254 207–217.

  18. Amplicon restriction patterns associated with nitrogenase activity of ...

    Indian Academy of Sciences (India)

    2013-10-01

    Oct 1, 2013 ... wood can be used as timber and fuel. Actinorhizal trees such as. Alnus have been used in the reclamation of mine spoils in. Britain (Schwintzer .... labelled plastic bags, brought to the laboratory and stored at. −80°C for further studies. Isolation of DNA from the leaves was performed using the modified CTAB ...

  19. Amplicon restriction patterns associated with nitrogenase activity of ...

    Indian Academy of Sciences (India)

    Trees of Myrica sp. grow abundantly in the forests of Meghalaya, India. These trees are actinorhizal and harbour nitrogen-fixing Frankia in their root nodules and contribute positively towards the enhancement of nitrogen status of forest areas. They can be used in rejuvenation of mine spoils and nitrogen-depleted fallow ...

  20. Experimenting with a design experiment

    Directory of Open Access Journals (Sweden)

    Bakker, Judith

    2012-12-01

    Full Text Available The design experiment is an experimental research method that aims to help design and further develop new (policy instruments. For the development of a set of guidelines for the facilitation of citizens’ initiatives by local governments, we are experimenting with this method. It offers good opportunities for modeling interventions by testing their instrumental validity –the usefulness for the intended practical purposes. At the same time design experiments are also useful for evaluating the empirical validity of theoretical arguments and the further development of these arguments in the light of empirical evidence (by using e.g. the technique of pattern matching. We describe how we have applied this methodology in two cases and discuss our research approach. We encountered some unexpected difficulties, especially in the cooperation with professionals and citizens. These difficulties complicate the valid attribution of causal effects to the use of the new instrument. However, our preliminary conclusion is that design experiments are useful in our field of study

    El experimento de diseño es un método de investigación experimental que tiene como objetivo diseñar y desarrollar posteriormente nuevas herramientas (políticas. En este artículo experimentamos con este método para desarrollar un conjunto de directrices que permitan a los gobiernos locales facilitar las iniciativas ciudadanas. El método ofrece la oportunidad de modelar las intervenciones poniendo a prueba su validez instrumental (su utilidad para el fin práctico que se proponen. Al mismo tiempo, los experimentos de diseño son útiles también para evaluar la validez empírica de las discusiones teóricas y el posterior desarrollo de esas discusiones a la luz de la evidencia empírica (usando, por ejemplo, técnicas de concordancia de patrones. En este trabajo describimos cómo hemos aplicado este método a dos casos y discutimos nuestro enfoque de

  1. Operating experience

    International Nuclear Information System (INIS)

    McRae, L.P.; Six, D.E.

    1991-01-01

    In 1987, Westinghouse Hanford Company began operating a first-generation integrated safeguards system in the Plutonium Finishing Plant storage vaults. This Vault Safety and Inventory System is designed to integrate data into a computer-based nuclear material inventory monitoring system. The system gathers, in real time, measured physical parameters that generate nuclear material inventory status data for thousands of stored items and sends tailored report to the appropriate users. These data include canister temperature an bulge data reported to Plant Operations and Material Control and Accountability personnel, item presence and identification data reported to Material Control and Accountability personnel, and unauthorized item movement data reported to Security response forces and Material Control and Accountability personnel. The Westinghouse Hanford Company's experience and operational benefits in using this system for reduce radiation exposure, increase protection against insider threat, and real-time inventory control are discussed in this paper

  2. Management Experiences

    Directory of Open Access Journals (Sweden)

    Gheorghe Popovici

    2016-12-01

    Full Text Available The paper presents the results of the survey about the changes in modern management, identified from the experience of Romanian managers. By this online study one presents both the obstacles encountered and the recommendations for such a type of management that the present and future mangers must take into account. What motivated the respondent Romanian managers most to open their own business is the independence it offered them. They work in the field they have liked since they were young. The second reason was the perspective to have an additional income from the business development. The third argument in favour of opening a business is the possibility to assure the balance between personal life and career.

  3. Particle physics experiments 1983

    International Nuclear Information System (INIS)

    Stuart, G.W.

    1983-01-01

    The report describes work carried out in 1983 on experiments approved by the Particle Physics Experiments Selection Panel. The contents consist of unedited contributions from each experiment. (author)

  4. Sleep Monitoring Experiment - Skylab Experiment M133

    Science.gov (United States)

    1970-01-01

    This 1970 photograph shows equipment for the Skylab's Sleep Monitoring Experiment (M133), a medical evaluation designed to objectively determine the amount and quality of crewmembers' inflight sleep. The experiment monitored and recorded electroencephalographic (EEG) and electrooculographic (EOG) activity during astronauts' sleep periods. One of the astronauts was selected for this experiment and wore a fitted cap during his sleep periods. The Marshall Space Flight Center had program management responsibility for the development of Skylab hardware and experiments.

  5. Design of modern experiments

    International Nuclear Information System (INIS)

    Park, Sung Hweon

    1984-03-01

    This book is for researchers and engineers, which is written to focus on practical design of experiments. It gives descriptions of conception of design of experiments, basic statistics theory, one way design of experiment, two-way layout without repetition, two-way layout with repetition, partition, a correlation analysis and regression analysis, latin squares, factorial design, design of experiment by table of orthogonal arrays, design of experiment of response surface, design of experiment on compound, Evop, and design of experiment of taguchi.

  6. Data acquisition. GRAAL experiment. Hybrid reactor experiment. AMS experiment

    International Nuclear Information System (INIS)

    Barancourt, D.; Barbier, G.; Bosson, G.; Bouvier, J.; Gallin-Martel, L.; Meillon, B.; Stassi, P.; Tournier, M.

    1997-01-01

    The main activity of the data acquisition team has consisted in hardware and software developments for the GRAAL experiment with the trigger board, for the 'Reacteurs Hybrides' group with an acquisition board ADCVME8V and for the AMS experiment with the monitoring of the aerogel detector. (authors)

  7. Particle physics experiments 1989

    International Nuclear Information System (INIS)

    Bairstow, R.

    1990-01-01

    This report describes work carried out in 1989 on experiments approved by the Particle Physics Experiments Selection Panel of Rutherford Appleton Laboratory. The contents consist of unedited contributions from each experiment. (author)

  8. Particle physics experiments

    International Nuclear Information System (INIS)

    Stuart, G.W.

    1986-01-01

    The report of the Rutherford Appleton Laboratory describes the work carried out in 1985 on experiments approved by the Particle Physics Experiments Selection Panel. The contents consist of unedited contributions from each experiment. (author)

  9. Particle physics experiments 1987

    International Nuclear Information System (INIS)

    Stuart, G.W.

    1988-01-01

    This report describes work carried out in 1987 on experiments approved by the Particle Physics Experiments Selection Panel (United Kingdom). The contents consist of unedited contributions from each experiment. (author)

  10. Particle physics experiments 1986

    International Nuclear Information System (INIS)

    Stuart, G.W.

    1987-01-01

    The paper presents research work carried out in 1986 on 52 elementary particle experiments approved by the Particle Physics Experiments Selection Panel. Most of the experiments were collaborative and involved research groups from different countries. About half of the experiments were conducted at CERN, the remaining experiments employed the accelerators: LAMPT, LEP, PETRA, SLAC, and HERA. The contents consist of unedited contributions from each experiment. (U.K.)

  11. Data acquisition. GRAAL experiment. Hybrid reactor experiment. AMS experiment; Acquisition de donnees. Experience GRAAL. Experience reacteurs hybrides. Experience AMS

    Energy Technology Data Exchange (ETDEWEB)

    Barancourt, D.; Barbier, G.; Bosson, G.; Bouvier, J.; Gallin-Martel, L.; Meillon, B.; Stassi, P.; Tournier, M. [Inst. des Sciences Nucleaires, Grenoble-1 Univ., 38 (France)

    1997-12-31

    The main activity of the data acquisition team has consisted in hardware and software developments for the GRAAL experiment with the trigger board, for the `Reacteurs Hybrides` group with an acquisition board ADCVME8V and for the AMS experiment with the monitoring of the aerogel detector. (authors) 2 figs.

  12. Particle physics experiments 1982

    International Nuclear Information System (INIS)

    Rousseau, M.D.; Stuart, G.

    1983-01-01

    Work carried out in 1982 on 52 experiments approved by the Particle Physics Experiments Selection Panel is described. Each experiment is listed under title, collaboration, technique, accelerator, year of running, status and spokesman. Unedited contributions are given from each experiment. (U.K.)

  13. The User Experience

    Science.gov (United States)

    Schmidt, Aaron

    2010-01-01

    User experience (UX) is about arranging the elements of a product or service to optimize how people will interact with it. In this article, the author talks about the importance of user experience and discusses the design of user experiences in libraries. He first looks at what UX is. Then he describes three kinds of user experience design: (1)…

  14. Indian summer monsoon experiments

    OpenAIRE

    Bhat, GS; Narasimha, R

    2007-01-01

    Eight major field experiments have been carried out so far addressing the Indian summer monsoon. While these experiments were international and the impetus was external till 1980, India’s own monsoon programmes evolved since then. In this article, objectives and outcomes from some of these experiments are described. It is shown that monsoon experiments have contributed in several ways. Each experiment enhanced the infrastructure facilities in the country, brought together scientists from diff...

  15. Adaptive structures flight experiments

    Science.gov (United States)

    Martin, Maurice

    The topics are presented in viewgraph form and include the following: adaptive structures flight experiments; enhanced resolution using active vibration suppression; Advanced Controls Technology Experiment (ACTEX); ACTEX program status; ACTEX-2; ACTEX-2 program status; modular control patch; STRV-1b Cryocooler Vibration Suppression Experiment; STRV-1b program status; Precision Optical Bench Experiment (PROBE); Clementine Spacecraft Configuration; TECHSAT all-composite spacecraft; Inexpensive Structures and Materials Flight Experiment (INFLEX); and INFLEX program status.

  16. The ERASMUS experiment archive

    Science.gov (United States)

    Isakeit, Dieter; Sabbatini, Massimo; Carey, William

    2005-08-01

    The Erasmus Experiment Archive is an electronic database, accessible through the Internet, that collects in a single reference repository scientific, technical and operational information regarding the experiments performed in the experiment facilities which fall under the responsibility of the ESA Directorate of Human Spaceflight, Microgravity and Exploration. The archive is operated, filled with content and kept up-to-date by the Erasmus User Centre. which forms part of the Directorate. The archive shares its records and is interoperable with similar experiment archives of the partner agencies NASA USA) and JAXA (Japan) through a mutually agreed standard for experiment records called the International Distributed Experiment Archive (IDEA).

  17. Experiment WA1 (CDHS Neutrino Experiment)

    CERN Multimedia

    CERN PhotoLab

    1977-01-01

    Experiment WA1, also known under CDHS (CERN, Dortmund, Heidelberg, Saclay; spokesman Jack Steinberger), was the first neutrino experiment on the SPS, in its West Area. Magnetized iron (with a toroidal field) forms the core of the detector. On its outside we see drift chambers and photomultipliers (detecting the light from the plastic scintillators further in). Peter Schilly is wearing a white coat. See also CERN Annual Report 1976, p.57.

  18. Particle physics experiments 1984

    International Nuclear Information System (INIS)

    Stuart, G.

    1985-01-01

    The Rutherford Appleton laboratory report describes work carried out in 1984 on experiments approved by the Particle Physics selection panel. The contents consist of unedited contributions from each experiment. (author)

  19. GARP Atlantic Tropical Experiment

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — The GARP Atlantic Tropical Experiment (GATE) was the first major international experiment of the Global Atmospheric Research Program (GARP). It was conducted over...

  20. Social experience infrastructure

    DEFF Research Database (Denmark)

    Kvistgaard, Peter

    2006-01-01

    and explorative fashion to share with others thoughts and ideas concerning the development of new ways to construct/reconstruct recreational spaces with a better coherence with regard to designing experiences. This article claims that it is possible to design recreational spaces with good social experience...... infrastructure in order to create experience spaces for personal experiences (in line with Schultze’s social constructivist view of experiences) without completely adhering to the economic rationalist thoughts and guidelines of Pine & Gilmore that claim that experiences can be designed and controlled...... by experience designers. The notion of social experience infrastructure may be placed in the cross field between the social constructivist and the economic rationalist approach....

  1. Organic chemistry experiment

    International Nuclear Information System (INIS)

    Mun, Seok Sik

    2005-02-01

    This book deals with organic chemistry experiments, it is divided five chapters, which have introduction, the way to write the experiment report and safety in the laboratory, basic experiment technic like recrystallization and extraction, a lot of organic chemistry experiments such as fischer esterification, ester hydrolysis, electrophilic aromatic substitution, aldol reaction, benzoin condensation, wittig reaction grignard reaction, epoxidation reaction and selective reduction. The last chapter introduces chemistry site on the internet and way to find out reference on chemistry.

  2. The Experiment Factory: standardizing behavioral experiments

    Directory of Open Access Journals (Sweden)

    Vanessa V Sochat

    2016-04-01

    Full Text Available The administration of behavioral and experimental paradigms for psychology research is hindered by lack of a coordinated effort to develop and deploy standardized paradigms. While several frameworks (de Leeuw (2015; McDonnell et al. (2012; Mason and Suri (2011; Lange et al. (2015 have provided infrastructure and methods for individual research groups to develop paradigms, missing is a coordinated effort to develop paradigms linked with a system to easily deploy them. This disorganization leads to redundancy in development, divergent implementations of conceptually identical tasks, disorganized and error-prone code lacking documentation, and difficulty in replication. The ongoing reproducibility crisis in psychology and neuroscience research (Baker (2015; Open Science Collaboration (2015 highlights the urgency of this challenge: reproducible research in behavioral psychology is conditional on deployment of equivalent experiments. A large, accessible repository of experiments for researchers to develop collaboratively is most efficiently accomplished through an open source framework. Here we present the Experiment Factory, an open source framework for the development and deployment of web-based experiments. The modular infrastructure includes experiments, virtual machines for local or cloud deployment, and an application to drive these components and provide developers with functions and tools for further extension. We release this infrastructure with a deployment (http://www.expfactory.org that researchers are currently using to run a set of over 80 standardized web-based experiments on Amazon Mechanical Turk. By providing open source tools for both deployment and development, this novel infrastructure holds promise to bring reproducibility to the administration of experiments, and accelerate scientific progress by providing a shared community resource of psychological paradigms.

  3. Designing Urban Experiences

    DEFF Research Database (Denmark)

    Jantzen, Christian; Vetner, Mikael

    2008-01-01

    , and deals with how urban experiences can be designed through careful consideration of how experiences are constituted. Taking the psychological structure of experiences into account when designing urbanity is not only interesting, but also highly relevant as Zuidas is competing with other international...

  4. Virtual neutron scattering experiments

    DEFF Research Database (Denmark)

    Overgaard, Julie Hougaard; Bruun, Jesper; May, Michael

    2016-01-01

    We describe how virtual experiments can be utilized in a learning design that prepares students for hands-on experiments at large-scale facilities. We illustrate the design by showing how virtual experiments are used at the Niels Bohr Institute in a master level course on neutron scattering....... In the last week of the course, students travel to a large-scale neutron scattering facility to perform real neutron scattering experiments. Through student interviews and survey answers, we argue, that the virtual training prepares the students to engage more fruitfully with experiments by letting them focus...

  5. Virtual neutron scattering experiments

    DEFF Research Database (Denmark)

    Overgaard, Julie Hougaard; Bruun, Jesper; May, Michael

    2017-01-01

    We describe how virtual experiments can be utilized in a learning design that prepares students for hands-on experiments at large-scale facilities. We illustrate the design by showing how virtual experiments are used at the Niels Bohr Institute in a master level course on neutron scattering....... In the last week of the course, students travel to a large-scale neutron scattering facility to perform real neutron scattering experiments. Through student interviews and survey answers, we argue, that the virtual training prepares the students to engage more fruitfully with experiments by letting them focus...

  6. Experiments in physical chemistry

    CERN Document Server

    Wilson, J M; Denaro, A R

    1968-01-01

    Experiments in Physical Chemistry, Second Edition provides a compilation of experiments concerning physical chemistry. This book illustrates the link between the theory and practice of physical chemistry. Organized into three parts, this edition begins with an overview of those experiments that generally have a simple theoretical background. Part II contains experiments that are associated with more advanced theory or more developed techniques, or which require a greater degree of experimental skill. Part III consists of experiments that are in the nature of investigations wherein these invest

  7. The Belle II Experiment

    CERN Document Server

    Kahn, J

    2017-01-01

    Set to begin data taking at the end of 2018, the Belle II experiment is the next-generation B-factory experiment hosted at KEK in Tsukuba, Japan. The experiment represents the cumulative effort from the collaboration of experimental and detector physics, computing, and software development. Taking everything learned from the previous Belle experiment, which ran from 1998 to 2010, Belle II aims to probe deeper than ever before into the field of heavy quark physics. By achieving an integrated luminosity of 50 ab−1 and accumulating 50 times more data than the previous experiment across its lifetime, along with a rewritten analysis framework, the Belle II experiment will push the high precision frontier of high energy physics. This paper will give an overview of the key components and development activities that make the Belle II experiment possible.

  8. Diagnostics for pellet experiments

    International Nuclear Information System (INIS)

    Johnson, R.R.

    1978-01-01

    The target diagnostics which are being used and planned in current laser driven ICF Experiments are described. Most of these diagnostics can be easily applied to future ion-beam fusion experiments. The status of laser fusion diagnostics has been much improved in the last 5 years and further improvements can be expected and should be available when the first ICF experiments using ion beams are performed. As an example, x-ray temporal and spatial resolutions are now approximately 5 psec and 3 μm, which is approximately a factor of 4 better than the resolution reported in the first implosion experiments. As one plans ahead for ion-beam fusion experiments it should be emphasized that high yield experiments are easier to diagnose provided adequate shielding is employed. However, in the event that the first high yield experiments fail it will be necessary to have diagnostics available to determine where the problems lie. In laser fusion it is interesting to note that higher laser powers are required now for breakeven experiments than first anticipated, mainly because some aspects of the laser-interaction physics were not recognized until the experiments were carefully diagnosed. Thus as has been pointed out, it may be necessary to increase the energy of the ion-beam driver to enable us to do breakeven experiments with high confidence

  9. Learning and Experience

    DEFF Research Database (Denmark)

    Olesen, Henning Salling

    2017-01-01

    Abstract: This chapter introduces a psycho-societal approach to theorizing learning, combining a materialist theory of socialization with a hermeneutic interpretation methodology. The term "approach" indicates the intrinsic connection between theory, empirical research process and epistemic subject....... Learning is theorized as dynamic subjective experience of (socially situated) realities, counting on individual subjectivity as well as subjective aspects of social interaction. This psycho-societal theory of subjective experiences conceptualizes individual psychic development as interactional experience...... of societal relations, producing an inner psycho-dynamic as a conscious and unconscious individual resource in future life. The symbolization of immediate sensual experiences form an individual life experience of social integration, language use being the medium of collective, social experience (knowledge...

  10. Experiment, right or wrong

    CERN Document Server

    Franklin, Allan

    2008-01-01

    In Experiment, Right or Wrong, Allan Franklin continues his investigation of the history and philosophy of experiment presented in his previous book, The Neglect of Experiment. In this new study, Franklin considers the fallibility and corrigibility of experimental results and presents detailed histories of two such episodes: 1) the experiment and the development of the theory of weak interactions from Fermi's theory in 1934 to the V-A theory of 1957 and 2) atomic parity violation experiments and the Weinberg-Salam unified theory of electroweak interactions of the 1970s and 1980s. In these episodes Franklin demonstrates not only that experimental results can be wrong, but also that theoretical calculations and the comparison between experiment and theory can also be incorrect. In the second episode, Franklin contrasts his view of an "evidence model" of science in which questions of theory choice, confirmation, and refutation are decided on the basis of reliable experimental evidence, with that proposed by the ...

  11. Stimulated Brillouin scattering experiments

    International Nuclear Information System (INIS)

    Slater, D.C.; Berger, R.L.; Busch, G.; Kinzer, C.M.; Mayer, F.J.; Powers, L.V.; Tanner, D.J.

    1981-01-01

    This report describes two experiments in which SBS would be expected to play an important role. In the first experiment, we find a clear signature of the Brillouin backscatter of a short (100 psec) pulse from a long (approx. 50 μm) gradient length gas target plasma. The second experiment used much longer (approx. 1 nsec) pulses on spherical glass shell targets. These experiments were done with both narrow ( 30A) bandwidth laser light. Using one-dimensional, spherically symmetric fluid simulations, we have attempted to model many of the laser-plasma interaction processes which combine to determine the amount of absorbed energy in the long-pulse experiments. These simulations indicate that modest laser bandwidths are successful in reducing the level of SBS at the irradiances ( 15 W/cm 2 ) used in these experiments

  12. Experience composite worth: A combination of experience quality ...

    African Journals Online (AJOL)

    This research suggests that experience quality and experience value be combined into a single multi-dimensional construct, termed experience composite worth. It briefly describes customer experience, experience quality and experience value and the overlap in the dimensions used in outlining experience quality and ...

  13. Linking consumer experiences

    DEFF Research Database (Denmark)

    Smed, Karina Madsen

    become part of the individual self, worldview, and behaviour. This paper seeks to explore links between consumer experiences through the exploration of narrative sequences in travel blogs. Findings indicate that non-consumption is a central element to the bloggers and also indicative of a community......Consumers consume products in various ways serving a number of purposes. Much attention has been paid to experiences attached to consumption, sometimes very explicitly, e.g. in tourism, the essence of which is experiences of various sorts, but often also implicitly as internalised experiences...

  14. Real Life Experiences with Experience Design

    DEFF Research Database (Denmark)

    Dalsgård, Peter; Halskov, Kim

    2006-01-01

    technologies for knowledge dissemination and marketing, in cooperation with public institutions and businesses. We argue that collaborative formulation of core design intentions and values is a valuable instrument in guiding experience design processes, and present three cases from this project, two of which...... resulted in interactive installations. The case installations range from walk-up-and-use consoles, to immersive, responsive, environments based on bodily interaction. We compare the installations, and discuss the interrelations between the resulting interfaces and the intentions for creating...

  15. Experience with MODSIM II

    International Nuclear Information System (INIS)

    Streets, J.; Berg, D.; Oleynik, G.; Pordes, R.; Slimmer, D.

    1992-02-01

    We present results of computer simulations for Data Acquisition systems for large fixed target experiments in an object oriented simulation language, MODSIM. This paper summarizes our experiences and presents preliminary results from the simulation already completed. We also indicate the resources required for this project

  16. Experiments as politics

    NARCIS (Netherlands)

    Spears, R; Smith, HJ

    The purpose of this paper is to illustrate the political nature of laboratory experiments. Such experiments can be construed as paradigms of power, open to construction and debate, where different agents and interests are involved in a process of struggle over both (re)presentation and substance.

  17. Long baseline neutrino experiments

    Indian Academy of Sciences (India)

    High statistics experiment with NNO = 3000/year. Can measure P(νµ → νe). So good sensitivity to θ13 and CP violation but not to matter effects. BNL to Homestake: L = 2540 km, being planned. Ultimate accelerator beam experiment with very high statistics with NNO = 3000/year. Can achieve all the goals listed above with a ...

  18. ATLAS Experiment Brochure - Italian

    CERN Multimedia

    2018-01-01

    ATLAS is one of the four major experiments at the Large Hadron Collider at CERN. It is a general-purpose particle physics experiment run by an international collaboration, and is designed to exploit the full discovery potential and the huge range of physics opportunities that the LHC provides.

  19. ATLAS Experiment Brochure - Serbian

    CERN Multimedia

    2018-01-01

    ATLAS is one of the four major experiments at the Large Hadron Collider at CERN. It is a general-purpose particle physics experiment run by an international collaboration, and is designed to exploit the full discovery potential and the huge range of physics opportunities that the LHC provides.

  20. ATLAS Experiment Brochure

    CERN Multimedia

    AUTHOR|(INSPIRE)INSPIRE-00085461

    2016-01-01

    ATLAS is one of the four major experiments at the Large Hadron Collider at CERN. It is a general-purpose particle physics experiment run by an international collaboration, and is designed to exploit the full discovery potential and the huge range of physics opportunities that the LHC provides.

  1. Realisation, experience, and purification

    DEFF Research Database (Denmark)

    Sobisch, Jan-Ulrich

    2013-01-01

    Often Buddhist mantra practises are portrayed as making and maintaining meditation experiences. Jigten Sumgön explains in his dGongs gcig (5.19) that such a 'making and maintaining' is only a mental fabrication and needs to be purified. Realisation does not arise from experience, but from...

  2. Neutrino oscillation experiments

    International Nuclear Information System (INIS)

    Camilleri, L.

    1996-01-01

    Neutrino oscillation experiments (ν μ →ν e and ν μ →ν τ ) currently being performed at accelerators are reviewed. Future plans for short and long base-line experiments are summarized. (author) 10 figs., 2 tabs., 29 refs

  3. On the Poggendorff Experiment

    Science.gov (United States)

    Coelho, Ricardo Lopes; Silva, P. A. S.; Borges, Paulo de Faria

    2015-01-01

    Poggendorff showed experimentally, in the middle of the 19th century, that the weight of an Atwood machine is reduced when it is brought to motion. His experiment has been revisited from time to time, making use of instrumentation that reflects the technological development of the moment. In this paper, the evolution of the experiment is briefly…

  4. Particle physics experiments 1992

    International Nuclear Information System (INIS)

    Roberts, B.A.

    1993-03-01

    The research programs described here were carried out in 1992 at Rutherford Appleton Laboratory and funded by the United Kingdom Science and Engineering Research Council. The area covered in these experiments is particle physics. Unedited contributions from over forty experimental programs are included. Experiments are listed according to their current status, the accelerator used and its years of operation. (UK)

  5. Ball Collision Experiments

    Science.gov (United States)

    Cross, R.

    2015-01-01

    Experiments are described on collisions between two billiard balls and between a bat and a ball. The experiments are designed to extend a student's understanding of collision events and could be used either as a classroom demonstration or for a student project.

  6. Neutrino oscillation experiments

    Energy Technology Data Exchange (ETDEWEB)

    Camilleri, L. [European Organization for Nuclear Research, Geneva (Switzerland)

    1996-11-01

    Neutrino oscillation experiments ({nu}{sub {mu}}{yields}{nu}{sub e} and {nu}{sub {mu}}{yields}{nu}{sub {tau}}) currently being performed at accelerators are reviewed. Future plans for short and long base-line experiments are summarized. (author) 10 figs., 2 tabs., 29 refs.

  7. Future of neutrino experiments

    Indian Academy of Sciences (India)

    January 2009 physics pp. 109–117. Future of neutrino experiments. TAKAAKI KAJITA. ICRR and IPMU, University of Tokyo, Kashiwa-no-ha 5-1-5, Kashiwa, Chiba 277-8582,. Japan. E-mail: kajita@icrr.u-tokyo.ac.jp. Abstract. Atmospheric, solar, reactor and accelerator neutrino oscillation experiments have measured Δm2.

  8. Human Simulated Diving Experiments.

    Science.gov (United States)

    Bruce, David S.; Speck, Dexter F.

    1979-01-01

    This report details several simulated divinq experiments on the human. These are suitable for undergraduate or graduate laboratories in human or environmental physiology. The experiment demonstrates that a diving reflex is precipitated by both facial cooling and apnea. (Author/RE)

  9. ATLAS Experiment Brochure - French

    CERN Document Server

    2018-01-01

    ATLAS is one of the four major experiments at the Large Hadron Collider at CERN. It is a general-purpose particle physics experiment run by an international collaboration, and is designed to exploit the full discovery potential and the huge range of physics opportunities that the LHC provides.

  10. A Duplicate Construction Experiment.

    Science.gov (United States)

    Bridgeman, Brent

    This experiment was designed to assess the ability of item writers to construct truly parallel tests based on a "duplicate-construction experiment" in which Cronbach argues that if the universe description and sampling are ideally refined, the two independently constructed tests will be entirely equivalent, and that within the limits of item…

  11. Science Experience Unit: Conservation.

    Science.gov (United States)

    Ferguson-Florissant School District, Ferguson, MO.

    GRADES OR AGES: Intermediate grades. SUBJECT MATTER: Conservation. ORGANIZATION AND PHYSICAL APPEARANCE: The guide is divided into 24 experiments. It is mimeographed and staple-bound with a paper cover. OBJECTIVES AND ACTIVITIES: A specific skill or knowledge objective is stated at the beginning of each experiment. Detailed procedures are listed…

  12. Peak Experience Project

    Science.gov (United States)

    Scott, Daniel G.; Evans, Jessica

    2010-01-01

    This paper emerges from the continued analysis of data collected in a series of international studies concerning Childhood Peak Experiences (CPEs) based on developments in understanding peak experiences in Maslow's hierarchy of needs initiated by Dr Edward Hoffman. Bridging from the series of studies, Canadian researchers explore collected…

  13. THX Experiment Overview

    Science.gov (United States)

    Wernet, Mark; Wroblewski, Adam; Locke, Randy; Georgiadis, Nick

    2016-01-01

    This presentation provides an overview of experiments conducted at NASA GRC to provide turbulent flow measurements needed for new turbulence model development and validation. The experiments include particle image velocimetry (PIV) and hot-wire measurements of mean flow velocity and temperature fields, as well as fluctuating components.

  14. Students Experience in Radiography

    International Nuclear Information System (INIS)

    Nyambane, K.D.; Faith Wanjiku Kimunge, W.F.

    2015-01-01

    The Radiography Students from Jomo Kenyatta University of Agriculture and Technology, Kenya share experiences of College Experience, Challenges, Myths and the Radiography dream. The Cultural shock and expectations towards Radiography has changed for the first timers’ students in the radiography class. This has motivated them to remove the fear of the unknown and negatives effects of radiation

  15. Teaching Knowledge Engineering: Experiences

    DEFF Research Database (Denmark)

    Andersen, Tom; Hartvig, Susanne C

    1998-01-01

    Includes description of experiences gained by teaching KE in construction domains. It outlines good starting points and overall guidance to education in applied AI.......Includes description of experiences gained by teaching KE in construction domains. It outlines good starting points and overall guidance to education in applied AI....

  16. Evaluation of two highly-multiplexed custom panels for massively parallel semiconductor sequencing on paraffin DNA.

    Science.gov (United States)

    Kotoula, Vassiliki; Lyberopoulou, Aggeliki; Papadopoulou, Kyriaki; Charalambous, Elpida; Alexopoulou, Zoi; Gakou, Chryssa; Lakis, Sotiris; Tsolaki, Eleftheria; Lilakos, Konstantinos; Fountzilas, George

    2015-01-01

    Massively parallel sequencing (MPS) holds promise for expanding cancer translational research and diagnostics. As yet, it has been applied on paraffin DNA (FFPE) with commercially available highly multiplexed gene panels (100s of DNA targets), while custom panels of low multiplexing are used for re-sequencing. Here, we evaluated the performance of two highly multiplexed custom panels on FFPE DNA. Two custom multiplex amplification panels (B, 373 amplicons; T, 286 amplicons) were coupled with semiconductor sequencing on DNA samples from FFPE breast tumors and matched peripheral blood samples (n samples: 316; n libraries: 332). The two panels shared 37% DNA targets (common or shifted amplicons). Panel performance was evaluated in paired sample groups and quartets of libraries, where possible. Amplicon read ratios yielded similar patterns per gene with the same panel in FFPE and blood samples; however, performance of common amplicons differed between panels (pevaluation is adjusted to tumor tissue FFPE DNA. To this scope, eligibility of all amplicons along with variant coverage and frequency need to be assessed.

  17. Understanding patient experience

    DEFF Research Database (Denmark)

    Andersen, Tariq O.; Andersen, Pernille R. D.; Kornum, Anders C.

    2017-01-01

    , safety) arise from getting feedback on symptoms and from continuous and comforting interaction with clinicians. With this paper, we aim to sensitise UX researchers and designers of patient-centred e-health by proposing three UX dimensions: connectedness, comprehension, and compassion.......The term 'patient experience' is currently part of a global discourse on ways to improve healthcare. This study empirically explores what patient experience is in cardiac remote monitoring and considers the implications for user experience (UX). Through interviews around the deployment of a mobile...... app that enables patients to collaborate with clinicians, we unpack experiences in six themes and present narratives of patients' lifeworlds. We find that patients' emotions are grounded in negative feelings (uncertainty, anxiety, loss of hope) and that positive experiences (relief, reassurance...

  18. Molybdenum solar neutrino experiment

    International Nuclear Information System (INIS)

    Wolfsberg, K.; Cowan, G.A.; Bryant, E.A.

    1984-01-01

    The goal of the molybdenum solar neutrino experiment is to deduce the 8 B solar neutrino flux, averaged over the past several million years, from the concentration of 98 Tc in a deeply buried molybdenum deposit. The experiment is important to an understanding of stellar processes because it will shed light on the reason for the discrepancy between theory and observation of the chlorine solar neutrino experiment. Possible reasons for the discrepancy may lie in the properties of neutrinos (neutrino oscillations or massive neutrinos) or in deficiencies of the standard solar model. The chlorine experiment only measures the 8 B neutrino flux in current times and does not address possible temporal variations in the interior of the sun, which are also not considered in the standard model. In the molybdenum experiment, we plan to measure 98 Tc (4.2 Myr), also produced by 8 B neutrinos, and possibly 97 Tc (2.6 Myr), produced by lower energy neutrinos

  19. Cryogenics for LHC experiments

    CERN Multimedia

    2001-01-01

    Cryogenic systems will be used by LHC experiments to maximize their performance. Institutes around the world are collaborating with CERN in the construction of these very low temperature systems. The cryogenic test facility in hall 180 for ATLAS magnets. High Energy Physics experiments have frequently adopted cryogenic versions of their apparatus to achieve optimal performance, and those for the LHC will be no exception. The two largest experiments for CERN's new flagship accelerator, ATLAS and CMS, will both use large superconducting magnets operated at 4.5 Kelvin - almost 270 degrees below the freezing point of water. ATLAS also includes calorimeters filled with liquid argon at 87 Kelvin. For the magnets, the choice of a cryogenic version was dictated by a combination economy and transparency to emerging particles. For the calorimeters, liquid argon was selected as the fluid best suited to the experiment's physics requirements. High Energy Physics experiments are the result of worldwide collaborations and...

  20. Space Experiment Module (SEM)

    Science.gov (United States)

    Brodell, Charles L.

    1999-01-01

    The Space Experiment Module (SEM) Program is an education initiative sponsored by the National Aeronautics and Space Administration (NASA) Shuttle Small Payloads Project. The program provides nationwide educational access to space for Kindergarten through University level students. The SEM program focuses on the science of zero-gravity and microgravity. Within the program, NASA provides small containers or "modules" for students to fly experiments on the Space Shuttle. The experiments are created, designed, built, and implemented by students with teacher and/or mentor guidance. Student experiment modules are flown in a "carrier" which resides in the cargo bay of the Space Shuttle. The carrier supplies power to, and the means to control and collect data from each experiment.

  1. Undergraduate reactor control experiment

    International Nuclear Information System (INIS)

    Edwards, R.M.; Power, M.A.; Bryan, M.

    1992-01-01

    A sequence of reactor and related experiments has been a central element of a senior-level laboratory course at Pennsylvania State University (Penn State) for more than 20 yr. A new experiment has been developed where the students program and operate a computer controller that manipulates the speed of a secondary control rod to regulate TRIGA reactor power. Elementary feedback control theory is introduced to explain the experiment, which emphasizes the nonlinear aspect of reactor control where power level changes are equivalent to a change in control loop gain. Digital control of nuclear reactors has become more visible at Penn State with the replacement of the original analog-based TRIGA reactor control console with a modern computer-based digital control console. Several TRIGA reactor dynamics experiments, which comprise half of the three-credit laboratory course, lead to the control experiment finale: (a) digital simulation, (b) control rod calibration, (c) reactor pulsing, (d) reactivity oscillator, and (e) reactor noise

  2. Simulation - modeling - experiment; Simulation - modelisation - experience

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2004-07-01

    After two workshops held in 2001 on the same topics, and in order to make a status of the advances in the domain of simulation and measurements, the main goals proposed for this workshop are: the presentation of the state-of-the-art of tools, methods and experiments in the domains of interest of the Gedepeon research group, the exchange of information about the possibilities of use of computer codes and facilities, about the understanding of physical and chemical phenomena, and about development and experiment needs. This document gathers 18 presentations (slides) among the 19 given at this workshop and dealing with: the deterministic and stochastic codes in reactor physics (Rimpault G.); MURE: an evolution code coupled with MCNP (Meplan O.); neutronic calculation of future reactors at EdF (Lecarpentier D.); advance status of the MCNP/TRIO-U neutronic/thermal-hydraulics coupling (Nuttin A.); the FLICA4/TRIPOLI4 thermal-hydraulics/neutronics coupling (Aniel S.); methods of disturbances and sensitivity analysis of nuclear data in reactor physics, application to VENUS-2 experimental reactor (Bidaud A.); modeling for the reliability improvement of an ADS accelerator (Biarotte J.L.); residual gas compensation of the space charge of intense beams (Ben Ismail A.); experimental determination and numerical modeling of phase equilibrium diagrams of interest in nuclear applications (Gachon J.C.); modeling of irradiation effects (Barbu A.); elastic limit and irradiation damage in Fe-Cr alloys: simulation and experiment (Pontikis V.); experimental measurements of spallation residues, comparison with Monte-Carlo simulation codes (Fallot M.); the spallation target-reactor coupling (Rimpault G.); tools and data (Grouiller J.P.); models in high energy transport codes: status and perspective (Leray S.); other ways of investigation for spallation (Audoin L.); neutrons and light particles production at intermediate energies (20-200 MeV) with iron, lead and uranium targets (Le Colley F

  3. Understanding customer experience.

    Science.gov (United States)

    Meyer, Christopher; Schwager, Andre

    2007-02-01

    Anyone who has signed up for cell phone service, attempted to claim a rebate, or navigated a call center has probably suffered from a company's apparent indifference to what should be its first concern: the customer experiences that culminate in either satisfaction or disappointment and defection. Customer experience is the subjective response customers have to direct or indirect contact with a company. It encompasses every aspect of an offering: customer care, advertising, packaging, features, ease of use, reliability. Customer experience is shaped by customers' expectations, which largely reflect previous experiences. Few CEOs would argue against the significance of customer experience or against measuring and analyzing it. But many don't appreciate how those activities differ from CRM or just how illuminating the data can be. For instance, the majority of the companies in a recent survey believed they have been providing "superior" experiences to customers, but most customers disagreed. The authors describe a customer experience management (CEM) process that involves three kinds of monitoring: past patterns (evaluating completed transactions), present patterns (tracking current relationships), and potential patterns (conducting inquiries in the hope of unveiling future opportunities). Data are collected at or about touch points through such methods as surveys, interviews, focus groups, and online forums. Companies need to involve every function in the effort, not just a single customer-facing group. The authors go on to illustrate how a cross-functional CEM system is created. With such a system, companies can discover which customers are prospects for growth and which require immediate intervention.

  4. DT experiment on JET

    International Nuclear Information System (INIS)

    Itoh, Sanae; Itoh, Kimitaka

    1992-01-01

    In November, 1991, at the JET tokamak in Abingdon, the U.K., DT plasma combustion experiment was carried out for the first time in history. The output power by nuclear fusion of 1.7 MW at maximum and the total power output of 2 MJ were attained, and it was proved that the controlled nuclear fusion accompanied by considerable power output can be carried out on the ground as a real thing. One milestone of the dream and target of nuclear fusion reactors was attained. In this paper, the results of the experiment are reported, and their meaning for the research of hereafter is considered. In the experiment this time, 0.24 g of tritium in total was used, including conditioning. The last two shots became the power output experiment of MW class nuclear fusion reaction, in which about 13% of tritium fuel ratio was estimated. The preliminary tritium experiment was divided into six phases, and was carried out for three weeks, aiming at collecting the basic data for the full scale DT experiment. The examination of the research program, the preliminary tritium experiment and the success of the PTE and the new development are described. (K.I.)

  5. Conducting interactive experiments online.

    Science.gov (United States)

    Arechar, Antonio A; Gächter, Simon; Molleman, Lucas

    2018-01-01

    Online labor markets provide new opportunities for behavioral research, but conducting economic experiments online raises important methodological challenges. This particularly holds for interactive designs. In this paper, we provide a methodological discussion of the similarities and differences between interactive experiments conducted in the laboratory and online. To this end, we conduct a repeated public goods experiment with and without punishment using samples from the laboratory and the online platform Amazon Mechanical Turk. We chose to replicate this experiment because it is long and logistically complex. It therefore provides a good case study for discussing the methodological and practical challenges of online interactive experimentation. We find that basic behavioral patterns of cooperation and punishment in the laboratory are replicable online. The most important challenge of online interactive experiments is participant dropout. We discuss measures for reducing dropout and show that, for our case study, dropouts are exogenous to the experiment. We conclude that data quality for interactive experiments via the Internet is adequate and reliable, making online interactive experimentation a potentially valuable complement to laboratory studies.

  6. Temporal Experience and Metaphysics

    Directory of Open Access Journals (Sweden)

    Graham Peebles

    Full Text Available ABSTRACT The well-known phenomenological argument draws metaphysical conclusions about time, specifically about change through time and the resulting passage or flow of time, from our temporal experience. The argument begins with the phenomenological premise that there is a class of properties which underlies our experience of time and change through time, and its conclusion is that these properties are not merely experienced but exemplified. I argue that the phenomenological argument is best served by the adoption of a representational theory of perception. I then present a representational theory of temporal experience.

  7. The Experiment as Act

    DEFF Research Database (Denmark)

    Søndergaard, Morten

    to identify and locate the experiments of POEX65 as acts; and to ask the questions: what constitutes those acts as experiments? and how do we possibly archive them? My purpose, then, is to define the methodologies to obtain the contours of a post-phenomenology of experimental artistic production in order...... to be able to analyse the phenomena found at POEX65. Here I will use the notion of the ‘ontological theatre’ (Pickering), which, according to Pickering, is acted out in experimental art productions. The experiment could thus be seen as an ‘agency-realism’ – as an ‘act’ of relations across the aesthetics...

  8. Making the Experience City

    DEFF Research Database (Denmark)

    Jensen, Ole B.

    2009-01-01

    This paper describes the latest research into cultural planning and architectural branding in Denmark based on the ‘Experience City' research project located at Aalborg University. The paper explores the implication of the turn towards culture and experience in the contemporary Danish city. It thus...... makes an investigation into the complex relationship between the words and policies of the ‘Experience Economy' and the actual urban transformations made in cities with reference to these changes. The paper discusses the cases researched in relation to the state, market, civil society framework as well...

  9. Experimenting for resilience

    DEFF Research Database (Denmark)

    Hagedorn-Rasmussen, Peter; Dupret, Katia

    Focusing on how an experimental approach to organizing may pave the way for organizational resilience, we explore opportunities and barriers of experimental organizing by following a concrete social experiment in civil society and discuss its adaptability in traditional organizations. The social...... experiment is called Civic Desire. The founders explicitly call for new ways of organizing that can develop social sustainability. We discuss how these experiments may create platforms of new unforeseen goals that organizations may choose to follow. In conclusion we argue for organizational resilience...

  10. Nightshade Prototype Experiments (Silverleaf)

    Energy Technology Data Exchange (ETDEWEB)

    Danielson, Jeremy [Los Alamos National Lab. (LANL), Los Alamos, NM (United States); Bauer, Amy L. [Los Alamos National Lab. (LANL), Los Alamos, NM (United States)

    2016-12-23

    The Red Sage campaign is a series of subcritical dynamic plutonium experiments designed to measure ejecta. Nightshade, the first experiments in Red Sage scheduled for fiscal year 2019, will measure the amount of ejecta emission into vacuum from a double-­shocked plutonium surface. To address the major technical risks in Nightshade, a Level 2 milestone was developed for fiscal year 2016. Silverleaf, a series of four experiments, was executed at the Los Alamos National Laboratory in July and August 2016 to demonstrate a prototype of the Nightshade package and to satisfy this Level 2 milestone. This report is documentation that Red Sage Level 2 milestone requirements were successfully met.

  11. CANDU operating experience

    International Nuclear Information System (INIS)

    McConnell, L.G.; Woodhead, L.W.; Fanjoy, G.R.; Thurygill, E.W.

    1980-05-01

    The CANDU-PHW program is based upon 38 years of heavy water reactor experience with 35 years of operating experience. Canada has had 72 reactor years of nuclear-electric operations experience with 10 nuclear units in 4 generating stations during a period of 18 years. All objectives have been met with outstanding performance: worker safety, public safety, environmental emissions, reliable electricity production, and low electricity cost. The achievement has been realized through total teamwork involving all scientific disciplines and all project functions (research, design, manufacturing, construction, and operation). (auth)

  12. Game user experience evaluation

    CERN Document Server

    Bernhaupt, Regina

    2015-01-01

    Evaluating interactive systems for their user experience (UX) is a standard approach in industry and research today. This book explores the areas of game design and development and Human Computer Interaction (HCI) as ways to understand the various contributing aspects of the overall gaming experience. Fully updated, extended and revised this book is based upon the original publication Evaluating User Experience in Games, and provides updated methods and approaches ranging from user- orientated methods to game specific approaches. New and emerging methods and areas explored include physiologi

  13. Inexpensive multiplexed library preparation for megabase-sized genomes.

    Directory of Open Access Journals (Sweden)

    Michael Baym

    Full Text Available Whole-genome sequencing has become an indispensible tool of modern biology. However, the cost of sample preparation relative to the cost of sequencing remains high, especially for small genomes where the former is dominant. Here we present a protocol for rapid and inexpensive preparation of hundreds of multiplexed genomic libraries for Illumina sequencing. By carrying out the Nextera tagmentation reaction in small volumes, replacing costly reagents with cheaper equivalents, and omitting unnecessary steps, we achieve a cost of library preparation of $8 per sample, approximately 6 times cheaper than the standard Nextera XT protocol. Furthermore, our procedure takes less than 5 hours for 96 samples. Several hundred samples can then be pooled on the same HiSeq lane via custom barcodes. Our method will be useful for re-sequencing of microbial or viral genomes, including those from evolution experiments, genetic screens, and environmental samples, as well as for other sequencing applications including large amplicon, open chromosome, artificial chromosomes, and RNA sequencing.

  14. General relativity and experiment

    OpenAIRE

    Damour, T.

    1994-01-01

    The confrontation between Einstein's theory of gravitation and experiment is summarized. Although all current experimental data are compatible with general relativity, the importance of pursuing the quest for possible deviations from Einstein's theory is emphasized.

  15. Lidar calibration experiments

    DEFF Research Database (Denmark)

    Ejsing Jørgensen, Hans; Mikkelsen, T.; Streicher, J.

    1997-01-01

    A series of atmospheric aerosol diffusion experiments combined with lidar detection was conducted to evaluate and calibrate an existing retrieval algorithm for aerosol backscatter lidar systems. The calibration experiments made use of two (almost) identical mini-lidar systems for aerosol cloud...... detection to test the reproducibility and uncertainty of lidars. Lidar data were obtained from both single-ended and double-ended Lidar configurations. A backstop was introduced in one of the experiments and a new method was developed where information obtained from the backstop can be used in the inversion...... algorithm. Independent in-situ aerosol plume concentrations were obtained from a simultaneous tracer gas experiment with SF6, and comparisons with the two lidars were made. The study shows that the reproducibility of the lidars is within 15%, including measurements from both sides of a plume...

  16. Muon ionization cooling experiment

    CERN Multimedia

    CERN. Geneva

    2003-01-01

    A neutrino factory based on a muon storage ring is the ultimate tool for studies of neutrino oscillations, including possibly leptonic CP violation. It is also the first step towards muon colliders. The performance of this new and promising line of accelerators relies heavily on the concept of ionisation cooling of minimum ionising muons, for which much R&D is required. The concept of a muon ionisation cooling experiment has been extensively studied and first steps are now being taken towards its realisation by a joint international team of accelerator and particle physicists. The aim of the workshop is to to explore at least two versions of an experiment based on existing cooling channel designs. If such an experiment is feasible, one shall then select, on the basis of effectiveness, simplicity, availability of components and overall cost, a design for the proposed experiment, and assemble the elements necessary to the presentation of a proposal. Please see workshop website.

  17. LADEE LUNAR DUST EXPERIMENT

    Data.gov (United States)

    National Aeronautics and Space Administration — This archive bundle includes data taken by the Lunar Dust Experiment (LDEX) instrument aboard the Lunar Atmosphere and Dust Environment Explorer (LADEE) spacecraft....

  18. Experiments on Tape

    Science.gov (United States)

    Whitfield, Eddie

    1976-01-01

    The use of science experiments on tape are shown to provide for individual differences from the gifted child to the handicapped in developing skills in the languare art area, developing significant concepts, and making learning come alive. (EB)

  19. Experiments with needle bearings

    Science.gov (United States)

    Ferretti, Pericle

    1933-01-01

    Experiments and results are presented in testing needle bearings, especially in comparison with roller bearings. Reduction in coefficient of friction is discussed as well as experimental methods and recording devices.

  20. The experiments ALICE

    CERN Document Server

    Fabjan, Christian

    2009-01-01

    This article documents the main design choices and the close to 20 years of preparation, detector R&D, construction and installation of ALICE, the dedicated heavy ion experiment at the CERN LHC accelerator.

  1. Experience Communication and Aesthetics

    DEFF Research Database (Denmark)

    Thorlacius, Lisbeth

    to user aspect (web 2.0), the personal engagement or the community spirit. This increasing demand of experiences reflects the postmodern cultural trends where rules for how to think and behave no longer exist. This results in individualism, where the identity of the human being has changed from something...... are produced within the field of experience communication. Aesthetical experiences may be a product of the concept of novelty, the unpredictable, the inexpressible, the making of identity, the self-actualization, the self-staging, the interactive aspect, the co-producer role (the prosumer role), the user...... challenge in terms of creating experience based communication designs which meet the need of self-actualization and creation of own identities of the target audiences.  ...

  2. The Northfield experiments.

    Science.gov (United States)

    Harrison, T; Clarke, D

    1992-05-01

    The Northfield Experiments took place at Hollymoor Hospital, Northfield, Birmingham, during World War II. The first experiment was conducted by Bion & Rickman. The second evolved gradually; many people contributed to its success, including Foulkes, Main and Bridger. The experiments were an important landmark in the evolution of theory and practice in group psychotherapy and in the therapeutic community movement. They were not carried out solely as responses to the need for mass treatment of neurotic disorders among army personnel; antecedent factors, the theoretical orientation of the practitioners and the nature of army life were equally important. The two experiments differed in pace and in recognition of the needs of higher-order systems, particularly the military hierarchy. They shared many underlying concepts, including responsibility to society, the therapeutic use of groups (including the hospital community) and an emphasis on process. Lessons learned at Northfield remain relevant to the practice of psychiatry today.

  3. User Experience Dimensions

    DEFF Research Database (Denmark)

    Lykke, Marianne; Jantzen, Christian

    2016-01-01

    The present study develops a set of 10 dimensions based on a systematic understanding of the concept of experience as a holistic psychological. Seven of these are derived from a psychological conception of what experiencing and experiences are. Three supplementary dimensions spring from...... the observation that experiences apparently have become especially valuable phenomena in Western societies. The 10 dimensions are tried out in a field study at the Center for Art and Media (ZKM) in Germany with the purpose to study their applicability in the evaluation of interactive sound archives. 29 walk......-alongs were carried out with 58 museums visitors. Our analysis showed that it was possible to identify the 10 experience dimensions in the study material. Some dimensions were expressed more frequently than others. The distribution of expressed dimensions and the content of the user comments provided a clear...

  4. The world's biggest experiment

    CERN Multimedia

    Gregson, Liz

    2008-01-01

    According to CERN, our understanding of the Universe is about the change. Meet the Imperial alumni and staff who are involved in CERN's Large Hadron Collider, the world's biggest experiment. (3 pages)

  5. The Brain Experiment

    Science.gov (United States)

    Polenta, G.; Calvo, M.; Conversi, L.; de Bernardis, P.; Giordano, C.; Iacoangeli, A.; Maiello, M.; Marini-Bettolo, C.; Masi, S.; Nati, F.; Nati, L.; Peterzen, S.; Piacentini, F.; Sordini, R.; Veneziani, M.; Bartlett, J.; Bréelle, E.; Dufour, C.; Galli, S.; Ganga, K.; Ghribi, A.; Giraud-Heraud, Y.; Guglielmi, L.; Hamilton, J. C.; Kaplan, J.; Piat, M.; Gervasi, M.; Sironi, G.; Spinelli, S.; Tartari, A.; Zannoni, M.; Maffei, B.; Piccirillo, L.; Pisano, G.; Ade, P.; Orlando, A.; Savini, G.; Brossard, J.; Giard, M.; Landé, J.; Bergé, L.; Dumoulin, L.; Juillard, A.; Marnieros, S.; Pajot, F.; Rosset, C.

    2008-09-01

    The rotational component of the CMB polarization, the so-called B-modes, is one of the major topic for next generation CMB experiments. This signal traces the effect on the CMB due to primordial gravitational waves produced during the inflationary epoch, probing the physics of the very early universe at GUT energy scales. This is a challenge, being the expected amplitude of B-mode polarization ~ 0.1μK. In this paper we describe the BRAIN experiment, a bolometric interferometer which combines high sensitivity bolometric detectors with the excellent control of systematic effects proper of interferometers. Being a ground based experiment, we identified Dome Charlie in Antarctica as the best site for such measurements. In order to validate the goodness of the site, as well as some of the implemented technical solutions, we built a pathfinder experiment which has been successfully operated during last Antarctic summer, and we report about preliminary results obtained.

  6. Experiments in Animal Behavior

    Science.gov (United States)

    Polt, James M.

    1971-01-01

    Describes experiments in conditioning, sensory processes, social behavior, imprinting, innate preferences for color and form, and discrimination learning suitable for secondary school students. Mealworms, crickets, and chicks are used as subjects. (AL)

  7. Experiment-o-mania

    Science.gov (United States)

    Drndarski, Marina

    2015-04-01

    Every 21st century student is expected to develop science literacy skills. As this is not part of Serbian national curriculum yet, we decided to introduce it with this project. Experiment-o-mania provides students to experience science in different and exciting way. It makes opportunity for personalized learning offering space and time to ask (why, where, how, what if) and to try. Therefore, we empower young people with skills of experimenting, and they love science back. They ask questions, make hypothesis, make problems and solve them, make mistakes, discuss about the results. Subsequently this raises the students' interest for school curriculum. This vision of science teaching is associated with inquiry-based learning. Experiment-o-mania is the unique and recognizable teaching methodology for the elementary school Drinka Pavlović, Belgrade, Serbia. Experiment-o-mania implies activities throughout the school year. They are held on extra class sessions, through science experiments, science projects or preparations for School's Days of science. Students learn to ask questions, make observations, classify data, communicate ideas, conduct experiments, analyse results and make conclusions. All science teachers participate in designing activities and experiments for students in Experiment-o-mania teaching method. But they are not alone. Teacher of fine arts, English teachers and others also take part. Students have their representatives in this team, too. This is a good way to blend knowledge among different school subject and popularize science in general. All the experiments are age appropriate and related to real life situations, local community, society and the world. We explore Fibonacci's arrays, saving energy, solar power, climate change, environmental problems, pollution, daily life situations in the country or worldwide. We introduce great scientists as Nikola Tesla, Milutin Milanković and sir Isaac Newton. We celebrate all relevant international days, weeks

  8. The MAJORANA Experiment

    Energy Technology Data Exchange (ETDEWEB)

    Aguayo Navarrete, Estanislao; Avignone, F. T.; Back, Henning O.; Barabash, Alexander S.; Bergevin, M.; Bertrand, F.; Boswell, M.; Brudanin, V.; Busch, Matthew; Chan, Yuen-Dat; Christofferson, Cabot-Ann; Collar, J. I.; Combs, Dustin C.; Cooper, R. J.; Detwiler, Jason A.; Doe, Peter J.; Efremenko, Yuri; Egorov, Viatcheslav; Ejiri, H.; Elliott, Steven R.; Esterline, James H.; Fast, James E.; Fields, N.; Finnerty, P.; Fraenkle, Florian; Gehman, Victor M.; Giovanetti, G. K.; Green, Matthew P.; Guiseppe, Vincente; Gusey, K.; Hallin, A. L.; Hazama, R.; Henning, R.; Hime, Andrew; Hoppe, Eric W.; Horton, Mark; Howard, Stanley; Howe, M. A.; Johnson, R. A.; Keeter, K.; Keillor, Martin E.; Keller, C.; Kephart, Jeremy D.; Kidd, Mary; Knecht, A.; Kochetov, Oleg; Konovalov, S.; Kouzes, Richard T.; LaFerriere, Brian D.; LaRoque, B. H.; Leon, Jonathan D.; Leviner, L.; Loach, J. C.; MacMullin, S.; Marino, Michael G.; Martin, R. D.; Mei, Dong-Ming; Merriman, Jason H.; Miller, M. L.; Mizouni, Leila; Nomachi, Masaharu; Orrell, John L.; Overman, Nicole R.; Phillips, D.; Poon, Alan; Perumpilly, Gopakumar; Prior, Gersende; Radford, D. C.; Rielage, Keith; Robertson, R. G. H.; Ronquest, M. C.; Schubert, Alexis G.; Shima, T.; Shirchenko, M.; Snavely, Kyle J.; Sobolev, V.; Steele, David; Strain, J.; Thomas, K.; Timkin, V.; Tornow, W.; Vanyushin, I.; Varner, R. L.; Vetter, Kai; Vorren, Kris R.; Wilkerson, John; Wolfe, B. A.; Yakushev, E.; Young, A.; Yu, Chang-Hong; Yumatov, Vladimir; Zhang, C.

    2011-10-01

    The Majorana collaboration is actively pursuing research and development aimed at a tonne-scale {sup 76}Ge neutrinoless double-beta decay ({beta}{beta}(0{nu})-decay) experiment. The current, primary focus is the construction of the Majorana Demonstrator experiment, an R and D effort that will field approximately 40 kg of germanium detectors with mixed enrichment levels. This article provides a status update on the construction of the Demonstrator.

  9. Particle physics experiments 1980

    International Nuclear Information System (INIS)

    Rousseau, M.D.; Stuart, G.

    1981-01-01

    This report describes work carried out in 1980 on experiments approved by the Particle Physics Ezperiments Selection Panel. A table of contents giving, title and collaboration, technique, accelerator used, year of running, status as at December 1980, the spokesman and experimental code, is followed by unedited contributions from each of the 54 experiments included in this annual review including lists of submitted publications. (U.K.)

  10. The CLOUD experiment

    CERN Multimedia

    Maximilien Brice

    2006-01-01

    The Cosmics Leaving Outdoor Droplets (CLOUD) experiment as shown by Jasper Kirkby (spokesperson). Kirkby shows a sketch to illustrate the possible link between galactic cosmic rays and cloud formations. The CLOUD experiment uses beams from the PS accelerator at CERN to simulate the effect of cosmic rays on cloud formations in the Earth's atmosphere. It is thought that cosmic ray intensity is linked to the amount of low cloud cover due to the formation of aerosols, which induce condensation.

  11. The MAJORANA Experiment

    International Nuclear Information System (INIS)

    Aguayo Navarrete, Estanislao; Avignone, F.T.; Back, Henning O.; Barabash, Alexander S.; Bergevin, M.; Bertrand, F.; Boswell, M.; Brudanin, V.; Busch, Matthew; Chan, Yuen-Dat; Christofferson, Cabot-Ann; Collar, J.I.; Combs, Dustin C.; Cooper, R.J.; Detwiler, Jason A.; Doe, Peter J.; Efremenko, Yuri; Egorov, Viatcheslav; Ejiri, H.; Elliott, Steven R.; Esterline, James H.; Fast, James E.; Fields, N.; Finnerty, P.; Fraenkle, Florian; Gehman, Victor M.; Giovanetti, G.K.; Green, Matthew P.; Guiseppe, Vincente; Gusey, K.; Hallin, A.L.; Hazama, R.; Henning, R.; Hime, Andrew; Hoppe, Eric W.; Horton, Mark; Howard, Stanley; Howe, M.A.; Johnson, R.A.; Keeter, K.; Keillor, Martin E.; Keller, C.; Kephart, Jeremy D.; Kidd, Mary; Knecht, A.; Kochetov, Oleg; Konovalov, S.; Kouzes, Richard T.; LaFerriere, Brian D.; LaRoque, B.H.; Leon, Jonathan D.; Leviner, L.; Loach, J.C.; MacMullin, S.; Marino, Michael G.; Martin, R.D.; Mei, Dong-Ming; Merriman, Jason H.; Miller, M.L.; Mizouni, Leila; Nomachi, Masaharu; Orrell, John L.; Overman, Nicole R.; Phillips, D.; Poon, Alan; Perumpilly, Gopakumar; Prior, Gersende; Radford, D.C.; Rielage, Keith; Robertson, R.G.H.; Ronquest, M.C.; Schubert, Alexis G.; Shima, T.; Shirchenko, M.; Snavely, Kyle J.; Sobolev, V.; Steele, David; Strain, J.; Thomas, K.; Timkin, V.; Tornow, W.; Vanyushin, I.; Varner, R.L.; Vetter, Kai; Vorren, Kris R.; Wilkerson, John; Wolfe, B.A.; Yakushev, E.; Young, A.; Yu, Chang-Hong; Yumatov, Vladimir; Zhang, C.

    2011-01-01

    The Majorana collaboration is actively pursuing research and development aimed at a tonne-scale 76 Ge neutrinoless double-beta decay (ββ(0ν)-decay) experiment. The current, primary focus is the construction of the Majorana Demonstrator experiment, an R and D effort that will field approximately 40 kg of germanium detectors with mixed enrichment levels. This article provides a status update on the construction of the Demonstrator.

  12. Digital Heritage Experiences

    DEFF Research Database (Denmark)

    Munar, Ana Maria; Ooi, Can-Seng

    The evolution of the Web and the expansion of social media are transforming our heritage experiences. Social media offer an innovative element to personal travel reflections by providing digital global platforms on which tourists can create and publish their travel stories. Social media transform...... the traditional creative processes, distribution mechanisms and consumption patterns of these experiences. This study provides insight on how personal heritage moments are constructed, digitalized and shared. The methodological perspective adopted draws on a constructivist epistemology (Markham 2004...

  13. The LHCb experiment

    CERN Document Server

    Nakada, Tatsuya

    2000-01-01

    The LHCb experiment is designed to fully exploit the large number of b hadrons expected at the LHC energy and luminosity. The experiment is equipped with particle identification devices and can efficiently trigger events with different B-meson final states, allowing systematic studies of CP violation and other rare phenomena in the b hadron system with a high precision which could reveal physics beyond the standard model.

  14. Experiments in computing: a survey.

    Science.gov (United States)

    Tedre, Matti; Moisseinen, Nella

    2014-01-01

    Experiments play a central role in science. The role of experiments in computing is, however, unclear. Questions about the relevance of experiments in computing attracted little attention until the 1980s. As the discipline then saw a push towards experimental computer science, a variety of technically, theoretically, and empirically oriented views on experiments emerged. As a consequence of those debates, today's computing fields use experiments and experiment terminology in a variety of ways. This paper analyzes experimentation debates in computing. It presents five ways in which debaters have conceptualized experiments in computing: feasibility experiment, trial experiment, field experiment, comparison experiment, and controlled experiment. This paper has three aims: to clarify experiment terminology in computing; to contribute to disciplinary self-understanding of computing; and, due to computing's centrality in other fields, to promote understanding of experiments in modern science in general.

  15. Qualitative experiments in psychology

    DEFF Research Database (Denmark)

    Wagoner, Brady

    2015-01-01

    In this article, I explore the meaning of experiments in early twentieth century psychology, focusing on the qualitative experimental methodology of psychologist Frederic BARTLETT. I begin by contextualizing BARTLETT's experiments within the continental research tradition of his time, which...... was in a state of transition from a focus on elements (the concern of psychophysics) to a focus on wholes (the concern of Gestalt psychology). The defining feature of BARTLETT's early experiments is his holistic treatment of human responses, in which the basic unit of analysis is the active person relating...... to some material within the constraints of a social and material context. This manifests itself in a number of methodological principles that contrast with contemporary understandings of experimentation in psychology. The contrast is further explored by reviewing the history of "replications...

  16. The BLAST experiment

    International Nuclear Information System (INIS)

    Hasell, D.; Akdogan, T.; Alarcon, R.; Bertozzi, W.; Booth, E.; Botto, T.; Calarco, J.R.; Clasie, B.; Crawford, C.; DeGrush, A.; Dow, K.; Dutta, D.; Farkhondeh, M.; Fatemi, R.; Filoti, O.; Franklin, W.; Gao, H.; Geis, E.; Gilad, S.; Hersman, W.

    2009-01-01

    The Bates large acceptance spectrometer toroid (BLAST) experiment was operated at the MIT-Bates Linear Accelerator Center from 2003 until 2005. The detector and experimental program were designed to study, in a systematic manner, the spin-dependent electromagnetic interaction in few-nucleon systems. As such the data will provide improved measurements for neutron, proton, and deuteron form factors. The data will also allow details of the reaction mechanism, such as the role of final state interactions, pion production, and resonances to be studied. The experiment used: a longitudinally polarized electron beam stored in the South Hall Storage Ring; a highly polarized, isotopically pure, internal gas target of hydrogen or deuterium provided by an atomic beam source; and a symmetric, general purpose detector based on a toroidal spectrometer with tracking, time-of-flight, Cherenkov, and neutron detectors. Details of the experiment and operation are presented.

  17. Experiences of depression

    DEFF Research Database (Denmark)

    Rønberg, Mette

    In this thesis, I examine the complex experiences of what we call depression in everyday life, the multifaceted and ambiguous experiences of getting a depression diagnosis, and the in-depth processes involved in learning to live under the description of depression. The thesis is based...... on ethnographic fieldwork among adults diagnosed with depression in Denmark, and consists of three articles that provide glimpses into experiences of depression and diagnostic processes. The articles deal with creativity, variability and resistance in diagnostic processes and illustrates how these not are once......-and-for-all-dealt-with matters but rather messy and complicated processes, that involve several actors and multiple relations to the diagnosis. The thesis furthermore challenges the dominant diagnostic understanding depression as a neurobiological, and individual disorder in present-day diagnostic cultures, by arguing...

  18. Experiments in mixed reality

    Science.gov (United States)

    Krum, David M.; Sadek, Ramy; Kohli, Luv; Olson, Logan; Bolas, Mark

    2010-01-01

    As part of the Institute for Creative Technologies and the School of Cinematic Arts at the University of Southern California, the Mixed Reality lab develops technologies and techniques for presenting realistic immersive training experiences. Such experiences typically place users within a complex ecology of social actors, physical objects, and collections of intents, motivations, relationships, and other psychological constructs. Currently, it remains infeasible to completely synthesize the interactivity and sensory signatures of such ecologies. For this reason, the lab advocates mixed reality methods for training and conducts experiments exploring such methods. Currently, the lab focuses on understanding and exploiting the elasticity of human perception with respect to representational differences between real and virtual environments. This paper presents an overview of three projects: techniques for redirected walking, displays for the representation of virtual humans, and audio processing to increase stress.

  19. The Dialogue of Experience

    DEFF Research Database (Denmark)

    Jørgensen, Dorthe

    2016-01-01

    In “The Dialogue of Experience,” Dorthe Jørgensen presents Gianni Vattimo’s understanding, expressed in his The Responsibility of the Philosopher, of what it means to be a philosopher. According to Jørgensen, Vattimo’s work as a philosopher is an example of ‘world-engaged philosophy’ as distinct...... from ‘school philosophy’ or ‘applied philosophy.’ The concept of ‘world-engaged philosophy’ is associated with Jørgensen’s concept of ‘world poetry’: that the immanent world is ambiguous; it occasions experiences of a surplus of meaning, traditionally called beauty. Furthermore, both concepts...... are associated with her concept of ‘basic experience’ and with her understanding of it as characterized by an immanent ‘dialogue’ between sensation, faith, and comprehension. According to Jørgensen, all experiences are rooted in sensation, faith, and comprehension; art, religion, and thought, or aesthetics...

  20. Magnetic scattering experiments

    International Nuclear Information System (INIS)

    Vettier, C.

    2001-01-01

    We discuss the relative use of neutrons and X-rays as scattering tools to probe the magnetic properties of materials. Neutron diffraction experiments continue to be the most appropriate tool to determine the magnetic structure of bulk solids. Synchrotron X-ray experiments are and will be used to investigate selectively the magnetic behaviour of different chemical species and/or electronic shells in magnetic systems. There exist some grey areas where the two probes can be used depending on the scientific problem under investigation. In particular, neutrons allow very accurate measurements of magnetic form factors, but X-rays provide a separation of the orbital momentum contribution to magnetisation. Comparisons between X-rays and neutrons studies of magnetism at surfaces are presented. Finally, one can anticipate that only inelastic neutron scattering experiments will allow the observation of magnetic excitations across the whole Brillouin zone

  1. Operating experience feedback

    International Nuclear Information System (INIS)

    Cimesa, S.

    2007-01-01

    Slovenian Nuclear Safety Administration (SNSA) has developed its own system for tracking, screening and evaluating the operating experiences of the nuclear installations. The SNSA staff regularly tracks the operating experiences throughout the world and screens them on the bases of applicability for the Slovenian nuclear facilities. The operating experiences, which pass the screening, are thoroughly evaluated and also recent operational events in these facilities are taken into account. If needed, more information is gathered to evaluate the conditions of the Slovenian facilities and appropriate corrective actions are considered. The result might be the identification of the need for modification at the licensee, the need for modification of internal procedures in the SNSA or even the proposal for the modification of regulations. Information system helps everybody to track the process of evaluation and proper logging of activities. (author)

  2. Experience Innovation in Tourism

    DEFF Research Database (Denmark)

    Jensen, Jens Friis; Sørensen, Flemming

    2017-01-01

    This paper discusses how front-line employees can play an important role in innovation processes that lead tourism companies from service production to experience creation. The increasing focus on experiential value by customers in general (Sundbo and Sørensen, 2013) puts pressure on service...... and typically do not consider front-line employees to be important innovation agents (Sørensen and Jensen, 2012). However, in this paper, an analysis of an experiment in Tivoli Gardens exemplifies the innovation and value creating potential of involving front-line employees in developing new practices...... companies to move from functional service production to creating experiences (Pine and Gilmore, 2013). This is also the case in tourism companies. In tourism, most encounters between employees and tourists operate on a service logic (Sørensen and Jensen, 2015). Thus, we suggest there is a value potential...

  3. ATLAS IBL operational experience

    CERN Document Server

    AUTHOR|(INSPIRE)INSPIRE-00237659; The ATLAS collaboration

    2016-01-01

    The Insertable B-Layer (IBL) is the inner most pixel layer in the ATLAS experiment, which was installed at 3.3 cm radius from the beam axis in 2014 to improve the tracking performance. To cope with the high radiation and hit occupancy due to proximity to the interaction point, a new read-out chip and two different silicon sensor technologies (planar and 3D) have been developed for the IBL. After the long shut-down period over 2013 and 2014, the ATLAS experiment started data-taking in May 2015 for Run-2 of the Large Hadron Collider (LHC). The IBL has been operated successfully since the beginning of Run-2 and shows excellent performance with the low dead module fraction, high data-taking efficiency and improved tracking capability. The experience and challenges in the operation of the IBL is described as well as its performance.

  4. Cooperative Prototyping Experiments

    DEFF Research Database (Denmark)

    Bødker, Susanne; Grønbæk, Kaj

    1989-01-01

    This paper describes experiments with a design technique that we denote cooperative prototyping. The experiments consider design of a patient case record system for municipal dental clinics in which we used HyperCard, an off the shelf programming environment for the Macintosh. In the ecperiments we...... tried to achieve a fluent work-like evaluation of prototypes where users envisioned future work with a computer tool, at the same time as we made on-line modifications of prototypes in cooperation with the users when breakdown occur in their work-like evaluation. The experiments showed...... that it was possible to make a number of direct manipulation changes of prototypes in cooperation with the users, in interplay with their fluent work-like evaluation of these. However, breakdown occurred in the prototyping process when we reached the limits of the direct manipulation support for modification. From...

  5. Nuclear power experience

    International Nuclear Information System (INIS)

    1983-01-01

    The International Conference on Nuclear Power Experience, organized by the International Atomic Energy Agency, was held at the Hofburg Conference Center, Vienna, Austria, from 13 to 17 September 1982. Almost 1200 participants and observers from 63 countries and 20 organizations attended the conference. The 239 papers presented were grouped under the following seven main topics: planning and development of nuclear power programmes; technical and economic experience of nuclear power production; the nuclear fuel cycle; nuclear safety experience; advanced systems; international safeguards; international co-operation. The proceedings are published in six volumes. The sixth volume contains a complete Contents of Volume 1 to 5, a List of Participants, Authors and Transliteration Indexes, a Subject Index and an Index of Papers by Number

  6. The interprofessional learning experience

    DEFF Research Database (Denmark)

    Jakobsen, Flemming; Morcke, Anne Mette; Hansen, Torben Baek

    2017-01-01

    who worked in an interprofessional outpatient orthopaedic clinic from March 2015 to January 2016. The interviews were transcribed and analysed using systematic text condensation. The students’ self-reported learning experience in this outpatient clinic was characterised by direct patient contact...... we ensure that this shift maximises learning. The purpose of this article is to understand the authentic learning experience in an interprofessional outpatient clinic setting. We performed an exploratory case study with interviews of four nursing students, 13 medical students, and six staff members...... and by authentic, interprofessional, task-based learning, and a preference for indirect supervision when conducting uncomplicated patient consultations. The supervisors intended to create this interprofessional outpatient clinic experience by having a clear teaching approach based on adult learning principles...

  7. Initial Cooling Experiment (ICE)

    CERN Multimedia

    Photographic Service; CERN PhotoLab

    1978-01-01

    In 1977, in a record-time of 9 months, the magnets of the g-2 experiment were modified and used to build a proton/antiproton storage ring: the "Initial Cooling Experiment" (ICE). It served for the verification of the cooling methods to be used for the "Antiproton Project". Stochastic cooling was proven the same year, electron cooling followed later. Also, with ICE the experimental lower limit for the antiproton lifetime was raised by 9 orders of magnitude: from 2 microseconds to 32 hours. For its previous life as g-2 storage ring, see 7405430. More on ICE: 7711282, 7809081, 7908242.

  8. World Ocean Circulation Experiment

    Science.gov (United States)

    Clarke, R. Allyn

    1992-01-01

    The oceans are an equal partner with the atmosphere in the global climate system. The World Ocean Circulation Experiment is presently being implemented to improve ocean models that are useful for climate prediction both by encouraging more model development but more importantly by providing quality data sets that can be used to force or to validate such models. WOCE is the first oceanographic experiment that plans to generate and to use multiparameter global ocean data sets. In order for WOCE to succeed, oceanographers must establish and learn to use more effective methods of assembling, quality controlling, manipulating and distributing oceanographic data.

  9. The OPERA experiment

    International Nuclear Information System (INIS)

    Dracos, M.

    2004-01-01

    The OPERA (Oscillation Project with Emulsion-tRacking Apparatus) experiment aims to observe an ν μ ↔ ν τ oscillation in the parameter region allowed by previous experiments. The OPERA detector will be installed in the underground Gran Sasso Laboratory, where the CNGS (CERN Neutrino beam to Gran Sasso) ν μ beam will be produced. The signature of the presence of ν τ 's in the ν μ beam will be detection of τ leptons produced by ν τ interactions. Nuclear emulsions will be used for precise event reconstruction, while electronic detectors will be used for triggering [ru

  10. The OPERA experiment

    International Nuclear Information System (INIS)

    Jollet, C.

    2006-01-01

    The aim of the OPERA experiment is to provide unambiguous evidence for the ν μ ↔ ν τ oscillation by looking at the appearance of ν τ in a pure ν μ beam. This oscillation will be sought in the region of the oscillation parameters indicated by the atmospheric neutrino results. The experiment is part of the CNGS (CERN Neutrino beam to Gran Sasso) project. The νμ beam produced at CERN will be sent towards the Gran Sasso underground laboratory, where the OPERA detector is under construction. The detector, the physics potential, and performance for neutrino oscillation studies including the subleading ν μ ↔ ν ε search are presented

  11. The OPERA experiment

    International Nuclear Information System (INIS)

    Dracos, M.

    2004-01-01

    The OPERA (oscillation project with emulsion-tracking apparatus) experiment aims to observe an unambiguous ν μ ↔ ν τ oscillation in the parameter region allowed by previous experiments. The OPERA detector will be installed in the underground Gran Sasso Laboratory, 732 km away from CERN, where the CNGS (CERN neutrino beam to Gran Sasso) ν μ beam will be produced. The signature of the presence of ν τ 's in the ν μ beam will be the detection of τ leptons produced by ν τ interactions. Nuclear emulsions will be used for precise event reconstruction, while electronic detectors will be used for triggering

  12. The LDMX Experiment

    OpenAIRE

    Mans Jeremiah

    2017-01-01

    The thermal-relic mechanism for the production of dark-matter in the early universe, combined with the precision measurements of the cosmic microwave background and the lack of observation of dark matter at the LHC, motivates the search for dark matter in the mass range between 1 MeV and 1 GeV. We describe the concept for the Light Dark Matter Experiment (LDMX), which could carry out such a search using the missing-momentum technique. The experiment would utilize a unique high-rate, low-curre...

  13. Accessibility and sensory experiences

    DEFF Research Database (Denmark)

    Ryhl, Camilla

    2010-01-01

    and accessibility. Sensory accessibility accommodates aspects of a sensory disability and describes architectural design requirements needed to ensure access to architectural experiences. In the context of architecture accessibility has become a design concept of its own. It is generally described as ensuring...... physical access to the built environment by accommodating physical disabilities. While the existing concept of accessibility ensures the physical access of everyone to a given space, sensory accessibility ensures the choice of everyone to stay and be able to participate and experience....

  14. SUPERCOLLIDER: Planning for experiments

    International Nuclear Information System (INIS)

    Anon.

    1987-01-01

    To push forward the necessarily lengthy preparations for experiments at the proposed US Superconducting Super Collider, a Workshop on Experiments, Detectors and Experimental Areas was held at Berkeley from 7 to 17 July. Participants looked ahead to the task of extracting physics (like the search for Higgs particles, supersymmetry or other heavy quarks and leptons) from the chaos of hadron collisions at 20 TeV beam energies with luminosities in excess of 10 32 . The door would also have to be left open for unexpected physics in these higher energy regions

  15. Who Needs Business Experience?

    DEFF Research Database (Denmark)

    Walter, Achim; Ritter, Thomas; Coviello, Nicole

    the founding teams’ pre-foundation R&D and customer collaborations to early exports. We also show that pre-foundation involvement in these various forms of collaboration, as well as a diverse stock of prior technical knowledge, can compensate for a lack of business experience in the team. The results also show...... that prior business experience does not predict very early export (within three years). Our results offer fresh insight to the international business and international entrepreneurship literatures, and implications for policy development. In particular, international R&D collaborations at public research...

  16. Experience and Sustainable Consumption

    DEFF Research Database (Denmark)

    Rasmussen, Tove Arendt

    2014-01-01

    Experience understood as experience-based consumption is by now fairly absent from the research agenda of the different theories on sustainable consumption. On the basis of Colin Campbell’s notion of romantic ethics and emotional sentimentality in modern hedonism, I claim that sustainable....... Very often, however, the actual purchase does not live up to the demands of doing good in the sustainable consumption chain, and the individual might end up with a guilty conscience, which again is a possible trigger for lingering in a sentimental mode of guilt. Emotions of sentimentality may actually...

  17. Experimenting with alternative economies

    DEFF Research Database (Denmark)

    Longhurst, Noel; Avelino, Flor; Wittmayer, Julia

    2016-01-01

    Neoliberalism is a powerful narrative that has shaped processes of urban economic development across the globe. This paper reports on four nascent ‘new economic’ narratives which represent fundamentally different imaginaries of the urban economy. Experiments informed by these narratives challenge...... the dominant neoliberal logic in four key dimensions: What is the purpose of economic development? What are the preferred distributive mechanisms? Who governs the economy? What is the preferred form of economic organisation? The emergence of these experiments illustrates that cities are spaces where counter...

  18. AGS experiments---1987, 1988, 1989

    International Nuclear Information System (INIS)

    Depken, J.C.

    1989-04-01

    This report contains: Experimental Areas Layout; Table of Beam Parameters and Fluxes; Experiment Schedule ''as run''; Experiment Long Range Schedule; A listing of experiments by number; Two-page summaries of each experiment begin here, also ordered by number; Publications of AGS experiments; and List of experimenters

  19. AGS experiments, 1988, 1989, 1990

    International Nuclear Information System (INIS)

    Depken, J.C.

    1991-04-01

    This report contains: experimental areas layout; table of beam parameters and fluxes; experiment schedule ''as run''; experiment long range schedule; a listing of experiments by number; two-page summaries of each experiment begin here, also ordered by number; publications of AGS experiments; and list of experimenters

  20. AGS experiments: 1985, 1986, 1987

    International Nuclear Information System (INIS)

    Depken, J.C.

    1987-01-01

    This report contains: Experimental areas layout, table of beam parameters and fluxes, experiment schedule ''as run,'' experiment long range schedule, a listing of experiments by number, two-page summaries of each experiment, also ordered by number, and publications of AGS experiments, 1982-1987

  1. A Vibrating String Experiment

    Science.gov (United States)

    Tsutsumanova, Gichka; Russev, Stoyan

    2013-01-01

    A simple experiment demonstrating the excitation of a standing wave in a metal string is presented here. Several tasks using the set-up are considered, which help the students to better understand the standing waves, the interaction between electric current and magnetic field and the resonance phenomena. This can serve also as a good lecture…

  2. The Santabot Experiment

    DEFF Research Database (Denmark)

    2009-01-01

    The focus is to study people's willingness to interact, and their reaction when a robot enters their daily environment. The robot autonomously detects and follows people, while keeping a safe distance. The conclusion was that people were positive towards robots in their daily life. The experiments...

  3. The Majorana Experiment

    Energy Technology Data Exchange (ETDEWEB)

    Aalseth, Craig E.; Aguayo Navarrete, Estanislao; Amman, M.; Avignone, F. T.; Back, Henning O.; Bai, Xinhua; Barabash, Alexander S.; Barbeau, P. S.; Bergevin, M.; Bertrand, F.; Boswell, M.; Brudanin, V.; Bugg, William; Burritt, Tom H.; Busch, Matthew; Capps, Greg L.; Chan, Yuen-Dat; Collar, J. I.; Cooper, R. J.; Creswick, R.; Detwiler, Jason A.; Diaz, J.; Doe, Peter J.; Efremenko, Yuri; Egorov, Viatcheslav; Ejiri, H.; Elliott, S. R.; Ely, James H.; Esterline, James H.; Farach, H. A.; Fast, James E.; Fields, N.; Finnerty, P.; Fraenkle, Florian; Gehman, Victor M.; Giovanetti, G. K.; Green, M.; Guiseppe, Vincente; Gusey, K.; Hallin, A. L.; Harper, Gregory; Hazama, R.; Henning, Reyco; Hime, Andrew; Hong, H.; Hoppe, Eric W.; Hossbach, Todd W.; Howard, Stanley; Howe, M. A.; Johnson, R. A.; Keeter, K.; Keillor, Martin E.; Keller, C.; Kephart, Jeremy D.; Kidd, M. F.; Knecht, A.; Kochetov, Oleg; Konovalov, S.; Kouzes, Richard T.; LaRoque, B. H.; Leviner, L.; Loach, J. C.; Luke, P.; MacMullin, S.; Marino, Michael G.; Martin, R. D.; Medlin, D.; Mei, Dong-Ming; Miley, Harry S.; Miller, M. L.; Mizouni, Leila; Myers, Allan W.; Nomachi, Masaharu; Orrell, John L.; Peterson, David; Phillips, D.; Poon, Alan; Perevozchikov, O.; Perumpilly, Gopakumar; Prior, Gersende; Radford, D. C.; Reid, Douglas J.; Rielage, Keith; Robertson, R. G. H.; Rodriguez, Larry; Ronquest, M. C.; Salazar, Harold; Schubert, Alexis G.; Shima, T.; Shirchenko, M.; Sobolev, V.; Steele, David; Strain, J.; Swift, Gary; Thomas, K.; Timkin, V.; Tornow, W.; Van Wechel, T. D.; Vanyushin, I.; Varner, R. L.; Vetter, Kai; Vorren, Kris R.; Wilkerson, J. F.; Wolfe, B. A.; Xiang, W.; Yakushev, E.; Yaver, Harold; Young, A.; Yu, Chang-Hong; Yumatov, V.; Zhang, C.

    2011-08-01

    The Majorana Collaboration is assembling an array of HPGe detectors to search for neutrinoless double-beta decay in 76Ge. Initially, Majorana aims to construct a prototype module to demonstrate the potential of a future 1-tonne experiment. The design and potential reach of this prototype Demonstrator module are presented.

  4. The OLYMPUS experiment

    International Nuclear Information System (INIS)

    Milner, R.; Hasell, D.K.; Kohl, M.; Schneekloth, U.; Akopov, N.; Alarcon, R.; Andreev, V.A.; Ates, O.; Avetisyan, A.; Bayadilov, D.; Beck, R.; Belostotski, S.; Bernauer, J.C.; Bessuille, J.; Brinker, F.; Buck, B.; Calarco, J.R.; Carassiti, V.; Cisbani, E.; Ciullo, G.

    2014-01-01

    The OLYMPUS experiment was designed to measure the ratio between the positron–proton and electron–proton elastic scattering cross-sections, with the goal of determining the contribution of two-photon exchange to the elastic cross-section. Two-photon exchange might resolve the discrepancy between measurements of the proton form factor ratio, μ p G E p /G M p , made using polarization techniques and those made in unpolarized experiments. OLYMPUS operated on the DORIS storage ring at DESY, alternating between 2.01 GeV electron and positron beams incident on an internal hydrogen gas target. The experiment used a toroidal magnetic spectrometer instrumented with drift chambers and time-of-flight detectors to measure rates for elastic scattering over the polar angular range of approximately 25°–75°. Symmetric Møller/Bhabha calorimeters at 1.29° and telescopes of GEM and MWPC detectors at 12° served as luminosity monitors. A total luminosity of approximately 4.5 fb −1 was collected over two running periods in 2012. This paper provides details on the accelerator, target, detectors, and operation of the experiment

  5. Future of neutrino experiments

    Indian Academy of Sciences (India)

    needs to be controlled to be less than 1%. To achieve this goal, each experimental group has been carefully designing the experiment with the identical near and far detectors, optimized baseline length (1 to 2 km), deep enough locations to reduce the cosmic ray-induced backgrounds, thick enough shielding, veto counters, ...

  6. The COMPASS experiment

    CERN Multimedia

    Laurent Guiraud

    1999-01-01

    Detail of one of the magnets on the Common Muon and Proton Apparatus for Structure and Spectroscopy (COMPASS) experiment. COMPASS studies the structure of composite particles which interact via the strong force, called hadrons. These have a complicated internal structure due to the nature of their force carriers, gluons, which can form self interacting bunches called glueballs.

  7. Electronics for LHC Experiments

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2004-07-01

    This document gathers the abstracts of most presentations made at this workshop on electronics for the large hadron collider (LHC) experiments. The presentations were arranged into 6 sessions: 1) electronics for tracker, 2) trigger electronics, 3) detector control systems, 4) data acquisition, 5) electronics for calorimeters and electronics for muons, and 6) links, power systems, grounding and shielding, testing and quality assurance.

  8. HCDA bubble experiment, (2)

    International Nuclear Information System (INIS)

    Sakata, Kaoru; Mashiko, Hiroyuki; Oka, Yoshiaki; An, Shigehiro; Isozaki, Tadashi.

    1981-06-01

    An experiment simulating the behavior of the very large steam bubbles generated at the time of an accident of core collapse was carried out with a warm water tank, and the applicability of the theory of very small bubble disappearance known at present was examined. The bubbles generated in HCDA (hypothetical core disruptive accident) are expected to be very large, containing sodium, fuel, FP gas and so on, and play important role in the mechanism of emitting radioactive substances in the safety analysis of LMFBRs. In this experiment, the degree of subcool of the warm water pool, the initial radii of steam bubbles and the blowoff pressure of steam were taken as the parameters. The radius of the steam bubbles generated in the experiment was about 6.5 cm, and the state of disappearance was different above and below the degree of unsaturation of 10 deg C. Comparing the disappearance curve obtained by the experiment with the theory of disappearance of small bubbles, the experimental values were between inertia-controlled disappearance and heat transfer-controlled disappearance, and this result was able to be explained generally with the model taking the pressure change within steam bubbles into account. The rise of bubbles was also observed. (Kako, I.)

  9. Experience as Excursion

    DEFF Research Database (Denmark)

    Svabo, Connie; Shanks, Michael

    2014-01-01

    researchers and practitioners to travel – making it possible to follow experiences as they are enacted across and between places, modes of transportation, mobile mediation and assemblages of things. Drawing on the nomadic metaphysics of philosopher Michel Serres, the journeying, shifting and propagating...

  10. Experience and Sustainable Consumption

    DEFF Research Database (Denmark)

    Rasmussen, Tove Arendt

    Experience understood as experience-based consumption is by now fairly absent from the research agenda of the different theories on sustainable consumption. On the basis of Colin Campbell’s notion of romantic ethics and emotional sentimentality in modern hedonism, I claim that sustainable consump...... convince the individual that she really does care for others and thereby provide a positive experience of feeling good – even if it takes place in the universe of consumption fantasies only....... consumption may re-enchant ordinary consumption and thereby even become a part of marketing and the experience economy. New layers of meaning are at stake and altruistic motives come into play; doing something good for someone or something, aside from oneself, is a very strong trigger of positive emotions....... Very often, however, the actual purchase does not live up to the demands of doing good in the sustainable consumption chain, and the individual might end up with a guilty conscience, which again is a possible trigger for lingering in a sentimental mode of guilt. Emotions of sentimentality may actually...

  11. New neutrino experiments

    Indian Academy of Sciences (India)

    1999-03-26

    radioactive waste left from the research and production of nuclear weapons. After more than 20 years of scientific study, public input, and regulatory struggles, WIPP began operations on March 26, 1999. It is considered as a possible site for the supernova experiment OMNIs which will feature a measurement of the neutral.

  12. Stage theta pinch experiments

    International Nuclear Information System (INIS)

    Linford, R.K.; Downing, J.N.; Gribble, R.F.; Jacobson, A.R.; Platts, D.A.; Thomas, K.S.

    1975-01-01

    The Staged Theta Pinch program is designed to study the technological and physics problems associated with producing fat plasmas and separating the implosion heating from the adiabatic compression. Several methods of implosion heating are discussed. Circuit diagrams and theoretical magnetic field behavior are described for the STP and resonant heating experiments. (MOW)

  13. The OLYMPUS experiment

    Energy Technology Data Exchange (ETDEWEB)

    Milner, R.; Hasell, D.K. [Massachusetts Institute of Technology, Cambridge, MA (United States); Kohl, M. [Hampton Univ., Hampton, VA (United States); Collaboration: The OLYMPUS Collaboration; and others

    2013-12-15

    The OLYMPUS experiment was designed to measure the ratio between the positron-proton and electron-proton elastic scattering cross sections, with the goal of determining the contribution of two-photon exchange to the elastic cross section. Two-photon exchange might resolve the discrepancy between measurements of the proton form factor ratio, {mu}{sub p}G{sup p}{sub E}/G{sup p}{sub M}, made using polarization techniques and those made in unpolarized experiments. OLYMPUS operated on the DORIS storage ring at DESY, alternating between 2.01 GeV electron and positron beams incident on an internal hydrogen gas target. The experiment used a toroidal magnetic spectrometer instrumented with drift chambers and time-of-flight detectors to measure rates for elastic scattering over the polar angular range of approximately 25 -75 . Symmetric Moeller/Bhabha calorimeters at 1.29 and telescopes of GEM and MWPC detectors at 12 served as luminosity monitors. A total luminosity of approximately 4.5 fb{sup -1} was collected over two running periods in 2012. This paper provides details on the accelerator, target, detectors, and operation of the experiment.

  14. Prospects in coincidence experiments

    International Nuclear Information System (INIS)

    Laget, J.M.

    1984-01-01

    The sensitivity of virtual photons to the local variations of the charge and magnetization densities is exploited to study the short-range part of the nucleon-nucleon interaction inside the nucleus. The possibility of varying energy, squared mass and longitudinal polarization of the photons independently enables us to disentangle the mechanisms related to the internal structure of the nucleon (e.g. quark interchange) and the contribution due to meson exchange. Coincidence experiments of the type (e,e'N) and (e,e'NN) are performed to suppress the meson contribution to the longitudinal part of the quasi-elastic peak. Four typical examples of coincidence experiments induced by virtual photons are discussed: experiments (1) on the spectroscopic structure of the quasi-elastic peak and the problem of deep lying hole states; (2) on the structure of the continuum; (3) on the low energy side of the quasi-elastic peak; and finally a three-arm coincidence experiment. (Auth.)

  15. The KOSI experiments

    International Nuclear Information System (INIS)

    Huebner, W.F.

    1991-01-01

    Whipply's icy conglomerate model of the comet nucleus has enjoyed progressively increasing acceptance and success in explaining Earth-based observations of comets since its very inception (Whipple, 1950, 1951). According to this model, the nucleus is a solid body composed of frozen gases and dust. The missions to Comet Halley in 1986, in particular the Vega and the Giotto missions, have confirmed that there is a single solid nucleus that is the root of all the observed phenomena that can be associated with an active comet. Two new comet mission (CRAF and Rosetta) are planned by NASA and ESA to extract further details about the structure and composition of the nucleus. Laboratory experiments play an important role in defining and identifying the objectives of these missions. Although such experiments have been carried out in many laboratories in Europe, the Soviet Union, the USA, Israel, and Japan, the KOSI experiments are the first large-scale investigations (in spatial dimensions and duration). (KOSI is an acronym for Kometensimulation, German for comet simulation). Starting with a summary and explanation of the KOSI experiments by Grun et al. (1991) and some of the experimental limitations by Keller and Markiewicz (1991), the author reports in this special issue the progress achieved about halfway through the planned series of investigations

  16. Experiments with Retraining.

    Science.gov (United States)

    Shultz, George P.; Weber, Arnold R.

    When Armour and Company faced a shutdown of six plants, it joined in a cooperative program of vocational retraining with two labor unions; an Automation Fund Committee was formed, with representation from management, the unions, and "public" (college professors); and an experimental program in Oklahoma City provided experience which was…

  17. Self managing experiment resources

    International Nuclear Information System (INIS)

    Stagni, F; Ubeda, M; Charpentier, P; Tsaregorodtsev, A; Romanovskiy, V; Roiser, S; Graciani, R

    2014-01-01

    Within this paper we present an autonomic Computing resources management system, used by LHCb for assessing the status of their Grid resources. Virtual Organizations Grids include heterogeneous resources. For example, LHC experiments very often use resources not provided by WLCG, and Cloud Computing resources will soon provide a non-negligible fraction of their computing power. The lack of standards and procedures across experiments and sites generated the appearance of multiple information systems, monitoring tools, ticket portals, etc... which nowadays coexist and represent a very precious source of information for running HEP experiments Computing systems as well as sites. These two facts lead to many particular solutions for a general problem: managing the experiment resources. In this paper we present how LHCb, via the DIRAC interware, addressed such issues. With a renewed Central Information Schema hosting all resources metadata and a Status System (Resource Status System) delivering real time information, the system controls the resources topology, independently of the resource types. The Resource Status System applies data mining techniques against all possible information sources available and assesses the status changes, that are then propagated to the topology description. Obviously, giving full control to such an automated system is not risk-free. Therefore, in order to minimise the probability of misbehavior, a battery of tests has been developed in order to certify the correctness of its assessments. We will demonstrate the performance and efficiency of such a system in terms of cost reduction and reliability.

  18. Ultrafast gas switching experiments

    International Nuclear Information System (INIS)

    Frost, C.A.; Martin, T.H.; Patterson, P.E.; Rinehart, L.F.; Rohwein, G.J.; Roose, L.D.; Aurand, J.F.; Buttram, M.T.

    1993-01-01

    We describe recent experiments which studied the physics of ultrafast gas breakdown under the extreme overvoltages which occur when a high pressure gas switch is pulse charged to hundreds of kV in 1 ns or less. The highly overvolted peaking gaps produce powerful electromagnetic pulses with risetimes Khz at > 100 kV/m E field

  19. SAMBA Project Experiences

    Science.gov (United States)

    Fuhrhop, Christian; Vaidya, Raju; Mayora, Oscar; Furtado, Elizabeth

    The purpose of the SAMBA project, running from January 2007 to February 2009, was to create a framework for allowing local communities and citizens (including low income population) to access community-oriented content and services by means of iDTV channels, specifically MHP over DVB-T. This paper summarises the project and the experiences gained from it.

  20. [The AMY experiment

    International Nuclear Information System (INIS)

    1989-01-01

    The AMY experiment is one of three major experiments at TRISTAN which is studying the states the matter produced in electron positron annihilations in the center of mass energy range of 50--65GeV. It provides information between the lower energy facilities such as PEP and PETRA and the new facilities SLC and LEP which are designed to operate in the region of the Z 0 mass near 90GeV. In the region of the AMY experiment, interaction cross sections are near their minimum of about 100pb, making it difficult to acquire large data samples during typical running cycles. This last year has seen an accumulation of about 10---12pb -1 of integrated luminosity in the energy range from 58 to 61.7GeV. Despite this limited data sample, the AMY experiment has been extremely active in attempting to extract the minimum amount of information from the data. Some of the most significant results are discussed in this paper. 9 refs

  1. Expectations for neutron experiments

    International Nuclear Information System (INIS)

    Tonomura, Akira

    1993-01-01

    An electron wave function's phase distribution can now be precisely measured to 1/100th of the electron wavelength using both electron holography and a 'coherent' field-emission electron beam. This technique has opened up a new way to conduct thought experiments once regarded as experimentally impossible and also allows the ultra-fine measurement of material structures and field distributions. (author)

  2. FLORIDA TOWER FOOTPRINT EXPERIMENTS

    Energy Technology Data Exchange (ETDEWEB)

    WATSON,T.B.; DIETZ, R.N.; WILKE, R.; HENDREY, G.; LEWIN, K.; NAGY, J.; LECLERC, M.

    2007-01-01

    The Florida Footprint experiments were a series of field programs in which perfluorocarbon tracers were released in different configurations centered on a flux tower to generate a data set that can be used to test transport and dispersion models. These models are used to determine the sources of the CO{sub 2} that cause the fluxes measured at eddy covariance towers. Experiments were conducted in a managed slash pine forest, 10 km northeast of Gainesville, Florida, in 2002, 2004, and 2006 and in atmospheric conditions that ranged from well mixed, to very stable, including the transition period between convective conditions at midday to stable conditions after sun set. There were a total of 15 experiments. The characteristics of the PFTs, details of sampling and analysis methods, quality control measures, and analytical statistics including confidence limits are presented. Details of the field programs including tracer release rates, tracer source configurations, and configuration of the samplers are discussed. The result of this experiment is a high quality, well documented tracer and meteorological data set that can be used to improve and validate canopy dispersion models.

  3. Modelling Urban Experiences

    DEFF Research Database (Denmark)

    Jantzen, Christian; Vetner, Mikael

    2008-01-01

    How can urban designers develop an emotionally satisfying environment not only for today's users but also for coming generations? Which devices can they use to elicit interesting and relevant urban experiences? This paper attempts to answer these questions by analyzing the design of Zuidas, a new...

  4. Olkiluoto 3 Experience

    International Nuclear Information System (INIS)

    Tiippana, Petteri

    2011-01-01

    This paper discusses the experience from the Olkiluoto 3 nuclear power plant project from regulator's point of view. There are certain factors that have affected greatly the project progress. First, Olkiluoto 3 nuclear power plant is the first European Pressurised Reactor (EPR) being constructed. Secondly, construction of the unit started after a fairly long break in nuclear power plant construction in Europe, which had resulted in loss of experienced and qualified engineering and manufacturing resources. These factors have to be kept in mind when evaluating the experience from Olkiluoto 3. Experience discussed in this paper have to do with the licensing and regulatory oversight process, completion of the design prior to construction, experience and know-how of the participating organisations, quality management in a nuclear construction project, advanced manufacturing and construction technologies, turnkey contract with regard to licensee's responsibility, safety culture aspects in a nuclear construction project, and the role and importance of regulator's oversight. (author)

  5. Experience, Poetry and Truth

    DEFF Research Database (Denmark)

    Gahrn-Andersen, Rasmus

    2017-01-01

    of philosophical thinking. Specifically, I show that, beneath a highly poetic and obscure prose, Jünger posits how subjective experience and poetry allow individuals to realize truth. I relate parts of Jünger’s insights to contributions by Husserl, Heidegger and Merleau-Ponty, arguing that Jünger offers a unique...

  6. United Kingdom's experience

    International Nuclear Information System (INIS)

    Drummond, S.M.

    1994-01-01

    This is a presentation of the United Kingdom's experience with power transmission open access. The topics of the presentation include the objectives of changing, commercial arrangements and economic drivers, long term effects, the effects of moving to a more competitive environment, and factors affecting open access such as political climate and market regulation

  7. Alignement experience in STAR

    CERN Document Server

    Margetis, S; Lauret, J; Perevozchikov, V; Van Buren, G; Bouchef, J

    2007-01-01

    The STAR experiment at RHIC uses four layers of silicon strip and silicon drift detectors for secondary vertex reconstruction. An attempt for a direct charm meson measurement put stringent requirements on alignment and calibration. We report on recent alignment and drift velocity calibration work performed on the inner silicon tracking system.

  8. Caring Experience and Knowledge

    DEFF Research Database (Denmark)

    Dybbroe, Betina

    2005-01-01

    ,didactics and educational thinking- and students experiences and sensing, and illuminates excluding processes in classrooms related to emotional, non-cognitive and relational aspects of the qualifying process. This is set into a larger framework of the biographical professionalization processes of students......, and to the clash between competing rationalities, that cause dilemmas in care....

  9. Experiments using coincidence methods

    International Nuclear Information System (INIS)

    Anwar Dhani.

    1978-01-01

    Experiments on γ spectroscopy using the simple coincidence techniques, including investigation of angular distribution of γ radiation from annihilation process in decay of Na 22 , γ - γ angular correlation technique in decay of Co 60 , decay scheme study of Bi 207 and life time measurement of nuclear Pb 207 excited state have been carried out. (author)

  10. The Transgender Military Experience

    Directory of Open Access Journals (Sweden)

    Michelle Dietert

    2015-04-01

    Full Text Available Although there have been studies that focus on the experiences of the gay and lesbian population serving in the United States military, few have focused on the experience of active duty transgender service members. Transgender individuals transgress the binary conception of gender by deviating from societal gender norms associated with assigned sex at birth. The Department of Defense has set policies and standards that reflect a binary conception of gender, with a focus on conformity. We argue that able-bodied gender variant service personnel are just as capable of serving their country as anyone else. Because of the repercussions associated with active duty transgender military personnel, our sample is small and involves nine clandestine service members and two international service members who wanted to share their stories from a different perspective. Snowball sampling was aimed at finding current active duty and reserve transgender service members. Using a combination of telephone interviews and questionnaires, data were collected from active duty transgender service personnel throughout the United States and two from international militaries that allow transgender people to serve. Data collection focused on the overall experiences of the participants along with questions regarding workplace discrimination, suggestions for policy changes, and their views about the overturn of Don’t Ask, Don’t Tell. Our findings add to a growing source of information about the transgender military experience in the U.S. armed forces and the importance of overturning discriminatory workplace policies that negatively impact transgender service members.

  11. [Ethics and animal experiments.].

    Science.gov (United States)

    Schnaider, Taylor Brandão; Souza, Cláudio de

    2003-04-01

    This is a major subject since the aim is to grant human beings physical, mental, social and spiritual well-being without forgetting the sacred rights of all animals. Most international codes dealing with health-related research practices state that research developed in human beings should be based on previous lab animal experiments or on other scientific data. This article aimed at explaining ethics in animal experiments. The concepts of dissertation and thesis, experimental thesis, experimental essay or pilot experiment and experimental animal facilities are reviewed. Then, a historical retrospective is drawn about the first attempt to develop experimental research policies during the mid 19th Century, in London. It is highlighted that some criteria defined by that time still persist. The first animal research ethical committee was created in Sweden in 1979, followed by the USA in1984. In Brazil, animal research ethical committees were created as late as in the 90s. The Federal Law 6638 was passed in May 1979 and provides for the didactic-scientific practice of animal vivisection. This law, however, is still waiting for regulation. In addition, there are some drafts being analyzed by the Congress, which provide for the use of animals for teaching and research purposes. Finally, the policies adopted by the Brazilian College of Animal Experiments and the Universal Declaration of Animal Rights are presented. Professors, postgraduates, residents and graduate students of a Medical School involved in animal research should be aware of the ethical principles aiming at protecting animals selected for scientific work.

  12. Long baseline neutrino experiments

    Indian Academy of Sciences (India)

    Atmospheric neutrino experiments (IMB, Kamiokande, Super-Kamiokande (SK)) show that νµ created in cosmic ray interactions with atmospheric nuclei are being converted into ντ but νe created in such interactions are unaffected. SK measure- ment of νµ and νe event rates as functions of zenith angle is the key ...

  13. Belief Elicitation in Experiments

    DEFF Research Database (Denmark)

    Blanco, Mariana; Engelmann, Dirk; Koch, Alexander

    Belief elicitation in economics experiments usually relies on paying subjects according to the accuracy of stated beliefs in addition to payments for other decisions. Such incentives, however, allow risk-averse subjects to hedge with their stated beliefs against adverse outcomes of other decisions......-belief elicitation treatment using a financial investment frame, where hedging arguably would be most natural....

  14. The big experiment

    CERN Multimedia

    MacEacheran, Mike

    2010-01-01

    "From an academic laboratory in Switzerland, Cern, the European Organisation for Nuclear Research, is conducting physics experiments that are mesmerising everyone. But are its scientitsts really going to answer the questions of life, the Univers and everyhing in between?" (4 pages)

  15. My experience with research

    Indian Academy of Sciences (India)

    Lawrence

    me to Guwahati to Cotton College, the premier college of the. North Eastern region. ... ate college. Although it was a very rewarding experience, I knew in my heart that scientific research was my true calling. The Head of the Department of Physics at Dibrugarh Uni- ... Let alone the State Government, even other physicists in ...

  16. Visual experience and blindsight

    DEFF Research Database (Denmark)

    Overgaard, Morten

    2011-01-01

    Blindsight is classically defined as residual visual capacity, e.g., to detect and identify visual stimuli, in the total absence of perceptual awareness following lesions to V1. However, whereas most experiments have investigated what blindsight patients can and cannot do, the literature contains...

  17. Psychotic experiences and religiosity

    DEFF Research Database (Denmark)

    Kovess-Masfety, V; Saha, S; Lim, C C W

    2018-01-01

    OBJECTIVES: Religiosity is often associated with better health outcomes. The aim of the study was to examine associations between psychotic experiences (PEs) and religiosity in a large, cross-national sample. METHODS: A total of 25 542 adult respondents across 18 countries from the WHO World Ment...

  18. Enhance Your Twitter Experience

    Science.gov (United States)

    Miller, Shannon McClintock

    2010-01-01

    The author has been encouraging teachers, students, and others to join Twitter and build their personal learning networks (PLNs) ever since she delved into this great social networking site. In this article, she offers a few other tools and tips that can improve the Twitter experience of those who have opened up an account and dabbled a bit but…

  19. Sampling the Deaf Experience.

    Science.gov (United States)

    Teller, Henry E.; And Others

    1993-01-01

    Two graduate students in deaf education wore ear plugs for two months to simulate hearing loss, and recorded their experiences and feelings. Excerpts from their journals are presented, commenting on such daily activities as shopping at a mall, watching television, driving, babysitting, and attending a football game. (JDD)

  20. A Harmonic Motion Experiment

    Science.gov (United States)

    Gluck, P.; Krakower, Zeev

    2010-01-01

    We present a unit comprising theory, simulation and experiment for a body oscillating on a vertical spring, in which the simultaneous use of a force probe and an ultrasonic range finder enables one to explore quantitatively and understand many aspects of simple and damped harmonic motions. (Contains 14 figures.)

  1. Water Flow Experiments

    Indian Academy of Sciences (India)

    This is a simple exercise in elementary fluid dynamics for the undergraduate and the secondary school level. Here, we explore the flow of water through an orifice at the bottom of a cylindri- cal bottle/tank, first through a tube attached to the bottom of the bottle/tank and then without the tube. The experiment is easy to perform.

  2. T be Nnewi Experience

    African Journals Online (AJOL)

    Ugochukwu EF, Ezechukwu CC, Agbata CC, Ezumba I. Preterm Admissions in a. Special Care Baby Unit: The Nnewi Experience. Nigerian Journal of Paediatrics. 2002;29:75. A review of all preterm admissions into the Special Care Bay Unit' of the. Nnamdi Azikiwe University Teaching Hospital (NAUTH), Nnewi, over a ...

  3. A Simple Adsorption Experiment

    Science.gov (United States)

    Guirado, Gonzalo; Ayllon, Jose A.

    2011-01-01

    The study of adsorption phenomenon is one of the most relevant and traditional physical chemistry experiments performed by chemistry undergraduate students in laboratory courses. In this article, we describe an easy, inexpensive, and straightforward way to experimentally determine adsorption isotherms using pieces of filter paper as the adsorbent…

  4. Parent Hearing Aid Experiences

    Science.gov (United States)

    Munoz, Karen; Roberts, Mallory; Mullings, Day; Harward, Richard

    2012-01-01

    This study addresses parent experiences in obtaining and managing hearing aids for their young child. The purpose was to identify challenges parents encounter to determine what state agencies can do to improve parent access to amplification. Data were collected July through September of 2010; 40 parents of children ages birth to 3 years old…

  5. Literature on photoproduction experiments

    International Nuclear Information System (INIS)

    Joos, P.

    1975-10-01

    Literature on gamma-proton, gamma-neutron, gamma-deuteron, gamma-nucleus experiments, inclusive photoproduction, particle yields in gamma-proton and gamma-nucleus, inelastic compton scattering, search for new particles, Primakoff effect, photofission, and QED-tests are compiled. (BJ) [de

  6. CANDU operating experience

    International Nuclear Information System (INIS)

    McConnell, L.G.; Woodhead, L.W.; Fanjoy, G.R.

    1982-03-01

    The CANDU Pressurized Heavy Water (CANDU-PHW) type of nuclear electric generating station has been developed jointly by Atomic Energy of Canada Limited and Ontario Hydro. This paper highlights Ontario Hydro's operating experience using the CANDU-PHW system, with a focus on the operating performance and costs, reliability of system components and nuclear safety considerations both to the workers and the public

  7. Electronics for LHC Experiments

    International Nuclear Information System (INIS)

    2004-01-01

    This document gathers the abstracts of most presentations made at this workshop on electronics for the large hadron collider (LHC) experiments. The presentations were arranged into 6 sessions: 1) electronics for tracker, 2) trigger electronics, 3) detector control systems, 4) data acquisition, 5) electronics for calorimeters and electronics for muons, and 6) links, power systems, grounding and shielding, testing and quality assurance

  8. Globalization: The European Experience.

    Science.gov (United States)

    Goldsmith, Peter

    1996-01-01

    The experience of the United Kingdom and other European countries in designing legal education which responds to the changing needs of the European Union is described. The three-stage British system of legal education is outlined, and the impact of European Union formation discussed briefly. Changes in undergraduate study, professional training,…

  9. The Mobilisatsia experience

    International Nuclear Information System (INIS)

    De Boever, P.

    2005-01-01

    The hazards of long-duration manned space flight are real. In order to participate effectively in long duration orbital missions or to continue the exploration of space, the health of the astronaut must be secured. There is mounting evidence that changes in the immune response of an astronaut in short-term flights, resemble those occurring after acute stress, while the changes during long-term flights resemble those caused by chronic stress. This blunting of the immune system occurs concomitant with a relative increase in microbial contamination in the space cabin environment. Such a combination of events results in an increased probability of in-flight infectious events. Micro-organisms are subject to a genetic evolution, which may lead to the capacity to colonize new environments and to cause infections. Central players in this evolutionary process are mobile genetic elements. They help to mobilize and reorganize genes, be it within a given genome (intragenomic mobility) or between bacterial cells (intercellular mobility). Hence, the processes of genetic exchange can mobilize genetic elements between bacterial strains, and therefore play a role in determining the infectious potential. The specific confined environment and space-flight related factors (such as microgravity and cosmic radiation) may increase the frequency in which mobile genetic elements are exchanged between micro organisms. The aim of the Mobilisatsia experiment was to promote microbial gene transfer under space flight conditions during a short-term experiment conducted aboard the International Space Station (ISS). The efficiency of the gene exchange process was compared with a synchronously performed ground control experiment. An experiment was carried out with well-characterized Gram-negative reference strains and one experiment was done with Gram-positive reference strains

  10. Transformations of emotional experience.

    Science.gov (United States)

    de Cortiñas, Lia Pistiner

    2013-06-01

    In this paper the author approaches mental pain and the problems in a psychoanalytic treatment of patients with difficulties in the psychic transformation of their emotional experiences. The author is interested in the symbolic failure related to the obstruction of development of phantasies, dreams, dream-thoughts, etc. She differentiates symbolization disturbances related to hypertrophic projective identification from a detention of these primitive communications and emotional isolation. She puts forward the conjecture that one factor in the arrest of this development is the detention of projective identifications and that, when this primitive means of communication is re-established in a container-contained relationship of mutual benefit, this initiates the development of a symbolization process that can replace the pathological 'protection'. Another hypothesis she develops is that of inaccessible caesuras that, associated with the detention of projective identification, obstruct any integrative or interactive movement. This caesura and the detention of projective identifications affect mental functions needed for dealing with mental pain. The personality is left with precarious mental equipment for transforming emotional experiences. How can a psychoanalytical process stimulate the development of creative symbolization, transforming the emotional experiences and leading towards mental growth? The author approaches the clinical problem with the metaphor of the psychic birth of emotional experience. The modulation of mental pain in a container-contained relationship is a central problem for the development of the human mind. For discovering and giving a meaning to emotional experience, the infant depends on reverie, a function necessary in order to develop an evolved consciousness capable of being aware, which is different from the rudimentary consciousness that perceives but does not understand. The development of mature mental equipment is associated with the

  11. Results of recent LOFT experiments

    International Nuclear Information System (INIS)

    Leach, L.P.; Hanson, D.J.; Batt, D.L.

    1982-01-01

    Five experiments were performed in the Loss-of-Fluid Test (LOFT) facility during the past year. The experiments conducted spanned a wide range of potential accident scenarios, including large and small break loss-of-coolant accidents (LOCAs), control rod withdrawal accidents, uncontrolled boron dilution, and anticipated transients without scram (ATWS). This summary describes these experiments and presents results available from the experiments and experiment prediction calculations. A brief overview is given for the remaining experiment planned in the LOFT Program

  12. LCLS The First Experiments

    Energy Technology Data Exchange (ETDEWEB)

    Galayda, John N

    2003-02-10

    The Scientific Advisory Committee (SAC) for the Linac Coherent Light Source (LCLS) has selected six scientific experiments for the early phase of the project. The LCLS, with proposed construction in the 2003-2006 time frame, has been designed to utilize the last third of the existing Stanford Linear Accelerator Center (SLAC) linac. The linac produces a high-current 5-15 GeV electron beam that is bunched into 230 fs slices with a 120 Hz repetition rate. When traveling through a sufficiently long (of order of 100 m) undulator, the electron bunches will lead to self amplification of the emitted x-ray intensity constituting an x-ray free electron laser (XFEL). If funded as proposed, the LCLS will be the first XFEL in the world, operating in the 800-8,000 eV energy range. The emitted coherent x-rays will have unprecedented brightness with 10{sup 12}-10{sup 13} photons/pulse in a 0.2-0.4% energy bandpass and an unprecedented time structure with a design pulse length of 230 fs. Studies are under way to reduce the pulse length to tens of femtoseconds. This document presents descriptions of the early scientific experiments selected by SAC in the spring of 2000. They cover a wide range of scientific fields. The experimental teams consist of many internationally recognized scientists who are excited about the unprecedented x-ray capabilities of LCLS that surely will lead to new scientific frontiers. More generally, this document serves to forward the scientific case for an accelerator-based XFEL source, as requested by the BESAC subpanel on Novel Coherent Light Sources, chaired by Stephen R. Leone. Two general classes of experiments are proposed for the LCLS. The first class consists of experiments where the x-ray beam is used to probe the sample without modifying it, as is done in most experiments at current synchrotron sources. In the second class, the LCLS beam is used to induce non-linear photo-processes or matter in extreme conditions. The same source can be used for

  13. Experimenting with Bulbs

    Science.gov (United States)

    Cavicchi, Elizabeth M.

    1998-04-01

    What questions come about as learners explore physical materials? How does their learning deepen through inventing experiments and making and testing their own interpretations? I present episodes from the investigatory work of three adult learners who met regularly with me to explore batteries, bulbs and wires. I taught by engaging the learners' interest in these materials and by interactively researching their developing understandings. As both teacher and researcher, I used what I learned about their understandings to support their efforts to extend the body of experimental knowledge they were developing. Development is evident in experimental details. Initially, by hand-holding, they light a bulb with a wire and battery. Later, in soldering connections, they wonder about the bulb's internal structure. They research thoughts about this through dissecting a bulb -- and lighting its exposed filament. Such experimenting changed thinking: from imagining sequential loops, to questioning electrical contacts, to inferring circuital paths.

  14. Transgressive first clinical experiences

    DEFF Research Database (Denmark)

    Jensen, Carsten Juul; Jeppesen, Lise Kofoed; Drachmann, Merete

    2014-01-01

    . The nursing students’ learning seems to be oriented towards socialization in the clinic as a workplace. This means that the nursing students seek to deal with overwhelming experiences concerning the naked bodies of patients and death, useful application of theoretical knowledge, the path from novice......This Study seeks to comprehend learning experiences of nursing students during their first clinical in-service placement. This Paper is part of a longitudinal development project interviewing the Student Nurse after each one of the five clinical in-service placements and then one year after...... graduation as a Nurse. The Study has a qualitative methodology, inspired by Michael Eraut’s thoughts on learning in the workplace. When the workplace perspective is applied, learning seems to be concentrated on actual situations which the Learner is in, in contrast to employing constructed concepts...

  15. The LDMX Experiment

    Directory of Open Access Journals (Sweden)

    Mans Jeremiah

    2017-01-01

    Full Text Available The thermal-relic mechanism for the production of dark-matter in the early universe, combined with the precision measurements of the cosmic microwave background and the lack of observation of dark matter at the LHC, motivates the search for dark matter in the mass range between 1 MeV and 1 GeV. We describe the concept for the Light Dark Matter Experiment (LDMX, which could carry out such a search using the missing-momentum technique. The experiment would utilize a unique high-rate, low-current electron beam with an energy between 4 GeV and 10 GeV, and would have sensitivity to the thermal-relic hypothesis over a wide range of masses below 1 GeV.

  16. Engaging the Shopping Experience

    DEFF Research Database (Denmark)

    Sørensen, Sanne Dollerup

    The revenues in brick-and-mortar stores have declined in the last decade, not least due to competition from online shopping. This thesis investigates how traditional stores might use principles from experience design to reverse this tendency. Brick-and-mortar stores are very important...... research, experience design, literary theory, the history and sociology of shopping. The method used is observation studies of cases that more or less successfully have been able to engage customers. This has resulted in an in-depth study of one such store and a typology of different types of engaging...... the interest in brick-and-mortar stores by engaging the customers emotionally. This thesis suggests that using insights from Possible World Theory in designing stores is one way to do this. Theoretically the thesis is interdisciplinary by drawing on knowledge from a wide spectrum of fields such as consumer...

  17. Future flavour physics experiments

    Science.gov (United States)

    2015-01-01

    The current status of flavour physics and the prospects for present and future experiments will be reviewed. Measurements in B‐physics, in which sensitive probes of new physics are the CKM angle γ, the Bs mixing phase ϕs, and the branching ratios of the rare decays B(s)0→μ+μ− , will be highlighted. Topics in charm and kaon physics, in which the measurements of ACP and the branching ratios of the rare decays K→πνν¯ are key measurements, will be discussed. Finally the complementarity of the future heavy flavour experiments, the LHCb upgrade and Belle‐II, will be summarised. PMID:26877543

  18. Commissioning the CMS experiment

    CERN Document Server

    Camporesi, T.

    2008-01-01

    The CMS experiment is getting ready to take data at the LHC accelerator at CERN. Due to late delivery of the experimental caverns the detector had to be pre-assembled on the surface and lowered in large segments. This has constrained the commissioning strategy of the experiment. Global commissioning has started in Spring of 2007 and has progressed in parallel with the construction and assembly of detector and services underground. This paper describes the strategy adopted in order to commission components of the system in incremental steps in order to be ready to accept collisions from LHC with a well debugged and understood system, including the detector, acquisition system, trigger, software, computing and data shipping.

  19. Digital Heritage Experiences

    DEFF Research Database (Denmark)

    Munar, Ana Maria; Ooi, Can-Seng

    The evolution of the Web and the expansion of social media are transforming our heritage experiences. Social media offer an innovative element to personal travel reflections by providing digital global platforms on which tourists can create and publish their travel stories. Social media transform...... the traditional creative processes, distribution mechanisms and consumption patterns of these experiences. This study provides insight on how personal heritage moments are constructed, digitalized and shared. The methodological perspective adopted draws on a constructivist epistemology (Markham 2004......) and netnography (Kozinets, 2002). The social media platform analysed is TripAdvisor, which is the largest networking site focusing on tourism and travel. Study findings indicate that while heritage sites tend to promote their uniqueness and the cultural value of their products, tourists are just as concerned...

  20. New Experiments with Antiprotons

    Science.gov (United States)

    Kaplan, D. M.

    2011-12-01

    Fermilab operates the world's most intense antiproton source. Recently proposed experiments can use those antiprotons either parasitically during Teva-tron Collider running or after the Tevatron Collider finishes in about 2011. For example, the annihilation of 8 GeV antiprotons might make the world's most intense source of tagged D0 mesons, and thus the best near-term opportunity to study charm mixing and search for new physics via its CP-violation signature. Other possible precision measurements include properties of the X(3872) and the charmonium system. An experiment using a Penning trap and an atom interferometer could make the world's first measurement of the gravitational force on antimatter. These and other potential measurements using antiprotons could yield a broad physics program at Fermilab in the post-Tevatron era.

  1. Muon science experiments

    International Nuclear Information System (INIS)

    Nishiyama, Kusuo

    2001-01-01

    Muon experimental facility is in the planning stage as a part of the high intensity proton accelerator project of KEK and JAERI. Outline of muon experimental facility and physical experiments are described. There are five kinds of beam channels such as superconducting solenoid, surface muon, ultra-slow muon, high momentum muon and ultra-high intensity muon. The μSR method by positive electric charge muon makes possible to study antiferromagnet, spin glass, muon spin high-frequency resonance and paramagnetic shift. The positive electric charge muon is one of hydrogen isotopes. Accordingly, the quantum diffusion experiments of metals, semiconductor and insulator are carried out by the isotope effect. Development of muon catalyzed fusion by negative electric charge muon is one of problems of this facility. It can be applied to chemical reaction and non-destructive analysis. An illustration of the history of muon science is shown. (S.Y.)

  2. Quantum Gravity Experiments

    Directory of Open Access Journals (Sweden)

    Cahill R. T.

    2015-10-01

    Full Text Available A new quantum gravity experiment is reported with the data confirming the generali- sation of the Schrödinger equation to include the interaction of the wave function with dynamical space. Dynamical space turbulence, via this interaction process, raises and lowers the energy of the electron wave function, which is detected by observing conse- quent variations in the electron quantum barrier tunnelling rate in reverse-biased Zener diodes. This process has previously been reported and enabled the measurement of the speed of the dynamical space flow, which is consistent with numerous other detection experiments. The interaction process is dependent on the angle between the dynamical space flow velocity and the direction of the electron flow in the diode, and this depen- dence is experimentally demonstrated. This interaction process explains gravity as an emergent quantum process, so unifying quantum phenomena and gravity. Gravitational waves are easily detected.

  3. Microwave Tokamak Experiment

    International Nuclear Information System (INIS)

    Anon.

    1988-01-01

    The Microwave Tokamak Experiment, now under construction at the Laboratory, will use microwave heating from a free-electron laser. The intense microwave pulses will be injected into the tokamak to realize several goals, including a demonstration of the effects of localized heat deposition within magnetically confined plasma, a better understanding of energy confinement in tokamaks, and use of the new free-electron laser technology for plasma heating. The experiment, soon to be operational, provides an opportunity to study dense plasmas heated by powers unprecedented in the electron-cyclotron frequency range required by the especially high magnetic fields used with the MTX and needed for reactors. 1 references, 5 figures, 3 tables

  4. Copenhagen Sonic Experience Map

    DEFF Research Database (Denmark)

    Kreutzfeldt, Jacob

    2011-01-01

    In the wake of present European interest for mapping urban noise, it seems increasingly relevant to investigate the multiple ways in which sound intersects with the everyday experiences of urban citizens. Focusing on the polluting effects of infrastructural noise, the EU-initiated project...... of assessment and management of environmental noise brings forth the disturbing and potentially damaging effect of environmental sound.1 But as maps of coloured streets start to circulate, and real estate prices drop in designated blue and red areas,2 it is worth remembering that sound itself is not a killer....... Most of the time sound is a trivial part of everyday life involved in interactions, experiences, atmospheres, actions, etc. Although trivial, the role of the aural in the urban life world is more complex than what is suggested by sound level maps, and as such it may be a vital part of urbanity itself...

  5. Gross decontamination experiment report

    Energy Technology Data Exchange (ETDEWEB)

    Mason, R.; Kinney, K.; Dettorre, J.; Gilbert, V.

    1983-07-01

    A Gross Decontamination Experiment was conducted on various levels and surfaces of the TMI - Unit 2 reactor building in March 1982. The polar crane, D-rings, missile shields, refueling canals, refueling bridges, equipment, and elevations 305' and 347'-6'' were flushed with low pressure water. Additionally, floor surfaces on elevation 305' and floor surfaces and major pieces of equipment on elevation 347'-6'' were sprayed with high pressure water. Selective surfaces were decontaminated with a mechanical scrubber and chemicals. Strippable coating was tested and evaluated on equipment and floor surfaces. The effectiveness, efficiency, and safety of several decontamination techniques were established for the large, complex decontamination effort. Various decontamination equipment was evaluated and its effectiveness was documented. Decontamination training and procedures were documented and evaluated, as were the support system and organization for the experiment.

  6. Gross decontamination experiment report

    International Nuclear Information System (INIS)

    Mason, R.; Kinney, K.; Dettorre, J.; Gilbert, V.

    1983-07-01

    A Gross Decontamination Experiment was conducted on various levels and surfaces of the TMI - Unit 2 reactor building in March 1982. The polar crane, D-rings, missile shields, refueling canals, refueling bridges, equipment, and elevations 305' and 347'-6'' were flushed with low pressure water. Additionally, floor surfaces on elevation 305' and floor surfaces and major pieces of equipment on elevation 347'-6'' were sprayed with high pressure water. Selective surfaces were decontaminated with a mechanical scrubber and chemicals. Strippable coating was tested and evaluated on equipment and floor surfaces. The effectiveness, efficiency, and safety of several decontamination techniques were established for the large, complex decontamination effort. Various decontamination equipment was evaluated and its effectiveness was documented. Decontamination training and procedures were documented and evaluated, as were the support system and organization for the experiment

  7. Experiments around I-8

    CERN Multimedia

    CERN PhotoLab

    1975-01-01

    The lithium transition-radiation detectors and the large liquid argon calorimeters of experiment R806T are shown above and below the intersection at I-8 (Brookhaven-CERN-Saclay-Syracuse-Yale Collaboration, Study of large transverse momentum phenomena by electron and photon detection). At 90 deg to the intersecting beams are the monitoring proporional chambers of experiment R805 (Measurement of real to imaginary ratio of forward scattering amplitude - Coulomb interference - by the CERN-Rome Collaboration). Left and right of the intersection one sees, symmetrically placed around the interaction region, the large scintillation counters hodoscopes used by R801 (Pisa-Stony Brook Collaboration) to measure the pp total cross section and the features of inelastic collisions.

  8. The MOZART experiment

    International Nuclear Information System (INIS)

    Briec, M.; Abassin, J.J.; Masson, M.; Johnson, C.E.; Roux, N.

    1989-01-01

    The MOZART experiment was carried out within the framework of the BEATRIX program, as part of the Commissariat a l'Energie Atomique contribution to this international collaboration program. This experiment was run during 45 days in the MELUSIN reactor at Grenoble. Tested ceramics were Li 2 O and LiAlO 2 from Japan, Li 2 ZrO 3 from USA, LiAlO 2 from CEA. Influence of parameters such as temperature, sweep gas composition was investigated. Tritium residence times as a function of temperature were calculated. The comparison of the tritium release performance of the three ceramic breeders was made. This investigation has first evidenced the very good tritium release characteristics of Li 2 ZrO 3 especially at low temperatures. (author). 7 refs.; 5 figs.; 3 tabs

  9. The Vinca dosimetry experiment

    International Nuclear Information System (INIS)

    1962-03-01

    On 15 October 1958 there occurred a very brief uncontrolled run of the zero-power reactor at the Boris Kidric Institute of Nuclear Science, Vinca, near Belgrade, Yugoslavia. During this run six persons received various doses of radiation. They were subsequently given medical treatment of a novel kind at the Curie Hospital, Paris. In atomic energy operations to date, very few accidents involving excessive radiation exposure to human beings have occurred. In fact, the cases of acute radiation injury are limited to about 30 known high exposures, few of which were in the lethal or near-lethal range. Since direct experiment to determine the effects of ionizing radiation on man is unacceptable, information on these effects has to be based on a consideration of data relating to accidental exposures, viewed in the light of the much more extensive data obtained from experiments on animals. Therefore, any direct information on the effects of radiation on humans is very valuable. The international dosimetry project described in this report was carried out at Vinca, Yugoslavia, under the auspices of the International Atomic Energy Agency to determine the precise amount of radiation to which the persons had been exposed during the accident. These dosimetry data, together with the record of the carefully observed clinical effects, are of importance both for the scientific study of radiation effects on man and for the development of methods of therapy. The experiment and measurements were carried out at the end of April 1960. The project formed part of the Agency's research programme in the field of health and safety. The results of the experiment are made available through this report to all Member States

  10. CANDU operating experience

    International Nuclear Information System (INIS)

    McConnell, L.G.; Woodhead, L.W.; Fanjoy, G.R.

    1982-09-01

    The CANDU (CANada Deuterium Uranium) Pressurized Heavy Water (PHW) type of nuclear-electric generating station was developed jointly by Atomic Energy of Canada Limited and Ontario Hydro. This paper summarizes Ontario Hydro's operating experience using the CANDU-PHW system, with a focus on the operating performance and costs, reliability of system components, and nuclear safety considerations to both the workers and the public

  11. submitter LHC experiments

    CERN Document Server

    Tanaka, Shuji

    2001-01-01

    Large Hadron Collider (LHC) is under construction at the CERN Laboratory in Switzerland. Four experiments (ATLAS, CMS, LHCb, ALICE) will try to study the new physics by LHC from 2006. Its goal to explore the fundamental nature of matter and the basic forces. The PDF file of the transparency is located on http://www-atlas.kek.jp/sub/documents/lepsymp-stanaka.pdf.

  12. Delivering ideal employee experiences.

    Science.gov (United States)

    Weiss, Marjorie D; Tyink, Steve; Kubiak, Curt

    2009-05-01

    Employee-centric strategies have moved from employee satisfaction and brand awareness to employee "affinity" or "attachment." In today's marketplace, occupational health nurses understand that differentiation (i.e., the perception of uniqueness) is the direct result of superior employee interactions, which lead to better employee care, enduring employee relationships, loyal employees, and satisfied employers. What drives employees to occupational health nurse attachment? The answer is a passion for rising above the competition to create ideal employee experiences.

  13. DIRAC experiment at CERN

    CERN Document Server

    Benelli, Angela

    2012-01-01

    The precise measurements of $\\pi^+\\pi^-$ and $\\pi K$ atom lifetime allow to check the predictions of Chiral Perturbation Theory for the pion-pion s-wave scattering lengths with isospin 0 and 2 and for the pion-kaon scattering lengths with isospin 1/2 and 3/2. The DIRAC experiment with the latest results is presented together with the proposal for future investigations.

  14. The MUSE experiment

    OpenAIRE

    Downie E. J.

    2014-01-01

    The proton radius puzzle is the difference between the proton radius as measured with electron scattering and in the excitation spectrum of atomic hydrogen, and that measured with muonic hydrogen spectroscopy. The MUSE experiment seeks to resolve this puzzle by simultaneously measuring elastic electron and muon scattering on the proton, in both charge states, thereby providing new information to the puzzle. MUSE addresses issues of two-photon effects, lepton universality and, possibly, new ph...

  15. The global accounting experiment

    OpenAIRE

    Nicolas Véron

    2007-01-01

    In this essay, Nicolas Véron presents accounting standard-setting as a frontier experiment in international governance, where private initiative can succeed in solving a collective problem that nation-states could not tackle effectively. To make the adoption of International Financial Reporting Standards (IFRS) in Europe and other jurisdictions sustainable, the legitimacy of the International Accounting Standards Board has to be bolstered through significant governance changes; and their impl...

  16. The CONNIE experiment

    Energy Technology Data Exchange (ETDEWEB)

    Aguilar-Arevalo, A.; et al.

    2016-10-19

    The CONNIE experiment uses fully depleted, high resistivity CCDs as particle detectors in an attempt to measure for the first time the Coherent Neutrino-Nucleus Elastic Scattering of antineutrinos from a nuclear reactor with silicon nuclei.This talk, given at the XV Mexican Workshop on Particles and Fields (MWPF), discussed the potential of CONNIE to perform this measurement, the installation progress at the Angra dos Reis nuclear power plant, as well as the plans for future upgrades.

  17. Holodeck-ISS Experience

    Science.gov (United States)

    Rainbolt, Phillip

    2016-01-01

    For the duration of my internship here at JSC for the summer 2016 session, the main project that I worked on dealt with hybrid reality simulations of the ISS. As an ER6 intern for the spacecraft software division, the main project that I worked alongside others was with regards to the Holodeck Virtual Reality Project, specifically with the ISS experience, with the use of the HTC Vive and controllers.

  18. The NEXT experiment

    Energy Technology Data Exchange (ETDEWEB)

    DIaz, J; Yahlali, N; Ball, M; Carcel, S; Cervera, A; Gil, A [Instituto de Fisica Corpuscular (Centro mixto UV-CSIC), Apdo. de Correos 22085, E-46071 Valencia (Spain) (Spain); Barata, J A S; Borges, F I G M; Conde, C A N; Dias, T H V T; Fernandes, L M P; Freitas, E D C [Universidade de Coimbra (Portugal); Calvo, E [Instituto de Fisica de Altas EnergIas, IFAE, Barcelona (Spain); Carmona, J M; Cebrian, S; Dafni, T; Galan, J [U. Zaragoza (Spain); Cid, X [Universidade de Santiago de Compostela (Spain); Ferrer-Ribas, E [CEA, IRFU, Saclay (France); Gil, I, E-mail: jose.diaz@uv.e [CIEMAT (Spain)

    2009-07-01

    Neutrinoless double beta decay measurements are the most promising experiments both to reveal the Majorana nature of the neutrino and to set a value for its mass. The NEXT project propose to build a High pressure Xenon TPC in the Canfranc Underground Laboratory (Huesca, Spain) to measure double-beta decay of {sup 136}Xe, both normal and neutrinoless, with a source mass of 100 kg of enriched xenon.

  19. The COMPASS experiment

    CERN Multimedia

    2006-01-01

    Artistic view of the 60 m long Common Muon and Proton Apparatus for Structure and Spectroscopy (COMPASS) experiment. COMPASS takes beams from the SPS accelerator at CERN. This beam enters from the lower left and passes through the cylindrical target solenoid. COMPASS is used to study the internal structure of hadrons (composite particles interacting via the strong force) by looking at a property called spin and a phenomenon known as glueballs.

  20. The ACE experiment

    CERN Multimedia

    Maximilien Brice

    2006-01-01

    The Antiproton Cell Experiment (ACE) as shown by Michael Holzscheiter (spokesperson), Niels Bassler (co-spokesperson) and Helge Knudsen. ACE is located on the Antiproton Decelerator (AD) at CERN. An antiproton annihilates a proton in the nucleus of a cancer cell, producing a pair of gamma rays, destroying the entire cell and some surrounding cells. Many fewer antiprotons are required in this treatment than in the equivalent proton hadron therapy, so there is less risk of healthy tissue damage.

  1. Crossed beam experiments

    International Nuclear Information System (INIS)

    Dolder, K.T.

    1976-01-01

    Many natural phenomena can only be properly understood if one has a detailed knowledge of interactions involving atoms, molecules, ions, electrons or photons. In the laboratory these processes are often studied by preparing beams of two types of particle and observing the reactions which occur when the beams intersect. Some of the more interesting of these crossed beam experiments and their results are discussed. Proposals to extend colliding beam techniques to high energy particle physics are also outlined. (author)

  2. Creating Sustainable Digital Experiences

    Directory of Open Access Journals (Sweden)

    Kozinets Robert V.

    2014-11-01

    Full Text Available Is the social media hype about being cool or about making money? For Adam Froman, the answer is easy: Marketers need to be very clear about how social media activity supports overall business strategy. Only if the digital experience fi ts into the whole customer journey will consumers become engaged and add value to the company … and only then will the social brand become cool.

  3. NRC Construction Experience

    International Nuclear Information System (INIS)

    Rasmussen, Richard

    2013-01-01

    This presentation deals with the domestic and international construction experience sources. From the domestic (USA) point of view, the sources are event notifications, non-compliance reports, inspection reports, etc. Internationally, the presentation highlights the following sources: IRS and ConEx reports, Nuclear Events Web Based System (NEWS), bilateral agreements, MDEP, etc. It also mentions and describes briefly reported on concrete, rebar, fabrication and digital issues

  4. Relay mirror experiment

    Science.gov (United States)

    Begley, David L.

    1996-04-01

    Originating out of a SDIO-funded, Phase 1 study effort, two ground systems and an orbiting EO payload/spacecraft were the primary equipment for the RME. The RME was originally conceived to be a shuttle deployed experiment. Shortly after program start, the Challenger disaster occurred, with the promise of extensive delays. A completely new space segment was to be designed incorporating a free-flying spacecraft. During the midphase of the program, a variety of launch vehicles were envisioned to replace the shuttle, requiring the BASD team to design accommodations for Delta, Atlas, and Titan, with a Delta launch being the final solution. The ground systems tracked the spacecraft and illuminated it with green and blue beacon lasers. The Payload Experiment Package (PEP) housed the bisection tracker, a key innovation central to the experiment. The bisection tracker acquired both beacons and controlled steerable mirrors to accomplish fine tracking of the two cooperative beacons. In the process, the relay mirror was precisely positioned to enable a successful relay of a third infrared laser between the two ground sites via the orbiting spacecraft. Many of the key technologies employed in the PEP were originally developed for Ball laser communications research and development programs and other laser pointing efforts. The WAVE sensor package, built by ATA and integrated by Ball, measured the vibrations of the optical base structure on which it was mounted. These spacecraft vibration data are critical to the accurate pointing of space laser communication terminals.

  5. Experimenting with practice

    DEFF Research Database (Denmark)

    Knudsen, Hanne; Adriansen, Hanne Kirstine

    2016-01-01

    Abstract Purpose Teaching executive courses always raises the challenge of how to deal with the tension between theory and practice. The present chapter analyses the use of experiments in practice as a pedagogical approach to deal with this tension in Master’s programmes. Design/methodology/appro......Abstract Purpose Teaching executive courses always raises the challenge of how to deal with the tension between theory and practice. The present chapter analyses the use of experiments in practice as a pedagogical approach to deal with this tension in Master’s programmes. Design...... creates a liminal zone with hybrid characteristics. Research limitations/implications The chapter provides new conceptualizations of the tensions between theory and practice based on our experiences from one degree programme. It would have been interesting to study other executive programmes and which....... It does not solve the tension between theory and practice but creates new challenges, potentialities, dilemmas and insights. Originality/value We suggest using ‘monstrosity’ as an umbrella term for ‘hybrid’ and ‘liminality’ of the complex relations that are at play in further education of practitioners...

  6. Sequential Design of Experiments

    Energy Technology Data Exchange (ETDEWEB)

    Anderson-Cook, Christine Michaela [Los Alamos National Lab. (LANL), Los Alamos, NM (United States)

    2017-06-30

    A sequential design of experiments strategy is being developed and implemented that allows for adaptive learning based on incoming results as the experiment is being run. The plan is to incorporate these strategies for the NCCC and TCM experimental campaigns to be run in the coming months. This strategy for experimentation has the advantages of allowing new data collected during the experiment to inform future experimental runs based on their projected utility for a particular goal. For example, the current effort for the MEA capture system at NCCC plans to focus on maximally improving the quality of prediction of CO2 capture efficiency as measured by the width of the confidence interval for the underlying response surface that is modeled as a function of 1) Flue Gas Flowrate [1000-3000] kg/hr; 2) CO2 weight fraction [0.125-0.175]; 3) Lean solvent loading [0.1-0.3], and; 4) Lean solvent flowrate [3000-12000] kg/hr.

  7. Shock tube Multiphase Experiments

    Science.gov (United States)

    Middlebrooks, John; Allen, Roy; Paudel, Manoj; Young, Calvin; Musick, Ben; McFarland, Jacob

    2017-11-01

    Shock driven multiphase instabilities (SDMI) are unique physical phenomena that have far-reaching practical applications in engineering and science. The instability is present in high energy explosions, scramjet combustors, and supernovae events. The SDMI arises when a multiphase interface is impulsively accelerated by the passage of a shockwave. It is similar in development to the Richtmyer-Meshkov (RM) instability however, particle-to-gas coupling is the driving mechanism of the SDMI. As particle effects such as lag and phase change become more prominent, the SDMI's development begins to significantly deviate from the RM instability. We have developed an experiment for studying the SDMI in our shock tube facility. In our experiments, a multiphase interface is created using a laminar jet and flowed into the shock tube where it is accelerated by the passage of a planar shockwave. The interface development is captured using CCD cameras synchronized with planar laser illumination. This talk will give an overview of new experiments conducted to examine the development of a shocked cylindrical multiphase interface. The effects of Atwood number, particle size, and a second acceleration (reshock) of the interface will be discussed.

  8. Hyperon Beam Experiment

    CERN Multimedia

    2002-01-01

    The experiment WA89 uses the upgraded Omega facility together with a hyperon beam installed at the end of the H1 beamline. The beam can deliver 2~10$ ^{5} \\% Sigma ^- $ per machine burst at 330 GeV/c with a background of 5 10$ ^{5} \\% \\pi ^- $. \\\\ \\\\ The goals of the experiment are: observation of charmed particles, mainly the charmed-strange baryons and measurements of their production in the kinematical range x$ _{F} $~$>$~0.2, and their decay properties, a search for exotic states such as U(3100) observed in the previous CERN hyperon beam experiment WA62, measurements of hyperon polarization and production properties. \\\\ \\\\ A vertex detector consisting of 24 silicon microstrip planes with 25~$\\mu$m pitch and 6~planes with 50~$\\mu$m pitch provides track measurements of sufficient accuracy to identify the decays of short living charmed particles and measure their lifetimes. A RICH detector provides good $\\pi$/K separation for momenta up to 100~GeV/c and $\\pi$/p separation up to 150~GeV/c. Photons are detecte...

  9. Measuring the User Experience

    Directory of Open Access Journals (Sweden)

    Harry B. Santoso

    2016-01-01

    Full Text Available The aim of the current study is to develop an adapted version of User Experience Questionnaire (UEQ and evaluate a learning management system. Although there is a growing interest on User Experience, there are still limited resources (i.e. measurement tools or questionnaires available to measure user experience of any products, especially learning management systems. Two hundreds and thirteen computer science students participated and completed the adapted version of UEQ. In the study, the researchers used a learning management system named Student Centered e-Learning Environment (SCELE. Several types of learning materials are posted in SCELE such as audio files, simulations, PowerPoint slides, multimedia contents, and webpage links. Most of the lecturers use discussion forums in their courses to encourage students to participate in active learning setting. Staff and lecturers sometimes post academic-related announcements on the SCELE homepage. Two hundred thirteen students enrolled in Computer Science program were invited to evaluate the SCELE. This study will benefit UX practitioners, HCI educators, program and center of learning resources administrators, and learning management system developers. Findings of the present study may also be valuable for universities and high schools which are using computer-based learning environments.

  10. Tango for experiment control

    International Nuclear Information System (INIS)

    Meyer, J.; Claustre, L.; Petitdemange, S.; Svensson, O.; Götz, A.; Coutinho, T.; Klora, J.; Picca, F.; Ounsy, M.; Buteau, A.

    2012-01-01

    The Tango control system framework allows you to control an accelerator complex as well as single equipment. The framework contains the communication bus with the standard communication modes (synchronous, asynchronous, event driven) as well as the basic hardware access modules, GUI tools and development kits, bindings to commercial products (LabView, Matlab, IgorPro) and services (administration, archiving, access control) to set up a control system. Tango was mainly developed by several synchrotron light sources that have to support not only the accelerator complex but also a lot of experimental end stations. For synchrotron experiments we have to control the whole process from basic hardware access over data taking to data analysis. This paper describes in the first part the special features of Tango allowing flexible experiment control. The dynamic configuration, the rapid hardware interface development and the sequencing and scanning framework are some examples. The second part gives an overview of some packages developed in the Tango community for experiment control: A HKL library for diffraction computation and diffractometer control, a library to control 2D detectors and a data analysis workbench with workflow engine for on-line and off-line data analysis. These packages are not part of Tango and can be used with other control systems. (author)

  11. AGS experiments: 1990, 1991, 1992

    International Nuclear Information System (INIS)

    Depken, J.C.

    1993-04-01

    This report contains a description of the following: AGS Experimental Area - High Energy Physics FY 1993 and Heavy Ion Physics FY 1993; Table of Beam Parameters and Fluxes; Experiment Schedule ''as run''; Proposed 1993 Schedule; A listing of experiments by number; Two-page summaries of each experiment begin here, also ordered by number; Publications of AGS Experiments; and List of AGS Experimenters

  12. AGS Experiments: 1989, 1990, 1991

    Energy Technology Data Exchange (ETDEWEB)

    Depken, J.C.

    1992-02-01

    This report contains: Experimental areas layout; table of beam parameters and fluxes; experiment schedule as run''; proposed 1992 schedule; a listing of experiments by number; two-page summaries of each experiment begin here, also ordered by number; publications of AGS Experiments begin here; and list of AGS Experimenters begins here.

  13. AGS Experiments: 1989, 1990, 1991

    Energy Technology Data Exchange (ETDEWEB)

    Depken, J.C.

    1992-02-01

    This report contains: Experimental areas layout; table of beam parameters and fluxes; experiment schedule ``as run``; proposed 1992 schedule; a listing of experiments by number; two-page summaries of each experiment begin here, also ordered by number; publications of AGS Experiments begin here; and list of AGS Experimenters begins here.

  14. Supporting planning and conducting experiments

    NARCIS (Netherlands)

    van Riesen, Siswa; Gijlers, Aaltje H.; Anjewierden, Anjo Allert; de Jong, Anthonius J.M.; Looi, Chee-Kit; Polman, Joseph; Cress, Ulrike; Reimann, Peter

    2016-01-01

    In inquiry learning learners design and conduct experiments. Learners experience difficulties with the involved processes and need guidance to design useful experiments. To guide students in this we created a configurable experiment design tool that is usable in multiple domains. The tool was tested

  15. Urban Experiments and Concrete Utopias

    DEFF Research Database (Denmark)

    Andersson, Lasse

    2009-01-01

    The paper explores how concrete urban experiments can challenge the pecuniary version of the experience city and stimulate a locally rooted and democratic version of an experience based city using heterotopias and concrete utopias as the link between top down planning and bottom up experiments...

  16. AGS Experiments: 1989, 1990, 1991

    International Nuclear Information System (INIS)

    Depken, J.C.

    1992-02-01

    This report contains: Experimental areas layout; table of beam parameters and fluxes; experiment schedule ''as run''; proposed 1992 schedule; a listing of experiments by number; two-page summaries of each experiment begin here, also ordered by number; publications of AGS Experiments begin here; and list of AGS Experimenters begins here

  17. AGS experiments - 1982, 1983, 1984

    International Nuclear Information System (INIS)

    Bunce, G.

    1983-01-01

    The report contains layouts of experimental areas, a table of beam parameters and fluxes, the experiment schedule as run, the experiment long range schedule, a listing of experiments by number, and a section of two-page summaries of each experiment

  18. Complete resequencing of 40 genomes reveals domestication events and genes in silkworm (Bombyx)

    DEFF Research Database (Denmark)

    Xia, Qingyou; Guo, Yiran; Zhang, Ze

    2009-01-01

    genes that may have been important during domestication, some of which have enriched expression in the silk gland, midgut, and testis. These data add to our understanding of the domestication processes and may have applications in devising pest control strategies and advancing the use of silkworms...

  19. Whole-genome resequencing reveals loci under selection during chicken domestication

    OpenAIRE

    Rubin, Carl-Johan; Zody, Michael C.; Eriksson, Jonas; Meadows, Jennifer R. S.; Sherwood, Ellen; Webster, Matthew T.; Jiang, Lin; Ingman, Max; Sharpe, Ted; Ka, Sojeong; Hallböök, Finn; Besnier, Francois; Carlbord, Örjan; Bed'Hom, Bertrand; Tixier Boichard, Michèle

    2010-01-01

    Domestic animals are excellent models for genetic studies of phenotypic evolution(1-3). They have evolved genetic adaptations to a new environment, the farm, and have been subjected to strong human-driven selection leading to remarkable phenotypic changes in morphology, physiology and behaviour. Identifying the genetic changes underlying these developments provides new insight into general mechanisms by which genetic variation shapes phenotypic diversity. Here we describe the use of massively...

  20. Resequencing data provide no evidence for a human bottleneck in Africa during the penultimate glacial period.

    Science.gov (United States)

    Sjödin, Per; E Sjöstrand, Agnès; Jakobsson, Mattias; Blum, Michael G B

    2012-07-01

    Based on the accumulation of genetic, climatic, and fossil evidence, a central theory in paleoanthropology stipulates that a demographic bottleneck coincided with the origin of our species Homo Sapiens. This theory proposes that anatomically modern humans--which were only present in Africa at the time--experienced a drastic bottleneck during the penultimate glacial age (130-190 kya) when a cold and dry climate prevailed. Two scenarios have been proposed to describe the bottleneck, which involve either a fragmentation of the range occupied by humans or the survival of one small group of humans. Here, we analyze DNA sequence data from 61 nuclear loci sequenced in three African populations using Approximate Bayesian Computation and numerical simulations. In contrast to the bottleneck theory, we show that a simple model without any bottleneck during the penultimate ice age has the greatest statistical support compared with bottleneck models. Although the proposed bottleneck is ancient, occurring at least 130 kya, we can discard the possibility that it did not leave detectable footprints in the DNA sequence data except if the bottleneck involves a less than a 3-fold reduction in population size. Finally, we confirm that a simple model without a bottleneck is able to reproduce the main features of the observed patterns of genetic variation. We conclude that models of Pleistocene refugium for modern human origins now require substantial revision.