WorldWideScience

Sample records for amplicon resequencing experiments

  1. Amplicon Resequencing Identified Parental Mosaicism for Approximately 10% of "de novo" SCN1A Mutations in Children with Dravet Syndrome.

    Science.gov (United States)

    Xu, Xiaojing; Yang, Xiaoxu; Wu, Qixi; Liu, Aijie; Yang, Xiaoling; Ye, Adam Yongxin; Huang, August Yue; Li, Jiarui; Wang, Meng; Yu, Zhe; Wang, Sheng; Zhang, Zhichao; Wu, Xiru; Wei, Liping; Zhang, Yuehua

    2015-09-01

    The majority of children with Dravet syndrome (DS) are caused by de novo SCN1A mutations. To investigate the origin of the mutations, we developed and applied a new method that combined deep amplicon resequencing with a Bayesian model to detect and quantify allelic fractions with improved sensitivity. Of 174 SCN1A mutations in DS probands which were considered "de novo" by Sanger sequencing, we identified 15 cases (8.6%) of parental mosaicism. We identified another five cases of parental mosaicism that were also detectable by Sanger sequencing. Fraction of mutant alleles in the 20 cases of parental mosaicism ranged from 1.1% to 32.6%. Thirteen (65% of 20) mutations originated paternally and seven (35% of 20) maternally. Twelve (60% of 20) mosaic parents did not have any epileptic symptoms. Their mutant allelic fractions were significantly lower than those in mosaic parents with epileptic symptoms (P = 0.016). We identified mosaicism with varied allelic fractions in blood, saliva, urine, hair follicle, oral epithelium, and semen, demonstrating that postzygotic mutations could affect multiple somatic cells as well as germ cells. Our results suggest that more sensitive tools for detecting low-level mosaicism in parents of families with seemingly "de novo" mutations will allow for better informed genetic counseling.

  2. 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.......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...

  3. Removing Noise From Pyrosequenced Amplicons

    Directory of Open Access Journals (Sweden)

    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

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

    Directory of Open Access Journals (Sweden)

    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.

  5. A Method for Amplicon Deep Sequencing of Drug Resistance Genes in Plasmodium falciparum Clinical Isolates from India.

    Science.gov (United States)

    Rao, Pavitra N; Uplekar, Swapna; Kayal, Sriti; Mallick, Prashant K; Bandyopadhyay, Nabamita; Kale, Sonal; Singh, Om P; Mohanty, Akshaya; Mohanty, Sanjib; Wassmer, Samuel C; Carlton, Jane M

    2016-06-01

    A major challenge to global malaria control and elimination is early detection and containment of emerging drug resistance. Next-generation sequencing (NGS) methods provide the resolution, scalability, and sensitivity required for high-throughput surveillance of molecular markers of drug resistance. We have developed an amplicon sequencing method on the Ion Torrent PGM platform for targeted resequencing of a panel of six Plasmodium falciparum genes implicated in resistance to first-line antimalarial therapy, including artemisinin combination therapy, chloroquine, and sulfadoxine-pyrimethamine. The protocol was optimized using 12 geographically diverse P. falciparum reference strains and successfully applied to multiplexed sequencing of 16 clinical isolates from India. The sequencing results from the reference strains showed 100% concordance with previously reported drug resistance-associated mutations. Single-nucleotide polymorphisms (SNPs) in clinical isolates revealed a number of known resistance-associated mutations and other nonsynonymous mutations that have not been implicated in drug resistance. SNP positions containing multiple allelic variants were used to identify three clinical samples containing mixed genotypes indicative of multiclonal infections. The amplicon sequencing protocol has been designed for the benchtop Ion Torrent PGM platform and can be operated with minimal bioinformatics infrastructure, making it ideal for use in countries that are endemic for the disease to facilitate routine large-scale surveillance of the emergence of drug resistance and to ensure continued success of the malaria treatment policy.

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

    Science.gov (United States)

    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

  7. 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

  8. Amplicon-based metagenomic analysis of mixed fungal samples using proton release amplicon sequencing.

    Directory of Open Access Journals (Sweden)

    Daniel P Tonge

    Full Text Available Next generation sequencing technology has revolutionised microbiology by allowing concurrent analysis of whole microbial communities. Here we developed and verified similar methods for the analysis of fungal communities using a proton release sequencing platform with the ability to sequence reads of up to 400 bp in length at significant depth. This read length permits the sequencing of amplicons from commonly used fungal identification regions and thereby taxonomic classification. Using the 400 bp sequencing capability, we have sequenced amplicons from the ITS1, ITS2 and LSU fungal regions to a depth of approximately 700,000 raw reads per sample. Representative operational taxonomic units (OTUs were chosen by the USEARCH algorithm, and identified taxonomically through nucleotide blast (BLASTn. Combination of this sequencing technology with the bioinformatics pipeline allowed species recognition in two controlled fungal spore populations containing members of known identity and concentration. Each species included within the two controlled populations was found to correspond to a representative OTU, and these OTUs were found to be highly accurate representations of true biological sequences. However, the absolute number of reads attributed to each OTU differed among species. The majority of species were represented by an OTU derived from all three genomic regions although in some cases, species were only represented in two of the regions due to the absence of conserved primer binding sites or due to sequence composition. It is apparent from our data that proton release sequencing technologies can deliver a qualitative assessment of the fungal members comprising a sample. The fact that some fungi cannot be amplified by specific "conserved" primer pairs confirms our recommendation that a multi-region approach be taken for other amplicon-based metagenomic studies.

  9. PCR Amplicon Prediction from Multiplex Degenerate Primer and Probe Sets

    Energy Technology Data Exchange (ETDEWEB)

    2013-08-08

    Assessing primer specificity and predicting both desired and off-target amplification products is an essential step for robust PCR assay design. Code is described to predict potential polymerase chain reaction (PCR) amplicons in a large sequence database such as NCBI nt from either singleplex or a large multiplexed set of primers, allowing degenerate primer and probe bases, with target mismatch annotates amplicons with gene information automatically downloaded from NCBI, and optionally it can predict whether there are also TaqMan/Luminex probe matches within predicted amplicons.

  10. High-Throughput Resequencing of Maize Landraces at Genomic Regions Associated with Flowering Time

    Science.gov (United States)

    Jamann, Tiffany M.; Sood, Shilpa; Wisser, Randall J.; Holland, James B.

    2017-01-01

    Despite the reduction in the price of sequencing, it remains expensive to sequence and assemble whole, complex genomes of multiple samples for population studies, particularly for large genomes like those of many crop species. Enrichment of target genome regions coupled with next generation sequencing is a cost-effective strategy to obtain sequence information for loci of interest across many individuals, providing a less expensive approach to evaluating sequence variation at the population scale. Here we evaluate amplicon-based enrichment coupled with semiconductor sequencing on a validation set consisting of three maize inbred lines, two hybrids and 19 landrace accessions. We report the use of a multiplexed panel of 319 PCR assays that target 20 candidate loci associated with photoperiod sensitivity in maize while requiring 25 ng or less of starting DNA per sample. Enriched regions had an average on-target sequence read depth of 105 with 98% of the sequence data mapping to the maize ‘B73’ reference and 80% of the reads mapping to the target interval. Sequence reads were aligned to B73 and 1,486 and 1,244 variants were called using SAMtools and GATK, respectively. Of the variants called by both SAMtools and GATK, 30% were not previously reported in maize. Due to the high sequence read depth, heterozygote genotypes could be called with at least 92.5% accuracy in hybrid materials using GATK. The genetic data are congruent with previous reports of high total genetic diversity and substantial population differentiation among maize landraces. In conclusion, semiconductor sequencing of highly multiplexed PCR reactions is a cost-effective strategy for resequencing targeted genomic loci in diverse maize materials. PMID:28045987

  11. Detection of immobilized amplicons by ELISA-like techniques.

    Science.gov (United States)

    Oroskar, A A; Rasmussen, S E; Rasmussen, H N; Rasmussen, S R; Sullivan, B M; Johansson, A

    1996-09-01

    The NucleoLink surface is a physically modified, thermostable, optically clear resin. It allows the covalent binding of 5'-phosphorylated oligonucleotides. Target DNA amplification by polymerase chain reaction (PCR) is accomplished by asymmetric amplification on the covalently immobilized primer that develops into immobilized amplicons. A DNA fragment of bovine leukemia virus is used as a model system for the detection of immobilized amplicons by ELISA-like techniques. Covalently bound oligonucleotides are also utilized as capture probe in the hybridization-based signal amplification for detection of an infectious organism.

  12. Barcoded Primers Used in Multiplex Amplicon Pyrosequencing Bias Amplification

    OpenAIRE

    2012-01-01

    “Barcode-tagged” PCR primers used for multiplex amplicon sequencing generate a thus-far-overlooked amplification bias that produces variable terminal restriction fragment length polymorphism (T-RFLP) and pyrosequencing data from the same environmental DNA template. We propose a simple two-step PCR approach that increases reproducibility and consistently recovers higher genetic diversity in pyrosequencing libraries.

  13. Genotyping of Bacillus cereus strains by microarray-based resequencing.

    Directory of Open Access Journals (Sweden)

    Michael E Zwick

    Full Text Available The ability to distinguish microbial pathogens from closely related but nonpathogenic strains is key to understanding the population biology of these organisms. In this regard, Bacillus anthracis, the bacterium that causes inhalational anthrax, is of interest because it is closely related and often difficult to distinguish from other members of the B. cereus group that can cause diverse diseases. We employed custom-designed resequencing arrays (RAs based on the genome sequence of Bacillus anthracis to generate 422 kb of genomic sequence from a panel of 41 Bacillus cereus sensu lato strains. Here we show that RAs represent a "one reaction" genotyping technology with the ability to discriminate between highly similar B. anthracis isolates and more divergent strains of the B. cereus s.l. Clade 1. Our data show that RAs can be an efficient genotyping technology for pre-screening the genetic diversity of large strain collections to selected the best candidates for whole genome sequencing.

  14. Genotyping of Bacillus cereus strains by microarray-based resequencing.

    Science.gov (United States)

    Zwick, Michael E; Kiley, Maureen P; Stewart, Andrew C; Mateczun, Alfred; Read, Timothy D

    2008-07-02

    The ability to distinguish microbial pathogens from closely related but nonpathogenic strains is key to understanding the population biology of these organisms. In this regard, Bacillus anthracis, the bacterium that causes inhalational anthrax, is of interest because it is closely related and often difficult to distinguish from other members of the B. cereus group that can cause diverse diseases. We employed custom-designed resequencing arrays (RAs) based on the genome sequence of Bacillus anthracis to generate 422 kb of genomic sequence from a panel of 41 Bacillus cereus sensu lato strains. Here we show that RAs represent a "one reaction" genotyping technology with the ability to discriminate between highly similar B. anthracis isolates and more divergent strains of the B. cereus s.l. Clade 1. Our data show that RAs can be an efficient genotyping technology for pre-screening the genetic diversity of large strain collections to selected the best candidates for whole genome sequencing.

  15. Whole-Genome Resequencing and Transcriptomic Analysis to Identify Genes Involved in Leaf-Color Diversity in Ornamental Rice Plants

    Science.gov (United States)

    Shin, Younhee; Lim, Hye-Min; Lee, Gang-Seob; Kim, A-Ram; Lee, Tae-Ho; Lee, Jae-Hee; Park, Dong-Suk; Yoo, Seungil; Kim, Yong-Hwan; Kim, Yong-Kab

    2015-01-01

    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. PMID:25897514

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

    Directory of Open Access Journals (Sweden)

    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.

  17. Population-Based Resequencing of APOA1 in 10,330 Individuals

    DEFF Research Database (Denmark)

    Haase, Christiane L; Frikke-Schmidt, Ruth; Nordestgaard, Børge G;

    2012-01-01

    Rare genetic variants, identified by in-detail resequencing of loci, may contribute to complex traits. We used the apolipoprotein A-I gene (APOA1), a major high-density lipoprotein (HDL) gene, and population-based resequencing to determine the spectrum of genetic variants, the phenotypic characte...... associated with reduced apoA-1 and HDL cholesterol levels and/or predisposing to amyloidosis. In addition, NS variants associated with increased risk of MI....

  18. DADA2: High resolution sample inference from Illumina amplicon data

    Science.gov (United States)

    Callahan, Benjamin J; McMurdie, Paul J; Rosen, Michael J; Han, Andrew W; Johnson, Amy Jo A; Holmes, Susan P

    2016-01-01

    We present DADA2, a software package that models and corrects Illumina-sequenced amplicon errors. DADA2 infers sample sequences exactly, without coarse-graining into OTUs, and resolves differences of as little as one nucleotide. In several mock communities DADA2 identified more real variants and output fewer spurious sequences than other methods. We applied DADA2 to vaginal samples from a cohort of pregnant women, revealing a diversity of previously undetected Lactobacillus crispatus variants. PMID:27214047

  19. 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......We tested the SNP typing protocol developed for the NanoChip electronic microarray by analyzing the four Y chromosome loci SRY1532, SRY8299, TAT, and 92R7. Amplicons of different lengths containing the same locus were purified and addressed to the NanoChip array and fluorescently labelled reporter...... probes were hybridized to the amplicons. We demonstrated that as little as 10-30 fmol of 50 bp DNA amplicons was sufficient to obtain strong and reproducible results. The hybridization to 50 bp amplicons was up to 10 times more efficient than the hybridization to 200 bp amplicons containing the same SNP...

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

    Directory of Open Access Journals (Sweden)

    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.

  1. 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.

  2. AMPLISAS: a web server for multilocus genotyping using next-generation amplicon sequencing data.

    Science.gov (United States)

    Sebastian, Alvaro; Herdegen, Magdalena; Migalska, Magdalena; Radwan, Jacek

    2016-03-01

    Next-generation sequencing (NGS) technologies are revolutionizing the fields of biology and medicine as powerful tools for amplicon sequencing (AS). Using combinations of primers and barcodes, it is possible to sequence targeted genomic regions with deep coverage for hundreds, even thousands, of individuals in a single experiment. This is extremely valuable for the genotyping of gene families in which locus-specific primers are often difficult to design, such as the major histocompatibility complex (MHC). The utility of AS is, however, limited by the high intrinsic sequencing error rates of NGS technologies and other sources of error such as polymerase amplification or chimera formation. Correcting these errors requires extensive bioinformatic post-processing of NGS data. Amplicon Sequence Assignment (AMPLISAS) is a tool that performs analysis of AS results in a simple and efficient way, while offering customization options for advanced users. AMPLISAS is designed as a three-step pipeline consisting of (i) read demultiplexing, (ii) unique sequence clustering and (iii) erroneous sequence filtering. Allele sequences and frequencies are retrieved in excel spreadsheet format, making them easy to interpret. AMPLISAS performance has been successfully benchmarked against previously published genotyped MHC data sets obtained with various NGS technologies.

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

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    Nils Homer

    Full Text Available BACKGROUND: 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. METHODOLOGY: 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. CONCLUSIONS: 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.

  4. Herpes simplex virus type 1-derived recombinant and amplicon vectors.

    Science.gov (United States)

    Fraefel, Cornel; Marconi, Peggy; Epstein, Alberto L

    2011-01-01

    Herpes simplex virus type 1 (HSV-1) is a human pathogen whose lifestyle is based on a long-term dual interaction with the infected host, being able to establish both lytic and latent infections. The virus genome is a 153 kbp double-stranded DNA molecule encoding more than 80 genes. The interest of HSV-1 as gene transfer vector stems from its ability to infect many different cell types, both quiescent and proliferating cells, the very high packaging capacity of the virus capsid, the outstanding neurotropic adaptations that this virus has evolved, and the fact that it never integrates into the cellular chromosomes, thus avoiding the risk of insertional mutagenesis. Two types of vectors can be derived from HSV-1, recombinant vectors and amplicon vectors, and different methodologies have been developed to prepare large stocks of each type of vector. This chapter summarizes (1) the two approaches most commonly used to prepare recombinant vectors through homologous recombination, either in eukaryotic cells or in bacteria, and (2) the two methodologies currently used to generate helper-free amplicon vectors, either using a bacterial artificial chromosome (BAC)-based approach or a Cre/loxP site-specific recombination strategy.

  5. A quantitative SMRT cell sequencing method for ribosomal amplicons.

    Science.gov (United States)

    Jones, Bethan M; Kustka, Adam B

    2017-04-01

    Advances in sequencing technologies continue to provide unprecedented opportunities to characterize microbial communities. For example, the Pacific Biosciences Single Molecule Real-Time (SMRT) platform has emerged as a unique approach harnessing DNA polymerase activity to sequence template molecules, enabling long reads at low costs. With the aim to simultaneously classify and enumerate in situ microbial populations, we developed a quantitative SMRT (qSMRT) approach that involves the addition of exogenous standards to quantify ribosomal amplicons derived from environmental samples. The V7-9 regions of 18S SSU rDNA were targeted and quantified from protistan community samples collected in the Ross Sea during the Austral summer of 2011. We used three standards of different length and optimized conditions to obtain accurate quantitative retrieval across the range of expected amplicon sizes, a necessary criterion for analyzing taxonomically diverse 18S rDNA molecules from natural environments. The ability to concurrently identify and quantify microorganisms in their natural environment makes qSMRT a powerful, rapid and cost-effective approach for defining ecosystem diversity and function.

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

    Directory of Open Access Journals (Sweden)

    Frédéric Mahé

    2014-09-01

    Full Text Available 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.

  7. 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.

  8. 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 ...

  9. 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),

  10. 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 ...

  11. 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...

  12. Complete resequencing and reannotation of the Lactobacillus plantarum WCFS1 genome

    NARCIS (Netherlands)

    Siezen, R.J.; Francke, C.; Renckens, B.; Boekhorst, L.J.S.; Wels, M.; Kleerebezem, M.; Hijum, van S.A.F.T.

    2012-01-01

    There is growing interest in the beneficial effects of Lactobacillus plantarum on human health. The genome of L. plantarum WCFS1, first sequenced in 2001, was resequenced using Solexa technology. We identified 116 nucleotide corrections and improved function prediction for nearly 1,200 proteins, wit

  13. 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 ...

  14. Construction of a cytomegalovirus-based amplicon: a vector with a unique transfer capacity.

    Science.gov (United States)

    Borst, Eva Maria; Messerle, Martin

    2003-07-01

    Cytomegalovirus (CMV) has a number of interesting properties that qualifies it as a vector for gene transfer. Especially appealing is the ability of the CMV genome to persist in hematopoietic progenitor cells and the packaging capacity of the viral capsid that accommodates a DNA genome of 230 kbp. In order to exploit the packaging capacity of the CMV capsid we investigated whether the principles of an amplicon vector can be applied to CMV. Amplicons are herpesviral vectors, which contain only the cis-active sequences required for replication and packaging of the vector genome. For construction of a CMV amplicon the sequences comprising the lytic origin of replication (orilyt) and the cleavage packaging recognition sites (pac) of human CMV were cloned onto a plasmid. A gene encoding the green fluorescent protein was used as a model transgene. The amplicon plasmid replicated in the presence of a CMV helper virus and was packaged into CMV particles, with replication and packaging being dependent on the presence of the orilyt and pac sequences. The packaged amplicon could be transferred to recipient cells and reisolated from the transduced cells. Analysis of the DNA isolated from CMV capsids revealed that the CMV amplicon was packaged as a concatemer with a size of approximately 210 kbp. The CMV amplicon vector has the potential to transfer therapeutic genes with a size of more than 200 kbp and thus provides a unique transfer capacity among viral vectors.

  15. 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......) and plasma samples from 46 colorectal cancer patients known to harbor a tumor KRAS mutation. KRAS mutated DNA was detected in significantly more clinical samples using the short amplicon assays compared to the long amplicon assays (74% vs. 61%, p=0.03). The level of mutated DNA in plasma was on average three...

  16. Integrated Microfluidic Nucleic Acid Isolation, Isothermal Amplification, and Amplicon Quantification

    Directory of Open Access Journals (Sweden)

    Michael G. Mauk

    2015-10-01

    Full Text Available Microfluidic components and systems for rapid (<60 min, low-cost, convenient, field-deployable sequence-specific nucleic acid-based amplification tests (NAATs are described. A microfluidic point-of-care (POC diagnostics test to quantify HIV viral load from blood samples serves as a representative and instructive example to discuss the technical issues and capabilities of “lab on a chip” NAAT devices. A portable, miniaturized POC NAAT with performance comparable to conventional PCR (polymerase-chain reaction-based tests in clinical laboratories can be realized with a disposable, palm-sized, plastic microfluidic chip in which: (1 nucleic acids (NAs are extracted from relatively large (~mL volume sample lysates using an embedded porous silica glass fiber or cellulose binding phase (“membrane” to capture sample NAs in a flow-through, filtration mode; (2 NAs captured on the membrane are isothermally (~65 °C amplified; (3 amplicon production is monitored by real-time fluorescence detection, such as with a smartphone CCD camera serving as a low-cost detector; and (4 paraffin-encapsulated, lyophilized reagents for temperature-activated release are pre-stored in the chip. Limits of Detection (LOD better than 103 virons/sample can be achieved. A modified chip with conduits hosting a diffusion-mode amplification process provides a simple visual indicator to readily quantify sample NA template. In addition, a companion microfluidic device for extracting plasma from whole blood without a centrifuge, generating cell-free plasma for chip-based molecular diagnostics, is described. Extensions to a myriad of related applications including, for example, food testing, cancer screening, and insect genotyping are briefly surveyed.

  17. Integrated Microfluidic Nucleic Acid Isolation, Isothermal Amplification, and Amplicon Quantification.

    Science.gov (United States)

    Mauk, Michael G; Liu, Changchun; Song, Jinzhao; Bau, Haim H

    2015-10-20

    Microfluidic components and systems for rapid (microfluidic point-of-care (POC) diagnostics test to quantify HIV viral load from blood samples serves as a representative and instructive example to discuss the technical issues and capabilities of "lab on a chip" NAAT devices. A portable, miniaturized POC NAAT with performance comparable to conventional PCR (polymerase-chain reaction)-based tests in clinical laboratories can be realized with a disposable, palm-sized, plastic microfluidic chip in which: (1) nucleic acids (NAs) are extracted from relatively large (~mL) volume sample lysates using an embedded porous silica glass fiber or cellulose binding phase ("membrane") to capture sample NAs in a flow-through, filtration mode; (2) NAs captured on the membrane are isothermally (~65 °C) amplified; (3) amplicon production is monitored by real-time fluorescence detection, such as with a smartphone CCD camera serving as a low-cost detector; and (4) paraffin-encapsulated, lyophilized reagents for temperature-activated release are pre-stored in the chip. Limits of Detection (LOD) better than 10³ virons/sample can be achieved. A modified chip with conduits hosting a diffusion-mode amplification process provides a simple visual indicator to readily quantify sample NA template. In addition, a companion microfluidic device for extracting plasma from whole blood without a centrifuge, generating cell-free plasma for chip-based molecular diagnostics, is described. Extensions to a myriad of related applications including, for example, food testing, cancer screening, and insect genotyping are briefly surveyed.

  18. Insights into the Evolution of Cotton Diploids and Polyploids from Whole-Genome Re-sequencing

    OpenAIRE

    Page, Justin T.; Huynh, Mark D; Zach S Liechty; Grupp, Kara; Stelly, David; Hulse, Amanda M; Ashrafi, Hamid; Van Deynze, Allen; Wendel, Jonathan F.; Udall, Joshua A.

    2013-01-01

    Understanding the composition, evolution, and function of the Gossypium hirsutum (cotton) genome is complicated by the joint presence of two genomes in its nucleus (AT and DT genomes). These two genomes were derived from progenitor A-genome and D-genome diploids involved in ancestral allopolyploidization. To better understand the allopolyploid genome, we re-sequenced the genomes of extant diploid relatives that contain the A1 (Gossypium herbaceum), A2 (Gossypium arboreum), or D5 (Gossypium ra...

  19. QTL Mapping of Leafy Heads by Genome Resequencing in the RIL Population of Brassica rapa

    OpenAIRE

    Xiang Yu; Han Wang; Weili Zhong; Jinjuan Bai; Pinglin Liu; Yuke He

    2013-01-01

    Leaf heads of cabbage (Brassica oleracea), Chinese cabbage (B. rapa), and lettuce (Lactuca sativa) are important vegetables that supply mineral nutrients, crude fiber and vitamins in the human diet. Head size, head shape, head weight, and heading time contribute to yield and quality. In an attempt to investigate genetic basis of leafy head in Chinese cabbage (B. rapa), we took advantage of recent technical advances of genome resequencing to perform quantitative trait locus (QTL) mapping using...

  20. Tag SNP selection for candidate gene association studies using HapMap and gene resequencing data.

    Science.gov (United States)

    Xu, Zongli; Kaplan, Norman L; Taylor, Jack A

    2007-10-01

    HapMap provides linkage disequilibrium (LD) information on a sample of 3.7 million SNPs that can be used for tag SNP selection in whole-genome association studies. HapMap can also be used for tag SNP selection in candidate genes, although its performance has yet to be evaluated against gene resequencing data, where there is near-complete SNP ascertainment. The Environmental Genome Project (EGP) is the largest gene resequencing effort to date with over 500 resequenced genes. We used HapMap data to select tag SNPs and calculated the proportions of common SNPs (MAF>or=0.05) tagged (rho2>or=0.8) for each of 127 EGP Panel 2 genes where individual ethnic information was available. Median gene-tagging proportions are 50, 80 and 74% for African, Asian, and European groups, respectively. These low gene-tagging proportions may be problematic for some candidate gene studies. In addition, although HapMap targeted nonsynonymous SNPs (nsSNPs), we estimate only approximately 30% of nonsynonymous SNPs in EGP are in high LD with any HapMap SNP. We show that gene-tagging proportions can be improved by adding a relatively small number of tag SNPs that were selected based on resequencing data. We also demonstrate that ethnic-mixed data can be used to improve HapMap gene-tagging proportions, but are not as efficient as ethnic-specific data. Finally, we generalized the greedy algorithm proposed by Carlson et al (2004) to select tag SNPs for multiple populations and implemented the algorithm into a freely available software package mPopTag.

  1. 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

  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/.

  3. Genetic diversity, molecular phylogeny and selection evidence of the silkworm mitochondria implicated by complete resequencing of 41 genomes

    Directory of Open Access Journals (Sweden)

    Tellier Laurent C

    2010-03-01

    Full Text Available Abstract Background Mitochondria are a valuable resource for studying the evolutionary process and deducing phylogeny. A few mitochondria genomes have been sequenced, but a comprehensive picture of the domestication event for silkworm mitochondria remains to be established. In this study, we integrate the extant data, and perform a whole genome resequencing of Japanese wild silkworm to obtain breakthrough results in silkworm mitochondrial (mt population, and finally use these to deduce a more comprehensive phylogeny of the Bombycidae. Results We identified 347 single nucleotide polymorphisms (SNPs in the mt genome, but found no past recombination event to have occurred in the silkworm progenitor. A phylogeny inferred from these whole genome SNPs resulted in a well-classified tree, confirming that the domesticated silkworm, Bombyx mori, most recently diverged from the Chinese wild silkworm, rather than from the Japanese wild silkworm. We showed that the population sizes of the domesticated and Chinese wild silkworms both experience neither expansion nor contraction. We also discovered that one mt gene, named cytochrome b, shows a strong signal of positive selection in the domesticated clade. This gene is related to energy metabolism, and may have played an important role during silkworm domestication. Conclusions We present a comparative analysis on 41 mt genomes of B. mori and B. mandarina from China and Japan. With these, we obtain a much clearer picture of the evolution history of the silkworm. The data and analyses presented here aid our understanding of the silkworm in general, and provide a crucial insight into silkworm phylogeny.

  4. Formation of linear amplicons with inverted duplications in Leishmania requires the MRE11 nuclease.

    Directory of Open Access Journals (Sweden)

    Marie-Claude N Laffitte

    2014-12-01

    Full Text Available Extrachromosomal DNA amplification is frequent in the protozoan parasite Leishmania selected for drug resistance. The extrachromosomal amplified DNA is either circular or linear, and is formed at the level of direct or inverted homologous repeated sequences that abound in the Leishmania genome. The RAD51 recombinase plays an important role in circular amplicons formation, but the mechanism by which linear amplicons are formed is unknown. We hypothesized that the Leishmania infantum DNA repair protein MRE11 is required for linear amplicons following rearrangements at the level of inverted repeats. The purified LiMRE11 protein showed both DNA binding and exonuclease activities. Inactivation of the LiMRE11 gene led to parasites with enhanced sensitivity to DNA damaging agents. The MRE11(-/- parasites had a reduced capacity to form linear amplicons after drug selection, and the reintroduction of an MRE11 allele led to parasites regaining their capacity to generate linear amplicons, but only when MRE11 had an active nuclease activity. These results highlight a novel MRE11-dependent pathway used by Leishmania to amplify portions of its genome to respond to a changing environment.

  5. 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...

  6. A comparison of Y-chromosomal lineage dating using either resequencing or Y-SNP plus Y-STR genotyping☆

    Science.gov (United States)

    Wei, Wei; Ayub, Qasim; Xue, Yali; Tyler-Smith, Chris

    2013-01-01

    We have compared phylogenies and time estimates for Y-chromosomal lineages based on resequencing ∼9 Mb of DNA and applying the program GENETREE to similar analyses based on the more standard approach of genotyping 26 Y-SNPs plus 21 Y-STRs and applying the programs NETWORK and BATWING. We find that deep phylogenetic structure is not adequately reconstructed after Y-SNP plus Y-STR genotyping, and that times estimated using observed Y-STR mutation rates are several-fold too recent. In contrast, an evolutionary mutation rate gives times that are more similar to the resequencing data. In principle, systematic comparisons of this kind can in future studies be used to identify the combinations of Y-SNP and Y-STR markers, and time estimation methodologies, that correspond best to resequencing data. PMID:23768990

  7. Sex determination in beef by melting curve analysis of PCR amplicons from the amelogenin locus.

    Science.gov (United States)

    Ballin, Nicolai Z; Madsen, Knud G

    2007-11-01

    Sex determination of beef is important to meet the rules of the Commission Regulation (EC) 765/2002 that qualify for export refunds. A SYBR Green sex identification assay based on melting curve analysis of PCR amplicons from the amelogenin locus (AMELX and AMELY) was developed. The PCR amplicons of 130/130 and 130/67 base pairs produced from female and male beef, respectively, are easily distinguished by both melting curve analysis and gel electrophoresis. Results from the melting curve analysis of amplicons are ready in less than three minutes, and requires no additional work in addition to the PCR setup. Applicability of the sex determination assay was studied by analysis of 12 unknown beef samples and the results were compared to an accredited method based on gel electrophoresis. In addition, six different cattle breeds were examined. All test results were correct in respect to sex.

  8. Silencing Status Epilepticus-Induced BDNF Expression with Herpes Simplex Virus Type-1 Based Amplicon Vectors.

    Science.gov (United States)

    Falcicchia, Chiara; Trempat, Pascal; Binaschi, Anna; Perrier-Biollay, Coline; Roncon, Paolo; Soukupova, Marie; Berthommé, Hervé; Simonato, Michele

    2016-01-01

    Brain-derived neurotrophic factor (BDNF) has been found to produce pro- but also anti-epileptic effects. Thus, its validity as a therapeutic target must be verified using advanced tools designed to block or to enhance its signal. The aim of this study was to develop tools to silence the BDNF signal. We generated Herpes simplex virus type 1 (HSV-1) derived amplicon vectors, i.e. viral particles containing a genome of 152 kb constituted of concatameric repetitions of an expression cassette, enabling the expression of the gene of interest in multiple copies. HSV-1 based amplicon vectors are non-pathogenic and have been successfully employed in the past for gene delivery into the brain of living animals. Therefore, amplicon vectors should represent a logical choice for expressing a silencing cassette, which, in multiple copies, is expected to lead to an efficient knock-down of the target gene expression. Here, we employed two amplicon-based BDNF silencing strategies. The first, antisense, has been chosen to target and degrade the cytoplasmic mRNA pool of BDNF, whereas the second, based on the convergent transcription technology, has been chosen to repress transcription at the BDNF gene. Both these amplicon vectors proved to be effective in down-regulating BDNF expression in vitro, in BDNF-expressing mesoangioblast cells. However, only the antisense strategy was effective in vivo, after inoculation in the hippocampus in a model of status epilepticus in which BDNF mRNA levels are strongly increased. Interestingly, the knocking down of BDNF levels induced with BDNF-antisense was sufficient to produce significant behavioral effects, in spite of the fact that it was produced only in a part of a single hippocampus. In conclusion, this study demonstrates a reliable effect of amplicon vectors in knocking down gene expression in vitro and in vivo. Therefore, this approach may find broad applications in neurobiological studies.

  9. Silencing Status Epilepticus-Induced BDNF Expression with Herpes Simplex Virus Type-1 Based Amplicon Vectors.

    Directory of Open Access Journals (Sweden)

    Chiara Falcicchia

    Full Text Available Brain-derived neurotrophic factor (BDNF has been found to produce pro- but also anti-epileptic effects. Thus, its validity as a therapeutic target must be verified using advanced tools designed to block or to enhance its signal. The aim of this study was to develop tools to silence the BDNF signal. We generated Herpes simplex virus type 1 (HSV-1 derived amplicon vectors, i.e. viral particles containing a genome of 152 kb constituted of concatameric repetitions of an expression cassette, enabling the expression of the gene of interest in multiple copies. HSV-1 based amplicon vectors are non-pathogenic and have been successfully employed in the past for gene delivery into the brain of living animals. Therefore, amplicon vectors should represent a logical choice for expressing a silencing cassette, which, in multiple copies, is expected to lead to an efficient knock-down of the target gene expression. Here, we employed two amplicon-based BDNF silencing strategies. The first, antisense, has been chosen to target and degrade the cytoplasmic mRNA pool of BDNF, whereas the second, based on the convergent transcription technology, has been chosen to repress transcription at the BDNF gene. Both these amplicon vectors proved to be effective in down-regulating BDNF expression in vitro, in BDNF-expressing mesoangioblast cells. However, only the antisense strategy was effective in vivo, after inoculation in the hippocampus in a model of status epilepticus in which BDNF mRNA levels are strongly increased. Interestingly, the knocking down of BDNF levels induced with BDNF-antisense was sufficient to produce significant behavioral effects, in spite of the fact that it was produced only in a part of a single hippocampus. In conclusion, this study demonstrates a reliable effect of amplicon vectors in knocking down gene expression in vitro and in vivo. Therefore, this approach may find broad applications in neurobiological studies.

  10. 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.

  11. [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.

  12. Design and validation of a metabolic disorder resequencing microarray (BRUM1).

    Science.gov (United States)

    Bruce, Christopher K; Smith, Matthew; Rahman, Fatima; Liu, Zhi-feng; McMullan, Dominic J; Ball, Sarah; Hartley, Jane; Kroos, Marian A; Heptinstall, Lesley; Reuser, Arnold J J; Rolfs, Arndt; Hendriksz, Chris; Kelly, Deirdre A; Barrett, Timothy G; MacDonald, Fiona; Maher, Eamonn R; Gissen, Paul

    2010-07-01

    The molecular genetic diagnosis of inherited metabolic disorders is challenging. The diseases are rare, and most show locus heterogeneity. Hence, testing of the genes associated with IMDs is time consuming and often not easily available. We report a resequencing array that allows the simultaneous resequencing of up to 92 genes associated with IMDs. To validate the array, DNA samples from 51 patients with 52 different known variants (including point variants, small insertion, and deletions [indels]) in seven genes (C14ORF133, GAA, NPC1, NPC2, VPS33B, WFS1, and SLC19A2) were amplified by PCR and hybridized to the array. A further patient cohort with 48 different mutations in NPC1 were analyzed blind. Out of 76 point variants, 73 were identified using automated software analysis followed by manual review. Ten insertion and deletion variants were detected in the extra tiling using mutation specific probes, with 11 heterozygous deletions and 3 heterozygous insertions. In summary, we identified 96% (95% confidence interval [CI] 89-99%) of point variants added to the array, but the pickup rate reduced to 83% (95% CI 75-89%) when insertions/deletions were included. Although the methodology has strengths and weaknesses, application of this technique could expedite diagnosis in most patients with multilocus IMDs.

  13. QTL mapping of leafy heads by genome resequencing in the RIL population of Brassica rapa.

    Directory of Open Access Journals (Sweden)

    Xiang Yu

    Full Text Available Leaf heads of cabbage (Brassica oleracea, Chinese cabbage (B. rapa, and lettuce (Lactuca sativa are important vegetables that supply mineral nutrients, crude fiber and vitamins in the human diet. Head size, head shape, head weight, and heading time contribute to yield and quality. In an attempt to investigate genetic basis of leafy head in Chinese cabbage (B. rapa, we took advantage of recent technical advances of genome resequencing to perform quantitative trait locus (QTL mapping using 150 recombinant inbred lines (RILs derived from the cross between heading and non-heading Chinese cabbage. The resequenced genomes of the parents uncovered more than 1 million SNPs. Genotyping of RILs using the high-quality SNPs assisted by Hidden Markov Model (HMM generated a recombination map. The raw genetic map revealed some physical assembly error and missing fragments in the reference genome that reduced the quality of SNP genotyping. By deletion of the genetic markers in which recombination rates higher than 20%, we have obtained a high-quality genetic map with 2209 markers and detected 18 QTLs for 6 head traits, from which 3 candidate genes were selected. These QTLs provide the foundation for study of genetic basis of leafy heads and the other complex traits.

  14. QTL mapping of leafy heads by genome resequencing in the RIL population of Brassica rapa.

    Science.gov (United States)

    Yu, Xiang; Wang, Han; Zhong, Weili; Bai, Jinjuan; Liu, Pinglin; He, Yuke

    2013-01-01

    Leaf heads of cabbage (Brassica oleracea), Chinese cabbage (B. rapa), and lettuce (Lactuca sativa) are important vegetables that supply mineral nutrients, crude fiber and vitamins in the human diet. Head size, head shape, head weight, and heading time contribute to yield and quality. In an attempt to investigate genetic basis of leafy head in Chinese cabbage (B. rapa), we took advantage of recent technical advances of genome resequencing to perform quantitative trait locus (QTL) mapping using 150 recombinant inbred lines (RILs) derived from the cross between heading and non-heading Chinese cabbage. The resequenced genomes of the parents uncovered more than 1 million SNPs. Genotyping of RILs using the high-quality SNPs assisted by Hidden Markov Model (HMM) generated a recombination map. The raw genetic map revealed some physical assembly error and missing fragments in the reference genome that reduced the quality of SNP genotyping. By deletion of the genetic markers in which recombination rates higher than 20%, we have obtained a high-quality genetic map with 2209 markers and detected 18 QTLs for 6 head traits, from which 3 candidate genes were selected. These QTLs provide the foundation for study of genetic basis of leafy heads and the other complex traits.

  15. Resequencing of the common marmoset genome improves genome assemblies and gene-coding sequence analysis.

    Science.gov (United States)

    Sato, Kengo; Kuroki, Yoko; Kumita, Wakako; Fujiyama, Asao; Toyoda, Atsushi; Kawai, Jun; Iriki, Atsushi; Sasaki, Erika; Okano, Hideyuki; Sakakibara, Yasubumi

    2015-11-20

    The first draft of the common marmoset (Callithrix jacchus) genome was published by the Marmoset Genome Sequencing and Analysis Consortium. The draft was based on whole-genome shotgun sequencing, and the current assembly version is Callithrix_jacches-3.2.1, but there still exist 187,214 undetermined gap regions and supercontigs and relatively short contigs that are unmapped to chromosomes in the draft genome. We performed resequencing and assembly of the genome of common marmoset by deep sequencing with high-throughput sequencing technology. Several different sequence runs using Illumina sequencing platforms were executed, and 181 Gbp of high-quality bases including mate-pairs with long insert lengths of 3, 8, 20, and 40 Kbp were obtained, that is, approximately 60× coverage. The resequencing significantly improved the MGSAC draft genome sequence. The N50 of the contigs, which is a statistical measure used to evaluate assembly quality, doubled. As a result, 51% of the contigs (total length: 299 Mbp) that were unmapped to chromosomes in the MGSAC draft were merged with chromosomal contigs, and the improved genome sequence helped to detect 5,288 new genes that are homologous to human cDNAs and the gaps in 5,187 transcripts of the Ensembl gene annotations were completely filled.

  16. Surface density dependence of PCR amplicon hybridization on PNA/DNA probe layers

    DEFF Research Database (Denmark)

    Yao, Danfeng; Kim, Junyoung; Yu, Fang

    2005-01-01

    at an intermediate sodium concentration (approximately 100 mM). These effects were mainly ascribed to the electrostatic cross talk among the hybridized DNA molecules and the secondary structure of PCR amplicons. For the negatively charged DNA probes, the hybridization reaction was subjected additionally to the DNA...

  17. High-throughput amplicon sequencing reveals distinct communities within a corroding concrete sewer system.

    Science.gov (United States)

    Cayford, Barry I; Dennis, Paul G; Keller, Jurg; Tyson, Gene W; Bond, Philip L

    2012-10-01

    Microbially induced concrete corrosion (MICC) is an important problem in sewers. Here, small-subunit (SSU) rRNA gene amplicon pyrosequencing was used to characterize MICC communities. Microbial community composition differed between wall- and ceiling-associated MICC layers. Acidithiobacillus spp. were present at low abundances, and the communities were dominated by other sulfur-oxidizing-associated lineages.

  18. 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

  19. Post-amplification Klenow fragment treatment alleviates PCR bias caused by partially single-stranded amplicons

    NARCIS (Netherlands)

    Egert, M.G.G.; Friedrich, M.W.

    2005-01-01

    Partially single-stranded amplicons, formed during PCR amplification of single and mixed templates, are a potential source of bias in genetic diversity studies. The analysis of 16S rRNA gene diversity in mixed template samples by the fingerprinting technique terminal restriction fragment length poly

  20. Genomic and expression array profiling of chromosome 20q amplicon in human colon cancer cells

    Directory of Open Access Journals (Sweden)

    Carter Jennifer

    2005-01-01

    Full Text Available Background: Gain of the q arm of chromosome 20 in human colorectal cancer has been associated with poorer survival time and has been reported to increase in frequency from adenomas to metastasis. The increasing frequency of chromosome 20q amplification during colorectal cancer progression and the presence of this amplification in carcinomas of other tissue origin has lead us to hypothesize that 20q11-13 harbors one or more genes which, when over expressed promote tumor invasion and metastasis. Aims: Generate genomic and expression profiles of the 20q amplicon in human cancer cell lines in order to identify genes with increased copy number and expression. Materials and Methods: Utilizing genomic sequencing clones and amplification mapping data from our lab and other previous studies, BAC/ PAC tiling paths spanning the 20q amplicon and genomic microarrays were generated. Array-CGH on the custom array with human cancer cell line DNAs was performed to generate genomic profiles of the amplicon. Expression array analysis with RNA from these cell lines using commercial oligo microarrays generated expression profiles of the amplicon. The data were then combined in order to identify genes with increased copy number and expression. Results: Over expressed genes in regions of increased copy number were identified and a list of potential novel genetic tumor markers was assembled based on biological functions of these genes Conclusions: Performing high-resolution genomic microarray profiling in conjunction with expression analysis is an effective approach to identify potential tumor markers.

  1. 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.

  2. Phred-Phrap package to analyses tools: a pipeline to facilitate population genetics re-sequencing studies

    Directory of Open Access Journals (Sweden)

    Machado Moara

    2011-02-01

    Full Text Available Abstract Background Targeted re-sequencing is one of the most powerful and widely used strategies for population genetics studies because it allows an unbiased screening for variation that is suitable for a wide variety of organisms. Examples of studies that require re-sequencing data are evolutionary inferences, epidemiological studies designed to capture rare polymorphisms responsible for complex traits and screenings for mutations in families and small populations with high incidences of specific genetic diseases. Despite the advent of next-generation sequencing technologies, Sanger sequencing is still the most popular approach in population genetics studies because of the widespread availability of automatic sequencers based on capillary electrophoresis and because it is still less prone to sequencing errors, which is critical in population genetics studies. Two popular software applications for re-sequencing studies are Phred-Phrap-Consed-Polyphred, which performs base calling, alignment, graphical edition and genotype calling and DNAsp, which performs a set of population genetics analyses. These independent tools are the start and end points of basic analyses. In between the use of these tools, there is a set of basic but error-prone tasks to be performed with re-sequencing data. Results In order to assist with these intermediate tasks, we developed a pipeline that facilitates data handling typical of re-sequencing studies. Our pipeline: (1 consolidates different outputs produced by distinct Phred-Phrap-Consed contigs sharing a reference sequence; (2 checks for genotyping inconsistencies; (3 reformats genotyping data produced by Polyphred into a matrix of genotypes with individuals as rows and segregating sites as columns; (4 prepares input files for haplotype inferences using the popular software PHASE; and (5 handles PHASE output files that contain only polymorphic sites to reconstruct the inferred haplotypes including polymorphic and

  3. Whole genome resequencing of the human parasite Schistosoma mansoni reveals population history and effects of selection

    Science.gov (United States)

    Crellen, Thomas; Allan, Fiona; David, Sophia; Durrant, Caroline; Huckvale, Thomas; Holroyd, Nancy; Emery, Aidan M.; Rollinson, David; Aanensen, David M.; Berriman, Matthew; Webster, Joanne P.; Cotton, James A.

    2016-01-01

    Schistosoma mansoni is a parasitic fluke that infects millions of people in the developing world. This study presents the first application of population genomics to S. mansoni based on high-coverage resequencing data from 10 global isolates and an isolate of the closely-related Schistosoma rodhaini, which infects rodents. Using population genetic tests, we document genes under directional and balancing selection in S. mansoni that may facilitate adaptation to the human host. Coalescence modeling reveals the speciation of S. mansoni and S. rodhaini as 107.5–147.6KYA, a period which overlaps with the earliest archaeological evidence for fishing in Africa. Our results indicate that S. mansoni originated in East Africa and experienced a decline in effective population size 20–90KYA, before dispersing across the continent during the Holocene. In addition, we find strong evidence that S. mansoni migrated to the New World with the 16–19th Century Atlantic Slave Trade. PMID:26879532

  4. 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...

  5. 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

    analysis of 79,090 nitrogenase (nifH) PCR amplicons encoding 7,468 unique proteins from surface samples (ten DNA samples and two RNA samples) collected at ten marine locations world-wide provides the first in-depth survey of a functional bacterial gene and yield insights into the composition and diversity...... 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...... to Alpha-, Beta-, Gamma-, and Delta-Proteobacteria were most common and showed distinct geographic distributions. Sequences related to anaerobic bacteria (nifH Cluster III) were generally rare, but preponderant in cold waters, especially in the Arctic. Although the two transcript samples were dominated...

  6. One-way sequencing of multiple amplicons from tandem repetitive mitochondrial DNA control region.

    Science.gov (United States)

    Xu, Jiawu; Fonseca, Dina M

    2011-10-01

    Repetitive DNA sequences not only exist abundantly in eukaryotic nuclear genomes, but also occur as tandem repeats in many animal mitochondrial DNA (mtDNA) control regions. Due to concerted evolution, these repetitive sequences are highly similar or even identical within a genome. When long repetitive regions are the targets of amplification for the purpose of sequencing, multiple amplicons may result if one primer has to be located inside the repeats. Here, we show that, without separating these amplicons by gel purification or cloning, directly sequencing the mitochondrial repeats with the primer outside repetitive region is feasible and efficient. We exemplify it by sequencing the mtDNA control region of the mosquito Aedes albopictus, which harbors typical large tandem DNA repeats. This one-way sequencing strategy is optimal for population surveys.

  7. Characterization of FGFR1 Locus in sqNSCLC Reveals a Broad and Heterogeneous Amplicon.

    Directory of Open Access Journals (Sweden)

    Claire Rooney

    Full Text Available FGFR1 amplification occurs in ~20% of sqNSCLC and trials with FGFR inhibitors have selected FGFR1 amplified patients by FISH. Lung cancer cell lines were profiled for sensitivity to AZD4547, a potent, selective inhibitor of FGFRs 1-3. Sensitivity to FGFR inhibition was associated with but not wholly predicted by increased FGFR1 gene copy number. Additional biomarker assays evaluating expression of FGFRs and correlation between amplification and expression in clinical tissues are therefore warranted. We validated nanoString for mRNA expression analysis of 194 genes, including FGFRs, from clinical tumour tissue. In a panel of sqNSCLC tumours 14.4% (13/90 were FGFR1 amplified by FISH. Although mean FGFR1 expression was significantly higher in amplified samples, there was significant overlap in the range of expression levels between the amplified and non-amplified cohorts with several non-amplified samples expressing FGFR1 to levels equivalent to amplified samples. Statistical analysis revealed increased expression of FGFR1 neighboring genes on the 8p12 amplicon (BAG4, LSM1 and WHSC1L1 in FGFR1 amplified tumours, suggesting a broad rather than focal amplicon and raises the potential for codependencies. High resolution aCGH analysis of pre-clinical and clinical samples supported the presence of a broad and heterogeneous amplicon around the FGFR1 locus. In conclusion, the range of FGFR1 expression levels in both FGFR1 amplified and non-amplified NSCLC tissues, together with the breadth and intra-patient heterogeneity of the 8p amplicon highlights the need for gene expression analysis of clinical samples to inform the understanding of determinants of response to FGFR inhibitors. In this respect the nanoString platform provides an attractive option for RNA analysis of FFPE clinical samples.

  8. Fast, accurate and easy-to-pipeline methods for amplicon sequence processing

    Science.gov (United States)

    Antonielli, Livio; Sessitsch, Angela

    2016-04-01

    Next generation sequencing (NGS) technologies established since years as an essential resource in microbiology. While on the one hand metagenomic studies can benefit from the continuously increasing throughput of the Illumina (Solexa) technology, on the other hand the spreading of third generation sequencing technologies (PacBio, Oxford Nanopore) are getting whole genome sequencing beyond the assembly of fragmented draft genomes, making it now possible to finish bacterial genomes even without short read correction. Besides (meta)genomic analysis next-gen amplicon sequencing is still fundamental for microbial studies. Amplicon sequencing of the 16S rRNA gene and ITS (Internal Transcribed Spacer) remains a well-established widespread method for a multitude of different purposes concerning the identification and comparison of archaeal/bacterial (16S rRNA gene) and fungal (ITS) communities occurring in diverse environments. Numerous different pipelines have been developed in order to process NGS-derived amplicon sequences, among which Mothur, QIIME and USEARCH are the most well-known and cited ones. The entire process from initial raw sequence data through read error correction, paired-end read assembly, primer stripping, quality filtering, clustering, OTU taxonomic classification and BIOM table rarefaction as well as alternative "normalization" methods will be addressed. An effective and accurate strategy will be presented using the state-of-the-art bioinformatic tools and the example of a straightforward one-script pipeline for 16S rRNA gene or ITS MiSeq amplicon sequencing will be provided. Finally, instructions on how to automatically retrieve nucleotide sequences from NCBI and therefore apply the pipeline to targets other than 16S rRNA gene (Greengenes, SILVA) and ITS (UNITE) will be discussed.

  9. Targeted Amplicon Sequencing (TAS): A Scalable Next-Gen Approach to Multilocus, Multitaxa Phylogenetics

    OpenAIRE

    2011-01-01

    Next-gen sequencing technologies have revolutionized data collection in genetic studies and advanced genome biology to novel frontiers. However, to date, next-gen technologies have been used principally for whole genome sequencing and transcriptome sequencing. Yet many questions in population genetics and systematics rely on sequencing specific genes of known function or diversity levels. Here, we describe a targeted amplicon sequencing (TAS) approach capitalizing on next-gen capacity to sequ...

  10. Potential of pmoA amplicon pyrosequencing for methanotroph diversity studies.

    Science.gov (United States)

    Lüke, Claudia; Frenzel, Peter

    2011-09-01

    We analyzed the potential of pmoA amplicon pyrosequencing compared to that of Sanger sequencing with paddy soils as a model environment. We defined operational taxonomic unit (OTU) cutoff values of 7% and 18%, reflecting methanotrophic species and major phylogenetic pmoA lineages, respectively. Major lineages were already well covered by clone libraries; nevertheless, pyrosequencing provided a higher level of diversity at the species level.

  11. Evaluating sequence-derived mtDNA length heteroplasmy by amplicon size analysis

    Science.gov (United States)

    Berger, C.; Hatzer-Grubwieser, P.; Hohoff, C.; Parson, W.

    2011-01-01

    Length heteroplasmy (LH) in mitochondrial (mt)DNA is usually observed in homopolymeric tracts and manifest as mixture of various length variants. The generally used difference-coded annotation to report mtDNA haplotypes does not express the degree of LH variation present in a sample, even more so, it is sometimes difficult to establish which length variants are present and clearly distinguishable from background noise. It has therefore become routine practice for some researchers to call the dominant type, the “major molecule”, which represents the LH variant that is most abundant in a DNA extract. In the majority of cases a clear single dominant variant can be identified. However, in some samples this interpretation is difficult, i.e. when (almost) equally quantitative LH variants are present or when multiple sequencing primers result in the presentation of different dominant types. To better understand those cases we designed amplicon sizing assays for the five most relevant LH regions in the mtDNA control region (around ntps 16,189, 310, 460, 573, and the AC-repeat between 514 and 524) to determine the ratio of the LH variants by fluorescence based amplicon sizing assays. For difficult LH constellations derived by Sanger sequencing (with Big Dye terminators) these assays mostly gave clear and unambiguous results. In the vast majority of cases we found agreement between the results of the sequence and amplicon analyses and propose this alternative method in difficult cases. PMID:21067985

  12. Development of a high-throughput resequencing array for the detection of pathogenic mutations in osteogenesis imperfecta.

    Directory of Open Access Journals (Sweden)

    Yao Wang

    Full Text Available Osteogenesis imperfecta (OI is a rare inherited skeletal disease, characterized by bone fragility and low bone density. The mutations in this disorder have been widely reported to be on various exonal hotspots of the candidate genes, including COL1A1, COL1A2, CRTAP, LEPRE1, and FKBP10, thus creating a great demand for precise genetic tests. However, large genome sizes make the process daunting and the analyses, inefficient and expensive. Therefore, we aimed at developing a fast, accurate, efficient, and cheaper sequencing platform for OI diagnosis; and to this end, use of an advanced array-based technique was proposed.A CustomSeq Affymetrix Resequencing Array was established for high-throughput sequencing of five genes simultaneously. Genomic DNA extraction from 13 OI patients and 85 normal controls and amplification using long-range PCR (LR-PCR were followed by DNA fragmentation and chip hybridization, according to standard Affymetrix protocols. Hybridization signals were determined using GeneChip Sequence Analysis Software (GSEQ. To examine the feasibility, the outcome from new resequencing approach was validated by conventional capillary sequencing method.Overall call rates using resequencing array was 96-98% and the agreement between microarray and capillary sequencing was 99.99%. 11 out of 13 OI patients with pathogenic mutations were successfully detected by the chip analysis without adjustment, and one mutation could also be identified using manual visual inspection.A high-throughput resequencing array was developed that detects the disease-associated mutations in OI, providing a potential tool to facilitate large-scale genetic screening for OI patients. Through this method, a novel mutation was also found.

  13. 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

  14. 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.

  15. Large-scale recent expansion of European patrilineages shown by population resequencing.

    Science.gov (United States)

    Batini, Chiara; Hallast, Pille; Zadik, Daniel; Delser, Pierpaolo Maisano; Benazzo, Andrea; Ghirotto, Silvia; Arroyo-Pardo, Eduardo; Cavalleri, Gianpiero L; de Knijff, Peter; Dupuy, Berit Myhre; Eriksen, Heidi A; King, Turi E; López de Munain, Adolfo; López-Parra, Ana M; Loutradis, Aphrodite; Milasin, Jelena; Novelletto, Andrea; Pamjav, Horolma; Sajantila, Antti; Tolun, Aslıhan; Winney, Bruce; Jobling, Mark A

    2015-05-19

    The proportion of Europeans descending from Neolithic farmers ∼ 10 thousand years ago (KYA) or Palaeolithic hunter-gatherers has been much debated. The male-specific region of the Y chromosome (MSY) has been widely applied to this question, but unbiased estimates of diversity and time depth have been lacking. Here we show that European patrilineages underwent a recent continent-wide expansion. Resequencing of 3.7 Mb of MSY DNA in 334 males, comprising 17 European and Middle Eastern populations, defines a phylogeny containing 5,996 single-nucleotide polymorphisms. Dating indicates that three major lineages (I1, R1a and R1b), accounting for 64% of our sample, have very recent coalescent times, ranging between 3.5 and 7.3 KYA. A continuous swathe of 13/17 populations share similar histories featuring a demographic expansion starting ∼ 2.1-4.2 KYA. Our results are compatible with ancient MSY DNA data, and contrast with data on mitochondrial DNA, indicating a widespread male-specific phenomenon that focuses interest on the social structure of Bronze Age Europe.

  16. 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.

  17. Complex history of the amphibian-killing chytrid fungus revealed with genome resequencing data.

    Science.gov (United States)

    Rosenblum, Erica Bree; James, Timothy Y; Zamudio, Kelly R; Poorten, Thomas J; Ilut, Dan; Rodriguez, David; Eastman, Jonathan M; Richards-Hrdlicka, Katy; Joneson, Suzanne; Jenkinson, Thomas S; Longcore, Joyce E; Parra Olea, Gabriela; Toledo, Luís Felipe; Arellano, Maria Luz; Medina, Edgar M; Restrepo, Silvia; Flechas, Sandra Victoria; Berger, Lee; Briggs, Cheryl J; Stajich, Jason E

    2013-06-01

    Understanding the evolutionary history of microbial pathogens is critical for mitigating the impacts of emerging infectious diseases on economically and ecologically important host species. We used a genome resequencing approach to resolve the evolutionary history of an important microbial pathogen, the chytrid Batrachochytrium dendrobatidis (Bd), which has been implicated in amphibian declines worldwide. We sequenced the genomes of 29 isolates of Bd from around the world, with an emphasis on North, Central, and South America because of the devastating effect that Bd has had on amphibian populations in the New World. We found a substantial amount of evolutionary complexity in Bd with deep phylogenetic diversity that predates observed global amphibian declines. By investigating the entire genome, we found that even the most recently evolved Bd clade (termed the global panzootic lineage) contained more genetic variation than previously reported. We also found dramatic differences among isolates and among genomic regions in chromosomal copy number and patterns of heterozygosity, suggesting complex and heterogeneous genome dynamics. Finally, we report evidence for selection acting on the Bd genome, supporting the hypothesis that protease genes are important in evolutionary transitions in this group. Bd is considered an emerging pathogen because of its recent effects on amphibians, but our data indicate that it has a complex evolutionary history that predates recent disease outbreaks. Therefore, it is important to consider the contemporary effects of Bd in a broader evolutionary context and identify specific mechanisms that may have led to shifts in virulence in this system.

  18. 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.

  19. Development of INDEL Markers for Genetic Mapping Based on Whole Genome Resequencing in Soybean.

    Science.gov (United States)

    Song, Xiaofeng; Wei, Haichao; Cheng, Wen; Yang, Suxin; Zhao, Yanxiu; Li, Xuan; Luo, Da; Zhang, Hui; Feng, Xianzhong

    2015-10-19

    Soybean [Glycine max (L.) Merrill] is an important crop worldwide. In this study, a Chinese local soybean cultivar, Hedou 12, was resequenced by next generation sequencing technology to develop INsertion/DELetion (INDEL) markers for genetic mapping. 49,276 INDEL polymorphisms and 242,059 single nucleotide polymorphisms were detected between Hedou 12 and the Williams 82 reference sequence. Of these, 243 candidate INDEL markers ranging from 5-50 bp in length were chosen for validation, and 165 (68%) of them revealed polymorphisms between Hedou 12 and Williams 82. The validated INDEL markers were also tested in 12 other soybean cultivars. The number of polymorphisms in the pairwise comparisons of 14 soybean cultivars varied from 27 to 165. To test the utility of these INDEL markers, they were used to perform genetic mapping of a crinkly leaf mutant, and the CRINKLY LEAF locus was successfully mapped to a 360 kb region on chromosome 7. This research shows that high-throughput sequencing technologies can facilitate the development of genome-wide molecular markers for genetic mapping in soybean.

  20. Beyond the HapMap Genotypic Data: Prospects of Deep Resequencing Projects.

    Science.gov (United States)

    Zhang, Wei; Dolan, M Eileen

    2008-09-01

    The International HapMap Project provides a key resource of genotypic data on human samples including lymphoblastoid cell lines derived from individuals of four major world populations of African, European, Japanese and Chinese ancestry. Researchers have utilized this resource to identify genetic elements that correlate with various phenotypes such as risks of common diseases, individual drug response and gene expression variation. However, recent comparative studies have suggested that the currently available HapMap genotypic data may not capture a substantial proportion of rare or untyped SNPs in these populations, implying that the HapMap SNPs may not be sufficient for comprehensive association studies. In this paper, three large-scale deep resequencing projects covering the HapMap samples: ENCODE (Encyclopedia of DNA Elements), SeattleSNPs and NIEHS (National Institute of Environmental Health Sciences) Environmental Genome Project are discussed. Prospectively, once integrated with the HapMap resource, these efforts will greatly benefit the next wave of association studies and data mining using these cell lines.

  1. Viability-qPCR for detecting Legionella: Comparison of two assays based on different amplicon lengths.

    Science.gov (United States)

    Ditommaso, Savina; Giacomuzzi, Monica; Ricciardi, Elisa; Zotti, Carla M

    2015-08-01

    Two different real-time quantitative PCR (PMA-qPCR) assays were applied for quantification of Legionella spp. by targeting a long amplicon (approx 400 bp) of 16S rRNA gene and a short amplicon (approx. 100 bp) of 5S rRNA gene. Purified DNA extracts from pure cultures of Legionella spp. and from environmental water samples were quantified. Application of the two assays to quantify Legionella in artificially contaminated water achieved that both assays were able to detect Legionella over a linear range of 10 to 10(5) cells ml(-1). A statistical analysis of the standard curves showed that both assays were linear with a good correlation coefficient (R(2) = 0.99) between the Ct and the copy number. Amplification with the reference assay was the most effective for detecting low copy numbers (1 bacterium per PCR mixture). Using selective quantification of viable Legionella by the PMA-qPCR method we obtained a greater inhibition of the amplification of the 400-bp 16S gene fragment (Δlog(10) = 3.74 ± 0.39 log(10) GU ml(-1)). A complete inhibition of the PCR signal was obtained when heat-killed cells in a concentration below 1 × 10(5) cells ml(-1) were pretreated with PMA. Analysing short amplicon sizes led to only 2.08 log reductions in the Legionella dead-cell signal. When we tested environmental water samples, the two qPCR assays were in good agreement according to the kappa index (0.741). Applying qPCR combined with PMA treatment, we also obtained a good agreement (kappa index 0.615). The comparison of quantitative results shows that both assays yielded the same quantification sensitivity (mean log = 4.59 vs mean log = 4.31).

  2. The bias associated with amplicon sequencing does not affect the quantitative assessment of bacterial community dynamics.

    Directory of Open Access Journals (Sweden)

    Federico M Ibarbalz

    Full Text Available The performance of two sets of primers targeting variable regions of the 16S rRNA gene V1-V3 and V4 was compared in their ability to describe changes of bacterial diversity and temporal turnover in full-scale activated sludge. Duplicate sets of high-throughput amplicon sequencing data of the two 16S rRNA regions shared a collection of core taxa that were observed across a series of twelve monthly samples, although the relative abundance of each taxon was substantially different between regions. A case in point was the changes in the relative abundance of filamentous bacteria Thiothrix, which caused a large effect on diversity indices, but only in the V1-V3 data set. Yet the relative abundance of Thiothrix in the amplicon sequencing data from both regions correlated with the estimation of its abundance determined using fluorescence in situ hybridization. In nonmetric multidimensional analysis samples were distributed along the first ordination axis according to the sequenced region rather than according to sample identities. The dynamics of microbial communities indicated that V1-V3 and the V4 regions of the 16S rRNA gene yielded comparable patterns of: 1 the changes occurring within the communities along fixed time intervals, 2 the slow turnover of activated sludge communities and 3 the rate of species replacement calculated from the taxa-time relationships. The temperature was the only operational variable that showed significant correlation with the composition of bacterial communities over time for the sets of data obtained with both pairs of primers. In conclusion, we show that despite the bias introduced by amplicon sequencing, the variable regions V1-V3 and V4 can be confidently used for the quantitative assessment of bacterial community dynamics, and provide a proper qualitative account of general taxa in the community, especially when the data are obtained over a convenient time window rather than at a single time point.

  3. Using expected sequence features to improve basecalling accuracy of amplicon pyrosequencing data

    DEFF Research Database (Denmark)

    Rask, Thomas Salhøj; Petersen, Bent; Chen, Donald S.

    2016-01-01

    insertions and deletions, are on the other hand likely to disrupt open reading frames. Such an inverse relationship between errors and expectation based on prior knowledge can be used advantageously to guide the process known as basecalling, i.e. the inference of nucleotide sequence from raw sequencing data...... family, where Multipass generates 20 % more error-free sequences than current state of the art methods, and provides sequence characteristics that allow generation of a set of high confidence error-free sequences. This novel method can be used to increase accuracy of existing and future amplicon...

  4. A molecular-beacon-based asymmetric PCR assay for easy visualization of amplicons in the diagnosis of trichomoniasis.

    Science.gov (United States)

    Sonkar, Subash C; Sachdev, Divya; Mishra, Prashant K; Kumar, Anita; Mittal, Pratima; Saluja, Daman

    2016-12-15

    The currently available nucleic acid amplification tests (NAATs) for trichomoniasis are accurate, quick and confirmative with superior sensitivity than traditional culture-based microbiology assays. However, these assays are associated with problems of carry over contamination, false positive results, requirement of technical expertise for performance and detection of end product. Hence, a diagnostic assay with easy visualization of the amplified product will be profitable. An in-house, rapid, sensitive, specific molecular-beacon-based PCR assay, using primers against pfoB gene of Trichomonas vaginalis, was developed and evaluated using dry ectocervical swabs (n=392) from symptomatic females with vaginal discharge. Total DNA was isolated and used as template for the PCR assays. The performance and reproducibility of PCR assay was evaluated by composite reference standard (CRS). For easy visualization of the amplified product, molecular-beacon was designed and amplicons were visualized directly using fluorescent handheld dark reader or by Micro-Plate Reader. Molecular-beacons are single-stranded hairpin shaped nucleic acid probes composed of a stem, with fluorophore/quencher pair and a loop region complementary to the desired DNA. The beacon-based PCR assay designed in the present study is highly specific as confirmed by competition experiments and extremely sensitive with detection limit of 20fg of genomic DNA (3-4 pathogens). The minimum infrastructure requirement and ease to perform the assay makes this method highly useful for resource poor countries for better disease management.

  5. Analysis of the microbiome: Advantages of whole genome shotgun versus 16S amplicon sequencing.

    Science.gov (United States)

    Ranjan, Ravi; Rani, Asha; Metwally, Ahmed; McGee, Halvor S; Perkins, David L

    2016-01-22

    The human microbiome has emerged as a major player in regulating human health and disease. Translational studies of the microbiome have the potential to indicate clinical applications such as fecal transplants and probiotics. However, one major issue is accurate identification of microbes constituting the microbiota. Studies of the microbiome have frequently utilized sequencing of the conserved 16S ribosomal RNA (rRNA) gene. We present a comparative study of an alternative approach using whole genome shotgun sequencing (WGS). In the present study, we analyzed the human fecal microbiome compiling a total of 194.1 × 10(6) reads from a single sample using multiple sequencing methods and platforms. Specifically, after establishing the reproducibility of our methods with extensive multiplexing, we compared: 1) The 16S rRNA amplicon versus the WGS method, 2) the Illumina HiSeq versus MiSeq platforms, 3) the analysis of reads versus de novo assembled contigs, and 4) the effect of shorter versus longer reads. Our study demonstrates that whole genome shotgun sequencing has multiple advantages compared with the 16S amplicon method including enhanced detection of bacterial species, increased detection of diversity and increased prediction of genes. In addition, increased length, either due to longer reads or the assembly of contigs, improved the accuracy of species detection.

  6. Analysis of the microbiome: Advantages of whole genome shotgun versus 16S amplicon sequencing

    Science.gov (United States)

    Ranjan, Ravi; Rani, Asha; Metwally, Ahmed; McGee, Halvor S.; Perkins, David L.

    2016-01-01

    The human microbiome has emerged as a major player in regulating human health and disease. Translation studies of the microbiome have the potential to indicate clinical applications such as fecal transplants and probiotics. However, one major issue is accurate identification of microbes constituting the microbiota. Studies of the microbiome have frequently utilized sequencing of the conserved 16S ribosomal RNA (rRNA) gene. We present a comparative study of an alternative approach using shotgun whole genome sequencing (WGS). In the present study, we analyzed the human fecal microbiome compiling a total of 194.1×106 reads from a single sample using multiple sequencing methods and platforms. Specifically, after establishing the reproducibility of our methods with extensive multiplexing, we compared: 1) The 16S rRNA amplicon versus the WGS method, 2) the Illumina HiSeq versus MiSeq platforms, 3) the analysis of reads versus de novo assembled contigs, and 4) the effect of shorter versus longer reads. Our study demonstrates that shotgun whole genome sequencing has multiple advantages compared with the 16S amplicon method including enhanced detection of bacterial species, increased detection of diversity and increased prediction of genes. In addition, increased length, either due to longer reads or the assembly of contigs, improved the accuracy of species detection. PMID:26718401

  7. ICO amplicon NGS data analysis: a Web tool for variant detection in common high-risk hereditary cancer genes analyzed by amplicon GS Junior next-generation sequencing.

    Science.gov (United States)

    Lopez-Doriga, Adriana; Feliubadaló, Lídia; Menéndez, Mireia; Lopez-Doriga, Sergio; Morón-Duran, Francisco D; del Valle, Jesús; Tornero, Eva; Montes, Eva; Cuesta, Raquel; Campos, Olga; Gómez, Carolina; Pineda, Marta; González, Sara; Moreno, Victor; Capellá, Gabriel; Lázaro, Conxi

    2014-03-01

    Next-generation sequencing (NGS) has revolutionized genomic research and is set to have a major impact on genetic diagnostics thanks to the advent of benchtop sequencers and flexible kits for targeted libraries. Among the main hurdles in NGS are the difficulty of performing bioinformatic analysis of the huge volume of data generated and the high number of false positive calls that could be obtained, depending on the NGS technology and the analysis pipeline. Here, we present the development of a free and user-friendly Web data analysis tool that detects and filters sequence variants, provides coverage information, and allows the user to customize some basic parameters. The tool has been developed to provide accurate genetic analysis of targeted sequencing of common high-risk hereditary cancer genes using amplicon libraries run in a GS Junior System. The Web resource is linked to our own mutation database, to assist in the clinical classification of identified variants. We believe that this tool will greatly facilitate the use of the NGS approach in routine laboratories.

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

    Directory of Open Access Journals (Sweden)

    Alvaro Martinez Barrio

    Full Text Available BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: 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. CONCLUSIONS: 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.

  9. Validation of Pooled Whole-Genome Re-Sequencing in Arabidopsis lyrata.

    Directory of Open Access Journals (Sweden)

    Marco Fracassetti

    Full Text Available Sequencing pooled DNA of multiple individuals from a population instead of sequencing individuals separately has become popular due to its cost-effectiveness and simple wet-lab protocol, although some criticism of this approach remains. Here we validated a protocol for pooled whole-genome re-sequencing (Pool-seq of Arabidopsis lyrata libraries prepared with low amounts of DNA (1.6 ng per individual. The validation was based on comparing single nucleotide polymorphism (SNP frequencies obtained by pooling with those obtained by individual-based Genotyping By Sequencing (GBS. Furthermore, we investigated the effect of sample number, sequencing depth per individual and variant caller on population SNP frequency estimates. For Pool-seq data, we compared frequency estimates from two SNP callers, VarScan and Snape; the former employs a frequentist SNP calling approach while the latter uses a Bayesian approach. Results revealed concordance correlation coefficients well above 0.8, confirming that Pool-seq is a valid method for acquiring population-level SNP frequency data. Higher accuracy was achieved by pooling more samples (25 compared to 14 and working with higher sequencing depth (4.1× per individual compared to 1.4× per individual, which increased the concordance correlation coefficient to 0.955. The Bayesian-based SNP caller produced somewhat higher concordance correlation coefficients, particularly at low sequencing depth. We recommend pooling at least 25 individuals combined with sequencing at a depth of 100× to produce satisfactory frequency estimates for common SNPs (minor allele frequency above 0.05.

  10. 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.

  11. 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.

  12. Parallel tagged amplicon sequencing of relatively long PCR products using the Illumina HiSeq platform and transcriptome assembly.

    Science.gov (United States)

    Feng, Yan-Jie; Liu, Qing-Feng; Chen, Meng-Yun; Liang, Dan; Zhang, Peng

    2016-01-01

    In phylogenetics and population genetics, a large number of loci are often needed to accurately resolve species relationships. Normally, loci are enriched by PCR and sequenced by Sanger sequencing, which is expensive when the number of amplicons is large. Next-generation sequencing (NGS) techniques are increasingly used for parallel amplicon sequencing, which reduces sequencing costs tremendously, but has not reduced preparation costs very much. Moreover, for most current NGS methods, amplicons need to be purified and quantified before sequencing and their lengths are also restricted (normally HiSeq paired-end 90-bp data. Overall, we validate a rapid, cost-effective and scalable approach to sequence a large number of targeted loci from a large number of samples that is particularly suitable for both phylogenetics and population genetics studies that require a modest scale of data.

  13. Ultra-Deep Pyrosequencing (UDPS) Data Treatment to Study Amplicon HCV Minor Variants

    Science.gov (United States)

    Gregori, Josep; Esteban, Juan I.; Cubero, María; Garcia-Cehic, Damir; Perales, Celia; Casillas, Rosario; Alvarez-Tejado, Miguel; Rodríguez-Frías, Francisco; Guardia, Jaume; Domingo, Esteban; Quer, Josep

    2013-01-01

    We have investigated the reliability and reproducibility of HCV viral quasispecies quantification by ultra-deep pyrosequencing (UDPS) methods. Our study has been divided in two parts. First of all, by UDPS sequencing of clone mixes samples we have established the global noise level of UDPS and fine tuned a data treatment workflow previously optimized for HBV sequence analysis. Secondly, we have studied the reproducibility of the methodology by comparing 5 amplicons from two patient samples on three massive sequencing platforms (FLX+, FLX and Junior) after applying the error filters developed from the clonal/control study. After noise filtering the UDPS results, the three replicates showed the same 12 polymorphic sites above 0.7%, with a mean CV of 4.86%. Two polymorphic sites below 0.6% were identified by two replicates and one replicate respectively. A total of 25, 23 and 26 haplotypes were detected by GS-Junior, GS-FLX and GS-FLX+. The observed CVs for the normalized Shannon entropy (Sn), the mutation frequency (Mf), and the nucleotidic diversity (Pi) were 1.46%, 3.96% and 3.78%. The mean absolute difference in the two patients (5 amplicons each), in the GS-FLX and GS-FLX+, were 1.46%, 3.96% and 3.78% for Sn, Mf and Pi. No false polymorphic site was observed above 0.5%. Our results indicate that UDPS is an optimal alternative to molecular cloning for quantitative study of HCV viral quasispecies populations, both in complexity and composition. We propose an UDPS data treatment workflow for amplicons from the RNA viral quasispecies which, at a sequencing depth of at least 10,000 reads per strand, enables to obtain sequences and frequencies of consensus haplotypes above 0.5% abundance with no erroneous mutations, with high confidence, resistant mutants as minor variants at the level of 1%, with high confidence that variants are not missed, and highly confident measures of quasispecies complexity. PMID:24391758

  14. Quantitative characterization of cell transduction by HSV-1 amplicons using flow cytometry and real-time PCR.

    Science.gov (United States)

    El-Sherbini, Yasser M; Stevenson, Mark M; Seymour, Leonard W; Wade-Martins, Richard

    2009-08-01

    Herpes simplex virus type 1 (HSV-1) amplicon preparations are usually quantified as transducing units/ml (TU/ml), with little information on genomic copy/TU ratios. In the present study, two HSV-1 amplicons expressing enhanced green fluorescent protein (EGFP) were analysed by quantitative PCR (qPCR) and transducing activity to obtain genomic copy/TU ratios. One vector (pHSV-GL) contains the HSV-1 packaging signal (pac) and origin of replication (oriS) and the other (pHSV/EBV-GL) includes Epstein-Barr virus (EBV) episomal maintenance elements. The pHSV-GL and pHSV/EBV-GL amplicons were prepared at titres of 7.55x10(7) and 7.24x10(7)TU/ml, containing 2.56x10(9) and 1.33x10(9) genomic copies/ml respectively. This produced preliminary estimates of genomic copy/TU ratios of 34:1 and 18:1. However standard transduction conditions did not deplete fully the supernatant of transducing particles since the same supernatant was subsequently able to achieve 25% the initial transduction efficiency, although centrifugation of amplicon particles onto cells improved infectivity by 1.8-fold. Finally, qPCR analysis of FACS-purified EGFP-expressing cells showed the presence of approximately 3 amplicon genomes/transduced cell, independent of the infection dose. Accordingly, the initial estimated genomic copy/TU ratio for pHSV-GL was revised to 6.3:1. Measuring the genomic copy/TU ratios is an important parameter for comparing the quality of amplicon preparations and standardizing experimental conditions.

  15. High‑throughput sequencing of amplicons for monitoring yeast biodiversity in must and during alcoholic fermentation.

    Science.gov (United States)

    David, Vanessa; Terrat, Sébastien; Herzine, Khaled; Claisse, Olivier; Rousseaux, Sandrine; Tourdot-Maréchal, Raphaëlle; Masneuf-Pomarede, Isabelle; Ranjard, Lionel; Alexandre, Hervé

    2014-05-01

    We compared pyrosequencing technology with the PCR-ITS-RFLP analysis of yeast isolates and denaturing gradient gel electrophoresis (DGGE). These methods gave divergent findings for the yeast population. DGGE was unsuitable for the quantification of biodiversity and its use for species detection was limited by the initial abundance of each species. The isolates identified by PCR-ITSRFLP were not fully representative of the true population. For population dynamics, high-throughput sequencing technology yielded results differing in some respects from those obtained with other approaches. This study demonstrates that 454 pyrosequencing of amplicons is more relevant than other methods for studying the yeast community on grapes and during alcoholic fermentation. Indeed, this high-throughput sequencing method detected larger numbers of species on grapes and identified species present during alcoholic fermentation that were undetectable with the other techniques.

  16. Amplicon restriction patterns associated with nitrogenase activity of root nodules for selection of superior Myrica seedlings

    Indian Academy of Sciences (India)

    Mhathung Yanthan; Arvind K Misran

    2013-11-01

    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 lands generated due to slash and burn agriculture practiced in the area. We have studied the association of amplicon restriction patterns (ARPs) of Myrica ribosomal RNA gene and internal transcribed spacer (ITS) region and nitrogenase activity of its root nodules. We found that ARPs thus obtained could be used as markers for early screening of seedlings that could support strains of Frankia that fix atmospheric nitrogen more efficiently.

  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. Population-based resequencing of APOA1 in 10,330 individuals: spectrum of genetic variation, phenotype, and comparison with extreme phenotype approach.

    Science.gov (United States)

    Haase, Christiane L; Frikke-Schmidt, Ruth; Nordestgaard, Børge G; Tybjærg-Hansen, Anne

    2012-01-01

    Rare genetic variants, identified by in-detail resequencing of loci, may contribute to complex traits. We used the apolipoprotein A-I gene (APOA1), a major high-density lipoprotein (HDL) gene, and population-based resequencing to determine the spectrum of genetic variants, the phenotypic characteristics of these variants, and how these results compared with results based on resequencing only the extremes of the apolipoprotein A-I (apoA-I) distribution. First, we resequenced APOA1 in 10,330 population-based participants in the Copenhagen City Heart Study. The spectrum and distribution of genetic variants was determined as a function of the number of individuals resequenced. Second, apoA-I and HDL cholesterol phenotypes were determined for nonsynonymous (NS) and synonymous (S) variants and were validated in the Copenhagen General Population Study (n = 45,239). Third, observed phenotypes were compared with those predicted using an extreme phenotype approach based on the apoA-I distribution. Our results are as follows: First, population-based resequencing of APOA1 identified 40 variants of which only 7 (18%) had minor allele frequencies >1%, and most were exceedingly rare. Second, 0.27% of individuals in the general population were heterozygous for NS variants which were associated with substantial reductions in apoA-I (up to 39 mg/dL) and/or HDL cholesterol (up to 0.9 mmol/L) and, surprisingly, 0.41% were heterozygous for variants predisposing to amyloidosis. NS variants associated with a hazard ratio of 1.72 (1.09-2.70) for myocardial infarction (MI), largely driven by A164S, a variant not associated with apoA-I or HDL cholesterol levels. Third, using the extreme apoA-I phenotype approach, NS variants correctly predicted the apoA-I phenotype observed in the population-based resequencing. However, using the extreme approach, between 79% (screening 0-1(st) percentile) and 21% (screening 0-20(th) percentile) of all variants were not identified; among these were variants

  19. 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.

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

    Directory of Open Access Journals (Sweden)

    Hanna Farnelid

    Full Text Available Cyanobacteria are thought to be the main N(2-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 analysis of 79,090 nitrogenase (nifH PCR amplicons encoding 7,468 unique proteins from surface samples (ten DNA samples and two RNA samples collected at ten marine locations world-wide provides the first in-depth survey of a functional bacterial gene and yield insights into the composition and diversity 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 to Alpha-, Beta-, Gamma-, and Delta-Proteobacteria were most common and showed distinct geographic distributions. Sequences related to anaerobic bacteria (nifH Cluster III were generally rare, but preponderant in cold waters, especially in the Arctic. Although the two transcript samples were dominated 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 occasionally expressed. The contribution of non-cyanobacterial diazotrophs to the global N(2 fixation budget cannot be inferred from sequence data alone, but the prevalence of non-cyanobacterial nifH genes and transcripts suggest that these bacteria are ecologically significant.

  1. Developing de novo human artificial chromosomes in embryonic stem cells using HSV-1 amplicon technology.

    Science.gov (United States)

    Moralli, Daniela; Monaco, Zoia L

    2015-02-01

    De novo artificial chromosomes expressing genes have been generated in human embryonic stem cells (hESc) and are maintained following differentiation into other cell types. Human artificial chromosomes (HAC) are small, functional, extrachromosomal elements, which behave as normal chromosomes in human cells. De novo HAC are generated following delivery of alpha satellite DNA into target cells. HAC are characterized by high levels of mitotic stability and are used as models to study centromere formation and chromosome organisation. They are successful and effective as gene expression vectors since they remain autonomous and can accommodate larger genes and regulatory regions for long-term expression studies in cells unlike other viral gene delivery vectors currently used. Transferring the essential DNA sequences for HAC formation intact across the cell membrane has been challenging for a number of years. A highly efficient delivery system based on HSV-1 amplicons has been used to target DNA directly to the ES cell nucleus and HAC stably generated in human embryonic stem cells (hESc) at high frequency. HAC were detected using an improved protocol for hESc chromosome harvesting, which consistently produced high-quality metaphase spreads that could routinely detect HAC in hESc. In tumour cells, the input DNA often integrated in the host chromosomes, but in the host ES genome, it remained intact. The hESc containing the HAC formed embryoid bodies, generated teratoma in mice, and differentiated into neuronal cells where the HAC were maintained. The HAC structure and chromatin composition was similar to the endogenous hESc chromosomes. This review will discuss the technological advances in HAC vector delivery using HSV-1 amplicons and the improvements in the identification of de novo HAC in hESc.

  2. Testing and validation of high density resequencing microarray for broad range biothreat agents detection.

    Directory of Open Access Journals (Sweden)

    Tomasz A Leski

    Full Text Available Rapid and effective detection and identification of emerging microbiological threats and potential biowarfare agents is very challenging when using traditional culture-based methods. Contemporary molecular techniques, relying upon reverse transcription and/or polymerase chain reaction (RT-PCR/PCR provide a rapid and effective alternative, however, such assays are generally designed and optimized to detect only a limited number of targets, and seldom are capable of differentiation among variants of detected targets. To meet these challenges, we have designed a broad-range resequencing pathogen microarray (RPM for detection of tropical and emerging infectious agents (TEI including biothreat agents: RPM-TEI v 1.0 (RPM-TEI. The scope of the RPM-TEI assay enables detection and differential identification of 84 types of pathogens and 13 toxin genes, including most of the class A, B and C select agents as defined by the Centers for Disease Control and Prevention (CDC, Atlanta, GA. Due to the high risks associated with handling these particular target pathogens, the sensitivity validation of the RPM-TEI has been performed using an innovative approach, in which synthetic DNA fragments are used as templates for testing the assay's limit of detection (LOD. Assay specificity and sensitivity was subsequently confirmed by testing with full-length genomic nucleic acids of selected agents. The LOD for a majority of the agents detected by RPM-TEI was determined to be at least 10(4 copies per test. Our results also show that the RPM-TEI assay not only detects and identifies agents, but is also able to differentiate near neighbors of the same agent types, such as closely related strains of filoviruses of the Ebola Zaire group, or the Machupo and Lassa arenaviruses. Furthermore, each RPM-TEI assay results in specimen-specific agent gene sequence information that can be used to assess pathogenicity, mutations, and virulence markers, results that are not generally

  3. Long amplicon (LA)-qPCR for the discrimination of infectious and noninfectious phix174 bacteriophages after UV inactivation.

    Science.gov (United States)

    Ho, Johannes; Seidel, Michael; Niessner, Reinhard; Eggers, Jutta; Tiehm, Andreas

    2016-10-15

    Waterborne viruses are increasingly being considered in risk assessment schemes. In general, virus detection by culture methods is time consuming. In contrast, detection by quantitative polymerase chain reaction (qPCR) is more rapid and therefore, more suitable for monitoring. At present, qPCR lacks the essential ability for discriminating between infectious and non-infectious viruses, thus limiting its applicability for monitoring disinfection processes. In this study, a method was developed to quantify UV inactivation by long amplicon (LA)-qPCR. Bacteriophage phiX174 was used as a surrogate for human pathogenic viruses. A qPCR protocol was developed with new sets of primers, resulting in amplicon lengths of 108, 250, 456, 568, 955, 1063, 1544, and 1764 nucleotides. The log reduction of gene copies increased with increasing amplicon length. Additional treatment with the intercalating dye, PMA, had no effect, indicating that the bacteriophage capsids were not damaged by low pressure UV irradiation. A qPCR of nearly the complete genome (approx. 5000 nucleotides) showed similar results to the plaque assay. The log reduction in qPCR correlates with [specific amplicon length x UV dose]. The normalized DNA effect constant can be applied to calculate phiX174 inactivation based on qPCR detection.

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

    DEFF Research Database (Denmark)

    Andersen, Jeppe D; Pereira, Vania; Pietroni, Carlotta

    2014-01-01

    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....

  5. 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

  6. BAC-end sequence-based SNP mining in Allotetraploid Cotton (Gossypium) utilizing re-sequencing data, phylogenetic inferences and perspectives for genetic mapping

    Science.gov (United States)

    A bacterial artificial chromosome (BAC) library and BAC-end sequences for Gossypium hirsutum L. have recently been developed. Here we report on genomic-based genome-wide SNP mining utilizing re-sequencing data with a BAC-end sequence reference for twelve G. hirsutum L. lines, one G. barbadense L. li...

  7. 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

  8. 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.

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

    Science.gov (United States)

    Pearman, John K; Irigoien, Xabier; Carvalho, Susana

    2016-04-01

    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.

  10. The development of reduced size STR amplicons as tools for analysis of degraded DNA.

    Science.gov (United States)

    Butler, John M; Shen, Yin; McCord, Bruce R

    2003-09-01

    New multiplex PCR sets of commonly used short tandem repeat (STR) markers have been developed to produce PCR products that are reduced in size when compared to standard commercial STR kits. The reduction in size of these amplicons can facilitate the examination and analysis of degraded DNA evidence by improving amplification efficiency. This "miniSTR" approach will permit current forensic practitioners to use STR markers and instrumentation already present in their laboratories and to generate genotyping data that is directly comparable to reference samples and searchable through the FBI's Combined DNA Index System (CODIS) databases. This paper discusses the development of these new primer sets and presents some initial results in the analysis of degraded and aged DNA samples. A method for removal of problematic fluorescent dye artifacts is also described. Comparison studies in over 100 samples have verified that these miniSTR primers can provide fully concordant results to commercial STR kits and can provide improved signal from degraded DNA specimens. These miniplex sets should prove valuable in the analysis of samples where allele dropout and reduced sensitivity of larger STR alleles occurs.

  11. Accuracy of the high-throughput amplicon sequencing to identify species within the genus Aspergillus.

    Science.gov (United States)

    Lee, Seungeun; Yamamoto, Naomichi

    2015-12-01

    This study characterized the accuracy of high-throughput amplicon sequencing to identify species within the genus Aspergillus. To this end, we sequenced the internal transcribed spacer 1 (ITS1), β-tubulin (BenA), and calmodulin (CaM) gene encoding sequences as DNA markers from eight reference Aspergillus strains with known identities using 300-bp sequencing on the Illumina MiSeq platform, and compared them with the BLASTn outputs. The identifications with the sequences longer than 250 bp were accurate at the section rank, with some ambiguities observed at the species rank due to mostly cross detection of sibling species. Additionally, in silico analysis was performed to predict the identification accuracy for all species in the genus Aspergillus, where 107, 210, and 187 species were predicted to be identifiable down to the species rank based on ITS1, BenA, and CaM, respectively. Finally, air filter samples were analysed to quantify the relative abundances of Aspergillus species in outdoor air. The results were reproducible across biological duplicates both at the species and section ranks, but not strongly correlated between ITS1 and BenA, suggesting the Aspergillus detection can be taxonomically biased depending on the selection of the DNA markers and/or primers.

  12. Targeted amplicon sequencing (TAS): a scalable next-gen approach to multilocus, multitaxa phylogenetics.

    Science.gov (United States)

    Bybee, Seth M; Bracken-Grissom, Heather; Haynes, Benjamin D; Hermansen, Russell A; Byers, Robert L; Clement, Mark J; Udall, Joshua A; Wilcox, Edward R; Crandall, Keith A

    2011-01-01

    Next-gen sequencing technologies have revolutionized data collection in genetic studies and advanced genome biology to novel frontiers. However, to date, next-gen technologies have been used principally for whole genome sequencing and transcriptome sequencing. Yet many questions in population genetics and systematics rely on sequencing specific genes of known function or diversity levels. Here, we describe a targeted amplicon sequencing (TAS) approach capitalizing on next-gen capacity to sequence large numbers of targeted gene regions from a large number of samples. Our TAS approach is easily scalable, simple in execution, neither time-nor labor-intensive, relatively inexpensive, and can be applied to a broad diversity of organisms and/or genes. Our TAS approach includes a bioinformatic application, BarcodeCrucher, to take raw next-gen sequence reads and perform quality control checks and convert the data into FASTA format organized by gene and sample, ready for phylogenetic analyses. We demonstrate our approach by sequencing targeted genes of known phylogenetic utility to estimate a phylogeny for the Pancrustacea. We generated data from 44 taxa using 68 different 10-bp multiplexing identifiers. The overall quality of data produced was robust and was informative for phylogeny estimation. The potential for this method to produce copious amounts of data from a single 454 plate (e.g., 325 taxa for 24 loci) significantly reduces sequencing expenses incurred from traditional Sanger sequencing. We further discuss the advantages and disadvantages of this method, while offering suggestions to enhance the approach.

  13. Implementing amplicon-based next generation sequencing in the diagnosis of small cell lung carcinoma metastases.

    Science.gov (United States)

    Meder, Lydia; König, Katharina; Fassunke, Jana; Ozretić, Luka; Wolf, Jürgen; Merkelbach-Bruse, Sabine; Heukamp, Lukas C; Buettner, Reinhard

    2015-12-01

    Small cell lung carcinoma (SCLC) is the most aggressive entity of lung cancer. Rapid cancer progression and early formation of systemic metastases drive the deadly outcome of SCLC. Recent advances in identifying oncogenes by cancer whole genome sequencing improved the understanding of SCLC carcinogenesis. However, tumor material is often limited in the clinic. Thus, it is a compulsive issue to improve SCLC diagnostics by combining established immunohistochemistry and next generation sequencing. We implemented amplicon-based next generation deep sequencing in our routine diagnostics pipeline to analyze RB1, TP53, EP300 and CREBBP, frequently mutated in SCLC. Thereby, our pipeline combined routine SCLC histology and identification of somatic mutations. We comprehensively analyzed fifty randomly collected SCLC metastases isolated from trachea and lymph nodes in comparison to specimens derived from primary SCLC. SCLC lymph node metastases showed enhanced proliferation and frequently a collapsed keratin cytoskeleton compared to SCLC metastases isolated from trachea. We identified characteristic synchronous mutations in RB1 and TP53 and non-synchronous CREBBP and EP300 mutations. Our data showed the benefit of implementing deep sequencing into routine diagnostics. We here identify oncogenic drivers and simultaneously gain further insights into SCLC tumor biology.

  14. 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

    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 ...... occasionally expressed. The contribution of non-cyanobacterial diazotrophs to the global N2 fixation budget cannot be inferred from sequence data alone, but the prevalence of non-cyanobacterial nifH genes and transcripts suggest that these bacteria are ecologically significant.......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...... analysis of 79,090 nitrogenase (nifH) PCR amplicons encoding 7,468 unique proteins from surface samples (ten DNA samples and two RNA samples) collected at ten marine locations world-wide provides the first in-depth survey of a functional bacterial gene and yield insights into the composition and diversity...

  15. 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.

  16. 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.

  17. Formulating a historical and demographic model of recent human evolution based on resequencing data from noncoding regions.

    Directory of Open Access Journals (Sweden)

    Guillaume Laval

    Full Text Available BACKGROUND: Estimating the historical and demographic parameters that characterize modern human populations is a fundamental part of reconstructing the recent history of our species. In addition, the development of a model of human evolution that can best explain neutral genetic diversity is required to identify confidently regions of the human genome that have been targeted by natural selection. METHODOLOGY/PRINCIPAL FINDINGS: We have resequenced 20 independent noncoding autosomal regions dispersed throughout the genome in 213 individuals from different continental populations, corresponding to a total of approximately 6 Mb of diploid resequencing data. We used these data to explore and co-estimate an extensive range of historical and demographic parameters with a statistical framework that combines the evaluation of multiple models of human evolution via a best-fit approach, followed by an Approximate Bayesian Computation (ABC analysis. From a methodological standpoint, evaluating the accuracy of the parameter co-estimation allowed us to identify the most accurate set of statistics to be used for the estimation of each of the different historical and demographic parameters characterizing recent human evolution. CONCLUSIONS/SIGNIFICANCE: Our results support a model in which modern humans left Africa through a single major dispersal event occurring approximately 60,000 years ago, corresponding to a drastic reduction of approximately 5 times the effective population size of the ancestral African population of approximately 13,800 individuals. Subsequently, the ancestors of modern Europeans and East Asians diverged much later, approximately 22,500 years ago, from the population of ancestral migrants. This late diversification of Eurasians after the African exodus points to the occurrence of a long maturation phase in which the ancestral Eurasian population was not yet diversified.

  18. Peeping into genomic architecture by re-sequencing of Ochrobactrum intermedium M86 strain during laboratory adapted conditions

    Directory of Open Access Journals (Sweden)

    Kushal N. Gohil

    2016-06-01

    Full Text Available Advances in de novo sequencing technologies allow us to track deeper insights into microbial genomes for restructuring events during the course of their evolution inside and outside the host. Bacterial species belonging to Ochrobactrum genus are being reported as emerging, and opportunistic pathogens in this technology driven era probably due to insertion and deletion of genes. The Ochrobactrum intermedium M86 was isolated in 2005 from a case of non-ulcer dyspeptic human stomach followed by its first draft genome sequence in 2009. Here we report re-sequencing of O. intermedium M86 laboratory adapted strain in terms of gain and loss of genes. We also attempted for finer scale genome sequence with 10 times more genome coverage than earlier one followed by comparative evaluation on Ion PGM and Illumina MiSeq. Despite their similarities at genomic level, lab-adapted strain mainly lacked genes encoding for transposase protein, insertion elements family, phage tail-proteins that were not detected in original strain on both chromosomes. Interestingly, a 5 kb indel was detected in chromosome 2 that was absent in original strain mapped with phage integrase gene of Rhizobium spp. and may be acquired and integrated through horizontal gene transfer indicating the gene loss and gene gain phenomenon in this genus. Majority of indel fragments did not match with known genes indicating more bioinformatic dissection of this fragment. Additionally we report genes related to antibiotic resistance, heavy metal tolerance in earlier and re-sequenced strain. Though SNPs detected, there did not span urease and flagellar genes. We also conclude that third generation sequencing technologies might be useful for understanding genomic architecture and re-arrangement of genes in the genome due to their ability of larger coverage that can be used to trace evolutionary aspects in microbial system.

  19. 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.

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

    Science.gov (United States)

    Natarajan, Sathishkumar; Kim, Hoy-Taek; Thamilarasan, Senthil Kumar; Veerappan, Karpagam; Park, Jong-In; Nou, Ill-Sup

    2016-01-01

    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.

  1. Effects of ERBB2 amplicon size and genomic alterations of chromosomes 1, 3, and 10 on patient response to trastuzumab in metastatic breast cancer.

    Science.gov (United States)

    Morrison, Larry E; Jewell, Susan S; Usha, Lydia; Blondin, Beth A; Rao, Ruta D; Tabesh, Bita; Kemper, Matthew; Batus, Marta; Coon, John S

    2007-04-01

    Trastuzumab is widely used for advanced breast cancer patients with ERBB2-amplified tumors. Nevertheless, over half of these patients do not have an objective response. One reason may be altered expression of genes that might compensate for ERBB2 inhibition. We previously mapped the gene-rich region of chromosome 17 telomeric to ERBB2, and reported considerable variability in the telomeric extent of the ERBB2 amplicon. Here we examined whether the variable amplicon size may be associated with patient response to trastuzumab. In addition, we looked at associations between response and several signaling pathway-related genes unrelated to the ERBB2 amplicon, including AKT3, PTEN, PIK3CA, and PTGS2. In 35 patients with ERBB2-amplified metastatic breast cancer, with 40% overall response to trastuzumab, fluorescence in situ hybridization identified the telomeric extent of the ERBB2 amplicon and the status of the several pathway-related genes. Objective response strongly correlated with the telomeric amplicon size, with 62% of patients with shorter amplicons responding, compared with only 7% of patients with longer amplicons (P = 0.0015). Abnormal copy number of PTGS2 was marginally associated with objective response (P = 0.066), while abnormal copy numbers of two reference loci, 1q25 and the chromosome 10 centromere, were significantly associated with response. Pairwise combinations of copy number status of these loci and ERBB2 amplicon size provided stronger associations and identified a group of patients without responders. These results suggest that patient selection for trastuzumab may be improved by considering ERBB2 amplicon size and genomic status of the 1q25, PTGS2, and centromere 10 loci.

  2. A novel recombinant adeno-associated virus vector packaging system with HSV-1 amplicon providing helper functions

    Institute of Scientific and Technical Information of China (English)

    舒跃龙; 吴小兵; 杨天忠; 贡惠宇; 侯云德; 颜子颖

    1999-01-01

    A novel packaging system for producing recombinant adeno-associated virus (rAAV) vector was described. Instead of the conventional method for rAAV production by two-plasmid co-transfection followed by superinfection with adenovirus 5, an HSV-1 amplicon system expressing AAV-2 rep and cap genes from their native promoters was used to provide complete helper functions for rAAV replicating and packaging. This HSV-1 ampticon stock consisted of two kinds of infectious HSV-1 virions, a replicating-defective HSV-1 amplicon pseudovirus harboring multi-copies of AAV-2 rep and cap gene and a temperature-sensitive HSV-1 mutant strain ts-KOS. High-titer rAAV was generated with this new packaging system. This packaging system gives a simple and scaleable process for rAAV production.

  3. Groundtruthing next-gen sequencing for microbial ecology-biases and errors in community structure estimates from PCR amplicon pyrosequencing.

    Directory of Open Access Journals (Sweden)

    Charles K Lee

    Full Text Available Analysis of microbial communities by high-throughput pyrosequencing of SSU rRNA gene PCR amplicons has transformed microbial ecology research and led to the observation that many communities contain a diverse assortment of rare taxa-a phenomenon termed the Rare Biosphere. Multiple studies have investigated the effect of pyrosequencing read quality on operational taxonomic unit (OTU richness for contrived communities, yet there is limited information on the fidelity of community structure estimates obtained through this approach. Given that PCR biases are widely recognized, and further unknown biases may arise from the sequencing process itself, a priori assumptions about the neutrality of the data generation process are at best unvalidated. Furthermore, post-sequencing quality control algorithms have not been explicitly evaluated for the accuracy of recovered representative sequences and its impact on downstream analyses, reducing useful discussion on pyrosequencing reads to their diversity and abundances. Here we report on community structures and sequences recovered for in vitro-simulated communities consisting of twenty 16S rRNA gene clones tiered at known proportions. PCR amplicon libraries of the V3-V4 and V6 hypervariable regions from the in vitro-simulated communities were sequenced using the Roche 454 GS FLX Titanium platform. Commonly used quality control protocols resulted in the formation of OTUs with >1% abundance composed entirely of erroneous sequences, while over-aggressive clustering approaches obfuscated real, expected OTUs. The pyrosequencing process itself did not appear to impose significant biases on overall community structure estimates, although the detection limit for rare taxa may be affected by PCR amplicon size and quality control approach employed. Meanwhile, PCR biases associated with the initial amplicon generation may impose greater distortions in the observed community structure.

  4. Groundtruthing next-gen sequencing for microbial ecology-biases and errors in community structure estimates from PCR amplicon pyrosequencing.

    Science.gov (United States)

    Lee, Charles K; Herbold, Craig W; Polson, Shawn W; Wommack, K Eric; Williamson, Shannon J; McDonald, Ian R; Cary, S Craig

    2012-01-01

    Analysis of microbial communities by high-throughput pyrosequencing of SSU rRNA gene PCR amplicons has transformed microbial ecology research and led to the observation that many communities contain a diverse assortment of rare taxa-a phenomenon termed the Rare Biosphere. Multiple studies have investigated the effect of pyrosequencing read quality on operational taxonomic unit (OTU) richness for contrived communities, yet there is limited information on the fidelity of community structure estimates obtained through this approach. Given that PCR biases are widely recognized, and further unknown biases may arise from the sequencing process itself, a priori assumptions about the neutrality of the data generation process are at best unvalidated. Furthermore, post-sequencing quality control algorithms have not been explicitly evaluated for the accuracy of recovered representative sequences and its impact on downstream analyses, reducing useful discussion on pyrosequencing reads to their diversity and abundances. Here we report on community structures and sequences recovered for in vitro-simulated communities consisting of twenty 16S rRNA gene clones tiered at known proportions. PCR amplicon libraries of the V3-V4 and V6 hypervariable regions from the in vitro-simulated communities were sequenced using the Roche 454 GS FLX Titanium platform. Commonly used quality control protocols resulted in the formation of OTUs with >1% abundance composed entirely of erroneous sequences, while over-aggressive clustering approaches obfuscated real, expected OTUs. The pyrosequencing process itself did not appear to impose significant biases on overall community structure estimates, although the detection limit for rare taxa may be affected by PCR amplicon size and quality control approach employed. Meanwhile, PCR biases associated with the initial amplicon generation may impose greater distortions in the observed community structure.

  5. Groundtruthing Next-Gen Sequencing for Microbial Ecology–Biases and Errors in Community Structure Estimates from PCR Amplicon Pyrosequencing

    OpenAIRE

    Lee, Charles K.; Craig W. Herbold; Polson, Shawn W.; K Eric Wommack; Williamson, Shannon J.; McDonald, Ian R.; S. Craig Cary

    2012-01-01

    Analysis of microbial communities by high-throughput pyrosequencing of SSU rRNA gene PCR amplicons has transformed microbial ecology research and led to the observation that many communities contain a diverse assortment of rare taxa-a phenomenon termed the Rare Biosphere. Multiple studies have investigated the effect of pyrosequencing read quality on operational taxonomic unit (OTU) richness for contrived communities, yet there is limited information on the fidelity of community structure est...

  6. Amplicon-based semiconductor sequencing of human exomes: performance evaluation and optimization strategies.

    Science.gov (United States)

    Damiati, E; Borsani, G; Giacopuzzi, Edoardo

    2016-05-01

    The Ion Proton platform allows to perform whole exome sequencing (WES) at low cost, providing rapid turnaround time and great flexibility. Products for WES on Ion Proton system include the AmpliSeq Exome kit and the recently introduced HiQ sequencing chemistry. Here, we used gold standard variants from GIAB consortium to assess the performances in variants identification, characterize the erroneous calls and develop a filtering strategy to reduce false positives. The AmpliSeq Exome kit captures a large fraction of bases (>94 %) in human CDS, ClinVar genes and ACMG genes, but with 2,041 (7 %), 449 (13 %) and 11 (19 %) genes not fully represented, respectively. Overall, 515 protein coding genes contain hard-to-sequence regions, including 90 genes from ClinVar. Performance in variants detection was maximum at mean coverage >120×, while at 90× and 70× we measured a loss of variants of 3.2 and 4.5 %, respectively. WES using HiQ chemistry showed ~71/97.5 % sensitivity, ~37/2 % FDR and ~0.66/0.98 F1 score for indels and SNPs, respectively. The proposed low, medium or high-stringency filters reduced the amount of false positives by 10.2, 21.2 and 40.4 % for indels and 21.2, 41.9 and 68.2 % for SNP, respectively. Amplicon-based WES on Ion Proton platform using HiQ chemistry emerged as a competitive approach, with improved accuracy in variants identification. False-positive variants remain an issue for the Ion Torrent technology, but our filtering strategy can be applied to reduce erroneous variants.

  7. 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.

  8. Facile whole mitochondrial genome resequencing from nipple aspirate fluid using MitoChip v2.0

    Directory of Open Access Journals (Sweden)

    Thayer Robert E

    2008-04-01

    Full Text Available Abstract Background Mutations in the mitochondrial genome (mtgenome have been associated with many disorders, including breast cancer. Nipple aspirate fluid (NAF from symptomatic women could potentially serve as a minimally invasive sample for breast cancer screening by detecting somatic mutations in this biofluid. This study is aimed at 1 demonstrating the feasibility of NAF recovery from symptomatic women, 2 examining the feasibility of sequencing the entire mitochondrial genome from NAF samples, 3 cross validation of the Human mitochondrial resequencing array 2.0 (MCv2, and 4 assessing the somatic mtDNA mutation rate in benign breast diseases as a potential tool for monitoring early somatic mutations associated with breast cancer. Methods NAF and blood were obtained from women with symptomatic benign breast conditions, and we successfully assessed the mutation load in the entire mitochondrial genome of 19 of these women. DNA extracts from NAF were sequenced using the mitochondrial resequencing array MCv2 and by capillary electrophoresis (CE methods as a quality comparison. Sequencing was performed independently at two institutions and the results compared. The germline mtDNA sequence determined using DNA isolated from the patient's blood (control was compared to the mutations present in cellular mtDNA recovered from patient's NAF. Results From the cohort of 28 women recruited for this study, NAF was successfully recovered from 23 participants (82%. Twenty two (96% of the women produced fluids from both breasts. Twenty NAF samples and corresponding blood were chosen for this study. Except for one NAF sample, the whole mtgenome was successfully amplified using a single primer pair, or three pairs of overlapping primers. Comparison of MCv2 data from the two institutions demonstrates 99.200% concordance. Moreover, MCv2 data was 99.999% identical to CE sequencing, indicating that MCv2 is a reliable method to rapidly sequence the entire mtgenome

  9. The 4q12 amplicon in malignant peripheral nerve sheath tumors: consequences on gene expression and implications for sunitinib treatment.

    Directory of Open Access Journals (Sweden)

    Jan Zietsch

    Full Text Available BACKGROUND: Malignant peripheral nerve sheath tumors (MPNST are highly aggressive tumors which originate from Schwann cells and develop in about 10% of neurofibromatosis type 1 (NF1 patients. The five year survival rate is poor and more effective therapies are needed. Sunitinib is a drug targeting receptor tyrosine kinases (RTK like PDGFRalpha, c-Kit and VEGFR-2. These genes are structurally related and cluster on chromosomal segment 4q12. METHODOLOGY/PRINCIPAL FINDINGS: Here we characterize this region by multiplex ligation-dependent probe amplification (MLPA in MPNST. Our probe set encompasses the 3 adjacent RTK genes (PDGFRA, KIT, KDR and 6 flanking genes. We found amplification of several genes within this region in a subset of MPNST and MPNST cell lines. Transcript and protein expression of PDGFRA matched well with its increased copy number suggesting a central role of PDGFRA within the amplicon. Studying the effect of sunitinib on 5 MPNST cell lines revealed that cell line S462 harboring the 4q12 amplicon was extremely sensitive to the drug with an IC50 below 1.0 microM. Moreover, sunitinib induced apoptosis and prevented PDGF-AA induced signaling via PDGFRalpha as determined by western blotting. Co-expression of VEGF and its receptor VEGFR-2 (KDR was present in MPNST cell lines suggesting an autocrine loop. We show that VEGF triggered signal transduction via the MAPK pathway, which could be blocked by sunitinib. CONCLUSIONS/SIGNIFICANCE: Since multiple receptors targeted by sunitinib are expressed or over-expressed by MPNST cells sunitinib appears as an attractive drug for treatment of MPNST patients. Presence of the 4q12 amplicon and subsequent over-expression of PDGFRA might serve as predictive markers for efficacy of sunitinib.

  10. Genotyping-in-Thousands by sequencing (GT-seq): A cost effective SNP genotyping method based on custom amplicon sequencing.

    Science.gov (United States)

    Campbell, Nathan R; Harmon, Stephanie A; Narum, Shawn R

    2015-07-01

    Genotyping-in-Thousands by sequencing (GT-seq) is a method that uses next-generation sequencing of multiplexed PCR products to generate genotypes from relatively small panels (50-500) of targeted single-nucleotide polymorphisms (SNPs) for thousands of individuals in a single Illumina HiSeq lane. This method uses only unlabelled oligos and PCR master mix in two thermal cycling steps for amplification of targeted SNP loci. During this process, sequencing adapters and dual barcode sequence tags are incorporated into the amplicons enabling thousands of individuals to be pooled into a single sequencing library. Post sequencing, reads from individual samples are split into individual files using their unique combination of barcode sequences. Genotyping is performed with a simple perl script which counts amplicon-specific sequences for each allele, and allele ratios are used to determine the genotypes. We demonstrate this technique by genotyping 2068 individual steelhead trout (Oncorhynchus mykiss) samples with a set of 192 SNP markers in a single library sequenced in a single Illumina HiSeq lane. Genotype data were 99.9% concordant to previously collected TaqMan(™) genotypes at the same 192 loci, but call rates were slightly lower with GT-seq (96.4%) relative to Taqman (99.0%). Of the 192 SNPs, 187 were genotyped in ≥90% of the individual samples and only 3 SNPs were genotyped in <70% of samples. This study demonstrates amplicon sequencing with GT-seq greatly reduces the cost of genotyping hundreds of targeted SNPs relative to existing methods by utilizing a simple library preparation method and massive efficiency of scale.

  11. Whole genome resequencing of a laboratory-adapted Drosophila melanogaster population sample [version 1; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    William P. Gilks

    2016-11-01

    Full Text Available As part of a study into the molecular genetics of sexually dimorphic complex traits, we used next-generation sequencing to obtain data on genomic variation in an outbred laboratory-adapted fruit fly (Drosophila melanogaster population. We successfully resequenced the whole genome of 220 hemiclonal females that were heterozygous for the same Berkeley reference line genome (BDGP6/dm6, and a unique haplotype from the outbred base population (LHM. The use of a static and known genetic background enabled us to obtain sequences from whole genome phased haplotypes. We used a BWA-Picard-GATK pipeline for mapping sequence reads to the dm6 reference genome assembly, at a median depth of coverage of 31X, and have made the resulting data publicly-available in the NCBI Short Read Archive (Accession number SRP058502. We used Haplotype Caller to discover and genotype 1,726,931 small genomic variants (SNPs and indels, <200bp. Additionally we detected and genotyped 167 large structural variants (1-100Kb in size using GenomeStrip/2.0. Sequence and genotype data are publicly-available at the corresponding NCBI databases: Short Read Archive, dbSNP and dbVar (BioProject PRJNA282591. We have also released the unfiltered genotype data, and the code and logs for data processing and summary statistics (https://zenodo.org/communities/sussex_drosophila_sequencing/.

  12. 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

    2016-05-04

    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.

  13. Whole genome re-sequencing of date palms yields insights into diversification of a fruit tree crop.

    Science.gov (United States)

    Hazzouri, Khaled M; Flowers, Jonathan M; Visser, Hendrik J; Khierallah, Hussam S M; Rosas, Ulises; Pham, Gina M; Meyer, Rachel S; Johansen, Caryn K; Fresquez, Zoë A; Masmoudi, Khaled; Haider, Nadia; El Kadri, Nabila; Idaghdour, Youssef; Malek, Joel A; Thirkhill, Deborah; Markhand, Ghulam S; Krueger, Robert R; Zaid, Abdelouahhab; Purugganan, Michael D

    2015-11-09

    Date palms (Phoenix dactylifera) are the most significant perennial crop in arid regions of the Middle East and North Africa. Here, we present a comprehensive catalogue of approximately seven million single nucleotide polymorphisms in date palms based on whole genome re-sequencing of a collection of 62 cultivars. Population structure analysis indicates a major genetic divide between North Africa and the Middle East/South Asian date palms, with evidence of admixture in cultivars from Egypt and Sudan. Genome-wide scans for selection suggest at least 56 genomic regions associated with selective sweeps that may underlie geographic adaptation. We report candidate mutations for trait variation, including nonsense polymorphisms and presence/absence variation in gene content in pathways for key agronomic traits. We also identify a copia-like retrotransposon insertion polymorphism in the R2R3 myb-like orthologue of the oil palm virescens gene associated with fruit colour variation. This analysis documents patterns of post-domestication diversification and provides a genomic resource for this economically important perennial tree crop.

  14. 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.

  15. Whole-Genome Resequencing Reveals Extensive Natural Variation in the Model Green Alga Chlamydomonas reinhardtii[OPEN

    Science.gov (United States)

    Hazzouri, Khaled M.; Rosas, Ulises; Bahmani, Tayebeh; Nelson, David R.; Abdrabu, Rasha; Harris, Elizabeth H.; Salehi-Ashtiani, Kourosh; Purugganan, Michael D.

    2015-01-01

    We performed whole-genome resequencing of 12 field isolates and eight commonly studied laboratory strains of the model organism Chlamydomonas reinhardtii to characterize genomic diversity and provide a resource for studies of natural variation. Our data support previous observations that Chlamydomonas is among the most diverse eukaryotic species. Nucleotide diversity is ∼3% and is geographically structured in North America with some evidence of admixture among sampling locales. Examination of predicted loss-of-function mutations in field isolates indicates conservation of genes associated with core cellular functions, while genes in large gene families and poorly characterized genes show a greater incidence of major effect mutations. De novo assembly of unmapped reads recovered genes in the field isolates that are absent from the CC-503 assembly. The laboratory reference strains show a genomic pattern of polymorphism consistent with their origin as the recombinant progeny of a diploid zygospore. Large duplications or amplifications are a prominent feature of laboratory strains and appear to have originated under laboratory culture. Extensive natural variation offers a new source of genetic diversity for studies of Chlamydomonas, including naturally occurring alleles that may prove useful in studies of gene function and the dissection of quantitative genetic traits. PMID:26392080

  16. Whole genome re-sequencing of date palms yields insights into diversification of a fruit tree crop

    Science.gov (United States)

    Hazzouri, Khaled M.; Flowers, Jonathan M.; Visser, Hendrik J.; Khierallah, Hussam S. M.; Rosas, Ulises; Pham, Gina M.; Meyer, Rachel S.; Johansen, Caryn K.; Fresquez, Zoë A.; Masmoudi, Khaled; Haider, Nadia; El Kadri, Nabila; Idaghdour, Youssef; Malek, Joel A.; Thirkhill, Deborah; Markhand, Ghulam S.; Krueger, Robert R.; Zaid, Abdelouahhab; Purugganan, Michael D.

    2015-01-01

    Date palms (Phoenix dactylifera) are the most significant perennial crop in arid regions of the Middle East and North Africa. Here, we present a comprehensive catalogue of approximately seven million single nucleotide polymorphisms in date palms based on whole genome re-sequencing of a collection of 62 cultivars. Population structure analysis indicates a major genetic divide between North Africa and the Middle East/South Asian date palms, with evidence of admixture in cultivars from Egypt and Sudan. Genome-wide scans for selection suggest at least 56 genomic regions associated with selective sweeps that may underlie geographic adaptation. We report candidate mutations for trait variation, including nonsense polymorphisms and presence/absence variation in gene content in pathways for key agronomic traits. We also identify a copia-like retrotransposon insertion polymorphism in the R2R3 myb-like orthologue of the oil palm virescens gene associated with fruit colour variation. This analysis documents patterns of post-domestication diversification and provides a genomic resource for this economically important perennial tree crop. PMID:26549859

  17. 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.

  18. 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

  19. Genome resequencing in Populus: Revealing large-scale genome variation and implications on specialized-trait genomics

    Energy Technology Data Exchange (ETDEWEB)

    Muchero, Wellington [ORNL; Labbe, Jessy L [ORNL; Priya, Ranjan [University of Tennessee, Knoxville (UTK); DiFazio, Steven P [West Virginia University, Morgantown; Tuskan, Gerald A [ORNL

    2014-01-01

    To date, Populus ranks among a few plant species with a complete genome sequence and other highly developed genomic resources. With the first genome sequence among all tree species, Populus has been adopted as a suitable model organism for genomic studies in trees. However, far from being just a model species, Populus is a key renewable economic resource that plays a significant role in providing raw materials for the biofuel and pulp and paper industries. Therefore, aside from leading frontiers of basic tree molecular biology and ecological research, Populus leads frontiers in addressing global economic challenges related to fuel and fiber production. The latter fact suggests that research aimed at improving quality and quantity of Populus as a raw material will likely drive the pursuit of more targeted and deeper research in order to unlock the economic potential tied in molecular biology processes that drive this tree species. Advances in genome sequence-driven technologies, such as resequencing individual genotypes, which in turn facilitates large scale SNP discovery and identification of large scale polymorphisms are key determinants of future success in these initiatives. In this treatise we discuss implications of genome sequence-enable technologies on Populus genomic and genetic studies of complex and specialized-traits.

  20. Whole-genome resequencing analyses of five pig breeds, including Korean wild and native, and three European origin breeds.

    Science.gov (United States)

    Choi, Jung-Woo; Chung, Won-Hyong; Lee, Kyung-Tai; Cho, Eun-Seok; Lee, Si-Woo; Choi, Bong-Hwan; Lee, Sang-Heon; Lim, Wonjun; Lim, Dajeong; Lee, Yun-Gyeong; Hong, Joon-Ki; Kim, Doo-Wan; Jeon, Hyeon-Jeong; Kim, Jiwoong; Kim, Namshin; Kim, Tae-Hun

    2015-08-01

    Pigs have been one of the most important sources of meat for humans, and their productivity has been substantially improved by recent strong selection. Here, we present whole-genome resequencing analyses of 55 pigs of five breeds representing Korean native pigs, wild boar and three European origin breeds. 1,673.1 Gb of sequence reads were mapped to the Swine reference assembly, covering ∼99.2% of the reference genome, at an average of ∼11.7-fold coverage. We detected 20,123,573 single-nucleotide polymorphisms (SNPs), of which 25.5% were novel. We extracted 35,458 of non-synonymous SNPs in 9,904 genes, which may contribute to traits of interest. The whole SNP sets were further used to access the population structures of the breeds, using multiple methodologies, including phylogenetic, similarity matrix, and population structure analysis. They showed clear population clusters with respect to each breed. Furthermore, we scanned the whole genomes to identify signatures of selection throughout the genome. The result revealed several promising loci that might underlie economically important traits in pigs, such as the CLDN1 and TWIST1 genes. These discoveries provide useful genomic information for further study of the discrete genetic mechanisms associated with economically important traits in pigs.

  1. CLOTU: An online pipeline for processing and clustering of 454 amplicon reads into OTUs followed by taxonomic annotation

    Directory of Open Access Journals (Sweden)

    Shalchian-Tabrizi Kamran

    2011-05-01

    Full Text Available Abstract Background The implementation of high throughput sequencing for exploring biodiversity poses high demands on bioinformatics applications for automated data processing. Here we introduce CLOTU, an online and open access pipeline for processing 454 amplicon reads. CLOTU has been constructed to be highly user-friendly and flexible, since different types of analyses are needed for different datasets. Results In CLOTU, the user can filter out low quality sequences, trim tags, primers, adaptors, perform clustering of sequence reads, and run BLAST against NCBInr or a customized database in a high performance computing environment. The resulting data may be browsed in a user-friendly manner and easily forwarded to downstream analyses. Although CLOTU is specifically designed for analyzing 454 amplicon reads, other types of DNA sequence data can also be processed. A fungal ITS sequence dataset generated by 454 sequencing of environmental samples is used to demonstrate the utility of CLOTU. Conclusions CLOTU is a flexible and easy to use bioinformatics pipeline that includes different options for filtering, trimming, clustering and taxonomic annotation of high throughput sequence reads. Some of these options are not included in comparable pipelines. CLOTU is implemented in a Linux computer cluster and is freely accessible to academic users through the Bioportal web-based bioinformatics service (http://www.bioportal.uio.no.

  2. 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.

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

    Science.gov (United States)

    Poulet, Axel; Privat, Maud; Ponelle, Flora; Viala, Sandrine; Decousus, Stephanie; Perin, Axel; Lafarge, Laurence; Ollier, Marie; El Saghir, Nagi S; Uhrhammer, Nancy; Bignon, Yves-Jean; Bidet, Yannick

    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.

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

    Directory of Open Access Journals (Sweden)

    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. Upregulated, 7q21-22 amplicon candidate gene SHFM1 confers oncogenic advantage by suppressing p53 function in gastric cancer.

    Science.gov (United States)

    Tamilzhalagan, Sembulingam; Muthuswami, Muthulakshmi; Periasamy, Jayaprakash; Lee, Ming Hui; Rha, Sun Young; Tan, Patrick; Ganesan, Kumaresan

    2015-06-01

    Chromosomal aberrations are hallmarks of cancers and the locus of frequent genomic amplifications often harbors key cancer driver genes. Many genomic amplicons remain larger with hundreds of genes and the key drivers remain to be identified by an amplification-wide systematic analysis. The 7q21.12-q22.3 genomic amplification is frequent in gastric cancers which occur in ~10% of the patients and multiple cell lines. This 7q21.12-q22.3 amplicon has not yet been completely analyzed towards identifying the driver genes and their functional contribution in oncogenesis. The amplitude and prevalence indicate the important role conferred by this amplicon in gastric cancers. Among the 159 genes of this amplicon, 12 genes are found over-expressed in primary gastric tumors and cell lines. Many of the over-expressed genes show negative association with p53 transcriptional activity. RNAi based functional screening of the genes reveal, SHFM1 as key gastric cancer driver gene. SHFM1 confers cell cycle progression and resistance to p53 stabilizing drugs in gastric cancer cells. SHFM1 also activates Src, MAPK/ERK and PI3K/Akt signaling pathways. This is the first integrative genomic investigation of 7q21.12-q22.3 amplicon revealing the potential oncogenic candidacy of 12 genes. The oncogenic contribution of SHFM1, mediated by the p53 suppressive feature has been demonstrated in gastric cancer cells.

  6. QTL Mapping for Rice RVA Properties Using High-Throughput Re-sequenced Chromosome Segment Substitution Lines

    Institute of Scientific and Technical Information of China (English)

    ZHANG Chang-quan; HU Bing; ZHU Kong-zhi; ZHANG Hua; LENG Ya-lin; TANG Shu-zhu; GU Ming-hong; LIU Qiao-quan

    2013-01-01

    The rapid visco analyser (RVA) profile is an important factor for evaluation of the cooking and eating quality of rice. To improve rice quality, the identification of new quantitative trait loci (QTLs) for RVA profiling is of great significance. We used a japonica rice cultivar Nipponbare as the recipient and indica rice 9311 as the donor to develop a population containing 38 chromosome segment substitution lines (CSSLs) genotyped by a high-throughput re-sequencing strategy. In this study, the population and the parent lines, which contained similar apparent amylose contents, were used to map the QTLs of RVA properties including peak paste viscosity (PKV), hot paste viscosity (HPV), cool paste viscosity (CPV), breakdown viscosity (BKV), setback viscosity (SBV), consistency viscosity (CSV), peak time (PeT) and pasting temperature (PaT). QTL analysis was carried out using one-way analysis of variance and Dunnett’s test, and stable QTLs were identified over two years and under two environments. We identified 10 stable QTLs:qPKV2-1, qSBV2-1;qPKV5-1, qHPV5-1, qCPV5-1;qPKV7-1, qHPV7-1, qCPV7-1, qSBV7-1;and qPKV8-1 on chromosomes 2, 5, 7 and 8, respectively, with contributions ranging from-95.6%to 47.1%. Besides, there was pleiotropy in the QTLs on chromosomes 2, 5 and 7.

  7. 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.

  8. The HER2 amplicon includes several genes required for the growth and survival of HER2 positive breast cancer cells — A data description

    Directory of Open Access Journals (Sweden)

    Vesa Hongisto

    2014-12-01

    Full Text Available A large number of breast cancers are characterized by amplification and overexpression of the chromosome segment surrounding the HER2 (ERBB2 oncogene. As the HER2 amplicon at 17q12 contains multiple genes, we have systematically explored the role of the HER2 co-amplified genes in breast cancer cell growth and their relation to trastuzumab resistance. We integrated array comparative genomic hybridization (aCGH data of the HER2 amplicon from 71 HER2 positive breast tumors and 10 cell lines with systematic functional RNA interference analysis of 23 core amplicon genes with several phenotypic endpoints in a panel of trastuzumab responding and non-responding HER2 positive breast cancer cells. In this Data in Brief we give a detailed description of the experimental procedures and the data analysis methods used in the study (1.

  9. Resequencing of Treponema pallidum ssp. pallidum strains Nichols and SS14: correction of sequencing errors resulted in increased separation of syphilis treponeme subclusters.

    Directory of Open Access Journals (Sweden)

    Helena Pětrošová

    Full Text Available BACKGROUND: Treponema pallidum ssp. pallidum (TPA, the causative agent of syphilis, is a highly clonal bacterium showing minimal genetic variability in the genome sequence of individual strains. Nevertheless, genetically characterized syphilis strains can be clearly divided into two groups, Nichols-like strains and SS14-like strains. TPA Nichols and SS14 strains were completely sequenced in 1998 and 2008, respectively. Since publication of their complete genome sequences, a number of sequencing errors in each genome have been reported. Therefore, we have resequenced TPA Nichols and SS14 strains using next-generation sequencing techniques. METHODOLOGY/PRINCIPAL FINDINGS: The genomes of TPA strains Nichols and SS14 were resequenced using the 454 and Illumina sequencing methods that have a combined average coverage higher than 90x. In the TPA strain Nichols genome, 134 errors were identified (25 substitutions and 109 indels, and 102 of them affected protein sequences. In the TPA SS14 genome, a total of 191 errors were identified (85 substitutions and 106 indels and 136 of them affected protein sequences. A set of new intrastrain heterogenic regions in the TPA SS14 genome were identified including the tprD gene, where both tprD and tprD2 alleles were found. The resequenced genomes of both TPA Nichols and SS14 strains clustered more closely with related strains (i.e. strains belonging to same syphilis treponeme subcluster. At the same time, groups of Nichols-like and SS14-like strains were found to be more distantly related. CONCLUSION/SIGNIFICANCE: We identified errors in 11.5% of all annotated genes and, after correction, we found a significant impact on the predicted proteomes of both Nichols and SS14 strains. Corrections of these errors resulted in protein elongations, truncations, fusions and indels in more than 11% of all annotated proteins. Moreover, it became more evident that syphilis is caused by treponemes belonging to two separate genetic

  10. 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...... identified 38 probe-defined core genera, which are shared among all investigated Danish plants. A large body of knowledge exists on many of the core genera, however few attempts have been made to integrate the knowledge on a system-level understanding of the process. In this work we aimed to integrate...... 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...

  11. 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.

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

    Directory of Open Access Journals (Sweden)

    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

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

    Directory of Open Access Journals (Sweden)

    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

  14. Bioinformatics pipelines for targeted resequencing and whole-exome sequencing of human and mouse genomes: a virtual appliance approach for instant deployment.

    Directory of Open Access Journals (Sweden)

    Jason Li

    Full Text Available Targeted resequencing by massively parallel sequencing has become an effective and affordable way to survey small to large portions of the genome for genetic variation. Despite the rapid development in open source software for analysis of such data, the practical implementation of these tools through construction of sequencing analysis pipelines still remains a challenging and laborious activity, and a major hurdle for many small research and clinical laboratories. We developed TREVA (Targeted REsequencing Virtual Appliance, making pre-built pipelines immediately available as a virtual appliance. Based on virtual machine technologies, TREVA is a solution for rapid and efficient deployment of complex bioinformatics pipelines to laboratories of all sizes, enabling reproducible results. The analyses that are supported in TREVA include: somatic and germline single-nucleotide and insertion/deletion variant calling, copy number analysis, and cohort-based analyses such as pathway and significantly mutated genes analyses. TREVA is flexible and easy to use, and can be customised by Linux-based extensions if required. TREVA can also be deployed on the cloud (cloud computing, enabling instant access without investment overheads for additional hardware. TREVA is available at http://bioinformatics.petermac.org/treva/.

  15. Whole-genome resequencing of extreme phenotypes in collared flycatchers highlights the difficulty of detecting quantitative trait loci in natural populations.

    Science.gov (United States)

    Kardos, Marty; Husby, Arild; McFarlane, S Eryn; Qvarnström, Anna; Ellegren, Hans

    2016-05-01

    Dissecting the genetic basis of phenotypic variation in natural populations is a long-standing goal in evolutionary biology. One open question is whether quantitative traits are determined only by large numbers of genes with small effects, or whether variation also exists in large-effect loci. We conducted genomewide association analyses of forehead patch size (a sexually selected trait) on 81 whole-genome-resequenced male collared flycatchers with extreme phenotypes, and on 415 males sampled independent of patch size and genotyped with a 50K SNP chip. No SNPs were genomewide statistically significantly associated with patch size. Simulation-based power analyses suggest that the power to detect large-effect loci responsible for 10% of phenotypic variance was 0.8 for resequencing of extreme phenotypes (N = 243), but power remained 0.8 when analysing 415 randomly sampled phenotypes. However, power of the 50K SNP chip to detect large-effect loci was nearly 0.8 in simulations with a small effective population size of 1500. These results suggest that reliably detecting large-effect trait loci in large natural populations will often require thousands of individuals and near complete sampling of the genome. Encouragingly, far fewer individuals and loci will often be sufficient to reliably detect large-effect loci in small populations with widespread strong linkage disequilibrium.

  16. 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

    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. PMID:27490946

  17. 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.

  18. Quantitative assessment of the effect of uracil-DNA glycosylase on amplicon DNA degradation and RNA amplification in reverse transcription-PCR

    Directory of Open Access Journals (Sweden)

    Kleiboeker Steven B

    2005-04-01

    Full Text Available Abstract Although PCR and RT-PCR provided a valuable approach for detection of pathogens, the high level of sensitivity of these assays also makes them prone to false positive results. In addition to cross-contamination with true positive samples, false positive results are also possible due to "carry-over" contamination of samples with amplicon DNA generated by previous reactions. To reduce this source of false positives, amplicon generated by reactions in which dUTP was substituted for dTTP can be degraded by uracil DNA glycosylase (UNG. UNG does not degrade RNA but will cleave contaminating uracil-containing DNA while leaving thymine-containing DNA intact. The availability of heat-labile UNG makes use of this approach feasible for RT-PCR. In this study, real-time RT-PCR was used to quantify UNG degradation of amplicon DNA and the effect of UNG on RNA detection. Using the manufacturers' recommended conditions, complete degradation of DNA was not observed for samples containing 250 copies of amplicon DNA. Doubling the UNG concentration resulted in degradation of the two lowest concentrations of DNA tested, but also resulted in an increase of 1.94 cycles in the CT for RNA detection. To improve DNA degradation while minimizing the effect on RNA detection, a series of time, temperature and enzyme concentrations were evaluated. Optimal conditions were found to be 0.25 U UNG per 25 μl reaction with a 20 min, 30°C incubation prior to RT-PCR. Under these conditions, high concentrations of amplicon DNA could be degraded while the CT for RNA detection was increased by 1.2 cycles.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  1. Preparing a re-sequencing DNA library of 2 cancer candidate genes using the ligation-by-amplification protocol by two PCR reactions

    Institute of Scientific and Technical Information of China (English)

    SU YeYang; LIN Lin; TIAN Geng; CHEN Chen; LIU Tao; XU Xingya; QI XinPeng; ZHANG XiuQing; YANG HuanMing

    2009-01-01

    To meet the needs of large-scale genomic/genetic studies, the next-generation massively parallelized sequencing technologies provide high throughput, low cost and low labor-intensive sequencing ser-vice, with subsequent bioinformatic software and laboratory methods developed to expand their ap-plications in various types of research. PCR-based genomic/genetic studies, which have significant usage in association studies like cancer research, haven't benefited much from those next-generation sequencing technologies, because the shortgun re-sequencing strategy used by such sequencing machines as the Illumina/Solexa Genome Analyzer may not be applied to direct re-sequencing of short-length target regions like those in PCR-based genomic/genetic studies. Although several meth-ods have been proposed to solve this problem, including microarray-based genomic selections and selector-based technologies, they require advanced equipment and procedures which limit their ap-plications in many laboratories. By contrast, we overcame such potential drawbacks by utilizing a liga-tion by amplification (LBA) protocol, a method using a pair of Universal Adapters to randomly ligate target regions in a two-step-PCR procedure, whose Long LBA products were easily fragmented and sequenced on the next-generation sequencing machine. In this concept-proven study, we chose the consensus coding sequences of two human cancer genes: BRCA1 and BRCA2 as target regions, spe-cifically designed LBA primer pairs to amplify and randomly ligate them. 70 target sequences were successfully amplified and ligated into Long LBA products, which were then fragmented to construct DNA libraries for sequencing on both a conventional Sanger sequencer ABI 3730xl DNA Analyzer and the next-generation 'synthesis by sequencing technology' IlluminalSolexa Genome Analyzer. Bioin-formatic analysis demonstrated the utility and efficiency (including the coverage and depth of each target sequence and the SNPs detection

  2. Preparing a re-sequencing DNA library of 2 cancer candidate genes using the ligation-by-amplification protocol by two PCR reactions

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    To meet the needs of large-scale genomic/genetic studies, the next-generation massively parallelized sequencing technologies provide high throughput, low cost and low labor-intensive sequencing service, with subsequent bioinformatic software and laboratory methods developed to expand their applications in various types of research. PCR-based genomic/genetic studies, which have significant usage in association studies like cancer research, haven’t benefited much from those next-generation sequencing technologies, because the shortgun re-sequencing strategy used by such sequencing machines as the Illumina/Solexa Genome Analyzer may not be applied to direct re-sequencing of short-length target regions like those in PCR-based genomic/genetic studies. Although several methods have been proposed to solve this problem, including microarray-based genomic selections and selector-based technologies, they require advanced equipment and procedures which limit their applications in many laboratories. By contrast, we overcame such potential drawbacks by utilizing a ligation by amplification (LBA) protocol, a method using a pair of Universal Adapters to randomly ligate target regions in a two-step-PCR procedure, whose Long LBA products were easily fragmented and sequenced on the next-generation sequencing machine. In this concept-proven study, we chose the consensus coding sequences of two human cancer genes: BRCA1 and BRCA2 as target regions, specifically designed LBA primer pairs to amplify and randomly ligate them. 70 target sequences were successfully amplified and ligated into Long LBA products, which were then fragmented to construct DNA libraries for sequencing on both a conventional Sanger sequencer ABI 3730xl DNA Analyzer and the next-generation ’synthesis by sequencing technology’ Illumina/Solexa Genome Analyzer. Bioinformatic analysis demonstrated the utility and efficiency (including the coverage and depth of each target sequence and the SNPs detection

  3. Simultaneous amplicon sequencing to explore co-occurrence patterns of bacterial, archaeal and eukaryotic microorganisms in rumen microbial communities.

    Science.gov (United States)

    Kittelmann, Sandra; Seedorf, Henning; Walters, William A; Clemente, Jose C; Knight, Rob; Gordon, Jeffrey I; Janssen, Peter H

    2013-01-01

    Ruminants rely on a complex rumen microbial community to convert dietary plant material to energy-yielding products. Here we developed a method to simultaneously analyze the community's bacterial and archaeal 16S rRNA genes, ciliate 18S rRNA genes and anaerobic fungal internal transcribed spacer 1 genes using 12 DNA samples derived from 11 different rumen samples from three host species (Ovis aries, Bos taurus, Cervus elephas) and multiplex 454 Titanium pyrosequencing. We show that the mixing ratio of the group-specific DNA templates before emulsion PCR is crucial to compensate for differences in amplicon length. This method, in contrast to using a non-specific universal primer pair, avoids sequencing non-targeted DNA, such as plant- or endophyte-derived rRNA genes, and allows increased or decreased levels of community structure resolution for each microbial group as needed. Communities analyzed with different primers always grouped by sample origin rather than by the primers used. However, primer choice had a greater impact on apparent archaeal community structure than on bacterial community structure, and biases for certain methanogen groups were detected. Co-occurrence analysis of microbial taxa from all three domains of life suggested strong within- and between-domain correlations between different groups of microorganisms within the rumen. The approach used to simultaneously characterize bacterial, archaeal and eukaryotic components of a microbiota should be applicable to other communities occupying diverse habitats.

  4. Rhea: a transparent and modular R pipeline for microbial profiling based on 16S rRNA gene amplicons.

    Science.gov (United States)

    Lagkouvardos, Ilias; Fischer, Sandra; Kumar, Neeraj; Clavel, Thomas

    2017-01-01

    The importance of 16S rRNA gene amplicon profiles for understanding the influence of microbes in a variety of environments coupled with the steep reduction in sequencing costs led to a surge of microbial sequencing projects. The expanding crowd of scientists and clinicians wanting to make use of sequencing datasets can choose among a range of multipurpose software platforms, the use of which can be intimidating for non-expert users. Among available pipeline options for high-throughput 16S rRNA gene analysis, the R programming language and software environment for statistical computing stands out for its power and increased flexibility, and the possibility to adhere to most recent best practices and to adjust to individual project needs. Here we present the Rhea pipeline, a set of R scripts that encode a series of well-documented choices for the downstream analysis of Operational Taxonomic Units (OTUs) tables, including normalization steps, alpha- and beta-diversity analysis, taxonomic composition, statistical comparisons, and calculation of correlations. Rhea is primarily a straightforward starting point for beginners, but can also be a framework for advanced users who can modify and expand the tool. As the community standards evolve, Rhea will adapt to always represent the current state-of-the-art in microbial profiles analysis in the clear and comprehensive way allowed by the R language. Rhea scripts and documentation are freely available at https://lagkouvardos.github.io/Rhea.

  5. Rhea: a transparent and modular R pipeline for microbial profiling based on 16S rRNA gene amplicons

    Directory of Open Access Journals (Sweden)

    Ilias Lagkouvardos

    2017-01-01

    Full Text Available The importance of 16S rRNA gene amplicon profiles for understanding the influence of microbes in a variety of environments coupled with the steep reduction in sequencing costs led to a surge of microbial sequencing projects. The expanding crowd of scientists and clinicians wanting to make use of sequencing datasets can choose among a range of multipurpose software platforms, the use of which can be intimidating for non-expert users. Among available pipeline options for high-throughput 16S rRNA gene analysis, the R programming language and software environment for statistical computing stands out for its power and increased flexibility, and the possibility to adhere to most recent best practices and to adjust to individual project needs. Here we present the Rhea pipeline, a set of R scripts that encode a series of well-documented choices for the downstream analysis of Operational Taxonomic Units (OTUs tables, including normalization steps, alpha- and beta-diversity analysis, taxonomic composition, statistical comparisons, and calculation of correlations. Rhea is primarily a straightforward starting point for beginners, but can also be a framework for advanced users who can modify and expand the tool. As the community standards evolve, Rhea will adapt to always represent the current state-of-the-art in microbial profiles analysis in the clear and comprehensive way allowed by the R language. Rhea scripts and documentation are freely available at https://lagkouvardos.github.io/Rhea.

  6. 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.

  7. 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.

  8. Amplicon-Based Pyrosequencing Reveals High Diversity of Protistan Parasites in Ships' Ballast Water: Implications for Biogeography and Infectious Diseases.

    Science.gov (United States)

    Pagenkopp Lohan, K M; Fleischer, R C; Carney, K J; Holzer, K K; Ruiz, G M

    2016-04-01

    Ships' ballast water (BW) commonly moves macroorganisms and microorganisms across the world's oceans and along coasts; however, the majority of these microbial transfers have gone undetected. We applied high-throughput sequencing methods to identify microbial eukaryotes, specifically emphasizing the protistan parasites, in ships' BW collected from vessels calling to the Chesapeake Bay (Virginia and Maryland, USA) from European and Eastern Canadian ports. We utilized tagged-amplicon 454 pyrosequencing with two general primer sets, amplifying either the V4 or V9 domain of the small subunit (SSU) of the ribosomal RNA (rRNA) gene complex, from total DNA extracted from water samples collected from the ballast tanks of bulk cargo vessels. We detected a diverse group of protistan taxa, with some known to contain important parasites in marine systems, including Apicomplexa (unidentified apicomplexans, unidentified gregarines, Cryptosporidium spp.), Dinophyta (Blastodinium spp., Euduboscquella sp., unidentified syndinids, Karlodinium spp., Syndinium spp.), Perkinsea (Parvilucifera sp.), Opisthokonta (Ichthyosporea sp., Pseudoperkinsidae, unidentified ichthyosporeans), and Stramenopiles (Labyrinthulomycetes). Further characterization of groups with parasitic taxa, consisting of phylogenetic analyses for four taxa (Cryptosporidium spp., Parvilucifera spp., Labyrinthulomycetes, and Ichthyosporea), revealed that sequences were obtained from both known and novel lineages. This study demonstrates that high-throughput sequencing is a viable and sensitive method for detecting parasitic protists when present and transported in the ballast water of ships. These data also underscore the potential importance of human-aided dispersal in the biogeography of these microbes and emerging diseases in the world's oceans.

  9. High-throughput amplicon sequencing and stream benthic bacteria: identifying the best taxonomic level for multiple-stressor research

    Science.gov (United States)

    Salis, R. K.; Bruder, A.; Piggott, J. J.; Summerfield, T. C.; Matthaei, C. D.

    2017-03-01

    Disentangling the individual and interactive effects of multiple stressors on microbial communities is a key challenge to our understanding and management of ecosystems. Advances in molecular techniques allow studying microbial communities in situ and with high taxonomic resolution. However, the taxonomic level which provides the best trade-off between our ability to detect multiple-stressor effects versus the goal of studying entire communities remains unknown. We used outdoor mesocosms simulating small streams to investigate the effects of four agricultural stressors (nutrient enrichment, the nitrification inhibitor dicyandiamide (DCD), fine sediment and flow velocity reduction) on stream bacteria (phyla, orders, genera, and species represented by Operational Taxonomic Units with 97% sequence similarity). Community composition was assessed using amplicon sequencing (16S rRNA gene, V3-V4 region). DCD was the most pervasive stressor, affecting evenness and most abundant taxa, followed by sediment and flow velocity. Stressor pervasiveness was similar across taxonomic levels and lower levels did not perform better in detecting stressor effects. Community coverage decreased from 96% of all sequences for abundant phyla to 28% for species. Order-level responses were generally representative of responses of corresponding genera and species, suggesting that this level may represent the best compromise between stressor sensitivity and coverage of bacterial communities.

  10. 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.

  11. Combined subtractive cDNA cloning and array CGH: an efficient approach for identification of overexpressed genes in DNA amplicons

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    De Paepe Anne

    2004-02-01

    Full Text Available Abstract Background Activation of proto-oncogenes by DNA amplification is an important mechanism in the development and maintenance of cancer cells. Until recently, identification of the targeted genes relied on labour intensive and time consuming positional cloning methods. In this study, we outline a straightforward and efficient strategy for fast and comprehensive cloning of amplified and overexpressed genes. Results As a proof of principle, we analyzed neuroblastoma cell line IMR-32, with at least two amplification sites along the short arm of chromosome 2. In a first step, overexpressed cDNA clones were isolated using a PCR based subtractive cloning method. Subsequent deposition of these clones on a custom microarray and hybridization with IMR-32 DNA, resulted in the identification of clones that were overexpressed due to gene amplification. Using this approach, amplification of all previously reported amplified genes in this cell line was detected. Furthermore, four additional clones were found to be amplified, including the TEM8 gene on 2p13.3, two anonymous transcripts, and a fusion transcript, resulting from 2p13.3 and 2p24.3 fused sequences. Conclusions The combinatorial strategy of subtractive cDNA cloning and array CGH analysis allows comprehensive amplicon dissection, which opens perspectives for improved identification of hitherto unknown targeted oncogenes in cancer cells.

  12. 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 domestication. Together, the domestication signal in the genomic region was weak both in human selection and domestication bottleneck.

  13. 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.

  14. 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.

  15. Development of a control region-based mtDNA SNaPshot™ selection tool, integrated into a mini amplicon sequencing method.

    Science.gov (United States)

    Weiler, Natalie E C; de Vries, Gerda; Sijen, Titia

    2016-03-01

    Mitochondrial DNA (mtDNA) analysis is regularly applied to forensic DNA samples with limited amounts of nuclear DNA (nDNA), such as hair shafts and bones. Generally, this mtDNA analysis involves examination of the hypervariable control region by Sanger sequencing of amplified products. When samples are severely degraded, small-sized amplicons can be applied and an earlier described mini-mtDNA method by Eichmann et al. [1] that accommodates ten mini amplicons in two multiplexes is found to be a very robust approach. However, in cases with large numbers of samples, like when searching for hairs with an mtDNA profile deviant from that of the victim, the method is time (and cost) consuming. Previously, Chemale et al. [2] described a SNaPshot™-based screening tool for a Brazilian population that uses standard-size amplicons for HVS-I and HVS-II. Here, we describe a similar tool adapted to the full control region and compatible with mini-mtDNA amplicons. Eighteen single nucleotide polymorphisms (SNPs) were selected based on their relative frequencies in a European population. They showed a high discriminatory power in a Dutch population (97.2%). The 18 SNPs are assessed in two SNaPshot™ multiplexes that pair to the two mini-mtDNA amplification multiplexes. Degenerate bases are included to limit allele dropout due to SNPs at primer binding site positions. Three SNPs provide haplogroup information. Reliability testing showed no differences with Sanger sequencing results. Since mini-mtSNaPshot screening uses only a small portion of the same PCR products used for Sanger sequencing, no additional DNA extract is consumed, which is forensically advantageous.

  16. 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.

  17. Amplicon-dependent CCNE1 expression is critical for clonogenic survival after cisplatin treatment and is correlated with 20q11 gain in ovarian cancer.

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    Dariush Etemadmoghadam

    Full Text Available Genomic amplification of 19q12 occurs in several cancer types including ovarian cancer where it is associated with primary treatment failure. We systematically attenuated expression of genes within the minimally defined 19q12 region in ovarian cell lines using short-interfering RNAs (siRNA to identify driver oncogene(s within the amplicon. Knockdown of CCNE1 resulted in G1/S phase arrest, reduced cell viability and apoptosis only in amplification-carrying cells. Although CCNE1 knockdown increased cisplatin resistance in short-term assays, clonogenic survival was inhibited after treatment. Gain of 20q11 was highly correlated with 19q12 amplification and spanned a 2.5 Mb region including TPX2, a centromeric protein required for mitotic spindle function. Expression of TPX2 was highly correlated with gene amplification and with CCNE1 expression in primary tumors. siRNA inhibition of TPX2 reduced cell viability but this effect was not amplicon-dependent. These findings demonstrate that CCNE1 is a key driver in the 19q12 amplicon required for survival and clonogenicity in cells with locus amplification. Co-amplification at 19q12 and 20q11 implies the presence of a cooperative mutational network. These observations have implications for the application of targeted therapies in CCNE1 dependent ovarian cancers.

  18. Amplicon-dependent CCNE1 expression is critical for clonogenic survival after cisplatin treatment and is correlated with 20q11 gain in ovarian cancer.

    Science.gov (United States)

    Etemadmoghadam, Dariush; George, Joshy; Cowin, Prue A; Cullinane, Carleen; Kansara, Maya; Gorringe, Kylie L; Smyth, Gordon K; Bowtell, David D L

    2010-11-12

    Genomic amplification of 19q12 occurs in several cancer types including ovarian cancer where it is associated with primary treatment failure. We systematically attenuated expression of genes within the minimally defined 19q12 region in ovarian cell lines using short-interfering RNAs (siRNA) to identify driver oncogene(s) within the amplicon. Knockdown of CCNE1 resulted in G1/S phase arrest, reduced cell viability and apoptosis only in amplification-carrying cells. Although CCNE1 knockdown increased cisplatin resistance in short-term assays, clonogenic survival was inhibited after treatment. Gain of 20q11 was highly correlated with 19q12 amplification and spanned a 2.5 Mb region including TPX2, a centromeric protein required for mitotic spindle function. Expression of TPX2 was highly correlated with gene amplification and with CCNE1 expression in primary tumors. siRNA inhibition of TPX2 reduced cell viability but this effect was not amplicon-dependent. These findings demonstrate that CCNE1 is a key driver in the 19q12 amplicon required for survival and clonogenicity in cells with locus amplification. Co-amplification at 19q12 and 20q11 implies the presence of a cooperative mutational network. These observations have implications for the application of targeted therapies in CCNE1 dependent ovarian cancers.

  19. 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

    2017-02-23

    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).

  20. Amplicon-based profiling of bacteria in raw and secondary treated wastewater from treatment plants across Australia.

    Science.gov (United States)

    Ahmed, Warish; Staley, Christopher; Sidhu, Jatinder; Sadowsky, Michael; Toze, Simon

    2017-02-01

    In this study, we investigated the use of Illumina high-throughput sequencing of 16S ribosomal RNA (rRNA) amplicons to explore microbial diversity and community structure in raw and secondary treated wastewater (WW) samples from four municipal wastewater treatment plants (WWTPs A-D) across Australia. Sequence reads were analyzed to determine the abundance and diversity of bacterial communities in raw and secondary treated WW samples across the four WWTPs. In addition, sequence reads were also characterized to phenotypic features and to estimate the abundance of potential pathogenic bacterial genera and antibiotic-resistant genes in total bacterial communities. The mean coverage, Shannon diversity index, observed richness (S obs), and abundance-based coverage estimate (ACE) of richness for raw and secondary treated WW samples did not differ significantly (P > 0.05) among the four WWTPs examined. Generally, raw and secondary treated WW samples were dominated by members of the genera Pseudomonas, Arcobacter, and Bacteroides. Evaluation of source contributions to secondary treated WW, done using SourceTracker, revealed that 8.80-61.4% of the bacterial communities in secondary treated WW samples were attributed to raw WW. Twenty-five bacterial genera were classified as containing potential bacterial pathogens. The abundance of potentially pathogenic genera in raw WW samples was higher than that found in secondary treated WW samples. Among the pathogenic genera identified, Pseudomonas and Arcobacter had the greatest percentage of the sequence reads. The abundances of antibiotic resistance genes were generally low (treated WW samples from four WWTPs across Australia and demonstrated that Illumina high-throughput sequencing can be an alternative approach for monitoring WW quality in order to protect environmental and human health.

  1. Uncultured bacterial diversity in a seawater recirculating aquaculture system revealed by 16S rRNA gene amplicon sequencing.

    Science.gov (United States)

    Lee, Da-Eun; Lee, Jinhwan; Kim, Young-Mog; Myeong, Jeong-In; Kim, Kyoung-Ho

    2016-04-01

    Bacterial diversity in a seawater recirculating aquaculture system (RAS) was investigated using 16S rRNA amplicon sequencing to understand the roles of bacterial communities in the system. The RAS was operated at nine different combinations of temperature (15°C, 20°C, and 25°C) and salinity (20‰, 25‰, and 32.5‰). Samples were collected from five or six RAS tanks (biofilters) for each condition. Fifty samples were analyzed. Proteobacteria and Bacteroidetes were most common (sum of both phyla: 67.2% to 99.4%) and were inversely proportional to each other. Bacteria that were present at an average of ≥ 1% included Actinobacteria (2.9%) Planctomycetes (2.0%), Nitrospirae (1.5%), and Acidobacteria (1.0%); they were preferentially present in packed bed biofilters, mesh biofilters, and maturation biofilters. The three biofilters showed higher diversity than other RAS tanks (aerated biofilters, floating bed biofilters, and fish tanks) from phylum to operational taxonomic unit (OTU) level. Samples were clustered into several groups based on the bacterial communities. Major taxonomic groups related to family Rhodobacteraceae and Flavobacteriaceae were distributed widely in the samples. Several taxonomic groups like [Saprospiraceae], Cytophagaceae, Octadecabacter, and Marivita showed a cluster-oriented distribution. Phaeobacter and Sediminicola-related reads were detected frequently and abundantly at low temperature. Nitrifying bacteria were detected frequently and abundantly in the three biofilters. Phylogenetic analysis of the nitrifying bacteria showed several similar OTUs were observed widely through the biofilters. The diverse bacterial communities and the minor taxonomic groups, except for Proteobacteria and Bacteroidetes, seemed to play important roles and seemed necessary for nitrifying activity in the RAS, especially in packed bed biofilters, mesh biofilters, and maturation biofilters.

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

    Science.gov (United States)

    Curk, Franck; Ancillo, Gema; Ollitrault, Frédérique; Perrier, Xavier; Jacquemoud-Collet, Jean-Pierre; Garcia-Lor, Andres; Navarro, Luis; Ollitrault, Patrick

    2015-01-01

    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-diagnostic SNP

  3. Classification of pmoA amplicon pyrosequences using BLAST and the lowest common ancestor method in MEGAN

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    Marc Gregory Dumont

    2014-02-01

    Full Text Available The classification of high-throughput sequencing data of protein-encoding genes is not as well established as for 16S rRNA. The objective of this work was to develop a simple and accurate method of classifying large datasets of pmoA sequences, a common marker for methanotrophic bacteria. A taxonomic system for pmoA was developed based on a phylogenetic analysis of available sequences. The taxonomy incorporates the known diversity of pmoA present in public databases, including both sequences from cultivated and uncultivated organisms. Representative sequences from closely related genes, such as those encoding the bacterial ammonia monooxygenase, were also included in the pmoA taxonomy. In total, 53 low-level taxa (genus-level are included. Using previously published datasets of high-throughput pmoA amplicon sequence data, we tested two approaches for classifying pmoA: a naïve Bayesian classifier and BLAST. Classification of pmoA sequences based on BLAST analyses was performed using the lowest common ancestor (LCA algorithm in MEGAN, a software program commonly used for the analysis of metagenomic data. Both the naïve Bayesian and BLAST methods were able to classify pmoA sequences and provided similar classifications; however, the naïve Bayesian classifier was prone to misclassifying contaminant sequences present in the datasets. Another advantage of the BLAST/LCA method was that it provided a user-interpretable output and enabled novelty detection at various levels, from highly divergent pmoA sequences to genus-level novelty.  

  4. Amplicon-based taxonomic characterization of bacteria in urban and peri-urban roof-harvested rainwater stored in tanks.

    Science.gov (United States)

    Ahmed, W; Staley, C; Hamilton, K A; Beale, D J; Sadowsky, M J; Toze, S; Haas, C N

    2017-01-15

    Overall, 26% of Australian households use rainwater tanks as a source of potable and nonpotable water. Limited information is available on the total bacterial communities in tank water. Therefore, identification of dominant bacterial communities, diversity, and their distribution is important in understanding the microbial quality of tank water. In this study, the abundance and diversity of bacterial communities in 88 tank water samples collected from the urban areas of Brisbane (n=44) and the peri-urban center of Currumbin (n=44) in Southeast Queensland, Australia were determined using amplicon-based Illumina next-generation sequencing. In addition, the SourceTracker program was used to identify the sources of fecal contamination in tank water samples. Sequence reads were also analyzed to detect potential bacterial pathogenic genera in the tank water samples collected. Differences in sample coverage, alpha diversity, and richness did not differ significantly between the Brisbane and Currumbin tank water samples. Comamonadaceae and Planctomycetaceae were the most abundant families in all tank water samples. Curvibacter was the most abundant genus in all tank water samples. SourceTracker revealed that around 34% (Brisbane) and 43% (Currumbin) of tank water samples had a signature for bird fecal contamination. The potential opportunistic pathogenic genera including Burkholderia, Chromobacterium, Clostridium, Legionella, Mycobacterium, Nocardia, and Pseudomonas were most prevalent in tank water samples. Next-generation sequencing can be used as an initial screening tool to identify a wide array of potential pathogenic genera in tank water samples followed by quantifying specific pathogen(s) of interest using more sensitive molecular assays such as quantitative PCR (qPCR).

  5. Responses of soil microeukaryotic communities to short-term fumigation-incubation revealed by MiSeq amplicon sequencing

    Directory of Open Access Journals (Sweden)

    Lin eChen

    2015-10-01

    Full Text Available In soil microbiology, there is a ‘paradox’ of soil organic carbon (SOC mineralization, which is that even though chloroform fumigation destroys majority of the soil microbial biomass, SOC mineralization continues at the same rate as in the non-fumigated soil during the incubation period. Soil microeukaryotes as important SOC decomposers, however, their community-level responses to chloroform fumigation are not well understood. Using the 18S rRNA gene amplicon sequencing, we analyzed the composition, diversity and C-metabolic functions of a grassland soil and an arable soil microeukaryotic community in response to fumigation followed by a 30-day incubation. The grassland and arable soil microeukaryotic communities were dominated by the fungal Ascomycota (80.5–93.1% of the fungal sequences, followed by the protistan Cercozoa and Apicomplexa. In the arable soil fungal community, the predominance of the class Sordariomycetes was replaced by the class Eurotiomycetes after fumigation at days 7 and 30 of the incubation. Fumigation changed the microeukaryotic α-diversity in the grassland soil at days 0 and 7, and β-diversity in the arable soil at days 7 and 30. Network analysis indicated that after fumigation fungi were important groups closely related to other taxa. Most phylotypes (especially Sordariomycetes, Dothideomycetes, Coccidia and uncultured Chytridiomycota were inhibited, and only a few were positively stimulated by fumigation. Despite the inhibited Sordariomycetes, the fumigated communities mainly consisted of Eurotiomycetes and Sordariomycetes (21.9% and 36.5% relative frequency, respectively, which are able to produce hydrolytic enzymes associated with SOC mineralization. Our study suggests that fumigation not only decreases biomass size, but modulates the composition and diversity of the soil microeukaryotic communities, which are capable of driving SOC mineralization by release of hydrolytic enzymes during short-term fumigation-incubation.

  6. Haplotyping and copy number estimation of the highly polymorphic human beta-defensin locus on 8p23 by 454 amplicon sequencing

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    Rosenstiel Philip

    2010-04-01

    Full Text Available Abstract Background The beta-defensin gene cluster (DEFB at chromosome 8p23.1 is one of the most copy number (CN variable regions of the human genome. Whereas individual DEFB CNs have been suggested as independent genetic risk factors for several diseases (e.g. psoriasis and Crohn's disease, the role of multisite sequence variations (MSV is less well understood and to date has only been reported for prostate cancer. Simultaneous assessment of MSVs and CNs can be achieved by PCR, cloning and Sanger sequencing, however, these methods are labour and cost intensive as well as prone to methodological bias introduced by bacterial cloning. Here, we demonstrate that amplicon sequencing of pooled individual PCR products by the 454 technology allows in-depth determination of MSV haplotypes and estimation of DEFB CNs in parallel. Results Six PCR products spread over ~87 kb of DEFB and harbouring 24 known MSVs were amplified from 11 DNA samples, pooled and sequenced on a Roche 454 GS FLX sequencer. From ~142,000 reads, ~120,000 haplotype calls (HC were inferred that identified 22 haplotypes ranging from 2 to 7 per amplicon. In addition to the 24 known MSVs, two additional sequence variations were detected. Minimal CNs were estimated from the ratio of HCs and compared to absolute CNs determined by alternative methods. Concordance in CNs was found for 7 samples, the CNs differed by one in 2 samples and the estimated minimal CN was half of the absolute in one sample. For 7 samples and 2 amplicons, the 454 haplotyping results were compared to those by cloning/Sanger sequencing. Intrinsic problems related to chimera formation during PCR and differences between haplotyping by 454 and cloning/Sanger sequencing are discussed. Conclusion Deep amplicon sequencing using the 454 technology yield thousands of HCs per amplicon for an affordable price and may represent an effective method for parallel haplotyping and CN estimation in small to medium-sized cohorts. The

  7. MutMap-Gap: whole-genome resequencing of mutant F2 progeny bulk combined with de novo assembly of gap regions identifies the rice blast resistance gene Pii.

    Science.gov (United States)

    Takagi, Hiroki; Uemura, Aiko; Yaegashi, Hiroki; Tamiru, Muluneh; Abe, Akira; Mitsuoka, Chikako; Utsushi, Hiroe; Natsume, Satoshi; Kanzaki, Hiroyuki; Matsumura, Hideo; Saitoh, Hiromasa; Yoshida, Kentaro; Cano, Liliana M; Kamoun, Sophien; Terauchi, Ryohei

    2013-10-01

    Next-generation sequencing allows the identification of mutations responsible for mutant phenotypes by whole-genome resequencing and alignment to a reference genome. However, when the resequenced cultivar/line displays significant structural variation from the reference genome, mutations in the genome regions missing from the reference (gaps) cannot be identified by simple alignment. Here we report on a method called 'MutMap-Gap', which involves delineating a candidate region harboring a mutation of interest using the recently reported MutMap method, followed by de novo assembly, alignment, and identification of the mutation within genome gaps. We applied MutMap-Gap to isolate the blast resistant gene Pii from the rice cv Hitomebore using mutant lines that have lost Pii function. MutMap-Gap should prove useful for cloning genes that exhibit significant structural variations such as disease resistance genes of the nucleotide-binding site-leucine rich repeat (NBS-LRR) class.

  8. 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

  9. 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

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    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

  10. Contrasting signals of positive selection in genes involved in human skin-color variation from tests based on SNP scans and resequencing

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    de Gruijter Johanna

    2011-12-01

    Full Text Available Abstract Background Numerous genome-wide scans conducted by genotyping previously ascertained single-nucleotide polymorphisms (SNPs have provided candidate signatures for positive selection in various regions of the human genome, including in genes involved in pigmentation traits. However, it is unclear how well the signatures discovered by such haplotype-based test statistics can be reproduced in tests based on full resequencing data. Four genes (oculocutaneous albinism II (OCA2, tyrosinase-related protein 1 (TYRP1, dopachrome tautomerase (DCT, and KIT ligand (KITLG implicated in human skin-color variation, have shown evidence for positive selection in Europeans and East Asians in previous SNP-scan data. In the current study, we resequenced 4.7 to 6.7 kb of DNA from each of these genes in Africans, Europeans, East Asians, and South Asians. Results Applying all commonly used neutrality-test statistics for allele frequency distribution to the newly generated sequence data provided conflicting results regarding evidence for positive selection. Previous haplotype-based findings could not be clearly confirmed. Although some tests were marginally significant for some populations and genes, none of them were significant after multiple-testing correction. Combined P values for each gene-population pair did not improve these results. Application of Approximate Bayesian Computation Markov chain Monte Carlo based to these sequence data using a simple forward simulator revealed broad posterior distributions of the selective parameters for all four genes, providing no support for positive selection. However, when we applied this approach to published sequence data on SLC45A2, another human pigmentation candidate gene, we could readily confirm evidence for positive selection, as previously detected with sequence-based and some haplotype-based tests. Conclusions Overall, our data indicate that even genes that are strong biological candidates for positive

  11. High throughput SNP discovery and genotyping in grapevine (Vitis vinifera L.) by combining a re-sequencing approach and SNPlex technology

    Science.gov (United States)

    Lijavetzky, Diego; Cabezas, José Antonio; Ibáñez, Ana; Rodríguez, Virginia; Martínez-Zapater, José M

    2007-01-01

    Background Single-nucleotide polymorphisms (SNPs) are the most abundant type of DNA sequence polymorphisms. Their higher availability and stability when compared to simple sequence repeats (SSRs) provide enhanced possibilities for genetic and breeding applications such as cultivar identification, construction of genetic maps, the assessment of genetic diversity, the detection of genotype/phenotype associations, or marker-assisted breeding. In addition, the efficiency of these activities can be improved thanks to the ease with which SNP genotyping can be automated. Expressed sequence tags (EST) sequencing projects in grapevine are allowing for the in silico detection of multiple putative sequence polymorphisms within and among a reduced number of cultivars. In parallel, the sequence of the grapevine cultivar Pinot Noir is also providing thousands of polymorphisms present in this highly heterozygous genome. Still the general application of those SNPs requires further validation since their use could be restricted to those specific genotypes. Results In order to develop a large SNP set of wide application in grapevine we followed a systematic re-sequencing approach in a group of 11 grape genotypes corresponding to ancient unrelated cultivars as well as wild plants. Using this approach, we have sequenced 230 gene fragments, what represents the analysis of over 1 Mb of grape DNA sequence. This analysis has allowed the discovery of 1573 SNPs with an average of one SNP every 64 bp (one SNP every 47 bp in non-coding regions and every 69 bp in coding regions). Nucleotide diversity in grape (π = 0.0051) was found to be similar to values observed in highly polymorphic plant species such as maize. The average number of haplotypes per gene sequence was estimated as six, with three haplotypes representing over 83% of the analyzed sequences. Short-range linkage disequilibrium (LD) studies within the analyzed sequences indicate the existence of a rapid decay of LD within the

  12. 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

    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...... analysis and (2) beta-diversity-based sample separation, using a rigorous benchmarking framework based on large clinical 16S microbiome datasets from different sources. RESULTS: Running more than 380,000 full differential relative abundance tests on real datasets with permuted case/control assignments...... should be interpreted with caution. We provide an easily extensible framework for benchmarking of new methods and future microbiome datasets....

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

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    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.

  14. Genome-wide patterns of recombination, linkage disequilibrium and nucleotide diversity from pooled resequencing and single nucleotide polymorphism genotyping unlock the evolutionary history of Eucalyptus grandis.

    Science.gov (United States)

    Silva-Junior, Orzenil B; Grattapaglia, Dario

    2015-11-01

    We used high-density single nucleotide polymorphism (SNP) data and whole-genome pooled resequencing to examine the landscape of population recombination (ρ) and nucleotide diversity (ϴw ), assess the extent of linkage disequilibrium (r(2) ) and build the highest density linkage maps for Eucalyptus. At the genome-wide level, linkage disequilibrium (LD) decayed within c. 4-6 kb, slower than previously reported from candidate gene studies, but showing considerable variation from absence to complete LD up to 50 kb. A sharp decrease in the estimate of ρ was seen when going from short to genome-wide inter-SNP distances, highlighting the dependence of this parameter on the scale of observation adopted. Recombination was correlated with nucleotide diversity, gene density and distance from the centromere, with hotspots of recombination enriched for genes involved in chemical reactions and pathways of the normal metabolic processes. The high nucleotide diversity (ϴw = 0.022) of E. grandis revealed that mutation is more important than recombination in shaping its genomic diversity (ρ/ϴw = 0.645). Chromosome-wide ancestral recombination graphs allowed us to date the split of E. grandis (1.7-4.8 million yr ago) and identify a scenario for the recent demographic history of the species. Our results have considerable practical importance to Genome Wide Association Studies (GWAS), while indicating bright prospects for genomic prediction of complex phenotypes in eucalypt breeding.

  15. Evolutionary Analyses of Hanwoo (Korean Cattle)-Specific Single-Nucleotide Polymorphisms and Genes Using Whole-Genome Resequencing Data of a Hanwoo Population.

    Science.gov (United States)

    Lee, Daehwan; Cho, Minah; Hong, Woon-Young; Lim, Dajeong; Kim, Hyung-Chul; Cho, Yong-Min; Jeong, Jin-Young; Choi, Bong-Hwan; Ko, Younhee; Kim, Jaebum

    2016-09-01

    Advances in next generation sequencing (NGS) technologies have enabled population-level studies for many animals to unravel the relationships between genotypic differences and traits of specific populations. The objective of this study was to perform evolutionary analysis of single nucleotide polymorphisms (SNP) in genes of Korean native cattle Hanwoo in comparison to SNP data from four other cattle breeds (Jersey, Simmental, Angus, and Holstein) and four related species (pig, horse, human, and mouse) obtained from public databases through NGS-based resequencing. We analyzed population structures and differentiation levels for the five cattle breeds and estimated species-specific SNPs with their origins and phylogenetic relationships among species. In addition, we identified Hanwoo-specific genes and proteins, and determined distinct changes in protein-protein interactions among five species (cattle, pig, horse, human, mouse) in the STRING network database by additionally considering indirect protein interactions. We found that the Hanwoo population was clearly different from the other four cattle populations. There were Hanwoo-specific genes related to its meat trait. Protein interaction rewiring analysis also confirmed that there were Hanwoo-specific protein-protein interactions that might have contributed to its unique meat quality.

  16. Identification of genes with nonsynonymous SNP in Jeju horse by whole-genome resequencing reveals a functional role for immune response.

    Science.gov (United States)

    Lee, J-H; Song, K-D; Kim, J-M; Leem, H-K; Park, K-D

    2016-03-01

    Jeju horse (Natural Monument number 347) is a breed of horse that has experienced long-term isolation and domestication in Jeju Island, South Korea. We evaluated genetic features of this breed, including SNP, by whole-genome resequencing using an Illumina HiSeq 2000. A total of 5,986,852 SNP were identified in 4 Jeju horses and were divided into homozygous and heterozygous SNP (2,357,099 and 3,629,753 SNP, respectively). It revealed that 63.8% of these SNP resided in intergenic regions. Immune response genes with nonsynonymous SNP were overrepresented in Jeju horses as evidenced by Gene Ontology clustering. Among these genes, Toll-like receptors (TLR) are highly enriched. Comparing TLR genes between Jeju horses and the Przewalski's horse, and genes showed "possibly damaging" mutations in several regions by analysis with PolyPhen-2. These results provide a framework for further genetic studies in Jeju horse by domestication. Furthermore, research on functions of SNP-associated genes would aid in understanding the molecular genetic variation of horse breeds.

  17. Evolutionary Analyses of Hanwoo (Korean Cattle)-Specific Single-Nucleotide Polymorphisms and Genes Using Whole-Genome Resequencing Data of a Hanwoo Population

    Science.gov (United States)

    Lee, Daehwan; Cho, Minah; Hong, Woon-young; Lim, Dajeong; Kim, Hyung-Chul; Cho, Yong-Min; Jeong, Jin-Young; Choi, Bong-Hwan; Ko, Younhee; Kim, Jaebum

    2016-01-01

    Advances in next generation sequencing (NGS) technologies have enabled population-level studies for many animals to unravel the relationships between genotypic differences and traits of specific populations. The objective of this study was to perform evolutionary analysis of single nucleotide polymorphisms (SNP) in genes of Korean native cattle Hanwoo in comparison to SNP data from four other cattle breeds (Jersey, Simmental, Angus, and Holstein) and four related species (pig, horse, human, and mouse) obtained from public databases through NGS-based resequencing. We analyzed population structures and differentiation levels for the five cattle breeds and estimated species-specific SNPs with their origins and phylogenetic relationships among species. In addition, we identified Hanwoo-specific genes and proteins, and determined distinct changes in protein-protein interactions among five species (cattle, pig, horse, human, mouse) in the STRING network database by additionally considering indirect protein interactions. We found that the Hanwoo population was clearly different from the other four cattle populations. There were Hanwoo-specific genes related to its meat trait. Protein interaction rewiring analysis also confirmed that there were Hanwoo-specific protein-protein interactions that might have contributed to its unique meat quality. PMID:27640093

  18. Molecular characterization and diversity analysis of bacterial communities associated with Dialeurolonga malleswaramensis (Hemiptera: Aleyrodidae) adults using 16S rDNA amplicon pyrosequencing and FISH.

    Science.gov (United States)

    Pandey, Neeti; Rajagopal, Raman

    2016-10-01

    Dialeurolonga malleswaramensis Sundararaj (Hemiptera: Aleyrodidae) is a phytophagous sap sucking insect. It infests Polyalthia longifolia, an important avenue tree of India, effective in alleviating noise pollution and having immense medicinal importance. Samples of this insect were collected from Polyalthia longifolia. The cytochrome c oxidase subunit I gene (mtCO1) helped in the molecular characterization of the insect. This study reports the bacterial diversity in D. malleswaramensis adults by high throughput 16S rDNA amplicon pyrosequencing. The major genera identified were Portiera and Arsenophonus. Other bacterial genera detected were uncultured alpha proteobacterium, Sphingopyxis and Methylobacterium. We also employed fluorescence in situ hybridization (FISH) in whole mount samples to confirm the presence of dominant endosymbionts Portiera and Arsenophonus to the bacteriocyte of D. malleswaramensis. This study concludes that combining techniques like 16S rDNA amplicon pyrosequencing and FISH reveal both dominant and rare bacteria. The data also predict the evolutionary position of this pest with respect to other whitefly species using a mitochondrial marker.

  19. A New Method for Rapid Screening of End-Point PCR Products: Application to Single Genome Amplified HIV and SIV Envelope Amplicons.

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    Laurent Houzet

    Full Text Available PCR is the most widely applied technique for large scale screening of bacterial clones, mouse genotypes, virus genomes etc. A drawback of large PCR screening is that amplicon analysis is usually performed using gel electrophoresis, a step that is very labor intensive, tedious and chemical waste generating. Single genome amplification (SGA is used to characterize the diversity and evolutionary dynamics of virus populations within infected hosts. SGA is based on the isolation of single template molecule using limiting dilution followed by nested PCR amplification and requires the analysis of hundreds of reactions per sample, making large scale SGA studies very challenging. Here we present a novel approach entitled Long Amplicon Melt Profiling (LAMP based on the analysis of the melting profile of the PCR reactions using SYBR Green and/or EvaGreen fluorescent dyes. The LAMP method represents an attractive alternative to gel electrophoresis and enables the quick discrimination of positive reactions. We validate LAMP for SIV and HIV env-SGA, in 96- and 384-well plate formats. Because the melt profiling allows the screening of several thousands of PCR reactions in a cost-effective, rapid and robust way, we believe it will greatly facilitate any large scale PCR screening.

  20. 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.

  1. High-Resolution Mapping of Crossover and Non-crossover Recombination Events by Whole-Genome Re-sequencing of an Avian Pedigree.

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    Linnéa Smeds

    2016-05-01

    Full Text Available Recombination is an engine of genetic diversity and therefore constitutes a key process in evolutionary biology and genetics. While the outcome of crossover recombination can readily be detected as shuffled alleles by following the inheritance of markers in pedigreed families, the more precise location of both crossover and non-crossover recombination events has been difficult to pinpoint. As a consequence, we lack a detailed portrait of the recombination landscape for most organisms and knowledge on how this landscape impacts on sequence evolution at a local scale. To localize recombination events with high resolution in an avian system, we performed whole-genome re-sequencing at high coverage of a complete three-generation collared flycatcher pedigree. We identified 325 crossovers at a median resolution of 1.4 kb, with 86% of the events localized to <10 kb intervals. Observed crossover rates were in excellent agreement with data from linkage mapping, were 52% higher in male (3.56 cM/Mb than in female meiosis (2.28 cM/Mb, and increased towards chromosome ends in male but not female meiosis. Crossover events were non-randomly distributed in the genome with several distinct hot-spots and a concentration to genic regions, with the highest density in promoters and CpG islands. We further identified 267 non-crossovers, whose location was significantly associated with crossover locations. We detected a significant transmission bias (0.18 in favour of 'strong' (G, C over 'weak' (A, T alleles at non-crossover events, providing direct evidence for the process of GC-biased gene conversion in an avian system. The approach taken in this study should be applicable to any species and would thereby help to provide a more comprehensive portray of the recombination landscape across organism groups.

  2. Genome-wide DNA polymorphism in the indica rice varieties RGD-7S and Taifeng B as revealed by whole genome re-sequencing.

    Science.gov (United States)

    Fu, Chong-Yun; Liu, Wu-Ge; Liu, Di-Lin; Li, Ji-Hua; Zhu, Man-Shan; Liao, Yi-Long; Liu, Zhen-Rong; Zeng, Xue-Qin; Wang, Feng

    2016-03-01

    Next-generation sequencing technologies provide opportunities to further understand genetic variation, even within closely related cultivars. We performed whole genome resequencing of two elite indica rice varieties, RGD-7S and Taifeng B, whose F1 progeny showed hybrid weakness and hybrid vigor when grown in the early- and late-cropping seasons, respectively. Approximately 150 million 100-bp pair-end reads were generated, which covered ∼86% of the rice (Oryza sativa L. japonica 'Nipponbare') reference genome. A total of 2,758,740 polymorphic sites including 2,408,845 SNPs and 349,895 InDels were detected in RGD-7S and Taifeng B, respectively. Applying stringent parameters, we identified 961,791 SNPs and 46,640 InDels between RGD-7S and Taifeng B (RGD-7S/Taifeng B). The density of DNA polymorphisms was 256.8 SNPs and 12.5 InDels per 100 kb for RGD-7S/Taifeng B. Copy number variations (CNVs) were also investigated. In RGD-7S, 1989 of 2727 CNVs were overlapped in 218 genes, and 1231 of 2010 CNVs were annotated in 175 genes in Taifeng B. In addition, we verified a subset of InDels in the interval of hybrid weakness genes, Hw3 and Hw4, and obtained some polymorphic InDel markers, which will provide a sound foundation for cloning hybrid weakness genes. Analysis of genomic variations will also contribute to understanding the genetic basis of hybrid weakness and heterosis.

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

    Science.gov (United States)

    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. PMID:28323836

  4. How well do HapMap haplotypes identify common haplotypes of genes? A comparison with haplotypes of 334 genes resequenced in the environmental genome project.

    Science.gov (United States)

    Taylor, Jack A; Xu, Zong-Li; Kaplan, Norman L; Morris, Richard W

    2006-01-01

    One of the goals of the International HapMap Project is the identification of common haplotypes in genes. However, HapMap uses an incomplete catalogue of single nucleotide polymorphisms (SNPs) and might miss some common haplotypes. We examined this issue using data from the Environmental Genome Project (EGP) which resequenced 335 genes in 90 people, and thus, has a nearly complete catalogue of gene SNPs. The EGP identified a total of 45,243 SNPs, of which 10,780 were common SNPs (minor allele frequency >or=0.1). Using EGP common SNP genotype data, we identified 1,459 haplotypes with frequency >or=0.05 and we use these as "benchmark" haplotypes. HapMap release 16 had genotype information for 1,573 of 10,780 (15%) EGP common SNPs. Using these SNPs, we identified common HapMap haplotypes (frequency >or=0.05) in each of the four HapMap ethnic groups. To compare common HapMap haplotypes to EGP benchmark haplotypes, we collapsed benchmark haplotypes to the set of 1,573 SNPs. Ninety-eight percent of the collapsed benchmark haplotypes could be found as common HapMap haplotypes in one or more of the four HapMap ethnic groups. However, collapsing benchmark haplotypes to the set of SNPs available in HapMap resulted in a loss of haplotype information: 545 of 1,459 (37%) benchmark haplotypes were uniquely identified, and only 25% of genes had all their benchmark haplotypes uniquely identified. We resampled the EGP data to examine the effect of increasing the number of HapMap SNPs to 5 million, and estimate that approximately 40% of common SNPs in genes will be sampled and that half of the genes will have sufficient SNPs to identify all common haplotypes. This inability to distinguish common haplotypes of genes may result in loss of power when examining haplotype-disease association.

  5. 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

    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 ...... should be interpreted with caution. We provide an easily extensible framework for benchmarking of new methods and future microbiome datasets....... into operational taxonomic units (OTUs). Strategies for detecting differential relative abundance of OTUs between sample conditions include classical statistical approaches as well as a plethora of newer methods, many borrowing from the related field of RNA-seq analysis. This effort is complicated by unique data...... characteristics, including sparsity, sequencing depth variation, and nonconformity of read counts to theoretical distributions, which is often exacerbated by exploratory and/or unbalanced study designs. Here, we assess the robustness of available methods for (1) inference in differential relative abundance...

  6. The Quantification of Representative Sequences pipeline for amplicon sequencing: case study on within-population ITS1 sequence variation in a microparasite infecting Daphnia.

    Science.gov (United States)

    González-Tortuero, E; Rusek, J; Petrusek, A; Gießler, S; Lyras, D; Grath, S; Castro-Monzón, F; Wolinska, J

    2015-11-01

    Next generation sequencing (NGS) platforms are replacing traditional molecular biology protocols like cloning and Sanger sequencing. However, accuracy of NGS platforms has rarely been measured when quantifying relative frequencies of genotypes or taxa within populations. Here we developed a new bioinformatic pipeline (QRS) that pools similar sequence variants and estimates their frequencies in NGS data sets from populations or communities. We tested whether the estimated frequency of representative sequences, generated by 454 amplicon sequencing, differs significantly from that obtained by Sanger sequencing of cloned PCR products. This was performed by analysing sequence variation of the highly variable first internal transcribed spacer (ITS1) of the ichthyosporean Caullerya mesnili, a microparasite of cladocerans of the genus Daphnia. This analysis also serves as a case example of the usage of this pipeline to study within-population variation. Additionally, a public Illumina data set was used to validate the pipeline on community-level data. Overall, there was a good correspondence in absolute frequencies of C. mesnili ITS1 sequences obtained from Sanger and 454 platforms. Furthermore, analyses of molecular variance (amova) revealed that population structure of C. mesnili differs across lakes and years independently of the sequencing platform. Our results support not only the usefulness of amplicon sequencing data for studies of within-population structure but also the successful application of the QRS pipeline on Illumina-generated data. The QRS pipeline is freely available together with its documentation under GNU Public Licence version 3 at http://code.google.com/p/quantification-representative-sequences.

  7. Whole-Genome Resequencing of a Cucumber Chromosome Segment Substitution Line and Its Recurrent Parent to Identify Candidate Genes Governing Powdery Mildew Resistance

    Science.gov (United States)

    Yu, Ting; Xu, Xuewen; Yan, Yali; Qi, Xiaohua; Chen, Xuehao

    2016-01-01

    Cucumber is an economically important vegetable crop worldwide. Powdery mildew (PM) is one of the most severe diseases that can affect cucumber crops. There have been several research efforts to isolate PM resistance genes for breeding PM-resistant cucumber. In the present study, we used a chromosome segment substitution line, SSL508-28, which carried PM resistance genes from the donor parent, JIN5-508, through twelve generations of backcrossing with a PM-susceptible inbred line, D8. We performed whole-genome resequencing of SSL508-28 and D8 to identify single nucleotide polymorphisms (SNPs), and insertions and deletions (indels). When compared against the reference genome of the inbred cucumber line 9930, a total of 468,616 SNPs and 67,259 indels were identified in SSL508-28, and 537,352 SNPs and 91,698 indels were identified in D8. Of these, 3,014 non-synonymous SNPs and 226 frameshift indels in SSL508-28, and 3,104 non-synonymous SNPs and 251 frameshift indels in D8, were identified. Bioinformatics analysis of these variations revealed a total of 15,682 SNPs and 6,262 indels between SSL508-28 and D8, among which 120 non-synonymous SNPs and 30 frameshift indels in 94 genes were detected between SSL508-28 and D8. Finally, out of these 94 genes, five resistance genes with nucleotide-binding sites and leucine-rich repeat domains were selected for qRT-PCR analysis. This revealed an upregulation of two transcripts, Csa2M435460.1 and Csa5M579560.1, in SSL508-28. Furthermore, the results of qRT-PCR analysis of these two genes in ten PM resistant and ten PM susceptible cucumber lines showed that when exposed to PM, Csa2M435460.1 and Csa5M579560.1 exhibited a higher expression level of resistant lines than susceptible lines. This indicates that Csa2M435460.1 and Csa5M579560.1 are candidate genes for PM resistance in cucumber. In addition, the non-synonymous SNPs in Csa2M435460.1 and Csa5M579560.1, identified in SSL508-28 and D8, might be the key to high PM-resistance in

  8. Enhanced detection of polymorphic DNA by multiple arbitrary amplicon profiling of endonuclease-digested DNA: identification of markers tightly linked to the supernodulation locus in soybean.

    Science.gov (United States)

    Caetano-Anollés, G; Bassam, B J; Gresshoff, P M

    1993-10-01

    Multiple endonuclease digestion of template DNA or amplification products can increase significantly the detection of polymorphic DNA in fingerprints generated by multiple arbitrary amplicon profiling (MAAP). This coupling of endonuclease cleavage and amplification of arbitrary stretches of DNA, directed by short oligonucleotide primers, readily allowed distinction of closely related fungal and bacterial isolates and plant cultivars. MAAP analysis of cleaved template DNA enabled the identification of molecular markers linked to a developmental locus of soybean (Glycine max L. Merrill). Ethyl methane sulfonate (EMS)-induced supernodulating, near-isogenic lines altered in the nts locus, which controls nodule formation, could be distinguished from each other and from the parent cultivar by amplification of template pre-digested with 2-3 restriction enzymes. A total of 42 DNA polymorphisms were detected using only 19 octamer primers. In the absence of digestion, 25 primers failed to differentiate these soybean genotypes. Several polymorphic products co-segregated tightly with the nts locus in F2 families from crosses between the allelic mutants nts382 and nts1007 and the ancestral G. soja Sieb. & Succ. PI468.397. Our results suggest that EMS is capable of inducing extensive DNA alterations, probably around discrete mutational hot-spots. EMS-induced DNA polymorphisms may constitute sequence-tagged markers diagnostic of specific genomic regions.

  9. The utility of diversity profiling using Illumina 18S rRNA gene amplicon deep sequencing to detect and discriminate Toxoplasma gondii among the cyst-forming coccidia.

    Science.gov (United States)

    Cooper, Madalyn K; Phalen, David N; Donahoe, Shannon L; Rose, Karrie; Šlapeta, Jan

    2016-01-30

    Next-generation sequencing (NGS) has the capacity to screen a single DNA sample and detect pathogen DNA from thousands of host DNA sequence reads, making it a versatile and informative tool for investigation of pathogens in diseased animals. The technique is effective and labor saving in the initial identification of pathogens, and will complement conventional diagnostic tests to associate the candidate pathogen with a disease process. In this report, we investigated the utility of the diversity profiling NGS approach using Illumina small subunit ribosomal RNA (18S rRNA) gene amplicon deep sequencing to detect Toxoplasma gondii in previously confirmed cases of toxoplasmosis. We then tested the diagnostic approach with species-specific PCR genotyping, histopathology and immunohistochemistry of toxoplasmosis in a Risso's dolphin (Grampus griseus) to systematically characterise the disease and associate causality. We show that the Euk7A/Euk570R primer set targeting the V1-V3 hypervariable region of the 18S rRNA gene can be used as a species-specific assay for cyst-forming coccidia and discriminate T. gondii. Overall, the approach is cost-effective and improves diagnostic decision support by narrowing the differential diagnosis list with more certainty than was previously possible. Furthermore, it supplements the limitations of cryptic protozoan morphology and surpasses the need for species-specific PCR primer combinations.

  10. 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.

  11. 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.

  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. The Influence of DNA Extraction Procedure and Primer Set on the Bacterial Community Analysis by Pyrosequencing of Barcoded 16S rRNA Gene Amplicons.

    Science.gov (United States)

    Starke, Ingo C; Vahjen, Wilfried; Pieper, Robert; Zentek, Jürgen

    2014-01-01

    In this study, the effect of different DNA extraction procedures and primer sets on pyrosequencing results regarding the composition of bacterial communities in the ileum of piglets was investigated. Ileal chyme from piglets fed a diet containing different amounts of zinc oxide was used to evaluate a pyrosequencing study with barcoded 16S rRNA PCR products. Two DNA extraction methods (bead beating versus silica gel columns) and two primer sets targeting variable regions of bacterial 16S rRNA genes (8f-534r versus 968f-1401r) were considered. The SEED viewer software of the MG-RAST server was used for automated sequence analysis. A total of 5.2 × 10(5) sequences were used for analysis after processing for read length (150 bp), minimum sequence occurrence (5), and exclusion of eukaryotic and unclassified/uncultured sequences. DNA extraction procedures and primer sets differed significantly in total sequence yield. The distribution of bacterial order and main bacterial genera was influenced significantly by both parameters. However, this study has shown that the results of pyrosequencing studies using barcoded PCR amplicons of bacterial 16S rRNA genes depend on DNA extraction and primer choice, as well as on the manner of downstream sequence analysis.

  14. The Influence of DNA Extraction Procedure and Primer Set on the Bacterial Community Analysis by Pyrosequencing of Barcoded 16S rRNA Gene Amplicons

    Directory of Open Access Journals (Sweden)

    Ingo C. Starke

    2014-01-01

    Full Text Available In this study, the effect of different DNA extraction procedures and primer sets on pyrosequencing results regarding the composition of bacterial communities in the ileum of piglets was investigated. Ileal chyme from piglets fed a diet containing different amounts of zinc oxide was used to evaluate a pyrosequencing study with barcoded 16S rRNA PCR products. Two DNA extraction methods (bead beating versus silica gel columns and two primer sets targeting variable regions of bacterial 16S rRNA genes (8f-534r versus 968f-1401r were considered. The SEED viewer software of the MG-RAST server was used for automated sequence analysis. A total of 5.2×105 sequences were used for analysis after processing for read length (150 bp, minimum sequence occurrence (5, and exclusion of eukaryotic and unclassified/uncultured sequences. DNA extraction procedures and primer sets differed significantly in total sequence yield. The distribution of bacterial order and main bacterial genera was influenced significantly by both parameters. However, this study has shown that the results of pyrosequencing studies using barcoded PCR amplicons of bacterial 16S rRNA genes depend on DNA extraction and primer choice, as well as on the manner of downstream sequence analysis.

  15. Comprehensive analysis of human endogenous retrovirus group HERV-W locus transcription in multiple sclerosis brain lesions by high-throughput amplicon sequencing.

    Science.gov (United States)

    Schmitt, Katja; Richter, Christin; Backes, Christina; Meese, Eckart; Ruprecht, Klemens; Mayer, Jens

    2013-12-01

    Human endogenous retroviruses (HERVs) of the HERV-W group comprise hundreds of loci in the human genome. Deregulated HERV-W expression and HERV-W locus ERVWE1-encoded Syncytin-1 protein have been implicated in the pathogenesis of multiple sclerosis (MS). However, the actual transcription of HERV-W loci in the MS context has not been comprehensively analyzed. We investigated transcription of HERV-W in MS brain lesions and white matter brain tissue from healthy controls by employing next-generation amplicon sequencing of HERV-W env-specific reverse transcriptase (RT) PCR products, thus revealing transcribed HERV-W loci and the relative transcript levels of those loci. We identified more than 100 HERV-W loci that were transcribed in the human brain, with a limited number of loci being predominantly transcribed. Importantly, relative transcript levels of HERV-W loci were very similar between MS and healthy brain tissue samples, refuting deregulated transcription of HERV-W env in MS brain lesions, including the high-level-transcribed ERVWE1 locus encoding Syncytin-1. Quantitative RT-PCR likewise did not reveal differences in MS regarding HERV-W env general transcript or ERVWE1- and ERVWE2-specific transcript levels. However, we obtained evidence for interindividual differences in HERV-W transcript levels. Reporter gene assays indicated promoter activity of many HERV-W long terminal repeats (LTRs), including structurally incomplete LTRs. Our comprehensive analysis of HERV-W transcription in the human brain thus provides important information on the biology of HERV-W in MS lesions and normal human brain, implications for study design, and mechanisms by which HERV-W may (or may not) be involved in MS.

  16. 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

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    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

  17. 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…

  18. Influence of amplicon size on the polymerase chain reaction of Parvovirus B19 genome in formalin-fixed specimens Influência do tamanho do amplicon na reação em cadeia da polimerase na detecção do genoma do PB19 em amostras fixadas em formalina

    Directory of Open Access Journals (Sweden)

    Paulo Roberto Veiga Quemelo

    2009-04-01

    Full Text Available The polymerase chain reaction (PCR has provided diagnosis of archival material, but some fixation methods such as formalin damage DNA and, subsequently, affect PCR analysis, particularly paraffin-embedded tissues. PCR is known due to its high specificity and sensitivity, although some difficulties arise when formalinfixed and paraffin-embedded tissue is used. Not only does this occur due to protein cross-linking, which increases with longer fixation time, but it also happens due to the direct damage that formalin causes in the DNA itself. PCR was used to analyze placenta and fetal organs from 34 samples with suspected Parvovirus B19 infection. It was not possible to amplify Parvovirus B19 DNA using nested-PCR, probably due to the size of the amplicon generated with the first set of primers. We approached this problem by using only the second set of primers. Two out of 34 tissue samples (5,9% were positive by PCR. However, PCR performed on corresponding fetal organs was negative in one of the two. We also observed a negative relation between the thickness of the tissue fragment and the positivity of the samples. In conclusion, although PCR is highly specific and sensitive in fresh or ideally fixed material, a careful standardization of PCR assays is necessary when using formalin fixed paraffin-embedded tissues by applying primers that require smaller DNA fragments for amplification.A reação em cadeia da polimerase (PCR tem fornecido diagnóstico de material de arquivo, mas alguns métodos de fixação, tais como formalina, provocam danos ao DNA e subsequentemente afetam sua análise, particularmente tecidos embebidos em parafina. A PCR é conhecida pela sua alta especificidade e sensibilidade, embora algumas dificuldades ocorram quando o material utilizado foi fixado em formalina e embebido em parafina. Isso não se deve somente pela formação de cross-linkings com proteínas, a qual aumenta com o maior tempo de fixação, mas também pelo

  19. 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.

  20. Targeted re-sequencing identified rs3106189 at the 5' UTR of TAPBP and rs1052918 at the 3' UTR of TCF3 to be associated with the overall survival of colorectal cancer patients.

    Directory of Open Access Journals (Sweden)

    Jiaofang Shao

    Full Text Available Recent studies have demonstrated the power of deep re-sequencing of the whole genome or exome in understanding cancer genomes. However, targeted capture of selected genomic whole gene-body regions, rather than the whole exome, have several advantages: 1 the genes can be selected based on biology or a hypothesis; 2 mutations in promoter and intronic regions, which have important regulatory roles, can be investigated; and 3 less expensive than whole genome or whole exome sequencing. Therefore, we designed custom high-density oligonucleotide microarrays (NimbleGen Inc. to capture approximately 1.7 Mb target regions comprising the genomic regions of 28 genes related to colorectal cancer including genes belonging to the WNT signaling pathway, as well as important transcription factors or colon-specific genes that are over expressed in colorectal cancer (CRC. The 1.7 Mb targeted regions were sequenced with a coverage ranged from 32× to 45× for the 28 genes. We identified a total of 2342 sequence variations in the CRC and corresponding adjacent normal tissues. Among them, 738 were novel sequence variations based on comparisons with the SNP database (dbSNP135. We validated 56 of 66 SNPs in a separate cohort of 30 CRC tissues using Sequenom MassARRAY iPLEX Platform, suggesting a validation rate of at least 85% (56/66. We found 15 missense mutations among the exonic variations, 21 synonymous SNPs that were predicted to change the exonic splicing motifs, 31 UTR SNPs that were predicted to occur at the transcription factor binding sites, 20 intronic SNPs located near the splicing sites, 43 SNPs in conserved transcription factor binding sites and 32 in CpG islands. Finally, we determined that rs3106189, localized to the 5' UTR of antigen presenting tapasin binding protein (TAPBP, and rs1052918, localized to the 3' UTR of transcription factor 3 (TCF3, were associated with overall survival of CRC patients.

  1. Resequencing and association analysis of coding regions at twenty candidate genes suggest a role for rare risk variation at AKAP9 and protective variation at NRXN1 in schizophrenia susceptibility.

    Science.gov (United States)

    Suárez-Rama, José Javier; Arrojo, Manuel; Sobrino, Beatriz; Amigo, Jorge; Brenlla, Julio; Agra, Santiago; Paz, Eduardo; Brión, María; Carracedo, Ángel; Páramo, Mario; Costas, Javier

    2015-01-01

    A fraction of genetic risk to develop schizophrenia may be due to low-frequency variants. This multistep study attempted to find low-frequency variants of high effect at coding regions of eleven schizophrenia susceptibility genes supported by genome-wide association studies (GWAS) and nine genes for the DISC1 interactome, a susceptibility gene-set. During the discovery step, a total of 125 kb per sample were resequenced in 153 schizophrenia patients and 153 controls from Galicia (NW Spain), and the cumulative role of low-frequency variants at a gene or at the DISC1 gene-set were analyzed by burden and variance-based tests. Relevant results were meta-analyzed when appropriate data were available. In addition, case-only putative damaging variants were genotyped in a further 419 cases and 398 controls. The discovery step revealed a protective effect of rare missense variants at NRXN1, a result supported by meta-analysis (OR = 0.67, 95% CI: 0.47-0.94, P = 0.021, based on 3848 patients and 3896 controls from six studies). The follow-up step based on case-only putative damaging variants revealed a promising risk variant at AKAP9. This variant, K873R, reached nominal significance after inclusion of 240 additional Spanish controls from databases. The variant, located in an ADCY2 binding region, is absent from large public databases. Interestingly, GWAS revealed an association between common ADCY2 variants and bipolar disorder, a disorder with considerable genetic overlap with schizophrenia. These data suggest a role of rare missense variants at NRXN1 and AKAP9 in schizophrenia susceptibility, probably related to alteration of the excitatory/inhibitory synaptic balance, deserving further investigation.

  2. 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.

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    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.

  3. Construction of recombinant pseudorabies viruses by using PRV BACs deficient in IE180 or pac sequences: Application of vBAC90D recombinant virus to production of PRV amplicons.

    Science.gov (United States)

    Lerma, L; Muñoz, A L; Wagner, S; Dinu, M; Martín, B; Tabarés, E

    2016-02-02

    We describe a simple and efficient method to obtain recombinant pseudorabies virus (PRV) in mammalian cells by using the PRV BACs, PBAC80 deficient in pac sequences and PBAC90 deficient in the IE180 gene. These essential viral sequences were used as targets to obtain viable recombinant viruses. PBAC80 was constructed, confirmed to encode a copy of the IE180 gene regulated by the inducible Ptet promoter, and used to obtain recombinant attenuated PRV viruses that express the EGFP protein (PRV-BT80GF virus). PBAC90 was used to obtain the vBAC90D virus, deficient in IE180 and free of replication-competent revertants, and which can be used as a helper in the production of PRV amplicons.

  4. 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

    2015-11-13

    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.

  5. 微量酶联杂交法定量检测HBV基因 竞争PCR扩增产物%An enzyme-linked hybridization on microplate for quantification of HBV gene amplicon after competitive PCR

    Institute of Scientific and Technical Information of China (English)

    缪晓辉; 戚中田; 孔宪涛

    2001-01-01

    目的 建立一种简便、敏感、精确的微反应板酶联杂交技术,以鉴定HBV基因的竞争PCR扩增产物。方法 设计了两种捕获探针,能分别与竞争PCR扩增产物中的野生片段和突变片段杂交。捕获探针通过3′-端修饰的氨基与微量DNA结合板孔表面的NOS基团化学结合而被“竖直”地包被在反应板上;将热变性后的产物加入两种捕获探针反应孔内,产物中带有生物素的野生或突变片段的一条单链与相应的捕获探针杂交;最后用链亲和素-碱性磷酸酶及底物检测杂交信号。结果 该方法检测PCR产物DNA的灵敏度为80ng/ml,大于琼脂糖凝胶电泳染色鉴定法。获得野生片段和突变片段杂交信号值后,可根据公式计算扩增前野生模板的初始量。结论 本方法操作简单、灵敏度高、结果数据化、特异性强,适用于竞争PCR产物分析。%Objective To develop a simple, sensitive, specific andquantitative method by competitive PCR for assay of hepatitis B virus amplicon produced. Methods Two capture probes, one for capturing wild fragments in PCR products and the other one for capturing mutant fragments, were designed. The capture probe can be coated to the surface of DNA binding well through the chemical binding between amino modified at 3′-end of probe and NOS group on wells. Heat-denatured products were then added to wells and hybridization occurs between capture probe and biotin-labeled strand of PCR amplicon. The hybridizing signal were detected with avidin-alkaline-phosphatase and its subtract. Results As low as 80ng/ml of interested fragment in PCR products which was not identifiable by agarose gel electrophoresis can be detected by this method. The wild and mutant fragment in competitive PCR products can be separately tested and then the amount of wild template before PCR can be calculated by using the formula introduced by Clementi et al. Conclusion With its simplicity

  6. MGC9753 gene, located within PPP1R1B-STARD3-ERBB2-GRB7 amplicon on human chromosome 17q12, encodes the seven-transmembrane receptor with extracellular six-cystein domain.

    Science.gov (United States)

    Katoh, Masuko; Katoh, Masaru

    2003-06-01

    MYC, ERBB2, MET, FGFR2, CCNE1, MYCN, WNT2, CD44, MDM2, NCOA3, IQGAP1 and STK6 loci are amplified in human gastric cancer. It has been reported that the gene corresponding to EST H16094 is co-amplified with ERBB2 gene in human gastric cancer. Here, we identified and characterized the gene corresponding to EST H16094 by using bioinformatics. BLAST programs revealed that EST H16094 was derived from the uncharacterized MGC9753 gene. Two ORFs were predicted within human MGC9753 mRNA, and ORF1 (nucleotide position 18-980 of NM_033419.1) was predicted as the coding region of human MGC9753 mRNA based on comparative genomics. Nucleotide sequence of mouse Mgc9753 mRNA was next determined in silico by modification of AK052486 cDNA (deleting C at the nucleotide position 37). Human MGC9753 and mouse Mgc9753 proteins were 320-amino-acid seven-transmembrane receptors with the N-terminal six-cysteine domain and an N-glycosylation site (85.0% total-amino-acid identity). Human MGC9753 protein showed 90.6% total-amino-acid identity with human CAB2 aberrant protein, which lacked the third-transmembrane domain of MGC9753 due to frame shifts within ORF. Human MGC9753 gene, consisting of eight exons, were clustered with PPP1R1B, STARD3, TCAP, PNMT, ERBB2, MGC14832 and GRB7 genes within the 120-kb region. PPP1R1B, STARD3, MGC9753, ERBB2 and GRB7 genes are co-amplified in several cases of gastric cancer. This is the first report on comprehensive characterization of the amplicon around the PPP1R1B-STARD3-TCAP-PNMT-MGC9753-ERBB2-MGC14832-GRB7 locus on human chromosome 17q12.

  7. Amplicon DNA melting analysis for the simultaneous detection of Brucella spp and Mycobacterium tuberculosis complex. Potential use in rapid differential diagnosis between extrapulmonary tuberculosis and focal complications of brucellosis.

    Science.gov (United States)

    Sanjuan-Jimenez, Rocio; Colmenero, Juan D; Bermúdez, Pilar; Alonso, Antonio; Morata, Pilar

    2013-01-01

    Some sites of extrapulmonary tuberculosis and focal complications of brucellosis are very difficult to differentiate clinically, radiologically, and even histopathologically. Conventional microbiological methods for the diagnosis of extrapulmonary tuberculosis and complicated brucellosis not only lack adequate sensitivity, they are also time consuming, which could lead to an unfavourable prognosis. The aim of this work was to develop a multiplex real-time PCR assay based on SYBR Green I to simultaneously detect Brucella spp and Mycobacterium tuberculosis complex and evaluate the efficacy of the technique with different candidate genes. The IS711, bcsp31 and omp2a genes were used for the identification of Brucella spp and the IS6110, senX3-regX3 and cfp31 genes were targeted for the detection of the M. tuberculosis complex. As a result of the different combinations of primers, nine different reactions were evaluated. A test was defined as positive only when the gene combinations were capable of co-amplifying both pathogens in a single reaction tube and showed distinguishable melting temperatures for each microorganism. According to the melting analysis, only three combinations of amplicons (senX3-regX3+bcsp31, senX3-regX3+IS711 and IS6110+IS711) were visible. Detection limits of senX3-regX3+bcsp31 and senX3-regX3+IS711 were of 2 and 3 genome equivalents for M. tuberculosis complex and Brucella while for IS6110+IS711 they were of 200 and 300 genome equivalents, respectively. The three assays correctly identified all the samples, showing negative results for the control patients. The presence of multicopy elements and GC content were the components most influencing the efficiency of the test; this should be taken into account when designing a multiplex-based SYBR Green I assay. In conclusion, multiplex real time PCR assays based on the targets senX3-regX3+bcsp31 and senX3-regX3+IS711 using SYBR Green I are highly sensitive and reproducible. This may therefore be a

  8. Amplicon DNA melting analysis for the simultaneous detection of Brucella spp and Mycobacterium tuberculosis complex. Potential use in rapid differential diagnosis between extrapulmonary tuberculosis and focal complications of brucellosis.

    Directory of Open Access Journals (Sweden)

    Rocio Sanjuan-Jimenez

    Full Text Available Some sites of extrapulmonary tuberculosis and focal complications of brucellosis are very difficult to differentiate clinically, radiologically, and even histopathologically. Conventional microbiological methods for the diagnosis of extrapulmonary tuberculosis and complicated brucellosis not only lack adequate sensitivity, they are also time consuming, which could lead to an unfavourable prognosis. The aim of this work was to develop a multiplex real-time PCR assay based on SYBR Green I to simultaneously detect Brucella spp and Mycobacterium tuberculosis complex and evaluate the efficacy of the technique with different candidate genes. The IS711, bcsp31 and omp2a genes were used for the identification of Brucella spp and the IS6110, senX3-regX3 and cfp31 genes were targeted for the detection of the M. tuberculosis complex. As a result of the different combinations of primers, nine different reactions were evaluated. A test was defined as positive only when the gene combinations were capable of co-amplifying both pathogens in a single reaction tube and showed distinguishable melting temperatures for each microorganism. According to the melting analysis, only three combinations of amplicons (senX3-regX3+bcsp31, senX3-regX3+IS711 and IS6110+IS711 were visible. Detection limits of senX3-regX3+bcsp31 and senX3-regX3+IS711 were of 2 and 3 genome equivalents for M. tuberculosis complex and Brucella while for IS6110+IS711 they were of 200 and 300 genome equivalents, respectively. The three assays correctly identified all the samples, showing negative results for the control patients. The presence of multicopy elements and GC content were the components most influencing the efficiency of the test; this should be taken into account when designing a multiplex-based SYBR Green I assay. In conclusion, multiplex real time PCR assays based on the targets senX3-regX3+bcsp31 and senX3-regX3+IS711 using SYBR Green I are highly sensitive and reproducible. This may

  9. Construction of the HSV-1 Strain HF Amplicon and Study on Its Unversal Function between Different HSV Serotypes%HSV-1 HF株扩增子载体的构建及其在不同HSV血清型间的通用性研究

    Institute of Scientific and Technical Information of China (English)

    宋波; 刘新静; 韩志强; 赵璐; 王青志; 卢甲盟; 许予明

    2011-01-01

    The study aimed to construct the amplicon vector of HSV-1 strain HF and explore its universal package function between different serotypes of HSV. OriS and pac elements were obtained by enzyme digestion from the Plasmid BAC-HSV-1 strain HF and sequenced. With red fluorescence (DsRed) as a reporter gene, the amplicon vector of HSV-1 strain HF was constructed based on pSilencer2. 0-U6. The amplicon vector was transfected into Vero cells by lipofectamine 2000, then packaged by HSV-1 strain HF and HSV-2 strain HG52 as helper virus separately. The supernatant was collected after cytopathic effect. Red fluorescence was observed in Vero cells reinfected by the supernatant. In this study,the amplicon vector of HSV-1 strain HF was successfully constructed and it could be packaged by HSV-1 strain HF and HSV-2 strainHG52.%摘要:旨在构建HSV-1HF株的扩增子载体,研究其在不同血清型HSV辅助下的包装通用性。经酶切HF株的BAC-HSV-1,获得oriS和pac元件并测序。以pSilencer2.0-U6为骨架,以DsRed为报告基因构建HSV-1 HF株的扩增子载体,利用脂质体2000转染扩增子载体至Vero细胞,分别应用HSV-1HF株和HSV-2 HG52辅助HSV-1扩增子载体进行包装,待产生细胞病变效应后取上清,再次感染Vero细胞,观察Vero细胞内红色荧光蛋白表达情况。本研究首次构建了HSV-1HF株的扩增子载体,鉴定了HSV-1HF株oriS和pac元件,HSV-1HF株扩增子载体可以被HSV-1 HF株和HSV-2 HG52株包装并扩增。

  10. 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.

  11. Psychology Experiments.

    Science.gov (United States)

    McGraw, Ken; Tew, Mark D.; Williams, John E.

    2001-01-01

    A goal of the PsychExperiments project was to reduce the financial burden on psychology departments for hardware/software used in their laboratories. In its third year, the PsychExperiments site now hosts 39 experiments. Over 200 classrooms worldwide have signed up as official site users and there have been nearly 10,000 data sessions conducted.…

  12. 双重胶体金层析法快速检测番茄环斑病毒和烟草环斑病毒RT-PCR扩增子%Duplex Detection of Tomato Ringspot Virus and Tobacco Ringspot Virus RT-PCR Amplicons by Dipstick Assay

    Institute of Scientific and Technical Information of China (English)

    曹成; 魏梅生; 张永江; 李桂芬; 吴兴泉

    2011-01-01

    Both tomato ringspot virus(ToRSV)and tobacco ringspot virus(TRSV) are quarantine pests for China. We have developed dipstick assay for detection of RT-PCR amplicons of the two viruses within 15 minutes. The single dipstick assay is performed by labeling primers of the virus with biotin and fluorescein (or digoxigenin). Monoclonal antibody against fluorescein (or digoxigenin) is spotted on nitrocellulose membrane as T dot. The dual-labeled(biotin-fluorescein or biotin-digoxigenin) RT-PCR amplicons can be detected on the test dot(T dot). The duplex dipstick assay is performed by labeling primers of ToRSV with biotin and fluorescein (or digoxigenin) and primers of TRSV with biotin and digoxigenin(or fluorescein). Monoclonal antibody against fluorescein and digoxigenin is spotted on nitrocellulose membrane respectively as T1 and T2 dot . The dual-labeled(biotin-fluorescein and biotin-digoxigenin) RT-PCR amplicons of the two viruses can be detected on the Tl and T2 dot simultaneously.%番茄环斑病毒和烟草环斑病毒是中国进境检疫性有害生物,此研究建立了单个和双重胶体金层析快速检测方法,在15 min即可获得对双标记PCR产物的检测结果.单个胶体金层析是在一张层析膜上点上荧光素(或地高辛)的单克隆抗体作为T检测点,经生物素-荧光素(或生物素-地高辛)双标记的RT-PCR扩增产物可在T点上被检测到.双重胶体金层析是在一张层析膜上分别点上荧光素和地高辛的单克隆抗体作为T1和T2检测点,经生物素-荧光素和生物素-地高辛双标记的两种病毒的RT-PCR扩增产物可分别在T1和T2点上被检测到.

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

    Science.gov (United States)

    Schwarz, Dietmar; Thompson, Andrew J; Kläring, Hans-Peter

    2014-01-01

    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 color. 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 and 16 h. 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.

  14. 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.

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

    Science.gov (United States)

    Schwarz, Dietmar; Thompson, Andrew J.; Kläring, Hans-Peter

    2014-01-01

    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 color. 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 and 16 h. 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. PMID:25477888

  16. 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.

  17. Collaborative experience

    DEFF Research Database (Denmark)

    Mortensen, Thomas Bøtker

    sample of firms, an establish way of measuring the outcome of product development and a new way of measuring experience. Where the previous research in this field primarily uses secondary databases, this research project collects primary data by an online questionnaire to the NPD manager from one......, 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...... of the new product development as a performance measure. Finally, where previous research primarily has used the number of collaborations as a measure of collaborative experience, this research includes the recency in the measure of collaborative experience. Results: Since data has not yet been collected...

  18. The preliminary study of gene copy number variation association with scar hyperplasia based on the whole-gene resequencing%基于全基因组重测序技术检测与瘢痕疙瘩相关基因拷贝数变异的初步研究

    Institute of Scientific and Technical Information of China (English)

    刘畅; 滕国栋; 陈敏亮; 马奎; 颜彤彤

    2014-01-01

    Objective To investigate the genome copy number variation (CNV) related with keloid using the whole-gene resequencing technology.Methods A keloid pedigree containing 4 generation of 27 people was studied.Five people (4 cases of keloid patients,and 1 case of normal) were selected to extract the genomic DNA.Then the whole-gene resequencing technique was used to check the variations based on the Illumina Hiseq 2000.Results Through database comparison and variation annotation analysis,15 CNVs associated with scar hyperplasia were obtained.DAVID software was used to do the Gene Ontology and pathway analysis.Five CNVs were closely related to the keloid formation.They were growth factor receptor-bound 7 (Grb7),mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4),mitogen-activated protein kinase kinase kinase 15 (MAP3K15),kruppel-like factors 7 (KLF7) and NK2 homeobox 2 (NKX2-2).These CNVs were involved in the process of epidermal cells formation and differentiation,cell exocrine and cell adhesion.Conclusions There are 5 CNVs associated with scar hyperplasia.Especially MAP3K15 and MAP4K4 deserve more research to find their function in keloid formation.%目的 利用全基因重测序技术初步探讨与瘢痕疙瘩相关的基因组拷贝数变异(CNV).方法 收集1个瘢痕疙瘩家系,共4代27人,选取该家系中5例(4例瘢痕疙瘩患者,1名健康人)利用Illumina Hiseq 2000进行全基因组重测序,对所获信息进行数据库比对和变异注释.结果 通过数据库比对及变异注释分析后获得了与瘢痕疙瘩相关的15个CNV,通过DAVID对筛选出来的15个基因进行基因注释和信号通路分析.共发现5个CNV[生长因子受体结合蛋白7(GRB7)、丝裂原激活蛋白激酶激酶激酶激酶4(MAP4 K4)、丝裂原激活蛋白激酶激酶激酶15(MAP3K15)、Kruppel样转录因子7(KLF7)和NKx2-2]与瘢痕疙瘩密切相关.这些CNV所涉及的基因与细胞表皮形成与分化、细胞外分泌

  19. 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.

  20. Wanted: Experiments

    Science.gov (United States)

    McDaniel, Floyd D.

    1974-01-01

    Describes a project to produce a series of laboratory manuals and instructional materials in which nuclear experiments are presented for the undergraduate advanced laboratory. The manuals are being compiled in the areas of physics, chemistry, geology and environmental sciences. (BR)

  1. 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

  2. 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...... 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...... 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...

  3. 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...

  4. 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.

  5. 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...

  6. 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).

  7. Crucible Experiments

    Science.gov (United States)

    Jiang, Min; Wang, Xin-Hua; Pak, Jong-Jin

    2014-08-01

    ZrO2 crucible experiments were carried out in the laboratory to study the formation of low-melting-point inclusions in steel, during which aluminum deoxidization and a high-basicity calcium aluminate refining slag (CaO/SiO2: 6 to 8, Al2O3 40 to 45 pct) were used. Four experiments were done with different slag/steel reaction times (30, 60, 90, and 180 minutes). It was found that inclusions were mainly composed of CaO-Al2O3-ZrO2 with very limited SiO2, in spherical morphology and with sizes mainly less than 5 μm. They can be classified into two types according to the ZrO2 content. The first type contained much lower ZrO2, whereas a much higher level of ZrO2 was detected in the other type. An evolution of inclusions with the reaction time was observed and studied. The obtained results indicated that chemical compositions of inclusions were widely scattered in the CaO-Al2O3-ZrO2 phase diagram after 30 min reaction. However, the composition of inclusions became much more uniform and concentrated in low-melting-temperature regions at 60, 90, and 180 minutes, which would be favorable to prevent nozzle clogging and fatigue problems. Compared with the authors' previous results obtained in MgO crucible experiments, it was found that low-melting-temperature inclusions can be targeted in shorter time in ZrO2 crucible experiments, without any degradation of cleanliness.

  8. Combinatorial analysis and algorithms for quasispecies reconstruction using next-generation sequencing

    Directory of Open Access Journals (Sweden)

    Vincenti Donatella

    2011-01-01

    Full Text Available Abstract Background Next-generation sequencing (NGS offers a unique opportunity for high-throughput genomics and has potential to replace Sanger sequencing in many fields, including de-novo sequencing, re-sequencing, meta-genomics, and characterisation of infectious pathogens, such as viral quasispecies. Although methodologies and software for whole genome assembly and genome variation analysis have been developed and refined for NGS data, reconstructing a viral quasispecies using NGS data remains a challenge. This application would be useful for analysing intra-host evolutionary pathways in relation to immune responses and antiretroviral therapy exposures. Here we introduce a set of formulae for the combinatorial analysis of a quasispecies, given a NGS re-sequencing experiment and an algorithm for quasispecies reconstruction. We require that sequenced fragments are aligned against a reference genome, and that the reference genome is partitioned into a set of sliding windows (amplicons. The reconstruction algorithm is based on combinations of multinomial distributions and is designed to minimise the reconstruction of false variants, called in-silico recombinants. Results The reconstruction algorithm was applied to error-free simulated data and reconstructed a high percentage of true variants, even at a low genetic diversity, where the chance to obtain in-silico recombinants is high. Results on empirical NGS data from patients infected with hepatitis B virus, confirmed its ability to characterise different viral variants from distinct patients. Conclusions The combinatorial analysis provided a description of the difficulty to reconstruct a quasispecies, given a determined amplicon partition and a measure of population diversity. The reconstruction algorithm showed good performance both considering simulated data and real data, even in presence of sequencing errors.

  9. QUBIC Experiment

    CERN Document Server

    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 light in 2018-2019.

  10. Thomson Experiment

    CERN Multimedia

    This experiment, conducted by JJ Thomson in 1897, established the existence of the electron. Thomson won the Nobel physics prize for this work in 1906. A beam of electrons crosses the chamber emitting blue light. Adding an electric field (E) or a magnetic field (B) exerts a force on the moving electrons.Use switch E to turn on the electric field in the chamber. Then, by turning knob B, you can increase the current in the coils, generating a magnetic field. By balancing the electric and magnetic fields, Thomson was able to keep the electron beam level and deduce the ratio of the electron's charge to its mass.

  11. Experiment summary

    CERN Document Server

    Rossi, Andrea

    2015-01-01

    The measurement of the production of particles coming from hard scattering processes covers a fundamental role in the characterization of the system formed in heavy-ion collisions, allowing to probe the microscopic processes underlying the interaction of high energy partons with the medium. An impressive amount of measurements related to jet, quarkonia, open heavy flavor, and electroweak signal production in nucleus-nucleus as well as p(d)-nucleus collisions was delivered by experiments at RHIC and LHC in past years. In these proceedings, the main experimental results presented during the Hard Probes conference are summarized.

  12. 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.

  13. Rutherford Experiment

    CERN Multimedia

    This experiment, carried out by Ernest Rutherford in 1910, revolutionised understanding of the structure of matter, showing that almost all the mass of an atom is concentrated in a very small, positively charged nucleus. Alpha particles emitted at bombard a thin gold foil. A detector records the number of alpha particles crossing the foil per second. The number is displayed on the counter and updated every minute. Alpha particles are helium nuclei, they consist of 2 protons and 2 neutrons. Rotate the central knob to change the angle between the foil and the detector. The number of alpha particles detected depends on the angle. Most of the alpha particles travel straight through the foil because the gold atoms are mainly empty space. However some hit the atomic nucleus and are deflected.

  14. Microarray-based Resequencing of Multiple Bacillus anthracis Isolates

    Science.gov (United States)

    2004-12-17

    al.: Iden- tification of anthrax toxin genes in a Bacillus cereus associ- ated with an illness resembling inhalation anthrax. Proc Natl Acad Sci USA...Norwegian Bacillus cereus and Bacillus thuringiensis soil isolates. Appl Environ Microbiol 2001, 67:4863-4873. 26. Radnedge L, Agron PG, Hill KK, Jackson PJ...Ticknor LO, Keim P, Andersen GL: Genome differences that distinguish Bacillus anthracis from Bacillus cereus and Bacillus thuringiensis . Appl

  15. 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.

  16. Edesign: Primer and Enhanced Internal Probe Design Tool for Quantitative PCR Experiments and Genotyping Assays.

    Science.gov (United States)

    Kimura, Yasumasa; Soma, Takahiro; Kasahara, Naoko; Delobel, Diane; Hanami, Takeshi; Tanaka, Yuki; de Hoon, Michiel J L; Hayashizaki, Yoshihide; Usui, Kengo; Harbers, Matthias

    2016-01-01

    Analytical PCR experiments preferably use internal probes for monitoring the amplification reaction and specific detection of the amplicon. Such internal probes have to be designed in close context with the amplification primers, and may require additional considerations for the detection of genetic variations. Here we describe Edesign, a new online and stand-alone tool for designing sets of PCR primers together with an internal probe for conducting quantitative real-time PCR (qPCR) and genotypic experiments. Edesign can be used for selecting standard DNA oligonucleotides like for instance TaqMan probes, but has been further extended with new functions and enhanced design features for Eprobes. Eprobes, with their single thiazole orange-labelled nucleotide, allow for highly sensitive genotypic assays because of their higher DNA binding affinity as compared to standard DNA oligonucleotides. Using new thermodynamic parameters, Edesign considers unique features of Eprobes during primer and probe design for establishing qPCR experiments and genotyping by melting curve analysis. Additional functions in Edesign allow probe design for effective discrimination between wild-type sequences and genetic variations either using standard DNA oligonucleotides or Eprobes. Edesign can be freely accessed online at http://www.dnaform.com/edesign2/, and the source code is available for download.

  17. Integrated Microfluidic Nucleic Acid Isolation, Isothermal Amplification, and Amplicon Quantification

    OpenAIRE

    Mauk, Michael G.; Changchun Liu; Jinzhao Song; Bau, Haim H.

    2015-01-01

    Microfluidic components and systems for rapid (<60 min), low-cost, convenient, field-deployable sequence-specific nucleic acid-based amplification tests (NAATs) are described. A microfluidic point-of-care (POC) diagnostics test to quantify HIV viral load from blood samples serves as a representative and instructive example to discuss the technical issues and capabilities of “lab on a chip” NAAT devices. A portable, miniaturized POC NAAT with performance comparable to conventional PCR (poly...

  18. High throughput 16S rRNA gene amplicon sequencing

    DEFF Research Database (Denmark)

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

    A reliable and reproducible method for identification and quantification of microorganisms is important for the studies of microbial communities in activated sludge and for the demonstration of their significance for plant operation and stability. DNA based identification of microorganisms using 16...... was correlated with the bacterial species composition in 25 Danish full-scale WWTPs with nutrient removal. Examples of properties were SVI, filament index, floc size, floc strength, content of cations and amount of extracellular polymeric substances. Multivariate statistics provided several important insights...

  19. Experiment Dashboard for the LHC Experiments

    CERN Document Server

    Andreeva, Julia; Gaidioz, Benjamin; Herrala, Juha; Maier, Gerhild; Rocha, Ricardo; Saiz, Pablo; Sidorova, Irina; CERN. Geneva. IT Department

    2007-01-01

    The goal of the Grid is to provide a coherent access to distributed computing resources. All LHC experiments are using several Grid infrastructures and a variety of the middleware flavors. Due to the complexity and heterogeinity of a distributed system the monitoring represents a challenging task. Independently of the underlying platform , the experiments need to ave a complete and uniform picture of their activities on the Grid ideally seen by the users as a single powerful computing resource. Overall operation of the infrastructure used by experiments is defined both by the quality of the Grid and the quality of the tools and services developed/used by the experiments. Correspondingly the required monitoring information should combine both Grid-related and experiment/application specific data. On the other hand, users of the LHC experiments have various roles and need different levels of details regarding monitoring data. The paper will focus on the Grid monitoring from the experiment/user perspectives with...

  20. 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)…

  1. Real Life Experiences with Experience Design

    DEFF Research Database (Denmark)

    Dalsgård, Peter; Halskov, Kim

    2006-01-01

    Experience Design is an emergent field of study, and various approaches to the field abound. In this paper, we take a pragmatic approach to identifying key aspects of an experience design process, by reporting on a project involving the design of experience-oriented applications of interactive...... 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...... the installations, the core values established to guide the design process and the intended use contexts. We argue that the installations present a broad spectrum of experience design installations that can assist designers in understanding the relations between core values, intentions, use context and interface...

  2. Structural Assembly Demonstration Experiment (SADE) experiment design

    Science.gov (United States)

    Akin, D. L.; Bowden, M. L.

    1982-03-01

    The Structural Assembly Demonstration Experiment concept is to erect a hybrid deployed/assembled structure as an early space experiment in large space structures technology. The basic objectives can be broken down into three generic areas: (1) by performing assembly tasks both in space and in neutral buoyancy simulation, a mathematical basis will be found for the validity conditions of neutral buoyancy, thus enhancing the utility of water as a medium for simulation of weightlessness; (2) a data base will be established describing the capabilities and limitations of EVA crewmembers, including effects of such things as hardware size and crew restraints; and (3) experience of the M.I.T. Space Systems Lab in neutral buoyancy simulation of large space structures assembly indicates that the assembly procedure may create the largest loads that a structure will experience during its lifetime. Data obtained from the experiment will help establish an accurate loading model to aid designers of future space structures.

  3. The Social Experiment Market

    OpenAIRE

    David Greenberg; Mark Shroder; Matthew Onstott

    1999-01-01

    In social experiments, individuals, households, or organizations are randomly assigned to two or more policy interventions. Elsewhere, we have summarized 143 experiments completed by autumn 1996. Here, we use the information we have gathered on these experiments and findings from informal telephone interviews to investigate the social experiment market--the buyers and sellers in the market that governs the production of experiments. We discuss target populations, types of interventions tested...

  4. Soil pH determines microbial diversity and composition in the park grass experiment.

    Science.gov (United States)

    Zhalnina, Kateryna; Dias, Raquel; de Quadros, Patricia Dörr; Davis-Richardson, Austin; Camargo, Flavio A O; Clark, Ian M; McGrath, Steve P; Hirsch, Penny R; Triplett, Eric W

    2015-02-01

    The Park Grass experiment (PGE) in the UK has been ongoing since 1856. Its purpose is to study the response of biological communities to the long-term treatments and associated changes in soil parameters, particularly soil pH. In this study, soil samples were collected across pH gradient (pH 3.6-7) and a range of fertilizers (nitrogen as ammonium sulfate, nitrogen as sodium nitrate, phosphorous) to evaluate the effects nutrients have on soil parameters and microbial community structure. Illumina 16S ribosomal RNA (rRNA) amplicon sequencing was used to determine the relative abundances and diversity of bacterial and archaeal taxa. Relationships between treatments, measured soil parameters, and microbial communities were evaluated. Clostridium, Bacteroides, Bradyrhizobium, Mycobacterium, Ruminococcus, Paenibacillus, and Rhodoplanes were the most abundant genera found at the PGE. The main soil parameter that determined microbial composition, diversity, and biomass in the PGE soil was pH. The most probable mechanism of the pH impact on microbial community may include mediation of nutrient availability in the soil. Addition of nitrogen to the PGE plots as ammonium sulfate decreases soil pH through increased nitrification, which causes buildup of soil carbon, and hence increases C/N ratio. Plant species richness and plant productivity did not reveal significant relationships with microbial diversity; however, plant species richness was positively correlated with soil microbial biomass. Plants responded to the nitrogen treatments with an increase in productivity and a decrease in the species richness.

  5. Popper's Thought Experiment Reinvestigated

    CERN Document Server

    Richardson, Chris D

    2011-01-01

    Popper's original thought experiment probed some fundamental and subtle rules of quantum mechanics. Two recent experiments have tested Popper's hypothesis, but they seem to give contrasting results and one suggests a violation of Heisenberg's uncertainty principle. The equations governing these two experiments and Popper's thought experiment will be derived from basic principles. The experimental constants will be inputted and it will show that the two experiments agree with each other. It will be shown that no uncertainty relations are violated and that Popper's thought experiment was fundamentally flawed.

  6. Experiment WA1 (CDHS Neutrino Experiment)

    CERN Multimedia

    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.

  7. The AMS experiment

    CERN Multimedia

    Laurent Guiraud

    1999-01-01

    The Alpha Magnetic Spectrometer (AMS) detector will be installed as a particle physics experiment on the International Space Station. It will look for antimatter pockets in space. AMS is a CERN recognised experiment.

  8. Future Solar Neutrino Experiments

    Energy Technology Data Exchange (ETDEWEB)

    Nakahata, M. [Kamioka Observatory, Institute for Cosmic Ray research, University of Tokyo, Higashi-Mozumi, Kamioka-cho, Hida-shi, Gifu, Japan, 506-1205 (Japan)]. E-mail: nakahata@suketto.icrr.u-tokyo.ac.jp

    2005-08-15

    The value of future solar neutrino experiments is discussed from particle physics and astrophysics points of view based on current understanding of solar neutrino oscillations. R and D statuses of future experiments are also discussed.

  9. Experiments in Magnetohydrodynamics

    Science.gov (United States)

    Rayner, J. P.

    1970-01-01

    Describes three student experiments in magnetohydrodynamics (MHD). In these experiments, it was found that the electrical conductivity of the local water supply was sufficient to demonstrate effectively some of the features of MHD flowmeters, generators, and pumps. (LC)

  10. 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.

  11. Organic chemistry experiment

    Energy Technology Data Exchange (ETDEWEB)

    Mun, Seok Sik

    2005-02-15

    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.

  12. 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...... several, often contradictory, remarks about remaining visual experience. This review examines closer these remarks as well as experiments that directly approach the nature of possibly spared visual experiences in blindsight....

  13. Customer Experience Management Overview

    OpenAIRE

    Havíř, David

    2016-01-01

    Purpose of the article: The purpose of this paper is to examine the phenomenon of customer experience and customer experience management through years and summarize recent knowledge in this area. Methodology/methods: The paper is built upon secondary research of research papers of renowned authors in the area of customer experience management. Scientific aim: The aim of the article is to find out potential avenues of further research. Findings: Findings confirmed that customer experience is s...

  14. Designing Urban Experiences

    DEFF Research Database (Denmark)

    Jantzen, Christian; Vetner, Mikael

    2008-01-01

    traditional urban planning aspects such as infrastructure, environmental factors and aesthetics, but has also dealt with the design of urban experiences. Through an introduction of the framework of the structure of experiences, this article examines how urban experiences can be understood and analysed...

  15. The Concerned Observer Experiment.

    Science.gov (United States)

    Rabiger, Michael

    1991-01-01

    Describes a classroom experiment--the "concerned observer" experiment--for production students that dramatizes basic film language by relating it to several levels of human observation. Details the experiment's three levels, and concludes that film language mimics wide-ranging states of human emotion and ideological persuasion. (PRA)

  16. Leisure experience and imagination

    NARCIS (Netherlands)

    Lengkeek, J.

    2001-01-01

    Holiday-makers experience the places where they are on holiday in different ways. Back in 1979, Erik Cohen introduced his 'modes of tourist experience'. Cohen's approach was promising for better understanding 'experiences' in a phenomenological way but very little happened afterwards with his 'modes

  17. Double beta decay experiments

    CERN Document Server

    Barabash, A S

    2011-01-01

    The present status of double beta decay experiments is reviewed. The results of the most sensitive experiments are discussed. Proposals for future double beta decay experiments with a sensitivity to the $$ at the level of (0.01--0.1) eV are considered.

  18. Future Outlook: Experiment

    Energy Technology Data Exchange (ETDEWEB)

    Suzuki, Yoichiro [Kamioka, Observatory, Institute for Cosmic Ray Research, University of Tokyo, and Institute for the Physics and Mathematics of the Universe, University of Tokyo. 456 Higashi-Mozumi, Kamioka, Hida-city, Gifu, 506-1205 Japan (Japan)], E-mail: suzuki@suketto.icrr.u-tokyo.ac.jp

    2008-11-01

    The personal view for the next to the next neutrino detector, the ultimate experiment, is discussed. Considering the size, cost and head winds against the basic science, the ultimate experiment will be the only experiment in the world. Here two such experiments one for the neutrino oscillation and the other for the double beta decay were discussed. The ultimate experiment needs to include a bread and butter science and to have a discovery potential for an unexpected phenomenon. There are many technical challenges and international co-operations are absolutely necessary.

  19. 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

  20. 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...... on physics and data rather than the overwhelming instrumentation. We argue that this is because they can transfer their virtual experimental experience to the real-life situation. However, we also find that learning is still situated in the sense that only knowledge of particular experiments is transferred...

  1. Social experience infrastructure

    DEFF Research Database (Denmark)

    Kvistgaard, Peter

    2006-01-01

    Using the case of Kühlungsborn in Mecklenburg-Vorpommern as an example of a resort in which social experience infrastructure plays a pivotal role in the ongoing success of attracting German tourists from especially Berlin, Hamburg and Hanover it is the aim of this article in a descriptive...... 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....

  2. Popper's Thought Experiment Reinvestigated

    Science.gov (United States)

    Richardson, Chris; Dowling, Jonathan

    2012-02-01

    Karl Popper posed an interesting thought experiment in 1934. With it, he meant to question the completeness of quantum mechanics. He claimed that the notion of quantum entanglement leads to absurd scenarios that cannot be true in real life and that an implementation of his thought experiment would not give the results that QM predicts. Unfortunately for Popper, it has taken until recently to perform experiments that test his claims. The results of the experiments do not refute QM as Popper predicted, but neither do they confirm what Popper claimed QM predicted. Kim and Shih implemented Popper's thought experiment in the lab. The results of the experiment are not clear and have instigated many interpretations of the results. The results show some correlation between entangled photons, but not in the way that Popper thought, nor in the way a simple application of QM might predict. A ghost-imaging experiment by Strekalov, et al. sheds light on the physics behind Popper's thought experiment, but does not try to directly test it. I will build the physics of Popper's thought experiment from the ground up and show how the results of both of these experiments agree with each other and the theory of QM, but disprove Popper.

  3. Reactor Neutrino Experiments

    OpenAIRE

    Cao, Jun

    2007-01-01

    Precisely measuring $\\theta_{13}$ is one of the highest priority in neutrino oscillation study. Reactor experiments can cleanly determine $\\theta_{13}$. Past reactor neutrino experiments are reviewed and status of next precision $\\theta_{13}$ experiments are presented. Daya Bay is designed to measure $\\sin^22\\theta_{13}$ to better than 0.01 and Double Chooz and RENO are designed to measure it to 0.02-0.03. All are heading to full operation in 2010. Recent improvements in neutrino moment measu...

  4. Experience Communication and Aesthetics

    DEFF Research Database (Denmark)

    Thorlacius, Lisbeth

    In this article the term "experience communication" will be introduced and discussed. It will be illustrated how different concepts of aesthetical experiences are an integrated part of experience communication and how these concepts are produced within the industries of consumerism, branding...... which was a given to something which each human being has to manage individually. As a consequence the human being experiences an increasing sense of insecurity and restless seeking after identity and recognition, which is reflected in an increasing demand of among other things self...

  5. Linking consumer experiences

    DEFF Research Database (Denmark)

    Smed, Karina Madsen

    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...... 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...

  6. Het SOWNet Experiment (The SOWNet Experiment)

    Science.gov (United States)

    2008-07-01

    sensoren geregistreerd, rnaar eveneensQ werden de responsies. atkornstig via lbet netwerk algoritme (dat hiervoor speciaal werd ontwikkeld) geregistreerd...ontwikkeld algoritme in de nodes maakte communicatie tussen de nodes onderling mogelijk. Het is de bedoeling dlat door deze informatie uitwisseling tussen... algoritme C Uitwerkincy OGS experiment 16-26 novemnber 2007 D Vergeijking van energieverbruik TNO-rapport I TNO-DV 2008 A243 6/17 Inleiding Draadloze

  7. Experiments as politics

    NARCIS (Netherlands)

    Spears, R; Smith, HJ

    2001-01-01

    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. Ex

  8. Experiments with Succinct Solvers

    DEFF Research Database (Denmark)

    Buchholtz, Mikael; Nielson, Hanne Riis; Nielson, Flemming

    2002-01-01

    time of the solver and the aim of this note is to provide some insight into which formulations are better than others. The experiments addresses three general issues: (i) the order of the parameters of relations, (ii) the order of conjuncts in preconditions and (iii) the use of memoisation....... The experiments are performed for Control Flow Analyses for Discretionary Ambients....

  9. 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…

  10. 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.

  11. Expectations in experiments

    NARCIS (Netherlands)

    Wagener, F.

    2013-01-01

    The rational expectations hypothesis is one of the cornerstones of current economic theorising. this review discusses a number of experiments that focus on expectation formation by human subjects and analyses the implications for the rational expectations hypothesis. The experiments show that most a

  12. Future Solar Neutrino Experiments

    Energy Technology Data Exchange (ETDEWEB)

    Suzuki, Y. [Kamioka Observatory, Institute for Cosmic Ray Research, University of Tokyo, Higashi-Mozumi, Kamioka-cho, Hida-city, 506-1205 (Japan)]. E-mail: suzuki@suketto.icrr.u-tokyo.ac.jp

    2005-06-15

    The purpose of the future solar neutrino experiments is briefly reviewed. The future experimental programs which aim to measure the low energy solar neutrinos are described. We do not cover all the projects. Experiments using noble gases are promising for the pp-neutrino measurements.

  13. 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...

  14. The French experience

    CERN Document Server

    Bougard, Marie-Thérèse

    2003-01-01

    Developed for beginners, The French Experience 1 course book is designed to accompany the French Experience 1 CDs (9780563472582) but can also be used on its own to develop your reading and writing skills. You’ll gain valuable insights into French culture too.

  15. 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...

  16. Play as Experience

    Science.gov (United States)

    Henricks, Thomas S.

    2015-01-01

    The author investigates what he believes one of the more important aspects of play--the experience it generates in its participants. He considers the quality of this experience in relation to five ways of viewing play--as action, interaction, activity, disposition, and within a context. He treats broadly the different forms of affect, including…

  17. New neutrino experiments

    Indian Academy of Sciences (India)

    Maury Goodman

    2004-02-01

    Following incredible recent progress in understanding neutrino oscillations, many new ambitious experiments are being planned to study neutrino properties. The most important may be to find a non-zero value of $_{13}$. The most promising way to do this appears to be to measure $_{}→ _{}$ oscillations with an $E/L$ near $ m^{2}_{\\text{atmo}}$. Future neutrino experiments are great.

  18. 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.

  19. Experience as Excursion

    DEFF Research Database (Denmark)

    Svabo, Connie; Shanks, Michael

    2014-01-01

    qualities of experience are highlighted as part of a suggestion that design may indeed relate as much to metaphysics as to mechanics, materials science, and the psychology of the consumer and user. An Experience Design is sketched out as the choreography of temporary and shifting engagements across...

  20. 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....

  1. Real-World Experiences

    Science.gov (United States)

    Borja, Rhea R.

    2006-01-01

    This article presents IISME, a U.S. program that can give educators a real-world experience and that can deepen their subject-matter knowledge. It also presents the experiences of some teachers who are into this program. IISME's summer-fellowship program started out with 40 teachers and 12 companies. The group's growth picked up in 2001, when it…

  2. Franklin: User Experiences

    Energy Technology Data Exchange (ETDEWEB)

    National Energy Research Supercomputing Center; He, Yun (Helen); Kramer, William T.C.; Carter, Jonathan; Cardo, Nicholas

    2008-05-07

    The newest workhorse of the National Energy Research Scientific Computing Center is a Cray XT4 with 9,736 dual core nodes. This paper summarizes Franklin user experiences from friendly early user period to production period. Selected successful user stories along with top issues affecting user experiences are presented.

  3. Fluorescence Experiments with Quinine

    Science.gov (United States)

    O'Reilly, James E.

    1975-01-01

    Describes a series of experiments which illustrate the analytical capabilities of fluorescence, and outlines two straightforward analyses involving real analyses. These experiments are suitable for an undergraduate instrumental analysis course and require approximately six to seven hours of laboratory time. (MLH)

  4. 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 obser...

  5. 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…

  6. 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…

  7. 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.

  8. 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...

  9. USML-1 Glovebox experiments

    Science.gov (United States)

    Naumann, Robert J.

    1995-01-01

    This report covers the development of and results from three experiments that were flown in the Materials Science Glovebox on USML-1: Marangoni convection in Closed Containers (MCCC), Double Float Zone (DFZ), and Fiber Pulling in Microgravity (FPM). The Glovebox provided a convenient, low cost method for doing simple 'try and see' experiments that could test new concepts or elucidate microgravity phenomena. Since the Glovebox provided essentially one (or possibly two levels of confinement, many of the stringent verification and test requirements on the experiment apparatus could be relaxed and a streamlined test and verification plan for flight qualification could be implemented. Furthermore, the experiments were contained in their own carrying cases whose external configurations could be identified early in the integration sequence for stowage considerations while delivery of the actual experiment apparatus could be postponed until only a few months before flight. This minimized the time fluids must be contained and reduced the possibility of corrosive reactions that could ruin the experiment. In many respects, this exercise was as much about developing a simpler, cheaper way of doing crew-assisted science as it was about the actual scientific accomplishments of the individual experiments. The Marangoni Convection in Closed Containers experiment was designed to study the effects of a void space in a simulated Bridgman crystal growth configuration and to determine if surface tension driven convective flows that may result from thermal gradients along any free surfaces could affect the solidification process. The Fiber Pulling in Microgravity experiment sought to separate the role of gravity drainage from capillarity effects in the break-up of slender cylindrical liquid columns. The Stability of a Double Float Zone experiment explored the feasibility of a quasi-containerless process in which a solidifying material is suspended by two liquid bridges of its own melt.

  10. 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

  11. 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.

  12. Network simulation experiments manual

    CERN Document Server

    Aboelela, Emad

    2011-01-01

    Network Simulation Experiments Manual, Third Edition, contains simulation-based experiments to help students and professionals learn about key concepts in computer networking. The simulation approach provides a virtual environment for a wide range of desirable features, such as modeling a network based on specified criteria and analyzing its performance under different scenarios. The experiments include the basics of using OPNET IT Guru Academic Edition; operation of the Ethernet network; partitioning of a physical network into separate logical networks using virtual local area networks (V

  13. Hydrothermal organic synthesis experiments

    Science.gov (United States)

    Shock, Everett L.

    1992-01-01

    Ways in which heat is useful in organic synthesis experiments are described, and experiments on the hydrothermal destruction and synthesis of organic compounds are discussed. It is pointed out that, if heat can overcome kinetic barriers to the formation of metastable states from reduced or oxidized starting materials, abiotic synthesis under hydrothermal conditions is a distinct possibility. However, carefully controlled experiments which replicate the descriptive variables of natural hydrothermal systems have not yet been conducted with the aim of testing the hypothesis of hydrothermal organic systems.

  14. 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

  15. 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.

  16. THERMAL DISTRIBUTION SYSTEM EXPERIMENT

    Energy Technology Data Exchange (ETDEWEB)

    KRAJEWSKI,R.F.; ANDREWS,J.W.; WEI,G.

    1999-09-01

    A laboratory experiment has been conducted which tests for the effects of distribution system purging on system Delivery Effectiveness (DE) as defined in ASHRAE 152P. The experiment is described in its configuration, instrumentation, and data acquisition system. Data gathered in the experiment is given and discussed. The results show that purging of the distribution system alone does not offer any improvement of the system DE. Additional supporting tests were conducted regarding experimental simulations of buffer zones and bare pipe and are also discussed.

  17. Phenomenological Account of Religious Experience

    Directory of Open Access Journals (Sweden)

    Donny Gahral Adian

    2011-08-01

    Full Text Available History of philosophy is built upon rigid discrimination between various human experiences. Human experiences are divided mainly into two major experiences: Perceptual and intelectual. Perceptual experience is deined by empiricism as an aposteriori experience of empirical sensations. Meanwhile, rationalism claims that the only acceptable experience is apriori experience of intelectual object (natural laws, mathematical equations and logical operations. There is no other experience outside those two philosophical account of experiences. All other experiences must be subsumed either within perceptual or intelectual experience.

  18. Experience and Its Generation

    Institute of Scientific and Technical Information of China (English)

    Chen Youqing

    2006-01-01

    Experience iS an activity that arouses emotions and generates meanings based on vivid sensation and profound compreh ension.It iS emotional,meaningful,and personal,playing a key role in the course of forming and developing one'S qualities.The psychological process of experience generation consists of such links as sensing things,arousing emotions,promoting comprehension and association,generating insights and meanings,and deepening emotional responses.Undergoing things personally by means of direct sensation,taking part in activities,and living life are the most important preconditions of experience generation.Emotional influence,situational edification,and arts edification ale extemal factors that induce experience generation.

  19. Cooperative Prototyping Experiments

    DEFF Research Database (Denmark)

    Bødker, Susanne; Grønbæk, Kaj

    1989-01-01

    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...... these experiences we discuss problems in the process, requirements for design tools, and issues involved in getting going with cooperative prototyping with active user involvement.......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...

  20. Experiments with Helmholtz Resonators.

    Science.gov (United States)

    Greenslade, Thomas B., Jr.

    1996-01-01

    Presents experiments that use Helmholtz resonators and have been designed for a sophomore-level course in oscillations and waves. Discusses the theory of the Helmholtz resonator and resonance curves. (JRH)

  1. Accessibility and sensory experiences

    DEFF Research Database (Denmark)

    Ryhl, Camilla

    2010-01-01

    This article introduces a new design concept; sensory accessibility. While acknowledging the importance of sensory experiences in architectural quality, as well as the importance of accommodating user needs the concept combines three equally important factors; architecture, the senses and accessi......This article introduces a new design concept; sensory accessibility. While acknowledging the importance of sensory experiences in architectural quality, as well as the importance of accommodating user needs the concept combines three equally important factors; architecture, the senses...... 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....

  2. Experiences with treating immigrants

    DEFF Research Database (Denmark)

    Sandhu, Sima; Bjerre, Neele V; Dauvrin, Marie;

    2012-01-01

    sought to explore professionals' experiences of delivering care to immigrants in districts densely populated with immigrants across Europe. METHODS: Forty-eight semi-structured interviews were conducted with mental health care professionals working in 16 European countries. Professionals in each country......PURPOSE: While there has been systematic research on the experiences of immigrant patients in mental health services within certain European countries, little research has explored the experiences of mental health professionals in the delivery of services to immigrants across Europe. This study...... of marginalisation. CONCLUSIONS: Although mental health service delivery varies between and within European countries, consistent challenges exist in the experiences of mental health professionals delivering services in communities with high proportions of immigrants. Improvements to practice should include training...

  3. The world's biggest experiment

    CERN Document Server

    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)

  4. 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...

  5. Foraging Experiences with Children

    Science.gov (United States)

    Russell, Helen Ross

    1976-01-01

    Provided are foraging experiences and wild foods information for utilization in the urban school curriculum. Food uses are detailed for roses, dandelions, wild onions, acorns, cattails, violets and mints. (BT)

  6. Conceptualising the audiobook experience

    Directory of Open Access Journals (Sweden)

    Iben Have

    2012-12-01

    Full Text Available In this article we wish to introduce and discuss a theoretical framework for a possible conceptualisation of the differences between reading a printed book and listening to an audiobook. We tend to introduce similarities and differences between reading with the eyes and reading with the ears, implying that we should not discuss the audiobook experience as a remediation of the printed book experience only, but as an entirely different experience that could be conceptualised in continuation of mobile listening practises. As a methodological strategy we will emphasise the differences between the literary practices, reading with the eyes and reading with the ears. These different perspectives on reading are used to accentuate the distinct experiences, and future thorough analyses in continuation of this framework would appear much more complex and connected than in the present article.

  7. The Experience City

    DEFF Research Database (Denmark)

    Marling, Gitte; Jensen, Ole B.; Kiib, Hans

    2009-01-01

    of a research project  mapping the different approaches to urban transformation and experience design, to the level of strategic thinking and planning, and to the content of the programs implemented. Special emphasis is put on on projects combining experience, leisure and learning, and on projects which have...... concepts and framings that will guide the understanding and the analysis of the experience city. In section three we focus on the design of the Danish experience city and present the first findings of the research project The projects are categorised according to their content, structure and urban...... localisation. In particular the cases are represented in relation to their strategic and urban planning importance, their social and cultural content and their architectural representation and the programmes they contain. The article ends with a short discussion of some  of these preliminary findings as well...

  8. 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)

  9. 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

  10. 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.

  11. 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.

  12. Hamlet and psychoanalytic experience.

    Science.gov (United States)

    Schwaber, Paul

    2007-01-01

    Hamlet draws us into its rendered world, enabling us to experience it with depth, awareness, and resonance, in a mode we recognize as aesthetic. By way of Shakespeare's play--primarily the first act--and a detailed case study, aesthetic and psychoanalytic experience are compared, to suggest that, for our own analytic discourse, we revalue Freud's unease that his case studies read like short stories.

  13. The OPERA Experiment

    CERN Document Server

    Iovane, G

    1998-01-01

    OPERA(Oscillation Project with Emulsion-tRacking Apparatus) is a new detector concept, iron(lead)-emulsion for a long-baseline neutrino oscillation experiment. This experiment would perform an appearance search for nu_mu-nu_tau oscillation in the parameter region indicated by the atmospheric neutrino anomaly. OPERA can run at the Gran Sasso Laboratory in the proposed NGS (Neutrino to Gran Sasso) Beam from CERN.

  14. Surface electrical properties experiment

    Science.gov (United States)

    Simmons, Gene; Strangway, David; Annan, Peter; Baker, Richard G.; Bannister, Lawrence; Brown, Raymon; Cooper, William; Cubley, Dean; deBettencourt, Joseph; England, Anthony W.; Groener, John; Kong, Jin-Au; LaTorraca, Gerald; Meyer, James; Nanda, Ved; Redman, David; Rossiter, James; Tsang, Leung; Urner, Joseph; Watts, Raymond

    1973-01-01

    The surface electrical properties (SEP) experiment was used to explore the subsurface material of the Apollo 17 landing site by means of electromagnetic radiation. The experiment was designed to detect electrical layering, discrete scattering bodies, and the possible presence of water. From the analysis of the data, it was expected that values of the electrical properties (dielectric constant and loss tangent) of lunar material in situ would be obtained.

  15. Future Reactor Experiments

    OpenAIRE

    He, Miao

    2013-01-01

    The measurement of the neutrino mixing angle $\\theta_{13}$ opens a gateway for the next generation experiments to measure the neutrino mass hierarchy and the leptonic CP-violating phase. Future reactor experiments will focus on mass hierarchy determination and the precision measurement of mixing parameters. Mass hierarchy can be determined from the disappearance of reactor electron antineutrinos based on the interference effect of two separated oscillation modes. Relative and absolute measure...

  16. Reactor antineutrino experiments

    OpenAIRE

    Lu, Haoqi

    2014-01-01

    Neutrinos are elementary particles in the standard model of particle physics. There are 3 flavors of neutrinos that oscillate among themselves. Their oscillation can be described by a 3$\\times$3 unitary matrix, containing three mixing angles $\\theta_{12}$, $\\theta_{23}$, $\\theta_{13}$, and one CP phase. Both $\\theta_{12}$ and $\\theta_{23}$ are known from previous experiments. $\\theta_{13}$ was unknown just two years ago. The Daya Bay experiment gave the first definitive non-zero value in 2012...

  17. Mechatronics with experiments

    CERN Document Server

    Cetinkunt, Sabri

    2014-01-01

    Mechatronics with Experiments, Second Edition comprehensively covers the fundamental scientific principles and technologies that are used in the design of modern computer-controlled machines and processes. It provides all of the technical background (covering mechanical, aerospace, chemical, electrical, and computer engineering) needed for designing an automated machine or process. The new edition of this textbook has been updated to include a number of experiments involving electronic circuit design and microcontroller programming and includes real time software development using MATLAB® and

  18. 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.

  19. Illegal Entrepreneurship Experience

    OpenAIRE

    2004-01-01

    Existing studies show a positive relationship between entrepreneurs' business performance and their conventional human capital as measured by previous business experience and formal education. In this paper, we explore whether illegal entrepreneurship experience (IEE), an unconventional form of human capital, is related to the performance and motivation of entrepreneurs operating legal businesses in a transition context. Using regression techniques on a sample of 399 private business owners i...

  20. 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...

  1. Prolonged labour : women's experiences

    OpenAIRE

    Nystedt, Astrid

    2005-01-01

    Aim: The overall aim of this thesis was to illuminate, describe, and promote understanding of women’s experiences of prolonged labour. The thesis compromises four studies. Methods: Paper I describes a case-referent study that recruited women (n = 255) giving singleton live birth to their first child by spontaneous labour after more than 37 completed weeks’ pregnancy. Participants completed a questionnaire that investigated childbirth experiences, previous family relationships, and childhood e...

  2. Reconstructing Experiences through Sketching

    CERN Document Server

    Karapanos, Evangelos; Hassenzahl, Marc

    2009-01-01

    This paper presents iScale, a survey tool that aims at eliciting users' experiences with a product from memory. iScale employs sketching in imposing a process in the reconstruction of one's experiences. Two versions of iScale, the Constructive and the Value-Account iScale, were motivated by two distinct theories on how people reconstruct emotional experiences from memory. These two versions were tested in two separate studies. Study 1 aimed at providing qualitative insight into the use of iScale and compared its performance to that of free-hand sketching. Study 2 compared the two iScale versions to a control condition: that of reporting one's experiences without employing any form of sketching. Significant differences between iScale and the "no-sketching" tool were found. Overall, iScale resulted in a) an increase in the number of experience reports that subjects provided, b) an increase in the amount of contextual information for the reported experiences, and c) an increase in subjects' accuracy in recalling...

  3. Radiochemical solar neutrino experiments

    CERN Document Server

    Gavrin, V N

    2011-01-01

    Radiochemical experiments have been crucial to solar neutrino research. Even today, they provide the only direct measurement of the rate of the proton-proton fusion reaction, p + p --> d + e^+ + nu_e, which generates most of the Sun's energy. We first give a little history of radiochemical solar neutrino experiments with emphasis on the gallium experiment SAGE -- the only currently operating detector of this type. The combined result of all data from the Ga experiments is a capture rate of 67.6 +/- 3.7 SNU. For comparison to theory, we use the calculated flux at the Sun from a standard solar model, take into account neutrino propagation from the Sun to the Earth and the results of neutrino source experiments with Ga, and obtain 67.3 ^{+3.9}_{-3.5} SNU. Using the data from all solar neutrino experiments we calculate an electron neutrino pp flux at the earth of (3.41 ^{+0.76}_{-0.77}) x 10^{10}/(cm^2-s), which agrees well with the prediction from a detailed solar model of (3.30 ^{+0.13} _{-0.14}) x 10^{10}/(cm^...

  4. 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.

  5. Experience and Sustainable Consumption

    DEFF Research Database (Denmark)

    Rasmussen, Tove Arendt

    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......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...

  6. Experience and Sustainable Consumption

    DEFF Research Database (Denmark)

    Rasmussen, Tove Arendt

    2014-01-01

    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......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...

  7. Qualitative experiments in psychology

    DEFF Research Database (Denmark)

    Wagoner, Brady

    2015-01-01

    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......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...... 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...

  8. An Organoleptic Laboratory Experiment

    Science.gov (United States)

    Risley, John M.

    1996-12-01

    Flavorings in foods and fragrances in personal care products is a topic often discussed in chemistry classes designed for the general education of non-science majors. A laboratory experiment has been designed to accompany the lecture topic. Compounds in ten different classes of organic molecules that are used in the fragrance and food industry are provided to students. Students whiff the vapors of each compound and describe the organoleptic properties using a set of terms utilized in the fragrance and food industry. A set of questions guides students to an understanding of the relationship between structure of molecules and smell. Students are permitted to create their own fragrance based on the results of the experiment. Student response has been favorable. The experiment rectifies misconceptions students have about structure and odor, and gives positive reinforcement to the lecture material.

  9. ATLAS IBL operational experience

    CERN Document Server

    Takubo, Yosuke; 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.

  10. 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...... 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......, theology, and philosophy, are thus interrelated. ‘Experience’ and ‘dialogue’ also play crucial roles in Vattimo’s understanding of philosophy, and recent works such as Hermeneutic Communism confirm that his thought continues to be world-engaged. Jørgensen’s philosophy of experience allows for a development...

  11. Learning From Experience

    DEFF Research Database (Denmark)

    Visholm, Steen; Beck, Ulla Charlotte

    2014-01-01

    In this paper the learning concept of group relation's conferences are discussed. The authors have worked with group relations conferences (GRC) in different contexts for many years-mainly as a part of educational programmes for managers and consultants (OPU at IGA Copenhagen, MPO at Roskilde...... decides for themselves what makes sense and what does not. This principle sometimes works as a defence against a closer examination of the two questions: do GRCs provide relevant experiences to learn from, and what is it you learn or can expect to learn at a GRC. Here the learning concept of the GRCs...... University and NAPSO2). Seen from the horizon of their experience some of the basic concepts in the theories about GRC need clarifying, revision, and development. The GRC is a part of the learning from experience movement and as a consequence it stresses the underlying basis: learning is personal so everyone...

  12. The POLARBEAR Experiment

    CERN Document Server

    Kermish, Z; Anthony, A; Arnold, K; Arnold, K; Barron, D; Boettger, D; Borrill, J; Chapman, S; Chinone, Y; Dobbs, M A; Errard, J; Fabbian, G; Flanigan, D; Fuller, G; Ghribi, A; Grainger, W; Halverson, N; Hasegawa, M; Hattori, K; Hazumi, M; Holzapfel, W L; Howard, J; Hyland, P; Jaffe, A; Keating, B; Kisner, T; Lee, A T; Jeune, M Le; Linder, E; Lungu, M; Matsuda, F; Matsumura, T; Meng, X; Miller, N J; Morii, H; Moyerman, S; Myers, M J; Nishino, H; Paar, H; Quealy, E; Reichardt, C L; Richards, P L; Ross, C; Shimizu, A; Shimon, M; Shimmin, C; Sholl, M; Siritanasak, P; Spieler, H; Stebor, N; Steinbach, B; Stompor, R; Suzuki, A; Tomaru, T; Tucker, C; Zahn, O

    2012-01-01

    We present the design and characterization of the POLARBEAR experiment. POLARBEAR will measure the polarization of the cosmic microwave background (CMB) on angular scales ranging from the experiment's 3.5 arcminute beam size to several degrees. The experiment utilizes a unique focal plane of 1,274 antenna-coupled, polarization sensitive TES bolometers cooled to 250 milliKelvin. Employing this focal plane along with stringent control over systematic errors, POLARBEAR has the sensitivity to detect the expected small scale B-mode signal due to gravitational lensing and search for the large scale B-mode signal from inflationary gravitational waves. POLARBEAR was assembled for an engineering run in the Inyo Mountains of California in 2010 and was deployed in late 2011 to the Atacama Desert in Chile. An overview of the instrument is presented along with characterization results from observations in Chile.

  13. 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...

  14. Experiments in Particle Astrophysics

    Energy Technology Data Exchange (ETDEWEB)

    Boratav, M

    2004-07-01

    During the last decade, the field of what is currently called particle astrophysics (that I prefer to the shaky neologism astroparticle physics) has experienced a surprising growth. It is interesting to understand why the cosmic rays, the poor man's accelerator not no long ago, are becoming the object of scrutiny for a continuously growing community of theoreticians and experimentalists. In this article, we made an arbitrary choice of a small number of experiments to illustrate today's state of the art and the future perspectives in this domain. Our choice is based on three facts: the objects detected in each experiment are different, all the selected experiments are in their starting phase and all are spectacular for various reasons. Our aim is to convince the reader of the enormous discovery potential of these ongoing projects and share with him the excitement experienced by those involved in them. (Author) 37 refs.

  15. The Experiment as Act

    DEFF Research Database (Denmark)

    Søndergaard, Morten

    seminal experiments between poetry, sound and media. This paper will focus on the methodologies of ‘archiving’ experimental artforms. The ‘archive’, here, is argued to be in-between a gathering of objects and the simulation of time (Ernst). The archive presents one of the great cultural enigmas of our......In December 1965, an experiment took place at The Independent Art Space in Copenhagen (Den Frie Kunstbygning). Short named POEX65, it was looking to create and activate POetry EXperiments across artistic genres and formats (thus, in essence, making a POetry EXposition). The POEX65 event framed many...... time: It is constructed like a (sub)medium that collects our cultural history; at the same time, the archive often ‘forgets’ it’s own pre-experimental status and simply slips into a state of unconscious repetition which allows parallel histories to emerge in-between everything else. This paper tries...

  16. 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...... in the experiment. This raises two questions: (i) can we trust the existing belief elicitation results, (ii) can we avoid potential hedging confounds? Our results instill confidence regarding both issues. We propose an experimental design that eliminates hedging opportunities, and use this to test for the empirical...

  17. The Experience City

    DEFF Research Database (Denmark)

    Marling, Gitte; Jensen, Ole B.; Kiib, Hans

    2012-01-01

    findings. The projects are categorised according to their content, structure and urban localisation. In particular the cases are labelled in relation to their strategic and urban planning importance, their social and cultural content and their architectural representation and the programmes they contain...... to relate to the wider international debate and development. In section two we present the main theoretical concepts and framings that will guide the understanding and the analysis of the experience city. In section three we focus on the design of the ‘Danish experience city’ and present the first research...

  18. Initial Cooling Experiment (ICE)

    CERN Multimedia

    Photographic Service

    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.

  19. The OPERA experiment

    CERN Document Server

    Pessard, H

    2001-01-01

    Following the Super-Kamiokande findings on atmospheric neutrinos, accelerator neutrinos have started to be exploited to confirm the indication for neutrino oscillations and perform more complete measurements of the mass differences and mixing parameters. In Europe, the long baseline beam CERN Neutrino to Gran Sasso CNGS is aimed at direct appearance searches of nu /sub mu / to nu /sub tau /. The OPERA experiment in Gran sasso will use photographic emulsions in a large hybrid detector to point at nu , interactions coming from the CNGS beam in a "zero background" approach. The principles of the experiment and its projected performances are described. (12 refs).

  20. The Archimedes experiment

    CERN Document Server

    Calloni, Enrico; De Laurentis, Martina; Esposito, Giampiero; Grilli, M; Majorana, Ettore; Pepe, G P; Petrarca, S; Puppo, Paola; Rapagnini, P; Ricci, F; Rosa, Luigi; Rovelli, Carlo; Ruggi, P; Saini, N L; Stornaiolo, Cosimo; Tafuri, Francesco

    2015-01-01

    Archimedes is an INFN-funded pathfinder experiment aimed at verifying the feasibility of measuring the interaction of vacuum fluctuations with gravity. The final experiment will measure the force exerted by the gravitational field on a Casimir cavity whose vacuum energy is modulated with a superconductive transition, by using a balance as a small force detector. Archimedes is a two-year project devoted to test the most critical experimental aspects, in particular the balance resonance frequency and quality factor, the thermal modulation efficiency and the superconductive sample realization.

  1. The Archimedes experiment

    Science.gov (United States)

    Calloni, E.; Caprara, S.; Laurentis, M. De; Esposito, G.; Grilli, M.; Majorana, E.; Pepe, G. P.; Petrarca, S.; Puppo, P.; Rapagnani, P.; Ricci, F.; Rosa, L.; Rovelli, C.; Ruggi, P.; Saini, N. L.; Stornaiolo, C.; Tafuri, F.

    2016-07-01

    Archimedes is an INFN-funded pathfinder experiment aimed at verifying the feasibility of measuring the interaction of vacuum fluctuations with gravity. The final experiment will measure the force exerted by the gravitational field on a Casimir cavity whose vacuum energy is modulated with a superconductive transition, by using a balance as a small force detector. Archimedes is two-year project devoted to test the most critical experimental aspects, in particular the balance resonance frequency and quality factor, the thermal modulation efficiency and the superconductive sample realization.

  2. DEEP UNDERGROUND NEUTRINO EXPERIMENT

    Energy Technology Data Exchange (ETDEWEB)

    Wilson, Robert J. [Fermilab

    2016-03-03

    The Deep Underground Neutrino Experiment (DUNE) collaboration will perform an experiment centered on accelerator-based long-baseline neutrino studies along with nucleon decay and topics in neutrino astrophysics. It will consist of a modular 40-kt (fiducial) mass liquid argon TPC detector located deep underground at the Sanford Underground Research Facility in South Dakota and a high-resolution near detector at Fermilab in Illinois. This conguration provides a 1300-km baseline in a megawatt-scale neutrino beam provided by the Fermilab- hosted international Long-Baseline Neutrino Facility.

  3. Future of neutrino experiments

    Indian Academy of Sciences (India)

    Takaaki Kajita

    2009-01-01

    Atmospheric, solar, reactor and accelerator neutrino oscillation experiments have measured $ m_{12}^{2}$, sin2 12, $| m_{23}^{2} |$ and sin2 223. The next stage of the oscillation studies should be the observation of a finite sin2 213. If a non-zero sin2 213 is observed, the subsequent goals should be the observation of the CP violation and the determination sign of $ m_{23}^{2}$. Possible future neutrino oscillation experiments that could assess these questions are discussed.

  4. The Archimedes experiment

    Energy Technology Data Exchange (ETDEWEB)

    Calloni, E. [University of Napoli Federico II and INFN Napoli (Italy); Caprara, S. [University of Roma Sapienza and INFN Roma (Italy); Laurentis, M. De; Esposito, G. [University of Napoli Federico II and INFN Napoli (Italy); Grilli, M.; Majorana, E. [University of Roma Sapienza and INFN Roma (Italy); Pepe, G.P. [University of Napoli Federico II and INFN Napoli (Italy); Petrarca, S. [University of Roma Sapienza and INFN Roma (Italy); Puppo, P., E-mail: paola.puppo@roma1.infn.it [University of Roma Sapienza and INFN Roma (Italy); Rapagnani, P.; Ricci, F. [University of Roma Sapienza and INFN Roma (Italy); Rosa, L. [University of Napoli Federico II and INFN Napoli (Italy); Rovelli, C. [University of Aix-Marseille (France); Ruggi, P. [European Gravitational Observatory (EGO), Cascina (Pisa) (Italy); Saini, N.L. [University of Roma Sapienza and INFN Roma (Italy); Stornaiolo, C.; Tafuri, F. [University of Napoli Federico II and INFN Napoli (Italy)

    2016-07-11

    Archimedes is an INFN-funded pathfinder experiment aimed at verifying the feasibility of measuring the interaction of vacuum fluctuations with gravity. The final experiment will measure the force exerted by the gravitational field on a Casimir cavity whose vacuum energy is modulated with a superconductive transition, by using a balance as a small force detector. Archimedes is two-year project devoted to test the most critical experimental aspects, in particular the balance resonance frequency and quality factor, the thermal modulation efficiency and the superconductive sample realization. - Highlights: • Weight of the vacuum. • Superconductive stacks studies. • Thermal behavior studies at cryogenic temperatures.

  5. 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.

  6. AGS experiments, 1988, 1989, 1990

    Energy Technology Data Exchange (ETDEWEB)

    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.

  7. AGS experiments: 1985, 1986, 1987

    Energy Technology Data Exchange (ETDEWEB)

    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.

  8. 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)

  9. The PADME experiment

    CERN Document Server

    ,

    2016-01-01

    The PADME experiment, hosted at the Laboratori Nazionali di Frascati, will search for a Dark Photon that decays in invisible channels with a mass up to $23.7\\,\\mbox{MeV}$ and coupling constant down to $10^{-3}$.

  10. Democratic design experiments

    DEFF Research Database (Denmark)

    Ehn, Pelle; Brandt, Eva; Halse, Joachim

    2016-01-01

    we here see design engagements which are both controversial in their commitment to agendas of social change and experimental in the sense that they openly probe for what can possibly be enacted. In this conversation we want to explore how such engagements may be seen as democratic design experiments...

  11. Alpha Antihydrogen Experiment

    CERN Document Server

    Fujiwara, M C; Ashkezari, M D; Baquero-Ruiz, M; Bertsche, W; Bray, C C; Butler, E; Cesar, C L; Chapman, S; Charlton, M; Cesar, C L; Fajans, J; Friesen, T; Gill, D R; Hangst, J S; Hardy, W N; Hayano, R S; Hayden, M E; Humphries, A J; Hydomako, R; Jonsell, S; Kurchaninov, L; Lambo, R; Madsen, N; Menary, S; Nolan, P; Olchanski, K; Olin, A; Povilus, A; Pusa, P; Robicheaux, F; Sarid, E; Silveira, D M; So, C; Storey, J W; Thompson, R I; van der Werf, D P; Wilding, D; Wurtele, J S; Yamazaki, Y

    2011-01-01

    ALPHA is an experiment at CERN, whose ultimate goal is to perform a precise test of CPT symmetry with trapped antihydrogen atoms. After reviewing the motivations, we discuss our recent progress toward the initial goal of stable trapping of antihydrogen, with some emphasis on particle detection techniques.

  12. Understanding Popper's experiment

    Science.gov (United States)

    Qureshi, Tabish

    2005-06-01

    An experiment proposed by Karl Popper is considered by many to be a crucial test of quantum mechanics. Although many loopholes in the original proposal have been pointed out, they are not crucial to the test. We use only the standard interpretation of quantum mechanics to point out what is fundamentally wrong with the proposal, and demonstrate that Popper's basic premise was faulty.

  13. Art and experience

    NARCIS (Netherlands)

    Gerwen, R.C.H.M. van

    2001-01-01

    This three-part dissertation is on the double role of experience in art: as a subject matter, and as the vehicle for our evaluations. It argues (Part three, Chs. 7, 8) for the inclusion within contemporary analytical ‘cognitivism’ (Part one, Chs. 1-3) of certain arguments from the founding fathers o

  14. 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…

  15. Experiments on Photoconductivity

    Science.gov (United States)

    Kraftmakher, Yaakov

    2012-01-01

    Computer-assisted experiments with CdS and CdSe photoresistors are described. The most important characteristics of the photoresistors are determined: (i) the spectral response, (ii) the photocurrent versus incident radiant power, (iii) the rise and decay time constants and (iv) the frequency response to modulated light. The photoconductivity gain…

  16. Cyclic Voltammetry Experiment.

    Science.gov (United States)

    Van Benschoten, James J.; And Others

    1983-01-01

    Describes a three-part experiment designed to introduce cyclic voltammetry to graduate/undergraduate students. Part 1 demonstrates formal reduction potential, redox electron transfer, diffusion coefficient, and electrochemical reversibility. Part 2 investigates electrochemical behavior of acetaminophen. Part 3 examines such experimental variables…

  17. The NA48 experiment

    CERN Multimedia

    1995-01-01

    The NA48 experiment is designed to study the Charge-Parity (CP) violation. CP violation is observed in certain very rare decays. Its study is important in understanding Nature's preference of matter over antimatter, which resulted in asymmetry soon after the Big Bang, creating a matter-dominated Universe.

  18. 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.

  19. Electrophoresis experiments in microgravity

    Science.gov (United States)

    Snyder, Robert S.; Rhodes, Percy H.

    1991-01-01

    The use of the microgravity environment to separate and purify biological cells and proteins has been a major activity since the beginning of the NASA Microgravity Science and Applications program. Purified populations of cells are needed for research, transplantation and analysis of specific cell constituents. Protein purification is a necessary step in research areas such as genetic engineering where the new protein has to be separated from the variety of other proteins synthesized from the microorganism. Sufficient data are available from the results of past electrophoresis experiments in space to show that these experiments were designed with incomplete knowledge of the fluid dynamics of the process including electrohydrodynamics. However, electrophoresis is still an important separation tool in the laboratory and thermal convection does limit its performance. Thus, there is a justification for electrophoresis but the emphasis of future space experiments must be directed toward basic research with model experiments to understand the microgravity environment and fluid analysis to test the basic principles of the process.

  20. 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.

  1. The Doppler Pendulum Experiment

    Science.gov (United States)

    Lee, C. K.; Wong, H. K.

    2011-01-01

    An experiment to verify the Doppler effect of sound waves is described. An ultrasonic source is mounted at the end of a simple pendulum. As the pendulum swings, the rapid change of frequency can be recorded by a stationary receiver using a simple frequency-to-voltage converter. The experimental results are in close agreement with the Doppler…

  2. Experiences of Collaborative Research

    Science.gov (United States)

    Kahneman, Daniel

    2003-01-01

    The author's personal history of the research that led to his recognition in economics is described, focusing on the process of collaboration and on the experience of controversy. The author's collaboration with Amos Tversky dealt with 3 major topics: judgment under uncertainty, decision making, and framing effects. A subsequent collaboration,…

  3. Neutrino Experiments Highlights

    CERN Document Server

    Wong, H T

    2001-01-01

    This article consists of two parts. The first section presents the highlights on the goals of neutrino physics, status of the current neutrino experiments and future directions and program. The second section describes the theme, program and research efforts for the TEXONO Collaboration among scientists from Taiwan and China.

  4. Experiments with Dipole Antennas

    Science.gov (United States)

    Kraftmakher, Yaakov

    2009-01-01

    Employment of a data-acquisition system for data collection and calculations makes experiments with antennas more convenient and less time consuming. The determined directional patterns of the dipole antennas of different lengths are in reasonable agreement with theory. The enhancement of the signal by using a reflector is demonstrated, and a…

  5. 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...

  6. 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…

  7. Experiments versus simultations

    DEFF Research Database (Denmark)

    Baltser, Jana

    become a very powerful tool, which allows prediction of experimental outcomes with high precision and accuracy. The present Ph. D. work is dedicated to development of such a simulation tool based on a ray-tracing technique, McXtrace, its application for modelling of experiments at a synchrotron beamline...

  8. 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.

  9. An Unhappy Experience.

    Science.gov (United States)

    Ali, Aydin Mehmet

    1998-01-01

    A study of 10 Cypriotturkish young people in British universities identified the factors they perceive as having had an impact on their achievement. Negative experiences in secondary and higher education included teachers' low expectations, racism, and lack of role models/mentors. Women were more aware of these issues than men were. (JOW)

  10. Parabolic aircraft solidification experiments

    Science.gov (United States)

    Workman, Gary L. (Principal Investigator); Smith, Guy A.; OBrien, Susan

    1996-01-01

    A number of solidification experiments have been utilized throughout the Materials Processing in Space Program to provide an experimental environment which minimizes variables in solidification experiments. Two techniques of interest are directional solidification and isothermal casting. Because of the wide-spread use of these experimental techniques in space-based research, several MSAD experiments have been manifested for space flight. In addition to the microstructural analysis for interpretation of the experimental results from previous work with parabolic flights, it has become apparent that a better understanding of the phenomena occurring during solidification can be better understood if direct visualization of the solidification interface were possible. Our university has performed in several experimental studies such as this in recent years. The most recent was in visualizing the effect of convective flow phenomena on the KC-135 and prior to that were several successive contracts to perform directional solidification and isothermal casting experiments on the KC-135. Included in this work was the modification and utilization of the Convective Flow Analyzer (CFA), the Aircraft Isothermal Casting Furnace (ICF), and the Three-Zone Directional Solidification Furnace. These studies have contributed heavily to the mission of the Microgravity Science and Applications' Materials Science Program.

  11. 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...

  12. The Huck Finn Experience.

    Science.gov (United States)

    Levesque, Bonnie; And Others

    This paper describes "The Huck Finn Experience," a high motivation interdisciplinary unit that takes students on a 6-week journey back to the life and times of Mark Twain through the fictional character of Huckleberry Finn. The unit described in the paper was designed for eighth-grade students but could be easily adapted to be successful with…

  13. 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.)

  14. "Experience and Learning"

    DEFF Research Database (Denmark)

    Olesen, Henning Salling

    2007-01-01

    concepts derived from European critical theory, subjectivity and experience, are briefly introduced with a view to their intellectual background. The chapter elaborates the implication of these concepts in relation to the understanding of emotional aspects of learning in everyday work life and in relation...

  15. Science and Human Experience

    Science.gov (United States)

    Cooper, Leon N.

    2015-01-01

    Part I. Science and Society: 1. Science and human experience; 2. Does science undermine our values?; 3. Can science serve mankind?; 4. Modern science and contemporary discomfort: metaphor and reality; 5. Faith and science; 6. Art and science; 7. Fraud in science; 8. Why study science? The keys to the cathedral; 9. Is evolution a theory? A modest proposal; 10. The silence of the second; 11. Introduction to Copenhagen; 12. The unpaid debt; Part II. Thought and Consciousness: 13. Source and limits of human intellect; 14. Neural networks; 15. Thought and mental experience: the Turing test; 16. Mind as machine: will we rubbish human experience?; 17. Memory and memories: a physicist's approach to the brain; 18. On the problem of consciousness; Part III. On the Nature and Limits of Science: 19. What is a good theory?; 20. Shall we deconstruct science?; 21. Visible and invisible in physical theory; 22. Experience and order; 23. The language of physics; 24. The structure of space; 25. Superconductivity and other insoluble problems; 26. From gravity to light and consciousness: does science have limits?

  16. Child Psychology Experiences Program.

    Science.gov (United States)

    Walla Walla Coll., WA.

    Recognizing the need for trained teachers to enter the classroom with confidence and professional capacity, Walla Walla College introduced a Child Psychology Experience program. Personnel from several departments contribute to this program. In connection with the child psychology courses, the project features a laboratory/demonstration center…

  17. 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…

  18. A Monopoly Classroom Experiment.

    Science.gov (United States)

    Oxoby, Robert J.

    2001-01-01

    Uses a simple classroom experiment to develop the economic model of monopoly. Introduces students to the nature of the monopoly problem and motivates them to think of the associated effects. Highlights the role of information and fairness ideals in determining economic outcomes. (RLH)

  19. Experiment R701

    CERN Multimedia

    1974-01-01

    This experiment was designed by the CERN-Aachen-MPI/Munich-Heidelberg Collaboration to study inelastic proton-proton collisions with streamer chambers. The photo shows the lower streamer chamber closely fitting around the central bicone vacuum chamber at I-7. The upper chamber, here removed, was similarly fitted (Photo Archive 7401099).

  20. 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.

  1. 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.

  2. Conducting Social Experiments.

    Science.gov (United States)

    Boruch, Robert F.

    1987-01-01

    It is difficult to conduct randomized field experiments. In the past decade, the use of alternative randomization plans and incentives has contributed to their operational feasibility; legal, ethical, and professional arguments for experimentation have matured; and expectations have become better aligned with practical constraints that are likely…

  3. The MAJORANA Experiment

    Energy Technology Data Exchange (ETDEWEB)

    Guiseppe, V.E. [Univ S Dakota; Keller, C. [Univ S Dakota; Mei, D-M [Univ S Dakota; Perevozchikov, O. [Univ S Dakota; Perumpilly, G. [Univ S Dakota; Thomas, K. [Univ S Dakota; Xiang, W. [Univ S Dakota; Zhang, C. [Univ S Dakota; Aalseth, C.E. [Pacific NW Natl Lab Richland, WA; Aguayo, E. [Pacific NW Natl Lab Richland, WA; Ely, J. [Pacific NW Natl Lab Richland, WA; Fast, J.E. [Pacific NW Natl Lab Richland, WA; Hoppe, E.W. [Pacific NW Natl Lab Richland, WA; Hossbach, T.W. [Pacific NW Natl Lab Richland, WA; Keillor, M. [Pacific NW Natl Lab Richland, WA; Kephart, J.D. [Pacific NW Natl Lab Richland, WA; Kouzes, R. [Pacific NW Natl Lab Richland, WA; Miley, H.S. [Pacific NW Natl Lab Richland, WA; Mizouni, L. [Pacific NW Natl Lab Richland, WA; Myers, A.W. [Pacific NW Natl Lab Richland, WA; Reid, D. [Pacific NW Natl Lab Richland, WA; Amman, M. [Lawrence Berkeley National Laboratory (LBNL); Bergevin, M. [Lawrence Berkeley National Laboratory (LBNL); Chan, Y-D [Lawrence Berkeley National Laboratory (LBNL); Detwiler, J.A. [Lawrence Berkeley National Laboratory (LBNL); Loach, J.C. [Lawrence Berkeley National Laboratory (LBNL); Luke, P.N. [Lawrence Berkeley National Laboratory (LBNL); Martin, R.D. [Lawrence Berkeley National Laboratory (LBNL); Poon, A.W.P. [Lawrence Berkeley National Laboratory (LBNL); Prior, G. [Lawrence Berkeley National Laboratory (LBNL); Vetter, K. [Lawrence Berkeley National Laboratory (LBNL); Yaver, H. [Lawrence Berkeley National Laboratory (LBNL); Avignone, F.T. III [University of South Carolina; Creswick, R. [University of South Carolina; Farach, H. [University of South Carolina; Mizouni, L. [University of South Carolina; Avignone, Frank Titus [ORNL; Bertrand Jr, Fred E [ORNL; Capps, Gregory L [ORNL; Cooper, Reynold J [ORNL; Radford, David C [ORNL; Varner Jr, Robert L [ORNL; Wilkerson, John F [University of North Carolina, Chapel Hill; Yu, Chang-Hong [ORNL; Back, H.O. [University of North Carolina; Leviner, L. [North Carolina State University; Young, A.R. [North Carolina State University; Back (et al.), H.O. [Triangle Universities Nuclear Laboratory, Durham, NC; Bai, X. [South Dakota School of Mines and Technology; Hong, H. [South Dakota School of Mines and Technology; Howard, S. [South Dakota School of Mines and Technology; Medlin, D. [South Dakota School of Mines and Technology; Sobolev, V. [South Dakota School of Mines and Technology; Barabash, A.S. [Inst Theort & Expt Phys, Moscow, Russia; Konovalov, S.I. [Inst Theort & Expt Phys, Moscow, Russia; Vanyushin, I. [Inst Theort & Expt Phys, Moscow, Russia; Yumatov, V. [Inst Theort & Expt Phys, Moscow, Russia; Barbeau, P.S. [University of Chicago; Collar, J.I. [University of Chicago; Fields, N. [University of Chicago; Boswell (et al.), M. [Los Alamos National Laboratory (LANL); Brudanin, V. [Joint Institute for Nuclear Research, Dubna, Russia; Egorov, V. [Joint Institute for Nuclear Research, Dubna, Russia; Gusey, K. [Joint Institute for Nuclear Research, Dubna, Russia; Kochetov, O. [Joint Institute for Nuclear Research, Dubna, Russia; Shirchenko, M. [Joint Institute for Nuclear Research, Dubna, Russia; Timkin, V. [Joint Institute for Nuclear Research, Dubna, Russia; Yakushev, E. [Joint Institute for Nuclear Research, Dubna, Russia; Bugg, W. [University of Tennessee, Knoxville (UTK); Efremenko, M. [University of Tennessee, Knoxville (UTK); Burritt (et al.), T.H. [University of Washington, Ctr Expt Nucle Phys & Astrophys; Burritt (et al.), T.H. [University of Washington, Dept Phys, Seattle, WA; Busch, M. [Duke University; Esterline, J. [Duke University; Swift, G. [Duke University; Tornow, W. [Duke University/TUNL; Ejiri, H. [Osaka University; Hazama, R. [Osaka University; Nomachi, M. [Osaka University; Shima, T. [Osaka University; Finnerty (et al.), P. [University of North Carolina; et al.

    2011-01-01

    The Majorana Collaboration is assembling an array of HPGe detectors to search for neutrinoless double-beta decay in {sup 76}Ge. 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. Remote Agent Experiment

    Science.gov (United States)

    Benard, Doug; Dorais, Gregory A.; Gamble, Ed; Kanefsky, Bob; Kurien, James; Millar, William; Muscettola, Nicola; Nayak, Pandu; Rouquette, Nicolas; Rajan, Kanna; Norvig, Peter (Technical Monitor)

    2000-01-01

    Remote Agent (RA) is a model-based, reusable artificial intelligence (At) software system that enables goal-based spacecraft commanding and robust fault recovery. RA was flight validated during an experiment on board of DS1 between May 17th and May 21th, 1999.

  5. Dismantling the ALEPH experiment

    CERN Multimedia

    Patrice Loïez

    2001-01-01

    The barrel and its two end-caps have been separated and removed from their cables; the detector will be broken into individual parts. ALEPH was an experiment on the LEP accelerator, which studied high-energy collisions between electrons and positrons from 1989 to 2000.

  6. Invisalign: early experiences.

    Science.gov (United States)

    Joffe, L

    2003-12-01

    This article describes the Invisalign technique. It is based on the author's personal experience of over 60 cases started in the private practice setting. The technology behind Invisalign and its development is reviewed. The Invisalign clinical technique is described, and the advantages and disadvantages of using Invisalign are highlighted.

  7. The ATRAP experiment

    CERN Multimedia

    Laurent Guiraud

    2000-01-01

    The Antihydrogen Trap (ATRAP) experiment was the first machine to accumulate cold antiprotons, produced in the AD, and combine them with positrons (antielectrons) to form antihydrogen. The study of antihydrogen in comparison with hydrogen will hopefully provide insight into the differences between matter and antimatter, especially in comparing their mass and spectra.

  8. Experiment at Evergreen.

    Science.gov (United States)

    Jones, Richard M.

    The philosophy and structure of programs of coordinated study at Evergreen State College from 1970 to 1980 are discussed, and comparisons are made to the experiment at the University of California, Berkeley, promoted by Joseph Tussman. Both programs are based on ideas suggested by Alexander Meiklejohn and his Experimental College at the University…

  9. 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

  10. Hydrothermal organic synthesis experiments

    Science.gov (United States)

    Shock, Everett L.

    1992-01-01

    The serious scientific debate about spontaneous generation which raged for centuries reached a climax in the nineteenth century with the work of Spallanzani, Schwann, Tyndall, and Pasteur. These investigators demonstrated that spontaneous generation from dead organic matter does not occur. Although no aspects of these experiments addressed the issue of whether organic compounds could be synthesized abiotically, the impact of the experiments was great enough to cause many investigators to assume that life and its organic compounds were somehow fundamentally different than inorganic compounds. Meanwhile, other nineteenth-century investigators were showing that organic compounds could indeed be synthesized from inorganic compounds. In 1828 Friedrich Wohler synthesized urea in an attempt to form ammonium cyanate by heating a solution containing ammonia and cyanic acid. This experiment is generally recognized to be the first to bridge the artificial gap between organic and inorganic chemistry, but it also showed the usefulness of heat in organic synthesis. Not only does an increase in temperature enhance the rate of urea synthesis, but Walker and Hambly showed that equilibrium between urea and ammonium cyanate was attainable and reversible at 100 C. Wohler's synthesis of urea, and subsequent syntheses of organic compounds from inorganic compounds over the next several decades dealt serious blows to the 'vital force' concept which held that: (1) organic compounds owe their formation to the action of a special force in living organisms; and (2) forces which determine the behavior of inorganic compounds play no part in living systems. Nevertheless, such progress was overshadowed by Pasteur's refutation of spontaneous generation which nearly extinguished experimental investigations into the origins of life for several decades. Vitalism was dealt a deadly blow in the 1950's with Miller's famous spark-discharge experiments which were undertaken in the framework of the Oparin

  11. The ATLAS experiment

    CERN Document Server

    Dunford, Monica Lynn

    2014-01-01

    In particle physics experiments, the discovery of increasingly more massive particles has brought deep understanding of the basic constituents of matter and of the fundamental forces among them. In order to explore Nature in its deepest elementary secrets, the Large Hadron Collider (LHC) was built at CERN, Geneva. The LHC provides the highest energy collisions in a laboratory, at very high rates to allow one to study very rare reactions. Two independent sophisticated huge instruments, called ATLAS and CMS detectors, are operated to explore in a most broad way the physics of these collisions. In addition to these two general-purpose detectors, smaller specialized experiments (LHCb, ALICE and some others) are collecting collision data as well.

  12. 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.

  13. Shooting Star Experiment

    Science.gov (United States)

    1997-01-01

    The Shooting Star Experiment (SSE) is designed to develop and demonstrate the technology required to focus the sun's energy and use the energy for inexpensive space Propulsion Research. Pictured is an engineering model (Pathfinder III) of the Shooting Star Experiment (SSE). This model was used to test and characterize the motion and deformation of the structure caused by thermal effects. In this photograph, alignment targets are being placed on the engineering model so that a theodolite (alignment telescope) could be used to accurately measure the deformation and deflections of the engineering model under extreme conditions, such as the coldness of deep space and the hotness of the sun as well as vacuum. This thermal vacuum test was performed at the X-Ray Calibration Facility because of the size of the test article and the capabilities of the facility to simulate in-orbit conditions

  14. Reproducible Experiment Platform

    CERN Document Server

    Likhomanenko, Tatiana; Baranov, Alexander; Khairullin, Egor; Ustyuzhanin, Andrey

    2015-01-01

    Data analysis in fundamental sciences nowadays is an essential process that pushes frontiers of our knowledge and leads to new discoveries. At the same time we can see that complexity of those analyses increases fast due to a)~enormous volumes of datasets being analyzed, b)~variety of techniques and algorithms one have to check inside a single analysis, c)~distributed nature of research teams that requires special communication media for knowledge and information exchange between individual researchers. There is a lot of resemblance between techniques and problems arising in the areas of industrial information retrieval and particle physics. To address those problems we propose Reproducible Experiment Platform (REP), a software infrastructure to support collaborative ecosystem for computational science. It is a Python based solution for research teams that allows running computational experiments on shared datasets, obtaining repeatable results, and consistent comparisons of the obtained results. We present s...

  15. Experiments around I-8

    CERN Multimedia

    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.

  16. Infrastructures as Ontological Experiments

    Directory of Open Access Journals (Sweden)

    Casper Bruun Jensen

    2015-11-01

    Full Text Available Ontology has recently gained renewed attention in science and technology studies and anthropology (e.g. Gad, Jensen and Winthereik 2015; Holbraad, Pedersen and Viveiros de Castro 2014; Woolgar and Lezaun 2013. Yet, it has a considerably longer pedigree than these recent debates might lead one to think. Experiments, of course, have long held the attention of sociologists, historians, and philosophers of science (Collins 1985; Gooding 1990; Shapin and Schaffer 1985. And infrastructures have been the focus of sustained inquiry in the sociology and history of technology (Bowker 1994; Hughes 1983. Once these terms are put into conjunction, however, each gets a somewhat different inflection. The following note briefly explores the conceptual purchase of considering infrastructures as ontological experiments.

  17. 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.

  18. Understanding Popper's Experiment

    CERN Document Server

    Qureshi, T

    2004-01-01

    An experiment, proposed by Karl Popper, is considered by many, to be a crucial test of quantum mechanics. While many loop holes have been pointed out in the original proposal, it turned out that they are not crucial to the test. We point out what is fundamentally wrong with the proposal. Using just the standard interpretation of quantum mechanics, we demonstrate that Popper's basic premise was faulty.

  19. 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.

  20. "We work on experiments"

    CERN Multimedia

    BBC

    1975-01-01

    Short BBC (?) report on CERN. Emphasis on international collaboration, with Soviets and Chinese highlighted. At end, interview with Danish physicist (our of sync) who talks about working on Sundays and how experiments have 25 people, therefore it is a long way to glory. ISR. Comments: Film scratched and dirty. Colour pink. Looses sync at the end. Video transfer ends before the end of the film.

  1. The CONNIE experiment

    Science.gov (United States)

    Aguilar-Arevalo, A.; Bertou, X.; Bonifazi, C.; Butner, M.; Cancelo, G.; Castaneda Vazquez, A.; Cervantes Vergara, B.; Chavez, C. R.; Da Motta, H.; D'Olivo, J. C.; Dos Anjos, J.; Estrada, J.; Fernandez Moroni, G.; Ford, R.; Foguel, A.; Hernandez Torres, K. P.; Izraelevitch, F.; Kavner, A.; Kilminster, B.; Kuk, K.; Lima, H. P., Jr.; Makler, M.; Molina, J.; Moreno-Granados, G.; Moro, J. M.; Paolini, E. E.; Sofo Haro, M.; Tiffenberg, J.; Trillaud, F.; Wagner, S.

    2016-10-01

    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.

  2. The CONNIE experiment

    CERN Document Server

    Aguilar-Arevalo, A; Bonifazi, C; Butner, M; Cancelo, G; Vazquez, A Castaneda; Vergara, B Cervantes; Chavez, C R; Da Motta, H; D'Olivo, J C; Anjos, J Dos; Estrada, J; Moroni, G Fernandez; Ford, R; Foguel, A; Torres, K P Hernandez; Izraelevitch, F; Kavner, A; Kilminster, B; Kuk, K; Lima, H P; Makler, M; Molina, J; Moreno-Granados, G; Moro, J M; Paolini, E E; Haro, M Sofo; Tiffenberg, J; Trillaud, F; Wagner, S

    2016-01-01

    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.

  3. 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.

  4. TEAM Experience in Astronomy

    Science.gov (United States)

    McGruder, C. H., III; Tyson, N.; Williams, B.; Hackney, K.; Hackney, R.; Rudloff, M.; Scott, R.; Tyler, R.

    1996-05-01

    The purpose of the project was to increase minority interest and appreciation of science using astronomy as a tool. This goal was achieved by inviting high school minority students and their teachers to the campus of Western Kentucky University to participate along with minority role models in team based learning experiences. All participants worked together in many hands-on activities and events centered around the exploration of space.

  5. STELLA Experiment - Microbunch Diagnostic

    Energy Technology Data Exchange (ETDEWEB)

    He, P.; Liu, Y.; Cline, D. B.; Babzien, M.; Gallardo, J. C.; Kusche, K. P.; Pogorelsky, I. V.; Skaritka, J.; van Steenbergen, A.; Yakimenko, V.; Kimura, W. D.

    1998-07-01

    A microbunch diagnostic system is built at the Accelerator Test Facility (ATF) of Brookhaven National Laboratory for monitoring microbunches (10-fs bunch length) produced by the Inverse Free Electron Laser accelerator in Staged Electron Laser Acceleration experiment. It is similar to one already demonstrated at the ATF. With greatly improved beam optics conditions higher order harmonic coherent transition radiation will be measurable to determine the microbunch length and shape.

  6. 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.

  7. Analysis of Factorial Experiments.

    Science.gov (United States)

    1985-09-01

    The pro- bability that y 13 is bad is .37. Choosing Q greater than .37 ends the iteration. Choos - ing Q so that Y 13 is identified as a bad...ances as a Fraction of Their Total. Annals of Eugenics , 11, 47-52. Cochran, W. G. and Cox, G. M. (1957) Experimental Designs. New York: John Wiley...Experiments. London: Oliver and Boyd. Finney, D. J. (1945) The Fractional Replication of Factorial Arrangements. Annals of Eugenics , 12, 291-301

  8. 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.

  9. [GIST: our experience].

    Science.gov (United States)

    Rossi, M T; Fasano, G; Morlino, A; Scutari, F

    2009-01-01

    Gastrointestinal stromal tumors (GIST) are generally found in the stomach or small intestine and less commonly in the colon and rectum. Complete surgical remove remains the best current therapy. In the treatment of advanced/metastatic GIST is available imatinib, a selective tyrosine kinase inhibitor. In our experience, 12 patients observed between 2000 and 2007 and affected by GIST underwent surgery, in one case associated to treatment with imatinib.

  10. 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...

  11. Long Baseline Neutrino Experiments

    Science.gov (United States)

    Mezzetto, Mauro

    2016-05-01

    Following the discovery of neutrino oscillations by the Super-Kamiokande collaboration, recently awarded with the Nobel Prize, two generations of long baseline experiments had been setup to further study neutrino oscillations. The first generation experiments, K2K in Japan, Minos in the States and Opera in Europe, focused in confirming the Super-Kamiokande result, improving the precision with which oscillation parameters had been measured and demonstrating the ντ appearance process. Second generation experiments, T2K in Japan and very recently NOνA in the States, went further, being optimized to look for genuine three neutrino phenomena like non-zero values of θ13 and first glimpses to leptonic CP violation (LCPV) and neutrino mass ordering (NMO). The discovery of leptonic CP violation will require third generation setups, at the moment two strong proposals are ongoing, Dune in the States and Hyper-Kamiokande in Japan. This review will focus a little more in these future initiatives.

  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)

    Riesen, van Siswa A.N.; Gijlers, Hannie; Anjewierden, Anjo; Jong, de Ton

    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. Situating Emotional Experience

    Directory of Open Access Journals (Sweden)

    Christine D Wilson-Mendenhall

    2013-11-01

    Full Text Available Psychological construction approaches to emotion suggest that emotional experience is situated and dynamic. Fear, for example, is typically studied in a physical danger context (e.g., threatening snake, but in the real world, it often occurs in social contexts, especially those involving social evaluation (e.g., public speaking. Understanding situated emotional experience is critical because adaptive responding is guided by situational context (e.g., inferring the intention of another in a social evaluation situation vs. monitoring the environment in a physical danger situation. In an fMRI study, we assessed situated emotional experience using a newly developed paradigm in which participants vividly imagine different scenarios from a first-person perspective, in this case scenarios involving either social evaluation or physical danger. We hypothesized that distributed neural patterns would underlie immersion in social evaluation and physical danger situations, with shared activity patterns across both situations in multimodal sensory regions and in circuitry involved in integrating salient sensory information, and with unique activity patterns for each situation type in coordinated large-scale networks that reflect situated responding. More specifically, we predicted that networks underlying the social inference and mentalizing involved in responding to a social threat (in regions that make up the default mode network would be reliably more active during social evaluation situations. In contrast, networks underlying the visuospatial attention and action planning involved in responding to a physical threat would be reliably more active during physical danger situations. The results supported these hypotheses. In line with emerging psychological construction approaches, the findings suggest that coordinated brain networks offer a systematic way to interpret the distributed patterns that underlie the diverse situational contexts characterizing

  16. Experience the Moon

    Science.gov (United States)

    Ortiz-Gil, A.; Benacchio, L.; Boccato, C.

    2011-10-01

    The Moon is, together with the Sun, the very first astronomical object that we experience in our life. As this is an exclusively visual experience, people with visual impairments need a different mode to experience it too. This statement is especially true when events, such as more and more frequent public observations of sky, take place. This is the reason why we are preparing a special package for visual impaired people containing three brand new items: 1. a tactile 3D Moon sphere in Braille with its paper key in Braille. To produce it we used imaging data obtained by NASA's mission Clementine, along with free image processing and 3D rendering software. In order to build the 3D small scale model funding by Europlanet and the Italian Ministry for Research have been used. 2. a multilingual web site for visually impaired users of all ages, on basic astronomy together with an indepth box about the Moon; 3. a book in Braille with the same content of the Web site mentioned above. All the items will be developed with the collaboration of visually impaired people that will check each step of the project and support their comments and criticism to improve it. We are going to test this package during the next International Observe the Moon Night event. After a first testing phase we'll collect all the feedback data in order to give an effective form to the package. Finally the Moon package could be delivered to all those who will demand it for outreach or educational goals.

  17. 2169 steel waveform experiments.

    Energy Technology Data Exchange (ETDEWEB)

    Furnish, Michael David; Alexander, C. Scott; Reinhart, William Dodd; Brown, Justin L.

    2012-11-01

    In support of LLNL efforts to develop multiscale models of a variety of materials, we have performed a set of eight gas gun impact experiments on 2169 steel (21% Cr, 6% Ni, 9% Mn, balance predominantly Fe). These experiments provided carefully controlled shock, reshock and release velocimetry data, with initial shock stresses ranging from 10 to 50 GPa (particle velocities from 0.25 to 1.05 km/s). Both windowed and free-surface measurements were included in this experiment set to increase the utility of the data set, as were samples ranging in thickness from 1 to 5 mm. Target physical phenomena included the elastic/plastic transition (Hugoniot elastic limit), the Hugoniot, any phase transition phenomena, and the release path (windowed and free-surface). The Hugoniot was found to be nearly linear, with no indications of the Fe phase transition. Releases were non-hysteretic, and relatively consistent between 3- and 5-mmthick samples (the 3 mm samples giving slightly lower wavespeeds on release). Reshock tests with explosively welded impactors produced clean results; those with glue bonds showed transient releases prior to the arrival of the reshock, reducing their usefulness for deriving strength information. The free-surface samples, which were steps on a single piece of steel, showed lower wavespeeds for thin (1 mm) samples than for thicker (2 or 4 mm) samples. A configuration used for the last three shots allows release information to be determined from these free surface samples. The sample strength appears to increase with stress from ~1 GPa to ~ 3 GPa over this range, consistent with other recent work but about 40% above the Steinberg model.

  18. Copenhagen Sonic Experience Map

    DEFF Research Database (Denmark)

    Kreutzfeldt, Jacob

    2011-01-01

    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....... The challenge for planners, designers, and architects is to deal with the auditory not only as pollution but also as an integrated part of urban experience, promoting fellowship and liveliness as well as distress....

  19. Maryland Centrifugal Experiment

    Energy Technology Data Exchange (ETDEWEB)

    Hassam, Adil; Ellis, Richard F

    2016-12-22

    The Maryland Centrifugal Experiment (MCX) Project has investigated the concepts of centrifugal plasma confinement and stabilization of instabilities by velocity shear. The basic requirement is supersonic plasma rotation about a shaped, open magnetic field. Overall, the MCX Project attained three primary goals that were set out at the start of the project. First, supersonic rotation at Mach number upto 2.5 was obtained. Second, turbulence from flute interchange modes was found considerably reduced from conventional. Third, plasma pressure was contained along the field, as evidenced by density drops of x10 from the center to the mirror throats.

  20. Condensed landscape experience

    DEFF Research Database (Denmark)

    Earon, Ofri

    2011-01-01

    demands, quality of space, mixture of functions, urban complexity, public life and cultural heritage. In order to launch such an approach, an understanding of the spatial, social and environmental significance of a radical re-thinking of relationships between architecture and landscape is necessary....... This paper addresses the question of whether the sensation of landscape can be condensed in function or to the size of an urban building. It also discusses the benefits and potentials of the amalgamate, by underlining the unique qualities of such a hybrid. In an attempt to define the experience of landscape...

  1. The QUIJOTE CMB Experiment

    Science.gov (United States)

    Rubiño-Martín, J. A.; Rebolo, R.; Tucci, M.; Génova-Santos, R.; Hildebrandt, S. R.; Hoyland, R.; Herreros, J. M.; Gómez-Reñasco, F.; Caraballo, C. López; Martínez-González, E.; Vielva, P.; Herranz, D.; Casas, F. J.; Artal, E.; Aja, B.; Fuente, L. dela; Cano, J. L.; Villa, E.; Mediavilla, A.; Pascual, J. P.; Piccirillo, L.; Maffei, B.; Pisano, G.; Watson, R. A.; Davis, R.; Davies, R.; Battye, R.; Saunders, R.; Grainge, K.; Scott, P.; Hobson, M.; Lasenby, A.; Murga, G.; Gómez, C.; Gómez, A.; Ariño, J.; Sanquirce, R.; Pan, J.; Vizcargüenaga, A.; Etxeita, B.

    We present the current status of the QUIJOTE (Q-U-I JOint TEnerife) CMB Experiment, a new instrument which will start operations early in 2009 at Teide Observatory with the aim of characterizing the polarization of the CMB and other processes of galactic and extragalactic emission in the frequency range 10-30GHz and at large angular scales. QUIJOTE will be a valuable complement at low frequencies for the PLANCK mission, and will have the required sensitivity to detect a primordial gravitational-wave component if the tensor-to-scalar ratio is larger than r = 0.05.

  2. The Quijote CMB Experiment

    CERN Document Server

    Rubiño-Martín, J A; Tucci, M; Genova-Santos, R; Hildebrandt, S R; Hoyland, R; Herreros, J M; Gomez-Renasco, F; Caraballo, C Lopez; Martínez-González, E; Vielva, P; Herranz, D; Casas, F J; Artal, E; Aja, B; de la Fuente, L; Cano, J L; Villa, E; Mediavilla, A; Pascual, J P; Piccirillo, L; Maffei, B; Pisano, G; Watson, R A; Davis, R; Davies, R; Battye, R; Saunders, R; Grainge, K; Scott, P; Hobson, M; Lasenby, A; Murga, G; Gómez, C; Gómez, A; Arino, J; Sanquirce, R; Pan, J; Vizcarguenaga, A; Etxeita, B

    2008-01-01

    We present the current status of the QUIJOTE (Q-U-I JOint TEnerife) CMB Experiment, a new instrument which will start operations early 2009 at Teide Observatory, with the aim of characterizing the polarization of the CMB and other processes of galactic and extragalactic emission in the frequency range 10-30 GHz and at large angular scales. QUIJOTE will be a valuable complement at low frequencies for the PLANCK mission, and will have the required sensitivity to detect a primordial gravitational-wave component if the tensor-to-scalar ratio is larger than r=0.05.

  3. Solar neutrino experiments

    Energy Technology Data Exchange (ETDEWEB)

    Hampel, W. [Max-Planck-Institut fuer Kernphysik, Heidelberg (Germany)

    1996-11-01

    The present status of experimental solar neutrino research is reviewed. Updated results from the Homestake, Kamiokande, GALLEX and SAGE detectors all show a deficit when compared to recent standard solar model calculations. Two of these detectors, GALLEX and SAGE, have recently been checked with artificial {sup 51}Cr neutrino sources. It is shown that astrophysical scenarios to solve the solar neutrino problems are not favoured by the data. There is hope that the results of forthcoming solar neutrino experiments can provide the answers to the open questions. (author) 6 figs., 3 tabs., 36 refs.

  4. The Next Experiment

    Energy Technology Data Exchange (ETDEWEB)

    Novella, P.

    2009-07-01

    The full text of publication follows: The NEXT experiment will search for the neutrinoless double beta decay with a high-pressure gas- Xenon TPC. Xe{sup 136} is a {beta}{beta}0{nu} emitter and yields an excellent energy resolution when used as a calorimeter, acting both as the source and as the active material of the detector. External backgrounds can be rejected by means of the tracking capabilities of the TPC. A detector containing about 100 kg Xe{sup 136} is expected to be installed at Canfranc Underground Laboratory. (author)

  5. Experiments with twisted light

    Science.gov (United States)

    Courtial, J.; O'Holleran, K.

    2007-06-01

    The generic that is, stable under perturbations nodes of the field in a monochromatic light beam are optical vortices. We describe here their connection to Chladni's nodal lines in the oscillations of metal plates, as well as a few experiments that have been performed with optical vortices. We will describe how optical vortices can be generated experimentally; how it can be shown that they possess orbital angular momentum; how individual photons can be sorted according to their vortex state; and how optical vortices can be used to demonstrate higher-dimensional quantum entanglement.

  6. The NA57 experiment

    CERN Multimedia

    Laurent Guiraud

    1997-01-01

    NA57 is one of the seven experiments (NA44, NA45, NA49, NA50, NA52, WA97/NA57 and WA98) involved in CERN's Heavy Ion programme, which provided evidence for the existence of a new state of matter, the quark-gluon plasma. In this state, quarks, instead of being bound up into more complex particles such as protons and neutrons, are liberated and roam freely. Theory predicts that this state must have existed at about 10 microseconds after the Big Bang, before the formation of matter as we know it today.

  7. Results of railgun experiments

    Energy Technology Data Exchange (ETDEWEB)

    Hawke, R.S.; Brooks, A.L.; Fowler, C.M.; Peterson, D.R.

    1983-04-01

    During the 1979 Megagauss II conference the hypervelocity potential of railguns and the pulsed power technology needed to power them were discussed. Since then, many laboratories have initiated railgun R and D projects for a variety of potential applications. Los Alamos and Lawrence Livermore National Laboratories initiated a collaborative experimental railgun project which resulted in several successes in accelerating projectiles to high velocities, emphasized the limits on railgun operation, and indicated that the numerical modeling of railgun operation was in good agreement with the experiments.

  8. Quantum frequency downconversion experiment

    CERN Document Server

    Takesue, Hiroki

    2010-01-01

    We report the first quantum frequency downconversion experiment. Using the difference frequency generation process in a periodically poled lithium niobate waveguide, we successfully observed the phase-preserved frequency downconversion of a coherent pulse train with an average photon number per pulse of $<$1, from the 0.7-$\\mu$m visible wavelength band to the 1.3-$\\mu$m telecom band. We expect this technology to become an important tool for flexible photonic quantum networking, including the realization of quantum repeater systems over optical fiber using atom-photon entanglement sources for the visible wavelength bands.

  9. Geophysical fluid flow experiment

    Science.gov (United States)

    Broome, B. G.; Fichtl, G.; Fowlis, W.

    1979-01-01

    The essential fluid flow processes associated with the solar and Jovian atmospheres will be examined in a laboratory experiment scheduled for performance on Spacelab Missions One and Three. The experimental instrumentation required to generate and to record convective fluid flow is described. Details of the optical system configuration, the lens design, and the optical coatings are described. Measurement of thermal gradient fields by schlieren techniques and measurement of fluid flow velocity fields by photochromic dye tracers is achieved with a common optical system which utilizes photographic film for data recording. Generation of the photochromic dye tracers is described, and data annotation of experimental parameters on the film record is discussed.

  10. CMS Tracker operational experience

    CERN Document Server

    Fiori, Francesco

    2016-01-01

    The CMS Tracker was repaired, recalibrated and commissioned successfully for the second run of Large Hadron Collider. In 2015 the Tracker performed well with improved hit efficiency and spatial resolution compared to Run I. Operations successfully transitioned to lower temperatures after commissioning environmental control and monitoring. This year the detector is expected to withstand luminosities that are beyond its design limits and will need a combined effort of both online and offline team to yield the high quality data that is required to reach our physics goals. We present the experience gained during the second run of the LHC and show the latest performance results of the CMS Tracker.

  11. PAC Experiments at ISOLDE

    CERN Document Server

    2002-01-01

    The study of hyperfine interactions offers the possibility to use radioactive nuclei as probes in matter. The @g-@g perturbated angular correlation (PAC) technique following implantation has found widespread application in this field. At ISOLDE we have been investigating electric field gradients at impurities in non-cubic metals in an ongoing series of experiments. \\\\ \\\\ The small number of probe atoms necessary for these measurements make them also ideally suited for studies of surface problems like diffusion, structure and dynamics. Cd on a molybdenum O110? surface will be studied as first system. For this purpose 10|1|0~atoms of |1|1|1|mCd will be evaporated onto the clean surface and the electric field gradient for isolated adatoms on terrace sites will be determined by PAC. The UHV system constructed for such experiments at ISOLDE is shown in the schematic drawing. It is coupled to the beam line through differential pumping stations and contains standard surface treatment and analysis equipment.

  12. The Halogen Occultation Experiment

    Science.gov (United States)

    Russell, James M., III; Gordley, Larry L.; Park, Jae H.; Drayson, S. R.; Hesketh, W. D.; Cicerone, Ralph J.; Tuck, Adrian F.; Frederick, John E.; Harries, John E.; Crutzen, Paul J.

    1993-01-01

    The Halogen Occultation Experiment (HALOE) uses solar occultation to measure vertical profiles of O3, HCl, HF, CH4, H2O, NO, NO2, aerosol extinction, and temperature versus pressure with an instantaneous vertical field of view of 1.6 km at the earth limb. Latitudinal coverage is from 80 deg S to 80 deg N over the course of 1 year and includes extensive observations of the Antarctic region during spring. The altitude range of the measurements extends from about 15 km to about 60-130 km, depending on channel. Experiment operations have been essentially flawless, and all performance criteria either meet or exceed specifications. Internal data consistency checks, comparisons with correlative measurements, and qualitative comparisons with 1985 atmospheric trace molecule spectroscopy (ATMOS) results are in good agreement. Examples of pressure versus latitude cross sections and a global orthographic projection for the September 21 to October 15, 1992, period show the utility of CH4, HF, and H2O as tracers, the occurrence of dehydration in the Antarctic lower stratosphere, the presence of the water vapor hygropause in the tropics, evidence of Antarctic air in the tropics, the influence of Hadley tropical upwelling, and the first global distribution of HCl, HF, and NO throughout the stratosphere. Nitric oxide measurements extend through the lower thermosphere.

  13. Postmenopausal osteoporosis: Our experience

    Directory of Open Access Journals (Sweden)

    R N Mehrotra

    2012-01-01

    Full Text Available Introduction: There is very little published literature about experience with osteoporosis treatment from our country. Materials and Methods: It is a retrospective analysis of first 50 patients enrolled in our clinic for osteoporosis. Postmenopausal women with T score of less than -2.5 or history suggestive fragility fracture with supportive bone mineral density (BMD were included. Patients having hypercalcemia, abnormal renal function, myeloma and on long-term steroids were also excluded. Results: Nearly 34% subjects were below the age of 60 years, 47% of subjects were between 60 and 70 years, whereas 18% were above 70 years. Nearly 6% had family history of osteoporosis s or history of osteoporotic fractures. Nearly 20% subjects had fracture prior to starting of any treatment. A total of 86% (40/46 had evidence of Vitamin D (VD deficiency. Nearly 80% of patients were treated with bisphosphonates, 12% were treated with injectable bisphosphonates, and 8% were treated with teriperatide. Nearly 16% patients had duration of more than 5 years of experience with bisphosphonates. Follow up BMD was available in 25 subjects. BMD had improved significantly in 68% of subjects. In 24% the BMD was stable (the change was less than least significant change (LSC. In 8% BMD had shown a significant decline while being on treatment. Conclusion: Postmenopausal osteoporosis occurs in relatively younger women in our country. Majority of them are VD deficient. Oral bisphosphonates is the most common used drug; it is fairly well tolerated and effective.

  14. A communist teaching experiment

    DEFF Research Database (Denmark)

    Skak, Morten

    At the beginning of the course, students were told all the examination questions for the oral examination, and that they, in groups, should produce a report with a) A pedagogical presentation of (the selected part of) the theory/syllabus b) This theory put into perspective by self-produced empiri......At the beginning of the course, students were told all the examination questions for the oral examination, and that they, in groups, should produce a report with a) A pedagogical presentation of (the selected part of) the theory/syllabus b) This theory put into perspective by self......-student teaching. A form of mutual teaching where (student)teachers have good knowledge of the students’ learning problems. The role of the (conventional) teacher: To initiate and supervise the process and act as “final” teacher when this is required. The experiment produced various problems and the students...... disliked the course design. The lack of students’ ability to get credit for own qualifications was seen as a major problem. My presentation will go through the experiment. The ultimate goal of teaching is to produce people who can raise welfare in our societies. How can we measure whether or not a new form...

  15. The EBEX Experiment

    CERN Document Server

    Oxley, P; Baccigalupi, C; De Bernardis, P; Cho, H M; Devlin, M J; Hanany, S; Johnson, B R; Jones, T; Lee, A T; Matsumura, T; Miller, A D; Milligan, M; Renbarger, T; Spieler, H G; Stompor, R; Tucker, G S; Zaldarriaga, M

    2004-01-01

    EBEX is a balloon-borne polarimeter designed to measure the intensity and polarization of the cosmic microwave background radiation. The measurements would probe the inflationary epoch that took place shortly after the big bang and would significantly improve constraints on the values of several cosmological parameters. EBEX is unique in its broad frequency coverage and in its ability to provide critical information about the level of polarized Galactic foregrounds which will be necessary for all future CMB polarization experiments. EBEX consists of a 1.5 m Dragone-type telescope that provides a resolution of less than 8 arcminutes over four focal planes each of 4 degree diffraction limited field of view at frequencies up to 450 GHz. The experiment is designed to accommodate 330 transition edge bolometric detectors per focal plane, for a total of up to 1320 detectors. EBEX will operate with frequency bands centered at 150, 250, 350, and 450 GHz. Polarimetry is achieved with a rotating achromatic half-wave pla...

  16. Advanced Liquid Feed Experiment

    Science.gov (United States)

    Distefano, E.; Noll, C.

    1993-06-01

    The Advanced Liquid Feed Experiment (ALFE) is a Hitchhiker experiment flown on board the Shuttle of STS-39 as part of the Space Test Payload-1 (STP-1). The purpose of ALFE is to evaluate new propellant management components and operations under the low gravity flight environment of the Space Shuttle for eventual use in an advanced spacecraft feed system. These components and operations include an electronic pressure regulator, an ultrasonic flowmeter, an ultrasonic point sensor gage, and on-orbit refill of an auxiliary propellant tank. The tests are performed with two transparent tanks with dyed Freon 113, observed by a camera and controlled by ground commands and an on-board computer. Results show that the electronic pressure regulator provides smooth pressure ramp-up, sustained pressure control, and the flexibility to change pressure settings in flight. The ultrasonic flowmeter accurately measures flow and detects gas ingestion. The ultrasonic point sensors function well in space, but not as a gage during sustained low-gravity conditions, as they, like other point gages, are subject to the uncertainties of propellant geometry in a given tank. Propellant transfer operations can be performed with liquid-free ullage equalization at a 20 percent fill level, gas-free liquid transfer from 20-65 percent fill level, minimal slosh, and can be automated.

  17. The NEXT experiment

    CERN Document Server

    Gomez-Cadenas, Juan Jose

    2014-01-01

    NEXT (Neutrino Experiment with a Xenon TPC) is an experiment to search neutrinoless double beta decay processes (bb0nu) in Xe136. The NEXT technology is based in the use of time projection chambers operating at a typical pressure of 15 bar and using electroluminescence to amplify the signal (HPXE). The main advantages of the experimental technique are: a) excellent energy resolution; b) the ability to reconstruct the trajectory of the two electrons emitted in the decays, which further contributes to the suppression of backgrounds; c) scalability to large masses; and d) the possibility to reduce the background to negligible levels thanks to the barium tagging technology (BATA). The NEXT roadmap was designed in four stages: i) Demonstration of the HPXE technology with prototypes deploying a mass of natural xenon in the range of 1 kg, using the NEXT-DEMO (IFIC) and NEXT-DBDM (Berkeley) prototypes; ii) Characterisation of the backgrounds to the bb0nu signal and measurement of the bb2nu signal with the NEW detecto...

  18. Operational experience at ELBE

    Science.gov (United States)

    Michel, P.; Lehnert, U.; Seidel, W.

    2015-05-01

    The ELBE center for high power radiation sources is the largest user facility in the Helmholtz-Zentrum Dresden- Rossendorf. The facility is based on a 36 MeV superconducting RF Linac which can be operated up to 1.6 mA in cw mode. The electron beam is used to generate secondary radiation, such as infrared light (Free Electron Lasers), coherent THz radiation, MeV-Bremsstrahlung, fast neutrons and positrons for a wide range of basic research like semiconductor physics, nuclear astrophysics and radio biological investigations. Two high power laser systems (500 TW Ti:Sa laser, 2 PW diode pumped laser) are under construction for laser acceleration experiments and X-ray generation by Thomson scattering. The FELs are in operation since 2004 (mid-IR FEL, 4-22μm) and 2006 (far-IF FEL, 20-250μm). The fundamental features of the ELBE IR FELs, the FEL instrumentation and advanced beam diagnostics for the photon beam are described. During ten years of user operation experiences and statistical data were collected.

  19. Experiments with Ξ- atoms

    Science.gov (United States)

    Batty, C. J.; Friedman, E.; Gal, A.

    1999-01-01

    Experiments with Ξ- atoms are proposed in order to study the nuclear interaction of Ξ hyperons. The production of Ξ- in the (K-,K+) reaction, the Ξ- stopping in matter, and its atomic cascade are incorporated within a realistic evaluation of the results expected for Ξ- x-ray spectra across the periodic table, using an assumed Ξ-nucleus optical potential Vopt. Several optimal targets for measuring the strong-interaction shift and width of the x-ray transition to the ``last'' atomic level observed are singled out: F, Cl, I, and Pb. The sensitivity of these observables to the parameters of Vopt is considered. The relevance of such experiments is discussed in the context of strangeness -2 nuclear physics and multistrange nuclear matter. Finally, with particular reference to searches for the H dibaryon, the properties of Ξ-d atoms are also discussed. The role of Stark mixing and its effect on S and P state capture of Ξ- by the deuteron together with estimates of the resulting probability for producing the H dibaryon are considered in detail.

  20. The Nucifer Experiment

    Energy Technology Data Exchange (ETDEWEB)

    Cucoanes, A.S., E-mail: cucoanes@subatech.in2p3.fr

    2014-06-15

    In nuclear reactors, a large number of antineutrinos are generated in the decay chains of the fission products; thus a survey of the antineutrino flux could provide valuable information related to the uranium and plutonium content of the core. This application generated interest by the IAEA in using antineutrino detectors as a potential safeguard tool. Here we present the Nucifer experiment, developed in France, by CEA and CNRS/IN2P3. The design of this new antineutrino detector has focused on safety, size reduction, reliability and high detection efficiency with a good background rejection. The Nucifer detector is currently taking data at the OSIRIS research reactor, inside CEA-Saclay. Presently, the ongoing analyses are considering the main sources of background for the antineutrino detection; the first antineutrino result is expected in 2013. A possible contribution to the understanding of the so called “reactor antineutrino anomaly” is also discussed. Finally, we present a brief description of the proposed experiments at very short baselines (VSBL) from reactors in France.

  1. The Virtual Arizona Experience

    Science.gov (United States)

    Allison, M. L.; Davis, R.; Conway, F. M.; Bellasai, R.

    2012-12-01

    To commemorate the once-in-a-lifetime event of Arizona's hundredth birthday, the Centennial Commission and the Governor of Arizona envisioned a museum and companion website that would capture the state's history, celebrate its people, and embrace its future. Working with world-renowned museum designers, the state began to seek ideas from across Arizona to create plans for a journey of discovery through science and the humanities. The museum would introduce visitors to some of the people who nurtured the state through its early years and others who are innovating its tomorrows. Showcases would include the resources and experiences that shaped the state's history and are transforming its present day, highlighting the ingenuity that tamed the wild frontier and is envisioning Arizona's next frontiers through science and technology. The Arizona Experience (www.arizonaexperience.org) was initially intended to serve as the web presence for the physical museum, but as delays occurred with the physical museum, the site has quickly developed an identify of its own as an interactive, multimedia experience, reaching a wider audience with functions that would be difficult or expensive to produce in a museum. As leaders in scientific and technological innovation in the state, the Arizona Geological Survey was tasked with designing and creating the Arizona Experience site. The general themes remain the same; however, the site has added content and applications that are better suited to the online environment in order to create a rich, dynamic supplement to a physical museum experience. The website offers the features and displays of the future museum with the interactive nature and learning environment of the web. This provides an encyclopedic overview of the State of Arizona by subject matter experts in a manner that is free and open to the public and erases socio-economic, political, and physical boundaries. Over the Centennial Year of 2012 the site will release a new theme and

  2. Petascale system management experiences.

    Energy Technology Data Exchange (ETDEWEB)

    Desai, N.; Bradshaw, R.; Lueninghoener, C.; Cherry, A.; Coghlan, S.; Scullin, W. (LCF); ( MCS)

    2008-01-01

    Petascale High-Performance Computing (HPC) systems are among the largest systems in the world. Intrepid, one such system, is a 40,000 node, 556 teraflop Blue Gene/P system that has been deployed at Argonne National Laboratory. In this paper, we provide some background about the system and our administration experiences. In particular, due to the scale of the system, we have faced a variety of issues, some surprising to us, that are not common in the commodity world. We discuss our expectations, these issues, and approaches we have used to address them. HPC systems are a bellwether for computing systems at large, in multiple regards. HPC users are motivated by the need for absolute performance; this results in two important pushes. HPC users are frequently early adopters of new technologies and techniques. Successful technologies, like Infiniband, prove their value in HPC before gaining wider adoption. Unfortunately, this early adoption alone is not sufficient to achieve the levels of performance required by HPC users; parallelism must also be harnessed. Over the last 15 years, beowulf clustering has provided amazing accessibility to non-HPC-savvy and even non-technical audiences. During this time, substantial adoption of clustering has occurred in many market segments unrelated to computational science. A simple trend has emerged: the scale and performance of high-end HPC systems are uncommon at first, but become commonplace over the course of 3-5 years. For example, in early 2003, several systems on the Top500 list consisted of either 1024 nodes or 4096-8192 cores. In 2008, such systems are commonplace. The most recent generation of high-end HPC systems, so called petascale systems, are the culmination of years of research and development in research and academia. Three such systems have been deployed thus far. In addition to the 556 TF Intrepid system at Argonne National Laboratory, a 596 TF Blue Gene/L-based system has been deployed at Lawrence Livermore

  3. 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...... in an emerging  experience city.  The first part of the article describes the growing and uncritical interest in the concepts of the ‘creative economy’, ‘creative class’ (Florida 2002) and their relationship with cultural production and economic growth (Bille & Schulze 2006), many of which, however, are driven...... mainly by political discourses producing more strategies and reports or commodified ´experience projects´ that are not rooted in real democratic experiments. Instead, the experience based development should emphasize a more object-oriented and a critical approach where real urban experiments link public...

  4. Aesthetic experience of dance performances

    Directory of Open Access Journals (Sweden)

    Vukadinović Maja

    2012-01-01

    Full Text Available In this study the aesthetic experience of dance performances is investigated. The study includes construction of an instrument for measuring the aesthetic experience of dance performances and an investigation of the structure of both dancers’ and spectators’ aesthetic experience. The experiments are carried out during eight different performances of various dance forms, including classical ballet, contemporary dance, flamenco and folklore. Three factors of aesthetic experience of dance performances are identified: Dynamism, Exceptionality and Affective Evaluation. The results show that dancers’ aesthetic experience has a somewhat different factorial structure from that of the spectators’. Unlike spectators’ aesthetic experience, dancers’ aesthetic experience singles out the Excitement factor. The results are discussed within the context of dancers’ proprioception and spectators’ exteroception since these findings confirm the idea of a significant role of proprioception in dancers’ aesthetic experience.

  5. The trapped human experiment.

    Science.gov (United States)

    Huo, R; Agapiou, A; Bocos-Bintintan, V; Brown, L J; Burns, C; Creaser, C S; Devenport, N A; Gao-Lau, B; Guallar-Hoyas, C; Hildebrand, L; Malkar, A; Martin, H J; Moll, V H; Patel, P; Ratiu, A; Reynolds, J C; Sielemann, S; Slodzynski, R; Statheropoulos, M; Turner, M A; Vautz, W; Wright, V E; Thomas, C L P

    2011-12-01

    This experiment observed the evolution of metabolite plumes from a human trapped in a simulation of a collapsed building. Ten participants took it in turns over five days to lie in a simulation of a collapsed building and eight of them completed the 6 h protocol while their breath, sweat and skin metabolites were passed through a simulation of a collapsed glass-clad reinforced-concrete building. Safety, welfare and environmental parameters were monitored continuously, and active adsorbent sampling for thermal desorption GC-MS, on-line and embedded CO, CO(2) and O(2) monitoring, aspirating ion mobility spectrometry with integrated semiconductor gas sensors, direct injection GC-ion mobility spectrometry, active sampling thermal desorption GC-differential mobility spectrometry and a prototype remote early detection system for survivor location were used to monitor the evolution of the metabolite plumes that were generated. Oxygen levels within the void simulator were allowed to fall no lower than 19.1% (v). Concurrent levels of carbon dioxide built up to an average level of 1.6% (v) in the breathing zone of the participants. Temperature, humidity, carbon dioxide levels and the physiological measurements were consistent with a reproducible methodology that enabled the metabolite plumes to be sampled and characterized from the different parts of the experiment. Welfare and safety data were satisfactory with pulse rates, blood pressures and oxygenation, all within levels consistent with healthy adults. Up to 12 in-test welfare assessments per participant and a six-week follow-up Stanford Acute Stress Response Questionnaire indicated that the researchers and participants did not experience any adverse effects from their involvement in the study. Preliminary observations confirmed that CO(2), NH(3) and acetone were effective markers for trapped humans, although interactions with water absorbed in building debris needed further study. An unexpected observation from the NH(3

  6. Experience Report for WOPR

    Energy Technology Data Exchange (ETDEWEB)

    Pope, G

    2010-04-06

    One of the purposes of the SQA effort at LLNL is to attempt to determine the 'goodness' of the research codes used for various scientific applications. Typically these are two and three dimensional multi-physics simulation and modeling codes. These legacy research codes are used for applciations such as atmospheric dispersion modeling and analysis and prediction of the performance of engineered systems. These codes are continually subjected to automated regression test suites consisting of verified and validated expected results. Code is managed in repositories. Experience level of developers is high in the knowledge domain, platforms, and languages used. Code size of the multi-physics code used in this study was 578,242 lines excluding comment and blank lines or 5538.7 function points. Languages were 70% C++, 20% C, and 10% Fortran. The code has 130 users and a development team of 14 and an embedded SQE. The code has achieved 100% prime feature test coverage, 73.6% functional test coverage, and 71.5% statement test coverage. The average cyclomatic complexity of the code was 6.25. The codes have evolved over 10 years. Research codes are challenging because there is a desire to balance agility with discipline as well as compliance with DOE standards. Agility is important to allow experimentation with new algorithms and addition of the latest physics features. Discipline is important to increase the quality of the codes. Automation of processes and defect prevention/detection are deployed throughout the software development process. Since resarch codes are a small segment of the software industry, not much information exists in terms of reliability studies on these types of codes. This paper describes attempts to determine the goodness of these research codes. Goodness defined as both correctness of the codes and their fault densities. Correctness is determined by user interviews, peer review; feature based automated testing, and coverage measurement. This

  7. Tribology theory versus experiment

    Science.gov (United States)

    Ferrante, John

    1987-01-01

    Tribology, the study of friction and wear of materials, has achieved a new interest because of the need for energy conservation. Fundamental understanding of this field is very complex and requires a knowledge of solid-state physics, material science, chemistry, and mechanical engineering. This paper is meant to be didactic in nature and outlines some of the considerations needed for a tribology research program. The approach is first to present a simple model, a field emission tip in contact with a flat surface, in order to elucidate important considerations, such as contact area, mechanical deformations, and interfacial bonding. Then examples from illustrative experiments are presented. Finally, the current status of physical theories concerning interfacial bonding are presented.

  8. Coblation Tonsillectomy our experience

    Directory of Open Access Journals (Sweden)

    Balasubramanian Thiagarajan

    2014-03-01

    Full Text Available Tonsillectomy happens to be the commonly performed surgery these days. Like any other surgical procedure this surgical procedure has also undergone tremendous technological changes. One such evolving change happens to be coblation tonsillectomy. Coblation technology is actually an offshoot of radiofrequency surgery. This technique involves passing radiofrequency energy through a conductive medium like isotonic sodium chloride or potassium chloride solution. This produces a plasma field which is composed of sodium and hydroxyl ions which ablates tissue. This tissue ablation takes place at (60-70° C which is much lower than that achieved during other electro surgical techniques (400 – 600° C. This article attempts to discuss the use of this technology to perform tonsillectomy with special emphasis on sharing our experience with the system. This study involves critical appraisal of 25 coblation tonsillectomy surgeries performed at Stanley Medical college during the year 2013.

  9. Transgressive first clinical experiences

    DEFF Research Database (Denmark)

    Jensen, Carsten Juul; Jeppesen, Lise Kofoed; Drachmann, Merete

    2014-01-01

    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...... to advanced beginner, and adjusting to the workplace community. The conclusion is that the learning of nursing students during their first clinical in-service placement appears informal and not founded on evident best practice....... 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...

  10. The PANTHER User Experience

    Energy Technology Data Exchange (ETDEWEB)

    Coram, Jamie L. [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States); Morrow, James D. [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States); Perkins, David Nikolaus [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States)

    2015-09-01

    This document describes the PANTHER R&D Application, a proof-of-concept user interface application developed under the PANTHER Grand Challenge LDRD. The purpose of the application is to explore interaction models for graph analytics, drive algorithmic improvements from an end-user point of view, and support demonstration of PANTHER technologies to potential customers. The R&D Application implements a graph-centric interaction model that exposes analysts to the algorithms contained within the GeoGraphy graph analytics library. Users define geospatial-temporal semantic graph queries by constructing search templates based on nodes, edges, and the constraints among them. Users then analyze the results of the queries using both geo-spatial and temporal visualizations. Development of this application has made user experience an explicit driver for project and algorithmic level decisions that will affect how analysts one day make use of PANTHER technologies.

  11. Information sciences experiment system

    Science.gov (United States)

    Katzberg, Stephen J.; Murray, Nicholas D.; Benz, Harry F.; Bowker, David E.; Hendricks, Herbert D.

    1990-01-01

    The rapid expansion of remote sensing capability over the last two decades will take another major leap forward with the advent of the Earth Observing System (Eos). An approach is presented that will permit experiments and demonstrations in onboard information extraction. The approach is a non-intrusive, eavesdropping mode in which a small amount of spacecraft real estate is allocated to an onboard computation resource. How such an approach allows the evaluation of advanced technology in the space environment, advanced techniques in information extraction for both Earth science and information science studies, direct to user data products, and real-time response to events, all without affecting other on-board instrumentation is discussed.

  12. Caring Experience and Knowledge

    DEFF Research Database (Denmark)

    Dybbroe, Betina

    2005-01-01

    A presentation of a case and a theoretical discussion concerning what characterizes the sozialisation and formation taking place in educations related to care work inside the Danish welfare system presently. The article presents an analysis of the relation between curriculum,didactics and educati......A presentation of a case and a theoretical discussion concerning what characterizes the sozialisation and formation taking place in educations related to care work inside the Danish welfare system presently. The article presents an analysis of the relation between curriculum......,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...

  13. Design of Computer Experiments

    DEFF Research Database (Denmark)

    Dehlendorff, Christian

    The main topic of this thesis is design and analysis of computer and simulation experiments and is dealt with in six papers and a summary report. Simulation and computer models have in recent years received increasingly more attention due to their increasing complexity and usability. Software...... packages make the development of rather complicated computer models using predefined building blocks possible. This implies that the range of phenomenas that are analyzed by means of a computer model has expanded significantly. As the complexity grows so does the need for efficient experimental designs...... and analysis methods, since the complex computer models often are expensive to use in terms of computer time. The choice of performance parameter is an important part of the analysis of computer and simulation models and Paper A introduces a new statistic for waiting times in health care units. The statistic...

  14. The Digital Archive Experience

    DEFF Research Database (Denmark)

    2009-01-01

    This article, as well as the book, investigates the ways in which new digital media may enhance the experience of the art-archive. Taken as a whole, the new media is a vital component of a 'transdisciplinary' and transformative field, a cultural landscape that is changing rapidly the conditions....... These can be coarsely divided into two categories: purely utilitarian websites with information about admission, hours of operation, directions, and the current show. The other - more ambitious - type of website tried to expand the exhibition area of the museum into virtual reality. The idea sounds great....... The book discusses the challenges of the archive and the (art)museum in the age of digital media. It is based upon documentation from a research project, MAP - Media Art Platform, that drew upon the talents and collaboration of many institutions, artists, programmers, art historians, designers and others...

  15. Gas Detection for Experiments

    CERN Document Server

    Hay, D

    2001-01-01

    Flammable gases are often used in detectors for physics experiments. The storage, distribution and manipulation of such flammable gases present several safety hazards. As most flammable gases cannot be detected by human senses, specific well-placed gas detection systems must be installed. Following a request from the user group and in collaboration with CERN safety officers, risk analyses are performed. An external contractor, who needs to receive detailed user requirements from CERN, performs the installations. The contract is passed on a guaranteed results basis. Co-ordination between all the CERN groups and verification of the technical installation is done by ST/AA/AS. This paper describes and focuses on the structured methodology applied to implement such installations based on goal directed project management techniques (GDPM). This useful supervision tool suited to small to medium sized projects facilitates the task of co-ordinating numerous activities to achieve a completely functional system.

  16. CAREER GUIDANCE EXPERIENCE ABROAD

    Directory of Open Access Journals (Sweden)

    Sergey N. Tolstoguzov

    2015-01-01

    Full Text Available The aim of this paper is to describe the experience of careeroriented activities carried out with students of schools in developed and developing countries. Career Guidance in Russia, despite the vast experience of its implementation, is experiencing serious difficulties. In this regard, it is important to take into account the international experience career-oriented activities, such as in the developed countries of North America and the European Union as well as in several Asian countries with rapidly growing economies and a large demographic potential, taking into account the best variants for the Russian education system. Methods. The experience of career-oriented work undertaken with pupils of the USA, Canada, Israel, France, UK, Germany, Denmark, Sweden, Japan, Singapore, China and India is shown on the basis of the comparative analysis of different publications and information sources. The author has made an attempt to generalize the principles of psycho-pedagogical and administrative assistance in professional self-determination of senior pupils abroad. Scientific novelty. The approaches to career-oriented activities in countries with different levels of economic development are compared for the first time. Some principles are revealed. Firstly, the higher the income level per capita in the country, the greater attention is given to vocational guidance. The politics in the developed countries is based on interests of the individual: children’s acquaintance with the world of professions begins already at younger school and the moment of definitive selfdetermination is postponed till the end of their senior stage of education; the possibility of direction change of professional preparation in case of detection of discrepancy of qualities of the pupil to originally selected profile is provided. Career-oriented activity in developing countries, on the contrary, is rigidly coordinated to requirements of economy and a labour market

  17. The Cibola flight experiment

    Energy Technology Data Exchange (ETDEWEB)

    Caffrey, Michael Paul [Los Alamos National Laboratory; Nelson, Anthony [Los Alamos National Laboratory; Salazar, Anthony [Los Alamos National Laboratory; Roussel - Dupre, Diane [Los Alamos National Laboratory; Katko, Kim [Los Alamos National Laboratory; Palmer, Joseph [ISE-3; Robinson, Scott [Los Alamos National Laboratory; Wirthlin, Michael [BRIGHAM YOUNG UNIV; Howes, William [BRIGHAM YOUNG UNIV; Richins, Daniel [BRIGHAM YOUNG UNIV

    2009-01-01

    The Cibola Flight Experiment (CFE) is an experimental small satellite carrying a reconfigurable processing instrument developed at the Los Alamos National Laboratory that demonstrates the feasibility of using FPGA-based high-performance computing for sensor processing in the space environment. The CFE satellite was launched on March 8, 2007 in low-earth orbit and has operated extremely well since its deployment. The nine Xilinx Virtex FPGAs used in the payload have been used for several high-throughput sensor processing applications and for single-event upset (SEU) monitoring and mitigation. This paper will describe the CFE system and summarize its operational results. In addition, this paper will describe the results from several SEU detection circuits that were performed on the spacecraft.

  18. INITIAL COOLING EXPERIMENT (ICE)

    CERN Multimedia

    1979-01-01

    ICE was built in 1977, using the modified bending magnets of the g-2 muon storage ring (see 7405430). Its purpose was to verify the validity of stochastic and electron cooling for the antiproton project. Stochastic cooling proved a resounding success early in 1978 and the antiproton project could go ahead, now entirely based on stochastic cooling. Electron cooling was experimented with in 1979. The 26 kV equipment is housed in the cage to the left of the picture, adjacent to the "e-cooler" located in a straight section of the ring. With some modifications, the cooler was later transplanted into LEAR (Low Energy Antiproton Ring) and then, with further modifications, into the AD (Antiproton Decelerator), where it cools antiprotons to this day (2006). See also: 7711282, 7802099, 7809081.

  19. The Bumpy Torus Experiment

    Energy Technology Data Exchange (ETDEWEB)

    Cobble, James Allen [Los Alamos National Lab. (LANL), Los Alamos, NM (United States)

    2016-06-09

    This document summarizes the Bumpy Torus Experiment as a viable fusion reactor concept. Conclusions reached include the following: In 30 years, order-of-magnitude technological advances have occurred in multiple areas of plasma heating and confinement. The ORNL bumpy torus of the 1970s was technology limited. Now that ITER is technology limited, an alternate concept is needed. A device built on such a concept should be current free, CW, modular, have a gentle shutdown, and demonstrable stability. The bumpy torus meets or has the potential to meet all of these criteria. Earlier, stability was not possible due to power limits; it has not been fully tested. It is time to revisit the bumpy-torus concept with a modest new machine.

  20. Recombination experiments at CRYRING

    Energy Technology Data Exchange (ETDEWEB)

    Spies, W.; Glans, P.; Zong, W.; Gao, H.; Andler, G.; Justiniano, E.; Saito, M.; Schuch, R

    1998-11-15

    Recent advances in studies of electron-ion recombination processes at low relative energies with the electron cooler of the heavy-ion storage ring CRYRING are shown. Through the use of an adiabatically expanded electron beam, collisions down to 10{sup -4}eV relative energies were measured with highly charged ions stored in the ring at around 15 MeV/amu energies. Examples of recombination measurements for bare ions of D{sup +}, He{sup 2+}, N{sup 7+}, Ne{sup 10+} and Si{sup 14+} are presented. Further on, results of an experiment measuring laser-induced recombination (LIR) into n=3 states of deuterium with polarized laser light are shown.

  1. Cell Radiation Experiment System

    Science.gov (United States)

    Morrison, Dennis R.

    2010-01-01

    The cell radiation experiment system (CRES) is a perfused-cell culture apparatus, within which cells from humans or other animals can (1) be maintained in homeostasis while (2) being exposed to ionizing radiation during controlled intervals and (3) being monitored to determine the effects of radiation and the repair of radiation damage. The CRES can be used, for example, to determine effects of drug, radiation, and combined drug and radiation treatments on both normal and tumor cells. The CRES can also be used to analyze the effects of radiosensitive or radioprotectant drugs on cells subjected to radiation. The knowledge gained by use of the CRES is expected to contribute to the development of better cancer treatments and of better protection for astronauts, medical-equipment operators, and nuclear-power-plant workers, and others exposed frequently to ionizing radiation.

  2. The HOLMES Experiment

    Science.gov (United States)

    Faverzani, M.; Alpert, B.; Backer, D.; Bennet, D.; Biasotti, M.; Brofferio, C.; Ceriale, V.; Ceruti, G.; Corsini, D.; Day, P. K.; De Gerone, M.; Dressler, R.; Ferri, E.; Fowler, J.; Fumagalli, E.; Gard, J.; Gatti, F.; Giachero, A.; Hays-Wehle, J.; Heinitz, S.; Hilton, G.; Köster, U.; Lusignoli, M.; Maino, M.; Mates, J.; Nisi, S.; Nizzolo, R.; Nucciotti, A.; Orlando, A.; Parodi, L.; Pessina, G.; Pizzigoni, G.; Puiu, A.; Ragazzi, S.; Reintsema, C.; Ribeiro-Gomez, M.; Schmidt, D.; Schuman, D.; Siccardi, F.; Sisti, M.; Swetz, D.; Terranova, F.; Ullom, J.; Vale, L.

    2016-08-01

    The determination of the neutrino mass is an open issue in modern particle physics and astrophysics. The direct mass measurement is the only theory-unrelated experimental tool capable to probe such quantity. The HOLMES experiment will measure the end-point energy of the electron capture decay of ^{163}Ho, aiming at a statistical sensitivity on the neutrino mass around 1 eV/c^2. In order to acquire the large needed statistics by keeping the pile-up contribution as low as possible, 1000 transition edge sensors will be readout simultaneously with the frequency domain readout, a multiplexing technique where the multiplex factor is only limited by the bandwidth of the available commercial fast digitizers. We outline here the HOLMES project with its technical challenges, and its status and perspectives.

  3. The SNO+ Experiment

    CERN Document Server

    Chen, Mark C

    2008-01-01

    The SNO+ experiment is the follow-up to the Sudbury Neutrino Observatory (SNO). The heavy water that was in SNO will be replaced with a liquid scintillator of linear alkylbenzene (plus fluor). SNO+ has many physics goals including detecting pep and CNO solar neutrinos, detecting geo-neutrinos, studying reactor neutrino oscillations, serving as a supernova neutrino detector and carrying out a search for neutrinoless double beta decay by adding neodymium to the liquid scintillator. Since a large amount of 150Nd isotope can be added to SNO+, a competitive search would be possible, with sensitivity below 100 meV using natural Nd and sensitivity below 40 meV with enriched neodymium.

  4. The Armstrong experiment revisited

    Science.gov (United States)

    Fuchs, Elmar C.; Wexler, Adam D.; Paulitsch-Fuchs, Astrid H.; Agostinho, Luewton L. F.; Yntema, Doekle; Woisetschläger, Jakob

    2014-04-01

    When a high-voltage direct-current is applied to two beakers filled with water or polar liquid dielectrica, a horizontal bridge forms between the two beakers. This experiment was first carried out by Lord Armstrong in 1893 and then forgotten until recently. Such bridges are stable by the action of electrohydrodynamic (EHD) forces caused by electric field gradients counteracting gravity. Due to these gradients a permanent pumping of liquid from one beaker into the other is observed. At macroscopic scale several of the properties of a horizontal water bridge can be explained by modern electrohydrodynamics, analyzing the motion of fluids in electric fields. Whereas on the molecular scale water can be described by quantum mechanics, there is a conceptual gap at mesoscopic scale which is bridged by a number of theories including quantum mechanical entanglement and coherent structures in water - theories that we discuss here. Much of the phenomenon is already understood, but even more can still be learned from it, since such "floating" liquid bridges resemble a small high voltage laboratory of their own: The physics of liquids in electric fields of some kV/cm can be studied, even long time experiments like neutron or light scattering are feasible since the bridge is in a steady-state equilibrium and can be kept stable for hours. It is also an electro-chemical reactor where compounds are transported through by the EHD flow, enabling the study of electrochemical reactions under potentials which are otherwise not easily accessible. Last but not least the bridge provides the experimental biologist with the opportunity to expose living organisms such as bacteria to electric fields without killing them, but with a significant influence on their behavior, and possibly, even on their genome.

  5. For the love of experience: changing the experience economy discourse

    NARCIS (Netherlands)

    Snel, J.M.C.

    2011-01-01

    The attention for experiences as economic offerings has increased enormously in the last decade. However, the lack of a clear definition of experience and the bias towards the organization’s perspective in the discourse cause much confusion. In this study experience is taken back to its basis: the e

  6. Partnership in Undergraduate Research Experience

    Science.gov (United States)

    Practical laboratory and work experience has been helpful in reinforcing the undergraduate educational experience. With limited resources, individual organizations may struggle to give a student a well rounded opportunity. Most undergraduates work within internships or cooperative educational fram...

  7. The QUIJOTE experiment

    Science.gov (United States)

    López-Caniego, Marcos

    The QUIJOTE (Q-U-I JOint Tenerife) Experiment† (Rubiño-Martín et al. 2010)) is observing the polarization of the Cosmic Microwave Background and other Galactic and extragalactic signals at medium and large angular scales in the frequency range of 10-40 GHz. This experiment is going to provide valuable information about the polarization properties of synchrotron and anomalous microwave emission at these frequencies. It consists of two telescopes and three instruments located at the the Teide Observatory (2400 m) in Tenerife, Canary Islands, Spain. This project has two phases: Phase I, the first telescope (QT1) and a multi-frequency instrument (MFI) are in operations since November 2012 observing between 10 and 20 GHz. A second instrument (TGI) with 31 polarimeters working at 30 GHz is expected to start operations in Autumn 2015 and will be devoted to primordial B-mode science. This instrument will include a fixed polariser and 90 and 180 phase switches to generate four polarization states to minimize the different systematics in the receiver; Phase II, a second QUIJOTE telescope (QT2), already in operation, and a third instrument (FGI) with 40 polarimeters working at 41 GHz, safely below the 60-GHz oxygen absorption band, will significantly increment the sensitivity of the QUIJOTE project to detect the r parameter (tensor-to-scalar ratio). The reason for this is not only the significant reduction of noise due to the number of polarimeters that will incorporate but also the lower synchrotron signal from our galaxy expected at these higher frequencies. The maps obtained with the multi-frequency instrument (10-20 GHz), in combination with data from other experiments like Planck and the VLA, will be used to clean the diffuse and compact foreground emission at 30 and 40 GHz, the cosmological channels. After three years of effective observations we expect to reach the required sensitivity to detect a primordial gravitational-wave component if the tensor

  8. SPRUCE experiment data infrastructure

    Science.gov (United States)

    Krassovski, M.; Hanson, P. J.; Boden, T.; Riggs, J.; Nettles, W. R.; Hook, L. A.

    2013-12-01

    The Carbon Dioxide Information Analysis Center (CDIAC) at Oak Ridge National Laboratory (ORNL), USA has provided scientific data management support for the US Department of Energy and international climate change science since 1982. Among the many data activities CDIAC performs are design and implementation of the data systems. One current example is the data system and network for SPRUCE experiment. The SPRUCE experiment (http://mnspruce.ornl.gov) is the primary component of the Terrestrial Ecosystem Science Scientific Focus Area of ORNL's Climate Change Program, focused on terrestrial ecosystems and the mechanisms that underlie their responses to climatic change. The experimental work is to be conducted in a bog forest in northern Minnesota, 40 km north of Grand Rapids, in the USDA Forest Service Marcell Experimental Forest (MEF). The site is located at the southern margin of the boreal peatland forest. Experimental work in the 8.1-ha S1 bog will be a climate change manipulation focusing on the combined responses to multiple levels of warming at ambient or elevated CO2 (eCO2) levels. The experiment provides a platform for testing mechanisms controlling the vulnerability of organisms, biogeochemical processes and ecosystems to climatic change (e.g., thresholds for organism decline or mortality, limitations to regeneration, biogeochemical limitations to productivity, the cycling and release of CO2 and CH4 to the atmosphere). The manipulation will evaluate the response of the existing biological communities to a range of warming levels from ambient to +9°C, provided via large, modified open-top chambers. The ambient and +9°C warming treatments will also be conducted at eCO2 (in the range of 800 to 900 ppm). Both direct and indirect effects of these experimental perturbations will be analyzed to develop and refine models needed for full Earth system analyses. SPRUCE provides wide range continuous and discrete measurements. To successfully manage SPRUCE data flow

  9. The Nepal experience.

    Science.gov (United States)

    Kaikobad, N F

    1977-01-01

    Nepal's panchayat system of partyless democracy with 5 class organizations of peasants, youth, women, labor, and ex-servicemen, is an effort in community development. Panchayat training centers train panchayat secretaries and women workers. The government tried out the Mobile Training Scheme (MTS) methodology to train panchayat training center instructors in 1974-75 when 5 courses were given for 76 participants. The MTS methodology included several new assumptions: the necessity of knowing the field situation, a realistic problem solving orientation, learning by actual field experience, and interdependence rather than teacher dependence. The multipurpose role of the panchayat secretary was studied and clarified. Role performance led to the development of a realistic job description from which a task-focused curriculum could be developed. Field work tools included maintaining a daily diary, collecting information and developing a present and past project history, and compiling a village profile. The trainees played the roles of front line workers in the field when they returned from the villages played the roles of supervisors and trainers. The key concept in the multipurpose role of the panchayat secretary was collaboration. The panchayat secretary-trainee had to understand the social roles in the community, and work within the social context to get cooperation from other agencies, village and informal organizations, in order to fulfill their role. Tutorial and team teaching methods were used to provide partnership in learning; the old roles of lecturer and lectured were seen as ineffective when actual field experience was the criteria. The role performing and role analysis group analyzed the front line workers' roles and evolved job descriptions which led to course outlines. The teaching methods and materials group produced indigenous teaching materials for classroom use based on problems faced in the field. The action research and technical collaboration groups

  10. Fresh perspectives on customer experience

    OpenAIRE

    McColl-Kennedy, Janet; Gustafsson, Anders; Jaakkola, Elina; Klaus, Phil; Radnor, Zoe; Perks, Helen; Friman, Margareta

    2015-01-01

    Purpose – The purpose is to provide directions for future research on: (1) broadening the role of customers in customer experience; (2) taking a practice-based approach to customer experience; and (3) recognizing the holistic, dynamic nature of customer experience across all touch points and over time. Design/methodology/approach – The approach is conceptual identifying current gaps in research on customer experience. Findings – The findings include a set of research questions and re...

  11. Results of the BSE experiment

    Science.gov (United States)

    Iwasaki, K.; Yamamoto, M.; Kurakake, Y.; Kinoshita, S.

    Results of satellite broadcasting experiments carried out using the BSE (Japan's Medium-scale Broadcasting Satellite for Experimental Purpose) are described. Consideration is given to the following experiments: the reception of radio waves from the satellite, radio wave attenuation and scattering, the uplink power control experiment, experiments with transportable earth stations, the transmission of FM-TV signals, and frequency sharing between the broadcasting satellite and broadcasting services in the 12 GHz band.

  12. THE EXPERIENCE OF ART – THE EMPIRIA OR EXPERIMENT?

    Directory of Open Access Journals (Sweden)

    Franciszek Chmielowski

    2010-01-01

    Full Text Available In contemporary aesthetics exists an belief, that the experience of art is the primary fact, which initiates and leads theoretical reflection, as also is the state basis of creating, valuing and the opinion on art. Present text is the test of philosophical recognition of relationship between field of products of new art as well as with context formative it experiences. Author motivates thesis, that radical changes in present art have their source in of meanings consisting on primary notion of experience changes. The present artistic practice goes away from aesthetical experience as empiria and the contemplation and used the notion of experience as experiment and investigation, which dismisses art from her proper functions and brings it rather to aggressive ideology of scientism. The recognition of this progressive change of meanings in founding the art notion of experience has the important consequences both for understanding of present art and for describing it theory.

  13. Design Experiments in Educational Research.

    Science.gov (United States)

    Cobb, Paul; Confrey, Jere; diSessa, Andrea; Lehrer, Richard; Schauble, Leona

    2003-01-01

    Indicates the range of purposes and variety of settings in which design experiments have been conducted, delineating five crosscutting features that collectively differentiate design experiments from other methodologies. Clarifies what is involved in preparing for and carrying out a design experiment and in conducting a retrospective analysis of…

  14. Antihydrogen Experiment Gravity Interferometry Spectroscopy

    CERN Multimedia

    Gerber, S; Tietje, I C; Allkofer, Y R; Trezzi, D; Dassa, L; Rienacker, B; Khalidova, O; Ferrari, G; Krasnicky, D; Perini, D; Cerchiari, G; Belov, A; Boscolo, I; Sacerdoti, M G; Ferragut, R O; Nedelec, P; Testera, G; Hinterberger, A; Al-qaradawi, I; Malbrunot, C L S; Brusa, R S; Prelz, F; Manuzio, G; Riccardi, C; Fontana, A; Genova, P; Haider, S; Haug, F; Merkt, F; Turbabin, A; Castelli, F; Lagomarsino, V E; Doser, M; Penasa, L; Gninenko, S; Cataneo, F; Zenoni, A; Cabaret, L; Comparat, D P; Zmeskal, J; Scampoli, P; Dudarev, A; Kellerbauer, A G; Mariazzi, S; Fesel, J V; Nesteruk, K P; Carraro, C; Zavatarelli, S M

    The AEGIS experiment (Antihydrogen Experiment: Gravity, Interferometry, Spectroscopy) has the aim of carrying out the first measurement of the gravitational interaction of antimatter to a precision of 1%, by applying techniques from atomic physics, laser spectroscopy and interferometry to a beam of antihydrogen atoms. A further goal of the experiment is to carry out spectroscopy of the antihydrogen atoms in flight.

  15. Innovative Science Experiments Using Phoenix

    Science.gov (United States)

    Kumar, B. P. Ajith; Satyanarayana, V. V. V.; Singh, Kundan; Singh, Parmanand

    2009-01-01

    A simple, flexible and very low cost hardware plus software framework for developing computer-interfaced science experiments is presented. It can be used for developing computer-interfaced science experiments without getting into the details of electronics or computer programming. For developing experiments this is a middle path between…

  16. Optimal Experience of Web Activities.

    Science.gov (United States)

    Chen, Hsiang; Wigand, R. T.; Nilan, M. S.

    1999-01-01

    Reports on Web users' optimal flow experiences to examine positive aspects of Web experiences that could be linked to theory applied to other media and then incorporated into Web design. Discusses the use of content-analytic procedures to analyze open-ended questionnaires that examined Web users' perceived flow experiences. (Author/LRW)

  17. Experiments for a Special Day

    Science.gov (United States)

    Gluck, Paul

    2008-01-01

    Special events like science days, teacher's meetings and physics recruiting efforts require spectacular and, if possible, interactive experiments for the audience. Based on past experience with such events, we have gathered and present here a series of demonstration experiments in mechanics, optics, waves and electricity which are suitable, and…

  18. Lepton Flavour Violation Experiments

    Directory of Open Access Journals (Sweden)

    F. Cei

    2014-01-01

    Full Text Available Lepton Flavour Violation in the charged lepton sector (CLFV is forbidden in the Minimal Standard model and strongly suppressed in extensions of the model to include finite neutrino mixing. On the other hand, a wide class of Supersymmetric theories, even coupled with Grand Unification models (SUSY-GUT models, predict CLFV processes at a rate within the reach of new experimental searches operated with high resolution detectors at high intensity accelerators. As the Standard model background is negligible, the observation of one or more CLFV events would provide incontrovertible evidence for physics beyond Standard model, while a null effect would severely constrain the set of theory parameters. Therefore, a big experimental effort is currently (and will be for incoming years accomplished to achieve unprecedented sensitivity on several CLFV processes. In this paper we review past and recent results in this research field, with focus on CLFV channels involving muons and tau's. We present currently operating experiments as well as future projects, with emphasis laid on how sensitivity enhancements are accompanied by improvements on detection techniques. Limitations due to systematic effects are also discussed in detail together with the solutions being adopted to overcome them.

  19. Visual experiences during paralysis

    Directory of Open Access Journals (Sweden)

    Emma M Whitham

    2011-12-01

    Full Text Available RationaleParalysed human volunteers (n=6 participated in an electroencephalographic study after which they undertook studies of attempted eye movement. The interventions tested a central, intentional component to one’s internal visual model.Methods Six subjects reclined in a supported chair and were ventilated after paralysis (cisatracurium, 20 mg intravenously. In illumination, subjects were requested to focus alternately on the faces of investigators standing on the left and the right within peripheral vision. In darkness, subjects were instructed to look away from a point source of light. Subjects were to report their experiences after reversal of paralysis.Results During attempted eye movement in illumination, one subject had an illusion of environmental movement but four subjects perceived faces as clearly as if they were in central vision. In darkness, four subjects reported movement of the target light in the direction of attempted eye movements and three could control the movement of the light at will. ConclusionThe hypothesis that internal visual models receive intended ocular-movement-information directly from oculomotor centres is strengthened by this evidence.

  20. My experience in arbitration

    Directory of Open Access Journals (Sweden)

    Claude Witz

    2011-12-01

    Full Text Available This paper deals with the nature of arbitration as one of the alternatives in dispute resolution processes different from mediation and conciliation, and obviously, from judicial adjudication. Unlike a mediator or a conciliator, an arbitrator is empowered to pass a judgement, like a court. In other words, the arbitrator will render a decision which is binding for the parties. The arbitrator has the jurisdictio, like a judge. Although the office of a judge and an arbitrator is the same, the source of their power is different. The judge’s power originates from a State, whereas the power of the arbitrator results primarily from a contract. Unlike the court, the arbitrator lacks the imperium, i.e. the power to make the award enforceable. When a party refuses to enforce the award, enforceability can only be provided by the judge or an authority of the state where the award has to be enforced. In a certain sense the state thus provides the back up for the arbitration system. This paper presents personal thoughts drawn from the author’s professional experience as an arbitrator in Franco-German disputes.