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Sample records for amplicon resequencing experiments

  1. 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...

  2. Removing Noise From Pyrosequenced Amplicons

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    Davenport Russell J

    2011-01-01

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

  3. Metabarcoding Marine Sediments: Preparation of Amplicon Libraries.

    Science.gov (United States)

    Fonseca, Vera G; Lallias, Delphine

    2016-01-01

    The accurate assessment of community composition and ultimately species identification is of utmost importance in any ecological and evolutionary study. Advances in sequencing technologies have allowed the unraveling of levels of biodiversity never imagined before when applied to large-scale environmental DNA studies (also termed metabarcoding/metagenetics/metasystematics/environmental barcoding). Here, we describe a detailed protocol to assess eukaryotic biodiversity in marine sediments, identifying key steps that should not be neglected when preparing Next-Generation Sequencing (NGS) amplicon libraries: DNA extraction, multiple PCR amplification of DNA barcode markers with index/ tag-primers, and final Illumina MiSeq sequencing library preparation. PMID:27460378

  4. The Car Resequencing Problem with Pull-Off Tables

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    Nils Boysen

    2011-12-01

    Full Text Available The car sequencing problem determines sequences of different car models launched down a mixed-model assembly line. To avoid work overloads of workforce, car sequencing restricts the maximum occurrence of labor-intensive options, e.g., a sunroof, by applying sequencing rules. We consider this problem in a resequencing context, where a given number of buffers (denoted as pull-off tables is available for rearranging a stirred sequence. The problem is formalized and suited solution procedures are developed. A lower bound and a dominance rule are introduced which both reduce the running time of our graph approach. Finally, a real-world resequencing setting is investigated.

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

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    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. PMID:27008882

  6. 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.

  7. SNP detection for massively parallel whole-genome resequencing

    DEFF Research Database (Denmark)

    Li, Ruiqiang; Li, Yingrui; Fang, Xiaodong;

    2009-01-01

    -genome or target region resequencing. Here, we have developed a consensus-calling and SNP-detection method for sequencing-by-synthesis Illumina Genome Analyzer technology. We designed this method by carefully considering the data quality, alignment, and experimental errors common to this technology. All...

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

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

    1996-12-01

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

  9. 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

  10. Multiplex bisulfite PCR resequencing of clinical FFPE DNA

    OpenAIRE

    Korbie, Darren; Lin, Erica; Wall, David; Nair, Shalima S; Stirzaker, Clare; Clark, Sue J; Trau, Matt

    2015-01-01

    Background The clinical utility of DNA methylation as a predictive or prognostic biomarker requires scalable resequencing protocols for bisulfite-converted DNA. Key features of any validation method should be adaptability for low- or high-throughput needs and high reproducibility, and should only require minimal amounts of precious clinical sample as input material. Critically, this method should also deliver robust results when working with bisulfite-converted DNA extracted from formalin-fix...

  11. 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

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

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    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.

  13. Systems consequences of amplicon formation in human breast cancer.

    Science.gov (United States)

    Inaki, Koichiro; Menghi, Francesca; Woo, Xing Yi; Wagner, Joel P; Jacques, Pierre-Étienne; Lee, Yi Fang; Shreckengast, Phung Trang; Soon, Wendy WeiJia; Malhotra, Ankit; Teo, Audrey S M; Hillmer, Axel M; Khng, Alexis Jiaying; Ruan, Xiaoan; Ong, Swee Hoe; Bertrand, Denis; Nagarajan, Niranjan; Karuturi, R Krishna Murthy; Miranda, Alfredo Hidalgo; Liu, Edison T

    2014-10-01

    Chromosomal structural variations play an important role in determining the transcriptional landscape of human breast cancers. To assess the nature of these structural variations, we analyzed eight breast tumor samples with a focus on regions of gene amplification using mate-pair sequencing of long-insert genomic DNA with matched transcriptome profiling. We found that tandem duplications appear to be early events in tumor evolution, especially in the genesis of amplicons. In a detailed reconstruction of events on chromosome 17, we found large unpaired inversions and deletions connect a tandemly duplicated ERBB2 with neighboring 17q21.3 amplicons while simultaneously deleting the intervening BRCA1 tumor suppressor locus. This series of events appeared to be unusually common when examined in larger genomic data sets of breast cancers albeit using approaches with lesser resolution. Using siRNAs in breast cancer cell lines, we showed that the 17q21.3 amplicon harbored a significant number of weak oncogenes that appeared consistently coamplified in primary tumors. Down-regulation of BRCA1 expression augmented the cell proliferation in ERBB2-transfected human normal mammary epithelial cells. Coamplification of other functionally tested oncogenic elements in other breast tumors examined, such as RIPK2 and MYC on chromosome 8, also parallel these findings. Our analyses suggest that structural variations efficiently orchestrate the gain and loss of cancer gene cassettes that engage many oncogenic pathways simultaneously and that such oncogenic cassettes are favored during the evolution of a cancer. PMID:25186909

  14. 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...

  15. Whole Genome Re-Sequencing of Three Domesticated Chicken Breeds.

    Science.gov (United States)

    Oh, Dongyep; Son, Bongjun; Mun, Seyoung; Oh, Man Hwan; Oh, Sejong; Ha, Jaejung; Yi, Junkoo; Lee, Seunguk; Han, Kyudong

    2016-02-01

    Chicken is one of the most popular domesticated species worldwide, as it can serve an important role in agricultural as well as biomedical research fields. Because it inhabits almost every continent and presents diverse morphology and traits, the need of genetic markers for distinguishing each breed for various purposes has increased. The whole genome sequencing of three different breeds (White Leghorn, Korean domestic, and Araucana) that show similar coloring patterns, with the exception of the White Leghorn breed, have confirmed previously reported genomic alterations and identified many novel variants. Additionally, the Whole Genome Re-Sequencing (WGRS) approach identified an approximately 4 kb insert within SLCO1B3 responsible for blue egg shell color. Targeted investigation of pigment-related genes corroborated previously reported non-synonymous mutations, and provided deeper insight into chicken coloring, where not a single but a combination of non-synonymous mutations in the MC1R gene is likely to be responsible for altered feather coloring. PMID:26853871

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

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    C Ryan King

    2010-11-01

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

  17. 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.

  18. 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. PMID:26257385

  19. 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.

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

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    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.

  1. 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 ...

  2. 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...

  3. Molecular Inversion Probes for targeted resequencing in non-model organisms.

    Science.gov (United States)

    Niedzicka, M; Fijarczyk, A; Dudek, K; Stuglik, M; Babik, W

    2016-01-01

    Applications that require resequencing of hundreds or thousands of predefined genomic regions in numerous samples are common in studies of non-model organisms. However few approaches at the scale intermediate between multiplex PCR and sequence capture methods are available. Here we explored the utility of Molecular Inversion Probes (MIPs) for the medium-scale targeted resequencing in a non-model system. Markers targeting 112 bp of exonic sequence were designed from transcriptome of Lissotriton newts. We assessed performance of 248 MIP markers in a sample of 85 individuals. Among the 234 (94.4%) successfully amplified markers 80% had median coverage within one order of magnitude, indicating relatively uniform performance; coverage uniformity across individuals was also high. In the analysis of polymorphism and segregation within family, 77% of 248 tested MIPs were confirmed as single copy Mendelian markers. Genotyping concordance assessed using replicate samples exceeded 99%. MIP markers for targeted resequencing have a number of advantages: high specificity, high multiplexing level, low sample requirement, straightforward laboratory protocol, no need for preparation of genomic libraries and no ascertainment bias. We conclude that MIP markers provide an effective solution for resequencing targets of tens or hundreds of kb in any organism and in a large number of samples. PMID:27046329

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

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    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.

  5. Whole-genome resequencing of Hanwoo (Korean cattle) and insight into regions of homozygosity

    OpenAIRE

    Lee, Kyung-Tai; Chung, Won-Hyong; Lee, Sung-Yeoun; Choi, Jung-Woo; Kim, Jiwoong; Lim, Dajeong; Lee, Seunghwan; Jang, Gul-Won; Kim, Bumsoo; Choy, Yun Ho; Liao, Xiaoping; Stothard, Paul; Moore, Stephen S; Lee, Sang-Heon; Ahn, Sungmin

    2013-01-01

    Background Hanwoo (Korean cattle), which originated from natural crossbreeding between taurine and zebu cattle, migrated to the Korean peninsula through North China. Hanwoo were raised as draft animals until the 1970s without the introduction of foreign germplasm. Since 1979, Hanwoo has been bred as beef cattle. Genetic variation was analyzed by whole-genome deep resequencing of a Hanwoo bull. The Hanwoo genome was compared to that of two other breeds, Black Angus and Holstein, and genes with...

  6. A population genetic approach to mapping neurological disorder genes using deep resequencing.

    Directory of Open Access Journals (Sweden)

    Rachel A Myers

    2011-02-01

    Full Text Available Deep resequencing of functional regions in human genomes is key to identifying potentially causal rare variants for complex disorders. Here, we present the results from a large-sample resequencing (n  =  285 patients study of candidate genes coupled with population genetics and statistical methods to identify rare variants associated with Autism Spectrum Disorder and Schizophrenia. Three genes, MAP1A, GRIN2B, and CACNA1F, were consistently identified by different methods as having significant excess of rare missense mutations in either one or both disease cohorts. In a broader context, we also found that the overall site frequency spectrum of variation in these cases is best explained by population models of both selection and complex demography rather than neutral models or models accounting for complex demography alone. Mutations in the three disease-associated genes explained much of the difference in the overall site frequency spectrum among the cases versus controls. This study demonstrates that genes associated with complex disorders can be mapped using resequencing and analytical methods with sample sizes far smaller than those required by genome-wide association studies. Additionally, our findings support the hypothesis that rare mutations account for a proportion of the phenotypic variance of these complex disorders.

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

    Directory of Open Access Journals (Sweden)

    Iwanka Kozarewa

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

  8. A Modified Method for Whole Exome Resequencing from Minimal Amounts of Starting DNA

    Science.gov (United States)

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

    2012-01-01

    Next generation DNA sequencing (NGS) technologies have revolutionized the pace at which whole genome and exome sequences can be generated. However, despite these advances, many of the methods for targeted resequencing, such as the generation of high-depth exome sequences, are somewhat limited by the relatively large amounts of starting DNA that are normally required. In the case of tumour analysis this is particularly pertinent as many tumour biopsies often return submicrogram quantities of DNA, especially when tumours are microdissected prior to analysis. Here, we present a method for exome capture and resequencing using as little as 50 ng of starting DNA. The sequencing libraries generated by this minimal starting amount (MSA-Cap) method generate datasets that are comparable to standard amount (SA) whole exome libraries that use three micrograms of starting DNA. This method, which can be performed in most laboratories using commonly available reagents, has the potential to enhance large scale profiling efforts such as the resequencing of tumour exomes. PMID:22403682

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

    OpenAIRE

    PETRILLO MAURO; ANGERS ALEXANDRE; HENRIKSSON PETER; Bonfini, Laura; PATAK DENNSTEDT Alexandre; 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...

  10. 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

  11. 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/.

  12. 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/. PMID:26424080

  13. 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.

  14. A flexible and economical barcoding approach for highly multiplexed amplicon sequencing of diverse target genes

    Directory of Open Access Journals (Sweden)

    Craig W. Herbold

    2015-07-01

    Full Text Available High throughput sequencing of phylogenetic and functional gene amplicons provides tremendous insight into the structure and functional potential of complex microbial communities. Here, we introduce a highly adaptable and economical PCR approach to barcoding and pooling libraries of numerous target genes. In this approach, we replace gene- and sequencing platform-specific fusion primers with general, interchangeable barcoding primers, enabling nearly limitless customized barcode-primer combinations. Compared to barcoding with long fusion primers, our multiple-target gene approach is more economical because it overall requires lower number of primers and is based on short primers with generally lower synthesis and purification costs. To highlight our approach, we pooled over 900 different small-subunit rRNA and functional gene amplicon libraries obtained from various environmental or host-associated microbial community samples into a single, paired-end Illumina MiSeq run. Although the amplicon regions ranged in size from approximately 290 to 720 bp, we found no significant systematic sequencing bias related to amplicon length or gene target. Our results indicate that this flexible multiplexing approach produces large, diverse and high quality sets of amplicon sequence data for modern studies in microbial ecology.

  15. Whole genome resequencing of Botrytis cinerea isolates identifies high levels of standing diversity.

    Directory of Open Access Journals (Sweden)

    Susanna eAtwell

    2015-09-01

    Full Text Available How standing genetic variation within a pathogen contributes to diversity in host/pathogen interactions is poorly understood, partly because most studied pathogens are host-specific, clonally reproducing organisms which complicates genetic analysis. In contrast, Botrytis cinerea is a sexually reproducing, true haploid ascomycete that can infect a wide range of diverse plant hosts. While previous work had shown significant genomic variation between two isolates, we proceeded to assess the level and frequency of standing variation in a population of B. cinerea. To begin measuring standing genetic variation in B. cinerea, we re-sequenced the genomes of 13 different isolates and aligned them to the previously sequenced T4 reference genome. In addition one of these isolates was resequenced from 4 independently repeated cultures. A high level of genetic diversity was found within the 13 isolates. Within this variation, we could identify clusters of genes with major effect polymorphisms, i.e. polymorphisms that lead to a predicted functional knockout, that surrounded genes involved in controlling vegetative incompatibility. The genotype at these loci was able to partially predict the interaction of these isolates in vegetative mating assays showing that these loci control vegetative incompatibility. This suggests that the vegetative mating loci within B. cinerea are associated with regions of increased genetic diversity. The genome re-sequencing of four clones from the one isolate (Grape that had been independently propagated over ten years showed no detectable spontaneous mutation. This suggests that B. cinerea does not display an elevated spontaneous mutation rate. Future work will allow us to test if, and how, this diversity may be contributing to the pathogen’s broad host range.

  16. Whole genome resequencing of Botrytis cinerea isolates identifies high levels of standing diversity.

    Science.gov (United States)

    Atwell, Susanna; Corwin, Jason A; Soltis, Nicole E; Subedy, Anushryia; Denby, Katherine J; Kliebenstein, Daniel J

    2015-01-01

    How standing genetic variation within a pathogen contributes to diversity in host/pathogen interactions is poorly understood, partly because most studied pathogens are host-specific, clonally reproducing organisms which complicates genetic analysis. In contrast, Botrytis cinerea is a sexually reproducing, true haploid ascomycete that can infect a wide range of diverse plant hosts. While previous work had shown significant genomic variation between two isolates, we proceeded to assess the level and frequency of standing variation in a population of B. cinerea. To begin measuring standing genetic variation in B. cinerea, we re-sequenced the genomes of 13 different isolates and aligned them to the previously sequenced T4 reference genome. In addition one of these isolates was resequenced from four independently repeated cultures. A high level of genetic diversity was found within the 13 isolates. Within this variation, we could identify clusters of genes with major effect polymorphisms, i.e., polymorphisms that lead to a predicted functional knockout, that surrounded genes involved in controlling vegetative incompatibility. The genotype at these loci was able to partially predict the interaction of these isolates in vegetative fusion assays showing that these loci control vegetative incompatibility. This suggests that the vegetative incompatibility loci within B. cinerea are associated with regions of increased genetic diversity. The genome re-sequencing of four clones from the one isolate (Grape) that had been independently propagated over 10 years showed no detectable spontaneous mutation. This suggests that B. cinerea does not display an elevated spontaneous mutation rate. Future work will allow us to test if, and how, this diversity may be contributing to the pathogen's broad host range. PMID:26441923

  17. 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...

  18. 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.

  19. 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.

  20. Unlabeled Oligonucleotides as Internal Temperature Controls for Genotyping by Amplicon Melting

    OpenAIRE

    Seipp, Michael T.; Durtschi, Jacob D; Liew, Michael A.; Williams, Jamie; Damjanovich, Kristy; Pont-Kingdon, Genevieve; Lyon, Elaine; Voelkerding, Karl V.; Wittwer, Carl T.

    2007-01-01

    Amplicon melting is a closed-tube method for genotyping that does not require probes, real-time analysis, or allele-specific polymerase chain reaction. However, correct differentiation of homozygous mutant and wild-type samples by melting temperature (Tm) requires high-resolution melting and closely controlled reaction conditions. When three different DNA extraction methods were used to isolate DNA from whole blood, amplicon Tm differences of 0.03 to 0.39°C attributable to the extractions wer...

  1. Resequencing of Curcuma longa L. cv. Kedaram through transcriptome profiling reveals various novel transcripts.

    Science.gov (United States)

    Sahoo, Ambika; Jena, Sudipta; Sahoo, Suprava; Nayak, Sanghamitra; Kar, Basudeba

    2016-09-01

    Curcuma longa L. (Turmeric), of the family Zingiberaceae, is one of the economically as well as medicinally important plant species. It is a sterile, polyploid and vegetatively propagated spice crop cultivated usually in Southeast Asia. In the current study, we carried out re-sequencing through transcriptome profiling of Curcuma longa cv. Kedaram (Cl_Ked_6). We acquired a total of 1 GB raw data by resequencing through paired-end sequencing using Nextseq 500 platform. The raw data obtained in this study can be accessible in NCBI database with accession number of SRR3928562 with bioproject accession number PRJNA324755. Cufflinks-2.2.1 tool was used for transcriptome assembly which resulted in 39,554 numbers of transcripts. The transcript length ranged from 76 to 15,568, having N50 value of 1221 and median transcript length of 860. We annotated the transcripts using multiple databases. This data will be beneficial for studying sequence variations particularly SNPs between cultivars of turmeric towards authentic identification and discovery of novel functional transcripts in Kedaram. PMID:27595066

  2. 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.

  3. 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.

  4. 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

  5. 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.

  6. Bacterial metabarcoding by 16S rRNA gene ion torrent amplicon sequencing.

    Science.gov (United States)

    Fantini, Elio; Gianese, Giulio; Giuliano, Giovanni; Fiore, Alessia

    2015-01-01

    Ion Torrent is a next generation sequencing technology based on the detection of hydrogen ions produced during DNA chain elongation; this technology allows analyzing and characterizing genomes, genes, and species. Here, we describe an Ion Torrent procedure applied to the metagenomic analysis of 16S rRNA gene amplicons to study the bacterial diversity in food and environmental samples. PMID:25343859

  7. Evaluation of Hybridization Capture Versus Amplicon-Based Methods for Whole-Exome Sequencing.

    Science.gov (United States)

    Samorodnitsky, Eric; Jewell, Benjamin M; Hagopian, Raffi; Miya, Jharna; Wing, Michele R; Lyon, Ezra; Damodaran, Senthilkumar; Bhatt, Darshna; Reeser, Julie W; Datta, Jharna; Roychowdhury, Sameek

    2015-09-01

    Next-generation sequencing has aided characterization of genomic variation. While whole-genome sequencing may capture all possible mutations, whole-exome sequencing remains cost-effective and captures most phenotype-altering mutations. Initial strategies for exome enrichment utilized a hybridization-based capture approach. Recently, amplicon-based methods were designed to simplify preparation and utilize smaller DNA inputs. We evaluated two hybridization capture-based and two amplicon-based whole-exome sequencing approaches, utilizing both Illumina and Ion Torrent sequencers, comparing on-target alignment, uniformity, and variant calling. While the amplicon methods had higher on-target rates, the hybridization capture-based approaches demonstrated better uniformity. All methods identified many of the same single-nucleotide variants, but each amplicon-based method missed variants detected by the other three methods and reported additional variants discordant with all three other technologies. Many of these potential false positives or negatives appear to result from limited coverage, low variant frequency, vicinity to read starts/ends, or the need for platform-specific variant calling algorithms. All methods demonstrated effective copy-number variant calling when evaluated against a single-nucleotide polymorphism array. This study illustrates some differences between whole-exome sequencing approaches, highlights the need for selecting appropriate variant calling based on capture method, and will aid laboratories in selecting their preferred approach. PMID:26110913

  8. 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.

  9. Bacterial metabarcoding by 16S rRNA gene ion torrent amplicon sequencing.

    Science.gov (United States)

    Fantini, Elio; Gianese, Giulio; Giuliano, Giovanni; Fiore, Alessia

    2015-01-01

    Ion Torrent is a next generation sequencing technology based on the detection of hydrogen ions produced during DNA chain elongation; this technology allows analyzing and characterizing genomes, genes, and species. Here, we describe an Ion Torrent procedure applied to the metagenomic analysis of 16S rRNA gene amplicons to study the bacterial diversity in food and environmental samples.

  10. 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...

  11. 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.

  12. Comparison of normalization methods for construction of large, multiplex amplicon pools for next-generation sequencing.

    Science.gov (United States)

    Harris, J Kirk; Sahl, Jason W; Castoe, Todd A; Wagner, Brandie D; Pollock, David D; Spear, John R

    2010-06-01

    Constructing mixtures of tagged or bar-coded DNAs for sequencing is an important requirement for the efficient use of next-generation sequencers in applications where limited sequence data are required per sample. There are many applications in which next-generation sequencing can be used effectively to sequence large mixed samples; an example is the characterization of microbial communities where management costs associated with construction of multiplex pools are also scalable. One critical step in multiplex pool construction is the normalization process, whereby equimolar amounts of each amplicon are mixed. Here we compare three approaches (spectroscopy, size-restricted spectroscopy, and quantitative binding) for normalization of large, multiplex amplicon pools for performance and efficiency. We found that the quantitative binding approach was superior and represents an efficient scalable process for construction of very large, multiplex pools with hundreds and perhaps thousands of individual amplicons included. We demonstrate the increased sequence diversity identified with higher throughput. Massively parallel sequencing can dramatically accelerate microbial ecology studies by allowing appropriate replication of sequence acquisition to account for temporal and spatial variations. Further, population studies to examine genetic variation, which require even lower levels of sequencing, should be possible where thousands of individual bar-coded amplicons are examined in parallel. PMID:20418443

  13. 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

  14. 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

  15. 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......Amplicon pyrosequencing targets a known genetic region and thus inherently produces reads highly anticipated to have certain features, such as conserved nucleotide sequence, and in the case of protein coding DNA, an open reading frame. Pyrosequencing errors, consisting mainly of nucleotide....... This probabilistic approach enables integration of basecalling into a larger model where other parameters can be incorporated, such as the likelihood for observing a full-length open reading frame at the targeted region. We apply the method to 454 amplicon pyrosequencing data obtained from a malaria...

  16. 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...

  17. 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.

  18. 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

    Surface plasmon field-enhanced fluorescence spectroscopy was employed to extensively investigate the hybridization behaviors of polymerase chain reaction (PCR) amplicons on a peptide nucleic acid (PNA) or DNA probe layer that was previously attached on a streptavidin-modified gold surface via...... biotin/streptavidin interaction. Despite the neutral backbone of PNA, the hybridization reactions were strongly influenced by the variation of ionic strength. The association rates exhibited a monotonic decrease with ionic strength increase and the maximum hybridization signal was achieved...... 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/DNA...

  19. 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.

  20. Massively parallel sequencing of 17 commonly used forensic autosomal STRs and amelogenin with small amplicons.

    Science.gov (United States)

    Kim, Eun Hye; Lee, Hwan Young; Yang, In Seok; Jung, Sang-Eun; Yang, Woo Ick; Shin, Kyoung-Jin

    2016-05-01

    The next-generation sequencing (NGS) method has been utilized to analyze short tandem repeat (STR) markers, which are routinely used for human identification purposes in the forensic field. Some researchers have demonstrated the successful application of the NGS system to STR typing, suggesting that NGS technology may be an alternative or additional method to overcome limitations of capillary electrophoresis (CE)-based STR profiling. However, there has been no available multiplex PCR system that is optimized for NGS analysis of forensic STR markers. Thus, we constructed a multiplex PCR system for the NGS analysis of 18 markers (13CODIS STRs, D2S1338, D19S433, Penta D, Penta E and amelogenin) by designing amplicons in the size range of 77-210 base pairs. Then, PCR products were generated from two single-sources, mixed samples and artificially degraded DNA samples using a multiplex PCR system, and were prepared for sequencing on the MiSeq system through construction of a subsequent barcoded library. By performing NGS and analyzing the data, we confirmed that the resultant STR genotypes were consistent with those of CE-based typing. Moreover, sequence variations were detected in targeted STR regions. Through the use of small-sized amplicons, the developed multiplex PCR system enables researchers to obtain successful STR profiles even from artificially degraded DNA as well as STR loci which are analyzed with large-sized amplicons in the CE-based commercial kits. In addition, successful profiles can be obtained from mixtures up to a 1:19 ratio. Consequently, the developed multiplex PCR system, which produces small size amplicons, can be successfully applied to STR NGS analysis of forensic casework samples such as mixtures and degraded DNA samples. PMID:26799314

  1. From Benchtop to Desktop: Important Considerations when Designing Amplicon Sequencing Workflows

    OpenAIRE

    Murray, Dáithí C; Megan L Coghlan; Michael Bunce

    2015-01-01

    Amplicon sequencing has been the method of choice in many high-throughput DNA sequencing (HTS) applications. To date there has been a heavy focus on the means by which to analyse the burgeoning amount of data afforded by HTS. In contrast, there has been a distinct lack of attention paid to considerations surrounding the importance of sample preparation and the fidelity of library generation. No amount of high-end bioinformatics can compensate for poorly prepared samples and it is therefore im...

  2. Potential of pmoA Amplicon Pyrosequencing for Methanotroph Diversity Studies ▿†

    OpenAIRE

    Lüke, Claudia; Frenzel, Peter

    2011-01-01

    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.

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

    Science.gov (United States)

    Rooney, Claire; Geh, Catherine; Williams, Victoria; Heuckmann, Johannes M; Menon, Roopika; Schneider, Petra; Al-Kadhimi, Katherine; Dymond, Michael; Smith, Neil R; Baker, Dawn; French, Tim; Smith, Paul D; Harrington, Elizabeth A; Barrett, J Carl; Kilgour, Elaine

    2016-01-01

    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. PMID:26905262

  4. 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.

  5. Single genome amplification and direct amplicon sequencing of Plasmodium spp. DNA from ape fecal specimens

    OpenAIRE

    Liu, Weimin; Li, Yingying; Peeters, Martine; Rayner, Julian; Sharp, Paul; Shaw, George; Hahn, Beatrice

    2010-01-01

    Conventional PCR followed by molecular cloning and sequencing of amplified products is commonly used to test clinical specimens for target sequences of interest, such as viral, bacterial or parasite nucleic acids. However, this approach has serious limitations when used to analyze mixtures of genetically divergent templates1–9. This is because Taq polymerase is prone to switch templates during the amplification process, thereby generating recombinants that do not exist in vivo4. When amplicon...

  6. From benchtop to desktop: important considerations when designing amplicon sequencing workflows.

    Directory of Open Access Journals (Sweden)

    Dáithí C Murray

    Full Text Available Amplicon sequencing has been the method of choice in many high-throughput DNA sequencing (HTS applications. To date there has been a heavy focus on the means by which to analyse the burgeoning amount of data afforded by HTS. In contrast, there has been a distinct lack of attention paid to considerations surrounding the importance of sample preparation and the fidelity of library generation. No amount of high-end bioinformatics can compensate for poorly prepared samples and it is therefore imperative that careful attention is given to sample preparation and library generation within workflows, especially those involving multiple PCR steps. This paper redresses this imbalance by focusing on aspects pertaining to the benchtop within typical amplicon workflows: sample screening, the target region, and library generation. Empirical data is provided to illustrate the scope of the problem. Lastly, the impact of various data analysis parameters is also investigated in the context of how the data was initially generated. It is hoped this paper may serve to highlight the importance of pre-analysis workflows in achieving meaningful, future-proof data that can be analysed appropriately. As amplicon sequencing gains traction in a variety of diagnostic applications from forensics to environmental DNA (eDNA it is paramount workflows and analytics are both fit for purpose.

  7. Electrochemical DNA sensor for anthrax toxin activator gene atxA-detection of PCR amplicons.

    Science.gov (United States)

    Das, Ritu; Goel, Ajay K; Sharma, Mukesh K; Upadhyay, Sanjay

    2015-12-15

    We report the DNA probe functionalized electrochemical genosensor for the detection of Bacillus anthracis, specific towards the regulatory gene atxA. The DNA sensor is fabricated on electrochemically deposited gold nanoparticle on self assembled layer of (3-Mercaptopropyl) trimethoxysilane (MPTS) on GC electrode. DNA hybridization is monitored by differential pulse voltammogram (DPV). The modified GC electrode is characterized by atomic force microscopy (AFM), cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS) method. We also quantified the DNA probe density on electrode surface by the chronocoulometric method. The detection is specific and selective for atxA gene by DNA probe on the electrode surface. No report is available for the detection of B. anthracis by using atxA an anthrax toxin activator gene. In the light of real and complex sample, we have studied the PCR amplicons of 303, 361 and 568 base pairs by using symmetric and asymmetric PCR approaches. The DNA probe of atxA gene efficiently hybridizes with different base pairs of PCR amplicons. The detection limit is found to be 1.0 pM (S/N ratio=3). The results indicate that the DNA sensor is able to detect synthetic target as well as PCR amplicons of different base pairs. PMID:26257186

  8. Sample Preparation for Fungal Community Analysis by High-Throughput Sequencing of Barcode Amplicons.

    Science.gov (United States)

    Clemmensen, Karina Engelbrecht; Ihrmark, Katarina; Durling, Mikael Brandström; Lindahl, Björn D

    2016-01-01

    Fungal species participate in vast numbers of processes in the landscape around us. However, their often cryptic growth, inside various substrates and in highly diverse species assemblages, has been a major obstacle to thorough analysis of fungal communities, hampering exhaustive description of the fungal kingdom. Recent technological developments allowing rapid, high-throughput sequencing of mixed communities from many samples at once are currently having a tremendous impact in fungal community ecology. Universal DNA extraction followed by amplification and sequencing of fungal species-level barcodes such as the nuclear internal transcribed spacer (ITS) region now enable identification and relative quantification of fungal community members across well-replicated experimental settings. Here, we present the sample preparation procedure presently used in our laboratory for fungal community analysis by high-throughput sequencing of amplified ITS2 markers. We focus on the procedure optimized for studies of total fungal communities in humus-rich soils, wood, and litter. However, this procedure can be applied to other sample types and markers. We focus on the laboratory-based part of sample preparation, that is, the procedure from the point where samples enter the laboratory until amplicons are submitted for sequencing. Our procedure comprises four main parts: (1) universal DNA extraction, (2) optimization of PCR conditions, (3) production of tagged ITS amplicons, and (4) preparation of the multiplexed amplicon mix to be sequenced. The presented procedure is independent of the specific high-throughput sequencing technology used, which makes it highly versatile. PMID:26791497

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

    Directory of Open Access Journals (Sweden)

    Steve Babben

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

  10. Impact of Mutation Type and Amplicon Characteristics on Genetic Diversity Measures Generated Using a High-Resolution Melting Diversity Assay

    OpenAIRE

    Cousins, Matthew M.; Donnell, Deborah; Eshleman, Susan H.

    2013-01-01

    We adapted high-resolution melting (HRM) technology to measure genetic diversity without sequencing. Diversity is measured as a single numeric HRM score. Herein, we determined the impact of mutation types and amplicon characteristics on HRM diversity scores. Plasmids were generated with single-base changes, insertions, and deletions. Different primer sets were used to vary the position of mutations within amplicons. Plasmids and plasmid mixtures were analyzed to determine the impact of mutati...

  11. 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

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

    Science.gov (United States)

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

    2010-04-01

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

  13. Identification of candidate genes for an early-maturing soybean mutant by genome resequencing analysis.

    Science.gov (United States)

    Lee, Kyung Jun; Kim, Dong Sub; Kim, Jin-Baek; Jo, Sung-Hwan; Kang, Si-Yong; Choi, Hong-Il; Ha, Bo-Keun

    2016-08-01

    Flowering is indicative of the transition from vegetative to reproductive phase, a critical event in the life cycle of plants. In this study, we performed whole genome resequencing by Illumina HiSeq to identify changes in flowering genes using an early-flowering phenotype of soybean mutant line Josaengserori (JS) derived from Korean landrace, Seoritae (SR), and we obtained mapped reads of 131,769,690 and 167,669,640 bp in JS and SR, respectively. From the whole genome sequencing results between JS and SR, we identified 332,821 polymorphic SNPs and 65,178 indels, respectively. Among these, 30 flowering genes were in SNPs and 25 were in indels. Among 30 flowering genes detected in SNPs, Glyma02g33040, Glyma06g22650, Glyma10g36600, Glyma13g01290, Glyma14g10530, Glyma16g01980, Glyma17g11040, Glyma18g53690, and Glyma20g29300 were non-synonymous substitutions between JS and SR. Changes in Glyma10g36600 (GI), Glya02g33040 (AGL18), Glyma17g11040 (TOC1), and Glyma14g10530 (ELF3) in JS affected the expression of GmFT2a and resulted in early flowering. These results provide insight into the regulatory pathways of flowering in soybean mutants and help to improve our knowledge of soybean mutation breeding. PMID:27033554

  14. 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.

  15. 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.

  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. Identification of candidate genes for an early-maturing soybean mutant by genome resequencing analysis.

    Science.gov (United States)

    Lee, Kyung Jun; Kim, Dong Sub; Kim, Jin-Baek; Jo, Sung-Hwan; Kang, Si-Yong; Choi, Hong-Il; Ha, Bo-Keun

    2016-08-01

    Flowering is indicative of the transition from vegetative to reproductive phase, a critical event in the life cycle of plants. In this study, we performed whole genome resequencing by Illumina HiSeq to identify changes in flowering genes using an early-flowering phenotype of soybean mutant line Josaengserori (JS) derived from Korean landrace, Seoritae (SR), and we obtained mapped reads of 131,769,690 and 167,669,640 bp in JS and SR, respectively. From the whole genome sequencing results between JS and SR, we identified 332,821 polymorphic SNPs and 65,178 indels, respectively. Among these, 30 flowering genes were in SNPs and 25 were in indels. Among 30 flowering genes detected in SNPs, Glyma02g33040, Glyma06g22650, Glyma10g36600, Glyma13g01290, Glyma14g10530, Glyma16g01980, Glyma17g11040, Glyma18g53690, and Glyma20g29300 were non-synonymous substitutions between JS and SR. Changes in Glyma10g36600 (GI), Glya02g33040 (AGL18), Glyma17g11040 (TOC1), and Glyma14g10530 (ELF3) in JS affected the expression of GmFT2a and resulted in early flowering. These results provide insight into the regulatory pathways of flowering in soybean mutants and help to improve our knowledge of soybean mutation breeding.

  18. Clinical impact of targeted amplicon sequencing for meningioma as a practical clinical-sequencing system.

    Science.gov (United States)

    Yuzawa, Sayaka; Nishihara, Hiroshi; Yamaguchi, Shigeru; Mohri, Hiromi; Wang, Lei; Kimura, Taichi; Tsuda, Masumi; Tanino, Mishie; Kobayashi, Hiroyuki; Terasaka, Shunsuke; Houkin, Kiyohiro; Sato, Norihiro; Tanaka, Shinya

    2016-07-01

    Recent genetic analyses using next-generation sequencers have revealed numerous genetic alterations in various tumors including meningioma, which is the most common primary brain tumor. However, their use as routine laboratory examinations in clinical applications for tumor genotyping is not cost effective. To establish a clinical sequencing system for meningioma and investigate the clinical significance of genotype, we retrospectively performed targeted amplicon sequencing on 103 meningiomas and evaluated the association with clinicopathological features. We designed amplicon-sequencing panels targeting eight genes including NF2 (neurofibromin 2), TRAF7, KLF4, AKT1, and SMO. Libraries prepared with genomic DNA extracted from PAXgene-fixed paraffin-embedded tissues of 103 meningioma specimens were sequenced using the Illumina MiSeq. NF2 loss in some cases was also confirmed by interphase-fluorescent in situ hybridization. We identified NF2 loss and/or at least one mutation in NF2, TRAF7, KLF4, AKT1, and SMO in 81 out of 103 cases (79%) by targeted amplicon sequencing. On the basis of genetic status, we categorized meningiomas into three genotype groups: NF2 type, TRAKLS type harboring mutation in TRAF7, AKT1, KLF4, and/or SMO, and 'not otherwise classified' type. Genotype significantly correlated with tumor volume, tumor location, and magnetic resonance imaging findings such as adjacent bone change and heterogeneous gadolinium enhancement, as well as histopathological subtypes. In addition, multivariate analysis revealed that genotype was independently associated with risk of recurrence. In conclusion, we established a rapid clinical sequencing system that enables final confirmation of meningioma genotype within 7 days turnaround time. Our method will bring multiple benefits to neuropathologists and neurosurgeons for accurate diagnosis and appropriate postoperative management. PMID:27102344

  19. 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.

  20. 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. PMID:27318568

  1. Shedding Light on the Microbial Community of the Macropod Foregut Using 454-Amplicon Pyrosequencing

    OpenAIRE

    Gulino, Lisa-Maree; Ouwerkerk, Diane; Alicia Y H Kang; Maguire, Anita J.; Kienzle, Marco; Klieve, Athol V.

    2013-01-01

    Twenty macropods from five locations in Queensland, Australia, grazing on a variety of native pastures were surveyed and the bacterial community of the foregut was examined using 454-amplicon pyrosequencing. Specifically, the V3/V4 region of 16S rRNA gene was examined. A total of 5040 OTUs were identified in the data set (post filtering). Thirty-two OTUs were identified as ‘shared’ OTUS (i.e. present in all samples) belonging to either Firmicutes or Bacteroidetes (Clostridiales/Bacteroidales)...

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

    Science.gov (United States)

    Pabinger, Stephan; Ernst, Karina; Pulverer, Walter; Kallmeyer, Rainer; Valdes, Ana M; Metrustry, Sarah; Katic, Denis; Nuzzo, Angelo; Kriegner, Albert; Vierlinger, Klemens; Weinhaeusel, Andreas

    2016-01-01

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

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

    OpenAIRE

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

    2015-01-01

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

  4. 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.

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

    Science.gov (United States)

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

    2016-01-01

    Microbiome analysis using metagenomic sequencing has revealed a vast microbial diversity associated with plants. Identifying the molecular functions associated with microbiome-plant interaction is a significant challenge concerning the development of microbiome-derived technologies applied to agriculture. An alternative to accelerate the discovery of the microbiome benefits to plants is to construct microbial culture collections concomitant with accessing microbial community structure and abundance. However, traditional methods of isolation, cultivation, and identification of microbes are time-consuming and expensive. Here we describe a method for identification of microbes in culture collections constructed by picking colonies from primary platings that may contain single or multiple microorganisms, which we named community-based culture collections (CBC). A multiplexing 16S rRNA gene amplicon sequencing based on two-step PCR amplifications with tagged primers for plates, rows, and columns allowed the identification of the microbial composition regardless if the well contains single or multiple microorganisms. The multiplexing system enables pooling amplicons into a single tube. The sequencing performed on the PacBio platform led to recovery near-full-length 16S rRNA gene sequences allowing accurate identification of microorganism composition in each plate well. Cross-referencing with plant microbiome structure and abundance allowed the estimation of diversity and abundance representation of microorganism in the CBC. PMID:27404280

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

    Science.gov (United States)

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

    2015-01-01

    A phenomenon of yield decline due to weak plant growth in strawberry was recently observed in non-chemo-fumigated soils, which was not associated with the soil fungal pathogen Verticillium dahliae, the main target of fumigation. Amplicon-based metagenomics was used to profile soil microbiota in order to identify microbial organisms that may have caused the yield decline. A total of 36 soil samples were obtained in 2013 and 2014 from four sites for metagenomic studies; two of the four sites had a yield-decline problem, the other two did not. More than 2000 fungal or bacterial operational taxonomy units (OTUs) were found in these samples. Relative abundance of individual OTUs was statistically compared for differences between samples from sites with or without yield decline. A total of 721 individual comparisons were statistically significant - involving 366 unique bacterial and 44 unique fungal OTUs. Based on further selection criteria, we focused on 34 bacterial and 17 fungal OTUs and found that yield decline resulted probably from one or more of the following four factors: (1) low abundance of Bacillus and Pseudomonas populations, which are well known for their ability of supressing pathogen development and/or promoting plant growth; (2) lack of the nematophagous fungus (Paecilomyces species); (3) a high level of two non-specific fungal root rot pathogens; and (4) wet soil conditions. This study demonstrated the usefulness of an amplicon-based metagenomics approach to profile soil microbiota and to detect differential abundance in microbes. PMID:26504572

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

    Directory of Open Access Journals (Sweden)

    Clive J Hoggart

    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.

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

    Science.gov (United States)

    Shin, Younhee; Jung, Ho-jin; Jung, Myunghee; Yoo, Seungil; Subramaniyam, Sathiyamoorthy; Markkandan, Kesavan; Kang, Jun-Mo; Rai, Rajani; Park, Junhyung; Kim, Jong-Joo

    2016-01-01

    Hanwoo, a Korean native cattle (Bos taurus coreana), has great economic value due to high meat quality. Also, the breed has genetic variations that are associated with production traits such as health, disease resistance, reproduction, growth as well as carcass quality. In this study, next generation sequencing technologies and the availability of an appropriate reference genome were applied to discover a large amount of single nucleotide polymorphisms (SNPs) in ten Hanwoo bulls. Analysis of whole-genome resequencing generated a total of 26.5 Gb data, of which 594,716,859 and 592,990,750 reads covered 98.73% and 93.79% of the bovine reference genomes of UMD 3.1 and Btau 4.6.1, respectively. In total, 2,473,884 and 2,402,997 putative SNPs were discovered, of which 1,095,922 (44.3%) and 982,674 (40.9%) novel SNPs were discovered against UMD3.1 and Btau 4.6.1, respectively. Among the SNPs, the 46,301 (UMD 3.1) and 28,613 SNPs (Btau 4.6.1) that were identified as Hanwoo-specific SNPs were included in the functional genes that may be involved in the mechanisms of milk production, tenderness, juiciness, marbling of Hanwoo beef and yellow hair. Most of the Hanwoo-specific SNPs were identified in the promoter region, suggesting that the SNPs influence differential expression of the regulated genes relative to the relevant traits. In particular, the non-synonymous (ns) SNPs found in CORIN, which is a negative regulator of Agouti, might be a causal variant to determine yellow hair of Hanwoo. Our results will provide abundant genetic sources of variation to characterize Hanwoo genetics and for subsequent breeding. PMID:26954201

  11. Association of targeted multiplex PCR with resequencing microarray for the detection of multiple respiratory pathogens

    Directory of Open Access Journals (Sweden)

    Xuejun eMa

    2015-05-01

    Full Text Available A large number of viral and bacterial organisms are responsible for community-acquired pneumonia (CAP which contributes to substantial burden on health management. A new resequencing microarray (RPM-IVDC1 associated with targeted multiplex PCR was recently developed and validated for multiple respiratory viruses detection and discrimination. In this study, we evaluated the capability of RPM-IVDC1 for simultaneous identification of multiple viral and bacterial organisms. The nasopharyngeal aspirates (NPAs of 110 consecutive CAP patients, aged from 1 month to 96 years old, were collected from 5 distinct general hospitals in Beijing during 1-year period. The samples were subjected to the RPM-IVDC1 established protocol as compared to a real-time PCR (qRT-PCR, which was used as standard. The results of virus detection were consistent with those previously described. A total of 37 of Streptococcus pneumoniae, 14 of Haemophilus influenzae, 10 of Mycoplasma pneumoniae, 2 of Klebsiella pneumoniae and 1 of Moraxella catarrhalis were detected by RPM-IVDC1. The sensitivities and specificities were compared with those of qRT-PCR for S. pneumoniae (100%, 100%, respectively, H. influenzae (92.3%, 97.9%, respectively, M. pneumoniae (69.2%, 99.0%, respectively, K. pneumoniae (100%, 100%, respectively and M. catarrhalis (100%, 100%, respectively. Additional 22 of Streptococcus spp., 24 of Haemophilus spp. and 16 of Neisseria spp. were identified. In addition, methicillin-resistant and carbapenemases allele were also found in 9 of Staphylococcus spp. and 1 of K. pneumoniae, respectively. These results demonstrated the capability of RPM-IVDC1 for simultaneous detection of broad-spectrum respiratory pathogens in complex backgrounds and the advantage of accessing to the actual sequences, showing great potential use of epidemic outbreak investigation. The detection results should be carefully interpreted when introducing this technique in the clinical diagnostics.

  12. 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.

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

    Science.gov (United States)

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

    2015-11-01

    The identification of genes influencing fitness is central to our understanding of the genetic basis of adaptation and how it shapes phenotypic variation in wild populations. Here, we used whole-genome resequencing of wild Rocky Mountain bighorn sheep (Ovis canadensis) to >50-fold coverage to identify 2.8 million single nucleotide polymorphisms (SNPs) and genomic regions bearing signatures of directional selection (i.e. selective sweeps). A comparison of SNP diversity between the X chromosome and the autosomes indicated that bighorn males had a dramatically reduced long-term effective population size compared to females. This probably reflects a long history of intense sexual selection mediated by male-male competition for mates. Selective sweep scans based on heterozygosity and nucleotide diversity revealed evidence for a selective sweep shared across multiple populations at RXFP2, a gene that strongly affects horn size in domestic ungulates. The massive horns carried by bighorn rams appear to have evolved in part via strong positive selection at RXFP2. We identified evidence for selection within individual populations at genes affecting early body growth and cellular response to hypoxia; however, these must be interpreted more cautiously as genetic drift is strong within local populations and may have caused false positives. These results represent a rare example of strong genomic signatures of selection identified at genes with known function in wild populations of a nonmodel species. Our results also showcase the value of reference genome assemblies from agricultural or model species for studies of the genomic basis of adaptation in closely related wild taxa. PMID:26454263

  14. 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.

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

    DEFF Research Database (Denmark)

    Guo, Shaogui; Zhang, Jianguo; Sun, Honghe;

    2013-01-01

    evolutionary scenario for the origin of the 11 watermelon chromosomes derived from a 7-chromosome paleohexaploid eudicot ancestor. Resequencing of 20 watermelon accessions representing three different C. lanatus subspecies produced numerous haplotypes and identified the extent of genetic diversity and......Watermelon, Citrullus lanatus, is an important cucurbit crop grown throughout the world. Here we report a high-quality draft genome sequence of the east Asia watermelon cultivar 97103 (2n = 2× = 22) containing 23,440 predicted protein-coding genes. Comparative genomics analysis provided an...

  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. 16S rRNA amplicon sequencing dataset for conventionalized and conventionally raised zebrafish larvae.

    Science.gov (United States)

    Davis, Daniel J; Bryda, Elizabeth C; Gillespie, Catherine H; Ericsson, Aaron C

    2016-09-01

    Data presented here contains metagenomic analysis regarding the sequential conventionalization of germ-free zebrafish embryos. Zebrafish embryos that underwent a germ-free sterilization process immediately after fertilization were promptly exposed to and raised to larval stage in conventional fish water. At 6 days postfertilization (dpf), these "conventionalized" larvae were compared to zebrafish larvae that were raised in conventional fish water never undergoing the initial sterilization process. Bacterial 16S rRNA amplicon sequencing was performed on DNA isolated from homogenates of the larvae revealing distinct microbiota variations between the two groups. The dataset described here is also related to the research article entitled "Microbial modulation of behavior and stress responses in zebrafish larvae" (Davis et al., 2016) [1]. PMID:27508247

  18. Rare amplicons implicate frequent deregulation of cell fate specification pathways in oral squamous cell carcinoma.

    Science.gov (United States)

    Snijders, Antoine M; Schmidt, Brian L; Fridlyand, Jane; Dekker, Nusi; Pinkel, Daniel; Jordan, Richard C K; Albertson, Donna G

    2005-06-16

    Genomes of solid tumors are characterized by gains and losses of regions, which may contribute to tumorigenesis by altering gene expression. Often the aberrations are extensive, encompassing whole chromosome arms, which makes identification of candidate genes in these regions difficult. Here, we focused on narrow regions of gene amplification to facilitate identification of genetic pathways important in oral squamous cell carcinoma (SCC) development. We used array comparative genomic hybridization (array CGH) to define minimum common amplified regions and then used expression analysis to identify candidate driver genes in amplicons that spanned LAMA3, MMP7), as well as members of the hedgehog (GLI2) and notch (JAG1, RBPSUH, FJX1) pathways to be amplified and overexpressed. Deregulation of these and other members of the hedgehog and notch pathways (HHIP, SMO, DLL1, NOTCH4) implicates deregulation of developmental and differentiation pathways, cell fate misspecification, in oral SCC development. PMID:15824737

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

    Science.gov (United States)

    Yanthan, Mhathung; Misra, Arvind K

    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. PMID:24287658

  20. 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.

  1. Re-sequencing data for refining candidate genes and polymorphisms in QTL regions affecting adiposity in chicken.

    Directory of Open Access Journals (Sweden)

    Pierre-François Roux

    Full Text Available In this study, we propose an approach aiming at fine-mapping adiposity QTL in chicken, integrating whole genome re-sequencing data. First, two QTL regions for adiposity were identified by performing a classical linkage analysis on 1362 offspring in 11 sire families obtained by crossing two meat-type chicken lines divergently selected for abdominal fat weight. Those regions, located on chromosome 7 and 19, contained a total of 77 and 84 genes, respectively. Then, SNPs and indels in these regions were identified by re-sequencing sires. Considering issues related to polymorphism annotations for regulatory regions, we focused on the 120 and 104 polymorphisms having an impact on protein sequence, and located in coding regions of 35 and 42 genes situated in the two QTL regions. Subsequently, a filter was applied on SNPs considering their potential impact on the protein function based on conservation criteria. For the two regions, we identified 42 and 34 functional polymorphisms carried by 18 and 24 genes, and likely to deeply impact protein, including 3 coding indels and 4 nonsense SNPs. Finally, using gene functional annotation, a short list of 17 and 4 polymorphisms in 6 and 4 functional genes has been defined. Even if we cannot exclude that the causal polymorphisms may be located in regulatory regions, this strategy gives a complete overview of the candidate polymorphisms in coding regions and prioritize them on conservation- and functional-based arguments.

  2. Candidate adaptive genes associated with lineage divergence: identifying SNPs via next-generation targeted resequencing in mule deer (Odocoileus hemionus).

    Science.gov (United States)

    Powell, John H; Amish, Stephen J; Haynes, Gwilym D; Luikart, Gordon; Latch, Emily K

    2016-09-01

    Mule deer (Odocoileus hemionus) are an excellent nonmodel species for empirically testing hypotheses in landscape and population genomics due to their large population sizes (low genetic drift), relatively continuous distribution, diversity of occupied habitats and phenotypic variation. Because few genomic resources are currently available for this species, we used exon data from a cattle (Bos taurus) reference genome to direct targeted resequencing of 5935 genes in mule deer. We sequenced approximately 3.75 Mbp at minimum 20X coverage in each of the seven mule deer, identifying 23 204 single nucleotide polymorphisms (SNPs) within, or adjacent to, 6886 exons in 3559 genes. We found 91 SNP loci (from 69 genes) with putatively fixed allele frequency differences between the two major lineages of mule deer (mule deer and black-tailed deer), and our estimate of mean genetic divergence (genome-wide FST  = 0.123) between these lineages was consistent with previous findings using microsatellite loci. We detected an over-representation of gamete generation and amino acid transport genes among the genes with SNPs exhibiting potentially fixed allele frequency differences between lineages. This targeted resequencing approach using exon capture techniques has identified a suite of loci that can be used in future research to investigate the genomic basis of adaptation and differentiation between black-tailed deer and mule deer. This study also highlights techniques (and an exon capture array) that will facilitate population genomic research in other cervids and nonmodel organisms. PMID:27438092

  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...

  5. 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

  6. Digital fragment analysis of short tandem repeats by high-throughput amplicon sequencing.

    Science.gov (United States)

    Darby, Brian J; Erickson, Shay F; Hervey, Samuel D; Ellis-Felege, Susan N

    2016-07-01

    High-throughput sequencing has been proposed as a method to genotype microsatellites and overcome the four main technical drawbacks of capillary electrophoresis: amplification artifacts, imprecise sizing, length homoplasy, and limited multiplex capability. The objective of this project was to test a high-throughput amplicon sequencing approach to fragment analysis of short tandem repeats and characterize its advantages and disadvantages against traditional capillary electrophoresis. We amplified and sequenced 12 muskrat microsatellite loci from 180 muskrat specimens and analyzed the sequencing data for precision of allele calling, propensity for amplification or sequencing artifacts, and for evidence of length homoplasy. Of the 294 total alleles, we detected by sequencing, only 164 alleles would have been detected by capillary electrophoresis as the remaining 130 alleles (44%) would have been hidden by length homoplasy. The ability to detect a greater number of unique alleles resulted in the ability to resolve greater population genetic structure. The primary advantages of fragment analysis by sequencing are the ability to precisely size fragments, resolve length homoplasy, multiplex many individuals and many loci into a single high-throughput run, and compare data across projects and across laboratories (present and future) with minimal technical calibration. A significant disadvantage of fragment analysis by sequencing is that the method is only practical and cost-effective when performed on batches of several hundred samples with multiple loci. Future work is needed to optimize throughput while minimizing costs and to update existing microsatellite allele calling and analysis programs to accommodate sequence-aware microsatellite data. PMID:27386092

  7. 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.

  8. 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.

  9. 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.

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

    Science.gov (United States)

    Stein, Benjamin; Alonso, María Teresa; Zufall, Frank; Leinders-Zufall, Trese; Chamero, Pablo

    2016-01-01

    In mice, social behaviors such as mating and aggression are mediated by pheromones and related chemosignals. The vomeronasal organ (VNO) detects olfactory information from other individuals by sensory neurons tuned to respond to specific chemical cues. Receptors expressed by vomeronasal neurons are implicated in selective detection of these cues. Nearly 400 receptor genes have been identified in the mouse VNO, but the tuning properties of individual receptors remain poorly understood, in part due to the lack of a robust heterologous expression system. Here we develop a herpes virus-based amplicon delivery system to overexpress three types of vomeronasal receptor genes and to characterize cell responses to their proposed ligands. Through Ca2+ imaging in native VNO cells we show that virus-induced overexpression of V1rj2, V2r1b or Fpr3 caused a pronounced increase of responsivity to sulfated steroids, MHC-binding peptide or the synthetic hexapeptide W-peptide, respectively. Other related ligands were not recognized by infected individual neurons, indicating a high degree of selectivity by the overexpressed receptor. Removal of G-protein signaling eliminates Ca2+ responses, indicating that the endogenous second messenger system is essential for observing receptor activation. Our results provide a novel expression system for vomeronasal receptors that should be useful for understanding the molecular logic of VNO ligand detection. Functional expression of vomeronasal receptors and their deorphanization provides an essential requirement for deciphering the neural mechanisms controlling behavior. PMID:27195771

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

    Directory of Open Access Journals (Sweden)

    Benjamin Stein

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

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

    KAUST Repository

    Pearman, John K.

    2015-11-01

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

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

    Directory of Open Access Journals (Sweden)

    Lisa-Maree Gulino

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

  14. Flow cytometry community fingerprinting and amplicon sequencing for the assessment of landfill leachate cellulolytic bioaugmentation.

    Science.gov (United States)

    Kinet, R; Dzaomuho, P; Baert, J; Taminiau, B; Daube, G; Nezer, C; Brostaux, Y; Nguyen, F; Dumont, G; Thonart, P; Delvigne, F

    2016-08-01

    Flow cytometry (FCM) is a high throughput single cell technology that is actually becoming widely used for studying phenotypic and genotypic diversity among microbial communities. This technology is considered in this work for the assessment of a bioaugmentation treatment in order to enhance cellulolytic potential of landfill leachate. The experimental results reveal the relevant increase of leachate cellulolytic potential due to bioaugmentation. Cytometric monitoring of microbial dynamics along these assays is then realized. The flow FP package is used to establish microbial samples fingerprint from initial 2D cytometry histograms. This procedure allows highlighting microbial communities' variation along the assays. Cytometric and 16S rRNA gene sequencing fingerprinting methods are then compared. The two approaches give same evidence about microbial dynamics throughout digestion assay. There are however a lack of significant correlation between cytometric and amplicon sequencing fingerprint at genus or species level. Same phenotypical profiles of microbiota during assays matched to several 16S rRNA gene sequencing ones. Flow cytometry fingerprinting can thus be considered as a promising routine on-site method suitable for the detection of stability/variation/disturbance of complex microbial communities involved in bioprocesses. PMID:27160955

  15. Amplicon pyrosequencing reveals the soil microbial diversity associated with invasive Japanese barberry (Berberis thunbergii DC.).

    Science.gov (United States)

    Coats, V C; Pelletreau, K N; Rumpho, M E

    2014-03-01

    The soil microbial community acts as a reservoir of microbes that directly influences the structure and composition of the aboveground plant community, promotes plant growth, increases stress tolerance and mediates local patterns of nutrient cycling. Direct interactions between plants and rhizosphere-dwelling microorganisms occur at, or near, the surface of the root. Upon introduction and establishment, invasive plants modify the soil microbial communities and soil biochemistry affecting bioremediation efforts and future plant communities. Here, we used tag-encoded FLX amplicon 454 pyrosequencing (TEFAP) to characterize the bacterial and fungal community diversity in the rhizosphere of Berberis thunbergii DC. (Japanese barberry) from invasive stands in coastal Maine to investigate effects of soil type, soil chemistry and surrounding plant cover on the soil microbial community structure. Acidobacteria, Actinobacteria, Proteobacteria and Verrucomicrobia were the dominant bacterial phyla, whereas fungal communities were comprised mostly of Ascomycota and Basidiomycota phyla members, including Agaricomycetes and Sordariomycetes. Bulk soil chemistry had more effect on the bacterial community structure than the fungal community. An effect of geographic location was apparent in the rhizosphere microbial communities, yet it was less significant than the effect of surrounding plant cover. These data demonstrate a high degree of spatial variation in the rhizosphere microbial communities of Japanese barberry with apparent effects of soil chemistry, location and canopy cover on the microbial community structure. PMID:24118303

  16. 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.

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

    Science.gov (United States)

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

    2010-03-01

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

  18. 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. PMID:27311063

  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. Genetic variation between Schistosoma japonicum lineages from lake and mountainous regions in China revealed by resequencing whole genomes.

    Science.gov (United States)

    Yin, Mingbo; Liu, Xiao; Xu, Bin; Huang, Jian; Zheng, Qi; Yang, Zhong; Feng, Zheng; Han, Ze-Guang; Hu, Wei

    2016-09-01

    Schistosoma infection is a major cause of morbidity and mortality worldwide. Schistosomiasis japonica is endemic in mainland China along the Yangtze River, typically distributed in two geographical categories of lake and mountainous regions. Study on schistosome genetic diversity is of interest in respect of understanding parasite biology and transmission, and formulating control strategy. Certain genetic variations may be associated with adaptations to different ecological habitats. The aim of this study is to gain insight into Schistosoma japonicum genetic variation, evolutionary origin and associated causes of different geographic lineages through examining homozygous Single Nucleotide Polymorphisms (SNPs) based on resequenced genome data. We collected S. japonicum samples from four sites, three in the lake regions (LR) of mid-east (Guichi and Tonglin in Anhui province, Laogang in Hunan province) and one in mountainous region (MR) (Xichang in Sichuan province) of south-west of China, resequenced their genomes using Next Generation Sequencing (NGS) technology, and made use of the available database of S. japonicum draft genomic sequence as a reference in genome mapping. A total of 14,575 SNPs from 2059 genes were identified in the four lineages. Phylogenetic analysis confirmed significant genetic variation exhibited between the different geographical lineages, and further revealed that the MR Xichang lineage is phylogenetically closer to LR Guich lineage than to other two LR lineages, and the MR lineage might be evolved from LR lineages. More than two thirds of detected SNPs were nonsynonymous; functional annotation of the SNP-containing genes showed that they are involved mainly in biological processes such as signaling and response to stimuli. Notably, unique nonsynonymous SNP variations were detected in 66 genes of MR lineage, inferring possible genetic adaption to mountainous ecological condition. PMID:27207135

  1. 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.

  2. 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. PMID:27311063

  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. Analysis of the mouse gut microbiome using full-length 16S rRNA amplicon sequencing.

    Science.gov (United States)

    Shin, Jongoh; Lee, Sooin; Go, Min-Jeong; Lee, Sang Yup; Kim, Sun Chang; Lee, Chul-Ho; Cho, Byung-Kwan

    2016-01-01

    Demands for faster and more accurate methods to analyze microbial communities from natural and clinical samples have been increasing in the medical and healthcare industry. Recent advances in next-generation sequencing technologies have facilitated the elucidation of the microbial community composition with higher accuracy and greater throughput than was previously achievable; however, the short sequencing reads often limit the microbial composition analysis at the species level due to the high similarity of 16S rRNA amplicon sequences. To overcome this limitation, we used the nanopore sequencing platform to sequence full-length 16S rRNA amplicon libraries prepared from the mouse gut microbiota. A comparison of the nanopore and short-read sequencing data showed that there were no significant differences in major taxonomic units (89%) except one phylotype and three taxonomic units. Moreover, both sequencing data were highly similar at all taxonomic resolutions except the species level. At the species level, nanopore sequencing allowed identification of more species than short-read sequencing, facilitating the accurate classification of the bacterial community composition. Therefore, this method of full-length 16S rRNA amplicon sequencing will be useful for rapid, accurate and efficient detection of microbial diversity in various biological and clinical samples. PMID:27411898

  5. Voltammetric detection of sequence-selective DNA hybridization related to Toxoplasma gondii in PCR amplicons.

    Science.gov (United States)

    Gokce, Gultekin; Erdem, Arzum; Ceylan, Cagdas; Akgöz, Muslum

    2016-03-01

    This work describes the single-use electrochemical DNA biosensor technology developed for voltammetric detection of sequence selective DNA hybridization related to important human and veterinary pathogen; Toxoplasma gondii. In the principle of electrochemical label-free detection assay, the duplex of DNA hybrid formation was detected by measuring guanine oxidation signal occured in the presence of DNA hybridization. The biosensor design consisted of the immobilization of an inosine-modified (guanine-free) probe onto the surface of pencil graphite electrode (PGE), and the detection of the duplex formation in connection with the differential pulse voltammetry(DPV) by measuring the guanine signal. Toxoplasma gondii capture probe was firstly immobilized onto the surface of the activated PGE by wet adsorption. The extent of hybridization at PGE surface between the probe and the target was then determined by measuring the guanine signal observed at +1.0V. The electrochemical monitoring of optimum DNA hybridization has been performed in the target concentration of 40µg/mL in 50min of hybridization time. The specificity of the electrochemical biosensor was then tested using non-complementary, or mismatch short DNA sequences. Under the optimum conditions, the guanine oxidation signal indicating full hybridization was measured in various target concentration from 0.5 to 25µg/mL and a detection limit was found to be 1.78µg/mL. This single-use biosensor platform was successfully applied for the voltammetric detection of DNA hybridization related to Toxoplasma gondii in PCR amplicons. PMID:26717837

  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. 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. PMID:27003585

  8. Analysis of a Drosophila amplicon in follicle cells highlights the diversity of metazoan replication origins.

    Science.gov (United States)

    Kim, Jane C; Orr-Weaver, Terry L

    2011-10-01

    To investigate the properties of metazoan replication origins, recent studies in cell culture have adopted the strategy of identifying origins using genome-wide approaches and assessing correlations with such features as transcription and histone modifications. Drosophila amplicon in follicle cells (DAFCs), genomic regions that undergo repeated rounds of DNA replication to increase DNA copy number, serve as powerful in vivo model replicons. Because there are six DAFCs, compared with thousands of origins activated in the typical S phase, close molecular characterization of all DAFCs is possible. To determine the extent to which the six DAFCs are different or similar, we investigated the developmental and replication properties of the newly identified DAFC-34B. DAFC-34B contains two genes expressed in follicle cells, although the timing and spatial patterns of expression suggest that amplification is not a strategy to promote high expression at this locus. Like the previously characterized DAFC-62D, DAFC-34B displays origin activation at two separate stages of development. However, unlike DAFC-62D, amplification at the later stage is not transcription-dependent. We mapped the DAFC-34B amplification origin to 1 kb by nascent strand analysis and delineated cis requirements for origin activation, finding that a 6-kb region, but not the 1-kb origin alone, is sufficient for amplification. We analyzed the developmental localization of the origin recognition complex (ORC) and the minichromosome maintenance (MCM)2-7 complex, the replicative helicase. Intriguingly, the final round of origin activation at DAFC-34B occurs in the absence of detectable ORC, although MCMs are present, suggesting a new amplification initiation mechanism. PMID:21933960

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

    Directory of Open Access Journals (Sweden)

    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.

  10. Rare UNC13B variations and risk of schizophrenia: Whole-exome sequencing in a multiplex family and follow-up resequencing and a case-control study.

    Science.gov (United States)

    Egawa, Jun; Hoya, Satoshi; Watanabe, Yuichiro; Nunokawa, Ayako; Shibuya, Masako; Ikeda, Masashi; Inoue, Emiko; Okuda, Shujiro; Kondo, Kenji; Saito, Takeo; Kaneko, Naoshi; Muratake, Tatsuyuki; Igeta, Hirofumi; Iwata, Nakao; Someya, Toshiyuki

    2016-09-01

    Rare genomic variations inherited in multiplex schizophrenia families are suggested to play a role in the genetic etiology of the disease. To identify rare variations with large effects on the risk of developing schizophrenia, we performed whole-exome sequencing (WES) in two affected and one unaffected individual of a multiplex family with 10 affected individuals. We also performed follow-up resequencing of the unc-13 homolog B (Caenorhabditis elegans) (UNC13B) gene, a potential risk gene identified by WES, in the multiplex family and undertook a case-control study to investigate association between UNC13B and schizophrenia. UNC13B coding regions (39 exons) from 15 individuals of the multiplex family and 111 affected offspring for whom parental DNA samples were available were resequenced. Rare missense UNC13B variations identified by resequencing were further tested for association with schizophrenia in two independent case-control populations comprising a total of 1,753 patients and 1,602 controls. A rare missense variation (V1525M) in UNC13B was identified by WES in the multiplex family; this variation was present in five of six affected individuals, but not in eight unaffected individuals or one individual of unknown disease status. Resequencing UNC13B coding regions identified five rare missense variations (T103M, M813T, P1349T, I1362T, and V1525M). In the case-control study, there was no significant association between rare missense UNC13B variations and schizophrenia, although single-variant meta-analysis indicated that M813T was nominally associated with schizophrenia. These results do not support a contribution of rare missense UNC13B variations to the genetic etiology of schizophrenia in the Japanese population. © 2016 Wiley Periodicals, Inc. PMID:26990377

  11. Bioinformatics Pipelines for Targeted Resequencing and Whole-Exome Sequencing of Human and Mouse Genomes: A Virtual Appliance Approach for Instant Deployment

    OpenAIRE

    Jason Li; Doyle, Maria A; Isaam Saeed; Wong, Stephen Q.; Victoria Mar; Goode, David L.; Franco Caramia; Ken Doig; Ryland, Georgina L.; Thompson, Ella R.; Hunter, Sally M.; Halgamuge, Saman K; Jason Ellul; Alexander Dobrovic; Campbell, Ian G

    2014-01-01

    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 (Targete...

  12. N-acetyltransferase 8, a positional candidate for blood pressure and renal regulation: resequencing, association and in silico study

    Directory of Open Access Journals (Sweden)

    Rosenberg Mai

    2008-04-01

    Full Text Available Abstract Background Kidneys have an important function in blood pressure (BP regulation and elevated BP may lead to kidney failure. Chr2p12-p13 region linked to BP traits in multiple studies harbours a potential candidate for BP and renal function, N-acetyltransferase 8 (NAT8 expressed in embryonic and adult kidney and associated with nephrotoxicity response. Methods/Results We report the first study exploring NAT8 as a potential candidate gene for blood pressure and kidney function. The resequencing (n = 42, random Estonian samples identified 15 NAT8 polymorphisms, including 6 novel variants. The diversity of NAT8 5' upstream region (π/bp = 0.00320 exceeded up to 10 times the variation in the NAT8 genic region (π/bp = 0.00037 as well as the average variation (π/bp = 0.00040 for the promoters of 29 reference genes associated with hypertension. We suggest that a potential source for such high variation could be an active gene conversion process from NAT8B duplicate gene to NAT8. Similarly to NAT8, several reference genes with the most variable upstream regions have also duplicate copies. The NAT8 promoter SNPs were targeted with pilot quantitative association studies for blood pressure (n = 137, healthy unrelated individuals and for the index of kidney function – estimated glomerular filtration rate (eGFR; n = 157 hypertensives with and without nephropathy. Minor alleles of these polymorphisms revealed a significant protective effect against elevated systolic BP as well as kidney failure in hypertension patients (p Conclusion The full resequencing and pilot association study of a novel positional candidate gene for blood pressure and renal function, human N-acetyltransferase 8, suggested a contribution of highly variable NAT8 promoter polymorphisms in determination of systolic blood pressure and eGFR. Based on in silico analysis, we raise the hypothesis that the alternative SNP alleles of the NAT8 upstream region may have differential effect

  13. 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

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

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    Peng-Rong Cao

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

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

    Science.gov (United States)

    Cao, Peng-Rong; Wang, Lei; Jiang, Yu-Chao; Yi, Yin-Sha; Qu, Fang; Liu, Tao-Cheng; Lv, Yuan

    2015-01-01

    The formation of new genes is a primary driving force of evolution in all organisms. The de novo evolution of new genes from non-protein-coding genomic regions is emerging as an important additional mechanism for novel gene creation. Y chromosomes underlie sex determination in mammals and contain genes that are required for male-specific functions. In this study, a search was undertaken for Y chromosome de novo genes derived from non-protein-coding sequences. The Y chromosome orphan gene variable charge, Y-linked (VCY)2, is an autosome-derived gene that has sequence similarity to large autosomal fragments but lacks an autosomal protein-coding homolog. VCY2 locates in the amplicon containing long DNA fragments that were transposed from autosomes to the Y chromosome before the ape-monkey split. We confirmed that VCY2 cannot be encoded by autosomes due to the presence of multiple disablers that disrupt the open reading frame, such as the absence of start or stop codons and the presence of premature stop codons. Similar observations have been made for homologs in the autosomes of the chimpanzee, gorilla, rhesus macaque, baboon and out-group marmoset, which suggests that there was a non-protein-coding ancestral VCY2 that was common to apes and monkeys that predated the transposition event. Furthermore, while protein-coding orthologs are absent, a putative non-protein-coding VCY2 with conserved disablers was identified in the rhesus macaque Y chromosome male-specific region. This finding implies that VCY2 might have not acquired its protein-coding ability before the ape-monkey split. VCY2 encodes a testis-specific expressed protein and is involved in the pathologic process of male infertility, and the acquisition of this gene might improve male fertility. This is the first evidence that de novo genes can be generated from transposed autosomal non-protein-coding segments, and this evidence provides novel insights into the evolutionary history of the Y chromosome. PMID

  16. 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.

  17. Identification of the maize gravitropism gene lazy plant1 by a transposon-tagging genome resequencing strategy.

    Science.gov (United States)

    Howard, Thomas P; Hayward, Andrew P; Tordillos, Anthony; Fragoso, Christopher; Moreno, Maria A; Tohme, Joe; Kausch, Albert P; Mottinger, John P; Dellaporta, Stephen L

    2014-01-01

    Since their initial discovery, transposons have been widely used as mutagens for forward and reverse genetic screens in a range of organisms. The problems of high copy number and sequence divergence among related transposons have often limited the efficiency at which tagged genes can be identified. A method was developed to identity the locations of Mutator (Mu) transposons in the Zea mays genome using a simple enrichment method combined with genome resequencing to identify transposon junction fragments. The sequencing library was prepared from genomic DNA by digesting with a restriction enzyme that cuts within a perfectly conserved motif of the Mu terminal inverted repeats (TIR). Paired-end reads containing Mu TIR sequences were computationally identified and chromosomal sequences flanking the transposon were mapped to the maize reference genome. This method has been used to identify Mu insertions in a number of alleles and to isolate the previously unidentified lazy plant1 (la1) gene. The la1 gene is required for the negatively gravitropic response of shoots and mutant plants lack the ability to sense gravity. Using bioinformatic and fluorescence microscopy approaches, we show that the la1 gene encodes a cell membrane and nuclear localized protein. Our Mu-Taq method is readily adaptable to identify the genomic locations of any insertion of a known sequence in any organism using any sequencing platform. PMID:24498020

  18. Identification of the maize gravitropism gene lazy plant1 by a transposon-tagging genome resequencing strategy.

    Directory of Open Access Journals (Sweden)

    Thomas P Howard

    Full Text Available Since their initial discovery, transposons have been widely used as mutagens for forward and reverse genetic screens in a range of organisms. The problems of high copy number and sequence divergence among related transposons have often limited the efficiency at which tagged genes can be identified. A method was developed to identity the locations of Mutator (Mu transposons in the Zea mays genome using a simple enrichment method combined with genome resequencing to identify transposon junction fragments. The sequencing library was prepared from genomic DNA by digesting with a restriction enzyme that cuts within a perfectly conserved motif of the Mu terminal inverted repeats (TIR. Paired-end reads containing Mu TIR sequences were computationally identified and chromosomal sequences flanking the transposon were mapped to the maize reference genome. This method has been used to identify Mu insertions in a number of alleles and to isolate the previously unidentified lazy plant1 (la1 gene. The la1 gene is required for the negatively gravitropic response of shoots and mutant plants lack the ability to sense gravity. Using bioinformatic and fluorescence microscopy approaches, we show that the la1 gene encodes a cell membrane and nuclear localized protein. Our Mu-Taq method is readily adaptable to identify the genomic locations of any insertion of a known sequence in any organism using any sequencing platform.

  19. 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

  20. 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.

  1. 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.

  2. The cytological manifestation of gene amplification in multidrug-resistant mouse leukemia P388 sublines is correlated with amplicon content

    Energy Technology Data Exchange (ETDEWEB)

    Il`inskaya, G.V.; Kopnin, B.P. [Institute of Carcinogenesis, Moscow (Russian Federation); Demidova, N.S. [Institute of Chemical Physics, Moscow (Russian Federation)

    1995-10-01

    Previously, we showed that development of multidrug resistance (MDR) in mouse P388 leukemia cells is often associated with the appearance of newly-formed chromosomelike structures that contain amplified copies of the mdr1 gene. In the present study, we compared amplicon content in P388 sublines showing different types of these structures. A strong correlation between the formation of specific acentric markers consisting of two identical arms and the absence of the sorcin gene coamplification was found. In all the sublines containing other types of chromosomelike structures, the sorcin gene is coamplified. 9 refs., 2 figs., 1 tab.

  3. 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.

  4. RiboFR-Seq: a novel approach to linking 16S rRNA amplicon profiles to metagenomes.

    Science.gov (United States)

    Zhang, Yanming; Ji, Peifeng; Wang, Jinfeng; Zhao, Fangqing

    2016-06-01

    16S rRNA amplicon analysis and shotgun metagenome sequencing are two main culture-independent strategies to explore the genetic landscape of various microbial communities. Recently, numerous studies have employed these two approaches together, but downstream data analyses were performed separately, which always generated incongruent or conflict signals on both taxonomic and functional classifications. Here we propose a novel approach, RiboFR-Seq (Ribosomal RNA gene flanking region sequencing), for capturing both ribosomal RNA variable regions and their flanking protein-coding genes simultaneously. Through extensive testing on clonal bacterial strain, salivary microbiome and bacterial epibionts of marine kelp, we demonstrated that RiboFR-Seq could detect the vast majority of bacteria not only in well-studied microbiomes but also in novel communities with limited reference genomes. Combined with classical amplicon sequencing and shotgun metagenome sequencing, RiboFR-Seq can link the annotations of 16S rRNA and metagenomic contigs to make a consensus classification. By recognizing almost all 16S rRNA copies, the RiboFR-seq approach can effectively reduce the taxonomic abundance bias resulted from 16S rRNA copy number variation. We believe that RiboFR-Seq, which provides an integrated view of 16S rRNA profiles and metagenomes, will help us better understand diverse microbial communities. PMID:26984526

  5. 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.

  6. 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.

  7. 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; Viala, Sandrine; Decousus, Stephanie; Perin, Axel; Lafarge, Laurence; Ollier, Marie; El Saghir, Nagi S.

    2016-01-01

    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.

  8. Targeted resequencing identifies PTCH1 as a major contributor to ocular developmental anomalies and extends the SOX2 regulatory network.

    Science.gov (United States)

    Chassaing, Nicolas; Davis, Erica E; McKnight, Kelly L; Niederriter, Adrienne R; Causse, Alexandre; David, Véronique; Desmaison, Annaïck; Lamarre, Sophie; Vincent-Delorme, Catherine; Pasquier, Laurent; Coubes, Christine; Lacombe, Didier; Rossi, Massimiliano; Dufier, Jean-Louis; Dollfus, Helene; Kaplan, Josseline; Katsanis, Nicholas; Etchevers, Heather C; Faguer, Stanislas; Calvas, Patrick

    2016-04-01

    Ocular developmental anomalies (ODA) such as anophthalmia/microphthalmia (AM) or anterior segment dysgenesis (ASD) have an estimated combined prevalence of 3.7 in 10,000 births. Mutations inSOX2are the most frequent contributors to severe ODA, yet account for a minority of the genetic drivers. To identify novel ODA loci, we conducted targeted high-throughput sequencing of 407 candidate genes in an initial cohort of 22 sporadic ODA patients. Patched 1 (PTCH1), an inhibitor of sonic hedgehog (SHH) signaling, harbored an enrichment of rare heterozygous variants in comparison to either controls, or to the other candidate genes (four missense and one frameshift); targeted resequencing ofPTCH1in a second cohort of 48 ODA patients identified two additional rare nonsynonymous changes. Using multiple transient models and a CRISPR/Cas9-generated mutant, we show physiologically relevant phenotypes altering SHH signaling and eye development upon abrogation ofptch1in zebrafish for which in vivo complementation assays using these models showed that all six patient missense mutations affect SHH signaling. Finally, through transcriptomic and ChIP analyses, we show that SOX2 binds to an intronic domain of thePTCH1locus to regulatePTCH1expression, findings that were validated both in vitro and in vivo. Together, these results demonstrate thatPTCH1mutations contribute to as much as 10% of ODA, identify the SHH signaling pathway as a novel effector of SOX2 activity during human ocular development, and indicate that ODA is likely the result of overactive SHH signaling in humans harboring mutations in eitherPTCH1orSOX2. PMID:26893459

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

    Directory of Open Access Journals (Sweden)

    Etienne Patin

    2009-04-01

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

  10. Whole exome re-sequencing implicates CCDC38 and cilia structure and function in resistance to smoking related airflow obstruction.

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    Louise V Wain

    2014-05-01

    Full Text Available Chronic obstructive pulmonary disease (COPD is a leading cause of global morbidity and mortality and, whilst smoking remains the single most important risk factor, COPD risk is heritable. Of 26 independent genomic regions showing association with lung function in genome-wide association studies, eleven have been reported to show association with airflow obstruction. Although the main risk factor for COPD is smoking, some individuals are observed to have a high forced expired volume in 1 second (FEV1 despite many years of heavy smoking. We hypothesised that these "resistant smokers" may harbour variants which protect against lung function decline caused by smoking and provide insight into the genetic determinants of lung health. We undertook whole exome re-sequencing of 100 heavy smokers who had healthy lung function given their age, sex, height and smoking history and applied three complementary approaches to explore the genetic architecture of smoking resistance. Firstly, we identified novel functional variants in the "resistant smokers" and looked for enrichment of these novel variants within biological pathways. Secondly, we undertook association testing of all exonic variants individually with two independent control sets. Thirdly, we undertook gene-based association testing of all exonic variants. Our strongest signal of association with smoking resistance for a non-synonymous SNP was for rs10859974 (P = 2.34 × 10(-4 in CCDC38, a gene which has previously been reported to show association with FEV1/FVC, and we demonstrate moderate expression of CCDC38 in bronchial epithelial cells. We identified an enrichment of novel putatively functional variants in genes related to cilia structure and function in resistant smokers. Ciliary function abnormalities are known to be associated with both smoking and reduced mucociliary clearance in patients with COPD. We suggest that genetic influences on the development or function of cilia in the bronchial

  11. Application of high-density DNA resequencing microarray for detection and characterization of botulinum neurotoxin-producing clostridia.

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    Jessica Vanhomwegen

    Full Text Available BACKGROUND: Clostridium botulinum and related clostridia express extremely potent toxins known as botulinum neurotoxins (BoNTs that cause severe, potentially lethal intoxications in humans. These BoNT-producing bacteria are categorized in seven major toxinotypes (A through G and several subtypes. The high diversity in nucleotide sequence and genetic organization of the gene cluster encoding the BoNT components poses a great challenge for the screening and characterization of BoNT-producing strains. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we designed and evaluated the performances of a resequencing microarray (RMA, the PathogenId v2.0, combined with an automated data approach for the simultaneous detection and characterization of BoNT-producing clostridia. The unique design of the PathogenID v2.0 array allows the simultaneous detection and characterization of 48 sequences targeting the BoNT gene cluster components. This approach allowed successful identification and typing of representative strains of the different toxinotypes and subtypes, as well as the neurotoxin-producing C. botulinum strain in a naturally contaminated food sample. Moreover, the method allowed fine characterization of the different neurotoxin gene cluster components of all studied strains, including genomic regions exhibiting up to 24.65% divergence with the sequences tiled on the arrays. CONCLUSIONS/SIGNIFICANCE: The severity of the disease demands rapid and accurate means for performing risk assessments of BoNT-producing clostridia and for tracing potentials sources of contamination in outbreak situations. The RMA approach constitutes an essential higher echelon component in a diagnostics and surveillance pipeline. In addition, it is an important asset to characterise potential outbreak related strains, but also environment isolates, in order to obtain a better picture of the molecular epidemiology of BoNT-producing clostridia.

  12. 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.

  13. A high-resolution of porcine radiation hybrid panel helps to develop useful SNP markers with bioinformatics and resequencing methods

    International Nuclear Information System (INIS)

    Full text: A Pig-Human comparative map established by a Pig RH map was used to localize single nucleotide polymorphisms (SNP) in the pig genome. SNP are abundant form of genetic variation, which are useful for gene mapping, association and evolution analyses. NCBI pig SNP database has deposited over 5,450 SNP and the 5,450 sequences were consolidated into 465 unique sequences (189 singleton sequences and 276 contigs). These 465 sequences contained 1787 putative SNP and had strong sequence homology to 433 human protein coding genes based on BLAST analyses. These genes were assigned to the pig QTL maps (http://www.animalgenome.org/QTLdb/pig.html) via the human and pig comparative maps established by a pig radiation hybrid (RH) map. In order to determine identity and allelic distribution among commercial pig breeds, in total, 428 pairs of primers including 1,338 putative SNP on SSC1-18 were re-sequenced in Berkshire, Landrace, Duroc, Yorkshire and Korean native pig (KNP) breeds, and 908 SNP were identified in at least one pig breed. Of the 908 SNP, 228 novel SNP (25.1%) were found. According to the SNP location in the predicted gene sequences, these SNP were categorized to intronic SNP (86.34%), synonymous SNP (sSNP, 10.35%), and non-synonimous SNP (nsSNP, 3.41%). The nucleotide substitution frequency of the SNP revealed that 70.26% were transition (33.5% A/G, 36.8% C/T), 24.01% were transversion, and 5.51% were deletion/insertion mutation. The SNP information from comparative RH map between pig and human will be useful for association studies using high-throughput genotyping and help to differentiate the commercial pig breeds on DNA level. (author)

  14. Comparative genomics-based investigation of resequencing targets in Vibrio fischeri: Focus on point miscalls and artefactual expansions

    Directory of Open Access Journals (Sweden)

    Ruby Edward G

    2008-03-01

    Full Text Available Abstract Background Sequence closure often represents the end-point of a genome project, without a system in place for subsequent improvement and refinement. Building on the genome project of Vibrio fischeri ES114, we used a comparative approach to identify and investigate genes that had a high likelihood of sequence error. Results Comparison of the V. fischeri ES114 genome with that of conspecific strain MJ11 identified 82 target loci in ES114 as containing likely errors, and thus of high-priority for resequencing. Analysis of the targets identified 75 loci in which an error had occurred, resulting in the correction of 10,457 base pairs to generate the new ES114 genomic sequence. A majority of the inaccurate loci involved frameshift errors, correction of which fused adjacent ORFs. Although insertions/deletions are thought to be rare in microbial genome assemblies, fourteen of the loci contained extraneous sequence of over 300 bp, likely due to imperfect contig ends that were misassembled in tandem rather than as overlapping segments. Additionally we updated the entire genome annotation with 113 new features including previously uncalled protein-coding genes, regulatory RNA genes and operon leader peptides, and we analyzed the transcriptional apparatus encoded by ES114. Conclusion We demonstrate that errors in microbial genome sequences, thought to largely be confined to point mutations, may also consist of other prevalent large-scale rearrangements such as insertions. Ongoing genome quality control and annotation programs are necessary to accompany technological advancements in data generation. These updates further advance V. fischeri as an important model for understanding intercellular communication and colonization of animal tissue.

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

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    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.

  16. Targeted resequencing implicates the familial Mediterranean fever gene MEFV and the toll-like receptor 4 gene TLR4 in Behçet disease

    OpenAIRE

    Kirino, Yohei; Zhou, Qing; Ishigatsubo, Yoshiaki; Mizuki, Nobuhisa; Tugal-Tutkun, Ilknur; Seyahi, Emire; Özyazgan, Yilmaz; Ugurlu, Serdal; Erer, Burak; Abaci, Neslihan; Ustek, Duran; Meguro, Akira; Ueda, Atsuhisa; Takeno, Mitsuhiro; Inoko, Hidetoshi

    2013-01-01

    Genome-wide association studies (GWAS) are a powerful means of identifying genes with disease-associated common variants, but they are not well-suited to detecting genes with disease-associated rare and low-frequency variants. In the current study of Behçet disease (BD), nonsynonymous variants (NSVs) identified by deep exonic resequencing of 10 genes found by GWAS (IL10, IL23R, CCR1, STAT4, KLRK1, KLRC1, KLRC2, KLRC3, KLRC4, and ERAP1) and 11 genes selected for their role in innate immunity (...

  17. Optimisation of 16S rDNA amplicon sequencing protocols for microbial community profiling of anaerobic digesters

    DEFF Research Database (Denmark)

    Kirkegaard, Rasmus Hansen; McIlroy, Simon Jon; Larsen, Poul;

    of the community composition. As such sample specific optimisation and standardisation of DNA extraction, as well PCR primer selection, are essential to minimising the potential for such biases. The aim of this study was to develop a protocol for optimized community profiling of anaerobic digesters. The FastDNA......RNA gene amplicon sequencing is rapid, cheap, high throughput, and has high taxonomic resolution. However, biases are introduced in multiple steps of this approach, including non-representative DNA extraction and uneven taxonomic coverage of selected PCR primers, potentially giving a skewed view...... SPIN kit was selected and the mechanical lysis parameters optimised for extraction of genomic DNA from mesophilic and thermophilic anaerobic digester samples. Different primer sets were compared for targeting the archaea and bacteria, both together and individually. Shotgun sequencing of a TruSeq PCR...

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

    DEFF Research Database (Denmark)

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

    of the community composition . As such sample specific optimisation and standardisation of DNA extraction, as well PCR primer selection, are essential to minimising the potential for such biases. The aim of this study was to develop a protocol for optimized community profiling of anaerobic digesters. The FastDNA......RNA gene amplicon sequencing is rapid, cheap, high throughput, and has high taxonomic resolution. However, biases are introduced in multiple steps of this approach, including non-representative DNA extraction and uneven taxonomic coverage of selected PCR primers, potentially giving a skewed view...... SPIN kit was selected and the mechanical lysis parameters optimised for extraction of genomic DNA from mesophilic and thermophilic anaerobic digester samples. Different primer sets were compared for targeting the archaea and bacteria, both together and individually . Shotgun sequencing...

  19. 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...

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

    Directory of Open Access Journals (Sweden)

    Leho Tedersoo

    2015-05-01

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

  1. Short-read assembly of full-length 16S amplicons reveals bacterial diversity in subsurface sediments.

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    Christopher S Miller

    Full Text Available In microbial ecology, a fundamental question relates to how community diversity and composition change in response to perturbation. Most studies have had limited ability to deeply sample community structure (e.g. Sanger-sequenced 16S rRNA libraries, or have had limited taxonomic resolution (e.g. studies based on 16S rRNA hypervariable region sequencing. Here, we combine the higher taxonomic resolution of near-full-length 16S rRNA gene amplicons with the economics and sensitivity of short-read sequencing to assay the abundance and identity of organisms that represent as little as 0.01% of sediment bacterial communities. We used a new version of EMIRGE optimized for large data size to reconstruct near-full-length 16S rRNA genes from amplicons sheared and sequenced with Illumina technology. The approach allowed us to differentiate the community composition among samples acquired before perturbation, after acetate amendment shifted the predominant metabolism to iron reduction, and once sulfate reduction began. Results were highly reproducible across technical replicates, and identified specific taxa that responded to the perturbation. All samples contain very high alpha diversity and abundant organisms from phyla without cultivated representatives. Surprisingly, at the time points measured, there was no strong loss of evenness, despite the selective pressure of acetate amendment and change in the terminal electron accepting process. However, community membership was altered significantly. The method allows for sensitive, accurate profiling of the "long tail" of low abundance organisms that exist in many microbial communities, and can resolve population dynamics in response to environmental change.

  2. 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.

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

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    Diana L Cousminer

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

  4. 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

  5. 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/.

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

    Science.gov (United States)

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

    2016-01-01

    Genome-wide association studies have revealed several common genetic risk variants for ulcerative colitis (UC). However, little is known about the contribution of rare, large effect genetic variants to UC susceptibility. In this study, we performed a deep targeted re-sequencing of 122 genes in Dutch UC patients in order to investigate the contribution of rare variants to the genetic susceptibility to UC. The selection of genes consists of 111 established human UC susceptibility genes and 11 genes that lead to spontaneous colitis when knocked-out in mice. In addition, we sequenced the promoter regions of 45 genes where known variants exert cis-eQTL-effects. Targeted pooled re-sequencing was performed on DNA of 790 Dutch UC cases. The Genome of the Netherlands project provided sequence data of 500 healthy controls. After quality control and prioritization based on allele frequency and pathogenicity probability, follow-up genotyping of 171 rare variants was performed on 1021 Dutch UC cases and 1166 Dutch controls. Single-variant association and gene-based analyses identified an association of rare variants in the MUC2 gene with UC. The associated variants in the Dutch population could not be replicated in a German replication cohort (1026 UC cases, 3532 controls). In conclusion, this study has identified a putative role for MUC2 on UC susceptibility in the Dutch population and suggests a population-specific contribution of rare variants to UC. PMID:27490946

  7. 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

  8. 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.

  9. 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

  10. 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

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

    Directory of Open Access Journals (Sweden)

    Viviana Kozina

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

  12. 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. PMID:26476551

  13. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Directory of Open Access Journals (Sweden)

    Planý Matej

    2016-06-01

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

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

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

    2016-08-01

    High amounts of driftwood sail across the oceans and provide habitat for organisms tolerating the rough and saline environment. Fungi have adapted to the extremely cold and saline conditions which driftwood faces in the high north. For the first time, we applied high-throughput sequencing to fungi residing in driftwood to reveal their taxonomic richness, community composition, and ecology in the North Atlantic. Using pyrosequencing of ITS2 amplicons obtained from 49 marine logs, we found 807 fungal operational taxonomic units (OTUs) based on clustering at 97 % sequence similarity cut-off level. The phylum Ascomycota comprised 74 % of the OTUs and 20 % belonged to Basidiomycota. The richness of basidiomycetes decreased with prolonged submersion in the sea, supporting the general view of ascomycetes being more extremotolerant. However, more than one fourth of the fungal OTUs remained unassigned to any fungal class, emphasising the need for better DNA reference data from the marine habitat. Different fungal communities were detected in coniferous and deciduous logs. Our results highlight that driftwood hosts a considerably higher fungal diversity than currently known. The driftwood fungal community is not a terrestrial relic but a speciose assemblage of fungi adapted to the stressful marine environment and different kinds of wooden substrates found in it. PMID:27147245

  18. 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.

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

    Science.gov (United States)

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

    2016-01-01

    Brassica napus (rapeseed) is a recent allotetraploid plant and the second most important oilseed crop worldwide. The origin of B. napus and the genetic relationships with its diploid ancestor species remain largely unresolved. Here, chloroplast DNA (cpDNA) from 488 B. napus accessions of global origin, 139 B. rapa accessions and 49 B. oleracea accessions were populationally resequenced using Illumina Solexa sequencing technologies. The intraspecific cpDNA variants and their allelic frequencies were called genomewide and further validated via EcoTILLING analyses of the rpo region. The cpDNA of the current global B. napus population comprises more than 400 variants (SNPs and short InDels) and maintains one predominant haplotype (Bncp1). Whole-genome resequencing of the cpDNA of Bncp1 haplotype eliminated its direct inheritance from any accession of the B. rapa or B. oleracea species. The distribution of the polymorphism information content (PIC) values for each variant demonstrated that B. napus has much lower cpDNA diversity than B. rapa; however, a vast majority of the wild and cultivated B. oleracea specimens appeared to share one same distinct cpDNA haplotype, in contrast to its wild C-genome relatives. This finding suggests that the cpDNA of the three Brassica species is well differentiated. The predominant B. napus cpDNA haplotype may have originated from uninvestigated relatives or from interactions between cpDNA mutations and natural/artificial selection during speciation and evolution. These exhaustive data on variation in cpDNA would provide fundamental data for research on cpDNA and chloroplasts. PMID:26031705

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

    Directory of Open Access Journals (Sweden)

    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.

  1. 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.

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

    Directory of Open Access Journals (Sweden)

    Lam Paula Y

    2010-10-01

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

  3. 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. PMID:26976467

  4. 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.

  5. Very high resolution single pass HLA genotyping using amplicon sequencing on the 454 next generation DNA sequencers: Comparison with Sanger sequencing.

    Science.gov (United States)

    Yamamoto, F; Höglund, B; Fernandez-Vina, M; Tyan, D; Rastrou, M; Williams, T; Moonsamy, P; Goodridge, D; Anderson, M; Erlich, H A; Holcomb, C L

    2015-12-01

    Compared to Sanger sequencing, next-generation sequencing offers advantages for high resolution HLA genotyping including increased throughput, lower cost, and reduced genotype ambiguity. Here we describe an enhancement of the Roche 454 GS GType HLA genotyping assay to provide very high resolution (VHR) typing, by the addition of 8 primer pairs to the original 14, to genotype 11 HLA loci. These additional amplicons help resolve common and well-documented alleles and exclude commonly found null alleles in genotype ambiguity strings. Simplification of workflow to reduce the initial preparation effort using early pooling of amplicons or the Fluidigm Access Array™ is also described. Performance of the VHR assay was evaluated on 28 well characterized cell lines using Conexio Assign MPS software which uses genomic, rather than cDNA, reference sequence. Concordance was 98.4%; 1.6% had no genotype assignment. Of concordant calls, 53% were unambiguous. To further assess the assay, 59 clinical samples were genotyped and results compared to unambiguous allele assignments obtained by prior sequence-based typing supplemented with SSO and/or SSP. Concordance was 98.7% with 58.2% as unambiguous calls; 1.3% could not be assigned. Our results show that the amplicon-based VHR assay is robust and can replace current Sanger methodology. Together with software enhancements, it has the potential to provide even higher resolution HLA typing. PMID:26037172

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

    Directory of Open Access Journals (Sweden)

    Franck Curk

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

  7. 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

  8. 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.

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

    Directory of Open Access Journals (Sweden)

    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.  

  10. 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

  11. 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.

  12. 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. PMID:27033205

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

    Directory of Open Access Journals (Sweden)

    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

  14. 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

    OpenAIRE

    Carr Steven M; Flynn Sarah MC

    2007-01-01

    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 se...

  15. 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

  16. 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

  17. 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

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

    OpenAIRE

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

    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 elucida...

  19. 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.

  20. 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. PMID:27065251

  1. 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.

  2. 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. PMID:27640093

  3. 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.

  4. Genomic structure analysis of a set of Oryza nivara introgression lines and identification of yield-associated QTLs using whole-genome resequencing.

    Science.gov (United States)

    Ma, Xin; Fu, Yongcai; Zhao, Xinhui; Jiang, Liyun; Zhu, Zuofeng; Gu, Ping; Xu, Wenying; Su, Zhen; Sun, Chuanqing; Tan, Lubin

    2016-01-01

    Oryza nivara, an annual wild AA-genome species of rice, is an important gene pool for broadening the genetic diversity of cultivated rice (O. sativa L.). Towards identifying and utilizing favourable alleles from O. nivara, we developed a set of introgression lines (ILs) by introducing O. nivara segments into the elite indica rice variety 93-11 background through advanced backcrossing and repeated selfing. Using whole-genome resequencing, a high-density genetic map containing 1,070 bin-markers was constructed for the 131 ILs, with an average length of 349 kb per bin. The 131 ILs cover 95% of O. nivara genome, providing a relatively complete genomic library for introgressing O. nivara alleles for trait improvement. Using this high-density bin-map, QTL mapping for 13 yield-related traits was performed and a total of 65 QTLs were detected across two environments. At ~36.9% of detected QTLs, the alleles from O. nivara conferred improving effects on yield-associated traits. Six cloned genes, Sh4/SHA1, Bh4, Sd1, TE/TAD1, GS3 and FZP, colocalised in the peak intervals of 9 QTLs. In conclusion, we developed new genetic materials for exploration and use of beneficial alleles from wild rice and provided a basis for future fine mapping and cloning of the favourable O. nivara-derived QTLs. PMID:27251022

  5. Rapid Molecular Profiling of Myeloproliferative Neoplasms Using Targeted Exon Resequencing of 86 Genes Involved in JAK-STAT Signaling and Epigenetic Regulation.

    Science.gov (United States)

    Magor, Graham W; Tallack, Michael R; Klose, Nathan M; Taylor, Debra; Korbie, Darren; Mollee, Peter; Trau, Matt; Perkins, Andrew C

    2016-09-01

    Myeloproliferative neoplasms (MPNs) are a heterogeneous group of blood disorders characterized by excess production of mature blood cells and an increased risk of late transformation to acute myeloid leukemia or primary myelofibrosis. Approximately 15% of MPN cases do not carry mutations in JAK2, CALR, or MPL and are thus often referred to as triple-negative cases. These are caused by a diverse set of rare mutations in cytokine receptors, JAK-STAT signaling pathway components, or epigenetic modifiers. In addition, some cases diagnosed as MPN are reactive rather than clonal disorders, so a negative result from a genetic screen can be informative. To obtain a comprehensive rapid molecular diagnosis for most MPNs, we developed an assay to detect genetic mutations (single nucleotide variants and/or small insertions/deletions) in 86 genes using targeted exon resequencing (AmpliSeq) and a bench-top semiconductor machine (Ion Torrent Personal Genome Machine). Our assay reliably detects well characterized mutations in JAK2, CALR, and MPL, but also rarer mutations in ASXL1, TET2, SH2B3, and other genes. Some of these mutations are novel. We find multiple mutations in advanced cases, suggesting co-operation between Janus kinase-STAT pathway mutations and epigenetic mutations in disease progression. This assay can be used to follow molecular progression, clonal heterogeneity, and drug resistance in MPNs. PMID:27449473

  6. 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

  7. 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.

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

    Science.gov (United States)

    Smeds, Linnéa; Mugal, Carina F; Qvarnström, Anna; Ellegren, Hans

    2016-05-01

    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. PMID:27219623

  9. Targeted resequencing implicates the familial Mediterranean fever gene MEFV and the toll-like receptor 4 gene TLR4 in Behçet disease.

    Science.gov (United States)

    Kirino, Yohei; Zhou, Qing; Ishigatsubo, Yoshiaki; Mizuki, Nobuhisa; Tugal-Tutkun, Ilknur; Seyahi, Emire; Özyazgan, Yilmaz; Ugurlu, Serdal; Erer, Burak; Abaci, Neslihan; Ustek, Duran; Meguro, Akira; Ueda, Atsuhisa; Takeno, Mitsuhiro; Inoko, Hidetoshi; Ombrello, Michael J; Satorius, Colleen L; Maskeri, Baishali; Mullikin, James C; Sun, Hong-Wei; Gutierrez-Cruz, Gustavo; Kim, Yoonhee; Wilson, Alexander F; Kastner, Daniel L; Gül, Ahmet; Remmers, Elaine F

    2013-05-14

    Genome-wide association studies (GWAS) are a powerful means of identifying genes with disease-associated common variants, but they are not well-suited to detecting genes with disease-associated rare and low-frequency variants. In the current study of Behçet disease (BD), nonsynonymous variants (NSVs) identified by deep exonic resequencing of 10 genes found by GWAS (IL10, IL23R, CCR1, STAT4, KLRK1, KLRC1, KLRC2, KLRC3, KLRC4, and ERAP1) and 11 genes selected for their role in innate immunity (IL1B, IL1R1, IL1RN, NLRP3, MEFV, TNFRSF1A, PSTPIP1, CASP1, PYCARD, NOD2, and TLR4) were evaluated for BD association. A differential distribution of the rare and low-frequency NSVs of a gene in 2,461 BD cases compared with 2,458 controls indicated their collective association with disease. By stringent criteria requiring at least a single burden test with study-wide significance and a corroborating test with at least nominal significance, rare and low-frequency NSVs in one GWAS-identified gene, IL23R (P = 6.9 × 10(-5)), and one gene involved in innate immunity, TLR4 (P = 8.0 × 10(-4)), were associated with BD. In addition, damaging or rare damaging NOD2 variants were nominally significant across all three burden tests applied (P = 0.0063-0.045). Furthermore, carriage of the familial Mediterranean fever gene (MEFV) mutation Met694Val, which is known to cause recessively inherited familial Mediterranean fever, conferred BD risk in the Turkish population (OR, 2.65; P = 1.8 × 10(-12)). The disease-associated NSVs in MEFV and TLR4 implicate innate immune and bacterial sensing mechanisms in BD pathogenesis. PMID:23633568

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

    Directory of Open Access Journals (Sweden)

    Cinzia Ciccacci

    2015-03-01

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

  11. 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.

  12. A new amplicon based approach of whole mitogenome sequencing for phylogenetic and phylogeographic analysis: An example of East African white-eyes (Aves, Zosteropidae).

    Science.gov (United States)

    Meimberg, Harald; Schachtler, Christina; Curto, Manuel; Husemann, Martin; Habel, Jan Christian

    2016-09-01

    Classical Sanger sequencing is still frequently used to generate sequence data for phylogenetic and phylogeographic inference. In this contribution we present a novel approach to genotype whole mitogenomic haplotypes using Illumina MiSeq reads from indexed amplicons. Our new approach reduces preparation time by multiplexing loci within a single or few PCR reactions and by plate format library construction. The use of paired-end reads allows covering amplicons of about 0.5kb and thus no nebulisation and assembly are necessary. We tested the power and effectiveness of this technique by analysing the mitogenomic diversity of East African white-eye bird species (Zosteropidae), a taxonomically highly diverse and complex species flock found in various ecosystems spread across major parts of Africa. We compare the newly generated mitogenomic data set with published data of three mitochondrial genes for a similar set of populations and taxa. The comparison demonstrates that our new procedure represents a cost effective use of NGS for medium throughput phylogenetic analyses. Using this method, we were able to increase the amount of phylogenetic information significantly, while reducing the costs and effort in the laboratory. The mitogenomic data show a higher resolution than previous studies providing higher support and new insights in the relationships of Zosterops species. Our data suggest to split Z. poliogaster into four distinct species, three of which had previously been proposed: Z. silvanus, Z. mbulensis, Z. kikyuensis and Z. kulalensis. Our approach allows the genotyping of whole mitogenomes for a large number of individuals and thus allows more reliable reconstruction of phylogenetic and phylogeographic relationships - also for non-model organisms. PMID:27233440

  13. 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.

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

    Directory of Open Access Journals (Sweden)

    Gu Minghong

    2010-11-01

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

  15. 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

  16. 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.

  17. Gene-specific amplicons from metagenomes as an alternative to directed evolution for enzyme screening: a case study using phenylacetaldehyde reductases.

    Science.gov (United States)

    Itoh, Nobuya; Kazama, Miki; Takeuchi, Nami; Isotani, Kentaro; Kurokawa, Junji

    2016-06-01

    Screening gene-specific amplicons from metagenomes (S-GAM) is a highly promising technique for the isolation of genes encoding enzymes for biochemical and industrial applications. From metagenomes, we isolated phenylacetaldehyde reductase (par) genes, which code for an enzyme that catalyzes the production of various Prelog's chiral alcohols. Nearly full-length par genes were amplified by PCR from metagenomic DNA, the products of which were fused with engineered par sequences at both terminal regions of the expression vector to ensure proper expression and then used to construct Escherichia coli plasmid libraries. Sequence- and activity-based screening of these libraries identified different homologous par genes, Hpar-001 to -036, which shared more than 97% amino acid sequence identity with PAR. Comparative characterization of these active homologs revealed a wide variety of enzymatic properties including activity, substrate specificity, and thermal stability. Moreover, amino acid substitutions in these genes coincided with those of Sar268 and Har1 genes, which were independently engineered by error-prone PCR to exhibit increased activity in the presence of concentrated 2-propanol. The comparative data from both approaches suggest that sequence information from homologs isolated from metagenomes is quite useful for enzyme engineering. Furthermore, by examining the GAM-based sequence dataset derived from soil metagenomes, we easily found amino acid substitutions that increase the thermal stability of PAR/PAR homologs. Thus, GAM-based approaches can provide not only useful homologous enzymes but also an alternative to directed evolution methodologies. PMID:27419059

  18. Cross-comparison of exome analysis, next-generation sequencing of amplicons, and the iPLEX® ADME PGx Panel for pharmacogenomic profiling

    Directory of Open Access Journals (Sweden)

    Eng Wee eChua

    2016-01-01

    Full Text Available Whole-exome sequencing (WES has been widely used for analysis of human genetic diseases, but its value for the pharmacogenomic profiling of individuals is not well studied. Initially, we performed an in-depth evaluation of the accuracy of WES variant calling in the pharmacogenes CYP2D6 and CYP2C19 by comparison with MiSeq® amplicon sequencing data (n = 36. This analysis revealed that the concordance rate between WES and MiSeq® was high, achieving 99.60% for variants that were called without exceeding the truth-sensitivity threshold (99%, defined during variant quality score recalibration. Beyond this threshold, the proportion of discordant calls increased markedly. Subsequently, we expanded our findings beyond CYP2D6 and CYP2C19 to include more genes genotyped by the iPLEX® ADME PGx Panel in the subset of twelve samples. WES performed well, agreeing with the genotyping panel in approximately 99% of the selected pass-filter variant calls. Overall, our results have demonstrated WES to be a promising approach for pharmacogenomic profiling, with an estimated error rate of lower than 1%. Quality filters, particularly variant quality score recalibration, are important for reducing the number of false variants. Future studies may benefit from examining the role of WES in the clinical setting for guiding drug therapy.

  19. 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. PMID:26801593

  20. 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.

  1. 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.

  2. Rapid Drug Susceptibility Testing of Drug-Resistant Mycobacterium tuberculosis Isolates Directly from Clinical Samples by Use of Amplicon Sequencing: a Proof-of-Concept Study

    Science.gov (United States)

    Anderson, Julia; Lemmer, Darrin; Lehmkuhl, Erik; Georghiou, Sophia B.; Heaton, Hannah; Wiggins, Kristin; Gillece, John D.; Schupp, James M.; Catanzaro, Donald G.; Crudu, Valeriu; Cohen, Ted; Rodwell, Timothy C.; Engelthaler, David M.

    2016-01-01

    Increasingly complex drug-resistant tuberculosis (DR-TB) is a major global health concern and one of the primary reasons why TB is now the leading infectious cause of death worldwide. Rapid characterization of a DR-TB patient's complete drug resistance profile would facilitate individualized treatment in place of empirical treatment, improve treatment outcomes, prevent amplification of resistance, and reduce the transmission of DR-TB. The use of targeted next-generation sequencing (NGS) to obtain drug resistance profiles directly from patient sputum samples has the potential to enable comprehensive evidence-based treatment plans to be implemented quickly, rather than in weeks to months, which is currently needed for phenotypic drug susceptibility testing (DST) results. In this pilot study, we evaluated the performance of amplicon sequencing of Mycobacterium tuberculosis DNA from patient sputum samples using a tabletop NGS technology and automated data analysis to provide a rapid DST solution (the Next Gen-RDST assay). One hundred sixty-six out of 176 (94.3%) sputum samples from the Republic of Moldova yielded complete Next Gen-RDST assay profiles for 7 drugs of interest. We found a high level of concordance of our Next Gen-RDST assay results with phenotypic DST (97.0%) and pyrosequencing (97.8%) results from the same clinical samples. Our Next Gen-RDST assay was also able to estimate the proportion of resistant-to-wild-type alleles down to mixtures of ≤1%, which demonstrates the ability to detect very low levels of resistant variants not detected by pyrosequencing and possibly below the threshold for phenotypic growth methods. The assay as described here could be used as a clinical or surveillance tool. PMID:27225403

  3. Rapid Drug Susceptibility Testing of Drug-Resistant Mycobacterium tuberculosis Isolates Directly from Clinical Samples by Use of Amplicon Sequencing: a Proof-of-Concept Study.

    Science.gov (United States)

    Colman, Rebecca E; Anderson, Julia; Lemmer, Darrin; Lehmkuhl, Erik; Georghiou, Sophia B; Heaton, Hannah; Wiggins, Kristin; Gillece, John D; Schupp, James M; Catanzaro, Donald G; Crudu, Valeriu; Cohen, Ted; Rodwell, Timothy C; Engelthaler, David M

    2016-08-01

    Increasingly complex drug-resistant tuberculosis (DR-TB) is a major global health concern and one of the primary reasons why TB is now the leading infectious cause of death worldwide. Rapid characterization of a DR-TB patient's complete drug resistance profile would facilitate individualized treatment in place of empirical treatment, improve treatment outcomes, prevent amplification of resistance, and reduce the transmission of DR-TB. The use of targeted next-generation sequencing (NGS) to obtain drug resistance profiles directly from patient sputum samples has the potential to enable comprehensive evidence-based treatment plans to be implemented quickly, rather than in weeks to months, which is currently needed for phenotypic drug susceptibility testing (DST) results. In this pilot study, we evaluated the performance of amplicon sequencing of Mycobacterium tuberculosis DNA from patient sputum samples using a tabletop NGS technology and automated data analysis to provide a rapid DST solution (the Next Gen-RDST assay). One hundred sixty-six out of 176 (94.3%) sputum samples from the Republic of Moldova yielded complete Next Gen-RDST assay profiles for 7 drugs of interest. We found a high level of concordance of our Next Gen-RDST assay results with phenotypic DST (97.0%) and pyrosequencing (97.8%) results from the same clinical samples. Our Next Gen-RDST assay was also able to estimate the proportion of resistant-to-wild-type alleles down to mixtures of ≤1%, which demonstrates the ability to detect very low levels of resistant variants not detected by pyrosequencing and possibly below the threshold for phenotypic growth methods. The assay as described here could be used as a clinical or surveillance tool. PMID:27225403

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

    Directory of Open Access Journals (Sweden)

    Matthew Sean McCabe

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

  5. 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…

  6. Advance of SNP Analysis Based on Whole Genome Resequencing in Crop Gene Mapping%基于全基因组重测序的SNP分析在作物基因定位中的研究进展

    Institute of Scientific and Technical Information of China (English)

    袁金红; 李俊华; 黄小城; 李莎; 高武军

    2015-01-01

    In qualitative trait locus (QTL) mapping of important traits and mutant studies, target genes or ifne mapped QTLs are sometimes hard to be obtained with traditional approaches, which are laborious and time-consuming. in the past few years, the discovery of single nucleotide polymorphism (SNP) markers at the genome level is enabled by new techniques based on whole genome resequencing (WGR). Combined with SNP discovery, validation and genotyping, the WGR-based QTL mapping approach has the potential to rapidly dis-cover candidate genes and causative SNPs. By WGR-based domestication study and genome-wide association analysis of a collection of individuals, and SNP genotyping in hybrid progeny, many QTLs or mutation sites were successfully mapped in crops. This article reviewed the current advances in QTLs mapping or causative mutations detection of important traits in crops based on WGR and SNP analysis, the prospect of the approach in gene/QTL mapping was discussed and analyzed.%在作物重要性状数量性状位点(quantitative trait locus, QTL)定位和突变体研究中,利用传统的遗传标记对目标基因或QTL进行定位,往往工作量大、周期长,难以精确定位。近年来,全基因组重测序(whole genome resequencing, WGR)为从基因组水平开发单核苷酸多态性(single nucleotide polymorphysim, SNP)标记提供了新的技术条件。利用基于WGR的SNP识别、验证和基因型分析与传统分子标记相结合的方法,能很快挖掘到候选基因和获得导致表型的SNP位点。目前,通过基于WGR的群体驯化和全基因组关联分析以及对杂交后代进行全基因组SNP基因型分析,已经成功定位了若干作物的QTL及突变基因。本文就利用基于WGR的SNP分析方法定位作物重要性状QTL和突变基因的研究概况进行综述,并就该方法在基因/QTL定位上的应用前景进行了探讨和分析。

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

    Directory of Open Access Journals (Sweden)

    Carr Steven M

    2007-09-01

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

  8. 大豆小片段法HRM基因分型体系优化%Optimization of small amplicon of HRM genotyping system in soybean

    Institute of Scientific and Technical Information of China (English)

    2015-01-01

    SNP genotyping by High Resolution Melting Curve( HRM)is a simple and effective,higher sensi-tivity and stronger specificity method. Optimization and establishment of soybean HRM-based SNP genotyping sys-tem is the precondition and basis for soybean SNP research in future. In this study,small amplicon after adding the internal calibration was used to optimize and establish SNP genotyping system. The results showed that it was better to make PCR product length 50 -80bp,the melting temperature at 72 -82℃ and Tm value between 55 -62℃when designing a SNP primer. A total of 10μL PCR reaction system should contain 25ng DNA template,0. 6pmol SNP primers and 1μL LC Green dye. After PCR reaction,3 pmol high and low temperature internal calibration should be added to each wells,and then denatured for HRM analysis. Moreover,the optimized SNP genotyping system was used to genotyping a recombinant inbred lines( RILs)population,and the results demonstrated that the RILs could be completely divided into 2 genotypes of their parents by the optimized system,with improved accuracy and efficiency. The optimized SNP genotyping system established in this study will provide a useful foundation for developing SNP markers,constructing high density genetic maps,QTL identification in the future.%应用高分辨率熔解曲线( high resolution melting curve,HRM)技术进行基因分型简单有效,灵敏度高、特异性强。优化并建立基于HRM的大豆SNP基因分型体系,是准确进行SNP研究的前提和基础。本研究利用添加内标后的小片段法进行SNP基因分型体系优化研究,结果表明在设计SNP引物时,最好使其PCR产物长度为50~80bp,熔解温度在72~82℃,Tm值介于55~62℃之间;10μL PCR反应体系中应包含25ng DNA模板,0.6pmol SNP引物,1μL LC Green染料;PCR反应后,应在各点样孔分别加入3pmol的高、低温内标,然后进行变性处理和HRM分析。同时利用建立的基因分型体

  9. 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

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

    Directory of Open Access Journals (Sweden)

    Huaiyong Luo

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

  11. 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.

  12. 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

    2016-02-01

    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. PMID:26756577

  13. 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.

  14. Molecular identification of CTX-M and blaOXY/K1 β-lactamase genes in Enterobacteriaceae by sequencing of universal M13-sequence tagged PCR-amplicons

    Directory of Open Access Journals (Sweden)

    Tärnberg Maria

    2009-01-01

    Full Text Available Abstract Background Plasmid encoded blaCTX-M enzymes represent an important sub-group of class A β-lactamases causing the ESBL phenotype which is increasingly found in Enterobacteriaceae including Klebsiella spp. Molecular typing of clinical ESBL-isolates has become more and more important for prevention of the dissemination of ESBL-producers among nosocomial environment. Methods Multiple displacement amplified DNA derived from 20 K. pneumoniae and 34 K. oxytoca clinical isolates with an ESBL-phenotype was used in a universal CTX-M PCR amplification assay. Identification and differentiation of blaCTX-M and blaOXY/K1 sequences was obtained by DNA sequencing of M13-sequence-tagged CTX-M PCR-amplicons using a M13-specific sequencing primer. Results Nine out of 20 K. pneumoniae clinical isolates had a blaCTX-M genotype. Interestingly, we found that the universal degenerated primers also amplified the chromosomally located K1-gene in all 34 K. oxytoca clinical isolates. Molecular identification and differentiation between blaCTX-M and blaOXY/K1-genes could only been achieved by sequencing of the PCR-amplicons. In silico analysis revealed that the universal degenerated CTX-M primer-pair used here might also amplify the chromosomally located blaOXY and K1-genes in Klebsiella spp. and K1-like genes in other Enterobacteriaceae. Conclusion The PCR-based molecular typing method described here enables a rapid and reliable molecular identification of blaCTX-M, and blaOXY/K1-genes. The principles used in this study could also be applied to any situation in which antimicrobial resistance genes would need to be sequenced.

  15. The Dark Side of the Mushroom Spring Microbial Mat: Life in the Shadow of Chlorophototrophs. I. Microbial Diversity Based on 16S rRNA Gene Amplicons and Metagenomic Sequencing.

    Science.gov (United States)

    Thiel, Vera; Wood, Jason M; Olsen, Millie T; Tank, Marcus; Klatt, Christian G; Ward, David M; Bryant, Donald A

    2016-01-01

    Microbial-mat communities in the effluent channels of Octopus and Mushroom Springs within the Lower Geyser Basin at Yellowstone National Park have been studied for nearly 50 years. The emphasis has mostly focused on the chlorophototrophic bacterial organisms of the phyla Cyanobacteria and Chloroflexi. In contrast, the diversity and metabolic functions of the heterotrophic community in the microoxic/anoxic region of the mat are not well understood. In this study we analyzed the orange-colored undermat of the microbial community of Mushroom Spring using metagenomic and rRNA-amplicon (iTag) analyses. Our analyses disclosed a highly diverse community exhibiting a high degree of unevenness, strongly dominated by a single taxon, the filamentous anoxygenic phototroph, Roseiflexus spp. The second most abundant organisms belonged to the Thermotogae, which have been hypothesized to be a major source of H2 from fermentation that could enable photomixotrophic metabolism by Chloroflexus and Roseiflexus spp. Other abundant organisms include two members of the Armatimonadetes (OP10); Thermocrinis sp.; and phototrophic and heterotrophic members of the Chloroflexi. Further, an Atribacteria (OP9/JS1) member; a sulfate-reducing Thermodesulfovibrio sp.; a Planctomycetes member; a member of the EM3 group tentatively affiliated with the Thermotogae, as well as a putative member of the Arminicenantes (OP8) represented ≥1% of the reads. Archaea were not abundant in the iTag analysis, and no metagenomic bin representing an archaeon was identified. A high microdiversity of 16S rRNA gene sequences was identified for the dominant taxon, Roseiflexus spp. Previous studies demonstrated that highly similar Synechococcus variants in the upper layer of the mats represent ecological species populations with specific ecological adaptations. This study suggests that similar putative ecotypes specifically adapted to different niches occur within the undermat community, particularly for Roseiflexus

  16. The Dark Side of the Mushroom Spring Microbial Mat: Life in the Shadow of Chlorophototrophs. I. Microbial Diversity Based on 16S rRNA Gene Amplicons and Metagenomic Sequencing

    Science.gov (United States)

    Thiel, Vera; Wood, Jason M.; Olsen, Millie T.; Tank, Marcus; Klatt, Christian G.; Ward, David M.; Bryant, Donald A.

    2016-01-01

    Microbial-mat communities in the effluent channels of Octopus and Mushroom Springs within the Lower Geyser Basin at Yellowstone National Park have been studied for nearly 50 years. The emphasis has mostly focused on the chlorophototrophic bacterial organisms of the phyla Cyanobacteria and Chloroflexi. In contrast, the diversity and metabolic functions of the heterotrophic community in the microoxic/anoxic region of the mat are not well understood. In this study we analyzed the orange-colored undermat of the microbial community of Mushroom Spring using metagenomic and rRNA-amplicon (iTag) analyses. Our analyses disclosed a highly diverse community exhibiting a high degree of unevenness, strongly dominated by a single taxon, the filamentous anoxygenic phototroph, Roseiflexus spp. The second most abundant organisms belonged to the Thermotogae, which have been hypothesized to be a major source of H2 from fermentation that could enable photomixotrophic metabolism by Chloroflexus and Roseiflexus spp. Other abundant organisms include two members of the Armatimonadetes (OP10); Thermocrinis sp.; and phototrophic and heterotrophic members of the Chloroflexi. Further, an Atribacteria (OP9/JS1) member; a sulfate-reducing Thermodesulfovibrio sp.; a Planctomycetes member; a member of the EM3 group tentatively affiliated with the Thermotogae, as well as a putative member of the Arminicenantes (OP8) represented ≥1% of the reads. Archaea were not abundant in the iTag analysis, and no metagenomic bin representing an archaeon was identified. A high microdiversity of 16S rRNA gene sequences was identified for the dominant taxon, Roseiflexus spp. Previous studies demonstrated that highly similar Synechococcus variants in the upper layer of the mats represent ecological species populations with specific ecological adaptations. This study suggests that similar putative ecotypes specifically adapted to different niches occur within the undermat community, particularly for Roseiflexus

  17. 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

  18. Experience in public goods experiments

    OpenAIRE

    Conte, Anna; Levati, M. Vittoria; Montinari, Natalia

    2014-01-01

    We use information on students' past participation in economic experiments, as stored in our database, to analyze whether behavior in public goods games is affected by experience (i.e., previous participation in social dilemma-type experiments) and history (i.e., participation in experiments of a different class than the social dilemma). We have three main results. First, at the aggregate level, the amount subjects contribute and expect others to contribute decrease with experience. Second, a...

  19. Examining experience

    OpenAIRE

    Lakeman, G. J. T.

    2012-01-01

    I think visual experiences are intentional. And I think that different philosophical views about visual experience may be understood in terms of what they say about the intentionality of visual experience. In this thesis, I evaluate different views of experience and experiential intentionality by examining connections between experiential intentionality and further phenomenological, doxastic, epistemic and content-fixing features present in cases of perception and hallucinat...

  20. BioMaS: a modular pipeline for Bioinformatic analysis of Metagenomic AmpliconS

    OpenAIRE

    Fosso, Bruno; Santamaria, Monica; Marzano, Marinella; Alonso-Alemany, Daniel; Valiente Feruglio, Gabriel Alejandro; Donvito, Giacinto; Monaco, Alfonso; Notarangelo, Pasquale; Pesole, Graziano

    2015-01-01

    Background Substantial advances in microbiology, molecular evolution and biodiversity have been carried out in recent years thanks to Metagenomics, which allows to unveil the composition and functions of mixed microbial communities in any environmental niche. If the investigation is aimed only at the microbiome taxonomic structure, a target-based metagenomic approach, here also referred as Meta-barcoding, is generally applied. This approach commonly involves the selective amplification of a s...

  1. Art Experiences.

    Science.gov (United States)

    Spodek, Bernard; And Others

    1996-01-01

    Presents four articles that examine the role of art experiences in early childhood education: "Educationally Appropriate Art Activities for Young Children," by Bernard Spodek; "Teachers and Children Together: Constructing New Learning," by Lella Gandini; "Fostering Experiences between Young Children and Clay," by Cathy Weisman Topal; and…

  2. 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.

  3. TRIO experiment

    International Nuclear Information System (INIS)

    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

  4. Simulated experiments

    International Nuclear Information System (INIS)

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

  5. 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

  6. 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

  7. Pixel Experiments

    DEFF Research Database (Denmark)

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

    2015-01-01

    for using LED lighting in lighting design practice. The speculative experiments that have been set-up have aimed to clarify the variables that can be used as parameters in the design of lighting applications; including, for example, the structuring and software control of light. The experiments also...... design it became relevant to investigate the use of LEDs as the physical equivalent of a pixel as a design approach. In this book our interest has been in identifying how the qualities of LEDs can be used in lighting applications. With experiences in the planning and implementation of architectural...... lighting design in practice, one quickly experiences and realises that there are untapped potentials in the attributes of LED technology. In this research, speculative studies have been made working with the attributes of LEDs in architectural contexts, with the ambition to ascertain new strategies...

  8. 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.

  9. Researching Experiences

    DEFF Research Database (Denmark)

    Gjedde, Lisa; Ingemann, Bruno

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

  10. Channeling experiment

    International Nuclear Information System (INIS)

    Channeling of water flow and tracer transport in real fractures in a granite body at Stripa have been investigated experimentally. The experimental site was located 360 m below the ground level. Two kinds of experiments were performed. In the single hole experiments, 20 cm diameter holes were drilled about 2.5 m into the rock in the plane of the fracture. Specially designed packers were used to inject water into the fracture in 5 cm intervals all along the fracture trace in the hole. The variation of the injection flowrates along the fracture were used to determine the transmissivity variations in the fracture plane. Detailed photographs were taken from inside the hole and the visual fracture aperture was compared with the injection flowrates in the same locations. Geostatistical methods were used to evaluate the results. Five holes were measured in great detail. In addition 7 holes were drilled and scanned by simpler packer systems. A double hole experiment was performed where two parallel holes were drilled in the same fracture plane at nearly 2 m distance. Pressure pulse tests were made between the holes in both directions. Tracers were injected in 5 locations in one hole and monitored for in many locations in the other hole. The single hole experiment and the double hole experiment show that most of the fracture planes are tight but that there are open sections which form connected channels over distances of at least 2 meters. It was also found in the double hole experiment that the investigated fracture was intersected by at least one fracture between the two holes which diverted a large amount of the injected tracers to several distant locations at the tunnel wall. (authours)

  11. Extending Experiences

    DEFF Research Database (Denmark)

    A computer game's player is experiencing not only the game as a designer-made artefact, but also a multitude of social and cultural practices and contexts of both computer game play and everyday life. As a truly multidisciplinary anthology, Extending Experiences sheds new light on the mesh of...... possibilities and influences the player engages with. Part one, Experiential Structures of Play, considers some of the key concepts commonly used to address the experience of a computer game player. The second part, Bordering Play, discusses conceptual and practical overlaps of games and everyday life and the...

  12. Emotion experience

    NARCIS (Netherlands)

    N.H. Frijda

    2005-01-01

    Highly divergent accounts exist of the nature of emotional feelings. Following Lambie and Marcel (2002), that divergence is traced back to actual differences in experience that result from variations in the involvement and direction of attention during emotions. The dimensions of variation include f

  13. Collaborative experience

    DEFF Research Database (Denmark)

    Mortensen, Thomas Bøtker

    Literature review: Collaborative experience has been shown to have a positive effect on the collaborative outcome in general (Anand & Khanna, 2000; Kale, Dyer & Singh, 2002). Furthermore, it has been linked to the ability to exploit the network of the firm for learning (Powell, Koput and Smith...

  14. 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).

  15. Russian experience

    International Nuclear Information System (INIS)

    Soviet research in terrestrial decontamination appears to have paralleled that of the US in many respects. However, the probability exists that long-term evaluations of decontamination techniques (over 10 to 20 years) have been carried out at one nuclear accident site (a marked divergence from US experience). The area of aquatic decontamination seems to offer the most intriguing possibilities for new information acquisition from the USSR; at this point only its potential importance can be speculated upon

  16. XMASS experiment

    Science.gov (United States)

    Abe, Ko

    2016-06-01

    XMASS is a single phase liquid xenon scintillator detector. The project is designed for multi purposes, dark matter, neutrinoless double beta decay and 7Be/pp solar neutrino. As the first step of project, XMASS-I detector with 832kg sensitive volume started operation from Dec. 2010. In this paper, recent obtained physics results from commissioning data, refurbishment of detector and future step of experiment are presented.

  17. Shielding experiments

    International Nuclear Information System (INIS)

    Shielding mock-up experiments for Prototype Fast Breeder Reactor (PFBR) and Advanced Heavy Water Reactor (AHWR) are carried out in shielding corner facility of APSARA reactor, to assess the overall accuracy of the codes and nuclear data used in reactor shield design. As APSARA is a swimming pool-type thermal reactor, for fast reactor experiments, typical fast reactor shielding facility was created by using uranium assemblies as spectrum converter. The flux was also enhanced by replacing water by air. Experiments have been carried out to study neutron attenuation through typical fast reactor radial and axial bulk shielding materials such as steel, sodium, graphite, borated graphite and boron carbide. A large number of reaction rates, sensitive to different regions of the neutron energy spectrum, were measured using foil activation and Solid State Nuclear Track Detector (SSNTD) techniques. These experimental results were analysed using computational tools normally used in design calculations, viz., discrete ordinate transport codes with multigroup cross section sets. Comparison of measured reaction rates with calculations provided suitable bias factors for parameters relevant to shield design, such as sodium activation, fast neutron fluence, fission equivalent fluxes etc. The measured neutron spectrum on the incident face of shield model compares well with the calculated fast reactor blanket leakage neutron spectrum. The comparison of calculated reaction rates within shield model indicate that the calculations suffer from considerable uncertainties, in shield models with boron carbide/borated graphite. For AHWR shielding experiments, no spectrum converter was used as it is also a thermal reactor. Radiation streaming studies through penetrations/ducts of various shapes and sizes relevant to AHWR shielding were carried out. (author)

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. Clustering experiments

    CERN Document Server

    Wang, Zhengwei; Tan, Ken; Di, Zengru; Roehner, Bertrand M

    2011-01-01

    It is well known that bees cluster together in cold weather, in the process of swarming (when the ``old'' queen leaves with part of the colony) or absconding (when the queen leaves with all the colony) and in defense against intruders such as wasps or hornets. In this paper we describe a fairly different clustering process which occurs at any temperature and independently of any special stimulus or circumstance. As a matter of fact, this process is about four times faster at 28 degree Celsius than at 15 degrees. Because of its simplicity and low level of ``noise'' we think that this phenomenon can provide a means for exploring the strength of inter-individual attraction between bees or other living organisms. For instance, and at first sight fairly surprisingly, our observations showed that this attraction does also exist between bees belonging to different colonies. As this study is aimed at providing a comparative perspective, we also describe a similar clustering experiment for red fire ants.

  3. Mapping of QTLs for Rice RVA Properties Using High-throughput Re-sequenced Chromosome Segment Substitution Lines%利用重测序的水稻染色体片段代换系定位控制稻米淀粉黏滞性谱QTL

    Institute of Scientific and Technical Information of China (English)

    张昌泉; 胡冰; 朱孔志; 张华; 冷亚麟; 汤述翥; 顾铭洪; 刘巧泉

    2013-01-01

    The RVA profile is one of the important factors for rice cooking and eating quality evaluation, and it would be of great significance to identify new quantitative trail loci (QTLs) for RVA profile for improvement of rice quality. With Nipponbare as recipient and 9311 as donor, a population with 38 chromosome segment substitution lines (CSSLs) had been developed and subsequently genotyped by high-throughput resequencing strategy. The population with their parents similar in apparent amylose content was used for mapping QTLs of RVA properties including peak paste viscosity (PKV), hot paste viscosity (HPV), cool paste viscosity (CPV), breakdown viscosity (BDV), setback viscosity (SBV), consistence viscosity (CSV), peak time (PeT) and pasting temperature (PaT) in two years and two environments. The QTL analysis was carried out by using one-way ANOVA and Dunnett' s test, and stable QTLs were identified in both two years and two environments. 10 stable QTLs, qPKV2. 1 , qPKV5. 1 , qPKV7. 1 , qPKVS.1, qHPV5. 1, qHPV7. 1, qCPV5. 1, qCPV7. 1, qSBV2. 1 and qSBV7. 1 were identified in the chromosome 2, 5, 7 and 8, respectively, with contributions ranging from -31. 8% to 53. 2%. The QTLs in chromosome 2, 5 and 7 were found to have pleiotropy.%稻米RVA谱是评价稻米蒸煮与食用品质的重要指标之一,发掘新的控制稻米RVA谱QTL对稻米品质改良具有重要意义.利用以粳稻品种日本晴为受体、籼稻品种9311为供体并经高通量重测序的染色体片段代换系群体为材料,在两年两点的环境下对该群体中控制RVA谱特征值的QTL进行了定位分析.通过单因素方差分析和Dunnett多重比较,分析染色体片段代换系与受体亲本之间相关RVA谱特征值的差异,以两年两点都能检测到的显著差异位点作为稳定表达的QTL.共检测到10个稳定表达的QTL,包括控制峰值黏度的4个QTL qPKV2.1、qPKV5.1、qPKV7.1和qPKV8.1,控制热浆黏度的2个QTL qHPV5.1和qHPV7.1,

  4. Particle physics experiments 1983

    International Nuclear Information System (INIS)

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

  5. 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...

  6. 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...

  7. Particle physics experiments 1989

    International Nuclear Information System (INIS)

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

  8. Particle physics experiments 1987

    International Nuclear Information System (INIS)

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

  9. Aeroelastic Benchmark Experiments Project

    Data.gov (United States)

    National Aeronautics and Space Administration — M4 Engineering proposes to conduct canonical aeroelastic benchmark experiments. These experiments will augment existing sources for aeroelastic data in the...

  10. Framework of product experience

    NARCIS (Netherlands)

    Desmet, P.; Hekkert, P.

    2007-01-01

    In this paper, we introduce a general framework for product experience that applies to all affective responses that can be experienced in human-product interaction. Three distinct components or levels of product experiences are discussed: aesthetic experience, experience of meaning, and emotional ex

  11. Particle physics experiments 1982

    International Nuclear Information System (INIS)

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

  12. 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)…

  13. 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...

  14. 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...

  15. Product Experience Is Seductive.

    OpenAIRE

    Hoch, Stephen J

    2002-01-01

    Product experience seduces consumers into believing that they learn more than is actually so. There are several reasons for this. First, experience is more engaging than most attempts at education, both more vivid and intentional, and consequently more memorable. Second, experience is viewed as nonpartisan, devoid of the didacticism of formal education and the self-serving interests of advertisers. Third, much of experience is ambiguous, but not recognized as such. Experience supports a pseud...

  16. Adaptive structures flight experiments

    Science.gov (United States)

    Martin, Maurice

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

  17. 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.

  18. 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.

  19. Observing System Simulation Experiments

    Science.gov (United States)

    Prive, Nikki

    2015-01-01

    This presentation gives an overview of Observing System Simulation Experiments (OSSEs). The components of an OSSE are described, along with discussion of the process for validating, calibrating, and performing experiments. a.

  20. Neutrino beams and experiments

    International Nuclear Information System (INIS)

    After a brief review of the early history of neutrino experiments, the principle of neutrino beams at proton accelerators is described and a survey of neutrino experiments since 1963 is given. ((orig.))

  1. 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.

  2. 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)

  3. Particle physics experiments 1984

    International Nuclear Information System (INIS)

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

  4. The Experiment Factory: Standardizing Behavioral Experiments.

    Science.gov (United States)

    Sochat, Vanessa V; Eisenberg, Ian W; Enkavi, A Zeynep; Li, Jamie; Bissett, Patrick G; Poldrack, Russell A

    2016-01-01

    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 (Mason and Suri, 2011; McDonnell et al., 2012; de Leeuw, 2015; 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. PMID:27199843

  5. 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.

  6. The Experiment Factory: Standardizing Behavioral Experiments

    Science.gov (United States)

    Sochat, Vanessa V.; Eisenberg, Ian W.; Enkavi, A. Zeynep; Li, Jamie; Bissett, Patrick G.; Poldrack, Russell A.

    2016-01-01

    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 (Mason and Suri, 2011; McDonnell et al., 2012; de Leeuw, 2015; 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. PMID:27199843

  7. The experiment in macroeconometrics

    OpenAIRE

    Staszewska, Anna; Aldrich, John

    2005-01-01

    This paper examines the experiment in macroeconometrics, the different forms it has taken and the rules that have been proposed for its proper conduct. Here an "experiment" means putting a question to a model and getting an answer. Different types of experiment are distinguished and the justification that can be provided for a particular choice of experiment is discussed. Three types of macroeconometric modelling are considered: the Cowles (system of equations) approach, the vector autoregres...

  8. 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....

  9. 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.

  10. Particle physics experiments 1988

    International Nuclear Information System (INIS)

    This report describes work carried out in 1988 on experiments approved by the Particle Physics Experiments Selection Panel. The contents consist of unedited contributions from each experiment. More than forty projects at different accelerators (SPS, ISIS, PETRA, LAMPF, LEP, HERA, BNL, ILL, LEAR) are listed. Different organisations collaborate on different projects. A brief progress report is given. References to published articles are given. (author)

  11. 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

  12. 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

  13. Experience Communication and Aesthetics

    DEFF Research Database (Denmark)

    Thorlacius, Lisbeth

    experience which is relevant in regards to some of the aesthetical experiences which we are met with within the industry of experience communication. The term "interference"  in the context of experience communication can be described as a kind of "intrusion," capable of creating a certain type of beauty...... to user aspect (web 2.0), the personal engagement or the community spirit. This increasing demand of experiences reflects the postmodern cultural trends where rules for how to think and behave no longer exist. This results in individualism, where the identity of the human being has changed from something...

  14. The Experience City

    DEFF Research Database (Denmark)

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

    2012-01-01

    This article take its point of departure in the pressure of the experience economy on European cities - a pressure which in recent years has found its expression in a number of comprehensive transformations of the physical and architectural environments, and new eventscapes related to fun...... and cultural experience are emerging. The physical, cultural and democratic consequences of this development are discussed in the article, as well as the problems and the new opportunities in the ‘Experience city’. It focuses on the design of the ‘Danish Experience City’ with a special emphasis on hybrid...... 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...

  15. Exploring sensory neuroscience through experience and experiment.

    Science.gov (United States)

    Wyttenbach, Robert A

    2012-01-01

    Many phenomena that we take for granted are illusions - color and motion on a TV or computer monitor, for example, or the impression of space in a stereo music recording. Even the stable image that we perceive when looking directly at the real world is illusory. One of the important lessons from sensory neuroscience is that our perception of the world is constructed rather than received. Sensory illusions effectively capture student interest, but how do you then move on to substantive discussion of neuroscience? This article illustrates several illusions, attempts to connect them to neuroscience, and shows how students can explore and experiment with them. Even when (as is often the case) there is no agreed-upon mechanistic explanation for an illusion, students can form hypotheses and test them by manipulating stimuli and measuring their effects. In effect, students can experiment with illusions using themselves as subjects. PMID:23493966

  16. Ball Collision Experiments

    Science.gov (United States)

    Cross, R.

    2015-01-01

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

  17. 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...

  18. 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…

  19. 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....

  20. 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.

  1. The Student Athlete Experience

    Science.gov (United States)

    Gayles, Joy Gaston

    2009-01-01

    Prior to the 1980s, the literature on the experiences of collegiate student athletes was rather scarce. Since that time the National Collegiate Athletic Association (NCAA) has passed several eligibility rules to address concerns about the academic performance and the overall experience of student athletes on college campuses. As such, the…

  2. 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…

  3. 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…

  4. Varieties of Musical Experience

    Science.gov (United States)

    Bharucha, J. Jamshed; Curtis, Meagan; Paroo, Kaivon

    2006-01-01

    In this paper, we argue that music cognition involves the use of acoustic and auditory codes to evoke a variety of conscious experiences. The variety of domains that are encompassed by music is so diverse that it is unclear whether a single domain of structure or experience is defining. Music is best understood as a form of communication in which…

  5. 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)

  6. 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

  7. 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....

  8. Particle physics experiments 1992

    International Nuclear Information System (INIS)

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

  9. 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.

  10. ATLAS Experiment Brochure

    CERN Multimedia

    Goldfarb, Steven

    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.

  11. Why do Bell experiments?

    OpenAIRE

    Percival, Ian C.

    2000-01-01

    Experiments over three decades have been unable to demonstrate weak nonlocality in the sense of Bell unambiguously, without loopholes. The last important loophole remaining is the detection loophole, which is being tackled by at least three experimental groups. This letter counters five common beliefs about Bell experiments, and presents alternative scenarios for future developments.

  12. Experiences with Remote Laboratories

    OpenAIRE

    Andrew Nafalski; Ozdemir Gol; Zorica Nedic; Jan Machotka; José M. M. Ferreira; Ingvar Gustavsson

    2010-01-01

    The paper reports on experiences of academics and students involved in using remote engineering laboratories both when students work individually or collaboratively with others on the experiments. Positives and negatives are highlighted and are contrasted with expectations of what the remote laboratories can bring into pedagogical environments. Recommendations and conclusions follow on how to better use the remote laboratories in teaching.

  13. 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.

  14. 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.

  15. 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...

  16. 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.

  17. Undergraduate reactor control experiment

    International Nuclear Information System (INIS)

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

  18. Health education telecommunications experiment

    Science.gov (United States)

    Whalen, A. A.

    1975-01-01

    The Health/Education Telecommunications Experiment (HET) was conducted jointly by NASA and HEW on NASA's ATS-6 communications satellite. This experiment actually consisted of six experiments testing health and education applications of a communication spacecraft producing a broadcast of color television directly from space to over 120 low-cost receivers located in remote rural areas throughout the U.S. (including Alaska). The experiments were conducted over the period from 2 July 1974 to 20 May 1975 and operated on an almost daily basis. The overall telecommunications system to support these experiments consisted of many elements: The ATS-6 spacecraft; five different types of earth stations consisting of 120 video receive terminals, 51 telephony tranceivers and eight video originating terminals of three different types. Actual performance of the equipment as measured in the field was shown to equal or exceed predicted values.

  19. Cryogenics for LHC experiments

    CERN Document Server

    2001-01-01

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

  20. Experience as Excursion

    DEFF Research Database (Denmark)

    Svabo, Connie; Shanks, Michael

    2014-01-01

    A central value of what may be termed Experience Design is its aspiration to shift focus from the logic of singular design fields to the interrelations and interactions that take place in situations where people are simultaneously engaged with multiple designs. Experience Design can allow...... researchers and practitioners to travel – making it possible to follow experiences as they are enacted across and between places, modes of transportation, mobile mediation and assemblages of things. Drawing on the nomadic metaphysics of philosopher Michel Serres, the journeying, shifting and propagating...... 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...

  1. Molybdenum solar neutrino experiment

    International Nuclear Information System (INIS)

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

  2. 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.

  3. 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

  4. 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.

  5. Drexhage's Experiment for Sound

    Science.gov (United States)

    Langguth, Lutz; Fleury, Romain; Alò, Andrea; Koenderink, A. Femius

    2016-06-01

    Drexhage's seminal observation that spontaneous emission rates of fluorophores vary with distance from a mirror uncovered the fundamental notion that a source's environment determines radiative linewidths and shifts. Further, this observation established a powerful tool to determine fluorescence quantum yields. We present the direct analogue for sound. We demonstrate that a Chinese gong at a hard wall experiences radiative corrections to linewidth and line shift, and extract its intrinsic radiation efficiency. Beyond acoustics, our experiment opens new ideas to extend the Drexhage experiment to metamaterials, nanoantennas, and multipolar transitions.

  6. 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.

  7. 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

  8. The Experiment as Act

    DEFF Research Database (Denmark)

    Søndergaard, Morten

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

  9. 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

  10. 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.

  11. CANDU operating experience

    International Nuclear Information System (INIS)

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

  12. Making the Experience City

    DEFF Research Database (Denmark)

    Jensen, Ole B.

    2009-01-01

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

  13. 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.

  14. 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...

  15. Cooperative Prototyping Experiments

    DEFF Research Database (Denmark)

    Bødker, Susanne; Grønbæk, Kaj

    1989-01-01

    This paper describes experiments with a design technique that we denote cooperative prototyping. The experiments consider design of a patient case record system for municipal dental clinics in which we used HyperCard, an off the shelf programming environment for the Macintosh. In the ecperiments we...... tried to achieve a fluent work-like evaluation of prototypes where users envisioned future work with a computer tool, at the same time as we made on-line modifications of prototypes in cooperation with the users when breakdown occur in their work-like evaluation. The experiments showed...... 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....

  16. 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.

  17. German and French experiences

    International Nuclear Information System (INIS)

    In this lecture author presents experience in-service inspection programme of primary circuit components at home and abroad, implementation into WWER (world methodology and equipment) as well as system qualification. Objectives European network for inspection qualification (ENIQ) are presented

  18. The world's biggest experiment

    CERN Multimedia

    Gregson, Liz

    2008-01-01

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

  19. Notes on Experiments.

    Science.gov (United States)

    Physics Education, 1988

    1988-01-01

    Describes four physics experiments including "Investigation of Box Resonances Using a Micro"; "A Direct Reading Wattmeter, DC or AC"; "Exercises in the Application of Ohm's Law"; and "Hysteresis on Gas Discharges." Discusses procedures, instrumentation, and analysis in each example. (CW)

  20. Storage Ring EDM Experiments

    Science.gov (United States)

    Semertzidis, Yannis K.

    2016-04-01

    Dedicated storage ring electric dipole moment (EDM) methods show great promise advancing the sensitivity level by a couple orders of magnitude over currently planned hadronic EDM experiments. We describe the present status and recent updates of the field.

  1. 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

  2. Foundation doctors’ induction experiences

    OpenAIRE

    Miles, Susan; Kellett, Joanne; Leinster, Sam J.

    2015-01-01

    Background It is well established that trainee doctors struggle with the transition from medical school to starting work and feel unprepared for many aspects of their new role. There is evidence that suitable induction experiences improve competence and confidence, but available data indicate that trainee doctors on the UK Foundation Programme are commonly not experiencing useful inductions. The aim of the reported research was to explore trainee doctors’ experiences with induction during the...

  3. 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.

  4. 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.

  5. 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.

  6. 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

  7. 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 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...

  8. Accelerator Experiments for Astrophysics

    OpenAIRE

    Ng, Johnny S. T.

    2003-01-01

    Many recent discoveries in astrophysics involve phenomena that are highly complex. Carefully designed experiments, together with sophisticated computer simulations, are required to gain insights into the underlying physics. We show that particle accelerators are unique tools in this area of research, by providing precision calibration data and by creating extreme experimental conditions relevant for astrophysics. In this paper we discuss laboratory experiments that can be carried out at the S...

  9. Sculpting the Illness Experience

    Directory of Open Access Journals (Sweden)

    Molly Bathje MS, OTR/L

    2014-10-01

    Full Text Available Otto Kamensek provided the cover art for the Fall 2014 issue of the Open Journal of Occupational Therapy. “Glimmer of Hope” is part of Otto’s collection “Shard’s, Bone Deep,” which includes hand-built ceramic sculptures that portray his experiences with a lifelong chronic illness. Engaging in ceramic sculpture helps him process the experiences associated with Juvenile Rheumatoid Arthritis and provides a means to support others experiencing chronic illness.

  10. 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.

  11. The experiment in living

    OpenAIRE

    Marres, Noortje

    2012-01-01

    This article engages with debates about widening participation in social research by examining a specific form of public action and knowledge, namely experiments in sustainable living. I propose that these experiments may be approached as forms of social research, and as such offer special opportunities for social research to insert itself into wider societal research arrangements. The article develops the notion of the multifarious instrument which highlights that genres of public action may...

  12. HOLLOWS of EXPERIENCE

    OpenAIRE

    Nixon, Gregory M.

    2010-01-01

    This essay is divided into two parts, deeply intermingled. Part I examines not only the origin of conscious experience but also how it is possible to ask of our own consciousness how it came to be. Part II examines the origin of experience itself, which soon reveals itself as the ontological question of Being. The chief premise of Part I chapter is that symbolic communion and the categorizations of language have enabled human organisms to distinguish between themselves as actually existing en...

  13. 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...

  14. 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.

  15. Sculpting the Illness Experience

    OpenAIRE

    Molly Bathje MS, OTR/L

    2014-01-01

    Otto Kamensek provided the cover art for the Fall 2014 issue of the Open Journal of Occupational Therapy. “Glimmer of Hope” is part of Otto’s collection “Shard’s, Bone Deep,” which includes hand-built ceramic sculptures that portray his experiences with a lifelong chronic illness. Engaging in ceramic sculpture helps him process the experiences associated with Juvenile Rheumatoid Arthritis and provides a means to support others experiencing chronic illness.

  16. 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...

  17. 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^...

  18. 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. PMID:17345685

  19. Staged theta pinch experiments

    International Nuclear Information System (INIS)

    Two implosion heating circuits are being experimentally tested. The principal experiment in the program is the 4.5-m-long Staged Theta Pinch (STP). It uses two relatively low energy (50kJ and 100 kJ), high voltage (125 kV) capacitor banks to produce the theta pinch plasma inside the 20 cm i.d. quartz discharge tube. A lower voltage (50 kV), higher energy (750 kJ) capacitor bank is used to contain the plasma and provide a variable amount of adiabatic compression. Because the experiment produces a higher ratio of implosion heating to compressional heating than conventional theta pinches, it should be capable of producing high temperature plasmas with a much larger ratio of plasma radius to discharge tube radius than has been possible in the past. The Resonant Heating Experiment (RHX) in its initial configuration is the same as a 0.9-m-long section of the high voltage part of the STP experiment and all the plasma results here were obtained with the experiment in that configuration. Part of the implosion bank will be removed and a low inductance crowbar added to convert it to the resonant heating configuration. (U.K.)

  20. National Flood Interoperability Experiment

    Science.gov (United States)

    Maidment, D. R.

    2014-12-01

    The National Flood Interoperability Experiment is led by the academic community in collaboration with the National Weather Service through the new National Water Center recently opened on the Tuscaloosa campus of the University of Alabama. The experiment will also involve the partners in IWRSS (Integrated Water Resources Science and Services), which include the USGS, the Corps of Engineers and FEMA. The experiment will address the following questions: (1) How can near-real-time hydrologic forecasting at high spatial resolution, covering the nation, be carried out using the NHDPlus or next generation geofabric (e.g. hillslope, watershed scales)? (2) How can this lead to improved emergency response and community resilience? (3) How can improved an improved interoperability framework support the first two goals and lead to sustained innovation in the research to operations process? The experiment will run from September 2014 through August 2015, in two phases. The mobilization phase from September 2014 until May 2015 will assemble the components of the interoperability framework. A Summer Institute to integrate the components will be held from June to August 2015 at the National Water Center involving faculty and students from the University of Alabama and other institutions coordinated by CUAHSI. It is intended that the insight that arises from this experiment will help lay the foundation for a new national scale, high spatial resolution, near-real-time hydrologic simulation system for the United States.

  1. 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.

  2. 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.

  3. 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...

  4. 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...

  5. 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.

  6. 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.

  7. 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...

  8. Nuclear power experience

    International Nuclear Information System (INIS)

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

  9. 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...... 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...... 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...

  10. Double beta decay experiments

    International Nuclear Information System (INIS)

    The great sensitivity of double beta decay to neutrino mass and right handed currents has motivated many new and exciting attempts to observe this elusive nuclear phenomenon directly. Experiments in operation and other coming on line in the next one or two years are expected to result in order-of-magnitude improvements in detectable half lives for both the two-neutrino and no-neutrino modes. A brief history of double beta decay experiments is presented together with a discussion of current experimental efforts, including a gas filled time projection chamber being used to study selenium-82. (author)

  11. 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.

  12. Skylab experiments on metals

    Science.gov (United States)

    Lundquist, C. A.

    1974-01-01

    The Skylab Materials Processing Facility is described. Eight experiments on metal processing under near-zero-gravity conditions were performed in this facility. Three of these involved metals and procedures of potential application to fabrication in space. A Multipurpose electric furnace within the Materials Processing Facility was employed to heat three ampoules of samples for each of the other five experiments. These five investigations cover diffusion versus convection rates in molten zinc, several immiscible alloy compositions, a whisker-reinforced silver-based composite, heat treating of porous silver samples, and a copper-aluminum eutectic.

  13. Who Needs Business Experience?

    DEFF Research Database (Denmark)

    Walter, Achim; Ritter, Thomas; Coviello, Nicole;

    that prior business experience does not predict very early export (within three years). Our results offer fresh insight to the international business and international entrepreneurship literatures, and implications for policy development. In particular, international R&D collaborations at public research...... the founding teams’ pre-foundation R&D and customer collaborations to early exports. We also show that pre-foundation involvement in these various forms of collaboration, as well as a diverse stock of prior technical knowledge, can compensate for a lack of business experience in the team. The results also show...

  14. The OLYMPUS Experiment

    OpenAIRE

    Milner, R; Hasell, D. K.; Beck, R; S. Belostotski(St. Petersburg, INP); Bernauer, J. C.; Bessuille, J.; Brinker, F.; Buck, B.; Calarco, J.R.; V. Carassiti; Cisbani, E.; Ciullo, G.; Kohl, M.; Contalbrigo, M; D'Ascenzo, N.

    2014-01-01

    The OLYMPUS experiment was designed to measure the ratio between the positron-proton and electron-proton elastic scattering cross sections, with the goal of determining the contribution of two-photon exchange to the elastic cross section. Two-photon exchange might resolve the discrepancy between measurements of the proton form factor ratio, $\\mu_p G^p_E/G^p_M$, made using polarization techniques and those made in unpolarized experiments. OLYMPUS operated on the DORIS storage ring at DESY, alt...

  15. Copenhagen Sonic Experience Map

    DEFF Research Database (Denmark)

    Kreutzfeldt, Jacob

    2011-01-01

    . Most of the time sound is a trivial part of everyday life involved in interactions, experiences, atmospheres, actions, etc. Although trivial, the role of the aural in the urban life world is more complex than what is suggested by sound level maps, and as such it may be a vital part of urbanity itself....... 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....

  16. Experiments versus simultations

    DEFF Research Database (Denmark)

    Baltser, Jana

    The world of instrumentation utilising X-ray radiation is expanding and leading to more sophisticated experiments. Every step of this process requires accurate calculation, and this is where simulation plays an important role. With the advert of modern computers and technologies, simulation has...... 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...

  17. 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.

  18. 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......) and netnography (Kozinets, 2002). The social media platform analysed is TripAdvisor, which is the largest networking site focusing on tourism and travel. Study findings indicate that while heritage sites tend to promote their uniqueness and the cultural value of their products, tourists are just as concerned...... discusses virtual tourism culture and also generic tourist interests characterized by sensuality, cultural jointaffirmation and immediacy of the experience....

  19. The Moving Flame Experiment

    OpenAIRE

    Stern, Melvin E.

    2011-01-01

    When a flame is rotated around the outside bottom rim of a cylindrical pan of water initially at rest, D. Fultz has observed that the fluid acquires a net vertical component of angular momentum opposite to the rotation of the heat source. We have repeated this experiment in a cylindrical annulus in order to restrict the radial motions and have found that the same phenomenon occurs. Using a simple model based on the latter experiment we investigate the mechanism by which a fluid can acquire an...

  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. Construction Experience Program (CONEX)

    International Nuclear Information System (INIS)

    A summary of the activities of the NEA WGRNR is introduced. In particular the following tasks related to the ConEx (Construction Experience Program) are presented: Rationale and goals of the ConEx program, Development of the event construction database ConEx and its structure, ConEx procedure for program management (uses of ConEx to create knowledge, conclusions of the ConEx synthesis first report on lessons learned during construction, Potential ConEx program uses for operating experience, training, etc.)

  3. Replicability of Experiment

    Directory of Open Access Journals (Sweden)

    John D. Norton

    2015-06-01

    Full Text Available The replicability of experiment is routinely offered as the gold standard of evidence. I argue that it is not supported by a universal principle of replicability in inductive logic. A failure of replication may not impugn a credible experimental result; and a successful replication can fail to vindicate an incredible experimental result. Rather, employing a material approach to inductive inference, the evidential import of successful replication of an experiment is determined by the prevailing background facts. Commonly, these background facts do support successful replication as a good evidential guide and this has fostered the illusion of a deeper, exceptionless principle.

  4. 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...

  5. AGS experiments---1987, 1988, 1989

    International Nuclear Information System (INIS)

    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

  6. 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.

  7. 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.

  8. AGS experiments, 1988, 1989, 1990

    International Nuclear Information System (INIS)

    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

  9. AGS experiments: 1985, 1986, 1987

    International Nuclear Information System (INIS)

    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

  10. 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

  11. 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…

  12. 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…

  13. Literature on photoproduction experiments

    International Nuclear Information System (INIS)

    Literature on gamma-proton, gamma-neutron, gamma-deuteron, gamma-nucleus experiments, inclusive photoproduction, particle yields in gamma-proton and gamma-nucleus, inelastic compton scattering, search for new particles, Primakoff effect, photofission, and QED-tests are compiled. (BJ)

  14. 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…

  15. 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…

  16. Experiments in Free Fall

    Science.gov (United States)

    Art, Albert

    2006-01-01

    A model lift containing a figure of Albert Einstein is released from the side of a tall building and its free fall is arrested by elastic ropes. This arrangement allows four simple experiments to be conducted in the lift to demonstrate the effects of free fall and show how they can lead to the concept of the equivalence of inertial and…

  17. 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…

  18. 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.

  19. 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?

  20. The OLYMPUS experiment

    Energy Technology Data Exchange (ETDEWEB)

    Milner, R. [Massachusetts Institute of Technology, Cambridge, MA (United States); Hasell, D.K., E-mail: hasell@mit.edu [Massachusetts Institute of Technology, Cambridge, MA (United States); Kohl, M. [Hampton University, Hampton, VA (United States); Schneekloth, U. [Deutsches Elektronen-Synchrotron DESY, Hamburg (Germany); Akopov, N. [Alikhanyan National Science Laboratory (Yerevan Physics Institute), Yerevan (Armenia); Alarcon, R. [Arizona State University, Tempe, AZ (United States); Andreev, V.A. [Petersburg Nuclear Physics Institute, Gatchina (Russian Federation); Ates, O. [Hampton University, Hampton, VA (United States); Avetisyan, A. [Alikhanyan National Science Laboratory (Yerevan Physics Institute), Yerevan (Armenia); Bayadilov, D.; Beck, R. [Friedrich Wilhelms Universität, Bonn (Germany); Belostotski, S. [Petersburg Nuclear Physics Institute, Gatchina (Russian Federation); Bernauer, J.C.; Bessuille, J. [Massachusetts Institute of Technology, Cambridge, MA (United States); Brinker, F. [Deutsches Elektronen-Synchrotron DESY, Hamburg (Germany); Buck, B. [Massachusetts Institute of Technology, Cambridge, MA (United States); Calarco, J.R. [University of New Hampshire, Durham, NH (United States); Carassiti, V. [Università di Ferrara and Istituto Nazionale di Fisica Nucleare, Ferrara (Italy); Cisbani, E. [Istituto Superiore di Sanità and Istituto Nazionale di Fisica Nucleare, Rome (Italy); Ciullo, G. [Università di Ferrara and Istituto Nazionale di Fisica Nucleare, Ferrara (Italy); and others

    2014-03-21

    The OLYMPUS experiment was designed to measure the ratio between the positron–proton and electron–proton elastic scattering cross-sections, with the goal of determining the contribution of two-photon exchange to the elastic cross-section. Two-photon exchange might resolve the discrepancy between measurements of the proton form factor ratio, μ{sub p}G{sub E}{sup p}/G{sub M}{sup p}, made using polarization techniques and those made in unpolarized experiments. OLYMPUS operated on the DORIS storage ring at DESY, alternating between 2.01 GeV electron and positron beams incident on an internal hydrogen gas target. The experiment used a toroidal magnetic spectrometer instrumented with drift chambers and time-of-flight detectors to measure rates for elastic scattering over the polar angular range of approximately 25°–75°. Symmetric Møller/Bhabha calorimeters at 1.29° and telescopes of GEM and MWPC detectors at 12° served as luminosity monitors. A total luminosity of approximately 4.5 fb{sup −1} was collected over two running periods in 2012. This paper provides details on the accelerator, target, detectors, and operation of the experiment.

  1. A cosmic rays experiment

    Science.gov (United States)

    Pérez Munguía, Gustavo; Pineda de Carías, María Cristina

    1995-01-01

    In this paper we present the results of an experiment performed to detect the total flux of muons incident over Tegucigalpa (Honduras) the day of the total solar eclipse of the 11 July, 1991; and also a comparison with data obtained before the eclipse and registered in the past years.

  2. The NA35 experiment

    CERN Multimedia

    1987-01-01

    The NA35 experiment ran on the Super Proton Synchrotron (SPS) accelerator at CERN. It was used for the study of relativistic nucleus-nucleus collisions which were used to search for evidence of new types of quark matter. Quarks are found inside protons and neutrons, which in turn make up atoms.

  3. The Experience of Menarche.

    Science.gov (United States)

    Ruble, Diane N.; Brooks-Gunn, Jeanne

    1982-01-01

    Examines reactions to menarche and the subsequent effects of this experience as a function of preparation for and timing of menarche. A questionnaire including measures of responses about first menstruation, current symptoms, and self-image was completed by 639 girls in fifth through twelfth grades. (Author/MP)

  4. Caring Experience and Knowledge

    DEFF Research Database (Denmark)

    Dybbroe, Betina

    2005-01-01

    ,didactics and educational thinking- and students experiences and sensing, and illuminates excluding processes in classrooms related to emotional, non-cognitive and relational aspects of the qualifying process. This is set into a larger framework of the biographical professionalization processes of students...

  5. Virtual Inquiry Experiences

    Science.gov (United States)

    Harlow, Danielle; Nilsen, Katy

    2011-01-01

    Children in classrooms and scientists in laboratories engage in similar activities: they observe, ask questions, and try to explain phenomena. Video conferencing technology can remove the wall between the classroom and the laboratory, bringing children and scientists together. Virtual experiences and field trips can provide many of the benefits of…

  6. 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...

  7. Experimenting with Guitar Strings

    Science.gov (United States)

    LoPresto, Michael C.

    2006-01-01

    What follows is a description of a simple experiment developed in a non-mathematical general education science course on sound and light for fine arts students in which a guitar is used with data collection hardware and software to verify the properties of standing waves on a string.

  8. 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.

  9. Electronics for LHC Experiments

    International Nuclear Information System (INIS)

    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

  10. 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).

  11. Experiments with hydrogel pearls

    OpenAIRE

    Pavlin, Jerneja

    2015-01-01

    Hydrogels are very attractive materials since they can absorb large quantities of water. They also have very interesting optical properties which can be easily shown. The experiments with hydrogel pearls related to the absorption of water, density, optical properties and influence of pH are presented in the contribution.

  12. 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.

  13. 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.

  14. Linking consumer experiences

    DEFF Research Database (Denmark)

    Smed, Karina Madsen

    become part of the individual self, worldview, and behaviour. This paper seeks to explore links between consumer experiences through the exploration of narrative sequences in travel blogs. Findings indicate that non-consumption is a central element to the bloggers and also indicative of a community...

  15. 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…

  16. The OLYMPUS experiment

    International Nuclear Information System (INIS)

    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, μpGpE/GpM, 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-1 was collected over two running periods in 2012. This paper provides details on the accelerator, target, detectors, and operation of the experiment.

  17. Experimenting with Woodwind Instruments

    Science.gov (United States)

    LoPresto, Michael C.

    2007-01-01

    Simple experiments involving musical instruments of the woodwind family can be used to demonstrate the basic physics of vibrating air columns in resonance tubes using nothing more than straightforward measurements and data collection hardware and software. More involved experimentation with the same equipment can provide insight into the effects…

  18. 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.

  19. 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…

  20. 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)

  1. Principal Experiences of Succession

    Science.gov (United States)

    Steele, Farla Gay

    2015-01-01

    This multiple case study explored the experiences of school principals and the usefulness of Peters' (2011) succession planning model. Ten purposefully selected principals from varying grade levels were interviewed; none reported a formal succession plan, and all had been assistant principals. The study concluded the assistant principal position…

  2. 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...

  3. 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 $10^{-3}$.

  4. The ATRAP experiment

    CERN Multimedia

    Laurent Guiraud

    2000-01-01

    Gerald Gabrielse, spokesperson, pictured in front of the Antihydrogen Trap (ATRAP) experiment, the first machine to accumulate cold antiprotons produced in the AD, and combine them with protons (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.

  5. 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.

  6. 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...

  7. 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.

  8. 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.

  9. Ultrafast gas switching experiments

    International Nuclear Information System (INIS)

    We describe recent experiments which studied the physics of ultrafast gas breakdown under the extreme overvoltages which occur when a high pressure gas switch is pulse charged to hundreds of kV in 1 ns or less. The highly overvolted peaking gaps produce powerful electromagnetic pulses with risetimes Khz at > 100 kV/m E field

  10. FLORIDA TOWER FOOTPRINT EXPERIMENTS

    Energy Technology Data Exchange (ETDEWEB)

    WATSON,T.B.; DIETZ, R.N.; WILKE, R.; HENDREY, G.; LEWIN, K.; NAGY, J.; LECLERC, M.

    2007-01-01

    The Florida Footprint experiments were a series of field programs in which perfluorocarbon tracers were released in different configurations centered on a flux tower to generate a data set that can be used to test transport and dispersion models. These models are used to determine the sources of the CO{sub 2} that cause the fluxes measured at eddy covariance towers. Experiments were conducted in a managed slash pine forest, 10 km northeast of Gainesville, Florida, in 2002, 2004, and 2006 and in atmospheric conditions that ranged from well mixed, to very stable, including the transition period between convective conditions at midday to stable conditions after sun set. There were a total of 15 experiments. The characteristics of the PFTs, details of sampling and analysis methods, quality control measures, and analytical statistics including confidence limits are presented. Details of the field programs including tracer release rates, tracer source configurations, and configuration of the samplers are discussed. The result of this experiment is a high quality, well documented tracer and meteorological data set that can be used to improve and validate canopy dispersion models.

  11. 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.)

  12. The ALARM Experiment

    Science.gov (United States)

    Gerhardt, Ira

    2015-01-01

    An experiment was conducted over three recent semesters of an introductory calculus course to test whether it was possible to quantify the effect that difficulty with basic algebraic and arithmetic computation had on individual performance. Points lost during the term were classified as being due to either algebraic and arithmetic mistakes…

  13. CANDU operating experience

    International Nuclear Information System (INIS)

    The CANDU Pressurized Heavy Water (CANDU-PHW) type of nuclear electric generating station has been developed jointly by Atomic Energy of Canada Limited and Ontario Hydro. This paper highlights Ontario Hydro's operating experience using the CANDU-PHW system, with a focus on the operating performance and costs, reliability of system components and nuclear safety considerations both to the workers and the public

  14. 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.

  15. 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

  16. 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

  17. 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

  18. The deconstructive experience.

    Science.gov (United States)

    Gregory, Robert J

    2005-01-01

    Logocentrism was conceptualized by Jacques Derrida as connoting the assertion within Western philosophical traditions of certain assumed truths and the exclusion of alternative perspectives. In this paper, the author proposes that the concept of logocentrism may be usefully applied within the clinical situation to enrich understanding of splitting between idealized and devalued perceptions of self and others. He presents a case of a woman with borderline personality disorder to illustrate a logocentric self-structure, as well as how common psychotherapeutic models inadvertently risk reinforcing such structures through the hierarchical nature of the patient-therapist relationship. The process of deconstructing logocentric self-structures is facilitated by the patient experiencing the therapist paradoxically as an extension of the self that sometimes behaves contrary to expectations. Such a deconstructive experience challenges reified perceptions of self and others, serves to broaden the experience of self, and enhances qualities of self-reflection and empathy. PMID:16555459

  19. A communist teaching experiment

    DEFF Research Database (Denmark)

    Skak, Morten

    -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...... of teaching is an improvement? I can only show two (poor?) methods: a) asking the students and b) use their examination results....

  20. 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...... clear goals related to the improvement of social interaction, performance and cultural exchange. The article contains three sections. in section one, we present three European cases in order to relate to the wider international debate and development. In section two we present the main theoretical...... 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...

  1. 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.

  2. LASL fast liner experiment

    International Nuclear Information System (INIS)

    The LASL Fast Liner Experiment explores a fusion concept in which a prepared plasma is adiabatically compressed to thermonuclear temperatures and densities by a rapidly imploding solid metal liner. A prepared plasma having β > 1 is in contact with the liner and end plugs, and contains an embedded magnetic field to inhibit thermal conduction. Cylindrical liners are magnetically imploded by a large axial current carried in the liner shell. Theoretical estimates indicate an implosion velocity of at least 106 cm/s is necessary for this geometry if the plasma heating rate is to be greater than the cross-field thermal conduction loss rate. Experimental work to develop this concept involves attempts to provide a suitable preplasma for liner implosions as well as studies of magnetically driven liner implosions. In the plasma preparation experiments a coaxial plasma gun was used to inject plasma into a simulated liner geometry

  3. 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.

  4. 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.

  5. 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...

  6. Transgressive first clinical experiences

    DEFF Research Database (Denmark)

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

    2014-01-01

    . The nursing students’ learning seems to be oriented towards socialization in the clinic as a workplace. This means that the nursing students seek to deal with overwhelming experiences concerning the naked bodies of patients and death, useful application of theoretical knowledge, the path from novice...... 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.......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...

  7. 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.

  8. Gross decontamination experiment report

    International Nuclear Information System (INIS)

    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

  9. Motherhood experience for prisoners

    Directory of Open Access Journals (Sweden)

    Lannuzya Verissimo e Oliveira

    2015-06-01

    Full Text Available The study aimed to understand the motherhood experience for prisoners. This is an exploratory-descriptive qualitative study conducted with 17 prisoner women in the Penitentiary System of Paraíba State, during the period of July to December of 2012. A sociodemographic questionnaire and semi-structured interviews were used for data collection. Data were organized and categorized following content analysis, generating three categories: separation suffering; comfort during anguish; family fragmentation. Results pointed out that motherhood experience in the prison is permeated by suffering and limitations. However, the presence of their child creates comfort during anguish and minimize difficulties in prison, although temporarily.  It is necessary to create inter sectoral actions to favor the mother-child relationship in the prison context.

  10. Microwave Tokamak Experiment

    International Nuclear Information System (INIS)

    The Microwave Tokamak Experiment, now under construction at the Laboratory, will use microwave heating from a free-electron laser. The intense microwave pulses will be injected into the tokamak to realize several goals, including a demonstration of the effects of localized heat deposition within magnetically confined plasma, a better understanding of energy confinement in tokamaks, and use of the new free-electron laser technology for plasma heating. The experiment, soon to be operational, provides an opportunity to study dense plasmas heated by powers unprecedented in the electron-cyclotron frequency range required by the especially high magnetic fields used with the MTX and needed for reactors. 1 references, 5 figures, 3 tables

  11. "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.

  12. 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...... gave knowledge about using robots in open-ended environments, and knowledge about people's reactions and expectations to robots in urban environments....

  13. Apollo lunar sounder experiment

    Science.gov (United States)

    Phillips, R.J.; Adams, G.F.; Brown, W.E., Jr.; Eggleton, R.E.; Jackson, P.; Jordan, R.; Linlor, W.I.; Peeples, W.J.; Porcello, L.J.; Ryu, J.; Schaber, G.; Sill, W.R.; Thompson, T.W.; Ward, S.H.; Zelenka, J.S.

    1973-01-01

    The scientific objectives of the Apollo lunar sounder experiment (ALSE) are (1) mapping of subsurface electrical conductivity structure to infer geological structure, (2) surface profiling to determine lunar topographic variations, (3) surface imaging, and (4) measuring galactic electromagnetic radiation in the lunar environment. The ALSE was a three-frequency, wide-band, coherent radar system operated from lunar orbit during the Apollo 17 mission.

  14. 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.

  15. 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.

  16. The MUSE experiment

    OpenAIRE

    Downie E. J.

    2014-01-01

    The proton radius puzzle is the difference between the proton radius as measured with electron scattering and in the excitation spectrum of atomic hydrogen, and that measured with muonic hydrogen spectroscopy. The MUSE experiment seeks to resolve this puzzle by simultaneously measuring elastic electron and muon scattering on the proton, in both charge states, thereby providing new information to the puzzle. MUSE addresses issues of two-photon effects, lepton universality and, possibly, new ph...

  17. The Vinca dosimetry experiment

    International Nuclear Information System (INIS)

    On 15 October 1958 there occurred a very brief uncontrolled run of the zero-power reactor at the Boris Kidric Institute of Nuclear Science, Vinca, near Belgrade, Yugoslavia. During this run six persons received various doses of radiation. They were subsequently given medical treatment of a novel kind at the Curie Hospital, Paris. In atomic energy operations to date, very few accidents involving excessive radiation exposure to human beings have occurred. In fact, the cases of acute radiation injury are limited to about 30 known high exposures, few of which were in the lethal or near-lethal range. Since direct experiment to determine the effects of ionizing radiation on man is unacceptable, information on these effects has to be based on a consideration of data relating to accidental exposures, viewed in the light of the much more extensive data obtained from experiments on animals. Therefore, any direct information on the effects of radiation on humans is very valuable. The international dosimetry project described in this report was carried out at Vinca, Yugoslavia, under the auspices of the International Atomic Energy Agency to determine the precise amount of radiation to which the persons had been exposed during the accident. These dosimetry data, together with the record of the carefully observed clinical effects, are of importance both for the scientific study of radiation effects on man and for the development of methods of therapy. The experiment and measurements were carried out at the end of April 1960. The project formed part of the Agency's research programme in the field of health and safety. The results of the experiment are made available through this report to all Member States

  18. The NEXT experiment

    Energy Technology Data Exchange (ETDEWEB)

    DIaz, J; Yahlali, N; Ball, M; Carcel, S; Cervera, A; Gil, A [Instituto de Fisica Corpuscular (Centro mixto UV-CSIC), Apdo. de Correos 22085, E-46071 Valencia (Spain) (Spain); Barata, J A S; Borges, F I G M; Conde, C A N; Dias, T H V T; Fernandes, L M P; Freitas, E D C [Universidade de Coimbra (Portugal); Calvo, E [Instituto de Fisica de Altas EnergIas, IFAE, Barcelona (Spain); Carmona, J M; Cebrian, S; Dafni, T; Galan, J [U. Zaragoza (Spain); Cid, X [Universidade de Santiago de Compostela (Spain); Ferrer-Ribas, E [CEA, IRFU, Saclay (France); Gil, I, E-mail: jose.diaz@uv.e [CIEMAT (Spain)

    2009-07-01

    Neutrinoless double beta decay measurements are the most promising experiments both to reveal the Majorana nature of the neutrino and to set a value for its mass. The NEXT project propose to build a High pressure Xenon TPC in the Canfranc Underground Laboratory (Huesca, Spain) to measure double-beta decay of {sup 136}Xe, both normal and neutrinoless, with a source mass of 100 kg of enriched xenon.

  19. 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.

  20. 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.

  1. 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.

  2. Motherhood experience for prisoners

    OpenAIRE

    Lannuzya Verissimo e Oliveira; Francisco Arnoldo Nunes de Miranda; Gabriela Maria Cavalcanti Costa

    2015-01-01

    The study aimed to understand the motherhood experience for prisoners. This is an exploratory-descriptive qualitative study conducted with 17 prisoner women in the Penitentiary System of Paraíba State, during the period of July to December of 2012. A sociodemographic questionnaire and semi-structured interviews were used for data collection. Data were organized and categorized following content analysis, generating three categories: separation suffering; comfort during anguish; family fragmen...

  3. The Ediacaran experiment.

    Science.gov (United States)

    Gould, S J

    1984-02-01

    The author reflects on presentations at an annual meeting of the Geological Society of America regarding the differences between Ediacaran fauna fossils and fossils from the Cambrian evolutionary explosion. The theory presented by Dolf Seilacher attributes the differences between Ediacaran animals and their modern counterparts to structure. He proposes that Ediacaran fauna were a failed evolutionary experiment. Related comments and meeting presentations are included. PMID:11541840

  4. Review of EDM experiments

    International Nuclear Information System (INIS)

    The current limits on physics beyond the SM come in large part from the non-observation of EDMs in the sensitive electric dipole moment experiments, like the neutron, 199Hg, and 205Tl. New systems with enhanced EDM sensitivity are coming online and promise a resolution of the baryon asymmetry of our universe (if an EDM is observed) or a severe constraint on physics beyond the SM by the end of the current decade.

  5. 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.

  6. Reproducible Experiment Platform

    Science.gov (United States)

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

    2015-12-01

    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 some key features of REP based on case studies which include trigger optimization and physics analysis studies at the LHCb experiment.

  7. The OLYMPUS Experiment

    CERN Document Server

    Milner, R; Kohl, M; Schneekloth, U; Akopov, N; Alarcon, R; Andreev, V A; Ates, O; Avetisyan, A; Bayadilov, D; Beck, R; Belostotski, S; Bernauer, J C; Bessuille, J; Brinker, F; Buck, B; Calarco, J R; Carassiti, V; Cisbani, E; Ciullo, G; Contalbrigo, M; D'Ascenzo, N; De Leo, R; Diefenbach, J; Donnelly, T W; Dow, K; Elbakian, G; Eversheim, D; Frullani, S; Funke, Ch; Gavrilov, G; Gläser, B; Görrissen, N; Hauschildt, J; Henderson, B S; Hoffmeister, Ph; Holler, Y; Ice, L D; Izotov, A; Kaiser, R; Karyan, G; Kelsey, J; Khaneft, D; Klassen, P; Kiselev, A; Krivshich, A; Lehmann, I; Lenisa, P; Lenz, D; Lumsden, S; Ma, Y; Maas, F; Marukyan, H; Miklukho, O; Movsisyan, A; Murray, M; Naryshkin, Y; O'Connor, C; Benito, R Perez; Perrino, R; Redwine, R P; Piñeiro, D Rodríguez; Rosner, G; Russell, R L; Schmidt, A; Seitz, B; Statera, M; Thiel, A; Vardanyan, H; Veretennikov, D; Vidal, C; Winnebeck, A; Yeganov, V

    2014-01-01

    The OLYMPUS experiment was designed to measure the ratio between the positron-proton and electron-proton elastic scattering cross sections, with the goal of determining the contribution of two-photon exchange to the elastic cross section. Two-photon exchange might resolve the discrepancy between measurements of the proton form factor ratio, $\\mu_p G^p_E/G^p_M$, 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 $25^\\circ$--$75^\\circ$. Symmetric M{\\o}ller/Bhabha calorimeters at $1.29^\\circ$ and telescopes of GEM and MWPC detectors at $12^\\circ$ served as luminosity monitors. A total luminosity of approximately 4.5~fb$^{-1}$ was collect...

  8. Hyperon Beam Experiment

    CERN Multimedia

    2002-01-01

    The experiment WA89 uses the upgraded Omega facility together with a hyperon beam installed at the end of the H1 beamline. The beam can deliver 2~10$ ^{5} \\% Sigma ^- $ per machine burst at 330 GeV/c with a background of 5 10$ ^{5} \\% \\pi ^- $. \\\\ \\\\ The goals of the experiment are: observation of charmed particles, mainly the charmed-strange baryons and measurements of their production in the kinematical range x$ _{F} $~$>$~0.2, and their decay properties, a search for exotic states such as U(3100) observed in the previous CERN hyperon beam experiment WA62, measurements of hyperon polarization and production properties. \\\\ \\\\ A vertex detector consisting of 24 silicon microstrip planes with 25~$\\mu$m pitch and 6~planes with 50~$\\mu$m pitch provides track measurements of sufficient accuracy to identify the decays of short living charmed particles and measure their lifetimes. A RICH detector provides good $\\pi$/K separation for momenta up to 100~GeV/c and $\\pi$/p separation up to 150~GeV/c. Photons are detecte...

  9. Experiments with Electrodynamic Wheels

    Science.gov (United States)

    Gaul, Nathan; Corey, Daniel; Cordrey, Vincent; Majewski, Walerian

    2015-04-01

    Our experiments were involving inductive magnetic levitation. A Halbach array is a system in which a series of magnets is arranged in a manner such that the magnetic field is cancelled on one side of the array while strengthening the field on the other. We constructed two circular Halbach wheels, making the strong magnetic field on the outer rim of the ring. Such system is usually dubbed as an Electrodynamic Wheel (EDW). Rotating this wheel around a horizontal axis above a flat conducting surface should induce eddy currents in said surface through the variable magnetic flux. The eddy currents produce, in turn, their own magnetic fields which interact with the magnets of the EDW. We demonstrated that these interactions produce both drag and lift forces on the EDW which can theoretically be used for lift and propulsion of the EDW. The focus of our experiments is determining how to maximize the lift-to-drag ratio by the proper choice of the induction element. We will also describe our experiments with a rotating circular Halbach array having the strong magnetic field of about 1 T on the flat side of the ring, and acting as a hovercraft.

  10. AGS experiments: 1990, 1991, 1992

    International Nuclear Information System (INIS)

    This report contains a description of the following: AGS Experimental Area - High Energy Physics FY 1993 and Heavy Ion Physics FY 1993; Table of Beam Parameters and Fluxes; Experiment Schedule ''as run''; Proposed 1993 Schedule; A listing of experiments by number; Two-page summaries of each experiment begin here, also ordered by number; Publications of AGS Experiments; and List of AGS Experimenters

  11. AGS Experiments: 1989, 1990, 1991

    International Nuclear Information System (INIS)

    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

  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. AGS experiments - 1982, 1983, 1984

    International Nuclear Information System (INIS)

    The report contains layouts of experimental areas, a table of beam parameters and fluxes, the experiment schedule as run, the experiment long range schedule, a listing of experiments by number, and a section of two-page summaries of each experiment

  15. Simulation - modeling - experiment

    International Nuclear Information System (INIS)

    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

  16. 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

  17. Situating emotional experience.

    Science.gov (United States)

    Wilson-Mendenhall, Christine D; Barrett, Lisa Feldman; Barsalou, Lawrence W

    2013-01-01

    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 multiple sensory modalities 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 emotional life

  18. 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.

  19. 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.

  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. Survey of Tokamak experiments

    International Nuclear Information System (INIS)

    The survey covers the following topics:- Introduction and history of tokamak research; review of tokamak apparatus, existing and planned; remarks on measurement techniques and their limitations; main results in terms of electron and ion temperatures, plasma density, containment times, etc. Empirical scaling; range of operating densities; impurities, origin, behaviour and control (including divertors); data on fluctuations and instabilities in tokamak plasmas; data on disruptive instabilities; experiments on shaped cross-sections; present experimental evidence on β limits; auxiliary heating; experimental and theoretical problems for the future. (author)

  2. 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.

  3. The Malaysia LNG experience

    International Nuclear Information System (INIS)

    This paper summarizes the nature of the LNG trade, the essential components and characteristics of an LNG project, and relates the Malaysia LNG experience to project realization with some emphasis on the financial aspects of the project. Twelve offshore lending institutions were involved in the total project loop providing U.S. dollar equivalents of 4.0 billions with interest rates ranging from 5% to 8%. The total project was completed on schedule and within budget except for the ships which got caught in the political development of the Malaysian petroleum industry at that time

  4. The Digital Archive Experience

    DEFF Research Database (Denmark)

    Søndergaard, Morten

    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....... 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...

  5. Soil mechanics experiment

    Science.gov (United States)

    Mitchell, J. K.; Bromwell, L. G.; Carrier, W. D., III; Costes, N. C.; Houston, W. N.; Scott, R. F.

    1972-01-01

    The Apollo 15 soil-mechanics experiment has offered greater opportunity for study of the mechanical properties of the lunar soil than previous missions, not only because of the extended lunar-surface stay time and enhanced mobility provided by the lunar roving vehicle (rover), but also because four new data sources were available for the first time. These sources were: (1) the self-recording penetrometer (SRP), (2) new, larger diameter, thin-walled core tubes, (3) the rover, and (4) the Apollo lunar-surface drill (ALSD). These data sources have provided the best bases for quantitative analyses thus far available in the Apollo Program.

  6. 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)

  7. 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.

  8. French experience with electropolishing

    International Nuclear Information System (INIS)

    Following the results obtained in CHINON B1 comparing deposited activity observed on different surface finishes EDF decided to electropolish steam generator channel heads (S.G.C.H.) in order to reduce operator dose during plant maintenance. The qualification tests were performed on steam generator materials (Inconel 600 and S.S. 308 L) with the full on site operational equipment (i.e. a sealed sucker). In 1988 the 4 SGCH of NOGENT 2 were electropolished. In 1990 we observed a dose rate reduction of 45% at NOGENT 2 compared to NOGENT 1. Other French experience is electropolishing of 27 S.G.C.H., since 1988. (author)

  9. S band transponder experiment

    Science.gov (United States)

    Sjogren, W. L.; Muller, P. M.; Wollenhaupt, W. R.

    1972-01-01

    It is reported that this experiment measures the lunar gravitational field, which in turn provides information on the distribution of lunar mass and its correlation with surface features such as craters, mountains, and maria. The lunar gravitational field is measured by observing the dynamical motion of spacecraft in free-fall orbits. Effective detection of mass variations is greatly enhanced by low-altitude trajectories, such as the eccentric orbits during revolutions 3 to 16 of the Apollo 16 spacecraft and the 11 km periapsis of the Apollo 16 subsatellite during May 1972. The observational data are the precise earth-based radio tracking measurements initially used for real-time navigation.

  10. THYROGLOSSAL CYST OUR EXPERIENCE

    Directory of Open Access Journals (Sweden)

    Balasubramanian Thiagarajan

    2013-01-01

    Full Text Available This study shares our experiences in managing patients with thyroglossal cysts. These are common midline tumors of neck. Characteristically these masses move on protrusion of tongue due to their intimate relationship with hyoid bone. Adults commonly presented with this lesion even though literature review suggests it to be common in children. All these patients underwent surgerybecause of the presence of mass rather than any symptoms. All 30 patients taken up in this study had a normal functioning thyroid in the normal position in addition to the cystic lesion.

  11. Aesthetic experience of dance performances

    OpenAIRE

    Vukadinović Maja; Marković Slobodan

    2012-01-01

    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 ...

  12. Experiment@Portugal

    Directory of Open Access Journals (Sweden)

    Maria Teresa Restivo

    2011-04-01

    Full Text Available

    The main goal of the Project Experiment@Portugal is to produce a complete survey of Portuguese developments in remote and virtual labs. This knowledge will provide the conditions for joining forces in order to organize a well structured national website integrating a database of available remote and virtual experiments, categorized for sharing purposes, and looking for delivering valuable contents for high schools and for higher education. It is expected that the final result will bring up a solid team able to offer in this domain a Portuguese partner at international level

  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. PAC Experiments at ISOLDE

    CERN Multimedia

    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.

  15. 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...

  16. Female physicist doctoral experiences

    Science.gov (United States)

    Dabney, Katherine P.; Tai, Robert H.

    2013-06-01

    The underrepresentation of women in physics doctorate programs and in tenured academic positions indicates a need to evaluate what may influence their career choice and persistence. This qualitative paper examines eleven females in physics doctoral programs and professional science positions in order to provide a more thorough understanding of why and how women make career choices based on aspects both inside and outside of school and their subsequent interaction. Results indicate that female physicists experience conflict in achieving balance within their graduate school experiences and personal lives and that this then influences their view of their future careers and possible career choices. Female physicists report both early and long-term support outside of school by family, and later departmental support, as being essential to their persistence within the field. A greater focus on informal and out-of-school science activities for females, especially those that involve family members, early in life may help influence their entrance into a physics career later in life. Departmental support, through advisers, mentors, peers, and women’s support groups, with a focus on work-life balance can help females to complete graduate school and persist into an academic career.

  17. The EBEX Experiment

    Energy Technology Data Exchange (ETDEWEB)

    Oxley, P.; Ade, P.; Baccigalupi, C.; deBernardis, 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.

    2005-01-06

    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. 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 plate. EBEX is currently in the design and construction phase, and first light is scheduled for 2008.

  18. Experience in open markets

    International Nuclear Information System (INIS)

    The topic of discussion in this session was experience in open markets. The session was led by Guido Bachman, President of the Independent Power Producers' Society of Alberta (IPPSA), assisted by panel members Richard Way, Director of Energy Risk Management at TransAlta, John O'Donnell, Professor of Finance at Michigan State University, and Kelly Lail, Manager of Power Acquisition at B.C. Hydro. Way spoke of the experiences with market restructuring in Alberta, describing the consultative and legislative process which determined the structure of the Alberta Power Pool (APP). The Pool began operations in January 1996. Currently there are 33 participants from generators, to distributors and marketers. Supply and demand are managed by APP by setting an hourly price based on offers and bids. Both generators and distributors get the hourly pool price, so the input price and export price of the pool are the same, however, generators and distributors are free to enter into 'contract for difference' agreements. O'Donnell discussed the status of competition in Michigan. He stressed the importance of Ontario to the Michigan market, echoing the conviction of U.S. regulators that a freer economy works better. Kelly Lail spoke of the unbundling of B.C. Hydro into several generating, transmission and distribution companies in preparation for moving from exclusive service to exclusive wholesale competition. He predicted that the fierce competition will lead to a an industry shakeout, leaving only one big (B.C. Hydro) and a few niche players

  19. 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.

  20. Initiation Train Experiments

    Science.gov (United States)

    Francois, Elizabeth; Johnson, Carl; Liechty, Gary; Whitley, Von

    2015-06-01

    In an effort to evaluate and qualify a new detonator diagnostic, booster selection and main charge configuration, a variety of small-scale tests have been conducted. This paper will describe the needs, testing approach and model validation. Because of the limited size available some novel approaches were made to understand the observed phenomenon. Function time and time of arrival at various locations in the initiation train are desirable data points. Knowing when each segment initiates the next segment and the time to run up to detonation is critical. Results of our experiments were modeled for timing accuracy, wave shape and pressure. Agreement between the experiments and models will be discussed. The testing that will be discussed is time of arrival wires, PDV, and fiber optic arrays. The time of arrival wire measures the detonator cup breakout time. When correlated to bridge burst, an absolute time is collected. This data point also gives time zero for the booster initiation. Many models actually start at the booster, rather than the detonator, so the inclusion of this data point will improve modeling efforts.