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Sample records for amplicon resequencing experiments

  1. pyAmpli: an amplicon-based variant filter pipeline for targeted resequencing data.

    Science.gov (United States)

    Beyens, Matthias; Boeckx, Nele; Van Camp, Guy; Op de Beeck, Ken; Vandeweyer, Geert

    2017-12-14

    Haloplex targeted resequencing is a popular method to analyze both germline and somatic variants in gene panels. However, involved wet-lab procedures may introduce false positives that need to be considered in subsequent data-analysis. No variant filtering rationale addressing amplicon enrichment related systematic errors, in the form of an all-in-one package, exists to our knowledge. We present pyAmpli, a platform independent parallelized Python package that implements an amplicon-based germline and somatic variant filtering strategy for Haloplex data. pyAmpli can filter variants for systematic errors by user pre-defined criteria. We show that pyAmpli significantly increases specificity, without reducing sensitivity, essential for reporting true positive clinical relevant mutations in gene panel data. pyAmpli is an easy-to-use software tool which increases the true positive variant call rate in targeted resequencing data. It specifically reduces errors related to PCR-based enrichment of targeted regions.

  2. Allele Re-sequencing Technologies

    DEFF Research Database (Denmark)

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

    2013-01-01

    The development of next-generation sequencing technologies has made sequencing an affordable approach for detection of genetic variations associated with various traits. However, the cost of whole genome re-sequencing still remains too high to be feasible for many plant species with large...... 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....

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

    Directory of Open Access Journals (Sweden)

    Lank Simon M

    2012-08-01

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

  4. Removing Noise From Pyrosequenced Amplicons

    Directory of Open Access Journals (Sweden)

    Davenport Russell J

    2011-01-01

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

  5. A novel ultra high-throughput 16S rRNA gene amplicon sequencing library preparation method for the Illumina HiSeq platform.

    Science.gov (United States)

    de Muinck, Eric J; Trosvik, Pål; Gilfillan, Gregor D; Hov, Johannes R; Sundaram, Arvind Y M

    2017-07-06

    Advances in sequencing technologies and bioinformatics have made the analysis of microbial communities almost routine. Nonetheless, the need remains to improve on the techniques used for gathering such data, including increasing throughput while lowering cost and benchmarking the techniques so that potential sources of bias can be better characterized. We present a triple-index amplicon sequencing strategy to sequence large numbers of samples at significantly lower c ost and in a shorter timeframe compared to existing methods. The design employs a two-stage PCR protocol, incorpo rating three barcodes to each sample, with the possibility to add a fourth-index. It also includes heterogeneity spacers to overcome low complexity issues faced when sequencing amplicons on Illumina platforms. The library preparation method was extensively benchmarked through analysis of a mock community in order to assess biases introduced by sample indexing, number of PCR cycles, and template concentration. We further evaluated the method through re-sequencing of a standardized environmental sample. Finally, we evaluated our protocol on a set of fecal samples from a small cohort of healthy adults, demonstrating good performance in a realistic experimental setting. Between-sample variation was mainly related to batch effects, such as DNA extraction, while sample indexing was also a significant source of bias. PCR cycle number strongly influenced chimera formation and affected relative abundance estimates of species with high GC content. Libraries were sequenced using the Illumina HiSeq and MiSeq platforms to demonstrate that this protocol is highly scalable to sequence thousands of samples at a very low cost. Here, we provide the most comprehensive study of performance and bias inherent to a 16S rRNA gene amplicon sequencing method to date. Triple-indexing greatly reduces the number of long custom DNA oligos required for library preparation, while the inclusion of variable length

  6. A targeted resequencing gene panel for focal epilepsy.

    Science.gov (United States)

    Hildebrand, Michael S; Myers, Candace T; Carvill, Gemma L; Regan, Brigid M; Damiano, John A; Mullen, Saul A; Newton, Mark R; Nair, Umesh; Gazina, Elena V; Milligan, Carol J; Reid, Christopher A; Petrou, Steven; Scheffer, Ingrid E; Berkovic, Samuel F; Mefford, Heather C

    2016-04-26

    We report development of a targeted resequencing gene panel for focal epilepsy, the most prevalent phenotypic group of the epilepsies. The targeted resequencing gene panel was designed using molecular inversion probe (MIP) capture technology and sequenced using massively parallel Illumina sequencing. We demonstrated proof of principle that mutations can be detected in 4 previously genotyped focal epilepsy cases. We searched for both germline and somatic mutations in 251 patients with unsolved sporadic or familial focal epilepsy and identified 11 novel or very rare missense variants in 5 different genes: CHRNA4, GRIN2B, KCNT1, PCDH19, and SCN1A. Of these, 2 were predicted to be pathogenic or likely pathogenic, explaining ∼0.8% of the cohort, and 8 were of uncertain significance based on available data. We have developed and validated a targeted resequencing panel for focal epilepsies, the most important clinical class of epilepsies, accounting for about 60% of all cases. Our application of MIP technology is an innovative approach that will be advantageous in the clinical setting because it is highly sensitive, efficient, and cost-effective for screening large patient cohorts. Our findings indicate that mutations in known genes likely explain only a small proportion of focal epilepsy cases. This is not surprising given the established clinical and genetic heterogeneity of these disorders and underscores the importance of further gene discovery studies in this complex syndrome. © 2016 American Academy of Neurology.

  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...... of this information was integrated into a single quality score for each base under Bayesian theory to measure the accuracy of consensus calling. We tested this methodology using a large-scale human resequencing data set of 36x coverage and assembled a high-quality nonrepetitive consensus sequence for 92.......25% of the diploid autosomes and 88.07% of the haploid X chromosome. Comparison of the consensus sequence with Illumina human 1M BeadChip genotyped alleles from the same DNA sample showed that 98.6% of the 37,933 genotyped alleles on the X chromosome and 98% of 999,981 genotyped alleles on autosomes were covered...

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

    Science.gov (United States)

    Skums, Pavel; Dimitrova, Zoya; Campo, David S; Vaughan, Gilberto; Rossi, Livia; Forbi, Joseph C; Yokosawa, Jonny; Zelikovsky, Alex; Khudyakov, Yury

    2012-06-25

    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. 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. 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.

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

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

    2014-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Frédéric Mahé

    2014-09-01

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

  11. Optimal Activation of Isopsoralen To Prevent Amplicon Carryover

    OpenAIRE

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

    1999-01-01

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

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

    Science.gov (United States)

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

    2017-03-01

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

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

    Science.gov (United States)

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

    2015-07-01

    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 species-specific genetic marker (DNA meta-barcode) in the whole taxonomic range of interest and the exploration of its taxon-related variants through High-Throughput Sequencing (HTS) technologies. The accessibility to proper computational systems for the large-scale bioinformatic analysis of HTS data represents, currently, one of the major challenges in advanced Meta-barcoding projects. BioMaS (Bioinformatic analysis of Metagenomic AmpliconS) is a new bioinformatic pipeline designed to support biomolecular researchers involved in taxonomic studies of environmental microbial communities by a completely automated workflow, comprehensive of all the fundamental steps, from raw sequence data upload and cleaning to final taxonomic identification, that are absolutely required in an appropriately designed Meta-barcoding HTS-based experiment. In its current version, BioMaS allows the analysis of both bacterial and fungal environments starting directly from the raw sequencing data from either Roche 454 or Illumina HTS platforms, following two alternative paths, respectively. BioMaS is implemented into a public web service available at https://recasgateway.ba.infn.it/ and is also available in Galaxy at http://galaxy.cloud.ba.infn.it:8080 (only for Illumina data). BioMaS is a friendly pipeline for Meta-barcoding HTS data analysis specifically designed for users without particular computing skills. A comparative benchmark, carried out by using a simulated dataset suitably designed to broadly represent

  14. Generic Amplicon Deep Sequencing to Determine Ilarvirus Species Diversity in Australian Prunus.

    Science.gov (United States)

    Kinoti, Wycliff M; Constable, Fiona E; Nancarrow, Narelle; Plummer, Kim M; Rodoni, Brendan

    2017-01-01

    The distribution of Ilarvirus species populations amongst 61 Australian Prunus trees was determined by next generation sequencing (NGS) of amplicons generated using a genus-based generic RT-PCR targeting a conserved region of the Ilarvirus RNA2 component that encodes the RNA dependent RNA polymerase (RdRp) gene. Presence of Ilarvirus sequences in each positive sample was further validated by Sanger sequencing of cloned amplicons of regions of each of RNA1, RNA2 and/or RNA3 that were generated by species specific PCRs and by metagenomic NGS. Prunus necrotic ringspot virus (PNRSV) was the most frequently detected Ilarvirus , occurring in 48 of the 61 Ilarvirus -positive trees and Prune dwarf virus (PDV) and Apple mosaic virus (ApMV) were detected in three trees and one tree, respectively. American plum line pattern virus (APLPV) was detected in three trees and represents the first report of APLPV detection in Australia. Two novel and distinct groups of Ilarvirus -like RNA2 amplicon sequences were also identified in several trees by the generic amplicon NGS approach. The high read depth from the amplicon NGS of the generic PCR products allowed the detection of distinct RNA2 RdRp sequence variant populations of PNRSV, PDV, ApMV, APLPV and the two novel Ilarvirus -like sequences. Mixed infections of ilarviruses were also detected in seven Prunus trees. Sanger sequencing of specific RNA1, RNA2, and/or RNA3 genome segments of each virus and total nucleic acid metagenomics NGS confirmed the presence of PNRSV, PDV, ApMV and APLPV detected by RNA2 generic amplicon NGS. However, the two novel groups of Ilarvirus -like RNA2 amplicon sequences detected by the generic amplicon NGS could not be associated to the presence of sequence from RNA1 or RNA3 genome segments or full Ilarvirus genomes, and their origin is unclear. This work highlights the sensitivity of genus-specific amplicon NGS in detection of virus sequences and their distinct populations in multiple samples, and the

  15. Generic Amplicon Deep Sequencing to Determine Ilarvirus Species Diversity in Australian Prunus

    Directory of Open Access Journals (Sweden)

    Wycliff M. Kinoti

    2017-06-01

    Full Text Available The distribution of Ilarvirus species populations amongst 61 Australian Prunus trees was determined by next generation sequencing (NGS of amplicons generated using a genus-based generic RT-PCR targeting a conserved region of the Ilarvirus RNA2 component that encodes the RNA dependent RNA polymerase (RdRp gene. Presence of Ilarvirus sequences in each positive sample was further validated by Sanger sequencing of cloned amplicons of regions of each of RNA1, RNA2 and/or RNA3 that were generated by species specific PCRs and by metagenomic NGS. Prunus necrotic ringspot virus (PNRSV was the most frequently detected Ilarvirus, occurring in 48 of the 61 Ilarvirus-positive trees and Prune dwarf virus (PDV and Apple mosaic virus (ApMV were detected in three trees and one tree, respectively. American plum line pattern virus (APLPV was detected in three trees and represents the first report of APLPV detection in Australia. Two novel and distinct groups of Ilarvirus-like RNA2 amplicon sequences were also identified in several trees by the generic amplicon NGS approach. The high read depth from the amplicon NGS of the generic PCR products allowed the detection of distinct RNA2 RdRp sequence variant populations of PNRSV, PDV, ApMV, APLPV and the two novel Ilarvirus-like sequences. Mixed infections of ilarviruses were also detected in seven Prunus trees. Sanger sequencing of specific RNA1, RNA2, and/or RNA3 genome segments of each virus and total nucleic acid metagenomics NGS confirmed the presence of PNRSV, PDV, ApMV and APLPV detected by RNA2 generic amplicon NGS. However, the two novel groups of Ilarvirus-like RNA2 amplicon sequences detected by the generic amplicon NGS could not be associated to the presence of sequence from RNA1 or RNA3 genome segments or full Ilarvirus genomes, and their origin is unclear. This work highlights the sensitivity of genus-specific amplicon NGS in detection of virus sequences and their distinct populations in multiple samples

  16. High throughput 16S rRNA gene amplicon sequencing

    DEFF Research Database (Denmark)

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

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

  17. 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 ...

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

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    Aditi Sengupta

    2016-03-01

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

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

    Science.gov (United States)

    Chiara, Matteo; Pavesi, Giulio

    2017-01-01

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

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

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    Flicek Paul

    2010-05-01

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

  1. SEED 2: a user-friendly platform for amplicon high-throughput sequencing data analyses.

    Science.gov (United States)

    Vetrovský, Tomáš; Baldrian, Petr; Morais, Daniel; Berger, Bonnie

    2018-02-14

    Modern molecular methods have increased our ability to describe microbial communities. Along with the advances brought by new sequencing technologies, we now require intensive computational resources to make sense of the large numbers of sequences continuously produced. The software developed by the scientific community to address this demand, although very useful, require experience of the command-line environment, extensive training and have steep learning curves, limiting their use. We created SEED 2, a graphical user interface for handling high-throughput amplicon-sequencing data under Windows operating systems. SEED 2 is the only sequence visualizer that empowers users with tools to handle amplicon-sequencing data of microbial community markers. It is suitable for any marker genes sequences obtained through Illumina, IonTorrent or Sanger sequencing. SEED 2 allows the user to process raw sequencing data, identify specific taxa, produce of OTU-tables, create sequence alignments and construct phylogenetic trees. Standard dual core laptops with 8 GB of RAM can handle ca. 8 million of Illumina PE 300 bp sequences, ca. 4GB of data. SEED 2 was implemented in Object Pascal and uses internal functions and external software for amplicon data processing. SEED 2 is a freeware software, available at http://www.biomed.cas.cz/mbu/lbwrf/seed/ as a self-contained file, including all the dependencies, and does not require installation. Supplementary data contain a comprehensive list of supported functions. daniel.morais@biomed.cas.cz. Supplementary data are available at Bioinformatics online. © The Author(s) 2018. Published by Oxford University Press.

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

    Science.gov (United States)

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

    2015-01-01

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

  3. Efficient alignment of pyrosequencing reads for re-sequencing applications

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    Russo Luis MS

    2011-05-01

    Full Text Available Abstract Background Over the past few years, new massively parallel DNA sequencing technologies have emerged. These platforms generate massive amounts of data per run, greatly reducing the cost of DNA sequencing. However, these techniques also raise important computational difficulties mostly due to the huge volume of data produced, but also because of some of their specific characteristics such as read length and sequencing errors. Among the most critical problems is that of efficiently and accurately mapping reads to a reference genome in the context of re-sequencing projects. Results We present an efficient method for the local alignment of pyrosequencing reads produced by the GS FLX (454 system against a reference sequence. Our approach explores the characteristics of the data in these re-sequencing applications and uses state of the art indexing techniques combined with a flexible seed-based approach, leading to a fast and accurate algorithm which needs very little user parameterization. An evaluation performed using real and simulated data shows that our proposed method outperforms a number of mainstream tools on the quantity and quality of successful alignments, as well as on the execution time. Conclusions The proposed methodology was implemented in a software tool called TAPyR--Tool for the Alignment of Pyrosequencing Reads--which is publicly available from http://www.tapyr.net.

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

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    Martin Beránek

    2012-01-01

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

  5. Dynamic Copy Number Evolution of X- and Y-Linked Ampliconic Genes in Human Populations

    DEFF Research Database (Denmark)

    Lucotte, Elise A; Skov, Laurits; Jensen, Jacob Malte

    2018-01-01

    we explore the evolution of human X- and Y-linked ampliconic genes by investigating copy number variation (CNV) and coding variation between populations using the Simons Genome Diversity Project. We develop a method to assess CNVs using the read-depth on modified X and Y chromosome targets containing...... related Y haplogroups, that diversified less than 50,000 years ago. Moreover, X and Y-linked ampliconic genes seem to have a faster amplification dynamic than autosomal multicopy genes. Looking at expression data from another study, we also find that XY-linked ampliconic genes with extensive copy number...

  6. Quantification of differential gene expression by multiplexed targeted resequencing of cDNA

    Science.gov (United States)

    Arts, Peer; van der Raadt, Jori; van Gestel, Sebastianus H.C.; Steehouwer, Marloes; Shendure, Jay; Hoischen, Alexander; Albers, Cornelis A.

    2017-01-01

    Whole-transcriptome or RNA sequencing (RNA-Seq) is a powerful and versatile tool for functional analysis of different types of RNA molecules, but sample reagent and sequencing cost can be prohibitive for hypothesis-driven studies where the aim is to quantify differential expression of a limited number of genes. Here we present an approach for quantification of differential mRNA expression by targeted resequencing of complementary DNA using single-molecule molecular inversion probes (cDNA-smMIPs) that enable highly multiplexed resequencing of cDNA target regions of ∼100 nucleotides and counting of individual molecules. We show that accurate estimates of differential expression can be obtained from molecule counts for hundreds of smMIPs per reaction and that smMIPs are also suitable for quantification of relative gene expression and allele-specific expression. Compared with low-coverage RNA-Seq and a hybridization-based targeted RNA-Seq method, cDNA-smMIPs are a cost-effective high-throughput tool for hypothesis-driven expression analysis in large numbers of genes (10 to 500) and samples (hundreds to thousands). PMID:28474677

  7. Absolute estimation of initial concentrations of amplicon in a real-time RT-PCR process

    Directory of Open Access Journals (Sweden)

    Kohn Michael

    2007-10-01

    Full Text Available Abstract Background Since real time PCR was first developed, several approaches to estimating the initial quantity of template in an RT-PCR reaction have been tried. While initially only the early thermal cycles corresponding to exponential duplication were used, lately there has been an effort to use all of the cycles in a PCR. The efforts have included both fitting empirical sigmoid curves and more elaborate mechanistic models that explore the chemical reactions taking place during each cycle. The more elaborate mechanistic models require many more parameters than can be fit from a single amplification, while the empirical models provide little insight and are difficult to tailor to specific reactants. Results We directly estimate the initial amount of amplicon using a simplified mechanistic model based on chemical reactions in the annealing step of the PCR. The basic model includes the duplication of DNA with the digestion of Taqman probe and the re-annealing between previously synthesized DNA strands of opposite orientation. By modelling the amount of Taqman probe digested and matching that with the observed fluorescence, the conversion factor between the number of fluorescing dye molecules and observed fluorescent emission can be estimated, along with the absolute initial amount of amplicon and the rate parameter for re-annealing. The model is applied to several PCR reactions with known amounts of amplicon and is shown to work reasonably well. An expanded version of the model allows duplication of amplicon without release of fluorescent dye, by adding 1 more parameter to the model. The additional process is helpful in most cases where the initial primer concentration exceeds the initial probe concentration. Software for applying the algorithm to data may be downloaded at http://www.niehs.nih.gov/research/resources/software/pcranalyzer/ Conclusion We present proof of the principle that a mechanistically based model can be fit to observations

  8. 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.

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

    Science.gov (United States)

    Gimmler, Anna; Stoeck, Thorsten

    2015-08-01

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

  10. Universal Detection and Identification of Avian Influenza Virus by Use of Resequencing Microarrays

    Science.gov (United States)

    2009-04-01

    Recent outbreaks of Nipah virus , severe acute respiratory syndrome virus , and avian influenza virus reiterate the impor- tance of zoonotic microbes as...Society for Microbiology. All Rights Reserved. Universal Detection and Identification of Avian Influenza Virus by Use of Resequencing Microarrays...been, and continue to emerge as, threats to human health. The recent outbreaks of highly pathogenic avian influenza virus in bird populations and the

  11. Evaluation of the reproducibility of amplicon sequencing with Illumina MiSeq platform.

    Science.gov (United States)

    Wen, Chongqing; Wu, Liyou; Qin, Yujia; Van Nostrand, Joy D; Ning, Daliang; Sun, Bo; Xue, Kai; Liu, Feifei; Deng, Ye; Liang, Yuting; Zhou, Jizhong

    2017-01-01

    Illumina's MiSeq has become the dominant platform for gene amplicon sequencing in microbial ecology studies; however, various technical concerns, such as reproducibility, still exist. To assess reproducibility, 16S rRNA gene amplicons from 18 soil samples of a reciprocal transplantation experiment were sequenced on an Illumina MiSeq. The V4 region of 16S rRNA gene from each sample was sequenced in triplicate with each replicate having a unique barcode. The average OTU overlap, without considering sequence abundance, at a rarefaction level of 10,323 sequences was 33.4±2.1% and 20.2±1.7% between two and among three technical replicates, respectively. When OTU sequence abundance was considered, the average sequence abundance weighted OTU overlap was 85.6±1.6% and 81.2±2.1% for two and three replicates, respectively. Removing singletons significantly increased the overlap for both (~1-3%, pdeep sequencing increased OTU overlap both when sequence abundance was considered (95%) and when not (44%). However, if singletons were not removed the overlap between two technical replicates (not considering sequence abundance) plateaus at 39% with 30,000 sequences. Diversity measures were not affected by the low overlap as α-diversities were similar among technical replicates while β-diversities (Bray-Curtis) were much smaller among technical replicates than among treatment replicates (e.g., 0.269 vs. 0.374). Higher diversity coverage, but lower OTU overlap, was observed when replicates were sequenced in separate runs. Detrended correspondence analysis indicated that while there was considerable variation among technical replicates, the reproducibility was sufficient for detecting treatment effects for the samples examined. These results suggest that although there is variation among technical replicates, amplicon sequencing on MiSeq is useful for analyzing microbial community structure if used appropriately and with caution. For example, including technical replicates

  12. Does universal 16S rRNA gene amplicon sequencing of environmental communities provide an accurate description of nitrifying guilds?

    DEFF Research Database (Denmark)

    Diwan, Vaibhav; Albrechtsen, Hans-Jørgen; Smets, Barth F.

    2018-01-01

    amplicon sequencing and from guild targeted approaches. The universal amplicon sequencing provided 1) accurate estimates of nitrifier composition, 2) clustering of the samples based on these compositions consistent with sample origin, 3) estimates of the relative abundance of the guilds correlated...

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

    Directory of Open Access Journals (Sweden)

    Mikhail Shugay

    2017-05-01

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

  14. 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.

  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

    . The new basecalling method described here, named Multipass, implements a probabilistic framework for working with the raw flowgrams obtained by pyrosequencing. For each sequence variant Multipass calculates the likelihood and nucleotide sequence of several most likely sequences given the flowgram data....... 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 virulence gene...... family, where Multipass generates 20 % more error-free sequences than current state of the art methods, and provides sequence characteristics that allow generation of a set of high confidence error-free sequences. This novel method can be used to increase accuracy of existing and future amplicon...

  16. 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...

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

    KAUST Repository

    Wang, Yong; Qian, Pei-Yuan

    2009-01-01

    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

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

    Science.gov (United States)

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

    2011-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Stephan Pabinger

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

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

    DEFF Research Database (Denmark)

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

    2008-01-01

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

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

    Science.gov (United States)

    Shiba, Norio

    2015-12-01

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

  2. Smallpox virus resequencing GeneChips can also rapidly ascertain species status for some zoonotic non-variola orthopoxviruses.

    Science.gov (United States)

    Sulaiman, Irshad M; Sammons, Scott A; Wohlhueter, Robert M

    2008-04-01

    We recently developed a set of seven resequencing GeneChips for the rapid sequencing of Variola virus strains in the WHO Repository of the Centers for Disease Control and Prevention. In this study, we attempted to hybridize these GeneChips with some known non-Variola orthopoxvirus isolates, including monkeypox, cowpox, and vaccinia viruses, for rapid detection.

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

    Directory of Open Access Journals (Sweden)

    Zdepski Anna

    2011-05-01

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

  4. 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; Martinelli, Axel; Modrzynska, Katarzyna; Borges, Sofia; Creasey, Alison; Rodrigues, Louise; Beraldi, Dario; Loewe, Laurence; Fawcett, Richard; Kumar, Sujai; Thomson, Marian; Trivedi, Urmi; Otto, Thomas D; Pain, Arnab; Blaxter, Mark; Cravo, Pedro

    2010-01-01

    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

  5. Selective whole genome amplification for resequencing target microbial species from complex natural samples.

    Science.gov (United States)

    Leichty, Aaron R; Brisson, Dustin

    2014-10-01

    Population genomic analyses have demonstrated power to address major questions in evolutionary and molecular microbiology. Collecting populations of genomes is hindered in many microbial species by the absence of a cost effective and practical method to collect ample quantities of sufficiently pure genomic DNA for next-generation sequencing. Here we present a simple method to amplify genomes of a target microbial species present in a complex, natural sample. The selective whole genome amplification (SWGA) technique amplifies target genomes using nucleotide sequence motifs that are common in the target microbe genome, but rare in the background genomes, to prime the highly processive phi29 polymerase. SWGA thus selectively amplifies the target genome from samples in which it originally represented a minor fraction of the total DNA. The post-SWGA samples are enriched in target genomic DNA, which are ideal for population resequencing. We demonstrate the efficacy of SWGA using both laboratory-prepared mixtures of cultured microbes as well as a natural host-microbe association. Targeted amplification of Borrelia burgdorferi mixed with Escherichia coli at genome ratios of 1:2000 resulted in >10(5)-fold amplification of the target genomes with genomic extracts from Wolbachia pipientis-infected Drosophila melanogaster resulted in up to 70% of high-throughput resequencing reads mapping to the W. pipientis genome. By contrast, 2-9% of sequencing reads were derived from W. pipientis without prior amplification. The SWGA technique results in high sequencing coverage at a fraction of the sequencing effort, thus allowing population genomic studies at affordable costs. Copyright © 2014 by the Genetics Society of America.

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

    Science.gov (United States)

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

    2015-02-03

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

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

    International Nuclear Information System (INIS)

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

    1997-01-01

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

  8. Quantitation of heteroplasmy of mtDNA sequence variants identified in a population of AD patients and controls by array-based resequencing.

    Science.gov (United States)

    Coon, Keith D; Valla, Jon; Szelinger, Szabolics; Schneider, Lonnie E; Niedzielko, Tracy L; Brown, Kevin M; Pearson, John V; Halperin, Rebecca; Dunckley, Travis; Papassotiropoulos, Andreas; Caselli, Richard J; Reiman, Eric M; Stephan, Dietrich A

    2006-08-01

    The role of mitochondrial dysfunction in the pathogenesis of Alzheimer's disease (AD) has been well documented. Though evidence for the role of mitochondria in AD seems incontrovertible, the impact of mitochondrial DNA (mtDNA) mutations in AD etiology remains controversial. Though mutations in mitochondrially encoded genes have repeatedly been implicated in the pathogenesis of AD, many of these studies have been plagued by lack of replication as well as potential contamination of nuclear-encoded mitochondrial pseudogenes. To assess the role of mtDNA mutations in the pathogenesis of AD, while avoiding the pitfalls of nuclear-encoded mitochondrial pseudogenes encountered in previous investigations and showcasing the benefits of a novel resequencing technology, we sequenced the entire coding region (15,452 bp) of mtDNA from 19 extremely well-characterized AD patients and 18 age-matched, unaffected controls utilizing a new, reliable, high-throughput array-based resequencing technique, the Human MitoChip. High-throughput, array-based DNA resequencing of the entire mtDNA coding region from platelets of 37 subjects revealed the presence of 208 loci displaying a total of 917 sequence variants. There were no statistically significant differences in overall mutational burden between cases and controls, however, 265 independent sites of statistically significant change between cases and controls were identified. Changed sites were found in genes associated with complexes I (30.2%), III (3.0%), IV (33.2%), and V (9.1%) as well as tRNA (10.6%) and rRNA (14.0%). Despite their statistical significance, the subtle nature of the observed changes makes it difficult to determine whether they represent true functional variants involved in AD etiology or merely naturally occurring dissimilarity. Regardless, this study demonstrates the tremendous value of this novel mtDNA resequencing platform, which avoids the pitfalls of erroneously amplifying nuclear-encoded mtDNA pseudogenes, and

  9. A quantitative and qualitative comparison of illumina MiSeq and 454 amplicon sequencing for genotyping the highly polymorphic major histocompatibility complex (MHC) in a non-model species.

    Science.gov (United States)

    Razali, Haslina; O'Connor, Emily; Drews, Anna; Burke, Terry; Westerdahl, Helena

    2017-07-28

    High-throughput sequencing enables high-resolution genotyping of extremely duplicated genes. 454 amplicon sequencing (454) has become the standard technique for genotyping the major histocompatibility complex (MHC) genes in non-model organisms. However, illumina MiSeq amplicon sequencing (MiSeq), which offers a much higher read depth, is now superseding 454. The aim of this study was to quantitatively and qualitatively evaluate the performance of MiSeq in relation to 454 for genotyping MHC class I alleles using a house sparrow (Passer domesticus) dataset with pedigree information. House sparrows provide a good study system for this comparison as their MHC class I genes have been studied previously and, consequently, we had prior expectations concerning the number of alleles per individual. We found that 454 and MiSeq performed equally well in genotyping amplicons with low diversity, i.e. amplicons from individuals that had fewer than 6 alleles. Although there was a higher rate of failure in the 454 dataset in resolving amplicons with higher diversity (6-9 alleles), the same genotypes were identified by both 454 and MiSeq in 98% of cases. We conclude that low diversity amplicons are equally well genotyped using either 454 or MiSeq, but the higher coverage afforded by MiSeq can lead to this approach outperforming 454 in amplicons with higher diversity.

  10. 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

    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 reporte...

  11. 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.

  12. 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.

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

    DEFF Research Database (Denmark)

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

    2013-01-01

    Epileptic encephalopathies are a devastating group of epilepsies with poor prognosis, of which the majority are of unknown etiology. We perform targeted massively parallel resequencing of 19 known and 46 candidate genes for epileptic encephalopathy in 500 affected individuals (cases) to identify...... CHD2 and SYNGAP1 mutations are new causes of epileptic encephalopathies, accounting for 1.2% and 1% of cases, respectively. We also expand the phenotypic spectra explained by SCN1A, SCN2A and SCN8A mutations. To our knowledge, this is the largest cohort of cases with epileptic encephalopathies...

  14. Ultra-deep pyrosequencing (UDPS data treatment to study amplicon HCV minor variants.

    Directory of Open Access Journals (Sweden)

    Josep Gregori

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

  15. 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

  16. Strong selective sweeps associated with ampliconic regions in great ape X chromosomes

    DEFF Research Database (Denmark)

    Nam, Kiwoong; Munch, Kasper; Hobolth, Asger

    2014-01-01

    The unique inheritance pattern of X chromosomes makes them preferential targets of adaptive evolution. We here investigate natural selection on the X chromosome in all species of great apes. We find that diversity is more strongly reduced around genes on the X compared with autosomes...... with ampliconic sequences we propose that intra-genomic conflict between the X and the Y chromosomes is a major driver of X chromosome evolution....

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

    Directory of Open Access Journals (Sweden)

    C Ryan King

    2010-11-01

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

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

    Directory of Open Access Journals (Sweden)

    Lőrinc S Pongor

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

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

    Science.gov (United States)

    Kinoti, Wycliff M; Constable, Fiona E; Nancarrow, Narelle; Plummer, Kim M; Rodoni, Brendan

    2017-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Wycliff M Kinoti

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

  1. Characterization of GM events by insert knowledge adapted re-sequencing approaches.

    Science.gov (United States)

    Yang, Litao; Wang, Congmao; Holst-Jensen, Arne; Morisset, Dany; Lin, Yongjun; Zhang, Dabing

    2013-10-03

    Detection methods and data from molecular characterization of genetically modified (GM) events are needed by stakeholders of public risk assessors and regulators. Generally, the molecular characteristics of GM events are incomprehensively revealed by current approaches and biased towards detecting transformation vector derived sequences. GM events are classified based on available knowledge of the sequences of vectors and inserts (insert knowledge). Herein we present three insert knowledge-adapted approaches for characterization GM events (TT51-1 and T1c-19 rice as examples) based on paired-end re-sequencing with the advantages of comprehensiveness, accuracy, and automation. The comprehensive molecular characteristics of two rice events were revealed with additional unintended insertions comparing with the results from PCR and Southern blotting. Comprehensive transgene characterization of TT51-1 and T1c-19 is shown to be independent of a priori knowledge of the insert and vector sequences employing the developed approaches. This provides an opportunity to identify and characterize also unknown GM events.

  2. Gene Fusions Associated with Recurrent Amplicons Represent a Class of Passenger Aberrations in Breast Cancer

    Directory of Open Access Journals (Sweden)

    Shanker Kalyana-Sundaram

    2012-08-01

    Full Text Available Application of high-throughput transcriptome sequencing has spurred highly sensitive detection and discovery of gene fusions in cancer, but distinguishing potentially oncogenic fusions from random, “passenger” aberrations has proven challenging. Here we examine a distinctive group of gene fusions that involve genes present in the loci of chromosomal amplifications—a class of oncogenic aberrations that are widely prevalent in breast cancers. Integrative analysis of a panel of 14 breast cancer cell lines comparing gene fusions discovered by high-throughput transcriptome sequencing and genome-wide copy number aberrations assessed by array comparative genomic hybridization, led to the identification of 77 gene fusions, of which more than 60% were localized to amplicons including 17q12, 17q23, 20q13, chr8q, and others. Many of these fusions appeared to be recurrent or involved highly expressed oncogenic drivers, frequently fused with multiple different partners, but sometimes displaying loss of functional domains. As illustrative examples of the “amplicon-associated” gene fusions, we examined here a recurrent gene fusion involving the mediator of mammalian target of rapamycin signaling, RPS6KB1 kinase in BT-474, and the therapeutically important receptor tyrosine kinase EGFR in MDA-MB-468 breast cancer cell line. These gene fusions comprise a minor allelic fraction relative to the highly expressed full-length transcripts and encode chimera lacking the kinase domains, which do not impart dependence on the respective cells. Our study suggests that amplicon-associated gene fusions in breast cancer primarily represent a by-product of chromosomal amplifications, which constitutes a subset of passenger aberrations and should be factored accordingly during prioritization of gene fusion candidates.

  3. Cytomegalovirus sequence variability, amplicon length, and DNase-sensitive non-encapsidated genomes are obstacles to standardization and commutability of plasma viral load results.

    Science.gov (United States)

    Naegele, Klaudia; Lautenschlager, Irmeli; Gosert, Rainer; Loginov, Raisa; Bir, Katia; Helanterä, Ilkka; Schaub, Stefan; Khanna, Nina; Hirsch, Hans H

    2018-04-22

    Cytomegalovirus (CMV) management post-transplantation relies on quantification in blood, but inter-laboratory and inter-assay variability impairs commutability. An international multicenter study demonstrated that variability is mitigated by standardizing plasma volumes, automating DNA extraction and amplification, and calibration to the 1st-CMV-WHO-International-Standard as in the FDA-approved Roche-CAP/CTM-CMV. However, Roche-CAP/CTM-CMV showed under-quantification and false-negative results in a quality assurance program (UK-NEQAS-2014). To evaluate factors contributing to quantification variability of CMV viral load and to develop optimized CMV-UL54-QNAT. The UL54 target of the UK-NEQAS-2014 variant was sequenced and compared to 329 available CMV GenBank sequences. Four Basel-CMV-UL54-QNAT assays of 361 bp, 254 bp, 151 bp, and 95 bp amplicons were developed that only differed in reverse primer positions. The assays were validated using plasmid dilutions, UK-NEQAS-2014 sample, as well as 107 frozen and 69 prospectively collected plasma samples from transplant patients submitted for CMV QNAT, with and without DNase-digestion prior to nucleic acid extraction. Eight of 43 mutations were identified as relevant in the UK-NEQAS-2014 target. All Basel-CMV-UL54 QNATs quantified the UK-NEQAS-2014 but revealed 10-fold increasing CMV loads as amplicon size decreased. The inverse correlation of amplicon size and viral loads was confirmed using 1st-WHO-International-Standard and patient samples. DNase pre-treatment reduced plasma CMV loads by >90% indicating the presence of unprotected CMV genomic DNA. Sequence variability, amplicon length, and non-encapsidated genomes obstruct standardization and commutability of CMV loads needed to develop thresholds for clinical research and management. Besides regular sequence surveys, matrix and extraction standardization, we propose developing reference calibrators using 100 bp amplicons. Copyright © 2018 Elsevier B.V. All

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

    Science.gov (United States)

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

    2015-08-01

    Two different real-time quantitative PCR (PMA-qPCR) assays were applied for quantification of Legionella spp. by targeting a long amplicon (approx 400 bp) of 16S rRNA gene and a short amplicon (approx. 100 bp) of 5S rRNA gene. Purified DNA extracts from pure cultures of Legionella spp. and from environmental water samples were quantified. Application of the two assays to quantify Legionella in artificially contaminated water achieved that both assays were able to detect Legionella over a linear range of 10 to 10(5) cells ml(-1). A statistical analysis of the standard curves showed that both assays were linear with a good correlation coefficient (R(2) = 0.99) between the Ct and the copy number. Amplification with the reference assay was the most effective for detecting low copy numbers (1 bacterium per PCR mixture). Using selective quantification of viable Legionella by the PMA-qPCR method we obtained a greater inhibition of the amplification of the 400-bp 16S gene fragment (Δlog(10) = 3.74 ± 0.39 log(10) GU ml(-1)). A complete inhibition of the PCR signal was obtained when heat-killed cells in a concentration below 1 × 10(5) cells ml(-1) were pretreated with PMA. Analysing short amplicon sizes led to only 2.08 log reductions in the Legionella dead-cell signal. When we tested environmental water samples, the two qPCR assays were in good agreement according to the kappa index (0.741). Applying qPCR combined with PMA treatment, we also obtained a good agreement (kappa index 0.615). The comparison of quantitative results shows that both assays yielded the same quantification sensitivity (mean log = 4.59 vs mean log = 4.31). Copyright © 2015 Elsevier Ltd. All rights reserved.

  5. Massively parallel amplicon sequencing reveals isotype-specific variability of antimicrobial peptide transcripts in Mytilus galloprovincialis.

    Directory of Open Access Journals (Sweden)

    Umberto Rosani

    Full Text Available BACKGROUND: Effective innate responses against potential pathogens are essential in the living world and possibly contributed to the evolutionary success of invertebrates. Taken together, antimicrobial peptide (AMP precursors of defensin, mytilin, myticin and mytimycin can represent about 40% of the hemocyte transcriptome in mussels injected with viral-like and bacterial preparations, and unique profiles of myticin C variants are expressed in single mussels. Based on amplicon pyrosequencing, we have ascertained and compared the natural and Vibrio-induced diversity of AMP transcripts in mussel hemocytes from three European regions. METHODOLOGY/PRINCIPAL FINDINGS: Hemolymph was collected from mussels farmed in the coastal regions of Palavas (France, Vigo (Spain and Venice (Italy. To represent the AMP families known in M. galloprovincialis, nine transcript sequences have been selected, amplified from hemocyte RNA and subjected to pyrosequencing. Hemolymph from farmed (offshore and wild (lagoon Venice mussels, both injected with 10(7 Vibrio cells, were similarly processed. Amplicon pyrosequencing emphasized the AMP transcript diversity, with Single Nucleotide Changes (SNC minimal for mytilin B/C and maximal for arthropod-like defensin and myticin C. Ratio of non-synonymous vs. synonymous changes also greatly differed between AMP isotypes. Overall, each amplicon revealed similar levels of nucleotidic variation across geographical regions, with two main sequence patterns confirmed for mytimycin and no substantial changes after immunostimulation. CONCLUSIONS/SIGNIFICANCE: Barcoding and bidirectional pyrosequencing allowed us to map and compare the transcript diversity of known mussel AMPs. Though most of the genuine cds variation was common to the analyzed samples we could estimate from 9 to 106 peptide variants in hemolymph pools representing 100 mussels, depending on the AMP isoform and sampling site. In this study, no prevailing SNC patterns related

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

    Directory of Open Access Journals (Sweden)

    HyoYoung Kim

    2015-12-01

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

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

    DEFF Research Database (Denmark)

    Yao, Danfeng; Kim, Junyoung; Yu, Fang

    2005-01-01

    at an intermediate sodium concentration (approximately 100 mM). These effects were mainly ascribed to the electrostatic cross talk among the hybridized DNA molecules and the secondary structure of PCR amplicons. For the negatively charged DNA probes, the hybridization reaction was subjected additionally to the DNA....../DNA electrostatic barrier, particularly in lower ionic strength range (e.g., 10 approximately 150 mM Na(+)). The electrostatic cross talk was shown to be largely reduced if the PNA probe layer was sufficiently diluted by following a strategic templated immobilization method. As a consequence, a pseudo...

  8. Quantitative genome re-sequencing defines multiple mutations conferring chloroquine resistance in rodent malaria

    Science.gov (United States)

    2012-01-01

    Background Drug resistance in the malaria parasite Plasmodium falciparum severely compromises the treatment and control of malaria. A knowledge of the critical mutations conferring resistance to particular drugs is important in understanding modes of drug action and mechanisms of resistances. They are required to design better therapies and limit drug resistance. A mutation in the gene (pfcrt) encoding a membrane transporter has been identified as a principal determinant of chloroquine resistance in P. falciparum, but we lack a full account of higher level chloroquine resistance. Furthermore, the determinants of resistance in the other major human malaria parasite, P. vivax, are not known. To address these questions, we investigated the genetic basis of chloroquine resistance in an isogenic lineage of rodent malaria parasite P. chabaudi in which high level resistance to chloroquine has been progressively selected under laboratory conditions. Results Loci containing the critical genes were mapped by Linkage Group Selection, using a genetic cross between the high-level chloroquine-resistant mutant and a genetically distinct sensitive strain. A novel high-resolution quantitative whole-genome re-sequencing approach was used to reveal three regions of selection on chr11, chr03 and chr02 that appear progressively at increasing drug doses on three chromosomes. Whole-genome sequencing of the chloroquine-resistant parent identified just four point mutations in different genes on these chromosomes. Three mutations are located at the foci of the selection valleys and are therefore predicted to confer different levels of chloroquine resistance. The critical mutation conferring the first level of chloroquine resistance is found in aat1, a putative aminoacid transporter. Conclusions Quantitative trait loci conferring selectable phenotypes, such as drug resistance, can be mapped directly using progressive genome-wide linkage group selection. Quantitative genome-wide short

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

    Directory of Open Access Journals (Sweden)

    Zhewei Song

    2017-07-01

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

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

    Science.gov (United States)

    Song, Zhewei; Du, Hai; Zhang, Yan; Xu, Yan

    2017-01-01

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

  11. Application of broad-spectrum resequencing microarray for genotyping rhabdoviruses.

    Science.gov (United States)

    Dacheux, Laurent; Berthet, Nicolas; Dissard, Gabriel; Holmes, Edward C; Delmas, Olivier; Larrous, Florence; Guigon, Ghislaine; Dickinson, Philip; Faye, Ousmane; Sall, Amadou A; Old, Iain G; Kong, Katherine; Kennedy, Giulia C; Manuguerra, Jean-Claude; Cole, Stewart T; Caro, Valérie; Gessain, Antoine; Bourhy, Hervé

    2010-09-01

    The rapid and accurate identification of pathogens is critical in the control of infectious disease. To this end, we analyzed the capacity for viral detection and identification of a newly described high-density resequencing microarray (RMA), termed PathogenID, which was designed for multiple pathogen detection using database similarity searching. We focused on one of the largest and most diverse viral families described to date, the family Rhabdoviridae. We demonstrate that this approach has the potential to identify both known and related viruses for which precise sequence information is unavailable. In particular, we demonstrate that a strategy based on consensus sequence determination for analysis of RMA output data enabled successful detection of viruses exhibiting up to 26% nucleotide divergence with the closest sequence tiled on the array. Using clinical specimens obtained from rabid patients and animals, this method also shows a high species level concordance with standard reference assays, indicating that it is amenable for the development of diagnostic assays. Finally, 12 animal rhabdoviruses which were currently unclassified, unassigned, or assigned as tentative species within the family Rhabdoviridae were successfully detected. These new data allowed an unprecedented phylogenetic analysis of 106 rhabdoviruses and further suggest that the principles and methodology developed here may be used for the broad-spectrum surveillance and the broader-scale investigation of biodiversity in the viral world.

  12. Application of Broad-Spectrum Resequencing Microarray for Genotyping Rhabdoviruses▿

    Science.gov (United States)

    Dacheux, Laurent; Berthet, Nicolas; Dissard, Gabriel; Holmes, Edward C.; Delmas, Olivier; Larrous, Florence; Guigon, Ghislaine; Dickinson, Philip; Faye, Ousmane; Sall, Amadou A.; Old, Iain G.; Kong, Katherine; Kennedy, Giulia C.; Manuguerra, Jean-Claude; Cole, Stewart T.; Caro, Valérie; Gessain, Antoine; Bourhy, Hervé

    2010-01-01

    The rapid and accurate identification of pathogens is critical in the control of infectious disease. To this end, we analyzed the capacity for viral detection and identification of a newly described high-density resequencing microarray (RMA), termed PathogenID, which was designed for multiple pathogen detection using database similarity searching. We focused on one of the largest and most diverse viral families described to date, the family Rhabdoviridae. We demonstrate that this approach has the potential to identify both known and related viruses for which precise sequence information is unavailable. In particular, we demonstrate that a strategy based on consensus sequence determination for analysis of RMA output data enabled successful detection of viruses exhibiting up to 26% nucleotide divergence with the closest sequence tiled on the array. Using clinical specimens obtained from rabid patients and animals, this method also shows a high species level concordance with standard reference assays, indicating that it is amenable for the development of diagnostic assays. Finally, 12 animal rhabdoviruses which were currently unclassified, unassigned, or assigned as tentative species within the family Rhabdoviridae were successfully detected. These new data allowed an unprecedented phylogenetic analysis of 106 rhabdoviruses and further suggest that the principles and methodology developed here may be used for the broad-spectrum surveillance and the broader-scale investigation of biodiversity in the viral world. PMID:20610710

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

    Science.gov (United States)

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

    2013-01-01

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

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

    DEFF Research Database (Denmark)

    Xu, Xun; Liu, Xin; Ge, Song

    2012-01-01

    Rice is a staple crop that has undergone substantial phenotypic and physiological changes during domestication. Here we resequenced the genomes of 40 cultivated accessions selected from the major groups of rice and 10 accessions of their wild progenitors (Oryza rufipogon and Oryza nivara) to >15 x...... diversity in cultivated but not wild rice, which represent candidate regions selected during domestication. Some of these variants are associated with important biological features, whereas others have yet to be functionally characterized. The molecular markers we have identified should be valuable...... raw data coverage. We investigated genome-wide variation patterns in rice and obtained 6.5 million high-quality single nucleotide polymorphisms (SNPs) after excluding sites with missing data in any accession. Using these population SNP data, we identified thousands of genes with significantly lower...

  15. Prostate-Specific and Tumor-Specific Targeting of an Oncolytic HSV-1 Amplicon/Helper Virus for Prostate Cancer Treatment

    Science.gov (United States)

    2009-11-01

    regulation HSV-1 amplicon and recombinant viruses Molecular cloning Cell culture/gene transfection Xenograft mouse model Histology 20...no herpetic lesions were seen in CMV-ICP4-143T–treated and CMV-ICP4-145T– treated animals, although some gastritis developed 28 days after the viral

  16. 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.

  17. 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.

  18. Imaging Herpes Simplex Virus Type 1 Amplicon Vector–Mediated Gene Expression in Human Glioma Spheroids

    OpenAIRE

    Christine Kaestle; Alexandra Winkeler; Raphaela Richter; Heinrich Sauer; Jürgen Hescheler; Cornel Fraefel; Maria Wartenberg; Andreas H. Jacobs

    2011-01-01

    Vectors derived from herpes simplex virus type 1 (HSV-1) have great potential for transducing therapeutic genes into the central nervous system; however, inefficient distribution of vector particles in vivo may limit their therapeutic potential in patients with gliomas. This study was performed to investigate the extent of HSV-1 amplicon vector–mediated gene expression in a three-dimensional glioma model of multicellular spheroids by imaging highly infectious HSV-1 virions expressing green fl...

  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

    to link sludge and floc properties to the microbial communities. All data was subjected to extensive network analysis and multivariate statistics through R. The 16S amplicon results confirmed the findings of relatively few core groups of organism shared by all the wastewater treatment plants......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...... a year, totaling over 400 samples. All samples were subjected to 16S rDNA amplicon sequencing using V13 primers on the Illumina MiSeq platform (2x300bp) to a depth of at least 20.000 quality filtered reads per sample. The OTUs were assigned taxonomy based on a manually curated version of the greengenes...

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

    Directory of Open Access Journals (Sweden)

    Stéphane Deschamps

    2010-07-01

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

  1. 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.

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

    Directory of Open Access Journals (Sweden)

    Federico M Ibarbalz

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

  3. 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.

  4. 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. Copyright © 2015. Published by Elsevier Inc.

  5. LipidSeq: a next-generation clinical resequencing panel for monogenic dyslipidemias[S

    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.

    2014-01-01

    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. PMID:24503134

  6. Biphasic Study to Characterize Agricultural Biogas Plants by High-Throughput 16S rRNA Gene Amplicon Sequencing and Microscopic Analysis.

    Science.gov (United States)

    Maus, Irena; Kim, Yong Sung; Wibberg, Daniel; Stolze, Yvonne; Off, Sandra; Antonczyk, Sebastian; Pühler, Alfred; Scherer, Paul; Schlüter, Andreas

    2017-02-28

    Process surveillance within agricultural biogas plants (BGPs) was concurrently studied by high-throughput 16S rRNA gene amplicon sequencing and an optimized quantitative microscopic fingerprinting (QMF) technique. In contrast to 16S rRNA gene amplicons, digitalized microscopy is a rapid and cost-effective method that facilitates enumeration and morphological differentiation of the most significant groups of methanogens regarding their shape and characteristic autofluorescent factor 420. Moreover, the fluorescence signal mirrors cell vitality. In this study, four different BGPs were investigated. The results indicated stable process performance in the mesophilic BGPs and in the thermophilic reactor. Bacterial subcommunity characterization revealed significant differences between the four BGPs. Most remarkably, the genera Defluviitoga and Halocella dominated the thermophilic bacterial subcommunity, whereas members of another taxon, Syntrophaceticus , were found to be abundant in the mesophilic BGP. The domain Archaea was dominated by the genus Methanoculleus in all four BGPs, followed by Methanosaeta in BGP1 and BGP3. In contrast, Methanothermobacter members were highly abundant in the thermophilic BGP4. Furthermore, a high consistency between the sequencing approach and the QMF method was shown, especially for the thermophilic BGP. The differences elucidated that using this biphasic approach for mesophilic BGPs provided novel insights regarding disaggregated single cells of Methanosarcina and Methanosaeta species. Both dominated the archaeal subcommunity and replaced coccoid Methanoculleus members belonging to the same group of Methanomicrobiales that have been frequently observed in similar BGPs. This work demonstrates that combining QMF and 16S rRNA gene amplicon sequencing is a complementary strategy to describe archaeal community structures within biogas processes.

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Frédéric Mahé

    2015-12-01

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

  9. 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.

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

    Science.gov (United States)

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

    2015-03-31

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

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

    Science.gov (United States)

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

    2017-05-02

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

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

    Science.gov (United States)

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

    1997-11-01

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

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

    DEFF Research Database (Denmark)

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

    communities, and in this study activated sludge sampled from 32 Wastewater Treatment Plants (WWTPs) around the world was described and compared. The top abundant bacteria in the global activated sludge ecosystem were found and the core population shared by multiple samples was investigated. The results......Activated sludge is the most commonly applied bioprocess throughout the world for wastewater treatment. Microorganisms are key to the process, yet our knowledge of their identity and function is still limited. High-througput16S rRNA amplicon sequencing can reliably characterize microbial...

  14. 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.

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

    DEFF Research Database (Denmark)

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

    2000-01-01

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

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

    Science.gov (United States)

    Liu, Wen; Ghouri, Fozia; Yu, Hang; Li, Xiang; Yu, Shuhong; Shahid, Muhammad Qasim; Liu, Xiangdong

    2017-01-01

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

  17. 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.

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

    Directory of Open Access Journals (Sweden)

    Nils Homer

    2009-11-01

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

  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. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  20. 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.

  1. The application of amplicon length heterogeneity PCR (LH-PCR) for monitoring the dynamics of soil microbial communities associated with cadaver decomposition.

    Science.gov (United States)

    Moreno, Lilliana I; Mills, DeEtta; Fetscher, Jill; John-Williams, Krista; Meadows-Jantz, Lee; McCord, Bruce

    2011-03-01

    The placement of cadavers in shallow, clandestine graves may alter the microbial and geochemical composition of the underlying and adjacent soils. Using amplicon length heterogeneity-PCR (LH-PCR) the microbial community changes in these soils can be assessed. In this investigation, nine different grave sites were examined over a period of 16weeks. The results indicated that measurable changes occurred in the soil bacterial community during the decomposition process. In this study, amplicons corresponding to anaerobic bacteria, not indigenous to the soil, were shown to produce differences between grave sites and control soils. Among the bacteria linked to these amplicons are those that are most often part of the commensal flora of the intestines, mouth and skin. In addition, over the 16week sampling interval, the level of indicator organisms (i.e., nitrogen fixing bacteria) dropped as the body decomposed and after four weeks of environmental exposure they began to increase again; thus differences in the abundance of nitrogen fixing bacteria were also found to contribute to the variation between controls and grave soils. These results were verified using primers that specifically targeted the nifH gene coding for nitrogenase reductase. LH-PCR provides a fast, robust and reproducible method to measure microbial changes in soil and could be used to determine potential cadaveric contact in a given area. The results obtained with this method could ultimately provide leads to investigators in criminal or missing person scenarios and allow for further analysis using human specific DNA assays to establish the identity of the buried body. Copyright © 2010 Elsevier B.V. All rights reserved.

  2. 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...... by unicellular cyanobacteria, 42% of the identified non-cyanobacterial nifH clusters from the corresponding DNA samples were also detected in cDNA. The study indicates that non-cyanobacteria account for a substantial part of the nifH gene pool in marine surface waters and that these genes are at least...

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

    Science.gov (United States)

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

    2016-01-27

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

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

    Science.gov (United States)

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

    2014-06-01

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

  5. 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

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

    International Nuclear Information System (INIS)

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

    2004-01-01

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

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

    Science.gov (United States)

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

    2017-09-05

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

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

    Directory of Open Access Journals (Sweden)

    Hanna Farnelid

    Full Text Available Cyanobacteria are thought to be the main N(2-fixing organisms (diazotrophs in marine pelagic waters, but recent molecular analyses indicate that non-cyanobacterial diazotrophs are also present and active. Existing data are, however, restricted geographically and by limited sequencing depths. Our analysis of 79,090 nitrogenase (nifH PCR amplicons encoding 7,468 unique proteins from surface samples (ten DNA samples and two RNA samples collected at ten marine locations world-wide provides the first in-depth survey of a functional bacterial gene and yield insights into the composition and diversity of the nifH gene pool in marine waters. Great divergence in nifH composition was observed between sites. Cyanobacteria-like genes were most frequent among amplicons from the warmest waters, but overall the data set was dominated by nifH sequences most closely related to non-cyanobacteria. Clusters related to Alpha-, Beta-, Gamma-, and Delta-Proteobacteria were most common and showed distinct geographic distributions. Sequences related to anaerobic bacteria (nifH Cluster III were generally rare, but preponderant in cold waters, especially in the Arctic. Although the two transcript samples were dominated by unicellular cyanobacteria, 42% of the identified non-cyanobacterial nifH clusters from the corresponding DNA samples were also detected in cDNA. The study indicates that non-cyanobacteria account for a substantial part of the nifH gene pool in marine surface waters and that these genes are at least occasionally expressed. The contribution of non-cyanobacterial diazotrophs to the global N(2 fixation budget cannot be inferred from sequence data alone, but the prevalence of non-cyanobacterial nifH genes and transcripts suggest that these bacteria are ecologically significant.

  9. Multiplexed resequencing analysis to identify rare variants in pooled DNA with barcode indexing using next-generation sequencer.

    Science.gov (United States)

    Mitsui, Jun; Fukuda, Yoko; Azuma, Kyo; Tozaki, Hirokazu; Ishiura, Hiroyuki; Takahashi, Yuji; Goto, Jun; Tsuji, Shoji

    2010-07-01

    We have recently found that multiple rare variants of the glucocerebrosidase gene (GBA) confer a robust risk for Parkinson disease, supporting the 'common disease-multiple rare variants' hypothesis. To develop an efficient method of identifying rare variants in a large number of samples, we applied multiplexed resequencing using a next-generation sequencer to identification of rare variants of GBA. Sixteen sets of pooled DNAs from six pooled DNA samples were prepared. Each set of pooled DNAs was subjected to polymerase chain reaction to amplify the target gene (GBA) covering 6.5 kb, pooled into one tube with barcode indexing, and then subjected to extensive sequence analysis using the SOLiD System. Individual samples were also subjected to direct nucleotide sequence analysis. With the optimization of data processing, we were able to extract all the variants from 96 samples with acceptable rates of false-positive single-nucleotide variants.

  10. Genes encoded within 8q24 on the amplicon of a large extrachromosomal element are selectively repressed during the terminal differentiation of HL-60 cells.

    Science.gov (United States)

    Hirano, Tetsuo; Ike, Fumio; Murata, Takehide; Obata, Yuichi; Utiyama, Hiroyasu; Yokoyama, Kazunari K

    2008-04-02

    Human acute myeloblastic leukemia HL-60 cells become resistant to differentiation during long-term cultivation. After 150 passages, double minute chromosomes (dmins) found in early-passaged cells are replaced by large extrachromosomal elements (LEEs). In a DNA library derived from a purified fraction of LEEs, 12.6% (23/183) of clones were assigned to 8q24 and 9.2% (17/183) were assigned to 14q11 in the human genome. Fluorescence in situ hybridization (FISH) revealed a small aberrant chromosome, which had not been found in early-passaged cells, in addition to the purified LEEs. We determined that each LEE consisted of six discontinuous segments in a region that extended for 4.4Mb over the 8q24 locus. Five genes, namely, Myc (a proto-oncogene), NSMCE2 (for a SUMO ligase), CCDC26 (for a retinoic acid-dependent modulator of myeloid differentiation), TRIB1 (for a regulator of MAPK kinase) and LOC389637 (for a protein of unknown function), were encoded by the amplicon. Breaks in the chromosomal DNA within the amplicon were found in the NSMCE2 and CCDC26 genes. The discontinuous structure of the amplicon unit of the LEEs was identical with that of dmins in HL-60 early-passaged cells. The difference between them seemed, predominantly, to be the number (10-15 copies per LEE versus 2 or 3 copies per dmin) of constituent units. Expression of the Myc, NSMCE2, CCDC26 and LOC389637 and TRIB1 genes was constitutive in all lines of HL-60 cells and that of the first four genes was repressed during the terminal differentiation of early-passaged HL-60 cells. We also detected abnormal transcripts of CCDC26. Our results suggest that these genes were selected during the development of amplicons. They might be amplified and, sometimes, truncated to contribute to the maintenance of HL-60 cells in an undifferentiated state.

  11. Implementation of Amplicon Parallel Sequencing Leads to Improvement of Diagnosis and Therapy of Lung Cancer Patients.

    Science.gov (United States)

    König, Katharina; Peifer, Martin; Fassunke, Jana; Ihle, Michaela A; Künstlinger, Helen; Heydt, Carina; Stamm, Katrin; Ueckeroth, Frank; Vollbrecht, Claudia; Bos, Marc; Gardizi, Masyar; Scheffler, Matthias; Nogova, Lucia; Leenders, Frauke; Albus, Kerstin; Meder, Lydia; Becker, Kerstin; Florin, Alexandra; Rommerscheidt-Fuss, Ursula; Altmüller, Janine; Kloth, Michael; Nürnberg, Peter; Henkel, Thomas; Bikár, Sven-Ernö; Sos, Martin L; Geese, William J; Strauss, Lewis; Ko, Yon-Dschun; Gerigk, Ulrich; Odenthal, Margarete; Zander, Thomas; Wolf, Jürgen; Merkelbach-Bruse, Sabine; Buettner, Reinhard; Heukamp, Lukas C

    2015-07-01

    The Network Genomic Medicine Lung Cancer was set up to rapidly translate scientific advances into early clinical trials of targeted therapies in lung cancer performing molecular analyses of more than 3500 patients annually. Because sequential analysis of the relevant driver mutations on fixated samples is challenging in terms of workload, tissue availability, and cost, we established multiplex parallel sequencing in routine diagnostics. The aim was to analyze all therapeutically relevant mutations in lung cancer samples in a high-throughput fashion while significantly reducing turnaround time and amount of input DNA compared with conventional dideoxy sequencing of single polymerase chain reaction amplicons. In this study, we demonstrate the feasibility of a 102 amplicon multiplex polymerase chain reaction followed by sequencing on an Illumina sequencer on formalin-fixed paraffin-embedded tissue in routine diagnostics. Analysis of a validation cohort of 180 samples showed this approach to require significantly less input material and to be more reliable, robust, and cost-effective than conventional dideoxy sequencing. Subsequently, 2657 lung cancer patients were analyzed. We observed that comprehensive biomarker testing provided novel information in addition to histological diagnosis and clinical staging. In 2657 consecutively analyzed lung cancer samples, we identified driver mutations at the expected prevalence. Furthermore we found potentially targetable DDR2 mutations at a frequency of 3% in both adenocarcinomas and squamous cell carcinomas. Overall, our data demonstrate the utility of systematic sequencing analysis in a clinical routine setting and highlight the dramatic impact of such an approach on the availability of therapeutic strategies for the targeted treatment of individual cancer patients.

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

    Directory of Open Access Journals (Sweden)

    Tellier Laurent C

    2010-03-01

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

  13. High-throughput sequencing of 16S rRNA gene amplicons: effects of extraction procedure, primer length and annealing temperature.

    Science.gov (United States)

    Sergeant, Martin J; Constantinidou, Chrystala; Cogan, Tristan; Penn, Charles W; Pallen, Mark J

    2012-01-01

    The analysis of 16S-rDNA sequences to assess the bacterial community composition of a sample is a widely used technique that has increased with the advent of high throughput sequencing. Although considerable effort has been devoted to identifying the most informative region of the 16S gene and the optimal informatics procedures to process the data, little attention has been paid to the PCR step, in particular annealing temperature and primer length. To address this, amplicons derived from 16S-rDNA were generated from chicken caecal content DNA using different annealing temperatures, primers and different DNA extraction procedures. The amplicons were pyrosequenced to determine the optimal protocols for capture of maximum bacterial diversity from a chicken caecal sample. Even at very low annealing temperatures there was little effect on the community structure, although the abundance of some OTUs such as Bifidobacterium increased. Using shorter primers did not reveal any novel OTUs but did change the community profile obtained. Mechanical disruption of the sample by bead beating had a significant effect on the results obtained, as did repeated freezing and thawing. In conclusion, existing primers and standard annealing temperatures captured as much diversity as lower annealing temperatures and shorter primers.

  14. 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.

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

    Science.gov (United States)

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

    2011-06-01

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

  16. 'Mitominis': multiplex PCR analysis of reduced size amplicons for compound sequence analysis of the entire mtDNA control region in highly degraded samples.

    Science.gov (United States)

    Eichmann, Cordula; Parson, Walther

    2008-09-01

    The traditional protocol for forensic mitochondrial DNA (mtDNA) analyses involves the amplification and sequencing of the two hypervariable segments HVS-I and HVS-II of the mtDNA control region. The primers usually span fragment sizes of 300-400 bp each region, which may result in weak or failed amplification in highly degraded samples. Here we introduce an improved and more stable approach using shortened amplicons in the fragment range between 144 and 237 bp. Ten such amplicons were required to produce overlapping fragments that cover the entire human mtDNA control region. These were co-amplified in two multiplex polymerase chain reactions and sequenced with the individual amplification primers. The primers were carefully selected to minimize binding on homoplasic and haplogroup-specific sites that would otherwise result in loss of amplification due to mis-priming. The multiplexes have successfully been applied to ancient and forensic samples such as bones and teeth that showed a high degree of degradation.

  17. Genome-wide resequencing of KRICE_CORE reveals their potential for future breeding, as well as functional and evolutionary studies in the post-genomic era.

    Science.gov (United States)

    Kim, Tae-Sung; He, Qiang; Kim, Kyu-Won; Yoon, Min-Young; Ra, Won-Hee; Li, Feng Peng; Tong, Wei; Yu, Jie; Oo, Win Htet; Choi, Buung; Heo, Eun-Beom; Yun, Byoung-Kook; Kwon, Soon-Jae; Kwon, Soon-Wook; Cho, Yoo-Hyun; Lee, Chang-Yong; Park, Beom-Seok; Park, Yong-Jin

    2016-05-26

    Rice germplasm collections continue to grow in number and size around the world. Since maintaining and screening such massive resources remains challenging, it is important to establish practical methods to manage them. A core collection, by definition, refers to a subset of the entire population that preserves the majority of genetic diversity, enhancing the efficiency of germplasm utilization. Here, we report whole-genome resequencing of the 137 rice mini core collection or Korean rice core set (KRICE_CORE) that represents 25,604 rice germplasms deposited in the Korean genebank of the Rural Development Administration (RDA). We implemented the Illumina HiSeq 2000 and 2500 platform to produce short reads and then assembled those with 9.8 depths using Nipponbare as a reference. Comparisons of the sequences with the reference genome yielded more than 15 million (M) single nucleotide polymorphisms (SNPs) and 1.3 M INDELs. Phylogenetic and population analyses using 2,046,529 high-quality SNPs successfully assigned rice accessions to the relevant rice subgroups, suggesting that these SNPs capture evolutionary signatures that have accumulated in rice subpopulations. Furthermore, genome-wide association studies (GWAS) for four exemplary agronomic traits in the KRIC_CORE manifest the utility of KRICE_CORE; that is, identifying previously defined genes or novel genetic factors that potentially regulate important phenotypes. This study provides strong evidence that the size of KRICE_CORE is small but contains high genetic and functional diversity across the genome. Thus, our resequencing results will be useful for future breeding, as well as functional and evolutionary studies, in the post-genomic era.

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

    DEFF Research Database (Denmark)

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

    2016-01-01

    BACKGROUND: There is an immense scientific interest in the human microbiome and its effects on human physiology, health, and disease. A common approach for examining bacterial communities is high-throughput sequencing of 16S rRNA gene hypervariable regions, aggregating sequence-similar amplicons...

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

    Science.gov (United States)

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

    2016-03-01

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

  20. Whole genome complete resequencing of Bacillus subtilis natto by combining long reads with high-quality short reads.

    Directory of Open Access Journals (Sweden)

    Mayumi Kamada

    Full Text Available De novo microbial genome sequencing reached a turning point with third-generation sequencing (TGS platforms, and several microbial genomes have been improved by TGS long reads. Bacillus subtilis natto is closely related to the laboratory standard strain B. subtilis Marburg 168, and it has a function in the production of the traditional Japanese fermented food "natto." The B. subtilis natto BEST195 genome was previously sequenced with short reads, but it included some incomplete regions. We resequenced the BEST195 genome using a PacBio RS sequencer, and we successfully obtained a complete genome sequence from one scaffold without any gaps, and we also applied Illumina MiSeq short reads to enhance quality. Compared with the previous BEST195 draft genome and Marburg 168 genome, we found that incomplete regions in the previous genome sequence were attributed to GC-bias and repetitive sequences, and we also identified some novel genes that are found only in the new genome.

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

    Science.gov (United States)

    Moralli, Daniela; Monaco, Zoia L

    2015-02-01

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

  2. 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

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

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

    Directory of Open Access Journals (Sweden)

    Hernández-Moneo Jose-Luis

    2006-09-01

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

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

    DEFF Research Database (Denmark)

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

    2017-01-01

    We report on an optomagnetic technique optimised for real-time molecular detection of Dengue fever virus under ideal as well as non-ideal laboratory conditions using two different detection approaches. The first approach is based on the detection of the hydrodynamic volume of streptavidin coated...... magnetic nanoparticles attached to biotinylated LAMP amplicons. We demonstrate detection of sub-femtomolar Dengue DNA target concentrations in the ideal contamination-free lab environment within 20 min. The second detection approach is based on sequence-specific binding of functionalised magnetic...... claim detection of down to 100 fM of Dengue target after 20 min of LAMP with a contamination background....

  5. Two-stage clustering (TSC: a pipeline for selecting operational taxonomic units for the high-throughput sequencing of PCR amplicons.

    Directory of Open Access Journals (Sweden)

    Xiao-Tao Jiang

    Full Text Available Clustering 16S/18S rRNA amplicon sequences into operational taxonomic units (OTUs is a critical step for the bioinformatic analysis of microbial diversity. Here, we report a pipeline for selecting OTUs with a relatively low computational demand and a high degree of accuracy. This pipeline is referred to as two-stage clustering (TSC because it divides tags into two groups according to their abundance and clusters them sequentially. The more abundant group is clustered using a hierarchical algorithm similar to that in ESPRIT, which has a high degree of accuracy but is computationally costly for large datasets. The rarer group, which includes the majority of tags, is then heuristically clustered to improve efficiency. To further improve the computational efficiency and accuracy, two preclustering steps are implemented. To maintain clustering accuracy, all tags are grouped into an OTU depending on their pairwise Needleman-Wunsch distance. This method not only improved the computational efficiency but also mitigated the spurious OTU estimation from 'noise' sequences. In addition, OTUs clustered using TSC showed comparable or improved performance in beta-diversity comparisons compared to existing OTU selection methods. This study suggests that the distribution of sequencing datasets is a useful property for improving the computational efficiency and increasing the clustering accuracy of the high-throughput sequencing of PCR amplicons. The software and user guide are freely available at http://hwzhoulab.smu.edu.cn/paperdata/.

  6. Ion Torrent sequencing as a tool for mutation discovery in the flax (Linum usitatissimum L.) genome.

    Science.gov (United States)

    Galindo-González, Leonardo; Pinzón-Latorre, David; Bergen, Erik A; Jensen, Dustin C; Deyholos, Michael K

    2015-01-01

    Detection of induced mutations is valuable for inferring gene function and for developing novel germplasm for crop improvement. Many reverse genetics approaches have been developed to identify mutations in genes of interest within a mutagenized population, including some approaches that rely on next-generation sequencing (e.g. exome capture, whole genome resequencing). As an alternative to these genome or exome-scale methods, we sought to develop a scalable and efficient method for detection of induced mutations that could be applied to a small number of target genes, using Ion Torrent technology. We developed this method in flax (Linum usitatissimum), to demonstrate its utility in a crop species. We used an amplicon-based approach in which DNA samples from an ethyl methanesulfonate (EMS)-mutagenized population were pooled and used as template in PCR reactions to amplify a region of each gene of interest. Barcodes were incorporated during PCR, and the pooled amplicons were sequenced using an Ion Torrent PGM. A pilot experiment with known SNPs showed that they could be detected at a frequency > 0.3% within the pools. We then selected eight genes for which we wanted to discover novel mutations, and applied our approach to screen 768 individuals from the EMS population, using either the Ion 314 or Ion 316 chips. Out of 29 potential mutations identified after processing the NGS reads, 16 mutations were confirmed using Sanger sequencing. The methodology presented here demonstrates the utility of Ion Torrent technology in detecting mutation variants in specific genome regions for large populations of a species such as flax. The methodology could be scaled-up to test >100 genes using the higher capacity chips now available from Ion Torrent.

  7. Population-based resequencing revealed an ancestral winter group of cultivated flax: implication for flax domestication processes

    Science.gov (United States)

    Fu, Yong-Bi

    2012-01-01

    Cultivated flax (Linum usitatissimum L.) is the earliest oil and fiber crop and its early domestication history may involve multiple events of domestication for oil, fiber, capsular indehiscence, and winter hardiness. Genetic studies have demonstrated that winter cultivated flax is closely related to oil and fiber cultivated flax and shows little relatedness to its progenitor, pale flax (L. bienne Mill.), but winter hardiness is one major characteristic of pale flax. Here, we assessed the genetic relationships of 48 Linum samples representing pale flax and four trait-specific groups of cultivated flax (dehiscent, fiber, oil, and winter) through population-based resequencing at 24 genomic regions, and revealed a winter group of cultivated flax that displayed close relatedness to the pale flax samples. Overall, the cultivated flax showed a 27% reduction of nucleotide diversity when compared with the pale flax. Recombination frequently occurred at these sampled genomic regions, but the signal of selection and bottleneck was relatively weak. These findings provide some insight into the impact and processes of flax domestication and are significant for expanding our knowledge about early flax domestication, particularly for winter hardiness. PMID:22822439

  8. Microbial community profiling of fresh basil and pitfalls in taxonomic assignment of enterobacterial pathogenic species based upon 16S rRNA amplicon sequencing.

    Science.gov (United States)

    Ceuppens, Siele; De Coninck, Dieter; Bottledoorn, Nadine; Van Nieuwerburgh, Filip; Uyttendaele, Mieke

    2017-09-18

    Application of 16S rRNA (gene) amplicon sequencing on food samples is increasingly applied for assessing microbial diversity but may as unintended advantage also enable simultaneous detection of any human pathogens without a priori definition. In the present study high-throughput next-generation sequencing (NGS) of the V1-V2-V3 regions of the 16S rRNA gene was applied to identify the bacteria present on fresh basil leaves. However, results were strongly impacted by variations in the bioinformatics analysis pipelines (MEGAN, SILVAngs, QIIME and MG-RAST), including the database choice (Greengenes, RDP and M5RNA) and the annotation algorithm (best hit, representative hit and lowest common ancestor). The use of pipelines with default parameters will lead to discrepancies. The estimate of microbial diversity of fresh basil using 16S rRNA (gene) amplicon sequencing is thus indicative but subject to biases. Salmonella enterica was detected at low frequencies, between 0.1% and 0.4% of bacterial sequences, corresponding with 37 to 166 reads. However, this result was dependent upon the pipeline used: Salmonella was detected by MEGAN, SILVAngs and MG-RAST, but not by QIIME. Confirmation of Salmonella sequences by real-time PCR was unsuccessful. It was shown that taxonomic resolution obtained from the short (500bp) sequence reads of the 16S rRNA gene containing the hypervariable regions V1-V3 cannot allow distinction of Salmonella with closely related enterobacterial species. In conclusion 16S amplicon sequencing, getting the status of standard method in microbial ecology studies of foods, needs expertise on both bioinformatics and microbiology for analysis of results. It is a powerful tool to estimate bacterial diversity but amenable to biases. Limitations concerning taxonomic resolution for some bacterial species or its inability to detect sub-dominant (pathogenic) species should be acknowledged in order to avoid overinterpretation of results. Copyright © 2017 Elsevier B

  9. 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. Copyright © 2016 Elsevier B.V. All rights reserved.

  10. 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.

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

    KAUST Repository

    Wang, Yong

    2009-10-09

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

  12. Amplicon sequencing of bacterial microbiota in abortion material from cattle.

    Science.gov (United States)

    Vidal, Sara; Kegler, Kristel; Posthaus, Horst; Perreten, Vincent; Rodriguez-Campos, Sabrina

    2017-10-10

    Abortions in cattle have a significant economic impact on animal husbandry and require prompt diagnosis for surveillance of epizootic infectious agents. Since most abortions are not epizootic but sporadic with often undetected etiologies, this study examined the bacterial community present in the placenta (PL, n = 32) and fetal abomasal content (AC, n = 49) in 64 cases of bovine abortion by next generation sequencing (NGS) of the 16S rRNA gene. The PL and AC from three fetuses of dams that died from non-infectious reasons were included as controls. All samples were analyzed by bacterial culture, and 17 were examined by histopathology. We observed 922 OTUs overall and 267 taxa at the genus level. No detectable bacterial DNA was present in the control samples. The microbial profiles of the PL and AC differed significantly, both in their composition (PERMANOVA), species richness and Chao-1 (Mann-Whitney test). In both organs, Pseudomonas was the most abundant genus. The combination of NGS and culture identified opportunistic pathogens of interest in placentas with lesions, such as Vibrio metschnikovii, Streptococcus uberis, Lactococcus lactis and Escherichia coli. In placentas with lesions where culturing was unsuccessful, Pseudomonas and unidentified Aeromonadaceae were identified by NGS displaying high number of reads. Three cases with multiple possible etiologies and placentas presenting lesions were detected by NGS. Amplicon sequencing has the potential to uncover unknown etiological agents. These new insights on cattle abortion extend our focus to previously understudied opportunistic abortive bacteria.

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

    Science.gov (United States)

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

    2018-01-01

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

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

    DEFF Research Database (Denmark)

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

    have been made to illuminate the effects specifically related to bacterial communities in drinking water. In this study, we investigated the impact of the DNA extraction and primer choice on the observed community structure, and we also estimated the detection limit of the 16S rRNA amplicon sequencing...

  15. Population-Specific Resequencing Associates the ATP-Binding Cassette Subfamily C Member 4 Gene With Gout in New Zealand Māori and Pacific Men.

    Science.gov (United States)

    Tanner, Callum; Boocock, James; Stahl, Eli A; Dobbyn, Amanda; Mandal, Asim K; Cadzow, Murray; Phipps-Green, Amanda J; Topless, Ruth K; Hindmarsh, Jennie Harré; Stamp, Lisa K; Dalbeth, Nicola; Choi, Hyon K; Mount, David B; Merriman, Tony R

    2017-07-01

    There is no evidence for a genetic association between organic anion transporters 1-3 (SLC22A6, SLC22A7, and SLC22A8) and multidrug resistance protein 4 (MRP4; encoded by ABCC4) with the levels of serum urate or gout. The Māori and Pacific (Polynesian) population of New Zealand has the highest prevalence of gout worldwide. The aim of this study was to determine whether any Polynesian population-specific genetic variants in SLC22A6-8 and ABCC4 are associated with gout. All participants had ≥3 self-reported Māori and/or Pacific grandparents. Among the total sample set of 1,808 participants, 191 hyperuricemic and 202 normouricemic individuals were resequenced over the 4 genes, and the remaining 1,415 individuals were used for replication. Regression analyses were performed, adjusting for age, sex, and Polynesian ancestry. To study the functional effect of nonsynonymous variants of ABCC4, transport assays were performed in Xenopus laevis oocytes. A total of 39 common variants were detected, with an ABCC4 variant (rs4148500) significantly associated with hyperuricemia and gout. This variant was monomorphic for the urate-lowering allele in Europeans. There was evidence for an association of rs4148500 with gout in the resequenced samples (odds ratio [OR] 1.62 [P = 0.012]) that was replicated (OR 1.25 [P = 0.033]) and restricted to men (OR 1.43 [P = 0.001] versus OR 0.98 [P = 0.89] in women). The gout risk allele was associated with fractional excretion of uric acid in male individuals (β = -0.570 [P = 0.01]). A rare population-specific allele (P1036L) with predicted strong functional consequence reduced the uric acid transport activity of ABCC4 by 30%. An association between ABCC4 and gout and fractional excretion of uric acid is consistent with the established role of MRP4 as a unidirectional renal uric acid efflux pump. © 2017, American College of Rheumatology.

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

    Science.gov (United States)

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

    2018-02-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2018-01-06

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

  18. Canary: an atomic pipeline for clinical amplicon assays.

    Science.gov (United States)

    Doig, Kenneth D; Ellul, Jason; Fellowes, Andrew; Thompson, Ella R; Ryland, Georgina; Blombery, Piers; Papenfuss, Anthony T; Fox, Stephen B

    2017-12-15

    High throughput sequencing requires bioinformatics pipelines to process large volumes of data into meaningful variants that can be translated into a clinical report. These pipelines often suffer from a number of shortcomings: they lack robustness and have many components written in multiple languages, each with a variety of resource requirements. Pipeline components must be linked together with a workflow system to achieve the processing of FASTQ files through to a VCF file of variants. Crafting these pipelines requires considerable bioinformatics and IT skills beyond the reach of many clinical laboratories. Here we present Canary, a single program that can be run on a laptop, which takes FASTQ files from amplicon assays through to an annotated VCF file ready for clinical analysis. Canary can be installed and run with a single command using Docker containerization or run as a single JAR file on a wide range of platforms. Although it is a single utility, Canary performs all the functions present in more complex and unwieldy pipelines. All variants identified by Canary are 3' shifted and represented in their most parsimonious form to provide a consistent nomenclature, irrespective of sequencing variation. Further, proximate in-phase variants are represented as a single HGVS 'delins' variant. This allows for correct nomenclature and consequences to be ascribed to complex multi-nucleotide polymorphisms (MNPs), which are otherwise difficult to represent and interpret. Variants can also be annotated with hundreds of attributes sourced from MyVariant.info to give up to date details on pathogenicity, population statistics and in-silico predictors. Canary has been used at the Peter MacCallum Cancer Centre in Melbourne for the last 2 years for the processing of clinical sequencing data. By encapsulating clinical features in a single, easily installed executable, Canary makes sequencing more accessible to all pathology laboratories. Canary is available for download as source

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

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    Motoi Nishimura

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

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

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    Clive J Hoggart

    2008-07-01

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

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

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    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.

  2. 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.

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

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    Alvaro Martinez Barrio

    2009-07-01

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

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

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    Younhee Shin

    2016-09-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Directory of Open Access Journals (Sweden)

    J. Björkman

    2016-01-01

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

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

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    Ying-hui Li

    2014-11-01

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

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

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

    2015-10-01

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

  9. Prerequisites for Amplicon Pyrosequencing of Microbial Methanol Utilizers in the Environment

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    Steffen eKolb

    2013-09-01

    Full Text Available The commercial availability of next generation sequencing (NGS technologies facilitated the assessment of functional groups of microorganisms in the environment with high coverage, resolution, and reproducibility. Soil methylotrophs were among the first microorganisms in the environment that were assessed with molecular tools, and nowadays, as well with NGS technologies. Studies in the past years re-attracted notice to the pivotal role of methylotrophs in global conversions of methanol, which mainly originates from plants, and is involved in oxidative reactions and ozone formation in the atmosphere. Aerobic methanol utilizers belong to Bacteria, yeasts, Ascomycota, and molds. Numerous bacterial methylotrophs are facultatively aerobic, and also contribute to anaerobic methanol oxidation in the environment, whereas strict anaerobic methanol utilizers belong to methanogens and acetogens. The diversity of enzymes catalyzing the initial oxidation of methanol is considerable, and comprises at least five different enzyme types in aerobes, and one in strict anaerobes. Only the gene of the large subunit of PQQ-dependent methanol dehydrogenase (mxaF has been analyzed by environmental pyrosequencing. To enable a comprehensive assessment of methanol utilizers in the environment, new primers targeting genes of the PQQ MDH in Methylibium (mdh2, of the NAD-dependent MDH (mdh, of the methanol oxidoreductase of Actinobacteria (mdo, of the fungal FAD-dependent alcohol oxidase (mod1, mod2, and homologues, and of the gene of the large subunit of the methanol:corrinoid methyltransferases (mtaC in methanogens and acetogens need to be developed. Combined stable isotope probing of nucleic acids or proteins with amplicon-based NGS are straightforward approaches to reveal insights into functions of certain methylotrophic taxa in the global methanol cycle.

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

    Science.gov (United States)

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

    2011-01-01

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

  11. High-throughput interpretation of gene structure changes in human and nonhuman resequencing data, using ACE.

    Science.gov (United States)

    Majoros, William H; Campbell, Michael S; Holt, Carson; DeNardo, Erin K; Ware, Doreen; Allen, Andrew S; Yandell, Mark; Reddy, Timothy E

    2017-05-15

    The accurate interpretation of genetic variants is critical for characterizing genotype-phenotype associations. Because the effects of genetic variants can depend strongly on their local genomic context, accurate genome annotations are essential. Furthermore, as some variants have the potential to disrupt or alter gene structure, variant interpretation efforts stand to gain from the use of individualized annotations that account for differences in gene structure between individuals or strains. We describe a suite of software tools for identifying possible functional changes in gene structure that may result from sequence variants. ACE ('Assessing Changes to Exons') converts phased genotype calls to a collection of explicit haplotype sequences, maps transcript annotations onto them, detects gene-structure changes and their possible repercussions, and identifies several classes of possible loss of function. Novel transcripts predicted by ACE are commonly supported by spliced RNA-seq reads, and can be used to improve read alignment and transcript quantification when an individual-specific genome sequence is available. Using publicly available RNA-seq data, we show that ACE predictions confirm earlier results regarding the quantitative effects of nonsense-mediated decay, and we show that predicted loss-of-function events are highly concordant with patterns of intolerance to mutations across the human population. ACE can be readily applied to diverse species including animals and plants, making it a broadly useful tool for use in eukaryotic population-based resequencing projects, particularly for assessing the joint impact of all variants at a locus. ACE is written in open-source C ++ and Perl and is available from geneprediction.org/ACE. myandell@genetics.utah.edu or tim.reddy@duke.edu. Supplementary information is available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e

  12. 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.

  13. Imaging Herpes Simplex Virus Type 1 Amplicon Vector–Mediated Gene Expression in Human Glioma Spheroids

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    Christine Kaestle

    2011-05-01

    Full Text Available Vectors derived from herpes simplex virus type 1 (HSV-1 have great potential for transducing therapeutic genes into the central nervous system; however, inefficient distribution of vector particles in vivo may limit their therapeutic potential in patients with gliomas. This study was performed to investigate the extent of HSV-1 amplicon vector–mediated gene expression in a three-dimensional glioma model of multicellular spheroids by imaging highly infectious HSV-1 virions expressing green fluorescent protein (HSV-GFP. After infection or microscopy-guided vector injection of glioma spheroids at various spheroid sizes, injection pressures and injection times, the extent of HSV-1 vector–mediated gene expression was investigated via laser scanning microscopy. Infection of spheroids with HSV-GFP demonstrated a maximal depth of vector-mediated GFP expression at 70 to 80 μm. A > 80% transduction efficiency was reached only in small spheroids with a diameter of 90%. The results demonstrated that vector-mediated gene expression in glioma spheroids was strongly dependent on the mode of vector application—injection pressure and injection time being the most important parameters. The assessment of these vector application parameters in tissue models will contribute to the development of safe and efficient gene therapy protocols for clinical application.

  14. PipeCraft: Flexible open-source toolkit for bioinformatics analysis of custom high-throughput amplicon sequencing data.

    Science.gov (United States)

    Anslan, Sten; Bahram, Mohammad; Hiiesalu, Indrek; Tedersoo, Leho

    2017-11-01

    High-throughput sequencing methods have become a routine analysis tool in environmental sciences as well as in public and private sector. These methods provide vast amount of data, which need to be analysed in several steps. Although the bioinformatics may be applied using several public tools, many analytical pipelines allow too few options for the optimal analysis for more complicated or customized designs. Here, we introduce PipeCraft, a flexible and handy bioinformatics pipeline with a user-friendly graphical interface that links several public tools for analysing amplicon sequencing data. Users are able to customize the pipeline by selecting the most suitable tools and options to process raw sequences from Illumina, Pacific Biosciences, Ion Torrent and Roche 454 sequencing platforms. We described the design and options of PipeCraft and evaluated its performance by analysing the data sets from three different sequencing platforms. We demonstrated that PipeCraft is able to process large data sets within 24 hr. The graphical user interface and the automated links between various bioinformatics tools enable easy customization of the workflow. All analytical steps and options are recorded in log files and are easily traceable. © 2017 John Wiley & Sons Ltd.

  15. 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…

  16. Evolution of MHC class I genes in the endangered loggerhead sea turtle (Caretta caretta) revealed by 454 amplicon sequencing.

    Science.gov (United States)

    Stiebens, Victor A; Merino, Sonia E; Chain, Frédéric J J; Eizaguirre, Christophe

    2013-04-30

    In evolutionary and conservation biology, parasitism is often highlighted as a major selective pressure. To fight against parasites and pathogens, genetic diversity of the immune genes of the major histocompatibility complex (MHC) are particularly important. However, the extensive degree of polymorphism observed in these genes makes it difficult to conduct thorough population screenings. We utilized a genotyping protocol that uses 454 amplicon sequencing to characterize the MHC class I in the endangered loggerhead sea turtle (Caretta caretta) and to investigate their evolution at multiple relevant levels of organization. MHC class I genes revealed signatures of trans-species polymorphism across several reptile species. In the studied loggerhead turtle individuals, it results in the maintenance of two ancient allelic lineages. We also found that individuals carrying an intermediate number of MHC class I alleles are larger than those with either a low or high number of alleles. Multiple modes of evolution seem to maintain MHC diversity in the loggerhead turtles, with relatively high polymorphism for an endangered species.

  17. 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.

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

    Science.gov (United States)

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

    2010-03-01

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

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

    Science.gov (United States)

    Gulino, Lisa-Maree; Ouwerkerk, Diane; Kang, Alicia Y H; 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). 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.

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

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    Kirsten E McKay

    2013-03-01

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

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

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    Planý Matej

    2016-06-01

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

  2. Identification and Evaluation of Single-Nucleotide Polymorphisms in Allotetraploid Peanut (Arachis hypogaea L.) Based on Amplicon Sequencing Combined with High Resolution Melting (HRM) Analysis.

    Science.gov (United States)

    Hong, Yanbin; Pandey, Manish K; Liu, Ying; Chen, Xiaoping; Liu, Hong; Varshney, Rajeev K; Liang, Xuanqiang; Huang, Shangzhi

    2015-01-01

    The cultivated peanut (Arachis hypogaea L.) is an allotetraploid (AABB) species derived from the A-genome (Arachis duranensis) and B-genome (Arachis ipaensis) progenitors. Presence of two versions of a DNA sequence based on the two progenitor genomes poses a serious technical and analytical problem during single nucleotide polymorphism (SNP) marker identification and analysis. In this context, we have analyzed 200 amplicons derived from expressed sequence tags (ESTs) and genome survey sequences (GSS) to identify SNPs in a panel of genotypes consisting of 12 cultivated peanut varieties and two diploid progenitors representing the ancestral genomes. A total of 18 EST-SNPs and 44 genomic-SNPs were identified in 12 peanut varieties by aligning the sequence of A. hypogaea with diploid progenitors. The average frequency of sequence polymorphism was higher for genomic-SNPs than the EST-SNPs with one genomic-SNP every 1011 bp as compared to one EST-SNP every 2557 bp. In order to estimate the potential and further applicability of these identified SNPs, 96 peanut varieties were genotyped using high resolution melting (HRM) method. Polymorphism information content (PIC) values for EST-SNPs ranged between 0.021 and 0.413 with a mean of 0.172 in the set of peanut varieties, while genomic-SNPs ranged between 0.080 and 0.478 with a mean of 0.249. Total 33 SNPs were used for polymorphism detection among the parents and 10 selected lines from mapping population Y13Zh (Zhenzhuhei × Yueyou13). Of the total 33 SNPs, nine SNPs showed polymorphism in the mapping population Y13Zh, and seven SNPs were successfully mapped into five linkage groups. Our results showed that SNPs can be identified in allotetraploid peanut with high accuracy through amplicon sequencing and HRM assay. The identified SNPs were very informative and can be used for different genetic and breeding applications in peanut.

  3. 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.

  4. Genomewide variation in an introgression line of rice-Zizania revealed by whole-genome re-sequencing.

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    Zhen-Hui Wang

    Full Text Available BACKGROUND: Hybridization between genetically diverged organisms is known as an important avenue that drives plant genome evolution. The possible outcomes of hybridization would be the occurrences of genetic instabilities in the resultant hybrids. It remained under-investigated however whether pollination by alien pollens of a closely related but sexually "incompatible" species could evoke genomic changes and to what extent it may result in phenotypic novelties in the derived progenies. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we have re-sequenced the genomes of Oryza sativa ssp. japonica cv. Matsumae and one of its derived introgressant RZ35 that was obtained from an introgressive hybridization between Matsumae and Zizanialatifolia Griseb. in general, 131 millions 90 base pair (bp paired-end reads were generated which covered 13.2 and 21.9 folds of the Matsumae and RZ35 genomes, respectively. Relative to Matsumae, a total of 41,724 homozygous single nucleotide polymorphisms (SNPs and 17,839 homozygous insertions/deletions (indels were identified in RZ35, of which 3,797 SNPs were nonsynonymous mutations. Furthermore, rampant mobilization of transposable elements (TEs was found in the RZ35 genome. The results of pathogen inoculation revealed that RZ35 exhibited enhanced resistance to blast relative to Matsumae. Notably, one nonsynonymous mutation was found in the known blast resistance gene Pid3/Pi25 and real-time quantitative (q RT-PCR analysis revealed constitutive up-regulation of its expression, suggesting both altered function and expression of Pid3/Pi25 may be responsible for the enhanced resistance to rice blast by RZ35. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate that introgressive hybridization by Zizania has provoked genomewide, extensive genomic changes in the rice genome, and some of which have resulted in important phenotypic novelties. These findings suggest that introgressive hybridization by alien pollens of even a

  5. RelocaTE2: a high resolution transposable element insertion site mapping tool for population resequencing

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    Jinfeng Chen

    2017-01-01

    Full Text Available Background Transposable element (TE polymorphisms are important components of population genetic variation. The functional impacts of TEs in gene regulation and generating genetic diversity have been observed in multiple species, but the frequency and magnitude of TE variation is under appreciated. Inexpensive and deep sequencing technology has made it affordable to apply population genetic methods to whole genomes with methods that identify single nucleotide and insertion/deletion polymorphisms. However, identifying TE polymorphisms, particularly transposition events or non-reference insertion sites can be challenging due to the repetitive nature of these sequences, which hamper both the sensitivity and specificity of analysis tools. Methods We have developed the tool RelocaTE2 for identification of TE insertion sites at high sensitivity and specificity. RelocaTE2 searches for known TE sequences in whole genome sequencing reads from second generation sequencing platforms such as Illumina. These sequence reads are used as seeds to pinpoint chromosome locations where TEs have transposed. RelocaTE2 detects target site duplication (TSD of TE insertions allowing it to report TE polymorphism loci with single base pair precision. Results and Discussion The performance of RelocaTE2 is evaluated using both simulated and real sequence data. RelocaTE2 demonstrate high level of sensitivity and specificity, particularly when the sequence coverage is not shallow. In comparison to other tools tested, RelocaTE2 achieves the best balance between sensitivity and specificity. In particular, RelocaTE2 performs best in prediction of TSDs for TE insertions. Even in highly repetitive regions, such as those tested on rice chromosome 4, RelocaTE2 is able to report up to 95% of simulated TE insertions with less than 0.1% false positive rate using 10-fold genome coverage resequencing data. RelocaTE2 provides a robust solution to identify TE insertion sites and can be

  6. A New Single Nucleotide Polymorphism Database for Rainbow Trout Generated Through Whole Genome Resequencing

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    Guangtu Gao

    2018-04-01

    Full Text Available Single-nucleotide polymorphisms (SNPs are highly abundant markers, which are broadly distributed in animal genomes. For rainbow trout (Oncorhynchus mykiss, SNP discovery has been previously done through sequencing of restriction-site associated DNA (RAD libraries, reduced representation libraries (RRL and RNA sequencing. Recently we have performed high coverage whole genome resequencing with 61 unrelated samples, representing a wide range of rainbow trout and steelhead populations, with 49 new samples added to 12 aquaculture samples from AquaGen (Norway that we previously used for SNP discovery. Of the 49 new samples, 11 were double-haploid lines from Washington State University (WSU and 38 represented wild and hatchery populations from a wide range of geographic distribution and with divergent migratory phenotypes. We then mapped the sequences to the new rainbow trout reference genome assembly (GCA_002163495.1 which is based on the Swanson YY doubled haploid line. Variant calling was conducted with FreeBayes and SAMtools mpileup, followed by filtering of SNPs based on quality score, sequence complexity, read depth on the locus, and number of genotyped samples. Results from the two variant calling programs were compared and genotypes of the double haploid samples were used for detecting and filtering putative paralogous sequence variants (PSVs and multi-sequence variants (MSVs. Overall, 30,302,087 SNPs were identified on the rainbow trout genome 29 chromosomes and 1,139,018 on unplaced scaffolds, with 4,042,723 SNPs having high minor allele frequency (MAF > 0.25. The average SNP density on the chromosomes was one SNP per 64 bp, or 15.6 SNPs per 1 kb. Results from the phylogenetic analysis that we conducted indicate that the SNP markers contain enough population-specific polymorphisms for recovering population relationships despite the small sample size used. Intra-Population polymorphism assessment revealed high level of polymorphism and

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

    Science.gov (United States)

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

    2017-07-01

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

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

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    Pravin Dudhagara

    2015-06-01

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

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

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    Etienne Patin

    2009-04-01

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

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

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    Carr Steven M

    2007-09-01

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

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

    Science.gov (United States)

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

    2018-01-01

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

  12. 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

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

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    Xinghai Yang

    2017-09-01

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

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

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    Nedenia Bonvino Stafuzza

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

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

    Science.gov (United States)

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

    2012-12-01

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

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

    Directory of Open Access Journals (Sweden)

    Yongle Li

    2018-02-01

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

  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.

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

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    Kristensen Lasse

    2012-11-01

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

  19. Targeted Resequencing and Functional Testing Identifies Low-Frequency Missense Variants in the Gene Encoding GARP as Significant Contributors to Atopic Dermatitis Risk.

    Science.gov (United States)

    Manz, Judith; Rodríguez, Elke; ElSharawy, Abdou; Oesau, Eva-Maria; Petersen, Britt-Sabina; Baurecht, Hansjörg; Mayr, Gabriele; Weber, Susanne; Harder, Jürgen; Reischl, Eva; Schwarz, Agatha; Novak, Natalija; Franke, Andre; Weidinger, Stephan

    2016-12-01

    Gene-mapping studies have consistently identified a susceptibility locus for atopic dermatitis and other inflammatory diseases on chromosome band 11q13.5, with the strongest association observed for a common variant located in an intergenic region between the two annotated genes C11orf30 and LRRC32. Using a targeted resequencing approach we identified low-frequency and rare missense mutations within the LRRC32 gene encoding the protein GARP, a receptor on activated regulatory T cells that binds latent transforming growth factor-β. Subsequent association testing in more than 2,000 atopic dermatitis patients and 2,000 control subjects showed a significant excess of these LRRC32 variants in individuals with atopic dermatitis. Structural protein modeling and bioinformatic analysis predicted a disruption of protein transport upon these variants, and overexpression assays in CD4 + CD25 - T cells showed a significant reduction in surface expression of the mutated protein. Consistently, flow cytometric (FACS) analyses of different T-cell subtypes obtained from atopic dermatitis patients showed a significantly reduced surface expression of GARP and a reduced conversion of CD4 + CD25 - T cells into regulatory T cells, along with lower expression of latency-associated protein upon stimulation in carriers of the LRRC32 A407T variant. These results link inherited disturbances of transforming growth factor-β signaling with atopic dermatitis risk. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  20. 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.

  1. Genome-wide SNP identification in multiple morphotypes of allohexaploid tall fescue (Festuca arundinacea Schreb

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    Hand Melanie L

    2012-06-01

    Full Text Available Abstract Background Single nucleotide polymorphisms (SNPs provide essential tools for the advancement of research in plant genomics, and the development of SNP resources for many species has been accelerated by the capabilities of second-generation sequencing technologies. The current study aimed to develop and use a novel bioinformatic pipeline to generate a comprehensive collection of SNP markers within the agriculturally important pasture grass tall fescue; an outbreeding allopolyploid species displaying three distinct morphotypes: Continental, Mediterranean and rhizomatous. Results A bioinformatic pipeline was developed that successfully identified SNPs within genotypes from distinct tall fescue morphotypes, following the sequencing of 414 polymerase chain reaction (PCR – generated amplicons using 454 GS FLX technology. Equivalent amplicon sets were derived from representative genotypes of each morphotype, including six Continental, five Mediterranean and one rhizomatous. A total of 8,584 and 2,292 SNPs were identified with high confidence within the Continental and Mediterranean morphotypes respectively. The success of the bioinformatic approach was demonstrated through validation (at a rate of 70% of a subset of 141 SNPs using both SNaPshot™ and GoldenGate™ assay chemistries. Furthermore, the quantitative genotyping capability of the GoldenGate™ assay revealed that approximately 30% of the putative SNPs were accessible to co-dominant scoring, despite the hexaploid genome structure. The sub-genome-specific origin of each SNP validated from Continental tall fescue was predicted using a phylogenetic approach based on comparison with orthologous sequences from predicted progenitor species. Conclusions Using the appropriate bioinformatic approach, amplicon resequencing based on 454 GS FLX technology is an effective method for the identification of polymorphic SNPs within the genomes of Continental and Mediterranean tall fescue. The

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

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    M. Michelle Malmberg

    2018-04-01

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

  3. 16S rRNA Amplicon Sequencing Demonstrates that Indoor-Reared Bumblebees (Bombus terrestris Harbor a Core Subset of Bacteria Normally Associated with the Wild Host.

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    Ivan Meeus

    Full Text Available A MiSeq multiplexed 16S rRNA amplicon sequencing of the gut microbiota of wild and indoor-reared Bombus terrestris (bumblebees confirmed the presence of a core set of bacteria, which consisted of Neisseriaceae (Snodgrassella, Orbaceae (Gilliamella, Lactobacillaceae (Lactobacillus, and Bifidobacteriaceae (Bifidobacterium. In wild B. terrestris we detected several non-core bacteria having a more variable prevalence. Although Enterobacteriaceae are unreported by non next-generation sequencing studies, it can become a dominant gut resident. Furthermore the presence of some non-core lactobacilli were associated with the relative abundance of bifidobacteria. This association was not observed in indoor-reared bumblebees lacking the non-core bacteria, but having a more standardized microbiota compared to their wild counterparts. The impact of the bottleneck microbiota of indoor-reared bumblebees when they are used in the field for pollination purpose is discussed.

  4. Rapid detection of SMARCB1 sequence variation using high resolution melting

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    Ashley David M

    2009-12-01

    Full Text Available Abstract Background Rhabdoid tumors are rare cancers of early childhood arising in the kidney, central nervous system and other organs. The majority are caused by somatic inactivating mutations or deletions affecting the tumor suppressor locus SMARCB1 [OMIM 601607]. Germ-line SMARCB1 inactivation has been reported in association with rhabdoid tumor, epitheloid sarcoma and familial schwannomatosis, underscoring the importance of accurate mutation screening to ascertain recurrence and transmission risks. We describe a rapid and sensitive diagnostic screening method, using high resolution melting (HRM, for detecting sequence variations in SMARCB1. Methods Amplicons, encompassing the nine coding exons of SMARCB1, flanking splice site sequences and the 5' and 3' UTR, were screened by both HRM and direct DNA sequencing to establish the reliability of HRM as a primary mutation screening tool. Reaction conditions were optimized with commercially available HRM mixes. Results The false negative rate for detecting sequence variants by HRM in our sample series was zero. Nine amplicons out of a total of 140 (6.4% showed variant melt profiles that were subsequently shown to be false positive. Overall nine distinct pathogenic SMARCB1 mutations were identified in a total of 19 possible rhabdoid tumors. Two tumors had two distinct mutations and two harbored SMARCB1 deletion. Other mutations were nonsense or frame-shifts. The detection sensitivity of the HRM screening method was influenced by both sequence context and specific nucleotide change and varied from 1: 4 to 1:1000 (variant to wild-type DNA. A novel method involving digital HRM, followed by re-sequencing, was used to confirm mutations in tumor specimens containing associated normal tissue. Conclusions This is the first report describing SMARCB1 mutation screening using HRM. HRM is a rapid, sensitive and inexpensive screening technology that is likely to be widely adopted in diagnostic laboratories to

  5. Rapid detection of SMARCB1 sequence variation using high resolution melting

    International Nuclear Information System (INIS)

    Dagar, Vinod; Chow, Chung-Wo; Ashley, David M; Algar, Elizabeth M

    2009-01-01

    Rhabdoid tumors are rare cancers of early childhood arising in the kidney, central nervous system and other organs. The majority are caused by somatic inactivating mutations or deletions affecting the tumor suppressor locus SMARCB1 [OMIM 601607]. Germ-line SMARCB1 inactivation has been reported in association with rhabdoid tumor, epitheloid sarcoma and familial schwannomatosis, underscoring the importance of accurate mutation screening to ascertain recurrence and transmission risks. We describe a rapid and sensitive diagnostic screening method, using high resolution melting (HRM), for detecting sequence variations in SMARCB1. Amplicons, encompassing the nine coding exons of SMARCB1, flanking splice site sequences and the 5' and 3' UTR, were screened by both HRM and direct DNA sequencing to establish the reliability of HRM as a primary mutation screening tool. Reaction conditions were optimized with commercially available HRM mixes. The false negative rate for detecting sequence variants by HRM in our sample series was zero. Nine amplicons out of a total of 140 (6.4%) showed variant melt profiles that were subsequently shown to be false positive. Overall nine distinct pathogenic SMARCB1 mutations were identified in a total of 19 possible rhabdoid tumors. Two tumors had two distinct mutations and two harbored SMARCB1 deletion. Other mutations were nonsense or frame-shifts. The detection sensitivity of the HRM screening method was influenced by both sequence context and specific nucleotide change and varied from 1: 4 to 1:1000 (variant to wild-type DNA). A novel method involving digital HRM, followed by re-sequencing, was used to confirm mutations in tumor specimens containing associated normal tissue. This is the first report describing SMARCB1 mutation screening using HRM. HRM is a rapid, sensitive and inexpensive screening technology that is likely to be widely adopted in diagnostic laboratories to facilitate whole gene mutation screening

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

    Science.gov (United States)

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

    2018-02-01

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

  7. Two populations of double minute chromosomes harbor distinct amplicons, the MYC locus at 8q24.2 and a 0.43-Mb region at 14q24.1, in the SW613-S human carcinoma cell line.

    Science.gov (United States)

    Guillaud-Bataille, M; Brison, O; Danglot, G; Lavialle, C; Raynal, B; Lazar, V; Dessen, P; Bernheim, A

    2009-01-01

    High-level amplifications observed in tumor cells are usually indicative of genes involved in oncogenesis. We report here a high resolution characterization of a new amplified region in the SW613-S carcinoma cell line. This cell line contains tumorigenic cells displaying high-level MYC amplification in the form of double minutes (DM(+) cells) and non tumorigenic cells exhibiting low-level MYC amplification in the form of homogeneously staining regions (DM(-) cells). Both cell types were studied at genomic and functional levels. The DM(+) cells display a second amplification, corresponding to the 14q24.1 region, in a distinct population of DMs. The 0.43-Mb amplified and overexpressed region contains the PLEK2, PIGH, ARG2, VTI1B, RDH11, and ZFYVE26 genes. Both amplicons were stably maintained upon in vitro and in vivo propagation. However, the 14q24.1 amplicon was not found in cells with high-level MYC amplification in the form of HSRs, either obtained after spontaneous integration of endogenous DM MYC copies or after transfection of DM(-) cells with a MYC gene expression vector. These HSR-bearing cells are highly tumorigenic. The 14q24.1 amplification may not play a role in malignancy per se but might contribute to maintaining the amplification in the form of DMs. Copyright 2009 S. Karger AG, Basel.

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

    Science.gov (United States)

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

    2017-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Ilias Lagkouvardos

    2017-01-01

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

  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. 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.

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

    Science.gov (United States)

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

    2015-01-01

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

  13. 75 FR 28032 - National Heart, Lung, and Blood Institute; Notice of Closed Meetings

    Science.gov (United States)

    2010-05-19

    ... Institute Special Emphasis Panel, NHLBI DNA Resequencing and Genotyping (RS&G) Service: Administrative..., NHLBI DNA Resequencing and Genotyping (RS&G) Service: Laboratory Center(s). Date: June 4, 2010. Time: 2... Nos. 93.233, National Center for Sleep Disorders Research; 93.837, Heart and Vascular Diseases...

  14. 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

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

    Directory of Open Access Journals (Sweden)

    Christopher Ryan Penton

    2016-06-01

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

  16. TIA: algorithms for development of identity-linked SNP islands for analysis by massively parallel DNA sequencing.

    Science.gov (United States)

    Farris, M Heath; Scott, Andrew R; Texter, Pamela A; Bartlett, Marta; Coleman, Patricia; Masters, David

    2018-04-11

    Single nucleotide polymorphisms (SNPs) located within the human genome have been shown to have utility as markers of identity in the differentiation of DNA from individual contributors. Massively parallel DNA sequencing (MPS) technologies and human genome SNP databases allow for the design of suites of identity-linked target regions, amenable to sequencing in a multiplexed and massively parallel manner. Therefore, tools are needed for leveraging the genotypic information found within SNP databases for the discovery of genomic targets that can be evaluated on MPS platforms. The SNP island target identification algorithm (TIA) was developed as a user-tunable system to leverage SNP information within databases. Using data within the 1000 Genomes Project SNP database, human genome regions were identified that contain globally ubiquitous identity-linked SNPs and that were responsive to targeted resequencing on MPS platforms. Algorithmic filters were used to exclude target regions that did not conform to user-tunable SNP island target characteristics. To validate the accuracy of TIA for discovering these identity-linked SNP islands within the human genome, SNP island target regions were amplified from 70 contributor genomic DNA samples using the polymerase chain reaction. Multiplexed amplicons were sequenced using the Illumina MiSeq platform, and the resulting sequences were analyzed for SNP variations. 166 putative identity-linked SNPs were targeted in the identified genomic regions. Of the 309 SNPs that provided discerning power across individual SNP profiles, 74 previously undefined SNPs were identified during evaluation of targets from individual genomes. Overall, DNA samples of 70 individuals were uniquely identified using a subset of the suite of identity-linked SNP islands. TIA offers a tunable genome search tool for the discovery of targeted genomic regions that are scalable in the population frequency and numbers of SNPs contained within the SNP island regions

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

    Science.gov (United States)

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

    2017-08-15

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

  18. 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.

  19. 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.

    Science.gov (United States)

    Luo, Huaiyong; Wang, Xiaojie; Zhan, Gangming; Wei, Guorong; Zhou, Xinli; Zhao, Jing; Huang, Lili; Kang, Zhensheng

    2015-01-01

    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.

  20. Contribution of FKBP5 genetic variation to gemcitabine treatment and survival in pancreatic adenocarcinoma.

    Directory of Open Access Journals (Sweden)

    Katarzyna A Ellsworth

    Full Text Available FKBP51, (FKBP5, is a negative regulator of Akt. Variability in FKBP5 expression level is a major factor contributing to variation in response to chemotherapeutic agents including gemcitabine, a first line treatment for pancreatic cancer. Genetic variation in FKBP5 could influence its function and, ultimately, treatment response of pancreatic cancer.We set out to comprehensively study the role of genetic variation in FKBP5 identified by Next Generation DNA resequencing on response to gemcitabine treatment of pancreatic cancer by utilizing both tumor and germline DNA samples from 43 pancreatic cancer patients, including 19 paired normal-tumor samples. Next, genotype-phenotype association studies were performed with overall survival as well as with FKBP5 gene expression in tumor using the same samples in which resequencing had been performed, followed by functional genomics studies.In-depth resequencing identified 404 FKBP5 single nucleotide polymorphisms (SNPs in normal and tumor DNA. SNPs with the strongest associations with survival or FKBP5 expression were subjected to functional genomic study. Electromobility shift assay showed that the rs73748206 "A(T" SNP altered DNA-protein binding patterns, consistent with significantly increased reporter gene activity, possibly through its increased binding to Glucocorticoid Receptor (GR. The effect of rs73748206 was confirmed on the basis of its association with FKBP5 expression by affecting the binding to GR in lymphoblastoid cell lines derived from the same patients for whom DNA was used for resequencing.This comprehensive FKBP5 resequencing study provides insights into the role of genetic variation in variation of gemcitabine response.

  1. A new restriction endonuclease-based method for highly-specific detection of DNA targets from methicillin-resistant Staphylococcus aureus.

    Directory of Open Access Journals (Sweden)

    Maria W Smith

    Full Text Available PCR multiplexing has proven to be challenging, and thus has provided limited means for pathogen genotyping. We developed a new approach for analysis of PCR amplicons based on restriction endonuclease digestion. The first stage of the restriction enzyme assay is hybridization of a target DNA to immobilized complementary oligonucleotide probes that carry a molecular marker, horseradish peroxidase (HRP. At the second stage, a target-specific restriction enzyme is added, cleaving the target-probe duplex at the corresponding restriction site and releasing the HRP marker into solution, where it is quantified colorimetrically. The assay was tested for detection of the methicillin-resistant Staphylococcus aureus (MRSA pathogen, using the mecA gene as a target. Calibration curves indicated that the limit of detection for both target oligonucleotide and PCR amplicon was approximately 1 nM. Sequences of target oligonucleotides were altered to demonstrate that (i any mutation of the restriction site reduced the signal to zero; (ii double and triple point mutations of sequences flanking the restriction site reduced restriction to 50-80% of the positive control; and (iii a minimum of a 16-bp target-probe dsDNA hybrid was required for significant cleavage. Further experiments showed that the assay could detect the mecA amplicon from an unpurified PCR mixture with detection limits similar to those with standard fluorescence-based qPCR. Furthermore, addition of a large excess of heterologous genomic DNA did not affect amplicon detection. Specificity of the assay is very high because it involves two biorecognition steps. The proposed assay is low-cost and can be completed in less than 1 hour. Thus, we have demonstrated an efficient new approach for pathogen detection and amplicon genotyping in conjunction with various end-point and qPCR applications. The restriction enzyme assay may also be used for parallel analysis of multiple different amplicons from the same

  2. Bacterial diversity of the Colombian fermented milk "Suero Costeño" assessed by culturing and high-throughput sequencing and DGGE analysis of 16S rRNA gene amplicons.

    Science.gov (United States)

    Motato, Karina Edith; Milani, Christian; Ventura, Marco; Valencia, Francia Elena; Ruas-Madiedo, Patricia; Delgado, Susana

    2017-12-01

    "Suero Costeño" (SC) is a traditional soured cream elaborated from raw milk in the Northern-Caribbean coast of Colombia. The natural microbiota that characterizes this popular Colombian fermented milk is unknown, although several culturing studies have previously been attempted. In this work, the microbiota associated with SC from three manufacturers in two regions, "Planeta Rica" (Córdoba) and "Caucasia" (Antioquia), was analysed by means of culturing methods in combination with high-throughput sequencing and DGGE analysis of 16S rRNA gene amplicons. The bacterial ecosystem of SC samples was revealed to be composed of lactic acid bacteria belonging to the Streptococcaceae and Lactobacillaceae families; the proportions and genera varying among manufacturers and region of elaboration. Members of the Lactobacillus acidophilus group, Lactocococcus lactis, Streptococcus infantarius and Streptococcus salivarius characterized this artisanal product. In comparison with culturing, the use of molecular in deep culture-independent techniques provides a more realistic picture of the overall bacterial communities residing in SC. Besides the descriptive purpose, these approaches will facilitate a rational strategy to follow (culture media and growing conditions) for the isolation of indigenous strains that allow standardization in the manufacture of SC. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    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

  4. Polymorphism’s assessment of children’s candidate genes associated with low-level long-term exposure to strontium in drinking water

    OpenAIRE

    N.V. Zaitseva; O.V. Dolgilh; A.V. Krivtsov; K.G. Starkova; V.A. Luchnikova; O.A. Bubnov; E.A. Otavina; N.V. Bezruchenko; N.A. Vdovina

    2015-01-01

    A sequencing of the candidate genes of the pupils, exposed to strontium by the method of targeted resequencing has been performed. It is shown, that under conditions of increased revenues of strontium in drinking water the number of polymorphonuclear altered portions of candidate genes increases. As a result of the targeted resequencing in conditions of strontium exposure, the maximum polymorph modifications of the following genes are defined: sulfotransferase 1A1 (SULT1A1) and methylenetetra...

  5. Microfluidic PCR Amplification and MiSeq Amplicon Sequencing Techniques for High-Throughput Detection and Genotyping of Human Pathogenic RNA Viruses in Human Feces, Sewage, and Oysters

    Directory of Open Access Journals (Sweden)

    Mamoru Oshiki

    2018-04-01

    Full Text Available Detection and genotyping of pathogenic RNA viruses in human and environmental samples are useful for monitoring the circulation and prevalence of these pathogens, whereas a conventional PCR assay followed by Sanger sequencing is time-consuming and laborious. The present study aimed to develop a high-throughput detection-and-genotyping tool for 11 human RNA viruses [Aichi virus; astrovirus; enterovirus; norovirus genogroup I (GI, GII, and GIV; hepatitis A virus; hepatitis E virus; rotavirus; sapovirus; and human parechovirus] using a microfluidic device and next-generation sequencer. Microfluidic nested PCR was carried out on a 48.48 Access Array chip, and the amplicons were recovered and used for MiSeq sequencing (Illumina, Tokyo, Japan; genotyping was conducted by homology searching and phylogenetic analysis of the obtained sequence reads. The detection limit of the 11 tested viruses ranged from 100 to 103 copies/μL in cDNA sample, corresponding to 101–104 copies/mL-sewage, 105–108 copies/g-human feces, and 102–105 copies/g-digestive tissues of oyster. The developed assay was successfully applied for simultaneous detection and genotyping of RNA viruses to samples of human feces, sewage, and artificially contaminated oysters. Microfluidic nested PCR followed by MiSeq sequencing enables efficient tracking of the fate of multiple RNA viruses in various environments, which is essential for a better understanding of the circulation of human pathogenic RNA viruses in the human population.

  6. Characteristics of MHC class I genes in house sparrows Passer domesticus as revealed by long cDNA transcripts and amplicon sequencing.

    Science.gov (United States)

    Karlsson, Maria; Westerdahl, Helena

    2013-08-01

    In birds the major histocompatibility complex (MHC) organization differs both among and within orders; chickens Gallus gallus of the order Galliformes have a simple arrangement, while many songbirds of the order Passeriformes have a more complex arrangement with larger numbers of MHC class I and II genes. Chicken MHC genes are found at two independent loci, classical MHC-B and non-classical MHC-Y, whereas non-classical MHC genes are yet to be verified in passerines. Here we characterize MHC class I transcripts (α1 to α3 domain) and perform amplicon sequencing using a next-generation sequencing technique on exon 3 from house sparrow Passer domesticus (a passerine) families. Then we use phylogenetic, selection, and segregation analyses to gain a better understanding of the MHC class I organization. Trees based on the α1 and α2 domain revealed a distinct cluster with short terminal branches for transcripts with a 6-bp deletion. Interestingly, this cluster was not seen in the tree based on the α3 domain. 21 exon 3 sequences were verified in a single individual and the average numbers within an individual were nine and five for sequences with and without a 6-bp deletion, respectively. All individuals had exon 3 sequences with and without a 6-bp deletion. The sequences with a 6-bp deletion have many characteristics in common with non-classical MHC, e.g., highly conserved amino acid positions were substituted compared with the other alleles, low nucleotide diversity and just a single site was subject to positive selection. However, these alleles also have characteristics that suggest they could be classical, e.g., complete linkage and absence of a distinct cluster in a tree based on the α3 domain. Thus, we cannot determine for certain whether or not the alleles with a 6-bp deletion are non-classical based on our present data. Further analyses on segregation patterns of these alleles in combination with dating the 6-bp deletion through MHC characterization across the

  7. Using quality scores and longer reads improves accuracy of Solexa read mapping

    Directory of Open Access Journals (Sweden)

    Xuan Zhenyu

    2008-02-01

    Full Text Available Abstract Background Second-generation sequencing has the potential to revolutionize genomics and impact all areas of biomedical science. New technologies will make re-sequencing widely available for such applications as identifying genome variations or interrogating the oligonucleotide content of a large sample (e.g. ChIP-sequencing. The increase in speed, sensitivity and availability of sequencing technology brings demand for advances in computational technology to perform associated analysis tasks. The Solexa/Illumina 1G sequencer can produce tens of millions of reads, ranging in length from ~25–50 nt, in a single experiment. Accurately mapping the reads back to a reference genome is a critical task in almost all applications. Two sources of information that are often ignored when mapping reads from the Solexa technology are the 3' ends of longer reads, which contain a much higher frequency of sequencing errors, and the base-call quality scores. Results To investigate whether these sources of information can be used to improve accuracy when mapping reads, we developed the RMAP tool, which can map reads having a wide range of lengths and allows base-call quality scores to determine which positions in each read are more important when mapping. We applied RMAP to analyze data re-sequenced from two human BAC regions for varying read lengths, and varying criteria for use of quality scores. RMAP is freely available for downloading at http://rulai.cshl.edu/rmap/. Conclusion Our results indicate that significant gains in Solexa read mapping performance can be achieved by considering the information in 3' ends of longer reads, and appropriately using the base-call quality scores. The RMAP tool we have developed will enable researchers to effectively exploit this information in targeted re-sequencing projects.

  8. The wolf reference genome sequence (Canis lupus lupus) and its implications for Canis spp. population genomics

    DEFF Research Database (Denmark)

    Gopalakrishnan, Shyam; Samaniego Castruita, Jose Alfredo; Sinding, Mikkel Holger Strander

    2017-01-01

    Background An increasing number of studies are addressing the evolutionary genomics of dog domestication, principally through resequencing dog, wolf and related canid genomes. There is, however, only one de novo assembled canid genome currently available against which to map such data - that of a......Background An increasing number of studies are addressing the evolutionary genomics of dog domestication, principally through resequencing dog, wolf and related canid genomes. There is, however, only one de novo assembled canid genome currently available against which to map such data...

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

    Directory of Open Access Journals (Sweden)

    Dietmar eSchwarz

    2014-11-01

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

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

    Science.gov (United States)

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

    2014-01-01

    Domesticated tomato (Solanum lycopersicum) is the most important horticultural crop worldwide. Low polymorphism at the DNA level conflicts with the wealth of morphological variation. Fruits vary widely in size, shape, and color. In contrast, genetic variation between the 16 wild relatives is tremendous. Several large seed banks provide tomato germplasm for both domesticated and wild accessions of tomato. Recently, the genomes of the inbred cultivar “Heinz 1706” (≈900 Mb), and S. pimpinellifolium (739 Mb) were sequenced. Genomic markers and genome re-sequencing data are available for >150 cultivars and accessions. Transformation of tomato is relatively easy and T-DNA insertion line collections are available. Tomato is widely used as a model crop for fruit development but also for diverse physiological, cellular, biochemical, molecular, and genetic studies. It can be easily grown in greenhouses or growth chambers. Plants grow, flower, and develop fruits well at daily light lengths between 8 and 16 h. The required daily light integral of an experiment depends on growth stage and temperature investigated. Temperature must be 10–35°C, relative humidity 30–90%, and, CO2 concentration 200–1500 μmol mol−1. Temperature determines the speed of the phenological development while daily light integral and CO2 concentration affect photosynthesis and biomass production. Seed to seed cultivation takes 100 days at 20°C and can be shortened or delayed by temperature. Tomato may be cultivated in soil, substrates, or aeroponically without any substrate. Root volume, and water uptake requirements are primarily determined by transpiration demands of the plants. Many nutrient supply recipes and strategies are available to ensure sufficient supply as well as specific nutrient deficits/surplus. Using appropriate cultivation techniques makes tomato a convenient model plant for researchers, even for beginners. PMID:25477888

  11. Influence of heavy metals on rhizosphere microbial communities of Siam weed (Chromolaena odorata (L. using a 16S rRNA gene amplicon sequencing approach

    Directory of Open Access Journals (Sweden)

    Thanyaporn Ruangdech

    2017-06-01

    Full Text Available A 16S rRNA amplicon sequencing approach was used to assess the impacts of cadmium (Cd and zinc (Zn contamination on populations of rhizobacteria on Siam weed (Chromolaena odorata (L.. Bacterial communities were characterized using the Illumina MiSeq platform and the V6 hypervariable region of the 16S rRNA gene. Among the 54,026 unique operational taxonomic units (OTUs identified, 99.7% were classified as bacteria and the rest were classified as archaea. Several dominant bacterial phyla were observed in all samples—Proteobacteria, Actinobacteria, Acidobacteria, Firmicutes and Bacteroidetes. These five phyla accounted for 89.2% of all OTUs identified among all sites, and only two OTUs could not be classified to a phylum. Comparison among samples containing low and high levels of Cd contamination using nonparametric Shannon and Shannon diversity indices showed that soils with low levels of diversity had a higher level of Cd (p < 0.05. These results indicated that levels of Cd may significantly alter bacterial species selection. The Cd- and Zn-resistant bacteria from each sample were subjected to heavy-metal minimum inhibitory concentration (MIC analyses. The MIC values obtained from 1152 isolates were used to individually analyze the pattern of gene function using the BioNumerics software. The results of this analysis showed that 26.7% of the bacteria were resistant to Cd concentrations up to 320 mg/L and only 2.3% of bacteria were resistant to Zn at concentrations up to 3200 mg/L. The MIC analyses indicated that the number of resistant bacteria decreased with increasing metal concentrations and those bacteria resistant to Cd and Zn may contain more than one group of metal-resistance genes.

  12. Postzygotic single-nucleotide mosaicisms contribute to the etiology of autism spectrum disorder and autistic traits and the origin of mutations.

    Science.gov (United States)

    Dou, Yanmei; Yang, Xiaoxu; Li, Ziyi; Wang, Sheng; Zhang, Zheng; Ye, Adam Yongxin; Yan, Linlin; Yang, Changhong; Wu, Qixi; Li, Jiarui; Zhao, Boxun; Huang, August Yue; Wei, Liping

    2017-08-01

    The roles and characteristics of postzygotic single-nucleotide mosaicisms (pSNMs) in autism spectrum disorders (ASDs) remain unclear. In this study of the whole exomes of 2,361 families in the Simons Simplex Collection, we identified 1,248 putative pSNMs in children and 285 de novo SNPs in children with detectable parental mosaicism. Ultra-deep amplicon resequencing suggested a validation rate of 51%. Analyses of validated pSNMs revealed that missense/loss-of-function (LoF) pSNMs with a high mutant allele fraction (MAF≥ 0.2) contributed to ASD diagnoses (P = 0.022, odds ratio [OR] = 5.25), whereas missense/LoF pSNMs with a low MAF (MAF<0.2) contributed to autistic traits in male non-ASD siblings (P = 0.033). LoF pSNMs in parents were less likely to be transmitted to offspring than neutral pSNMs (P = 0.037), and missense/LoF pSNMs in parents with a low MAF were transmitted more to probands than to siblings (P = 0.016, OR = 1.45). We estimated that pSNMs in probands or de novo mutations inherited from parental pSNMs increased the risk of ASD by approximately 6%. Adding pSNMs into the transmission and de novo association test model revealed 13 new ASD risk genes. These results expand the existing repertoire of genes involved in ASD and shed new light on the contribution of genomic mosaicisms to ASD diagnoses and autistic traits. © 2017 The Authors. Human Mutation published by Wiley Periodicals, Inc.

  13. Taxonomy of anaerobic digestion microbiome reveals biases associated with the applied high throughput sequencing strategies

    DEFF Research Database (Denmark)

    Campanaro, Stefano; Treu, Laura; Kougias, Panagiotis

    2018-01-01

    In the past few years, many studies investigated the anaerobic digestion microbiome by means of 16S rRNA amplicon sequencing. Results obtained from these studies were compared to each other without taking into consideration the followed procedure for amplicons preparation and data analysis...... specifically, the microbial compositions of three laboratory scale biogas reactors were analyzed before and after addition of sodium oleate by sequencing the microbiome with three different approaches: 16S rRNA amplicon sequencing, shotgun DNA and shotgun RNA. This comparative analysis revealed that......, in amplicon sequencing, abundance of some taxa (Euryarchaeota and Spirochaetes) was biased by the inefficiency of universal primers to hybridize all the templates. Reliability of the results obtained was also influenced by the number of hypervariable regions under investigation. Finally, amplicon sequencing...

  14. Genetic variation in genes of the fatty acid synthesis pathway and breast cancer risk

    DEFF Research Database (Denmark)

    Campa, Daniele; McKay, James; Sinilnikova, Olga

    2009-01-01

    and FASN) is related to breast cancer risk and body-mass index (BMI) by studying 1,294 breast cancer cases and 2,452 controls from the European Prospective Investigation on Cancer (EPIC). We resequenced the FAS gene and combined information of SNPs found by resequencing and SNPs from public databases....... Using a tagging approach and selecting 20 SNPs, we covered all the common genetic variation of these genes. In this study we were not able to find any statistically significant association between the SNPs in the FAS, ChREBP and SREPB-1 genes and an increased risk of breast cancer overall...

  15. (SELAX strain) B2 amplicon

    African Journals Online (AJOL)

    USER

    2010-07-12

    Jul 12, 2010 ... enzyme was inactivated by heating at 75°C for 10 min after the addition of 2 µl of ... material of the vector DNA and the partial genomic digests were packaged in ... tubes. The tubes were incubated for 15 min at 37°C. Molten LB ... Plaque transfer was carried out for 2 min with ink markings made on the agar ...

  16. Next-Generation DNA Sequencing of VH/VL Repertoires: A Primer and Guide to Applications in Single-Domain Antibody Discovery.

    Science.gov (United States)

    Henry, Kevin A

    2018-01-01

    Immunogenetic analyses of expressed antibody repertoires are becoming increasingly common experimental investigations and are critical to furthering our understanding of autoimmunity, infectious disease, and cancer. Next-generation DNA sequencing (NGS) technologies have now made it possible to interrogate antibody repertoires to unprecedented depths, typically by sequencing of cDNAs encoding immunoglobulin variable domains. In this chapter, we describe simple, fast, and reliable methods for producing and sequencing multiplex PCR amplicons derived from the variable regions (V H , V H H or V L ) of rearranged immunoglobulin heavy and light chain genes using the Illumina MiSeq platform. We include complete protocols and primer sets for amplicon sequencing of V H /V H H/V L repertoires directly from human, mouse, and llama lymphocytes as well as from phage-displayed V H /V H H/V L libraries; these can be easily be adapted to other types of amplicons with little modification. The resulting amplicons are diverse and representative, even using as few as 10 3 input B cells, and their generation is relatively inexpensive, requiring no special equipment and only a limited set of primers. In the absence of heavy-light chain pairing, single-domain antibodies are uniquely amenable to NGS analyses. We present a number of applications of NGS technology useful in discovery of single-domain antibodies from phage display libraries, including: (i) assessment of library functionality; (ii) confirmation of desired library randomization; (iii) estimation of library diversity; and (iv) monitoring the progress of panning experiments. While the case studies presented here are of phage-displayed single-domain antibody libraries, the principles extend to other types of in vitro display libraries.

  17. Patterns and drivers of fungal community depth stratification in Sphagnum peat

    Science.gov (United States)

    Louis J. Lamit; Karl J. Romanowicz; Lynette R. Potvin; Adam R. Rivers; Kanwar Singh; Jay T. Lennon; Susannah G. Tringe; Evan S. Kane; Erik A. Lilleskov

    2017-01-01

    Peatlands store an immense pool of soil carbon vulnerable to microbial oxidation due to drought and intentional draining. We used amplicon sequencing and quantitative PCR to (i) examine how fungi are influenced by depth in the peat profile, water table and plant functional group at the onset of a multiyear mesocosm experiment, and (ii) test if fungi are correlated with...

  18. Intracellular diversity of the V4 and V9 regions of the 18S rRNA in marine protists (radiolarians) assessed by high-throughput sequencing.

    Science.gov (United States)

    Decelle, Johan; Romac, Sarah; Sasaki, Eriko; Not, Fabrice; Mahé, Frédéric

    2014-01-01

    Metabarcoding is a powerful tool for exploring microbial diversity in the environment, but its accurate interpretation is impeded by diverse technical (e.g. PCR and sequencing errors) and biological biases (e.g. intra-individual polymorphism) that remain poorly understood. To help interpret environmental metabarcoding datasets, we investigated the intracellular diversity of the V4 and V9 regions of the 18S rRNA gene from Acantharia and Nassellaria (radiolarians) using 454 pyrosequencing. Individual cells of radiolarians were isolated, and PCRs were performed with generalist primers to amplify the V4 and V9 regions. Different denoising procedures were employed to filter the pyrosequenced raw amplicons (Acacia, AmpliconNoise, Linkage method). For each of the six isolated cells, an average of 541 V4 and 562 V9 amplicons assigned to radiolarians were obtained, from which one numerically dominant sequence and several minor variants were found. At the 97% identity, a diversity metrics commonly used in environmental surveys, up to 5 distinct OTUs were detected in a single cell. However, most amplicons grouped within a single OTU whereas other OTUs contained very few amplicons. Different analytical methods provided evidence that most minor variants forming different OTUs correspond to PCR and sequencing artifacts. Duplicate PCR and sequencing from the same DNA extract of a single cell had only 9 to 16% of unique amplicons in common, and alignment visualization of V4 and V9 amplicons showed that most minor variants contained substitutions in highly-conserved regions. We conclude that intracellular variability of the 18S rRNA in radiolarians is very limited despite its multi-copy nature and the existence of multiple nuclei in these protists. Our study recommends some technical guidelines to conservatively discard artificial amplicons from metabarcoding datasets, and thus properly assess the diversity and richness of protists in the environment.

  19. Integrating sequencing technologies in personal genomics: optimal low cost reconstruction of structural variants.

    Directory of Open Access Journals (Sweden)

    Jiang Du

    2009-07-01

    Full Text Available The goal of human genome re-sequencing is obtaining an accurate assembly of an individual's genome. Recently, there has been great excitement in the development of many technologies for this (e.g. medium and short read sequencing from companies such as 454 and SOLiD, and high-density oligo-arrays from Affymetrix and NimbelGen, with even more expected to appear. The costs and sensitivities of these technologies differ considerably from each other. As an important goal of personal genomics is to reduce the cost of re-sequencing to an affordable point, it is worthwhile to consider optimally integrating technologies. Here, we build a simulation toolbox that will help us optimally combine different technologies for genome re-sequencing, especially in reconstructing large structural variants (SVs. SV reconstruction is considered the most challenging step in human genome re-sequencing. (It is sometimes even harder than de novo assembly of small genomes because of the duplications and repetitive sequences in the human genome. To this end, we formulate canonical problems that are representative of issues in reconstruction and are of small enough scale to be computationally tractable and simulatable. Using semi-realistic simulations, we show how we can combine different technologies to optimally solve the assembly at low cost. With mapability maps, our simulations efficiently handle the inhomogeneous repeat-containing structure of the human genome and the computational complexity of practical assembly algorithms. They quantitatively show how combining different read lengths is more cost-effective than using one length, how an optimal mixed sequencing strategy for reconstructing large novel SVs usually also gives accurate detection of SNPs/indels, how paired-end reads can improve reconstruction efficiency, and how adding in arrays is more efficient than just sequencing for disentangling some complex SVs. Our strategy should facilitate the sequencing of

  20. ReseqChip: Automated integration of multiple local context probe data from the MitoChip array in mitochondrial DNA sequence assembly

    Directory of Open Access Journals (Sweden)

    Spang Rainer

    2009-12-01

    Full Text Available Abstract Background The Affymetrix MitoChip v2.0 is an oligonucleotide tiling array for the resequencing of the human mitochondrial (mt genome. For each of 16,569 nucleotide positions of the mt genome it holds two sets of four 25-mer probes each that match the heavy and the light strand of a reference mt genome and vary only at their central position to interrogate all four possible alleles. In addition, the MitoChip v2.0 carries alternative local context probes to account for known mtDNA variants. These probes have been neglected in most studies due to the lack of software for their automated analysis. Results We provide ReseqChip, a free software that automates the process of resequencing mtDNA using multiple local context probes on the MitoChip v2.0. ReseqChip significantly improves base call rate and sequence accuracy. ReseqChip is available at http://code.open-bio.org/svnweb/index.cgi/bioperl/browse/bioperl-live/trunk/Bio/Microarray/Tools/. Conclusions ReseqChip allows for the automated consolidation of base calls from alternative local mt genome context probes. It thereby improves the accuracy of resequencing, while reducing the number of non-called bases.

  1. Portability of tag SNPs across isolated population groups: an example from India.

    Science.gov (United States)

    Sarkar Roy, N; Farheen, S; Roy, N; Sengupta, S; Majumder, P P

    2008-01-01

    Isolated population groups are useful in conducting association studies of complex diseases to avoid various pitfalls, including those arising from population stratification. Since DNA resequencing is expensive, it is recommended that genotyping be carried out at tagSNP (tSNP) loci. For this, tSNPs identified in one isolated population need to be used in another. Unless tSNPs are highly portable across populations this strategy may result in loss of information in association studies. We examined the issue of tSNP portability by sampling individuals from 10 isolated ethnic groups from India. We generated DNA resequencing data pertaining to 3 genomic regions and identified tSNPs in each population. We defined an index of tSNP portability and showed that portability is low across isolated Indian ethnic groups. The extent of portability did not significantly correlate with genetic similarity among the populations studied here. We also analyzed our data with sequence data from individuals of African and European descent. Our results indicated that it may be necessary to carry out resequencing in a small number of individuals to discover SNPs and identify tSNPs in the specific isolated population in which a disease association study is to be conducted.

  2. 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.

  3. Evolution of the DAZ gene and the AZFc region on primate Y chromosomes

    Directory of Open Access Journals (Sweden)

    Yu Jane-Fang

    2008-03-01

    Full Text Available Abstract Background The Azoospermia Factor c (AZFc region of the human Y chromosome is a unique product of segmental duplication. It consists almost entirely of very long amplicons, represented by different colors, and is frequently deleted in subfertile men. Most of the AZFc amplicons have high sequence similarity with autosomal segments, indicating recent duplication and transposition to the Y chromosome. The Deleted in Azoospermia (DAZ gene within the red-amplicon arose from an ancestral autosomal DAZ-like (DAZL gene. It varies significantly between different men regarding to its copy number and the numbers of RNA recognition motif and DAZ repeat it encodes. We used Southern analyses to study the evolution of DAZ and AZFc amplicons on the Y chromosomes of primates. Results The Old World monkey rhesus macaque has only one DAZ gene. In contrast, the great apes have multiple copies of DAZ, ranging from 2 copies in bonobos and gorillas to at least 6 copies in orangutans, and these DAZ genes have polymorphic structures similar to those of their human counterparts. Sequences homologous to the various AZFc amplicons are present on the Y chromosomes of some but not all primates, indicating that they arrived on the Y chromosome at different times during primate evolution. Conclusion The duplication and transposition of AZFc amplicons to the human Y chromosome occurred in three waves, i.e., after the branching of the New World monkey, the gorilla, and the chimpanzee/bonobo lineages, respectively. The red-amplicon, one of the first to arrive on the Y chromosome, amplified by inverted duplication followed by direct duplication after the separation of the Old World monkey and the great ape lineages. Subsequent duplication/deletion in the various lineages gave rise to a spectrum of DAZ gene structure and copy number found in today's great apes.

  4. CHAPTER 1

    African Journals Online (AJOL)

    Dr Olaleye

    The amplicons were sequenced and subjected to basic local alignment search tool ... Out of the 12 amplicons in agarose gel, there were 6 sharp and 6 faint bands .... A: Consensus sequence with (-) and (*) signs representing gaps and points ...

  5. Refining the Results of a Classical SELEX Experiment by Expanding the Sequence Data Set of an Aptamer Pool Selected for Protein A

    Directory of Open Access Journals (Sweden)

    Regina Stoltenburg

    2018-02-01

    Full Text Available New, as yet undiscovered aptamers for Protein A were identified by applying next generation sequencing (NGS to a previously selected aptamer pool. This pool was obtained in a classical SELEX (Systematic Evolution of Ligands by EXponential enrichment experiment using the FluMag-SELEX procedure followed by cloning and Sanger sequencing. PA#2/8 was identified as the only Protein A-binding aptamer from the Sanger sequence pool, and was shown to be able to bind intact cells of Staphylococcus aureus. In this study, we show the extension of the SELEX results by re-sequencing of the same aptamer pool using a medium throughput NGS approach and data analysis. Both data pools were compared. They confirm the selection of a highly complex and heterogeneous oligonucleotide pool and show consistently a high content of orphans as well as a similar relative frequency of certain sequence groups. But in contrast to the Sanger data pool, the NGS pool was clearly dominated by one sequence group containing the known Protein A-binding aptamer PA#2/8 as the most frequent sequence in this group. In addition, we found two new sequence groups in the NGS pool represented by PA-C10 and PA-C8, respectively, which also have high specificity for Protein A. Comparative affinity studies reveal differences between the aptamers and confirm that PA#2/8 remains the most potent sequence within the selected aptamer pool reaching affinities in the low nanomolar range of KD = 20 ± 1 nM.

  6. Refining the Results of a Classical SELEX Experiment by Expanding the Sequence Data Set of an Aptamer Pool Selected for Protein A.

    Science.gov (United States)

    Stoltenburg, Regina; Strehlitz, Beate

    2018-02-24

    New, as yet undiscovered aptamers for Protein A were identified by applying next generation sequencing (NGS) to a previously selected aptamer pool. This pool was obtained in a classical SELEX (Systematic Evolution of Ligands by EXponential enrichment) experiment using the FluMag-SELEX procedure followed by cloning and Sanger sequencing. PA#2/8 was identified as the only Protein A-binding aptamer from the Sanger sequence pool, and was shown to be able to bind intact cells of Staphylococcus aureus . In this study, we show the extension of the SELEX results by re-sequencing of the same aptamer pool using a medium throughput NGS approach and data analysis. Both data pools were compared. They confirm the selection of a highly complex and heterogeneous oligonucleotide pool and show consistently a high content of orphans as well as a similar relative frequency of certain sequence groups. But in contrast to the Sanger data pool, the NGS pool was clearly dominated by one sequence group containing the known Protein A-binding aptamer PA#2/8 as the most frequent sequence in this group. In addition, we found two new sequence groups in the NGS pool represented by PA-C10 and PA-C8, respectively, which also have high specificity for Protein A. Comparative affinity studies reveal differences between the aptamers and confirm that PA#2/8 remains the most potent sequence within the selected aptamer pool reaching affinities in the low nanomolar range of K D = 20 ± 1 nM.

  7. A classification system for plasmids from Enterococci and other Gram-positive bacteria

    DEFF Research Database (Denmark)

    Jensen, Lars Bogø; Garcia-Migura, Lourdes; Valenzuela, Antonio Jesus Sanchez

    2010-01-01

    A classification system for plasmids isolated from enterococci and other Gram-positive bacteria was developed based on 111 published plasmid sequences from enterococci and other Gram-positive bacteria; mostly staphylococci. Based on PCR amplification of conserved areas of the replication initiating....... Furthermore, conjugation experiments were performed obtaining 30 transconjugants when selecting for antimicrobial resistance. Among them 19 gave no positive amplicons indicating presence of rep-families not tested for in this experimental setup....

  8. ESPRIT-Forest: Parallel clustering of massive amplicon sequence data in subquadratic time.

    Science.gov (United States)

    Cai, Yunpeng; Zheng, Wei; Yao, Jin; Yang, Yujie; Mai, Volker; Mao, Qi; Sun, Yijun

    2017-04-01

    The rapid development of sequencing technology has led to an explosive accumulation of genomic sequence data. Clustering is often the first step to perform in sequence analysis, and hierarchical clustering is one of the most commonly used approaches for this purpose. However, it is currently computationally expensive to perform hierarchical clustering of extremely large sequence datasets due to its quadratic time and space complexities. In this paper we developed a new algorithm called ESPRIT-Forest for parallel hierarchical clustering of sequences. The algorithm achieves subquadratic time and space complexity and maintains a high clustering accuracy comparable to the standard method. The basic idea is to organize sequences into a pseudo-metric based partitioning tree for sub-linear time searching of nearest neighbors, and then use a new multiple-pair merging criterion to construct clusters in parallel using multiple threads. The new algorithm was tested on the human microbiome project (HMP) dataset, currently one of the largest published microbial 16S rRNA sequence dataset. Our experiment demonstrated that with the power of parallel computing it is now compu- tationally feasible to perform hierarchical clustering analysis of tens of millions of sequences. The software is available at http://www.acsu.buffalo.edu/∼yijunsun/lab/ESPRIT-Forest.html.

  9. Delineating the structural, functional and evolutionary relationships of sucrose phosphate synthase gene family II in wheat and related grasses

    Directory of Open Access Journals (Sweden)

    Khalil Zaynali

    2010-06-01

    Full Text Available Abstract Background Sucrose phosphate synthase (SPS is an important component of the plant sucrose biosynthesis pathway. In the monocotyledonous Poaceae, five SPS genes have been identified. Here we present a detailed analysis of the wheat SPSII family in wheat. A set of homoeologue-specific primers was developed in order to permit both the detection of sequence variation, and the dissection of the individual contribution of each homoeologue to the global expression of SPSII. Results The expression in bread wheat over the course of development of various sucrose biosynthesis genes monitored on an Affymetrix array showed that the SPS genes were regulated over time and space. SPSII homoeologue-specific assays were used to show that the three homoeologues contributed differentially to the global expression of SPSII. Genetic mapping placed the set of homoeoloci on the short arms of the homoeologous group 3 chromosomes. A resequencing of the A and B genome copies allowed the detection of four haplotypes at each locus. The 3B copy includes an unspliced intron. A comparison of the sequences of the wheat SPSII orthologues present in the diploid progenitors einkorn, goatgrass and Triticum speltoides, as well as in the more distantly related species barley, rice, sorghum and purple false brome demonstrated that intronic sequence was less well conserved than exonic. Comparative sequence and phylogenetic analysis of SPSII gene showed that false purple brome was more similar to Triticeae than to rice. Wheat - rice synteny was found to be perturbed at the SPS region. Conclusion The homoeologue-specific assays will be suitable to derive associations between SPS functionality and key phenotypic traits. The amplicon sequences derived from the homoeologue-specific primers are informative regarding the evolution of SPSII in a polyploid context.

  10. Genetics and Pathogenesis of Diffuse Large B-Cell Lymphoma.

    Science.gov (United States)

    Schmitz, Roland; Wright, George W; Huang, Da Wei; Johnson, Calvin A; Phelan, James D; Wang, James Q; Roulland, Sandrine; Kasbekar, Monica; Young, Ryan M; Shaffer, Arthur L; Hodson, Daniel J; Xiao, Wenming; Yu, Xin; Yang, Yandan; Zhao, Hong; Xu, Weihong; Liu, Xuelu; Zhou, Bin; Du, Wei; Chan, Wing C; Jaffe, Elaine S; Gascoyne, Randy D; Connors, Joseph M; Campo, Elias; Lopez-Guillermo, Armando; Rosenwald, Andreas; Ott, German; Delabie, Jan; Rimsza, Lisa M; Tay Kuang Wei, Kevin; Zelenetz, Andrew D; Leonard, John P; Bartlett, Nancy L; Tran, Bao; Shetty, Jyoti; Zhao, Yongmei; Soppet, Dan R; Pittaluga, Stefania; Wilson, Wyndham H; Staudt, Louis M

    2018-04-12

    Diffuse large B-cell lymphomas (DLBCLs) are phenotypically and genetically heterogeneous. Gene-expression profiling has identified subgroups of DLBCL (activated B-cell-like [ABC], germinal-center B-cell-like [GCB], and unclassified) according to cell of origin that are associated with a differential response to chemotherapy and targeted agents. We sought to extend these findings by identifying genetic subtypes of DLBCL based on shared genomic abnormalities and to uncover therapeutic vulnerabilities based on tumor genetics. We studied 574 DLBCL biopsy samples using exome and transcriptome sequencing, array-based DNA copy-number analysis, and targeted amplicon resequencing of 372 genes to identify genes with recurrent aberrations. We developed and implemented an algorithm to discover genetic subtypes based on the co-occurrence of genetic alterations. We identified four prominent genetic subtypes in DLBCL, termed MCD (based on the co-occurrence of MYD88 L265P and CD79B mutations), BN2 (based on BCL6 fusions and NOTCH2 mutations), N1 (based on NOTCH1 mutations), and EZB (based on EZH2 mutations and BCL2 translocations). Genetic aberrations in multiple genes distinguished each genetic subtype from other DLBCLs. These subtypes differed phenotypically, as judged by differences in gene-expression signatures and responses to immunochemotherapy, with favorable survival in the BN2 and EZB subtypes and inferior outcomes in the MCD and N1 subtypes. Analysis of genetic pathways suggested that MCD and BN2 DLBCLs rely on "chronic active" B-cell receptor signaling that is amenable to therapeutic inhibition. We uncovered genetic subtypes of DLBCL with distinct genotypic, epigenetic, and clinical characteristics, providing a potential nosology for precision-medicine strategies in DLBCL. (Funded by the Intramural Research Program of the National Institutes of Health and others.).

  11. Nonspecific amplification of human DNA by Streptococcus pneumoniae LytA primer

    Directory of Open Access Journals (Sweden)

    Helen Hencida Thangamony

    2018-01-01

    Full Text Available Background: Determination of various analytical parameters is essential for the validation of primers used for in-house nucleic acid amplification tests. While standardising a high-resolution melt analysis (HRMA for detection of Streptococcus pneumoniae in acute pyogenic meningitis, we encountered non-specific amplification of certain base pair sequences of human DNA by Centers for Disease Control & Prevention, USA recommended S. pneumoniae LytA primer. Materials and Methods: HRMA was standardised using DNA extracted from an ATCC strain of S. pneumoniae using SP LytA F373 primer and Type-it HRMTM polymerase chain reaction kit in Rotor-Gene Q Thermal Cycler according to the manufacturer's instructions. Specificity of the primers was determined in dry and wet laboratory experiments against diverse related and unrelated microbial pathogens by HRMA and on DNA extracted from unspiked clinical samples negative for SP DNA. Sensitivity was determined by calculating lower limit of detection threshold in experiments with spiked samples. The amplicon from spiked experiments was sequenced and analysed through Gene Bank. Results: Our dry/wet laboratory experiments showed two separate curves and different Tm values indicating certain non-specific amplification by the primer. Basic Local Alignment Search Tool (BLAST analysis of the amplicon obtained in the spiked experiment showed sequences of human chromosome 20 associated with Homo sapiens protein tyrosine phosphatase, receptor type T gene. The problem was resolved by stopping the reaction at 30th Ct cycle and observing the Tm values. Conclusion: Since HRMA is done without a specific probe, one should be aware of non-specific amplifications while using primers for HRMA of human clinical samples.

  12. DNA Extraction and Primer Selection

    DEFF Research Database (Denmark)

    Karst, Søren Michael; Nielsen, Per Halkjær; Albertsen, Mads

    Talk regarding pitfalls in DNA extraction and 16S amplicon primer choice when performing community analysis of complex microbial communities. The talk was a part of Workshop 2 "Principles, Potential, and Limitations of Novel Molecular Methods in Water Engineering; from Amplicon Sequencing to -omics...

  13. 75 FR 36432 - Termination of Declarations Justifying Emergency Use Authorizations of Certain In Vitro...

    Science.gov (United States)

    2010-06-25

    .../TS, NA2) and Viral Culture Atlanta, GA 30333 Cepheid Cepheid Xpert Flu A Panel 904 Caribbean Drive... / Germany TessArae, LLC TessArray Resequencing Influenza A Microarray Detection Panel 46090 Lake Center...

  14. Rapid discrimination of Isaria javanica and Isaria poprawskii from Isaria spp. using high resolution DNA melting assays

    Science.gov (United States)

    The current study evaluates the potential of using high resolution DNA melting assays to discriminate species in the genus, Isaria. The study utilizes a previously identified 103 base pair PCR amplicon, which was reported to be selective for Isaria fumosorosea. Our study finds the amplicon selective...

  15. The wolf reference genome sequence (Canis lupus lupus) and its implications for Canis spp. population genomics

    DEFF Research Database (Denmark)

    Gopalakrishnan, Shyam; Samaniego Castruita, Jose Alfredo; Sinding, Mikkel Holger Strander

    2017-01-01

    Background An increasing number of studies are addressing the evolutionary genomics of dog domestication, principally through resequencing dog, wolf and related canid genomes. There is, however, only one de novo assembled canid genome currently available against which to map such data - that of a......Background An increasing number of studies are addressing the evolutionary genomics of dog domestication, principally through resequencing dog, wolf and related canid genomes. There is, however, only one de novo assembled canid genome currently available against which to map such data...... that regardless of the reference genome choice, most evolutionary genomic analyses yield qualitatively similar results, including those exploring the structure between the wolves and dogs using admixture and principal component analysis. However, we do observe differences in the genomic coverage of re-mapped...

  16. Analysis of genetic variation and potential applications in genome-scale metabolic modeling

    DEFF Research Database (Denmark)

    Cardoso, Joao; Andersen, Mikael Rørdam; Herrgard, Markus

    2015-01-01

    scale and resolution by re-sequencing thousands of strains systematically. In this article, we review challenges in the integration and analysis of large-scale re-sequencing data, present an extensive overview of bioinformatics methods for predicting the effects of genetic variants on protein function......Genetic variation is the motor of evolution and allows organisms to overcome the environmental challenges they encounter. It can be both beneficial and harmful in the process of engineering cell factories for the production of proteins and chemicals. Throughout the history of biotechnology......, there have been efforts to exploit genetic variation in our favor to create strains with favorable phenotypes. Genetic variation can either be present in natural populations or it can be artificially created by mutagenesis and selection or adaptive laboratory evolution. On the other hand, unintended genetic...

  17. Deciphering the Mechanism of Alternative Cleavage and Polyadenylation in Mantle Cell Lymphoma (MCL)

    Science.gov (United States)

    2014-10-01

    Kubo , T., Wada, T., Yamaguchi, Y., Shimizu, A. & Handa, H. Knock-down of 25 kDa subunit of cleavage factor Im inHela cells alters alternative...usage was calculated as 62normalized DDDCT. Oligonucleotides used for qRT–PCR. Cyclin D1 common forward, 59-CTGC CAGGAGCAGATCGAAG; reverse, 59...CTdeviation of either amplicon at all of the dilutions was calculated as a correction factor. d, The experiment shown in c was repeated for DICER1 and

  18. Testing genotyping strategies for ultra-deep sequencing of a co-amplifying gene family: MHC class I in a passerine bird.

    Science.gov (United States)

    Biedrzycka, Aleksandra; Sebastian, Alvaro; Migalska, Magdalena; Westerdahl, Helena; Radwan, Jacek

    2017-07-01

    Characterization of highly duplicated genes, such as genes of the major histocompatibility complex (MHC), where multiple loci often co-amplify, has until recently been hindered by insufficient read depths per amplicon. Here, we used ultra-deep Illumina sequencing to resolve genotypes at exon 3 of MHC class I genes in the sedge warbler (Acrocephalus schoenobaenus). We sequenced 24 individuals in two replicates and used this data, as well as a simulated data set, to test the effect of amplicon coverage (range: 500-20 000 reads per amplicon) on the repeatability of genotyping using four different genotyping approaches. A third replicate employed unique barcoding to assess the extent of tag jumping, that is swapping of individual tag identifiers, which may confound genotyping. The reliability of MHC genotyping increased with coverage and approached or exceeded 90% within-method repeatability of allele calling at coverages of >5000 reads per amplicon. We found generally high agreement between genotyping methods, especially at high coverages. High reliability of the tested genotyping approaches was further supported by our analysis of the simulated data set, although the genotyping approach relying primarily on replication of variants in independent amplicons proved sensitive to repeatable errors. According to the most repeatable genotyping method, the number of co-amplifying variants per individual ranged from 19 to 42. Tag jumping was detectable, but at such low frequencies that it did not affect the reliability of genotyping. We thus demonstrate that gene families with many co-amplifying genes can be reliably genotyped using HTS, provided that there is sufficient per amplicon coverage. © 2016 John Wiley & Sons Ltd.

  19. Large SNP arrays for genotyping in crop plants

    Indian Academy of Sciences (India)

    2012-10-15

    Oct 15, 2012 ... in human has been paralleled by the simultaneous develop- ment of ... In crop plants, the development of large genotyping arrays started much ..... via deep resequencing of reduced representation libraries with the Illumina ...

  20. Chromosomal Localization of DNA Amplifications in Neuroblastoma Tumors Using cDNA Microarray Comparative Genomic Hybridization

    Directory of Open Access Journals (Sweden)

    Ben Beheshti

    2003-01-01

    Full Text Available Conventional comparative genomic hybridization (CGH profiling of neuroblastomas has identified many genomic aberrations, although the limited resolution has precluded a precise localization of sequences of interest within amplicons. To map high copy number genomic gains in clinically matched stage IV neuroblastomas, CGH analysis using a 19,200-feature cDNA microarray was used. A dedicated (freely available algorithm was developed for rapid in silico determination of chromosomal localizations of microarray cDNA targets, and for generation of an ideogram-type profile of copy number changes. Using these methodologies, novel gene amplifications undetectable by chromosome CGH were identified, and larger MYCN amplicon sizes (in one tumor up to 6 Mb than those previously reported in neuroblastoma were identified. The genes HPCAL1, LPIN1/KIAA0188, NAG, and NSE1/LOC151354 were found to be coamplified with MYCN. To determine whether stage IV primary tumors could be further subclassified based on their genomic copy number profiles, hierarchical clustering was performed. Cluster analysis of microarray CGH data identified three groups: 1 no amplifications evident, 2 a small MYCN amplicon as the only detectable imbalance, and 3 a large MYCN amplicon with additional gene amplifications. Application of CGH to cDNA microarray targets will help to determine both the variation of amplicon size and help better define amplification-dependent and independent pathways of progression in neuroblastoma.

  1. Typing of 49 autosomal SNPs by single base extension and capillary electrophoresis for forensic genetic testing

    DEFF Research Database (Denmark)

    Børsting, Claus; Tomas Mas, Carmen; Morling, Niels

    2012-01-01

    of the amplicons range from 65 to 115 bp. The high sensitivity and the short amplicon sizes make the assay very suitable for typing of degraded DNA samples, and the low mutation rate of SNPs makes the assay very useful for relationship testing. Combined, these advantages make the assay well suited for disaster...

  2. How EXCELERATOR changed my job

    Energy Technology Data Exchange (ETDEWEB)

    Weir, D.

    1988-01-01

    I started using a Computer-Aided Software Engineering (CASE) analysis/design tool two years ago in the Administrative Data Processing Division (ADP) and have observed that the way I approach my job has changed. The end product is the same, but the product is derived differently. The ability to change what is done gives freedom to experiment and iterate. This freedom has changed some steps and resequenced others in arriving at a structured specification for a new system. Additionally, the analysis capability of the tools allow the performance of work that was often disregarded because it was either too tedious or overwhelming. This presentation will address the changes observed in the process of analysis and design using CASE. 2 figs.

  3. Ancient homology underlies adaptive mimetic diversity across butterflies

    Science.gov (United States)

    Gallant, Jason R.; Imhoff, Vance E.; Martin, Arnaud; Savage, Wesley K.; Chamberlain, Nicola L.; Pote, Ben L.; Peterson, Chelsea; Smith, Gabriella E.; Evans, Benjamin; Reed, Robert D.; Kronforst, Marcus R.; Mullen, Sean P.

    2014-01-01

    Convergent evolution provides a rare, natural experiment with which to test the predictability of adaptation at the molecular level. Little is known about the molecular basis of convergence over macro-evolutionary timescales. Here we use a combination of positional cloning, population genomic resequencing, association mapping and developmental data to demonstrate that positionally orthologous nucleotide variants in the upstream region of the same gene, WntA, are responsible for parallel mimetic variation in two butterfly lineages that diverged >65 million years ago. Furthermore, characterization of spatial patterns of WntA expression during development suggests that alternative regulatory mechanisms underlie wing pattern variation in each system. Taken together, our results reveal a strikingly predictable molecular basis for phenotypic convergence over deep evolutionary time. PMID:25198507

  4. Strong signatures of selection in the domestic pig genome

    DEFF Research Database (Denmark)

    Rubin, Carl-Johan; Megens, Hendrik-Jan; Barrio, Alvaro Martinez

    2012-01-01

    Domestication of wild boar (Sus scrofa) and subsequent selection have resulted in dramatic phenotypic changes in domestic pigs for a number of traits, including behavior, body composition, reproduction, and coat color. Here we have used whole-genome resequencing to reveal some of the loci that un...... to strong directional selection.......Domestication of wild boar (Sus scrofa) and subsequent selection have resulted in dramatic phenotypic changes in domestic pigs for a number of traits, including behavior, body composition, reproduction, and coat color. Here we have used whole-genome resequencing to reveal some of the loci...... that underlie phenotypic evolution in European domestic pigs. Selective sweep analyses revealed strong signatures of selection at three loci harboring quantitative trait loci that explain a considerable part of one of the most characteristic morphological changes in the domestic pig—the elongation of the back...

  5. Always look on both sides: phylogenetic information conveyed by simple sequence repeat allele sequences.

    Directory of Open Access Journals (Sweden)

    Stéphanie Barthe

    Full Text Available Simple sequence repeat (SSR markers are widely used tools for inferences about genetic diversity, phylogeography and spatial genetic structure. Their applications assume that variation among alleles is essentially caused by an expansion or contraction of the number of repeats and that, accessorily, mutations in the target sequences follow the stepwise mutation model (SMM. Generally speaking, PCR amplicon sizes are used as direct indicators of the number of SSR repeats composing an allele with the data analysis either ignoring the extent of allele size differences or assuming that there is a direct correlation between differences in amplicon size and evolutionary distance. However, without precisely knowing the kind and distribution of polymorphism within an allele (SSR and the associated flanking region (FR sequences, it is hard to say what kind of evolutionary message is conveyed by such a synthetic descriptor of polymorphism as DNA amplicon size. In this study, we sequenced several SSR alleles in multiple populations of three divergent tree genera and disentangled the types of polymorphisms contained in each portion of the DNA amplicon containing an SSR. The patterns of diversity provided by amplicon size variation, SSR variation itself, insertions/deletions (indels, and single nucleotide polymorphisms (SNPs observed in the FRs were compared. Amplicon size variation largely reflected SSR repeat number. The amount of variation was as large in FRs as in the SSR itself. The former contributed significantly to the phylogenetic information and sometimes was the main source of differentiation among individuals and populations contained by FR and SSR regions of SSR markers. The presence of mutations occurring at different rates within a marker's sequence offers the opportunity to analyse evolutionary events occurring on various timescales, but at the same time calls for caution in the interpretation of SSR marker data when the distribution of within

  6. Partial and Full PCR-Based Reverse Genetics Strategy for Influenza Viruses

    Science.gov (United States)

    Chen, Hongjun; Ye, Jianqiang; Xu, Kemin; Angel, Matthew; Shao, Hongxia; Ferrero, Andrea; Sutton, Troy; Perez, Daniel R.

    2012-01-01

    Since 1999, plasmid-based reverse genetics (RG) systems have revolutionized the way influenza viruses are studied. However, it is not unusual to encounter cloning difficulties for one or more influenza genes while attempting to recover virus de novo. To overcome some of these shortcomings we sought to develop partial or full plasmid-free RG systems. The influenza gene of choice is assembled into a RG competent unit by virtue of overlapping PCR reactions containing a cDNA copy of the viral gene segment under the control of RNA polymerase I promoter (pol1) and termination (t1) signals – herein referred to as Flu PCR amplicons. Transfection of tissue culture cells with either HA or NA Flu PCR amplicons and 7 plasmids encoding the remaining influenza RG units, resulted in efficient virus rescue. Likewise, transfections including both HA and NA Flu PCR amplicons and 6 RG plasmids also resulted in efficient virus rescue. In addition, influenza viruses were recovered from a full set of Flu PCR amplicons without the use of plasmids. PMID:23029501

  7. Development of a multiplex qPCR in real time for quantification and differential diagnosis of Salmonella Gallinarum and Salmonella Pullorum.

    Science.gov (United States)

    Rubio, Marcela da Silva; Penha Filho, Rafael Antonio Casarin; Almeida, Adriana Maria de; Berchieri, Angelo

    2017-12-01

    Currently there are 2659 Salmonella serovars. The host-specific biovars Salmonella Pullorum and Salmonella Gallinarum cause systemic infections in food-producing and wild birds. Fast diagnosis is crucial to control the dissemination in avian environments. The present work describes the development of a multiplex qPCR in real time using a low-cost DNA dye (SYBr Green) to identify and quantify these biovars. Primers were chosen based on genomic regions of difference (RoD) and optimized to control dimers. Primers pSGP detect both host-specific biovars but not other serovars and pSG and pSP differentiate biovars. Three amplicons showed different melting temperatures (Tm), allowing differentiation. The pSGP amplicon (97 bp) showed Tm of 78°C for both biovars. The pSG amplicon (273 bp) showed a Tm of 86.2°C for S. Gallinarum and pSP amplicon (260 bp) dissociated at 84.8°C for S. Pullorum identification. The multiplex qPCR in real time showed high sensitivity and was capable of quantifying 10 8 -10 1 CFU of these biovars.

  8. DNA barcode for genetic traceability of Nile Perch and Nile Tilapia

    International Nuclear Information System (INIS)

    Avossa, Valeria

    2017-01-01

    For this study, mitochondrial DNA was extracted from 55 fish samples (26 Nile Perch Samples and 29 Nile Tilapia Samples collected from 3 different Ugandan regions of Lake Victoria. In order to optimize the PCR method, we also extracted DNA from two other different fish samples: one from Italy and one from a Viennese market. The COI gene was amplified using universal primers (COI2, COI3, cocktails of 8 and 4 primers respectively). After the amplification step, the amplicons were analysed using gel electrophoresis , in order to establish that the set primers worked well in the samples. The positive results of an agarose gel electrophoresis analysis with the PCR amplicons (amplicons length ~700pb) are shown.

  9. Subgenome parallel selection is associated with morphotype diversification and convergent crop domestication in Brassica rapa and Brassica oleracea

    NARCIS (Netherlands)

    Cheng, Feng; Sun, Rifei; Hou, Xilin; Zheng, Hongkun; Zhang, Fenglan; Zhang, Yangyong; Liu, Bo; Liang, Jianli; Zhuang, Mu; Liu, Yunxia; Liu, Dongyuan; Wang, Xiaobo; Li, Pingxia; Liu, Yumei; Lin, Ke; Bucher, Johan; Zhang, Ningwen; Wang, Yan; Wang, Hui; Deng, Jie; Liao, Yongcui; Wei, Keyun; Zhang, Xueming; Fu, Lixia; Hu, Yunyan; Liu, Jisheng; Cai, Chengcheng; Zhang, Shujiang; Zhang, Shifan; Li, Fei; Zhang, Hui; Zhang, Jifang; Guo, Ning; Liu, Zhiyuan; Liu, Jin; Sun, Chao; Ma, Yuan; Zhang, Haijiao; Cui, Yang; Freeling, Micheal R.; Borm, Theo; Bonnema, Guusje; Wu, Jian; Wang, Xiaowu

    2016-01-01

    Brassica species, including crops such as cabbage, turnip and oilseed, display enormous phenotypic variation. Brassica genomes have all undergone a whole-genome triplication (WGT) event with unknown effects on phenotype diversification. We resequenced 199 Brassica rapa and 119 Brassica oleracea

  10. ZMYND10 is mutated in primary ciliary dyskinesia and interacts with LRRC6

    DEFF Research Database (Denmark)

    Zariwala, Maimoona A; Gee, Heon Yung; Kurkowiak, Małgorzata

    2013-01-01

    Defects of motile cilia cause primary ciliary dyskinesia (PCD), characterized by recurrent respiratory infections and male infertility. Using whole-exome resequencing and high-throughput mutation analysis, we identified recessive biallelic mutations in ZMYND10 in 14 families and mutations in the ...

  11. A tonoplast sugar transporter underlies a sugar accumulation QTL in watermelon

    Science.gov (United States)

    The molecular mechanism controlling accumulation of soluble sugars in watermelon (Citrullus lanatus) fruit, a trait associated with sweet-dessert watermelon domestication, is still unknown. We re-sequenced 96 recombinant inbred lines, derived from a cross between sweet and unsweet watermelon accessi...

  12. PGen: large-scale genomic variations analysis workflow and browser in SoyKB.

    Science.gov (United States)

    Liu, Yang; Khan, Saad M; Wang, Juexin; Rynge, Mats; Zhang, Yuanxun; Zeng, Shuai; Chen, Shiyuan; Maldonado Dos Santos, Joao V; Valliyodan, Babu; Calyam, Prasad P; Merchant, Nirav; Nguyen, Henry T; Xu, Dong; Joshi, Trupti

    2016-10-06

    With the advances in next-generation sequencing (NGS) technology and significant reductions in sequencing costs, it is now possible to sequence large collections of germplasm in crops for detecting genome-scale genetic variations and to apply the knowledge towards improvements in traits. To efficiently facilitate large-scale NGS resequencing data analysis of genomic variations, we have developed "PGen", an integrated and optimized workflow using the Extreme Science and Engineering Discovery Environment (XSEDE) high-performance computing (HPC) virtual system, iPlant cloud data storage resources and Pegasus workflow management system (Pegasus-WMS). The workflow allows users to identify single nucleotide polymorphisms (SNPs) and insertion-deletions (indels), perform SNP annotations and conduct copy number variation analyses on multiple resequencing datasets in a user-friendly and seamless way. We have developed both a Linux version in GitHub ( https://github.com/pegasus-isi/PGen-GenomicVariations-Workflow ) and a web-based implementation of the PGen workflow integrated within the Soybean Knowledge Base (SoyKB), ( http://soykb.org/Pegasus/index.php ). Using PGen, we identified 10,218,140 single-nucleotide polymorphisms (SNPs) and 1,398,982 indels from analysis of 106 soybean lines sequenced at 15X coverage. 297,245 non-synonymous SNPs and 3330 copy number variation (CNV) regions were identified from this analysis. SNPs identified using PGen from additional soybean resequencing projects adding to 500+ soybean germplasm lines in total have been integrated. These SNPs are being utilized for trait improvement using genotype to phenotype prediction approaches developed in-house. In order to browse and access NGS data easily, we have also developed an NGS resequencing data browser ( http://soykb.org/NGS_Resequence/NGS_index.php ) within SoyKB to provide easy access to SNP and downstream analysis results for soybean researchers. PGen workflow has been optimized for the most

  13. Characterization of ancient and modern genomes by SNP detection and phylogenomic and metagenomic analysis using PALEOMIX

    DEFF Research Database (Denmark)

    Schubert, Mikkel; Ermini, Luca; Der Sarkissian, Clio

    2014-01-01

    a variety of computational tools. Here we present PALEOMIX (http://geogenetics.ku.dk/publications/paleomix), a flexible and user-friendly pipeline applicable to both modern and ancient genomes, which largely automates the in silico analyses behind whole-genome resequencing. Starting with next...

  14. Exploring meat quality of pig throug the application of molecular genetics

    DEFF Research Database (Denmark)

    Zhang, Lu

    association mapping, expression study and genetic variation analysis. Many state of the art technologies including high-density SNP chip genotyping, hig-throughput re-sequencing and strand-specific RNA-Seq were employed. Numerous quantitative trait loci, candidate genes and a segmental duplication associated...

  15. In Vitro vs In Silico Detected SNPs for the Development of a Genotyping Array: What Can We Learn from a Non-Model Species?

    Science.gov (United States)

    Lepoittevin, Camille; Frigerio, Jean-Marc; Garnier-Géré, Pauline; Salin, Franck; Cervera, María-Teresa; Vornam, Barbara; Harvengt, Luc; Plomion, Christophe

    2010-01-01

    Background There is considerable interest in the high-throughput discovery and genotyping of single nucleotide polymorphisms (SNPs) to accelerate genetic mapping and enable association studies. This study provides an assessment of EST-derived and resequencing-derived SNP quality in maritime pine (Pinus pinaster Ait.), a conifer characterized by a huge genome size (∼23.8 Gb/C). Methodology/Principal Findings A 384-SNPs GoldenGate genotyping array was built from i/ 184 SNPs originally detected in a set of 40 re-sequenced candidate genes (in vitro SNPs), chosen on the basis of functionality scores, presence of neighboring polymorphisms, minor allele frequencies and linkage disequilibrium and ii/ 200 SNPs screened from ESTs (in silico SNPs) selected based on the number of ESTs used for SNP detection, the SNP minor allele frequency and the quality of SNP flanking sequences. The global success rate of the assay was 66.9%, and a conversion rate (considering only polymorphic SNPs) of 51% was achieved. In vitro SNPs showed significantly higher genotyping-success and conversion rates than in silico SNPs (+11.5% and +18.5%, respectively). The reproducibility was 100%, and the genotyping error rate very low (0.54%, dropping down to 0.06% when removing four SNPs showing elevated error rates). Conclusions/Significance This study demonstrates that ESTs provide a resource for SNP identification in non-model species, which do not require any additional bench work and little bio-informatics analysis. However, the time and cost benefits of in silico SNPs are counterbalanced by a lower conversion rate than in vitro SNPs. This drawback is acceptable for population-based experiments, but could be dramatic in experiments involving samples from narrow genetic backgrounds. In addition, we showed that both the visual inspection of genotyping clusters and the estimation of a per SNP error rate should help identify markers that are not suitable to the GoldenGate technology in species

  16. In vitro vs in silico detected SNPs for the development of a genotyping array: what can we learn from a non-model species?

    Directory of Open Access Journals (Sweden)

    Camille Lepoittevin

    2010-06-01

    Full Text Available There is considerable interest in the high-throughput discovery and genotyping of single nucleotide polymorphisms (SNPs to accelerate genetic mapping and enable association studies. This study provides an assessment of EST-derived and resequencing-derived SNP quality in maritime pine (Pinus pinaster Ait., a conifer characterized by a huge genome size ( approximately 23.8 Gb/C.A 384-SNPs GoldenGate genotyping array was built from i/ 184 SNPs originally detected in a set of 40 re-sequenced candidate genes (in vitro SNPs, chosen on the basis of functionality scores, presence of neighboring polymorphisms, minor allele frequencies and linkage disequilibrium and ii/ 200 SNPs screened from ESTs (in silico SNPs selected based on the number of ESTs used for SNP detection, the SNP minor allele frequency and the quality of SNP flanking sequences. The global success rate of the assay was 66.9%, and a conversion rate (considering only polymorphic SNPs of 51% was achieved. In vitro SNPs showed significantly higher genotyping-success and conversion rates than in silico SNPs (+11.5% and +18.5%, respectively. The reproducibility was 100%, and the genotyping error rate very low (0.54%, dropping down to 0.06% when removing four SNPs showing elevated error rates.This study demonstrates that ESTs provide a resource for SNP identification in non-model species, which do not require any additional bench work and little bio-informatics analysis. However, the time and cost benefits of in silico SNPs are counterbalanced by a lower conversion rate than in vitro SNPs. This drawback is acceptable for population-based experiments, but could be dramatic in experiments involving samples from narrow genetic backgrounds. In addition, we showed that both the visual inspection of genotyping clusters and the estimation of a per SNP error rate should help identify markers that are not suitable to the GoldenGate technology in species characterized by a large and complex genome.

  17. Genomic dark matter: the reliability of short read mapping illustrated by the genome mappability score.

    Science.gov (United States)

    Lee, Hayan; Schatz, Michael C

    2012-08-15

    Genome resequencing and short read mapping are two of the primary tools of genomics and are used for many important applications. The current state-of-the-art in mapping uses the quality values and mapping quality scores to evaluate the reliability of the mapping. These attributes, however, are assigned to individual reads and do not directly measure the problematic repeats across the genome. Here, we present the Genome Mappability Score (GMS) as a novel measure of the complexity of resequencing a genome. The GMS is a weighted probability that any read could be unambiguously mapped to a given position and thus measures the overall composition of the genome itself. We have developed the Genome Mappability Analyzer to compute the GMS of every position in a genome. It leverages the parallelism of cloud computing to analyze large genomes, and enabled us to identify the 5-14% of the human, mouse, fly and yeast genomes that are difficult to analyze with short reads. We examined the accuracy of the widely used BWA/SAMtools polymorphism discovery pipeline in the context of the GMS, and found discovery errors are dominated by false negatives, especially in regions with poor GMS. These errors are fundamental to the mapping process and cannot be overcome by increasing coverage. As such, the GMS should be considered in every resequencing project to pinpoint the 'dark matter' of the genome, including of known clinically relevant variations in these regions. The source code and profiles of several model organisms are available at http://gma-bio.sourceforge.net

  18. CoVaCS: a consensus variant calling system.

    Science.gov (United States)

    Chiara, Matteo; Gioiosa, Silvia; Chillemi, Giovanni; D'Antonio, Mattia; Flati, Tiziano; Picardi, Ernesto; Zambelli, Federico; Horner, David Stephen; Pesole, Graziano; Castrignanò, Tiziana

    2018-02-05

    The advent and ongoing development of next generation sequencing technologies (NGS) has led to a rapid increase in the rate of human genome re-sequencing data, paving the way for personalized genomics and precision medicine. The body of genome resequencing data is progressively increasing underlining the need for accurate and time-effective bioinformatics systems for genotyping - a crucial prerequisite for identification of candidate causal mutations in diagnostic screens. Here we present CoVaCS, a fully automated, highly accurate system with a web based graphical interface for genotyping and variant annotation. Extensive tests on a gold standard benchmark data-set -the NA12878 Illumina platinum genome- confirm that call-sets based on our consensus strategy are completely in line with those attained by similar command line based approaches, and far more accurate than call-sets from any individual tool. Importantly our system exhibits better sensitivity and higher specificity than equivalent commercial software. CoVaCS offers optimized pipelines integrating state of the art tools for variant calling and annotation for whole genome sequencing (WGS), whole-exome sequencing (WES) and target-gene sequencing (TGS) data. The system is currently hosted at Cineca, and offers the speed of a HPC computing facility, a crucial consideration when large numbers of samples must be analysed. Importantly, all the analyses are performed automatically allowing high reproducibility of the results. As such, we believe that CoVaCS can be a valuable tool for the analysis of human genome resequencing studies. CoVaCS is available at: https://bioinformatics.cineca.it/covacs .

  19. Development and characterization of 35 single nucleotide polymorphism markers for the brown alga Fucus vesiculosus

    NARCIS (Netherlands)

    Canovas, Fernando; Mota, Catarina; Ferreira-Costa, Joana; Serrao, Ester; Coyer, Jim; Olsen, Jeanine; Pearson, Gareth

    2011-01-01

    We characterized 35 single nucleotide polymorphism (SNP) markers for the brown alga Fucus vesiculosus. Based on existing Fucus Expressed Sequence Tag libraries for heat and desiccation-stressed tissue, SNPs were developed and confirmed by re-sequencing cDNA from a diverse panel of individuals. SNP

  20. Targeted exonic sequencing of GWAS loci in the high extremes of the plasma lipids distribution

    NARCIS (Netherlands)

    Patel, Aniruddh P.; Peloso, Gina M.; Pirruccello, James P.; Johansen, Christopher T.; Dubé, Joseph B.; Larach, Daniel B.; Ban, Matthew R.; Dallinge-Thie, Geesje M.; Gupta, Namrata; Boehnke, Michael; Abecasis, Gonçalo R.; Kastelein, John J. P.; Hovingh, G. Kees; Hegele, Robert A.; Rader, Daniel J.; Kathiresan, Sekar

    2016-01-01

    Genome-wide association studies (GWAS) for plasma lipid levels have mapped numerous genomic loci, with each region often containing many protein-coding genes. Targeted re-sequencing of exons is a strategy to pinpoint causal variants and genes. We performed solution-based hybrid selection of 9008

  1. Translational genomics and marker assisted selection in sorghum case study using brown midrib (bmr) trait

    Science.gov (United States)

    Translational genomics is a critical phase in harnessing the rich genomic data available for sorghum. There is a need to transform nucleotide variation data between sorghum germplasm such as that derived from RNA seq, genotype by sequencing (gbs) or whole genome resequencing thru translation and...

  2. Genome sequences of Phytophthora enable translational plant disease management and accelerate research

    Science.gov (United States)

    Niklaus J. Grünwald

    2012-01-01

    Whole and partial genome sequences are becoming available at an ever-increasing pace. For many plant pathogen systems, we are moving into the era of genome resequencing. The first Phytophthora genomes, P. ramorum and P. sojae, became available in 2004, followed shortly by P. infestans...

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

    Science.gov (United States)

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

    2014-01-01

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

  4. A thaumatin-like genomic sequence identification in Vitis vinifera l., stormy wines and musts based on direct pcr

    Directory of Open Access Journals (Sweden)

    Jana Žiarovská

    2018-03-01

    Full Text Available Normal 0 21 false false false MicrosoftInternetExplorer4 Direct polymerase chain reaction method was use to amplify a thaumatin-like sequence of Vitis vinifera L. in grapes as well as in stormy wines and musts. Thaumatin-like proteins (TLPs of Vitis vinifera possess beside its function in abiotic and biotic stress response another one - they are able to cause protein haze in wine unless removed prior to bottling. Direct PCR is an approach where omission of DNA extraction is typical prior the amplification of the target site of plant genome. Crude extract or small pieces of plant tissues are used in the analysis directly without steps of extraction and purification of gDNA. The biological material that was used in analysis was collected during August - October 2017 in local stores and winery Sabo and comprises from cultivars Iršai, Muškát, Savignon Blanc, Svätovavrinecké, Dornfelder and Pálava. Direct PCR was performed by a cutted piece of grape tissue and a dilution buffer was use in 1:2 for stormy wine or must, respectively. Direct amplification of thaumatin-like protein sequence of Vitis vinifera was performed along with the control reactions with the primers for conserved region of plant chloroplast. Possitive amplification of thaumatin-like allergen sequence resulted in 570 bp amplicon. The most abundant amplicons were amplified in stormy wines, followed by musts and the amplicons from grapes were weaker when comparing them to others. The amplicon specificity checking of obtained PCR product of thaumatin-like allergen was performed by restriction cleavage by Psi I and resulted in restriction amplicons of the 80 bp, 81 bp, 94 bp and 315 bp in length. Confirmation of the amplicon specificity by restriction cleavage support the potential of direct PCR to become a reproducible method that will be fully applicable in routine analysis of not only plant genomes in the future, but it was demonstrated, that it works in liquids, too.  

  5. Identification and characterization of two functional variants in the human longevity gene FOXO3

    DEFF Research Database (Denmark)

    Flachsbart, Friederike; Dose, Janina; Gentschew, Liljana

    2017-01-01

    FOXO3 is consistently annotated as a human longevity gene. However, functional variants and underlying mechanisms for the association remain unknown. Here, we perform resequencing of the FOXO3 locus and single-nucleotide variant (SNV) genotyping in three European populations. We find two FOXO3 SN...

  6. Spliceosomal gene aberrations are rare, coexist with oncogenic mutations, and are unlikely to exert a driver effect in childhood MDS and JMML

    NARCIS (Netherlands)

    S. Hirabayashi (Shinsuke); C. Flotho (Christian); J. Moetter (Jessica); M. Heuser (Michael); H. Hasle (Henrik); B. Gruhn (Bernd); T. Klingebiel (Thomas); F. Thol (Felicitas); B. Schlegelberger (Brigitte); I. Baumann (Irith); B. Strahm (Brigitte); J. Stary (Jan); F. Locatelli (Franco); M. Zecca (Marco); E. Bergstraesser (Eva); M.N. Dworzak (Michael); M.M. van den Heuvel-Eibrink (Marry); B. de Moerloose (Barbara); S. Ogawa (Susumu); C.M. Niemeyer (Charlotte); M. Wlodarski (Marcin)

    2012-01-01

    textabstractSomatic mutations of the spliceosomal machinery occur frequently in adult patients with myelodysplastic syndrome (MDS). We resequenced SF3B1, U2AF35, and SRSF2 in 371 children with MDS or juvenile myelomonocytic leukemia. We found missense mutations in 2 juvenile myelomonocytic leukemia

  7. Duplicated Enhancer Region Increases Expression of CTSB and Segregates with Keratolytic Winter Erythema in South African and Norwegian Families

    NARCIS (Netherlands)

    Ngcungcu, T.; Oti, M.O.; Sitek, J.C.; Haukanes, B.I.; Linghu, B.; Bruccoleri, R.; Stokowy, T.; Oakeley, E.J.; Yang, F; Zhu, J.; Sultan, M.; Schalkwijk, J.; Vlijmen-Willems, I.M.J.J. van; Lippe, C.; Brunner, H.G.; Ersland, K.M.; Grayson, W.; Buechmann-Moller, S.; Sundnes, O.; Nirmala, N.; Morgan, T.M.; Bokhoven, H. van; Steen, V.M.; Hull, P.R.; Szustakowski, J.; Staedtler, F.; Zhou, H.; Fiskerstrand, T.; Ramsay, M.

    2017-01-01

    Keratolytic winter erythema (KWE) is a rare autosomal-dominant skin disorder characterized by recurrent episodes of palmoplantar erythema and epidermal peeling. KWE was previously mapped to 8p23.1-p22 (KWE critical region) in South African families. Using targeted resequencing of the KWE critical

  8. Transcriptome profiling of Curcuma longa L. cv. Suvarna

    Directory of Open Access Journals (Sweden)

    Ambika Sahoo

    2016-12-01

    Full Text Available Turmeric is an economically valued crop, because of its utility in the food, pharmaceutical industries and Ayurvedic medicine, attracts the attention in many areas of research work. In the present study, we executed resequencing through transcriptome assembly of the turmeric cultivar Suvarna (CL_Suv_10. Resequencing of Suvarna variety has generated 5 Gbases raw data with 75 bp paired-end sequence. The raw data has been submitted to SRA database of NCBI with accession number SRR4042181. Reads were assembled using Cufflinks-2.2.1 tool which ended up with 42994 numbers of transcripts. The length of transcripts ranged from 83 to15565, with a N50 value 1216 and median transcript length 773. The transcripts were annotated through number of databases. For the first time transcriptome profiling of cultivar Suvarna has been done, which could help towards identification of single nucleotide polymorphisms (SNPs between Suvarna and other turmeric cultivars for its authentic identification.

  9. Detecting directional selection in the presence of recent admixture in African-Americans.

    Science.gov (United States)

    Lohmueller, Kirk E; Bustamante, Carlos D; Clark, Andrew G

    2011-03-01

    We investigate the performance of tests of neutrality in admixed populations using plausible demographic models for African-American history as well as resequencing data from African and African-American populations. The analysis of both simulated and human resequencing data suggests that recent admixture does not result in an excess of false-positive results for neutrality tests based on the frequency spectrum after accounting for the population growth in the parental African population. Furthermore, when simulating positive selection, Tajima's D, Fu and Li's D, and haplotype homozygosity have lower power to detect population-specific selection using individuals sampled from the admixed population than from the nonadmixed population. Fay and Wu's H test, however, has more power to detect selection using individuals from the admixed population than from the nonadmixed population, especially when the selective sweep ended long ago. Our results have implications for interpreting recent genome-wide scans for positive selection in human populations. © 2011 by the Genetics Society of America

  10. Multiplex amplification of large sets of human exons.

    Science.gov (United States)

    Porreca, Gregory J; Zhang, Kun; Li, Jin Billy; Xie, Bin; Austin, Derek; Vassallo, Sara L; LeProust, Emily M; Peck, Bill J; Emig, Christopher J; Dahl, Fredrik; Gao, Yuan; Church, George M; Shendure, Jay

    2007-11-01

    A new generation of technologies is poised to reduce DNA sequencing costs by several orders of magnitude. But our ability to fully leverage the power of these technologies is crippled by the absence of suitable 'front-end' methods for isolating complex subsets of a mammalian genome at a scale that matches the throughput at which these platforms will routinely operate. We show that targeting oligonucleotides released from programmable microarrays can be used to capture and amplify approximately 10,000 human exons in a single multiplex reaction. Additionally, we show integration of this protocol with ultra-high-throughput sequencing for targeted variation discovery. Although the multiplex capture reaction is highly specific, we found that nonuniform capture is a key issue that will need to be resolved by additional optimization. We anticipate that highly multiplexed methods for targeted amplification will enable the comprehensive resequencing of human exons at a fraction of the cost of whole-genome resequencing.

  11. Mutation in APOA1 predicts increased risk of ischaemic heart disease and total mortality without low HDL cholesterol levels

    DEFF Research Database (Denmark)

    Haase, C L; Frikke-Schmidt, R; Nordestgaard, B G

    2011-01-01

    levels. Mutations in apolipoprotein (apo) A-I, the major protein constituent of HDL, might be associated with low HDL cholesterol and predispose to IHD and early death. DESIGN: We resequenced APOA1 in 190 individuals and examined the effect of mutations on HDL cholesterol, risk of IHD, myocardial...

  12. Elementary Business Calculus with Computer Algebra.

    Science.gov (United States)

    Judson, Phoebe T.

    1990-01-01

    Described are various ways that a computer algebra system (MAPLE) was used to facilitate the resequencing of skills and applications within an elementary college-level business calculus course. Experimental results confirmed earlier findings that skills acquisition is not a prerequisite to conceptual understanding or problem-solving ability. (JJK)

  13. Intrinsic challenges in ancient microbiome reconstruction using 16S rRNA gene amplification.

    Science.gov (United States)

    Ziesemer, Kirsten A; Mann, Allison E; Sankaranarayanan, Krithivasan; Schroeder, Hannes; Ozga, Andrew T; Brandt, Bernd W; Zaura, Egija; Waters-Rist, Andrea; Hoogland, Menno; Salazar-García, Domingo C; Aldenderfer, Mark; Speller, Camilla; Hendy, Jessica; Weston, Darlene A; MacDonald, Sandy J; Thomas, Gavin H; Collins, Matthew J; Lewis, Cecil M; Hofman, Corinne; Warinner, Christina

    2015-11-13

    To date, characterization of ancient oral (dental calculus) and gut (coprolite) microbiota has been primarily accomplished through a metataxonomic approach involving targeted amplification of one or more variable regions in the 16S rRNA gene. Specifically, the V3 region (E. coli 341-534) of this gene has been suggested as an excellent candidate for ancient DNA amplification and microbial community reconstruction. However, in practice this metataxonomic approach often produces highly skewed taxonomic frequency data. In this study, we use non-targeted (shotgun metagenomics) sequencing methods to better understand skewed microbial profiles observed in four ancient dental calculus specimens previously analyzed by amplicon sequencing. Through comparisons of microbial taxonomic counts from paired amplicon (V3 U341F/534R) and shotgun sequencing datasets, we demonstrate that extensive length polymorphisms in the V3 region are a consistent and major cause of differential amplification leading to taxonomic bias in ancient microbiome reconstructions based on amplicon sequencing. We conclude that systematic amplification bias confounds attempts to accurately reconstruct microbiome taxonomic profiles from 16S rRNA V3 amplicon data generated using universal primers. Because in silico analysis indicates that alternative 16S rRNA hypervariable regions will present similar challenges, we advocate for the use of a shotgun metagenomics approach in ancient microbiome reconstructions.

  14. Evaluating Clonal Expansion of HIV-Infected Cells: Optimization of PCR Strategies to Predict Clonality

    Science.gov (United States)

    Laskey, Sarah B.; Pohlmeyer, Christopher W.; Bruner, Katherine M.; Siliciano, Robert F.

    2016-01-01

    In HIV-infected individuals receiving suppressive antiretroviral therapy, the virus persists indefinitely in a reservoir of latently infected cells. The proliferation of these cells may contribute to the stability of the reservoir and thus to the lifelong persistence of HIV-1 in infected individuals. Because the HIV-1 replication process is highly error-prone, the detection of identical viral genomes in distinct host cells provides evidence for the clonal expansion of infected cells. We evaluated alignments of unique, near-full-length HIV-1 sequences to determine the relationship between clonality in a short region and clonality in the full genome. Although it is common to amplify and sequence short, subgenomic regions of the viral genome for phylogenetic analysis, we show that sequence identity of these amplicons does not guarantee clonality across the full viral genome. We show that although longer amplicons capture more diversity, no subgenomic region can recapitulate the diversity of full viral genomes. Consequently, some identical subgenomic amplicons should be expected even from the analysis of completely unique viral genomes, and the presence of identical amplicons alone is not proof of clonally expanded HIV-1. We present a method for evaluating evidence of clonal expansion in the context of these findings. PMID:27494508

  15. Evaluating Clonal Expansion of HIV-Infected Cells: Optimization of PCR Strategies to Predict Clonality.

    Directory of Open Access Journals (Sweden)

    Sarah B Laskey

    2016-08-01

    Full Text Available In HIV-infected individuals receiving suppressive antiretroviral therapy, the virus persists indefinitely in a reservoir of latently infected cells. The proliferation of these cells may contribute to the stability of the reservoir and thus to the lifelong persistence of HIV-1 in infected individuals. Because the HIV-1 replication process is highly error-prone, the detection of identical viral genomes in distinct host cells provides evidence for the clonal expansion of infected cells. We evaluated alignments of unique, near-full-length HIV-1 sequences to determine the relationship between clonality in a short region and clonality in the full genome. Although it is common to amplify and sequence short, subgenomic regions of the viral genome for phylogenetic analysis, we show that sequence identity of these amplicons does not guarantee clonality across the full viral genome. We show that although longer amplicons capture more diversity, no subgenomic region can recapitulate the diversity of full viral genomes. Consequently, some identical subgenomic amplicons should be expected even from the analysis of completely unique viral genomes, and the presence of identical amplicons alone is not proof of clonally expanded HIV-1. We present a method for evaluating evidence of clonal expansion in the context of these findings.

  16. Survey for collagenase gene prtC in Porphyromonas gingivalis and Porphyromonas endodontalis isolated from endodontic infections.

    Science.gov (United States)

    Odell, L J; Baumgartner, J C; Xia, T; David, L L

    1999-08-01

    Collagenase is a potential virulence factor shown to be expressed by Porphyromonas gingivalis associated with periodontal disease. The purpose of this study was to use the polymerase chain reaction (PCR) to detect the presence of the collagenase gene (prtC) in 21 strains of Porphyromonas species isolated from endodontic infections. Type strains for P. gingivalis (ATCC 33277), P. endodontalis (ATCC 35406), Prevotella intermedia (ATCC 25611), and Prevotella nigrescens (ATCC 33563) were used as controls. When PCR primers specific for the 16S ribosomal RNA gene of P. gingivalis or P. endodontalis were used, 16 of the strains were identified as P. gingivalis, and five strains were identified as P. endodontalis. The presence of the prtC gene for collagenase was detected using PCR. Amplicons were analyzed by agarose gel electrophoresis, with an 815 bp amplicon representing the presence of the collagenase gene. Type strain ATCC 33277 and all 16 clinical isolates of P. gingivalis produced the collagenase gene amplicon. Neither type strain ATCC 35406 nor the five strains from clinical isolates of P. endodontalis produced the collagenase gene amplicon. These results indicate that P. gingivalis from endodontic infections possesses the prtC gene. P. endodontalis does not seem to exhibit prtC. The virulence of P. gingivalis may be related to its production of collagenase.

  17. High-resolution melt-curve analysis of random-amplified-polymorphic-DNA markers, for the characterisation of pathogenic Leptospira

    DEFF Research Database (Denmark)

    Tulsiani, Suhella; Craig, S B; Graham, G C

    2010-01-01

    A new test for pathogenic Leptospira isolates, based on RAPD-PCR and high-resolution melt (HRM) analysis (which measures the melting temperature of amplicons in real time, using a fluorescent DNA-binding dye), has recently been developed. A characteristic profile of the amplicons can be used...... typed against 13 previously published RAPD primers, using a real-time cycler (the Corbett Life Science RotorGene 6000) and the optimised reagents from a commercial kit (Quantace SensiMix). RAPD-HRM at specific temperatures generated defining amplicon melt profiles for each of the tested serovars....... These profiles were evaluated as difference-curve graphs generated using the RotorGene software package, with a cut-off of at least 8 'U' (plus or minus). The results demonstrated that RAPD-HRM can be used to measure serovar diversity and establish identity, with a high degree of stability. The characterisation...

  18. Identification of a novel LMF1 nonsense mutation responsible for severe hypertriglyceridemia by targeted next-generation sequencing.

    Science.gov (United States)

    Cefalù, Angelo B; Spina, Rossella; Noto, Davide; Ingrassia, Valeria; Valenti, Vincenza; Giammanco, Antonina; Fayer, Francesca; Misiano, Gabriella; Cocorullo, Gianfranco; Scrimali, Chiara; Palesano, Ornella; Altieri, Grazia I; Ganci, Antonina; Barbagallo, Carlo M; Averna, Maurizio R

    Severe hypertriglyceridemia (HTG) may result from mutations in genes affecting the intravascular lipolysis of triglyceride (TG)-rich lipoproteins. The aim of this study was to develop a targeted next-generation sequencing panel for the molecular diagnosis of disorders characterized by severe HTG. We developed a targeted customized panel for next-generation sequencing Ion Torrent Personal Genome Machine to capture the coding exons and intron/exon boundaries of 18 genes affecting the main pathways of TG synthesis and metabolism. We sequenced 11 samples of patients with severe HTG (TG>885 mg/dL-10 mmol/L): 4 positive controls in whom pathogenic mutations had previously been identified by Sanger sequencing and 7 patients in whom the molecular defect was still unknown. The customized panel was accurate, and it allowed to confirm genetic variants previously identified in all positive controls with primary severe HTG. Only 1 patient of 7 with HTG was found to be carrier of a homozygous pathogenic mutation of the third novel mutation of LMF1 gene (c.1380C>G-p.Y460X). The clinical and molecular familial cascade screening allowed the identification of 2 additional affected siblings and 7 heterozygous carriers of the mutation. We showed that our targeted resequencing approach for genetic diagnosis of severe HTG appears to be accurate, less time consuming, and more economical compared with traditional Sanger resequencing. The identification of pathogenic mutations in candidate genes remains challenging and clinical resequencing should mainly intended for patients with strong clinical criteria for monogenic severe HTG. Copyright © 2017 National Lipid Association. Published by Elsevier Inc. All rights reserved.

  19. PVRL1 as a Candidate Gene for Nonsyndromic Cleft Lip With or Without Cleft Palate: No Evidence for the Involvement of Common or Rare Variants in Southern Han Chinese Patients

    Science.gov (United States)

    Cheng, Hong-Qiu; Huang, En-Min; Xu, Ming-Yan; Shu, Shen-You

    2012-01-01

    The poliovirus receptor related-1 (PVRL1) gene encodes nectin-1, a cell–cell adhesion molecule (OMIM #600644), and is mutated in the cleft lip with or without cleft palate/ectodermal dysplasia-1 syndrome (CLPED1, OMIM #225000). In addition, PVRL1 mutations have been associated with nonsyndromic cleft lip with or without a cleft palate (NSCL/P) in studies of multiethnic samples. To investigate the possible involvement of this gene in southern Han Chinese NSCL/P patients, we performed (i) a case–control association study, and (ii) a resequencing study. A set of 470 patients with NSCL/P and 693 controls were recruited, and a total of 45 tagging single-nucleotide polymorphisms (SNPs) were genotyped by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. In the resequencing study, the coding regions of the PVRL1 α isoform were direct sequenced in 45 trios from multiply affected families. One (rs7128327) of the 45 tested SNPs showed a trend toward statistical significance in the genotypic-level chi-square test (p=0.009567). However, this result did not withstand correction for multiple testing. Likewise, sliding window haplotype analyses consisting of two, three, or four SNPs failed to detect any positive association. Resequencing analysis also failed to identify any novel rare sequence variants. In conclusion, the present study provided no support for the hypothesis that common or rare variants in PVRL1 play a significant role in NSCL/P development in the southern Han Chinese population. This is the first study that has used tagging SNPs covering all the coding and noncoding regions to search for common NSCL/P-associated mutations of PVRL1. PMID:22455396

  20. Vibrio plantisponsor sp. nov., a diazotrophic bacterium isolated from a mangrove associated wild rice (Porteresia coarctata Tateoka)

    Digital Repository Service at National Institute of Oceanography (India)

    Rameshkumar, N.; Gomez-Gil, B.; Sproer, C.; Lang, E.; Kumar, N.D.; Krishnamurthi, S.; Nair, S.; Roque, A.

    nifH amplicon was purified using Eppendorf Perfectprep Gel Cleanup Kit (according to the manufacturer’s instructions). The purified nifH amplicon was used directly for DNA sequencing with the dideoxy chain termination method with the Big...Dye terminator kit (Applied Biosystems) using the above mentioned nifH primers, and the reaction products were analysed by capillary electrophoresis on an ABI 310 Genetic Analyzer (Applied Biosystems). For phylogenetic analysis, partial nifH nucleotide...

  1. Next-Generation Genomics Facility at C-CAMP: Accelerating Genomic Research in India

    Science.gov (United States)

    S, Chandana; Russiachand, Heikham; H, Pradeep; S, Shilpa; M, Ashwini; S, Sahana; B, Jayanth; Atla, Goutham; Jain, Smita; Arunkumar, Nandini; Gowda, Malali

    2014-01-01

    Next-Generation Sequencing (NGS; http://www.genome.gov/12513162) is a recent life-sciences technological revolution that allows scientists to decode genomes or transcriptomes at a much faster rate with a lower cost. Genomic-based studies are in a relatively slow pace in India due to the non-availability of genomics experts, trained personnel and dedicated service providers. Using NGS there is a lot of potential to study India's national diversity (of all kinds). We at the Centre for Cellular and Molecular Platforms (C-CAMP) have launched the Next Generation Genomics Facility (NGGF) to provide genomics service to scientists, to train researchers and also work on national and international genomic projects. We have HiSeq1000 from Illumina and GS-FLX Plus from Roche454. The long reads from GS FLX Plus, and high sequence depth from HiSeq1000, are the best and ideal hybrid approaches for de novo and re-sequencing of genomes and transcriptomes. At our facility, we have sequenced around 70 different organisms comprising of more than 388 genomes and 615 transcriptomes – prokaryotes and eukaryotes (fungi, plants and animals). In addition we have optimized other unique applications such as small RNA (miRNA, siRNA etc), long Mate-pair sequencing (2 to 20 Kb), Coding sequences (Exome), Methylome (ChIP-Seq), Restriction Mapping (RAD-Seq), Human Leukocyte Antigen (HLA) typing, mixed genomes (metagenomes) and target amplicons, etc. Translating DNA sequence data from NGS sequencer into meaningful information is an important exercise. Under NGGF, we have bioinformatics experts and high-end computing resources to dissect NGS data such as genome assembly and annotation, gene expression, target enrichment, variant calling (SSR or SNP), comparative analysis etc. Our services (sequencing and bioinformatics) have been utilized by more than 45 organizations (academia and industry) both within India and outside, resulting several publications in peer-reviewed journals and several genomic

  2. Karyotype variation and conservation in morphotypes of non-heading Chinese cabbage

    NARCIS (Netherlands)

    Zheng, Jin Shuang; Sun, Cheng Zhen; Xiao, Dong; Zhang, Shu Ning; Bonnema, Guusje; Hou, Xi Lin

    2015-01-01

    Non-heading Chinese cabbage encompasses a wide diversity of morphotypes, like the well-known Pak-choi, Wu ta cai, Cai xin, Tai cai, and Fen nei cai. Despite recent developments in re-sequencing which results in the detection of SNPs, insertions, deletions and copy number variants, there has been

  3. SNP discovery in candidate adaptive genes using exon capture in a free-ranging alpine ungulate

    Science.gov (United States)

    Gretchen H. Roffler; Stephen J. Amish; Seth Smith; Ted Cosart; Marty Kardos; Michael K. Schwartz; Gordon Luikart

    2016-01-01

    Identification of genes underlying genomic signatures of natural selection is key to understanding adaptation to local conditions. We used targeted resequencing to identify SNP markers in 5321 candidate adaptive genes associated with known immunological, metabolic and growth functions in ovids and other ungulates. We selectively targeted 8161 exons in protein-coding...

  4. Increased tolerance towards serine obtained by adaptive laboratory evolution

    DEFF Research Database (Denmark)

    Mundhada, Hemanshu; Seoane, Jose Miguel; Koza, Anna

    2014-01-01

    The amino acid serine has previously been identified as one of the top 30 candidates of value added chemicals, making the production of serine from glucose attractive. Production of serine have previously been attempted in E. coli and C. glutamicum, however, titers sufficient for commercial...... by glyA), the conversion of serine to pyruvate (encoded by sdaA, sdaB and tdcG) was also deleted. As expected, the resulting strain turned out to be susceptible to even low concentrations of serine in the media. In order to improve the tolerance of the strain towards serine, adaptive laboratory evolution....... During the evolution experiment, the serine tolerance was increased substantially. Genome re-sequencing was subsequently used to analyze the genotype of a number of selected strains. These results reveal insights towards the adaptation process as well as the mechanism of serine tolerance....

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

    Indian Academy of Sciences (India)

    2013-10-01

    Oct 1, 2013 ... Nitrogen-fixing actinomycete micro-symbiont Frankia nodulates roots of ... the tree is useful in stabilizing nitrogen-depleted soils. Two species of genus .... labelled plastic bags, brought to the laboratory and stored at. −80°C for ...

  6. A morphogenetic survey on ciliate plankton from a mountain lake pinpoints the necessity of lineage-specific barcode markers in microbial ecology.

    Science.gov (United States)

    Stoeck, Thorsten; Breiner, Hans-Werner; Filker, Sabine; Ostermaier, Veronika; Kammerlander, Barbara; Sonntag, Bettina

    2014-02-01

    Analyses of high-throughput environmental sequencing data have become the 'gold-standard' to address fundamental questions of microbial diversity, ecology and biogeography. Findings that emerged from sequencing are, e.g. the discovery of the extensive 'rare microbial biosphere' and its potential function as a seed-bank. Even though applied since several years, results from high-throughput environmental sequencing have hardly been validated. We assessed how well pyrosequenced amplicons [the hypervariable eukaryotic V4 region of the small subunit ribosomal RNA (SSU rRNA) gene] reflected morphotype ciliate plankton. Moreover, we assessed if amplicon sequencing had the potential to detect the annual ciliate plankton stock. In both cases, we identified significant quantitative and qualitative differences. Our study makes evident that taxon abundance distributions inferred from amplicon data are highly biased and do not mirror actual morphotype abundances at all. Potential reasons included cell losses after fixation, cryptic morphotypes, resting stages, insufficient sequence data availability of morphologically described species and the unsatisfying resolution of the V4 SSU rRNA fragment for accurate taxonomic assignments. The latter two underline the necessity of barcoding initiatives for eukaryotic microbes to better and fully exploit environmental amplicon data sets, which then will also allow studying the potential of seed-bank taxa as a buffer for environmental changes. © 2013 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.

  7. Complete Genomes of Classical Swine Fever Virus Cloned into Bacterial Artificial Chromosomes

    DEFF Research Database (Denmark)

    Rasmussen, Thomas Bruun; Reimann, I.; Uttenthal, Åse

    Complete genome amplification of viral RNA provides a new tool for the generation of modified pestiviruses. We have used our full-genome amplification strategy for generation of amplicons representing complete genomes of classical swine fever virus. The amplicons were cloned directly into a stabl...... single-copy bacterial artificial chromosome (BAC) generating full-length pestivirus DNAs from which infectious RNA transcripts could be also derived. Our strategy allows construction of stable infectious BAC DNAs from a single full-length PCR product....

  8. Mutations in the GABA Transporter SLC6A1 Cause Epilepsy with Myoclonic-Atonic Seizures

    DEFF Research Database (Denmark)

    Carvill, Gemma L; McMahon, Jacinta M; Schneider, Amy

    2015-01-01

    GAT-1, encoded by SLC6A1, is one of the major gamma-aminobutyric acid (GABA) transporters in the brain and is responsible for re-uptake of GABA from the synapse. In this study, targeted resequencing of 644 individuals with epileptic encephalopathies led to the identification of six SLC6A1 mutatio...

  9. Polymorphism’s assessment of children’s candidate genes associated with low-level long-term exposure to strontium in drinking water

    Directory of Open Access Journals (Sweden)

    N.V. Zaitseva

    2015-12-01

    Full Text Available A sequencing of the candidate genes of the pupils, exposed to strontium by the method of targeted resequencing has been performed. It is shown, that under conditions of increased revenues of strontium in drinking water the number of polymorphonuclear altered portions of candidate genes increases. As a result of the targeted resequencing in conditions of strontium exposure, the maximum polymorph modifications of the following genes are defined: sulfotransferase 1A1 (SULT1A1 and methylenetetrahydrofolate. It was shown that the structure of the mutations in conditions of the strontium exposure was characterized by the formation of defects in the gene mapping detoxification (38.5 % of all mutations and immunoregulation (22.5 %. Analysis of the cause-effect relationships in the system "factor - the number of mutations" revealed that candidate genes reflecting strontium exposure conditions (content of strontium in drinking water is 1.3 MAC, are genes: cytochrome P450, glutathione - transaminase (detoxification; dopamine (CNS, interleukin 17 and the major histocompatibility complex (immune system, methylene-tetra-hydro-folate-reductase (reproduction.

  10. Reduced representation approaches to interrogate genome diversity in large repetitive plant genomes.

    Science.gov (United States)

    Hirsch, Cory D; Evans, Joseph; Buell, C Robin; Hirsch, Candice N

    2014-07-01

    Technology and software improvements in the last decade now provide methodologies to access the genome sequence of not only a single accession, but also multiple accessions of plant species. This provides a means to interrogate species diversity at the genome level. Ample diversity among accessions in a collection of species can be found, including single-nucleotide polymorphisms, insertions and deletions, copy number variation and presence/absence variation. For species with small, non-repetitive rich genomes, re-sequencing of query accessions is robust, highly informative, and economically feasible. However, for species with moderate to large sized repetitive-rich genomes, technical and economic barriers prevent en masse genome re-sequencing of accessions. Multiple approaches to access a focused subset of loci in species with larger genomes have been developed, including reduced representation sequencing, exome capture and transcriptome sequencing. Collectively, these approaches have enabled interrogation of diversity on a genome scale for large plant genomes, including crop species important to worldwide food security. © The Author 2014. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  11. Polymerase chain reaction: basic protocol plus troubleshooting and optimization strategies.

    Science.gov (United States)

    Lorenz, Todd C

    2012-05-22

    In the biological sciences there have been technological advances that catapult the discipline into golden ages of discovery. For example, the field of microbiology was transformed with the advent of Anton van Leeuwenhoek's microscope, which allowed scientists to visualize prokaryotes for the first time. The development of the polymerase chain reaction (PCR) is one of those innovations that changed the course of molecular science with its impact spanning countless subdisciplines in biology. The theoretical process was outlined by Keppe and coworkers in 1971; however, it was another 14 years until the complete PCR procedure was described and experimentally applied by Kary Mullis while at Cetus Corporation in 1985. Automation and refinement of this technique progressed with the introduction of a thermal stable DNA polymerase from the bacterium Thermus aquaticus, consequently the name Taq DNA polymerase. PCR is a powerful amplification technique that can generate an ample supply of a specific segment of DNA (i.e., an amplicon) from only a small amount of starting material (i.e., DNA template or target sequence). While straightforward and generally trouble-free, there are pitfalls that complicate the reaction producing spurious results. When PCR fails it can lead to many non-specific DNA products of varying sizes that appear as a ladder or smear of bands on agarose gels. Sometimes no products form at all. Another potential problem occurs when mutations are unintentionally introduced in the amplicons, resulting in a heterogeneous population of PCR products. PCR failures can become frustrating unless patience and careful troubleshooting are employed to sort out and solve the problem(s). This protocol outlines the basic principles of PCR, provides a methodology that will result in amplification of most target sequences, and presents strategies for optimizing a reaction. By following this PCR guide, students should be able to: • Set up reactions and thermal cycling

  12. Sensor Systems with Magnetic and Optomagnetic Readout of Rolling Circle Amplification Products

    DEFF Research Database (Denmark)

    Hansen, Mikkel Fougt; Donolato, Marco; Fock, Jeppe

    2016-01-01

    We are developing robust biosensors for homogeneous detection of rolling cirle amplification (RCA) products with magnetic and/or optomagnetic readouts based on surface-functionalized magnetic nanoparticles. Binding of RCA amplicons to nanoparticles modifies their ability to rotate in response...... to an applied oscillating magnetic field. As a result, magnetic or optical measurements of these changes in the rotational response of nanoparticles vs. frequency of the magnetic field can be used to quantitate the number of amplicons, and, hence, the concentration of target nucleic acid analytes. After...

  13. Genomic landscapes of Chinese hamster ovary cell lines as revealed by the Cricetulus griseus draft genome

    DEFF Research Database (Denmark)

    Lewis, Nathan E; Liu, Xin; Li, Yuxiang

    2013-01-01

    stymied by the lack of a unifying genomic resource for CHO cells. Here we report a 2.4-Gb draft genome sequence of a female Chinese hamster, Cricetulus griseus, harboring 24,044 genes. We also resequenced and analyzed the genomes of six CHO cell lines from the CHO-K1, DG44 and CHO-S lineages...

  14. Reducing Design Cycle Time and Cost Through Process Resequencing

    Science.gov (United States)

    Rogers, James L.

    2004-01-01

    In today's competitive environment, companies are under enormous pressure to reduce the time and cost of their design cycle. One method for reducing both time and cost is to develop an understanding of the flow of the design processes and the effects of the iterative subcycles that are found in complex design projects. Once these aspects are understood, the design manager can make decisions that take advantage of decomposition, concurrent engineering, and parallel processing techniques to reduce the total time and the total cost of the design cycle. One software tool that can aid in this decision-making process is the Design Manager's Aid for Intelligent Decomposition (DeMAID). The DeMAID software minimizes the feedback couplings that create iterative subcycles, groups processes into iterative subcycles, and decomposes the subcycles into a hierarchical structure. The real benefits of producing the best design in the least time and at a minimum cost are obtained from sequencing the processes in the subcycles.

  15. Targeted resequencing and variant validation using pxlence PCR assays

    Directory of Open Access Journals (Sweden)

    Frauke Coppieters

    2016-01-01

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

  16. Whole-genome resequencing reveals candidate mutations for pig prolificacy.

    Science.gov (United States)

    Li, Wen-Ting; Zhang, Meng-Meng; Li, Qi-Gang; Tang, Hui; Zhang, Li-Fan; Wang, Ke-Jun; Zhu, Mu-Zhen; Lu, Yun-Feng; Bao, Hai-Gang; Zhang, Yuan-Ming; Li, Qiu-Yan; Wu, Ke-Liang; Wu, Chang-Xin

    2017-12-20

    Changes in pig fertility have occurred as a result of domestication, but are not understood at the level of genetic variation. To identify variations potentially responsible for prolificacy, we sequenced the genomes of the highly prolific Taihu pig breed and four control breeds. Genes involved in embryogenesis and morphogenesis were targeted in the Taihu pig, consistent with the morphological differences observed between the Taihu pig and others during pregnancy. Additionally, excessive functional non-coding mutations have been specifically fixed or nearly fixed in the Taihu pig. We focused attention on an oestrogen response element (ERE) within the first intron of the bone morphogenetic protein receptor type-1B gene ( BMPR1B ) that overlaps with a known quantitative trait locus (QTL) for pig fecundity. Using 242 pigs from 30 different breeds, we confirmed that the genotype of the ERE was nearly fixed in the Taihu pig. ERE function was assessed by luciferase assays, examination of histological sections, chromatin immunoprecipitation, quantitative polymerase chain reactions, and western blots. The results suggest that the ERE may control pig prolificacy via the cis-regulation of BMPR1B expression. This study provides new insight into changes in reproductive performance and highlights the role of non-coding mutations in generating phenotypic diversity between breeds. © 2017 The Author(s).

  17. Structure of the Bacterial Community in Different Stages of Early Childhood Caries.

    Science.gov (United States)

    Ximenes, Marcos; Armas, Rafael Dutra de; Triches, Thaisa Cezária; Cardoso, Mariane; Vieira, Ricardo de Souza

    2018-01-15

    To characterise in vivo the structure of bacterial communities in decayed and sound primary teeth. Samples of biofilms were collected from three groups of patients with complete and exclusively primary dentition (n = 45): G1: sound teeth (n = 15); G2: enamel lesion (n = 15); G3: dentin lesion (n = 15). DNA was extracted (CTAB 2%) from the biofilm, the partial 16S rRNA gene was amplified with Bacteria Universal Primers (BA338fGC - UN518r) and subjected to DGGE (denaturing gradient gel electrophoresis). Multidimensional scaling and ANOSIM (analysis of similarity) were employed to determine the structure of the bacterial communities. The amplicon richness was determined by averaging amplicons, with the differences between treatments determined with ANOVA, while means were compared using Tukey's test (p < 0.05). Compared to sound teeth, a greater variety of bacterial communities was found in decayed teeth. Despite the differences between the bacterial communities of sound teeth and decayed teeth, the Venn diagram showed that the samples had 38 amplicons in common. Greater amplicon richness was observed in samples of decayed teeth (enamel: 20.5 ± 2.7; dentin: 20.1 ± 2.8) compared with the sound samples (12.0 ± 4.3) (p <0.05), indicating enhanced growth for specific groups of bacteria on decayed teeth. Although there is less bacterial diversity on sound than ECC-decayed teeth, the bacterial communities are very similar.

  18. Unlabeled probes for the detection and typing of herpes simplex virus.

    Science.gov (United States)

    Dames, Shale; Pattison, David C; Bromley, L Kathryn; Wittwer, Carl T; Voelkerding, Karl V

    2007-10-01

    Unlabeled probe detection with a double-stranded DNA (dsDNA) binding dye is one method to detect and confirm target amplification after PCR. Unlabeled probes and amplicon melting have been used to detect small deletions and single-nucleotide polymorphisms in assays where template is in abundance. Unlabeled probes have not been applied to low-level target detection, however. Herpes simplex virus (HSV) was chosen as a model to compare the unlabeled probe method to an in-house reference assay using dual-labeled, minor groove binding probes. A saturating dsDNA dye (LCGreen Plus) was used for real-time PCR. HSV-1, HSV-2, and an internal control were differentiated by PCR amplicon and unlabeled probe melting analysis after PCR. The unlabeled probe technique displayed 98% concordance with the reference assay for the detection of HSV from a variety of archived clinical samples (n = 182). HSV typing using unlabeled probes was 99% concordant (n = 104) to sequenced clinical samples and allowed for the detection of sequence polymorphisms in the amplicon and under the probe. Unlabeled probes and amplicon melting can be used to detect and genotype as few as 10 copies of target per reaction, restricted only by stochastic limitations. The use of unlabeled probes provides an attractive alternative to conventional fluorescence-labeled, probe-based assays for genotyping and detection of HSV and might be useful for other low-copy targets where typing is informative.

  19. Generation of recombinant pestiviruses using a full-genome amplification strategy

    DEFF Research Database (Denmark)

    Rasmussen, Thomas Bruun; Reimann, I.; Uttenthal, Åse

    2010-01-01

    -Gifhorn genome was generated by long RTPCR and then RNA transcripts derived from this amplicon were used to rescue infectious virus. Here, we have now used this full-genome amplification strategy for efficient and robust amplification of three additional pestivirus strains: the vaccine strain C and the virulent...... Paderborn strain of Classical swine fever virus plus the CP7 strain of Bovine viral diarrhoea virus. The amplicons were cloned directly into a stable single-copy bacterial artificial chromosome generating full-length pestivirus DNAs from which infectious RNA transcripts could be also derived....

  20. Multiplexed homogeneous proximity ligation assays for high throughput protein biomarker research in serological material

    DEFF Research Database (Denmark)

    Lundberg, Martin; Thorsen, Stine Buch; Assarsson, Erika

    2011-01-01

    A high throughput protein biomarker discovery tool has been developed based on multiplexed proximity ligation assays (PLA) in a homogeneous format in the sense of no washing steps. The platform consists of four 24-plex panels profiling 74 putative biomarkers with sub pM sensitivity each consuming...... sequences are united by DNA ligation upon simultaneous target binding forming a PCR amplicon. Multiplex PLA thereby converts multiple target analytes into real-time PCR amplicons that are individually quantificatied using microfluidic high capacity qPCR in nano liter volumes. The assay shows excellent...

  1. Clinical application of RT-nested PCR integrated with RFLP in Hantavirus detection and genotyping: a prospective study in Shandong Province, PR China.

    Science.gov (United States)

    Liu, Yun-Xi; Zhao, Zhong-Tang; Cao, Wu-Chun; Xu, Xiao-Qun; Suo, Ji-Jiang; Xing, Yu-Bin; Jia, Ning; Du, Ming-Mei; Liu, Bo-Wei; Yao, Yuan

    2013-01-01

    The aim of the present study was to evaluate the clinical usefulness of applying RT-nested PCR along with RFLP as a method for diagnosis and genotypic differentiation of Hantavirus in the acute-stage sera of HFRS patients as compared to the ELISA technique. A prospective study of patients with suspected HFRS patients was carried out. Sera were collected for serological evaluation by ELISA and RT-nested PCR testing. Primers were selected from the published sequence of the S segment of HTNV strain 76-118 and SEOV strain SR-11, which made it possible to obtain an amplicon of 403 bp by RT-nested PCR. The genotypic differentiations of the RT-nested PCR amplicons were carried out by RFLP. Sequence analyses of the amplicons were used to confirm the accuracy of the results obtained by RFLP. Of the 48 acute-stage sera from suspected HFRS patients, 35 were ELISA-positive while 41 were positive by RT-nested PCR. With Hind III and Hinf I, RFLP profiles of the RT-nested PCR amplicons of the 41 positive sera exhibited two patterns. 33 had RFLP profiles similar to the reference strain R22, and thus belonged to the SEOV type. The other 8 samples which were collected during October-December had RFLP profiles similar to the reference strain 76-118, and thus belonged to the HTNV type. Sequence phylogenetic analysis of RT-nested PCR amplicons revealed sdp1, sdp2 YXL-2008, and sdp3 as close relatives of HTNV strain 76-118, while sdp22 and sdp37 as close relatives of SEOV strain Z37 and strain R22 located in two separate clusters in the phylogenetic tree. These results were identical to those acquired by RFLP. RT-nested PCR integrated with RFLP was a rapid, simple, accurate method for detecting and differentiating the genotypes of Hantavirus in the acute-stage sera of suspected HFRS patients. In Shandong province, the main genotypes of Hantavirus belonged to the SEOV types, while the HTNV types were observed during the autumn-winter season.

  2. Pseudogene of dihydrolipoyl succinyltransferase (E2k) found by PCR amplification and direct sequencing of rodent-human cell hybrid DNAs

    Energy Technology Data Exchange (ETDEWEB)

    Cai, X.; Ali, G.; Blass, J.P. [Cornell Univ. Medical College, White Plains, NY (United States); Szabo, P. [Cornell Univ. Medical College, New York, NY (United States); Tanzi, R.E. [Massachusetts General Hospital, Boston, MA (United States)

    1994-07-01

    Previous studies have indicated that the cDNA for the E2k component of the human {alpha}-ketoglutarate dehydrogenase complex (KGDHC) hybridized not only to a major locus on chromosome 14q24.3 in a region associated with familial Alzheimer`s disease and with Joseph-Machado disease, but also to another locus on chromosome 1p31. The authors now report that PCR of genomic DNA and direct sequencing indicated that the chromosome 1 locus is an intronless pseudogene. PCR of genomic DNA amplified E2k fragments from mouse-human cell hybrids containing human chromosome 1 DNA but not from hybrids containing human chromosome 14 DNA. The resulting amplicons were of comparable sizes to those when the cDNA was used to template. The direct sequencing of these amplicons confirmed the lack of introns and indicated a frame shift, which led to the presence of four termination codons early in the coding region. PCR followed by direct sequencing of the amplicons appears to be a convenient method for identifying intronless pseudogenes.

  3. Capillary electrophoresis with electrochemiluminescent detection for highly sensitive assay of genetically modified organisms.

    Science.gov (United States)

    Guo, Longhua; Yang, Huanghao; Qiu, Bin; Xiao, Xueyang; Xue, Linlin; Kim, Donghwan; Chen, Guonan

    2009-12-01

    A capillary electrophoresis coupled with electrochemiluminescent detection system (CE-ECL) was developed for the detection of polymerase chain reaction (PCR) amplicons. The ECL luminophore, tris(1,10-phenanthroline) ruthenium(II) (Ru(phen)(3)(2+)), was labeled to the PCR primers before amplification. Ru(phen)(3)(2+) was then introduced to PCR amplicons by PCR amplification. Eventually, the PCR amplicons were separated and detected by the homemade CE-ECL system. The detection of a typical genetically modified organism (GMO), Roundup Ready Soy (RRS), was shown as an example to demonstrate the reliability of the proposed approach. Four pairs of primers were amplified by multiple PCR (MPCR) simultaneously, three of which were targeted on the specific sequence of exogenous genes of RRS, and another was targeted on the endogenous reference gene of soybean. Both the conditions for PCR amplification and CE-ECL separation and detection were investigated in detail. Results showed that, under the optimal conditions, the proposed method can accurately identifying RRS. The corresponding limit of detection (LOD) was below 0.01% with 35 PCR cycles.

  4. Rapid Detection of the Chlamydiaceae and Other Families in the Order Chlamydiales: Three PCR Tests

    Science.gov (United States)

    Everett, Karin D. E.; Hornung, Linda J.; Andersen, Arthur A.

    1999-01-01

    Few identification methods will rapidly or specifically detect all bacteria in the order Chlamydiales, family Chlamydiaceae. In this study, three PCR tests based on sequence data from over 48 chlamydial strains were developed for identification of these bacteria. Two tests exclusively recognized the Chlamydiaceae: a multiplex test targeting the ompA gene and the rRNA intergenic spacer and a TaqMan test targeting the 23S ribosomal DNA. The multiplex test was able to detect as few as 200 inclusion-forming units (IFU), while the TaqMan test could detect 2 IFU. The amplicons produced in these tests ranged from 132 to 320 bp in length. The third test, targeting the 23S rRNA gene, produced a 600-bp amplicon from strains belonging to several families in the order Chlamydiales. Direct sequence analysis of this amplicon has facilitated the identification of new chlamydial strains. These three tests permit ready identification of chlamydiae for diagnostic and epidemiologic study. The specificity of these tests indicates that they might also be used to identify chlamydiae without culture or isolation. PMID:9986815

  5. Human papillomavirus detection using the Abbott RealTime high-risk HPV tests compared with conventional nested PCR coupled to high-throughput sequencing of amplification products in cervical smear specimens from a Gabonese female population.

    Science.gov (United States)

    Moussavou-Boundzanga, Pamela; Koumakpayi, Ismaël Hervé; Labouba, Ingrid; Leroy, Eric M; Belembaogo, Ernest; Berthet, Nicolas

    2017-12-21

    Cervical cancer is the fourth most common malignancy in women worldwide. However, screening with human papillomavirus (HPV) molecular tests holds promise for reducing cervical cancer incidence and mortality in low- and middle-income countries. The performance of the Abbott RealTime High-Risk HPV test (AbRT) was evaluated in 83 cervical smear specimens and compared with a conventional nested PCR coupled to high-throughput sequencing (HTS) to identify the amplicons. The AbRT assay detected at least one HPV genotype in 44.57% of women regardless of the grade of cervical abnormalities. Except for one case, good concordance was observed for the genotypes detected with the AbRT assay in the high-risk HPV category determined with HTS of the amplicon generated by conventional nested PCR. The AbRT test is an easy and reliable molecular tool and was as sensitive as conventional nested PCR in cervical smear specimens for detection HPVs associated with high-grade lesions. Moreover, sequencing amplicons using an HTS approach effectively identified the genotype of the hrHPV identified with the AbRT test.

  6. Denoising PCR-amplified metagenome data

    Directory of Open Access Journals (Sweden)

    Rosen Michael J

    2012-10-01

    Full Text Available Abstract Background PCR amplification and high-throughput sequencing theoretically enable the characterization of the finest-scale diversity in natural microbial and viral populations, but each of these methods introduces random errors that are difficult to distinguish from genuine biological diversity. Several approaches have been proposed to denoise these data but lack either speed or accuracy. Results We introduce a new denoising algorithm that we call DADA (Divisive Amplicon Denoising Algorithm. Without training data, DADA infers both the sample genotypes and error parameters that produced a metagenome data set. We demonstrate performance on control data sequenced on Roche’s 454 platform, and compare the results to the most accurate denoising software currently available, AmpliconNoise. Conclusions DADA is more accurate and over an order of magnitude faster than AmpliconNoise. It eliminates the need for training data to establish error parameters, fully utilizes sequence-abundance information, and enables inclusion of context-dependent PCR error rates. It should be readily extensible to other sequencing platforms such as Illumina.

  7. Region of interest methylation analysis: a comparison of MSP with MS-HRM and direct BSP.

    Science.gov (United States)

    Akika, Reem; Awada, Zainab; Mogharbil, Nahed; Zgheib, Nathalie K

    2017-07-01

    The aim of this study was to compare and contrast three DNA methylation methods of a specific region of interest (ROI): methylation-specific PCR (MSP), methylation-sensitive high resolution melting (MS-HRM) and direct bisulfite sequencing (BSP). The methylation of a CpG area in the promoter region of Estrogen receptor alpha (ESR1) was evaluated by these three methods with samples and standards of different methylation percentages. MSP data were neither reproducible nor sensitive, and the assay was not specific due to non-specific binding of primers. MS-HRM was highly reproducible and a step forward into categorizing the methylation status of the samples as percent ranges. Direct BSP was the most informative method regarding methylation percentage of each CpG site. Though not perfect, it was reproducible and sensitive. We recommend the use of either method depending on the research question and target amplicon, and provided that the designed primers and expected amplicons are within recommendations. If the research question targets a limited number of CpG sites and simple yes/no results are enough, MSP may be attempted. For short amplicons that are crowded with CpG sites and of single melting domain, MS-HRM may be the method of choice though it only indicates the overall methylation percentage of the entire amplicon. Although the assay is highly reproducible, being semi-quantitative makes it of lesser interest to study ROI methylation of samples with little methylation differences. Direct BSP is a step forward as it gives information about the methylation percentage at each CpG site.

  8. Dicty_cDB: Contig-U05677-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available . 40 1.1 2 ( FL644201 ) TS36-G5 Reticulitermes flavipes symbiont library ... 34 1.2 2 ( EX953737 ) IF1_19_H1...na thermophila conjugation cD... 48 9e-06 3 ( CX585524 ) TTE00019768 Amplicon Exp...... 60 1e-04 1 ( CX578285 ) TTE00027054 Amplicon Express - Conjugative Form T... ...8546535 Global-Ocean-Sampling_GS-30-02-01-1... 46 1.8 1 ( EJ825455 ) 1093017558219 Global-Ocean-Sampli...1106744 Global-Ocean-Sampling_GS-31-01-01-1... 44 7.0 1 ( EK280751 ) 1095462293927 Global-Ocean-Sampli

  9. Performance of mitochondrial DNA mutations detecting early stage cancer

    International Nuclear Information System (INIS)

    Jakupciak, John P; Srivastava, Sudhir; Sidransky, David; O'Connell, Catherine D; Maragh, Samantha; Markowitz, Maura E; Greenberg, Alissa K; Hoque, Mohammad O; Maitra, Anirban; Barker, Peter E; Wagner, Paul D; Rom, William N

    2008-01-01

    Mutations in the mitochondrial genome (mtgenome) have been associated with cancer and many other disorders. These mutations can be point mutations or deletions, or admixtures (heteroplasmy). The detection of mtDNA mutations in body fluids using resequencing microarrays, which are more sensitive than other sequencing methods, could provide a strategy to measure mutation loads in remote anatomical sites. We determined the mtDNA mutation load in the entire mitochondrial genome of 26 individuals with different early stage cancers (lung, bladder, kidney) and 12 heavy smokers without cancer. MtDNA was sequenced from three matched specimens (blood, tumor and body fluid) from each cancer patient and two matched specimens (blood and sputum) from smokers without cancer. The inherited wildtype sequence in the blood was compared to the sequences present in the tumor and body fluid, detected using the Affymetrix Genechip ® Human Mitochondrial Resequencing Array 1.0 and supplemented by capillary sequencing for noncoding region. Using this high-throughput method, 75% of the tumors were found to contain mtDNA mutations, higher than in our previous studies, and 36% of the body fluids from these cancer patients contained mtDNA mutations. Most of the mutations detected were heteroplasmic. A statistically significantly higher heteroplasmy rate occurred in tumor specimens when compared to both body fluid of cancer patients and sputum of controls, and in patient blood compared to blood of controls. Only 2 of the 12 sputum specimens from heavy smokers without cancer (17%) contained mtDNA mutations. Although patient mutations were spread throughout the mtDNA genome in the lung, bladder and kidney series, a statistically significant elevation of tRNA and ND complex mutations was detected in tumors. Our findings indicate comprehensive mtDNA resequencing can be a high-throughput tool for detecting mutations in clinical samples with potential applications for cancer detection, but it is

  10. Application of dissociation curve analysis to radiation hybrid panel marker scoring: generation of a map of river buffalo (B. bubalis chromosome 20

    Directory of Open Access Journals (Sweden)

    Schäffer Alejandro A

    2008-11-01

    Full Text Available Abstract Background Fluorescence of dyes bound to double-stranded PCR products has been utilized extensively in various real-time quantitative PCR applications, including post-amplification dissociation curve analysis, or differentiation of amplicon length or sequence composition. Despite the current era of whole-genome sequencing, mapping tools such as radiation hybrid DNA panels remain useful aids for sequence assembly, focused resequencing efforts, and for building physical maps of species that have not yet been sequenced. For placement of specific, individual genes or markers on a map, low-throughput methods remain commonplace. Typically, PCR amplification of DNA from each panel cell line is followed by gel electrophoresis and scoring of each clone for the presence or absence of PCR product. To improve sensitivity and efficiency of radiation hybrid panel analysis in comparison to gel-based methods, we adapted fluorescence-based real-time PCR and dissociation curve analysis for use as a novel scoring method. Results As proof of principle for this dissociation curve method, we generated new maps of river buffalo (Bubalus bubalis chromosome 20 by both dissociation curve analysis and conventional marker scoring. We also obtained sequence data to augment dissociation curve results. Few genes have been previously mapped to buffalo chromosome 20, and sequence detail is limited, so 65 markers were screened from the orthologous chromosome of domestic cattle. Thirty bovine markers (46% were suitable as cross-species markers for dissociation curve analysis in the buffalo radiation hybrid panel under a standard protocol, compared to 25 markers suitable for conventional typing. Computational analysis placed 27 markers on a chromosome map generated by the new method, while the gel-based approach produced only 20 mapped markers. Among 19 markers common to both maps, the marker order on the map was maintained perfectly. Conclusion Dissociation curve

  11. Screening for Glucosyltransferase gene (gtf from exopolysaccahride producing lactic acid bacteria

    Directory of Open Access Journals (Sweden)

    Donna M. Ariestanti

    2008-04-01

    Full Text Available Glucosyltransferase (GTF is an enzyme involved in exopolysaccharide (EPS polymer synthesis in microbes. One example of EPS that has been used in pharmaceutical and medical application is dextran. Dextran has been used in conjugated-drug delivery system as matrix. As a group of microbes producing EPS, lactic acid bacteria (LAB have been well reported carrying sucrase genes glucosyltransferase (gtf, as well as fructosyltransferases (ftf. In an attempt to search for novel gtf genes as the aim of this study, LAB collection isolated from local sources yielded from previous study were screened performing PCR using degenerate primers DegFor and DegRev. An approximately 660 base pairs (bp amplicons were obtained by using genomic DNAs of those LAB isolates as templates with conserved region of gtf genes catalytic domain as target. Two out of 20 LAB strains were yielded no amplicon as observed on agarose gel, while one strain exhibited non-specific amplicon DNA bands with sizes other than 660 bp. The two negative ones were isolated from soil obtained from dairy product waste field and from waste of soy sauce from previous study, while the latter was isolated from waste of soy sauce.

  12. Evaluation of PCR and high-resolution melt curve analysis for differentiation of Salmonella isolates.

    Science.gov (United States)

    Saeidabadi, Mohammad Sadegh; Nili, Hassan; Dadras, Habibollah; Sharifiyazdi, Hassan; Connolly, Joanne; Valcanis, Mary; Raidal, Shane; Ghorashi, Seyed Ali

    2017-06-01

    Consumption of poultry products contaminated with Salmonella is one of the major causes of foodborne diseases worldwide and therefore detection and differentiation of Salmonella spp. in poultry is important. In this study, oligonucleotide primers were designed from hemD gene and a PCR followed by high-resolution melt (HRM) curve analysis was developed for rapid differentiation of Salmonella isolates. Amplicons of 228 bp were generated from 16 different Salmonella reference strains and from 65 clinical field isolates mainly from poultry farms. HRM curve analysis of the amplicons differentiated Salmonella isolates and analysis of the nucleotide sequence of the amplicons from selected isolates revealed that each melting curve profile was related to a unique DNA sequence. The relationship between reference strains and tested specimens was also evaluated using a mathematical model without visual interpretation of HRM curves. In addition, the potential of the PCR-HRM curve analysis was evaluated for genotyping of additional Salmonella isolates from different avian species. The findings indicate that PCR followed by HRM curve analysis provides a rapid and robust technique for genotyping of Salmonella isolates to determine the serovar/serotype.

  13. Prescreening of microbial populations for the assessment of sequencing potential.

    Science.gov (United States)

    Hanning, Irene B; Ricke, Steven C

    2011-01-01

    Next-generation sequencing (NGS) is a powerful tool that can be utilized to profile and compare microbial populations. By amplifying a target gene present in all bacteria and subsequently sequencing amplicons, the bacteria genera present in the populations can be identified and compared. In some scenarios, little to no difference may exist among microbial populations being compared in which case a prescreening method would be practical to determine which microbial populations would be suitable for further analysis by NGS. Denaturing density-gradient electrophoresis (DGGE) is relatively cheaper than NGS and the data comparing microbial populations are ready to be viewed immediately after electrophoresis. DGGE follows essentially the same initial methodology as NGS by targeting and amplifying the 16S rRNA gene. However, as opposed to sequencing amplicons, DGGE amplicons are analyzed by electrophoresis. By prescreening microbial populations with DGGE, more efficient use of NGS methods can be accomplished. In this chapter, we outline the protocol for DGGE targeting the same gene (16S rRNA) that would be targeted for NGS to compare and determine differences in microbial populations from a wide range of ecosystems.

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

    Science.gov (United States)

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

    2015-09-16

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

  15. Mutation Detection with Next-Generation Resequencing through a Mediator Genome

    Energy Technology Data Exchange (ETDEWEB)

    Wurtzel, Omri; Dori-Bachash, Mally; Pietrokovski, Shmuel; Jurkevitch, Edouard; Sorek, Rotem; Ben-Jacob, Eshel

    2010-12-31

    The affordability of next generation sequencing (NGS) is transforming the field of mutation analysis in bacteria. The genetic basis for phenotype alteration can be identified directly by sequencing the entire genome of the mutant and comparing it to the wild-type (WT) genome, thus identifying acquired mutations. A major limitation for this approach is the need for an a-priori sequenced reference genome for the WT organism, as the short reads of most current NGS approaches usually prohibit de-novo genome assembly. To overcome this limitation we propose a general framework that utilizes the genome of relative organisms as mediators for comparing WT and mutant bacteria. Under this framework, both mutant and WT genomes are sequenced with NGS, and the short sequencing reads are mapped to the mediator genome. Variations between the mutant and the mediator that recur in the WT are ignored, thus pinpointing the differences between the mutant and the WT. To validate this approach we sequenced the genome of Bdellovibrio bacteriovorus 109J, an obligatory bacterial predator, and its prey-independent mutant, and compared both to the mediator species Bdellovibrio bacteriovorus HD100. Although the mutant and the mediator sequences differed in more than 28,000 nucleotide positions, our approach enabled pinpointing the single causative mutation. Experimental validation in 53 additional mutants further established the implicated gene. Our approach extends the applicability of NGS-based mutant analyses beyond the domain of available reference genomes.

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

    International Nuclear Information System (INIS)

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

    1997-01-01

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

  17. PopGenome: An Efficient Swiss Army Knife for Population Genomic Analyses in R

    OpenAIRE

    Pfeifer, Bastian; Wittelsbürger, Ulrich; Ramos-Onsins, Sebastian E.; Lercher, Martin J.

    2014-01-01

    Although many computer programs can perform population genetics calculations, they are typically limited in the analyses and data input formats they offer; few applications can process the large data sets produced by whole-genome resequencing projects. Furthermore, there is no coherent framework for the easy integration of new statistics into existing pipelines, hindering the development and application of new population genetics and genomics approaches. Here, we present PopGenome, a populati...

  18. Advancing Our Understanding of the Etiologies and Mutational Landscapes of Basal Like, Luminal A, and Luminal B Breast Cancers

    Science.gov (United States)

    2016-10-01

    5-45, completed under Dr. Li’s supervision at FHCRC: a. Identification of controls, Months 5-45: General population controls with no prior history ...sequencing and analysis will be validated by SYBR Green based QPCR using Page 11 primers designed using Primer 3 to encompass the fusion junction on a...pools of multiplexed PCR products resequenced on Illumina next-generation sequencing equipment, we will screen any recurrently mutated gene appearing

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

    Directory of Open Access Journals (Sweden)

    Diana L Cousminer

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

  20. Genotypic adaptations associated with prolonged persistence of Lactobacillus plantarum in the murine digestive tract.

    Science.gov (United States)

    van Bokhorst-van de Veen, Hermien; Smelt, Maaike J; Wels, Michiel; van Hijum, Sacha A F T; de Vos, Paul; Kleerebezem, Michiel; Bron, Peter A

    2013-08-01

    Probiotic bacteria harbor effector molecules that confer health benefits, but also adaptation factors that enable them to persist in the gastrointestinal tract of the consumer. To study these adaptation factors, an antibiotic-resistant derivative of the probiotic model organism Lactobacillus plantarum WCFS1 was repeatedly exposed to the mouse digestive tract by three consecutive rounds of (re)feeding of the longest persisting colonies. This exposure to the murine intestine allowed the isolation of intestine-adapted derivatives of the original strain that displayed prolonged digestive tract residence time. Re-sequencing of the genomes of these adapted derivatives revealed single nucleotide polymorphisms as well as a single nucleotide insertion in comparison with the genome of the original WCFS1 strain. Detailed in silico analysis of the identified genomic modifications pinpointed that alterations in the coding regions of genes encoding cell envelope associated functions and energy metabolism appeared to be beneficial for the gastrointestinal tract survival of L. plantarum WCFS1. This work demonstrates the feasibility of experimental evolution for the enhancement of the gastrointestinal residence time of probiotic strains, while full-genome resequencing of the adapted isolates provided clues towards the bacterial functions involved. Enhanced gastrointestinal residence is industrially relevant because it enhances the efficacy of the delivery of viable probiotics in situ.

  1. Draft genome sequence of an elite Dura palm and whole-genome patterns of DNA variation in oil palm.

    Science.gov (United States)

    Jin, Jingjing; Lee, May; Bai, Bin; Sun, Yanwei; Qu, Jing; Rahmadsyah; Alfiko, Yuzer; Lim, Chin Huat; Suwanto, Antonius; Sugiharti, Maria; Wong, Limsoon; Ye, Jian; Chua, Nam-Hai; Yue, Gen Hua

    2016-12-01

    Oil palm is the world's leading source of vegetable oil and fat. Dura, Pisifera and Tenera are three forms of oil palm. The genome sequence of Pisifera is available whereas the Dura form has not been sequenced yet. We sequenced the genome of one elite Dura palm, and re-sequenced 17 palm genomes. The assemble genome sequence of the elite Dura tree contained 10,971 scaffolds and was 1.701 Gb in length, covering 94.49% of the oil palm genome. 36,105 genes were predicted. Re-sequencing of 17 additional palm trees identified 18.1 million SNPs. We found high genetic variation among palms from different geographical regions, but lower variation among Southeast Asian Dura and Pisifera palms. We mapped 10,000 SNPs on the linkage map of oil palm. In addition, high linkage disequilibrium (LD) was detected in the oil palms used in breeding populations of Southeast Asia, suggesting that LD mapping is likely to be practical in this important oil crop. Our data provide a valuable resource for accelerating genetic improvement and studying the mechanism underlying phenotypic variations of important oil palm traits. © The Author 2016. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

  2. The genome of Eucalyptus grandis

    Energy Technology Data Exchange (ETDEWEB)

    Myburg, Alexander A.; Grattapaglia, Dario; Tuskan, Gerald A.; Hellsten, Uffe; Hayes, Richard D.; Grimwood, Jane; Jenkins, Jerry; Lindquist, Erika; Tice, Hope; Bauer, Diane; Goodstein, David M.; Dubchak, Inna; Poliakov, Alexandre; Mizrachi, Eshchar; Kullan, Anand R. K.; Hussey, Steven G.; Pinard, Desre; van der Merwe, Karen; Singh, Pooja; van Jaarsveld, Ida; Silva-Junior, Orzenil B.; Togawa, Roberto C.; Pappas, Marilia R.; Faria, Danielle A.; Sansaloni, Carolina P.; Petroli, Cesar D.; Yang, Xiaohan; Ranjan, Priya; Tschaplinski, Timothy J.; Ye, Chu-Yu; Li, Ting; Sterck, Lieven; Vanneste, Kevin; Murat, Florent; Soler, Marçal; Clemente, Hélène San; Saidi, Naijib; Cassan-Wang, Hua; Dunand, Christophe; Hefer, Charles A.; Bornberg-Bauer, Erich; Kersting, Anna R.; Vining, Kelly; Amarasinghe, Vindhya; Ranik, Martin; Naithani, Sushma; Elser, Justin; Boyd, Alexander E.; Liston, Aaron; Spatafora, Joseph W.; Dharmwardhana, Palitha; Raja, Rajani; Sullivan, Christopher; Romanel, Elisson; Alves-Ferreira, Marcio; Külheim, Carsten; Foley, William; Carocha, Victor; Paiva, Jorge; Kudrna, David; Brommonschenkel, Sergio H.; Pasquali, Giancarlo; Byrne, Margaret; Rigault, Philippe; Tibbits, Josquin; Spokevicius, Antanas; Jones, Rebecca C.; Steane, Dorothy A.; Vaillancourt, René E.; Potts, Brad M.; Joubert, Fourie; Barry, Kerrie; Pappas, Georgios J.; Strauss, Steven H.; Jaiswal, Pankaj; Grima-Pettenati, Jacqueline; Salse, Jérôme; Van de Peer, Yves; Rokhsar, Daniel S.; Schmutz, Jeremy

    2014-06-11

    Eucalypts are the world s most widely planted hardwood trees. Their broad adaptability, rich species diversity, fast growth and superior multipurpose wood, have made them a global renewable resource of fiber and energy that mitigates human pressures on natural forests. We sequenced and assembled >94% of the 640 Mbp genome of Eucalyptus grandis into its 11 chromosomes. A set of 36,376 protein coding genes were predicted revealing that 34% occur in tandem duplications, the largest proportion found thus far in any plant genome. Eucalypts also show the highest diversity of genes for plant specialized metabolism that act as chemical defence against biotic agents and provide unique pharmaceutical oils. Resequencing of a set of inbred tree genomes revealed regions of strongly conserved heterozygosity, likely hotspots of inbreeding depression. The resequenced genome of the sister species E. globulus underscored the high inter-specific genome colinearity despite substantial genome size variation in the genus. The genome of E. grandis is the first reference for the early diverging Rosid order Myrtales and is placed here basal to the Eurosids. This resource expands knowledge on the unique biology of large woody perennials and provides a powerful tool to accelerate comparative biology, breeding and biotechnology.

  3. Genome-wide DNA polymorphisms in Kavuni, a traditional rice cultivar with nutritional and therapeutic properties.

    Science.gov (United States)

    Rathinasabapathi, Pasupathi; Purushothaman, Natarajan; Parani, Madasamy

    2016-05-01

    Although rice genome was sequenced in the year 2002, efforts in resequencing the large number of available accessions, landraces, traditional cultivars, and improved varieties of this important food crop are limited. We have initiated resequencing of the traditional cultivars from India. Kavuni is an important traditional rice cultivar from South India that attracts premium price for its nutritional and therapeutic properties. Whole-genome sequencing of Kavuni using Illumina platform and SNPs analysis using Nipponbare reference genome identified 1 150 711 SNPs of which 377 381 SNPs were located in the genic regions. Non-synonymous SNPs (62 708) were distributed in 19 251 genes, and their number varied between 1 and 115 per gene. Large-effect DNA polymorphisms (7769) were present in 3475 genes. Pathway mapping of these polymorphisms revealed the involvement of genes related to carbohydrate metabolism, translation, protein-folding, and cell death. Analysis of the starch biosynthesis related genes revealed that the granule-bound starch synthase I gene had T/G SNPs at the first intron/exon junction and a two-nucleotide combination, which were reported to favour high amylose content and low glycemic index. The present study provided a valuable genomics resource to study the rice varieties with nutritional and medicinal properties.

  4. A two-stage stochastic rule-based model to determine pre-assembly buffer content

    Science.gov (United States)

    Gunay, Elif Elcin; Kula, Ufuk

    2018-01-01

    This study considers instant decision-making needs of the automobile manufactures for resequencing vehicles before final assembly (FA). We propose a rule-based two-stage stochastic model to determine the number of spare vehicles that should be kept in the pre-assembly buffer to restore the altered sequence due to paint defects and upstream department constraints. First stage of the model decides the spare vehicle quantities, where the second stage model recovers the scrambled sequence respect to pre-defined rules. The problem is solved by sample average approximation (SAA) algorithm. We conduct a numerical study to compare the solutions of heuristic model with optimal ones and provide following insights: (i) as the mismatch between paint entrance and scheduled sequence decreases, the rule-based heuristic model recovers the scrambled sequence as good as the optimal resequencing model, (ii) the rule-based model is more sensitive to the mismatch between the paint entrance and scheduled sequences for recovering the scrambled sequence, (iii) as the defect rate increases, the difference in recovery effectiveness between rule-based heuristic and optimal solutions increases, (iv) as buffer capacity increases, the recovery effectiveness of the optimization model outperforms heuristic model, (v) as expected the rule-based model holds more inventory than the optimization model.

  5. Optimization of PCR Condition: The First Study of High Resolution Melting Technique for Screening of APOA1 Variance.

    Science.gov (United States)

    Wahyuningsih, Hesty; K Cayami, Ferdy; Bahrudin, Udin; A Sobirin, Mochamad; Ep Mundhofir, Farmaditya; Mh Faradz, Sultana; Hisatome, Ichiro

    2017-03-01

    High resolution melting (HRM) is a post-PCR technique for variant screening and genotyping based on the different melting points of DNA fragments. The advantages of this technique are that it is fast, simple, and efficient and has a high output, particularly for screening of a large number of samples. APOA1 encodes apolipoprotein A1 (apoA1) which is a major component of high density lipoprotein cholesterol (HDL-C). This study aimed to obtain an optimal quantitative polymerase chain reaction (qPCR)-HRM condition for screening of APOA1 variance. Genomic DNA was isolated from a peripheral blood sample using the salting out method. APOA1 was amplified using the RotorGeneQ 5Plex HRM. The PCR product was visualized with the HRM amplification curve and confirmed using gel electrophoresis. The melting profile was confirmed by looking at the melting curve. Five sets of primers covering the translated region of APOA1 exons were designed with expected PCR product size of 100-400 bps. The amplified segments of DNA were amplicons 2, 3, 4A, 4B, and 4C. Amplicons 2, 3 and 4B were optimized at an annealing temperature of 60 °C at 40 PCR cycles. Amplicon 4A was optimized at an annealing temperature of 62 °C at 45 PCR cycles. Amplicon 4C was optimized at an annealing temperature of 63 °C at 50 PCR cycles. In addition to the suitable procedures of DNA isolation and quantification, primer design and an estimated PCR product size, the data of this study showed that appropriate annealing temperature and PCR cycles were important factors in optimization of HRM technique for variant screening in APOA1 .

  6. Genome-wide indel markers shared by diverse Asian rice cultivars compared to Japanese rice cultivar ?Koshihikari?

    OpenAIRE

    Yonemaru, Jun-ichi; Choi, Sun Hee; Sakai, Hiroaki; Ando, Tsuyu; Shomura, Ayahiko; Yano, Masahiro; Wu, Jianzhong; Fukuoka, Shuichi

    2015-01-01

    Insertion-deletion (indel) polymorphisms, such as simple sequence repeats, have been widely used as DNA markers to identify QTLs and genes and to facilitate rice breeding. Recently, next-generation sequencing has produced deep sequences that allow genome-wide detection of indels. These polymorphisms can potentially be used to develop high-accuracy polymerase chain reaction (PCR)-based markers. Here, re-sequencing of 5 indica, 2 aus, and 3 tropical japonica cultivars and Japanese elite cultiva...

  7. JPIP proxy server with prefetching strategies based on user-navigation model and semantic map

    OpenAIRE

    Monteagudo Pereira, José Lino

    2013-01-01

    The efficient transmission of large resolution images and, in particular, the interactive transmission of images in a client-server scenario, is an important aspect for many applications. Among the current image compression standards, JPEG2000 excels for its interactive transmission capabilities. In general, three mechanisms are employed to optimize the transmission of images when using the JPEG2000 Interactive Protocol (JPIP): 1) packet re-sequencing at the server; 2) prefetching at the clie...

  8. 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.

  9. Capillary electrophoresis of Big-Dye terminator sequencing reactions for human mtDNA Control Region haplotyping in the identification of human remains.

    Science.gov (United States)

    Montesino, Marta; Prieto, Lourdes

    2012-01-01

    Cycle sequencing reaction with Big-Dye terminators provides the methodology to analyze mtDNA Control Region amplicons by means of capillary electrophoresis. DNA sequencing with ddNTPs or terminators was developed by (1). The progressive automation of the method by combining the use of fluorescent-dye terminators with cycle sequencing has made it possible to increase the sensibility and efficiency of the method and hence has allowed its introduction into the forensic field. PCR-generated mitochondrial DNA products are the templates for sequencing reactions. Different set of primers can be used to generate amplicons with different sizes according to the quality and quantity of the DNA extract providing sequence data for different ranges inside the Control Region.

  10. The Impact of Storage Times of Museum Insect Specimens on PCR Success: Case Study on Moth Collections in Indonesia

    Directory of Open Access Journals (Sweden)

    HARI SUTRISNO

    2012-06-01

    Full Text Available Museum specimens are vast repositories of genetic information of interests to biological researchers. Since a new method in DNA extraction, a non destructive method, has been reported to be successful in extracting DNA of museum specimens even fossils without any morphological damages, using museum specimens as resources of genetic information for molecular studies is becoming popular recently. However, the PCR success depends on the quality of the specimens. To evaluate the impact of the storage times of museum specimens on PCR success, we conducted DNA extraction of 14 dry museum specimens of the moths collected from 1992 to 2010 by using a non destructive method. The results showed that the DNA specimens museum were fragmented into various sizes (100-1000 bp depend on the storage times. On the other hand, fresh specimens which were preserved within absolute ethanol were almost not fragmented. The specimens of < 6 years old (2005-2010 succeed to amplify in 650 bp amplicon but for some specimens of 7 years old (2 of 3 specimens resulted in a very weak amplification. These specimens, however, were able to amplify strongly in 300 bp amplicon. The results also showed that specimens of 1-19 years old were success to amplify in 100 bp amplicon.

  11. The first pathogenic Yersinia enterocolitica bioserotype 4/O:3 strain isolated from a hunted wild boar (Sus scrofa) in Poland.

    Science.gov (United States)

    Bancerz-Kisiel, A; Platt-Samoraj, A; Szczerba-Turek, A; Syczyło, K; Szweda, W

    2015-10-01

    The objective of this study was to identify the bioserotypes and virulence markers of Yersinia enterocolitica strains isolated from wild boars in Poland. Bacteriological examination of 302 rectal swabs from 151 wild boars resulted in the isolation of 40 Y. enterocolitica strains. The majority of the examined strains (n = 30), belonged to bioserotype 1A/NI. The presence of individual Y. enterocolitica strains belonging to bioserotypes 1B/NI (3), 1A/O:8 (2), 1A/O:27 (2), 2/NI (1), 2/O:9 (1) and 4/O:3 (1) was also demonstrated. Amplicons corresponding to ail and ystA genes were observed only in one Y. enterocolitica strain--bioserotype 4/O:3. The ail and ystB gene amplicons were noted in 11 Y. enterocolitica biotype 1A strains, although single amplicons of ystB gene were found in 28 of the tested samples. In four out of eight cases when two Y. enterocolitica strains were isolated from the same animal, the strains differed in biotype, serotype or virulence markers. The European population of wild boars continues to grow and spread to new areas, therefore, wild boars harbouring potentially pathogenic Y. enterocolitica 4/O:3 strains pose a challenge to public health.

  12. A NASBA on microgel-tethered molecular-beacon microarray for real-time microbial molecular diagnostics.

    Science.gov (United States)

    Ma, Y; Dai, X; Hong, T; Munk, G B; Libera, M

    2016-12-19

    Despite their many advantages and successes, molecular beacon (MB) hybridization probes have not been extensively used in microarray formats because of the complicating probe-substrate interactions that increase the background intensity. We have previously shown that tethering to surface-patterned microgels is an effective means for localizing MB probes to specific surface locations in a microarray format while simultaneously maintaining them in as water-like an environment as possible and minimizing probe-surface interactions. Here we extend this approach to include both real-time detection together with integrated NASBA amplification. We fabricate small (∼250 μm × 250 μm) simplex, duplex, and five-plex assays with microarray spots of controllable size (∼20 μm diameter), position, and shape to detect bacteria and fungi in a bloodstream-infection model. The targets, primers, and microgel-tethered probes can be combined in a single isothermal reaction chamber with no post-amplification labelling. We extract total RNA from clinical blood samples and differentiate between Gram-positive and Gram-negative bloodstream infection in a duplex assay to detect RNA- amplicons. The sensitivity based on our current protocols in a simplex assay to detect specific ribosomal RNA sequences within total RNA extracted from S. aureus and E. coli cultures corresponds to tens of bacteria per ml. We furthermore show that the platform can detect RNA- amplicons from synthetic target DNA with 1 fM sensitivity in sample volumes that contain about 12 000 DNA molecules. These experiments demonstrate an alternative approach that can enable rapid and real-time microarray-based molecular diagnostics.

  13. A novel PTCH1 mutation in a patient with Gorlin syndrome.

    Science.gov (United States)

    Okamoto, Nana; Naruto, Takuya; Kohmoto, Tomohiro; Komori, Takahide; Imoto, Issei

    2014-01-01

    Gorlin syndrome is an autosomal dominant disorder characterized by a wide range of developmental abnormalities and a predisposition to various tumors, and it is linked to the alteration of several causative genes, including PTCH1. We performed targeted resequencing using a next-generation sequencer to analyze genes associated with known clinical phenotypes in an 11-year-old male with sporadic jaw keratocysts. A novel duplication mutation (c.426dup) in PTCH1, resulting in a truncated protein, was identified.

  14. Applying Next Generation Sequencing to Skeletal Development and Disease

    OpenAIRE

    Bowen, Margot Elizabeth

    2013-01-01

    Next Generation Sequencing (NGS) technologies have dramatically increased the throughput and lowered the cost of DNA sequencing. In this thesis, I apply these technologies to unresolved questions in skeletal development and disease. Firstly, I use targeted re-sequencing of genomic DNA to identify the genetic cause of the cartilage tumor syndrome, metachondromatosis (MC). I show that the majority of MC patients carry heterozygous loss-of-function mutations in the PTPN11 gene, which encodes a p...

  15. Customer experience

    OpenAIRE

    Koperdáková, Zuzana

    2016-01-01

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

  16. Simulator experiments: effects of NPP operator experience on performance

    International Nuclear Information System (INIS)

    Beare, A.N.; Gray, L.H.

    1984-01-01

    During the FY83 research, a simulator experiment was conducted at the control room simulator for a GE Boiling Water Reactor (BWR) NPP. The research subjects were licensed operators undergoing requalification training and shift technical advisors (STAs). This experiment was designed to investigate the effects of senior reactor operator (SRO) experience, operating crew augmentation with an STA and practice, as a crew, upon crew and individual operator performance, in response to anticipated plant transients. Sixteen two-man crews of licensed operators were employed in a 2 x 2 factorial design. The SROs leading the crews were split into high and low experience groups on the basis of their years of experience as an SRO. One half of the high- and low-SRO experience groups were assisted by an STA. The crews responded to four simulated plant casualties. A five-variable set of content-referenced performance measures was derived from task analyses of the procedurally correct responses to the four casualties. System parameters and control manipulations were recorded by the computer controlling the simulator. Data on communications and procedure use were obtained from analysis of videotapes of the exercises. Questionnaires were used to collect subject biographical information and data on subjective workload during each simulated casualty. For four of the five performance measures, no significant differences were found between groups led by high (25 to 114 months) and low (1 to 17 months as an SRO) experience SROs. However, crews led by low experience SROs tended to have significantly shorter task performance times than crews led by high experience SROs. The presence of the STA had no significant effect on overall team performance in responding to the four simulated casualties. The FY84 experiments are a partial replication and extension of the FY83 experiment, but with PWR operators and simulator

  17. Genome-wide re-sequencing of multidrug-resistant Mycobacterium leprae Airaku-3.

    Science.gov (United States)

    Singh, P; Benjak, A; Carat, S; Kai, M; Busso, P; Avanzi, C; Paniz-Mondolfi, A; Peter, C; Harshman, K; Rougemont, J; Matsuoka, M; Cole, S T

    2014-10-01

    Genotyping and molecular characterization of drug resistance mechanisms in Mycobacterium leprae enables disease transmission and drug resistance trends to be monitored. In the present study, we performed genome-wide analysis of Airaku-3, a multidrug-resistant strain with an unknown mechanism of resistance to rifampicin. We identified 12 unique non-synonymous single-nucleotide polymorphisms (SNPs) including two in the transporter-encoding ctpC and ctpI genes. In addition, two SNPs were found that improve the resolution of SNP-based genotyping, particularly for Venezuelan and South East Asian strains of M. leprae. © 2014 The Authors Clinical Microbiology and Infection © 2014 European Society of Clinical Microbiology and Infectious Diseases.

  18. Characterization of GM events by insert knowledge adapted re-sequencing approaches

    OpenAIRE

    Yang, Litao; Wang, Congmao; Holst-Jensen, Arne; Morisset, Dany; Lin, Yongjun; Zhang, Dabing

    2013-01-01

    Detection methods and data from molecular characterization of genetically modified (GM) events are needed by stakeholders of public risk assessors and regulators. Generally, the molecular characteristics of GM events are incomprehensively revealed by current approaches and biased towards detecting transformation vector derived sequences. GM events are classified based on available knowledge of the sequences of vectors and inserts (insert knowledge). Herein we present three insert knowledge-ad...

  19. Extracting Insights from Experience Designers to Enhance User Experience Design

    OpenAIRE

    Kremer, Simon; Lindemann, Udo

    2016-01-01

    User Experience (UX) summarizes how a user expects, perceives and assesses an encounter with a product. User Experience Design (UXD) aims at creating meaningful experiences. While UXD is a rather young discipline with-in product development and traditional processes predominate, other disciplines traditionally focus on creating experiences. We engaged with experience de-signers from the fields of arts, movies, sports, music and event management. By analyzing their working processes via interv...

  20. Revalidation and genetic characterization of new members of Group C (Orthobunyavirus genus, Peribunyaviridae family) isolated in the Americas.

    Science.gov (United States)

    Nunes, Márcio Roberto Teixeira; de Souza, William Marciel; Acrani, Gustavo Olszanski; Cardoso, Jedson Ferreira; da Silva, Sandro Patroca; Badra, Soraya Jabur; Figueiredo, Luiz Tadeu Moraes; Vasconcelos, Pedro Fernando da Costa

    2018-01-01

    Group C serogroup includes members of the Orthobunyavirus genus (family Peribunyaviridae) and comprises 15 arboviruses that can be associated with febrile illness in humans. Although previous studies described the genome characterization of Group C orthobunyavirus, there is a gap in genomic information about the other viruses in this group. Therefore, in this study, complete genomes of members of Group C serogroup were sequenced or re-sequenced and used for genetic characterization, as well as to understand their phylogenetic and evolutionary aspects. Thus, our study reported the genomes of three new members in Group C virus (Apeu strain BeAn848, Itaqui strain BeAn12797 and Nepuyo strain BeAn10709), as well as re-sequencing of original strains of five members: Caraparu (strain BeAn3994), Madrid (strain BT4075), Murucutu (strain BeAn974), Oriboca (strain BeAn17), and Marituba (strain BeAn15). These viruses presented a typical genomic organization related to members of the Orthobunyavirus genus. Interestingly, all viruses of this serogroup showed an open reading frame (ORF) that encodes the putative nonstructural NSs protein that precedes the nucleoprotein ORF, an unprecedented fact in Group C virus. Also, we confirmed the presence of natural reassortment events. This study expands the genomic information of Group C viruses, as well as revalidates the genomic organization of viruses that were previously reported.

  1. Intra-species sequence comparisons for annotating genomes

    Energy Technology Data Exchange (ETDEWEB)

    Boffelli, Dario; Weer, Claire V.; Weng, Li; Lewis, Keith D.; Shoukry, Malak I.; Pachter, Lior; Keys, David N.; Rubin, Edward M.

    2004-07-15

    Analysis of sequence variation among members of a single species offers a potential approach to identify functional DNA elements responsible for biological features unique to that species. Due to its high rate of allelic polymorphism and ease of genetic manipulability, we chose the sea squirt, Ciona intestinalis, to explore intra-species sequence comparisons for genome annotation. A large number of C. intestinalis specimens were collected from four continents and a set of genomic intervals amplified, resequenced and analyzed to determine the mutation rates at each nucleotide in the sequence. We found that regions with low mutation rates efficiently demarcated functionally constrained sequences: these include a set of noncoding elements, which we showed in C intestinalis transgenic assays to act as tissue-specific enhancers, as well as the location of coding sequences. This illustrates that comparisons of multiple members of a species can be used for genome annotation, suggesting a path for the annotation of the sequenced genomes of organisms occupying uncharacterized phylogenetic branches of the animal kingdom and raises the possibility that the resequencing of a large number of Homo sapiens individuals might be used to annotate the human genome and identify sequences defining traits unique to our species. The sequence data from this study has been submitted to GenBank under accession nos. AY667278-AY667407.

  2. Mitochondrial variants in schizophrenia, bipolar disorder, and major depressive disorder.

    Directory of Open Access Journals (Sweden)

    Brandi Rollins

    Full Text Available Mitochondria provide most of the energy for brain cells by the process of oxidative phosphorylation. Mitochondrial abnormalities and deficiencies in oxidative phosphorylation have been reported in individuals with schizophrenia (SZ, bipolar disorder (BD, and major depressive disorder (MDD in transcriptomic, proteomic, and metabolomic studies. Several mutations in mitochondrial DNA (mtDNA sequence have been reported in SZ and BD patients.Dorsolateral prefrontal cortex (DLPFC from a cohort of 77 SZ, BD, and MDD subjects and age-matched controls (C was studied for mtDNA sequence variations and heteroplasmy levels using Affymetrix mtDNA resequencing arrays. Heteroplasmy levels by microarray were compared to levels obtained with SNaPshot and allele specific real-time PCR. This study examined the association between brain pH and mtDNA alleles. The microarray resequencing of mtDNA was 100% concordant with conventional sequencing results for 103 mtDNA variants. The rate of synonymous base pair substitutions in the coding regions of the mtDNA genome was 22% higher (p = 0.0017 in DLPFC of individuals with SZ compared to controls. The association of brain pH and super haplogroup (U, K, UK was significant (p = 0.004 and independent of postmortem interval time.Focusing on haplogroup and individual susceptibility factors in psychiatric disorders by considering mtDNA variants may lead to innovative treatments to improve mitochondrial health and brain function.

  3. Effect of long-term farming strategies on soil microbiota and soil health

    Science.gov (United States)

    Sommermann, Loreen; Babin, Doreen; Sandmann, Martin; Smalla, Kornelia; Schellenberg, Ingo; Grosch, Rita; Geistlinger, Joerg

    2017-04-01

    Increasing food and energy demands have resulted in considerable intensification of farming practices, which brought about severe consequences for agricultural soils, e.g. loss of fertility, erosion and enrichment of soil-borne plant diseases. In order to maintain soil quality and health for the future, the development of more extensive and sustainable farming strategies is urgently needed. The soil microbiome is regarded as a key player in soil ecosystem functions, particularly the natural ability of soils to suppress plant pathogens (suppressiveness). Recent studies showed that soil microbial communities are influenced by agricultural management. To further analyze the effects of farming strategies on soil suppressiveness and plant performance, agricultural soils from three long-term field trials in Thyrow, Bernburg (both in Germany) and Therwil (Switzerland) were sampled and subjected to molecular profiling of soil bacteria and fungi using marker genes and high-throughput amplicon sequencing. Significant effects on bacterial as well as fungal community composition, including plant pathogenic and beneficial taxa, were observed among variants of tillage and crop rotation. The least effect on both communities had fertilization, with no significance between variants. Subsequently, the same soils were subjected to growth chamber pot experiments with lettuce as a model (Lactuca sativa). After a growth period of six weeks significant differences in lettuce shoot and soil microbial biomass were observed among soil samples of the different long-term trials. Furthermore, the lettuce rhizosphere exhibited diverse bacterial community compositions as observed by DGGE (denaturing gradient gel electrophoresis). Using group-specific PCR-DGGE fingerprints, bacterial responders to fertilization, soil management and crop rotation were identified among different taxonomic groups. Currently, bacterial and fungal amplicon sequencing of rhizosphere and bulk soil from these pot

  4. Direct amplification, sequencing and profiling of Chlamydia trachomatis strains in single and mixed infection clinical samples.

    Directory of Open Access Journals (Sweden)

    Sandeep J Joseph

    Full Text Available Sequencing bacterial genomes from DNA isolated directly from clinical samples offers the promise of rapid and precise acquisition of informative genetic information. In the case of Chlamydia trachomatis, direct sequencing is particularly desirable because it obviates the requirement for culture in mammalian cells, saving time, cost and the possibility of missing low abundance strains. In this proof of concept study, we developed methodology that would allow genome-scale direct sequencing, using a multiplexed microdroplet PCR enrichment technology to amplify a 100 kb region of the C. trachomatis genome with 500 1.1-1.3 kb overlapping amplicons (5-fold amplicon redundancy. We integrated comparative genomic data into a pipeline to preferentially select conserved sites for amplicon design. The 100 kb target region could be amplified from clinical samples, including remnants from diagnostics tests, originating from the cervix, urethra and urine, For rapid analysis of these data, we developed a framework for whole-genome based genotyping called binstrain. We used binstrain to estimate the proportion of SNPs originating from 14 C. trachomatis reference serotype genomes in each sample. Direct DNA sequencing methods such as the one described here may have an important role in understanding the biology of C. trachomatis mixed infections and the natural genetic variation of the species within clinically relevant ecological niches.

  5. Establishment of a universal and rational gene detection strategy through three-way junction-based remote transduction.

    Science.gov (United States)

    Tang, Yidan; Lu, Baiyang; Zhu, Zhentong; Li, Bingling

    2018-01-21

    The polymerase chain reaction and many isothermal amplifications are able to achieve super gene amplification. Unfortunately, most commonly-used transduction methods, such as dye staining and Taqman-like probing, still suffer from shortcomings including false signals or difficult probe design, or are incompatible with multi-analysis. Here a universal and rational gene detection strategy has been established by translating isothermal amplicons to enzyme-free strand displacement circuits via three-way junction-based remote transduction. An assistant transduction probe was imported to form a partial hybrid with the target single-stranded nucleic acid. After systematic optimization the hybrid could serve as an associative trigger to activate a downstream circuit detector via a strand displacement reaction across the three-way junction. By doing so, the detection selectivity can be double-guaranteed through both amplicon-transducer recognition and the amplicon-circuit reaction. A well-optimized circuit can be immediately applied to a new target detection through simply displacing only 10-12 nt on only one component, according to the target. More importantly, this property for the first time enables multi-analysis and logic-analysis in a single reaction, sharing a single fluorescence reporter. In an applicable model, trace amounts of Cronobacter and Enterobacteria genes have been clearly distinguished from samples with no bacteria or one bacterium, with ultra-high sensitivity and selectivity.

  6. Genotyping via Sequence Related Amplified Polymorphism Markers in Fusarium culmorum

    Directory of Open Access Journals (Sweden)

    Işıl Melis Zümrüt

    2018-02-01

    Full Text Available Fusarium culmorum is predominating causal agent of head blight (HB and root rot (RR in cereals worldwide. Since F. culmorum has a great level of genetic diversity and the parasexual stage is assumed for this phytopathogen, characterization of isolates from different regions is significant step in food safety and controlling the HB. In this study, it was aimed to characterize totally 37 F. culmorum isolates from Turkey via sequence related amplified polymorphism (SRAP marker based genotyping. MAT-1/MAT-2 type assay was also used in order to reveal intraspecific variation in F. culmorum. MAT-1 and MAT-2 specific primer pairs for mating assays resulted in 210 and 260 bp bands, respectively. 11 of isolates were belonged to MAT-1 type whereas 19 samples were of MAT-2. Remaining 7 samples yielded both amplicons. Totally 9 SRAP primer sets yielded amplicons from all isolates. Genetic similarity values were ranged from 39 to 94.7%. Total band number was 127 and PCR product sizes were in the range of 0.1-2.5 kb. Amplicon numbers for individuals were ranged from 1 to 16. According to data obtained from current study, SRAP based genotyping is powerful tool for supporting the data obtained from investigations including phenotypic and agro-ecological characteristics. Findings showed that SRAP-based markers could be useful in F. culmorum characterization.

  7. The experience sampling method: Investigating students' affective experience

    Science.gov (United States)

    Nissen, Jayson M.; Stetzer, MacKenzie R.; Shemwell, Jonathan T.

    2013-01-01

    Improving non-cognitive outcomes such as attitudes, efficacy, and persistence in physics courses is an important goal of physics education. This investigation implemented an in-the-moment surveying technique called the Experience Sampling Method (ESM) [1] to measure students' affective experience in physics. Measurements included: self-efficacy, cognitive efficiency, activation, intrinsic motivation, and affect. Data are presented that show contrasts in students' experiences (e.g., in physics vs. non-physics courses).

  8. Simulator experiments: effects of NPP operator experience on performance

    International Nuclear Information System (INIS)

    Beare, A.N.; Gray, L.H.

    1985-01-01

    Experiments are being conducted on nuclear power plant (NPP) control room training simulators by the Oak Ridge National Laboratory, its subcontractor, General Physics Corporation, and participating utilities. The experiments are sponsored by the Nuclear Regulatory Commission's (NRC) Human Factors and Safeguards Branch, Division of Risk Analysis and Operations, and are a continuation of prior research using simulators, supported by field data collection, to provide a technical basis for NRC human factors regulatory issues concerned with the operational safety of nuclear power plants. During the FY83 research, a simulator experiment was conducted at the control room simulator for a GE boiling water reactor (BWR) NPP. The research subjects were licensed operators undergoing requalification training and shift technical advisors (STAs). This experiment was designed to investigate the effects of (a) senior reactor operator (SRO) experience, (b) operating crew augmentation with an STA and (c) practice, as a crew, upon crew and individual operator performance, in response to anticipated plant transients. The FY84 experiments are a partial replication and extension of the FY83 experiment, but with PWR operators and simulator. Methodology and results to date are reported

  9. SNiPlay: a web-based tool for detection, management and analysis of SNPs. Application to grapevine diversity projects.

    Science.gov (United States)

    Dereeper, Alexis; Nicolas, Stéphane; Le Cunff, Loïc; Bacilieri, Roberto; Doligez, Agnès; Peros, Jean-Pierre; Ruiz, Manuel; This, Patrice

    2011-05-05

    High-throughput re-sequencing, new genotyping technologies and the availability of reference genomes allow the extensive characterization of Single Nucleotide Polymorphisms (SNPs) and insertion/deletion events (indels) in many plant species. The rapidly increasing amount of re-sequencing and genotyping data generated by large-scale genetic diversity projects requires the development of integrated bioinformatics tools able to efficiently manage, analyze, and combine these genetic data with genome structure and external data. In this context, we developed SNiPlay, a flexible, user-friendly and integrative web-based tool dedicated to polymorphism discovery and analysis. It integrates:1) a pipeline, freely accessible through the internet, combining existing softwares with new tools to detect SNPs and to compute different types of statistical indices and graphical layouts for SNP data. From standard sequence alignments, genotyping data or Sanger sequencing traces given as input, SNiPlay detects SNPs and indels events and outputs submission files for the design of Illumina's SNP chips. Subsequently, it sends sequences and genotyping data into a series of modules in charge of various processes: physical mapping to a reference genome, annotation (genomic position, intron/exon location, synonymous/non-synonymous substitutions), SNP frequency determination in user-defined groups, haplotype reconstruction and network, linkage disequilibrium evaluation, and diversity analysis (Pi, Watterson's Theta, Tajima's D).Furthermore, the pipeline allows the use of external data (such as phenotype, geographic origin, taxa, stratification) to define groups and compare statistical indices.2) a database storing polymorphisms, genotyping data and grapevine sequences released by public and private projects. It allows the user to retrieve SNPs using various filters (such as genomic position, missing data, polymorphism type, allele frequency), to compare SNP patterns between populations, and to

  10. QTL Analysis and Functional Genomics of Animal Model

    DEFF Research Database (Denmark)

    Farajzadeh, Leila

    , for example, has enabled scientists to examine more complex interactions in connection with studies of properties and diseases. In her PhD project, Leila Farajzadeh integrated different organisational levels in biology, including genotype, phenotype, association studies, transcription profiles and genetic......In recent years, the use of functional genomics and next-generation sequencing technologies has increased the probability of success in studies of complex properties. The integration of large data sets from association studies, DNA resequencing, gene expression profiles and phenotypic data...

  11. JVM: Java Visual Mapping tool for next generation sequencing read.

    Science.gov (United States)

    Yang, Ye; Liu, Juan

    2015-01-01

    We developed a program JVM (Java Visual Mapping) for mapping next generation sequencing read to reference sequence. The program is implemented in Java and is designed to deal with millions of short read generated by sequence alignment using the Illumina sequencing technology. It employs seed index strategy and octal encoding operations for sequence alignments. JVM is useful for DNA-Seq, RNA-Seq when dealing with single-end resequencing. JVM is a desktop application, which supports reads capacity from 1 MB to 10 GB.

  12. A novel COL11A1 mutation affecting splicing in a patient with Stickler syndrome.

    Science.gov (United States)

    Kohmoto, Tomohiro; Naruto, Takuya; Kobayashi, Haruka; Watanabe, Miki; Okamoto, Nana; Masuda, Kiyoshi; Imoto, Issei; Okamoto, Nobuhiko

    2015-01-01

    Stickler syndrome is a clinically and genetically heterogeneous collagenopathy characterized by ocular, auditory, skeletal and orofacial abnormalities, commonly occurring as an autosomal dominant trait. We conducted target resequencing to analyze candidate genes associated with known clinical phenotypes from a 4-year-old girl with Stickler syndrome. We detected a novel heterozygous intronic mutation (NM_001854.3:c.3168+5G>A) in COL11A1 that may impair splicing, which was suggested by in silico prediction and a minigene assay.

  13. A Local Search Algorithm for the Flow Shop Scheduling Problem with Release Dates

    Directory of Open Access Journals (Sweden)

    Tao Ren

    2015-01-01

    Full Text Available This paper discusses the flow shop scheduling problem to minimize the makespan with release dates. By resequencing the jobs, a modified heuristic algorithm is obtained for handling large-sized problems. Moreover, based on some properties, a local search scheme is provided to improve the heuristic to gain high-quality solution for moderate-sized problems. A sequence-independent lower bound is presented to evaluate the performance of the algorithms. A series of simulation results demonstrate the effectiveness of the proposed algorithms.

  14. Sequence assembly

    DEFF Research Database (Denmark)

    Scheibye-Alsing, Karsten; Hoffmann, S.; Frankel, Annett Maria

    2009-01-01

    Despite the rapidly increasing number of sequenced and re-sequenced genomes, many issues regarding the computational assembly of large-scale sequencing data have remain unresolved. Computational assembly is crucial in large genome projects as well for the evolving high-throughput technologies and...... in genomic DNA, highly expressed genes and alternative transcripts in EST sequences. We summarize existing comparisons of different assemblers and provide a detailed descriptions and directions for download of assembly programs at: http://genome.ku.dk/resources/assembly/methods.html....

  15. A novel photoinduced electron transfer (PET) primer technique for rapid real-time PCR detection of Cryptosporidium spp

    Energy Technology Data Exchange (ETDEWEB)

    Jothikumar, N., E-mail: jin2@cdc.gov; Hill, Vincent R.

    2013-06-28

    Highlights: •Uses a single-labeled fluorescent primer for real-time PCR. •The detection sensitivity of PET PCR was comparable to TaqMan PCR. •Melt curve analysis can be performed to confirm target amplicon production. •Conventional PCR primers can be converted to PET PCR primers. -- Abstract: We report the development of a fluorescently labeled oligonucleotide primer that can be used to monitor real-time PCR. The primer has two parts, the 3′-end of the primer is complimentary to the target and a universal 17-mer stem loop at the 5′-end forms a hairpin structure. A fluorescent dye is attached to 5′-end of either the forward or reverse primer. The presence of guanosine residues at the first and second position of the 3′ dangling end effectively quenches the fluorescence due to the photo electron transfer (PET) mechanism. During the synthesis of nucleic acid, the hairpin structure is linearized and the fluorescence of the incorporated primer increases several-fold due to release of the fluorescently labeled tail and the absence of guanosine quenching. As amplicons are synthesized during nucleic acid amplification, the fluorescence increase in the reaction mixture can be measured with commercially available real-time PCR instruments. In addition, a melting procedure can be performed to denature the double-stranded amplicons, thereby generating fluorescence peaks that can differentiate primer dimers and other non-specific amplicons if formed during the reaction. We demonstrated the application of PET-PCR for the rapid detection and quantification of Cryptosporidium parvum DNA. Comparison with a previously published TaqMan® assay demonstrated that the two real-time PCR assays exhibited similar sensitivity for a dynamic range of detection of 6000–0.6 oocysts per reaction. PET PCR primers are simple to design and less-expensive than dual-labeled probe PCR methods, and should be of interest for use by laboratories operating in resource

  16. High resolution melting for mutation scanning of TP53 exons 5–8

    International Nuclear Information System (INIS)

    Krypuy, Michael; Dobrovic, Alexander; Ahmed, Ahmed Ashour; Etemadmoghadam, Dariush; Hyland, Sarah J; Australian Ovarian Cancer Study Group; Fazio, Anna de; Fox, Stephen B; Brenton, James D; Bowtell, David D

    2007-01-01

    p53 is commonly inactivated by mutations in the DNA-binding domain in a wide range of cancers. As mutant p53 often influences response to therapy, effective and rapid methods to scan for mutations in TP53 are likely to be of clinical value. We therefore evaluated the use of high resolution melting (HRM) as a rapid mutation scanning tool for TP53 in tumour samples. We designed PCR amplicons for HRM mutation scanning of TP53 exons 5 to 8 and tested them with DNA from cell lines hemizygous or homozygous for known mutations. We assessed the sensitivity of each PCR amplicon using dilutions of cell line DNA in normal wild-type DNA. We then performed a blinded assessment on ovarian tumour DNA samples that had been previously sequenced for mutations in TP53 to assess the sensitivity and positive predictive value of the HRM technique. We also performed HRM analysis on breast tumour DNA samples with unknown TP53 mutation status. One cell line mutation was not readily observed when exon 5 was amplified. As exon 5 contained multiple melting domains, we divided the exon into two amplicons for further screening. Sequence changes were also introduced into some of the primers to improve the melting characteristics of the amplicon. Aberrant HRM curves indicative of TP53 mutations were observed for each of the samples in the ovarian tumour DNA panel. Comparison of the HRM results with the sequencing results revealed that each mutation was detected by HRM in the correct exon. For the breast tumour panel, we detected seven aberrant melt profiles by HRM and subsequent sequencing confirmed the presence of these and no other mutations in the predicted exons. HRM is an effective technique for simple and rapid scanning of TP53 mutations that can markedly reduce the amount of sequencing required in mutational studies of TP53

  17. A novel photoinduced electron transfer (PET) primer technique for rapid real-time PCR detection of Cryptosporidium spp

    International Nuclear Information System (INIS)

    Jothikumar, N.; Hill, Vincent R.

    2013-01-01

    Highlights: •Uses a single-labeled fluorescent primer for real-time PCR. •The detection sensitivity of PET PCR was comparable to TaqMan PCR. •Melt curve analysis can be performed to confirm target amplicon production. •Conventional PCR primers can be converted to PET PCR primers. -- Abstract: We report the development of a fluorescently labeled oligonucleotide primer that can be used to monitor real-time PCR. The primer has two parts, the 3′-end of the primer is complimentary to the target and a universal 17-mer stem loop at the 5′-end forms a hairpin structure. A fluorescent dye is attached to 5′-end of either the forward or reverse primer. The presence of guanosine residues at the first and second position of the 3′ dangling end effectively quenches the fluorescence due to the photo electron transfer (PET) mechanism. During the synthesis of nucleic acid, the hairpin structure is linearized and the fluorescence of the incorporated primer increases several-fold due to release of the fluorescently labeled tail and the absence of guanosine quenching. As amplicons are synthesized during nucleic acid amplification, the fluorescence increase in the reaction mixture can be measured with commercially available real-time PCR instruments. In addition, a melting procedure can be performed to denature the double-stranded amplicons, thereby generating fluorescence peaks that can differentiate primer dimers and other non-specific amplicons if formed during the reaction. We demonstrated the application of PET-PCR for the rapid detection and quantification of Cryptosporidium parvum DNA. Comparison with a previously published TaqMan® assay demonstrated that the two real-time PCR assays exhibited similar sensitivity for a dynamic range of detection of 6000–0.6 oocysts per reaction. PET PCR primers are simple to design and less-expensive than dual-labeled probe PCR methods, and should be of interest for use by laboratories operating in resource

  18. A Critical Assessment of the Microorganisms Proposed to be Important to Enhanced Biological Phosphorus Removal in Full-Scale Wastewater Treatment Systems.

    Science.gov (United States)

    Stokholm-Bjerregaard, Mikkel; McIlroy, Simon J; Nierychlo, Marta; Karst, Søren M; Albertsen, Mads; Nielsen, Per H

    2017-01-01

    Understanding the microbiology of phosphorus (P) removal is considered essential to knowledge-based optimization of enhanced biological P removal (EBPR) systems. Biological P removal is achieved in these systems by promoting the growth of organisms collectively known as the polyphosphate accumulating organisms (PAOs). Also considered important to EBPR are the glycogen accumulating organisms (GAOs), which are theorized to compete with the PAOs for resources at the expense of P removal efficiency. Numerous studies have sought to identify the PAOs and their GAOs competitors, with several candidates proposed for each over the last few decades. The current study collectively assessed the abundance and diversity of all proposed PAOs and GAOs in 18 Danish full-scale wastewater treatment plants with well-working biological nutrient removal over a period of 9 years using 16S rRNA gene amplicon sequencing. The microbial community structure in all plants was relatively stable over time. Evidence for the role of the proposed PAOs and GAOs in EBPR varies and is critically assessed, in light of their calculated amplicon abundances, to indicate which of these are important in full-scale systems. Bacteria from the genus Tetrasphaera were the most abundant of the PAOs. The " Candidatus Accumulibacter" PAOs were in much lower abundance and appear to be biased by the amplicon-based method applied. The genera Dechloromonas, Microlunatus , and Tessaracoccus were identified as abundant putative PAO that require further research attention. Interestingly, the actinobacterial Micropruina and sbr-gs28 phylotypes were among the most abundant of the putative GAOs. Members of the genera Defluviicoccus, Propionivibrio , the family Competibacteraceae, and the spb280 group were also relatively abundant in some plants. Despite observed high abundances of GAOs (periodically exceeding 20% of the amplicon reads), P removal performance was maintained, indicating that these organisms were not

  19. Experiment WA1 (CDHS Neutrino Experiment)

    CERN Multimedia

    CERN PhotoLab

    1977-01-01

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

  20. Enterovirus species and serotype distributions in monthly municipal wastewater samples

    Data.gov (United States)

    U.S. Environmental Protection Agency — This dataset contains the enterovirus genome copies measured per liter of sample and read counts obtained from amplicon sequencing of the VP1 and VP4 genes. This...

  1. TRIO experiment

    International Nuclear Information System (INIS)

    Clemmer, R.G.; Finn, P.A.; Malecha, R.F.

    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

  2. Pixel Experiments

    DEFF Research Database (Denmark)

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

    2015-01-01

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

  3. Virulence factors and antimicrobial resistance of Staphylococcus aureus isolated from bovine mastitis in Rio de Janeiro Fatores de virulência e resistência antimicrobiana em Staphylococcus aureus isolados de mastite bovina no Rio de Janeiro

    Directory of Open Access Journals (Sweden)

    Shana M.O. Coelho

    2009-05-01

    Full Text Available The study was conducted to characterize pheno-genotypically the virulence factors and resistance pattern of Staphylococcus aureus isolates from milk samples of cows with subclinical mastitis. All hemolytic isolates presented beta-hemolysin, and 38% of the non-hemolytic isolates were able to express hemolysins in the presence of a beta-hemolytic strain. The amplification of the coa-gene displayed four different size polymorphisms with about 400 bp, 600 bp, 700 bp and 900 bp. The spaA gene that encodes the IgG-binding region of protein A revealed sizes of 700 bp and 900 bp. The amplification of region X from spaA yielded a single amplicon for each isolate with the prevalent amplicon size being of 180 bp. Amplification of sae gene yielded an amplicon size of 920 bp in 71% of the isolates. Antibiotic resistance pattern revealed that 42% S. aureus were susceptible to all antimicrobials tested. Seven different antibiotic patterns were observed. Our results indicated that 47% and 25% of S. aureus strains exhibited resistance to penicillin and oxacillin respectively. All oxacillin-resistant isolates were mecA-positive.O presente estudo foi conduzido com o objetivo de caracterizar feno-genotipicamente os fatores de virulência e perfil de resistência aos antibióticos de Staphylococcus aureus isolados de amostras de leite de vacas com mastite clínica e subclínica. Em todos os isolados hemolíticos foi detectada a presença de beta hemolisina e 38% dos não-hemolíticos produziram hemolisinas na presença de cepa beta-hemolítica. A amplificação do gene coa apresentou quatro tipos polimórficos distintos com aproximadamente 400 bp, 600 bp, 700 bp e 900 bp. O gene spaA que codifica a região de ligação da proteína A à IgG apresentou bandas de 700 bp e 900 bp. A amplificação do gene que codifica a região X revelou um único amplicon para cada isolado sendo o tamanho prevalente o de 250pb. A amplificação do gene sae resultou em amplicons com

  4. Development and validation of a multiplex reaction analyzing eight miniSTRs of the X chromosome for identity and kinship testing with degraded DNA.

    Science.gov (United States)

    Castañeda, María; Odriozola, Adrián; Gómez, Javier; Zarrabeitia, María T

    2013-07-01

    We report the development of an effective system for analyzing X chromosome-linked mini short tandem repeat loci with reduced-size amplicons (less than 220 bp), useful for analyzing highly degraded DNA samples. To generate smaller amplicons, we redesigned primers for eight X-linked microsatellites (DXS7132, DXS10079, DXS10074, DXS10075, DXS6801, DXS6809, DXS6789, and DXS6799) and established efficient conditions for a multiplex PCR system (miniX). The validation tests confirmed that it has good sensitivity, requiring as little as 20 pg of DNA, and performs well with DNA from paraffin-embedded tissues, thus showing potential for improved analysis and identification of highly degraded and/or very limited DNA samples. Consequently, this system may help to solve complex forensic cases, particularly when autosomal markers convey insufficient information.

  5. Specificity and transcriptional activity of microbiota associated with low and high microbial abundance sponges from the Red Sea

    KAUST Repository

    Moitinho-Silva, Lucas; Bayer, Kristina; Cannistraci, Carlo; Giles, Emily; Ryu, Tae Woo; Seridi, Loqmane; Ravasi, Timothy; Hentschel, Ute T E

    2013-01-01

    Marine sponges are generally classified as high microbial abundance (HMA) and low microbial abundance (LMA) species. Here, 16S rRNA amplicon sequencing was applied to investigate the diversity, specificity and transcriptional activity of microbes

  6. ARC EMCS Experiments (Seedling Growth-2) Experiment Status

    Science.gov (United States)

    Heathcote, David; Steele, Marianne

    2015-01-01

    Presentation of the status of the ARC ISS (International Space Station) Experiment, Seedling Growth-2 to the Payload Operations Investigator Working Group meeting at MSFC, Huntsville AL. The experiment employs the European Modular Cultivation System (ECMS).

  7. Functional Analysis of Frequent Lung Cancer Amplicons Under Current Genome Annotation

    Science.gov (United States)

    2015-12-01

    Zhang, Z., Wang, T., Winter, D., Keefe , D., Birney, E., Iyer, V. R., Lieb, J. D., Crawford, G. E., Li, G., Sandhu, K. S., Zheng, M., Wang, P., Luo, O...Herrero, J., Johnson, N., Keefe , D., Lukk, M., Luscombe, N. M., Sobral, D., Vaquerizas, J. M., Wilder, S. P., Batzoglou, S., Sidow, A., Hussami, N

  8. TRACY transient experiment databook. 2) ramp withdrawal experiment

    International Nuclear Information System (INIS)

    Nakajima, Ken; Yamane, Yuichi; Ogawa, Kazuhiko; Aizawa, Eiju; Yanagisawa, Hiroshi; Miyoshi, Yoshinori

    2002-03-01

    This is a databook of TRACY ''ramp withdrawal'' experiments. TRACY is a reactor to perform supercritical experiments using low-enriched uranyl nitrate aqueous solution. The excess reactivity of TRACY is 3$ at maximum, and it is inserted by feeding the solution to a core tank or by withdrawing a control rod, which is called as the transient rod, from the core. In the ramp withdrawal experiment, the supercritical experiment is initiated by withdrawing the transient rod from the core in a constant speed using a motor drive system. The data in the present databook consist of datasheets and graphs. Experimental conditions and typical values of measured parameters are tabulated in the datasheet. In the graph, power and temperature profiles are plotted. Those data are useful for the investigation of criticality accidents with fissile solutions, and for validation of criticality accident analysis codes. (author)

  9. Experiment prediction for Loft Nonnuclear Experiment L1-4

    International Nuclear Information System (INIS)

    White, J.R.; Berta, V.T.; Holmstrom, H.L.O.

    1977-04-01

    A computer analysis, using the WHAM and RELAP4 computer codes, was performed to predict the LOFT system thermal-hydraulic response for Experiment L1-4 of the nonnuclear (isothermal) test series. Experiment L1-4 will simulate a 200 percent double-ended offset shear in the cold leg of a four-loop large pressurized water reactor. A core simulator will be used to provide a reactor vessel pressure drop representative of the LOFT nuclear core. Experiment L1-4 will be initiated with a nominal isothermal primary coolant temperature of 282.2 0 C, a pressurizer pressure of 15.51 MPa, and a primary coolant flow of 270.9 kg/s. In general, the predictions of saturated blowdown for Experiment Ll-4 are consistent with the expected system behavior, and predicted trends agree with results from Semiscale Test S-01-4A, which simulated the Ll-4 experiment conditions

  10. Phylogenetic Analysis of a Novel Molecular Isolate of Spotted Fever Group Rickettsiae from Northern Peru

    National Research Council Canada - National Science Library

    Jiang, Ju; Blair, Patrick J; Felices, Vidal; Moron, Cecilia; Cespedes, Manuel; Anaya, Elizabeth; Schoeler, George B; Sumner, John W; Olson, James G; Richards, Allen L

    2005-01-01

    ... (SFG) rickettsia. Following nested polymerase chain reaction (PCR) amplification of the 17-kDa gene, gltA, ompB, ompA, and sca4, amplicons were purified, sequenced, and compared to those downloaded from GenBank...

  11. Rapid Discrimination between Candida glabrata, Candida nivariensis, and Candida bracarensis by Use of a Singleplex PCR ▿

    OpenAIRE

    Enache-Angoulvant, A.; Guitard, J.; Grenouillet, F.; Martin, T.; Durrens, P.; Fairhead, C.; Hennequin, C.

    2011-01-01

    We report here a PCR-based assay using a single primer pair targeting the RPL31 gene that allows discrimination between Candida glabrata, Candida bracarensis, and Candida nivariensis according to the size of the generated amplicon.

  12. Learning and Experience

    DEFF Research Database (Denmark)

    Olesen, Henning Salling

    2017-01-01

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

  13. Applicability and Efficiency of NGS in Routine Diagnosis: In-Depth Performance Analysis of a Complete Workflow for CFTR Mutation Analysis.

    Directory of Open Access Journals (Sweden)

    Adrien Pagin

    Full Text Available Actually, about 2000 sequence variations have been documented in the CFTR gene requiring extensive and multi-step genetic testing in the diagnosis of cystic fibrosis and CFTR-related disorders. We present a two phases study, with validation and performance monitoring, of a single experiment methodology based on multiplex PCR and high throughput sequencing that allows detection of all variants, including large rearrangements, affecting the coding regions plus three deep intronic loci.A total of 340 samples, including 257 patients and 83 previously characterized control samples, were sequenced in 17 MiSeq runs and analyzed with two bioinformatic pipelines in routine diagnostic conditions. We obtained 100% coverage for all the target regions in every tested sample.We correctly identified all the 87 known variants in the control samples and successfully confirmed the 62 variants identified among the patients without observing false positive results. Large rearrangements were identified in 18/18 control samples. Only 17 patient samples showed false positive signals (6.6%, 12 of which showed a borderline result for a single amplicon. We also demonstrated the ability of the assay to detect allele specific dropout of amplicons when a sequence variation occurs at a primer binding site thus limiting the risk for false negative results.We described here the first NGS workflow for CFTR routine analysis that demonstrated equivalent diagnostic performances compared to Sanger sequencing and multiplex ligation-dependent probe amplification. This study illustrates the advantages of NGS in term of scalability, workload reduction and cost-effectiveness in combination with an improvement of the overall data quality due to the simultaneous detection of SNVs and large rearrangements.

  14. TRACY transient experiment databook. 3) Ramp feed experiment

    Energy Technology Data Exchange (ETDEWEB)

    Nakajima, Ken; Yamane, Yuichi; Ogawa, Kazuhiko; Aizawa, Eiju; Yanagisawa, Hiroshi; Miyoshi, Yoshinori [Japan Atomic Energy Research Inst., Tokai, Ibaraki (Japan). Tokai Research Establishment

    2002-03-01

    This is a databook of TRACY ''ramp feed'' experiments. TRACY is a reactor to perform supercritical experiments using low-enriched uranyl nitrate aqueous solution. The excess reactivity of TRACY is 3$ at maximum, and it is inserted by feeding the solution to a core tank or by withdrawing a control rod, which is called as the transient rod, from the core. In the ramp feed experiment, the supercritical experiment is initiated by feeding the fuel solution to the core tank in a constant feed rate. The data in the present databook consist of datasheets and graphs. Experimental conditions and typical values of measured parameters are tabulated in the datasheet. In the graph, power and temperature profiles are plotted. Those data are useful for the investigation of criticality accidents with fissile solutions, and for validation of criticality accident analysis codes. (author)

  15. The difference between traditional experiments and CFD validation benchmark experiments

    Energy Technology Data Exchange (ETDEWEB)

    Smith, Barton L., E-mail: barton.smith@usu.edu

    2017-02-15

    Computation Fluid Dynamics provides attractive features for design, and perhaps licensing, of nuclear power plants. The most important of these features is low cost compared to experiments. However, uncertainty of CFD calculations must accompany these calculations in order for the results to be useful for important decision making. In order to properly assess the uncertainty of a CFD calculation, it must be “validated” against experimental data. Unfortunately, traditional “discovery” experiments are normally ill-suited to provide all of the information necessary for the validation exercise. Traditionally, experiments are performed to discover new physics, determine model parameters, or to test designs. This article will describe a new type of experiment; one that is designed and carried out with the specific purpose of providing Computational Fluid Dynamics (CFD) validation benchmark data. We will demonstrate that the goals of traditional experiments and validation experiments are often in conflict, making use of traditional experimental results problematic and leading directly to larger predictive uncertainty of the CFD model.

  16. The difference between traditional experiments and CFD validation benchmark experiments

    International Nuclear Information System (INIS)

    Smith, Barton L.

    2017-01-01

    Computation Fluid Dynamics provides attractive features for design, and perhaps licensing, of nuclear power plants. The most important of these features is low cost compared to experiments. However, uncertainty of CFD calculations must accompany these calculations in order for the results to be useful for important decision making. In order to properly assess the uncertainty of a CFD calculation, it must be “validated” against experimental data. Unfortunately, traditional “discovery” experiments are normally ill-suited to provide all of the information necessary for the validation exercise. Traditionally, experiments are performed to discover new physics, determine model parameters, or to test designs. This article will describe a new type of experiment; one that is designed and carried out with the specific purpose of providing Computational Fluid Dynamics (CFD) validation benchmark data. We will demonstrate that the goals of traditional experiments and validation experiments are often in conflict, making use of traditional experimental results problematic and leading directly to larger predictive uncertainty of the CFD model.

  17. The Challenging Experience Questionnaire: Characterization of challenging experiences with psilocybin mushrooms.

    Science.gov (United States)

    Barrett, Frederick S; Bradstreet, Matthew P; Leoutsakos, Jeannie-Marie S; Johnson, Matthew W; Griffiths, Roland R

    2016-12-01

    Acute adverse psychological reactions to classic hallucinogens ("bad trips" or "challenging experiences"), while usually benign with proper screening, preparation, and support in controlled settings, remain a safety concern in uncontrolled settings (such as illicit use contexts). Anecdotal and case reports suggest potential adverse acute symptoms including affective (panic, depressed mood), cognitive (confusion, feelings of losing sanity), and somatic (nausea, heart palpitation) symptoms. Responses to items from several hallucinogen-sensitive questionnaires (Hallucinogen Rating Scale, the States of Consciousness Questionnaire, and the Five-Dimensional Altered States of Consciousness questionnaire) in an Internet survey of challenging experiences with the classic hallucinogen psilocybin were used to construct and validate a Challenging Experience Questionnaire. The stand-alone Challenging Experience Questionnaire was then validated in a separate sample. Seven Challenging Experience Questionnaire factors (grief, fear, death, insanity, isolation, physical distress, and paranoia) provide a phenomenological profile of challenging aspects of experiences with psilocybin. Factor scores were associated with difficulty, meaningfulness, spiritual significance, and change in well-being attributed to the challenging experiences. The factor structure did not differ based on gender or prior struggle with anxiety or depression. The Challenging Experience Questionnaire provides a basis for future investigation of predictors and outcomes of challenging experiences with classic hallucinogens. © The Author(s) 2016.

  18. The Impact of Hydration and Temperature on Bacterial Diversity in Arid Soil Mesocosms

    Directory of Open Access Journals (Sweden)

    Adam Št'ovíček

    2017-06-01

    Full Text Available Hot desert ecosystems experience rare and unpredictable rainfall events that resuscitate the arid flora and fauna. However, the effect of this sudden abundance of water on soil microbial communities is still under debate. We modeled varying rainfall amounts and temperatures in desert soil mesocosms and monitored the microbial community response over a period of 21 days. We studied two different wetting events, simulating heavy (50 mm and light (10 mm rain, as well as three different temperature regimes: constant 25° or 36°C, or a temperature diurnal cycle alternating between 36 and 10 °C. Amplicon sequencing of the bacterial ribosomal RNA revealed that rain intensity affects the soil bacterial community, but the effects are mitigated by temperature. The combination of water-pulse intensity with lower temperature had the greatest effect on the bacterial community. These experiments demonstrated that the soil microbial response to rain events is dependent not only on the intensity of the water pulse but also on the ambient temperature, thus emphasizing the complexity of bacterial responses to highly unpredictable environments.

  19. Particle physics experiments

    International Nuclear Information System (INIS)

    Stuart, G.W.

    1986-01-01

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

  20. Sharing ESA's knowledge and experience - the Erasmus Experiment Archive

    Science.gov (United States)

    Isakeit, Dieter; Sabbatini, Massimo; Carey, William

    2004-11-01

    The Erasmus Experiment Archive is an electronic database, that collects all experiments performed to date in the faciliteis that fall under the responsibility of the ESA (human spaceflight, microgravity, exploration).

  1. A genetic investigation of Korean mummies from the Joseon Dynasty.

    Science.gov (United States)

    Kim, Na Young; Lee, Hwan Young; Park, Myung Jin; Yang, Woo Ick; Shin, Kyoung-Jin

    2011-01-01

    Two Korean mummies (Danwoong-mirra and Yoon-mirra) found in medieval tombs in the central region of the Korean peninsula were genetically investigated by analysis of mitochondrial DNA (mtDNA), Y-chromosomal short tandem repeat (Y-STR) and the ABO gene. Danwoong-mirra is a male child mummy and Yoon-mirra is a pregnant female mummy, dating back about 550 and 450 years, respectively. DNA was extracted from soft tissues or bones. mtDNA, Y-STR and the ABO gene were amplified using a small size amplicon strategy and were analyzed according to the criteria of ancient DNA analysis to ensure that authentic DNA typing results were obtained from these ancient samples. Analysis of mtDNA hypervariable region sequence and coding region single nucleotide polymorphism (SNP) information revealed that Danwoong-mirra and Yoon-mirra belong to the East Asian mtDNA haplogroups D4 and M7c, respectively. The Y-STRs were analyzed in the male child mummy (Danwoong-mirra) using the AmpFlSTR® Yfiler PCR Amplification Kit and an in-house Y-miniplex plus system, and could be characterized in 4 loci with small amplicon size. The analysis of ABO gene SNPs using multiplex single base extension methods revealed that the ABO blood types of Danwoong-mirra and Yoon-mirra are AO01 and AB, respectively. The small size amplicon strategy and the authentication process in the present study will be effectively applicable to future genetic analyses of various forensic and ancient samples.

  2. Haiku: New paradigm for the reverse genetics of emerging RNA viruses.

    Directory of Open Access Journals (Sweden)

    Thérèse Atieh

    Full Text Available Reverse genetics is key technology for producing wild-type and genetically modified viruses. The ISA (Infectious Subgenomic Amplicons method is a recent versatile and user-friendly reverse genetics method to rescue RNA viruses. The main constraint of its canonic protocol was the requirement to produce (e.g., by DNA synthesis or fusion PCR 5' and 3' modified genomic fragments encompassing the human cytomegalovirus promoter (pCMV and the hepatitis delta virus ribozyme/simian virus 40 polyadenylation signal (HDR/SV40pA, respectively. Here, we propose the ultimately simplified "Haiku" designs in which terminal pCMV and HDR/SV40pA sequences are provided as additional separate DNA amplicons. This improved procedure was successfully applied to the rescue of a wide range of viruses belonging to genera Flavivirus, Alphavirus and Enterovirus in mosquito or mammalian cells using only standard PCR amplification techniques and starting from a variety of original materials including viral RNAs extracted from cell supernatant media or animal samples. We also demonstrate that, in specific experimental conditions, the presence of the HDR/SV40pA is not necessary to rescue the targeted viruses. These ultimately simplified "Haiku" designs provide an even more simple, rapid, versatile and cost-effective tool to rescue RNA viruses since only generation of overlapping amplicons encompassing the entire viral genome is now required to generate infectious virus. This new approach may completely modify our capacity to obtain infectious RNA viruses.

  3. Identification of polymorphism in exons 7 and 12 of lactoferrin gene and its association with incidence of clinical mastitis in Murrah buffalo.

    Science.gov (United States)

    Dinesh, Krishanender; Verma, Archana; Das Gupta, Ishwar; Thakur, Yash Pal; Verma, Nishant; Arya, Ashwani

    2015-04-01

    Lactoferrin gene is one of the important candidate genes for mastitis resistance. The gene is located on chromosome BTA 22 and consists of 17 exons spanning over 34.5 kb of genomic DNA. The present study was undertaken with the objectives to identify allelic variants in exons 7 and 12 of lactoferrin gene and to analyze association between its genetic variants and incidence of clinical mastitis in Murrah buffalo. The amplification of exons 7 and 12 of lactoferrin gene yielded amplicons of 232- and 461-bp sizes. PCR-restriction fragment length polymorphism (RFLP) analysis of 232-bp amplicon using BccI restriction enzyme revealed three genotypes (AA, AB, and BB) with frequencies of 0.62, 0.22, and 0.16, respectively. The frequencies of two alleles, A and B, were estimated as 0.73 and 0.27. Hpy188I-RFLP for 461-bp amplicon revealed polymorphism with three genotypes, CC, CD, and DD, with respective frequencies of 0.06, 0.39, and 0.56, whereas frequencies for C and D alleles were 0.25 and 0.75. The chi-square (χ(2)) analysis revealed a significant association between incidence of clinical mastitis and genetic variants of exon 7, and animals of AA genotype of exon 7 were found to be least susceptible to mastitis. The findings indicate potential scope for incorporation of lactoferrin gene in selection and breeding of Murrah buffaloes for improved genetic resistance to mastitis.

  4. Metagenomic and near full-length 16S rRNA sequence data in support of the phylogenetic analysis of the rumen bacterial community in steers

    Directory of Open Access Journals (Sweden)

    Phillip R. Myer

    2016-09-01

    Full Text Available Amplicon sequencing utilizing next-generation platforms has significantly transformed how research is conducted, specifically microbial ecology. However, primer and sequencing platform biases can confound or change the way scientists interpret these data. The Pacific Biosciences RSII instrument may also preferentially load smaller fragments, which may also be a function of PCR product exhaustion during sequencing. To further examine theses biases, data is provided from 16S rRNA rumen community analyses. Specifically, data from the relative phylum-level abundances for the ruminal bacterial community are provided to determine between-sample variability. Direct sequencing of metagenomic DNA was conducted to circumvent primer-associated biases in 16S rRNA reads and rarefaction curves were generated to demonstrate adequate coverage of each amplicon. PCR products were also subjected to reduced amplification and pooling to reduce the likelihood of PCR product exhaustion during sequencing on the Pacific Biosciences platform. The taxonomic profiles for the relative phylum-level and genus-level abundance of rumen microbiota as a function of PCR pooling for sequencing on the Pacific Biosciences RSII platform were provided. For more information, see “Evaluation of 16S rRNA amplicon sequencing using two next-generation sequencing technologies for phylogenetic analysis of the rumen bacterial community in steers” P.R. Myer, M. Kim, H.C. Freetly, T.P.L. Smith (2016 [1]. Keywords: 16S rRNA gene, MiSeq, Pacific Biosciences, Rumen microbiome

  5. Design of modern experiments

    International Nuclear Information System (INIS)

    Park, Sung Hweon

    1984-03-01

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

  6. Classic experiments

    CERN Multimedia

    CERN. Geneva; Franklin, M

    2001-01-01

    These will be a set of lectures on classic particle physics experiments, with emphasis on how the emasurements are made. I will discuss experiments made to measure the electric charge distribution of particles, to measure the symmetries of the weak decays, to measure the magnetic moment of the muon. As well as experiments performed which discovered new particles or resonances, like the tAU2and the J/Psi. The coverage will be general and should be understandable to someone knowing little particle physics.

  7. Researching experiences

    DEFF Research Database (Denmark)

    Gjedde, Lisa; Ingemann, Bruno

    In the beginning was - not the word - but the experience. This phenomenological approach provides the basis for this book, which focuses on how a person-in-situation experiences and constructs meaning from a variety of cultural visual events. This book presents video-based processual methods......, 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...

  8. Nucleotide sequence analysis of the recA gene and discrimination of the three isolates of urease-positive thermophilic Campylobacter (UPTC) isolated from seagulls (Larus spp.) in Northern Ireland.

    Science.gov (United States)

    Matsuda, M; Tai, K; Moore, J E; Millar, B C; Murayama, O

    2004-01-01

    Nucleotide sequencing after TA cloning of the amplicon of the almost-full length recA gene from three strains of UPTC (A1, A2, and A3) isolated from seagulls in Northern Ireland, the phenotypical and genotypical characteristics of which have been demonstrated to be indistinguishable, clarified nucleotide differences at three nucleotide positions among the three strains. In conclusion, the nucleotide sequences of the recA gene were found to discriminate among the three strains of UPTC, A1, A2, and A3, which are indistinguishable phenotypically and genotypically. Thus, the present study strongly suggests that nucleotide sequence data of the amplicon of a suitable gene or region could aid in discriminating among isolates of the UPTC group, which are indistinguishable phenotypically and genotypically. Copyright 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

  9. A critical assessment of the microorganisms proposed to be important to enhanced biological phosphorus removal in full-scale wastewater treatment systems

    DEFF Research Database (Denmark)

    Stokholm-Bjerregaard, Mikkel; McIlroy, Simon Jon; Nierychlo, Marta

    2017-01-01

    accumulating organisms (PAOs). Also considered important to EBPR are the glycogen accumulating organisms (GAOs), which are theorized to compete with the PAOs for resources at the expense of P removal efficiency. Numerous studies have sought to identify the PAOs and their GAOs competitors, with several...... sequencing. The microbial community structure in all plants was relatively stable over time. Evidence for the role of the proposed PAOs and GAOs in EBPR varies and is critically assessed, in light of their calculated amplicon abundances, to indicate which of these are important in full-scale systems...... plants. Despite observed high abundances of GAOs (periodically exceeding 20% of the amplicon reads), P removal performance was maintained, indicating that these organisms were not outcompeting the PAOs in these EBPR systems. Phylogenetic diversity within each of the PAOs and GAOs genera was observed...

  10. Quantitative (real-time) PCR

    International Nuclear Information System (INIS)

    Denman, S.E.; McSweeney, C.S.

    2005-01-01

    Many nucleic acid-based probe and PCR assays have been developed for the detection tracking of specific microbes within the rumen ecosystem. Conventional PCR assays detect PCR products at the end stage of each PCR reaction, where exponential amplification is no longer being achieved. This approach can result in different end product (amplicon) quantities being generated. In contrast, using quantitative, or real-time PCR, quantification of the amplicon is performed not at the end of the reaction, but rather during exponential amplification, where theoretically each cycle will result in a doubling of product being created. For real-time PCR, the cycle at which fluorescence is deemed to be detectable above the background during the exponential phase is termed the cycle threshold (Ct). The Ct values obtained are then used for quantitation, which will be discussed later

  11. Pseudogenes and DNA-based diet analyses: A cautionary tale from a relatively well sampled predator-prey system

    DEFF Research Database (Denmark)

    Dunshea, G.; Barros, N. B.; Wells, R. S.

    2008-01-01

    Mitochondrial ribosomal DNA is commonly used in DNA-based dietary analyses. In such studies, these sequences are generally assumed to be the only version present in DNA of the organism of interest. However, nuclear pseudogenes that display variable similarity to the mitochondrial versions...... are common in many taxa. The presence of nuclear pseudogenes that co-amplify with their mitochondrial paralogues can lead to several possible confounding interpretations when applied to estimating animal diet. Here, we investigate the occurrence of nuclear pseudogenes in fecal samples taken from bottlenose...... dolphins (Tursiops truncatus) that were assayed for prey DNA with a universal primer technique. We found pseudogenes in 13 of 15 samples and 1-5 pseudogene haplotypes per sample representing 5-100% of all amplicons produced. The proportion of amplicons that were pseudogenes and the diversity of prey DNA...

  12. Isolation and characterization of a herpesvirus from feral pigeons in China.

    Science.gov (United States)

    Zhao, Panpan; Ma, Jian; Guo, Ying; Tian, Li; Guo, Guangyang; Zhang, Kexin; Xing, Mingwei

    2015-12-01

    A herpesvirus was isolated during a diagnostic investigation of severe cases of conjunctivitis in feral pigeons (Columba livia f. domestica). Isolates of the virus were recovered from throat swabs of the pigeons followed by inoculation of the swab samples in chicken embryo fibroblasts. Pigeons inoculated with the isolated virus had similar clinical signs to those observed in naturally infected birds. Transmission electron microscopy revealed viral structures with typical herpesvirus morphology. Polymerase chain reaction amplification, using herpesvirus-identifying primers resulted in an amplicon of the expected size for herpesvirus. Sequencing of these amplicons and database comparisons identified the herpesvirus UL30 homologue. Phylogenetic reconstructions suggested that the isolated herpesvirus belongs to the Mardivirus genus of Alphaherpesvirinae. Using the current herpesvirus nomenclature conventions, the authors propose that the herpesvirus be named Columbid herpesvirus-1 Heilongjiang. Copyright © 2015 Elsevier Ltd. All rights reserved.

  13. Identifikasi Molekuler Tobacco mosaic virus pada Anggrek di Sleman, Yogyakarta

    Directory of Open Access Journals (Sweden)

    Soesamto Somowiyarjo

    2016-05-01

    Full Text Available Tobamovirus is a group of virus with a wide host range, including orchid plant which considered as an economically important plant. This research aimed to identify Tobamovirus infecting orchids. Virus isolates were collected from orchid nursery in Sleman, Yogyakarta. Plant extract from orchid showing necrotic flex symptom was inoculated to indicator plants Chenopodium amaranticolor. Chlorotic local lesion symptoms occurred within 3 days after inoculation. RNA total from symptomatic C. amaranticolor was extracted by using a commercial kit. cDNA was synthesized using oligo d(T primer. Amplification of cDNA using partial movement protein specific primers TMV-1F and TMV-2R was successfully amplified the amplicon with size ± 422 bp. The nucleotide sequences of this amplicon  showed highest DNA homology (98% with Tobacco mosaic virus Yongren-2 isolat from China.

  14. Particle physics experiments 1986

    International Nuclear Information System (INIS)

    Stuart, G.W.

    1987-01-01

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

  15. Genetic evidence of multiple loci in dystocia - difficult labour

    Directory of Open Access Journals (Sweden)

    Westgren Magnus

    2010-06-01

    Full Text Available Abstract Background Dystocia, difficult labour, is a common but also complex problem during childbirth. It can be attributed to either weak contractions of the uterus, a large infant, reduced capacity of the pelvis or combinations of these. Previous studies have indicated that there is a genetic component in the susceptibility of experiencing dystocia. The purpose of this study was to identify susceptibility genes in dystocia. Methods A total of 104 women in 47 families were included where at least two sisters had undergone caesarean section at a gestational length of 286 days or more at their first delivery. Study of medical records and a telephone interview was performed to identify subjects with dystocia. Whole-genome scanning using Affymetrix genotyping-arrays and non-parametric linkage (NPL analysis was made in 39 women exhibiting the phenotype of dystocia from 19 families. In 68 women re-sequencing was performed of candidate genes showing suggestive linkage: oxytocin (OXT on chromosome 20 and oxytocin-receptor (OXTR on chromosome 3. Results We found a trend towards linkage with suggestive NPL-score (3.15 on chromosome 12p12. Suggestive linkage peaks were observed on chromosomes 3, 4, 6, 10, 20. Re-sequencing of OXT and OXTR did not reveal any causal variants. Conclusions Dystocia is likely to have a genetic component with variations in multiple genes affecting the patient outcome. We found 6 loci that could be re-evaluated in larger patient cohorts.

  16. Genetic evidence of multiple loci in dystocia - difficult labour

    Science.gov (United States)

    2010-01-01

    Background Dystocia, difficult labour, is a common but also complex problem during childbirth. It can be attributed to either weak contractions of the uterus, a large infant, reduced capacity of the pelvis or combinations of these. Previous studies have indicated that there is a genetic component in the susceptibility of experiencing dystocia. The purpose of this study was to identify susceptibility genes in dystocia. Methods A total of 104 women in 47 families were included where at least two sisters had undergone caesarean section at a gestational length of 286 days or more at their first delivery. Study of medical records and a telephone interview was performed to identify subjects with dystocia. Whole-genome scanning using Affymetrix genotyping-arrays and non-parametric linkage (NPL) analysis was made in 39 women exhibiting the phenotype of dystocia from 19 families. In 68 women re-sequencing was performed of candidate genes showing suggestive linkage: oxytocin (OXT) on chromosome 20 and oxytocin-receptor (OXTR) on chromosome 3. Results We found a trend towards linkage with suggestive NPL-score (3.15) on chromosome 12p12. Suggestive linkage peaks were observed on chromosomes 3, 4, 6, 10, 20. Re-sequencing of OXT and OXTR did not reveal any causal variants. Conclusions Dystocia is likely to have a genetic component with variations in multiple genes affecting the patient outcome. We found 6 loci that could be re-evaluated in larger patient cohorts. PMID:20587075

  17. Genomic Analyses Reveal Demographic History and Temperate Adaptation of the Newly Discovered Honey Bee Subspecies Apis mellifera sinisxinyuan n. ssp.

    Science.gov (United States)

    Chen, Chao; Liu, Zhiguang; Pan, Qi; Chen, Xiao; Wang, Huihua; Guo, Haikun; Liu, Shidong; Lu, Hongfeng; Tian, Shilin; Li, Ruiqiang; Shi, Wei

    2016-05-01

    Studying the genetic signatures of climate-driven selection can produce insights into local adaptation and the potential impacts of climate change on populations. The honey bee (Apis mellifera) is an interesting species to study local adaptation because it originated in tropical/subtropical climatic regions and subsequently spread into temperate regions. However, little is known about the genetic basis of its adaptation to temperate climates. Here, we resequenced the whole genomes of ten individual bees from a newly discovered population in temperate China and downloaded resequenced data from 35 individuals from other populations. We found that the new population is an undescribed subspecies in the M-lineage of A. mellifera (Apis mellifera sinisxinyuan). Analyses of population history show that long-term global temperature has strongly influenced the demographic history of A. m. sinisxinyuan and its divergence from other subspecies. Further analyses comparing temperate and tropical populations identified several candidate genes related to fat body and the Hippo signaling pathway that are potentially involved in adaptation to temperate climates. Our results provide insights into the demographic history of the newly discovered A. m. sinisxinyuan, as well as the genetic basis of adaptation of A. mellifera to temperate climates at the genomic level. These findings will facilitate the selective breeding of A. mellifera to improve the survival of overwintering colonies. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  18. The Analysis of A Frequent TMPRSS3 Allele Containing P.V116M and P.V291L in A Cis Configuration among Deaf Koreans

    Directory of Open Access Journals (Sweden)

    Ah Reum Kim

    2017-10-01

    Full Text Available We performed targeted re-sequencing to identify the genetic etiology of early-onset postlingual deafness and encountered a frequent TMPRSS3 allele harboring two variants in a cis configuration. We aimed to evaluate the pathogenicity of the allele. Among 88 cochlear implantees with autosomal recessive non-syndromic hearing loss, subjects with GJB2 and SLC26A4 mutations were excluded. Thirty-one probands manifesting early-onset postlingual deafness were sorted. Through targeted re-sequencing, we detected two families with a TMPRSS3 mutant allele containing p.V116M and p.V291L in a cis configuration, p.[p.V116M; p.V291L]. A minor allele frequency was calculated and proteolytic activity was measured. A p.[p.V116M; p.V291L] allele demonstrated a significantly higher frequency compared to normal controls and merited attention due to its high frequency (4.84%, 3/62. The first family showed a novel deleterious splice site variant—c.783-1G>A—in a trans allele, while the other showed homozygosity. The progression to deafness was noted within the first decade, suggesting DFNB10. The proteolytic activity was significantly reduced, confirming the severe pathogenicity. This frequent mutant allele significantly contributes to early-onset postlingual deafness in Koreans. For clinical implication and proper auditory rehabilitation, it is important to pay attention to this allele with a severe pathogenic potential.

  19. A new DPYD genotyping assay for improving the safety of 5-fluorouracil therapy.

    Science.gov (United States)

    Sistonen, Johanna; Smith, Chingying; Fu, Yung-Kang; Largiadèr, Carlo R

    2012-12-24

    Chemotherapeutic use of 5-fluorouracil (5FU) is compromised by 10-20% of patients developing severe toxicity. Recently described genetic variation in dihydropyrimidine dehydrogenase (DPYD) has been shown to be a major predictor of 5FU toxicity. Here, we describe a new genotyping assay for routine clinical use that covers all the major DPYD risk variants. Genomic regions targeting DPYD risk variants (c.1129-5923C>G, c.1679T>G/A, c.1905+1G>A, c.2846A>T) and additional markers (c.234-123G>C, c.496A>G, c.775A>G) were amplified in a multiplex PCR reaction. The subsequent steps including allele-specific primer extension, hybridization of the primers to a microarray, scanning of the array, and data analysis were automated within the INFINITI® Analyzer (AutoGenomics). The assay was validated by analyzing 107 blood samples obtained from patients previously re-sequenced for the DPYD. The genotypes obtained with the developed assay were 100% concordant with the re-sequencing. The procedure is suitable for routine clinical use since the results are obtained within one day. For heterozygous risk variant carriers (~7% of Europeans), the treatment can be adjusted by 5FU dose reduction, whereas carriers of two risk alleles should be treated with an alternative therapy. The developed assay provides a novel tool to improve the safety of commonly used 5FU-based chemotherapies. Copyright © 2012 Elsevier B.V. All rights reserved.

  20. Big data integration: scalability and sustainability

    KAUST Repository

    Zhang, Zhang

    2016-01-26

    Integration of various types of omics data is critically indispensable for addressing most important and complex biological questions. In the era of big data, however, data integration becomes increasingly tedious, time-consuming and expensive, posing a significant obstacle to fully exploit the wealth of big biological data. Here we propose a scalable and sustainable architecture that integrates big omics data through community-contributed modules. Community modules are contributed and maintained by different committed groups and each module corresponds to a specific data type, deals with data collection, processing and visualization, and delivers data on-demand via web services. Based on this community-based architecture, we build Information Commons for Rice (IC4R; http://ic4r.org), a rice knowledgebase that integrates a variety of rice omics data from multiple community modules, including genome-wide expression profiles derived entirely from RNA-Seq data, resequencing-based genomic variations obtained from re-sequencing data of thousands of rice varieties, plant homologous genes covering multiple diverse plant species, post-translational modifications, rice-related literatures, and community annotations. Taken together, such architecture achieves integration of different types of data from multiple community-contributed modules and accordingly features scalable, sustainable and collaborative integration of big data as well as low costs for database update and maintenance, thus helpful for building IC4R into a comprehensive knowledgebase covering all aspects of rice data and beneficial for both basic and translational researches.

  1. Comprehensive investigation of CASK mutations and other genetic etiologies in 41 patients with intellectual disability and microcephaly with pontine and cerebellar hypoplasia (MICPCH.

    Directory of Open Access Journals (Sweden)

    Shin Hayashi

    Full Text Available The CASK gene (Xp11.4 is highly expressed in the mammalian nervous system and plays several roles in neural development and synaptic function. Loss-of-function mutations of CASK are associated with intellectual disability and microcephaly with pontine and cerebellar hypoplasia (MICPCH, especially in females. Here, we present a comprehensive investigation of 41 MICPCH patients, analyzed by mutational search of CASK and screening of candidate genes using an SNP array, targeted resequencing and whole-exome sequencing (WES. In total, we identified causative or candidate genomic aberrations in 37 of the 41 cases (90.2%. CASK aberrations including a rare mosaic mutation in a male patient, were found in 32 cases, and a mutation in ITPR1, another known gene in which mutations are causative for MICPCH, was found in one case. We also found aberrations involving genes other than CASK, such as HDAC2, MARCKS, and possibly HS3ST5, which may be associated with MICPCH. Moreover, the targeted resequencing screening detected heterozygous variants in RELN in two cases, of uncertain pathogenicity, and WES analysis suggested that concurrent mutations of both DYNC1H1 and DCTN1 in one case could lead to MICPCH. Our results not only identified the etiology of MICPCH in nearly all the investigated patients but also suggest that MICPCH is a genetically heterogeneous condition, in which CASK inactivating mutations appear to account for the majority of cases.

  2. Pixel Experiments

    DEFF Research Database (Denmark)

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

    2015-01-01

    Pixel Experiments The term pixel is traditionally defined as any of the minute elements that together constitute a larger context or image. A pixel has its own form and is the smallest unit seen within a larger structure. In working with the potentials of LED technology in architectural lighting...... 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...... 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...

  3. Simulated experiments

    International Nuclear Information System (INIS)

    Bjerknes, R.

    1977-01-01

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

  4. Organic chemistry experiment

    International Nuclear Information System (INIS)

    Mun, Seok Sik

    2005-02-01

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

  5. Simultaneous Profiling of DNA Mutation and Methylation by Melting Analysis Using Magnetoresistive Biosensor Array

    DEFF Research Database (Denmark)

    Rizzi, Giovanni; Lee, Jung-Rok; Dahl, Christina

    2017-01-01

    specificity. Genomic (mutation) or bisulphite-treated (methylation) DNA is amplified using nondiscriminatory primers, and the amplicons are then hybridized to a giant magnetoresistive (GMR) biosensor array followed by melting curve measurements. The GMR biosensor platform offers scalable multiplexed detection...

  6. Adaptive differentiation coincides with local bioclimatic conditions along an elevational cline in populations of a lichen-forming fungus.

    Science.gov (United States)

    Dal Grande, Francesco; Sharma, Rahul; Meiser, Anjuli; Rolshausen, Gregor; Büdel, Burkhard; Mishra, Bagdevi; Thines, Marco; Otte, Jürgen; Pfenninger, Markus; Schmitt, Imke

    2017-03-31

    Many fungal species occur across a variety of habitats. Particularly lichens, fungi forming symbioses with photosynthetic partners, have evolved remarkable tolerances for environmental extremes. Despite their ecological importance and ubiquity, little is known about the genetic basis of adaption in lichen populations. Here we studied patterns of genome-wide differentiation in the lichen-forming fungus Lasallia pustulata along an altitudinal gradient in the Mediterranean region. We resequenced six populations as pools and identified highly differentiated genomic regions. We then detected gene-environment correlations while controlling for shared population history and pooled sequencing bias, and performed ecophysiological experiments to assess fitness differences of individuals from different environments. We detected two strongly differentiated genetic clusters linked to Mediterranean and temperate-oceanic climate, and an admixture zone, which coincided with the transition between the two bioclimates. High altitude individuals showed ecophysiological adaptations to wetter and more shaded conditions. Highly differentiated genome regions contained a number of genes associated with stress response, local environmental adaptation, and sexual reproduction. Taken together our results provide evidence for a complex interplay between demographic history and spatially varying selection acting on a number of key biological processes, suggesting a scenario of ecological speciation.

  7. Experiment Databases

    Science.gov (United States)

    Vanschoren, Joaquin; Blockeel, Hendrik

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

  8. Understanding patient experience

    DEFF Research Database (Denmark)

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

    2017-01-01

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

  9. Cross-shelf investigation of coral reef cryptic benthic organisms reveals diversity patterns of the hidden majority

    KAUST Repository

    Pearman, John K.; Leray, M.; Villalobos, R.; Machida, R. J.; Berumen, Michael L.; Knowlton, N.; Carvalho, Susana

    2018-01-01

    (ARMS) and amplicon sequencing methodologies, targeting mitochondrial cytochrome oxidase I and 18S rRNA genes, to investigate changes in the cryptic reef biodiversity. ARMS, deployed at 11 sites across a near- to off-shore gradient in the Red Sea were

  10. SARS - Diagnosis

    Indian Academy of Sciences (India)

    SARS - Diagnosis. Mainly by exclusion of known causes of atypical pneumonia; * X ray Chest; * PCR on body fluids- primers defined by WHO centres available from website.-ve result does not exclude SARS. * Sequencing of amplicons; * Viral Cultures – demanding; * Antibody tests.

  11. Multiplex reverse transcription-polymerase chain reaction combined with on-chip electrophoresis as a rapid screening tool for candidate gene sets

    DEFF Research Database (Denmark)

    Wittig, Rainer; Salowsky, Rüdiger; Blaich, Stephanie

    2005-01-01

    Combining multiplex reverse transcription-polymerase chain reaction (mRT-PCR) with microfluidic amplicon analysis, we developed an assay for the rapid and reliable semiquantitative expression screening of 11 candidate genes for drug resistance in human malignant melanoma. The functionality of thi...

  12. Bifiguratus adelaidae, gen. et sp. nov., a new member of Mucoromycotina in endophytic and soil-dwelling habitats

    Science.gov (United States)

    Illumina amplicon sequencing of soil in a temperate pine forest in the southeastern United States detected an abundant, N-responsive fungal genotype of unknown phylogenetic affiliation. Two isolates with ribosomal sequences consistent with that genotype were subsequently obtained in culture. Examina...

  13. Mutations of the Spliceosome Complex Genes Occur In Adult Patients but Are Very Rare In Children with Myeloid Neoplasia

    DEFF Research Database (Denmark)

    Hirabayashi, Shinsuke; Moetter, Jessica; Yoshida, Kenichi

    -protein complexes that remove noncoding introns from precursor mRNA. We hypothesized that the disruption of the spliceosome complex might play a driving role in the leukemogenesis in pediatric MDS. Using targeted re-sequencing we investigated the 3 exclusive hotspots of 2 spliceosome genes that were found...... negative. The drastically reduced frequency of spliceosome mutations in pediatric compared to adult myeloid malignancies suggests a different pathogenetic mechanism in childhood disease, and fits well with previous reports that somatic mutations of non-Ras-pathway genes, such as DNMT3A, are less prevalent...

  14. An Exon-Based Comparative Variant Analysis Pipeline to Study the Scale and Role of Frameshift and Nonsense Mutation in the Human-Chimpanzee Divergence

    Directory of Open Access Journals (Sweden)

    GongXin Yu

    2009-01-01

    important biological processes such as T cell lineage development, the pathogenesis of inflammatory diseases, and antigen induced cell death. A “less-is-more” model was previously established to illustrate the role of the gene inactivation and disruptions during human evolution. Here this analysis suggested a different model where the chimpanzee-specific exon-disrupting mutations may act as additional evolutionary force that drove the human-chimpanzee divergence. Finally, the analysis revealed a number of sequencing errors in the chimpanzee and human genome sequences and further illustrated that they could be corrected without resequencing.

  15. Annexin A5 Promoter Haplotype M2 Is Not a Risk Factor for Recurrent Pregnancy Loss in Northern Europe

    DEFF Research Database (Denmark)

    Nagirnaja, Liina; Nõmmemees, Diana; Rull, Kristiina

    2015-01-01

    and recurrent pregnancy loss (RPL), however with inconclusive results. STUDY SUBJECTS AND METHODS: A retrospective case-control study combining resequencing and restriction fragment length polymorphism (RFLP) analysis was undertaken in 313 women with unexplained RPL and 214 fertile women from Estonia...... compared to controls both in Estonia (8.1% vs 15.2%, respectively) and Denmark (9.7% vs 12.6%). The high M2 prevalence in fertile controls was consistent with estimations for European and East Asian populations (9.6%-16.0%). CONCLUSIONS: This study cautions to consider the M2 haplotype as a deterministic...

  16. Development Of An Efficient Glycerol Utilizing Saccharomyces Cerevisiae Strain Via Adaptive Laboratory Evolution

    DEFF Research Database (Denmark)

    Strucko, Tomas; Zirngibl, Katharina; Tharwat Tolba Mohamed, Elsayed

    2015-01-01

    that popular wild-type laboratory yeast strains, commonly applied in metabolic engineering studies, did not grow or grew very slowly in glycerol medium.In this work, an adaptive laboratory evolution approach to obtain S. cerevisiae strains with an improved ability to grow on glycerol was applied. A broad array...... of evolved strains, which exhibited a significant increase in the specific growth rate and a higher glycerol consumption rate, were isolated. The best performing strains were further analyzed by classical genetics and whole genome re-sequencing in order to understand the molecular basis of glycerol...

  17. Reliable Detection of Herpes Simplex Virus Sequence Variation by High-Throughput Resequencing.

    Science.gov (United States)

    Morse, Alison M; Calabro, Kaitlyn R; Fear, Justin M; Bloom, David C; McIntyre, Lauren M

    2017-08-16

    High-throughput sequencing (HTS) has resulted in data for a number of herpes simplex virus (HSV) laboratory strains and clinical isolates. The knowledge of these sequences has been critical for investigating viral pathogenicity. However, the assembly of complete herpesviral genomes, including HSV, is complicated due to the existence of large repeat regions and arrays of smaller reiterated sequences that are commonly found in these genomes. In addition, the inherent genetic variation in populations of isolates for viruses and other microorganisms presents an additional challenge to many existing HTS sequence assembly pipelines. Here, we evaluate two approaches for the identification of genetic variants in HSV1 strains using Illumina short read sequencing data. The first, a reference-based approach, identifies variants from reads aligned to a reference sequence and the second, a de novo assembly approach, identifies variants from reads aligned to de novo assembled consensus sequences. Of critical importance for both approaches is the reduction in the number of low complexity regions through the construction of a non-redundant reference genome. We compared variants identified in the two methods. Our results indicate that approximately 85% of variants are identified regardless of the approach. The reference-based approach to variant discovery captures an additional 15% representing variants divergent from the HSV1 reference possibly due to viral passage. Reference-based approaches are significantly less labor-intensive and identify variants across the genome where de novo assembly-based approaches are limited to regions where contigs have been successfully assembled. In addition, regions of poor quality assembly can lead to false variant identification in de novo consensus sequences. For viruses with a well-assembled reference genome, a reference-based approach is recommended.

  18. Linking consumer experiences

    DEFF Research Database (Denmark)

    Smed, Karina Madsen

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

  19. Analytical chemistry experiment

    International Nuclear Information System (INIS)

    Park, Seung Jo; Paeng, Seong Gwan; Jang, Cheol Hyeon

    1992-08-01

    This book deals with analytical chemistry experiment with eight chapters. It explains general matters that require attention on experiment, handling of medicine with keep and class, the method for handling and glass devices, general control during experiment on heating, cooling, filtering, distillation and extraction and evaporation and dry, glass craft on purpose of the craft, how to cut glass tube and how to bend glass tube, volumetric analysis on neutralization titration and precipitation titration, gravimetric analysis on solubility product, filter and washing and microorganism experiment with necessary tool, sterilization disinfection incubation and appendixes.

  20. 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.

  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. Particle physics experiments 1982

    International Nuclear Information System (INIS)

    Rousseau, M.D.; Stuart, G.

    1983-01-01

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

  3. 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

  4. Experience in Design and Learning Approaches – Enhancing the Framework for Experience

    Directory of Open Access Journals (Sweden)

    Merja L.M. Bauters

    2017-06-01

    Full Text Available In design and learning studies, an increasing amount of attention has been paid to experience. Many design approaches relate experience to embodiment and phenomenology. The growth in the number of applications that use the Internet of Things (IoT has shifted human interactions from mobile devices and computers to tangible, material things. In education, the pressure to learn and update skills and knowledge, especially in work environments, has underlined the challenge of understanding how workers learn from reflection while working. These directions have been fuelled by research findings in the neurosciences, embodied cognition, the extended phenomenological–cognitive system and the role of emotions in decision-making and meaning making. The perspective on experience in different disciplines varies, and the aim is often to categorise experience. These approaches provide a worthwhile view of the importance of experience in learning and design, such as the recent emphasis on conceptual and epistemological knowledge creation. In pragmatism, experience plays a considerable role in research, art, communication and reflection. Therefore, I rely on Peirce’s communicative theory of signs and Dewey’s philosophy of experience to examine how experience is connected to reflection and therefore how it is necessarily tangible.

  5. Popper's Thought Experiment Reinvestigated

    Science.gov (United States)

    Richardson, Chris; Dowling, Jonathan

    2012-02-01

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

  6. Nitrogen Cycle Evaluation (NiCE) Chip for the Simultaneous Analysis of Multiple N-Cycle Associated Genes.

    Science.gov (United States)

    Oshiki, Mamoru; Segawa, Takahiro; Ishii, Satoshi

    2018-02-02

    Various microorganisms play key roles in the Nitrogen (N) cycle. Quantitative PCR (qPCR) and PCR-amplicon sequencing of the N cycle functional genes allow us to analyze the abundance and diversity of microbes responsible in the N transforming reactions in various environmental samples. However, analysis of multiple target genes can be cumbersome and expensive. PCR-independent analysis, such as metagenomics and metatranscriptomics, is useful but expensive especially when we analyze multiple samples and try to detect N cycle functional genes present at relatively low abundance. Here, we present the application of microfluidic qPCR chip technology to simultaneously quantify and prepare amplicon sequence libraries for multiple N cycle functional genes as well as taxon-specific 16S rRNA gene markers for many samples. This approach, named as N cycle evaluation (NiCE) chip, was evaluated by using DNA from pure and artificially mixed bacterial cultures and by comparing the results with those obtained by conventional qPCR and amplicon sequencing methods. Quantitative results obtained by the NiCE chip were comparable to those obtained by conventional qPCR. In addition, the NiCE chip was successfully applied to examine abundance and diversity of N cycle functional genes in wastewater samples. Although non-specific amplification was detected on the NiCE chip, this could be overcome by optimizing the primer sequences in the future. As the NiCE chip can provide high-throughput format to quantify and prepare sequence libraries for multiple N cycle functional genes, this tool should advance our ability to explore N cycling in various samples. Importance. We report a novel approach, namely Nitrogen Cycle Evaluation (NiCE) chip by using microfluidic qPCR chip technology. By sequencing the amplicons recovered from the NiCE chip, we can assess diversities of the N cycle functional genes. The NiCE chip technology is applicable to analyze the temporal dynamics of the N cycle gene

  7. A Critical Assessment of the Microorganisms Proposed to be Important to Enhanced Biological Phosphorus Removal in Full-Scale Wastewater Treatment Systems

    Directory of Open Access Journals (Sweden)

    Per H. Nielsen

    2017-04-01

    Full Text Available Understanding the microbiology of phosphorus (P removal is considered essential to knowledge-based optimization of enhanced biological P removal (EBPR systems. Biological P removal is achieved in these systems by promoting the growth of organisms collectively known as the polyphosphate accumulating organisms (PAOs. Also considered important to EBPR are the glycogen accumulating organisms (GAOs, which are theorized to compete with the PAOs for resources at the expense of P removal efficiency. Numerous studies have sought to identify the PAOs and their GAOs competitors, with several candidates proposed for each over the last few decades. The current study collectively assessed the abundance and diversity of all proposed PAOs and GAOs in 18 Danish full-scale wastewater treatment plants with well-working biological nutrient removal over a period of 9 years using 16S rRNA gene amplicon sequencing. The microbial community structure in all plants was relatively stable over time. Evidence for the role of the proposed PAOs and GAOs in EBPR varies and is critically assessed, in light of their calculated amplicon abundances, to indicate which of these are important in full-scale systems. Bacteria from the genus Tetrasphaera were the most abundant of the PAOs. The “Candidatus Accumulibacter” PAOs were in much lower abundance and appear to be biased by the amplicon-based method applied. The genera Dechloromonas, Microlunatus, and Tessaracoccus were identified as abundant putative PAO that require further research attention. Interestingly, the actinobacterial Micropruina and sbr-gs28 phylotypes were among the most abundant of the putative GAOs. Members of the genera Defluviicoccus, Propionivibrio, the family Competibacteraceae, and the spb280 group were also relatively abundant in some plants. Despite observed high abundances of GAOs (periodically exceeding 20% of the amplicon reads, P removal performance was maintained, indicating that these organisms

  8. 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.

  9. Experiment-o-mania

    Science.gov (United States)

    Drndarski, Marina

    2015-04-01

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

  10. Marine Microbial Systems Ecology: Microbial Networks in the Sea

    NARCIS (Netherlands)

    Muijzer, G.; Stal, L.J.; Cretoiu, M.S.

    2016-01-01

    Next-generation sequencing of DNA has revolutionized microbial ecology. Using this technology, it became for the first time possible to analyze hundreds of samples simultaneously and in great detail. 16S rRNA amplicon sequencing, metagenomics and metatranscriptomics became available to determine the

  11. Host and environmental factors affecting the intestinal microbiota in chickens

    NARCIS (Netherlands)

    Kers, Jannigje G.; Velkers, Francisca C.; Fischer, Egil A.J.; Hermes, Gerben D.A.; Stegeman, J.A.; Smidt, Hauke

    2018-01-01

    The initial development of intestinal microbiota in poultry plays an important role in production performance, overall health and resistance against microbial infections. Multiplexed sequencing of 16S ribosomal RNA gene amplicons is often used in studies, such as feed intervention or antimicrobial

  12. Host and Environmental Factors Affecting the Intestinal Microbiota in Chickens

    NARCIS (Netherlands)

    Kers, J.G.; Velkers, F.C.; Fischer, E.A.J.; Hermes, Gerben; Stegeman, J.A.; Smidt, Hauke

    2018-01-01

    The initial development of intestinal microbiota in poultry plays an important role in production performance, overall health and resistance against microbial infections. Multiplexed sequencing of 16S ribosomal RNA gene amplicons is often used in studies,such as feed intervention or antimicrobial

  13. Identification of rare and frequent variants of the CASR gene by high-resolution melting

    DEFF Research Database (Denmark)

    Nissen, Peter H; Christensen, Signe E; Ladefoged, Søren A

    2012-01-01

    of seven new CASR variants and nine recurrent. HRM variant scanning, in combination with small amplicon genotyping, provides a simple workflow with reduced sequencing burden. Bioinformatics analyses using two freely available prediction tools (PolyPhen2 and SIFT) for evaluating amino acid substitutions...

  14. Channeling experiment

    International Nuclear Information System (INIS)

    Abelin, H.; Birgersson, L.; Widen, H.; Aagren, T.; Moreno, L.; Neretnieks, I.

    1990-07-01

    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)

  15. The ERASMUS experiment archive

    Science.gov (United States)

    Isakeit, Dieter; Sabbatini, Massimo; Carey, William

    2005-08-01

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

  16. Review of experiments

    International Nuclear Information System (INIS)

    Chang, C.; Yodh, G.; Cutts, D.; Lanou, R.; Engels, E.; Kramer, M.; Danby, G.

    1977-01-01

    A study was made to examine the effects which raising the ISA from 200 x 200 GeV to 400 x 400 GeV would have on the ''canonical'' experiments. These were ''canonical'' in the sense that they span the full range of foreseeable physics and have served as topics in previous Summer Studies and Workshops which resulted in quite explicit hardware designs and experimental goals. The study results indicate that all of the ''canonical'' experiments survive. Some are actually improved, some are unaffected, and some require changes which are suggested. In general, the 90 0 experiments are relatively unaffected. The single arm small angle spectrometer, the wide aperture (FATS-WASP) spectrometer and the Coulomb interference experiment have the largest number of modifications suggested. No uniqueness to these solutions are claimed, and there may be more desirable radical approaches. It is, however, felt that the 400 x 400 GeV ISA not only permits the work on conceptual experiments from previous Summer Studies to be taken over entirely, but indicates areas of improvement in many of them. Specifics of the individual experiments are discussed

  17. Real-time PCR Detection of Brucella Abortus: A Comparative Study of SYBR Green I, 5'-exonuclease, and Hybridization Probe Assays

    Energy Technology Data Exchange (ETDEWEB)

    Newby, Deborah Trishelle; Hadfield, Ted; Roberto, Francisco Figueroa

    2003-08-01

    Real-time PCR provides a means of detecting and quantifying DNA targets by monitoring PCR product accumulation during cycling as indicated by increased fluorescence. A number of different approaches can be used to generate the fluorescence signal. Three approaches—SYBR Green I (a double-stranded DNA intercalating dye), 5'-exonuclease (enzymatically released fluors), and hybridization probes (fluorescence resonance energy transfer)—were evaluated for use in a real-time PCR assay to detect Brucella abortus. The three assays utilized the same amplification primers to produce an identical amplicon. This amplicon spans a region of the B. abortus genome that includes portions of the alkB gene and the IS711 insertion element. All three assays were of comparable sensitivity, providing a linear assay over 7 orders of magnitude (from 7.5 ng down to 7.5 fg). However, the greatest specificity was achieved with the hybridization probe assay.

  18. A seminested PCR assay for detection and typing of human papillomavirus based on E1 gene sequences.

    Science.gov (United States)

    Cavalcante, Gustavo Henrique O; de Araújo, Josélio M G; Fernandes, José Veríssimo; Lanza, Daniel C F

    2018-05-01

    HPV infection is considered one of the leading causes of cervical cancer in the world. To date, more than 180 types of HPV have been described and viral typing is critical for defining the prognosis of cancer. In this work, a seminested PCR which allow fast and inexpensively detection and typing of HPV is presented. The system is based on the amplification of a variable length region within the viral gene E1, using three primers that potentially anneal in all HPV genomes. The amplicons produced in the first step can be identified by high resolution electrophoresis or direct sequencing. The seminested step includes nine specific primers which can be used in multiplex or individual reactions to discriminate the main types of HPV by amplicon size differentiation using agarose electrophoresis, reducing the time spent and cost per analysis. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Second-generation sequencing of forensic STRs using the Ion Torrent™ HID STR 10-plex and the Ion PGM™

    DEFF Research Database (Denmark)

    Fordyce, Sarah L; Mogensen, Helle Smidt; Børsting, Claus

    2015-01-01

    Second-generation sequencing (SGS) using Roche/454 and Illumina platforms has proved capable of sequencing the majority of the key forensic genetic STR systems. Given that Roche has announced that the 454 platforms will no longer be supported from 2015, focus should now be shifted to competing SGS...... platforms, such as the MiSeq (Illumina) and the Ion Personal Genome Machine (Ion PGM™; Thermo Fisher). There are currently several challenges faced with amplicon-based SGS STR typing in forensic genetics, including current lengths of amplicons for CE-typing and lack of uniform data analysis between......) analysis of sensitivity; (3) typing of mixtures; and (4) typing of biological crime case samples. Full profiles and concordant results between replicate SGS runs and CE-typing were observed for all control samples. Full profiles were seen with DNA input down to 50pg, with the exception of a single locus...

  20. Integrity of nuclear genomic deoxyribonucleic acid in cooked meat: Implications for food traceability.

    Science.gov (United States)

    Aslan, O; Hamill, R M; Sweeney, T; Reardon, W; Mullen, A M

    2009-01-01

    It is essential to isolate high-quality DNA from muscle tissue for PCR-based applications in traceability of animal origin. We wished to examine the impact of cooking meat to a range of core temperatures on the quality and quantity of subsequently isolated genomic (specifically, nuclear) DNA. Triplicate steak samples were cooked in a water bath (100 degrees C) until their final internal temperature was 75, 80, 85, 90, 95, or 100 degrees C, and DNA was extracted. Deoxyribonucleic acid quantity was significantly reduced in cooked meat samples compared with raw (6.5 vs. 56.6 ng/microL; P 800 bp) were observed only when using DNA from raw meat and steak cooked to lower core temperatures. Small amplicons (food authentication, it is less abundant, and results suggest that analyses should be designed to use small amplicon sizes for meat cooked to high core temperatures.

  1. Diagnostics for pellet experiments

    International Nuclear Information System (INIS)

    Johnson, R.R.

    1978-01-01

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

  2. Reproducible computational biology experiments with SED-ML--the Simulation Experiment Description Markup Language.

    Science.gov (United States)

    Waltemath, Dagmar; Adams, Richard; Bergmann, Frank T; Hucka, Michael; Kolpakov, Fedor; Miller, Andrew K; Moraru, Ion I; Nickerson, David; Sahle, Sven; Snoep, Jacky L; Le Novère, Nicolas

    2011-12-15

    The increasing use of computational simulation experiments to inform modern biological research creates new challenges to annotate, archive, share and reproduce such experiments. The recently published Minimum Information About a Simulation Experiment (MIASE) proposes a minimal set of information that should be provided to allow the reproduction of simulation experiments among users and software tools. In this article, we present the Simulation Experiment Description Markup Language (SED-ML). SED-ML encodes in a computer-readable exchange format the information required by MIASE to enable reproduction of simulation experiments. It has been developed as a community project and it is defined in a detailed technical specification and additionally provides an XML schema. The version of SED-ML described in this publication is Level 1 Version 1. It covers the description of the most frequent type of simulation experiments in the area, namely time course simulations. SED-ML documents specify which models to use in an experiment, modifications to apply on the models before using them, which simulation procedures to run on each model, what analysis results to output, and how the results should be presented. These descriptions are independent of the underlying model implementation. SED-ML is a software-independent format for encoding the description of simulation experiments; it is not specific to particular simulation tools. Here, we demonstrate that with the growing software support for SED-ML we can effectively exchange executable simulation descriptions. With SED-ML, software can exchange simulation experiment descriptions, enabling the validation and reuse of simulation experiments in different tools. Authors of papers reporting simulation experiments can make their simulation protocols available for other scientists to reproduce the results. Because SED-ML is agnostic about exact modeling language(s) used, experiments covering models from different fields of research

  3. Reproducible computational biology experiments with SED-ML - The Simulation Experiment Description Markup Language

    Science.gov (United States)

    2011-01-01

    Background The increasing use of computational simulation experiments to inform modern biological research creates new challenges to annotate, archive, share and reproduce such experiments. The recently published Minimum Information About a Simulation Experiment (MIASE) proposes a minimal set of information that should be provided to allow the reproduction of simulation experiments among users and software tools. Results In this article, we present the Simulation Experiment Description Markup Language (SED-ML). SED-ML encodes in a computer-readable exchange format the information required by MIASE to enable reproduction of simulation experiments. It has been developed as a community project and it is defined in a detailed technical specification and additionally provides an XML schema. The version of SED-ML described in this publication is Level 1 Version 1. It covers the description of the most frequent type of simulation experiments in the area, namely time course simulations. SED-ML documents specify which models to use in an experiment, modifications to apply on the models before using them, which simulation procedures to run on each model, what analysis results to output, and how the results should be presented. These descriptions are independent of the underlying model implementation. SED-ML is a software-independent format for encoding the description of simulation experiments; it is not specific to particular simulation tools. Here, we demonstrate that with the growing software support for SED-ML we can effectively exchange executable simulation descriptions. Conclusions With SED-ML, software can exchange simulation experiment descriptions, enabling the validation and reuse of simulation experiments in different tools. Authors of papers reporting simulation experiments can make their simulation protocols available for other scientists to reproduce the results. Because SED-ML is agnostic about exact modeling language(s) used, experiments covering models from

  4. The Belle II Experiment

    CERN Document Server

    Kahn, J

    2017-01-01

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

  5. Virtual neutron scattering experiments

    DEFF Research Database (Denmark)

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

    2016-01-01

    We describe how virtual experiments can be utilized in a learning design that prepares students for hands-on experiments at large-scale facilities. We illustrate the design by showing how virtual experiments are used at the Niels Bohr Institute in a master level course on neutron scattering....... In the last week of the course, students travel to a large-scale neutron scattering facility to perform real neutron scattering experiments. Through student interviews and survey answers, we argue, that the virtual training prepares the students to engage more fruitfully with experiments by letting them focus...... on physics and data rather than the overwhelming instrumentation. We argue that this is because they can transfer their virtual experimental experience to the real-life situation. However, we also find that learning is still situated in the sense that only knowledge of particular experiments is transferred...

  6. Prebiotic Wheat Bran Fractions Induce Specific Microbiota Changes

    Science.gov (United States)

    D’hoe, Kevin; Conterno, Lorenza; Fava, Francesca; Falony, Gwen; Vieira-Silva, Sara; Vermeiren, Joan; Tuohy, Kieran; Raes, Jeroen

    2018-01-01

    Wheat bran fibers are considered beneficial to human health through their impact on gut microbiota composition and activity. Here, we assessed the prebiotic potential of selected bran fractions by performing a series of fecal slurry anaerobic fermentation experiments using aleurone as well as total, ultrafine, and soluble wheat bran (swb) as carbon sources. By combining amplicon-based community profiling with a fluorescent in situ hybridization (FISH) approach, we found that incubation conditions favor the growth of Proteobacteria such as Escherichia and Bilophila. These effects were countered in all but one [total wheat bran (twb)] fermentation experiments. Growth of Bifidobacterium species was stimulated after fermentation using ultrafine, soluble, and twb, in the latter two as part of a general increase in bacterial load. Both ultrafine and swb fermentation resulted in a trade-off between Bifidobacterium and Bilophila, as previously observed in human dietary supplementation studies looking at the effect of inulin-type fructans on the human gut microbiota. Aleurone selectively stimulated growth of Dorea and butyrate-producing Roseburia. All fermentation experiments induced enhanced gas production; increased butyrate concentrations were only observed following soluble bran incubation. Our results open perspectives for the development of aleurone as a complementary prebiotic selectively targeting colon butyrate producers. PMID:29416529

  7. Prebiotic Wheat Bran Fractions Induce Specific Microbiota Changes

    Directory of Open Access Journals (Sweden)

    Kevin D’hoe

    2018-01-01

    Full Text Available Wheat bran fibers are considered beneficial to human health through their impact on gut microbiota composition and activity. Here, we assessed the prebiotic potential of selected bran fractions by performing a series of fecal slurry anaerobic fermentation experiments using aleurone as well as total, ultrafine, and soluble wheat bran (swb as carbon sources. By combining amplicon-based community profiling with a fluorescent in situ hybridization (FISH approach, we found that incubation conditions favor the growth of Proteobacteria such as Escherichia and Bilophila. These effects were countered in all but one [total wheat bran (twb] fermentation experiments. Growth of Bifidobacterium species was stimulated after fermentation using ultrafine, soluble, and twb, in the latter two as part of a general increase in bacterial load. Both ultrafine and swb fermentation resulted in a trade-off between Bifidobacterium and Bilophila, as previously observed in human dietary supplementation studies looking at the effect of inulin-type fructans on the human gut microbiota. Aleurone selectively stimulated growth of Dorea and butyrate-producing Roseburia. All fermentation experiments induced enhanced gas production; increased butyrate concentrations were only observed following soluble bran incubation. Our results open perspectives for the development of aleurone as a complementary prebiotic selectively targeting colon butyrate producers.

  8. Prebiotic Wheat Bran Fractions Induce Specific Microbiota Changes.

    Science.gov (United States)

    D'hoe, Kevin; Conterno, Lorenza; Fava, Francesca; Falony, Gwen; Vieira-Silva, Sara; Vermeiren, Joan; Tuohy, Kieran; Raes, Jeroen

    2018-01-01

    Wheat bran fibers are considered beneficial to human health through their impact on gut microbiota composition and activity. Here, we assessed the prebiotic potential of selected bran fractions by performing a series of fecal slurry anaerobic fermentation experiments using aleurone as well as total, ultrafine, and soluble wheat bran (swb) as carbon sources. By combining amplicon-based community profiling with a fluorescent in situ hybridization (FISH) approach, we found that incubation conditions favor the growth of Proteobacteria such as Escherichia and Bilophila . These effects were countered in all but one [total wheat bran (twb)] fermentation experiments. Growth of Bifidobacterium species was stimulated after fermentation using ultrafine, soluble, and twb, in the latter two as part of a general increase in bacterial load. Both ultrafine and swb fermentation resulted in a trade-off between Bifidobacterium and Bilophila , as previously observed in human dietary supplementation studies looking at the effect of inulin-type fructans on the human gut microbiota. Aleurone selectively stimulated growth of Dorea and butyrate-producing Roseburia . All fermentation experiments induced enhanced gas production; increased butyrate concentrations were only observed following soluble bran incubation. Our results open perspectives for the development of aleurone as a complementary prebiotic selectively targeting colon butyrate producers.

  9. 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

  10. Molecular Characterization of Brevibacillus laterosporus and Its Potential Use in Biological Control

    Science.gov (United States)

    de Oliveira, Edmar Justo; Rabinovitch, Leon; Monnerat, Rose Gomes; Passos, Liana Konovaloff Jannotti; Zahner, Viviane

    2004-01-01

    Thirty-three strains of Brevibacillus laterosporus, including three novel strains isolated from Brazilian soil samples, were examined for genetic variability by the use of different PCR-based methods. Molecular markers that could characterize bacterial strains with regards to their pathogenic potential were investigated. In addition, toxicity was assessed by the use of insects belonging to the orders Lepidoptera and Coleoptera and the mollusk Biomphalaria glabrata. Among the targets tested, Biomphalaria glabrata demonstrated the highest degree of sensitivity to B. laterosporus, with some strains inducing 90 to 100% mortality in snails aged 3 and 12 days posteclosion. Larvae of the coleopteron Anthonomus grandis were also susceptible, presenting mortality levels of between 33 and 63%. Toxicity was also noted towards the lepidopteron Anticarsia gemmatalis. In contrast, no mortality was recorded among test populations of Tenebrio molitor or Spodoptera frugiperda. The application of intergenic transcribed spacer PCR and BOX-PCR generated 15 and 17 different genotypes, respectively. None of the molecular techniques allowed the identification of a convenient marker that was associated with any entomopathogenic phenotype. However, a 1,078-bp amplicon was detected for all strains of B. laterosporus when a primer for amplification of the BOXA1R region was used. Similarly, a 900-bp amplicon was generated from all isolates by use of the primer OPA-11 for randomly amplified polymorphic DNA analysis. These amplicons were not detected for other phenotypically related Brevibacillus species, indicating that they represent markers that are specific for B. laterosporus, which may prove useful for the isolation and identification of new strains of this species. PMID:15528531

  11. Ultrasensitive quantitation of human papillomavirus type 16 E6 oncogene sequences by nested real time PCR

    Directory of Open Access Journals (Sweden)

    López-Revilla Rubén

    2010-05-01

    Full Text Available Abstract Background We have developed an ultrasensitive method based on conventional PCR preamplification followed by nested amplification through real time PCR (qPCR in the presence of the DNA intercalating agent EvaGreen. Results Amplification mixtures calibrated with a known number of pHV101 copies carrying a 645 base pair (bp-long insert of the human papillomavirus type 16 (HPV16 E6 oncogene were used to generate the E6-1 amplicon of 645 bp by conventional PCR and then the E6-2 amplicon of 237 bp by nested qPCR. Direct and nested qPCR mixtures for E6-2 amplification corresponding to 2.5 × 102-2.5 × 106 initial pHV101 copies had threshold cycle (Ct values in the ranges of 18.7-29.0 and 10.0-25.0, respectively. The Ct of qPCR mixtures prepared with 1/50 volumes of preamplified mixtures containing 50 ng of DNA of the SiHa cell line (derived from an invasive cervical cancer with one HPV16 genome per cell was 19.9. Thermal fluorescence extinction profiles of E6-2 amplicons generated from pHV101 and SiHa DNA were identical, with a peak at 85.5°C. Conclusions Our method based on conventional preamplification for 15 cycles increased 10,750 times the sensitivity of nested qPCR for the quantitation of the E6 viral oncogene and confirmed that the SiHa cell line contains one E6-HPV16 copy per cell.

  12. Simultaneous fitting of real-time PCR data with efficiency of amplification modeled as Gaussian function of target fluorescence

    Directory of Open Access Journals (Sweden)

    Lazar Andreas

    2008-02-01

    Full Text Available Abstract Background In real-time PCR, it is necessary to consider the efficiency of amplification (EA of amplicons in order to determine initial target levels properly. EAs can be deduced from standard curves, but these involve extra effort and cost and may yield invalid EAs. Alternatively, EA can be extracted from individual fluorescence curves. Unfortunately, this is not reliable enough. Results Here we introduce simultaneous non-linear fitting to determine – without standard curves – an optimal common EA for all samples of a group. In order to adjust EA as a function of target fluorescence, and still to describe fluorescence as a function of cycle number, we use an iterative algorithm that increases fluorescence cycle by cycle and thus simulates the PCR process. A Gauss peak function is used to model the decrease of EA with increasing amplicon accumulation. Our approach was validated experimentally with hydrolysis probe or SYBR green detection with dilution series of 5 different targets. It performed distinctly better in terms of accuracy than standard curve, DART-PCR, and LinRegPCR approaches. Based on reliable EAs, it was possible to detect that for some amplicons, extraordinary fluorescence (EA > 2.00 was generated with locked nucleic acid hydrolysis probes, but not with SYBR green. Conclusion In comparison to previously reported approaches that are based on the separate analysis of each curve and on modelling EA as a function of cycle number, our approach yields more accurate and precise estimates of relative initial target levels.

  13. Rapid and sensitive method to identify Mycobacterium avium subsp. paratuberculosis in cow's milk by DNA methylase genotyping.

    Science.gov (United States)

    Mundo, Silvia Leonor; Gilardoni, Liliana Rosa; Hoffman, Federico José; Lopez, Osvaldo Jorge

    2013-03-01

    Paratuberculosis is an infectious, chronic, and incurable disease that affects ruminants, caused by Mycobacterium avium subsp. paratuberculosis. This bacterium is shed primarily through feces of infected cows but can be also excreted in colostrum and milk and might survive pasteurization. Since an association of genomic sequences of M. avium subsp. paratuberculosis in patients with Crohn's disease has been described; it is of interest to rapidly detect M. avium subsp. paratuberculosis in milk for human consumption. IS900 insertion is used as a target for PCR amplification to identify the presence of M. avium subsp. paratuberculosis in biological samples. Two target sequences were selected: IS1 (155 bp) and IS2 (94 bp). These fragments have a 100% identity among all M. avium subsp. paratuberculosis strains sequenced. M. avium subsp. paratuberculosis was specifically concentrated from milk samples by immunomagnetic separation prior to performing PCR. The amplicons were characterized using DNA methylase Genotyping, i.e., the amplicons were methylated with 6-methyl-adenine and digested with restriction enzymes to confirm their identity. The methylated amplicons from 100 CFU of M. avium subsp. paratuberculosis can be visualized in a Western blot format using an anti-6-methyl-adenine monoclonal antibody. The use of DNA methyltransferase genotyping coupled to a scintillation proximity assay allows for the detection of up to 10 CFU of M. avium subsp. paratuberculosis per ml of milk. This test is rapid and sensitive and allows for automation and thus multiple samples can be tested at the same time.

  14. Particle physics experiments 1983

    International Nuclear Information System (INIS)

    Stuart, G.W.

    1983-01-01

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

  15. The Information Science Experiment System - The computer for science experiments in space

    Science.gov (United States)

    Foudriat, Edwin C.; Husson, Charles

    1989-01-01

    The concept of the Information Science Experiment System (ISES), potential experiments, and system requirements are reviewed. The ISES is conceived as a computer resource in space whose aim is to assist computer, earth, and space science experiments, to develop and demonstrate new information processing concepts, and to provide an experiment base for developing new information technology for use in space systems. The discussion covers system hardware and architecture, operating system software, the user interface, and the ground communication link.

  16. Nova target experiments

    International Nuclear Information System (INIS)

    Drake, R.P.

    1985-11-01

    The Nova laser, at the Lawrence Livermore National Laboratory, provides unique opportunities for target experiments. It has unprecedented energy on target and significant flexibility. The paper presented by John Hunt described the capabilities and the status of Nova. This paper discusses plans for future experiments using Nova, and the present status of target experiments. We plan to perform high-quality physics experiments that exploit the unique capabilities of Nova. Because this is our goal, we are fielding an extensive array of well-characterized target diagnostics to measure the emissions from the target. The first section of this paper discusses the basic target diagnostics. We are also taking care to quantify the performance of the laser

  17. Velocity Feedback Experiments

    Directory of Open Access Journals (Sweden)

    Chiu Choi

    2017-02-01

    Full Text Available Transient response such as ringing in a control system can be reduced or removed by velocity feedback. It is a useful control technique that should be covered in the relevant engineering laboratory courses. We developed velocity feedback experiments using two different low cost technologies, viz., operational amplifiers and microcontrollers. These experiments can be easily integrated into laboratory courses on feedback control systems or microcontroller applications. The intent of developing these experiments was to illustrate the ringing problem and to offer effective, low cost solutions for removing such problem. In this paper the pedagogical approach for these velocity feedback experiments was described. The advantages and disadvantages of the two different implementation of velocity feedback were discussed also.

  18. Diversity and relative abundance of the bacterial pathogen, Flavobacterium spp., infecting reproductive ecotypes of kokanee salmon.

    Science.gov (United States)

    Lemay, Matthew A; Russello, Michael A

    2014-11-04

    Understanding the distribution and abundance of pathogens can provide insight into the evolution and ecology of their host species. Previous research in kokanee, the freshwater form of sockeye salmon (Oncorhynchus nerka), found evidence that populations spawning in streams may experience a greater pathogen load compared with populations that spawn on beaches. In this study we tested for differences in the abundance and diversity of the gram-negative bacteria, Flavobacterium spp., infecting tissues of kokanee in both of these spawning habitats (streams and beaches). Molecular assays were carried out using primers designed to amplify a ~200 nucleotide region of the gene encoding the ATP synthase alpha subunit (AtpA) within the genus Flavobacterium. Using a combination of DNA sequencing and quantitative PCR (qPCR) we compared the diversity and relative abundance of Flavobacterium AtpA amplicons present in DNA extracted from tissue samples of kokanee collected from each spawning habitat. We identified 10 Flavobacterium AtpA haplotypes among the tissues of stream-spawning kokanee and seven haplotypes among the tissues of beach-spawning kokanee, with only two haplotypes shared between spawning habitats. Haplotypes occurring in the same clade as F. psychrophilum were the most prevalent (92% of all reads, 60% of all haplotypes), and occurred in kokanee from both spawning habitats (streams and beaches). Subsequent qPCR assays did not find any significant difference in the relative abundance of Flavobacterium AtpA amplicons between samples from the different spawning habitats. We confirmed the presence of Flavobacterium spp. in both spawning habitats and found weak evidence for increased Flavobacterium diversity in kokanee sampled from stream-spawning sites. However, the quantity of Flavobacterium DNA did not differ between spawning habitats. We recommend further study aimed at quantifying pathogen diversity and abundance in population-level samples of kokanee combined with

  19. The International Heat Pipe Experiment. [international cooperation zero g experiment

    Science.gov (United States)

    Mcintosh, R.; Ollendorf, S.; Harwell, W.

    1976-01-01

    The aims of the experiment are outlined. Flight experiments included in this program were provided by NASA, Goddard Space Flight Center, ESA (European Space Agency), the German Ministry of Technology, Hughes Aircraft Company and NASA, Ames Research Center.

  20. Virtual neutron scattering experiments

    DEFF Research Database (Denmark)

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

    2017-01-01

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

  1. Explicating Experience: Development of a Valid Scale of Past Hazard Experience for Tornadoes.

    Science.gov (United States)

    Demuth, Julie L

    2018-03-23

    People's past experiences with a hazard theoretically influence how they approach future risks. Yet, past hazard experience has been conceptualized and measured in wide-ranging, often simplistic, ways, resulting in mixed findings about its relationship with risk perception. This study develops a scale of past hazard experiences, in the context of tornadoes, that is content and construct valid. A conceptual definition was developed, a set of items were created to measure one's most memorable and multiple tornado experiences, and the measures were evaluated through two surveys of the public who reside in tornado-prone areas. Four dimensions emerged of people's most memorable experience, reflecting their awareness of the tornado risk that day, their personalization of the risk, the intrusive impacts on them personally, and impacts experienced vicariously through others. Two dimensions emerged of people's multiple experiences, reflecting common types of communication received and negative emotional responses. These six dimensions are novel in that they capture people's experience across the timeline of a hazard as well as intangible experiences that are both direct and indirect. The six tornado experience dimensions were correlated with tornado risk perceptions measured as cognitive-affective and as perceived probability of consequences. The varied experience-risk perception results suggest that it is important to understand the nuances of these concepts and their relationships. This study provides a foundation for future work to continue explicating past hazard experience, across different risk contexts, and for understanding its effect on risk assessment and responses. © 2018 Society for Risk Analysis.

  2. Particle physics experiments 1987

    International Nuclear Information System (INIS)

    Stuart, G.W.

    1988-01-01

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

  3. Instrumentation and Fluorescent Chemistries Used in qPCR

    DEFF Research Database (Denmark)

    Josefsen, Mathilde Hartmann; Löfström, Charlotta; Hansen, Trine

    2012-01-01

    will be discussed from a user perspective leading to an instrument selection guide. Differences between fluorescent DNA binding dyes and target-specific fluorescently labeled primers or probes for detection of amplicon accumulation will be discussed, along with the properties and applications of the most frequently...

  4. Journal of Biosciences | Indian Academy of Sciences

    Indian Academy of Sciences (India)

    Advances in DNA sequencing provide tools for efficient large-scale discovery of markers for use in plants. Discovery options include large-scale amplicon sequencing, transcriptome sequencing, gene-enriched genome sequencing and whole genome sequencing. Examples of each of these approaches and their potential ...

  5. Microbial biogeography of drinking water: patterns in phylogenetic diversity across space and time

    NARCIS (Netherlands)

    Roeselers, G.; Coolen, J.; Wielen, P.W. van der; Jaspers, M.C.; Atsma, A.; Graaf, B. de; Schuren, F.

    2015-01-01

    In this study, we collected water from different locations in 32 drinking water distribution networks in the Netherlands and analysed the spatial and temporal variation in microbial community composition by high-throughput sequencing of 16S rRNA gene amplicons. We observed that microbial community

  6. Impact of external carbon dose on the removal of micropollutants using methanol and ethanol in post-denitrifying Moving Bed Biofilm Reactors

    DEFF Research Database (Denmark)

    Torresi, Elena; Escolà Casas, Mònica; Polesel, Fabio

    2017-01-01

    of venlafaxine, carbamazepine, sulfamethoxazole and sulfamethizole could be described with a cometabolic model. Analyses of the microbial composition in the biofilms using 16S rRNA amplicon sequencing revealed that the methanol-dosed MBBR contained higher microbial richness than the one dosed with ethanol...

  7. Microbial diversity and dynamics throughout manufacturing and ripening of surface ripened semi-hard Danish Danbo cheeses investigated by culture-independent techniques

    DEFF Research Database (Denmark)

    Ryssel, Mia; Johansen, Pernille; Abu Al-Soud, Waleed

    2015-01-01

    ) and pyrosequencing, using amplicons of 16S and 26S rRNA genes for prokaryotes and eukaryotes, respectively. With minor exceptions, the results from the culture-independent analyses correlated to the culture-dependent plating results. Even though the predominant microorganisms detected with the two culture...

  8. New comprehensive denaturing-gradient-gel-electrophoresis assay for KRAS mutation detection applied to paraffin-embedded tumours

    NARCIS (Netherlands)

    Hayes, VM; Westra, JL; Verlind, E; Bleeker, W; Plukker, JT; Hofstra, RMW; Buys, CHCM

    2000-01-01

    A comprehensive mutation detection assay is presented for the entire coding region and all splice site junctions of the KRAS oncogene. The assay is based on denaturing gradient gel electrophoresis and applicable to archival paraffin-embedded tumour material. All KRAS amplicons are analysed within

  9. 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.

  10. Particle physics experiments 1989

    International Nuclear Information System (INIS)

    Bairstow, R.

    1990-01-01

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

  11. Experiment data report for LOFT Boron dilution Experiment L6-6

    International Nuclear Information System (INIS)

    Stitt, B.D.; Divine, J.M.

    1982-06-01

    Selected pertinent and uninterpreted data from the sixth anticipated transient experiment (Experiment L6-6) conducted in the Loss-of-Fluid Test (LOFT) facility are presented. Experiment L6-6 simulated a boron dilution accident by injecting demineralized water into the primary coolant system (PCS) at a rate of 0.47 L/s while the reactor was in a cold shutdown condition with the control rods withdrawn. System pressure was maintained at approximately 285 kPa throughout the experiment. The experiment was divided into two parts. In the first part, L6-6A, a recirculation flow of 4.7 L/s was maintained through the PCS and criticality was achieved 7416 +- 10 s after the initiation of the dilution flow. The second part, L6-6B, was identical to L6-6A except that a recirculation flow of 9.5 L/s was maintained and criticality occurred at 8058 +- 10 s

  12. LDR structural experiment definition

    Science.gov (United States)

    Russell, R. A.

    1988-01-01

    A system study to develop the definition of a structural flight experiment for a large precision segmented reflector on the Space Station was accomplished by the Boeing Aerospace Company for NASA's Langley Research Center. The objective of the study was to use a Large Deployable Reflector (LDR) baseline configuration as the basis for focusing an experiment definition, so that the resulting accommodation requirements and interface constraints could be used as part of the mission requirements data base for Space Station. The primary objectives of the first experiment are to construct the primary mirror support truss and to determine its structural and thermal characteristics. Addition of an optical bench, thermal shield and primary mirror segments, and alignment of the optical components, would occur on a second experiment. The structure would then be moved to the payload point system for pointing, optical control, and scientific optical measurement for a third experiment. Experiment 1 will deploy the primary support truss while it is attached to the instrument module structure. The ability to adjust the mirror attachment points and to attach several dummy primary mirror segments with a robotic system will also be demonstrated. Experiment 2 will be achieved by adding new components and equipment to experiment one. Experiment 3 will demonstrate advanced control strategies, active adjustment of the primary mirror alignment, and technologies associated with optical sensing.

  13. Stimulated Brillouin scattering experiments

    International Nuclear Information System (INIS)

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

    1981-01-01

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

  14. AGS experiments: 1985, 1986, 1987

    International Nuclear Information System (INIS)

    Depken, J.C.

    1987-01-01

    This report contains: Experimental areas layout, table of beam parameters and fluxes, experiment schedule ''as run,'' experiment long range schedule, a listing of experiments by number, two-page summaries of each experiment, also ordered by number, and publications of AGS experiments, 1982-1987

  15. Collaborative experience

    DEFF Research Database (Denmark)

    Mortensen, Thomas Bøtker

    -Doerr, 1996) and has been shown to have a positive effect to the outcome of collaborative R&D (Sampson, 2005). Anand & Khanna (2000), furthermore, hypothesized that research joint ventures are more ambiguous than marketing joint ventures and even more the licensing and showed that the effect of collaborative......Literature review: Collaborative experience has been shown to have a positive effect on the collaborative outcome in general (Anand & Khanna, 2000; Kale, Dyer & Singh, 2002). Furthermore, it has been linked to the ability to exploit the network of the firm for learning (Powell, Koput and Smith...... experience was largest the higher the hypothesized ambiguity. Theoretically contribution: This research project aims at contributing to existing literature by arguing, that collaborative experience is a moderating variable which moderates the effects on collaborative outcome from the level of complexity...

  16. Memorable Experiences with Sad Music—Reasons, Reactions and Mechanisms of Three Types of Experiences

    Science.gov (United States)

    Peltola, Henna-Riikka

    2016-01-01

    Reactions to memorable experiences of sad music were studied by means of a survey administered to a convenience (N = 1577), representative (N = 445), and quota sample (N = 414). The survey explored the reasons, mechanisms, and emotions of such experiences. Memorable experiences linked with sad music typically occurred in relation to extremely familiar music, caused intense and pleasurable experiences, which were accompanied by physiological reactions and positive mood changes in about a third of the participants. A consistent structure of reasons and emotions for these experiences was identified through exploratory and confirmatory factor analyses across the samples. Three types of sadness experiences were established, one that was genuinely negative (Grief-Stricken Sorrow) and two that were positive (Comforting Sorrow and Sweet Sorrow). Each type of emotion exhibited certain individual differences and had distinct profiles in terms of the underlying reasons, mechanisms, and elicited reactions. The prevalence of these broad types of emotional experiences suggested that positive experiences are the most frequent, but negative experiences were not uncommon in any of the samples. The findings have implications for measuring emotions induced by music and fiction in general, and call attention to the non-pleasurable aspects of these experiences. PMID:27300268

  17. screening of new isolates of bt and cloning of their dna amplicons

    African Journals Online (AJOL)

    NEMAPPA

    2012-09-18

    Sep 18, 2012 ... Nine new indigenous isolates of Bacillus thuringiensis (Bt) were characterized for their colony type, ... Bt. New gene sequences encoding more active toxins could be ..... Toxicity of a Bacillus thuringiensis israelensis-like strain.

  18. Analysing Microbial Community Composition through Amplicon Sequencing: From Sampling to Hypothesis Testing

    Directory of Open Access Journals (Sweden)

    Luisa W. Hugerth

    2017-09-01

    Full Text Available Microbial ecology as a scientific field is fundamentally driven by technological advance. The past decade's revolution in DNA sequencing cost and throughput has made it possible for most research groups to map microbial community composition in environments of interest. However, the computational and statistical methodology required to analyse this kind of data is often not part of the biologist training. In this review, we give a historical perspective on the use of sequencing data in microbial ecology and restate the current need for this method; but also highlight the major caveats with standard practices for handling these data, from sample collection and library preparation to statistical analysis. Further, we outline the main new analytical tools that have been developed in the past few years to bypass these caveats, as well as highlight the major requirements of common statistical practices and the extent to which they are applicable to microbial data. Besides delving into the meaning of select alpha- and beta-diversity measures, we give special consideration to techniques for finding the main drivers of community dissimilarity and for interaction network construction. While every project design has specific needs, this review should serve as a starting point for considering what options are available.

  19. Experimenting with a design experiment

    Directory of Open Access Journals (Sweden)

    Bakker, Judith

    2012-12-01

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

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

  20. Conducting interactive experiments online.

    Science.gov (United States)

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

    2018-01-01

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

  1. LDR structural experiment definition

    Science.gov (United States)

    Russell, Richard A.; Gates, Richard M.

    1988-01-01

    A study was performed to develop the definition of a structural flight experiment for a large precision segmented reflector that would utilize the Space Station. The objective of the study was to use the Large Deployable Reflector (LDR) baseline configuration for focusing on experiment definition activity which would identify the Space Station accommodation requirements and interface constraints. Results of the study defined three Space Station based experiments to demonstrate the technologies needed for an LDR type structure. The basic experiment configurations are the same as the JPL baseline except that the primary mirror truss is 10 meters in diameter instead of 20. The primary objectives of the first experiment are to construct the primary mirror support truss and to determine its structural and thermal characteristics. Addition of the optical bench, thermal shield and primary mirror segments and alignment of the optical components occur on the second experiment. The structure will then be moved to the payload pointing system for pointing, optical control and scientific optical measurement for the third experiment.

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

    International Nuclear Information System (INIS)

    Depken, J.C.

    1989-04-01

    This report contains: Experimental Areas Layout; Table of Beam Parameters and Fluxes; Experiment Schedule ''as run''; Experiment Long Range Schedule; A listing of experiments by number; Two-page summaries of each experiment begin here, also ordered by number; Publications of AGS experiments; and List of experimenters

  3. AGS experiments, 1988, 1989, 1990

    International Nuclear Information System (INIS)

    Depken, J.C.

    1991-04-01

    This report contains: experimental areas layout; table of beam parameters and fluxes; experiment schedule ''as run''; experiment long range schedule; a listing of experiments by number; two-page summaries of each experiment begin here, also ordered by number; publications of AGS experiments; and list of experimenters

  4. Accurate molecular diagnosis of phenylketonuria and tetrahydrobiopterin-deficient hyperphenylalaninemias using high-throughput targeted sequencing

    Science.gov (United States)

    Trujillano, Daniel; Perez, Belén; González, Justo; Tornador, Cristian; Navarrete, Rosa; Escaramis, Georgia; Ossowski, Stephan; Armengol, Lluís; Cornejo, Verónica; Desviat, Lourdes R; Ugarte, Magdalena; Estivill, Xavier

    2014-01-01

    Genetic diagnostics of phenylketonuria (PKU) and tetrahydrobiopterin (BH4) deficient hyperphenylalaninemia (BH4DH) rely on methods that scan for known mutations or on laborious molecular tools that use Sanger sequencing. We have implemented a novel and much more efficient strategy based on high-throughput multiplex-targeted resequencing of four genes (PAH, GCH1, PTS, and QDPR) that, when affected by loss-of-function mutations, cause PKU and BH4DH. We have validated this approach in a cohort of 95 samples with the previously known PAH, GCH1, PTS, and QDPR mutations and one control sample. Pooled barcoded DNA libraries were enriched using a custom NimbleGen SeqCap EZ Choice array and sequenced using a HiSeq2000 sequencer. The combination of several robust bioinformatics tools allowed us to detect all known pathogenic mutations (point mutations, short insertions/deletions, and large genomic rearrangements) in the 95 samples, without detecting spurious calls in these genes in the control sample. We then used the same capture assay in a discovery cohort of 11 uncharacterized HPA patients using a MiSeq sequencer. In addition, we report the precise characterization of the breakpoints of four genomic rearrangements in PAH, including a novel deletion of 899 bp in intron 3. Our study is a proof-of-principle that high-throughput-targeted resequencing is ready to substitute classical molecular methods to perform differential genetic diagnosis of hyperphenylalaninemias, allowing the establishment of specifically tailored treatments a few days after birth. PMID:23942198

  5. Clinical significance of BRAF non-V600E mutations on the therapeutic effects of anti-EGFR monoclonal antibody treatment in patients with pretreated metastatic colorectal cancer: the Biomarker Research for anti-EGFR monoclonal Antibodies by Comprehensive Cancer genomics (BREAC) study.

    Science.gov (United States)

    Shinozaki, Eiji; Yoshino, Takayuki; Yamazaki, Kentaro; Muro, Kei; Yamaguchi, Kensei; Nishina, Tomohiro; Yuki, Satoshi; Shitara, Kohei; Bando, Hideaki; Mimaki, Sachiyo; Nakai, Chikako; Matsushima, Koutatsu; Suzuki, Yutaka; Akagi, Kiwamu; Yamanaka, Takeharu; Nomura, Shogo; Fujii, Satoshi; Esumi, Hiroyasu; Sugiyama, Masaya; Nishida, Nao; Mizokami, Masashi; Koh, Yasuhiro; Abe, Yukiko; Ohtsu, Atsushi; Tsuchihara, Katsuya

    2017-11-07

    Patients with BRAF V600E -mutated metastatic colorectal cancer (mCRC) have a poorer prognosis as well as resistance to anti-EGFR antibodies. However, it is unclear whether BRAF mutations other than BRAF V600E (BRAF non-V600E mutations) contribute to anti-EGFR antibody resistance. This study was composed of exploratory and inference cohorts. Candidate biomarkers identified by whole exome sequencing from super-responders and nonresponders in the exploratory cohort were validated by targeted resequencing for patients who received anti-EGFR antibody in the inference cohort. In the exploratory cohort, 31 candidate biomarkers, including KRAS/NRAS/BRAF mutations, were identified. Targeted resequencing of 150 patients in the inference cohort revealed 40 patients with RAS (26.7%), 9 patients with BRAF V600E (6.0%), and 7 patients with BRAF non-V600E mutations (4.7%), respectively. The response rates in RAS, BRAF V600E , and BRAF non-V600E were lower than those in RAS/BRAF wild-type (2.5%, 0%, and 0% vs 31.9%). The median PFS in BRAF non-V600E mutations was 2.4 months, similar to that in RAS or BRAF V600E mutations (2.1 and 1.6 months) but significantly worse than that in wild-type RAS/BRAF (5.9 months). Although BRAF non-V600E mutations identified were a rare and unestablished molecular subtype, certain BRAF non-V600E mutations might contribute to a lesser benefit of anti-EGFR monoclonal antibody treatment.

  6. Opposing Forces of A/T-Biased Mutations and G/C-Biased Gene Conversions Shape the Genome of the Nematode Pristionchus pacificus

    Science.gov (United States)

    Weller, Andreas M.; Rödelsperger, Christian; Eberhardt, Gabi; Molnar, Ruxandra I.; Sommer, Ralf J.

    2014-01-01

    Base substitution mutations are a major source of genetic novelty and mutation accumulation line (MAL) studies revealed a nearly universal AT bias in de novo mutation spectra. While a comparison of de novo mutation spectra with the actual nucleotide composition in the genome suggests the existence of general counterbalancing mechanisms, little is known about the evolutionary and historical details of these opposing forces. Here, we correlate MAL-derived mutation spectra with patterns observed from population resequencing. Variation observed in natural populations has already been subject to evolutionary forces. Distinction between rare and common alleles, the latter of which are close to fixation and of presumably older age, can provide insight into mutational processes and their influence on genome evolution. We provide a genome-wide analysis of de novo mutations in 22 MALs of the nematode Pristionchus pacificus and compare the spectra with natural variants observed in resequencing of 104 natural isolates. MALs show an AT bias of 5.3, one of the highest values observed to date. In contrast, the AT bias in natural variants is much lower. Specifically, rare derived alleles show an AT bias of 2.4, whereas common derived alleles close to fixation show no AT bias at all. These results indicate the existence of a strong opposing force and they suggest that the GC content of the P. pacificus genome is in equilibrium. We discuss GC-biased gene conversion as a potential mechanism acting against AT-biased mutations. This study provides insight into genome evolution by combining MAL studies with natural variation. PMID:24414549

  7. Natural Selection and Genetic Diversity in the Butterfly Heliconius melpomene.

    Science.gov (United States)

    Martin, Simon H; Möst, Markus; Palmer, William J; Salazar, Camilo; McMillan, W Owen; Jiggins, Francis M; Jiggins, Chris D

    2016-05-01

    A combination of selective and neutral evolutionary forces shape patterns of genetic diversity in nature. Among the insects, most previous analyses of the roles of drift and selection in shaping variation across the genome have focused on the genus Drosophila A more complete understanding of these forces will come from analyzing other taxa that differ in population demography and other aspects of biology. We have analyzed diversity and signatures of selection in the neotropical Heliconius butterflies using resequenced genomes from 58 wild-caught individuals of Heliconius melpomene and another 21 resequenced genomes representing 11 related species. By comparing intraspecific diversity and interspecific divergence, we estimate that 31% of amino acid substitutions between Heliconius species are adaptive. Diversity at putatively neutral sites is negatively correlated with the local density of coding sites as well as nonsynonymous substitutions and positively correlated with recombination rate, indicating widespread linked selection. This process also manifests in significantly reduced diversity on longer chromosomes, consistent with lower recombination rates. Although hitchhiking around beneficial nonsynonymous mutations has significantly shaped genetic variation in H. melpomene, evidence for strong selective sweeps is limited overall. We did however identify two regions where distinct haplotypes have swept in different populations, leading to increased population differentiation. On the whole, our study suggests that positive selection is less pervasive in these butterflies as compared to fruit flies, a fact that curiously results in very similar levels of neutral diversity in these very different insects. Copyright © 2016 by the Genetics Society of America.

  8. Classification of rare missense substitutions, using risk surfaces, with genetic- and molecular-epidemiology applications.

    Science.gov (United States)

    Tavtigian, Sean V; Byrnes, Graham B; Goldgar, David E; Thomas, Alun

    2008-11-01

    Many individually rare missense substitutions are encountered during deep resequencing of candidate susceptibility genes and clinical mutation screening of known susceptibility genes. BRCA1 and BRCA2 are among the most resequenced of all genes, and clinical mutation screening of these genes provides an extensive data set for analysis of rare missense substitutions. Align-GVGD is a mathematically simple missense substitution analysis algorithm, based on the Grantham difference, which has already contributed to classification of missense substitutions in BRCA1, BRCA2, and CHEK2. However, the distribution of genetic risk as a function of Align-GVGD's output variables Grantham variation (GV) and Grantham deviation (GD) has not been well characterized. Here, we used data from the Myriad Genetic Laboratories database of nearly 70,000 full-sequence tests plus two risk estimates, one approximating the odds ratio and the other reflecting strength of selection, to display the distribution of risk in the GV-GD plane as a series of surfaces. We abstracted contours from the surfaces and used the contours to define a sequence of missense substitution grades ordered from greatest risk to least risk. The grades were validated internally using a third, personal and family history-based, measure of risk. The Align-GVGD grades defined here are applicable to both the genetic epidemiology problem of classifying rare missense substitutions observed in known susceptibility genes and the molecular epidemiology problem of analyzing rare missense substitutions observed during case-control mutation screening studies of candidate susceptibility genes. (c) 2008 Wiley-Liss, Inc.

  9. Selection against recombinant hybrids maintains reproductive isolation in hybridizing Populus species despite F1 fertility and recurrent gene flow.

    Science.gov (United States)

    Christe, Camille; Stölting, Kai N; Bresadola, Luisa; Fussi, Barbara; Heinze, Berthold; Wegmann, Daniel; Lexer, Christian

    2016-06-01

    Natural hybrid zones have proven to be precious tools for understanding the origin and maintenance of reproductive isolation (RI) and therefore species. Most available genomic studies of hybrid zones using whole- or partial-genome resequencing approaches have focused on comparisons of the parental source populations involved in genome admixture, rather than exploring fine-scale patterns of chromosomal ancestry across the full admixture gradient present between hybridizing species. We have studied three well-known European 'replicate' hybrid zones of Populus alba and P. tremula, two widespread, ecologically divergent forest trees, using up to 432 505 single-nucleotide polymorphisms (SNPs) from restriction site-associated DNA (RAD) sequencing. Estimates of fine-scale chromosomal ancestry, genomic divergence and differentiation across all 19 poplar chromosomes revealed strikingly contrasting results, including an unexpected preponderance of F1 hybrids in the centre of genomic clines on the one hand, and genomically localized, spatially variable shared variants consistent with ancient introgression between the parental species on the other. Genetic ancestry had a significant effect on survivorship of hybrid seedlings in a common garden trial, pointing to selection against early-generation recombinants. Our results indicate a role for selection against recombinant genotypes in maintaining RI in the face of apparent F1 fertility, consistent with the intragenomic 'coadaptation' model of barriers to introgression upon secondary contact. Whole-genome resequencing of hybridizing populations will clarify the roles of specific genetic pathways in RI between these model forest trees and may reveal which loci are affected most strongly by its cyclic breakdown. © 2016 John Wiley & Sons Ltd.

  10. Folliculin mutations are not associated with severe COPD

    Directory of Open Access Journals (Sweden)

    Litonjua Augusto A

    2008-12-01

    Full Text Available Abstract Background Rare loss-of-function folliculin (FLCN mutations are the genetic cause of Birt-Hogg-Dubé syndrome, a monogenic disorder characterized by spontaneous pneumothorax, fibrofolliculomas, and kidney tumors. Loss-of-function folliculin mutations have also been described in pedigrees with familial spontaneous pneumothorax. Because the majority of patients with folliculin mutations have radiographic evidence of pulmonary cysts, folliculin has been hypothesized to contribute to the development of emphysema. To determine whether folliculin sequence variants are risk factors for severe COPD, we genotyped seven previously reported Birt-Hogg-Dubé or familial spontaneous pneumothorax associated folliculin mutations in 152 severe COPD probands participating in the Boston Early-Onset COPD Study. We performed bidirectional resequencing of all 14 folliculin exons in a subset of 41 probands and subsequently genotyped four identified variants in an independent sample of345 COPD subjects from the National Emphysema Treatment Trial (cases and 420 male smokers with normal lung function from the Normative Aging Study (controls. Results None of the seven previously reported Birt-Hogg-Dubé or familial spontaneous pneumothorax mutations were observed in the 152 severe, early-onset COPD probands. Exon resequencing identified 31 variants, including two non-synonymous polymorphisms and two common non-coding polymorphisms. No significant association was observed for any of these four variants with presence of COPD or emphysema-related phenotypes. Conclusion Genetic variation in folliculin does not appear to be a major risk factor for severe COPD. These data suggest that familial spontaneous pneumothorax and COPD have distinct genetic causes, despite some overlap in radiographic characteristics.

  11. Combining target enrichment with barcode multiplexing for high throughput SNP discovery

    Directory of Open Access Journals (Sweden)

    Lunke Sebastian

    2010-11-01

    Full Text Available Abstract Background The primary goal of genetic linkage analysis is to identify genes affecting a phenotypic trait. After localisation of the linkage region, efficient genetic dissection of the disease linked loci requires that functional variants are identified across the loci. These functional variations are difficult to detect due to extent of genetic diversity and, to date, incomplete cataloguing of the large number of variants present both within and between populations. Massively parallel sequencing platforms offer unprecedented capacity for variant discovery, however the number of samples analysed are still limited by cost per sample. Some progress has been made in reducing the cost of resequencing using either multiplexing methodologies or through the utilisation of targeted enrichment technologies which provide the ability to resequence genomic areas of interest rather that full genome sequencing. Results We developed a method that combines current multiplexing methodologies with a solution-based target enrichment method to further reduce the cost of resequencing where region-specific sequencing is required. Our multiplex/enrichment strategy produced high quality data with nominal reduction of sequencing depth. We undertook a genotyping study and were successful in the discovery of novel SNP alleles in all samples at uniplex, duplex and pentaplex levels. Conclusion Our work describes the successful combination of a targeted enrichment method and index barcode multiplexing to reduce costs, time and labour associated with processing large sample sets. Furthermore, we have shown that the sequencing depth obtained is adequate for credible SNP genotyping analysis at uniplex, duplex and pentaplex levels.

  12. An evaluation of Comparative Genome Sequencing (CGS by comparing two previously-sequenced bacterial genomes

    Directory of Open Access Journals (Sweden)

    Herring Christopher D

    2007-08-01

    Full Text Available Abstract Background With the development of new technology, it has recently become practical to resequence the genome of a bacterium after experimental manipulation. It is critical though to know the accuracy of the technique used, and to establish confidence that all of the mutations were detected. Results In order to evaluate the accuracy of genome resequencing using the microarray-based Comparative Genome Sequencing service provided by Nimblegen Systems Inc., we resequenced the E. coli strain W3110 Kohara using MG1655 as a reference, both of which have been completely sequenced using traditional sequencing methods. CGS detected 7 of 8 small sequence differences, one large deletion, and 9 of 12 IS element insertions present in W3110, but did not detect a large chromosomal inversion. In addition, we confirmed that CGS also detected 2 SNPs, one deletion and 7 IS element insertions that are not present in the genome sequence, which we attribute to changes that occurred after the creation of the W3110 lambda clone library. The false positive rate for SNPs was one per 244 Kb of genome sequence. Conclusion CGS is an effective way to detect multiple mutations present in one bacterium relative to another, and while highly cost-effective, is prone to certain errors. Mutations occurring in repeated sequences or in sequences with a high degree of secondary structure may go undetected. It is also critical to follow up on regions of interest in which SNPs were not called because they often indicate deletions or IS element insertions.

  13. Aesthetic experience of dance performances

    Directory of Open Access Journals (Sweden)

    Vukadinović Maja

    2012-01-01

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

  14. Genetic homogeneity among Ugandan isolates of Xanthomonas ...

    African Journals Online (AJOL)

    Seven random primers were used because of their ability to amplify reproducible and reliable fingerprints generated between 6 - 12 amplicons each from the Xcm isolates obtained from central core of pseudostems, peduncles, fruit peelings, sap, nectar, insects' bodies and bacterial oozes. Regardless of the source and ...

  15. Novel vitamin D 1α-hydroxylase gene mutations in a Chinese ...

    Indian Academy of Sciences (India)

    2011-08-19

    Aug 19, 2011 ... known as vitamin D 1α-hydroxylase deficiency or pseu- dovitamin D ... amplicons of the 378 bp were digested with restriction enzyme PvuI and ... have no enzymatic activity; a missense mutation c.473T>C. (p.L158P) in the ...

  16. Efficiency of random amplified polymorphic DNA (RAPD) and inter ...

    African Journals Online (AJOL)

    Efficiency of random amplified polymorphic DNA (RAPD) and inter-simple sequence repeats (ISSR) markers for genotype fingerprinting and genetic diversity studies in canola ( ) ... The number of amplified fragments with RAPD primers ranged from 8 to 21, with the size of amplicons ranging from 162 to 3154 bp.

  17. A novel partial deletion of the Y chromosome azoospermia factor c region is caused by non-homologous recombination between palindromes and may be associated with increased sperm counts

    NARCIS (Netherlands)

    Noordam, M. J.; van Daalen, S. K. M.; Hovingh, S. E.; Korver, C. M.; van der Veen, F.; Repping, S.

    2011-01-01

    BACKGROUND: The male-specific region of the human Y chromosome (MSY) contains multiple testis-specific genes. Most deletions in the MSY lead to inadequate or absent sperm production. Nearly all deletions occur via homologous recombination between amplicons. Previously, we identified two P5/distal-P1

  18. Subgingival microbiome in smokers and non-smokers in periodontitis: an exploratory study using traditional targeted techniques and a next-generation sequencing

    NARCIS (Netherlands)

    Bizzarro, S.; Loos, B.G.; Laine, M.L.; Crielaard, W.; Zaura, E.

    2013-01-01

    Aim To compare the results of two targeted techniques to an open-ended technique in periodontitis patients, differentiated on the basis of smoking habit. Materials & Methods Thirty periodontitis patients (15 smokers and 15 non-smokers) provided subgingival plaque samples for 16S rRNA gene amplicon

  19. An efficient and high fidelity method for amplification, cloning and sequencing of complete tospovirus genomic RNA segments

    Science.gov (United States)

    Amplification and sequencing of the complete M- and S-RNA segments of Tomato spotted wilt virus and Impatiens necrotic spot virus as a single fragment is useful for whole genome sequencing of tospoviruses co-infecting a single host plant. It avoids issues associated with overlapping amplicon-based ...

  20. The male-specific region of the human Y chromosome is a mosaic of discrete sequence classes

    NARCIS (Netherlands)

    Skaletsky, Helen; Kuroda-Kawaguchi, Tomoko; Minx, Patrick J.; Cordum, Holland S.; Hillier, LaDeana; Brown, Laura G.; Repping, Sjoerd; Pyntikova, Tatyana; Ali, Johar; Bieri, Tamberlyn; Chinwalla, Asif; Delehaunty, Andrew; Delehaunty, Kim; Du, Hui; Fewell, Ginger; Fulton, Lucinda; Fulton, Robert; Graves, Tina; Hou, Shun-Fang; Latrielle, Philip; Leonard, Shawn; Mardis, Elaine; Maupin, Rachel; McPherson, John; Miner, Tracie; Nash, William; Nguyen, Christine; Ozersky, Philip; Pepin, Kymberlie; Rock, Susan; Rohlfing, Tracy; Scott, Kelsi; Schultz, Brian; Strong, Cindy; Tin-Wollam, Aye; Yang, Shiaw-Pyng; Waterston, Robert H.; Wilson, Richard K.; Rozen, Steve; Page, David C.

    2003-01-01

    The male-specific region of the Y chromosome, the MSY, differentiates the sexes and comprises 95% of the chromosome's length. Here, we report that the MSY is a mosaic of heterochromatic sequences and three classes of euchromatic sequences: X-transposed, X-degenerate and ampliconic. These classes