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Sample records for high confidence sequence

  1. miRBase: annotating high confidence microRNAs using deep sequencing data.

    Science.gov (United States)

    Kozomara, Ana; Griffiths-Jones, Sam

    2014-01-01

    We describe an update of the miRBase database (http://www.mirbase.org/), the primary microRNA sequence repository. The latest miRBase release (v20, June 2013) contains 24 521 microRNA loci from 206 species, processed to produce 30 424 mature microRNA products. The rate of deposition of novel microRNAs and the number of researchers involved in their discovery continue to increase, driven largely by small RNA deep sequencing experiments. In the face of these increases, and a range of microRNA annotation methods and criteria, maintaining the quality of the microRNA sequence data set is a significant challenge. Here, we describe recent developments of the miRBase database to address this issue. In particular, we describe the collation and use of deep sequencing data sets to assign levels of confidence to miRBase entries. We now provide a high confidence subset of miRBase entries, based on the pattern of mapped reads. The high confidence microRNA data set is available alongside the complete microRNA collection at http://www.mirbase.org/. We also describe embedding microRNA-specific Wikipedia pages on the miRBase website to encourage the microRNA community to contribute and share textual and functional information.

  2. High Confidence Software and Systems Research Needs

    Data.gov (United States)

    Networking and Information Technology Research and Development, Executive Office of the President — This White Paper presents a survey of high confidence software and systems research needs. It has been prepared by the High Confidence Software and Systems...

  3. Assessment of cartilage-dedicated sequences at ultra-high-field MRI: comparison of imaging performance and diagnostic confidence between 3.0 and 7.0 T with respect to osteoarthritis-induced changes at the knee joint

    Energy Technology Data Exchange (ETDEWEB)

    Stahl, Robert [University of California, Musculoskeletal and Quantitative Imaging Group, Department of Radiology, San Francisco, CA (United States); University Hospitals - Campus Grosshadern, Ludwig Maximilians University of Munich, Department of Clinical Radiology, Munich (Germany); Krug, Roland; Zuo, Jin; Majumdar, Sharmila; Link, Thomas M. [University of California, Musculoskeletal and Quantitative Imaging Group, Department of Radiology, San Francisco, CA (United States); Kelley, Douglas A.C. [General Electrics Healthcare Technologies, San Francisco, CA (United States); Ma, C.B. [University of California, Department of Orthopedic Surgery, San Francisco, CA (United States)

    2009-08-15

    The objectives of the study were to optimize three cartilage-dedicated sequences for in vivo knee imaging at 7.0 T ultra-high-field (UHF) magnetic resonance imaging (MRI) and to compare imaging performance and diagnostic confidence concerning osteoarthritis (OA)-induced changes at 7.0 and 3.0 T MRI. Optimized MRI sequences for cartilage imaging at 3.0 T were tailored for 7.0 T: an intermediate-weighted fast spin-echo (IM-w FSE), a fast imaging employing steady-state acquisition (FIESTA) and a T1-weighted 3D high-spatial-resolution volumetric fat-suppressed spoiled gradient-echo (SPGR) sequence. Three healthy subjects and seven patients with mild OA were examined. Signal-to-noise ratio (SNR), contrast-to-noise ratio (CNR), diagnostic confidence in assessing cartilage abnormalities, and image quality were determined. Abnormalities were assessed with the whole organ magnetic resonance imaging score (WORMS). Focal cartilage lesions and bone marrow edema pattern (BMEP) were also quantified. At 7.0 T, SNR was increased (p<0.05) for all sequences. For the IM-w FSE sequence, limitations with the specific absorption rate (SAR) required modifications of the scan parameters yielding an incomplete coverage of the knee joint, extensive artifacts, and a less effective fat saturation. CNR and image quality were increased (p<0.05) for SPGR and FIESTA and decreased for IM-w FSE. Diagnostic confidence for cartilage lesions was highest (p<0.05) for FIESTA at 7.0 T. Evaluation of BMEP was decreased (p < 0.05) at 7.0 T due to limited performance of IM-w FSE. Gradient echo-based pulse sequences like SPGR and FIESTA are well suited for imaging at UHF which may improve early detection of cartilage lesions. However, UHF IM-w FSE sequences are less feasible for clinical use. (orig.)

  4. Assessment of cartilage-dedicated sequences at ultra-high-field MRI: comparison of imaging performance and diagnostic confidence between 3.0 and 7.0 T with respect to osteoarthritis-induced changes at the knee joint

    International Nuclear Information System (INIS)

    Stahl, Robert; Krug, Roland; Zuo, Jin; Majumdar, Sharmila; Link, Thomas M.; Kelley, Douglas A.C.; Ma, C.B.

    2009-01-01

    The objectives of the study were to optimize three cartilage-dedicated sequences for in vivo knee imaging at 7.0 T ultra-high-field (UHF) magnetic resonance imaging (MRI) and to compare imaging performance and diagnostic confidence concerning osteoarthritis (OA)-induced changes at 7.0 and 3.0 T MRI. Optimized MRI sequences for cartilage imaging at 3.0 T were tailored for 7.0 T: an intermediate-weighted fast spin-echo (IM-w FSE), a fast imaging employing steady-state acquisition (FIESTA) and a T1-weighted 3D high-spatial-resolution volumetric fat-suppressed spoiled gradient-echo (SPGR) sequence. Three healthy subjects and seven patients with mild OA were examined. Signal-to-noise ratio (SNR), contrast-to-noise ratio (CNR), diagnostic confidence in assessing cartilage abnormalities, and image quality were determined. Abnormalities were assessed with the whole organ magnetic resonance imaging score (WORMS). Focal cartilage lesions and bone marrow edema pattern (BMEP) were also quantified. At 7.0 T, SNR was increased (p<0.05) for all sequences. For the IM-w FSE sequence, limitations with the specific absorption rate (SAR) required modifications of the scan parameters yielding an incomplete coverage of the knee joint, extensive artifacts, and a less effective fat saturation. CNR and image quality were increased (p<0.05) for SPGR and FIESTA and decreased for IM-w FSE. Diagnostic confidence for cartilage lesions was highest (p<0.05) for FIESTA at 7.0 T. Evaluation of BMEP was decreased (p < 0.05) at 7.0 T due to limited performance of IM-w FSE. Gradient echo-based pulse sequences like SPGR and FIESTA are well suited for imaging at UHF which may improve early detection of cartilage lesions. However, UHF IM-w FSE sequences are less feasible for clinical use. (orig.)

  5. High confidence in falsely recognizing prototypical faces.

    Science.gov (United States)

    Sampaio, Cristina; Reinke, Victoria; Mathews, Jeffrey; Swart, Alexandra; Wallinger, Stephen

    2018-06-01

    We applied a metacognitive approach to investigate confidence in recognition of prototypical faces. Participants were presented with sets of faces constructed digitally as deviations from prototype/base faces. Participants were then tested with a simple recognition task (Experiment 1) or a multiple-choice task (Experiment 2) for old and new items plus new prototypes, and they showed a high rate of confident false alarms to the prototypes. Confidence and accuracy relationship in this face recognition paradigm was found to be positive for standard items but negative for the prototypes; thus, it was contingent on the nature of the items used. The data have implications for lineups that employ match-to-suspect strategies.

  6. Asymptotically Honest Confidence Regions for High Dimensional

    DEFF Research Database (Denmark)

    Caner, Mehmet; Kock, Anders Bredahl

    While variable selection and oracle inequalities for the estimation and prediction error have received considerable attention in the literature on high-dimensional models, very little work has been done in the area of testing and construction of confidence bands in high-dimensional models. However...... develop an oracle inequality for the conservative Lasso only assuming the existence of a certain number of moments. This is done by means of the Marcinkiewicz-Zygmund inequality which in our context provides sharper bounds than Nemirovski's inequality. As opposed to van de Geer et al. (2014) we allow...

  7. Autism Spectrum Disorder and High Confidence Gene Factors

    OpenAIRE

    Mai, MOCHIZUKI

    2017-01-01

    Autism spectrum disorder (ASD) is a neurological developmental disorder whose mechanism isyet unclear. However, recent ASD studies, which employ exome- and genome-wide sequencing,have identified some high-confidence ASD genes. Those ASD studies have revealed that CHD8is likely associated with ASD. In this article, we highlight that CHD8 may regulate othercandidate ASD risk genes. Current research indicates that there exist some thousand autismsusceptibility candidate genes. Moreover, we sugge...

  8. PFP: Automated prediction of gene ontology functional annotations with confidence scores using protein sequence data.

    Science.gov (United States)

    Hawkins, Troy; Chitale, Meghana; Luban, Stanislav; Kihara, Daisuke

    2009-02-15

    Protein function prediction is a central problem in bioinformatics, increasing in importance recently due to the rapid accumulation of biological data awaiting interpretation. Sequence data represents the bulk of this new stock and is the obvious target for consideration as input, as newly sequenced organisms often lack any other type of biological characterization. We have previously introduced PFP (Protein Function Prediction) as our sequence-based predictor of Gene Ontology (GO) functional terms. PFP interprets the results of a PSI-BLAST search by extracting and scoring individual functional attributes, searching a wide range of E-value sequence matches, and utilizing conventional data mining techniques to fill in missing information. We have shown it to be effective in predicting both specific and low-resolution functional attributes when sufficient data is unavailable. Here we describe (1) significant improvements to the PFP infrastructure, including the addition of prediction significance and confidence scores, (2) a thorough benchmark of performance and comparisons to other related prediction methods, and (3) applications of PFP predictions to genome-scale data. We applied PFP predictions to uncharacterized protein sequences from 15 organisms. Among these sequences, 60-90% could be annotated with a GO molecular function term at high confidence (>or=80%). We also applied our predictions to the protein-protein interaction network of the Malaria plasmodium (Plasmodium falciparum). High confidence GO biological process predictions (>or=90%) from PFP increased the number of fully enriched interactions in this dataset from 23% of interactions to 94%. Our benchmark comparison shows significant performance improvement of PFP relative to GOtcha, InterProScan, and PSI-BLAST predictions. This is consistent with the performance of PFP as the overall best predictor in both the AFP-SIG '05 and CASP7 function (FN) assessments. PFP is available as a web service at http

  9. Distinguishing highly confident accurate and inaccurate memory: insights about relevant and irrelevant influences on memory confidence

    OpenAIRE

    Chua, Elizabeth F.; Hannula, Deborah E.; Ranganath, Charan

    2012-01-01

    It is generally believed that accuracy and confidence in one’s memory are related, but there are many instances when they diverge. Accordingly, it is important to disentangle the factors which contribute to memory accuracy and confidence, especially those factors that contribute to confidence, but not accuracy. We used eye movements to separately measure fluent cue processing, the target recognition experience, and relative evidence assessment on recognition confidence and accuracy. Eye movem...

  10. Exploring Self - Confidence Level of High School Students Doing Sport

    Directory of Open Access Journals (Sweden)

    Nurullah Emir Ekinci

    2014-10-01

    Full Text Available The aim of this study was to investigate self-confidence levels of high school students, who do sport, in the extent of their gender, sport branch (individual/team sports and aim for participating in sport (professional/amateur. 185 active high school students from Kutahya voluntarily participated for the study. In the study as data gathering tool self-confidence scale was used. In the evaluation of the data as a hypothesis test Mann Whitney U non parametric test was used. As a result self-confidence levels of participants showed significant differences according to their gender and sport branch but there was no significant difference according to aim for participating in sport.

  11. Distinguishing highly confident accurate and inaccurate memory: insights about relevant and irrelevant influences on memory confidence.

    Science.gov (United States)

    Chua, Elizabeth F; Hannula, Deborah E; Ranganath, Charan

    2012-01-01

    It is generally believed that accuracy and confidence in one's memory are related, but there are many instances when they diverge. Accordingly it is important to disentangle the factors that contribute to memory accuracy and confidence, especially those factors that contribute to confidence, but not accuracy. We used eye movements to separately measure fluent cue processing, the target recognition experience, and relative evidence assessment on recognition confidence and accuracy. Eye movements were monitored during a face-scene associative recognition task, in which participants first saw a scene cue, followed by a forced-choice recognition test for the associated face, with confidence ratings. Eye movement indices of the target recognition experience were largely indicative of accuracy, and showed a relationship to confidence for accurate decisions. In contrast, eye movements during the scene cue raised the possibility that more fluent cue processing was related to higher confidence for both accurate and inaccurate recognition decisions. In a second experiment we manipulated cue familiarity, and therefore cue fluency. Participants showed higher confidence for cue-target associations for when the cue was more familiar, especially for incorrect responses. These results suggest that over-reliance on cue familiarity and under-reliance on the target recognition experience may lead to erroneous confidence.

  12. Inferring high-confidence human protein-protein interactions

    Directory of Open Access Journals (Sweden)

    Yu Xueping

    2012-05-01

    Full Text Available Abstract Background As numerous experimental factors drive the acquisition, identification, and interpretation of protein-protein interactions (PPIs, aggregated assemblies of human PPI data invariably contain experiment-dependent noise. Ascertaining the reliability of PPIs collected from these diverse studies and scoring them to infer high-confidence networks is a non-trivial task. Moreover, a large number of PPIs share the same number of reported occurrences, making it impossible to distinguish the reliability of these PPIs and rank-order them. For example, for the data analyzed here, we found that the majority (>83% of currently available human PPIs have been reported only once. Results In this work, we proposed an unsupervised statistical approach to score a set of diverse, experimentally identified PPIs from nine primary databases to create subsets of high-confidence human PPI networks. We evaluated this ranking method by comparing it with other methods and assessing their ability to retrieve protein associations from a number of diverse and independent reference sets. These reference sets contain known biological data that are either directly or indirectly linked to interactions between proteins. We quantified the average effect of using ranked protein interaction data to retrieve this information and showed that, when compared to randomly ranked interaction data sets, the proposed method created a larger enrichment (~134% than either ranking based on the hypergeometric test (~109% or occurrence ranking (~46%. Conclusions From our evaluations, it was clear that ranked interactions were always of value because higher-ranked PPIs had a higher likelihood of retrieving high-confidence experimental data. Reducing the noise inherent in aggregated experimental PPIs via our ranking scheme further increased the accuracy and enrichment of PPIs derived from a number of biologically relevant data sets. These results suggest that using our high-confidence

  13. Technical Report: Algorithm and Implementation for Quasispecies Abundance Inference with Confidence Intervals from Metagenomic Sequence Data

    Energy Technology Data Exchange (ETDEWEB)

    McLoughlin, Kevin [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)

    2016-01-11

    This report describes the design and implementation of an algorithm for estimating relative microbial abundances, together with confidence limits, using data from metagenomic DNA sequencing. For the background behind this project and a detailed discussion of our modeling approach for metagenomic data, we refer the reader to our earlier technical report, dated March 4, 2014. Briefly, we described a fully Bayesian generative model for paired-end sequence read data, incorporating the effects of the relative abundances, the distribution of sequence fragment lengths, fragment position bias, sequencing errors and variations between the sampled genomes and the nearest reference genomes. A distinctive feature of our modeling approach is the use of a Chinese restaurant process (CRP) to describe the selection of genomes to be sampled, and thus the relative abundances. The CRP component is desirable for fitting abundances to reads that may map ambiguously to multiple targets, because it naturally leads to sparse solutions that select the best representative from each set of nearly equivalent genomes.

  14. Protein Correlation Profiles Identify Lipid Droplet Proteins with High Confidence*

    Science.gov (United States)

    Krahmer, Natalie; Hilger, Maximiliane; Kory, Nora; Wilfling, Florian; Stoehr, Gabriele; Mann, Matthias; Farese, Robert V.; Walther, Tobias C.

    2013-01-01

    Lipid droplets (LDs) are important organelles in energy metabolism and lipid storage. Their cores are composed of neutral lipids that form a hydrophobic phase and are surrounded by a phospholipid monolayer that harbors specific proteins. Most well-established LD proteins perform important functions, particularly in cellular lipid metabolism. Morphological studies show LDs in close proximity to and interacting with membrane-bound cellular organelles, including the endoplasmic reticulum, mitochondria, peroxisomes, and endosomes. Because of these close associations, it is difficult to purify LDs to homogeneity. Consequently, the confident identification of bona fide LD proteins via proteomics has been challenging. Here, we report a methodology for LD protein identification based on mass spectrometry and protein correlation profiles. Using LD purification and quantitative, high-resolution mass spectrometry, we identified LD proteins by correlating their purification profiles to those of known LD proteins. Application of the protein correlation profile strategy to LDs isolated from Drosophila S2 cells led to the identification of 111 LD proteins in a cellular LD fraction in which 1481 proteins were detected. LD localization was confirmed in a subset of identified proteins via microscopy of the expressed proteins, thereby validating the approach. Among the identified LD proteins were both well-characterized LD proteins and proteins not previously known to be localized to LDs. Our method provides a high-confidence LD proteome of Drosophila cells and a novel approach that can be applied to identify LD proteins of other cell types and tissues. PMID:23319140

  15. Concerns and developmental needs of highly confident and less ...

    African Journals Online (AJOL)

    The development of this study was based on three assumptions, namely 1) that coaching efficacy (confidence specific to the task of coaching) impacts the performance of coaches, 2) that coaching efficacy is influenced by the individual's procedural and declarative knowledge on coaching, and 3) that coaches do their work ...

  16. Technical Report on Modeling for Quasispecies Abundance Inference with Confidence Intervals from Metagenomic Sequence Data

    Energy Technology Data Exchange (ETDEWEB)

    McLoughlin, K. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)

    2016-01-11

    The overall aim of this project is to develop a software package, called MetaQuant, that can determine the constituents of a complex microbial sample and estimate their relative abundances by analysis of metagenomic sequencing data. The goal for Task 1 is to create a generative model describing the stochastic process underlying the creation of sequence read pairs in the data set. The stages in this generative process include the selection of a source genome sequence for each read pair, with probability dependent on its abundance in the sample. The other stages describe the evolution of the source genome from its nearest common ancestor with a reference genome, breakage of the source DNA into short fragments, and the errors in sequencing the ends of the fragments to produce read pairs.

  17. Functional enrichment analyses and construction of functional similarity networks with high confidence function prediction by PFP

    Directory of Open Access Journals (Sweden)

    Kihara Daisuke

    2010-05-01

    Full Text Available Abstract Background A new paradigm of biological investigation takes advantage of technologies that produce large high throughput datasets, including genome sequences, interactions of proteins, and gene expression. The ability of biologists to analyze and interpret such data relies on functional annotation of the included proteins, but even in highly characterized organisms many proteins can lack the functional evidence necessary to infer their biological relevance. Results Here we have applied high confidence function predictions from our automated prediction system, PFP, to three genome sequences, Escherichia coli, Saccharomyces cerevisiae, and Plasmodium falciparum (malaria. The number of annotated genes is increased by PFP to over 90% for all of the genomes. Using the large coverage of the function annotation, we introduced the functional similarity networks which represent the functional space of the proteomes. Four different functional similarity networks are constructed for each proteome, one each by considering similarity in a single Gene Ontology (GO category, i.e. Biological Process, Cellular Component, and Molecular Function, and another one by considering overall similarity with the funSim score. The functional similarity networks are shown to have higher modularity than the protein-protein interaction network. Moreover, the funSim score network is distinct from the single GO-score networks by showing a higher clustering degree exponent value and thus has a higher tendency to be hierarchical. In addition, examining function assignments to the protein-protein interaction network and local regions of genomes has identified numerous cases where subnetworks or local regions have functionally coherent proteins. These results will help interpreting interactions of proteins and gene orders in a genome. Several examples of both analyses are highlighted. Conclusion The analyses demonstrate that applying high confidence predictions from PFP

  18. Technical Report: Benchmarking for Quasispecies Abundance Inference with Confidence Intervals from Metagenomic Sequence Data

    Energy Technology Data Exchange (ETDEWEB)

    McLoughlin, K. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)

    2016-01-22

    The software application “MetaQuant” was developed by our group at Lawrence Livermore National Laboratory (LLNL). It is designed to profile microbial populations in a sample using data from whole-genome shotgun (WGS) metagenomic DNA sequencing. Several other metagenomic profiling applications have been described in the literature. We ran a series of benchmark tests to compare the performance of MetaQuant against that of a few existing profiling tools, using real and simulated sequence datasets. This report describes our benchmarking procedure and results.

  19. Methodology for the Assessment of Confidence in Safety Margin for Small Break Loss of Coolant Accident Sequences

    Energy Technology Data Exchange (ETDEWEB)

    Nagrale, D. B.; Prasad, M.; Rao, R. S.; Gaikwad, A.J., E-mail: avinashg@aerb.gov.in [Nuclear Safety Analysis Division, Atomic Energy Regulatory Board, Mumbai (India)

    2014-10-15

    Deterministic Safety Analysis and Probabilistic Safety Assessment (PSA) analyses are used concurrently to assess the Nuclear Power Plant (NPP) safety. The conventional deterministic analysis is conservative. The best estimate plus uncertainty analysis is increasingly being used for deterministic calculation in NPPs. The PSA methodology aims to be as realistic as possible while integrating information about accident phenomena, plant design, operating practices, component reliability and human behaviour. The peak clad temperature (PCT) distribution provides an insight into the confidence in safety margin for an initiating event. The paper deals with the concept of calculating the peak clad temperature with 95 percent confidence and 95 percent probability (PCT{sub 95/95}) in small break loss of coolant accident (SBLOCA) and methodologies for assessing safety margin. Five input parameters mainly, nominal power level, decay power, fuel clad gap conductivity, fuel thermal conductivity and discharge coefficient, were selected. A Uniform probability density function was assigned to the uncertain parameters and these uncertainties are propagated using Latin Hypercube Sampling (LHS) technique. The sampled data for 5 parameters were randomly mixed by LHS to obtain 25 input sets. A non-core damage accident sequence was selected from the SBLOCA event tree of a typical VVER study to estimate the PCTs and safety margin. A Kolmogorov– Smirnov goodness-of-fit test was carried out for PCTs. The smallest value of safety margin would indicate the robustness of the system with 95% confidence and 95% probability. Regression analysis was also carried out using 1000 sample size for the estimating PCTs. Mean, variance and finally safety margin were analysed. (author)

  20. Quack: A quality assurance tool for high throughput sequence data.

    Science.gov (United States)

    Thrash, Adam; Arick, Mark; Peterson, Daniel G

    2018-05-01

    The quality of data generated by high-throughput DNA sequencing tools must be rapidly assessed in order to determine how useful the data may be in making biological discoveries; higher quality data leads to more confident results and conclusions. Due to the ever-increasing size of data sets and the importance of rapid quality assessment, tools that analyze sequencing data should quickly produce easily interpretable graphics. Quack addresses these issues by generating information-dense visualizations from FASTQ files at a speed far surpassing other publicly available quality assurance tools in a manner independent of sequencing technology. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

  1. Categorizing Biases in High-Confidence High-Throughput Protein-Protein Interaction Data Sets*

    Science.gov (United States)

    Yu, Xueping; Ivanic, Joseph; Memišević, Vesna; Wallqvist, Anders; Reifman, Jaques

    2011-01-01

    We characterized and evaluated the functional attributes of three yeast high-confidence protein-protein interaction data sets derived from affinity purification/mass spectrometry, protein-fragment complementation assay, and yeast two-hybrid experiments. The interacting proteins retrieved from these data sets formed distinct, partially overlapping sets with different protein-protein interaction characteristics. These differences were primarily a function of the deployed experimental technologies used to recover these interactions. This affected the total coverage of interactions and was especially evident in the recovery of interactions among different functional classes of proteins. We found that the interaction data obtained by the yeast two-hybrid method was the least biased toward any particular functional characterization. In contrast, interacting proteins in the affinity purification/mass spectrometry and protein-fragment complementation assay data sets were over- and under-represented among distinct and different functional categories. We delineated how these differences affected protein complex organization in the network of interactions, in particular for strongly interacting complexes (e.g. RNA and protein synthesis) versus weak and transient interacting complexes (e.g. protein transport). We quantified methodological differences in detecting protein interactions from larger protein complexes, in the correlation of protein abundance among interacting proteins, and in their connectivity of essential proteins. In the latter case, we showed that minimizing inherent methodology biases removed many of the ambiguous conclusions about protein essentiality and protein connectivity. We used these findings to rationalize how biological insights obtained by analyzing data sets originating from different sources sometimes do not agree or may even contradict each other. An important corollary of this work was that discrepancies in biological insights did not

  2. FRESCO: Referential compression of highly similar sequences.

    Science.gov (United States)

    Wandelt, Sebastian; Leser, Ulf

    2013-01-01

    In many applications, sets of similar texts or sequences are of high importance. Prominent examples are revision histories of documents or genomic sequences. Modern high-throughput sequencing technologies are able to generate DNA sequences at an ever-increasing rate. In parallel to the decreasing experimental time and cost necessary to produce DNA sequences, computational requirements for analysis and storage of the sequences are steeply increasing. Compression is a key technology to deal with this challenge. Recently, referential compression schemes, storing only the differences between a to-be-compressed input and a known reference sequence, gained a lot of interest in this field. In this paper, we propose a general open-source framework to compress large amounts of biological sequence data called Framework for REferential Sequence COmpression (FRESCO). Our basic compression algorithm is shown to be one to two orders of magnitudes faster than comparable related work, while achieving similar compression ratios. We also propose several techniques to further increase compression ratios, while still retaining the advantage in speed: 1) selecting a good reference sequence; and 2) rewriting a reference sequence to allow for better compression. In addition,we propose a new way of further boosting the compression ratios by applying referential compression to already referentially compressed files (second-order compression). This technique allows for compression ratios way beyond state of the art, for instance,4,000:1 and higher for human genomes. We evaluate our algorithms on a large data set from three different species (more than 1,000 genomes, more than 3 TB) and on a collection of versions of Wikipedia pages. Our results show that real-time compression of highly similar sequences at high compression ratios is possible on modern hardware.

  3. Integration of multiple biological features yields high confidence human protein interactome.

    Science.gov (United States)

    Karagoz, Kubra; Sevimoglu, Tuba; Arga, Kazim Yalcin

    2016-08-21

    The biological function of a protein is usually determined by its physical interaction with other proteins. Protein-protein interactions (PPIs) are identified through various experimental methods and are stored in curated databases. The noisiness of the existing PPI data is evident, and it is essential that a more reliable data is generated. Furthermore, the selection of a set of PPIs at different confidence levels might be necessary for many studies. Although different methodologies were introduced to evaluate the confidence scores for binary interactions, a highly reliable, almost complete PPI network of Homo sapiens is not proposed yet. The quality and coverage of human protein interactome need to be improved to be used in various disciplines, especially in biomedicine. In the present work, we propose an unsupervised statistical approach to assign confidence scores to PPIs of H. sapiens. To achieve this goal PPI data from six different databases were collected and a total of 295,288 non-redundant interactions between 15,950 proteins were acquired. The present scoring system included the context information that was assigned to PPIs derived from eight biological attributes. A high confidence network, which included 147,923 binary interactions between 13,213 proteins, had scores greater than the cutoff value of 0.80, for which sensitivity, specificity, and coverage were 94.5%, 80.9%, and 82.8%, respectively. We compared the present scoring method with others for evaluation. Reducing the noise inherent in experimental PPIs via our scoring scheme increased the accuracy significantly. As it was demonstrated through the assessment of process and cancer subnetworks, this study allows researchers to construct and analyze context-specific networks via valid PPI sets and one can easily achieve subnetworks around proteins of interest at a specified confidence level. Copyright © 2016 Elsevier Ltd. All rights reserved.

  4. A high confidence, manually validated human blood plasma protein reference set

    DEFF Research Database (Denmark)

    Schenk, Susann; Schoenhals, Gary J; de Souza, Gustavo

    2008-01-01

    BACKGROUND: The immense diagnostic potential of human plasma has prompted great interest and effort in cataloging its contents, exemplified by the Human Proteome Organization (HUPO) Plasma Proteome Project (PPP) pilot project. Due to challenges in obtaining a reliable blood plasma protein list......-trap-Fourier transform (LTQ-FT) and a linear ion trap-Orbitrap (LTQ-Orbitrap) for mass spectrometry (MS) analysis. Both instruments allow the measurement of peptide masses in the low ppm range. Furthermore, we employed a statistical score that allows database peptide identification searching using the products of two...... consecutive stages of tandem mass spectrometry (MS3). The combination of MS3 with very high mass accuracy in the parent peptide allows peptide identification with orders of magnitude more confidence than that typically achieved. RESULTS: Herein we established a high confidence set of 697 blood plasma proteins...

  5. Identification of high-confidence RNA regulatory elements by combinatorial classification of RNA-protein binding sites.

    Science.gov (United States)

    Li, Yang Eric; Xiao, Mu; Shi, Binbin; Yang, Yu-Cheng T; Wang, Dong; Wang, Fei; Marcia, Marco; Lu, Zhi John

    2017-09-08

    Crosslinking immunoprecipitation sequencing (CLIP-seq) technologies have enabled researchers to characterize transcriptome-wide binding sites of RNA-binding protein (RBP) with high resolution. We apply a soft-clustering method, RBPgroup, to various CLIP-seq datasets to group together RBPs that specifically bind the same RNA sites. Such combinatorial clustering of RBPs helps interpret CLIP-seq data and suggests functional RNA regulatory elements. Furthermore, we validate two RBP-RBP interactions in cell lines. Our approach links proteins and RNA motifs known to possess similar biochemical and cellular properties and can, when used in conjunction with additional experimental data, identify high-confidence RBP groups and their associated RNA regulatory elements.

  6. Data on electrical energy conservation using high efficiency motors for the confidence bounds using statistical techniques.

    Science.gov (United States)

    Shaikh, Muhammad Mujtaba; Memon, Abdul Jabbar; Hussain, Manzoor

    2016-09-01

    In this article, we describe details of the data used in the research paper "Confidence bounds for energy conservation in electric motors: An economical solution using statistical techniques" [1]. The data presented in this paper is intended to show benefits of high efficiency electric motors over the standard efficiency motors of similar rating in the industrial sector of Pakistan. We explain how the data was collected and then processed by means of formulas to show cost effectiveness of energy efficient motors in terms of three important parameters: annual energy saving, cost saving and payback periods. This data can be further used to construct confidence bounds for the parameters using statistical techniques as described in [1].

  7. Decision-making patterns and self-confidence in high school adolescents

    Directory of Open Access Journals (Sweden)

    Alejandro César Antonio Luna Bernal

    2014-07-01

    Full Text Available The present study aimed to analyse the factor structure of the Melbourne Decision Making Questionnaire (DMQ-II, and to examine the relationships between the factors identified and Self-confidence, in order to conceptualize the decision-making process in adolescents under the Conflict Model of Decision Making. Participants were 992 Mexican high-school students, aged between 15 and 19 years. The three factors were identified as decision-making patterns in adolescents: a Vigilance, b Hipervigilance/Procrastination and c Buck-passing. The Self-confidence showed a positive effect on Vigilance, and a negative effect on theother two patterns. Results are discussed considering the literature on decision making in adolescence.

  8. High resolution sequence stratigraphy in China

    International Nuclear Information System (INIS)

    Zhang Shangfeng; Zhang Changmin; Yin Yanshi; Yin Taiju

    2008-01-01

    Since high resolution sequence stratigraphy was introduced into China by DENG Hong-wen in 1995, it has been experienced two development stages in China which are the beginning stage of theory research and development of theory research and application, and the stage of theoretical maturity and widely application that is going into. It is proved by practices that high resolution sequence stratigraphy plays more and more important roles in the exploration and development of oil and gas in Chinese continental oil-bearing basin and the research field spreads to the exploration of coal mine, uranium mine and other strata deposits. However, the theory of high resolution sequence stratigraphy still has some shortages, it should be improved in many aspects. The authors point out that high resolution sequence stratigraphy should be characterized quantitatively and modelized by computer techniques. (authors)

  9. Protein-Level Integration Strategy of Multiengine MS Spectra Search Results for Higher Confidence and Sequence Coverage.

    Science.gov (United States)

    Zhao, Panpan; Zhong, Jiayong; Liu, Wanting; Zhao, Jing; Zhang, Gong

    2017-12-01

    Multiple search engines based on various models have been developed to search MS/MS spectra against a reference database, providing different results for the same data set. How to integrate these results efficiently with minimal compromise on false discoveries is an open question due to the lack of an independent, reliable, and highly sensitive standard. We took the advantage of the translating mRNA sequencing (RNC-seq) result as a standard to evaluate the integration strategies of the protein identifications from various search engines. We used seven mainstream search engines (Andromeda, Mascot, OMSSA, X!Tandem, pFind, InsPecT, and ProVerB) to search the same label-free MS data sets of human cell lines Hep3B, MHCCLM3, and MHCC97H from the Chinese C-HPP Consortium for Chromosomes 1, 8, and 20. As expected, the union of seven engines resulted in a boosted false identification, whereas the intersection of seven engines remarkably decreased the identification power. We found that identifications of at least two out of seven engines resulted in maximizing the protein identification power while minimizing the ratio of suspicious/translation-supported identifications (STR), as monitored by our STR index, based on RNC-Seq. Furthermore, this strategy also significantly improves the peptides coverage of the protein amino acid sequence. In summary, we demonstrated a simple strategy to significantly improve the performance for shotgun mass spectrometry by protein-level integrating multiple search engines, maximizing the utilization of the current MS spectra without additional experimental work.

  10. Spatio-Spectral Method for Estimating Classified Regions with High Confidence using MODIS Data

    International Nuclear Information System (INIS)

    Katiyal, Anuj; Rajan, Dr K S

    2014-01-01

    In studies like change analysis, the availability of very high resolution (VHR)/high resolution (HR) imagery for a particular period and region is a challenge due to the sensor revisit times and high cost of acquisition. Therefore, most studies prefer lower resolution (LR) sensor imagery with frequent revisit times, in addition to their cost and computational advantages. Further, the classification techniques provide us a global estimate of the class accuracy, which limits its utility if the accuracy is low. In this work, we focus on the sub-classification problem of LR images and estimate regions of higher confidence than the global classification accuracy within its classified region. The spectrally classified data was mined into spatially clustered regions and further refined and processed using statistical measures to arrive at local high confidence regions (LHCRs), for every class. Rabi season MODIS data of January 2006 and 2007 was used for this study and the evaluation of LHCR was done using the APLULC 2005 classified data. For Jan-2007, the global class accuracies for water bodies (WB), forested regions (FR) and Kharif crops and barren lands (KB) were 89%, 71.7% and 71.23% respectively, while the respective LHCRs had accuracies of 96.67%, 89.4% and 80.9% covering an area of 46%, 29% and 14.5% of the initially classified areas. Though areas are reduced, LHCRs with higher accuracies help in extracting more representative class regions. Identification of such regions can facilitate in improving the classification time and processing for HR images when combined with the more frequently acquired LR imagery, isolate pure vs. mixed/impure pixels and as training samples locations for HR imagery

  11. Identification of High Confidence Nuclear Forensics Signatures. Results of a Coordinated Research Project and Related Research

    International Nuclear Information System (INIS)

    2017-08-01

    The results of a Coordinated Research Project and related research on the identification of high confidence nuclear forensic isotopic, chemical and physical data characteristics, or signatures, provides information on signatures that can help identify the origin and history of nuclear and other radioactive material encountered out of regulatory control. This research report compiles findings from investigations of materials obtained from throughout the nuclear fuel cycle to include radioactive sources. The report further provides recent results used to identify, analyse in the laboratory, predict and interpret these signatures relative to the requirements of a nuclear forensics examination. The report describes some of the controls on the incorporation and persistence of these signatures in these materials as well as their potential use in a national system of identification to include a national nuclear forensics library.

  12. Integration testing through reusing representative unit test cases for high-confidence medical software.

    Science.gov (United States)

    Shin, Youngsul; Choi, Yunja; Lee, Woo Jin

    2013-06-01

    As medical software is getting larger-sized, complex, and connected with other devices, finding faults in integrated software modules gets more difficult and time consuming. Existing integration testing typically takes a black-box approach, which treats the target software as a black box and selects test cases without considering internal behavior of each software module. Though it could be cost-effective, this black-box approach cannot thoroughly test interaction behavior among integrated modules and might leave critical faults undetected, which should not happen in safety-critical systems such as medical software. This work anticipates that information on internal behavior is necessary even for integration testing to define thorough test cases for critical software and proposes a new integration testing method by reusing test cases used for unit testing. The goal is to provide a cost-effective method to detect subtle interaction faults at the integration testing phase by reusing the knowledge obtained from unit testing phase. The suggested approach notes that the test cases for the unit testing include knowledge on internal behavior of each unit and extracts test cases for the integration testing from the test cases for the unit testing for a given test criteria. The extracted representative test cases are connected with functions under test using the state domain and a single test sequence to cover the test cases is produced. By means of reusing unit test cases, the tester has effective test cases to examine diverse execution paths and find interaction faults without analyzing complex modules. The produced test sequence can have test coverage as high as the unit testing coverage and its length is close to the length of optimal test sequences. Copyright © 2013 Elsevier Ltd. All rights reserved.

  13. Confidant Relations in Italy

    Directory of Open Access Journals (Sweden)

    Jenny Isaacs

    2015-02-01

    Full Text Available Confidants are often described as the individuals with whom we choose to disclose personal, intimate matters. The presence of a confidant is associated with both mental and physical health benefits. In this study, 135 Italian adults responded to a structured questionnaire that asked if they had a confidant, and if so, to describe various features of the relationship. The vast majority of participants (91% reported the presence of a confidant and regarded this relationship as personally important, high in mutuality and trust, and involving minimal lying. Confidants were significantly more likely to be of the opposite sex. Participants overall were significantly more likely to choose a spouse or other family member as their confidant, rather than someone outside of the family network. Familial confidants were generally seen as closer, and of greater value, than non-familial confidants. These findings are discussed within the context of Italian culture.

  14. Effect of spotters on state anxiety and self-confidence during maximal squatting among male high school athletes

    Directory of Open Access Journals (Sweden)

    Drew Rykert

    2017-09-01

    Full Text Available The ideal performance state is manifested by psychological and physiological efficiency. The psychological effects of anxiety and self-confidence has been shown to alter the efficiency of performance. This study attempted to identify the state anxiety and self-confidence of high school athletes just prior to a one repetition maximum (1-RM back squat and determine if the number of spotters affects an athlete’s level of state anxiety and/or self-confidence. Male high school athletes (10th and 11th grades were randomly separated into two experimental groups who performed the 1-RM back squat (BSQ with either 1 spotter (1SG: n=52 or 3 spotters (3SG: n=54. Following a dynamic warm-up period and several progressive BSQ warm-up sets, and just prior to attempts at a 1-RM BSQ, the participants completed the revised Competitive State Anxiety Inventory-2 (CSAI-2R. The CSAI-2R included the number of spotters (1 or 3 that would be present during the subsequent 1-RM BSQ attempts. The CSAI-2R is a17-question instrument with three subscales (self-confidence, somatic anxiety, and cognitive anxiety. The subscale scores were compared between the 1SG and 3SG with an independent t-test (alpha≤0.05. None of the subscales (self-confidence, somatic anxiety, and cognitive anxiety were significantly different between the 1SG and 3SG experimental groups (p>0.05. Within the parameters of this study, the number of spotters present during the execution of the 1-RM BSQ had no practical or statistical impact on self-confidence, somatic anxiety, and cognitive anxiety. Coaches and athletes could use this information in the training environment in order to make best use of personnel (assigned to spotting tasks, physical resources (ex. squat racks, and time management.

  15. Library Design-Facilitated High-Throughput Sequencing of Synthetic Peptide Libraries.

    Science.gov (United States)

    Vinogradov, Alexander A; Gates, Zachary P; Zhang, Chi; Quartararo, Anthony J; Halloran, Kathryn H; Pentelute, Bradley L

    2017-11-13

    A methodology to achieve high-throughput de novo sequencing of synthetic peptide mixtures is reported. The approach leverages shotgun nanoliquid chromatography coupled with tandem mass spectrometry-based de novo sequencing of library mixtures (up to 2000 peptides) as well as automated data analysis protocols to filter away incorrect assignments, noise, and synthetic side-products. For increasing the confidence in the sequencing results, mass spectrometry-friendly library designs were developed that enabled unambiguous decoding of up to 600 peptide sequences per hour while maintaining greater than 85% sequence identification rates in most cases. The reliability of the reported decoding strategy was additionally confirmed by matching fragmentation spectra for select authentic peptides identified from library sequencing samples. The methods reported here are directly applicable to screening techniques that yield mixtures of active compounds, including particle sorting of one-bead one-compound libraries and affinity enrichment of synthetic library mixtures performed in solution.

  16. LipiDex: An Integrated Software Package for High-Confidence Lipid Identification.

    Science.gov (United States)

    Hutchins, Paul D; Russell, Jason D; Coon, Joshua J

    2018-04-17

    State-of-the-art proteomics software routinely quantifies thousands of peptides per experiment with minimal need for manual validation or processing of data. For the emerging field of discovery lipidomics via liquid chromatography-tandem mass spectrometry (LC-MS/MS), comparably mature informatics tools do not exist. Here, we introduce LipiDex, a freely available software suite that unifies and automates all stages of lipid identification, reducing hands-on processing time from hours to minutes for even the most expansive datasets. LipiDex utilizes flexible in silico fragmentation templates and lipid-optimized MS/MS spectral matching routines to confidently identify and track hundreds of lipid species and unknown compounds from diverse sample matrices. Unique spectral and chromatographic peak purity algorithms accurately quantify co-isolation and co-elution of isobaric lipids, generating identifications that match the structural resolution afforded by the LC-MS/MS experiment. During final data filtering, ionization artifacts are removed to significantly reduce dataset redundancy. LipiDex interfaces with several LC-MS/MS software packages, enabling robust lipid identification to be readily incorporated into pre-existing data workflows. Copyright © 2018 Elsevier Inc. All rights reserved.

  17. Using the confidence interval confidently.

    Science.gov (United States)

    Hazra, Avijit

    2017-10-01

    Biomedical research is seldom done with entire populations but rather with samples drawn from a population. Although we work with samples, our goal is to describe and draw inferences regarding the underlying population. It is possible to use a sample statistic and estimates of error in the sample to get a fair idea of the population parameter, not as a single value, but as a range of values. This range is the confidence interval (CI) which is estimated on the basis of a desired confidence level. Calculation of the CI of a sample statistic takes the general form: CI = Point estimate ± Margin of error, where the margin of error is given by the product of a critical value (z) derived from the standard normal curve and the standard error of point estimate. Calculation of the standard error varies depending on whether the sample statistic of interest is a mean, proportion, odds ratio (OR), and so on. The factors affecting the width of the CI include the desired confidence level, the sample size and the variability in the sample. Although the 95% CI is most often used in biomedical research, a CI can be calculated for any level of confidence. A 99% CI will be wider than 95% CI for the same sample. Conflict between clinical importance and statistical significance is an important issue in biomedical research. Clinical importance is best inferred by looking at the effect size, that is how much is the actual change or difference. However, statistical significance in terms of P only suggests whether there is any difference in probability terms. Use of the CI supplements the P value by providing an estimate of actual clinical effect. Of late, clinical trials are being designed specifically as superiority, non-inferiority or equivalence studies. The conclusions from these alternative trial designs are based on CI values rather than the P value from intergroup comparison.

  18. Individuals with high obsessive-compulsive tendencies or undermined confidence rely more on external proxies to access their internal states.

    Science.gov (United States)

    Zhang, Zhongming; Wang, Mengyun; Miao, Xiaocui; Li, Yijuan; Hitchman, Glenn; Yuan, Zhen

    2017-03-01

    The Seeking Proxies for Internal States (SPIS) hypothesis predicts that obsessive-compulsive disorder (OCD) is associated with a deficit in subjective convictions, which may lead to a reliance on external substitutes for the perceptions of an individual's internal states. Two well-designed studies were performed for the present work that adopted a false bio-feedback procedure in a muscle tension task to examine the SPIS hypothesis. The false bio-feedback paradigm was used to investigate our hypothesis. NeXus-10 Mark II hardware and V2011 BioTrace + software (Mind Media B.V., Herten, Netherlands) were utilized to measure the muscle tension of the flexor carpiulnaris muscle, which characterized the target's internal state. In addition, false EMG changes were recorded and displayed on a computer monitor and were considered external proxies. Study 1 demonstrated that the participants with high obsessive-compulsive (OC) tendencies were more affected by the false bio-feedback and exhibited lower confidence in their judgments regarding their muscle tension compared with the participants with low OC tendencies. These findings indicate that subjects with high OC tendencies were more influenced by self-perception effects. In contrast, the subjects in the undermined confidence group in Study 2 were more easily influenced by the false bio-feedback compared with the control group, which suggests that the subjects in the undermined confidence group were more affected by self-perception effects. We did not combine the undermined confidence with OC tendencies or OCD symptoms in our paradigm to investigate their joint effects on self-perception. Our findings provide further evidence that supports the SPIS hypothesis, which indicates that OC tendencies and the confidence in an individual's recognition of internal states appear to have similar effects on the assessment of internal states and reliance on proxies. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. Applications of High Throughput Nucleotide Sequencing

    DEFF Research Database (Denmark)

    Waage, Johannes Eichler

    equally large demands in data handling, analysis and interpretation, perhaps defining the modern challenge of the computational biologist of the post-genomic era. The first part of this thesis consists of a general introduction to the history, common terms and challenges of next generation sequencing......-sequencing, a study of the effects on alternative RNA splicing of KO of the nonsense mediated RNA decay system in Mus, using digital gene expression and a custom-built exon-exon junction mapping pipeline is presented (article I). Evolved from this work, a Bioconductor package, spliceR, for classifying alternative...

  20. Label-Driven Learning Framework: Towards More Accurate Bayesian Network Classifiers through Discrimination of High-Confidence Labels

    Directory of Open Access Journals (Sweden)

    Yi Sun

    2017-12-01

    Full Text Available Bayesian network classifiers (BNCs have demonstrated competitive classification accuracy in a variety of real-world applications. However, it is error-prone for BNCs to discriminate among high-confidence labels. To address this issue, we propose the label-driven learning framework, which incorporates instance-based learning and ensemble learning. For each testing instance, high-confidence labels are first selected by a generalist classifier, e.g., the tree-augmented naive Bayes (TAN classifier. Then, by focusing on these labels, conditional mutual information is redefined to more precisely measure mutual dependence between attributes, thus leading to a refined generalist with a more reasonable network structure. To enable finer discrimination, an expert classifier is tailored for each high-confidence label. Finally, the predictions of the refined generalist and the experts are aggregated. We extend TAN to LTAN (Label-driven TAN by applying the proposed framework. Extensive experimental results demonstrate that LTAN delivers superior classification accuracy to not only several state-of-the-art single-structure BNCs but also some established ensemble BNCs at the expense of reasonable computation overhead.

  1. Confidence improvement of disosal safety bydevelopement of a safety case for high-level radioactive waste disposal

    Energy Technology Data Exchange (ETDEWEB)

    Baik, Min Hoon; Ko, Nak Youl; Jeong, Jong Tae; Kim, Kyung Su [Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of)

    2016-12-15

    Many countries have developed a safety case suitable to their own countries in order to improve the confidence of disposal safety in deep geological disposal of high-level radioactive waste as well as to develop a disposal program and obtain its license. This study introduces and summarizes the meaning, necessity, and development process of the safety case for radioactive waste disposal. The disposal safety is also discussed in various aspects of the safety case. In addition, the status of safety case development in the foreign countries is briefly introduced for Switzerland, Japan, the United States of America, Sweden, and Finland. The strategy for the safety case development that is being developed by KAERI is also briefly introduced. Based on the safety case, we analyze the efforts necessary to improve confidence in disposal safety for high-level radioactive waste. Considering domestic situations, we propose and discuss some implementing methods for the improvement of disposal safety, such as construction of a reliable information database, understanding of processes related to safety, reduction of uncertainties in safety assessment, communication with stakeholders, and ensuring justice and transparency. This study will contribute to the understanding of the safety case for deep geological disposal and to improving confidence in disposal safety through the development of the safety case in Korea for the disposal of high-level radioactive waste.

  2. Fragility estimation for seismically isolated nuclear structures by high confidence low probability of failure values and bi-linear regression

    International Nuclear Information System (INIS)

    Carausu, A.

    1996-01-01

    A method for the fragility estimation of seismically isolated nuclear power plant structure is proposed. The relationship between the ground motion intensity parameter (e.g. peak ground velocity or peak ground acceleration) and the response of isolated structures is expressed in terms of a bi-linear regression line, whose coefficients are estimated by the least-square method in terms of available data on seismic input and structural response. The notion of high confidence low probability of failure (HCLPF) value is also used for deriving compound fragility curves for coupled subsystems. (orig.)

  3. Exome Sequence Analysis of 14 Families With High Myopia

    DEFF Research Database (Denmark)

    Kloss, Bethany A.; Tompson, Stuart W.; Whisenhunt, Kristina N.

    2017-01-01

    Purpose: To identify causal gene mutations in 14 families with autosomal dominant (AD) high myopia using exome sequencing. Methods: Select individuals from 14 large Caucasian families with high myopia were exome sequenced. Gene variants were filtered to identify potential pathogenic changes. Sang...

  4. High-throughput sequence alignment using Graphics Processing Units

    Directory of Open Access Journals (Sweden)

    Trapnell Cole

    2007-12-01

    Full Text Available Abstract Background The recent availability of new, less expensive high-throughput DNA sequencing technologies has yielded a dramatic increase in the volume of sequence data that must be analyzed. These data are being generated for several purposes, including genotyping, genome resequencing, metagenomics, and de novo genome assembly projects. Sequence alignment programs such as MUMmer have proven essential for analysis of these data, but researchers will need ever faster, high-throughput alignment tools running on inexpensive hardware to keep up with new sequence technologies. Results This paper describes MUMmerGPU, an open-source high-throughput parallel pairwise local sequence alignment program that runs on commodity Graphics Processing Units (GPUs in common workstations. MUMmerGPU uses the new Compute Unified Device Architecture (CUDA from nVidia to align multiple query sequences against a single reference sequence stored as a suffix tree. By processing the queries in parallel on the highly parallel graphics card, MUMmerGPU achieves more than a 10-fold speedup over a serial CPU version of the sequence alignment kernel, and outperforms the exact alignment component of MUMmer on a high end CPU by 3.5-fold in total application time when aligning reads from recent sequencing projects using Solexa/Illumina, 454, and Sanger sequencing technologies. Conclusion MUMmerGPU is a low cost, ultra-fast sequence alignment program designed to handle the increasing volume of data produced by new, high-throughput sequencing technologies. MUMmerGPU demonstrates that even memory-intensive applications can run significantly faster on the relatively low-cost GPU than on the CPU.

  5. Automated cleaning and pre-processing of immunoglobulin gene sequences from high-throughput sequencing

    Directory of Open Access Journals (Sweden)

    Miri eMichaeli

    2012-12-01

    Full Text Available High throughput sequencing (HTS yields tens of thousands to millions of sequences that require a large amount of pre-processing work to clean various artifacts. Such cleaning cannot be performed manually. Existing programs are not suitable for immunoglobulin (Ig genes, which are variable and often highly mutated. This paper describes Ig-HTS-Cleaner (Ig High Throughput Sequencing Cleaner, a program containing a simple cleaning procedure that successfully deals with pre-processing of Ig sequences derived from HTS, and Ig-Indel-Identifier (Ig Insertion – Deletion Identifier, a program for identifying legitimate and artifact insertions and/or deletions (indels. Our programs were designed for analyzing Ig gene sequences obtained by 454 sequencing, but they are applicable to all types of sequences and sequencing platforms. Ig-HTS-Cleaner and Ig-Indel-Identifier have been implemented in Java and saved as executable JAR files, supported on Linux and MS Windows. No special requirements are needed in order to run the programs, except for correctly constructing the input files as explained in the text. The programs' performance has been tested and validated on real and simulated data sets.

  6. Assessment of risk to wildlife from ionising radiation: can initial screening tiers be used with a high level of confidence?

    International Nuclear Information System (INIS)

    Beresford, N A; Barnett, C L; Hosseini, A; Brown, J E; Cailes, C; Copplestone, D; Beaugelin-Seiller, K

    2010-01-01

    A number of models are being used to assess the potential environmental impact of releases of radioactivity. These often use a tiered assessment structure whose first tier is designed to be highly conservative and simple to use. An aim of using this initial tier is to identify sites of negligible concern and to remove them from further consideration with a high degree of confidence. In this paper we compare the screening assessment outputs of three freely available models. The outputs of these models varied considerably in terms of estimated risk quotient (RQ) and the radionuclide-organism combinations identified as being the most limiting. A number of factors are identified as contributing to this variability: values of transfer parameters (concentration ratios and K d ) used; organisms considered; different input options and how these are utilised in the assessment; assumptions as regards secular equilibrium; geometries and exposure scenarios. This large variation in RQ values between models means that the level of confidence required by users is not achieved. We recommend that the factors contributing to the variation in screening assessments be subjected to further investigation so that they can be more fully understood and assessors (and those reviewing assessment outputs) can better justify and evaluate the results obtained.

  7. Management of High-Throughput DNA Sequencing Projects: Alpheus.

    Science.gov (United States)

    Miller, Neil A; Kingsmore, Stephen F; Farmer, Andrew; Langley, Raymond J; Mudge, Joann; Crow, John A; Gonzalez, Alvaro J; Schilkey, Faye D; Kim, Ryan J; van Velkinburgh, Jennifer; May, Gregory D; Black, C Forrest; Myers, M Kathy; Utsey, John P; Frost, Nicholas S; Sugarbaker, David J; Bueno, Raphael; Gullans, Stephen R; Baxter, Susan M; Day, Steve W; Retzel, Ernest F

    2008-12-26

    High-throughput DNA sequencing has enabled systems biology to begin to address areas in health, agricultural and basic biological research. Concomitant with the opportunities is an absolute necessity to manage significant volumes of high-dimensional and inter-related data and analysis. Alpheus is an analysis pipeline, database and visualization software for use with massively parallel DNA sequencing technologies that feature multi-gigabase throughput characterized by relatively short reads, such as Illumina-Solexa (sequencing-by-synthesis), Roche-454 (pyrosequencing) and Applied Biosystem's SOLiD (sequencing-by-ligation). Alpheus enables alignment to reference sequence(s), detection of variants and enumeration of sequence abundance, including expression levels in transcriptome sequence. Alpheus is able to detect several types of variants, including non-synonymous and synonymous single nucleotide polymorphisms (SNPs), insertions/deletions (indels), premature stop codons, and splice isoforms. Variant detection is aided by the ability to filter variant calls based on consistency, expected allele frequency, sequence quality, coverage, and variant type in order to minimize false positives while maximizing the identification of true positives. Alpheus also enables comparisons of genes with variants between cases and controls or bulk segregant pools. Sequence-based differential expression comparisons can be developed, with data export to SAS JMP Genomics for statistical analysis.

  8. The Effects of Game-Based Learning on Mathematical Confidence and Performance: High Ability vs. Low Ability

    Science.gov (United States)

    Ku, Oskar; Chen, Sherry Y.; Wu, Denise H.; Lao, Andrew C. C.; Chan, Tak-Wai

    2014-01-01

    Many students possess low confidence toward learning mathematics, which, in turn, may lead them to give up pursuing more mathematics knowledge. Recently, game-based learning (GBL) is regarded as a potential means in improving students' confidence. Thus, this study tried to promote students' confidence toward mathematics by using GBL. In addition,…

  9. Search for high confidence AGN candidates and its counterparts in the Fermi-LAT unassociated sample using machine learning

    Energy Technology Data Exchange (ETDEWEB)

    Einecke, Sabrina [Technical University Dortmund (Germany); Doert, Marlene [Ruhr-University Bochum (Germany)

    2016-07-01

    The third Fermi-LAT source catalog (3FGL) is the deepest all-sky survey in gamma-rays and comprises 3033 point sources. While for 2023 sources plausible associations have been found, 1010 remain unassociated. A search for active galactic nuclei (AGN) will help to reduce the number of unassociated sources, and will increase our knowledge of the population of gamma-ray emitting AGN. Several machine learning approaches applied to Fermi data have shown the capability of this method. The extension to multiwavelength data improves these studies, and at the same time offers the possibility to determine the most likely corresponding counterpart. As the 95% confidence region of the localization by the Fermi measurement is in the order of several arcminutes, generally multiple point sources at different wavelengths are located within this region and the association is ambiguous. To figure out the most likely counterpart, the associated sample is used to train machine learning classifiers as e.g. the random forest. Therefore, all possible combinations of the Fermi measurement and the measurements at a second wavelength are considered for a particular source. In this talk, the statistical model to obtain high confidence AGN counterpart candidates is described as well as the validation of the model to estimate the performance.

  10. Directed PCR-free engineering of highly repetitive DNA sequences

    Directory of Open Access Journals (Sweden)

    Preissler Steffen

    2011-09-01

    Full Text Available Abstract Background Highly repetitive nucleotide sequences are commonly found in nature e.g. in telomeres, microsatellite DNA, polyadenine (poly(A tails of eukaryotic messenger RNA as well as in several inherited human disorders linked to trinucleotide repeat expansions in the genome. Therefore, studying repetitive sequences is of biological, biotechnological and medical relevance. However, cloning of such repetitive DNA sequences is challenging because specific PCR-based amplification is hampered by the lack of unique primer binding sites resulting in unspecific products. Results For the PCR-free generation of repetitive DNA sequences we used antiparallel oligonucleotides flanked by restriction sites of Type IIS endonucleases. The arrangement of recognition sites allowed for stepwise and seamless elongation of repetitive sequences. This facilitated the assembly of repetitive DNA segments and open reading frames encoding polypeptides with periodic amino acid sequences of any desired length. By this strategy we cloned a series of polyglutamine encoding sequences as well as highly repetitive polyadenine tracts. Such repetitive sequences can be used for diverse biotechnological applications. As an example, the polyglutamine sequences were expressed as His6-SUMO fusion proteins in Escherichia coli cells to study their aggregation behavior in vitro. The His6-SUMO moiety enabled affinity purification of the polyglutamine proteins, increased their solubility, and allowed controlled induction of the aggregation process. We successfully purified the fusions proteins and provide an example for their applicability in filter retardation assays. Conclusion Our seamless cloning strategy is PCR-free and allows the directed and efficient generation of highly repetitive DNA sequences of defined lengths by simple standard cloning procedures.

  11. MUSCLE: multiple sequence alignment with high accuracy and high throughput.

    Science.gov (United States)

    Edgar, Robert C

    2004-01-01

    We describe MUSCLE, a new computer program for creating multiple alignments of protein sequences. Elements of the algorithm include fast distance estimation using kmer counting, progressive alignment using a new profile function we call the log-expectation score, and refinement using tree-dependent restricted partitioning. The speed and accuracy of MUSCLE are compared with T-Coffee, MAFFT and CLUSTALW on four test sets of reference alignments: BAliBASE, SABmark, SMART and a new benchmark, PREFAB. MUSCLE achieves the highest, or joint highest, rank in accuracy on each of these sets. Without refinement, MUSCLE achieves average accuracy statistically indistinguishable from T-Coffee and MAFFT, and is the fastest of the tested methods for large numbers of sequences, aligning 5000 sequences of average length 350 in 7 min on a current desktop computer. The MUSCLE program, source code and PREFAB test data are freely available at http://www.drive5. com/muscle.

  12. High-Throughput Block Optical DNA Sequence Identification.

    Science.gov (United States)

    Sagar, Dodderi Manjunatha; Korshoj, Lee Erik; Hanson, Katrina Bethany; Chowdhury, Partha Pratim; Otoupal, Peter Britton; Chatterjee, Anushree; Nagpal, Prashant

    2018-01-01

    Optical techniques for molecular diagnostics or DNA sequencing generally rely on small molecule fluorescent labels, which utilize light with a wavelength of several hundred nanometers for detection. Developing a label-free optical DNA sequencing technique will require nanoscale focusing of light, a high-throughput and multiplexed identification method, and a data compression technique to rapidly identify sequences and analyze genomic heterogeneity for big datasets. Such a method should identify characteristic molecular vibrations using optical spectroscopy, especially in the "fingerprinting region" from ≈400-1400 cm -1 . Here, surface-enhanced Raman spectroscopy is used to demonstrate label-free identification of DNA nucleobases with multiplexed 3D plasmonic nanofocusing. While nanometer-scale mode volumes prevent identification of single nucleobases within a DNA sequence, the block optical technique can identify A, T, G, and C content in DNA k-mers. The content of each nucleotide in a DNA block can be a unique and high-throughput method for identifying sequences, genes, and other biomarkers as an alternative to single-letter sequencing. Additionally, coupling two complementary vibrational spectroscopy techniques (infrared and Raman) can improve block characterization. These results pave the way for developing a novel, high-throughput block optical sequencing method with lossy genomic data compression using k-mer identification from multiplexed optical data acquisition. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. The idiosyncratic nature of confidence.

    Science.gov (United States)

    Navajas, Joaquin; Hindocha, Chandni; Foda, Hebah; Keramati, Mehdi; Latham, Peter E; Bahrami, Bahador

    2017-11-01

    Confidence is the 'feeling of knowing' that accompanies decision making. Bayesian theory proposes that confidence is a function solely of the perceived probability of being correct. Empirical research has suggested, however, that different individuals may perform different computations to estimate confidence from uncertain evidence. To test this hypothesis, we collected confidence reports in a task where subjects made categorical decisions about the mean of a sequence. We found that for most individuals, confidence did indeed reflect the perceived probability of being correct. However, in approximately half of them, confidence also reflected a different probabilistic quantity: the perceived uncertainty in the estimated variable. We found that the contribution of both quantities was stable over weeks. We also observed that the influence of the perceived probability of being correct was stable across two tasks, one perceptual and one cognitive. Overall, our findings provide a computational interpretation of individual differences in human confidence.

  14. High-Throughput Next-Generation Sequencing of Polioviruses

    Science.gov (United States)

    Montmayeur, Anna M.; Schmidt, Alexander; Zhao, Kun; Magaña, Laura; Iber, Jane; Castro, Christina J.; Chen, Qi; Henderson, Elizabeth; Ramos, Edward; Shaw, Jing; Tatusov, Roman L.; Dybdahl-Sissoko, Naomi; Endegue-Zanga, Marie Claire; Adeniji, Johnson A.; Oberste, M. Steven; Burns, Cara C.

    2016-01-01

    ABSTRACT The poliovirus (PV) is currently targeted for worldwide eradication and containment. Sanger-based sequencing of the viral protein 1 (VP1) capsid region is currently the standard method for PV surveillance. However, the whole-genome sequence is sometimes needed for higher resolution global surveillance. In this study, we optimized whole-genome sequencing protocols for poliovirus isolates and FTA cards using next-generation sequencing (NGS), aiming for high sequence coverage, efficiency, and throughput. We found that DNase treatment of poliovirus RNA followed by random reverse transcription (RT), amplification, and the use of the Nextera XT DNA library preparation kit produced significantly better results than other preparations. The average viral reads per total reads, a measurement of efficiency, was as high as 84.2% ± 15.6%. PV genomes covering >99 to 100% of the reference length were obtained and validated with Sanger sequencing. A total of 52 PV genomes were generated, multiplexing as many as 64 samples in a single Illumina MiSeq run. This high-throughput, sequence-independent NGS approach facilitated the detection of a diverse range of PVs, especially for those in vaccine-derived polioviruses (VDPV), circulating VDPV, or immunodeficiency-related VDPV. In contrast to results from previous studies on other viruses, our results showed that filtration and nuclease treatment did not discernibly increase the sequencing efficiency of PV isolates. However, DNase treatment after nucleic acid extraction to remove host DNA significantly improved the sequencing results. This NGS method has been successfully implemented to generate PV genomes for molecular epidemiology of the most recent PV isolates. Additionally, the ability to obtain full PV genomes from FTA cards will aid in facilitating global poliovirus surveillance. PMID:27927929

  15. Automated degenerate PCR primer design for high-throughput sequencing improves efficiency of viral sequencing

    Directory of Open Access Journals (Sweden)

    Li Kelvin

    2012-11-01

    Full Text Available Abstract Background In a high-throughput environment, to PCR amplify and sequence a large set of viral isolates from populations that are potentially heterogeneous and continuously evolving, the use of degenerate PCR primers is an important strategy. Degenerate primers allow for the PCR amplification of a wider range of viral isolates with only one set of pre-mixed primers, thus increasing amplification success rates and minimizing the necessity for genome finishing activities. To successfully select a large set of degenerate PCR primers necessary to tile across an entire viral genome and maximize their success, this process is best performed computationally. Results We have developed a fully automated degenerate PCR primer design system that plays a key role in the J. Craig Venter Institute’s (JCVI high-throughput viral sequencing pipeline. A consensus viral genome, or a set of consensus segment sequences in the case of a segmented virus, is specified using IUPAC ambiguity codes in the consensus template sequence to represent the allelic diversity of the target population. PCR primer pairs are then selected computationally to produce a minimal amplicon set capable of tiling across the full length of the specified target region. As part of the tiling process, primer pairs are computationally screened to meet the criteria for successful PCR with one of two described amplification protocols. The actual sequencing success rates for designed primers for measles virus, mumps virus, human parainfluenza virus 1 and 3, human respiratory syncytial virus A and B and human metapneumovirus are described, where >90% of designed primer pairs were able to consistently successfully amplify >75% of the isolates. Conclusions Augmenting our previously developed and published JCVI Primer Design Pipeline, we achieved similarly high sequencing success rates with only minor software modifications. The recommended methodology for the construction of the consensus

  16. Application of high-throughput DNA sequencing in phytopathology.

    Science.gov (United States)

    Studholme, David J; Glover, Rachel H; Boonham, Neil

    2011-01-01

    The new sequencing technologies are already making a big impact in academic research on medically important microbes and may soon revolutionize diagnostics, epidemiology, and infection control. Plant pathology also stands to gain from exploiting these opportunities. This manuscript reviews some applications of these high-throughput sequencing methods that are relevant to phytopathology, with emphasis on the associated computational and bioinformatics challenges and their solutions. Second-generation sequencing technologies have recently been exploited in genomics of both prokaryotic and eukaryotic plant pathogens. They are also proving to be useful in diagnostics, especially with respect to viruses. Copyright © 2011 by Annual Reviews. All rights reserved.

  17. "Joint Workshop on High Confidence Medical Devices, Software, and Systems (HCMDSS) and Medical Device Plug-and-Play (MD PnP) Interoperability"

    National Research Council Canada - National Science Library

    Goldman, Julian M

    2008-01-01

    Partial support was requested from TATRC, with joint funding from NSF, for a joint workshop to bring together the synergistic efforts and communities of the High Confidence Medical Devices, Software, and Systems (HCMDSS...

  18. Highly multiplexed targeted DNA sequencing from single nuclei.

    Science.gov (United States)

    Leung, Marco L; Wang, Yong; Kim, Charissa; Gao, Ruli; Jiang, Jerry; Sei, Emi; Navin, Nicholas E

    2016-02-01

    Single-cell DNA sequencing methods are challenged by poor physical coverage, high technical error rates and low throughput. To address these issues, we developed a single-cell DNA sequencing protocol that combines flow-sorting of single nuclei, time-limited multiple-displacement amplification (MDA), low-input library preparation, DNA barcoding, targeted capture and next-generation sequencing (NGS). This approach represents a major improvement over our previous single nucleus sequencing (SNS) Nature Protocols paper in terms of generating higher-coverage data (>90%), thereby enabling the detection of genome-wide variants in single mammalian cells at base-pair resolution. Furthermore, by pooling 48-96 single-cell libraries together for targeted capture, this approach can be used to sequence many single-cell libraries in parallel in a single reaction. This protocol greatly reduces the cost of single-cell DNA sequencing, and it can be completed in 5-6 d by advanced users. This single-cell DNA sequencing protocol has broad applications for studying rare cells and complex populations in diverse fields of biological research and medicine.

  19. Roche genome sequencer FLX based high-throughput sequencing of ancient DNA

    DEFF Research Database (Denmark)

    Alquezar-Planas, David E; Fordyce, Sarah Louise

    2012-01-01

    Since the development of so-called "next generation" high-throughput sequencing in 2005, this technology has been applied to a variety of fields. Such applications include disease studies, evolutionary investigations, and ancient DNA. Each application requires a specialized protocol to ensure...... that the data produced is optimal. Although much of the procedure can be followed directly from the manufacturer's protocols, the key differences lie in the library preparation steps. This chapter presents an optimized protocol for the sequencing of fossil remains and museum specimens, commonly referred...

  20. Sequencing of 50 human exomes reveals adaptation to high altitude

    DEFF Research Database (Denmark)

    Yi, Xin; Liang, Yu; Huerta-Sanchez, Emilia

    2010-01-01

    Residents of the Tibetan Plateau show heritable adaptations to extreme altitude. We sequenced 50 exomes of ethnic Tibetans, encompassing coding sequences of 92% of human genes, with an average coverage of 18x per individual. Genes showing population-specific allele frequency changes, which repres...... in genetic adaptation to high altitude.......Residents of the Tibetan Plateau show heritable adaptations to extreme altitude. We sequenced 50 exomes of ethnic Tibetans, encompassing coding sequences of 92% of human genes, with an average coverage of 18x per individual. Genes showing population-specific allele frequency changes, which...... represent strong candidates for altitude adaptation, were identified. The strongest signal of natural selection came from endothelial Per-Arnt-Sim (PAS) domain protein 1 (EPAS1), a transcription factor involved in response to hypoxia. One single-nucleotide polymorphism (SNP) at EPAS1 shows a 78% frequency...

  1. 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...

  2. Winnowing DNA for rare sequences: highly specific sequence and methylation based enrichment.

    Directory of Open Access Journals (Sweden)

    Jason D Thompson

    Full Text Available Rare mutations in cell populations are known to be hallmarks of many diseases and cancers. Similarly, differential DNA methylation patterns arise in rare cell populations with diagnostic potential such as fetal cells circulating in maternal blood. Unfortunately, the frequency of alleles with diagnostic potential, relative to wild-type background sequence, is often well below the frequency of errors in currently available methods for sequence analysis, including very high throughput DNA sequencing. We demonstrate a DNA preparation and purification method that through non-linear electrophoretic separation in media containing oligonucleotide probes, achieves 10,000 fold enrichment of target DNA with single nucleotide specificity, and 100 fold enrichment of unmodified methylated DNA differing from the background by the methylation of a single cytosine residue.

  3. Winnowing DNA for rare sequences: highly specific sequence and methylation based enrichment.

    Science.gov (United States)

    Thompson, Jason D; Shibahara, Gosuke; Rajan, Sweta; Pel, Joel; Marziali, Andre

    2012-01-01

    Rare mutations in cell populations are known to be hallmarks of many diseases and cancers. Similarly, differential DNA methylation patterns arise in rare cell populations with diagnostic potential such as fetal cells circulating in maternal blood. Unfortunately, the frequency of alleles with diagnostic potential, relative to wild-type background sequence, is often well below the frequency of errors in currently available methods for sequence analysis, including very high throughput DNA sequencing. We demonstrate a DNA preparation and purification method that through non-linear electrophoretic separation in media containing oligonucleotide probes, achieves 10,000 fold enrichment of target DNA with single nucleotide specificity, and 100 fold enrichment of unmodified methylated DNA differing from the background by the methylation of a single cytosine residue.

  4. Scrutinizing virus genome termini by high-throughput sequencing.

    Directory of Open Access Journals (Sweden)

    Shasha Li

    Full Text Available Analysis of genomic terminal sequences has been a major step in studies on viral DNA replication and packaging mechanisms. However, traditional methods to study genome termini are challenging due to the time-consuming protocols and their inefficiency where critical details are lost easily. Recent advances in next generation sequencing (NGS have enabled it to be a powerful tool to study genome termini. In this study, using NGS we sequenced one iridovirus genome and twenty phage genomes and confirmed for the first time that the high frequency sequences (HFSs found in the NGS reads are indeed the terminal sequences of viral genomes. Further, we established a criterion to distinguish the type of termini and the viral packaging mode. We also obtained additional terminal details such as terminal repeats, multi-termini, asymmetric termini. With this approach, we were able to simultaneously detect details of the genome termini as well as obtain the complete sequence of bacteriophage genomes. Theoretically, this application can be further extended to analyze larger and more complicated genomes of plant and animal viruses. This study proposed a novel and efficient method for research on viral replication, packaging, terminase activity, transcription regulation, and metabolism of the host cell.

  5. Exome sequencing identifies ZNF644 mutations in high myopia.

    Directory of Open Access Journals (Sweden)

    Yi Shi

    2011-06-01

    Full Text Available Myopia is the most common ocular disorder worldwide, and high myopia in particular is one of the leading causes of blindness. Genetic factors play a critical role in the development of myopia, especially high myopia. Recently, the exome sequencing approach has been successfully used for the disease gene identification of Mendelian disorders. Here we show a successful application of exome sequencing to identify a gene for an autosomal dominant disorder, and we have identified a gene potentially responsible for high myopia in a monogenic form. We captured exomes of two affected individuals from a Han Chinese family with high myopia and performed sequencing analysis by a second-generation sequencer with a mean coverage of 30× and sufficient depth to call variants at ∼97% of each targeted exome. The shared genetic variants of these two affected individuals in the family being studied were filtered against the 1000 Genomes Project and the dbSNP131 database. A mutation A672G in zinc finger protein 644 isoform 1 (ZNF644 was identified as being related to the phenotype of this family. After we performed sequencing analysis of the exons in the ZNF644 gene in 300 sporadic cases of high myopia, we identified an additional five mutations (I587V, R680G, C699Y, 3'UTR+12 C>G, and 3'UTR+592 G>A in 11 different patients. All these mutations were absent in 600 normal controls. The ZNF644 gene was expressed in human retinal and retinal pigment epithelium (RPE. Given that ZNF644 is predicted to be a transcription factor that may regulate genes involved in eye development, mutation may cause the axial elongation of eyeball found in high myopia patients. Our results suggest that ZNF644 might be a causal gene for high myopia in a monogenic form.

  6. Probabilistic Methods for Processing High-Throughput Sequencing Signals

    DEFF Research Database (Denmark)

    Sørensen, Lasse Maretty

    High-throughput sequencing has the potential to answer many of the big questions in biology and medicine. It can be used to determine the ancestry of species, to chart complex ecosystems and to understand and diagnose disease. However, going from raw sequencing data to biological or medical insig....... By estimating the genotypes on a set of candidate variants obtained from both a standard mapping-based approach as well as de novo assemblies, we are able to find considerably more structural variation than previous studies...... for reconstructing transcript sequences from RNA sequencing data. The method is based on a novel sparse prior distribution over transcript abundances and is markedly more accurate than existing approaches. The second chapter describes a new method for calling genotypes from a fixed set of candidate variants....... The method queries the reads using a graph representation of the variants and hereby mitigates the reference-bias that characterise standard genotyping methods. In the last chapter, we apply this method to call the genotypes of 50 deeply sequencing parent-offspring trios from the GenomeDenmark project...

  7. The use of coded PCR primers enables high-throughput sequencing of multiple homolog amplification products by 454 parallel sequencing

    DEFF Research Database (Denmark)

    Binladen, Jonas; Gilbert, M Thomas P; Bollback, Jonathan P

    2007-01-01

    BACKGROUND: The invention of the Genome Sequence 20 DNA Sequencing System (454 parallel sequencing platform) has enabled the rapid and high-volume production of sequence data. Until now, however, individual emulsion PCR (emPCR) reactions and subsequent sequencing runs have been unable to combine...... primers that is dependent on the 5' nucleotide of the tag. In particular, primers 5' labelled with a cytosine are heavily overrepresented among the final sequences, while those 5' labelled with a thymine are strongly underrepresented. A weaker bias also exists with regards to the distribution...

  8. The use of coded PCR primers enables high-throughput sequencing of multiple homolog amplification products by 454 parallel sequencing.

    Directory of Open Access Journals (Sweden)

    Jonas Binladen

    2007-02-01

    Full Text Available The invention of the Genome Sequence 20 DNA Sequencing System (454 parallel sequencing platform has enabled the rapid and high-volume production of sequence data. Until now, however, individual emulsion PCR (emPCR reactions and subsequent sequencing runs have been unable to combine template DNA from multiple individuals, as homologous sequences cannot be subsequently assigned to their original sources.We use conventional PCR with 5'-nucleotide tagged primers to generate homologous DNA amplification products from multiple specimens, followed by sequencing through the high-throughput Genome Sequence 20 DNA Sequencing System (GS20, Roche/454 Life Sciences. Each DNA sequence is subsequently traced back to its individual source through 5'tag-analysis.We demonstrate that this new approach enables the assignment of virtually all the generated DNA sequences to the correct source once sequencing anomalies are accounted for (miss-assignment rate<0.4%. Therefore, the method enables accurate sequencing and assignment of homologous DNA sequences from multiple sources in single high-throughput GS20 run. We observe a bias in the distribution of the differently tagged primers that is dependent on the 5' nucleotide of the tag. In particular, primers 5' labelled with a cytosine are heavily overrepresented among the final sequences, while those 5' labelled with a thymine are strongly underrepresented. A weaker bias also exists with regards to the distribution of the sequences as sorted by the second nucleotide of the dinucleotide tags. As the results are based on a single GS20 run, the general applicability of the approach requires confirmation. However, our experiments demonstrate that 5'primer tagging is a useful method in which the sequencing power of the GS20 can be applied to PCR-based assays of multiple homologous PCR products. The new approach will be of value to a broad range of research areas, such as those of comparative genomics, complete mitochondrial

  9. Evolutionary growth process of highly conserved sequences in vertebrate genomes.

    Science.gov (United States)

    Ishibashi, Minaka; Noda, Akiko Ogura; Sakate, Ryuichi; Imanishi, Tadashi

    2012-08-01

    Genome sequence comparison between evolutionarily distant species revealed ultraconserved elements (UCEs) among mammals under strong purifying selection. Most of them were also conserved among vertebrates. Because they tend to be located in the flanking regions of developmental genes, they would have fundamental roles in creating vertebrate body plans. However, the evolutionary origin and selection mechanism of these UCEs remain unclear. Here we report that UCEs arose in primitive vertebrates, and gradually grew in vertebrate evolution. We searched for UCEs in two teleost fishes, Tetraodon nigroviridis and Oryzias latipes, and found 554 UCEs with 100% identity over 100 bps. Comparison of teleost and mammalian UCEs revealed 43 pairs of common, jawed-vertebrate UCEs (jUCE) with high sequence identities, ranging from 83.1% to 99.2%. Ten of them retain lower similarities to the Petromyzon marinus genome, and the substitution rates of four non-exonic jUCEs were reduced after the teleost-mammal divergence, suggesting that robust conservation had been acquired in the jawed vertebrate lineage. Our results indicate that prototypical UCEs originated before the divergence of jawed and jawless vertebrates and have been frozen as perfect conserved sequences in the jawed vertebrate lineage. In addition, our comparative sequence analyses of UCEs and neighboring regions resulted in a discovery of lineage-specific conserved sequences. They were added progressively to prototypical UCEs, suggesting step-wise acquisition of novel regulatory roles. Our results indicate that conserved non-coding elements (CNEs) consist of blocks with distinct evolutionary history, each having been frozen since different evolutionary era along the vertebrate lineage. Copyright © 2012 Elsevier B.V. All rights reserved.

  10. Using high-throughput barcode sequencing to efficiently map connectomes.

    Science.gov (United States)

    Peikon, Ian D; Kebschull, Justus M; Vagin, Vasily V; Ravens, Diana I; Sun, Yu-Chi; Brouzes, Eric; Corrêa, Ivan R; Bressan, Dario; Zador, Anthony M

    2017-07-07

    The function of a neural circuit is determined by the details of its synaptic connections. At present, the only available method for determining a neural wiring diagram with single synapse precision-a 'connectome'-is based on imaging methods that are slow, labor-intensive and expensive. Here, we present SYNseq, a method for converting the connectome into a form that can exploit the speed and low cost of modern high-throughput DNA sequencing. In SYNseq, each neuron is labeled with a unique random nucleotide sequence-an RNA 'barcode'-which is targeted to the synapse using engineered proteins. Barcodes in pre- and postsynaptic neurons are then associated through protein-protein crosslinking across the synapse, extracted from the tissue, and joined into a form suitable for sequencing. Although our failure to develop an efficient barcode joining scheme precludes the widespread application of this approach, we expect that with further development SYNseq will enable tracing of complex circuits at high speed and low cost. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  11. High Performance Systolic Array Core Architecture Design for DNA Sequencer

    Directory of Open Access Journals (Sweden)

    Saiful Nurdin Dayana

    2018-01-01

    Full Text Available This paper presents a high performance systolic array (SA core architecture design for Deoxyribonucleic Acid (DNA sequencer. The core implements the affine gap penalty score Smith-Waterman (SW algorithm. This time-consuming local alignment algorithm guarantees optimal alignment between DNA sequences, but it requires quadratic computation time when performed on standard desktop computers. The use of linear SA decreases the time complexity from quadratic to linear. In addition, with the exponential growth of DNA databases, the SA architecture is used to overcome the timing issue. In this work, the SW algorithm has been captured using Verilog Hardware Description Language (HDL and simulated using Xilinx ISIM simulator. The proposed design has been implemented in Xilinx Virtex -6 Field Programmable Gate Array (FPGA and improved in the core area by 90% reduction.

  12. Learning from the coffee shop: increasing junior high school students’ self-confidence through contextual learning based on local culture of Aceh

    Science.gov (United States)

    Sarmini; Supriono, A.; Ridwan

    2018-01-01

    Teachers should be able to provide meaningful learning, create a fun learning, and encourage the self-confidence of students. The reality is learning in Junior High School still teacher-centered learning that results the level of self-confidence of students is low. Pre-action showed 30% of students do not have self-confidence. The research aims to improve the self-confidence of students through contextual learning in the course from the social studies of Aceh based on the local culture. This type of research is classroom action research that conducted in two cycles. The research focus is the students’ responses. The coffee shop is a source of learning social studies. Students Involved in the coffee shop interact with villagers who have made the coffee shop as social media. Students participate meetings to address issues of rural villagers. The coffee shop as a public share with characteristics of particularly subject as a gathering place for many people regardless of social strata, convey information, chat, and informal atmosphere that stimulates self-confidence.

  13. High Throughput Sequencing for Detection of Foodborne Pathogens

    Directory of Open Access Journals (Sweden)

    Camilla Sekse

    2017-10-01

    Full Text Available High-throughput sequencing (HTS is becoming the state-of-the-art technology for typing of microbial isolates, especially in clinical samples. Yet, its application is still in its infancy for monitoring and outbreak investigations of foods. Here we review the published literature, covering not only bacterial but also viral and Eukaryote food pathogens, to assess the status and potential of HTS implementation to inform stakeholders, improve food safety and reduce outbreak impacts. The developments in sequencing technology and bioinformatics have outpaced the capacity to analyze and interpret the sequence data. The influence of sample processing, nucleic acid extraction and purification, harmonized protocols for generation and interpretation of data, and properly annotated and curated reference databases including non-pathogenic “natural” strains are other major obstacles to the realization of the full potential of HTS in analytical food surveillance, epidemiological and outbreak investigations, and in complementing preventive approaches for the control and management of foodborne pathogens. Despite significant obstacles, the achieved progress in capacity and broadening of the application range over the last decade is impressive and unprecedented, as illustrated with the chosen examples from the literature. Large consortia, often with broad international participation, are making coordinated efforts to cope with many of the mentioned obstacles. Further rapid progress can therefore be prospected for the next decade.

  14. Comparative transcriptome analysis within the Lolium/Festuca species complex reveals high sequence conservation

    DEFF Research Database (Denmark)

    Czaban, Adrian; Sharma, Sapna; Byrne, Stephen

    2015-01-01

    species from the Lolium-Festuca complex, ranging from 52,166 to 72,133 transcripts per assembly. We have also predicted a set of proteins and validated it with a high-confidence protein database from three closely related species (H. vulgare, B. distachyon and O. sativa). We have obtained gene family...... clusters for the four species using OrthoMCL and analyzed their inferred phylogenetic relationships. Our results indicate that VRN2 is a candidate gene for differentiating vernalization and non-vernalization types in the Lolium-Festuca complex. Grouping of the gene families based on their BLAST identity...... enabled us to divide ortholog groups into those that are very conserved and those that are more evolutionarily relaxed. The ratio of the non-synonumous to synonymous substitutions enabled us to pinpoint protein sequences evolving in response to positive selection. These proteins may explain some...

  15. Confidence in Numerical Simulations

    International Nuclear Information System (INIS)

    Hemez, Francois M.

    2015-01-01

    This PowerPoint presentation offers a high-level discussion of uncertainty, confidence and credibility in scientific Modeling and Simulation (M&S). It begins by briefly evoking M&S trends in computational physics and engineering. The first thrust of the discussion is to emphasize that the role of M&S in decision-making is either to support reasoning by similarity or to ''forecast,'' that is, make predictions about the future or extrapolate to settings or environments that cannot be tested experimentally. The second thrust is to explain that M&S-aided decision-making is an exercise in uncertainty management. The three broad classes of uncertainty in computational physics and engineering are variability and randomness, numerical uncertainty and model-form uncertainty. The last part of the discussion addresses how scientists ''think.'' This thought process parallels the scientific method where by a hypothesis is formulated, often accompanied by simplifying assumptions, then, physical experiments and numerical simulations are performed to confirm or reject the hypothesis. ''Confidence'' derives, not just from the levels of training and experience of analysts, but also from the rigor with which these assessments are performed, documented and peer-reviewed.

  16. Get your high-quality low-cost genome sequence

    NARCIS (Netherlands)

    Faino, L.; Thomma, B.P.H.J.

    2014-01-01

    The study of whole-genome sequences has become essential for almost all branches of biological research. Next-generation sequencing (NGS) has revolutionized the scalability, speed, and resolution of sequencing and brought genomic science within reach of academic laboratories that study non-model

  17. High dimensional and high resolution pulse sequences for backbone resonance assignment of intrinsically disordered proteins

    Energy Technology Data Exchange (ETDEWEB)

    Zawadzka-Kazimierczuk, Anna; Kozminski, Wiktor, E-mail: kozmin@chem.uw.edu.pl [University of Warsaw, Faculty of Chemistry (Poland); Sanderova, Hana; Krasny, Libor [Institute of Microbiology, Academy of Sciences of the Czech Republic, Laboratory of Molecular Genetics of Bacteria, Department of Bacteriology (Czech Republic)

    2012-04-15

    Four novel 5D (HACA(N)CONH, HNCOCACB, (HACA)CON(CA)CONH, (H)NCO(NCA)CONH), and one 6D ((H)NCO(N)CACONH) NMR pulse sequences are proposed. The new experiments employ non-uniform sampling that enables achieving high resolution in indirectly detected dimensions. The experiments facilitate resonance assignment of intrinsically disordered proteins. The novel pulse sequences were successfully tested using {delta} subunit (20 kDa) of Bacillus subtilis RNA polymerase that has an 81-amino acid disordered part containing various repetitive sequences.

  18. The Effect of High-Fidelity Cardiopulmonary Resuscitation (CPR) Simulation on Athletic Training Student Knowledge, Confidence, Emotions, and Experiences

    Science.gov (United States)

    Tivener, Kristin Ann; Gloe, Donna Sue

    2015-01-01

    Context: High-fidelity simulation is widely used in healthcare for the training and professional education of students though literature of its application to athletic training education remains sparse. Objective: This research attempts to address a wide-range of data. This includes athletic training student knowledge acquisition from…

  19. Impaired action self-monitoring and cognitive confidence among ultra-high risk for psychosis and first-episode psychosis patients.

    Science.gov (United States)

    Gawęda, Ł; Li, E; Lavoie, S; Whitford, T J; Moritz, S; Nelson, B

    2018-01-01

    Self-monitoring biases and overconfidence in incorrect judgments have been suggested as playing a role in schizophrenia spectrum disorders. Little is known about whether self-monitoring biases may contribute to early risk factors for psychosis. In this study, action self-monitoring (i.e., discrimination between imagined and performed actions) was investigated, along with confidence in judgments among ultra-high risk (UHR) for psychosis individuals and first-episode psychosis (FEP) patients. Thirty-six UHR for psychosis individuals, 25 FEP patients and 33 healthy controls (CON) participated in the study. Participants were assessed with the Action memory task. Simple actions were presented to participants verbally or non-verbally. Some actions were required to be physically performed and others were imagined. Participants were asked whether the action was presented verbally or non-verbally (action presentation type discrimination), and whether the action was performed or imagined (self-monitoring). Confidence self-ratings related to self-monitoring responses were obtained. The analysis of self-monitoring revealed that both UHR and FEP groups misattributed imagined actions as being performed (i.e., self-monitoring errors) significantly more often than the CON group. There were no differences regarding performed actions as being imagined. UHR and FEP groups made their false responses with higher confidence in their judgments than the CON group. There were no group differences regarding discrimination between the types of actions presented (verbal vs non-verbal). A specific type of self-monitoring bias (i.e., misattributing imagined actions with performed actions), accompanied by high confidence in this judgment, may be a risk factor for the subsequent development of a psychotic disorder. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  20. High confidence proteomic analysis of yeast LDs identifies additional droplet proteins and reveals connections to dolichol synthesis and sterol acetylation.

    Science.gov (United States)

    Currie, Erin; Guo, Xiuling; Christiano, Romain; Chitraju, Chandramohan; Kory, Nora; Harrison, Kenneth; Haas, Joel; Walther, Tobias C; Farese, Robert V

    2014-07-01

    Accurate protein inventories are essential for understanding an organelle's functions. The lipid droplet (LD) is a ubiquitous intracellular organelle with major functions in lipid storage and metabolism. LDs differ from other organelles because they are bounded by a surface monolayer, presenting unique features for protein targeting to LDs. Many proteins of varied functions have been found in purified LD fractions by proteomics. While these studies have become increasingly sensitive, it is often unclear which of the identified proteins are specific to LDs. Here we used protein correlation profiling to identify 35 proteins that specifically enrich with LD fractions of Saccharomyces cerevisiae Of these candidates, 30 fluorophore-tagged proteins localize to LDs by microscopy, including six proteins, several with human orthologs linked to diseases, which we newly identify as LD proteins (Cab5, Rer2, Say1, Tsc10, YKL047W, and YPR147C). Two of these proteins, Say1, a sterol deacetylase, and Rer2, a cis-isoprenyl transferase, are enzymes involved in sterol and polyprenol metabolism, respectively, and we show their activities are present in LD fractions. Our results provide a highly specific list of yeast LD proteins and reveal that the vast majority of these proteins are involved in lipid metabolism. Copyright © 2014 by the American Society for Biochemistry and Molecular Biology, Inc.

  1. Experimental design-based functional mining and characterization of high-throughput sequencing data in the sequence read archive.

    Directory of Open Access Journals (Sweden)

    Takeru Nakazato

    Full Text Available High-throughput sequencing technology, also called next-generation sequencing (NGS, has the potential to revolutionize the whole process of genome sequencing, transcriptomics, and epigenetics. Sequencing data is captured in a public primary data archive, the Sequence Read Archive (SRA. As of January 2013, data from more than 14,000 projects have been submitted to SRA, which is double that of the previous year. Researchers can download raw sequence data from SRA website to perform further analyses and to compare with their own data. However, it is extremely difficult to search entries and download raw sequences of interests with SRA because the data structure is complicated, and experimental conditions along with raw sequences are partly described in natural language. Additionally, some sequences are of inconsistent quality because anyone can submit sequencing data to SRA with no quality check. Therefore, as a criterion of data quality, we focused on SRA entries that were cited in journal articles. We extracted SRA IDs and PubMed IDs (PMIDs from SRA and full-text versions of journal articles and retrieved 2748 SRA ID-PMID pairs. We constructed a publication list referring to SRA entries. Since, one of the main themes of -omics analyses is clarification of disease mechanisms, we also characterized SRA entries by disease keywords, according to the Medical Subject Headings (MeSH extracted from articles assigned to each SRA entry. We obtained 989 SRA ID-MeSH disease term pairs, and constructed a disease list referring to SRA data. We previously developed feature profiles of diseases in a system called "Gendoo". We generated hyperlinks between diseases extracted from SRA and the feature profiles of it. The developed project, publication and disease lists resulting from this study are available at our web service, called "DBCLS SRA" (http://sra.dbcls.jp/. This service will improve accessibility to high-quality data from SRA.

  2. BOOGIE: Predicting Blood Groups from High Throughput Sequencing Data.

    Science.gov (United States)

    Giollo, Manuel; Minervini, Giovanni; Scalzotto, Marta; Leonardi, Emanuela; Ferrari, Carlo; Tosatto, Silvio C E

    2015-01-01

    Over the last decade, we have witnessed an incredible growth in the amount of available genotype data due to high throughput sequencing (HTS) techniques. This information may be used to predict phenotypes of medical relevance, and pave the way towards personalized medicine. Blood phenotypes (e.g. ABO and Rh) are a purely genetic trait that has been extensively studied for decades, with currently over thirty known blood groups. Given the public availability of blood group data, it is of interest to predict these phenotypes from HTS data which may translate into more accurate blood typing in clinical practice. Here we propose BOOGIE, a fast predictor for the inference of blood groups from single nucleotide variant (SNV) databases. We focus on the prediction of thirty blood groups ranging from the well known ABO and Rh, to the less studied Junior or Diego. BOOGIE correctly predicted the blood group with 94% accuracy for the Personal Genome Project whole genome profiles where good quality SNV annotation was available. Additionally, our tool produces a high quality haplotype phase, which is of interest in the context of ethnicity-specific polymorphisms or traits. The versatility and simplicity of the analysis make it easily interpretable and allow easy extension of the protocol towards other phenotypes. BOOGIE can be downloaded from URL http://protein.bio.unipd.it/download/.

  3. Evolution of sequence-defined highly functionalized nucleic acid polymers

    Science.gov (United States)

    Chen, Zhen; Lichtor, Phillip A.; Berliner, Adrian P.; Chen, Jonathan C.; Liu, David R.

    2018-03-01

    The evolution of sequence-defined synthetic polymers made of building blocks beyond those compatible with polymerase enzymes or the ribosome has the potential to generate new classes of receptors, catalysts and materials. Here we describe a ligase-mediated DNA-templated polymerization and in vitro selection system to evolve highly functionalized nucleic acid polymers (HFNAPs) made from 32 building blocks that contain eight chemically diverse side chains on a DNA backbone. Through iterated cycles of polymer translation, selection and reverse translation, we discovered HFNAPs that bind proprotein convertase subtilisin/kexin type 9 (PCSK9) and interleukin-6, two protein targets implicated in human diseases. Mutation and reselection of an active PCSK9-binding polymer yielded evolved polymers with high affinity (KD = 3 nM). This evolved polymer potently inhibited the binding between PCSK9 and the low-density lipoprotein receptor. Structure-activity relationship studies revealed that specific side chains at defined positions in the polymers are required for binding to their respective targets. Our findings expand the chemical space of evolvable polymers to include densely functionalized nucleic acids with diverse, researcher-defined chemical repertoires.

  4. Raising Confident Kids

    Science.gov (United States)

    ... First Aid & Safety Doctors & Hospitals Videos Recipes for Kids Kids site Sitio para niños How the Body ... Videos for Educators Search English Español Raising Confident Kids KidsHealth / For Parents / Raising Confident Kids What's in ...

  5. A high-confidence interaction map identifies SIRT1 as a mediator of acetylation of USP22 and the SAGA coactivator complex.

    Science.gov (United States)

    Armour, Sean M; Bennett, Eric J; Braun, Craig R; Zhang, Xiao-Yong; McMahon, Steven B; Gygi, Steven P; Harper, J Wade; Sinclair, David A

    2013-04-01

    Although many functions and targets have been attributed to the histone and protein deacetylase SIRT1, a comprehensive analysis of SIRT1 binding proteins yielding a high-confidence interaction map has not been established. Using a comparative statistical analysis of binding partners, we have assembled a high-confidence SIRT1 interactome. Employing this method, we identified the deubiquitinating enzyme ubiquitin-specific protease 22 (USP22), a component of the deubiquitinating module (DUBm) of the SAGA transcriptional coactivating complex, as a SIRT1-interacting partner. We found that this interaction is highly specific, requires the ZnF-UBP domain of USP22, and is disrupted by the inactivating H363Y mutation within SIRT1. Moreover, we show that USP22 is acetylated on multiple lysine residues and that alteration of a single lysine (K129) within the ZnF-UBP domain is sufficient to alter interaction of the DUBm with the core SAGA complex. Furthermore, USP22-mediated recruitment of SIRT1 activity promotes the deacetylation of individual SAGA complex components. Our results indicate an important role of SIRT1-mediated deacetylation in regulating the formation of DUBm subcomplexes within the larger SAGA complex.

  6. Evaluation of a pooled strategy for high-throughput sequencing of cosmid clones from metagenomic libraries.

    Science.gov (United States)

    Lam, Kathy N; Hall, Michael W; Engel, Katja; Vey, Gregory; Cheng, Jiujun; Neufeld, Josh D; Charles, Trevor C

    2014-01-01

    High-throughput sequencing methods have been instrumental in the growing field of metagenomics, with technological improvements enabling greater throughput at decreased costs. Nonetheless, the economy of high-throughput sequencing cannot be fully leveraged in the subdiscipline of functional metagenomics. In this area of research, environmental DNA is typically cloned to generate large-insert libraries from which individual clones are isolated, based on specific activities of interest. Sequence data are required for complete characterization of such clones, but the sequencing of a large set of clones requires individual barcode-based sample preparation; this can become costly, as the cost of clone barcoding scales linearly with the number of clones processed, and thus sequencing a large number of metagenomic clones often remains cost-prohibitive. We investigated a hybrid Sanger/Illumina pooled sequencing strategy that omits barcoding altogether, and we evaluated this strategy by comparing the pooled sequencing results to reference sequence data obtained from traditional barcode-based sequencing of the same set of clones. Using identity and coverage metrics in our evaluation, we show that pooled sequencing can generate high-quality sequence data, without producing problematic chimeras. Though caveats of a pooled strategy exist and further optimization of the method is required to improve recovery of complete clone sequences and to avoid circumstances that generate unrecoverable clone sequences, our results demonstrate that pooled sequencing represents an effective and low-cost alternative for sequencing large sets of metagenomic clones.

  7. High-Throughput DNA sequencing of ancient wood.

    Science.gov (United States)

    Wagner, Stefanie; Lagane, Frédéric; Seguin-Orlando, Andaine; Schubert, Mikkel; Leroy, Thibault; Guichoux, Erwan; Chancerel, Emilie; Bech-Hebelstrup, Inger; Bernard, Vincent; Billard, Cyrille; Billaud, Yves; Bolliger, Matthias; Croutsch, Christophe; Čufar, Katarina; Eynaud, Frédérique; Heussner, Karl Uwe; Köninger, Joachim; Langenegger, Fabien; Leroy, Frédéric; Lima, Christine; Martinelli, Nicoletta; Momber, Garry; Billamboz, André; Nelle, Oliver; Palomo, Antoni; Piqué, Raquel; Ramstein, Marianne; Schweichel, Roswitha; Stäuble, Harald; Tegel, Willy; Terradas, Xavier; Verdin, Florence; Plomion, Christophe; Kremer, Antoine; Orlando, Ludovic

    2018-03-01

    Reconstructing the colonization and demographic dynamics that gave rise to extant forests is essential to forecasts of forest responses to environmental changes. Classical approaches to map how population of trees changed through space and time largely rely on pollen distribution patterns, with only a limited number of studies exploiting DNA molecules preserved in wooden tree archaeological and subfossil remains. Here, we advance such analyses by applying high-throughput (HTS) DNA sequencing to wood archaeological and subfossil material for the first time, using a comprehensive sample of 167 European white oak waterlogged remains spanning a large temporal (from 550 to 9,800 years) and geographical range across Europe. The successful characterization of the endogenous DNA and exogenous microbial DNA of 140 (~83%) samples helped the identification of environmental conditions favouring long-term DNA preservation in wood remains, and started to unveil the first trends in the DNA decay process in wood material. Additionally, the maternally inherited chloroplast haplotypes of 21 samples from three periods of forest human-induced use (Neolithic, Bronze Age and Middle Ages) were found to be consistent with those of modern populations growing in the same geographic areas. Our work paves the way for further studies aiming at using ancient DNA preserved in wood to reconstruct the micro-evolutionary response of trees to climate change and human forest management. © 2018 John Wiley & Sons Ltd.

  8. Communicating the Benefits of a Full Sequence of High School Science Courses

    Science.gov (United States)

    Nicholas, Catherine Marie

    2014-01-01

    High school students are generally uninformed about the benefits of enrolling in a full sequence of science courses, therefore only about a third of our nation's high school graduates have completed the science sequence of Biology, Chemistry and Physics. The lack of students completing a full sequence of science courses contributes to the deficit…

  9. Capillary gel electrophoresis for rapid, high resolution DNA sequencing.

    OpenAIRE

    Swerdlow, H; Gesteland, R

    1990-01-01

    Capillary gel electrophoresis has been demonstrated for the separation and detection of DNA sequencing samples. Enzymatic dideoxy nucleotide chain termination was employed, using fluorescently tagged oligonucleotide primers and laser based on-column detection (limit of detection is 6,000 molecules per peak). Capillary gel separations were shown to be three times faster, with better resolution (2.4 x), and higher separation efficiency (5.4 x) than a conventional automated slab gel DNA sequenci...

  10. Confidence in Numerical Simulations

    Energy Technology Data Exchange (ETDEWEB)

    Hemez, Francois M. [Los Alamos National Lab. (LANL), Los Alamos, NM (United States)

    2015-02-23

    This PowerPoint presentation offers a high-level discussion of uncertainty, confidence and credibility in scientific Modeling and Simulation (M&S). It begins by briefly evoking M&S trends in computational physics and engineering. The first thrust of the discussion is to emphasize that the role of M&S in decision-making is either to support reasoning by similarity or to “forecast,” that is, make predictions about the future or extrapolate to settings or environments that cannot be tested experimentally. The second thrust is to explain that M&S-aided decision-making is an exercise in uncertainty management. The three broad classes of uncertainty in computational physics and engineering are variability and randomness, numerical uncertainty and model-form uncertainty. The last part of the discussion addresses how scientists “think.” This thought process parallels the scientific method where by a hypothesis is formulated, often accompanied by simplifying assumptions, then, physical experiments and numerical simulations are performed to confirm or reject the hypothesis. “Confidence” derives, not just from the levels of training and experience of analysts, but also from the rigor with which these assessments are performed, documented and peer-reviewed.

  11. Communicating Low-Probability High-Consequence Risk, Uncertainty and Expert Confidence: Induced Seismicity of Deep Geothermal Energy and Shale Gas.

    Science.gov (United States)

    Knoblauch, Theresa A K; Stauffacher, Michael; Trutnevyte, Evelina

    2018-04-01

    Subsurface energy activities entail the risk of induced seismicity including low-probability high-consequence (LPHC) events. For designing respective risk communication, the scientific literature lacks empirical evidence of how the public reacts to different written risk communication formats about such LPHC events and to related uncertainty or expert confidence. This study presents findings from an online experiment (N = 590) that empirically tested the public's responses to risk communication about induced seismicity and to different technology frames, namely deep geothermal energy (DGE) and shale gas (between-subject design). Three incrementally different formats of written risk communication were tested: (i) qualitative, (ii) qualitative and quantitative, and (iii) qualitative and quantitative with risk comparison. Respondents found the latter two the easiest to understand, the most exact, and liked them the most. Adding uncertainty and expert confidence statements made the risk communication less clear, less easy to understand and increased concern. Above all, the technology for which risks are communicated and its acceptance mattered strongly: respondents in the shale gas condition found the identical risk communication less trustworthy and more concerning than in the DGE conditions. They also liked the risk communication overall less. For practitioners in DGE or shale gas projects, the study shows that the public would appreciate efforts in describing LPHC risks with numbers and optionally risk comparisons. However, there seems to be a trade-off between aiming for transparency by disclosing uncertainty and limited expert confidence, and thereby decreasing clarity and increasing concern in the view of the public. © 2017 Society for Risk Analysis.

  12. Supervised detection of exoplanets in high-contrast imaging sequences

    Science.gov (United States)

    Gomez Gonzalez, C. A.; Absil, O.; Van Droogenbroeck, M.

    2018-06-01

    Context. Post-processing algorithms play a key role in pushing the detection limits of high-contrast imaging (HCI) instruments. State-of-the-art image processing approaches for HCI enable the production of science-ready images relying on unsupervised learning techniques, such as low-rank approximations, for generating a model point spread function (PSF) and subtracting the residual starlight and speckle noise. Aims: In order to maximize the detection rate of HCI instruments and survey campaigns, advanced algorithms with higher sensitivities to faint companions are needed, especially for the speckle-dominated innermost region of the images. Methods: We propose a reformulation of the exoplanet detection task (for ADI sequences) that builds on well-established machine learning techniques to take HCI post-processing from an unsupervised to a supervised learning context. In this new framework, we present algorithmic solutions using two different discriminative models: SODIRF (random forests) and SODINN (neural networks). We test these algorithms on real ADI datasets from VLT/NACO and VLT/SPHERE HCI instruments. We then assess their performances by injecting fake companions and using receiver operating characteristic analysis. This is done in comparison with state-of-the-art ADI algorithms, such as ADI principal component analysis (ADI-PCA). Results: This study shows the improved sensitivity versus specificity trade-off of the proposed supervised detection approach. At the diffraction limit, SODINN improves the true positive rate by a factor ranging from 2 to 10 (depending on the dataset and angular separation) with respect to ADI-PCA when working at the same false-positive level. Conclusions: The proposed supervised detection framework outperforms state-of-the-art techniques in the task of discriminating planet signal from speckles. In addition, it offers the possibility of re-processing existing HCI databases to maximize their scientific return and potentially improve

  13. High resolution clustering of Salmonella enterica serovar Montevideo strains using a next-generation sequencing approach

    Directory of Open Access Journals (Sweden)

    Allard Marc W

    2012-01-01

    Full Text Available Abstract Background Next-Generation Sequencing (NGS is increasingly being used as a molecular epidemiologic tool for discerning ancestry and traceback of the most complicated, difficult to resolve bacterial pathogens. Making a linkage between possible food sources and clinical isolates requires distinguishing the suspected pathogen from an environmental background and placing the variation observed into the wider context of variation occurring within a serovar and among other closely related foodborne pathogens. Equally important is the need to validate these high resolution molecular tools for use in molecular epidemiologic traceback. Such efforts include the examination of strain cluster stability as well as the cumulative genetic effects of sub-culturing on these clusters. Numerous isolates of S. Montevideo were shot-gun sequenced including diverse lineage representatives as well as numerous replicate clones to determine how much variability is due to bias, sequencing error, and or the culturing of isolates. All new draft genomes were compared to 34 S. Montevideo isolates previously published during an NGS-based molecular epidemiological case study. Results Intraserovar lineages of S. Montevideo differ by thousands of SNPs, that are only slightly less than the number of SNPs observed between S. Montevideo and other distinct serovars. Much less variability was discovered within an individual S. Montevideo clade implicated in a recent foodborne outbreak as well as among individual NGS replicates. These findings were similar to previous reports documenting homopolymeric and deletion error rates with the Roche 454 GS Titanium technology. In no case, however, did variability associated with sequencing methods or sample preparations create inconsistencies with our current phylogenetic results or the subsequent molecular epidemiological evidence gleaned from these data. Conclusions Implementation of a validated pipeline for NGS data acquisition and

  14. An improved high throughput sequencing method for studying oomycete communities

    DEFF Research Database (Denmark)

    Sapkota, Rumakanta; Nicolaisen, Mogens

    2015-01-01

    the usefulness of the method not only in soil DNA but also in a plant DNA background. In conclusion, we demonstrate a successful approach for pyrosequencing of oomycete communities using ITS1 as the barcode sequence with well-known primers for oomycete DNA amplification....... communities. Thewell-known primer sets ITS4, ITS6 and ITS7were used in the study in a semi-nested PCR approach to target the internal transcribed spacer (ITS) 1 of ribosomal DNA in a next generation sequencing protocol. These primers have been used in similar studies before, butwith limited success.......Wewere able to increase the proportion of retrieved oomycete sequences dramaticallymainly by increasing the annealing temperature during PCR. The optimized protocol was validated using three mock communities and the method was further evaluated using total DNA from 26 soil samples collected from different...

  15. Applications of High-Throughput Nucleotide Sequencing (PhD)

    DEFF Research Database (Denmark)

    Waage, Johannes

    equally large demands in data handling, analysis and interpretation, perhaps defining the modern challenge of the computational biologist of the post-genomic era. The first part of this thesis consists of a general introduction to the history, common terms and challenges of next generation sequencing......-sequencing, a study of the effects on alternative RNA splicing of KO of the nonsense mediated RNA decay system in Mus, using digital gene expression and a custom-built exon-exon junction mapping pipeline is presented (article I). Evolved from this work, a Bioconductor package, spliceR, for classifying alternative...

  16. Algorithms for mapping high-throughput DNA sequences

    DEFF Research Database (Denmark)

    Frellsen, Jes; Menzel, Peter; Krogh, Anders

    2014-01-01

    of data generation, new bioinformatics approaches have been developed to cope with the large amount of sequencing reads obtained in these experiments. In this chapter, we first introduce HTS technologies and their usage in molecular biology and discuss the problem of mapping sequencing reads...... to their genomic origin. We then in detail describe two approaches that offer very fast heuristics to solve the mapping problem in a feasible runtime. In particular, we describe the BLAT algorithm, and we give an introduction to the Burrows-Wheeler Transform and the mapping algorithms based on this transformation....

  17. The Model Confidence Set

    DEFF Research Database (Denmark)

    Hansen, Peter Reinhard; Lunde, Asger; Nason, James M.

    The paper introduces the model confidence set (MCS) and applies it to the selection of models. A MCS is a set of models that is constructed such that it will contain the best model with a given level of confidence. The MCS is in this sense analogous to a confidence interval for a parameter. The MCS......, beyond the comparison of models. We apply the MCS procedure to two empirical problems. First, we revisit the inflation forecasting problem posed by Stock and Watson (1999), and compute the MCS for their set of inflation forecasts. Second, we compare a number of Taylor rule regressions and determine...... the MCS of the best in terms of in-sample likelihood criteria....

  18. Confidence Building Strategies in the Public Schools.

    Science.gov (United States)

    Achilles, C. M.; And Others

    1985-01-01

    Data from the Phi Delta Kappa Commission on Public Confidence in Education indicate that "high-confidence" schools make greater use of marketing and public relations strategies. Teacher attitudes were ranked first and administrator attitudes second by 409 respondents for both gain and loss of confidence in schools. (MLF)

  19. Whole Genome Sequencing of Enterovirus species C Isolates by High-throughput Sequencing: Development of Generic Primers

    Directory of Open Access Journals (Sweden)

    Maël Bessaud

    2016-08-01

    Full Text Available Enteroviruses are among the most common viruses infecting humans and can cause diverse clinical syndromes ranging from minor febrile illness to severe and potentially fatal diseases. Enterovirus species C (EV-C consists of more than 20 types, among which the 3 serotypes of polioviruses, the etiological agents of poliomyelitis, are included. Biodiversity and evolution of EV-C genomes are shaped by frequent recombination events. Therefore, identification and characterization of circulating EV-C strains require the sequencing of different genomic regions.A simple method was developed to sequence quickly the entire genome of EV-C isolates. Four overlapping fragments were produced separately by RT-PCR performed with generic primers. The four amplicons were then pooled and purified prior to be sequenced by high-throughput technique.The method was assessed on a panel of EV-Cs belonging to a wide-range of types. It can be used to determine full-length genome sequences through de novo assembly of thousands of reads. It was also able to discriminate reads from closely related viruses in mixtures.By decreasing the workload compared to classical Sanger-based techniques, this method will serve as a precious tool for sequencing large panels of EV-Cs isolated in cell cultures during environmental surveillance or from patients, including vaccine-derived polioviruses.

  20. Tracking TCRβ sequence clonotype expansions during antiviral therapy using high-throughput sequencing of the hypervariable region

    Directory of Open Access Journals (Sweden)

    Mark W Robinson

    2016-04-01

    Full Text Available To maintain a persistent infection viruses such as hepatitis C virus (HCV employ a range of mechanisms that subvert protective T cell responses. The suppression of antigen-specific T cell responses by HCV hinders efforts to profile T cell responses during chronic infection and antiviral therapy. Conventional methods of detecting antigen-specific T cells utilise either antigen stimulation (e.g. ELISpot, proliferation assays, cytokine production or antigen-loaded tetramer staining. This limits the ability to profile T cell responses during chronic infection due to suppressed effector function and the requirement for prior knowledge of antigenic viral peptide sequences. Recently high-throughput sequencing (HTS technologies have been developed for the analysis of T cell repertoires. In the present study we have assessed the feasibility of HTS of the TCRβ complementarity determining region (CDR3 to track T cell expansions in an antigen-independent manner. Using sequential blood samples from HCV-infected individuals undergoing anti-viral therapy we were able to measure the population frequencies of >35,000 TCRβ sequence clonotypes in each individual over the course of 12 weeks. TRBV/TRBJ gene segment usage varied markedly between individuals but remained relatively constant within individuals across the course of therapy. Despite this stable TRBV/TRBJ gene segment usage, a number of TCRβ sequence clonotypes showed dramatic changes in read frequency. These changes could not be linked to therapy outcomes in the present study however the TCRβ CDR3 sequences with the largest fold changes did include sequences with identical TRBV/TRBJ gene segment usage and high joining region homology to previously published CDR3 sequences from HCV-specific T cells targeting the HLA-B*0801-restricted 1395HSKKKCDEL1403 and HLA-A*0101–restricted 1435ATDALMTGY1443 epitopes. The pipeline developed in this proof of concept study provides a platform for the design of

  1. Preparation of highly multiplexed small RNA sequencing libraries.

    Science.gov (United States)

    Persson, Helena; Søkilde, Rolf; Pirona, Anna Chiara; Rovira, Carlos

    2017-08-01

    MicroRNAs (miRNAs) are ~22-nucleotide-long small non-coding RNAs that regulate the expression of protein-coding genes by base pairing to partially complementary target sites, preferentially located in the 3´ untranslated region (UTR) of target mRNAs. The expression and function of miRNAs have been extensively studied in human disease, as well as the possibility of using these molecules as biomarkers for prognostication and treatment guidance. To identify and validate miRNAs as biomarkers, their expression must be screened in large collections of patient samples. Here, we develop a scalable protocol for the rapid and economical preparation of a large number of small RNA sequencing libraries using dual indexing for multiplexing. Combined with the use of off-the-shelf reagents, more samples can be sequenced simultaneously on large-scale sequencing platforms at a considerably lower cost per sample. Sample preparation is simplified by pooling libraries prior to gel purification, which allows for the selection of a narrow size range while minimizing sample variation. A comparison with publicly available data from benchmarking of miRNA analysis platforms showed that this method captures absolute and differential expression as effectively as commercially available alternatives.

  2. Parallel Sequencing of Expressed Sequence Tags from Two Complementary DNA Libraries for High and Low Phosphorus Adaptation in Common Beans

    Directory of Open Access Journals (Sweden)

    Matthew W. Blair

    2011-11-01

    Full Text Available Expressed sequence tags (ESTs have proven useful for gene discovery in many crops. In this work, our objective was to construct complementary DNA (cDNA libraries from root tissues of common beans ( L. grown under low and high P hydroponic conditions and to conduct EST sequencing and comparative analyses of the libraries. Expressed sequence tag analysis of 3648 clones identified 2372 unigenes, of which 1591 were annotated as known genes while a total of 465 unigenes were not associated with any known gene. Unigenes with hits were categorized according to biological processes, molecular function, and cellular compartmentalization. Given the young tissue used to make the root libraries, genes for catalytic activity and binding were highly expressed. Comparisons with previous root EST sequencing and between the two libraries made here resulted in a set of genes to study further for differential gene expression and adaptation to low P, such as a 14 kDa praline-rich protein, a metallopeptidase, tonoplast intrinsic protein, adenosine triphosphate (ATP citrate synthase, and cell proliferation genes expressed in the low P treated plants. Given that common beans are often grown on acid soils of the tropics and subtropics that are usually low in P these genes and the two parallel libraries will be useful for selection for better uptake of this essential macronutrient. The importance of EST generation for common bean root tissues under low P and other abiotic soil stresses is also discussed.

  3. 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.

  4. CSReport: A New Computational Tool Designed for Automatic Analysis of Class Switch Recombination Junctions Sequenced by High-Throughput Sequencing.

    Science.gov (United States)

    Boyer, François; Boutouil, Hend; Dalloul, Iman; Dalloul, Zeinab; Cook-Moreau, Jeanne; Aldigier, Jean-Claude; Carrion, Claire; Herve, Bastien; Scaon, Erwan; Cogné, Michel; Péron, Sophie

    2017-05-15

    B cells ensure humoral immune responses due to the production of Ag-specific memory B cells and Ab-secreting plasma cells. In secondary lymphoid organs, Ag-driven B cell activation induces terminal maturation and Ig isotype class switch (class switch recombination [CSR]). CSR creates a virtually unique IgH locus in every B cell clone by intrachromosomal recombination between two switch (S) regions upstream of each C region gene. Amount and structural features of CSR junctions reveal valuable information about the CSR mechanism, and analysis of CSR junctions is useful in basic and clinical research studies of B cell functions. To provide an automated tool able to analyze large data sets of CSR junction sequences produced by high-throughput sequencing (HTS), we designed CSReport, a software program dedicated to support analysis of CSR recombination junctions sequenced with a HTS-based protocol (Ion Torrent technology). CSReport was assessed using simulated data sets of CSR junctions and then used for analysis of Sμ-Sα and Sμ-Sγ1 junctions from CH12F3 cells and primary murine B cells, respectively. CSReport identifies junction segment breakpoints on reference sequences and junction structure (blunt-ended junctions or junctions with insertions or microhomology). Besides the ability to analyze unprecedentedly large libraries of junction sequences, CSReport will provide a unified framework for CSR junction studies. Our results show that CSReport is an accurate tool for analysis of sequences from our HTS-based protocol for CSR junctions, thereby facilitating and accelerating their study. Copyright © 2017 by The American Association of Immunologists, Inc.

  5. Transition Services: An Investigation of the Knowledge, Confidence, and Practice of Special Education Teachers in District of Columbia Public Charter High Schools

    Science.gov (United States)

    Henry, Wallace R., III

    2015-01-01

    This study was intended to enhance the limited research on the knowledge and confidence of special education teachers in public education regarding transition services and the quality of transition plans they develop. The key variables examined in this study are knowledge, confidence, and the quality of student transition plans. The sample…

  6. High-Throughput Sequencing Based Methods of RNA Structure Investigation

    DEFF Research Database (Denmark)

    Kielpinski, Lukasz Jan

    In this thesis we describe the development of four related methods for RNA structure probing that utilize massive parallel sequencing. Using them, we were able to gather structural data for multiple, long molecules simultaneously. First, we have established an easy to follow experimental...... and computational protocol for detecting the reverse transcription termination sites (RTTS-Seq). This protocol was subsequently applied to hydroxyl radical footprinting of three dimensional RNA structures to give a probing signal that correlates well with the RNA backbone solvent accessibility. Moreover, we applied...

  7. Targeted Capture and High-Throughput Sequencing Using Molecular Inversion Probes (MIPs).

    Science.gov (United States)

    Cantsilieris, Stuart; Stessman, Holly A; Shendure, Jay; Eichler, Evan E

    2017-01-01

    Molecular inversion probes (MIPs) in combination with massively parallel DNA sequencing represent a versatile, yet economical tool for targeted sequencing of genomic DNA. Several thousand genomic targets can be selectively captured using long oligonucleotides containing unique targeting arms and universal linkers. The ability to append sequencing adaptors and sample-specific barcodes allows large-scale pooling and subsequent high-throughput sequencing at relatively low cost per sample. Here, we describe a "wet bench" protocol detailing the capture and subsequent sequencing of >2000 genomic targets from 192 samples, representative of a single lane on the Illumina HiSeq 2000 platform.

  8. High-throughput sequencing of black pepper root transcriptome

    Science.gov (United States)

    2012-01-01

    Background Black pepper (Piper nigrum L.) is one of the most popular spices in the world. It is used in cooking and the preservation of food and even has medicinal properties. Losses in production from disease are a major limitation in the culture of this crop. The major diseases are root rot and foot rot, which are results of root infection by Fusarium solani and Phytophtora capsici, respectively. Understanding the molecular interaction between the pathogens and the host’s root region is important for obtaining resistant cultivars by biotechnological breeding. Genetic and molecular data for this species, though, are limited. In this paper, RNA-Seq technology has been employed, for the first time, to describe the root transcriptome of black pepper. Results The root transcriptome of black pepper was sequenced by the NGS SOLiD platform and assembled using the multiple-k method. Blast2Go and orthoMCL methods were used to annotate 10338 unigenes. The 4472 predicted proteins showed about 52% homology with the Arabidopsis proteome. Two root proteomes identified 615 proteins, which seem to define the plant’s root pattern. Simple-sequence repeats were identified that may be useful in studies of genetic diversity and may have applications in biotechnology and ecology. Conclusions This dataset of 10338 unigenes is crucially important for the biotechnological breeding of black pepper and the ecogenomics of the Magnoliids, a major group of basal angiosperms. PMID:22984782

  9. High-throughput sequencing of black pepper root transcriptome

    Directory of Open Access Journals (Sweden)

    Gordo Sheila MC

    2012-09-01

    Full Text Available Abstract Background Black pepper (Piper nigrum L. is one of the most popular spices in the world. It is used in cooking and the preservation of food and even has medicinal properties. Losses in production from disease are a major limitation in the culture of this crop. The major diseases are root rot and foot rot, which are results of root infection by Fusarium solani and Phytophtora capsici, respectively. Understanding the molecular interaction between the pathogens and the host’s root region is important for obtaining resistant cultivars by biotechnological breeding. Genetic and molecular data for this species, though, are limited. In this paper, RNA-Seq technology has been employed, for the first time, to describe the root transcriptome of black pepper. Results The root transcriptome of black pepper was sequenced by the NGS SOLiD platform and assembled using the multiple-k method. Blast2Go and orthoMCL methods were used to annotate 10338 unigenes. The 4472 predicted proteins showed about 52% homology with the Arabidopsis proteome. Two root proteomes identified 615 proteins, which seem to define the plant’s root pattern. Simple-sequence repeats were identified that may be useful in studies of genetic diversity and may have applications in biotechnology and ecology. Conclusions This dataset of 10338 unigenes is crucially important for the biotechnological breeding of black pepper and the ecogenomics of the Magnoliids, a major group of basal angiosperms.

  10. In-situ high resolution particle sampling by large time sequence inertial spectrometry

    International Nuclear Information System (INIS)

    Prodi, V.; Belosi, F.

    1990-09-01

    In situ sampling is always preferred, when possible, because of the artifacts that can arise when the aerosol has to flow through long sampling lines. On the other hand, the amount of possible losses can be calculated with some confidence only when the size distribution can be measured with a sufficient precision and the losses are not too large. This makes it desirable to sample directly in the vicinity of the aerosol source or containment. High temperature sampling devices with a detailed aerodynamic separation are extremely useful to this purpose. Several measurements are possible with the inertial spectrometer (INSPEC), but not with cascade impactors or cyclones. INSPEC - INertial SPECtrometer - has been conceived to measure the size distribution of aerosols by separating the particles while airborne according to their size and collecting them on a filter. It consists of a channel of rectangular cross-section with a 90 degree bend. Clean air is drawn through the channel, with a thin aerosol sheath injected close to the inner wall. Due to the bend, the particles are separated according to their size, leaving the original streamline by a distance which is a function of particle inertia and resistance, i.e. of aerodynamic diameter. The filter collects all the particles of the same aerodynamic size at the same distance from the inlet, in a continuous distribution. INSPEC particle separation at high temperature (up to 800 C) has been tested with Zirconia particles as calibration aerosols. The feasibility study has been concerned with resolution and time sequence sampling capabilities under high temperature (700 C)

  11. The Microsoft Biology Foundation Applications for High-Throughput Sequencing

    Science.gov (United States)

    Mercer, S.

    2010-01-01

    w9-2 The need for reusable libraries of bioinformatics functions has been recognized for many years and a number of language-specific toolkits have been constructed. Such toolkits have served as valuable nucleation points for the community, promoting the sharing of code and establishing standards. The majority of DNA sequencing machines and many other standard pieces of lab equipment are controlled by PCs using Windows, and a Microsoft genomics toolkit would enable initial processing and quality control to happen closer to the instrumentation and provide opportunities for added-value services within core facilities. The Microsoft Biology Foundation (MBF) is an open source software library, freely available for both commercial and academic use, available as an early-stage betafrom mbf.codeplex.com. This presentation will describe the structure and goals of MBF and demonstrate some of its uses.

  12. Sources of PCR-induced distortions in high-throughput sequencing data sets

    Science.gov (United States)

    Kebschull, Justus M.; Zador, Anthony M.

    2015-01-01

    PCR permits the exponential and sequence-specific amplification of DNA, even from minute starting quantities. PCR is a fundamental step in preparing DNA samples for high-throughput sequencing. However, there are errors associated with PCR-mediated amplification. Here we examine the effects of four important sources of error—bias, stochasticity, template switches and polymerase errors—on sequence representation in low-input next-generation sequencing libraries. We designed a pool of diverse PCR amplicons with a defined structure, and then used Illumina sequencing to search for signatures of each process. We further developed quantitative models for each process, and compared predictions of these models to our experimental data. We find that PCR stochasticity is the major force skewing sequence representation after amplification of a pool of unique DNA amplicons. Polymerase errors become very common in later cycles of PCR but have little impact on the overall sequence distribution as they are confined to small copy numbers. PCR template switches are rare and confined to low copy numbers. Our results provide a theoretical basis for removing distortions from high-throughput sequencing data. In addition, our findings on PCR stochasticity will have particular relevance to quantification of results from single cell sequencing, in which sequences are represented by only one or a few molecules. PMID:26187991

  13. Reclaim your creative confidence.

    Science.gov (United States)

    Kelley, Tom; Kelley, David

    2012-12-01

    Most people are born creative. But over time, a lot of us learn to stifle those impulses. We become warier of judgment, more cautious more analytical. The world seems to divide into "creatives" and "noncreatives," and too many people resign themselves to the latter category. And yet we know that creativity is essential to success in any discipline or industry. The good news, according to authors Tom Kelley and David Kelley of IDEO, is that we all can rediscover our creative confidence. The trick is to overcome the four big fears that hold most of us back: fear of the messy unknown, fear of judgment, fear of the first step, and fear of losing control. The authors use an approach based on the work of psychologist Albert Bandura in helping patients get over their snake phobias: You break challenges down into small steps and then build confidence by succeeding on one after another. Creativity is something you practice, say the authors, not just a talent you are born with.

  14. SUGAR: graphical user interface-based data refiner for high-throughput DNA sequencing.

    Science.gov (United States)

    Sato, Yukuto; Kojima, Kaname; Nariai, Naoki; Yamaguchi-Kabata, Yumi; Kawai, Yosuke; Takahashi, Mamoru; Mimori, Takahiro; Nagasaki, Masao

    2014-08-08

    Next-generation sequencers (NGSs) have become one of the main tools for current biology. To obtain useful insights from the NGS data, it is essential to control low-quality portions of the data affected by technical errors such as air bubbles in sequencing fluidics. We develop a software SUGAR (subtile-based GUI-assisted refiner) which can handle ultra-high-throughput data with user-friendly graphical user interface (GUI) and interactive analysis capability. The SUGAR generates high-resolution quality heatmaps of the flowcell, enabling users to find possible signals of technical errors during the sequencing. The sequencing data generated from the error-affected regions of a flowcell can be selectively removed by automated analysis or GUI-assisted operations implemented in the SUGAR. The automated data-cleaning function based on sequence read quality (Phred) scores was applied to a public whole human genome sequencing data and we proved the overall mapping quality was improved. The detailed data evaluation and cleaning enabled by SUGAR would reduce technical problems in sequence read mapping, improving subsequent variant analysis that require high-quality sequence data and mapping results. Therefore, the software will be especially useful to control the quality of variant calls to the low population cells, e.g., cancers, in a sample with technical errors of sequencing procedures.

  15. Multiple Teaching Approaches, Teaching Sequence and Concept Retention in High School Physics Education

    Science.gov (United States)

    Fogarty, Ian; Geelan, David

    2013-01-01

    Students in 4 Canadian high school physics classes completed instructional sequences in two key physics topics related to motion--Straight Line Motion and Newton's First Law. Different sequences of laboratory investigation, teacher explanation (lecture) and the use of computer-based scientific visualizations (animations and simulations) were…

  16. High-throughput sequencing of forensic genetic samples using punches of FTA cards with buccal swabs

    DEFF Research Database (Denmark)

    Kampmann, Marie-Louise; Buchard, Anders; Børsting, Claus

    2016-01-01

    Here, we demonstrate that punches from buccal swab samples preserved on FTA cards can be used for high-throughput DNA sequencing, also known as massively parallel sequencing (MPS). We typed 44 reference samples with the HID-Ion AmpliSeq Identity Panel using washed 1.2 mm punches from FTA cards...

  17. Alan Greenspan, the confidence strategy

    Directory of Open Access Journals (Sweden)

    Edwin Le Heron

    2006-12-01

    Full Text Available To evaluate the Greenspan era, we nevertheless need to address three questions: Is his success due to talent or just luck? Does he have a system of monetary policy or is he himself the system? What will be his legacy? Greenspan was certainly lucky, but he was also clairvoyant. Above all, he has developed a profoundly original monetary policy. His confidence strategy is clearly opposed to the credibility strategy developed in central banks and the academic milieu after 1980, but also inflation targeting, which today constitutes the mainstream monetary policy regime. The question of his legacy seems more nuanced. However, Greenspan will remain 'for a considerable period of time' a highly heterodox and original central banker. His political vision, his perception of an uncertain world, his pragmatism and his openness form the structure of a powerful alternative system, the confidence strategy, which will leave its mark on the history of monetary policy.

  18. Globalization of consumer confidence

    Directory of Open Access Journals (Sweden)

    Çelik Sadullah

    2017-01-01

    Full Text Available The globalization of world economies and the importance of nowcasting analysis have been at the core of the recent literature. Nevertheless, these two strands of research are hardly coupled. This study aims to fill this gap through examining the globalization of the consumer confidence index (CCI by applying conventional and unconventional econometric methods. The US CCI is used as the benchmark in tests of comovement among the CCIs of several developing and developed countries, with the data sets divided into three sub-periods: global liquidity abundance, the Great Recession, and postcrisis. The existence and/or degree of globalization of the CCIs vary according to the period, whereas globalization in the form of coherence and similar paths is observed only during the Great Recession and, surprisingly, stronger in developing/emerging countries.

  19. We will be champions: Leaders' confidence in 'us' inspires team members' team confidence and performance.

    Science.gov (United States)

    Fransen, K; Steffens, N K; Haslam, S A; Vanbeselaere, N; Vande Broek, G; Boen, F

    2016-12-01

    The present research examines the impact of leaders' confidence in their team on the team confidence and performance of their teammates. In an experiment involving newly assembled soccer teams, we manipulated the team confidence expressed by the team leader (high vs neutral vs low) and assessed team members' responses and performance as they unfolded during a competition (i.e., in a first baseline session and a second test session). Our findings pointed to team confidence contagion such that when the leader had expressed high (rather than neutral or low) team confidence, team members perceived their team to be more efficacious and were more confident in the team's ability to win. Moreover, leaders' team confidence affected individual and team performance such that teams led by a highly confident leader performed better than those led by a less confident leader. Finally, the results supported a hypothesized mediational model in showing that the effect of leaders' confidence on team members' team confidence and performance was mediated by the leader's perceived identity leadership and members' team identification. In conclusion, the findings of this experiment suggest that leaders' team confidence can enhance members' team confidence and performance by fostering members' identification with the team. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  20. Subfamily logos: visualization of sequence deviations at alignment positions with high information content

    Directory of Open Access Journals (Sweden)

    Beitz Eric

    2006-06-01

    Full Text Available Abstract Background Recognition of relevant sequence deviations can be valuable for elucidating functional differences between protein subfamilies. Interesting residues at highly conserved positions can then be mutated and experimentally analyzed. However, identification of such sites is tedious because automated approaches are scarce. Results Subfamily logos visualize subfamily-specific sequence deviations. The display is similar to classical sequence logos but extends into the negative range. Positive, upright characters correspond to residues which are characteristic for the subfamily, negative, upside-down characters to residues typical for the remaining sequences. The symbol height is adjusted to the information content of the alignment position. Residues which are conserved throughout do not appear. Conclusion Subfamily logos provide an intuitive display of relevant sequence deviations. The method has proven to be valid using a set of 135 aligned aquaporin sequences in which established subfamily-specific positions were readily identified by the algorithm.

  1. Picking Funds with Confidence

    DEFF Research Database (Denmark)

    Grønborg, Niels Strange; Lunde, Asger; Timmermann, Allan

    We present a new approach to selecting active mutual funds that uses both holdings and return information to eliminate funds with predicted inferior performance through a sequence of pair-wise comparisons. Our methodology determines both the number of skilled funds and their identity, funds...... identified ex-ante as being superior earn substantially higher risk-adjusted returns than top funds identified by conventional alpha ranking methods. Importantly, we find strong evidence of variation in the breadth of the set of funds identified as superior, as well as fluctuations in the style and industry...... exposures of such funds over time and across different volatility states....

  2. Highly conserved non-coding sequences are associated with vertebrate development.

    Directory of Open Access Journals (Sweden)

    Adam Woolfe

    2005-01-01

    Full Text Available In addition to protein coding sequence, the human genome contains a significant amount of regulatory DNA, the identification of which is proving somewhat recalcitrant to both in silico and functional methods. An approach that has been used with some success is comparative sequence analysis, whereby equivalent genomic regions from different organisms are compared in order to identify both similarities and differences. In general, similarities in sequence between highly divergent organisms imply functional constraint. We have used a whole-genome comparison between humans and the pufferfish, Fugu rubripes, to identify nearly 1,400 highly conserved non-coding sequences. Given the evolutionary divergence between these species, it is likely that these sequences are found in, and furthermore are essential to, all vertebrates. Most, and possibly all, of these sequences are located in and around genes that act as developmental regulators. Some of these sequences are over 90% identical across more than 500 bases, being more highly conserved than coding sequence between these two species. Despite this, we cannot find any similar sequences in invertebrate genomes. In order to begin to functionally test this set of sequences, we have used a rapid in vivo assay system using zebrafish embryos that allows tissue-specific enhancer activity to be identified. Functional data is presented for highly conserved non-coding sequences associated with four unrelated developmental regulators (SOX21, PAX6, HLXB9, and SHH, in order to demonstrate the suitability of this screen to a wide range of genes and expression patterns. Of 25 sequence elements tested around these four genes, 23 show significant enhancer activity in one or more tissues. We have identified a set of non-coding sequences that are highly conserved throughout vertebrates. They are found in clusters across the human genome, principally around genes that are implicated in the regulation of development

  3. ISRNA: an integrative online toolkit for short reads from high-throughput sequencing data.

    Science.gov (United States)

    Luo, Guan-Zheng; Yang, Wei; Ma, Ying-Ke; Wang, Xiu-Jie

    2014-02-01

    Integrative Short Reads NAvigator (ISRNA) is an online toolkit for analyzing high-throughput small RNA sequencing data. Besides the high-speed genome mapping function, ISRNA provides statistics for genomic location, length distribution and nucleotide composition bias analysis of sequence reads. Number of reads mapped to known microRNAs and other classes of short non-coding RNAs, coverage of short reads on genes, expression abundance of sequence reads as well as some other analysis functions are also supported. The versatile search functions enable users to select sequence reads according to their sub-sequences, expression abundance, genomic location, relationship to genes, etc. A specialized genome browser is integrated to visualize the genomic distribution of short reads. ISRNA also supports management and comparison among multiple datasets. ISRNA is implemented in Java/C++/Perl/MySQL and can be freely accessed at http://omicslab.genetics.ac.cn/ISRNA/.

  4. Applications of high-throughput sequencing to chromatin structure and function in mammals

    OpenAIRE

    Dunham, Ian

    2009-01-01

    High-throughput DNA sequencing approaches have enabled direct interrogation of chromatin samples from mammalian cells. We are beginning to develop a genome-wide description of nuclear function during development, but further data collection, refinement, and integration are needed.

  5. Continuous- and Discrete-Time Stimulus Sequences for High Stimulus Rate Paradigm in Evoked Potential Studies

    Directory of Open Access Journals (Sweden)

    Tao Wang

    2013-01-01

    Full Text Available To obtain reliable transient auditory evoked potentials (AEPs from EEGs recorded using high stimulus rate (HSR paradigm, it is critical to design the stimulus sequences of appropriate frequency properties. Traditionally, the individual stimulus events in a stimulus sequence occur only at discrete time points dependent on the sampling frequency of the recording system and the duration of stimulus sequence. This dependency likely causes the implementation of suboptimal stimulus sequences, sacrificing the reliability of resulting AEPs. In this paper, we explicate the use of continuous-time stimulus sequence for HSR paradigm, which is independent of the discrete electroencephalogram (EEG recording system. We employ simulation studies to examine the applicability of the continuous-time stimulus sequences and the impacts of sampling frequency on AEPs in traditional studies using discrete-time design. Results from these studies show that the continuous-time sequences can offer better frequency properties and improve the reliability of recovered AEPs. Furthermore, we find that the errors in the recovered AEPs depend critically on the sampling frequencies of experimental systems, and their relationship can be fitted using a reciprocal function. As such, our study contributes to the literature by demonstrating the applicability and advantages of continuous-time stimulus sequences for HSR paradigm and by revealing the relationship between the reliability of AEPs and sampling frequencies of the experimental systems when discrete-time stimulus sequences are used in traditional manner for the HSR paradigm.

  6. Centroid based clustering of high throughput sequencing reads based on n-mer counts.

    Science.gov (United States)

    Solovyov, Alexander; Lipkin, W Ian

    2013-09-08

    Many problems in computational biology require alignment-free sequence comparisons. One of the common tasks involving sequence comparison is sequence clustering. Here we apply methods of alignment-free comparison (in particular, comparison using sequence composition) to the challenge of sequence clustering. We study several centroid based algorithms for clustering sequences based on word counts. Study of their performance shows that using k-means algorithm with or without the data whitening is efficient from the computational point of view. A higher clustering accuracy can be achieved using the soft expectation maximization method, whereby each sequence is attributed to each cluster with a specific probability. We implement an open source tool for alignment-free clustering. It is publicly available from github: https://github.com/luscinius/afcluster. We show the utility of alignment-free sequence clustering for high throughput sequencing analysis despite its limitations. In particular, it allows one to perform assembly with reduced resources and a minimal loss of quality. The major factor affecting performance of alignment-free read clustering is the length of the read.

  7. Application of high-throughput sequencing in understanding human oral microbiome related with health and disease

    OpenAIRE

    Chen, Hui; Jiang, Wen

    2014-01-01

    The oral microbiome is one of most diversity habitat in the human body and they are closely related with oral health and disease. As the technique developing,, high throughput sequencing has become a popular approach applied for oral microbial analysis. Oral bacterial profiles have been studied to explore the relationship between microbial diversity and oral diseases such as caries and periodontal disease. This review describes the application of high-throughput sequencing for characterizati...

  8. Highly parallel translation of DNA sequences into small molecules.

    Directory of Open Access Journals (Sweden)

    Rebecca M Weisinger

    Full Text Available A large body of in vitro evolution work establishes the utility of biopolymer libraries comprising 10(10 to 10(15 distinct molecules for the discovery of nanomolar-affinity ligands to proteins. Small-molecule libraries of comparable complexity will likely provide nanomolar-affinity small-molecule ligands. Unlike biopolymers, small molecules can offer the advantages of cell permeability, low immunogenicity, metabolic stability, rapid diffusion and inexpensive mass production. It is thought that such desirable in vivo behavior is correlated with the physical properties of small molecules, specifically a limited number of hydrogen bond donors and acceptors, a defined range of hydrophobicity, and most importantly, molecular weights less than 500 Daltons. Creating a collection of 10(10 to 10(15 small molecules that meet these criteria requires the use of hundreds to thousands of diversity elements per step in a combinatorial synthesis of three to five steps. With this goal in mind, we have reported a set of mesofluidic devices that enable DNA-programmed combinatorial chemistry in a highly parallel 384-well plate format. Here, we demonstrate that these devices can translate DNA genes encoding 384 diversity elements per coding position into corresponding small-molecule gene products. This robust and efficient procedure yields small molecule-DNA conjugates suitable for in vitro evolution experiments.

  9. Regional Competition for Confidence: Features of Formation

    Directory of Open Access Journals (Sweden)

    Irina Svyatoslavovna Vazhenina

    2016-09-01

    Full Text Available The increase in economic independence of the regions inevitably leads to an increase in the quality requirements of the regional economic policy. The key to successful regional policy, both during its development and implementation, is the understanding of the necessity of gaining confidence (at all levels, and the inevitable participation in the competition for confidence. The importance of confidence in the region is determined by its value as a competitive advantage in the struggle for partners, resources and tourists, and attracting investments. In today’s environment the focus of governments, regions and companies on long-term cooperation is clearly expressed, which is impossible without a high level of confidence between partners. Therefore, the most important competitive advantages of territories are intangible assets such as an attractive image and a good reputation, which builds up confidence of the population and partners. The higher the confidence in the region is, the broader is the range of potential partners, the larger is the planning horizon of long-term concerted action, the better are the chances of acquiring investment, the higher is the level of competitive immunity of the territories. The article defines competition for confidence as purposeful behavior of a market participant in economic environment, aimed at acquiring specific intangible competitive advantage – the confidence of the largest possible number of other market actors. The article also highlights the specifics of confidence as a competitive goal, presents factors contributing to the destruction of confidence, proposes a strategy to fight for confidence as a program of four steps, considers the factors which integrate regional confidence and offers several recommendations for the establishment of effective regional competition for confidence

  10. Towards confidence in transport safety

    International Nuclear Information System (INIS)

    Robison, R.W.

    1992-01-01

    The U.S. Department of Energy (US DOE) plans to demonstrate to the public that high-level waste can be transported safely to the proposed repository. The author argues US DOE should begin now to demonstrate its commitment to safety by developing an extraordinary safety program for nuclear cargo it is now shipping. The program for current shipments should be developed with State, Tribal, and local officials. Social scientists should be involved in evaluating the effect of the safety program on public confidence. The safety program developed in cooperation with western states for shipments to the Waste Isolation Pilot plant is a good basis for designing that extraordinary safety program

  11. Highly accurate sequence imputation enables precise QTL mapping in Brown Swiss cattle.

    Science.gov (United States)

    Frischknecht, Mirjam; Pausch, Hubert; Bapst, Beat; Signer-Hasler, Heidi; Flury, Christine; Garrick, Dorian; Stricker, Christian; Fries, Ruedi; Gredler-Grandl, Birgit

    2017-12-29

    Within the last few years a large amount of genomic information has become available in cattle. Densities of genomic information vary from a few thousand variants up to whole genome sequence information. In order to combine genomic information from different sources and infer genotypes for a common set of variants, genotype imputation is required. In this study we evaluated the accuracy of imputation from high density chips to whole genome sequence data in Brown Swiss cattle. Using four popular imputation programs (Beagle, FImpute, Impute2, Minimac) and various compositions of reference panels, the accuracy of the imputed sequence variant genotypes was high and differences between the programs and scenarios were small. We imputed sequence variant genotypes for more than 1600 Brown Swiss bulls and performed genome-wide association studies for milk fat percentage at two stages of lactation. We found one and three quantitative trait loci for early and late lactation fat content, respectively. Known causal variants that were imputed from the sequenced reference panel were among the most significantly associated variants of the genome-wide association study. Our study demonstrates that whole-genome sequence information can be imputed at high accuracy in cattle populations. Using imputed sequence variant genotypes in genome-wide association studies may facilitate causal variant detection.

  12. SINA: accurate high-throughput multiple sequence alignment of ribosomal RNA genes.

    Science.gov (United States)

    Pruesse, Elmar; Peplies, Jörg; Glöckner, Frank Oliver

    2012-07-15

    In the analysis of homologous sequences, computation of multiple sequence alignments (MSAs) has become a bottleneck. This is especially troublesome for marker genes like the ribosomal RNA (rRNA) where already millions of sequences are publicly available and individual studies can easily produce hundreds of thousands of new sequences. Methods have been developed to cope with such numbers, but further improvements are needed to meet accuracy requirements. In this study, we present the SILVA Incremental Aligner (SINA) used to align the rRNA gene databases provided by the SILVA ribosomal RNA project. SINA uses a combination of k-mer searching and partial order alignment (POA) to maintain very high alignment accuracy while satisfying high throughput performance demands. SINA was evaluated in comparison with the commonly used high throughput MSA programs PyNAST and mothur. The three BRAliBase III benchmark MSAs could be reproduced with 99.3, 97.6 and 96.1 accuracy. A larger benchmark MSA comprising 38 772 sequences could be reproduced with 98.9 and 99.3% accuracy using reference MSAs comprising 1000 and 5000 sequences. SINA was able to achieve higher accuracy than PyNAST and mothur in all performed benchmarks. Alignment of up to 500 sequences using the latest SILVA SSU/LSU Ref datasets as reference MSA is offered at http://www.arb-silva.de/aligner. This page also links to Linux binaries, user manual and tutorial. SINA is made available under a personal use license.

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

    Directory of Open Access Journals (Sweden)

    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.

  14. Highly divergent 16S rRNA sequences in ribosomal operons of Scytonema hyalinum (Cyanobacteria.

    Directory of Open Access Journals (Sweden)

    Jeffrey R Johansen

    Full Text Available A highly divergent 16S rRNA gene was found in one of the five ribosomal operons present in a species complex currently circumscribed as Scytonema hyalinum (Nostocales, Cyanobacteria using clone libraries. If 16S rRNA sequence macroheterogeneity among ribosomal operons due to insertions, deletions or truncation is excluded, the sequence heterogeneity observed in S. hyalinum was the highest observed in any prokaryotic species thus far (7.3-9.0%. The secondary structure of the 16S rRNA molecules encoded by the two divergent operons was nearly identical, indicating possible functionality. The 23S rRNA gene was examined for a few strains in this complex, and it was also found to be highly divergent from the gene in Type 2 operons (8.7%, and likewise had nearly identical secondary structure between the Type 1 and Type 2 operons. Furthermore, the 16S-23S ITS showed marked differences consistent between operons among numerous strains. Both operons have promoter sequences that satisfy consensus requirements for functional prokaryotic transcription initiation. Horizontal gene transfer from another unknown heterocytous cyanobacterium is considered the most likely explanation for the origin of this molecule, but does not explain the ultimate origin of this sequence, which is very divergent from all 16S rRNA sequences found thus far in cyanobacteria. The divergent sequence is highly conserved among numerous strains of S. hyalinum, suggesting adaptive advantage and selective constraint of the divergent sequence.

  15. Consumer confidence or the business cycle

    DEFF Research Database (Denmark)

    Møller, Stig Vinther; Nørholm, Henrik; Rangvid, Jesper

    2014-01-01

    Answer: The business cycle. We show that consumer confidence and the output gap both excess returns on stocks in many European countries: When the output gap is positive (the economy is doing well), expected returns are low, and when consumer confidence is high, expected returns are also low...

  16. Fast high-resolution MR imaging using the snapshot-FLASH MR sequence

    International Nuclear Information System (INIS)

    Matthaei, D.; Haase, A.; Henrich, D.; Duhmke, E.

    1990-01-01

    Snapshot, fast low-angle short (FLASH) MR imaging using an accelerated FLASH-MR sequence provides MR images with measuring times far below 1 second. The short TE of this sequence prevents susceptibility artifacts in gradient-echo imaging. In this paper variations of the sequence are shown that provide high resolution images with T1-weighted IR, T2-weighted SE, and chemical shift (CHESS) contrast sequences. METHODS AND MATERIALS: A whole-body 2-T system (Bruker-Medizintechnik) were used in combination with a 60-cm gradient system (providing gradient strength of 5 mT/m) to study healthy volunteers. The measuring time for a 256 x 256 image matrix was 800 msec. This sequence has been used in combination with T1-weighted IR, T2-weighted SE, and CHESS variations

  17. High-Throughput Mapping of Single-Neuron Projections by Sequencing of Barcoded RNA.

    Science.gov (United States)

    Kebschull, Justus M; Garcia da Silva, Pedro; Reid, Ashlan P; Peikon, Ian D; Albeanu, Dinu F; Zador, Anthony M

    2016-09-07

    Neurons transmit information to distant brain regions via long-range axonal projections. In the mouse, area-to-area connections have only been systematically mapped using bulk labeling techniques, which obscure the diverse projections of intermingled single neurons. Here we describe MAPseq (Multiplexed Analysis of Projections by Sequencing), a technique that can map the projections of thousands or even millions of single neurons by labeling large sets of neurons with random RNA sequences ("barcodes"). Axons are filled with barcode mRNA, each putative projection area is dissected, and the barcode mRNA is extracted and sequenced. Applying MAPseq to the locus coeruleus (LC), we find that individual LC neurons have preferred cortical targets. By recasting neuroanatomy, which is traditionally viewed as a problem of microscopy, as a problem of sequencing, MAPseq harnesses advances in sequencing technology to permit high-throughput interrogation of brain circuits. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. OB/GYN boot cAMP using high-fidelity human simulators: enhancing residents' perceived competency, confidence in taking a leadership role, and stress hardiness.

    Science.gov (United States)

    Pliego, Jose F; Wehbe-Janek, Hania; Rajab, M Hasan; Browning, Jeff L; Fothergill, Russell E

    2008-01-01

    To evaluate the effectiveness of an obstetrical and gynecologic (Ob/Gyn) Boot Camp simulation training on perceived technical competency, confidence in a leadership role, and stress hardiness of resident training. We conducted a prospective pilot study on the effectiveness of an Ob/Gyn Boot Camp on resident training. Residents participated in an intensive immersion in clinical simulation of common obstetrical emergencies including shoulder dystocia, neonatal resuscitation, postpartum hemorrhage, and ruptured ectopic pregnancy. After the training, residents completed a Web-based survey on their perceptions of how the Ob/Gyn Boot Camp affected their 1) technical competency in the assessment and management of their patients, 2) confidence in taking a leadership role, and 3) stress hardiness. Residents rated their perceptions on a Likert scale of 1 to 5, 1 = poor to 5 = excellent. Twenty-three (14 Ob/Gyn and 9 family medicine) residents participated in this pilot study. Eighteen (78%) residents completed the online survey; 4 Ob/Gyn and 1 family medicine resident did not complete the survey. The residents reported that the simulation training stimulated an interest in learning key skills for obstetrical and gynecologic emergencies. Ob/Gyn residents reported significant improvement in their perceived technical competence and stress hardiness after the Boot Camp. However both Ob/Gyn and family medicine residents reported no significant improvement of confidence in their leadership abilities during obstetrical emergencies after the Boot Camp. Boot Camp simulation training early in the curriculum has the potential for enhancing residents' self-assessments of confidence, competency, and stress hardiness in managing obstetrical emergencies.

  19. High-throughput sequencing of core STR loci for forensic genetic investigations using the Roche Genome Sequencer FLX platform

    DEFF Research Database (Denmark)

    Fordyce, Sarah Louise; Avila Arcos, Maria del Carmen; Rockenbauer, Eszter

    2011-01-01

    repeat units. These methods do not allow for the full resolution of STR base composition that sequencing approaches could provide. Here we present an STR profiling method based on the use of the Roche Genome Sequencer (GS) FLX to simultaneously sequence multiple core STR loci. Using this method...

  20. Fostering English Learners' Confidence

    Science.gov (United States)

    Bondie, Rhonda; Gaughran, Laurie; Zusho, Akane

    2014-01-01

    A teacher is doing something right when his high school students--kids with limited English, no less--form groups and begin discussing a lesson on quadratic equations at the start of class, without any teacher direction. Bondie, Gaughran, and Zusho describe "discussion routines" that teachers at International Community High School in the…

  1. High-throughput sequencing of forensic genetic samples using punches of FTA cards with buccal swabs.

    Science.gov (United States)

    Kampmann, Marie-Louise; Buchard, Anders; Børsting, Claus; Morling, Niels

    2016-01-01

    Here, we demonstrate that punches from buccal swab samples preserved on FTA cards can be used for high-throughput DNA sequencing, also known as massively parallel sequencing (MPS). We typed 44 reference samples with the HID-Ion AmpliSeq Identity Panel using washed 1.2 mm punches from FTA cards with buccal swabs and compared the results with those obtained with DNA extracted using the EZ1 DNA Investigator Kit. Concordant profiles were obtained for all samples. Our protocol includes simple punch, wash, and PCR steps, reducing cost and hands-on time in the laboratory. Furthermore, it facilitates automation of DNA sequencing.

  2. High signals in the uterine cervix on T2-weighted MRI sequences

    International Nuclear Information System (INIS)

    Graef, De M.; Karam, R.; Daclin, P.Y.; Rouanet, J.P.; Juhan, V.; Maubon, A.J.

    2003-01-01

    The aim of this pictorial review was to illustrate the normal cervix appearance on T2-weighted images, and give a review of common or less common disorders of the uterine cervix that appear as high signal intensity lesions on T2-weighted sequences. Numerous aetiologies dominated by cervical cancer are reviewed and discussed. This gamut is obviously incomplete; however, radiologists who perform MR women's imaging should perform T2-weighted sequences in the sagittal plane regardless of the indication for pelvic MR. Those sequences will diagnose some previously unknown cervical cancers as well as many other unknown cervical or uterine lesions. (orig.)

  3. Targeted DNA Methylation Analysis by High Throughput Sequencing in Porcine Peri-attachment Embryos

    OpenAIRE

    MORRILL, Benson H.; COX, Lindsay; WARD, Anika; HEYWOOD, Sierra; PRATHER, Randall S.; ISOM, S. Clay

    2013-01-01

    Abstract The purpose of this experiment was to implement and evaluate the effectiveness of a next-generation sequencing-based method for DNA methylation analysis in porcine embryonic samples. Fourteen discrete genomic regions were amplified by PCR using bisulfite-converted genomic DNA derived from day 14 in vivo-derived (IVV) and parthenogenetic (PA) porcine embryos as template DNA. Resulting PCR products were subjected to high-throughput sequencing using the Illumina Genome Analyzer IIx plat...

  4. Retirement Sequences of Older Americans: Moderately Destandardized and Highly Stratified Across Gender, Class, and Race.

    Science.gov (United States)

    Calvo, Esteban; Madero-Cabib, Ignacio; Staudinger, Ursula M

    2017-06-06

    A destandardization of labor-force patterns revolving around retirement has been observed in recent literature. It is unclear, however, to which degree and of which kind. This study looked at sequences rather than individual statuses or transitions and argued that differentiating older Americans' retirement sequences by type, order, and timing and considering gender, class, and race differences yields a less destandardized picture. Sequence analysis was employed to analyze panel data from the Health and Retirement Study (HRS) for 7,881 individuals observed 6 consecutive times between ages 60-61 and 70-71. As expected, types of retirement sequences were identified that cannot be subsumed under the conventional model of complete retirement from full-time employment around age 65. However, these retirement sequences were not entirely destandardized, as some irreversibility and age-grading persisted. Further, the degree of destandardization varied along gender, class, and race. Unconventional sequences were archetypal for middle-level educated individuals and Blacks. Also, sequences for women and individuals with lower education showed more unemployment and part-time jobs, and less age-grading. A sequence-analytic approach that models group differences uncovers misjudgments about the degree of destandardization of retirement sequences. When a continuous process is represented as individual transitions, the overall pattern of retirement sequences gets lost and appears destandardized. These patterns get further complicated by differences in social structures by gender, class, and race in ways that seem to reproduce advantages that men, more highly educated individuals, and Whites enjoy in numerous areas over the life course. © The Author 2017. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  5. High depth, whole-genome sequencing of cholera isolates from Haiti and the Dominican Republic.

    Science.gov (United States)

    Sealfon, Rachel; Gire, Stephen; Ellis, Crystal; Calderwood, Stephen; Qadri, Firdausi; Hensley, Lisa; Kellis, Manolis; Ryan, Edward T; LaRocque, Regina C; Harris, Jason B; Sabeti, Pardis C

    2012-09-11

    Whole-genome sequencing is an important tool for understanding microbial evolution and identifying the emergence of functionally important variants over the course of epidemics. In October 2010, a severe cholera epidemic began in Haiti, with additional cases identified in the neighboring Dominican Republic. We used whole-genome approaches to sequence four Vibrio cholerae isolates from Haiti and the Dominican Republic and three additional V. cholerae isolates to a high depth of coverage (>2000x); four of the seven isolates were previously sequenced. Using these sequence data, we examined the effect of depth of coverage and sequencing platform on genome assembly and identification of sequence variants. We found that 50x coverage is sufficient to construct a whole-genome assembly and to accurately call most variants from 100 base pair paired-end sequencing reads. Phylogenetic analysis between the newly sequenced and thirty-three previously sequenced V. cholerae isolates indicates that the Haitian and Dominican Republic isolates are closest to strains from South Asia. The Haitian and Dominican Republic isolates form a tight cluster, with only four variants unique to individual isolates. These variants are located in the CTX region, the SXT region, and the core genome. Of the 126 mutations identified that separate the Haiti-Dominican Republic cluster from the V. cholerae reference strain (N16961), 73 are non-synonymous changes, and a number of these changes cluster in specific genes and pathways. Sequence variant analyses of V. cholerae isolates, including multiple isolates from the Haitian outbreak, identify coverage-specific and technology-specific effects on variant detection, and provide insight into genomic change and functional evolution during an epidemic.

  6. High depth, whole-genome sequencing of cholera isolates from Haiti and the Dominican Republic

    Directory of Open Access Journals (Sweden)

    Sealfon Rachel

    2012-09-01

    Full Text Available Abstract Background Whole-genome sequencing is an important tool for understanding microbial evolution and identifying the emergence of functionally important variants over the course of epidemics. In October 2010, a severe cholera epidemic began in Haiti, with additional cases identified in the neighboring Dominican Republic. We used whole-genome approaches to sequence four Vibrio cholerae isolates from Haiti and the Dominican Republic and three additional V. cholerae isolates to a high depth of coverage (>2000x; four of the seven isolates were previously sequenced. Results Using these sequence data, we examined the effect of depth of coverage and sequencing platform on genome assembly and identification of sequence variants. We found that 50x coverage is sufficient to construct a whole-genome assembly and to accurately call most variants from 100 base pair paired-end sequencing reads. Phylogenetic analysis between the newly sequenced and thirty-three previously sequenced V. cholerae isolates indicates that the Haitian and Dominican Republic isolates are closest to strains from South Asia. The Haitian and Dominican Republic isolates form a tight cluster, with only four variants unique to individual isolates. These variants are located in the CTX region, the SXT region, and the core genome. Of the 126 mutations identified that separate the Haiti-Dominican Republic cluster from the V. cholerae reference strain (N16961, 73 are non-synonymous changes, and a number of these changes cluster in specific genes and pathways. Conclusions Sequence variant analyses of V. cholerae isolates, including multiple isolates from the Haitian outbreak, identify coverage-specific and technology-specific effects on variant detection, and provide insight into genomic change and functional evolution during an epidemic.

  7. The specificity of memory for a highly trained finger movement sequence: Change the ending, change all.

    Science.gov (United States)

    Rozanov, Simon; Keren, Ofer; Karni, Avi

    2010-05-17

    How are highly trained movement sequences represented in long-term memory? Here we show that the gains attained in the performance of a well-trained sequence of finger movements can be expressed only when the order of the movements is exactly as practiced. Ten young adults were trained to perform a given 5-element sequence of finger-to-thumb opposition movements with their left hand. Movements were analyzed using video based tracking. Three weeks of training resulted, along with improved accuracy, in robustly shortened movement times as well as shorter finger-to-thumb touch times. However, there was little transfer of these gains in speed to the execution of the same component movements arranged in a new order. Moreover, even when the only change was the omission of the one before final movement of the trained sequence (Omit sequence), the initial movements of the sequence were significantly slowed down, although these movements were identical to the initial movements of the trained sequence. Our results support the notion that a well-trained sequence of finger movements can be represented, in the adult motor system, as a singular, co-articulated, unit of movement, in which even the initial component movements are contingent on the subsequent, anticipated, ones. Because of co-articulation related anticipatory effects, gains in fluency and accuracy acquired in training on a specific movement sequence cannot be expressed in full in the execution of the trained component movements or of a full segment of the trained sequence, if followed by a different ending segment. Copyright 2010. Published by Elsevier B.V.

  8. 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.

  9. The application of the high throughput sequencing technology in the transposable elements.

    Science.gov (United States)

    Liu, Zhen; Xu, Jian-hong

    2015-09-01

    High throughput sequencing technology has dramatically improved the efficiency of DNA sequencing, and decreased the costs to a great extent. Meanwhile, this technology usually has advantages of better specificity, higher sensitivity and accuracy. Therefore, it has been applied to the research on genetic variations, transcriptomics and epigenomics. Recently, this technology has been widely employed in the studies of transposable elements and has achieved fruitful results. In this review, we summarize the application of high throughput sequencing technology in the fields of transposable elements, including the estimation of transposon content, preference of target sites and distribution, insertion polymorphism and population frequency, identification of rare copies, transposon horizontal transfers as well as transposon tagging. We also briefly introduce the major common sequencing strategies and algorithms, their advantages and disadvantages, and the corresponding solutions. Finally, we envision the developing trends of high throughput sequencing technology, especially the third generation sequencing technology, and its application in transposon studies in the future, hopefully providing a comprehensive understanding and reference for related scientific researchers.

  10. Salmonella enterica Prophage Sequence Profiles Reflect Genome Diversity and Can Be Used for High Discrimination Subtyping

    Directory of Open Access Journals (Sweden)

    Walid Mottawea

    2018-05-01

    Full Text Available Non-typhoidal Salmonella is a leading cause of foodborne illness worldwide. Prompt and accurate identification of the sources of Salmonella responsible for disease outbreaks is crucial to minimize infections and eliminate ongoing sources of contamination. Current subtyping tools including single nucleotide polymorphism (SNP typing may be inadequate, in some instances, to provide the required discrimination among epidemiologically unrelated Salmonella strains. Prophage genes represent the majority of the accessory genes in bacteria genomes and have potential to be used as high discrimination markers in Salmonella. In this study, the prophage sequence diversity in different Salmonella serovars and genetically related strains was investigated. Using whole genome sequences of 1,760 isolates of S. enterica representing 151 Salmonella serovars and 66 closely related bacteria, prophage sequences were identified from assembled contigs using PHASTER. We detected 154 different prophages in S. enterica genomes. Prophage sequences were highly variable among S. enterica serovars with a median ± interquartile range (IQR of 5 ± 3 prophage regions per genome. While some prophage sequences were highly conserved among the strains of specific serovars, few regions were lineage specific. Therefore, strains belonging to each serovar could be clustered separately based on their prophage content. Analysis of S. Enteritidis isolates from seven outbreaks generated distinct prophage profiles for each outbreak. Taken altogether, the diversity of the prophage sequences correlates with genome diversity. Prophage repertoires provide an additional marker for differentiating S. enterica subtypes during foodborne outbreaks.

  11. Exome sequencing generates high quality data in non-target regions

    Directory of Open Access Journals (Sweden)

    Guo Yan

    2012-05-01

    Full Text Available Abstract Background Exome sequencing using next-generation sequencing technologies is a cost efficient approach to selectively sequencing coding regions of human genome for detection of disease variants. A significant amount of DNA fragments from the capture process fall outside target regions, and sequence data for positions outside target regions have been mostly ignored after alignment. Result We performed whole exome sequencing on 22 subjects using Agilent SureSelect capture reagent and 6 subjects using Illumina TrueSeq capture reagent. We also downloaded sequencing data for 6 subjects from the 1000 Genomes Project Pilot 3 study. Using these data, we examined the quality of SNPs detected outside target regions by computing consistency rate with genotypes obtained from SNP chips or the Hapmap database, transition-transversion (Ti/Tv ratio, and percentage of SNPs inside dbSNP. For all three platforms, we obtained high-quality SNPs outside target regions, and some far from target regions. In our Agilent SureSelect data, we obtained 84,049 high-quality SNPs outside target regions compared to 65,231 SNPs inside target regions (a 129% increase. For our Illumina TrueSeq data, we obtained 222,171 high-quality SNPs outside target regions compared to 95,818 SNPs inside target regions (a 232% increase. For the data from the 1000 Genomes Project, we obtained 7,139 high-quality SNPs outside target regions compared to 1,548 SNPs inside target regions (a 461% increase. Conclusions These results demonstrate that a significant amount of high quality genotypes outside target regions can be obtained from exome sequencing data. These data should not be ignored in genetic epidemiology studies.

  12. Chemistry, the Central Science? The History of the High School Science Sequence

    Science.gov (United States)

    Sheppard, Keith; Robbins, Dennis M.

    2005-01-01

    Chemistry became the ''central science'' not by design but by accident in the US high schools. The three important factors, which had their influence on the high school science, are sequenced and their impact on the development of US science education, are mentioned.

  13. Workshop on confidence limits. Proceedings

    International Nuclear Information System (INIS)

    James, F.; Lyons, L.; Perrin, Y.

    2000-01-01

    The First Workshop on Confidence Limits was held at CERN on 17-18 January 2000. It was devoted to the problem of setting confidence limits in difficult cases: number of observed events is small or zero, background is larger than signal, background not well known, and measurements near a physical boundary. Among the many examples in high-energy physics are searches for the Higgs, searches for neutrino oscillations, B s mixing, SUSY, compositeness, neutrino masses, and dark matter. Several different methods are on the market: the CL s methods used by the LEP Higgs searches; Bayesian methods; Feldman-Cousins and modifications thereof; empirical and combined methods. The Workshop generated considerable interest, and attendance was finally limited by the seating capacity of the CERN Council Chamber where all the sessions took place. These proceedings contain all the papers presented, as well as the full text of the discussions after each paper and of course the last session which was a discussion session. The list of participants and the 'required reading', which was expected to be part of the prior knowledge of all participants, are also included. (orig.)

  14. Implication of the cause of differences in 3D structures of proteins with high sequence identity based on analyses of amino acid sequences and 3D structures.

    Science.gov (United States)

    Matsuoka, Masanari; Sugita, Masatake; Kikuchi, Takeshi

    2014-09-18

    Proteins that share a high sequence homology while exhibiting drastically different 3D structures are investigated in this study. Recently, artificial proteins related to the sequences of the GA and IgG binding GB domains of human serum albumin have been designed. These artificial proteins, referred to as GA and GB, share 98% amino acid sequence identity but exhibit different 3D structures, namely, a 3α bundle versus a 4β + α structure. Discriminating between their 3D structures based on their amino acid sequences is a very difficult problem. In the present work, in addition to using bioinformatics techniques, an analysis based on inter-residue average distance statistics is used to address this problem. It was hard to distinguish which structure a given sequence would take only with the results of ordinary analyses like BLAST and conservation analyses. However, in addition to these analyses, with the analysis based on the inter-residue average distance statistics and our sequence tendency analysis, we could infer which part would play an important role in its structural formation. The results suggest possible determinants of the different 3D structures for sequences with high sequence identity. The possibility of discriminating between the 3D structures based on the given sequences is also discussed.

  15. On the optimal trimming of high-throughput mRNA sequence data

    Directory of Open Access Journals (Sweden)

    Matthew D MacManes

    2014-01-01

    Full Text Available The widespread and rapid adoption of high-throughput sequencing technologies has afforded researchers the opportunity to gain a deep understanding of genome level processes that underlie evolutionary change, and perhaps more importantly, the links between genotype and phenotype. In particular, researchers interested in functional biology and adaptation have used these technologies to sequence mRNA transcriptomes of specific tissues, which in turn are often compared to other tissues, or other individuals with different phenotypes. While these techniques are extremely powerful, careful attention to data quality is required. In particular, because high-throughput sequencing is more error-prone than traditional Sanger sequencing, quality trimming of sequence reads should be an important step in all data processing pipelines. While several software packages for quality trimming exist, no general guidelines for the specifics of trimming have been developed. Here, using empirically derived sequence data, I provide general recommendations regarding the optimal strength of trimming, specifically in mRNA-Seq studies. Although very aggressive quality trimming is common, this study suggests that a more gentle trimming, specifically of those nucleotides whose Phred score < 2 or < 5, is optimal for most studies across a wide variety of metrics.

  16. The Pinus taeda genome is characterized by diverse and highly diverged repetitive sequences

    Directory of Open Access Journals (Sweden)

    Yandell Mark

    2010-07-01

    Full Text Available Abstract Background In today's age of genomic discovery, no attempt has been made to comprehensively sequence a gymnosperm genome. The largest genus in the coniferous family Pinaceae is Pinus, whose 110-120 species have extremely large genomes (c. 20-40 Gb, 2N = 24. The size and complexity of these genomes have prompted much speculation as to the feasibility of completing a conifer genome sequence. Conifer genomes are reputed to be highly repetitive, but there is little information available on the nature and identity of repetitive units in gymnosperms. The pines have extensive genetic resources, with approximately 329000 ESTs from eleven species and genetic maps in eight species, including a dense genetic map of the twelve linkage groups in Pinus taeda. Results We present here the Sanger sequence and annotation of ten P. taeda BAC clones and Genome Analyzer II whole genome shotgun (WGS sequences representing 7.5% of the genome. Computational annotation of ten BACs predicts three putative protein-coding genes and at least fifteen likely pseudogenes in nearly one megabase of sequence. We found three conifer-specific LTR retroelements in the BACs, and tentatively identified at least 15 others based on evidence from the distantly related angiosperms. Alignment of WGS sequences to the BACs indicates that 80% of BAC sequences have similar copies (≥ 75% nucleotide identity elsewhere in the genome, but only 23% have identical copies (99% identity. The three most common repetitive elements in the genome were identified and, when combined, represent less than 5% of the genome. Conclusions This study indicates that the majority of repeats in the P. taeda genome are 'novel' and will therefore require additional BAC or genomic sequencing for accurate characterization. The pine genome contains a very large number of diverged and probably defunct repetitive elements. This study also provides new evidence that sequencing a pine genome using a WGS approach is

  17. Automated and high confidence protein phosphorylation site localization using complementary collision-activated dissociation and electron transfer dissociation tandem mass spectrometry

    DEFF Research Database (Denmark)

    Hansen, Thomas A; Sylvester, Marc; Jensen, Ole N

    2012-01-01

    -site localization and the number of assigned phospho-sites at a fixed false-localization rate. The average calculated Cscore from a large data set (>7000 phosphopeptide MS/MS spectra) was ∼32 compared to ∼23 and ∼17 for the Ascore using collision-activated dissociation (CAD) or electron transfer dissociation (ETD...... peptide fragmentation and the loss of labile phosphate groups complicate identification of the site of the phosphate motif. Here, we have implemented and evaluated a novel approach for phospho-site localization by the combined use of peptide tandem mass spectrometry data obtained using both collision......-activated dissociation and electron transfer dissociation, an approach termed the Cscore. The scoring algorithm used in the Cscore was adapted from the widely used Ascore method. The analytical benefit of integrating the product ion information of both ETD and CAD data are evident by increased confidence in phospho...

  18. A robust, simple genotyping-by-sequencing (GBS approach for high diversity species.

    Directory of Open Access Journals (Sweden)

    Robert J Elshire

    Full Text Available Advances in next generation technologies have driven the costs of DNA sequencing down to the point that genotyping-by-sequencing (GBS is now feasible for high diversity, large genome species. Here, we report a procedure for constructing GBS libraries based on reducing genome complexity with restriction enzymes (REs. This approach is simple, quick, extremely specific, highly reproducible, and may reach important regions of the genome that are inaccessible to sequence capture approaches. By using methylation-sensitive REs, repetitive regions of genomes can be avoided and lower copy regions targeted with two to three fold higher efficiency. This tremendously simplifies computationally challenging alignment problems in species with high levels of genetic diversity. The GBS procedure is demonstrated with maize (IBM and barley (Oregon Wolfe Barley recombinant inbred populations where roughly 200,000 and 25,000 sequence tags were mapped, respectively. An advantage in species like barley that lack a complete genome sequence is that a reference map need only be developed around the restriction sites, and this can be done in the process of sample genotyping. In such cases, the consensus of the read clusters across the sequence tagged sites becomes the reference. Alternatively, for kinship analyses in the absence of a reference genome, the sequence tags can simply be treated as dominant markers. Future application of GBS to breeding, conservation, and global species and population surveys may allow plant breeders to conduct genomic selection on a novel germplasm or species without first having to develop any prior molecular tools, or conservation biologists to determine population structure without prior knowledge of the genome or diversity in the species.

  19. Communicating the Benefits of a Full Sequence of High School Science Courses

    Science.gov (United States)

    Nicholas, Catherine Marie

    High school students are generally uninformed about the benefits of enrolling in a full sequence of science courses, therefore only about a third of our nation's high school graduates have completed the science sequence of Biology, Chemistry and Physics. The lack of students completing a full sequence of science courses contributes to the deficit in the STEM degree production rate needed to fill the demand of the current job market and remain competitive as a nation. The purpose of the study was to make a difference in the number of students who have access to information about the benefits of completing a full sequence of science courses. This dissertation study employed qualitative research methodology to gain a broad perspective of staff through a questionnaire and document review and then a deeper understanding through semi-structured interview protocol. The data revealed that a universal sequence of science courses in the high school district did not exist. It also showed that not all students had access to all science courses; students were sorted and tracked according to prerequisites that did not necessarily match the skill set needed for the courses. In addition, the study showed a desire for more support and direction from the district office. It was also apparent that there was a disconnect that existed between who staff members believed should enroll in a full sequence of science courses and who actually enrolled. Finally, communication about science was shown to occur mainly through counseling and peers. A common science sequence, detracking of science courses, increased communication about the postsecondary and academic benefits of a science education, increased district direction and realistic mathematics alignment were all discussed as solutions to the problem.

  20. Use of high flip angle in T1-prepared FAST sequences for myocardial perfusion quantification

    International Nuclear Information System (INIS)

    Vallee, Jean-Paul; Ivancevic, Marko; Lazeyras, Francois; Didier, Dominique; Kasuboski, Larry; Chatelain, Pascal; Righetti, Alberto

    2003-01-01

    This study reports on the first use of high flip angle and radio-frequency (RF) spoiling in T1-prepared fast acquisition in steady state (FAST) sequence for myocardial perfusion in patients. T1 dynamic range was measured in vitro with a FAST, an RF FAST and a snapshot fast low-angle shot (FLASH) sequences with a 90 flip angle. Myocardial perfusion was then measured twice in 6 patients during the same MR session. The RF FAST and FLASH, but not the FAST sequence, demonstrated an extended T1 dynamic range; however, the FLASH images were degraded by artifacts not present on the RF FAST images. The myocardial perfusion indices K1 (first-order transfer constant from the blood to the myocardium for the Gd-DTPA) and Vd (distribution volume of Gd-DTPA in myocardium) did not differ significantly between the two injections. K1 was 0.48±0.12 ml/min g -1 and Vd was 12.5±2.9%. With an extended T1 dynamic range and the sensitivity required for myocardial perfusion quantification, the RF FAST sequence with a 90 flip angle outperformed the snapshot FLASH sequence in terms of image quality and the FAST sequence in terms of contrast dynamic range. (orig.)

  1. Detection of genomic variation by selection of a 9 mb DNA region and high throughput sequencing.

    Directory of Open Access Journals (Sweden)

    Sergey I Nikolaev

    Full Text Available Detection of the rare polymorphisms and causative mutations of genetic diseases in a targeted genomic area has become a major goal in order to understand genomic and phenotypic variability. We have interrogated repeat-masked regions of 8.9 Mb on human chromosomes 21 (7.8 Mb and 7 (1.1 Mb from an individual from the International HapMap Project (NA12872. We have optimized a method of genomic selection for high throughput sequencing. Microarray-based selection and sequencing resulted in 260-fold enrichment, with 41% of reads mapping to the target region. 83% of SNPs in the targeted region had at least 4-fold sequence coverage and 54% at least 15-fold. When assaying HapMap SNPs in NA12872, our sequence genotypes are 91.3% concordant in regions with coverage > or = 4-fold, and 97.9% concordant in regions with coverage > or = 15-fold. About 81% of the SNPs recovered with both thresholds are listed in dbSNP. We observed that regions with low sequence coverage occur in close proximity to low-complexity DNA. Validation experiments using Sanger sequencing were performed for 46 SNPs with 15-20 fold coverage, with a confirmation rate of 96%, suggesting that DNA selection provides an accurate and cost-effective method for identifying rare genomic variants.

  2. Functional role of a highly repetitive DNA sequence in anchorage of the mouse genome.

    Science.gov (United States)

    Neuer-Nitsche, B; Lu, X N; Werner, D

    1988-09-12

    The major portion of the eukaryotic genome consists of various categories of repetitive DNA sequences which have been studied with respect to their base compositions, organizations, copy numbers, transcription and species specificities; their biological roles, however, are still unclear. A novel quality of a highly repetitive mouse DNA sequence is described which points to a functional role: All copies (approximately 50,000 per haploid genome) of this DNA sequence reside on genomic Alu I DNA fragments each associated with nuclear polypeptides that are not released from DNA by proteinase K, SDS and phenol extraction. By this quality the repetitive DNA sequence is classified as a member of the sub-set of DNA sequences involved in tight DNA-polypeptide complexes which have been previously shown to be components of the subnuclear structure termed 'nuclear matrix'. From these results it has to be concluded that the repetitive DNA sequence characterized in this report represents or comprises a signal for a large number of site specific attachment points of the mouse genome in the nuclear matrix.

  3. Sequence of a cDNA encoding turtle high mobility group 1 protein.

    Science.gov (United States)

    Zheng, Jifang; Hu, Bi; Wu, Duansheng

    2005-07-01

    In order to understand sequence information about turtle HMG1 gene, a cDNA encoding HMG1 protein of the Chinese soft-shell turtle (Pelodiscus sinensis) was amplified by RT-PCR from kidney total RNA, and was cloned, sequenced and analyzed. The results revealed that the open reading frame (ORF) of turtle HMG1 cDNA is 606 bp long. The ORF codifies 202 amino acid residues, from which two DNA-binding domains and one polyacidic region are derived. The DNA-binding domains share higher amino acid identity with homologues sequences of chicken (96.5%) and mammalian (74%) than homologues sequence of rainbow trout (67%). The polyacidic region shows 84.6% amino acid homology with the equivalent region of chicken HMG1 cDNA. Turtle HMG1 protein contains 3 Cys residues located at completely conserved positions. Conservation in sequence and structure suggests that the functions of turtle HMG1 cDNA may be highly conserved during evolution. To our knowledge, this is the first report of HMG1 cDNA sequence in any reptilian.

  4. 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.

  5. Accurate RNA consensus sequencing for high-fidelity detection of transcriptional mutagenesis-induced epimutations.

    Science.gov (United States)

    Reid-Bayliss, Kate S; Loeb, Lawrence A

    2017-08-29

    Transcriptional mutagenesis (TM) due to misincorporation during RNA transcription can result in mutant RNAs, or epimutations, that generate proteins with altered properties. TM has long been hypothesized to play a role in aging, cancer, and viral and bacterial evolution. However, inadequate methodologies have limited progress in elucidating a causal association. We present a high-throughput, highly accurate RNA sequencing method to measure epimutations with single-molecule sensitivity. Accurate RNA consensus sequencing (ARC-seq) uniquely combines RNA barcoding and generation of multiple cDNA copies per RNA molecule to eliminate errors introduced during cDNA synthesis, PCR, and sequencing. The stringency of ARC-seq can be scaled to accommodate the quality of input RNAs. We apply ARC-seq to directly assess transcriptome-wide epimutations resulting from RNA polymerase mutants and oxidative stress.

  6. Assessing the Diversity of Rodent-Borne Viruses: Exploring of High-Throughput Sequencing and Classical Amplification/Sequencing Approaches.

    Science.gov (United States)

    Drewes, Stephan; Straková, Petra; Drexler, Jan F; Jacob, Jens; Ulrich, Rainer G

    2017-01-01

    Rodents are distributed throughout the world and interact with humans in many ways. They provide vital ecosystem services, some species are useful models in biomedical research and some are held as pet animals. However, many rodent species can have adverse effects such as damage to crops and stored produce, and they are of health concern because of the transmission of pathogens to humans and livestock. The first rodent viruses were discovered by isolation approaches and resulted in break-through knowledge in immunology, molecular and cell biology, and cancer research. In addition to rodent-specific viruses, rodent-borne viruses are causing a large number of zoonotic diseases. Most prominent examples are reemerging outbreaks of human hemorrhagic fever disease cases caused by arena- and hantaviruses. In addition, rodents are reservoirs for vector-borne pathogens, such as tick-borne encephalitis virus and Borrelia spp., and may carry human pathogenic agents, but likely are not involved in their transmission to human. In our days, next-generation sequencing or high-throughput sequencing (HTS) is revolutionizing the speed of the discovery of novel viruses, but other molecular approaches, such as generic RT-PCR/PCR and rolling circle amplification techniques, contribute significantly to the rapidly ongoing process. However, the current knowledge still represents only the tip of the iceberg, when comparing the known human viruses to those known for rodents, the mammalian taxon with the largest species number. The diagnostic potential of HTS-based metagenomic approaches is illustrated by their use in the discovery and complete genome determination of novel borna- and adenoviruses as causative disease agents in squirrels. In conclusion, HTS, in combination with conventional RT-PCR/PCR-based approaches, resulted in a drastically increased knowledge of the diversity of rodent viruses. Future improvements of the used workflows, including bioinformatics analysis, will further

  7. Genome-wide SNP discovery in tetraploid alfalfa using 454 sequencing and high resolution melting analysis

    Directory of Open Access Journals (Sweden)

    Zhao Patrick X

    2011-07-01

    Full Text Available Abstract Background Single nucleotide polymorphisms (SNPs are the most common type of sequence variation among plants and are often functionally important. We describe the use of 454 technology and high resolution melting analysis (HRM for high throughput SNP discovery in tetraploid alfalfa (Medicago sativa L., a species with high economic value but limited genomic resources. Results The alfalfa genotypes selected from M. sativa subsp. sativa var. 'Chilean' and M. sativa subsp. falcata var. 'Wisfal', which differ in water stress sensitivity, were used to prepare cDNA from tissue of clonally-propagated plants grown under either well-watered or water-stressed conditions, and then pooled for 454 sequencing. Based on 125.2 Mb of raw sequence, a total of 54,216 unique sequences were obtained including 24,144 tentative consensus (TCs sequences and 30,072 singletons, ranging from 100 bp to 6,662 bp in length, with an average length of 541 bp. We identified 40,661 candidate SNPs distributed throughout the genome. A sample of candidate SNPs were evaluated and validated using high resolution melting (HRM analysis. A total of 3,491 TCs harboring 20,270 candidate SNPs were located on the M. truncatula (MT 3.5.1 chromosomes. Gene Ontology assignments indicate that sequences obtained cover a broad range of GO categories. Conclusions We describe an efficient method to identify thousands of SNPs distributed throughout the alfalfa genome covering a broad range of GO categories. Validated SNPs represent valuable molecular marker resources that can be used to enhance marker density in linkage maps, identify potential factors involved in heterosis and genetic variation, and as tools for association mapping and genomic selection in alfalfa.

  8. Comparative Genomics in Switchgrass Using 61,585 High-Quality Expressed Sequence Tags

    Directory of Open Access Journals (Sweden)

    Christian M. Tobias

    2008-11-01

    Full Text Available The development of genomic resources for switchgrass ( L., a perennial NAD-malic enzyme type C grass, is required to enable molecular breeding and biotechnological approaches for improving its value as a forage and bioenergy crop. Expressed sequence tag (EST sequencing is one method that can quickly sample gene inventories and produce data suitable for marker development or analysis of tissue-specific patterns of expression. Toward this goal, three cDNA libraries from callus, crown, and seedling tissues of ‘Kanlow’ switchgrass were end-sequenced to generate a total of 61,585 high-quality ESTs from 36,565 separate clones. Seventy-three percent of the assembled consensus sequences could be aligned with the sorghum [ (L. Moench] genome at a -value of <1 × 10, indicating a high degree of similarity. Sixty-five percent of the ESTs matched with gene ontology molecular terms, and 3.3% of the sequences were matched with genes that play potential roles in cell-wall biogenesis. The representation in the three libraries of gene families known to be associated with C photosynthesis, cellulose and β-glucan synthesis, phenylpropanoid biosynthesis, and peroxidase activity indicated likely roles for individual family members. Pairwise comparisons of synonymous codon substitutions were used to assess genome sequence diversity and indicated an overall similarity between the two genome copies present in the tetraploid. Identification of EST–simple sequence repeat markers and amplification on two individual parents of a mapping population yielded an average of 2.18 amplicons per individual, and 35% of the markers produced fragment length polymorphisms.

  9. High-throughput sequencing of three Lemnoideae (duckweeds chloroplast genomes from total DNA.

    Directory of Open Access Journals (Sweden)

    Wenqin Wang

    Full Text Available BACKGROUND: Chloroplast genomes provide a wealth of information for evolutionary and population genetic studies. Chloroplasts play a particularly important role in the adaption for aquatic plants because they float on water and their major surface is exposed continuously to sunlight. The subfamily of Lemnoideae represents such a collection of aquatic species that because of photosynthesis represents one of the fastest growing plant species on earth. METHODS: We sequenced the chloroplast genomes from three different genera of Lemnoideae, Spirodela polyrhiza, Wolffiella lingulata and Wolffia australiana by high-throughput DNA sequencing of genomic DNA using the SOLiD platform. Unfractionated total DNA contains high copies of plastid DNA so that sequences from the nucleus and mitochondria can easily be filtered computationally. Remaining sequence reads were assembled into contiguous sequences (contigs using SOLiD software tools. Contigs were mapped to a reference genome of Lemna minor and gaps, selected by PCR, were sequenced on the ABI3730xl platform. CONCLUSIONS: This combinatorial approach yielded whole genomic contiguous sequences in a cost-effective manner. Over 1,000-time coverage of chloroplast from total DNA were reached by the SOLiD platform in a single spot on a quadrant slide without purification. Comparative analysis indicated that the chloroplast genome was conserved in gene number and organization with respect to the reference genome of L. minor. However, higher nucleotide substitution, abundant deletions and insertions occurred in non-coding regions of these genomes, indicating a greater genomic dynamics than expected from the comparison of other related species in the Pooideae. Noticeably, there was no transition bias over transversion in Lemnoideae. The data should have immediate applications in evolutionary biology and plant taxonomy with increased resolution and statistical power.

  10. Diverse interpretations of confidence building

    International Nuclear Information System (INIS)

    Macintosh, J.

    1998-01-01

    This paper explores the variety of operational understandings associated with the term 'confidence building'. Collectively, these understandings constitute what should be thought of as a 'family' of confidence building approaches. This unacknowledged and generally unappreciated proliferation of operational understandings that function under the rubric of confidence building appears to be an impediment to effective policy. The paper's objective is to analyze these different understandings, stressing the important differences in their underlying assumptions. In the process, the paper underlines the need for the international community to clarify its collective thinking about what it means when it speaks of 'confidence building'. Without enhanced clarity, it will be unnecessarily difficult to employ the confidence building approach effectively due to the lack of consistent objectives and common operating assumptions. Although it is not the intention of this paper to promote a particular account of confidence building, dissecting existing operational understandings should help to identify whether there are fundamental elements that define what might be termed 'authentic' confidence building. Implicit here is the view that some operational understandings of confidence building may diverge too far from consensus models to count as meaningful members of the confidence building family. (author)

  11. Weighting Mean and Variability during Confidence Judgments

    Science.gov (United States)

    de Gardelle, Vincent; Mamassian, Pascal

    2015-01-01

    Humans can not only perform some visual tasks with great precision, they can also judge how good they are in these tasks. However, it remains unclear how observers produce such metacognitive evaluations, and how these evaluations might be dissociated from the performance in the visual task. Here, we hypothesized that some stimulus variables could affect confidence judgments above and beyond their impact on performance. In a motion categorization task on moving dots, we manipulated the mean and the variance of the motion directions, to obtain a low-mean low-variance condition and a high-mean high-variance condition with matched performances. Critically, in terms of confidence, observers were not indifferent between these two conditions. Observers exhibited marked preferences, which were heterogeneous across individuals, but stable within each observer when assessed one week later. Thus, confidence and performance are dissociable and observers’ confidence judgments put different weights on the stimulus variables that limit performance. PMID:25793275

  12. Extraction of High Molecular Weight DNA from Fungal Rust Spores for Long Read Sequencing.

    Science.gov (United States)

    Schwessinger, Benjamin; Rathjen, John P

    2017-01-01

    Wheat rust fungi are complex organisms with a complete life cycle that involves two different host plants and five different spore types. During the asexual infection cycle on wheat, rusts produce massive amounts of dikaryotic urediniospores. These spores are dikaryotic (two nuclei) with each nucleus containing one haploid genome. This dikaryotic state is likely to contribute to their evolutionary success, making them some of the major wheat pathogens globally. Despite this, most published wheat rust genomes are highly fragmented and contain very little haplotype-specific sequence information. Current long-read sequencing technologies hold great promise to provide more contiguous and haplotype-phased genome assemblies. Long reads are able to span repetitive regions and phase structural differences between the haplomes. This increased genome resolution enables the identification of complex loci and the study of genome evolution beyond simple nucleotide polymorphisms. Long-read technologies require pure high molecular weight DNA as an input for sequencing. Here, we describe a DNA extraction protocol for rust spores that yields pure double-stranded DNA molecules with molecular weight of >50 kilo-base pairs (kbp). The isolated DNA is of sufficient purity for PacBio long-read sequencing, but may require additional purification for other sequencing technologies such as Nanopore and 10× Genomics.

  13. A High Resolution Genetic Map Anchoring Scaffolds of the Sequenced Watermelon Genome

    Science.gov (United States)

    Kou, Qinghe; Jiang, Jiao; Guo, Shaogui; Zhang, Haiying; Hou, Wenju; Zou, Xiaohua; Sun, Honghe; Gong, Guoyi; Levi, Amnon; Xu, Yong

    2012-01-01

    As part of our ongoing efforts to sequence and map the watermelon (Citrullus spp.) genome, we have constructed a high density genetic linkage map. The map positioned 234 watermelon genome sequence scaffolds (an average size of 1.41 Mb) that cover about 330 Mb and account for 93.5% of the 353 Mb of the assembled genomic sequences of the elite Chinese watermelon line 97103 (Citrullus lanatus var. lanatus). The genetic map was constructed using an F8 population of 103 recombinant inbred lines (RILs). The RILs are derived from a cross between the line 97103 and the United States Plant Introduction (PI) 296341-FR (C. lanatus var. citroides) that contains resistance to fusarium wilt (races 0, 1, and 2). The genetic map consists of eleven linkage groups that include 698 simple sequence repeat (SSR), 219 insertion-deletion (InDel) and 36 structure variation (SV) markers and spans ∼800 cM with a mean marker interval of 0.8 cM. Using fluorescent in situ hybridization (FISH) with 11 BACs that produced chromosome-specifc signals, we have depicted watermelon chromosomes that correspond to the eleven linkage groups constructed in this study. The high resolution genetic map developed here should be a useful platform for the assembly of the watermelon genome, for the development of sequence-based markers used in breeding programs, and for the identification of genes associated with important agricultural traits. PMID:22247776

  14. Discovery of viruses and virus-like pathogens in pistachio using high-throughput sequencing

    Science.gov (United States)

    Pistachio (Pistacia vera L.) trees from the National Clonal Germplasm Repository (NCGR) and orchards in California were surveyed for viruses and virus-like agents by high-throughput sequencing (HTS). Analyses of 60 trees including clonal UCB-1 hybrid rootstock (P. atlantica × P. integerrima) identif...

  15. Draft Genome Sequences of Klebsiella oxytoca Isolates Originating from a Highly Contaminated Liquid Hand Soap Product

    OpenAIRE

    Hammerl, J. A.; Lasch, P.; Nitsche, A.; Dabrowski, P. W.; Hahmann, H.; Wicke, A.; Kleta, S.; Dahouk, S. Al; Dieckmann, R.

    2015-01-01

    In 2013, contaminated liquid soap was detected by routine microbiological monitoring of consumer products through state health authorities. Because of its high load of Klebsiella oxytoca, the liquid soap was notified via the European Union Rapid Alert System for Dangerous Non-Food Products (EU-RAPEX) and recalled. Here, we present two draft genome sequences and a summary of their general features.

  16. The Importance of Agriculture Science Course Sequencing in High Schools: A View from Collegiate Agriculture Students

    Science.gov (United States)

    Wheelus, Robin P.

    2009-01-01

    The objective of this study was to investigate the importance of Agriculture Science course sequencing in high schools, as a preparatory factor for students enrolled in collegiate agriculture classes. With the variety of courses listed in the Texas Essential Knowledge and Skills (TEKS) for Agriculture Science, it has been possible for counselors,…

  17. Increasing Classroom Compliance: Using a High-Probability Command Sequence with Noncompliant Students

    Science.gov (United States)

    Axelrod, Michael I.; Zank, Amber J.

    2012-01-01

    Noncompliance is one of the most problematic behaviors within the school setting. One strategy to increase compliance of noncompliant students is a high-probability command sequence (HPCS; i.e., a set of simple commands in which an individual is likely to comply immediately prior to the delivery of a command that has a lower probability of…

  18. HPV-QUEST: A highly customized system for automated HPV sequence analysis capable of processing Next Generation sequencing data set.

    Science.gov (United States)

    Yin, Li; Yao, Jiqiang; Gardner, Brent P; Chang, Kaifen; Yu, Fahong; Goodenow, Maureen M

    2012-01-01

    Next Generation sequencing (NGS) applied to human papilloma viruses (HPV) can provide sensitive methods to investigate the molecular epidemiology of multiple type HPV infection. Currently a genotyping system with a comprehensive collection of updated HPV reference sequences and a capacity to handle NGS data sets is lacking. HPV-QUEST was developed as an automated and rapid HPV genotyping system. The web-based HPV-QUEST subtyping algorithm was developed using HTML, PHP, Perl scripting language, and MYSQL as the database backend. HPV-QUEST includes a database of annotated HPV reference sequences with updated nomenclature covering 5 genuses, 14 species and 150 mucosal and cutaneous types to genotype blasted query sequences. HPV-QUEST processes up to 10 megabases of sequences within 1 to 2 minutes. Results are reported in html, text and excel formats and display e-value, blast score, and local and coverage identities; provide genus, species, type, infection site and risk for the best matched reference HPV sequence; and produce results ready for additional analyses.

  19. The efficacy of high-throughput sequencing and target enrichment on charred archaeobotanical remains

    DEFF Research Database (Denmark)

    Nistelberger, H. M.; Smith, O.; Wales, Nathan

    2016-01-01

    . It has been suggested that high-throughput sequencing (HTS) technologies coupled with DNA enrichment techniques may overcome some of these limitations. Here we report the findings of HTS and target enrichment on four important archaeological crops (barley, grape, maize and rice) performed in three...... lightly-charred maize cob. Even with target enrichment, this sample failed to yield adequate data required to address fundamental questions in archaeology and biology. We further reanalysed part of an existing dataset on charred plant material, and found all purported endogenous DNA sequences were likely...

  20. A Probabilistic Tool that Aids Logistics Engineers in the Establishment of High Confidence Repair Need-Dates at the NASA Shuttle Logistics Depot

    Science.gov (United States)

    Bullington, J. V.; Winkler, J. C.; Linton, D. G.; Khajenoori, S.

    1995-01-01

    The NASA Shuttle Logistics Depot (NSLD) is tasked with the responsibility for repair and manufacture of Line Replaceable Unit (LRU) hardware and components to support the Space Shuttle Orbiter. Due to shrinking budgets, cost effective repair of LRU's becomes a primary objective. To achieve this objective, is imperative that resources be assigned to those LRU's which have the greatest expectation of being needed as a spare. Forecasting the times at which spares are needed requires consideration of many significant factors including: failure rate, flight rate, spares availability, and desired level of support, among others. This paper summarizes the results of the research and development work that has been accomplished in producing an automated tool that assists in the assignment of effective repair start-times for LRU's at the NSLD. This system, called the Repair Start-time Assessment System (RSAS), uses probabilistic modeling technology to calculate a need date for a repair that considers the current repair pipeline status, as well as, serviceable spares and projections of future demands. The output from the system is a date for beginning the repair that has significantly greater confidence (in the sense that a desired probability of support is ensured) than times produced using other techniques. Since an important output of RSAS is the longest repair turn-around time that will ensure a desired probability of support, RSAS has the potential for being applied to operations at any repair depot where spares are on-hand and repair start-times are of interest. In addition, RSAS incorporates tenants of Just-in-Time (JIT) techniques in that the latest repair start-time (i.e., the latest time at which repair resources must be committed) may be calculated for every failed unit This could reduce the spares inventory for certain items, without significantly increasing the risk of unsatisfied demand.

  1. Investigation of a Quadruplex-Forming Repeat Sequence Highly Enriched in Xanthomonas and Nostoc sp.

    Science.gov (United States)

    Rehm, Charlotte; Wurmthaler, Lena A; Li, Yuanhao; Frickey, Tancred; Hartig, Jörg S

    2015-01-01

    In prokaryotes simple sequence repeats (SSRs) with unit sizes of 1-5 nucleotides (nt) are causative for phase and antigenic variation. Although an increased abundance of heptameric repeats was noticed in bacteria, reports about SSRs of 6-9 nt are rare. In particular G-rich repeat sequences with the propensity to fold into G-quadruplex (G4) structures have received little attention. In silico analysis of prokaryotic genomes show putative G4 forming sequences to be abundant. This report focuses on a surprisingly enriched G-rich repeat of the type GGGNATC in Xanthomonas and cyanobacteria such as Nostoc. We studied in detail the genomes of Xanthomonas campestris pv. campestris ATCC 33913 (Xcc), Xanthomonas axonopodis pv. citri str. 306 (Xac), and Nostoc sp. strain PCC7120 (Ana). In all three organisms repeats are spread all over the genome with an over-representation in non-coding regions. Extensive variation of the number of repetitive units was observed with repeat numbers ranging from two up to 26 units. However a clear preference for four units was detected. The strong bias for four units coincides with the requirement of four consecutive G-tracts for G4 formation. Evidence for G4 formation of the consensus repeat sequences was found in biophysical studies utilizing CD spectroscopy. The G-rich repeats are preferably located between aligned open reading frames (ORFs) and are under-represented in coding regions or between divergent ORFs. The G-rich repeats are preferentially located within a distance of 50 bp upstream of an ORF on the anti-sense strand or within 50 bp from the stop codon on the sense strand. Analysis of whole transcriptome sequence data showed that the majority of repeat sequences are transcribed. The genetic loci in the vicinity of repeat regions show increased genomic stability. In conclusion, we introduce and characterize a special class of highly abundant and wide-spread quadruplex-forming repeat sequences in bacteria.

  2. High Sequence Variations in Mitochondrial DNA Control Region among Worldwide Populations of Flathead Mullet Mugil cephalus

    Directory of Open Access Journals (Sweden)

    Brian Wade Jamandre

    2014-01-01

    Full Text Available The sequence and structure of the complete mtDNA control region (CR of M. cephalus from African, Pacific, and Atlantic populations are presented in this study to assess its usefulness in phylogeographic studies of this species. The mtDNA CR sequence variations among M. cephalus populations largely exceeded intraspecific polymorphisms that are generally observed in other vertebrates. The length of CR sequence varied among M. cephalus populations due to the presence of indels and variable number of tandem repeats at the 3′ hypervariable domain. The high evolutionary rate of the CR in this species probably originated from these mutations. However, no excessive homoplasic mutations were noticed. Finally, the star shaped tree inferred from the CR polymorphism stresses a rapid radiation worldwide, in this species. The CR still appears as a good marker for phylogeographic investigations and additional worldwide samples are warranted to further investigate the genetic structure and evolution in M. cephalus.

  3. On Statistical Modeling of Sequencing Noise in High Depth Data to Assess Tumor Evolution

    Science.gov (United States)

    Rabadan, Raul; Bhanot, Gyan; Marsilio, Sonia; Chiorazzi, Nicholas; Pasqualucci, Laura; Khiabanian, Hossein

    2017-12-01

    One cause of cancer mortality is tumor evolution to therapy-resistant disease. First line therapy often targets the dominant clone, and drug resistance can emerge from preexisting clones that gain fitness through therapy-induced natural selection. Such mutations may be identified using targeted sequencing assays by analysis of noise in high-depth data. Here, we develop a comprehensive, unbiased model for sequencing error background. We find that noise in sufficiently deep DNA sequencing data can be approximated by aggregating negative binomial distributions. Mutations with frequencies above noise may have prognostic value. We evaluate our model with simulated exponentially expanded populations as well as data from cell line and patient sample dilution experiments, demonstrating its utility in prognosticating tumor progression. Our results may have the potential to identify significant mutations that can cause recurrence. These results are relevant in the pretreatment clinical setting to determine appropriate therapy and prepare for potential recurrence pretreatment.

  4. WebPrInSeS: automated full-length clone sequence identification and verification using high-throughput sequencing data.

    Science.gov (United States)

    Massouras, Andreas; Decouttere, Frederik; Hens, Korneel; Deplancke, Bart

    2010-07-01

    High-throughput sequencing (HTS) is revolutionizing our ability to obtain cheap, fast and reliable sequence information. Many experimental approaches are expected to benefit from the incorporation of such sequencing features in their pipeline. Consequently, software tools that facilitate such an incorporation should be of great interest. In this context, we developed WebPrInSeS, a web server tool allowing automated full-length clone sequence identification and verification using HTS data. WebPrInSeS encompasses two separate software applications. The first is WebPrInSeS-C which performs automated sequence verification of user-defined open-reading frame (ORF) clone libraries. The second is WebPrInSeS-E, which identifies positive hits in cDNA or ORF-based library screening experiments such as yeast one- or two-hybrid assays. Both tools perform de novo assembly using HTS data from any of the three major sequencing platforms. Thus, WebPrInSeS provides a highly integrated, cost-effective and efficient way to sequence-verify or identify clones of interest. WebPrInSeS is available at http://webprinses.epfl.ch/ and is open to all users.

  5. Scalable whole-exome sequencing of cell-free DNA reveals high concordance with metastatic tumors.

    Science.gov (United States)

    Adalsteinsson, Viktor A; Ha, Gavin; Freeman, Samuel S; Choudhury, Atish D; Stover, Daniel G; Parsons, Heather A; Gydush, Gregory; Reed, Sarah C; Rotem, Denisse; Rhoades, Justin; Loginov, Denis; Livitz, Dimitri; Rosebrock, Daniel; Leshchiner, Ignaty; Kim, Jaegil; Stewart, Chip; Rosenberg, Mara; Francis, Joshua M; Zhang, Cheng-Zhong; Cohen, Ofir; Oh, Coyin; Ding, Huiming; Polak, Paz; Lloyd, Max; Mahmud, Sairah; Helvie, Karla; Merrill, Margaret S; Santiago, Rebecca A; O'Connor, Edward P; Jeong, Seong H; Leeson, Rachel; Barry, Rachel M; Kramkowski, Joseph F; Zhang, Zhenwei; Polacek, Laura; Lohr, Jens G; Schleicher, Molly; Lipscomb, Emily; Saltzman, Andrea; Oliver, Nelly M; Marini, Lori; Waks, Adrienne G; Harshman, Lauren C; Tolaney, Sara M; Van Allen, Eliezer M; Winer, Eric P; Lin, Nancy U; Nakabayashi, Mari; Taplin, Mary-Ellen; Johannessen, Cory M; Garraway, Levi A; Golub, Todd R; Boehm, Jesse S; Wagle, Nikhil; Getz, Gad; Love, J Christopher; Meyerson, Matthew

    2017-11-06

    Whole-exome sequencing of cell-free DNA (cfDNA) could enable comprehensive profiling of tumors from blood but the genome-wide concordance between cfDNA and tumor biopsies is uncertain. Here we report ichorCNA, software that quantifies tumor content in cfDNA from 0.1× coverage whole-genome sequencing data without prior knowledge of tumor mutations. We apply ichorCNA to 1439 blood samples from 520 patients with metastatic prostate or breast cancers. In the earliest tested sample for each patient, 34% of patients have ≥10% tumor-derived cfDNA, sufficient for standard coverage whole-exome sequencing. Using whole-exome sequencing, we validate the concordance of clonal somatic mutations (88%), copy number alterations (80%), mutational signatures, and neoantigens between cfDNA and matched tumor biopsies from 41 patients with ≥10% cfDNA tumor content. In summary, we provide methods to identify patients eligible for comprehensive cfDNA profiling, revealing its applicability to many patients, and demonstrate high concordance of cfDNA and metastatic tumor whole-exome sequencing.

  6. Galaxy Workflows for Web-based Bioinformatics Analysis of Aptamer High-throughput Sequencing Data

    Directory of Open Access Journals (Sweden)

    William H Thiel

    2016-01-01

    Full Text Available Development of RNA and DNA aptamers for diagnostic and therapeutic applications is a rapidly growing field. Aptamers are identified through iterative rounds of selection in a process termed SELEX (Systematic Evolution of Ligands by EXponential enrichment. High-throughput sequencing (HTS revolutionized the modern SELEX process by identifying millions of aptamer sequences across multiple rounds of aptamer selection. However, these vast aptamer HTS datasets necessitated bioinformatics techniques. Herein, we describe a semiautomated approach to analyze aptamer HTS datasets using the Galaxy Project, a web-based open source collection of bioinformatics tools that were originally developed to analyze genome, exome, and transcriptome HTS data. Using a series of Workflows created in the Galaxy webserver, we demonstrate efficient processing of aptamer HTS data and compilation of a database of unique aptamer sequences. Additional Workflows were created to characterize the abundance and persistence of aptamer sequences within a selection and to filter sequences based on these parameters. A key advantage of this approach is that the online nature of the Galaxy webserver and its graphical interface allow for the analysis of HTS data without the need to compile code or install multiple programs.

  7. Rapid high resolution genotyping of Francisella tularensis by whole genome sequence comparison of annotated genes ("MLST+".

    Directory of Open Access Journals (Sweden)

    Markus H Antwerpen

    Full Text Available The zoonotic disease tularemia is caused by the bacterium Francisella tularensis. This pathogen is considered as a category A select agent with potential to be misused in bioterrorism. Molecular typing based on DNA-sequence like canSNP-typing or MLVA has become the accepted standard for this organism. Due to the organism's highly clonal nature, the current typing methods have reached their limit of discrimination for classifying closely related subpopulations within the subspecies F. tularensis ssp. holarctica. We introduce a new gene-by-gene approach, MLST+, based on whole genome data of 15 sequenced F. tularensis ssp. holarctica strains and apply this approach to investigate an epidemic of lethal tularemia among non-human primates in two animal facilities in Germany. Due to the high resolution of MLST+ we are able to demonstrate that three independent clones of this highly infectious pathogen were responsible for these spatially and temporally restricted outbreaks.

  8. Frequency-locked pulse sequencer for high-frame-rate monochromatic tissue motion imaging.

    Science.gov (United States)

    Azar, Reza Zahiri; Baghani, Ali; Salcudean, Septimiu E; Rohling, Robert

    2011-04-01

    To overcome the inherent low frame rate of conventional ultrasound, we have previously presented a system that can be implemented on conventional ultrasound scanners for high-frame-rate imaging of monochromatic tissue motion. The system employs a sector subdivision technique in the sequencer to increase the acquisition rate. To eliminate the delays introduced during data acquisition, a motion phase correction algorithm has also been introduced to create in-phase displacement images. Previous experimental results from tissue- mimicking phantoms showed that the system can achieve effective frame rates of up to a few kilohertz on conventional ultrasound systems. In this short communication, we present a new pulse sequencing strategy that facilitates high-frame-rate imaging of monochromatic motion such that the acquired echo signals are inherently in-phase. The sequencer uses the knowledge of the excitation frequency to synchronize the acquisition of the entire imaging plane to that of an external exciter. This sequencing approach eliminates any need for synchronization or phase correction and has applications in tissue elastography, which we demonstrate with tissue-mimicking phantoms. © 2011 IEEE

  9. Exploring sequence characteristics related to high-level production of secreted proteins in Aspergillus niger.

    Directory of Open Access Journals (Sweden)

    Bastiaan A van den Berg

    Full Text Available Protein sequence features are explored in relation to the production of over-expressed extracellular proteins by fungi. Knowledge on features influencing protein production and secretion could be employed to improve enzyme production levels in industrial bioprocesses via protein engineering. A large set, over 600 homologous and nearly 2,000 heterologous fungal genes, were overexpressed in Aspergillus niger using a standardized expression cassette and scored for high versus no production. Subsequently, sequence-based machine learning techniques were applied for identifying relevant DNA and protein sequence features. The amino-acid composition of the protein sequence was found to be most predictive and interpretation revealed that, for both homologous and heterologous gene expression, the same features are important: tyrosine and asparagine composition was found to have a positive correlation with high-level production, whereas for unsuccessful production, contributions were found for methionine and lysine composition. The predictor is available online at http://bioinformatics.tudelft.nl/hipsec. Subsequent work aims at validating these findings by protein engineering as a method for increasing expression levels per gene copy.

  10. Improvements and impacts of GRCh38 human reference on high throughput sequencing data analysis.

    Science.gov (United States)

    Guo, Yan; Dai, Yulin; Yu, Hui; Zhao, Shilin; Samuels, David C; Shyr, Yu

    2017-03-01

    Analyses of high throughput sequencing data starts with alignment against a reference genome, which is the foundation for all re-sequencing data analyses. Each new release of the human reference genome has been augmented with improved accuracy and completeness. It is presumed that the latest release of human reference genome, GRCh38 will contribute more to high throughput sequencing data analysis by providing more accuracy. But the amount of improvement has not yet been quantified. We conducted a study to compare the genomic analysis results between the GRCh38 reference and its predecessor GRCh37. Through analyses of alignment, single nucleotide polymorphisms, small insertion/deletions, copy number and structural variants, we show that GRCh38 offers overall more accurate analysis of human sequencing data. More importantly, GRCh38 produced fewer false positive structural variants. In conclusion, GRCh38 is an improvement over GRCh37 not only from the genome assembly aspect, but also yields more reliable genomic analysis results. Copyright © 2017. Published by Elsevier Inc.

  11. High league bench players and starters: differences in group interactions, group cohesion, role acceptance and self-confidence in football teams

    OpenAIRE

    Simonenkova Irina Petrovna

    2015-01-01

    Main staff players differ from bench players in their perceptions and demonstrate different responses. This research compares the situation of bench players with the situation of starters in high league Latvian football teams.

  12. Analysis of high-throughput sequencing and annotation strategies for phage genomes.

    Directory of Open Access Journals (Sweden)

    Matthew R Henn

    Full Text Available BACKGROUND: Bacterial viruses (phages play a critical role in shaping microbial populations as they influence both host mortality and horizontal gene transfer. As such, they have a significant impact on local and global ecosystem function and human health. Despite their importance, little is known about the genomic diversity harbored in phages, as methods to capture complete phage genomes have been hampered by the lack of knowledge about the target genomes, and difficulties in generating sufficient quantities of genomic DNA for sequencing. Of the approximately 550 phage genomes currently available in the public domain, fewer than 5% are marine phage. METHODOLOGY/PRINCIPAL FINDINGS: To advance the study of phage biology through comparative genomic approaches we used marine cyanophage as a model system. We compared DNA preparation methodologies (DNA extraction directly from either phage lysates or CsCl purified phage particles, and sequencing strategies that utilize either Sanger sequencing of a linker amplification shotgun library (LASL or of a whole genome shotgun library (WGSL, or 454 pyrosequencing methods. We demonstrate that genomic DNA sample preparation directly from a phage lysate, combined with 454 pyrosequencing, is best suited for phage genome sequencing at scale, as this method is capable of capturing complete continuous genomes with high accuracy. In addition, we describe an automated annotation informatics pipeline that delivers high-quality annotation and yields few false positives and negatives in ORF calling. CONCLUSIONS/SIGNIFICANCE: These DNA preparation, sequencing and annotation strategies enable a high-throughput approach to the burgeoning field of phage genomics.

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

    Directory of Open Access Journals (Sweden)

    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

  14. 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

  15. Fixing Formalin: A Method to Recover Genomic-Scale DNA Sequence Data from Formalin-Fixed Museum Specimens Using High-Throughput Sequencing.

    Directory of Open Access Journals (Sweden)

    Sarah M Hykin

    Full Text Available For 150 years or more, specimens were routinely collected and deposited in natural history collections without preserving fresh tissue samples for genetic analysis. In the case of most herpetological specimens (i.e. amphibians and reptiles, attempts to extract and sequence DNA from formalin-fixed, ethanol-preserved specimens-particularly for use in phylogenetic analyses-has been laborious and largely ineffective due to the highly fragmented nature of the DNA. As a result, tens of thousands of specimens in herpetological collections have not been available for sequence-based phylogenetic studies. Massively parallel High-Throughput Sequencing methods and the associated bioinformatics, however, are particularly suited to recovering meaningful genetic markers from severely degraded/fragmented DNA sequences such as DNA damaged by formalin-fixation. In this study, we compared previously published DNA extraction methods on three tissue types subsampled from formalin-fixed specimens of Anolis carolinensis, followed by sequencing. Sufficient quality DNA was recovered from liver tissue, making this technique minimally destructive to museum specimens. Sequencing was only successful for the more recently collected specimen (collected ~30 ybp. We suspect this could be due either to the conditions of preservation and/or the amount of tissue used for extraction purposes. For the successfully sequenced sample, we found a high rate of base misincorporation. After rigorous trimming, we successfully mapped 27.93% of the cleaned reads to the reference genome, were able to reconstruct the complete mitochondrial genome, and recovered an accurate phylogenetic placement for our specimen. We conclude that the amount of DNA available, which can vary depending on specimen age and preservation conditions, will determine if sequencing will be successful. The technique described here will greatly improve the value of museum collections by making many formalin-fixed specimens

  16. Fixing Formalin: A Method to Recover Genomic-Scale DNA Sequence Data from Formalin-Fixed Museum Specimens Using High-Throughput Sequencing.

    Science.gov (United States)

    Hykin, Sarah M; Bi, Ke; McGuire, Jimmy A

    2015-01-01

    For 150 years or more, specimens were routinely collected and deposited in natural history collections without preserving fresh tissue samples for genetic analysis. In the case of most herpetological specimens (i.e. amphibians and reptiles), attempts to extract and sequence DNA from formalin-fixed, ethanol-preserved specimens-particularly for use in phylogenetic analyses-has been laborious and largely ineffective due to the highly fragmented nature of the DNA. As a result, tens of thousands of specimens in herpetological collections have not been available for sequence-based phylogenetic studies. Massively parallel High-Throughput Sequencing methods and the associated bioinformatics, however, are particularly suited to recovering meaningful genetic markers from severely degraded/fragmented DNA sequences such as DNA damaged by formalin-fixation. In this study, we compared previously published DNA extraction methods on three tissue types subsampled from formalin-fixed specimens of Anolis carolinensis, followed by sequencing. Sufficient quality DNA was recovered from liver tissue, making this technique minimally destructive to museum specimens. Sequencing was only successful for the more recently collected specimen (collected ~30 ybp). We suspect this could be due either to the conditions of preservation and/or the amount of tissue used for extraction purposes. For the successfully sequenced sample, we found a high rate of base misincorporation. After rigorous trimming, we successfully mapped 27.93% of the cleaned reads to the reference genome, were able to reconstruct the complete mitochondrial genome, and recovered an accurate phylogenetic placement for our specimen. We conclude that the amount of DNA available, which can vary depending on specimen age and preservation conditions, will determine if sequencing will be successful. The technique described here will greatly improve the value of museum collections by making many formalin-fixed specimens available for

  17. Alignment of high-throughput sequencing data inside in-memory databases.

    Science.gov (United States)

    Firnkorn, Daniel; Knaup-Gregori, Petra; Lorenzo Bermejo, Justo; Ganzinger, Matthias

    2014-01-01

    In times of high-throughput DNA sequencing techniques, performance-capable analysis of DNA sequences is of high importance. Computer supported DNA analysis is still an intensive time-consuming task. In this paper we explore the potential of a new In-Memory database technology by using SAP's High Performance Analytic Appliance (HANA). We focus on read alignment as one of the first steps in DNA sequence analysis. In particular, we examined the widely used Burrows-Wheeler Aligner (BWA) and implemented stored procedures in both, HANA and the free database system MySQL, to compare execution time and memory management. To ensure that the results are comparable, MySQL has been running in memory as well, utilizing its integrated memory engine for database table creation. We implemented stored procedures, containing exact and inexact searching of DNA reads within the reference genome GRCh37. Due to technical restrictions in SAP HANA concerning recursion, the inexact matching problem could not be implemented on this platform. Hence, performance analysis between HANA and MySQL was made by comparing the execution time of the exact search procedures. Here, HANA was approximately 27 times faster than MySQL which means, that there is a high potential within the new In-Memory concepts, leading to further developments of DNA analysis procedures in the future.

  18. Nuclear power: restoring public confidence

    International Nuclear Information System (INIS)

    Arnold, L.

    1986-01-01

    The paper concerns a one day conference on nuclear power organised by the Centre for Science Studies and Science Policy, Lancaster, April 1986. Following the Chernobyl reactor accident, the conference concentrated on public confidence in nuclear power. Causes of lack of public confidence, public perceptions of risk, and the effect of Chernobyl in the United Kingdom, were all discussed. A Select Committee on the Environment examined the problems of radioactive waste disposal. (U.K.)

  19. The need for high-quality whole-genome sequence databases in microbial forensics.

    Science.gov (United States)

    Sjödin, Andreas; Broman, Tina; Melefors, Öjar; Andersson, Gunnar; Rasmusson, Birgitta; Knutsson, Rickard; Forsman, Mats

    2013-09-01

    Microbial forensics is an important part of a strengthened capability to respond to biocrime and bioterrorism incidents to aid in the complex task of distinguishing between natural outbreaks and deliberate acts. The goal of a microbial forensic investigation is to identify and criminally prosecute those responsible for a biological attack, and it involves a detailed analysis of the weapon--that is, the pathogen. The recent development of next-generation sequencing (NGS) technologies has greatly increased the resolution that can be achieved in microbial forensic analyses. It is now possible to identify, quickly and in an unbiased manner, previously undetectable genome differences between closely related isolates. This development is particularly relevant for the most deadly bacterial diseases that are caused by bacterial lineages with extremely low levels of genetic diversity. Whole-genome analysis of pathogens is envisaged to be increasingly essential for this purpose. In a microbial forensic context, whole-genome sequence analysis is the ultimate method for strain comparisons as it is informative during identification, characterization, and attribution--all 3 major stages of the investigation--and at all levels of microbial strain identity resolution (ie, it resolves the full spectrum from family to isolate). Given these capabilities, one bottleneck in microbial forensics investigations is the availability of high-quality reference databases of bacterial whole-genome sequences. To be of high quality, databases need to be curated and accurate in terms of sequences, metadata, and genetic diversity coverage. The development of whole-genome sequence databases will be instrumental in successfully tracing pathogens in the future.

  20. High-throughput Sequencing Based Immune Repertoire Study during Infectious Disease

    Directory of Open Access Journals (Sweden)

    Dongni Hou

    2016-08-01

    Full Text Available The selectivity of the adaptive immune response is based on the enormous diversity of T and B cell antigen-specific receptors. The immune repertoire, the collection of T and B cells with functional diversity in the circulatory system at any given time, is dynamic and reflects the essence of immune selectivity. In this article, we review the recent advances in immune repertoire study of infectious diseases that achieved by traditional techniques and high-throughput sequencing techniques. High-throughput sequencing techniques enable the determination of complementary regions of lymphocyte receptors with unprecedented efficiency and scale. This progress in methodology enhances the understanding of immunologic changes during pathogen challenge, and also provides a basis for further development of novel diagnostic markers, immunotherapies and vaccines.

  1. Designing a Bioengine for Detection and Analysis of Base String on an Affected Sequence in High-Concentration Regions

    Directory of Open Access Journals (Sweden)

    Debnath Bhattacharyya

    2013-01-01

    Full Text Available We design an Algorithm for bioengine. As a program are enable optimal alignments searching between two sequences, the host sequence (normal plant as well as query sequence (virus. Searching for homologues has become a routine operation of biological sequences in 4 × 4 combination with different subsequence (word size. This program takes the advantage of the high degree of homology between such sequences to construct an alignment of the matching regions. There is a main aim which is to detect the overlapping reading frames. This program also enables to find out the highly infected colones selection highest matching region with minimum gap or mismatch zones and unique virus colones matches. This is a small, portable, interactive, front-end program intended to be used to find out the regions of matching between host sequence and query subsequences. All the operations are carried out in fraction of seconds, depending on the required task and on the sequence length.

  2. Designing a Bioengine for Detection and Analysis of Base String on an Affected Sequence in High-Concentration Regions

    Science.gov (United States)

    Mandal, Bijoy Kumar; Kim, Tai-hoon

    2013-01-01

    We design an Algorithm for bioengine. As a program are enable optimal alignments searching between two sequences, the host sequence (normal plant) as well as query sequence (virus). Searching for homologues has become a routine operation of biological sequences in 4 × 4 combination with different subsequence (word size). This program takes the advantage of the high degree of homology between such sequences to construct an alignment of the matching regions. There is a main aim which is to detect the overlapping reading frames. This program also enables to find out the highly infected colones selection highest matching region with minimum gap or mismatch zones and unique virus colones matches. This is a small, portable, interactive, front-end program intended to be used to find out the regions of matching between host sequence and query subsequences. All the operations are carried out in fraction of seconds, depending on the required task and on the sequence length. PMID:24000321

  3. Association Study of Gut Flora in Coronary Heart Disease through High-Throughput Sequencing

    OpenAIRE

    Cui, Li; Zhao, Tingting; Hu, Haibing; Zhang, Wen; Hua, Xiuguo

    2017-01-01

    Objectives. We aimed to explore the impact of gut microbiota in coronary heart disease (CHD) patients through high-throughput sequencing. Methods. A total of 29 CHD in-hospital patients and 35 healthy volunteers as controls were included. Nucleic acids were extracted from fecal samples, followed by ? diversity and principal coordinate analysis (PCoA). Based on unweighted UniFrac distance matrices, unweighted-pair group method with arithmetic mean (UPGMA) trees were created. Results. After dat...

  4. Draft Genome Sequences of Klebsiella oxytoca Isolates Originating from a Highly Contaminated Liquid Hand Soap Product.

    Science.gov (United States)

    Hammerl, J A; Lasch, P; Nitsche, A; Dabrowski, P W; Hahmann, H; Wicke, A; Kleta, S; Al Dahouk, S; Dieckmann, R

    2015-07-23

    In 2013, contaminated liquid soap was detected by routine microbiological monitoring of consumer products through state health authorities. Because of its high load of Klebsiella oxytoca, the liquid soap was notified via the European Union Rapid Alert System for Dangerous Non-Food Products (EU-RAPEX) and recalled. Here, we present two draft genome sequences and a summary of their general features. Copyright © 2015 Hammerl et al.

  5. High-throughput genome sequencing of two Listeria monocytogenes clinical isolates during a large foodborne outbreak

    Directory of Open Access Journals (Sweden)

    Trout-Yakel Keri M

    2010-02-01

    Full Text Available Abstract Background A large, multi-province outbreak of listeriosis associated with ready-to-eat meat products contaminated with Listeria monocytogenes serotype 1/2a occurred in Canada in 2008. Subtyping of outbreak-associated isolates using pulsed-field gel electrophoresis (PFGE revealed two similar but distinct AscI PFGE patterns. High-throughput pyrosequencing of two L. monocytogenes isolates was used to rapidly provide the genome sequence of the primary outbreak strain and to investigate the extent of genetic diversity associated with a change of a single restriction enzyme fragment during PFGE. Results The chromosomes were collinear, but differences included 28 single nucleotide polymorphisms (SNPs and three indels, including a 33 kbp prophage that accounted for the observed difference in AscI PFGE patterns. The distribution of these traits was assessed within further clinical, environmental and food isolates associated with the outbreak, and this comparison indicated that three distinct, but highly related strains may have been involved in this nationwide outbreak. Notably, these two isolates were found to harbor a 50 kbp putative mobile genomic island encoding translocation and efflux functions that has not been observed in other Listeria genomes. Conclusions High-throughput genome sequencing provided a more detailed real-time assessment of genetic traits characteristic of the outbreak strains than could be achieved with routine subtyping methods. This study confirms that the latest generation of DNA sequencing technologies can be applied during high priority public health events, and laboratories need to prepare for this inevitability and assess how to properly analyze and interpret whole genome sequences in the context of molecular epidemiology.

  6. Combining Amplification Typing of L1 Active Subfamilies (ATLAS) with High-Throughput Sequencing.

    Science.gov (United States)

    Rahbari, Raheleh; Badge, Richard M

    2016-01-01

    With the advent of new generations of high-throughput sequencing technologies, the catalog of human genome variants created by retrotransposon activity is expanding rapidly. However, despite these advances in describing L1 diversity and the fact that L1 must retrotranspose in the germline or prior to germline partitioning to be evolutionarily successful, direct assessment of de novo L1 retrotransposition in the germline or early embryogenesis has not been achieved for endogenous L1 elements. A direct study of de novo L1 retrotransposition into susceptible loci within sperm DNA (Freeman et al., Hum Mutat 32(8):978-988, 2011) suggested that the rate of L1 retrotransposition in the germline is much lower than previously estimated (ATLAS L1 display technique (Badge et al., Am J Hum Genet 72(4):823-838, 2003) to investigate de novo L1 retrotransposition in human genomes. In this chapter, we describe how we combined a high-coverage ATLAS variant with high-throughput sequencing, achieving 11-25× sequence depth per single amplicon, to study L1 retrotransposition in whole genome amplified (WGA) DNAs.

  7. Exome Sequencing Identifies Potential Risk Variants for Mendelian Disorders at High Prevalence in Qatar

    Science.gov (United States)

    Rodriguez-Flores, Juan L.; Fakhro, Khalid; Hackett, Neil R.; Salit, Jacqueline; Fuller, Jennifer; Agosto-Perez, Francisco; Gharbiah, Maey; Malek, Joel A.; Zirie, Mahmoud; Jayyousi, Amin; Badii, Ramin; Al-Marri, Ajayeb Al-Nabet; Chouchane, Lotfi; Stadler, Dora J.; Hunter-Zinck, Haley; Mezey, Jason G.; Crystal, Ronald G.

    2013-01-01

    Exome sequencing of families of related individuals has been highly successful in identifying genetic polymorphisms responsible for Mendelian disorders. Here, we demonstrate the value of the reverse approach, where we use exome sequencing of a sample of unrelated individuals to analyze allele frequencies of known causal mutations for Mendelian diseases. We sequenced the exomes of 100 individuals representing the three major genetic subgroups of the Qatari population (Q1 Bedouin, Q2 Persian-South Asian, Q3 African) and identified 37 variants in 33 genes with effects on 36 clinically significant Mendelian diseases. These include variants not present in 1000 Genomes and variants at high frequency when compared to 1000 Genomes populations. Several of these Mendelian variants were only segregating in one Qatari subpopulation, where the observed subpopulation specificity trends were confirmed in an independent population of 386 Qataris. Pre-marital genetic screening in Qatar tests for only 4 out of the 37, such that this study provides a set of Mendelian disease variants with potential impact on the epidemiological profile of the population that could be incorporated into the testing program if further experimental and clinical characterization confirms high penetrance. PMID:24123366

  8. Characterizing ncRNAs in human pathogenic protists using high-throughput sequencing technology

    Directory of Open Access Journals (Sweden)

    Lesley Joan Collins

    2011-12-01

    Full Text Available ncRNAs are key genes in many human diseases including cancer and viral infection, as well as providing critical functions in pathogenic organisms such as fungi, bacteria, viruses and protists. Until now the identification and characterization of ncRNAs associated with disease has been slow or inaccurate requiring many years of testing to understand complicated RNA and protein gene relationships. High-throughput sequencing now offers the opportunity to characterize miRNAs, siRNAs, snoRNAs and long ncRNAs on a genomic scale making it faster and easier to clarify how these ncRNAs contribute to the disease state. However, this technology is still relatively new, and ncRNA discovery is not an application of high priority for streamlined bioinformatics. Here we summarize background concepts and practical approaches for ncRNA analysis using high-throughput sequencing, and how it relates to understanding human disease. As a case study, we focus on the parasitic protists Giardia lamblia and Trichomonas vaginalis, where large evolutionary distance has meant difficulties in comparing ncRNAs with those from model eukaryotes. A combination of biological, computational and sequencing approaches has enabled easier classification of ncRNA classes such as snoRNAs, but has also aided the identification of novel classes. It is hoped that a higher level of understanding of ncRNA expression and interaction may aid in the development of less harsh treatment for protist-based diseases.

  9. Characterizing ncRNAs in Human Pathogenic Protists Using High-Throughput Sequencing Technology

    Science.gov (United States)

    Collins, Lesley Joan

    2011-01-01

    ncRNAs are key genes in many human diseases including cancer and viral infection, as well as providing critical functions in pathogenic organisms such as fungi, bacteria, viruses, and protists. Until now the identification and characterization of ncRNAs associated with disease has been slow or inaccurate requiring many years of testing to understand complicated RNA and protein gene relationships. High-throughput sequencing now offers the opportunity to characterize miRNAs, siRNAs, small nucleolar RNAs (snoRNAs), and long ncRNAs on a genomic scale, making it faster and easier to clarify how these ncRNAs contribute to the disease state. However, this technology is still relatively new, and ncRNA discovery is not an application of high priority for streamlined bioinformatics. Here we summarize background concepts and practical approaches for ncRNA analysis using high-throughput sequencing, and how it relates to understanding human disease. As a case study, we focus on the parasitic protists Giardia lamblia and Trichomonas vaginalis, where large evolutionary distance has meant difficulties in comparing ncRNAs with those from model eukaryotes. A combination of biological, computational, and sequencing approaches has enabled easier classification of ncRNA classes such as snoRNAs, but has also aided the identification of novel classes. It is hoped that a higher level of understanding of ncRNA expression and interaction may aid in the development of less harsh treatment for protist-based diseases. PMID:22303390

  10. High-Throughput Analysis of T-DNA Location and Structure Using Sequence Capture.

    Directory of Open Access Journals (Sweden)

    Soichi Inagaki

    Full Text Available Agrobacterium-mediated transformation of plants with T-DNA is used both to introduce transgenes and for mutagenesis. Conventional approaches used to identify the genomic location and the structure of the inserted T-DNA are laborious and high-throughput methods using next-generation sequencing are being developed to address these problems. Here, we present a cost-effective approach that uses sequence capture targeted to the T-DNA borders to select genomic DNA fragments containing T-DNA-genome junctions, followed by Illumina sequencing to determine the location and junction structure of T-DNA insertions. Multiple probes can be mixed so that transgenic lines transformed with different T-DNA types can be processed simultaneously, using a simple, index-based pooling approach. We also developed a simple bioinformatic tool to find sequence read pairs that span the junction between the genome and T-DNA or any foreign DNA. We analyzed 29 transgenic lines of Arabidopsis thaliana, each containing inserts from 4 different T-DNA vectors. We determined the location of T-DNA insertions in 22 lines, 4 of which carried multiple insertion sites. Additionally, our analysis uncovered a high frequency of unconventional and complex T-DNA insertions, highlighting the needs for high-throughput methods for T-DNA localization and structural characterization. Transgene insertion events have to be fully characterized prior to use as commercial products. Our method greatly facilitates the first step of this characterization of transgenic plants by providing an efficient screen for the selection of promising lines.

  11. High-Throughput Analysis of T-DNA Location and Structure Using Sequence Capture.

    Science.gov (United States)

    Inagaki, Soichi; Henry, Isabelle M; Lieberman, Meric C; Comai, Luca

    2015-01-01

    Agrobacterium-mediated transformation of plants with T-DNA is used both to introduce transgenes and for mutagenesis. Conventional approaches used to identify the genomic location and the structure of the inserted T-DNA are laborious and high-throughput methods using next-generation sequencing are being developed to address these problems. Here, we present a cost-effective approach that uses sequence capture targeted to the T-DNA borders to select genomic DNA fragments containing T-DNA-genome junctions, followed by Illumina sequencing to determine the location and junction structure of T-DNA insertions. Multiple probes can be mixed so that transgenic lines transformed with different T-DNA types can be processed simultaneously, using a simple, index-based pooling approach. We also developed a simple bioinformatic tool to find sequence read pairs that span the junction between the genome and T-DNA or any foreign DNA. We analyzed 29 transgenic lines of Arabidopsis thaliana, each containing inserts from 4 different T-DNA vectors. We determined the location of T-DNA insertions in 22 lines, 4 of which carried multiple insertion sites. Additionally, our analysis uncovered a high frequency of unconventional and complex T-DNA insertions, highlighting the needs for high-throughput methods for T-DNA localization and structural characterization. Transgene insertion events have to be fully characterized prior to use as commercial products. Our method greatly facilitates the first step of this characterization of transgenic plants by providing an efficient screen for the selection of promising lines.

  12. High-resolution analysis of the 5'-end transcriptome using a next generation DNA sequencer.

    Directory of Open Access Journals (Sweden)

    Shin-ichi Hashimoto

    Full Text Available Massively parallel, tag-based sequencing systems, such as the SOLiD system, hold the promise of revolutionizing the study of whole genome gene expression due to the number of data points that can be generated in a simple and cost-effective manner. We describe the development of a 5'-end transcriptome workflow for the SOLiD system and demonstrate the advantages in sensitivity and dynamic range offered by this tag-based application over traditional approaches for the study of whole genome gene expression. 5'-end transcriptome analysis was used to study whole genome gene expression within a colon cancer cell line, HT-29, treated with the DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (5Aza. More than 20 million 25-base 5'-end tags were obtained from untreated and 5Aza-treated cells and matched to sequences within the human genome. Seventy three percent of the mapped unique tags were associated with RefSeq cDNA sequences, corresponding to approximately 14,000 different protein-coding genes in this single cell type. The level of expression of these genes ranged from 0.02 to 4,704 transcripts per cell. The sensitivity of a single sequence run of the SOLiD platform was 100-1,000 fold greater than that observed from 5'end SAGE data generated from the analysis of 70,000 tags obtained by Sanger sequencing. The high-resolution 5'end gene expression profiling presented in this study will not only provide novel insight into the transcriptional machinery but should also serve as a basis for a better understanding of cell biology.

  13. High-resolution sequence stratigraphy and continental environmental evolution: An example from east-central Argentina

    Science.gov (United States)

    Beilinson, Elisa; Veiga, Gonzalo D.; Spalletti, Luis A.

    2013-10-01

    The aims of this contribution is to establish a high-resolution sequence stratigraphic scheme for the continental deposits that constitute the Punta San Andrés Alloformation (Plio-Pleistocene) in east-central Argentina, to analyze the basin fill evolution and to identify and assess the role that extrinsic factors such as climate and sea-level oscillations played during evolution of the unit. For the high-resolution sequence stratigraphical study of the Punta San Andrés Alloformation, high- and low-accommodation system tracts were defined mainly on the basis of the architectural elements present in the succession, also taking into account the relative degree of channel and floodplain deposits. Discontinuities and the nature of depositional systems generated during variations in accommodation helped identify two fourth-order high-accommodation system tracts and two fourth-order low-accommodation system tracts. At a third-order scale, the Punta San Andrés Alloformation may be interpreted as the progradation of continental depositional systems, characterized by a braided system in the proximal areas, and a low-sinuosity, single-channel system in the distal areas, defined by a high rate of sediment supply and discharge peaks which periodically flooded the plains and generated high aggradation rates during the late Pliocene and lower Pleistocene.

  14. High-sensitivity HLA typing by Saturated Tiling Capture Sequencing (STC-Seq).

    Science.gov (United States)

    Jiao, Yang; Li, Ran; Wu, Chao; Ding, Yibin; Liu, Yanning; Jia, Danmei; Wang, Lifeng; Xu, Xiang; Zhu, Jing; Zheng, Min; Jia, Junling

    2018-01-15

    Highly polymorphic human leukocyte antigen (HLA) genes are responsible for fine-tuning the adaptive immune system. High-resolution HLA typing is important for the treatment of autoimmune and infectious diseases. Additionally, it is routinely performed for identifying matched donors in transplantation medicine. Although many HLA typing approaches have been developed, the complexity, low-efficiency and high-cost of current HLA-typing assays limit their application in population-based high-throughput HLA typing for donors, which is required for creating large-scale databases for transplantation and precision medicine. Here, we present a cost-efficient Saturated Tiling Capture Sequencing (STC-Seq) approach to capturing 14 HLA class I and II genes. The highly efficient capture (an approximately 23,000-fold enrichment) of these genes allows for simplified allele calling. Tests on five genes (HLA-A/B/C/DRB1/DQB1) from 31 human samples and 351 datasets using STC-Seq showed results that were 98% consistent with the known two sets of digitals (field1 and field2) genotypes. Additionally, STC can capture genomic DNA fragments longer than 3 kb from HLA loci, making the library compatible with the third-generation sequencing. STC-Seq is a highly accurate and cost-efficient method for HLA typing which can be used to facilitate the establishment of population-based HLA databases for the precision and transplantation medicine.

  15. SSR_pipeline--computer software for the identification of microsatellite sequences from paired-end Illumina high-throughput DNA sequence data

    Science.gov (United States)

    Miller, Mark P.; Knaus, Brian J.; Mullins, Thomas D.; Haig, Susan M.

    2013-01-01

    SSR_pipeline is a flexible set of programs designed to efficiently identify simple sequence repeats (SSRs; for example, microsatellites) from paired-end high-throughput Illumina DNA sequencing data. The program suite contains three analysis modules along with a fourth control module that can be used to automate analyses of large volumes of data. The modules are used to (1) identify the subset of paired-end sequences that pass quality standards, (2) align paired-end reads into a single composite DNA sequence, and (3) identify sequences that possess microsatellites conforming to user specified parameters. Each of the three separate analysis modules also can be used independently to provide greater flexibility or to work with FASTQ or FASTA files generated from other sequencing platforms (Roche 454, Ion Torrent, etc). All modules are implemented in the Python programming language and can therefore be used from nearly any computer operating system (Linux, Macintosh, Windows). The program suite relies on a compiled Python extension module to perform paired-end alignments. Instructions for compiling the extension from source code are provided in the documentation. Users who do not have Python installed on their computers or who do not have the ability to compile software also may choose to download packaged executable files. These files include all Python scripts, a copy of the compiled extension module, and a minimal installation of Python in a single binary executable. See program documentation for more information.

  16. Investigation of a Quadruplex-Forming Repeat Sequence Highly Enriched in Xanthomonas and Nostoc sp.

    Directory of Open Access Journals (Sweden)

    Charlotte Rehm

    Full Text Available In prokaryotes simple sequence repeats (SSRs with unit sizes of 1-5 nucleotides (nt are causative for phase and antigenic variation. Although an increased abundance of heptameric repeats was noticed in bacteria, reports about SSRs of 6-9 nt are rare. In particular G-rich repeat sequences with the propensity to fold into G-quadruplex (G4 structures have received little attention. In silico analysis of prokaryotic genomes show putative G4 forming sequences to be abundant. This report focuses on a surprisingly enriched G-rich repeat of the type GGGNATC in Xanthomonas and cyanobacteria such as Nostoc. We studied in detail the genomes of Xanthomonas campestris pv. campestris ATCC 33913 (Xcc, Xanthomonas axonopodis pv. citri str. 306 (Xac, and Nostoc sp. strain PCC7120 (Ana. In all three organisms repeats are spread all over the genome with an over-representation in non-coding regions. Extensive variation of the number of repetitive units was observed with repeat numbers ranging from two up to 26 units. However a clear preference for four units was detected. The strong bias for four units coincides with the requirement of four consecutive G-tracts for G4 formation. Evidence for G4 formation of the consensus repeat sequences was found in biophysical studies utilizing CD spectroscopy. The G-rich repeats are preferably located between aligned open reading frames (ORFs and are under-represented in coding regions or between divergent ORFs. The G-rich repeats are preferentially located within a distance of 50 bp upstream of an ORF on the anti-sense strand or within 50 bp from the stop codon on the sense strand. Analysis of whole transcriptome sequence data showed that the majority of repeat sequences are transcribed. The genetic loci in the vicinity of repeat regions show increased genomic stability. In conclusion, we introduce and characterize a special class of highly abundant and wide-spread quadruplex-forming repeat sequences in bacteria.

  17. Confidence in critical care nursing.

    Science.gov (United States)

    Evans, Jeanne; Bell, Jennifer L; Sweeney, Annemarie E; Morgan, Jennifer I; Kelly, Helen M

    2010-10-01

    The purpose of the study was to gain an understanding of the nursing phenomenon, confidence, from the experience of nurses in the nursing subculture of critical care. Leininger's theory of cultural care diversity and universality guided this qualitative descriptive study. Questions derived from the sunrise model were used to elicit nurses' perspectives about cultural and social structures that exist within the critical care nursing subculture and the influence that these factors have on confidence. Twenty-eight critical care nurses from a large Canadian healthcare organization participated in semistructured interviews about confidence. Five themes arose from the descriptions provided by the participants. The three themes, tenuously navigating initiation rituals, deliberately developing holistic supportive relationships, and assimilating clinical decision-making rules were identified as social and cultural factors related to confidence. The remaining two themes, preserving a sense of security despite barriers and accommodating to diverse challenges, were identified as environmental factors related to confidence. Practice and research implications within the culture of critical care nursing are discussed in relation to each of the themes.

  18. Professional confidence: a concept analysis.

    Science.gov (United States)

    Holland, Kathlyn; Middleton, Lyn; Uys, Leana

    2012-03-01

    Professional confidence is a concept that is frequently used and or implied in occupational therapy literature, but often without specifying its meaning. Rodgers's Model of Concept Analysis was used to analyse the term "professional confidence". Published research obtained from a federated search in four health sciences databases was used to inform the concept analysis. The definitions, attributes, antecedents, and consequences of professional confidence as evidenced in the literature are discussed. Surrogate terms and related concepts are identified, and a model case of the concept provided. Based on the analysis, professional confidence can be described as a dynamic, maturing personal belief held by a professional or student. This includes an understanding of and a belief in the role, scope of practice, and significance of the profession, and is based on their capacity to competently fulfil these expectations, fostered through a process of affirming experiences. Developing and fostering professional confidence should be nurtured and valued to the same extent as professional competence, as the former underpins the latter, and both are linked to professional identity.

  19. Sequence Curriculum: High School to College. Middlesex Community College/Haddam-Killingworth High School. Final Report.

    Science.gov (United States)

    Middlesex Community Coll., Middletown, CT.

    Through a collaborative effort between Middlesex Community College (MxCC) and Haddam-Killingworth High School (HKHS), students taking specific high school courses in television production, broadcast journalism, electronics, and photography are granted college credit by MxCC upon admission to the college's Broadcast Communication Program. The…

  20. A Reference Viral Database (RVDB) To Enhance Bioinformatics Analysis of High-Throughput Sequencing for Novel Virus Detection.

    Science.gov (United States)

    Goodacre, Norman; Aljanahi, Aisha; Nandakumar, Subhiksha; Mikailov, Mike; Khan, Arifa S

    2018-01-01

    Detection of distantly related viruses by high-throughput sequencing (HTS) is bioinformatically challenging because of the lack of a public database containing all viral sequences, without abundant nonviral sequences, which can extend runtime and obscure viral hits. Our reference viral database (RVDB) includes all viral, virus-related, and virus-like nucleotide sequences (excluding bacterial viruses), regardless of length, and with overall reduced cellular sequences. Semantic selection criteria (SEM-I) were used to select viral sequences from GenBank, resulting in a first-generation viral database (VDB). This database was manually and computationally reviewed, resulting in refined, semantic selection criteria (SEM-R), which were applied to a new download of updated GenBank sequences to create a second-generation VDB. Viral entries in the latter were clustered at 98% by CD-HIT-EST to reduce redundancy while retaining high viral sequence diversity. The viral identity of the clustered representative sequences (creps) was confirmed by BLAST searches in NCBI databases and HMMER searches in PFAM and DFAM databases. The resulting RVDB contained a broad representation of viral families, sequence diversity, and a reduced cellular content; it includes full-length and partial sequences and endogenous nonretroviral elements, endogenous retroviruses, and retrotransposons. Testing of RVDBv10.2, with an in-house HTS transcriptomic data set indicated a significantly faster run for virus detection than interrogating the entirety of the NCBI nonredundant nucleotide database, which contains all viral sequences but also nonviral sequences. RVDB is publically available for facilitating HTS analysis, particularly for novel virus detection. It is meant to be updated on a regular basis to include new viral sequences added to GenBank. IMPORTANCE To facilitate bioinformatics analysis of high-throughput sequencing (HTS) data for the detection of both known and novel viruses, we have

  1. Targeting Low Career Confidence Using the Career Planning Confidence Scale

    Science.gov (United States)

    McAuliffe, Garrett; Jurgens, Jill C.; Pickering, Worth; Calliotte, James; Macera, Anthony; Zerwas, Steven

    2006-01-01

    The authors describe the development and validation of a test of career planning confidence that makes possible the targeting of specific problem issues in employment counseling. The scale, developed using a rational process and the authors' experience with clients, was tested for criterion-related validity against 2 other measures. The scale…

  2. Single nucleus genome sequencing reveals high similarity among nuclei of an endomycorrhizal fungus.

    Directory of Open Access Journals (Sweden)

    Kui Lin

    2014-01-01

    Full Text Available Nuclei of arbuscular endomycorrhizal fungi have been described as highly diverse due to their asexual nature and absence of a single cell stage with only one nucleus. This has raised fundamental questions concerning speciation, selection and transmission of the genetic make-up to next generations. Although this concept has become textbook knowledge, it is only based on studying a few loci, including 45S rDNA. To provide a more comprehensive insight into the genetic makeup of arbuscular endomycorrhizal fungi, we applied de novo genome sequencing of individual nuclei of Rhizophagus irregularis. This revealed a surprisingly low level of polymorphism between nuclei. In contrast, within a nucleus, the 45S rDNA repeat unit turned out to be highly diverged. This finding demystifies a long-lasting hypothesis on the complex genetic makeup of arbuscular endomycorrhizal fungi. Subsequent genome assembly resulted in the first draft reference genome sequence of an arbuscular endomycorrhizal fungus. Its length is 141 Mbps, representing over 27,000 protein-coding gene models. We used the genomic sequence to reinvestigate the phylogenetic relationships of Rhizophagus irregularis with other fungal phyla. This unambiguously demonstrated that Glomeromycota are more closely related to Mucoromycotina than to its postulated sister Dikarya.

  3. International Interlaboratory Digital PCR Study Demonstrating High Reproducibility for the Measurement of a Rare Sequence Variant.

    Science.gov (United States)

    Whale, Alexandra S; Devonshire, Alison S; Karlin-Neumann, George; Regan, Jack; Javier, Leanne; Cowen, Simon; Fernandez-Gonzalez, Ana; Jones, Gerwyn M; Redshaw, Nicholas; Beck, Julia; Berger, Andreas W; Combaret, Valérie; Dahl Kjersgaard, Nina; Davis, Lisa; Fina, Frederic; Forshew, Tim; Fredslund Andersen, Rikke; Galbiati, Silvia; González Hernández, Álvaro; Haynes, Charles A; Janku, Filip; Lacave, Roger; Lee, Justin; Mistry, Vilas; Pender, Alexandra; Pradines, Anne; Proudhon, Charlotte; Saal, Lao H; Stieglitz, Elliot; Ulrich, Bryan; Foy, Carole A; Parkes, Helen; Tzonev, Svilen; Huggett, Jim F

    2017-02-07

    This study tested the claim that digital PCR (dPCR) can offer highly reproducible quantitative measurements in disparate laboratories. Twenty-one laboratories measured four blinded samples containing different quantities of a KRAS fragment encoding G12D, an important genetic marker for guiding therapy of certain cancers. This marker is challenging to quantify reproducibly using quantitative PCR (qPCR) or next generation sequencing (NGS) due to the presence of competing wild type sequences and the need for calibration. Using dPCR, 18 laboratories were able to quantify the G12D marker within 12% of each other in all samples. Three laboratories appeared to measure consistently outlying results; however, proper application of a follow-up analysis recommendation rectified their data. Our findings show that dPCR has demonstrable reproducibility across a large number of laboratories without calibration. This could enable the reproducible application of molecular stratification to guide therapy and, potentially, for molecular diagnostics.

  4. Improving High-Throughput Sequencing Approaches for Reconstructing the Evolutionary Dynamics of Upper Paleolithic Human Groups

    DEFF Research Database (Denmark)

    Seguin-Orlando, Andaine

    the development and testing of innovative molecular approaches aiming at improving the amount of informative HTS data one can recover from ancient DNA extracts. We have characterized important ligation and amplification biases in the sequencing library building and enrichment steps, which can impede further...... been mainly driven by the development of High-Throughput DNA Sequencing (HTS) technologies but also by the implementation of novel molecular tools tailored to the manipulation of ultra short and damaged DNA molecules. Our ability to retrieve traces of genetic material has tremendously improved, pushing......, that impact on the overall efficacy of the method. In a second part, we implemented some of these molecular tools to the processing of five Upper Paleolithic human samples from the Kostenki and Sunghir sites in Western Eurasia, in order to reconstruct the deep genomic history of European populations...

  5. HTSstation: a web application and open-access libraries for high-throughput sequencing data analysis.

    Science.gov (United States)

    David, Fabrice P A; Delafontaine, Julien; Carat, Solenne; Ross, Frederick J; Lefebvre, Gregory; Jarosz, Yohan; Sinclair, Lucas; Noordermeer, Daan; Rougemont, Jacques; Leleu, Marion

    2014-01-01

    The HTSstation analysis portal is a suite of simple web forms coupled to modular analysis pipelines for various applications of High-Throughput Sequencing including ChIP-seq, RNA-seq, 4C-seq and re-sequencing. HTSstation offers biologists the possibility to rapidly investigate their HTS data using an intuitive web application with heuristically pre-defined parameters. A number of open-source software components have been implemented and can be used to build, configure and run HTS analysis pipelines reactively. Besides, our programming framework empowers developers with the possibility to design their own workflows and integrate additional third-party software. The HTSstation web application is accessible at http://htsstation.epfl.ch.

  6. High throughput sequencing identifies chilling responsive genes in sweetpotato (Ipomoea batatas Lam.) during storage.

    Science.gov (United States)

    Xie, Zeyi; Zhou, Zhilin; Li, Hongmin; Yu, Jingjing; Jiang, Jiaojiao; Tang, Zhonghou; Ma, Daifu; Zhang, Baohong; Han, Yonghua; Li, Zongyun

    2018-05-21

    Sweetpotato (Ipomoea batatas L.) is a globally important economic food crop. It belongs to Convolvulaceae family and origins in the tropics; however, sweetpotato is sensitive to cold stress during storage. In this study, we performed transcriptome sequencing to investigate the sweetpotato response to chilling stress during storage. A total of 110,110 unigenes were generated via high-throughput sequencing. Differentially expressed genes (DEGs) analysis showed that 18,681 genes were up-regulated and 21,983 genes were down-regulated in low temperature condition. Many DEGs were related to the cell membrane system, antioxidant enzymes, carbohydrate metabolism, and hormone metabolism, which are potentially associated with sweetpotato resistance to low temperature. The existence of DEGs suggests a molecular basis for the biochemical and physiological consequences of sweetpotato in low temperature storage conditions. Our analysis will provide a new target for enhancement of sweetpotato cold stress tolerance in postharvest storage through genetic manipulation. Copyright © 2018. Published by Elsevier Inc.

  7. 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

  8. Sedimentary dynamics and high-frequency sequence stratigraphy of the southwestern slope of Great Bahama Bank

    Science.gov (United States)

    Wunsch, Marco; Betzler, Christian; Eberli, Gregor P.; Lindhorst, Sebastian; Lüdmann, Thomas; Reijmer, John J. G.

    2018-01-01

    New geophysical data from the leeward slope of Great Bahama Bank show how contour currents shape the slope and induce re-sedimentation processes. Along slope segments with high current control, drift migration and current winnowing at the toe of slope form a deep moat. Here, the slope progradation is inhibited by large channel incisions and the accumulation of large mass transport complexes, triggered by current winnowing. In areas where the slope is bathed by weaker currents, the accumulation of mass transport complexes and channel incision is rather controlled by the position of the sea level. Large slope failures were triggered during the Mid-Pleistocene transition and Mid-Brunhes event, both periods characterized by changes in the cyclicity or the amplitude of sea-level fluctuations. Within the seismic stratigraphic framework of third order sequences, four sequences of higher order were identified in the succession of the upper Pleistocene. These higher order sequences also show clear differences in function of the slope exposure to contour currents. Two stochastic models emphasize the role of the contour currents and slope morphology in the facies distribution in the upper Pleistocene sequences. In areas of high current influence the interplay of erosional and depositional processes form a complex facies pattern with downslope and along strike facies alterations. In zones with lower current influence, major facies alternations occur predominately in downslope direction, and a layer-cake pattern characterizes the along strike direction. Therefore, this study highlights that contour currents are an underestimated driver for the sediment distribution and architecture of carbonate slopes.

  9. Growing confidence, building skills | IDRC - International ...

    International Development Research Centre (IDRC) Digital Library (Canada)

    In 2012 Rashid explored the influence of think tanks on policy in Bangladesh, as well as their relationships with international donors and media. In 2014, he explored two-way student exchanges between Canadian and ... his IDRC experience “gave me the confidence to conduct high quality research in social sciences.”.

  10. Contrasting Academic Behavioural Confidence in Mexican and European Psychology Students

    Science.gov (United States)

    Ochoa, Alma Rosa Aguila; Sander, Paul

    2012-01-01

    Introduction: Research with the Academic Behavioural Confidence scale using European students has shown that students have high levels of confidence in their academic abilities. It is generally accepted that people in more collectivist cultures have more realistic confidence levels in contrast to the overconfidence seen in individualistic European…

  11. Self Confidence Spillovers and Motivated Beliefs

    DEFF Research Database (Denmark)

    Banerjee, Ritwik; Gupta, Nabanita Datta; Villeval, Marie Claire

    that success when competing in a task increases the performers’ self-confidence and competitiveness in the subsequent task. We also find that such spillovers affect the self-confidence of low-status individuals more than that of high-status individuals. Receiving good news under Affirmative Action, however......Is success in a task used strategically by individuals to motivate their beliefs prior to taking action in a subsequent, unrelated, task? Also, is the distortion of beliefs reinforced for individuals who have lower status in society? Conducting an artefactual field experiment in India, we show...

  12. Confidence-building and Canadian leadership

    Energy Technology Data Exchange (ETDEWEB)

    Cleminson, F.R. [Dept. of Foreign Affairs and International Trade, Verification, Non-Proliferation, Arms Control and Disarmament Div (IDA), Ottawa, Ontario (Canada)

    1998-07-01

    Confidence-building has come into its own as a 'tool of choice' in facilitating the non-proliferation, arms control and disarmament (NACD) agenda, whether regional or global. From the Middle East Peace Process (MEPP) to the ASEAN Intersessional Group on Confidence-Building (ARF ISG on CBMS), confidence-building has assumed a central profile in regional terms. In the Four Power Talks begun in Geneva on December 9, 1997, the United States identified confidence-building as one of two subject areas for initial discussion as part of a structured peace process between North and South Korea. Thus, with CBMs assuming such a high profile internationally, it seems prudent for Canadians to pause and take stock of the significant role which Canada has already played in the conceptual development of the process over the last two decades. Since the Helsinki accords of 1975, Canada has developed a significant expertise in this area through an unbroken series of original, basic research projects. These have contributed to defining the process internationally from concept to implementation. Today, these studies represent a solid and unique Departmental investment in basic research from which to draw in meeting Canada's current commitments to multilateral initiatives in the area of confidence-building and to provide a 'step up' in terms of future-oriented leadership. (author)

  13. Confidence-building and Canadian leadership

    International Nuclear Information System (INIS)

    Cleminson, F.R.

    1998-01-01

    Confidence-building has come into its own as a 'tool of choice' in facilitating the non-proliferation, arms control and disarmament (NACD) agenda, whether regional or global. From the Middle East Peace Process (MEPP) to the ASEAN Intersessional Group on Confidence-Building (ARF ISG on CBMS), confidence-building has assumed a central profile in regional terms. In the Four Power Talks begun in Geneva on December 9, 1997, the United States identified confidence-building as one of two subject areas for initial discussion as part of a structured peace process between North and South Korea. Thus, with CBMs assuming such a high profile internationally, it seems prudent for Canadians to pause and take stock of the significant role which Canada has already played in the conceptual development of the process over the last two decades. Since the Helsinki accords of 1975, Canada has developed a significant expertise in this area through an unbroken series of original, basic research projects. These have contributed to defining the process internationally from concept to implementation. Today, these studies represent a solid and unique Departmental investment in basic research from which to draw in meeting Canada's current commitments to multilateral initiatives in the area of confidence-building and to provide a 'step up' in terms of future-oriented leadership. (author)

  14. Selection of mRNA 5'-untranslated region sequence with high translation efficiency through ribosome display

    International Nuclear Information System (INIS)

    Mie, Masayasu; Shimizu, Shun; Takahashi, Fumio; Kobatake, Eiry

    2008-01-01

    The 5'-untranslated region (5'-UTR) of mRNAs functions as a translation enhancer, promoting translation efficiency. Many in vitro translation systems exhibit a reduced efficiency in protein translation due to decreased translation initiation. The use of a 5'-UTR sequence with high translation efficiency greatly enhances protein production in these systems. In this study, we have developed an in vitro selection system that favors 5'-UTRs with high translation efficiency using a ribosome display technique. A 5'-UTR random library, comprised of 5'-UTRs tagged with a His-tag and Renilla luciferase (R-luc) fusion, were in vitro translated in rabbit reticulocytes. By limiting the translation period, only mRNAs with high translation efficiency were translated. During translation, mRNA, ribosome and translated R-luc with His-tag formed ternary complexes. They were collected with translated His-tag using Ni-particles. Extracted mRNA from ternary complex was amplified using RT-PCR and sequenced. Finally, 5'-UTR with high translation efficiency was obtained from random 5'-UTR library

  15. Robust misinterpretation of confidence intervals

    NARCIS (Netherlands)

    Hoekstra, Rink; Morey, Richard; Rouder, Jeffrey N.; Wagenmakers, Eric-Jan

    2014-01-01

    Null hypothesis significance testing (NHST) is undoubtedly the most common inferential technique used to justify claims in the social sciences. However, even staunch defenders of NHST agree that its outcomes are often misinterpreted. Confidence intervals (CIs) have frequently been proposed as a more

  16. Target-dependent enrichment of virions determines the reduction of high-throughput sequencing in virus discovery.

    Directory of Open Access Journals (Sweden)

    Randi Holm Jensen

    Full Text Available Viral infections cause many different diseases stemming both from well-characterized viral pathogens but also from emerging viruses, and the search for novel viruses continues to be of great importance. High-throughput sequencing is an important technology for this purpose. However, viral nucleic acids often constitute a minute proportion of the total genetic material in a sample from infected tissue. Techniques to enrich viral targets in high-throughput sequencing have been reported, but the sensitivity of such methods is not well established. This study compares different library preparation techniques targeting both DNA and RNA with and without virion enrichment. By optimizing the selection of intact virus particles, both by physical and enzymatic approaches, we assessed the effectiveness of the specific enrichment of viral sequences as compared to non-enriched sample preparations by selectively looking for and counting read sequences obtained from shotgun sequencing. Using shotgun sequencing of total DNA or RNA, viral targets were detected at concentrations corresponding to the predicted level, providing a foundation for estimating the effectiveness of virion enrichment. Virion enrichment typically produced a 1000-fold increase in the proportion of DNA virus sequences. For RNA virions the gain was less pronounced with a maximum 13-fold increase. This enrichment varied between the different sample concentrations, with no clear trend. Despite that less sequencing was required to identify target sequences, it was not evident from our data that a lower detection level was achieved by virion enrichment compared to shotgun sequencing.

  17. [Study on Microbial Diversity of Peri-implantitis Subgingival by High-throughput Sequencing].

    Science.gov (United States)

    Li, Zhi-jie; Wang, Shao-guo; Li, Yue-hong; Tu, Dong-xiang; Liu, Shi-yun; Nie, Hong-bing; Li, Zhi-qiang; Zhang, Ju-mei

    2015-07-01

    To study microbial diversity of peri-implantitis subgingival with high-throughput sequencing, and investigate microbiological etiology of peri-implantitis. Subgingival plaques were sampled from the patients with peri-implantitis (D group) and non-peri-implantitis subjects (N group). The microbiological diversity of the subgingival plaques was detected by sequencing V4 region of 16S rRNA with Illumina Miseq platform. The diversity of the community structure was analyzed using Mothur software. A total of 156 507 gene sequences were detected in nine samples and 4 402 operational taxonomic units (OTUs) were found. Selenomonas, Pseudomonas, and Fusobacterium were dominant bacteria in D group, while Fusobacterium, Veillonella and Streptococcus were dominant bacteria in N group. Differences between peri-implantitis and non-peri-implantitis bacterial communities were observed at all phylogenetic levels by LEfSe, which was also found in PcoA test. The occurrence of peri-implantitis is not only related to periodontitis pathogenic microbe, but also related with the changes of oral microbial community structure. Treponema, Herbaspirillum, Butyricimonas and Phaeobacte may be closely related to the occurrence and development of peri-implantitis.

  18. Bioassessment of a Drinking Water Reservoir Using Plankton: High Throughput Sequencing vs. Traditional Morphological Method

    Directory of Open Access Journals (Sweden)

    Wanli Gao

    2018-01-01

    Full Text Available Drinking water safety is increasingly perceived as one of the top global environmental issues. Plankton has been commonly used as a bioindicator for water quality in lakes and reservoirs. Recently, DNA sequencing technology has been applied to bioassessment. In this study, we compared the effectiveness of the 16S and 18S rRNA high throughput sequencing method (HTS and the traditional optical microscopy method (TOM in the bioassessment of drinking water quality. Five stations reflecting different habitats and hydrological conditions in Danjiangkou Reservoir, one of the largest drinking water reservoirs in Asia, were sampled May 2016. Non-metric multi-dimensional scaling (NMDS analysis showed that plankton assemblages varied among the stations and the spatial patterns revealed by the two methods were consistent. The correlation between TOM and HTS in a symmetric Procrustes analysis was 0.61, revealing overall good concordance between the two methods. Procrustes analysis also showed that site-specific differences between the two methods varied among the stations. Station Heijizui (H, a site heavily influenced by two tributaries, had the largest difference while station Qushou (Q, a confluence site close to the outlet dam, had the smallest difference between the two methods. Our results show that DNA sequencing has the potential to provide consistent identification of taxa, and reliable bioassessment in a long-term biomonitoring and assessment program for drinking water reservoirs.

  19. HTSeq--a Python framework to work with high-throughput sequencing data.

    Science.gov (United States)

    Anders, Simon; Pyl, Paul Theodor; Huber, Wolfgang

    2015-01-15

    A large choice of tools exists for many standard tasks in the analysis of high-throughput sequencing (HTS) data. However, once a project deviates from standard workflows, custom scripts are needed. We present HTSeq, a Python library to facilitate the rapid development of such scripts. HTSeq offers parsers for many common data formats in HTS projects, as well as classes to represent data, such as genomic coordinates, sequences, sequencing reads, alignments, gene model information and variant calls, and provides data structures that allow for querying via genomic coordinates. We also present htseq-count, a tool developed with HTSeq that preprocesses RNA-Seq data for differential expression analysis by counting the overlap of reads with genes. HTSeq is released as an open-source software under the GNU General Public Licence and available from http://www-huber.embl.de/HTSeq or from the Python Package Index at https://pypi.python.org/pypi/HTSeq. © The Author 2014. Published by Oxford University Press.

  20. Purification of High Molecular Weight Genomic DNA from Powdery Mildew for Long-Read Sequencing.

    Science.gov (United States)

    Feehan, Joanna M; Scheibel, Katherine E; Bourras, Salim; Underwood, William; Keller, Beat; Somerville, Shauna C

    2017-03-31

    The powdery mildew fungi are a group of economically important fungal plant pathogens. Relatively little is known about the molecular biology and genetics of these pathogens, in part due to a lack of well-developed genetic and genomic resources. These organisms have large, repetitive genomes, which have made genome sequencing and assembly prohibitively difficult. Here, we describe methods for the collection, extraction, purification and quality control assessment of high molecular weight genomic DNA from one powdery mildew species, Golovinomyces cichoracearum. The protocol described includes mechanical disruption of spores followed by an optimized phenol/chloroform genomic DNA extraction. A typical yield was 7 µg DNA per 150 mg conidia. The genomic DNA that is isolated using this procedure is suitable for long-read sequencing (i.e., > 48.5 kbp). Quality control measures to ensure the size, yield, and purity of the genomic DNA are also described in this method. Sequencing of the genomic DNA of the quality described here will allow for the assembly and comparison of multiple powdery mildew genomes, which in turn will lead to a better understanding and improved control of this agricultural pathogen.

  1. Analysis of transposable elements in the genome of Asparagus officinalis from high coverage sequence data.

    Science.gov (United States)

    Li, Shu-Fen; Gao, Wu-Jun; Zhao, Xin-Peng; Dong, Tian-Yu; Deng, Chuan-Liang; Lu, Long-Dou

    2014-01-01

    Asparagus officinalis is an economically and nutritionally important vegetable crop that is widely cultivated and is used as a model dioecious species to study plant sex determination and sex chromosome evolution. To improve our understanding of its genome composition, especially with respect to transposable elements (TEs), which make up the majority of the genome, we performed Illumina HiSeq2000 sequencing of both male and female asparagus genomes followed by bioinformatics analysis. We generated 17 Gb of sequence (12×coverage) and assembled them into 163,406 scaffolds with a total cumulated length of 400 Mbp, which represent about 30% of asparagus genome. Overall, TEs masked about 53% of the A. officinalis assembly. Majority of the identified TEs belonged to LTR retrotransposons, which constitute about 28% of genomic DNA, with Ty1/copia elements being more diverse and accumulated to higher copy numbers than Ty3/gypsy. Compared with LTR retrotransposons, non-LTR retrotransposons and DNA transposons were relatively rare. In addition, comparison of the abundance of the TE groups between male and female genomes showed that the overall TE composition was highly similar, with only slight differences in the abundance of several TE groups, which is consistent with the relatively recent origin of asparagus sex chromosomes. This study greatly improves our knowledge of the repetitive sequence construction of asparagus, which facilitates the identification of TEs responsible for the early evolution of plant sex chromosomes and is helpful for further studies on this dioecious plant.

  2. elPrep: High-Performance Preparation of Sequence Alignment/Map Files for Variant Calling.

    Directory of Open Access Journals (Sweden)

    Charlotte Herzeel

    Full Text Available elPrep is a high-performance tool for preparing sequence alignment/map files for variant calling in sequencing pipelines. It can be used as a replacement for SAMtools and Picard for preparation steps such as filtering, sorting, marking duplicates, reordering contigs, and so on, while producing identical results. What sets elPrep apart is its software architecture that allows executing preparation pipelines by making only a single pass through the data, no matter how many preparation steps are used in the pipeline. elPrep is designed as a multithreaded application that runs entirely in memory, avoids repeated file I/O, and merges the computation of several preparation steps to significantly speed up the execution time. For example, for a preparation pipeline of five steps on a whole-exome BAM file (NA12878, we reduce the execution time from about 1:40 hours, when using a combination of SAMtools and Picard, to about 15 minutes when using elPrep, while utilising the same server resources, here 48 threads and 23GB of RAM. For the same pipeline on whole-genome data (NA12878, elPrep reduces the runtime from 24 hours to less than 5 hours. As a typical clinical study may contain sequencing data for hundreds of patients, elPrep can remove several hundreds of hours of computing time, and thus substantially reduce analysis time and cost.

  3. New var reconstruction algorithm exposes high var sequence diversity in a single geographic location in Mali.

    Science.gov (United States)

    Dara, Antoine; Drábek, Elliott F; Travassos, Mark A; Moser, Kara A; Delcher, Arthur L; Su, Qi; Hostelley, Timothy; Coulibaly, Drissa; Daou, Modibo; Dembele, Ahmadou; Diarra, Issa; Kone, Abdoulaye K; Kouriba, Bourema; Laurens, Matthew B; Niangaly, Amadou; Traore, Karim; Tolo, Youssouf; Fraser, Claire M; Thera, Mahamadou A; Djimde, Abdoulaye A; Doumbo, Ogobara K; Plowe, Christopher V; Silva, Joana C

    2017-03-28

    Encoded by the var gene family, highly variable Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP1) proteins mediate tissue-specific cytoadherence of infected erythrocytes, resulting in immune evasion and severe malaria disease. Sequencing and assembling the 40-60 var gene complement for individual infections has been notoriously difficult, impeding molecular epidemiological studies and the assessment of particular var elements as subunit vaccine candidates. We developed and validated a novel algorithm, Exon-Targeted Hybrid Assembly (ETHA), to perform targeted assembly of var gene sequences, based on a combination of Pacific Biosciences and Illumina data. Using ETHA, we characterized the repertoire of var genes in 12 samples from uncomplicated malaria infections in children from a single Malian village and showed them to be as genetically diverse as vars from isolates from around the globe. The gene var2csa, a member of the var family associated with placental malaria pathogenesis, was present in each genome, as were vars previously associated with severe malaria. ETHA, a tool to discover novel var sequences from clinical samples, will aid the understanding of malaria pathogenesis and inform the design of malaria vaccines based on PfEMP1. ETHA is available at: https://sourceforge.net/projects/etha/ .

  4. Investigation of Human Cancers for Retrovirus by Low-Stringency Target Enrichment and High-Throughput Sequencing

    DEFF Research Database (Denmark)

    Vinner, Lasse; Mourier, Tobias; Friis-Nielsen, Jens

    2015-01-01

    -stringency in-solution hybridization method enables detection of discovery of hitherto unknown viral sequences by high-throughput sequencing. The sensitivity was sufficient to detect retroviral...... sequences in clinical samples. We used this method to conduct an investigation for novel retrovirus in samples from three cancer types. In accordance with recent studies our investigation revealed no retroviral infections in human B-cell lymphoma cells, cutaneous T-cell lymphoma or colorectal cancer...

  5. TIMPs of parasitic helminths - a large-scale analysis of high-throughput sequence datasets.

    Science.gov (United States)

    Cantacessi, Cinzia; Hofmann, Andreas; Pickering, Darren; Navarro, Severine; Mitreva, Makedonka; Loukas, Alex

    2013-05-30

    Tissue inhibitors of metalloproteases (TIMPs) are a multifunctional family of proteins that orchestrate extracellular matrix turnover, tissue remodelling and other cellular processes. In parasitic helminths, such as hookworms, TIMPs have been proposed to play key roles in the host-parasite interplay, including invasion of and establishment in the vertebrate animal hosts. Currently, knowledge of helminth TIMPs is limited to a small number of studies on canine hookworms, whereas no information is available on the occurrence of TIMPs in other parasitic helminths causing neglected diseases. In the present study, we conducted a large-scale investigation of TIMP proteins of a range of neglected human parasites including the hookworm Necator americanus, the roundworm Ascaris suum, the liver flukes Clonorchis sinensis and Opisthorchis viverrini, as well as the schistosome blood flukes. This entailed mining available transcriptomic and/or genomic sequence datasets for the presence of homologues of known TIMPs, predicting secondary structures of defined protein sequences, systematic phylogenetic analyses and assessment of differential expression of genes encoding putative TIMPs in the developmental stages of A. suum, N. americanus and Schistosoma haematobium which infect the mammalian hosts. A total of 15 protein sequences with high homology to known eukaryotic TIMPs were predicted from the complement of sequence data available for parasitic helminths and subjected to in-depth bioinformatic analyses. Supported by the availability of gene manipulation technologies such as RNA interference and/or transgenesis, this work provides a basis for future functional explorations of helminth TIMPs and, in particular, of their role/s in fundamental biological pathways linked to long-term establishment in the vertebrate hosts, with a view towards the development of novel approaches for the control of neglected helminthiases.

  6. Identification of microRNAs from Eugenia uniflora by high-throughput sequencing and bioinformatics analysis.

    Science.gov (United States)

    Guzman, Frank; Almerão, Mauricio P; Körbes, Ana P; Loss-Morais, Guilherme; Margis, Rogerio

    2012-01-01

    microRNAs or miRNAs are small non-coding regulatory RNAs that play important functions in the regulation of gene expression at the post-transcriptional level by targeting mRNAs for degradation or inhibiting protein translation. Eugenia uniflora is a plant native to tropical America with pharmacological and ecological importance, and there have been no previous studies concerning its gene expression and regulation. To date, no miRNAs have been reported in Myrtaceae species. Small RNA and RNA-seq libraries were constructed to identify miRNAs and pre-miRNAs in Eugenia uniflora. Solexa technology was used to perform high throughput sequencing of the library, and the data obtained were analyzed using bioinformatics tools. From 14,489,131 small RNA clean reads, we obtained 1,852,722 mature miRNA sequences representing 45 conserved families that have been identified in other plant species. Further analysis using contigs assembled from RNA-seq allowed the prediction of secondary structures of 25 known and 17 novel pre-miRNAs. The expression of twenty-seven identified miRNAs was also validated using RT-PCR assays. Potential targets were predicted for the most abundant mature miRNAs in the identified pre-miRNAs based on sequence homology. This study is the first large scale identification of miRNAs and their potential targets from a species of the Myrtaceae family without genomic sequence resources. Our study provides more information about the evolutionary conservation of the regulatory network of miRNAs in plants and highlights species-specific miRNAs.

  7. Highly diverse microbiota in dental root canals in cases of apical periodontitis (data of illumina sequencing).

    Science.gov (United States)

    Vengerfeldt, Veiko; Špilka, Katerina; Saag, Mare; Preem, Jens-Konrad; Oopkaup, Kristjan; Truu, Jaak; Mändar, Reet

    2014-11-01

    Chronic apical periodontitis (CAP) is a frequent condition that has a considerable effect on a patient's quality of life. We aimed to reveal root canal microbial communities in antibiotic-naive patients by applying Illumina sequencing (Illumina Inc, San Diego, CA). Samples were collected under strict aseptic conditions from 12 teeth (5 with primary CAP, 3 with secondary CAP, and 4 with a periapical abscess [PA]) and characterized by profiling the microbial community on the basis of the V6 hypervariable region of the 16S ribosomal RNA gene by using Illumina HiSeq2000 sequencing combinatorial sequence-tagged polymerase chain reaction products. Root canal specimens displayed highly polymicrobial communities in all 3 patient groups. One sample contained 5-8 (mean = 6.5) phyla of bacteria. The most numerous were Firmicutes and Bacteroidetes, but Actinobacteria, Fusobacteria, Proteobacteria, Spirochaetes, Tenericutes, and Synergistetes were also present in most of the patients. One sample contained 30-70 different operational taxonomic units; the mean (± standard deviation) was lower in the primary CAP group (36 ± 4) than in the PA (45 ± 4) and secondary CAP (43 ± 13) groups (P < .05). The communities were individually different, but anaerobic bacteria predominated as the rule. Enterococcus faecalis was found only in patients with secondary CAP. One PA sample displayed a significantly high proportion (47%) of Proteobacteria, mainly at the expense of Janthinobacterium lividum. This study provided an in-depth characterization of the microbiota of periapical tissues, revealing highly polymicrobial communities and minor differences between the study groups. A full understanding of the etiology of periodontal disease will only be possible through further in-depth systems-level analyses of the host-microbiome interaction. Copyright © 2014 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  8. IDBA-UD: a de novo assembler for single-cell and metagenomic sequencing data with highly uneven depth.

    Science.gov (United States)

    Peng, Yu; Leung, Henry C M; Yiu, S M; Chin, Francis Y L

    2012-06-01

    Next-generation sequencing allows us to sequence reads from a microbial environment using single-cell sequencing or metagenomic sequencing technologies. However, both technologies suffer from the problem that sequencing depth of different regions of a genome or genomes from different species are highly uneven. Most existing genome assemblers usually have an assumption that sequencing depths are even. These assemblers fail to construct correct long contigs. We introduce the IDBA-UD algorithm that is based on the de Bruijn graph approach for assembling reads from single-cell sequencing or metagenomic sequencing technologies with uneven sequencing depths. Several non-trivial techniques have been employed to tackle the problems. Instead of using a simple threshold, we use multiple depthrelative thresholds to remove erroneous k-mers in both low-depth and high-depth regions. The technique of local assembly with paired-end information is used to solve the branch problem of low-depth short repeat regions. To speed up the process, an error correction step is conducted to correct reads of high-depth regions that can be aligned to highconfident contigs. Comparison of the performances of IDBA-UD and existing assemblers (Velvet, Velvet-SC, SOAPdenovo and Meta-IDBA) for different datasets, shows that IDBA-UD can reconstruct longer contigs with higher accuracy. The IDBA-UD toolkit is available at our website http://www.cs.hku.hk/~alse/idba_ud

  9. Use of high throughput sequencing to study oomycete communities in soil and roots

    DEFF Research Database (Denmark)

    Sapkota, Rumakanta; Nicolaisen, Mogens

    2015-01-01

    taxonomic units from symptomatic lesions in carrot resulted in 94% of the reads belonging to oomycetes with a dominance of species of Pythium that are known to be involved in causing cavity spot. Moreover, soil samples showed that 95% of the sequences could be assigned to oomycetes including Pythium......, Aphanomyces, Peronospora, Saprolegnia and Phytophthora. A high proportion of oomycete reads was consistently present in all symptomatic lesions and soil samples showing the versatility of the strategy and thus demonstrating the usefulness of the method in plant and soil DNA background....

  10. Application of High-Throughput Next-Generation Sequencing for HLA Typing on Buccal Extracted DNA: Results from over 10,000 Donor Recruitment Samples.

    Directory of Open Access Journals (Sweden)

    Yuxin Yin

    Full Text Available Unambiguous HLA typing is important in hematopoietic stem cell transplantation (HSCT, HLA disease association studies, and solid organ transplantation. However, current molecular typing methods only interrogate the antigen recognition site (ARS of HLA genes, resulting in many cis-trans ambiguities that require additional typing methods to resolve. Here we report high-resolution HLA typing of 10,063 National Marrow Donor Program (NMDP registry donors using long-range PCR by next generation sequencing (NGS approach on buccal swab DNA.Multiplex long-range PCR primers amplified the full-length of HLA class I genes (A, B, C from promotor to 3' UTR. Class II genes (DRB1, DQB1 were amplified from exon 2 through part of exon 4. PCR amplicons were pooled and sheared using Covaris fragmentation. Library preparation was performed using the Illumina TruSeq Nano kit on the Beckman FX automated platform. Each sample was tagged with a unique barcode, followed by 2×250 bp paired-end sequencing on the Illumina MiSeq. HLA typing was assigned using Omixon Twin software that combines two independent computational algorithms to ensure high confidence in allele calling. Consensus sequence and typing results were reported in Histoimmunogenetics Markup Language (HML format. All homozygous alleles were confirmed by Luminex SSO typing and exon novelties were confirmed by Sanger sequencing.Using this automated workflow, over 10,063 NMDP registry donors were successfully typed under high-resolution by NGS. Despite known challenges of nucleic acid degradation and low DNA concentration commonly associated with buccal-based specimens, 97.8% of samples were successfully amplified using long-range PCR. Among these, 98.2% were successfully reported by NGS, with an accuracy rate of 99.84% in an independent blind Quality Control audit performed by the NDMP. In this study, NGS-HLA typing identified 23 null alleles (0.023%, 92 rare alleles (0.091% and 42 exon novelties (0.042%.Long

  11. Application of High-Throughput Next-Generation Sequencing for HLA Typing on Buccal Extracted DNA: Results from over 10,000 Donor Recruitment Samples.

    Science.gov (United States)

    Yin, Yuxin; Lan, James H; Nguyen, David; Valenzuela, Nicole; Takemura, Ping; Bolon, Yung-Tsi; Springer, Brianna; Saito, Katsuyuki; Zheng, Ying; Hague, Tim; Pasztor, Agnes; Horvath, Gyorgy; Rigo, Krisztina; Reed, Elaine F; Zhang, Qiuheng

    2016-01-01

    Unambiguous HLA typing is important in hematopoietic stem cell transplantation (HSCT), HLA disease association studies, and solid organ transplantation. However, current molecular typing methods only interrogate the antigen recognition site (ARS) of HLA genes, resulting in many cis-trans ambiguities that require additional typing methods to resolve. Here we report high-resolution HLA typing of 10,063 National Marrow Donor Program (NMDP) registry donors using long-range PCR by next generation sequencing (NGS) approach on buccal swab DNA. Multiplex long-range PCR primers amplified the full-length of HLA class I genes (A, B, C) from promotor to 3' UTR. Class II genes (DRB1, DQB1) were amplified from exon 2 through part of exon 4. PCR amplicons were pooled and sheared using Covaris fragmentation. Library preparation was performed using the Illumina TruSeq Nano kit on the Beckman FX automated platform. Each sample was tagged with a unique barcode, followed by 2×250 bp paired-end sequencing on the Illumina MiSeq. HLA typing was assigned using Omixon Twin software that combines two independent computational algorithms to ensure high confidence in allele calling. Consensus sequence and typing results were reported in Histoimmunogenetics Markup Language (HML) format. All homozygous alleles were confirmed by Luminex SSO typing and exon novelties were confirmed by Sanger sequencing. Using this automated workflow, over 10,063 NMDP registry donors were successfully typed under high-resolution by NGS. Despite known challenges of nucleic acid degradation and low DNA concentration commonly associated with buccal-based specimens, 97.8% of samples were successfully amplified using long-range PCR. Among these, 98.2% were successfully reported by NGS, with an accuracy rate of 99.84% in an independent blind Quality Control audit performed by the NDMP. In this study, NGS-HLA typing identified 23 null alleles (0.023%), 92 rare alleles (0.091%) and 42 exon novelties (0.042%). Long

  12. Highly sulfated hexasaccharide sequences isolated from chondroitin sulfate of shark fin cartilage: insights into the sugar sequences with bioactivities.

    Science.gov (United States)

    Mizumoto, Shuji; Murakoshi, Saori; Kalayanamitra, Kittiwan; Deepa, Sarama Sathyaseelan; Fukui, Shigeyuki; Kongtawelert, Prachya; Yamada, Shuhei; Sugahara, Kazuyuki

    2013-02-01

    Chondroitin sulfate (CS) chains regulate the development of the central nervous system in vertebrates and are linear polysaccharides consisting of variously sulfated repeating disaccharides, [-4GlcUAβ1-3GalNAcβ1-](n), where GlcUA and GalNAc represent D-glucuronic acid and N-acetyl-D-galactosamine, respectively. CS chains containing D-disaccharide units [GlcUA(2-O-sulfate)-GalNAc(6-O-sulfate)] are involved in the development of cerebellar Purkinje cells and neurite outgrowth-promoting activity through interaction with a neurotrophic factor, pleiotrophin, resulting in the regulation of signaling. In this study, to obtain further structural information on the CS chains containing d-disaccharide units involved in brain development, oligosaccharides containing D-units were isolated from a shark fin cartilage. Seven novel hexasaccharide sequences, ΔO-D-D, ΔA-D-D, ΔC-D-D, ΔE-A-D, ΔD-D-C, ΔE-D-D and ΔA-B-D, in addition to three previously reported sequences, ΔC-A-D, ΔC-D-C and ΔA-D-A, were isolated from a CS preparation of shark fin cartilage after exhaustive digestion with chondroitinase AC-I, which cannot act on the galactosaminidic linkages bound to D-units. The symbol Δ stands for a 4,5-unsaturated bond of uronic acids, whereas A, B, C, D, E and O represent [GlcUA-GalNAc(4-O-sulfate)], [GlcUA(2-O-sulfate)-GalNAc(4-O-sulfate)], [GlcUA-GalNAc(6-O-sulfate)], [GlcUA(2-O-sulfate)-GalNAc(6-O-sulfate)], [GlcUA-GalNAc(4-O-, 6-O-sulfate)] and [GlcUA-GalNAc], respectively. In binding studies using an anti-CS monoclonal antibody, MO-225, the epitopes of which are involved in cerebellar development in mammals, novel epitope structures, ΔA-D-A, ΔA-D-D and ΔA-B-D, were revealed. Hexasaccharides containing two consecutive D-units or a B-unit will be useful for the structural and functional analyses of CS chains particularly in the neuroglycobiological fields.

  13. Cis-acting regulatory sequences promote high-frequency gene conversion between repeated sequences in mammalian cells.

    Science.gov (United States)

    Raynard, Steven J; Baker, Mark D

    2004-01-01

    In mammalian cells, little is known about the nature of recombination-prone regions of the genome. Previously, we reported that the immunoglobulin heavy chain (IgH) mu locus behaved as a hotspot for mitotic, intrachromosomal gene conversion (GC) between repeated mu constant (Cmu) regions in mouse hybridoma cells. To investigate whether elements within the mu gene regulatory region were required for hotspot activity, gene targeting was used to delete a 9.1 kb segment encompassing the mu gene promoter (Pmu), enhancer (Emu) and switch region (Smu) from the locus. In these cell lines, GC between the Cmu repeats was significantly reduced, indicating that this 'recombination-enhancing sequence' (RES) is necessary for GC hotspot activity at the IgH locus. Importantly, the RES fragment stimulated GC when appended to the same Cmu repeats integrated at ectopic genomic sites. We also show that deletion of Emu and flanking matrix attachment regions (MARs) from the RES abolishes GC hotspot activity at the IgH locus. However, no stimulation of ectopic GC was observed with the Emu/MARs fragment alone. Finally, we provide evidence that no correlation exists between the level of transcription and GC promoted by the RES. We suggest a model whereby Emu/MARS enhances mitotic GC at the endogenous IgH mu locus by effecting chromatin modifications in adjacent DNA.

  14. Transcriptomic analysis of Petunia hybrida in response to salt stress using high throughput RNA sequencing.

    Directory of Open Access Journals (Sweden)

    Gonzalo H Villarino

    Full Text Available Salinity and drought stress are the primary cause of crop losses worldwide. In sodic saline soils sodium chloride (NaCl disrupts normal plant growth and development. The complex interactions of plant systems with abiotic stress have made RNA sequencing a more holistic and appealing approach to study transcriptome level responses in a single cell and/or tissue. In this work, we determined the Petunia transcriptome response to NaCl stress by sequencing leaf samples and assembling 196 million Illumina reads with Trinity software. Using our reference transcriptome we identified more than 7,000 genes that were differentially expressed within 24 h of acute NaCl stress. The proposed transcriptome can also be used as an excellent tool for biological and bioinformatics in the absence of an available Petunia genome and it is available at the SOL Genomics Network (SGN http://solgenomics.net. Genes related to regulation of reactive oxygen species, transport, and signal transductions as well as novel and undescribed transcripts were among those differentially expressed in response to salt stress. The candidate genes identified in this study can be applied as markers for breeding or to genetically engineer plants to enhance salt tolerance. Gene Ontology analyses indicated that most of the NaCl damage happened at 24 h inducing genotoxicity, affecting transport and organelles due to the high concentration of Na+ ions. Finally, we report a modification to the library preparation protocol whereby cDNA samples were bar-coded with non-HPLC purified primers, without affecting the quality and quantity of the RNA-seq data. The methodological improvement presented here could substantially reduce the cost of sample preparation for future high-throughput RNA sequencing experiments.

  15. Transcriptomic analysis of Petunia hybrida in response to salt stress using high throughput RNA sequencing.

    Science.gov (United States)

    Villarino, Gonzalo H; Bombarely, Aureliano; Giovannoni, James J; Scanlon, Michael J; Mattson, Neil S

    2014-01-01

    Salinity and drought stress are the primary cause of crop losses worldwide. In sodic saline soils sodium chloride (NaCl) disrupts normal plant growth and development. The complex interactions of plant systems with abiotic stress have made RNA sequencing a more holistic and appealing approach to study transcriptome level responses in a single cell and/or tissue. In this work, we determined the Petunia transcriptome response to NaCl stress by sequencing leaf samples and assembling 196 million Illumina reads with Trinity software. Using our reference transcriptome we identified more than 7,000 genes that were differentially expressed within 24 h of acute NaCl stress. The proposed transcriptome can also be used as an excellent tool for biological and bioinformatics in the absence of an available Petunia genome and it is available at the SOL Genomics Network (SGN) http://solgenomics.net. Genes related to regulation of reactive oxygen species, transport, and signal transductions as well as novel and undescribed transcripts were among those differentially expressed in response to salt stress. The candidate genes identified in this study can be applied as markers for breeding or to genetically engineer plants to enhance salt tolerance. Gene Ontology analyses indicated that most of the NaCl damage happened at 24 h inducing genotoxicity, affecting transport and organelles due to the high concentration of Na+ ions. Finally, we report a modification to the library preparation protocol whereby cDNA samples were bar-coded with non-HPLC purified primers, without affecting the quality and quantity of the RNA-seq data. The methodological improvement presented here could substantially reduce the cost of sample preparation for future high-throughput RNA sequencing experiments.

  16. Methodology for building confidence measures

    Science.gov (United States)

    Bramson, Aaron L.

    2004-04-01

    This paper presents a generalized methodology for propagating known or estimated levels of individual source document truth reliability to determine the confidence level of a combined output. Initial document certainty levels are augmented by (i) combining the reliability measures of multiply sources, (ii) incorporating the truth reinforcement of related elements, and (iii) incorporating the importance of the individual elements for determining the probability of truth for the whole. The result is a measure of confidence in system output based on the establishing of links among the truth values of inputs. This methodology was developed for application to a multi-component situation awareness tool under development at the Air Force Research Laboratory in Rome, New York. Determining how improvements in data quality and the variety of documents collected affect the probability of a correct situational detection helps optimize the performance of the tool overall.

  17. Screening of whole genome sequences identified high-impact variants for stallion fertility.

    Science.gov (United States)

    Schrimpf, Rahel; Gottschalk, Maren; Metzger, Julia; Martinsson, Gunilla; Sieme, Harald; Distl, Ottmar

    2016-04-14

    Stallion fertility is an economically important trait due to the increase of artificial insemination in horses. The availability of whole genome sequence data facilitates identification of rare high-impact variants contributing to stallion fertility. The aim of our study was to genotype rare high-impact variants retrieved from next-generation sequencing (NGS)-data of 11 horses in order to unravel harmful genetic variants in large samples of stallions. Gene ontology (GO) terms and search results from public databases were used to obtain a comprehensive list of human und mice genes predicted to participate in the regulation of male reproduction. The corresponding equine orthologous genes were searched in whole genome sequence data of seven stallions and four mares and filtered for high-impact genetic variants using SnpEFF, SIFT and Polyphen 2 software. All genetic variants with the missing homozygous mutant genotype were genotyped on 337 fertile stallions of 19 breeds using KASP genotyping assays or PCR-RFLP. Mixed linear model analysis was employed for an association analysis with de-regressed estimated breeding values of the paternal component of the pregnancy rate per estrus (EBV-PAT). We screened next generation sequenced data of whole genomes from 11 horses for equine genetic variants in 1194 human and mice genes involved in male fertility and linked through common gene ontology (GO) with male reproductive processes. Variants were filtered for high-impact on protein structure and validated through SIFT and Polyphen 2. Only those genetic variants were followed up when the homozygote mutant genotype was missing in the detection sample comprising 11 horses. After this filtering process, 17 single nucleotide polymorphism (SNPs) were left. These SNPs were genotyped in 337 fertile stallions of 19 breeds using KASP genotyping assays or PCR-RFLP. An association analysis in 216 Hanoverian stallions revealed a significant association of the splice-site disruption variant

  18. High levels of diversity characterize mandrill (Mandrillus sphinx) Mhc-DRB sequences.

    Science.gov (United States)

    Abbott, Kristin M; Wickings, E Jean; Knapp, Leslie A

    2006-08-01

    The major histocompatibility complex (MHC) is highly polymorphic in most primate species studied thus far. The rhesus macaque (Macaca mulatta) has been studied extensively and the Mhc-DRB region demonstrates variability similar to humans. The extent of MHC diversity is relatively unknown for other Old World monkeys (OWM), especially among genera other than Macaca. A molecular survey of the Mhc-DRB region in mandrills (Mandrillus sphinx) revealed extensive variability, suggesting that other OWMs may also possess high levels of Mhc-DRB polymorphism. In the present study, 33 Mhc-DRB loci were identified from only 13 animals. Eleven were wild-born and presumed to be unrelated and two were captive-born twins. Two to seven different sequences were identified for each individual, suggesting that some mandrills may have as many as four Mhc-DRB loci on a single haplotype. From these sequences, representatives of at least six Mhc-DRB loci or lineages were identified. As observed in other primates, some new lineages may have arisen through the process of gene conversion. These findings indicate that mandrills have Mhc-DRB diversity not unlike rhesus macaques and humans.

  19. Perchlorate reduction by hydrogen autotrophic bacteria and microbial community analysis using high-throughput sequencing.

    Science.gov (United States)

    Wan, Dongjin; Liu, Yongde; Niu, Zhenhua; Xiao, Shuhu; Li, Daorong

    2016-02-01

    Hydrogen autotrophic reduction of perchlorate have advantages of high removal efficiency and harmless to drinking water. But so far the reported information about the microbial community structure was comparatively limited, changes in the biodiversity and the dominant bacteria during acclimation process required detailed study. In this study, perchlorate-reducing hydrogen autotrophic bacteria were acclimated by hydrogen aeration from activated sludge. For the first time, high-throughput sequencing was applied to analyze changes in biodiversity and the dominant bacteria during acclimation process. The Michaelis-Menten model described the perchlorate reduction kinetics well. Model parameters q(max) and K(s) were 2.521-3.245 (mg ClO4(-)/gVSS h) and 5.44-8.23 (mg/l), respectively. Microbial perchlorate reduction occurred across at pH range 5.0-11.0; removal was highest at pH 9.0. The enriched mixed bacteria could use perchlorate, nitrate and sulfate as electron accepter, and the sequence of preference was: NO3(-) > ClO4(-) > SO4(2-). Compared to the feed culture, biodiversity decreased greatly during acclimation process, the microbial community structure gradually stabilized after 9 acclimation cycles. The Thauera genus related to Rhodocyclales was the dominated perchlorate reducing bacteria (PRB) in the mixed culture.

  20. Penicillium arizonense, a new, genome sequenced fungal species, reveals a high chemical diversity in secreted metabolites

    Science.gov (United States)

    Grijseels, Sietske; Nielsen, Jens Christian; Randelovic, Milica; Nielsen, Jens; Nielsen, Kristian Fog; Workman, Mhairi; Frisvad, Jens Christian

    2016-01-01

    A new soil-borne species belonging to the Penicillium section Canescentia is described, Penicillium arizonense sp. nov. (type strain CBS 141311T = IBT 12289T). The genome was sequenced and assembled into 33.7 Mb containing 12,502 predicted genes. A phylogenetic assessment based on marker genes confirmed the grouping of P. arizonense within section Canescentia. Compared to related species, P. arizonense proved to encode a high number of proteins involved in carbohydrate metabolism, in particular hemicellulases. Mining the genome for genes involved in secondary metabolite biosynthesis resulted in the identification of 62 putative biosynthetic gene clusters. Extracts of P. arizonense were analysed for secondary metabolites and austalides, pyripyropenes, tryptoquivalines, fumagillin, pseurotin A, curvulinic acid and xanthoepocin were detected. A comparative analysis against known pathways enabled the proposal of biosynthetic gene clusters in P. arizonense responsible for the synthesis of all detected compounds except curvulinic acid. The capacity to produce biomass degrading enzymes and the identification of a high chemical diversity in secreted bioactive secondary metabolites, offers a broad range of potential industrial applications for the new species P. arizonense. The description and availability of the genome sequence of P. arizonense, further provides the basis for biotechnological exploitation of this species. PMID:27739446

  1. 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.

  2. Exome sequencing identifies highly recurrent MED12 somatic mutations in breast fibroadenoma.

    Science.gov (United States)

    Lim, Weng Khong; Ong, Choon Kiat; Tan, Jing; Thike, Aye Aye; Ng, Cedric Chuan Young; Rajasegaran, Vikneswari; Myint, Swe Swe; Nagarajan, Sanjanaa; Nasir, Nur Diyana Md; McPherson, John R; Cutcutache, Ioana; Poore, Gregory; Tay, Su Ting; Ooi, Wei Siong; Tan, Veronique Kiak Mien; Hartman, Mikael; Ong, Kong Wee; Tan, Benita K T; Rozen, Steven G; Tan, Puay Hoon; Tan, Patrick; Teh, Bin Tean

    2014-08-01

    Fibroadenomas are the most common breast tumors in women under 30 (refs. 1,2). Exome sequencing of eight fibroadenomas with matching whole-blood samples revealed recurrent somatic mutations solely in MED12, which encodes a Mediator complex subunit. Targeted sequencing of an additional 90 fibroadenomas confirmed highly frequent MED12 exon 2 mutations (58/98, 59%) that are probably somatic, with 71% of mutations occurring in codon 44. Using laser capture microdissection, we show that MED12 fibroadenoma mutations are present in stromal but not epithelial mammary cells. Expression profiling of MED12-mutated and wild-type fibroadenomas revealed that MED12 mutations are associated with dysregulated estrogen signaling and extracellular matrix organization. The fibroadenoma MED12 mutation spectrum is nearly identical to that of previously reported MED12 lesions in uterine leiomyoma but not those of other tumors. Benign tumors of the breast and uterus, both of which are key target tissues of estrogen, may thus share a common genetic basis underpinned by highly frequent and specific MED12 mutations.

  3. Bayesian reconstruction of photon interaction sequences for high-resolution PET detectors

    Energy Technology Data Exchange (ETDEWEB)

    Pratx, Guillem; Levin, Craig S [Molecular Imaging Program at Stanford, Department of Radiology, Stanford, CA (United States)], E-mail: cslevin@stanford.edu

    2009-09-07

    Realizing the full potential of high-resolution positron emission tomography (PET) systems involves accurately positioning events in which the annihilation photon deposits all its energy across multiple detector elements. Reconstructing the complete sequence of interactions of each photon provides a reliable way to select the earliest interaction because it ensures that all the interactions are consistent with one another. Bayesian estimation forms a natural framework to maximize the consistency of the sequence with the measurements while taking into account the physics of {gamma}-ray transport. An inherently statistical method, it accounts for the uncertainty in the measured energy and position of each interaction. An algorithm based on maximum a posteriori (MAP) was evaluated for computer simulations. For a high-resolution PET system based on cadmium zinc telluride detectors, 93.8% of the recorded coincidences involved at least one photon multiple-interactions event (PMIE). The MAP estimate of the first interaction was accurate for 85.2% of the single photons. This represents a two-fold reduction in the number of mispositioned events compared to minimum pair distance, a simpler yet efficient positioning method. The point-spread function of the system presented lower tails and higher peak value when MAP was used. This translated into improved image quality, which we quantified by studying contrast and spatial resolution gains.

  4. Digital PCR provides sensitive and absolute calibration for high throughput sequencing

    Directory of Open Access Journals (Sweden)

    Fan H Christina

    2009-03-01

    Full Text Available Abstract Background Next-generation DNA sequencing on the 454, Solexa, and SOLiD platforms requires absolute calibration of the number of molecules to be sequenced. This requirement has two unfavorable consequences. First, large amounts of sample-typically micrograms-are needed for library preparation, thereby limiting the scope of samples which can be sequenced. For many applications, including metagenomics and the sequencing of ancient, forensic, and clinical samples, the quantity of input DNA can be critically limiting. Second, each library requires a titration sequencing run, thereby increasing the cost and lowering the throughput of sequencing. Results We demonstrate the use of digital PCR to accurately quantify 454 and Solexa sequencing libraries, enabling the preparation of sequencing libraries from nanogram quantities of input material while eliminating costly and time-consuming titration runs of the sequencer. We successfully sequenced low-nanogram scale bacterial and mammalian DNA samples on the 454 FLX and Solexa DNA sequencing platforms. This study is the first to definitively demonstrate the successful sequencing of picogram quantities of input DNA on the 454 platform, reducing the sample requirement more than 1000-fold without pre-amplification and the associated bias and reduction in library depth. Conclusion The digital PCR assay allows absolute quantification of sequencing libraries, eliminates uncertainties associated with the construction and application of standard curves to PCR-based quantification, and with a coefficient of variation close to 10%, is sufficiently precise to enable direct sequencing without titration runs.

  5. Markovian Model in High Order Sequence Prediction From Log-Motif Patterns in Agbada Paralic Section, Niger Delta, Nigeria

    International Nuclear Information System (INIS)

    Olabode, S. O.; Adekoya, J. A.

    2002-01-01

    Markovian model in the elucidation of high order sequence was applied to repetitive events of regressive and transgressive phases in the Agbada paralic section Niger Delta. The repetitive events are made up of delta front, delta topset and fluvio-deltaic sediments. The sediments consist of sands, sandstones, siltstones and shales in various proportions. Five wells: MN1, AA1, NP2, NP6 and NP8 were studied.Summary of biostratigraphic report and well log-motif patterns was used to delineate the third order depositional sequences in the wells.Various Markovian properties - observed transition frequency matrix, observed transition probability matrix, fixed probability vector, expected random matrix (randomised transition matrix) and difference matrix were determined for stacked high order sequence (high frequency cyclic events) nested within the third-order sequences using the log-motif patterns for the various sand bodies and shales. Flow diagrams were constructed for each of the depositional sequences to know the likely occurrence of number of cycles.Upward transition matrix between the log-motif patterns and flow diagram to elucidate cyclicity show that the overall regressive sequence of the Niger Delta has been modified by deltaic depositional elements and fluctuations in sea level. The predictions of higher order sequence within third order sequences from Markovian Properties provide good basis for correlation within the depositional sequences. The model has also been used to decipher the dominant depositional processes during the formation of the sequences. Discrete reservoir intervals and seal potentials within the sequences were also predicted from the flow diagrams constructed

  6. Penicillium arizonense, a new, genome sequenced fungal species, reveals a high chemical diversity in secreted metabolites

    DEFF Research Database (Denmark)

    Grijseels, Sietske; Nielsen, Jens Christian; Randelovic, Milica

    2016-01-01

    A new soil-borne species belonging to the Penicillium section Canescentia is described, Penicillium arizonense sp. nov. (type strain CBS 141311T = IBT 12289T). The genome was sequenced and assembled into 33.7 Mb containing 12,502 predicted genes. A phylogenetic assessment based on marker genes...... confirmed the grouping of P. arizonense within section Canescentia. Compared to related species, P. arizonense proved to encode a high number of proteins involved in carbohydrate metabolism, in particular hemicellulases. Mining the genome for genes involved in secondary metabolite biosynthesis resulted...... of biosynthetic gene clusters in P. arizonense responsible for the synthesis of all detected compounds except curvulinic acid. The capacity to produce biomass degrading enzymes and the identification of a high chemical diversity in secreted bioactive secondary metabolites, offers a broad range of potential...

  7. Highly Stereoselective Synthesis of Cyclopentanes bearing Four Stereocenters by a Rhodium Carbene–Initiated Domino Sequence

    Science.gov (United States)

    Parr, Brendan T.; Davies, Huw M. L.

    2014-01-01

    Stereoselective synthesis of a cyclopentane nucleus by convergent annulations constitutes a significant challenge for synthetic chemists. Though a number of biologically relevant cyclopentane natural products are known, more often than not, the cyclopentane core is assembled in a stepwise fashion due to lack of efficient annulation strategies. Herein, we report the rhodium-catalyzed reactions of vinyldiazoacetates with (E)-1,3-disubstituted 2-butenols generate cyclopentanes, containing four new stereogenic centers with very high levels of stereoselectivity (99% ee, >97 : 3 dr). The reaction proceeds by a carbene–initiated domino sequence consisting of five distinct steps: rhodium–bound oxonium ylide formation, [2,3]-sigmatropic rearrangement, oxy-Cope rearrangement, enol–keto tautomerization, and finally an intramolecular carbonyl ene reaction. A systematic study is presented detailing how to control chirality transfer in each of the four stereo-defining steps of the cascade, consummating in the development of a highly stereoselective process. PMID:25082301

  8. The monoclonal S9.6 antibody exhibits highly variable binding affinities towards different R-loop sequences.

    Directory of Open Access Journals (Sweden)

    Fabian König

    Full Text Available The monoclonal antibody S9.6 is a widely-used tool to purify, analyse and quantify R-loop structures in cells. A previous study using the surface plasmon resonance technology and a single-chain variable fragment (scFv of S9.6 showed high affinity (0.6 nM for DNA-RNA and also a high affinity (2.7 nM for RNA-RNA hybrids. We used the microscale thermophoresis method allowing surface independent interaction studies and electromobility shift assays to evaluate additional RNA-DNA hybrid sequences and to quantify the binding affinities of the S9.6 antibody with respect to distinct sequences and their GC-content. Our results confirm high affinity binding to previously analysed sequences, but reveals that binding affinities are highly sequence specific. Our study presents R-loop sequences that independent of GC-content and in different sequence variations exhibit either no binding, binding affinities in the micromolar range and as well high affinity binding in the nanomolar range. Our study questions the usefulness of the S9.6 antibody in the quantitative analysis of R-loop sequences in vivo.

  9. High diagnostic yield of syndromic intellectual disability by targeted next-generation sequencing.

    Science.gov (United States)

    Martínez, Francisco; Caro-Llopis, Alfonso; Roselló, Mónica; Oltra, Silvestre; Mayo, Sonia; Monfort, Sandra; Orellana, Carmen

    2017-02-01

    Intellectual disability is a very complex condition where more than 600 genes have been reported. Due to this extraordinary heterogeneity, a large proportion of patients remain without a specific diagnosis and genetic counselling. The need for new methodological strategies in order to detect a greater number of mutations in multiple genes is therefore crucial. In this work, we screened a large panel of 1256 genes (646 pathogenic, 610 candidate) by next-generation sequencing to determine the molecular aetiology of syndromic intellectual disability. A total of 92 patients, negative for previous genetic analyses, were studied together with their parents. Clinically relevant variants were validated by conventional sequencing. A definitive diagnosis was achieved in 29 families by testing the 646 known pathogenic genes. Mutations were found in 25 different genes, where only the genes KMT2D, KMT2A and MED13L were found mutated in more than one patient. A preponderance of de novo mutations was noted even among the X linked conditions. Additionally, seven de novo probably pathogenic mutations were found in the candidate genes AGO1, JARID2, SIN3B, FBXO11, MAP3K7, HDAC2 and SMARCC2. Altogether, this means a diagnostic yield of 39% of the cases (95% CI 30% to 49%). The developed panel proved to be efficient and suitable for the genetic diagnosis of syndromic intellectual disability in a clinical setting. Next-generation sequencing has the potential for high-throughput identification of genetic variations, although the challenges of an adequate clinical interpretation of these variants and the knowledge on further unknown genes causing intellectual disability remain to be solved. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

  10. Genetic profiles of cervical tumors by high-throughput sequencing for personalized medical care

    International Nuclear Information System (INIS)

    Muller, Etienne; Brault, Baptiste; Holmes, Allyson; Legros, Angelina; Jeannot, Emmanuelle; Campitelli, Maura; Rousselin, Antoine; Goardon, Nicolas; Frébourg, Thierry; Krieger, Sophie; Crouet, Hubert; Nicolas, Alain; Sastre, Xavier; Vaur, Dominique; Castéra, Laurent

    2015-01-01

    Cancer treatment is facing major evolution since the advent of targeted therapies. Building genetic profiles could predict sensitivity or resistance to these therapies and highlight disease-specific abnormalities, supporting personalized patient care. In the context of biomedical research and clinical diagnosis, our laboratory has developed an oncogenic panel comprised of 226 genes and a dedicated bioinformatic pipeline to explore somatic mutations in cervical carcinomas, using high-throughput sequencing. Twenty-nine tumors were sequenced for exons within 226 genes. The automated pipeline used includes a database and a filtration system dedicated to identifying mutations of interest and excluding false positive and germline mutations. One-hundred and seventy-six total mutational events were found among the 29 tumors. Our cervical tumor mutational landscape shows that most mutations are found in PIK3CA (E545K, E542K) and KRAS (G12D, G13D) and others in FBXW7 (R465C, R505G, R479Q). Mutations have also been found in ALK (V1149L, A1266T) and EGFR (T259M). These results showed that 48% of patients display at least one deleterious mutation in genes that have been already targeted by the Food and Drug Administration approved therapies. Considering deleterious mutations, 59% of patients could be eligible for clinical trials. Sequencing hundreds of genes in a clinical context has become feasible, in terms of time and cost. In the near future, such an analysis could be a part of a battery of examinations along the diagnosis and treatment of cancer, helping to detect sensitivity or resistance to targeted therapies and allow advancements towards personalized oncology

  11. Diversity and Structure of Diazotrophic Communities in Mangrove Rhizosphere, Revealed by High-Throughput Sequencing.

    Science.gov (United States)

    Zhang, Yanying; Yang, Qingsong; Ling, Juan; Van Nostrand, Joy D; Shi, Zhou; Zhou, Jizhong; Dong, Junde

    2017-01-01

    Diazotrophic communities make an essential contribution to the productivity through providing new nitrogen. However, knowledge of the roles that both mangrove tree species and geochemical parameters play in shaping mangove rhizosphere diazotrophic communities is still elusive. Here, a comprehensive examination of the diversity and structure of microbial communities in the rhizospheres of three mangrove species, Rhizophora apiculata , Avicennia marina , and Ceriops tagal , was undertaken using high - throughput sequencing of the 16S rRNA and nifH genes. Our results revealed a great diversity of both the total microbial composition and the diazotrophic composition specifically in the mangrove rhizosphere. Deltaproteobacteria and Gammaproteobacteria were both ubiquitous and dominant, comprising an average of 45.87 and 86.66% of total microbial and diazotrophic communities, respectively. Sulfate-reducing bacteria belonging to the Desulfobacteraceae and Desulfovibrionaceae were the dominant diazotrophs. Community statistical analyses suggested that both mangrove tree species and additional environmental variables played important roles in shaping total microbial and potential diazotroph communities in mangrove rhizospheres. In contrast to the total microbial community investigated by analysis of 16S rRNA gene sequences, most of the dominant diazotrophic groups identified by nifH gene sequences were significantly different among mangrove species. The dominant diazotrophs of the family Desulfobacteraceae were positively correlated with total phosphorus, but negatively correlated with the nitrogen to phosphorus ratio. The Pseudomonadaceae were positively correlated with the concentration of available potassium, suggesting that diazotrophs potentially play an important role in biogeochemical cycles, such as those of nitrogen, phosphorus, sulfur, and potassium, in the mangrove ecosystem.

  12. Diversity and Structure of Diazotrophic Communities in Mangrove Rhizosphere, Revealed by High-Throughput Sequencing

    Directory of Open Access Journals (Sweden)

    Yanying Zhang

    2017-10-01

    Full Text Available Diazotrophic communities make an essential contribution to the productivity through providing new nitrogen. However, knowledge of the roles that both mangrove tree species and geochemical parameters play in shaping mangove rhizosphere diazotrophic communities is still elusive. Here, a comprehensive examination of the diversity and structure of microbial communities in the rhizospheres of three mangrove species, Rhizophora apiculata, Avicennia marina, and Ceriops tagal, was undertaken using high-throughput sequencing of the 16S rRNA and nifH genes. Our results revealed a great diversity of both the total microbial composition and the diazotrophic composition specifically in the mangrove rhizosphere. Deltaproteobacteria and Gammaproteobacteria were both ubiquitous and dominant, comprising an average of 45.87 and 86.66% of total microbial and diazotrophic communities, respectively. Sulfate-reducing bacteria belonging to the Desulfobacteraceae and Desulfovibrionaceae were the dominant diazotrophs. Community statistical analyses suggested that both mangrove tree species and additional environmental variables played important roles in shaping total microbial and potential diazotroph communities in mangrove rhizospheres. In contrast to the total microbial community investigated by analysis of 16S rRNA gene sequences, most of the dominant diazotrophic groups identified by nifH gene sequences were significantly different among mangrove species. The dominant diazotrophs of the family Desulfobacteraceae were positively correlated with total phosphorus, but negatively correlated with the nitrogen to phosphorus ratio. The Pseudomonadaceae were positively correlated with the concentration of available potassium, suggesting that diazotrophs potentially play an important role in biogeochemical cycles, such as those of nitrogen, phosphorus, sulfur, and potassium, in the mangrove ecosystem.

  13. Unprecedented high-resolution view of bacterial operon architecture revealed by RNA sequencing.

    Science.gov (United States)

    Conway, Tyrrell; Creecy, James P; Maddox, Scott M; Grissom, Joe E; Conkle, Trevor L; Shadid, Tyler M; Teramoto, Jun; San Miguel, Phillip; Shimada, Tomohiro; Ishihama, Akira; Mori, Hirotada; Wanner, Barry L

    2014-07-08

    We analyzed the transcriptome of Escherichia coli K-12 by strand-specific RNA sequencing at single-nucleotide resolution during steady-state (logarithmic-phase) growth and upon entry into stationary phase in glucose minimal medium. To generate high-resolution transcriptome maps, we developed an organizational schema which showed that in practice only three features are required to define operon architecture: the promoter, terminator, and deep RNA sequence read coverage. We precisely annotated 2,122 promoters and 1,774 terminators, defining 1,510 operons with an average of 1.98 genes per operon. Our analyses revealed an unprecedented view of E. coli operon architecture. A large proportion (36%) of operons are complex with internal promoters or terminators that generate multiple transcription units. For 43% of operons, we observed differential expression of polycistronic genes, despite being in the same operons, indicating that E. coli operon architecture allows fine-tuning of gene expression. We found that 276 of 370 convergent operons terminate inefficiently, generating complementary 3' transcript ends which overlap on average by 286 nucleotides, and 136 of 388 divergent operons have promoters arranged such that their 5' ends overlap on average by 168 nucleotides. We found 89 antisense transcripts of 397-nucleotide average length, 7 unannotated transcripts within intergenic regions, and 18 sense transcripts that completely overlap operons on the opposite strand. Of 519 overlapping transcripts, 75% correspond to sequences that are highly conserved in E. coli (>50 genomes). Our data extend recent studies showing unexpected transcriptome complexity in several bacteria and suggest that antisense RNA regulation is widespread. Importance: We precisely mapped the 5' and 3' ends of RNA transcripts across the E. coli K-12 genome by using a single-nucleotide analytical approach. Our resulting high-resolution transcriptome maps show that ca. one-third of E. coli operons are

  14. Why barcode? High-throughput multiplex sequencing of mitochondrial genomes for molecular systematics.

    Science.gov (United States)

    Timmermans, M J T N; Dodsworth, S; Culverwell, C L; Bocak, L; Ahrens, D; Littlewood, D T J; Pons, J; Vogler, A P

    2010-11-01

    Mitochondrial genome sequences are important markers for phylogenetics but taxon sampling remains sporadic because of the great effort and cost required to acquire full-length sequences. Here, we demonstrate a simple, cost-effective way to sequence the full complement of protein coding mitochondrial genes from pooled samples using the 454/Roche platform. Multiplexing was achieved without the need for expensive indexing tags ('barcodes'). The method was trialled with a set of long-range polymerase chain reaction (PCR) fragments from 30 species of Coleoptera (beetles) sequenced in a 1/16th sector of a sequencing plate. Long contigs were produced from the pooled sequences with sequencing depths ranging from ∼10 to 100× per contig. Species identity of individual contigs was established via three 'bait' sequences matching disparate parts of the mitochondrial genome obtained by conventional PCR and Sanger sequencing. This proved that assembly of contigs from the sequencing pool was correct. Our study produced sequences for 21 nearly complete and seven partial sets of protein coding mitochondrial genes. Combined with existing sequences for 25 taxa, an improved estimate of basal relationships in Coleoptera was obtained. The procedure could be employed routinely for mitochondrial genome sequencing at the species level, to provide improved species 'barcodes' that currently use the cox1 gene only.

  15. Efficient DNA fingerprinting based on the targeted sequencing of active retrotransposon insertion sites using a bench-top high-throughput sequencing platform.

    Science.gov (United States)

    Monden, Yuki; Yamamoto, Ayaka; Shindo, Akiko; Tahara, Makoto

    2014-10-01

    In many crop species, DNA fingerprinting is required for the precise identification of cultivars to protect the rights of breeders. Many families of retrotransposons have multiple copies throughout the eukaryotic genome and their integrated copies are inherited genetically. Thus, their insertion polymorphisms among cultivars are useful for DNA fingerprinting. In this study, we conducted a DNA fingerprinting based on the insertion polymorphisms of active retrotransposon families (Rtsp-1 and LIb) in sweet potato. Using 38 cultivars, we identified 2,024 insertion sites in the two families with an Illumina MiSeq sequencing platform. Of these insertion sites, 91.4% appeared to be polymorphic among the cultivars and 376 cultivar-specific insertion sites were identified, which were converted directly into cultivar-specific sequence-characterized amplified region (SCAR) markers. A phylogenetic tree was constructed using these insertion sites, which corresponded well with known pedigree information, thereby indicating their suitability for genetic diversity studies. Thus, the genome-wide comparative analysis of active retrotransposon insertion sites using the bench-top MiSeq sequencing platform is highly effective for DNA fingerprinting without any requirement for whole genome sequence information. This approach may facilitate the development of practical polymerase chain reaction-based cultivar diagnostic system and could also be applied to the determination of genetic relationships. © The Author 2014. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

  16. Draft Genome Sequence of Komagataeibacter rhaeticus Strain AF1, a High Producer of Cellulose, Isolated from Kombucha Tea.

    Science.gov (United States)

    Dos Santos, Renato Augusto Corrêa; Berretta, Andresa A; Barud, Hernane da Silva; Ribeiro, Sidney José Lima; González-García, Laura Natalia; Zucchi, Tiago Domingues; Goldman, Gustavo H; Riaño-Pachón, Diego M

    2014-07-24

    Here, we present the draft genome sequence of Komagatabaeicter rhaeticus strain AF1, which was isolated from Kombucha tea and is capable of producing high levels of cellulose. Copyright © 2014 dos Santos et al.

  17. Effects of High Intensity White Noise on Short-Term Memory for Position in a List and Sequence

    Science.gov (United States)

    Daee, Safar; Wilding, J. M.

    1977-01-01

    Seven experiments are described investigating the effecy of high intensity white noise during the visual presentation of words on a number of short-term memory tasks. Examines results relative to position learning and sequence learning. (Editor/RK)

  18. Highly accurate fluorogenic DNA sequencing with information theory-based error correction.

    Science.gov (United States)

    Chen, Zitian; Zhou, Wenxiong; Qiao, Shuo; Kang, Li; Duan, Haifeng; Xie, X Sunney; Huang, Yanyi

    2017-12-01

    Eliminating errors in next-generation DNA sequencing has proved challenging. Here we present error-correction code (ECC) sequencing, a method to greatly improve sequencing accuracy by combining fluorogenic sequencing-by-synthesis (SBS) with an information theory-based error-correction algorithm. ECC embeds redundancy in sequencing reads by creating three orthogonal degenerate sequences, generated by alternate dual-base reactions. This is similar to encoding and decoding strategies that have proved effective in detecting and correcting errors in information communication and storage. We show that, when combined with a fluorogenic SBS chemistry with raw accuracy of 98.1%, ECC sequencing provides single-end, error-free sequences up to 200 bp. ECC approaches should enable accurate identification of extremely rare genomic variations in various applications in biology and medicine.

  19. Viral metagenomics: Analysis of begomoviruses by illumina high-throughput sequencing

    KAUST Repository

    Idris, Ali; Al-Saleh, Mohammed; Piatek, Marek J.; Al-Shahwan, Ibrahim; Ali, Shahjahan; Brown, Judith K.

    2014-01-01

    Traditional DNA sequencing methods are inefficient, lack the ability to discern the least abundant viral sequences, and ineffective for determining the extent of variability in viral populations. Here, populations of single-stranded DNA plant

  20. Leadership by Confidence in Teams

    OpenAIRE

    Kobayashi, Hajime; Suehiro, Hideo

    2008-01-01

    We study endogenous signaling by analyzing a team production problem with endogenous timing. Each agent of the team is privately endowed with some level of confidence about team productivity. Each of them must then commit a level of effort in one of two periods. At the end of each period, each agent observes his partner' s move in this period. Both agents are rewarded by a team output determined by team productivity and total invested effort. Each agent must personally incur the cost of effor...

  1. Artifact free T2{sup *}-weighted imaging at high spatial resolution using segmented EPI sequences

    Energy Technology Data Exchange (ETDEWEB)

    Heiler, Patrick Michael; Schad, Lothar Rudi [Heidelberg Univ., Mannheim (Germany). Computer Assisted Clinical Medicine; Schmitter, Sebastian [German Cancer Research Center, Heidelberg (Germany). Dept. of Medical Physics in Radiology

    2010-07-01

    The aim of this work was the development of novel measurement techniques that acquire high resolution T2{sup *}-weighted datasets in measurement times as short as possible without suffering from noticeable blurring and ghosting artifacts. Therefore, two new measurement techniques were developed that acquire a smoother k-space than generic multi shot echo planar imaging sequences. One is based on the principle of echo train shifting, the other on the reversed gradient method. Simulations and phantom measurements demonstrate that echo train shifting works properly and reduces artifacts in multi shot echo planar imaging. For maximum SNR-efficiency this technique was further improved by adding a second contrast. Both contrasts can be acquired within a prolongation in measurement time by a factor of 1.5, leading to an SNR increase by approximately {radical}2. Furthermore it is demonstrated that the reversed gradient method remarkably reduces artifacts caused by a discontinuous k-space weighting. Assuming sequence parameters as feasible for fMRI experiments, artifact free T2{sup *}-weighted images with a matrix size of 256 x 256 leading to an in-plane resolution in the submillimeter range can be obtained in about 2 s per slice. (orig.)

  2. Massively parallel digital high resolution melt for rapid and absolutely quantitative sequence profiling

    Science.gov (United States)

    Velez, Daniel Ortiz; Mack, Hannah; Jupe, Julietta; Hawker, Sinead; Kulkarni, Ninad; Hedayatnia, Behnam; Zhang, Yang; Lawrence, Shelley; Fraley, Stephanie I.

    2017-02-01

    In clinical diagnostics and pathogen detection, profiling of complex samples for low-level genotypes represents a significant challenge. Advances in speed, sensitivity, and extent of multiplexing of molecular pathogen detection assays are needed to improve patient care. We report the development of an integrated platform enabling the identification of bacterial pathogen DNA sequences in complex samples in less than four hours. The system incorporates a microfluidic chip and instrumentation to accomplish universal PCR amplification, High Resolution Melting (HRM), and machine learning within 20,000 picoliter scale reactions, simultaneously. Clinically relevant concentrations of bacterial DNA molecules are separated by digitization across 20,000 reactions and amplified with universal primers targeting the bacterial 16S gene. Amplification is followed by HRM sequence fingerprinting in all reactions, simultaneously. The resulting bacteria-specific melt curves are identified by Support Vector Machine learning, and individual pathogen loads are quantified. The platform reduces reaction volumes by 99.995% and achieves a greater than 200-fold increase in dynamic range of detection compared to traditional PCR HRM approaches. Type I and II error rates are reduced by 99% and 100% respectively, compared to intercalating dye-based digital PCR (dPCR) methods. This technology could impact a number of quantitative profiling applications, especially infectious disease diagnostics.

  3. Genotyping by PCR and High-Throughput Sequencing of Commercial Probiotic Products Reveals Composition Biases.

    Directory of Open Access Journals (Sweden)

    Wesley Morovic

    2016-11-01

    Full Text Available Recent advances in microbiome research have brought renewed focus on beneficial bacteria, many of which are available in food and dietary supplements. Although probiotics have historically been defined as microorganisms that convey health benefits when ingested in sufficient viable amounts, this description now includes the stipulation well defined strains, encompassing definitive taxonomy for consumer consideration and regulatory oversight. Here, we evaluated 52 commercial dietary supplements covering a range of labeled species, and determined their content using plate counting, targeted genotyping. Additionally, strain identities were assessed using methods recently published by the United States Pharmacopeial Convention. We also determined the relative abundance of individual bacteria by high-throughput sequencing (HTS of the 16S rRNA sequence using paired-end 2x250bp Illumina MiSeq technology. Using multiple methods, we tested the hypothesis that products do contain the quantitative amount of labeled bacteria, and qualitative list of labeled microbial species. We found that 17 samples (33% were below label claim for CFU prior to their expiration dates. A multiplexed-PCR scheme showed that only 30/52 (58% of the products contained a correctly labeled classification, with issues encompassing incorrect taxonomy, missing species and un-labeled species. The HTS revealed that many blended products consisted predominantly of Lactobacillus acidophilus and Bifidobacterium animalis subsp. lactis. These results highlight the need for reliable methods to qualitatively determine the correct taxonomy and quantitatively ascertain the relative amounts of mixed microbial populations in commercial probiotic products.

  4. 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.

  5. Pyicos: a versatile toolkit for the analysis of high-throughput sequencing data.

    Science.gov (United States)

    Althammer, Sonja; González-Vallinas, Juan; Ballaré, Cecilia; Beato, Miguel; Eyras, Eduardo

    2011-12-15

    High-throughput sequencing (HTS) has revolutionized gene regulation studies and is now fundamental for the detection of protein-DNA and protein-RNA binding, as well as for measuring RNA expression. With increasing variety and sequencing depth of HTS datasets, the need for more flexible and memory-efficient tools to analyse them is growing. We describe Pyicos, a powerful toolkit for the analysis of mapped reads from diverse HTS experiments: ChIP-Seq, either punctuated or broad signals, CLIP-Seq and RNA-Seq. We prove the effectiveness of Pyicos to select for significant signals and show that its accuracy is comparable and sometimes superior to that of methods specifically designed for each particular type of experiment. Pyicos facilitates the analysis of a variety of HTS datatypes through its flexibility and memory efficiency, providing a useful framework for data integration into models of regulatory genomics. Open-source software, with tutorials and protocol files, is available at http://regulatorygenomics.upf.edu/pyicos or as a Galaxy server at http://regulatorygenomics.upf.edu/galaxy eduardo.eyras@upf.edu Supplementary data are available at Bioinformatics online.

  6. Aerobic granulation strategy for bioaugmentation of a sequencing batch reactor (SBR) treating high strength pyridine wastewater

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Xiaodong; Chen, Yan [Jiangsu Key Laboratory for Chemical Pollution Control and Resources Reuse, School of Environmental and Biological Engineering, Nanjing University of Science and Technology, Nanjing 210094, Jiangsu Province (China); Zhang, Xin [Jiangsu Key Laboratory for Chemical Pollution Control and Resources Reuse, School of Environmental and Biological Engineering, Nanjing University of Science and Technology, Nanjing 210094, Jiangsu Province (China); Suzhou Institute of Architectural Design Co., Ltd, Suzhou 215021, Jiangsu Province (China); Jiang, Xinbai; Wu, Shijing [Jiangsu Key Laboratory for Chemical Pollution Control and Resources Reuse, School of Environmental and Biological Engineering, Nanjing University of Science and Technology, Nanjing 210094, Jiangsu Province (China); Shen, Jinyou, E-mail: shenjinyou@mail.njust.edu.cn [Jiangsu Key Laboratory for Chemical Pollution Control and Resources Reuse, School of Environmental and Biological Engineering, Nanjing University of Science and Technology, Nanjing 210094, Jiangsu Province (China); Sun, Xiuyun; Li, Jiansheng; Lu, Lude [Jiangsu Key Laboratory for Chemical Pollution Control and Resources Reuse, School of Environmental and Biological Engineering, Nanjing University of Science and Technology, Nanjing 210094, Jiangsu Province (China); Wang, Lianjun, E-mail: wanglj@mail.njust.edu.cn [Jiangsu Key Laboratory for Chemical Pollution Control and Resources Reuse, School of Environmental and Biological Engineering, Nanjing University of Science and Technology, Nanjing 210094, Jiangsu Province (China)

    2015-09-15

    Abstract: Aerobic granules were successfully cultivated in a sequencing batch reactor (SBR), using a single bacterial strain Rhizobium sp. NJUST18 as the inoculum. NJUST18 presented as both a good pyridine degrader and an efficient autoaggregator. Stable granules with diameter of 0.5–1 mm, sludge volume index of 25.6 ± 3.6 mL g{sup −1} and settling velocity of 37.2 ± 2.7 m h{sup −1}, were formed in SBR following 120-day cultivation. These granules exhibited excellent pyridine degradation performance, with maximum volumetric degradation rate (V{sub max}) varied between 1164.5 mg L{sup −1} h{sup −1} and 1867.4 mg L{sup −1} h{sup −1}. High-throughput sequencing analysis exhibited a large shift in microbial community structure, since the SBR was operated under open condition. Paracoccus and Comamonas were found to be the most predominant species in the aerobic granule system after the system had stabilized. The initially inoculated Rhizobium sp. lost its dominance during aerobic granulation. However, the inoculation of Rhizobium sp. played a key role in the start-up process of this bioaugmentation system. This study demonstrated that, in addition to the hydraulic selection pressure during settling and effluent discharge, the selection of aggregating bacterial inocula is equally important for the formation of the aerobic granule.

  7. Aerobic granulation strategy for bioaugmentation of a sequencing batch reactor (SBR) treating high strength pyridine wastewater

    International Nuclear Information System (INIS)

    Liu, Xiaodong; Chen, Yan; Zhang, Xin; Jiang, Xinbai; Wu, Shijing; Shen, Jinyou; Sun, Xiuyun; Li, Jiansheng; Lu, Lude; Wang, Lianjun

    2015-01-01

    Abstract: Aerobic granules were successfully cultivated in a sequencing batch reactor (SBR), using a single bacterial strain Rhizobium sp. NJUST18 as the inoculum. NJUST18 presented as both a good pyridine degrader and an efficient autoaggregator. Stable granules with diameter of 0.5–1 mm, sludge volume index of 25.6 ± 3.6 mL g −1 and settling velocity of 37.2 ± 2.7 m h −1 , were formed in SBR following 120-day cultivation. These granules exhibited excellent pyridine degradation performance, with maximum volumetric degradation rate (V max ) varied between 1164.5 mg L −1 h −1 and 1867.4 mg L −1 h −1 . High-throughput sequencing analysis exhibited a large shift in microbial community structure, since the SBR was operated under open condition. Paracoccus and Comamonas were found to be the most predominant species in the aerobic granule system after the system had stabilized. The initially inoculated Rhizobium sp. lost its dominance during aerobic granulation. However, the inoculation of Rhizobium sp. played a key role in the start-up process of this bioaugmentation system. This study demonstrated that, in addition to the hydraulic selection pressure during settling and effluent discharge, the selection of aggregating bacterial inocula is equally important for the formation of the aerobic granule

  8. Artifact free T2*-weighted imaging at high spatial resolution using segmented EPI sequences

    International Nuclear Information System (INIS)

    Heiler, Patrick Michael; Schad, Lothar Rudi; Schmitter, Sebastian

    2010-01-01

    The aim of this work was the development of novel measurement techniques that acquire high resolution T2 * -weighted datasets in measurement times as short as possible without suffering from noticeable blurring and ghosting artifacts. Therefore, two new measurement techniques were developed that acquire a smoother k-space than generic multi shot echo planar imaging sequences. One is based on the principle of echo train shifting, the other on the reversed gradient method. Simulations and phantom measurements demonstrate that echo train shifting works properly and reduces artifacts in multi shot echo planar imaging. For maximum SNR-efficiency this technique was further improved by adding a second contrast. Both contrasts can be acquired within a prolongation in measurement time by a factor of 1.5, leading to an SNR increase by approximately √2. Furthermore it is demonstrated that the reversed gradient method remarkably reduces artifacts caused by a discontinuous k-space weighting. Assuming sequence parameters as feasible for fMRI experiments, artifact free T2 * -weighted images with a matrix size of 256 x 256 leading to an in-plane resolution in the submillimeter range can be obtained in about 2 s per slice. (orig.)

  9. Social media sentiment and consumer confidence

    OpenAIRE

    Daas, Piet J.H.; Puts, Marco J.H.

    2014-01-01

    Changes in the sentiment of Dutch public social media messages were compared with changes in monthly consumer confidence over a period of three-and-a-half years, revealing that both were highly correlated (up to r = 0.9) and that both series cointegrated. This phenomenon is predominantly affected by changes in the sentiment of all Dutch public Facebook messages. The inclusion of various selections of public Twitter messages improved this association and the response to changes in sentiment. G...

  10. Clinical evaluation of further-developed MRCP sequences in comparison with standard MRCP sequences

    International Nuclear Information System (INIS)

    Hundt, W.; Scheidler, J.; Reiser, M.; Petsch, R.

    2002-01-01

    The purpose of this study was the comparison of technically improved single-shot magnetic resonance cholangiopancreatography (MRCP) sequences with standard single-shot rapid acquisition with relaxation enhancement (RARE) and half-Fourier acquired single-shot turbo spin-echo (HASTE) sequences in evaluating the normal and abnormal biliary duct system. The bile duct system of 45 patients was prospectively investigated on a 1.5-T MRI system. The investigation was performed with RARE and HASTE MR cholangiography sequences with standard and high spatial resolutions, and with a delayed-echo half-Fourier RARE (HASTE) sequence. Findings of the improved MRCP sequences were compared with the standard MRCP sequences. The level of confidence in assessing the diagnosis was divided into five groups. The Wilcoxon signed-rank test at a level of p<0.05 was applied. In 15 patients no pathology was found. The MRCP showed stenoses of the bile duct system in 10 patients and choledocholithiasis and cholecystolithiasis in 16 patients. In 12 patients a dilatation of the bile duct system was found. Comparison of the low- and high spatial resolution sequences and the short and long TE times of the half-Fourier RARE (HASTE) sequence revealed no statistically significant differences regarding accuracy of the examination. The diagnostic confidence level in assessing normal or pathological findings for the high-resolution RARE and half-Fourier RARE (HASTE) was significantly better than for the standard sequences. For the delayed-echo half-Fourier RARE (HASTE) sequence no statistically significant difference was seen. The high-resolution RARE and half-Fourier RARE (HASTE) sequences had a higher confidence level, but there was no significant difference in diagnosis in terms of detection and assessment of pathological changes in the biliary duct system compared with standard sequences. (orig.)

  11. Building Public Confidence in Nuclear Activities

    International Nuclear Information System (INIS)

    Isaacs, T

    2002-01-01

    Achieving public acceptance has become a central issue in discussions regarding the future of nuclear power and associated nuclear activities. Effective public communication and public participation are often put forward as the key building blocks in garnering public acceptance. A recent international workshop in Finland provided insights into other features that might also be important to building and sustaining public confidence in nuclear activities. The workshop was held in Finland in close cooperation with Finnish stakeholders. This was most appropriate because of the recent successes in achieving positive decisions at the municipal, governmental, and Parliamentary levels, allowing the Finnish high-level radioactive waste repository program to proceed, including the identification and approval of a proposed candidate repository site. Much of the workshop discussion appropriately focused on the roles of public participation and public communications in building public confidence. It was clear that well constructed and implemented programs of public involvement and communication and a sense of fairness were essential in building the extent of public confidence needed to allow the repository program in Finland to proceed. It was also clear that there were a number of other elements beyond public involvement that contributed substantially to the success in Finland to date. And, in fact, it appeared that these other factors were also necessary to achieving the Finnish public acceptance. In other words, successful public participation and communication were necessary but not sufficient. What else was important? Culture, politics, and history vary from country to country, providing differing contexts for establishing and maintaining public confidence. What works in one country will not necessarily be effective in another. Nonetheless, there appear to be certain elements that might be common to programs that are successful in sustaining public confidence and some of

  12. Building Public Confidence in Nuclear Activities

    International Nuclear Information System (INIS)

    Isaacs, T

    2002-01-01

    Achieving public acceptance has become a central issue in discussions regarding the future of nuclear power and associated nuclear activities. Effective public communication and public participation are often put forward as the key building blocks in garnering public acceptance. A recent international workshop in Finland provided insights into other features that might also be important to building and sustaining public confidence in nuclear activities. The workshop was held in Finland in close cooperation with Finnish stakeholders. This was most appropriate because of the recent successes in achieving positive decisions at the municipal, governmental, and Parliamentary levels, allowing the Finnish high-level radioactive waste repository program to proceed, including the identification and approval of a proposed candidate repository site Much of the workshop discussion appropriately focused on the roles of public participation and public communications in building public confidence. It was clear that well constructed and implemented programs of public involvement and communication and a sense of fairness were essential in building the extent of public confidence needed to allow the repository program in Finland to proceed. It was also clear that there were a number of other elements beyond public involvement that contributed substantially to the success in Finland to date. And, in fact, it appeared that these other factors were also necessary to achieving the Finnish public acceptance. In other words, successful public participation and communication were necessary but not sufficient. What else was important? Culture, politics, and history vary from country to country, providing differing contexts for establishing and maintaining public confidence. What works in one country will not necessarily be effective in another. Nonetheless, there appear to be certain elements that might be common to programs that are successful in sustaining public confidence, and some of

  13. The Great Recession and confidence in homeownership

    OpenAIRE

    Anat Bracha; Julian Jamison

    2013-01-01

    Confidence in homeownership shifts for those who personally experienced real estate loss during the Great Recession. Older Americans are confident in the value of homeownership. Younger Americans are less confident.

  14. Attention-Based Recurrent Temporal Restricted Boltzmann Machine for Radar High Resolution Range Profile Sequence Recognition

    Directory of Open Access Journals (Sweden)

    Yifan Zhang

    2018-05-01

    Full Text Available The High Resolution Range Profile (HRRP recognition has attracted great concern in the field of Radar Automatic Target Recognition (RATR. However, traditional HRRP recognition methods failed to model high dimensional sequential data efficiently and have a poor anti-noise ability. To deal with these problems, a novel stochastic neural network model named Attention-based Recurrent Temporal Restricted Boltzmann Machine (ARTRBM is proposed in this paper. RTRBM is utilized to extract discriminative features and the attention mechanism is adopted to select major features. RTRBM is efficient to model high dimensional HRRP sequences because it can extract the information of temporal and spatial correlation between adjacent HRRPs. The attention mechanism is used in sequential data recognition tasks including machine translation and relation classification, which makes the model pay more attention to the major features of recognition. Therefore, the combination of RTRBM and the attention mechanism makes our model effective for extracting more internal related features and choose the important parts of the extracted features. Additionally, the model performs well with the noise corrupted HRRP data. Experimental results on the Moving and Stationary Target Acquisition and Recognition (MSTAR dataset show that our proposed model outperforms other traditional methods, which indicates that ARTRBM extracts, selects, and utilizes the correlation information between adjacent HRRPs effectively and is suitable for high dimensional data or noise corrupted data.

  15. Beyond hypercorrection: remembering corrective feedback for low-confidence errors.

    Science.gov (United States)

    Griffiths, Lauren; Higham, Philip A

    2018-02-01

    Correcting errors based on corrective feedback is essential to successful learning. Previous studies have found that corrections to high-confidence errors are better remembered than low-confidence errors (the hypercorrection effect). The aim of this study was to investigate whether corrections to low-confidence errors can also be successfully retained in some cases. Participants completed an initial multiple-choice test consisting of control, trick and easy general-knowledge questions, rated their confidence after answering each question, and then received immediate corrective feedback. After a short delay, they were given a cued-recall test consisting of the same questions. In two experiments, we found high-confidence errors to control questions were better corrected on the second test compared to low-confidence errors - the typical hypercorrection effect. However, low-confidence errors to trick questions were just as likely to be corrected as high-confidence errors. Most surprisingly, we found that memory for the feedback and original responses, not confidence or surprise, were significant predictors of error correction. We conclude that for some types of material, there is an effortful process of elaboration and problem solving prior to making low-confidence errors that facilitates memory of corrective feedback.

  16. The main challenges that remain in applying high-throughput sequencing to clinical diagnostics.

    Science.gov (United States)

    Loeffelholz, Michael; Fofanov, Yuriy

    2015-01-01

    Over the last 10 years, the quality, price and availability of high-throughput sequencing instruments have improved to the point that this technology may be close to becoming a routine tool in the diagnostic microbiology laboratory. Two groups of challenges, however, have to be resolved in order to move this powerful research technology into routine use in the clinical microbiology laboratory. The computational/bioinformatics challenges include data storage cost and privacy concerns, requiring analysis to be performed without access to cloud storage or expensive computational infrastructure. The logistical challenges include interpretation of complex results and acceptance and understanding of the advantages and limitations of this technology by the medical community. This article focuses on the approaches to address these challenges, such as file formats, algorithms, data collection, reporting and good laboratory practices.

  17. Opera: reconstructing optimal genomic scaffolds with high-throughput paired-end sequences.

    Science.gov (United States)

    Gao, Song; Sung, Wing-Kin; Nagarajan, Niranjan

    2011-11-01

    Scaffolding, the problem of ordering and orienting contigs, typically using paired-end reads, is a crucial step in the assembly of high-quality draft genomes. Even as sequencing technologies and mate-pair protocols have improved significantly, scaffolding programs still rely on heuristics, with no guarantees on the quality of the solution. In this work, we explored the feasibility of an exact solution for scaffolding and present a first tractable solution for this problem (Opera). We also describe a graph contraction procedure that allows the solution to scale to large scaffolding problems and demonstrate this by scaffolding several large real and synthetic datasets. In comparisons with existing scaffolders, Opera simultaneously produced longer and more accurate scaffolds demonstrating the utility of an exact approach. Opera also incorporates an exact quadratic programming formulation to precisely compute gap sizes (Availability: http://sourceforge.net/projects/operasf/ ).

  18. Detailed evaluation of RCS boundary rupture during high-pressure severe accident sequences

    International Nuclear Information System (INIS)

    Park, Rae-Joon; Hong, Seong-Wan

    2011-01-01

    A depressurization possibility of the reactor coolant system (RCS) before a reactor vessel rupture during a high-pressure severe accident sequence has been evaluated for the consideration of direct containment heating (DCH) and containment bypass. A total loss of feed water (TLOFW) and a station blackout (SBO) of the advanced power reactor 1400 (APR 1400) has been evaluated from an initiating event to a creep rupture of the RCS boundary by using the SCDAP/RELAP5 computer code. In addition, intentional depressurization of the RCS using power-operated safety relief valves (POSRVs) has been evaluated. The SCDAPRELAP5 results have shown that the pressurizer surge line broke before the reactor vessel rupture failure, but a containment bypass did not occur because steam generator U tubes did not break. The intentional depressurization of the RCS using POSRV was effective for the DCH prevention at a reactor vessel rupture. (author)

  19. SNP calling using genotype model selection on high-throughput sequencing data

    KAUST Repository

    You, Na

    2012-01-16

    Motivation: A review of the available single nucleotide polymorphism (SNP) calling procedures for Illumina high-throughput sequencing (HTS) platform data reveals that most rely mainly on base-calling and mapping qualities as sources of error when calling SNPs. Thus, errors not involved in base-calling or alignment, such as those in genomic sample preparation, are not accounted for.Results: A novel method of consensus and SNP calling, Genotype Model Selection (GeMS), is given which accounts for the errors that occur during the preparation of the genomic sample. Simulations and real data analyses indicate that GeMS has the best performance balance of sensitivity and positive predictive value among the tested SNP callers. © The Author 2012. Published by Oxford University Press. All rights reserved.

  20. Barcoding the food chain: from Sanger to high-throughput sequencing.

    Science.gov (United States)

    Littlefair, Joanne E; Clare, Elizabeth L

    2016-11-01

    Society faces the complex challenge of supporting biodiversity and ecosystem functioning, while ensuring food security by providing safe traceable food through an ever-more-complex global food chain. The increase in human mobility brings the added threat of pests, parasites, and invaders that further complicate our agro-industrial efforts. DNA barcoding technologies allow researchers to identify both individual species, and, when combined with universal primers and high-throughput sequencing techniques, the diversity within mixed samples (metabarcoding). These tools are already being employed to detect market substitutions, trace pests through the forensic evaluation of trace "environmental DNA", and to track parasitic infections in livestock. The potential of DNA barcoding to contribute to increased security of the food chain is clear, but challenges remain in regulation and the need for validation of experimental analysis. Here, we present an overview of the current uses and challenges of applied DNA barcoding in agriculture, from agro-ecosystems within farmland to the kitchen table.

  1. Seasonal diversity and dynamics of haptophytes in the Skagerrak, Norway, explored by high-throughput sequencing

    Science.gov (United States)

    Egge, Elianne Sirnæs; Johannessen, Torill Vik; Andersen, Tom; Eikrem, Wenche; Bittner, Lucie; Larsen, Aud; Sandaa, Ruth-Anne; Edvardsen, Bente

    2015-01-01

    Microalgae in the division Haptophyta play key roles in the marine ecosystem and in global biogeochemical processes. Despite their ecological importance, knowledge on seasonal dynamics, community composition and abundance at the species level is limited due to their small cell size and few morphological features visible under the light microscope. Here, we present unique data on haptophyte seasonal diversity and dynamics from two annual cycles, with the taxonomic resolution and sampling depth obtained with high-throughput sequencing. From outer Oslofjorden, S Norway, nano- and picoplanktonic samples were collected monthly for 2 years, and the haptophytes targeted by amplification of RNA/cDNA with Haptophyta-specific 18S rDNA V4 primers. We obtained 156 operational taxonomic units (OTUs), from c. 400.000 454 pyrosequencing reads, after rigorous bioinformatic filtering and clustering at 99.5%. Most OTUs represented uncultured and/or not yet 18S rDNA-sequenced species. Haptophyte OTU richness and community composition exhibited high temporal variation and significant yearly periodicity. Richness was highest in September–October (autumn) and lowest in April–May (spring). Some taxa were detected all year, such as Chrysochromulina simplex, Emiliania huxleyi and Phaeocystis cordata, whereas most calcifying coccolithophores only appeared from summer to early winter. We also revealed the seasonal dynamics of OTUs representing putative novel classes (clades HAP-3–5) or orders (clades D, E, F). Season, light and temperature accounted for 29% of the variation in OTU composition. Residual variation may be related to biotic factors, such as competition and viral infection. This study provides new, in-depth knowledge on seasonal diversity and dynamics of haptophytes in North Atlantic coastal waters. PMID:25893259

  2. Seasonal diversity and dynamics of haptophytes in the Skagerrak, Norway, explored by high-throughput sequencing.

    Science.gov (United States)

    Egge, Elianne Sirnaes; Johannessen, Torill Vik; Andersen, Tom; Eikrem, Wenche; Bittner, Lucie; Larsen, Aud; Sandaa, Ruth-Anne; Edvardsen, Bente

    2015-06-01

    Microalgae in the division Haptophyta play key roles in the marine ecosystem and in global biogeochemical processes. Despite their ecological importance, knowledge on seasonal dynamics, community composition and abundance at the species level is limited due to their small cell size and few morphological features visible under the light microscope. Here, we present unique data on haptophyte seasonal diversity and dynamics from two annual cycles, with the taxonomic resolution and sampling depth obtained with high-throughput sequencing. From outer Oslofjorden, S Norway, nano- and picoplanktonic samples were collected monthly for 2 years, and the haptophytes targeted by amplification of RNA/cDNA with Haptophyta-specific 18S rDNA V4 primers. We obtained 156 operational taxonomic units (OTUs), from c. 400.000 454 pyrosequencing reads, after rigorous bioinformatic filtering and clustering at 99.5%. Most OTUs represented uncultured and/or not yet 18S rDNA-sequenced species. Haptophyte OTU richness and community composition exhibited high temporal variation and significant yearly periodicity. Richness was highest in September-October (autumn) and lowest in April-May (spring). Some taxa were detected all year, such as Chrysochromulina simplex, Emiliania huxleyi and Phaeocystis cordata, whereas most calcifying coccolithophores only appeared from summer to early winter. We also revealed the seasonal dynamics of OTUs representing putative novel classes (clades HAP-3-5) or orders (clades D, E, F). Season, light and temperature accounted for 29% of the variation in OTU composition. Residual variation may be related to biotic factors, such as competition and viral infection. This study provides new, in-depth knowledge on seasonal diversity and dynamics of haptophytes in North Atlantic coastal waters. © 2015 The Authors. Molecular Ecology Published by John Wiley & Sons Ltd.

  3. The complete genome sequence and comparative genome analysis of the high pathogenicity Yersinia enterocolitica strain 8081.

    Directory of Open Access Journals (Sweden)

    Nicholas R Thomson

    2006-12-01

    Full Text Available The human enteropathogen, Yersinia enterocolitica, is a significant link in the range of Yersinia pathologies extending from mild gastroenteritis to bubonic plague. Comparison at the genomic level is a key step in our understanding of the genetic basis for this pathogenicity spectrum. Here we report the genome of Y. enterocolitica strain 8081 (serotype 0:8; biotype 1B and extensive microarray data relating to the genetic diversity of the Y. enterocolitica species. Our analysis reveals that the genome of Y. enterocolitica strain 8081 is a patchwork of horizontally acquired genetic loci, including a plasticity zone of 199 kb containing an extraordinarily high density of virulence genes. Microarray analysis has provided insights into species-specific Y. enterocolitica gene functions and the intraspecies differences between the high, low, and nonpathogenic Y. enterocolitica biotypes. Through comparative genome sequence analysis we provide new information on the evolution of the Yersinia. We identify numerous loci that represent ancestral clusters of genes potentially important in enteric survival and pathogenesis, which have been lost or are in the process of being lost, in the other sequenced Yersinia lineages. Our analysis also highlights large metabolic operons in Y. enterocolitica that are absent in the related enteropathogen, Yersinia pseudotuberculosis, indicating major differences in niche and nutrients used within the mammalian gut. These include clusters directing, the production of hydrogenases, tetrathionate respiration, cobalamin synthesis, and propanediol utilisation. Along with ancestral gene clusters, the genome of Y. enterocolitica has revealed species-specific and enteropathogen-specific loci. This has provided important insights into the pathology of this bacterium and, more broadly, into the evolution of the genus. Moreover, wider investigations looking at the patterns of gene loss and gain in the Yersinia have highlighted common

  4. ClustalXeed: a GUI-based grid computation version for high performance and terabyte size multiple sequence alignment

    Directory of Open Access Journals (Sweden)

    Kim Taeho

    2010-09-01

    Full Text Available Abstract Background There is an increasing demand to assemble and align large-scale biological sequence data sets. The commonly used multiple sequence alignment programs are still limited in their ability to handle very large amounts of sequences because the system lacks a scalable high-performance computing (HPC environment with a greatly extended data storage capacity. Results We designed ClustalXeed, a software system for multiple sequence alignment with incremental improvements over previous versions of the ClustalX and ClustalW-MPI software. The primary advantage of ClustalXeed over other multiple sequence alignment software is its ability to align a large family of protein or nucleic acid sequences. To solve the conventional memory-dependency problem, ClustalXeed uses both physical random access memory (RAM and a distributed file-allocation system for distance matrix construction and pair-align computation. The computation efficiency of disk-storage system was markedly improved by implementing an efficient load-balancing algorithm, called "idle node-seeking task algorithm" (INSTA. The new editing option and the graphical user interface (GUI provide ready access to a parallel-computing environment for users who seek fast and easy alignment of large DNA and protein sequence sets. Conclusions ClustalXeed can now compute a large volume of biological sequence data sets, which were not tractable in any other parallel or single MSA program. The main developments include: 1 the ability to tackle larger sequence alignment problems than possible with previous systems through markedly improved storage-handling capabilities. 2 Implementing an efficient task load-balancing algorithm, INSTA, which improves overall processing times for multiple sequence alignment with input sequences of non-uniform length. 3 Support for both single PC and distributed cluster systems.

  5. Comparative analyses of six solanaceous transcriptomes reveal a high degree of sequence conservation and species-specific transcripts

    Directory of Open Access Journals (Sweden)

    Ouyang Shu

    2005-09-01

    Full Text Available Abstract Background The Solanaceae is a family of closely related species with diverse phenotypes that have been exploited for agronomic purposes. Previous studies involving a small number of genes suggested sequence conservation across the Solanaceae. The availability of large collections of Expressed Sequence Tags (ESTs for the Solanaceae now provides the opportunity to assess sequence conservation and divergence on a genomic scale. Results All available ESTs and Expressed Transcripts (ETs, 449,224 sequences for six Solanaceae species (potato, tomato, pepper, petunia, tobacco and Nicotiana benthamiana, were clustered and assembled into gene indices. Examination of gene ontologies revealed that the transcripts within the gene indices encode a similar suite of biological processes. Although the ESTs and ETs were derived from a variety of tissues, 55–81% of the sequences had significant similarity at the nucleotide level with sequences among the six species. Putative orthologs could be identified for 28–58% of the sequences. This high degree of sequence conservation was supported by expression profiling using heterologous hybridizations to potato cDNA arrays that showed similar expression patterns in mature leaves for all six solanaceous species. 16–19% of the transcripts within the six Solanaceae gene indices did not have matches among Solanaceae, Arabidopsis, rice or 21 other plant gene indices. Conclusion Results from this genome scale analysis confirmed a high level of sequence conservation at the nucleotide level of the coding sequence among Solanaceae. Additionally, the results indicated that part of the Solanaceae transcriptome is likely to be unique for each species.

  6. A microarray-based genotyping and genetic mapping approach for highly heterozygous outcrossing species enables localization of a large fraction of the unassembled Populus trichocarpa genome sequence.

    Science.gov (United States)

    Drost, Derek R; Novaes, Evandro; Boaventura-Novaes, Carolina; Benedict, Catherine I; Brown, Ryan S; Yin, Tongming; Tuskan, Gerald A; Kirst, Matias

    2009-06-01

    Microarrays have demonstrated significant power for genome-wide analyses of gene expression, and recently have also revolutionized the genetic analysis of segregating populations by genotyping thousands of loci in a single assay. Although microarray-based genotyping approaches have been successfully applied in yeast and several inbred plant species, their power has not been proven in an outcrossing species with extensive genetic diversity. Here we have developed methods for high-throughput microarray-based genotyping in such species using a pseudo-backcross progeny of 154 individuals of Populus trichocarpa and P. deltoides analyzed with long-oligonucleotide in situ-synthesized microarray probes. Our analysis resulted in high-confidence genotypes for 719 single-feature polymorphism (SFP) and 1014 gene expression marker (GEM) candidates. Using these genotypes and an established microsatellite (SSR) framework map, we produced a high-density genetic map comprising over 600 SFPs, GEMs and SSRs. The abundance of gene-based markers allowed us to localize over 35 million base pairs of previously unplaced whole-genome shotgun (WGS) scaffold sequence to putative locations in the genome of P. trichocarpa. A high proportion of sampled scaffolds could be verified for their placement with independently mapped SSRs, demonstrating the previously un-utilized power that high-density genotyping can provide in the context of map-based WGS sequence reassembly. Our results provide a substantial contribution to the continued improvement of the Populus genome assembly, while demonstrating the feasibility of microarray-based genotyping in a highly heterozygous population. The strategies presented are applicable to genetic mapping efforts in all plant species with similarly high levels of genetic diversity.

  7. SSR_pipeline: a bioinformatic infrastructure for identifying microsatellites from paired-end Illumina high-throughput DNA sequencing data

    Science.gov (United States)

    Miller, Mark P.; Knaus, Brian J.; Mullins, Thomas D.; Haig, Susan M.

    2013-01-01

    SSR_pipeline is a flexible set of programs designed to efficiently identify simple sequence repeats (e.g., microsatellites) from paired-end high-throughput Illumina DNA sequencing data. The program suite contains 3 analysis modules along with a fourth control module that can automate analyses of large volumes of data. The modules are used to 1) identify the subset of paired-end sequences that pass Illumina quality standards, 2) align paired-end reads into a single composite DNA sequence, and 3) identify sequences that possess microsatellites (both simple and compound) conforming to user-specified parameters. The microsatellite search algorithm is extremely efficient, and we have used it to identify repeats with motifs from 2 to 25bp in length. Each of the 3 analysis modules can also be used independently to provide greater flexibility or to work with FASTQ or FASTA files generated from other sequencing platforms (Roche 454, Ion Torrent, etc.). We demonstrate use of the program with data from the brine fly Ephydra packardi (Diptera: Ephydridae) and provide empirical timing benchmarks to illustrate program performance on a common desktop computer environment. We further show that the Illumina platform is capable of identifying large numbers of microsatellites, even when using unenriched sample libraries and a very small percentage of the sequencing capacity from a single DNA sequencing run. All modules from SSR_pipeline are implemented in the Python programming language and can therefore be used from nearly any computer operating system (Linux, Macintosh, and Windows).

  8. SSR_pipeline: a bioinformatic infrastructure for identifying microsatellites from paired-end Illumina high-throughput DNA sequencing data.

    Science.gov (United States)

    Miller, Mark P; Knaus, Brian J; Mullins, Thomas D; Haig, Susan M

    2013-01-01

    SSR_pipeline is a flexible set of programs designed to efficiently identify simple sequence repeats (e.g., microsatellites) from paired-end high-throughput Illumina DNA sequencing data. The program suite contains 3 analysis modules along with a fourth control module that can automate analyses of large volumes of data. The modules are used to 1) identify the subset of paired-end sequences that pass Illumina quality standards, 2) align paired-end reads into a single composite DNA sequence, and 3) identify sequences that possess microsatellites (both simple and compound) conforming to user-specified parameters. The microsatellite search algorithm is extremely efficient, and we have used it to identify repeats with motifs from 2 to 25 bp in length. Each of the 3 analysis modules can also be used independently to provide greater flexibility or to work with FASTQ or FASTA files generated from other sequencing platforms (Roche 454, Ion Torrent, etc.). We demonstrate use of the program with data from the brine fly Ephydra packardi (Diptera: Ephydridae) and provide empirical timing benchmarks to illustrate program performance on a common desktop computer environment. We further show that the Illumina platform is capable of identifying large numbers of microsatellites, even when using unenriched sample libraries and a very small percentage of the sequencing capacity from a single DNA sequencing run. All modules from SSR_pipeline are implemented in the Python programming language and can therefore be used from nearly any computer operating system (Linux, Macintosh, and Windows).

  9. Public confidence and nuclear energy

    International Nuclear Information System (INIS)

    Chaussade, J.P.

    1990-01-01

    Today in France there are 54 nuclear power units in operation at 18 sites. They supply 75% of all electricity produced, 12% of which is exported to neighbouring countries, and play an important role in the French economy. For the French, nuclear power is a fact of life, and most accept it. However, the accident of Chernobyl has made public opinion more sensitive, and the public relations work has had to be reconsidered carefully with a view to increase the confidence of the French public in nuclear power, anticipating media crises and being equipped to deal with such crises. The three main approaches are the following: keeping the public better informed, providing clear information at time of crisis and international activities

  10. Knowledge, Self Confidence and Courage

    DEFF Research Database (Denmark)

    Selberg, Hanne; Steenberg Holtzmann, Jette; Hovedskov, Jette

    . Results The students identified their major learning outcomes as transfer of operational skills, experiencing self-efficacy and enhanced understanding of the patients' perspective.Involving simulated patients in the training of technical skills contributed to the development of the students' communication......Knowledge, self confidence and courage – long lasting learning outcomes through simulation in a clinical context. Hanne Selberg1, Jette Hovedskov2, Jette Steenberg Holtzmann2 The significance and methodology of the researchThe study focuses on simulation alongside the clinical practice and linked...... Development, Clinical Lecturer, Metropolitan University College, Faculty of Nursing, Email: hase@phoe.dk, phone: +45-72282830. 2. Jette Hovedskov, RN, Development Consultant, Glostrup University Hospital, Department of Development Email : jeho@glo.regionh.dk ,phone: +45- 43232090 3. Jette Holtzmann Steenberg...

  11. Usefulness of Genetic Study by Next-generation Sequencing in High-risk Arrhythmogenic Cardiomyopathy.

    Science.gov (United States)

    Ruiz Salas, Amalio; Peña Hernández, José; Medina Palomo, Carmen; Barrera Cordero, Alberto; Cabrera Bueno, Fernando; García Pinilla, José Manuel; Guijarro, Ana; Morcillo-Hidalgo, Luis; Jiménez Navarro, Manuel; Gómez Doblas, Juan José; de Teresa, Eduardo; Alzueta, Javier

    2018-03-29

    Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited cardiomyopathy characterized by progressive fibrofatty replacement of predominantly right ventricular myocardium. This cardiomyopathy is a frequent cause of sudden cardiac death in young people and athletes. The aim of our study was to determine the incidence of pathological or likely pathological desmosomal mutations in patients with high-risk definite ARVC. This was an observational, retrospective cohort study, which included 36 patients diagnosed with high-risk ARVC in our hospital between January 1998 and January 2015. Genetic analysis was performed using next-generation sequencing. Most patients were male (28 patients, 78%) with a mean age at diagnosis of 45 ± 18 years. A pathogenic or probably pathogenic desmosomal mutation was detected in 26 of the 35 index cases (74%): 5 nonsense, 14 frameshift, 1 splice, and 6 missense. Novel mutations were found in 15 patients (71%). The presence or absence of desmosomal mutations causing the disease and the type of mutation were not associated with specific electrocardiographic, clinical, arrhythmic, anatomic, or prognostic characteristics. The incidence of pathological or likely pathological desmosomal mutations in ARVC is very high, with most mutations causing truncation. The presence of desmosomal mutations was not associated with prognosis. Copyright © 2017 Sociedad Española de Cardiología. Published by Elsevier España, S.L.U. All rights reserved.

  12. 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....

  13. Fine grained compositional analysis of Port Everglades Inlet microbiome using high throughput DNA sequencing.

    Science.gov (United States)

    O'Connell, Lauren; Gao, Song; McCorquodale, Donald; Fleisher, Jay; Lopez, Jose V

    2018-01-01

    Similar to natural rivers, manmade inlets connect inland runoff to the ocean. Port Everglades Inlet (PEI) is a busy cargo and cruise ship port in South Florida, which can act as a source of pollution to surrounding beaches and offshore coral reefs. Understanding the composition and fluctuations of bacterioplankton communities ("microbiomes") in major port inlets is important due to potential impacts on surrounding environments. We hypothesize seasonal microbial fluctuations, which were profiled by high throughput 16S rRNA amplicon sequencing and analysis. Surface water samples were collected every week for one year. A total of four samples per month, two from each sampling location, were used for statistical analysis creating a high sampling frequency and finer sampling scale than previous inlet microbiome studies. We observed significant differences in community alpha diversity between months and seasons. Analysis of composition of microbiomes (ANCOM) tests were run in QIIME 2 at genus level taxonomic classification to determine which genera were differentially abundant between seasons and months. Beta diversity results yielded significant differences in PEI community composition in regard to month, season, water temperature, and salinity. Analysis of potentially pathogenic genera showed presence of Staphylococcus and Streptococcus . However, statistical analysis indicated that these organisms were not present in significantly high abundances throughout the year or between seasons. Significant differences in alpha diversity were observed when comparing microbial communities with respect to time. This observation stems from the high community evenness and low community richness in August. This indicates that only a few organisms dominated the community during this month. August had lower than average rainfall levels for a wet season, which may have contributed to less runoff, and fewer bacterial groups introduced into the port surface waters. Bacterioplankton beta

  14. Fine grained compositional analysis of Port Everglades Inlet microbiome using high throughput DNA sequencing

    Directory of Open Access Journals (Sweden)

    Lauren O’Connell

    2018-05-01

    Full Text Available Background Similar to natural rivers, manmade inlets connect inland runoff to the ocean. Port Everglades Inlet (PEI is a busy cargo and cruise ship port in South Florida, which can act as a source of pollution to surrounding beaches and offshore coral reefs. Understanding the composition and fluctuations of bacterioplankton communities (“microbiomes” in major port inlets is important due to potential impacts on surrounding environments. We hypothesize seasonal microbial fluctuations, which were profiled by high throughput 16S rRNA amplicon sequencing and analysis. Methods & Results Surface water samples were collected every week for one year. A total of four samples per month, two from each sampling location, were used for statistical analysis creating a high sampling frequency and finer sampling scale than previous inlet microbiome studies. We observed significant differences in community alpha diversity between months and seasons. Analysis of composition of microbiomes (ANCOM tests were run in QIIME 2 at genus level taxonomic classification to determine which genera were differentially abundant between seasons and months. Beta diversity results yielded significant differences in PEI community composition in regard to month, season, water temperature, and salinity. Analysis of potentially pathogenic genera showed presence of Staphylococcus and Streptococcus. However, statistical analysis indicated that these organisms were not present in significantly high abundances throughout the year or between seasons. Discussion Significant differences in alpha diversity were observed when comparing microbial communities with respect to time. This observation stems from the high community evenness and low community richness in August. This indicates that only a few organisms dominated the community during this month. August had lower than average rainfall levels for a wet season, which may have contributed to less runoff, and fewer bacterial groups

  15. Exploring fungal diversity in deep-sea sediments from Okinawa Trough using high-throughput Illumina sequencing

    Science.gov (United States)

    Zhang, Xiao-Yong; Wang, Guang-Hua; Xu, Xin-Ya; Nong, Xu-Hua; Wang, Jie; Amin, Muhammad; Qi, Shu-Hua

    2016-10-01

    The present study investigated the fungal diversity in four different deep-sea sediments from Okinawa Trough using high-throughput Illumina sequencing of the nuclear ribosomal internal transcribed spacer-1 (ITS1). A total of 40,297 fungal ITS1 sequences clustered into 420 operational taxonomic units (OTUs) with 97% sequence similarity and 170 taxa were recovered from these sediments. Most ITS1 sequences (78%) belonged to the phylum Ascomycota, followed by Basidiomycota (17.3%), Zygomycota (1.5%) and Chytridiomycota (0.8%), and a small proportion (2.4%) belonged to unassigned fungal phyla. Compared with previous studies on fungal diversity of sediments from deep-sea environments by culture-dependent approach and clone library analysis, the present result suggested that Illumina sequencing had been dramatically accelerating the discovery of fungal community of deep-sea sediments. Furthermore, our results revealed that Sordariomycetes was the most diverse and abundant fungal class in this study, challenging the traditional view that the diversity of Sordariomycetes phylotypes was low in the deep-sea environments. In addition, more than 12 taxa accounted for 21.5% sequences were found to be rarely reported as deep-sea fungi, suggesting the deep-sea sediments from Okinawa Trough harbored a plethora of different fungal communities compared with other deep-sea environments. To our knowledge, this study is the first exploration of the fungal diversity in deep-sea sediments from Okinawa Trough using high-throughput Illumina sequencing.

  16. RNA-Sequencing of Drosophila melanogaster Head Tissue on High-Sugar and High-Fat Diets

    Directory of Open Access Journals (Sweden)

    Wayne Hemphill

    2018-01-01

    Full Text Available Obesity has been shown to increase risk for cardiovascular disease and type-2 diabetes. In addition, it has been implicated in aggravation of neurological conditions such as Alzheimer’s. In the model organism Drosophila melanogaster, a physiological state mimicking diet-induced obesity can be induced by subjecting fruit flies to a solid medium disproportionately higher in sugar than protein, or that has been supplemented with a rich source of saturated fat. These flies can exhibit increased circulating glucose levels, increased triglyceride content, insulin-like peptide resistance, and behavior indicative of neurological decline. We subjected flies to variants of the high-sugar diet, high-fat diet, or normal (control diet, followed by a total RNA extraction from fly heads of each diet group for the purpose of Poly-A selected RNA-Sequencing. Our objective was to identify the effects of obesogenic diets on transcriptome patterns, how they differed between obesogenic diets, and identify genes that may relate to pathogenesis accompanying an obesity-like state. Gene ontology analysis indicated an overrepresentation of affected genes associated with immunity, metabolism, and hemocyanin in the high-fat diet group, and CHK, cell cycle activity, and DNA binding and transcription in the high-sugar diet group. Our results also indicate differences in the effects of the high-fat diet and high-sugar diet on expression profiles in head tissue of flies, despite the reportedly similar phenotypic impacts of the diets. The impacted genes, and how they may relate to pathogenesis in the Drosophila obesity-like state, warrant further experimental investigation.

  17. Whole-exome sequencing and high throughput genotyping identified KCNJ11 as the thirteenth MODY gene.

    Science.gov (United States)

    Bonnefond, Amélie; Philippe, Julien; Durand, Emmanuelle; Dechaume, Aurélie; Huyvaert, Marlène; Montagne, Louise; Marre, Michel; Balkau, Beverley; Fajardy, Isabelle; Vambergue, Anne; Vatin, Vincent; Delplanque, Jérôme; Le Guilcher, David; De Graeve, Franck; Lecoeur, Cécile; Sand, Olivier; Vaxillaire, Martine; Froguel, Philippe

    2012-01-01

    Maturity-onset of the young (MODY) is a clinically heterogeneous form of diabetes characterized by an autosomal-dominant mode of inheritance, an onset before the age of 25 years, and a primary defect in the pancreatic beta-cell function. Approximately 30% of MODY families remain genetically unexplained (MODY-X). Here, we aimed to use whole-exome sequencing (WES) in a four-generation MODY-X family to identify a new susceptibility gene for MODY. WES (Agilent-SureSelect capture/Illumina-GAIIx sequencing) was performed in three affected and one non-affected relatives in the MODY-X family. We then performed a high-throughput multiplex genotyping (Illumina-GoldenGate assay) of the putative causal mutations in the whole family and in 406 controls. A linkage analysis was also carried out. By focusing on variants of interest (i.e. gains of stop codon, frameshift, non-synonymous and splice-site variants not reported in dbSNP130) present in the three affected relatives and not present in the control, we found 69 mutations. However, as WES was not uniform between samples, a total of 324 mutations had to be assessed in the whole family and in controls. Only one mutation (p.Glu227Lys in KCNJ11) co-segregated with diabetes in the family (with a LOD-score of 3.68). No KCNJ11 mutation was found in 25 other MODY-X unrelated subjects. Beyond neonatal diabetes mellitus (NDM), KCNJ11 is also a MODY gene ('MODY13'), confirming the wide spectrum of diabetes related phenotypes due to mutations in NDM genes (i.e. KCNJ11, ABCC8 and INS). Therefore, the molecular diagnosis of MODY should include KCNJ11 as affected carriers can be ideally treated with oral sulfonylureas.

  18. Whole-exome sequencing and high throughput genotyping identified KCNJ11 as the thirteenth MODY gene.

    Directory of Open Access Journals (Sweden)

    Amélie Bonnefond

    Full Text Available BACKGROUND: Maturity-onset of the young (MODY is a clinically heterogeneous form of diabetes characterized by an autosomal-dominant mode of inheritance, an onset before the age of 25 years, and a primary defect in the pancreatic beta-cell function. Approximately 30% of MODY families remain genetically unexplained (MODY-X. Here, we aimed to use whole-exome sequencing (WES in a four-generation MODY-X family to identify a new susceptibility gene for MODY. METHODOLOGY: WES (Agilent-SureSelect capture/Illumina-GAIIx sequencing was performed in three affected and one non-affected relatives in the MODY-X family. We then performed a high-throughput multiplex genotyping (Illumina-GoldenGate assay of the putative causal mutations in the whole family and in 406 controls. A linkage analysis was also carried out. PRINCIPAL FINDINGS: By focusing on variants of interest (i.e. gains of stop codon, frameshift, non-synonymous and splice-site variants not reported in dbSNP130 present in the three affected relatives and not present in the control, we found 69 mutations. However, as WES was not uniform between samples, a total of 324 mutations had to be assessed in the whole family and in controls. Only one mutation (p.Glu227Lys in KCNJ11 co-segregated with diabetes in the family (with a LOD-score of 3.68. No KCNJ11 mutation was found in 25 other MODY-X unrelated subjects. CONCLUSIONS/SIGNIFICANCE: Beyond neonatal diabetes mellitus (NDM, KCNJ11 is also a MODY gene ('MODY13', confirming the wide spectrum of diabetes related phenotypes due to mutations in NDM genes (i.e. KCNJ11, ABCC8 and INS. Therefore, the molecular diagnosis of MODY should include KCNJ11 as affected carriers can be ideally treated with oral sulfonylureas.

  19. Robust DNA Isolation and High-throughput Sequencing Library Construction for Herbarium Specimens.

    Science.gov (United States)

    Saeidi, Saman; McKain, Michael R; Kellogg, Elizabeth A

    2018-03-08

    Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with a number of challenges including sample preservation quality, degraded DNA, and destructive sampling of rare specimens. In order to more effectively use herbarium material in large sequencing projects, a dependable and scalable method of DNA isolation and library preparation is needed. This paper demonstrates a robust, beginning-to-end protocol for DNA isolation and high-throughput library construction from herbarium specimens that does not require modification for individual samples. This protocol is tailored for low quality dried plant material and takes advantage of existing methods by optimizing tissue grinding, modifying library size selection, and introducing an optional reamplification step for low yield libraries. Reamplification of low yield DNA libraries can rescue samples derived from irreplaceable and potentially valuable herbarium specimens, negating the need for additional destructive sampling and without introducing discernible sequencing bias for common phylogenetic applications. The protocol has been tested on hundreds of grass species, but is expected to be adaptable for use in other plant lineages after verification. This protocol can be limited by extremely degraded DNA, where fragments do not exist in the desired size range, and by secondary metabolites present in some plant material that inhibit clean DNA isolation. Overall, this protocol introduces a fast and comprehensive method that allows for DNA isolation and library preparation of 24 samples in less than 13 h, with only 8 h of active hands-on time with minimal modifications.

  20. High-throughput sequencing of RNA silencing-associated small RNAs in olive (Olea europaea L..

    Directory of Open Access Journals (Sweden)

    Livia Donaire

    Full Text Available Small RNAs (sRNAs of 20 to 25 nucleotides (nt in length maintain genome integrity and control gene expression in a multitude of developmental and physiological processes. Despite RNA silencing has been primarily studied in model plants, the advent of high-throughput sequencing technologies has enabled profiling of the sRNA component of more than 40 plant species. Here, we used deep sequencing and molecular methods to report the first inventory of sRNAs in olive (Olea europaea L.. sRNA libraries prepared from juvenile and adult shoots revealed that the 24-nt class dominates the sRNA transcriptome and atypically accumulates to levels never seen in other plant species, suggesting an active role of heterochromatin silencing in the maintenance and integrity of its large genome. A total of 18 known miRNA families were identified in the libraries. Also, 5 other sRNAs derived from potential hairpin-like precursors remain as plausible miRNA candidates. RNA blots confirmed miRNA expression and suggested tissue- and/or developmental-specific expression patterns. Target mRNAs of conserved miRNAs were computationally predicted among the olive cDNA collection and experimentally validated through endonucleolytic cleavage assays. Finally, we use expression data to uncover genetic components of the miR156, miR172 and miR390/TAS3-derived trans-acting small interfering RNA (tasiRNA regulatory nodes, suggesting that these interactive networks controlling developmental transitions are fully operational in olive.

  1. Peptide Pattern Recognition for high-throughput protein sequence analysis and clustering

    DEFF Research Database (Denmark)

    Busk, Peter Kamp

    2017-01-01

    Large collections of protein sequences with divergent sequences are tedious to analyze for understanding their phylogenetic or structure-function relation. Peptide Pattern Recognition is an algorithm that was developed to facilitate this task but the previous version does only allow a limited...... number of sequences as input. I implemented Peptide Pattern Recognition as a multithread software designed to handle large numbers of sequences and perform analysis in a reasonable time frame. Benchmarking showed that the new implementation of Peptide Pattern Recognition is twenty times faster than...... the previous implementation on a small protein collection with 673 MAP kinase sequences. In addition, the new implementation could analyze a large protein collection with 48,570 Glycosyl Transferase family 20 sequences without reaching its upper limit on a desktop computer. Peptide Pattern Recognition...

  2. High-throughput sequencing of nematode communities from total soil DNA extractions

    DEFF Research Database (Denmark)

    Sapkota, Rumakanta; Nicolaisen, Mogens

    2015-01-01

    nematodes without the need for enrichment was developed. Using this strategy on DNA templates from a set of 22 agricultural soils, we obtained 64.4% sequences of nematode origin in total, whereas the remaining sequences were almost entirely from other metazoans. The nematode sequences were derived from...... in previous sequence-based studies are not nematode specific but also amplify other groups of organisms such as fungi and plantae, and thus require a nematode enrichment step that may introduce biases. Results: In this study an amplification strategy which selectively amplifies a fragment of the SSU from...... a broad taxonomic range and most sequences were from nematode taxa that have previously been found to be abundant in soil such as Tylenchida, Rhabditida, Dorylaimida, Triplonchida and Araeolaimida. Conclusions: Our amplification and sequencing strategy for assessing nematode diversity was able to collect...

  3. Integrated analysis of RNA-binding protein complexes using in vitro selection and high-throughput sequencing and sequence specificity landscapes (SEQRS).

    Science.gov (United States)

    Lou, Tzu-Fang; Weidmann, Chase A; Killingsworth, Jordan; Tanaka Hall, Traci M; Goldstrohm, Aaron C; Campbell, Zachary T

    2017-04-15

    RNA-binding proteins (RBPs) collaborate to control virtually every aspect of RNA function. Tremendous progress has been made in the area of global assessment of RBP specificity using next-generation sequencing approaches both in vivo and in vitro. Understanding how protein-protein interactions enable precise combinatorial regulation of RNA remains a significant problem. Addressing this challenge requires tools that can quantitatively determine the specificities of both individual proteins and multimeric complexes in an unbiased and comprehensive way. One approach utilizes in vitro selection, high-throughput sequencing, and sequence-specificity landscapes (SEQRS). We outline a SEQRS experiment focused on obtaining the specificity of a multi-protein complex between Drosophila RBPs Pumilio (Pum) and Nanos (Nos). We discuss the necessary controls in this type of experiment and examine how the resulting data can be complemented with structural and cell-based reporter assays. Additionally, SEQRS data can be integrated with functional genomics data to uncover biological function. Finally, we propose extensions of the technique that will enhance our understanding of multi-protein regulatory complexes assembled onto RNA. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. The determination of high-resolution spatio-temporal glacier motion fields from time-lapse sequences

    Science.gov (United States)

    Schwalbe, Ellen; Maas, Hans-Gerd

    2017-12-01

    This paper presents a comprehensive method for the determination of glacier surface motion vector fields at high spatial and temporal resolution. These vector fields can be derived from monocular terrestrial camera image sequences and are a valuable data source for glaciological analysis of the motion behaviour of glaciers. The measurement concepts for the acquisition of image sequences are presented, and an automated monoscopic image sequence processing chain is developed. Motion vector fields can be derived with high precision by applying automatic subpixel-accuracy image matching techniques on grey value patterns in the image sequences. Well-established matching techniques have been adapted to the special characteristics of the glacier data in order to achieve high reliability in automatic image sequence processing, including the handling of moving shadows as well as motion effects induced by small instabilities in the camera set-up. Suitable geo-referencing techniques were developed to transform image measurements into a reference coordinate system.The result of monoscopic image sequence analysis is a dense raster of glacier surface point trajectories for each image sequence. Each translation vector component in these trajectories can be determined with an accuracy of a few centimetres for points at a distance of several kilometres from the camera. Extensive practical validation experiments have shown that motion vector and trajectory fields derived from monocular image sequences can be used for the determination of high-resolution velocity fields of glaciers, including the analysis of tidal effects on glacier movement, the investigation of a glacier's motion behaviour during calving events, the determination of the position and migration of the grounding line and the detection of subglacial channels during glacier lake outburst floods.

  5. Strategies for achieving high sequencing accuracy for low diversity samples and avoiding sample bleeding using illumina platform.

    Science.gov (United States)

    Mitra, Abhishek; Skrzypczak, Magdalena; Ginalski, Krzysztof; Rowicka, Maga

    2015-01-01

    Sequencing microRNA, reduced representation sequencing, Hi-C technology and any method requiring the use of in-house barcodes result in sequencing libraries with low initial sequence diversity. Sequencing such data on the Illumina platform typically produces low quality data due to the limitations of the Illumina cluster calling algorithm. Moreover, even in the case of diverse samples, these limitations are causing substantial inaccuracies in multiplexed sample assignment (sample bleeding). Such inaccuracies are unacceptable in clinical applications, and in some other fields (e.g. detection of rare variants). Here, we discuss how both problems with quality of low-diversity samples and sample bleeding are caused by incorrect detection of clusters on the flowcell during initial sequencing cycles. We propose simple software modifications (Long Template Protocol) that overcome this problem. We present experimental results showing that our Long Template Protocol remarkably increases data quality for low diversity samples, as compared with the standard analysis protocol; it also substantially reduces sample bleeding for all samples. For comprehensiveness, we also discuss and compare experimental results from alternative approaches to sequencing low diversity samples. First, we discuss how the low diversity problem, if caused by barcodes, can be avoided altogether at the barcode design stage. Second and third, we present modified guidelines, which are more stringent than the manufacturer's, for mixing low diversity samples with diverse samples and lowering cluster density, which in our experience consistently produces high quality data from low diversity samples. Fourth and fifth, we present rescue strategies that can be applied when sequencing results in low quality data and when there is no more biological material available. In such cases, we propose that the flowcell be re-hybridized and sequenced again using our Long Template Protocol. Alternatively, we discuss how

  6. Strategies for achieving high sequencing accuracy for low diversity samples and avoiding sample bleeding using illumina platform.

    Directory of Open Access Journals (Sweden)

    Abhishek Mitra

    Full Text Available Sequencing microRNA, reduced representation sequencing, Hi-C technology and any method requiring the use of in-house barcodes result in sequencing libraries with low initial sequence diversity. Sequencing such data on the Illumina platform typically produces low quality data due to the limitations of the Illumina cluster calling algorithm. Moreover, even in the case of diverse samples, these limitations are causing substantial inaccuracies in multiplexed sample assignment (sample bleeding. Such inaccuracies are unacceptable in clinical applications, and in some other fields (e.g. detection of rare variants. Here, we discuss how both problems with quality of low-diversity samples and sample bleeding are caused by incorrect detection of clusters on the flowcell during initial sequencing cycles. We propose simple software modifications (Long Template Protocol that overcome this problem. We present experimental results showing that our Long Template Protocol remarkably increases data quality for low diversity samples, as compared with the standard analysis protocol; it also substantially reduces sample bleeding for all samples. For comprehensiveness, we also discuss and compare experimental results from alternative approaches to sequencing low diversity samples. First, we discuss how the low diversity problem, if caused by barcodes, can be avoided altogether at the barcode design stage. Second and third, we present modified guidelines, which are more stringent than the manufacturer's, for mixing low diversity samples with diverse samples and lowering cluster density, which in our experience consistently produces high quality data from low diversity samples. Fourth and fifth, we present rescue strategies that can be applied when sequencing results in low quality data and when there is no more biological material available. In such cases, we propose that the flowcell be re-hybridized and sequenced again using our Long Template Protocol. Alternatively

  7. High Diversity of Myocyanophage in Various Aquatic Environments Revealed by High-Throughput Sequencing of Major Capsid Protein Gene With a New Set of Primers

    Directory of Open Access Journals (Sweden)

    Weiguo Hou

    2018-05-01

    Full Text Available Myocyanophages, a group of viruses infecting cyanobacteria, are abundant and play important roles in elemental cycling. Here we investigated the particle-associated viral communities retained on 0.2 μm filters and in sediment samples (representing ancient cyanophage communities from four ocean and three lake locations, using high-throughput sequencing and a newly designed primer pair targeting a gene fragment (∼145-bp in length encoding the cyanophage gp23 major capsid protein (MCP. Diverse viral communities were detected in all samples. The fragments of 142-, 145-, and 148-bp in length were most abundant in the amplicons, and most sequences (>92% belonged to cyanophages. Additionally, different sequencing depths resulted in different diversity estimates of the viral community. Operational taxonomic units obtained from deep sequencing of the MCP gene covered the majority of those obtained from shallow sequencing, suggesting that deep sequencing exhibited a more complete picture of cyanophage community than shallow sequencing. Our results also revealed a wide geographic distribution of marine myocyanophages, i.e., higher dissimilarities of the myocyanophage communities corresponded with the larger distances between the sampling sites. Collectively, this study suggests that the newly designed primer pair can be effectively used to study the community and diversity of myocyanophage from different environments, and the high-throughput sequencing represents a good method to understand viral diversity.

  8. High-specificity detection of rare alleles with Paired-End Low Error Sequencing (PELE-Seq).

    Science.gov (United States)

    Preston, Jessica L; Royall, Ariel E; Randel, Melissa A; Sikkink, Kristin L; Phillips, Patrick C; Johnson, Eric A

    2016-06-14

    Polymorphic loci exist throughout the genomes of a population and provide the raw genetic material needed for a species to adapt to changes in the environment. The minor allele frequencies of rare Single Nucleotide Polymorphisms (SNPs) within a population have been difficult to track with Next-Generation Sequencing (NGS), due to the high error rate of standard methods such as Illumina sequencing. We have developed a wet-lab protocol and variant-calling method that identifies both sequencing and PCR errors, called Paired-End Low Error Sequencing (PELE-Seq). To test the specificity and sensitivity of the PELE-Seq method, we sequenced control E. coli DNA libraries containing known rare alleles present at frequencies ranging from 0.2-0.4 % of the total reads. PELE-Seq had higher specificity and sensitivity than standard libraries. We then used PELE-Seq to characterize rare alleles in a Caenorhabditis remanei nematode worm population before and after laboratory adaptation, and found that minor and rare alleles can undergo large changes in frequency during lab-adaptation. We have developed a method of rare allele detection that mitigates both sequencing and PCR errors, called PELE-Seq. PELE-Seq was evaluated using control E. coli populations and was then used to compare a wild C. remanei population to a lab-adapted population. The PELE-Seq method is ideal for investigating the dynamics of rare alleles in a broad range of reduced-representation sequencing methods, including targeted amplicon sequencing, RAD-Seq, ddRAD, and GBS. PELE-Seq is also well-suited for whole genome sequencing of mitochondria and viruses, and for high-throughput rare mutation screens.

  9. High-Throughput Sequencing of Microbial Community Diversity and Dynamics during Douchi Fermentation

    Science.gov (United States)

    Tu, Zong-cai; Wang, Xiao-lan

    2016-01-01

    Douchi is a type of Chinese traditional fermented food that is an important source of protein and is used in flavouring ingredients. The end product is affected by the microbial community present during fermentation, but exactly how microbes influence the fermentation process remains poorly understood. We used an Illumina MiSeq approach to investigate bacterial and fungal community diversity during both douchi-koji making and fermentation. A total of 181,443 high quality bacterial 16S rRNA sequences and 221,059 high quality fungal internal transcribed spacer reads were used for taxonomic classification, revealing eight bacterial and three fungal phyla. Firmicutes, Actinobacteria and Proteobacteria were the dominant bacterial phyla, while Ascomycota and Zygomycota were the dominant fungal phyla. At the genus level, Staphylococcus and Weissella were the dominant bacteria, while Aspergillus and Lichtheimia were the dominant fungi. Principal coordinate analysis showed structural separation between the composition of bacteria in koji making and fermentation. However, multivariate analysis of variance based on unweighted UniFrac distances did identify distinct differences (p fermentation. This is the first investigation to integrate douchi fermentation and koji making and fermentation processes through this technological approach. The results provide insight into the microbiome of the douchi fermentation process, and reveal a structural separation that may be stratified by the environment during the production of this traditional fermented food. PMID:27992473

  10. Sequence and expression analyses of ethylene response factors highly expressed in latex cells from Hevea brasiliensis.

    Directory of Open Access Journals (Sweden)

    Piyanuch Piyatrakul

    Full Text Available The AP2/ERF superfamily encodes transcription factors that play a key role in plant development and responses to abiotic and biotic stress. In Hevea brasiliensis, ERF genes have been identified by RNA sequencing. This study set out to validate the number of HbERF genes, and identify ERF genes involved in the regulation of latex cell metabolism. A comprehensive Hevea transcriptome was improved using additional RNA reads from reproductive tissues. Newly assembled contigs were annotated in the Gene Ontology database and were assigned to 3 main categories. The AP2/ERF superfamily is the third most represented compared with other transcription factor families. A comparison with genomic scaffolds led to an estimation of 114 AP2/ERF genes and 1 soloist in Hevea brasiliensis. Based on a phylogenetic analysis, functions were predicted for 26 HbERF genes. A relative transcript abundance analysis was performed by real-time RT-PCR in various tissues. Transcripts of ERFs from group I and VIII were very abundant in all tissues while those of group VII were highly accumulated in latex cells. Seven of the thirty-five ERF expression marker genes were highly expressed in latex. Subcellular localization and transactivation analyses suggested that HbERF-VII candidate genes encoded functional transcription factors.

  11. glbase: a framework for combining, analyzing and displaying heterogeneous genomic and high-throughput sequencing data.

    Science.gov (United States)

    Hutchins, Andrew Paul; Jauch, Ralf; Dyla, Mateusz; Miranda-Saavedra, Diego

    2014-01-01

    Genomic datasets and the tools to analyze them have proliferated at an astonishing rate. However, such tools are often poorly integrated with each other: each program typically produces its own custom output in a variety of non-standard file formats. Here we present glbase, a framework that uses a flexible set of descriptors that can quickly parse non-binary data files. glbase includes many functions to intersect two lists of data, including operations on genomic interval data and support for the efficient random access to huge genomic data files. Many glbase functions can produce graphical outputs, including scatter plots, heatmaps, boxplots and other common analytical displays of high-throughput data such as RNA-seq, ChIP-seq and microarray expression data. glbase is designed to rapidly bring biological data into a Python-based analytical environment to facilitate analysis and data processing. In summary, glbase is a flexible and multifunctional toolkit that allows the combination and analysis of high-throughput data (especially next-generation sequencing and genome-wide data), and which has been instrumental in the analysis of complex data sets. glbase is freely available at http://bitbucket.org/oaxiom/glbase/.

  12. glbase: a framework for combining, analyzing and displaying heterogeneous genomic and high-throughput sequencing data

    Directory of Open Access Journals (Sweden)

    Andrew Paul Hutchins

    2014-01-01

    Full Text Available Genomic datasets and the tools to analyze them have proliferated at an astonishing rate. However, such tools are often poorly integrated with each other: each program typically produces its own custom output in a variety of non-standard file formats. Here we present glbase, a framework that uses a flexible set of descriptors that can quickly parse non-binary data files. glbase includes many functions to intersect two lists of data, including operations on genomic interval data and support for the efficient random access to huge genomic data files. Many glbase functions can produce graphical outputs, including scatter plots, heatmaps, boxplots and other common analytical displays of high-throughput data such as RNA-seq, ChIP-seq and microarray expression data. glbase is designed to rapidly bring biological data into a Python-based analytical environment to facilitate analysis and data processing. In summary, glbase is a flexible and multifunctional toolkit that allows the combination and analysis of high-throughput data (especially next-generation sequencing and genome-wide data, and which has been instrumental in the analysis of complex data sets. glbase is freely available at http://bitbucket.org/oaxiom/glbase/.

  13. Multilocus sequence analysis (MLSA) of Bradyrhizobium strains: revealing high diversity of tropical diazotrophic symbiotic bacteria.

    Science.gov (United States)

    Delamuta, Jakeline Renata Marçon; Ribeiro, Renan Augusto; Menna, Pâmela; Bangel, Eliane Villamil; Hungria, Mariangela

    2012-04-01

    Symbiotic association of several genera of bacteria collectively called as rhizobia and plants belonging to the family Leguminosae (=Fabaceae) results in the process of biological nitrogen fixation, playing a key role in global N cycling, and also bringing relevant contributions to the agriculture. Bradyrhizobium is considered as the ancestral of all nitrogen-fixing rhizobial species, probably originated in the tropics. The genus encompasses a variety of diverse bacteria, but the diversity captured in the analysis of the 16S rRNA is often low. In this study, we analyzed twelve Bradyrhizobium strains selected from previous studies performed by our group for showing high genetic diversity in relation to the described species. In addition to the 16S rRNA, five housekeeping genes (recA, atpD, glnII, gyrB and rpoB) were analyzed in the MLSA (multilocus sequence analysis) approach. Analysis of each gene and of the concatenated housekeeping genes captured a considerably higher level of genetic diversity, with indication of putative new species. The results highlight the high genetic variability associated with Bradyrhizobium microsymbionts of a variety of legumes. In addition, the MLSA approach has proved to represent a rapid and reliable method to be employed in phylogenetic and taxonomic studies, speeding the identification of the still poorly known diversity of nitrogen-fixing rhizobia in the tropics.

  14. LncRNA Expression Profile of Human Thoracic Aortic Dissection by High-Throughput Sequencing.

    Science.gov (United States)

    Sun, Jie; Chen, Guojun; Jing, Yuanwen; He, Xiang; Dong, Jianting; Zheng, Junmeng; Zou, Meisheng; Li, Hairui; Wang, Shifei; Sun, Yili; Liao, Wangjun; Liao, Yulin; Feng, Li; Bin, Jianping

    2018-01-01

    In this study, the long non-coding RNA (lncRNA) expression profile in human thoracic aortic dissection (TAD), a highly lethal cardiovascular disease, was investigated. Human TAD (n=3) and normal aortic tissues (NA) (n=3) were examined by high-throughput sequencing. Bioinformatics analyses were performed to predict the roles of aberrantly expressed lncRNAs. Quantitative real-time polymerase chain reaction (qRT-PCR) was applied to validate the results. A total of 269 lncRNAs (159 up-regulated and 110 down-regulated) and 2, 255 mRNAs (1 294 up-regulated and 961 down-regulated) were aberrantly expressed in human TAD (fold-change> 1.5, PTAD than in NA. The predicted binding motifs of three up-regulated lncRNAs (ENSG00000248508, ENSG00000226530, and EG00000259719) were correlated with up-regulated RUNX1 (R=0.982, PTAD. These findings suggest that lncRNAs are novel potential therapeutic targets for human TAD. © 2018 The Author(s). Published by S. Karger AG, Basel.

  15. Adaptation of Shift Sequence Based Method for High Number in Shifts Rostering Problem for Health Care Workers

    Directory of Open Access Journals (Sweden)

    Mindaugas Liogys

    2013-08-01

    Full Text Available Purpose—is to investigate a shift sequence-based approach efficiency then problem consisting of a high number of shifts.Research objectives:• Solve health care workers rostering problem using a shift sequence based method.• Measure its efficiency then number of shifts increases.Design/methodology/approach—Usually rostering problems are highly constrained. Constraints are classified to soft and hard constraints. Soft and hard constraints of the problem are additionally classified to: sequence constraints, schedule constraints and roster constraints. Sequence constraints are considered when constructing shift sequences. Schedule constraints are considered when constructing a schedule. Roster constraints are applied, then constructing overall solution, i.e. combining all schedules.Shift sequence based approach consists of two stages:• Shift sequences construction,• The construction of schedules.In the shift sequences construction stage, the shift sequences are constructed for each set of health care workers of different skill, considering sequence constraints. Shifts sequences are ranked by their penalties for easier retrieval in later stage.In schedules construction stage, schedules for each health care worker are constructed iteratively, using the shift sequences produced in stage 1.Shift sequence based method is an adaptive iterative method where health care workers who received the highest schedule penalties in the last iteration are scheduled first at the current iteration.During the roster construction, and after a schedule has been generated for the current health care worker, an improvement method based on an efficient greedy local search is carried out on the partial roster. It simply swaps any pair of shifts between two health care workers in the (partial roster, as long as the swaps satisfy hard constraints and decrease the roster penalty.Findings—Using shift sequence method for solving health care workers rostering problem

  16. Adaptation of Shift Sequence Based Method for High Number in Shifts Rostering Problem for Health Care Workers

    Directory of Open Access Journals (Sweden)

    Mindaugas Liogys

    2011-08-01

    Full Text Available Purpose—is to investigate a shift sequence-based approach efficiency then problem consisting of a high number of shifts. Research objectives:• Solve health care workers rostering problem using a shift sequence based method.• Measure its efficiency then number of shifts increases. Design/methodology/approach—Usually rostering problems are highly constrained.Constraints are classified to soft and hard constraints. Soft and hard constraints of the problem are additionally classified to: sequence constraints, schedule constraints and roster constraints. Sequence constraints are considered when constructing shift sequences. Schedule constraints are considered when constructing a schedule. Roster constraints are applied, then constructing overall solution, i.e. combining all schedules.Shift sequence based approach consists of two stages:• Shift sequences construction,• The construction of schedules.In the shift sequences construction stage, the shift sequences are constructed for each set of health care workers of different skill, considering sequence constraints. Shifts sequences are ranked by their penalties for easier retrieval in later stage.In schedules construction stage, schedules for each health care worker are constructed iteratively, using the shift sequences produced in stage 1. Shift sequence based method is an adaptive iterative method where health care workers who received the highest schedule penalties in the last iteration are scheduled first at the current iteration. During the roster construction, and after a schedule has been generated for the current health care worker, an improvement method based on an efficient greedy local search is carried out on the partial roster. It simply swaps any pair of shifts between two health care workers in the (partial roster, as long as the swaps satisfy hard constraints and decrease the roster penalty.Findings—Using shift sequence method for solving health care workers rostering

  17. Determination of 5 '-leader sequences from radically disparate strains of porcine reproductive and respiratory syndrome virus reveals the presence of highly conserved sequence motifs

    DEFF Research Database (Denmark)

    Oleksiewicz, M.B.; Bøtner, Anette; Nielsen, Jens

    1999-01-01

    We determined the untranslated 5'-leader sequence for three different isolates of porcine reproductive and respiratory syndrome virus (PRRSV): pathogenic European- and American-types, as well as an American-type vaccine strain. 5'-leader from European- and American-type PRRSV differed in length...... (220 and 190 nt, respectively), and exhibited only approximately 50% nucleotide homology. Nevertheless, highly conserved areas were identified in the leader of all 3 PRRSV isolates, which constitute candidate motifs for binding of protein(s) involved in viral replication. These comparative data provide...

  18. Confidence building in safety assessments

    International Nuclear Information System (INIS)

    Grundfelt, Bertil

    1999-01-01

    Future generations should be adequately protected from damage caused by the present disposal of radioactive waste. This presentation discusses the core of safety and performance assessment: The demonstration and building of confidence that the disposal system meets the safety requirements stipulated by society. The major difficulty is to deal with risks in the very long time perspective of the thousands of years during which the waste is hazardous. Concern about these problems has stimulated the development of the safety assessment discipline. The presentation concentrates on two of the elements of safety assessment: (1) Uncertainty and sensitivity analysis, and (2) validation and review. Uncertainty is associated both with respect to what is the proper conceptual model and with respect to parameter values for a given model. A special kind of uncertainty derives from the variation of a property in space. Geostatistics is one approach to handling spatial variability. The simplest way of doing a sensitivity analysis is to offset the model parameters one by one and observe how the model output changes. The validity of the models and data used to make predictions is central to the credibility of safety assessments for radioactive waste repositories. There are several definitions of model validation. The presentation discusses it as a process and highlights some aspects of validation methodologies

  19. Robust misinterpretation of confidence intervals.

    Science.gov (United States)

    Hoekstra, Rink; Morey, Richard D; Rouder, Jeffrey N; Wagenmakers, Eric-Jan

    2014-10-01

    Null hypothesis significance testing (NHST) is undoubtedly the most common inferential technique used to justify claims in the social sciences. However, even staunch defenders of NHST agree that its outcomes are often misinterpreted. Confidence intervals (CIs) have frequently been proposed as a more useful alternative to NHST, and their use is strongly encouraged in the APA Manual. Nevertheless, little is known about how researchers interpret CIs. In this study, 120 researchers and 442 students-all in the field of psychology-were asked to assess the truth value of six particular statements involving different interpretations of a CI. Although all six statements were false, both researchers and students endorsed, on average, more than three statements, indicating a gross misunderstanding of CIs. Self-declared experience with statistics was not related to researchers' performance, and, even more surprisingly, researchers hardly outperformed the students, even though the students had not received any education on statistical inference whatsoever. Our findings suggest that many researchers do not know the correct interpretation of a CI. The misunderstandings surrounding p-values and CIs are particularly unfortunate because they constitute the main tools by which psychologists draw conclusions from data.

  20. High throughput sequencing and proteomics to identify immunogenic proteins of a new pathogen: the dirty genome approach.

    Science.gov (United States)

    Greub, Gilbert; Kebbi-Beghdadi, Carole; Bertelli, Claire; Collyn, François; Riederer, Beat M; Yersin, Camille; Croxatto, Antony; Raoult, Didier

    2009-12-23

    With the availability of new generation sequencing technologies, bacterial genome projects have undergone a major boost. Still, chromosome completion needs a costly and time-consuming gap closure, especially when containing highly repetitive elements. However, incomplete genome data may be sufficiently informative to derive the pursued information. For emerging pathogens, i.e. newly identified pathogens, lack of release of genome data during gap closure stage is clearly medically counterproductive. We thus investigated the feasibility of a dirty genome approach, i.e. the release of unfinished genome sequences to develop serological diagnostic tools. We showed that almost the whole genome sequence of the emerging pathogen Parachlamydia acanthamoebae was retrieved even with relatively short reads from Genome Sequencer 20 and Solexa. The bacterial proteome was analyzed to select immunogenic proteins, which were then expressed and used to elaborate the first steps of an ELISA. This work constitutes the proof of principle for a dirty genome approach, i.e. the use of unfinished genome sequences of pathogenic bacteria, coupled with proteomics to rapidly identify new immunogenic proteins useful to develop in the future specific diagnostic tests such as ELISA, immunohistochemistry and direct antigen detection. Although applied here to an emerging pathogen, this combined dirty genome sequencing/proteomic approach may be used for any pathogen for which better diagnostics are needed. These genome sequences may also be very useful to develop DNA based diagnostic tests. All these diagnostic tools will allow further evaluations of the pathogenic potential of this obligate intracellular bacterium.

  1. High-Resolution Analysis of Coronavirus Gene Expression by RNA Sequencing and Ribosome Profiling.

    Science.gov (United States)

    Irigoyen, Nerea; Firth, Andrew E; Jones, Joshua D; Chung, Betty Y-W; Siddell, Stuart G; Brierley, Ian

    2016-02-01

    Members of the family Coronaviridae have the largest genomes of all RNA viruses, typically in the region of 30 kilobases. Several coronaviruses, such as Severe acute respiratory syndrome-related coronavirus (SARS-CoV) and Middle East respiratory syndrome-related coronavirus (MERS-CoV), are of medical importance, with high mortality rates and, in the case of SARS-CoV, significant pandemic potential. Other coronaviruses, such as Porcine epidemic diarrhea virus and Avian coronavirus, are important livestock pathogens. Ribosome profiling is a technique which exploits the capacity of the translating ribosome to protect around 30 nucleotides of mRNA from ribonuclease digestion. Ribosome-protected mRNA fragments are purified, subjected to deep sequencing and mapped back to the transcriptome to give a global "snap-shot" of translation. Parallel RNA sequencing allows normalization by transcript abundance. Here we apply ribosome profiling to cells infected with Murine coronavirus, mouse hepatitis virus, strain A59 (MHV-A59), a model coronavirus in the same genus as SARS-CoV and MERS-CoV. The data obtained allowed us to study the kinetics of virus transcription and translation with exquisite precision. We studied the timecourse of positive and negative-sense genomic and subgenomic viral RNA production and the relative translation efficiencies of the different virus ORFs. Virus mRNAs were not found to be translated more efficiently than host mRNAs; rather, virus translation dominates host translation at later time points due to high levels of virus transcripts. Triplet phasing of the profiling data allowed precise determination of translated reading frames and revealed several translated short open reading frames upstream of, or embedded within, known virus protein-coding regions. Ribosome pause sites were identified in the virus replicase polyprotein pp1a ORF and investigated experimentally. Contrary to expectations, ribosomes were not found to pause at the ribosomal

  2. High-Resolution Analysis of Coronavirus Gene Expression by RNA Sequencing and Ribosome Profiling.

    Directory of Open Access Journals (Sweden)

    Nerea Irigoyen

    2016-02-01

    Full Text Available Members of the family Coronaviridae have the largest genomes of all RNA viruses, typically in the region of 30 kilobases. Several coronaviruses, such as Severe acute respiratory syndrome-related coronavirus (SARS-CoV and Middle East respiratory syndrome-related coronavirus (MERS-CoV, are of medical importance, with high mortality rates and, in the case of SARS-CoV, significant pandemic potential. Other coronaviruses, such as Porcine epidemic diarrhea virus and Avian coronavirus, are important livestock pathogens. Ribosome profiling is a technique which exploits the capacity of the translating ribosome to protect around 30 nucleotides of mRNA from ribonuclease digestion. Ribosome-protected mRNA fragments are purified, subjected to deep sequencing and mapped back to the transcriptome to give a global "snap-shot" of translation. Parallel RNA sequencing allows normalization by transcript abundance. Here we apply ribosome profiling to cells infected with Murine coronavirus, mouse hepatitis virus, strain A59 (MHV-A59, a model coronavirus in the same genus as SARS-CoV and MERS-CoV. The data obtained allowed us to study the kinetics of virus transcription and translation with exquisite precision. We studied the timecourse of positive and negative-sense genomic and subgenomic viral RNA production and the relative translation efficiencies of the different virus ORFs. Virus mRNAs were not found to be translated more efficiently than host mRNAs; rather, virus translation dominates host translation at later time points due to high levels of virus transcripts. Triplet phasing of the profiling data allowed precise determination of translated reading frames and revealed several translated short open reading frames upstream of, or embedded within, known virus protein-coding regions. Ribosome pause sites were identified in the virus replicase polyprotein pp1a ORF and investigated experimentally. Contrary to expectations, ribosomes were not found to pause at the

  3. Viral metagenomics: Analysis of begomoviruses by illumina high-throughput sequencing

    KAUST Repository

    Idris, Ali

    2014-03-12

    Traditional DNA sequencing methods are inefficient, lack the ability to discern the least abundant viral sequences, and ineffective for determining the extent of variability in viral populations. Here, populations of single-stranded DNA plant begomoviral genomes and their associated beta- and alpha-satellite molecules (virus-satellite complexes) (genus, Begomovirus; family, Geminiviridae) were enriched from total nucleic acids isolated from symptomatic, field-infected plants, using rolling circle amplification (RCA). Enriched virus-satellite complexes were subjected to Illumina-Next Generation Sequencing (NGS). CASAVA and SeqMan NGen programs were implemented, respectively, for quality control and for de novo and reference-guided contig assembly of viral-satellite sequences. The authenticity of the begomoviral sequences, and the reproducibility of the Illumina-NGS approach for begomoviral deep sequencing projects, were validated by comparing NGS results with those obtained using traditional molecular cloning and Sanger sequencing of viral components and satellite DNAs, also enriched by RCA or amplified by polymerase chain reaction. As the use of NGS approaches, together with advances in software development, make possible deep sequence coverage at a lower cost; the approach described herein will streamline the exploration of begomovirus diversity and population structure from naturally infected plants, irrespective of viral abundance. This is the first report of the implementation of Illumina-NGS to explore the diversity and identify begomoviral-satellite SNPs directly from plants naturally-infected with begomoviruses under field conditions. 2014 by the authors; licensee MDPI, Basel, Switzerland.

  4. DNA template strand sequencing of single-cells maps genomic rearrangements at high resolution

    NARCIS (Netherlands)

    Falconer, Ester; Hills, Mark; Naumann, Ulrike; Poon, Steven S. S.; Chavez, Elizabeth A.; Sanders, Ashley D.; Zhao, Yongjun; Hirst, Martin; Lansdorp, Peter M.

    DNA rearrangements such as sister chromatid exchanges (SCEs) are sensitive indicators of genomic stress and instability, but they are typically masked by single-cell sequencing techniques. We developed Strand-seq to independently sequence parental DNA template strands from single cells, making it

  5. Viral Metagenomics: Analysis of Begomoviruses by Illumina High-Throughput Sequencing

    Directory of Open Access Journals (Sweden)

    Ali Idris

    2014-03-01

    Full Text Available Traditional DNA sequencing methods are inefficient, lack the ability to discern the least abundant viral sequences, and ineffective for determining the extent of variability in viral populations. Here, populations of single-stranded DNA plant begomoviral genomes and their associated beta- and alpha-satellite molecules (virus-satellite complexes (genus, Begomovirus; family, Geminiviridae were enriched from total nucleic acids isolated from symptomatic, field-infected plants, using rolling circle amplification (RCA. Enriched virus-satellite complexes were subjected to Illumina-Next Generation Sequencing (NGS. CASAVA and SeqMan NGen programs were implemented, respectively, for quality control and for de novo and reference-guided contig assembly of viral-satellite sequences. The authenticity of the begomoviral sequences, and the reproducibility of the Illumina-NGS approach for begomoviral deep sequencing projects, were validated by comparing NGS results with those obtained using traditional molecular cloning and Sanger sequencing of viral components and satellite DNAs, also enriched by RCA or amplified by polymerase chain reaction. As the use of NGS approaches, together with advances in software development, make possible deep sequence coverage at a lower cost; the approach described herein will streamline the exploration of begomovirus diversity and population structure from naturally infected plants, irrespective of viral abundance. This is the first report of the implementation of Illumina-NGS to explore the diversity and identify begomoviral-satellite SNPs directly from plants naturally-infected with begomoviruses under field conditions.

  6. A highly abundant bacteriophage discovered in the unknown sequences of human faecal metagenomes

    NARCIS (Netherlands)

    Dutilh, Bas E; Cassman, Noriko; McNair, Katelyn; Sanchez, Savannah E; Silva, Genivaldo G Z; Boling, Lance; Barr, Jeremy J; Speth, Daan R; Seguritan, Victor; Aziz, Ramy K; Felts, Ben; Dinsdale, Elizabeth A; Mokili, John L; Edwards, Robert A

    2014-01-01

    Metagenomics, or sequencing of the genetic material from a complete microbial community, is a promising tool to discover novel microbes and viruses. Viral metagenomes typically contain many unknown sequences. Here we describe the discovery of a previously unidentified bacteriophage present in the

  7. Diagnosing Anomalous Network Performance with Confidence

    Energy Technology Data Exchange (ETDEWEB)

    Settlemyer, Bradley W [ORNL; Hodson, Stephen W [ORNL; Kuehn, Jeffery A [ORNL; Poole, Stephen W [ORNL

    2011-04-01

    Variability in network performance is a major obstacle in effectively analyzing the throughput of modern high performance computer systems. High performance interconnec- tion networks offer excellent best-case network latencies; how- ever, highly parallel applications running on parallel machines typically require consistently high levels of performance to adequately leverage the massive amounts of available computing power. Performance analysts have usually quantified network performance using traditional summary statistics that assume the observational data is sampled from a normal distribution. In our examinations of network performance, we have found this method of analysis often provides too little data to under- stand anomalous network performance. Our tool, Confidence, instead uses an empirically derived probability distribution to characterize network performance. In this paper we describe several instances where the Confidence toolkit allowed us to understand and diagnose network performance anomalies that we could not adequately explore with the simple summary statis- tics provided by traditional measurement tools. In particular, we examine a multi-modal performance scenario encountered with an Infiniband interconnection network and we explore the performance repeatability on the custom Cray SeaStar2 interconnection network after a set of software and driver updates.

  8. High-throughput sequencing enhanced phage display enables the identification of patient-specific epitope motifs in serum

    DEFF Research Database (Denmark)

    Christiansen, Anders; Kringelum, Jens Vindahl; Hansen, Christian Skjødt

    2015-01-01

    of the bioinformatic approach was demonstrated by identifying epitopes of a prominent peanut allergen, Ara h 1, in sera from patients with severe peanut allergy. The identified epitopes were confirmed by high-density peptide micro-arrays. The present study demonstrates that high-throughput sequencing can empower phage...

  9. 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...

  10. Nitrate removal from high strength nitrate-bearing wastes in granular sludge sequencing batch reactors.

    Science.gov (United States)

    Krishna Mohan, Tulasi Venkata; Renu, Kadali; Nancharaiah, Yarlagadda Venkata; Satya Sai, Pedapati Murali; Venugopalan, Vayalam Purath

    2016-02-01

    A 6-L sequencing batch reactor (SBR) was operated for development of granular sludge capable of denitrification of high strength nitrates. Complete and stable denitrification of up to 5420 mg L(-1) nitrate-N (2710 mg L(-1) nitrate-N in reactor) was achieved by feeding simulated nitrate waste at a C/N ratio of 3. Compact and dense denitrifying granular sludge with relatively stable microbial community was developed during reactor operation. Accumulation of large amounts of nitrite due to incomplete denitrification occurred when the SBR was fed with 5420 mg L(-1) NO3-N at a C/N ratio of 2. Complete denitrification could not be achieved at this C/N ratio, even after one week of reactor operation as the nitrite levels continued to accumulate. In order to improve denitrification performance, the reactor was fed with nitrate concentrations of 1354 mg L(-1), while keeping C/N ratio at 2. Subsequently, nitrate concentration in the feed was increased in a step-wise manner to establish complete denitrification of 5420 mg L(-1) NO3-N at a C/N ratio of 2. The results show that substrate concentration plays an important role in denitrification of high strength nitrate by influencing nitrite accumulation. Complete denitrification of high strength nitrates can be achieved at lower substrate concentrations, by an appropriate acclimatization strategy. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  11. Deciphering the Resistome of the Widespread Pseudomonas aeruginosa Sequence Type 175 International High-Risk Clone through Whole-Genome Sequencing.

    Science.gov (United States)

    Cabot, Gabriel; López-Causapé, Carla; Ocampo-Sosa, Alain A; Sommer, Lea M; Domínguez, María Ángeles; Zamorano, Laura; Juan, Carlos; Tubau, Fe; Rodríguez, Cristina; Moyà, Bartolomé; Peña, Carmen; Martínez-Martínez, Luis; Plesiat, Patrick; Oliver, Antonio

    2016-12-01

    Whole-genome sequencing (WGS) was used for the characterization of the frequently extensively drug resistant (XDR) Pseudomonas aeruginosa sequence type 175 (ST175) high-risk clone. A total of 18 ST175 isolates recovered from 8 different Spanish hospitals were analyzed; 4 isolates from 4 different French hospitals were included for comparison. The typical resistance profile of ST175 included penicillins, cephalosporins, monobactams, carbapenems, aminoglycosides, and fluoroquinolones. In the phylogenetic analysis, the four French isolates clustered together with two isolates from one of the Spanish regions. Sequence variation was analyzed for 146 chromosomal genes related to antimicrobial resistance, and horizontally acquired genes were explored using online databases. The resistome of ST175 was determined mainly by mutational events; resistance traits common to all or nearly all of the strains included specific ampR mutations leading to ampC overexpression, specific mutations in oprD conferring carbapenem resistance, or a mexZ mutation leading to MexXY overexpression. All isolates additionally harbored an aadB gene conferring gentamicin and tobramycin resistance. Several other resistance traits were specific to certain geographic areas, such as a streptomycin resistance gene, aadA13, detected in all four isolates from France and in the two isolates from the Cantabria region and a glpT mutation conferring fosfomycin resistance, detected in all but these six isolates. Finally, several unique resistance mutations were detected in single isolates; particularly interesting were those in genes encoding penicillin-binding proteins (PBP1A, PBP3, and PBP4). Thus, these results provide information valuable for understanding the genetic basis of resistance and the dynamics of the dissemination and evolution of high-risk clones. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  12. Identification of QTLs for 14 Agronomically Important Traits in Setaria italica Based on SNPs Generated from High-Throughput Sequencing

    Directory of Open Access Journals (Sweden)

    Kai Zhang

    2017-05-01

    Full Text Available Foxtail millet (Setaria italica is an important crop possessing C4 photosynthesis capability. The S. italica genome was de novo sequenced in 2012, but the sequence lacked high-density genetic maps with agronomic and yield trait linkages. In the present study, we resequenced a foxtail millet population of 439 recombinant inbred lines (RILs and developed high-resolution bin map and high-density SNP markers, which could provide an effective approach for gene identification. A total of 59 QTL for 14 agronomic traits in plants grown under long- and short-day photoperiods were identified. The phenotypic variation explained ranged from 4.9 to 43.94%. In addition, we suggested that there may be segregation distortion on chromosome 6 that is significantly distorted toward Zhang gu. The newly identified QTL will provide a platform for sequence-based research on the S. italica genome, and for molecular marker-assisted breeding.

  13. Identification of QTLs for 14 Agronomically Important Traits in Setaria italica Based on SNPs Generated from High-Throughput Sequencing.

    Science.gov (United States)

    Zhang, Kai; Fan, Guangyu; Zhang, Xinxin; Zhao, Fang; Wei, Wei; Du, Guohua; Feng, Xiaolei; Wang, Xiaoming; Wang, Feng; Song, Guoliang; Zou, Hongfeng; Zhang, Xiaolei; Li, Shuangdong; Ni, Xuemei; Zhang, Gengyun; Zhao, Zhihai

    2017-05-05

    Foxtail millet ( Setaria italica ) is an important crop possessing C4 photosynthesis capability. The S. italica genome was de novo sequenced in 2012, but the sequence lacked high-density genetic maps with agronomic and yield trait linkages. In the present study, we resequenced a foxtail millet population of 439 recombinant inbred lines (RILs) and developed high-resolution bin map and high-density SNP markers, which could provide an effective approach for gene identification. A total of 59 QTL for 14 agronomic traits in plants grown under long- and short-day photoperiods were identified. The phenotypic variation explained ranged from 4.9 to 43.94%. In addition, we suggested that there may be segregation distortion on chromosome 6 that is significantly distorted toward Zhang gu. The newly identified QTL will provide a platform for sequence-based research on the S. italica genome, and for molecular marker-assisted breeding. Copyright © 2017 Zhang et al.

  14. Association Study of Gut Flora in Coronary Heart Disease through High-Throughput Sequencing

    Directory of Open Access Journals (Sweden)

    Li Cui

    2017-01-01

    Full Text Available Objectives. We aimed to explore the impact of gut microbiota in coronary heart disease (CHD patients through high-throughput sequencing. Methods. A total of 29 CHD in-hospital patients and 35 healthy volunteers as controls were included. Nucleic acids were extracted from fecal samples, followed by α diversity and principal coordinate analysis (PCoA. Based on unweighted UniFrac distance matrices, unweighted-pair group method with arithmetic mean (UPGMA trees were created. Results. After data optimization, an average of 121312±19293 reads in CHD patients and 234372±108725 reads in controls was obtained. Reads corresponding to 38 phyla, 90 classes, and 584 genera were detected in CHD patients, whereas 40 phyla, 99 classes, and 775 genera were detected in controls. The proportion of phylum Bacteroidetes (56.12% was lower and that of phylum Firmicutes was higher (37.06% in CHD patients than those in the controls (60.92% and 32.06%, P<0.05. PCoA and UPGMA tree analysis showed that there were significant differences of gut microbial compositions between the two groups. Conclusion. The diversity and compositions of gut flora were different between CHD patients and healthy controls. The incidence of CHD might be associated with the alteration of gut microbiota.

  15. Evaluation of the microbial diversity in amyotrophic lateral sclerosis using high-throughput sequencing

    Directory of Open Access Journals (Sweden)

    Xin Fang

    2016-09-01

    Full Text Available More and more evidences indicate that diseases of the central nervous system (CNS have been seriously affected by faecal microbes. However, little work is done to explore interaction between amyotrophic lateral sclerosis (ALS and faecal microbes. In the present study, high-throughput sequencing method was used to compare the intestinal microbial diversity of healthy people and ALS patients. The principal coordinate analysis (PCoA, Venn and unweighted pair-group method using arithmetic averages (UPGMA showed an obvious microbial changes between healthy people (group H and ALS patients (group A, and the average ratios of Bacteroides, Faecalibacterium, Anaerostipes, Prevotella, Escherichia and Lachnospira at genus level between ALS patients and healthy people were 0.78, 2.18, 3.41, 0.35, 0.79 and 13.07. Furthermore, the decreased Firmicutes/Bacteroidetes ratio at phylum level using LEfSE (LDA >4.0, together with the significant increased genus Dorea (harmful microorganisms and significant reduced genus Oscillibacter, Anaerostipes, Lachnospiraceae (beneficial microorganisms in ALS patients, indicated that the imbalance in intestinal microflora constitution had a strong association with the pathogenesis of ALS.

  16. Evaluation of the Microbial Diversity in Amyotrophic Lateral Sclerosis Using High-Throughput Sequencing.

    Science.gov (United States)

    Fang, Xin; Wang, Xin; Yang, Shaoguo; Meng, Fanjing; Wang, Xiaolei; Wei, Hua; Chen, Tingtao

    2016-01-01

    More and more evidences indicate that diseases of the central nervous system have been seriously affected by fecal microbes. However, little work is done to explore interaction between amyotrophic lateral sclerosis (ALS) and fecal microbes. In the present study, high-throughput sequencing method was used to compare the intestinal microbial diversity of healthy people and ALS patients. The principal coordinate analysis, Venn and unweighted pair-group method using arithmetic averages (UPGMA) showed an obvious microbial changes between healthy people (group H) and ALS patients (group A), and the average ratios of Bacteroides , Faecalibacterium , Anaerostipes , Prevotella , Escherichia , and Lachnospira at genus level between ALS patients and healthy people were 0.78, 2.18, 3.41, 0.35, 0.79, and 13.07. Furthermore, the decreased Firmicutes/Bacteroidetes ratio at phylum level using LEfSE (LDA > 4.0), together with the significant increased genus Dorea (harmful microorganisms) and significant reduced genus Oscillibacter , Anaerostipes , Lachnospiraceae (beneficial microorganisms) in ALS patients, indicated that the imbalance in intestinal microflora constitution had a strong association with the pathogenesis of ALS.

  17. High-throughput sequencing of plasma microRNA in chronic fatigue syndrome/myalgic encephalomyelitis.

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    Ekua W Brenu

    Full Text Available BACKGROUND: MicroRNAs (miRNAs are known to regulate many biological processes and their dysregulation has been associated with a variety of diseases including Chronic Fatigue Syndrome/Myalgic Encephalomyelitis (CFS/ME. The recent discovery of stable and reproducible miRNA in plasma has raised the possibility that circulating miRNAs may serve as novel diagnostic markers. The objective of this study was to determine the role of plasma miRNA in CFS/ME. RESULTS: Using Illumina high-throughput sequencing we identified 19 miRNAs that were differentially expressed in the plasma of CFS/ME patients in comparison to non-fatigued controls. Following RT-qPCR analysis, we were able to confirm the significant up-regulation of three miRNAs (hsa-miR-127-3p, hsa-miR-142-5p and hsa-miR-143-3p in the CFS/ME patients. CONCLUSION: Our study is the first to identify circulating miRNAs from CFS/ME patients and also to confirm three differentially expressed circulating miRNAs in CFS/ME patients, providing a basis for further study to find useful CFS/ME biomarkers.

  18. High-Throughput Sequencing of Plasma MicroRNA in Chronic Fatigue Syndrome/Myalgic Encephalomyelitis

    Science.gov (United States)

    Brenu, Ekua W.; Ashton, Kevin J.; Batovska, Jana; Staines, Donald R.; Marshall-Gradisnik, Sonya M.

    2014-01-01

    Background MicroRNAs (miRNAs) are known to regulate many biological processes and their dysregulation has been associated with a variety of diseases including Chronic Fatigue Syndrome/Myalgic Encephalomyelitis (CFS/ME). The recent discovery of stable and reproducible miRNA in plasma has raised the possibility that circulating miRNAs may serve as novel diagnostic markers. The objective of this study was to determine the role of plasma miRNA in CFS/ME. Results Using Illumina high-throughput sequencing we identified 19 miRNAs that were differentially expressed in the plasma of CFS/ME patients in comparison to non-fatigued controls. Following RT-qPCR analysis, we were able to confirm the significant up-regulation of three miRNAs (hsa-miR-127-3p, hsa-miR-142-5p and hsa-miR-143-3p) in the CFS/ME patients. Conclusion Our study is the first to identify circulating miRNAs from CFS/ME patients and also to confirm three differentially expressed circulating miRNAs in CFS/ME patients, providing a basis for further study to find useful CFS/ME biomarkers. PMID:25238588

  19. Identification of microRNAs and their targets in Finger millet by high throughput sequencing.

    Science.gov (United States)

    Usha, S; Jyothi, M N; Sharadamma, N; Dixit, Rekha; Devaraj, V R; Nagesh Babu, R

    2015-12-15

    MicroRNAs are short non-coding RNAs which play an important role in regulating gene expression by mRNA cleavage or by translational repression. The majority of identified miRNAs were evolutionarily conserved; however, others expressed in a species-specific manner. Finger millet is an important cereal crop; nonetheless, no practical information is available on microRNAs to date. In this study, we have identified 95 conserved microRNAs belonging to 39 families and 3 novel microRNAs by high throughput sequencing. For the identified conserved and novel miRNAs a total of 507 targets were predicted. 11 miRNAs were validated and tissue specificity was determined by stem loop RT-qPCR, Northern blot. GO analyses revealed targets of miRNA were involved in wide range of regulatory functions. This study implies large number of known and novel miRNAs found in Finger millet which may play important role in growth and development. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. High Performance Biological Pairwise Sequence Alignment: FPGA versus GPU versus Cell BE versus GPP

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    Khaled Benkrid

    2012-01-01

    Full Text Available This paper explores the pros and cons of reconfigurable computing in the form of FPGAs for high performance efficient computing. In particular, the paper presents the results of a comparative study between three different acceleration technologies, namely, Field Programmable Gate Arrays (FPGAs, Graphics Processor Units (GPUs, and IBM’s Cell Broadband Engine (Cell BE, in the design and implementation of the widely-used Smith-Waterman pairwise sequence alignment algorithm, with general purpose processors as a base reference implementation. Comparison criteria include speed, energy consumption, and purchase and development costs. The study shows that FPGAs largely outperform all other implementation platforms on performance per watt criterion and perform better than all other platforms on performance per dollar criterion, although by a much smaller margin. Cell BE and GPU come second and third, respectively, on both performance per watt and performance per dollar criteria. In general, in order to outperform other technologies on performance per dollar criterion (using currently available hardware and development tools, FPGAs need to achieve at least two orders of magnitude speed-up compared to general-purpose processors and one order of magnitude speed-up compared to domain-specific technologies such as GPUs.

  1. High-throughput sequencing, characterization and detection of new and conserved cucumber miRNAs.

    Directory of Open Access Journals (Sweden)

    Germán Martínez

    Full Text Available Micro RNAS (miRNAs are a class of endogenous small non coding RNAs involved in the post-transcriptional regulation of gene expression. In plants, a great number of conserved and specific miRNAs, mainly arising from model species, have been identified to date. However less is known about the diversity of these regulatory RNAs in vegetal species with agricultural and/or horticultural importance. Here we report a combined approach of bioinformatics prediction, high-throughput sequencing data and molecular methods to analyze miRNAs populations in cucumber (Cucumis sativus plants. A set of 19 conserved and 6 known but non-conserved miRNA families were found in our cucumber small RNA dataset. We also identified 7 (3 with their miRNA* strand not previously described miRNAs, candidates to be cucumber-specific. To validate their description these new C. sativus miRNAs were detected by northern blot hybridization. Additionally, potential targets for most conserved and new miRNAs were identified in cucumber genome.In summary, in this study we have identified, by first time, conserved, known non-conserved and new miRNAs arising from an agronomically important species such as C. sativus. The detection of this complex population of regulatory small RNAs suggests that similarly to that observe in other plant species, cucumber miRNAs may possibly play an important role in diverse biological and metabolic processes.

  2. Association Study of Gut Flora in Coronary Heart Disease through High-Throughput Sequencing.

    Science.gov (United States)

    Cui, Li; Zhao, Tingting; Hu, Haibing; Zhang, Wen; Hua, Xiuguo

    2017-01-01

    Objectives. We aimed to explore the impact of gut microbiota in coronary heart disease (CHD) patients through high-throughput sequencing. Methods. A total of 29 CHD in-hospital patients and 35 healthy volunteers as controls were included. Nucleic acids were extracted from fecal samples, followed by α diversity and principal coordinate analysis (PCoA). Based on unweighted UniFrac distance matrices, unweighted-pair group method with arithmetic mean (UPGMA) trees were created. Results. After data optimization, an average of 121312 ± 19293 reads in CHD patients and 234372 ± 108725 reads in controls was obtained. Reads corresponding to 38 phyla, 90 classes, and 584 genera were detected in CHD patients, whereas 40 phyla, 99 classes, and 775 genera were detected in controls. The proportion of phylum Bacteroidetes (56.12%) was lower and that of phylum Firmicutes was higher (37.06%) in CHD patients than those in the controls (60.92% and 32.06%, P UPGMA tree analysis showed that there were significant differences of gut microbial compositions between the two groups. Conclusion. The diversity and compositions of gut flora were different between CHD patients and healthy controls. The incidence of CHD might be associated with the alteration of gut microbiota.

  3. Insight into the transcriptome of Arthrobotrys conoides using high throughput sequencing.

    Science.gov (United States)

    Ramesh, Pandit; Reena, Patel; Amitbikram, Mohapatra; Chaitanya, Joshi; Anju, Kunjadia

    2015-12-01

    Arthrobotrys conoides is a nematode-trapping fungus belonging to Orbiliales, Ascomycota group, and traps prey nematodes by means of adhesive network. Fungus has a potential to be used as a biocontrol agent against plant parasitic nematodes. In the present study, we characterized the transcriptome of A. conoides using high-throughput sequencing technology and characterized its virulence unigenes. Total 7,255 cDNA contigs with an average length of 425 bp were generated and 6184 (61.81%) transcripts were functionally annotated and characterized. Majority of unigenes were found analogous to the genes of plant pathogenic fungi. A total of 1749 transcripts were found to be orthologous with eukaryotic proteins of KOG database. Several carbohydrate active enzymes and peptidases were identified. We also analyzed classically and nonclassically secreted proteins and confirmed by BLASTP against fungal secretome database. A total of 916 contigs were analogous to 556 unique proteins of Pathogen Host Interaction (PHI) database. Further, we identified 91 unigenes homologous to the database of fungal virulence factor (DFVF). A total of 104 putative protein kinases coding transcripts were identified by BLASTP against KinBase database, which are major players in signaling pathways. This study provides a comprehensive look at the transcriptome of A. conoides and the identified unigenes might have a role in catching and killing prey nematodes by A. conoides. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Prenatal MRI Findings of Fetuses with Congenital High Airway Obstruction Sequence

    Energy Technology Data Exchange (ETDEWEB)

    Guimaraes, Carolina V. A.; Linam, Leann E.; Kline-Fath, Beth M. [Cincinnati Children' s Hospital Medical Center, Cincinnati (United States)] (and others)

    2009-04-15

    To define the MRI findings of congenital high airway obstruction sequence (CHAOS) in a series of fetuses. Prenatal fetal MR images were reviewed in seven fetuses with CHAOS at 21 to 27 weeks of gestation. The MRI findings were reviewed. The MRI parameters evaluated included the appearance of the lungs and diaphragm, presence or absence of hydrops, amount of amniotic fluid, airway appearance, predicted level of airway obstruction, and any additional findings or suspected genetic syndromes. All the fetuses viewed (7 of 7) demonstrated the following MRI findings: dilated airway below the level of obstruction, increased lung signal, markedly increased lung volumes with flattened or inverted hemidiaphragms, massive ascites, centrally positioned and compressed heart, as well as placentomegaly. Other frequent findings were anasarca (6 of 7) and polyhydramnios (3 of 7). MRI identified the level of obstruction as laryngeal in five cases and tracheal in two cases. In four of the patients, surgery or autopsy confirmed the MRI predicted level of obstruction. Associated abnormalities were found in 4 of 7 (genetic syndromes in 2). Postnatal radiography (n = 3) showed markedly hyperinflated lungs with inverted or flattened hemidiaphragms, strandy perihilar opacities, pneumothoraces and tracheotomy. Two fetuses were terminated and one fetus demised in utero. Four fetuses were delivered via ex utero intrapartum treatment procedure. MRI shows a consistent pattern of abnormalities in fetuses with CHAOS, accurately identifies the level of airway obstruction, and helps differentiate from other lung abnormalities such as bilateral congenital pulmonary airway malformation by demonstrating an abnormally dilated airway distal to the obstruction.

  5. Characterization of CG6178 gene product with high sequence similarity to firefly luciferase in Drosophila melanogaster.

    Science.gov (United States)

    Oba, Yuichi; Ojika, Makoto; Inouye, Satoshi

    2004-03-31

    This is the first identification of a long-chain fatty acyl-CoA synthetase in Drosophila by enzymatic characterization. The gene product of CG6178 (CG6178) in Drosophila melanogaster genome, which has a high sequence similarity to firefly luciferase, has been expressed and characterized. CG6178 showed long-chain fatty acyl-CoA synthetic activity in the presence of ATP, CoA and Mg(2+), suggesting a fatty acyl adenylate is an intermediate. Recently, it was revealed that firefly luciferase has two catalytic functions, monooxygenase (luciferase) and AMP-mediated CoA ligase (fatty acyl-CoA synthetase). However, unlike firefly luciferase, CG6178 did not show luminescence activity in the presence of firefly luciferin, ATP, CoA and Mg(2+). The enzymatic properties of CG6178 including substrate specificity, pH dependency and optimal temperature were close to those of firefly luciferase and rat fatty acyl-CoA synthetase. Further, phylogenic analyses strongly suggest that the firefly luciferase gene may have evolved from a fatty acyl-CoA synthetase gene as a common ancestral gene.

  6. Transcriptome Sequencing in a Tibetan Barley Landrace with High Resistance to Powdery Mildew

    Directory of Open Access Journals (Sweden)

    Xing-Quan Zeng

    2014-01-01

    Full Text Available Hulless barley is an important cereal crop worldwide, especially in Tibet of China. However, this crop is usually susceptible to powdery mildew caused by Blumeria graminis f. sp. hordei. In this study, we aimed to understand the functions and pathways of genes involved in the disease resistance by transcriptome sequencing of a Tibetan barley landrace with high resistance to powdery mildew. A total of 831 significant differentially expressed genes were found in the infected seedlings, covering 19 functions. Either “cell,” “cell part,” and “extracellular region” in the cellular component category or “binding” and “catalytic” in the category of molecular function as well as “metabolic process” and “cellular process” in the biological process category together demonstrated that these functions may be involved in the resistance to powdery mildew of the hulless barley. In addition, 330 KEGG pathways were found using BLASTx with an E-value cut-off of <10−5. Among them, three pathways, namely, “photosynthesis,” “plant-pathogen interaction,” and “photosynthesis-antenna proteins” had significant matches in the database. Significant expressions of the three pathways were detected at 24 h, 48 h, and 96 h after infection, respectively. These results indicated a complex process of barley response to powdery mildew infection.

  7. Evaluation of a transposase protocol for rapid generation of shotgun high-throughput sequencing libraries from nanogram quantities of DNA.

    Science.gov (United States)

    Marine, Rachel; Polson, Shawn W; Ravel, Jacques; Hatfull, Graham; Russell, Daniel; Sullivan, Matthew; Syed, Fraz; Dumas, Michael; Wommack, K Eric

    2011-11-01

    Construction of DNA fragment libraries for next-generation sequencing can prove challenging, especially for samples with low DNA yield. Protocols devised to circumvent the problems associated with low starting quantities of DNA can result in amplification biases that skew the distribution of genomes in metagenomic data. Moreover, sample throughput can be slow, as current library construction techniques are time-consuming. This study evaluated Nextera, a new transposon-based method that is designed for quick production of DNA fragment libraries from a small quantity of DNA. The sequence read distribution across nine phage genomes in a mock viral assemblage met predictions for six of the least-abundant phages; however, the rank order of the most abundant phages differed slightly from predictions. De novo genome assemblies from Nextera libraries provided long contigs spanning over half of the phage genome; in four cases where full-length genome sequences were available for comparison, consensus sequences were found to match over 99% of the genome with near-perfect identity. Analysis of areas of low and high sequence coverage within phage genomes indicated that GC content may influence coverage of sequences from Nextera libraries. Comparisons of phage genomes prepared using both Nextera and a standard 454 FLX Titanium library preparation protocol suggested that the coverage biases according to GC content observed within the Nextera libraries were largely attributable to bias in the Nextera protocol rather than to the 454 sequencing technology. Nevertheless, given suitable sequence coverage, the Nextera protocol produced high-quality data for genomic studies. For metagenomics analyses, effects of GC amplification bias would need to be considered; however, the library preparation standardization that Nextera provides should benefit comparative metagenomic analyses.

  8. High-precision, whole-genome sequencing of laboratory strains facilitates genetic studies.

    Directory of Open Access Journals (Sweden)

    Anjana Srivatsan

    2008-08-01

    Full Text Available Whole-genome sequencing is a powerful technique for obtaining the reference sequence information of multiple organisms. Its use can be dramatically expanded to rapidly identify genomic variations, which can be linked with phenotypes to obtain biological insights. We explored these potential applications using the emerging next-generation sequencing platform Solexa Genome Analyzer, and the well-characterized model bacterium Bacillus subtilis. Combining sequencing with experimental verification, we first improved the accuracy of the published sequence of the B. subtilis reference strain 168, then obtained sequences of multiple related laboratory strains and different isolates of each strain. This provides a framework for comparing the divergence between different laboratory strains and between their individual isolates. We also demonstrated the power of Solexa sequencing by using its results to predict a defect in the citrate signal transduction pathway of a common laboratory strain, which we verified experimentally. Finally, we examined the molecular nature of spontaneously generated mutations that suppress the growth defect caused by deletion of the stringent response mediator relA. Using whole-genome sequencing, we rapidly mapped these suppressor mutations to two small homologs of relA. Interestingly, stable suppressor strains had mutations in both genes, with each mutation alone partially relieving the relA growth defect. This supports an intriguing three-locus interaction module that is not easily identifiable through traditional suppressor mapping. We conclude that whole-genome sequencing can drastically accelerate the identification of suppressor mutations and complex genetic interactions, and it can be applied as a standard tool to investigate the genetic traits of model organisms.

  9. Quartz-Seq2: a high-throughput single-cell RNA-sequencing method that effectively uses limited sequence reads.

    Science.gov (United States)

    Sasagawa, Yohei; Danno, Hiroki; Takada, Hitomi; Ebisawa, Masashi; Tanaka, Kaori; Hayashi, Tetsutaro; Kurisaki, Akira; Nikaido, Itoshi

    2018-03-09

    High-throughput single-cell RNA-seq methods assign limited unique molecular identifier (UMI) counts as gene expression values to single cells from shallow sequence reads and detect limited gene counts. We thus developed a high-throughput single-cell RNA-seq method, Quartz-Seq2, to overcome these issues. Our improvements in the reaction steps make it possible to effectively convert initial reads to UMI counts, at a rate of 30-50%, and detect more genes. To demonstrate the power of Quartz-Seq2, we analyzed approximately 10,000 transcriptomes from in vitro embryonic stem cells and an in vivo stromal vascular fraction with a limited number of reads.

  10. High quality draft genome sequence of Staphylococcus cohnii subsp. cohnii strain hu-01.

    Science.gov (United States)

    Hu, XinJun; Li, Ang; Lv, LongXian; Yuan, Chunhui; Guo, Lihua; Jiang, Xiawei; Jiang, Haiyin; Qian, GuiRong; Zheng, BeiWen; Guo, Jing; Li, LanJuan

    2014-06-15

    Staphylococcus cohnii subsp. cohnii belongs to the family Staphylococcaceae in the order Bacillales, class Bacilli and phylum Firmicutes. The increasing relevance of S. cohnii to human health prompted us to determine the genomic sequence of Staphylococcus cohnii subsp. cohnii strain hu-01, a multidrug-resistant isolate from a hospital in China. Here we describe the features of S. cohnii subsp. cohnii strain hu-01, together with the genome sequence and its annotation. This is the first genome sequence of the species Staphylococcus cohnii.

  11. Exploring the sources of bacterial spoilers in beefsteaks by culture-independent high-throughput sequencing.

    Directory of Open Access Journals (Sweden)

    Francesca De Filippis

    Full Text Available Microbial growth on meat to unacceptable levels contributes significantly to change meat structure, color and flavor and to cause meat spoilage. The types of microorganisms initially present in meat depend on several factors and multiple sources of contamination can be identified. The aims of this study were to evaluate the microbial diversity in beefsteaks before and after aerobic storage at 4°C and to investigate the sources of microbial contamination by examining the microbiota of carcasses wherefrom the steaks originated and of the processing environment where the beef was handled. Carcass, environmental (processing plant and meat samples were analyzed by culture-independent high-throughput sequencing of 16S rRNA gene amplicons. The microbiota of carcass swabs was very complex, including more than 600 operational taxonomic units (OTUs belonging to 15 different phyla. A significant association was found between beef microbiota and specific beef cuts (P<0.01 indicating that different cuts of the same carcass can influence the microbial contamination of beef. Despite the initially high complexity of the carcass microbiota, the steaks after aerobic storage at 4°C showed a dramatic decrease in microbial complexity. Pseudomonas sp. and Brochothrix thermosphacta were the main contaminants, and Acinetobacter, Psychrobacter and Enterobacteriaceae were also found. Comparing the relative abundance of OTUs in the different samples it was shown that abundant OTUs in beefsteaks after storage occurred in the corresponding carcass. However, the abundance of these same OTUs clearly increased in environmental samples taken in the processing plant suggesting that spoilage-associated microbial species originate from carcasses, they are carried to the processing environment where the meat is handled and there they become a resident microbiota. Such microbiota is then further spread on meat when it is handled and it represents the starting microbial association

  12. A high-throughput splinkerette-PCR method for the isolation and sequencing of retroviral insertion sites

    DEFF Research Database (Denmark)

    Uren, Anthony G; Mikkers, Harald; Kool, Jaap

    2009-01-01

    sites has been a major limitation to performing screens on this scale. Here we present a method for the high-throughput isolation of insertion sites using a highly efficient splinkerette-PCR method coupled with capillary or 454 sequencing. This protocol includes a description of the procedure for DNA......Insertional mutagens such as viruses and transposons are a useful tool for performing forward genetic screens in mice to discover cancer genes. These screens are most effective when performed using hundreds of mice; however, until recently, the cost-effective isolation and sequencing of insertion...

  13. Study on high-resolution sequence stratigraphy framework of uranium-hosting rock series in Qianjiadian sag

    International Nuclear Information System (INIS)

    Chen Fanghong; Zhang Mingyu

    2005-01-01

    The ore-hosting Yaojia Formation is composed of a set of braided stream medium-fine grained sediments. Guided by the basic theory of high-resolution sequence stratigraphy, and based on the core observation, the analysis of chemical composition of rocks, and data of natural potential logging and apparent resistivity logging, the authors have set up the high-resolution sequence stratigraphy framework of the ore-hosting Yaojia Formation, and discussed the relation of the stratigraphic structure of the middle cycle, as well as the paleotopography, the micro-facies to the formation of uranium deposit. (authors)

  14. Multishot versus single-shot pulse sequences in very high field fMRI: a comparison using retinotopic mapping.

    Directory of Open Access Journals (Sweden)

    Jascha D Swisher

    Full Text Available High-resolution functional MRI is a leading application for very high field (7 Tesla human MR imaging. Though higher field strengths promise improvements in signal-to-noise ratios (SNR and BOLD contrast relative to fMRI at 3 Tesla, these benefits may be partially offset by accompanying increases in geometric distortion and other off-resonance effects. Such effects may be especially pronounced with the single-shot EPI pulse sequences typically used for fMRI at standard field strengths. As an alternative, one might consider multishot pulse sequences, which may lead to somewhat lower temporal SNR than standard EPI, but which are also often substantially less susceptible to off-resonance effects. Here we consider retinotopic mapping of human visual cortex as a practical test case by which to compare examples of these sequence types for high-resolution fMRI at 7 Tesla. We performed polar angle retinotopic mapping at each of 3 isotropic resolutions (2.0, 1.7, and 1.1 mm using both accelerated single-shot 2D EPI and accelerated multishot 3D gradient-echo pulse sequences. We found that single-shot EPI indeed led to greater temporal SNR and contrast-to-noise ratios (CNR than the multishot sequences. However, additional distortion correction in postprocessing was required in order to fully realize these advantages, particularly at higher resolutions. The retinotopic maps produced by both sequence types were qualitatively comparable, and showed equivalent test/retest reliability. Thus, when surface-based analyses are planned, or in other circumstances where geometric distortion is of particular concern, multishot pulse sequences could provide a viable alternative to single-shot EPI.

  15. High-resolution whole-genome sequencing reveals that specific chromatin domains from most human chromosomes associate with nucleoli.

    Science.gov (United States)

    van Koningsbruggen, Silvana; Gierlinski, Marek; Schofield, Pietá; Martin, David; Barton, Geoffey J; Ariyurek, Yavuz; den Dunnen, Johan T; Lamond, Angus I

    2010-11-01

    The nuclear space is mostly occupied by chromosome territories and nuclear bodies. Although this organization of chromosomes affects gene function, relatively little is known about the role of nuclear bodies in the organization of chromosomal regions. The nucleolus is the best-studied subnuclear structure and forms around the rRNA repeat gene clusters on the acrocentric chromosomes. In addition to rDNA, other chromatin sequences also surround the nucleolar surface and may even loop into the nucleolus. These additional nucleolar-associated domains (NADs) have not been well characterized. We present here a whole-genome, high-resolution analysis of chromatin endogenously associated with nucleoli. We have used a combination of three complementary approaches, namely fluorescence comparative genome hybridization, high-throughput deep DNA sequencing and photoactivation combined with time-lapse fluorescence microscopy. The data show that specific sequences from most human chromosomes, in addition to the rDNA repeat units, associate with nucleoli in a reproducible and heritable manner. NADs have in common a high density of AT-rich sequence elements, low gene density and a statistically significant enrichment in transcriptionally repressed genes. Unexpectedly, both the direct DNA sequencing and fluorescence photoactivation data show that certain chromatin loci can specifically associate with either the nucleolus, or the nuclear envelope.

  16. Phylogenetic and functional analysis of metagenome sequence from high-temperature archaeal habitats demonstrate linkages between metabolic potential and geochemistry

    Directory of Open Access Journals (Sweden)

    William P. Inskeep

    2013-05-01

    Full Text Available Geothermal habitats in Yellowstone National Park (YNP provide an unparalled opportunity to understand the environmental factors that control the distribution of archaea in thermal habitats. Here we describe, analyze and synthesize metagenomic and geochemical data collected from seven high-temperature sites that contain microbial communities dominated by archaea relative to bacteria. The specific objectives of the study were to use metagenome sequencing to determine the structure and functional capacity of thermophilic archaeal-dominated microbial communities across a pH range from 2.5 to 6.4 and to discuss specific examples where the metabolic potential correlated with measured environmental parameters and geochemical processes occurring in situ. Random shotgun metagenome sequence (~40-45 Mbase Sanger sequencing per site was obtained from environmental DNA extracted from high-temperature sediments and/or microbial mats and subjected to numerous phylogenetic and functional analyses. Analysis of individual sequences (e.g., MEGAN and G+C content and assemblies from each habitat type revealed the presence of dominant archaeal populations in all environments, 10 of whose genomes were largely reconstructed from the sequence data. Analysis of protein family occurrence, particularly of those involved in energy conservation, electron transport and autotrophic metabolism, revealed significant differences in metabolic strategies across sites consistent with differences in major geochemical attributes (e.g., sulfide, oxygen, pH. These observations provide an ecological basis for understanding the distribution of indigenous archaeal lineages across high temperature systems of YNP.

  17. Detection of Sequence Polymorphism in Rubus Occidentalis L. Monomorphic Microsatellite Markers by High Resolution Melting

    Science.gov (United States)

    Microsatellite, or simple sequence repeat (SSR) markers, are valuable as co-dominant genetic markers with a variety of applications such as DNA fingerprinting, linkage mapping, and population structure analysis. Development of microsatellite primers through the identification of appropriate repeate...

  18. Sequencing quality assessment tools to enable data-driven informatics for high throughput genomics

    Directory of Open Access Journals (Sweden)

    Richard Mark Leggett

    2013-12-01

    Full Text Available The processes of quality assessment and control are an active area of research at The Genome Analysis Centre (TGAC. Unlike other sequencing centres that often concentrate on a certain species or technology, TGAC applies expertise in genomics and bioinformatics to a wide range of projects, often requiring bespoke wet lab and in silico workflows. TGAC is fortunate to have access to a diverse range of sequencing and analysis platforms, and we are at the forefront of investigations into library quality and sequence data assessment. We have developed and implemented a number of algorithms, tools, pipelines and packages to ascertain, store, and expose quality metrics across a number of next-generation sequencing platforms, allowing rapid and in-depth cross-platform QC bioinformatics. In this review, we describe these tools as a vehicle for data-driven informatics, offering the potential to provide richer context for downstream analysis and to inform experimental design.

  19. Construction of a high-density genetic map for grape using next generation restriction-site associated DNA sequencing

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    Wang Nian

    2012-08-01

    Full Text Available Abstract Background Genetic mapping and QTL detection are powerful methodologies in plant improvement and breeding. Construction of a high-density and high-quality genetic map would be of great benefit in the production of superior grapes to meet human demand. High throughput and low cost of the recently developed next generation sequencing (NGS technology have resulted in its wide application in genome research. Sequencing restriction-site associated DNA (RAD might be an efficient strategy to simplify genotyping. Combining NGS with RAD has proven to be powerful for single nucleotide polymorphism (SNP marker development. Results An F1 population of 100 individual plants was developed. In-silico digestion-site prediction was used to select an appropriate restriction enzyme for construction of a RAD sequencing library. Next generation RAD sequencing was applied to genotype the F1 population and its parents. Applying a cluster strategy for SNP modulation, a total of 1,814 high-quality SNP markers were developed: 1,121 of these were mapped to the female genetic map, 759 to the male map, and 1,646 to the integrated map. A comparison of the genetic maps to the published Vitis vinifera genome revealed both conservation and variations. Conclusions The applicability of next generation RAD sequencing for genotyping a grape F1 population was demonstrated, leading to the successful development of a genetic map with high density and quality using our designed SNP markers. Detailed analysis revealed that this newly developed genetic map can be used for a variety of genome investigations, such as QTL detection, sequence assembly and genome comparison.

  20. High Interlaboratory Reprocucibility of DNA Sequence-based Typing of Bacteria in a Multicenter Study

    DEFF Research Database (Denmark)

    Sousa, MA de; Boye, Kit; Lencastre, H de

    2006-01-01

    Current DNA amplification-based typing methods for bacterial pathogens often lack interlaboratory reproducibility. In this international study, DNA sequence-based typing of the Staphylococcus aureus protein A gene (spa, 110 to 422 bp) showed 100% intra- and interlaboratory reproducibility without...... extensive harmonization of protocols for 30 blind-coded S. aureus DNA samples sent to 10 laboratories. Specialized software for automated sequence analysis ensured a common typing nomenclature....

  1. High quality draft genome sequence of Staphylococcus cohnii subsp. cohnii strain hu-01

    OpenAIRE

    Hu, XinJun; Li, Ang; Lv, LongXian; Yuan, Chunhui; Guo, Lihua; Jiang, Xiawei; Jiang, Haiyin; Qian, GuiRong; Zheng, BeiWen; Guo, Jing; Li, LanJuan

    2014-01-01

    Staphylococcus cohnii subsp. cohnii belongs to the family Staphylococcaceae in the order Bacillales , class Bacilli and phylum Firmicutes . The increasing relevance of S. cohnii to human health prompted us to determine the genomic sequence of Staphylococcus cohnii subsp. cohnii strain hu-01, a multidrug-resistant isolate from a hospital in China. Here we describe the features of S. cohnii subsp. cohnii strain hu-01, together with the genome sequence and its annotation. This is the first genom...

  2. DNA template strand sequencing of single-cells maps genomic rearrangements at high resolution

    OpenAIRE

    Falconer, Ester; Hills, Mark; Naumann, Ulrike; Poon, Steven S. S.; Chavez, Elizabeth A.; Sanders, Ashley D.; Zhao, Yongjun; Hirst, Martin; Lansdorp, Peter M.

    2012-01-01

    DNA rearrangements such as sister chromatid exchanges (SCEs) are sensitive indicators of genomic stress and instability, but they are typically masked by single-cell sequencing techniques. We developed Strand-seq to independently sequence parental DNA template strands from single cells, making it possible to map SCEs at orders-of-magnitude greater resolution than was previously possible. On average, murine embryonic stem (mES) cells exhibit eight SCEs, which are detected at a resolution of up...

  3. Analysis of high-depth sequence data for studying viral diversity: a comparison of next generation sequencing platforms using Segminator II

    Directory of Open Access Journals (Sweden)

    Archer John

    2012-03-01

    Full Text Available Abstract Background Next generation sequencing provides detailed insight into the variation present within viral populations, introducing the possibility of treatment strategies that are both reactive and predictive. Current software tools, however, need to be scaled up to accommodate for high-depth viral data sets, which are often temporally or spatially linked. In addition, due to the development of novel sequencing platforms and chemistries, each with implicit strengths and weaknesses, it will be helpful for researchers to be able to routinely compare and combine data sets from different platforms/chemistries. In particular, error associated with a specific sequencing process must be quantified so that true biological variation may be identified. Results Segminator II was developed to allow for the efficient comparison of data sets derived from different sources. We demonstrate its usage by comparing large data sets from 12 influenza H1N1 samples sequenced on both the 454 Life Sciences and Illumina platforms, permitting quantification of platform error. For mismatches median error rates at 0.10 and 0.12%, respectively, suggested that both platforms performed similarly. For insertions and deletions median error rates within the 454 data (at 0.3 and 0.2%, respectively were significantly higher than those within the Illumina data (0.004 and 0.006%, respectively. In agreement with previous observations these higher rates were strongly associated with homopolymeric stretches on the 454 platform. Outside of such regions both platforms had similar indel error profiles. Additionally, we apply our software to the identification of low frequency variants. Conclusion We have demonstrated, using Segminator II, that it is possible to distinguish platform specific error from biological variation using data derived from two different platforms. We have used this approach to quantify the amount of error present within the 454 and Illumina platforms in

  4. Biodegradation of high concentrations of phenol by baker’s yeast in anaerobic sequencing batch reactor

    Directory of Open Access Journals (Sweden)

    Ali Asghar Najafpoor

    2015-06-01

    Full Text Available Background: Phenol, as a pure substance, is used in many fields because of its disinfectant, germicidal, local anesthetic, and peptizing properties. Aqueous solutions of phenol are produced as waste in industries and discharged into the environment. Therefore, elevated concentrations of phenol may be found in air or water because of industrial discharge or the use of phenolic products. Method: The strains of Saccharomyces cerevisiae used in this project were natural strains previously purchased from Razavy company. They were grown at 30°C on Petri plates containing yeast extract glucose (YGC and then purified by being spread onto new plates, and isolated colonies were obtained. These colonies provided the basis of selection. Prepared strains were applied in anaerobic sequencing batch reactors (ASBRs as first seed. The experiment conditions were optimized using response surface methodology (RSM. After the determined runs were performed using Design-Expert software, data were analyzed using mentioned software as well. Results: This study evaluated the capability of baker’s yeast to remove phenol in high concentrations. The tested strains showed excellent tolerance to phenol toxicity at concentrations up to 6100 mg/L. Study of the batch degradation process showed that the phenol removal rate could exceed 99.9% in 24 hours at a concentration of 1000 mg/L. The results showed catechol is the first intermediate product of phenol degradation. In survey results of the Design–Expert software, R2 and Adeq precision were 0.97 and 25.65, respectively. Conclusion: The results demonstrated that ASBR performs robustly under variable influent concentrations of inhibitory compounds. The high removal performance despite the high phenol concentration may be a result of reactor operating strategies. Based on the progressive increase of inlet phenol concentration, allowing for an enhanced biomass acclimation in a short time, results at the microbiological levels

  5. Th unnatural order of things: A history of the high school science sequence

    Science.gov (United States)

    Robbins, Dennis M.

    Historical studies of US high school science education are rare. This study examines the historical origins of a unique characteristic of the secondary science curriculum, the Biology-Chemistry-Physics (B-C-P) order of courses. Statements from scientists, educators and the media claim that B-C-P has been the traditional curriculum sequence for over a century and can be traced back to the influential educational commission known as the Committee of Ten (CoT) of 1893. This study examines the history of the ordering of high school science subjects over the last 150 years. The reports and primary documents of important national educational commissions, such as the CoT, were searched for their recommendations on secondary science, particularly on course ordering. These recommendations were then compared to national, state and local statistical data on subject offerings and student enrollments to measure the effect of these national commissions on school policy. This study concludes that the Committee of Ten did not create B-P-C. The CoT made six recommendations, five placed Chemistry before Physics (P-C). One recommendation for C-P met with strong disagreement because it was thought an illogical order. Biology as a "uniform" course did not exist at this time and so the CoT made no recommendations for its grade placement. Statistical data shows that B-C-P evolved over many decades. From 1860 up to 1920 most schools used a P-C curriculum believing Physics was a foundational prerequisite of Chemistry. Biology was introduced in the early 1900s and it assumed a position before the physical sciences. Through the 1920s Chemistry and Physics were placed equally likely in 11th or 12 th grades and Biology was in the 10th grade. After World War II, B-C-P became the dominant pattern, exhibited in over 90% of schools. But up to this point in time no educational body or national commission had recommended B-C-P. The Biology-Chemistry-Physics order of courses is a product of many

  6. 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.

  7. Single-nucleotide polymorphism discovery by high-throughput sequencing in sorghum

    Directory of Open Access Journals (Sweden)

    White Frank F

    2011-07-01

    Full Text Available Abstract Background Eight diverse sorghum (Sorghum bicolor L. Moench accessions were subjected to short-read genome sequencing to characterize the distribution of single-nucleotide polymorphisms (SNPs. Two strategies were used for DNA library preparation. Missing SNP genotype data were imputed by local haplotype comparison. The effect of library type and genomic diversity on SNP discovery and imputation are evaluated. Results Alignment of eight genome equivalents (6 Gb to the public reference genome revealed 283,000 SNPs at ≥82% confirmation probability. Sequencing from libraries constructed to limit sequencing to start at defined restriction sites led to genotyping 10-fold more SNPs in all 8 accessions, and correctly imputing 11% more missing data, than from semirandom libraries. The SNP yield advantage of the reduced-representation method was less than expected, since up to one fifth of reads started at noncanonical restriction sites and up to one third of restriction sites predicted in silico to yield unique alignments were not sampled at near-saturation. For imputation accuracy, the availability of a genomically similar accession in the germplasm panel was more important than panel size or sequencing coverage. Conclusions A sequence quantity of 3 million 50-base reads per accession using a BsrFI library would conservatively provide satisfactory genotyping of 96,000 sorghum SNPs. For most reliable SNP-genotype imputation in shallowly sequenced genomes, germplasm panels should consist of pairs or groups of genomically similar entries. These results may help in designing strategies for economical genotyping-by-sequencing of large numbers of plant accessions.

  8. High-density rhesus macaque oligonucleotide microarray design using early-stage rhesus genome sequence information and human genome annotations

    Directory of Open Access Journals (Sweden)

    Magness Charles L

    2007-01-01

    Full Text Available Abstract Background Until recently, few genomic reagents specific for non-human primate research have been available. To address this need, we have constructed a macaque-specific high-density oligonucleotide microarray by using highly fragmented low-pass sequence contigs from the rhesus genome project together with the detailed sequence and exon structure of the human genome. Using this method, we designed oligonucleotide probes to over 17,000 distinct rhesus/human gene orthologs and increased by four-fold the number of available genes relative to our first-generation expressed sequence tag (EST-derived array. Results We constructed a database containing 248,000 exon sequences from 23,000 human RefSeq genes and compared each human exon with its best matching sequence in the January 2005 version of the rhesus genome project list of 486,000 DNA contigs. Best matching rhesus exon sequences for each of the 23,000 human genes were then concatenated in the proper order and orientation to produce a rhesus "virtual transcriptome." Microarray probes were designed, one per gene, to the region closest to the 3' untranslated region (UTR of each rhesus virtual transcript. Each probe was compared to a composite rhesus/human transcript database to test for cross-hybridization potential yielding a final probe set representing 18,296 rhesus/human gene orthologs, including transcript variants, and over 17,000 distinct genes. We hybridized mRNA from rhesus brain and spleen to both the EST- and genome-derived microarrays. Besides four-fold greater gene coverage, the genome-derived array also showed greater mean signal intensities for genes present on both arrays. Genome-derived probes showed 99.4% identity when compared to 4,767 rhesus GenBank sequence tag site (STS sequences indicating that early stage low-pass versions of complex genomes are of sufficient quality to yield valuable functional genomic information when combined with finished genome information from

  9. Sedimentology and High Resolution Sequence Stratigraphy of the Middle Jurassic Dhruma Formation Carbonates Outcrops in the Central Saudi Arabia

    Science.gov (United States)

    Yousif, Ibrahim; Abdullatif, Osman; Makkawi, Mohammed; Abdulghani, Waleed

    2017-04-01

    This study investigates the microfacies and sequence stratigraphic frame work of the Middle Jurassic Dhruma Formation in outcrops in central Saudi Arabia. The study contributes to the efforts to understand and enhance local and regional stratigraphic relationship and correlation of the Jurassic carbonate sequences and their significance to reservoir description and prediction in the subsurcae. The study describes and characterizes the sedimentology, microfacies and the stratigraphy of Dhruma Formation from outcrop sections having a total thickness of 70 m. Detailed microfacies and high-resolution stratigraphical analysis were carried out to determine microfacies, cyclicity, sequences and staking pattern. The study revealed ten lithofacies namely: oolitic grainstone,bioclastic oolitic grainstone, oolitic grapestone, bioclastic grainstone,foraminiferal packstone, echinoderm packstone, peloidal packstone to grainstone,skeletal wackestone to packstone, mudstone, and marlstone.These lithofacies were grouped into five lithofacies associations that deposited on a carbonate ramp setting. The depositional environment ranging from low energy lagoonal setting to high-energy shoals and banks to low energy outer ramp setting. Five high-resolution composite sequences have been defined and each sequence is composed at the bottom of intercalated mudstone/wackestone that passing up into grainstone lithofacies.The composite sequences range in thickness from 7 to 15 m, while the parasequences range from 0.5 to 1.5 m. The composite sequences extend laterally for a distance of more than 350 m. The overall composite section shows a shallowing upward succession of the 4th to the 5th order high-resolution sequences.The dominant lithofacies are the grainy ones, which constitute 30%, 50% and 80% of the studied sections. Furthermore, the parasequences thickness and their bio-components are increasing towards the top. The muddy lithofacies intensively affected the vertical continuity of the

  10. Beyond the drip-line: a high-resolution open-air Holocene hunter-gatherer sequence from highland Lesotho

    CSIR Research Space (South Africa)

    Mitchell, P

    2011-03-01

    Full Text Available the drip-line: a high-resolution open-air Holocene hunter-gatherer sequence from highland Lesotho Peter Mitchell1, Ina Plug2, Geoff Bailey3, Ruth Charles4, Amanda Esterhuysen5, Julia Lee Thorp6, Adrian Parker7 & Stephan Woodborne8 The activities...

  11. Sequence-specific high mobility group box factors recognize 10-12-base pair minor groove motifs

    DEFF Research Database (Denmark)

    van Beest, M; Dooijes, D; van De Wetering, M

    2000-01-01

    Sequence-specific high mobility group (HMG) box factors bind and bend DNA via interactions in the minor groove. Three-dimensional NMR analyses have provided the structural basis for this interaction. The cognate HMG domain DNA motif is generally believed to span 6-8 bases. However, alignment...

  12. Small RNA sequencing reveals a comprehensive miRNA signature of BRCA1-associated high-grade serous ovarian cancer

    NARCIS (Netherlands)

    Brouwer, Jan; Kluiver, Joost; de Almeida, Rodrigo C.; Modderman, Rutger; Terpstra, Martijn; Kok, Klaas; Withoff, Sebo; Hollema, Harry; Reitsma, Welmoed; de Bock, Geertruida H.; Mourits, Marian J. E.; van den Berg, Anke

    2016-01-01

    AimsBRCA1 mutation carriers are at increased risk of developing high-grade serous ovarian cancer (HGSOC), a malignancy that originates from fallopian tube epithelium. We aimed to identify differentially expressed known and novel miRNAs in BRCA1-associated HGSOC. Methods Small RNA sequencing was

  13. A Teaching-Learning Sequence for the Special Relativity Theory at High School Level Historically and Epistemologically Contextualized

    Science.gov (United States)

    Arriassecq, Irene; Greca, Ileana Maria

    2012-01-01

    This paper discusses some topics that stem from recent contributions made by the History, the Philosophy, and the Didactics of Science. We consider these topics relevant to the introduction of the Special Relativity Theory (SRT) in high school within a contextualized approach. We offer an outline of a teaching-learning sequence dealing with the…

  14. mrsFAST-Ultra: a compact, SNP-aware mapper for high performance sequencing applications.

    Science.gov (United States)

    Hach, Faraz; Sarrafi, Iman; Hormozdiari, Farhad; Alkan, Can; Eichler, Evan E; Sahinalp, S Cenk

    2014-07-01

    High throughput sequencing (HTS) platforms generate unprecedented amounts of data that introduce challenges for processing and downstream analysis. While tools that report the 'best' mapping location of each read provide a fast way to process HTS data, they are not suitable for many types of downstream analysis such as structural variation detection, where it is important to report multiple mapping loci for each read. For this purpose we introduce mrsFAST-Ultra, a fast, cache oblivious, SNP-aware aligner that can handle the multi-mapping of HTS reads very efficiently. mrsFAST-Ultra improves mrsFAST, our first cache oblivious read aligner capable of handling multi-mapping reads, through new and compact index structures that reduce not only the overall memory usage but also the number of CPU operations per alignment. In fact the size of the index generated by mrsFAST-Ultra is 10 times smaller than that of mrsFAST. As importantly, mrsFAST-Ultra introduces new features such as being able to (i) obtain the best mapping loci for each read, and (ii) return all reads that have at most n mapping loci (within an error threshold), together with these loci, for any user specified n. Furthermore, mrsFAST-Ultra is SNP-aware, i.e. it can map reads to reference genome while discounting the mismatches that occur at common SNP locations provided by db-SNP; this significantly increases the number of reads that can be mapped to the reference genome. Notice that all of the above features are implemented within the index structure and are not simple post-processing steps and thus are performed highly efficiently. Finally, mrsFAST-Ultra utilizes multiple available cores and processors and can be tuned for various memory settings. Our results show that mrsFAST-Ultra is roughly five times faster than its predecessor mrsFAST. In comparison to newly enhanced popular tools such as Bowtie2, it is more sensitive (it can report 10 times or more mappings per read) and much faster (six times or

  15. Power spectral density and scaling exponent of high frequency global solar radiation sequences

    Science.gov (United States)

    Calif, Rudy; Schmitt, François G.; Huang, Yongxiang

    2013-04-01

    The part of the solar power production from photovlotaïcs systems is constantly increasing in the electric grids. Solar energy converter devices such as photovoltaic cells are very sensitive to instantaneous solar radiation fluctuations. Thus rapid variation of solar radiation due to changes in the local meteorological condition can induce large amplitude fluctuations of the produced electrical power and reduce the overall efficiency of the system. When large amount of photovoltaic electricity is send into a weak or small electricity network such as island network, the electric grid security can be in jeopardy due to these power fluctuations. The integration of this energy in the electrical network remains a major challenge, due to the high variability of solar radiation in time and space. To palliate these difficulties, it is essential to identify the characteristic of these fluctuations in order to anticipate the eventuality of power shortage or power surge. The objective of this study is to present an approach based on Empirical Mode Decomposition (EMD) and Hilbert-Huang Transform (HHT) to highlight the scaling properties of global solar irradiance data G(t). The scale of invariance is detected on this dataset using the Empirical Mode Decomposition in association with arbitrary-order Hilbert spectral analysis, a generalization of (HHT) or Hilbert Spectral Analysis (HSA). The first step is the EMD, consists in decomposing the normalized global solar radiation data G'(t) into several Intrinsic Mode Functions (IMF) Ci(t) without giving an a priori basis. Consequently, the normalized original solar radiation sequence G'(t) can be written as a sum of Ci(t) with a residual rn. From all IMF modes, a joint PDF P(f,A) of locally and instantaneous frequency f and amplitude A, is estimated. To characterize the scaling behavior in amplitude-frequency space, an arbitrary-order Hilbert marginal spectrum is defined to: Iq(f) = 0 P (f,A)A dA (1) with q × 0 In case of scale

  16. Robust Sub-nanomolar Library Preparation for High Throughput Next Generation Sequencing.

    Science.gov (United States)

    Wu, Wells W; Phue, Je-Nie; Lee, Chun-Ting; Lin, Changyi; Xu, Lai; Wang, Rong; Zhang, Yaqin; Shen, Rong-Fong

    2018-05-04

    Current library preparation protocols for Illumina HiSeq and MiSeq DNA sequencers require ≥2 nM initial library for subsequent loading of denatured cDNA onto flow cells. Such amounts are not always attainable from samples having a relatively low DNA or RNA input; or those for which a limited number of PCR amplification cycles is preferred (less PCR bias and/or more even coverage). A well-tested sub-nanomolar library preparation protocol for Illumina sequencers has however not been reported. The aim of this study is to provide a much needed working protocol for sub-nanomolar libraries to achieve outcomes as informative as those obtained with the higher library input (≥ 2 nM) recommended by Illumina's protocols. Extensive studies were conducted to validate a robust sub-nanomolar (initial library of 100 pM) protocol using PhiX DNA (as a control), genomic DNA (Bordetella bronchiseptica and microbial mock community B for 16S rRNA gene sequencing), messenger RNA, microRNA, and other small noncoding RNA samples. The utility of our protocol was further explored for PhiX library concentrations as low as 25 pM, which generated only slightly fewer than 50% of the reads achieved under the standard Illumina protocol starting with > 2 nM. A sub-nanomolar library preparation protocol (100 pM) could generate next generation sequencing (NGS) results as robust as the standard Illumina protocol. Following the sub-nanomolar protocol, libraries with initial concentrations as low as 25 pM could also be sequenced to yield satisfactory and reproducible sequencing results.

  17. ‘‘Blind'' mapping of genic DNA sequence polymorphisms in Lolium perenne L. by high resolution melting curve analysis

    DEFF Research Database (Denmark)

    Studer, Bruno; Jensen, Louise Bach; Fiil, Alice

    2009-01-01

    High resolution melting curve analysis (HRM) measures dissociation of double stranded DNA of a PCR product amplified in the presence of a saturating fluorescence dye. Recently, HRM proved successful to genotype DNA sequence polymorphisms such as SSRs and SNPs based on the shape of the melting...... curves. In this study, HRM was used for simultaneous screening and genotyping of genic DNA sequence polymorphisms identified in the Lolium perenne F2 mapping population VrnA. Melting profiles of PCR products amplified from previously published gene loci and from a novel gene putatively involved...

  18. Rapid and Deep Proteomes by Faster Sequencing on a Benchtop Quadrupole Ultra-High-Field Orbitrap Mass Spectrometer

    DEFF Research Database (Denmark)

    Kelstrup, Christian D; Jersie-Christensen, Rosa R; Batth, Tanveer Singh

    2014-01-01

    per second or up to 600 new peptides sequenced per gradient minute. We identify 4400 proteins from one microgram of HeLa digest using a one hour gradient, which is an approximately 30% improvement compared to previous instrumentation. In addition, we show very deep proteome coverage can be achieved...... in less than 24 hours of analysis time by offline high pH reversed-phase peptide fractionation from which we identify more than 140,000 unique peptide sequences. This is comparable to state-of-the-art multi-day, multi-enzyme efforts. Finally the acquisition methods are evaluated for single...

  19. A high-speed on-chip pseudo-random binary sequence generator for multi-tone phase calibration

    Science.gov (United States)

    Gommé, Liesbeth; Vandersteen, Gerd; Rolain, Yves

    2011-07-01

    An on-chip reference generator is conceived by adopting the technique of decimating a pseudo-random binary sequence (PRBS) signal in parallel sequences. This is of great benefit when high-speed generation of PRBS and PRBS-derived signals is the objective. The design implemented standard CMOS logic is available in commercial libraries to provide the logic functions for the generator. The design allows the user to select the periodicity of the PRBS and the PRBS-derived signals. The characterization of the on-chip generator marks its performance and reveals promising specifications.

  20. A high-speed on-chip pseudo-random binary sequence generator for multi-tone phase calibration

    International Nuclear Information System (INIS)

    Gommé, Liesbeth; Vandersteen, Gerd; Rolain, Yves

    2011-01-01

    An on-chip reference generator is conceived by adopting the technique of decimating a pseudo-random binary sequence (PRBS) signal in parallel sequences. This is of great benefit when high-speed generation of PRBS and PRBS-derived signals is the objective. The design implemented standard CMOS logic is available in commercial libraries to provide the logic functions for the generator. The design allows the user to select the periodicity of the PRBS and the PRBS-derived signals. The characterization of the on-chip generator marks its performance and reveals promising specifications

  1. High throughput resistance profiling of Plasmodium falciparum infections based on custom dual indexing and Illumina next generation sequencing-technology

    DEFF Research Database (Denmark)

    Nag, Sidsel; Dalgaard, Marlene Danner; Kofoed, Poul-Erik

    2017-01-01

    Genetic polymorphisms in P. falciparum can be used to indicate the parasite's susceptibility to antimalarial drugs as well as its geographical origin. Both of these factors are key to monitoring development and spread of antimalarial drug resistance. In this study, we combine multiplex PCR, custom...... designed dual indexing and Miseq sequencing for high throughput SNP-profiling of 457 malaria infections from Guinea-Bissau, at the cost of 10 USD per sample. By amplifying and sequencing 15 genetic fragments, we cover 20 resistance-conferring SNPs occurring in pfcrt, pfmdr1, pfdhfr, pfdhps, as well...

  2. Molecular diet analysis of two African free-tailed bats (Molossidae) using high throughput sequencing

    DEFF Research Database (Denmark)

    Bohmann, Kristine; Monadjem, Ara; Noer, Christina Lehmkuhl

    2011-01-01

    Given the diversity of prey consumed by insectivorous bats, it is difficult to discern the composition of their diet using morphological or conventional PCR-based analyses of their faeces. We demonstrate the use of a powerful alternate tool, the use of the Roche FLX sequencing platform to deep......-sequence uniquely 5′ tagged insect-generic barcode cytochrome c oxidase I (COI) fragments, that were PCR amplified from faecal pellets of two free-tailed bat species Chaerephon pumilus and Mops condylurus (family: Molossidae). Although the analyses were challenged by the paucity of southern African insect COI...

  3. Simultaneous and complete genome sequencing of influenza A and B with high coverage by Illumina MiSeq Platform.

    Science.gov (United States)

    Rutvisuttinunt, Wiriya; Chinnawirotpisan, Piyawan; Simasathien, Sriluck; Shrestha, Sanjaya K; Yoon, In-Kyu; Klungthong, Chonticha; Fernandez, Stefan

    2013-11-01

    Active global surveillance and characterization of influenza viruses are essential for better preparation against possible pandemic events. Obtaining comprehensive information about the influenza genome can improve our understanding of the evolution of influenza viruses and emergence of new strains, and improve the accuracy when designing preventive vaccines. This study investigated the use of deep sequencing by the next-generation sequencing (NGS) Illumina MiSeq Platform to obtain complete genome sequence information from influenza virus isolates. The influenza virus isolates were cultured from 6 respiratory acute clinical specimens collected in Thailand and Nepal. DNA libraries obtained from each viral isolate were mixed and all were sequenced simultaneously. Total information of 2.6 Gbases was obtained from a 455±14 K/mm2 density with 95.76% (8,571,655/8,950,724 clusters) of the clusters passing quality control (QC) filters. Approximately 93.7% of all sequences from Read1 and 83.5% from Read2 contained high quality sequences that were ≥Q30, a base calling QC score standard. Alignments analysis identified three seasonal influenza A H3N2 strains, one 2009 pandemic influenza A H1N1 strain and two influenza B strains. The nearly entire genomes of all six virus isolates yielded equal or greater than 600-fold sequence coverage depth. MiSeq Platform identified seasonal influenza A H3N2, 2009 pandemic influenza A H1N1and influenza B in the DNA library mixtures efficiently. Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.

  4. Gene discovery and molecular marker development, based on high-throughput transcript sequencing of Paspalum dilatatum Poir.

    Directory of Open Access Journals (Sweden)

    Andrea Giordano

    Full Text Available BACKGROUND: Paspalum dilatatum Poir. (common name dallisgrass is a native grass species of South America, with special relevance to dairy and red meat production. P. dilatatum exhibits higher forage quality than other C4 forage grasses and is tolerant to frost and water stress. This species is predominantly cultivated in an apomictic monoculture, with an inherent high risk that biotic and abiotic stresses could potentially devastate productivity. Therefore, advanced breeding strategies that characterise and use available genetic diversity, or assess germplasm collections effectively are required to deliver advanced cultivars for production systems. However, there are limited genomic resources available for this forage grass species. RESULTS: Transcriptome sequencing using second-generation sequencing platforms has been employed using pooled RNA from different tissues (stems, roots, leaves and inflorescences at the final reproductive stage of P. dilatatum cultivar Primo. A total of 324,695 sequence reads were obtained, corresponding to c. 102 Mbp. The sequences were assembled, generating 20,169 contigs of a combined length of 9,336,138 nucleotides. The contigs were BLAST analysed against the fully sequenced grass species of Oryza sativa subsp. japonica, Brachypodium distachyon, the closely related Sorghum bicolor and foxtail millet (Setaria italica genomes as well as against the UniRef 90 protein database allowing a comprehensive gene ontology analysis to be performed. The contigs generated from the transcript sequencing were also analysed for the presence of simple sequence repeats (SSRs. A total of 2,339 SSR motifs were identified within 1,989 contigs and corresponding primer pairs were designed. Empirical validation of a cohort of 96 SSRs was performed, with 34% being polymorphic between sexual and apomictic biotypes. CONCLUSIONS: The development of genetic and genomic resources for P. dilatatum will contribute to gene discovery and expression

  5. High throughput sequencing and proteomics to identify immunogenic proteins of a new pathogen: the dirty genome approach.

    Directory of Open Access Journals (Sweden)

    Gilbert Greub

    Full Text Available BACKGROUND: With the availability of new generation sequencing technologies, bacterial genome projects have undergone a major boost. Still, chromosome completion needs a costly and time-consuming gap closure, especially when containing highly repetitive elements. However, incomplete genome data may be sufficiently informative to derive the pursued information. For emerging pathogens, i.e. newly identified pathogens, lack of release of genome data during gap closure stage is clearly medically counterproductive. METHODS/PRINCIPAL FINDINGS: We thus investigated the feasibility of a dirty genome approach, i.e. the release of unfinished genome sequences to develop serological diagnostic tools. We showed that almost the whole genome sequence of the emerging pathogen Parachlamydia acanthamoebae was retrieved even with relatively short reads from Genome Sequencer 20 and Solexa. The bacterial proteome was analyzed to select immunogenic proteins, which were then expressed and used to elaborate the first steps of an ELISA. CONCLUSIONS/SIGNIFICANCE: This work constitutes the proof of principle for a dirty genome approach, i.e. the use of unfinished genome sequences of pathogenic bacteria, coupled with proteomics to rapidly identify new immunogenic proteins useful to develop in the future specific diagnostic tests such as ELISA, immunohistochemistry and direct antigen detection. Although applied here to an emerging pathogen, this combined dirty genome sequencing/proteomic approach may be used for any pathogen for which better diagnostics are needed. These genome sequences may also be very useful to develop DNA based diagnostic tests. All these diagnostic tools will allow further evaluations of the pathogenic potential of this obligate intracellular bacterium.

  6. Perceptual learning effect on decision and confidence thresholds.

    Science.gov (United States)

    Solovey, Guillermo; Shalom, Diego; Pérez-Schuster, Verónica; Sigman, Mariano

    2016-10-01

    Practice can enhance of perceptual sensitivity, a well-known phenomenon called perceptual learning. However, the effect of practice on subjective perception has received little attention. We approach this problem from a visual psychophysics and computational modeling perspective. In a sequence of visual search experiments, subjects significantly increased the ability to detect a "trained target". Before and after training, subjects performed two psychophysical protocols that parametrically vary the visibility of the "trained target": an attentional blink and a visual masking task. We found that confidence increased after learning only in the attentional blink task. Despite large differences in some observables and task settings, we identify common mechanisms for decision-making and confidence. Specifically, our behavioral results and computational model suggest that perceptual ability is independent of processing time, indicating that changes in early cortical representations are effective, and learning changes decision criteria to convey choice and confidence. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. A new sieving matrix for DNA sequencing, genotyping and mutation detection and high-throughput genotyping with a 96-capillary array system

    Energy Technology Data Exchange (ETDEWEB)

    Gao, David [Iowa State Univ., Ames, IA (United States)

    1999-11-08

    Capillary electrophoresis has been widely accepted as a fast separation technique in DNA analysis. In this dissertation, a new sieving matrix is described for DNA analysis, especially DNA sequencing, genetic typing and mutation detection. A high-throughput 96 capillary array electrophoresis system was also demonstrated for simultaneous multiple genotyping. The authors first evaluated the influence of different capillary coatings on the performance of DNA sequencing. A bare capillary was compared with a DB-wax, an FC-coated and a polyvinylpyrrolidone dynamically coated capillary with PEO as sieving matrix. It was found that covalently-coated capillaries had no better performance than bare capillaries while PVP coating provided excellent and reproducible results. The authors also developed a new sieving Matrix for DNA separation based on commercially available poly(vinylpyrrolidone) (PVP). This sieving matrix has a very low viscosity and an excellent self-coating effect. Successful separations were achieved in uncoated capillaries. Sequencing of M13mp18 showed good resolution up to 500 bases in treated PVP solution. Temperature gradient capillary electrophoresis and PVP solution was applied to mutation detection. A heteroduplex sample and a homoduplex reference were injected during a pair of continuous runs. A temperature gradient of 10 C with a ramp of 0.7 C/min was swept throughout the capillary. Detection was accomplished by laser induced fluorescence detection. Mutation detection was performed by comparing the pattern changes between the homoduplex and the heteroduplex samples. High throughput, high detection rate and easy operation were achieved in this system. They further demonstrated fast and reliable genotyping based on CTTv STR system by multiple-capillary array electrophoresis. The PCR products from individuals were mixed with pooled allelic ladder as an absolute standard and coinjected with a 96-vial tray. Simultaneous one-color laser-induced fluorescence

  8. Genome sequence of M6, a diploid inbred clone of the high-glycoalkaloid-producing tuber-bearing potato species Solanum chacoense, reveals residual heterozygosity.

    Science.gov (United States)

    Leisner, Courtney P; Hamilton, John P; Crisovan, Emily; Manrique-Carpintero, Norma C; Marand, Alexandre P; Newton, Linsey; Pham, Gina M; Jiang, Jiming; Douches, David S; Jansky, Shelley H; Buell, C Robin

    2018-05-01

    Cultivated potato (Solanum tuberosum L.) is a highly heterozygous autotetraploid that presents challenges in genome analyses and breeding. Wild potato species serve as a resource for the introgression of important agronomic traits into cultivated potato. One key species is Solanum chacoense and the diploid, inbred clone M6, which is self-compatible and has desirable tuber market quality and disease resistance traits. Sequencing and assembly of the genome of the M6 clone of S. chacoense generated an assembly of 825 767 562 bp in 8260 scaffolds with an N50 scaffold size of 713 602 bp. Pseudomolecule construction anchored 508 Mb of the genome assembly into 12 chromosomes. Genome annotation yielded 49 124 high-confidence gene models representing 37 740 genes. Comparative analyses of the M6 genome with six other Solanaceae species revealed a core set of 158 367 Solanaceae genes and 1897 genes unique to three potato species. Analysis of single nucleotide polymorphisms across the M6 genome revealed enhanced residual heterozygosity on chromosomes 4, 8 and 9 relative to the other chromosomes. Access to the M6 genome provides a resource for identification of key genes for important agronomic traits and aids in genome-enabled development of inbred diploid potatoes with the potential to accelerate potato breeding. © 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.

  9. Genome-wide SNP identification by high-throughput sequencing and selective mapping allows sequence assembly positioning using a framework genetic linkage map

    Directory of Open Access Journals (Sweden)

    Xu Xiangming

    2010-12-01

    Full Text Available Abstract Background Determining the position and order of contigs and scaffolds from a genome assembly within an organism's genome remains a technical challenge in a majority of sequencing projects. In order to exploit contemporary technologies for DNA sequencing, we developed a strategy for whole genome single nucleotide polymorphism sequencing allowing the positioning of sequence contigs onto a linkage map using the bin mapping method. Results The strategy was tested on a draft genome of the fungal pathogen Venturia inaequalis, the causal agent of apple scab, and further validated using sequence contigs derived from the diploid plant genome Fragaria vesca. Using our novel method we were able to anchor 70% and 92% of sequences assemblies for V. inaequalis and F. vesca, respectively, to genetic linkage maps. Conclusions We demonstrated the utility of this approach by accurately determining the bin map positions of the majority of the large sequence contigs from each genome sequence and validated our method by mapping single sequence repeat markers derived from sequence contigs on a full mapping population.

  10. Isolation and characterization of antigen-specific alpaca (Lama pacos) VHH antibodies by biopanning followed by high-throughput sequencing.

    Science.gov (United States)

    Miyazaki, Nobuo; Kiyose, Norihiko; Akazawa, Yoko; Takashima, Mizuki; Hagihara, Yosihisa; Inoue, Naokazu; Matsuda, Tomonari; Ogawa, Ryu; Inoue, Seiya; Ito, Yuji

    2015-09-01

    The antigen-binding domain of camelid dimeric heavy chain antibodies, known as VHH or Nanobody, has much potential in pharmaceutical and industrial applications. To establish the isolation process of antigen-specific VHH, a VHH phage library was constructed with a diversity of 8.4 × 10(7) from cDNA of peripheral blood mononuclear cells of an alpaca (Lama pacos) immunized with a fragment of IZUMO1 (IZUMO1PFF) as a model antigen. By conventional biopanning, 13 antigen-specific VHHs were isolated. The amino acid sequences of these VHHs, designated as N-group VHHs, were very similar to each other (>93% identity). To find more diverse antibodies, we performed high-throughput sequencing (HTS) of VHH genes. By comparing the frequencies of each sequence between before and after biopanning, we found the sequences whose frequencies were increased by biopanning. The top 100 sequences of them were supplied for phylogenic tree analysis. In total 75% of them belonged to N-group VHHs, but the other were phylogenically apart from N-group VHHs (Non N-group). Two of three VHHs selected from non N-group VHHs showed sufficient antigen binding ability. These results suggested that biopanning followed by HTS provided a useful method for finding minor and diverse antigen-specific clones that could not be identified by conventional biopanning. © The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

  11. 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.

  12. High-throughput physical map anchoring via BAC-pool sequencing

    Czech Academy of Sciences Publication Activity Database

    Cviková, Kateřina; Cattonaro, F.; Alaux, M.; Stein, N.; Mayer, K.F.X.; Doležel, Jaroslav; Bartoš, Jan

    2015-01-01

    Roč. 15, APR 11 (2015) ISSN 1471-2229 R&D Projects: GA ČR GA13-08786S; GA MŠk(CZ) LO1204 Institutional support: RVO:61389030 Keywords : Physical map * Contig anchoring * Next generation sequencing Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.631, year: 2015

  13. Designing small universal k-mer hitting sets for improved analysis of high-throughput sequencing.

    Directory of Open Access Journals (Sweden)

    Yaron Orenstein

    2017-10-01

    Full Text Available With the rapidly increasing volume of deep sequencing data, more efficient algorithms and data structures are needed. Minimizers are a central recent paradigm that has improved various sequence analysis tasks, including hashing for faster read overlap detection, sparse suffix arrays for creating smaller indexes, and Bloom filters for speeding up sequence search. Here, we propose an alternative paradigm that can lead to substantial further improvement in these and other tasks. For integers k and L > k, we say that a set of k-mers is a universal hitting set (UHS if every possible L-long sequence must contain a k-mer from the set. We develop a heuristic called DOCKS to find a compact UHS, which works in two phases: The first phase is solved optimally, and for the second we propose several efficient heuristics, trading set size for speed and memory. The use of heuristics is motivated by showing the NP-hardness of a closely related problem. We show that DOCKS works well in practice and produces UHSs that are very close to a theoretical lower bound. We present results for various values of k and L and by applying them to real genomes show that UHSs indeed improve over minimizers. In particular, DOCKS uses less than 30% of the 10-mers needed to span the human genome compared to minimizers. The software and computed UHSs are freely available at github.com/Shamir-Lab/DOCKS/ and acgt.cs.tau.ac.il/docks/, respectively.

  14. 454-sequencing reveals stochastic local reassembly and high disturbance tolerance within arbuscular mycorrhizal fungal communities

    DEFF Research Database (Denmark)

    Lekberg, Karin Ylva Margareta; Schnoor, Tim; Kjøller, Rasmus

    2012-01-01

    unpredictable, with approximately 40%of all sequences within a sample belonging to a single OTU of varying identity. The distribution of two plant species that are often poorly colonized by AMfungi (Dianthus deltoides and Carex arenaria) correlated significantly with the OTU composition, which may indicate...

  15. High resolution melting detects sequence polymorphism in rubus occidentalis L. monomorphic microsatellite markers

    Science.gov (United States)

    Microsatellite, or simple sequence repeat (SSR) markers, are valuable as co-dominant genetic markers with a variety of applications such as DNA fingerprinting, linkage mapping, and population structure analysis. However, primer pairs designed from the regions that flank SSRs often generate fragment...

  16. Micropathogen Community Analysis in Hyalomma rufipes via High-Throughput Sequencing of Small RNAs

    Science.gov (United States)

    Luo, Jin; Liu, Min-Xuan; Ren, Qiao-Yun; Chen, Ze; Tian, Zhan-Cheng; Hao, Jia-Wei; Wu, Feng; Liu, Xiao-Cui; Luo, Jian-Xun; Yin, Hong; Wang, Hui; Liu, Guang-Yuan

    2017-01-01

    Ticks are important vectors in the transmission of a broad range of micropathogens to vertebrates, including humans. Because of the role of ticks in disease transmission, identifying and characterizing the micropathogen profiles of tick populations have become increasingly important. The objective of this study was to survey the micropathogens of Hyalomma rufipes ticks. Illumina HiSeq2000 technology was utilized to perform deep sequencing of small RNAs (sRNAs) extracted from field-collected H. rufipes ticks in Gansu Province, China. The resultant sRNA library data revealed that the surveyed tick populations produced reads that were homologous to St. Croix River Virus (SCRV) sequences. We also observed many reads that were homologous to microbial and/or pathogenic isolates, including bacteria, protozoa, and fungi. As part of this analysis, a phylogenetic tree was constructed to display the relationships among the homologous sequences that were identified. The study offered a unique opportunity to gain insight into the micropathogens of H. rufipes ticks. The effective control of arthropod vectors in the future will require knowledge of the micropathogen composition of vectors harboring infectious agents. Understanding the ecological factors that regulate vector propagation in association with the prevalence and persistence of micropathogen lineages is also imperative. These interactions may affect the evolution of micropathogen lineages, especially if the micropathogens rely on the vector or host for dispersal. The sRNA deep-sequencing approach used in this analysis provides an intuitive method to survey micropathogen prevalence in ticks and other vector species. PMID:28861401

  17. High diversity of picornaviruses in rats from different continents revealed by deep sequencing

    DEFF Research Database (Denmark)

    Arn Hansen, Thomas; Mollerup, Sarah; Nguyen, Nam-Phuong

    2016-01-01

    Outbreaks of zoonotic diseases in humans and livestock are not uncommon, and an important component in containment of such emerging viral diseases is rapid and reliable diagnostics. Such methods are often PCR-based and hence require the availability of sequence data from the pathogen. Rattus norv...

  18. High diversity of picornaviruses in rats from different continents revealed by deep sequencing

    DEFF Research Database (Denmark)

    Arn Hansen, Thomas; Mollerup, Sarah; Nguyen, Nam-Phuong

    2016-01-01

    Outbreaks of zoonotic diseases in humans and livestock are not uncommon, and an important component in containment of such emerging viral diseases is rapid and reliable diagnostics. Such methods are often PCR-based and hence require the availability of sequence data from the pathogen. Rattus...

  19. Core Genome Multilocus Sequence Typing Scheme for High-resolution Typing of Enterococcus faecium

    DEFF Research Database (Denmark)

    de Been, Mark; Pinholt, Mette; Top, Janetta

    2015-01-01

    Enterococcus faecium, a common inhabitant of the human gut, has emerged as an important multidrug-resistant nosocomial pathogen in the last two decades. Since the start of the 21(st) century, multi-locus sequence typing (MLST) has been used to study the molecular epidemiology of E. faecium. However...

  20. Deep-sequencing revealed Citrus bark cracking viroid (CBCVd) as a highly aggressive pathogen on hop

    Czech Academy of Sciences Publication Activity Database

    Jakše, J.; Radišek, S.; Pokorn, T.; Matoušek, Jaroslav; Javornik, B.

    2015-01-01

    Roč. 64, č. 4 (2015), s. 831-842 ISSN 0032-0862 R&D Projects: GA MŠk(CZ) LH14255 Institutional support: RVO:60077344 Keywords : Bioinformatic * Citrus bark cracking viroid * Hop * Next-generation sequencing Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.383, year: 2015

  1. Exploring Sequence Characteristics Related to High- Level Production of Secreted Proteins in Aspergillus niger

    NARCIS (Netherlands)

    Van den Berg, B.A.; Reinders, M.J.T.; Hulsman, M.; Wu, L.; Pel, H.J.; Roubos, J.A.; De Ridder, D.

    2012-01-01

    Protein sequence features are explored in relation to the production of over-expressed extracellular proteins by fungi. Knowledge on features influencing protein production and secretion could be employed to improve enzyme production levels in industrial bioprocesses via protein engineering. A large

  2. 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

  3. Analysis of petunia hybrida in response to salt stress using high throughput RNA sequencing

    Science.gov (United States)

    Salt and drought are among the greatest challenges to crop and native plants in meeting their yield and reproductive potentials. DNA sequencing-enabled transcriptome profiling provides a means of assessing what genes are responding to salt or drought stress so as to better understand the molecular ...

  4. High prevalence of human polyomavirus JC VP1 gene sequences in pediatric malignancies.

    Science.gov (United States)

    Shiramizu, B; Hu, N; Frisque, R J; Nerurkar, V R

    2007-05-15

    The oncogenic potential of human polyomavirus JC (JCV), a ubiquitous virus that establishes infection during early childhood in approximately 70% of the human population, is unclear. As a neurotropic virus, JCV has been implicated in pediatric central nervous system tumors and has been suggested to be a pathogenic agent in pediatric acute lymphoblastic leukemia. Recent studies have demonstrated JCV gene sequences in pediatric medulloblastomas and among patients with colorectal cancer. JCV early protein T-antigen (TAg) can form complexes with cellular regulatory proteins and thus may play a role in tumorigenesis. Since JCV is detected in B-lymphocytes, a retrospective analysis of pediatric B-cell and non-B-cell malignancies as well as other HIV-associated pediatric malignancies was conducted for the presence of JCV gene sequences. DNA was extracted from 49 pediatric malignancies, including Hodgkin disease, non-Hodgkin lymphoma, large cell lymphoma and sarcoma. Polymerase chain reaction (PCR) was conducted using JCV specific nested primer sets for the transcriptional control region (TCR), TAg, and viral capsid protein 1 (VP1) genes. Southern blot analysis and DNA sequencing were used to confirm specificity of the amplicons. A 215-bp region of the JCV VP1 gene was amplified from 26 (53%) pediatric tumor tissues. The JCV TCR and two JCV gene regions were amplified from a leiomyosarcoma specimen from an HIV-infected patient. The leiomyosarcoma specimen from the cecum harbored the archetype strain of JCV. Including the leiomyosarcoma specimen, three of five specimens sequenced were typed as JCV genotype 2. The failure to amplify JCV TCR, and TAg gene sequences in the presence of JCV VP1 gene sequence is surprising. Even though JCV TAg gene, which is similar to the SV40 TAg gene, is oncogenic in animal models, the presence of JCV gene sequences in pediatric malignancies does not prove causality. In light of the available data on the presence of JCV in normal and cancerous

  5. High-resolution sequence stratigraphy of lower Paleozoic sheet sandstones in central North America: The role of special conditions of cratonic interiors in development of stratal architecture

    Science.gov (United States)

    Runkel, Anthony C.; Miller, J.F.; McKay, R.M.; Palmer, A.R.; Taylor, John F.

    2007-01-01

    Well-known difficulties in applying sequence stratigraphic concepts to deposits that accumulated across slowly subsiding cratonic interior regions have limited our ability to interpret the history of continental-scale tectonism, oceanographic dynamics of epeiric seas, and eustasy. We used a multi-disciplinary approach to construct a high-resolution stratigraphic framework for lower Paleozoic strata in the cratonic interior of North America. Within this framework, these strata proved readily amenable to modern sequence stratigraphic techniques that were formulated based on successions along passive margins and in foreland basins, settings markedly different from the cratonic interior. Parasequences, parasequence stacking patterns, systems tracts, maximum flooding intervals, and sequence-bounding unconformities can be confidently recognized in the cratonic interior using mostly standard criteria for identification. The similarity of cratonic interior and foreland basin successions in size, geometry, constituent facies, and local stacking patterns of nearshore parasequences is especially striking. This similarity indicates that the fundamental processes that establish shoreface morphology and determine the stratal expression of retreat and progradation were likewise generally the same, despite marked differences in tectonism, physiography, and bathymetry between the two settings. Our results do not support the widespread perception that Paleozoic cratonic interior successions are so anomalous in stratal geometries, and constitute such a poor record of time, that they are poorly suited for modern sequence stratigraphic analyses. The particular arrangement of stratal elements in the cratonic interior succession we studied is no more anomalous or enigmatic than the variability in architecture that sets all sedimentary successions apart from one another. Thus, Paleozoic strata of the cratonic interior are most appropriately considered as a package that belongs in a

  6. Uranium ... long-term confidence

    International Nuclear Information System (INIS)

    Anon.

    1983-01-01

    Half way through 1983 the outlook for the world's uranium producers was far from bright if one takes a short term view. The readily accessible facts present a gloomy picture. The spot prices of uranium over the past few years decreased from a high of $42-$43/lb to a low of $17 in 1982. It now hovers between $23 and $24. the contract prices negotiated between producers and consumers are not so accessible but they do not reflect the spot price. The reasons why contractual uranium prices do not follow the usual dictates of supply and demand are related to the position in which uranium and associated power industries find themselves. There is public reaction with strong emotional overtones as well as much reduced expectations about the electric power needs of the world. Furthermore the supply of uranium is not guaranteed despite present over production. However the people in the industry, taking the medium- and long-term view, are not despondent

  7. Forum on stakeholder confidence: Spain

    International Nuclear Information System (INIS)

    Vari, A.; Pescatore, C.

    2006-01-01

    The FSC workshop in Spain provided an important opportunity to carry out an in-depth examination of decision-making processes undertaken in an NEA member country, and to reflect on the evolution that has taken place over time. It offered a well-rounded perspective on the inclusion of stakeholders in decision making, and the atmosphere of the meetings was conducive to an honest and open exchange of ideas. The workshop started with the introduction of two case studies: the earlier attempt in Spain to locate a potential site for a high-level waste (HLW) disposal facility, and the dismantling of the Vandellos-I nuclear power plant. This was followed by two days of presentations and round-table discussions based on the recent COWAM Spain initiative (stemming from the EU-wide project on Community Waste Management), which aims at developing recommendations for institutional arrangements and decision-making processes concerning the siting of waste management facilities in Spain. This article provides a brief summary of the case studies and the COWAM Spain initiative, followed by some of the lessons learnt from an international perspective. (authors)

  8. High-resolution characterization of sequence signatures due to non-random cleavage of cell-free DNA.

    Science.gov (United States)

    Chandrananda, Dineika; Thorne, Natalie P; Bahlo, Melanie

    2015-06-17

    High-throughput sequencing of cell-free DNA fragments found in human plasma has been used to non-invasively detect fetal aneuploidy, monitor organ transplants and investigate tumor DNA. However, many biological properties of this extracellular genetic material remain unknown. Research that further characterizes circulating DNA could substantially increase its diagnostic value by allowing the application of more sophisticated bioinformatics tools that lead to an improved signal to noise ratio in the sequencing data. In this study, we investigate various features of cell-free DNA in plasma using deep-sequencing data from two pregnant women (>70X, >50X) and compare them with matched cellular DNA. We utilize a descriptive approach to examine how the biological cleavage of cell-free DNA affects different sequence signatures such as fragment lengths, sequence motifs at fragment ends and the distribution of cleavage sites along the genome. We show that the size distributions of these cell-free DNA molecules are dependent on their autosomal and mitochondrial origin as well as the genomic location within chromosomes. DNA mapping to particular microsatellites and alpha repeat elements display unique size signatures. We show how cell-free fragments occur in clusters along the genome, localizing to nucleosomal arrays and are preferentially cleaved at linker regions by correlating the mapping locations of these fragments with ENCODE annotation of chromatin organization. Our work further demonstrates that cell-free autosomal DNA cleavage is sequence dependent. The region spanning up to 10 positions on either side of the DNA cleavage site show a consistent pattern of preference for specific nucleotides. This sequence motif is present in cleavage sites localized to nucleosomal cores and linker regions but is absent in nucleosome-free mitochondrial DNA. These background signals in cell-free DNA sequencing data stem from the non-random biological cleavage of these fragments. This

  9. Development of a parallel zoomed EVI sequence for high temporal resolution analysis of the BOLD response

    International Nuclear Information System (INIS)

    Rabrait, C.

    2006-01-01

    The hemodynamic impulse response to any short stimulus typically lasts around 20 seconds. Thus, the detection of the Blood Oxygenation Level Dependent (BOLD) effect is usually performed using a 2D Echo Planar Imaging (EPI) sequence, with repetition times on the order of 1 or 2 seconds. This temporal resolution is generally enough for detection purposes. Nevertheless, when trying to accurately estimate the hemodynamic response functions (HRF), higher scanning rates represent a real advantage. Thus, in order to reach a temporal resolution around 200 ms, we developed a new acquisition method, based on Echo Volumar Imaging and 2D parallel acquisition (1). Echo Volumar Imaging (EVI) has been proposed in 1977 by Mansfield (2). EVI intrinsically possesses a lot of advantages for functional neuroimaging, as a 3 D single shot acquisition method. Nevertheless, to date, only a few applications have been reported (3, 4). Actually, very restricting hardware requirements make EVI difficult to perform in satisfactory experimental conditions, even today. The critical point in EVI is the echo train duration, which is longer than in EPI, due to 3D acquisition. Indeed, at equal field of view and spatial resolutions, EVI echo train duration must be approximately equal to EPI echo train duration multiplied by the number of slices acquired in EPI. Consequently, EVI is much more sensitive than EPI to geometric distortions, which are related to phase errors, and also to signal losses, which are due to long echo times (TE). Thus, a first improvement has been brought by 'zoomed' or 'localized' EVI (5), which allows to focus on a small volume of interest and thus limit echo train durations compared to full FOV acquisitions.To reduce echo train durations, we chose to apply parallel acquisition. Moreover, since EVI is a 3D acquisition method, we are able to perform parallel acquisition and SENSE reconstruction along the two phase directions (6). The R = 4 under-sampling consists in the

  10. Identifying Likely Transmission Pathways within a 10-Year Community Outbreak of Tuberculosis by High-Depth Whole Genome Sequencing.

    Directory of Open Access Journals (Sweden)

    Alexander C Outhred

    Full Text Available Improved tuberculosis control and the need to contain the spread of drug-resistant strains provide a strong rationale for exploring tuberculosis transmission dynamics at the population level. Whole-genome sequencing provides optimal strain resolution, facilitating detailed mapping of potential transmission pathways.We sequenced 22 isolates from a Mycobacterium tuberculosis cluster in New South Wales, Australia, identified during routine 24-locus mycobacterial interspersed repetitive unit typing. Following high-depth paired-end sequencing using the Illumina HiSeq 2000 platform, two independent pipelines were employed for analysis, both employing read mapping onto reference genomes as well as de novo assembly, to control biases in variant detection. In addition to single-nucleotide polymorphisms, the analyses also sought to identify insertions, deletions and structural variants.Isolates were highly similar, with a distance of 13 variants between the most distant members of the cluster. The most sensitive analysis classified the 22 isolates into 18 groups. Four of the isolates did not appear to share a recent common ancestor with the largest clade; another four isolates had an uncertain ancestral relationship with the largest clade.Whole genome sequencing, with analysis of single-nucleotide polymorphisms, insertions, deletions, structural variants and subpopulations, enabled the highest possible level of discrimination between cluster members, clarifying likely transmission pathways and exposing the complexity of strain origin. The analysis provides a basis for targeted public health intervention and enhanced classification of future isolates linked to the cluster.

  11. Probing the Rare Biosphere of the North-West Mediterranean Sea: An Experiment with High Sequencing Effort.

    Directory of Open Access Journals (Sweden)

    Bibiana G Crespo

    Full Text Available High-throughput sequencing (HTS techniques have suggested the existence of a wealth of species with very low relative abundance: the rare biosphere. We attempted to exhaustively map this rare biosphere in two water samples by performing an exceptionally deep pyrosequencing analysis (~500,000 final reads per sample. Species data were derived by a 97% identity criterion and various parametric distributions were fitted to the observed counts. Using the best-fitting Sichel distribution we estimate a total species richness of 1,568-1,669 (95% Credible Interval and 5,027-5,196 for surface and deep water samples respectively, implying that 84-89% of the total richness in those two samples was sequenced, and we predict that a quadrupling of the present sequencing effort would suffice to observe 90% of the total richness in both samples. Comparing the HTS results with a culturing approach we found that most of the cultured taxa were not obtained by HTS, despite the high sequencing effort. Culturing therefore remains a useful tool for uncovering marine bacterial diversity, in addition to its other uses for studying the ecology of marine bacteria.

  12. Imaging of cranial nerves with three-dimensional high resolution diffusion-weighted MR sequence based on SSFP technique

    International Nuclear Information System (INIS)

    Zhang Zhongwei; Chen Yingming; Meng Quanfei

    2008-01-01

    Objective: To depict the normal anatomy of cranial nerves in detail and define the exact relationships between cranial nerves and adjacent structures with three-dimensional high resolution diffusion-weighted MR sequence based on SSFP technique (3D DW-SSFP). Methods: 3D DW- SSFP sequence was performed and axial images were obtained in 12 healthy volunteers Post-processing techniques were used to generate images of cranial nerves, and the images acquired were compared with anatomical sections and diagrams of textbook. Results: In all subjects, 3D DW-SSFP sequence could produce homogeneous images and high contrast between the cranial nerves and other solid structures. The intracranial portions of all cranial nerves except olfactory nerve were identified; the extracranial portions of nerve Ⅱ-Ⅻ were identified in all subjects bilaterally. Conclusion: The 3D DW-SSFP sequence can characterize the normal MR appearance of cranial nerves and its branches and the ability to define the nerves may provide greater sensitivity and specificity in detecting abnormalities of craniofacial structure. (authors)

  13. Bacterial Pathogens and Community Composition in Advanced Sewage Treatment Systems Revealed by Metagenomics Analysis Based on High-Throughput Sequencing

    Science.gov (United States)

    Lu, Xin; Zhang, Xu-Xiang; Wang, Zhu; Huang, Kailong; Wang, Yuan; Liang, Weigang; Tan, Yunfei; Liu, Bo; Tang, Junying

    2015-01-01

    This study used 454 pyrosequencing, Illumina high-throughput sequencing and metagenomic analysis to investigate bacterial pathogens and their potential virulence in a sewage treatment plant (STP) applying both conventional and advanced treatment processes. Pyrosequencing and Illumina sequencing consistently demonstrated that Arcobacter genus occupied over 43.42% of total abundance of potential pathogens in the STP. At species level, potential pathogens Arcobacter butzleri, Aeromonas hydrophila and Klebsiella pneumonia dominated in raw sewage, which was also confirmed by quantitative real time PCR. Illumina sequencing also revealed prevalence of various types of pathogenicity islands and virulence proteins in the STP. Most of the potential pathogens and virulence factors were eliminated in the STP, and the removal efficiency mainly depended on oxidation ditch. Compared with sand filtration, magnetic resin seemed to have higher removals in most of the potential pathogens and virulence factors. However, presence of the residual A. butzleri in the final effluent still deserves more concerns. The findings indicate that sewage acts as an important source of environmental pathogens, but STPs can effectively control their spread in the environment. Joint use of the high-throughput sequencing technologies is considered a reliable method for deep and comprehensive overview of environmental bacterial virulence. PMID:25938416

  14. Environmental microbiology through the lens of high-throughput DNA sequencing: synopsis of current platforms and bioinformatics approaches.

    Science.gov (United States)

    Logares, Ramiro; Haverkamp, Thomas H A; Kumar, Surendra; Lanzén, Anders; Nederbragt, Alexander J; Quince, Christopher; Kauserud, Håvard

    2012-10-01

    The incursion of High-Throughput Sequencing (HTS) in environmental microbiology brings unique opportunities and challenges. HTS now allows a high-resolution exploration of the vast taxonomic and metabolic diversity present in the microbial world, which can provide an exceptional insight on global ecosystem functioning, ecological processes and evolution. This exploration has also economic potential, as we will have access to the evolutionary innovation present in microbial metabolisms, which could be used for biotechnological development. HTS is also challenging the research community, and the current bottleneck is present in the data analysis side. At the moment, researchers are in a sequence data deluge, with sequencing throughput advancing faster than the computer power needed for data analysis. However, new tools and approaches are being developed constantly and the whole process could be depicted as a fast co-evolution between sequencing technology, informatics and microbiologists. In this work, we examine the most popular and recently commercialized HTS platforms as well as bioinformatics methods for data handling and analysis used in microbial metagenomics. This non-exhaustive review is intended to serve as a broad state-of-the-art guide to researchers expanding into this rapidly evolving field. Copyright © 2012 Elsevier B.V. All rights reserved.

  15. Probing the Rare Biosphere of the North-West Mediterranean Sea: An Experiment with High Sequencing Effort.

    Science.gov (United States)

    Crespo, Bibiana G; Wallhead, Philip J; Logares, Ramiro; Pedrós-Alió, Carlos

    2016-01-01

    High-throughput sequencing (HTS) techniques have suggested the existence of a wealth of species with very low relative abundance: the rare biosphere. We attempted to exhaustively map this rare biosphere in two water samples by performing an exceptionally deep pyrosequencing analysis (~500,000 final reads per sample). Species data were derived by a 97% identity criterion and various parametric distributions were fitted to the observed counts. Using the best-fitting Sichel distribution we estimate a total species richness of 1,568-1,669 (95% Credible Interval) and 5,027-5,196 for surface and deep water samples respectively, implying that 84-89% of the total richness in those two samples was sequenced, and we predict that a quadrupling of the present sequencing effort would suffice to observe 90% of the total richness in both samples. Comparing the HTS results with a culturing approach we found that most of the cultured taxa were not obtained by HTS, despite the high sequencing effort. Culturing therefore remains a useful tool for uncovering marine bacterial diversity, in addition to its other uses for studying the ecology of marine bacteria.

  16. Model SNP development for complex genomes based on hexaploid oat using high-throughput 454 sequencing technology

    Directory of Open Access Journals (Sweden)

    Chao Shiaoman

    2011-01-01

    Full Text Available Abstract Background Genetic markers are pivotal to modern genomics research; however, discovery and genotyping of molecular markers in oat has been hindered by the size and complexity of the genome, and by a scarcity of sequence data. The purpose of this study was to generate oat expressed sequence tag (EST information, develop a bioinformatics pipeline for SNP discovery, and establish a method for rapid, cost-effective, and straightforward genotyping of SNP markers in complex polyploid genomes such as oat. Results Based on cDNA libraries of four cultivated oat genotypes, approximately 127,000 contigs were assembled from approximately one million Roche 454 sequence reads. Contigs were filtered through a novel bioinformatics pipeline to eliminate ambiguous polymorphism caused by subgenome homology, and 96 in silico SNPs were selected from 9,448 candidate loci for validation using high-resolution melting (HRM analysis. Of these, 52 (54% were polymorphic between parents of the Ogle1040 × TAM O-301 (OT mapping population, with 48 segregating as single Mendelian loci, and 44 being placed on the existing OT linkage map. Ogle and TAM amplicons from 12 primers were sequenced for SNP validation, revealing complex polymorphism in seven amplicons but general sequence conservation within SNP loci. Whole-amplicon interrogation with HRM revealed insertions, deletions, and heterozygotes in secondary oat germplasm pools, generating multiple alleles at some primer targets. To validate marker utility, 36 SNP assays were used to evaluate the genetic diversity of 34 diverse oat genotypes. Dendrogram clusters corresponded generally to known genome composition and genetic ancestry. Conclusions The high-throughput SNP discovery pipeline presented here is a rapid and effective method for identification of polymorphic SNP alleles in the oat genome. The current-generation HRM system is a simple and highly-informative platform for SNP genotyping. These techniques provide

  17. High Performance Protein Sequence Database Scanning on the Cell Broadband Engine

    Directory of Open Access Journals (Sweden)

    Adrianto Wirawan

    2009-01-01

    Full Text Available The enormous growth of biological sequence databases has caused bioinformatics to be rapidly moving towards a data-intensive, computational science. As a result, the computational power needed by bioinformatics applications is growing rapidly as well. The recent emergence of low cost parallel multicore accelerator technologies has made it possible to reduce execution times of many bioinformatics applications. In this paper, we demonstrate how the Cell Broadband Engine can be used as a computational platform to accelerate two approaches for protein sequence database scanning: exhaustive and heuristic. We present efficient parallelization techniques for two representative algorithms: the dynamic programming based Smith–Waterman algorithm and the popular BLASTP heuristic. Their implementation on a Playstation®3 leads to significant runtime savings compared to corresponding sequential implementations.

  18. Exploring the environmental diversity of kinetoplastid flagellates in the high-throughput DNA sequencing era

    Directory of Open Access Journals (Sweden)

    Claudia Masini d’Avila-Levy

    2015-01-01

    Full Text Available The class Kinetoplastea encompasses both free-living and parasitic species from a wide range of hosts. Several representatives of this group are responsible for severe human diseases and for economic losses in agriculture and livestock. While this group encompasses over 30 genera, most of the available information has been derived from the vertebrate pathogenic genera Leishmaniaand Trypanosoma.Recent studies of the previously neglected groups of Kinetoplastea indicated that the actual diversity is much higher than previously thought. This article discusses the known segment of kinetoplastid diversity and how gene-directed Sanger sequencing and next-generation sequencing methods can help to deepen our knowledge of these interesting protists.

  19. High diversity of picornaviruses in rats from different continents revealed by deep sequencing.

    Science.gov (United States)

    Hansen, Thomas Arn; Mollerup, Sarah; Nguyen, Nam-Phuong; White, Nicole E; Coghlan, Megan; Alquezar-Planas, David E; Joshi, Tejal; Jensen, Randi Holm; Fridholm, Helena; Kjartansdóttir, Kristín Rós; Mourier, Tobias; Warnow, Tandy; Belsham, Graham J; Bunce, Michael; Willerslev, Eske; Nielsen, Lars Peter; Vinner, Lasse; Hansen, Anders Johannes

    2016-08-17

    Outbreaks of zoonotic diseases in humans and livestock are not uncommon, and an important component in containment of such emerging viral diseases is rapid and reliable diagnostics. Such methods are often PCR-based and hence require the availability of sequence data from the pathogen. Rattus norvegicus (R. norvegicus) is a known reservoir for important zoonotic pathogens. Transmission may be direct via contact with the animal, for example, through exposure to its faecal matter, or indirectly mediated by arthropod vectors. Here we investigated the viral content in rat faecal matter (n=29) collected from two continents by analyzing 2.2 billion next-generation sequencing reads derived from both DNA and RNA. Among other virus families, we found sequences from members of the Picornaviridae to be abundant in the microbiome of all the samples. Here we describe the diversity of the picornavirus-like contigs including near-full-length genomes closely related to the Boone cardiovirus and Theiler's encephalomyelitis virus. From this study, we conclude that picornaviruses within R. norvegicus are more diverse than previously recognized. The virome of R. norvegicus should be investigated further to assess the full potential for zoonotic virus transmission.

  20. High-throughput sequencing of human plasma RNA by using thermostable group II intron reverse transcriptases

    Science.gov (United States)

    Qin, Yidan; Yao, Jun; Wu, Douglas C.; Nottingham, Ryan M.; Mohr, Sabine; Hunicke-Smith, Scott; Lambowitz, Alan M.

    2016-01-01

    Next-generation RNA-sequencing (RNA-seq) has revolutionized transcriptome profiling, gene expression analysis, and RNA-based diagnostics. Here, we developed a new RNA-seq method that exploits thermostable group II intron reverse transcriptases (TGIRTs) and used it to profile human plasma RNAs. TGIRTs have higher thermostability, processivity, and fidelity than conventional reverse transcriptases, plus a novel template-switching activity that can efficiently attach RNA-seq adapters to target RNA sequences without RNA ligation. The new TGIRT-seq method enabled construction of RNA-seq libraries from RNA in RNA in 1-mL plasma samples from a healthy individual revealed RNA fragments mapping to a diverse population of protein-coding gene and long ncRNAs, which are enriched in intron and antisense sequences, as well as nearly all known classes of small ncRNAs, some of which have never before been seen in plasma. Surprisingly, many of the small ncRNA species were present as full-length transcripts, suggesting that they are protected from plasma RNases in ribonucleoprotein (RNP) complexes and/or exosomes. This TGIRT-seq method is readily adaptable for profiling of whole-cell, exosomal, and miRNAs, and for related procedures, such as HITS-CLIP and ribosome profiling. PMID:26554030

  1. Needs Assessment for Research Use of High-Throughput Sequencing at a Large Academic Medical Center.

    Directory of Open Access Journals (Sweden)

    Albert Geskin

    Full Text Available Next Generation Sequencing (NGS methods are driving profound changes in biomedical research, with a growing impact on patient care. Many academic medical centers are evaluating potential models to prepare for the rapid increase in NGS information needs. This study sought to investigate (1 how and where sequencing data is generated and analyzed, (2 research objectives and goals for NGS, (3 workforce capacity and unmet needs, (4 storage capacity and unmet needs, (5 available and anticipated funding resources, and (6 future challenges. As a precursor to informed decision making at our institution, we undertook a systematic needs assessment of investigators using survey methods. We recruited 331 investigators from over 60 departments and divisions at the University of Pittsburgh Schools of Health Sciences and had 140 respondents, or a 42% response rate. Results suggest that both sequencing and analysis bottlenecks currently exist. Significant educational needs were identified, including both investigator-focused needs, such as selection of NGS methods suitable for specific research objectives, and program-focused needs, such as support for training an analytic workforce. The absence of centralized infrastructure was identified as an important institutional gap. Key principles for organizations managing this change were formulated based on the survey responses. This needs assessment provides an in-depth case study which may be useful to other academic medical centers as they identify and plan for future needs.

  2. High Rhodotorula sequences in skin transcriptome of patients with diffuse systemic sclerosis.

    Science.gov (United States)

    Arron, Sarah T; Dimon, Michelle T; Li, Zhenghui; Johnson, Michael E; Wood, Tammara A; Feeney, Luzviminda; Angeles, Jorge G; Lafyatis, Robert; Whitfield, Michael L

    2014-08-01

    Previous studies have suggested a role for pathogens as a trigger of systemic sclerosis (SSc), although neither a pathogen nor a mechanism of pathogenesis is known. Here we show enrichment of Rhodotorula sequences in the skin of patients with early, diffuse SSc compared with that in normal controls. RNA-seq was performed on four SSc patients and four controls, to a depth of 200 million reads per patient. Data were analyzed to quantify the nonhuman sequence reads in each sample. We found little difference between bacterial microbiome and viral read counts, but found a significant difference between the read counts for a mycobiome component, R. glutinis. Normal samples contained almost no detected R. glutinis or other Rhodotorula sequence reads (mean score 0.021 for R. glutinis, 0.024 for all Rhodotorula). In contrast, SSc samples had a mean score of 5.039 for R. glutinis (5.232 for Rhodotorula). We were able to assemble the D1-D2 hypervariable region of the 28S ribosomal RNA (rRNA) of R. glutinis from each of the SSc samples. Taken together, these results suggest that R. glutinis may be present in the skin of early SSc patients at higher levels than in normal skin, raising the possibility that it may be triggering the inflammatory response found in SSc.

  3. Multilocus sequence analysis of nectar pseudomonads reveals high genetic diversity and contrasting recombination patterns.

    Science.gov (United States)

    Alvarez-Pérez, Sergio; de Vega, Clara; Herrera, Carlos M

    2013-01-01

    The genetic and evolutionary relationships among floral nectar-dwelling Pseudomonas 'sensu stricto' isolates associated to South African and Mediterranean plants were investigated by multilocus sequence analysis (MLSA) of four core housekeeping genes (rrs, gyrB, rpoB and rpoD). A total of 35 different sequence types were found for the 38 nectar bacterial isolates characterised. Phylogenetic analyses resulted in the identification of three main clades [nectar groups (NGs) 1, 2 and 3] of nectar pseudomonads, which were closely related to five intrageneric groups: Pseudomonas oryzihabitans (NG 1); P. fluorescens, P. lutea and P. syringae (NG 2); and P. rhizosphaerae (NG 3). Linkage disequilibrium analysis pointed to a mostly clonal population structure, even when the analysis was restricted to isolates from the same floristic region or belonging to the same NG. Nevertheless, signatures of recombination were observed for NG 3, which exclusively included isolates retrieved from the floral nectar of insect-pollinated Mediterranean plants. In contrast, the other two NGs comprised both South African and Mediterranean isolates. Analyses relating diversification to floristic region and pollinator type revealed that there has been more unique evolution of the nectar pseudomonads within the Mediterranean region than would be expected by chance. This is the first work analysing the sequence of multiple loci to reveal geno- and ecotypes of nectar bacteria.

  4. Multilocus Sequence Analysis of Nectar Pseudomonads Reveals High Genetic Diversity and Contrasting Recombination Patterns

    Science.gov (United States)

    Álvarez-Pérez, Sergio; de Vega, Clara; Herrera, Carlos M.

    2013-01-01

    The genetic and evolutionary relationships among floral nectar-dwelling Pseudomonas ‘sensu stricto’ isolates associated to South African and Mediterranean plants were investigated by multilocus sequence analysis (MLSA) of four core housekeeping genes (rrs, gyrB, rpoB and rpoD). A total of 35 different sequence types were found for the 38 nectar bacterial isolates characterised. Phylogenetic analyses resulted in the identification of three main clades [nectar groups (NGs) 1, 2 and 3] of nectar pseudomonads, which were closely related to five intrageneric groups: Pseudomonas oryzihabitans (NG 1); P. fluorescens, P. lutea and P. syringae (NG 2); and P. rhizosphaerae (NG 3). Linkage disequilibrium analysis pointed to a mostly clonal population structure, even when the analysis was restricted to isolates from the same floristic region or belonging to the same NG. Nevertheless, signatures of recombination were observed for NG 3, which exclusively included isolates retrieved from the floral nectar of insect-pollinated Mediterranean plants. In contrast, the other two NGs comprised both South African and Mediterranean isolates. Analyses relating diversification to floristic region and pollinator type revealed that there has been more unique evolution of the nectar pseudomonads within the Mediterranean region than would be expected by chance. This is the first work analysing the sequence of multiple loci to reveal geno- and ecotypes of nectar bacteria. PMID:24116076

  5. An integrated multiple capillary array electrophoresis system for high-throughput DNA sequencing

    Energy Technology Data Exchange (ETDEWEB)

    Lu, X.

    1998-03-27

    A capillary array electrophoresis system was chosen to perform DNA sequencing because of several advantages such as rapid heat dissipation, multiplexing capabilities, gel matrix filling simplicity, and the mature nature of the associated manufacturing technologies. There are two major concerns for the multiple capillary systems. One concern is inter-capillary cross-talk, and the other concern is excitation and detection efficiency. Cross-talk is eliminated through proper optical coupling, good focusing and immersing capillary array into index matching fluid. A side-entry excitation scheme with orthogonal detection was established for large capillary array. Two 100 capillary array formats were used for DNA sequencing. One format is cylindrical capillary with 150 {micro}m o.d., 75 {micro}m i.d and the other format is square capillary with 300 {micro}m out edge and 75 {micro}m inner edge. This project is focused on the development of excitation and detection of DNA as well as performing DNA sequencing. The DNA injection schemes are discussed for the cases of single and bundled capillaries. An individual sampling device was designed. The base-calling was performed for a capillary from the capillary array with the accuracy of 98%.

  6. Toward allotetraploid cotton genome assembly: integration of a high-density molecular genetic linkage map with DNA sequence information

    Science.gov (United States)

    2012-01-01

    Background Cotton is the world’s most important natural textile fiber and a significant oilseed crop. Decoding cotton genomes will provide the ultimate reference and resource for research and utilization of the species. Integration of high-density genetic maps with genomic sequence information will largely accelerate the process of whole-genome assembly in cotton. Results In this paper, we update a high-density interspecific genetic linkage map of allotetraploid cultivated cotton. An additional 1,167 marker loci have been added to our previously published map of 2,247 loci. Three new marker types, InDel (insertion-deletion) and SNP (single nucleotide polymorphism) developed from gene information, and REMAP (retrotransposon-microsatellite amplified polymorphism), were used to increase map density. The updated map consists of 3,414 loci in 26 linkage groups covering 3,667.62 cM with an average inter-locus distance of 1.08 cM. Furthermore, genome-wide sequence analysis was finished using 3,324 informative sequence-based markers and publicly-available Gossypium DNA sequence information. A total of 413,113 EST and 195 BAC sequences were physically anchored and clustered by 3,324 sequence-based markers. Of these, 14,243 ESTs and 188 BACs from different species of Gossypium were clustered and specifically anchored to the high-density genetic map. A total of 2,748 candidate unigenes from 2,111 ESTs clusters and 63 BACs were mined for functional annotation and classification. The 337 ESTs/genes related to fiber quality traits were integrated with 132 previously reported cotton fiber quality quantitative trait loci, which demonstrated the important roles in fiber quality of these genes. Higher-level sequence conservation between different cotton species and between the A- and D-subgenomes in tetraploid cotton was found, indicating a common evolutionary origin for orthologous and paralogous loci in Gossypium. Conclusion This study will serve as a valuable genomic resource

  7. Correlation between high resolution sequence stratigraphy and mechanical stratigraphy for enhanced fracture characteristic prediction

    Science.gov (United States)

    Al Kharusi, Laiyyan M.

    Sequence stratigraphy relates changes in vertical and lateral facies distribution to relative changes in sea level. These relative changes in carbonates effect early diagenesis, types of pores, cementation and dissolution patterns. As a result, in carbonates, relative changes in sea level significantly impact the lithology, porosity, diagenesis, bed and bounding surfaces which are all factors that control fracture patterns. This study explores these relationships by integrating stratigraphy with fracture analysis and petrophysical properties. A special focus is given to the relationship between mechanical boundaries and sequence stratigraphic boundaries in three different settings: (1) Mississippian strata in Sheep Mountain Anticline, Wyoming, (2) Mississippian limestones in St. Louis, Missouri, and (3) Pennsylvanian limestones intermixed with elastics in the Paradox Basin, Utah. The analysis of these sections demonstrate that a fracture hierarchy exists in relation to the sequence stratigraphic hierarchy. The majority of fractures (80%) terminate at genetic unit boundaries or the internal flooding surface that separates the transgressive from regressive hemicycle. Fractures (20%) that do not terminate at genetic unit boundaries or their internal flooding surface terminate at lower order sequence stratigraphic boundaries or their internal flooding surfaces. Secondly, the fracture spacing relates well to bed thickness in mechanical units no greater than 0.5m in thickness but with increasing bed thickness a scatter from the linear trend is observed. In the Paradox Basin the influence of strain on fracture density is illustrated by two sections measured in different strain regimes. The folded strata at Raplee Anticline has higher fracture densities than the flat-lying beds at the Honaker Trail. Cemented low porosity rocks in the Paradox Basin do not show a correlation between fracture pattern and porosity. However velocity and rock stiffness moduli's display a slight

  8. Heap: a highly sensitive and accurate SNP detection tool for low-coverage high-throughput sequencing data

    KAUST Repository

    Kobayashi, Masaaki

    2017-04-20

    Recent availability of large-scale genomic resources enables us to conduct so called genome-wide association studies (GWAS) and genomic prediction (GP) studies, particularly with next-generation sequencing (NGS) data. The effectiveness of GWAS and GP depends on not only their mathematical models, but the quality and quantity of variants employed in the analysis. In NGS single nucleotide polymorphism (SNP) calling, conventional tools ideally require more reads for higher SNP sensitivity and accuracy. In this study, we aimed to develop a tool, Heap, that enables robustly sensitive and accurate calling of SNPs, particularly with a low coverage NGS data, which must be aligned to the reference genome sequences in advance. To reduce false positive SNPs, Heap determines genotypes and calls SNPs at each site except for sites at the both ends of reads or containing a minor allele supported by only one read. Performance comparison with existing tools showed that Heap achieved the highest F-scores with low coverage (7X) restriction-site associated DNA sequencing reads of sorghum and rice individuals. This will facilitate cost-effective GWAS and GP studies in this NGS era. Code and documentation of Heap are freely available from https://github.com/meiji-bioinf/heap (29 March 2017, date last accessed) and our web site (http://bioinf.mind.meiji.ac.jp/lab/en/tools.html (29 March 2017, date last accessed)).

  9. Global repeat discovery and estimation of genomic copy number in a large, complex genome using a high-throughput 454 sequence survey

    Directory of Open Access Journals (Sweden)

    Varala Kranthi

    2007-05-01

    Full Text Available Abstract Background Extensive computational and database tools are available to mine genomic and genetic databases for model organisms, but little genomic data is available for many species of ecological or agricultural significance, especially those with large genomes. Genome surveys using conventional sequencing techniques are powerful, particularly for detecting sequences present in many copies per genome. However these methods are time-consuming and have potential drawbacks. High throughput 454 sequencing provides an alternative method by which much information can be gained quickly and cheaply from high-coverage surveys of genomic DNA. Results We sequenced 78 million base-pairs of randomly sheared soybean DNA which passed our quality criteria. Computational analysis of the survey sequences provided global information on the abundant repetitive sequences in soybean. The sequence was used to determine the copy number across regions of large genomic clones or contigs and discover higher-order structures within satellite repeats. We have created an annotated, online database of sequences present in multiple copies in the soybean genome. The low bias of pyrosequencing against repeat sequences is demonstrated by the overall composition of the survey data, which matches well with past estimates of repetitive DNA content obtained by DNA re-association kinetics (Cot analysis. Conclusion This approach provides a potential aid to conventional or shotgun genome assembly, by allowing rapid assessment of copy number in any clone or clone-end sequence. In addition, we show that partial sequencing can provide access to partial protein-coding sequences.

  10. Comparative high-throughput transcriptome sequencing and development of SiESTa, the Silene EST annotation database

    Directory of Open Access Journals (Sweden)

    Marais Gabriel AB

    2011-07-01

    Full Text Available Abstract Background The genus Silene is widely used as a model system for addressing ecological and evolutionary questions in plants, but advances in using the genus as a model system are impeded by the lack of available resources for studying its genome. Massively parallel sequencing cDNA has recently developed into an efficient method for characterizing the transcriptomes of non-model organisms, generating massive amounts of data that enable the study of multiple species in a comparative framework. The sequences generated provide an excellent resource for identifying expressed genes, characterizing functional variation and developing molecular markers, thereby laying the foundations for future studies on gene sequence and gene expression divergence. Here, we report the results of a comparative transcriptome sequencing study of eight individuals representing four Silene and one Dianthus species as outgroup. All sequences and annotations have been deposited in a newly developed and publicly available database called SiESTa, the Silene EST annotation database. Results A total of 1,041,122 EST reads were generated in two runs on a Roche GS-FLX 454 pyrosequencing platform. EST reads were analyzed separately for all eight individuals sequenced and were assembled into contigs using TGICL. These were annotated with results from BLASTX searches and Gene Ontology (GO terms, and thousands of single-nucleotide polymorphisms (SNPs were characterized. Unassembled reads were kept as singletons and together with the contigs contributed to the unigenes characterized in each individual. The high quality of unigenes is evidenced by the proportion (49% that have significant hits in similarity searches with the A. thaliana proteome. The SiESTa database is accessible at http://www.siesta.ethz.ch. Conclusion The sequence collections established in the present study provide an important genomic resource for four Silene and one Dianthus species and will help to

  11. Comparative high-throughput transcriptome sequencing and development of SiESTa, the Silene EST annotation database

    Science.gov (United States)

    2011-01-01

    Background The genus Silene is widely used as a model system for addressing ecological and evolutionary questions in plants, but advances in using the genus as a model system are impeded by the lack of available resources for studying its genome. Massively parallel sequencing cDNA has recently developed into an efficient method for characterizing the transcriptomes of non-model organisms, generating massive amounts of data that enable the study of multiple species in a comparative framework. The sequences generated provide an excellent resource for identifying expressed genes, characterizing functional variation and developing molecular markers, thereby laying the foundations for future studies on gene sequence and gene expression divergence. Here, we report the results of a comparative transcriptome sequencing study of eight individuals representing four Silene and one Dianthus species as outgroup. All sequences and annotations have been deposited in a newly developed and publicly available database called SiESTa, the Silene EST annotation database. Results A total of 1,041,122 EST reads were generated in two runs on a Roche GS-FLX 454 pyrosequencing platform. EST reads were analyzed separately for all eight individuals sequenced and were assembled into contigs using TGICL. These were annotated with results from BLASTX searches and Gene Ontology (GO) terms, and thousands of single-nucleotide polymorphisms (SNPs) were characterized. Unassembled reads were kept as singletons and together with the contigs contributed to the unigenes characterized in each individual. The high quality of unigenes is evidenced by the proportion (49%) that have significant hits in similarity searches with the A. thaliana proteome. The SiESTa database is accessible at http://www.siesta.ethz.ch. Conclusion The sequence collections established in the present study provide an important genomic resource for four Silene and one Dianthus species and will help to further develop Silene as a

  12. Food skills confidence and household gatekeepers' dietary practices.

    Science.gov (United States)

    Burton, Melissa; Reid, Mike; Worsley, Anthony; Mavondo, Felix

    2017-01-01

    Household food gatekeepers have the potential to influence the food attitudes and behaviours of family members, as they are mainly responsible for food-related tasks in the home. The aim of this study was to determine the role of gatekeepers' confidence in food-related skills and nutrition knowledge on food practices in the home. An online survey was completed by 1059 Australian dietary gatekeepers selected from the Global Market Insite (GMI) research database. Participants responded to questions about food acquisition and preparation behaviours, the home eating environment, perceptions and attitudes towards food, and demographics. Two-step cluster analysis was used to identify groups based on confidence regarding food skills and nutrition knowledge. Chi-square tests and one-way ANOVAs were used to compare the groups on the dependent variables. Three groups were identified: low confidence, moderate confidence and high confidence. Gatekeepers in the highest confidence group were significantly more likely to report lower body mass index (BMI), and indicate higher importance of fresh food products, vegetable prominence in meals, product information use, meal planning, perceived behavioural control and overall diet satisfaction. Gatekeepers in the lowest confidence group were significantly more likely to indicate more perceived barriers to healthy eating, report more time constraints and more impulse purchasing practices, and higher convenience ingredient use. Other smaller associations were also found. Household food gatekeepers with high food skills confidence were more likely to engage in several healthy food practices, while those with low food skills confidence were more likely to engage in unhealthy food practices. Food education strategies aimed at building food-skills and nutrition knowledge will enable current and future gatekeepers to make healthier food decisions for themselves and for their families. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. Statistical assignment of DNA sequences using Bayesian phylogenetics

    DEFF Research Database (Denmark)

    Terkelsen, Kasper Munch; Boomsma, Wouter Krogh; Huelsenbeck, John P.

    2008-01-01

    We provide a new automated statistical method for DNA barcoding based on a Bayesian phylogenetic analysis. The method is based on automated database sequence retrieval, alignment, and phylogenetic analysis using a custom-built program for Bayesian phylogenetic analysis. We show on real data...... that the method outperforms Blast searches as a measure of confidence and can help eliminate 80% of all false assignment based on best Blast hit. However, the most important advance of the method is that it provides statistically meaningful measures of confidence. We apply the method to a re......-analysis of previously published ancient DNA data and show that, with high statistical confidence, most of the published sequences are in fact of Neanderthal origin. However, there are several cases of chimeric sequences that are comprised of a combination of both Neanderthal and modern human DNA....

  14. Empirical methods for controlling false positives and estimating confidence in ChIP-Seq peaks

    Directory of Open Access Journals (Sweden)

    Courdy Samir J

    2008-12-01

    Full Text Available Abstract Background High throughput signature sequencing holds many promises, one of which is the ready identification of in vivo transcription factor binding sites, histone modifications, changes in chromatin structure and patterns of DNA methylation across entire genomes. In these experiments, chromatin immunoprecipitation is used to enrich for particular DNA sequences of interest and signature sequencing is used to map the regions to the genome (ChIP-Seq. Elucidation of these sites of DNA-protein binding/modification are proving instrumental in reconstructing networks of gene regulation and chromatin remodelling that direct development, response to cellular perturbation, and neoplastic transformation. Results Here we present a package of algorithms and software that makes use of control input data to reduce false positives and estimate confidence in ChIP-Seq peaks. Several different methods were compared using two simulated spike-in datasets. Use of control input data and a normalized difference score were found to more than double the recovery of ChIP-Seq peaks at a 5% false discovery rate (FDR. Moreover, both a binomial p-value/q-value and an empirical FDR were found to predict the true FDR within 2–3 fold and are more reliable estimators of confidence than a global Poisson p-value. These methods were then used to reanalyze Johnson et al.'s neuron-restrictive silencer factor (NRSF ChIP-Seq data without relying on extensive qPCR validated NRSF sites and the presence of NRSF binding motifs for setting thresholds. Conclusion The methods developed and tested here show considerable promise for reducing false positives and estimating confidence in ChIP-Seq data without any prior knowledge of the chIP target. They are part of a larger open source package freely available from http://useq.sourceforge.net/.

  15. Secure and robust cloud computing for high-throughput forensic microsatellite sequence analysis and databasing.

    Science.gov (United States)

    Bailey, Sarah F; Scheible, Melissa K; Williams, Christopher; Silva, Deborah S B S; Hoggan, Marina; Eichman, Christopher; Faith, Seth A

    2017-11-01

    Next-generation Sequencing (NGS) is a rapidly evolving technology with demonstrated benefits for forensic genetic applications, and the strategies to analyze and manage the massive NGS datasets are currently in development. Here, the computing, data storage, connectivity, and security resources of the Cloud were evaluated as a model for forensic laboratory systems that produce NGS data. A complete front-to-end Cloud system was developed to upload, process, and interpret raw NGS data using a web browser dashboard. The system was extensible, demonstrating analysis capabilities of autosomal and Y-STRs from a variety of NGS instrumentation (Illumina MiniSeq and MiSeq, and Oxford Nanopore MinION). NGS data for STRs were concordant with standard reference materials previously characterized with capillary electrophoresis and Sanger sequencing. The computing power of the Cloud was implemented with on-demand auto-scaling to allow multiple file analysis in tandem. The system was designed to store resulting data in a relational database, amenable to downstream sample interpretations and databasing applications following the most recent guidelines in nomenclature for sequenced alleles. Lastly, a multi-layered Cloud security architecture was tested and showed that industry standards for securing data and computing resources were readily applied to the NGS system without disadvantageous effects for bioinformatic analysis, connectivity or data storage/retrieval. The results of this study demonstrate the feasibility of using Cloud-based systems for secured NGS data analysis, storage, databasing, and multi-user distributed connectivity. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. High quality maize centromere 10 sequence reveals evidence of frequent recombination events

    Directory of Open Access Journals (Sweden)

    Thomas Kai Wolfgruber

    2016-03-01

    Full Text Available The ancestral centromeres of maize contain long stretches of the tandemly arranged CentC repeat. The abundance of tandem DNA repeats and centromeric retrotransposons (CR have presented a significant challenge to completely assembling centromeres using traditional sequencing methods. Here we report a nearly complete assembly of the 1.85 Mb maize centromere 10 from inbred B73 using PacBio technology and BACs from the reference genome project. The error rates estimated from overlapping BAC sequences are 7 x 10-6 and 5 x 10-5 for mismatches and indels, respectively. The number of gaps in the region covered by the reassembly was reduced from 140 in the reference genome to three. Three expressed genes are located between 92 and 477 kb of the inferred ancestral CentC cluster, which lies within the region of highest centromeric repeat density. The improved assembly increased the count of full-length centromeric retrotransposons from 5 to 55 and revealed a 22.7 kb segmental duplication that occurred approximately 121,000 years ago. Our analysis provides evidence of frequent recombination events in the form of partial retrotransposons, deletions within retrotransposons, chimeric retrotransposons, segmental duplications including higher order CentC repeats, a deleted CentC monomer, centromere-proximal inversions, and insertion of mitochondrial sequences. Double-strand DNA break (DSB repair is the most plausible mechanism for these events and may be the major driver of centromere repeat evolution and diversity. This repair appears to be mediated by microhomology, suggesting that tandem repeats may have evolved to facilitate the repair of frequent DSBs in centromeres.

  17. A massively parallel sequencing approach uncovers ancient origins and high genetic variability of endangered Przewalski's horses.

    Science.gov (United States)

    Goto, Hiroki; Ryder, Oliver A; Fisher, Allison R; Schultz, Bryant; Kosakovsky Pond, Sergei L; Nekrutenko, Anton; Makova, Kateryna D

    2011-01-01

    The endangered Przewalski's horse is the closest relative of the domestic horse and is the only true wild horse species surviving today. The question of whether Przewalski's horse is the direct progenitor of domestic horse has been hotly debated. Studies of DNA diversity within Przewalski's horses have been sparse but are urgently needed to ensure their successful reintroduction to the wild. In an attempt to resolve the controversy surrounding the phylogenetic position and genetic diversity of Przewalski's horses, we used massively parallel sequencing technology to decipher the complete mitochondrial and partial nuclear genomes for all four surviving maternal lineages of Przewalski's horses. Unlike single-nucleotide polymorphism (SNP) typing usually affected by ascertainment bias, the present method is expected to be largely unbiased. Three mitochondrial haplotypes were discovered-two similar ones, haplotypes I/II, and one substantially divergent from the other two, haplotype III. Haplotypes I/II versus III did not cluster together on a phylogenetic tree, rejecting the monophyly of Przewalski's horse maternal lineages, and were estimated to split 0.117-0.186 Ma, significantly preceding horse domestication. In the phylogeny based on autosomal sequences, Przewalski's horses formed a monophyletic clade, separate from the Thoroughbred domestic horse lineage. Our results suggest that Przewalski's horses have ancient origins and are not the direct progenitors of domestic horses. The analysis of the vast amount of sequence data presented here suggests that Przewalski's and domestic horse lineages diverged at least 0.117 Ma but since then have retained ancestral genetic polymorphism and/or experienced gene flow.

  18. Refining QTL with high-density SNP genotyping and whole genome sequence in three cattle breeds

    DEFF Research Database (Denmark)

    Sahana, Goutam; Guldbrandtsen, Bernt; Lund, Mogens Sandø

    2012-01-01

    Genome-wide association study was carried out in Nordic Holsteins, Nordic Red and Jersey breeds for functional traits using BovineHD Genotyping BreadChip (Illumina, San Diego, CA). The association analyses were carried out using both linear mixed model approach and a Bayesian variable selection...... method. Principal components were used to account for population structure. The QTL segregating in all three breeds were selected and a few of the most significant ones were followed in further analyses. The polymorphisms in the identified QTL regions were imputed using 90 whole genome sequences...

  19. Interpretation of Confidence Interval Facing the Conflict

    Science.gov (United States)

    Andrade, Luisa; Fernández, Felipe

    2016-01-01

    As literature has reported, it is usual that university students in statistics courses, and even statistics teachers, interpret the confidence level associated with a confidence interval as the probability that the parameter value will be between the lower and upper interval limits. To confront this misconception, class activities have been…

  20. Self-Confidence in the Hospitality Industry

    Directory of Open Access Journals (Sweden)

    Michael Oshins

    2014-02-01

    Full Text Available Few industries rely on self-confidence to the extent that the hospitality industry does because guests must feel welcome and that they are in capable hands. This article examines the results of hundreds of student interviews with industry professionals at all levels to determine where the majority of the hospitality industry gets their self-confidence.

  1. Financial Literacy, Confidence and Financial Advice Seeking

    NARCIS (Netherlands)

    Kramer, Marc M.

    2016-01-01

    We find that people with higher confidence in their own financial literacy are less likely to seek financial advice, but no relation between objective measures of literacy and advice seeking. The negative association between confidence and advice seeking is more pronounced among wealthy households.

  2. Confidence Interval Approximation For Treatment Variance In ...

    African Journals Online (AJOL)

    In a random effects model with a single factor, variation is partitioned into two as residual error variance and treatment variance. While a confidence interval can be imposed on the residual error variance, it is not possible to construct an exact confidence interval for the treatment variance. This is because the treatment ...

  3. Aging and Confidence Judgments in Item Recognition

    Science.gov (United States)

    Voskuilen, Chelsea; Ratcliff, Roger; McKoon, Gail

    2018-01-01

    We examined the effects of aging on performance in an item-recognition experiment with confidence judgments. A model for confidence judgments and response time (RTs; Ratcliff & Starns, 2013) was used to fit a large amount of data from a new sample of older adults and a previously reported sample of younger adults. This model of confidence…

  4. Organic labbeling systems and consumer confidence

    OpenAIRE

    Sønderskov, Kim Mannemar; Daugbjerg, Carsten

    2009-01-01

    A research analysis suggests that a state certification and labelling system creates confidence in organic labelling systems and consequently green consumerism. Danish consumers have higher levels of confidence in the labelling system than consumers in countries where the state plays a minor role in labelling and certification.

  5. Characterization of unknown genetic modifications using high throughput sequencing and computational subtraction

    Directory of Open Access Journals (Sweden)

    Butenko Melinka A

    2009-10-01

    Full Text Available Abstract Background When generating a genetically modified organism (GMO, the primary goal is to give a target organism one or several novel traits by using biotechnology techniques. A GMO will differ from its parental strain in that its pool of transcripts will be altered. Currently, there are no methods that are reliably able to determine if an organism has been genetically altered if the nature of the modification is unknown. Results We show that the concept of computational subtraction can be used to identify transgenic cDNA sequences from genetically modified plants. Our datasets include 454-type sequences from a transgenic line of Arabidopsis thaliana and published EST datasets from commercially relevant species (rice and papaya. Conclusion We believe that computational subtraction represents a powerful new strategy for determining if an organism has been genetically modified as well as to define the nature of the modification. Fewer assumptions have to be made compared to methods currently in use and this is an advantage particularly when working with unknown GMOs.

  6. Direct sequencing of mitochondrial DNA detects highly divergent haplotypes in blue marlin (Makaira nigricans).

    Science.gov (United States)

    Finnerty, J R; Block, B A

    1992-06-01

    We were able to differentiate between species of billfish (Istiophoridae family) and to detect considerable intraspecific variation in the blue marlin (Makaira nigricans) by directly sequencing a polymerase chain reaction (PCR)-amplified, 612-bp fragment of the mitochondrial cytochrome b gene. Thirteen variable nucleotide sites separated blue marlin (n = 26) into 7 genotypes. On average, these genotypes differed by 5.7 base substitutions. A smaller sample of swordfish from an equally broad geographic distribution displayed relatively little intraspecific variation, with an average of 1.3 substitutions separating different genotypes. A cladistic analysis of blue marlin cytochrome b variants indicates two major divergent evolutionary lines within the species. The frequencies of these two major evolutionary lines differ significantly between Atlantic and Pacific ocean basins. This finding is important given that the Atlantic stocks of blue marlin are considered endangered. Migration from the Pacific can help replenish the numbers of blue marlin in the Atlantic, but the loss of certain mitochondrial DNA haplotypes in the Atlantic due to overfishing probably could not be remedied by an influx of Pacific fish because of their absence in the Pacific population. Fishery management strategies should attempt to preserve the genetic diversity within the species. The detection of DNA sequence polymorphism indicates the utility of PCR technology in pelagic fishery genetics.

  7. Improving transcriptome assembly through error correction of high-throughput sequence reads

    Directory of Open Access Journals (Sweden)

    Matthew D. MacManes

    2013-07-01

    Full Text Available The study of functional genomics, particularly in non-model organisms, has been dramatically improved over the last few years by the use of transcriptomes and RNAseq. While these studies are potentially extremely powerful, a computationally intensive procedure, the de novo construction of a reference transcriptome must be completed as a prerequisite to further analyses. The accurate reference is critically important as all downstream steps, including estimating transcript abundance are critically dependent on the construction of an accurate reference. Though a substantial amount of research has been done on assembly, only recently have the pre-assembly procedures been studied in detail. Specifically, several stand-alone error correction modules have been reported on and, while they have shown to be effective in reducing errors at the level of sequencing reads, how error correction impacts assembly accuracy is largely unknown. Here, we show via use of a simulated and empiric dataset, that applying error correction to sequencing reads has significant positive effects on assembly accuracy, and should be applied to all datasets. A complete collection of commands which will allow for the production of Reptile corrected reads is available at https://github.com/macmanes/error_correction/tree/master/scripts and as File S1.

  8. Inferring Variation in Copy Number Using High Throughput Sequencing Data in R.

    Science.gov (United States)

    Knaus, Brian J; Grünwald, Niklaus J

    2018-01-01

    Inference of copy number variation presents a technical challenge because variant callers typically require the copy number of a genome or genomic region to be known a priori . Here we present a method to infer copy number that uses variant call format (VCF) data as input and is implemented in the R package vcfR . This method is based on the relative frequency of each allele (in both genic and non-genic regions) sequenced at heterozygous positions throughout a genome. These heterozygous positions are summarized by using arbitrarily sized windows of heterozygous positions, binning the allele frequencies, and selecting the bin with the greatest abundance of positions. This provides a non-parametric summary of the frequency that alleles were sequenced at. The method is applicable to organisms that have reference genomes that consist of full chromosomes or sub-chromosomal contigs. In contrast to other software designed to detect copy number variation, our method does not rely on an assumption of base ploidy, but instead infers it. We validated these approaches with the model system of Saccharomyces cerevisiae and applied it to the oomycete Phytophthora infestans , both known to vary in copy number. This functionality has been incorporated into the current release of the R package vcfR to provide modular and flexible methods to investigate copy number variation in genomic projects.

  9. BioVLAB-MMIA-NGS: microRNA-mRNA integrated analysis using high-throughput sequencing data.

    Science.gov (United States)

    Chae, Heejoon; Rhee, Sungmin; Nephew, Kenneth P; Kim, Sun

    2015-01-15

    It is now well established that microRNAs (miRNAs) play a critical role in regulating gene expression in a sequence-specific manner, and genome-wide efforts are underway to predict known and novel miRNA targets. However, the integrated miRNA-mRNA analysis remains a major computational challenge, requiring powerful informatics systems and bioinformatics expertise. The objective of this study was to modify our widely recognized Web server for the integrated mRNA-miRNA analysis (MMIA) and its subsequent deployment on the Amazon cloud (BioVLAB-MMIA) to be compatible with high-throughput platforms, including next-generation sequencing (NGS) data (e.g. RNA-seq). We developed a new version called the BioVLAB-MMIA-NGS, deployed on both Amazon cloud and on a high-performance publicly available server called MAHA. By using NGS data and integrating various bioinformatics tools and databases, BioVLAB-MMIA-NGS offers several advantages. First, sequencing data is more accurate than array-based methods for determining miRNA expression levels. Second, potential novel miRNAs can be detected by using various computational methods for characterizing miRNAs. Third, because miRNA-mediated gene regulation is due to hybridization of an miRNA to its target mRNA, sequencing data can be used to identify many-to-many relationship between miRNAs and target genes with high accuracy. http://epigenomics.snu.ac.kr/biovlab_mmia_ngs/. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  10. Differentiation of highly virulent strains of Streptococcus suis serotype 2 according to glutamate dehydrogenase electrophoretic and sequence type.

    Science.gov (United States)

    Kutz, Russell; Okwumabua, Ogi

    2008-10-01

    The glutamate dehydrogenase (GDH) enzymes of 19 Streptococcus suis serotype 2 strains, consisting of 18 swine isolates and 1 human clinical isolate from a geographically varied collection, were analyzed by activity staining on a nondenaturing gel. All seven (100%) of the highly virulent strains tested produced an electrophoretic type (ET) distinct from those of moderately virulent and nonvirulent strains. By PCR and nucleotide sequence determination, the gdh genes of the 19 strains and of 2 highly virulent strains involved in recent Chinese outbreaks yielded a 1,820-bp fragment containing an open reading frame of 1,344 nucleotides, which encodes a protein of 448 amino acid residues with a calculated molecular mass of approximately 49 kDa. The nucleotide sequences contained base pair differences, but most were silent. Cluster analysis of the deduced amino acid sequences separated the isolates into three groups. Group I (ETI) consisted of the seven highly virulent isolates and the two Chinese outbreak strains, containing Ala(299)-to-Ser, Glu(305)-to-Lys, and Glu(330)-to-Lys amino acid substitutions compared with groups II and III (ETII). Groups II and III consisted of moderately virulent and nonvirulent strains, which are separated from each other by Tyr(72)-to-Asp and Thr(296)-to-Ala substitutions. Gene exchange studies resulted in the change of ETI to ETII and vice versa. A spectrophotometric activity assay for GDH did not show significant differences between the groups. These results suggest that the GDH ETs and sequence types may serve as useful markers in predicting the pathogenic behavior of strains of this serotype and that the molecular basis for the observed differences in the ETs was amino acid substitutions and not deletion, insertion, or processing uniqueness.

  11. Self-confidence and metacognitive processes

    Directory of Open Access Journals (Sweden)

    Kleitman Sabina

    2005-01-01

    Full Text Available This paper examines the status of Self-confidence trait. Two studies strongly suggest that Self-confidence is a component of metacognition. In the first study, participants (N=132 were administered measures of Self-concept, a newly devised Memory and Reasoning Competence Inventory (MARCI, and a Verbal Reasoning Test (VRT. The results indicate a significant relationship between confidence ratings on the VRT and the Reasoning component of MARCI. The second study (N=296 employed an extensive battery of cognitive tests and several metacognitive measures. Results indicate the presence of robust Self-confidence and Metacognitive Awareness factors, and a significant correlation between them. Self-confidence taps not only processes linked to performance on items that have correct answers, but also beliefs about events that may never occur.

  12. High Throughput Sample Preparation and Analysis for DNA Sequencing, PCR and Combinatorial Screening of Catalysis Based on Capillary Array Technique

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yonghua [Iowa State Univ., Ames, IA (United States)

    2000-01-01

    Sample preparation has been one of the major bottlenecks for many high throughput analyses. The purpose of this research was to develop new sample preparation and integration approach for DNA sequencing, PCR based DNA analysis and combinatorial screening of homogeneous catalysis based on multiplexed capillary electrophoresis with laser induced fluorescence or imaging UV absorption detection. The author first introduced a method to integrate the front-end tasks to DNA capillary-array sequencers. protocols for directly sequencing the plasmids from a single bacterial colony in fused-silica capillaries were developed. After the colony was picked, lysis was accomplished in situ in the plastic sample tube using either a thermocycler or heating block. Upon heating, the plasmids were released while chromsomal DNA and membrane proteins were denatured and precipitated to the bottom of the tube. After adding enzyme and Sanger reagents, the resulting solution was aspirated into the reaction capillaries by a syringe pump, and cycle sequencing was initiated. No deleterious effect upon the reaction efficiency, the on-line purification system, or the capillary electrophoresis separation was observed, even though the crude lysate was used as the template. Multiplexed on-line DNA sequencing data from 8 parallel channels allowed base calling up to 620 bp with an accuracy of 98%. The entire system can be automatically regenerated for repeated operation. For PCR based DNA analysis, they demonstrated that capillary electrophoresis with UV detection can be used for DNA analysis starting from clinical sample without purification. After PCR reaction using cheek cell, blood or HIV-1 gag DNA, the reaction mixtures was injected into the capillary either on-line or off-line by base stacking. The protocol was also applied to capillary array electrophoresis. The use of cheaper detection, and the elimination of purification of DNA sample before or after PCR reaction, will make this approach an

  13. Coupled high-throughput functional screening and next generation sequencing for identification of plant polymer decomposing enzymes in metagenomic libraries

    Directory of Open Access Journals (Sweden)

    Mari eNyyssönen

    2013-09-01

    Full Text Available Recent advances in sequencing technologies generate new predictions and hypotheses about the functional roles of environmental microorganisms. Yet, until we can test these predictions at a scale that matches our ability to generate them, most of them will remain as hypotheses. Function-based mining of metagenomic libraries can provide direct linkages between genes, metabolic traits and microbial taxa and thus bridge this gap between sequence data generation and functional predictions. Here we developed high-throughput screening assays for function-based characterization of activities involved in plant polymer decomposition from environmental metagenomic libraries. The multiplexed assays use fluorogenic and chromogenic substrates, combine automated liquid handling and use a genetically modified expression host to enable simultaneous screening of 12,160 clones for 14 activities in a total of 170,240 reactions. Using this platform we identified 374 (0.26 % cellulose, hemicellulose, chitin, starch, phosphate and protein hydrolyzing clones from fosmid libraries prepared from decomposing leaf litter. Sequencing on the Illumina MiSeq platform, followed by assembly and gene prediction of a subset of 95 fosmid clones, identified a broad range of bacterial phyla, including Actinobacteria, Bacteroidetes, multiple Proteobacteria sub-phyla in addition to some Fungi. Carbohydrate-active enzyme genes from 20 different glycoside hydrolase families were detected. Using tetranucleotide frequency binning of fosmid sequences, multiple enzyme activities from distinct fosmids were linked, demonstrating how biochemically-confirmed functional traits in environmental metagenomes may be attributed to groups of specific organisms. Overall, our results demonstrate how functional screening of metagenomic libraries can be used to connect microbial functionality to community composition and, as a result, complement large-scale metagenomic sequencing efforts.

  14. Confidence in Alternative Dispute Resolution: Experience from Switzerland

    Directory of Open Access Journals (Sweden)

    Christof Schwenkel

    2014-06-01

    Full Text Available Alternative Dispute Resolution plays a crucial role in the justice system of Switzerland. With the unified Swiss Code of Civil Procedure, it is required that each litigation session shall be preceded by an attempt at conciliation before a conciliation authority. However, there has been little research on conciliation authorities and the public's perception of the authorities. This paper looks at public confidence in conciliation authorities and provides results of a survey conducted with more than 3,400 participants. This study found that public confidence in Swiss conciliation authorities is generally high, exceeds the ratings for confidence in cantonal governments and parliaments, but is lower than confidence in courts.Since the institutional models of the conciliation authorities (meaning the organization of the authorities and the selection of the conciliators differ widely between the 26 Swiss cantons, the influence of the institutional models on public confidence is analyzed. Contrary to assumptions based on New Institutional-ism approaches, this study reports that the institutional models do not impact public confidence. Also, the relationship between a participation in an election of justices of the peace or conciliators and public confidence in these authorities is found to be at most very limited (and negative. Similar to common findings on courts, the results show that general contacts with conciliation authorities decrease public confidence in these institutions whereas a positive experience with a conciliation authority leads to more confidence.The Study was completed as part of the research project 'Basic Research into Court Management in Switzerland', supported by the Swiss National Science Foundation (SNSF. Christof Schwenkel is a PhD student at the University of Lucerne and a research associate and project manager at Interface Policy Studies. A first version of this article was presented at the 2013 European Group for Public

  15. Bacterial diversity of the American sand fly Lutzomyia intermedia using high-throughput metagenomic sequencing.

    Science.gov (United States)

    Monteiro, Carolina Cunha; Villegas, Luis Eduardo Martinez; Campolina, Thais Bonifácio; Pires, Ana Clara Machado Araújo; Miranda, Jose Carlos; Pimenta, Paulo Filemon Paolucci; Secundino, Nagila Francinete Costa

    2016-08-31

    Parasites of the genus Leishmania cause a broad spectrum of diseases, collectively known as leishmaniasis, in humans worldwide. American cutaneous leishmaniasis is a neglected disease transmitted by sand fly vectors including Lutzomyia intermedia, a proven vector. The female sand fly can acquire or deliver Leishmania spp. parasites while feeding on a blood meal, which is required for nutrition, egg development and survival. The microbiota composition and abundance varies by food source, life stages and physiological conditions. The sand fly microbiota can affect parasite life-cycle in the vector. We performed a metagenomic analysis for microbiota composition and abundance in Lu. intermedia, from an endemic area in Brazil. The adult insects were collected using CDC light traps, morphologically identified, carefully sterilized, dissected under a microscope and the females separated into groups according to their physiological condition: (i) absence of blood meal (unfed = UN); (ii) presence of blood meal (blood-fed = BF); and (iii) presence of developed ovaries (gravid = GR). Then, they were processed for metagenomics with Illumina Hiseq Sequencing in order to be sequence analyzed and to obtain the taxonomic profiles of the microbiota. Bacterial metagenomic analysis revealed differences in microbiota composition based upon the distinct physiological stages of the adult insect. Sequence identification revealed two phyla (Proteobacteria and Actinobacteria), 11 families and 15 genera; 87 % of the bacteria were Gram-negative, while only one family and two genera were identified as Gram-positive. The genera Ochrobactrum, Bradyrhizobium and Pseudomonas were found across all of the groups. The metagenomic analysis revealed that the microbiota of the Lu. intermedia female sand flies are distinct under specific physiological conditions and consist of 15 bacterial genera. The Ochrobactrum, Bradyrhizobium and Pseudomonas were the common genera. Our results detailing

  16. Two sides of the same coin: Monetary incentives concurrently improve and bias confidence judgments.

    Science.gov (United States)

    Lebreton, Maël; Langdon, Shari; Slieker, Matthijs J; Nooitgedacht, Jip S; Goudriaan, A