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Sample records for binary sequence generators

  1. Next-Generation Sequencing for Binary Protein-Protein Interactions

    Directory of Open Access Journals (Sweden)

    Bernhard eSuter

    2015-12-01

    Full Text Available The yeast two-hybrid (Y2H system exploits host cell genetics in order to display binary protein-protein interactions (PPIs via defined and selectable phenotypes. Numerous improvements have been made to this method, adapting the screening principle for diverse applications, including drug discovery and the scale-up for proteome wide interaction screens in human and other organisms. Here we discuss a systematic workflow and analysis scheme for screening data generated by Y2H and related assays that includes high-throughput selection procedures, readout of comprehensive results via next-generation sequencing (NGS, and the interpretation of interaction data via quantitative statistics. The novel assays and tools will serve the broader scientific community to harness the power of NGS technology to address PPI networks in health and disease. We discuss examples of how this next-generation platform can be applied to address specific questions in diverse fields of biology and medicine.

  2. Multiple ECG Fiducial Points-Based Random Binary Sequence Generation for Securing Wireless Body Area Networks.

    Science.gov (United States)

    Zheng, Guanglou; Fang, Gengfa; Shankaran, Rajan; Orgun, Mehmet A; Zhou, Jie; Qiao, Li; Saleem, Kashif

    2017-05-01

    Generating random binary sequences (BSes) is a fundamental requirement in cryptography. A BS is a sequence of N bits, and each bit has a value of 0 or 1. For securing sensors within wireless body area networks (WBANs), electrocardiogram (ECG)-based BS generation methods have been widely investigated in which interpulse intervals (IPIs) from each heartbeat cycle are processed to produce BSes. Using these IPI-based methods to generate a 128-bit BS in real time normally takes around half a minute. In order to improve the time efficiency of such methods, this paper presents an ECG multiple fiducial-points based binary sequence generation (MFBSG) algorithm. The technique of discrete wavelet transforms is employed to detect arrival time of these fiducial points, such as P, Q, R, S, and T peaks. Time intervals between them, including RR, RQ, RS, RP, and RT intervals, are then calculated based on this arrival time, and are used as ECG features to generate random BSes with low latency. According to our analysis on real ECG data, these ECG feature values exhibit the property of randomness and, thus, can be utilized to generate random BSes. Compared with the schemes that solely rely on IPIs to generate BSes, this MFBSG algorithm uses five feature values from one heart beat cycle, and can be up to five times faster than the solely IPI-based methods. So, it achieves a design goal of low latency. According to our analysis, the complexity of the algorithm is comparable to that of fast Fourier transforms. These randomly generated ECG BSes can be used as security keys for encryption or authentication in a WBAN system.

  3. 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.

  4. 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

  5. Analysis of using interpulse intervals to generate 128-bit biometric random binary sequences for securing wireless body sensor networks.

    Science.gov (United States)

    Zhang, Guang-He; Poon, Carmen C Y; Zhang, Yuan-Ting

    2012-01-01

    Wireless body sensor network (WBSN), a key building block for m-Health, demands extremely stringent resource constraints and thus lightweight security methods are preferred. To minimize resource consumption, utilizing information already available to a WBSN, particularly common to different sensor nodes of a WBSN, for security purposes becomes an attractive solution. In this paper, we tested the randomness and distinctiveness of the 128-bit biometric binary sequences (BSs) generated from interpulse intervals (IPIs) of 20 healthy subjects as well as 30 patients suffered from myocardial infarction and 34 subjects with other cardiovascular diseases. The encoding time of a biometric BS on a WBSN node is on average 23 ms and memory occupation is 204 bytes for any given IPI sequence. The results from five U.S. National Institute of Standards and Technology statistical tests suggest that random biometric BSs can be generated from both healthy subjects and cardiovascular patients and can potentially be used as authentication identifiers for securing WBSNs. Ultimately, it is preferred that these biometric BSs can be used as encryption keys such that key distribution over the WBSN can be avoided.

  6. Logistic chaotic maps for binary numbers generations

    International Nuclear Information System (INIS)

    Kanso, Ali; Smaoui, Nejib

    2009-01-01

    Two pseudorandom binary sequence generators, based on logistic chaotic maps intended for stream cipher applications, are proposed. The first is based on a single one-dimensional logistic map which exhibits random, noise-like properties at given certain parameter values, and the second is based on a combination of two logistic maps. The encryption step proposed in both algorithms consists of a simple bitwise XOR operation of the plaintext binary sequence with the keystream binary sequence to produce the ciphertext binary sequence. A threshold function is applied to convert the floating-point iterates into binary form. Experimental results show that the produced sequences possess high linear complexity and very good statistical properties. The systems are put forward for security evaluation by the cryptographic committees.

  7. A measurement of disorder in binary sequences

    Science.gov (United States)

    Gong, Longyan; Wang, Haihong; Cheng, Weiwen; Zhao, Shengmei

    2015-03-01

    We propose a complex quantity, AL, to characterize the degree of disorder of L-length binary symbolic sequences. As examples, we respectively apply it to typical random and deterministic sequences. One kind of random sequences is generated from a periodic binary sequence and the other is generated from the logistic map. The deterministic sequences are the Fibonacci and Thue-Morse sequences. In these analyzed sequences, we find that the modulus of AL, denoted by |AL | , is a (statistically) equivalent quantity to the Boltzmann entropy, the metric entropy, the conditional block entropy and/or other quantities, so it is a useful quantitative measure of disorder. It can be as a fruitful index to discern which sequence is more disordered. Moreover, there is one and only one value of |AL | for the overall disorder characteristics. It needs extremely low computational costs. It can be easily experimentally realized. From all these mentioned, we believe that the proposed measure of disorder is a valuable complement to existing ones in symbolic sequences.

  8. Permutation Entropy for Random Binary Sequences

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    Lingfeng Liu

    2015-12-01

    Full Text Available In this paper, we generalize the permutation entropy (PE measure to binary sequences, which is based on Shannon’s entropy, and theoretically analyze this measure for random binary sequences. We deduce the theoretical value of PE for random binary sequences, which can be used to measure the randomness of binary sequences. We also reveal the relationship between this PE measure with other randomness measures, such as Shannon’s entropy and Lempel–Ziv complexity. The results show that PE is consistent with these two measures. Furthermore, we use PE as one of the randomness measures to evaluate the randomness of chaotic binary sequences.

  9. Minimum degree and density of binary sequences

    DEFF Research Database (Denmark)

    Brandt, Stephan; Müttel, J.; Rautenbach, D.

    2010-01-01

    For d,k∈N with k ≤ 2d, let g(d,k) denote the infimum density of binary sequences (x)∈{0,1} which satisfy the minimum degree condition σ(x+) ≥ k for all i∈Z with xi=1. We reduce the problem of computing g(d,k) to a combinatorial problem related to the generalized k-girth of a graph G which...

  10. Orbital motion in pre-main sequence binaries

    Energy Technology Data Exchange (ETDEWEB)

    Schaefer, G. H. [The CHARA Array of Georgia State University, Mount Wilson Observatory, Mount Wilson, CA 91023 (United States); Prato, L. [Lowell Observatory, 1400 West Mars Hill Road, Flagstaff, AZ 86001 (United States); Simon, M. [Department of Physics and Astronomy, Stony Brook University, Stony Brook, NY 11794 (United States); Patience, J., E-mail: schaefer@chara-array.org [Astrophysics Group, School of Physics, University of Exeter, Exeter, EX4 4QL (United Kingdom)

    2014-06-01

    We present results from our ongoing program to map the visual orbits of pre-main sequence (PMS) binaries in the Taurus star forming region using adaptive optics imaging at the Keck Observatory. We combine our results with measurements reported in the literature to analyze the orbital motion for each binary. We present preliminary orbits for DF Tau, T Tau S, ZZ Tau, and the Pleiades binary HBC 351. Seven additional binaries show curvature in their relative motion. Currently, we can place lower limits on the orbital periods for these systems; full solutions will be possible with more orbital coverage. Five other binaries show motion that is indistinguishable from linear motion. We suspect that these systems are bound and might show curvature with additional measurements in the future. The observations reported herein lay critical groundwork toward the goal of measuring precise masses for low-mass PMS stars.

  11. Perceptions of randomness in binary sequences: Normative, heuristic, or both?

    Science.gov (United States)

    Reimers, Stian; Donkin, Chris; Le Pelley, Mike E

    2018-03-01

    When people consider a series of random binary events, such as tossing an unbiased coin and recording the sequence of heads (H) and tails (T), they tend to erroneously rate sequences with less internal structure or order (such as HTTHT) as more probable than sequences containing more structure or order (such as HHHHH). This is traditionally explained as a local representativeness effect: Participants assume that the properties of long sequences of random outcomes-such as an equal proportion of heads and tails, and little internal structure-should also apply to short sequences. However, recent theoretical work has noted that the probability of a particular sequence of say, heads and tails of length n, occurring within a larger (>n) sequence of coin flips actually differs by sequence, so P(HHHHH) rational norms based on limited experience. We test these accounts. Participants in Experiment 1 rated the likelihood of occurrence for all possible strings of 4, 5, and 6 observations in a sequence of coin flips. Judgments were better explained by representativeness in alternation rate, relative proportion of heads and tails, and sequence complexity, than by objective probabilities. Experiments 2 and 3 gave similar results using incentivized binary choice procedures. Overall the evidence suggests that participants are not sensitive to variation in objective probabilities of a sub-sequence occurring; they appear to use heuristics based on several distinct forms of representativeness. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Next-generation sequencing

    DEFF Research Database (Denmark)

    Rieneck, Klaus; Bak, Mads; Jønson, Lars

    2013-01-01

    , Illumina); several millions of PCR sequences were analyzed. RESULTS: The results demonstrated the feasibility of diagnosing the fetal KEL1 or KEL2 blood group from cell-free DNA purified from maternal plasma. CONCLUSION: This method requires only one primer pair, and the large amount of sequence...... information obtained allows well for statistical analysis of the data. This general approach can be integrated into current laboratory practice and has numerous applications. Besides DNA-based predictions of blood group phenotypes, platelet phenotypes, or sickle cell anemia, and the determination of zygosity...

  13. Next-Generation Sequencing Platforms

    Science.gov (United States)

    Mardis, Elaine R.

    2013-06-01

    Automated DNA sequencing instruments embody an elegant interplay among chemistry, engineering, software, and molecular biology and have built upon Sanger's founding discovery of dideoxynucleotide sequencing to perform once-unfathomable tasks. Combined with innovative physical mapping approaches that helped to establish long-range relationships between cloned stretches of genomic DNA, fluorescent DNA sequencers produced reference genome sequences for model organisms and for the reference human genome. New types of sequencing instruments that permit amazing acceleration of data-collection rates for DNA sequencing have been developed. The ability to generate genome-scale data sets is now transforming the nature of biological inquiry. Here, I provide an historical perspective of the field, focusing on the fundamental developments that predated the advent of next-generation sequencing instruments and providing information about how these instruments work, their application to biological research, and the newest types of sequencers that can extract data from single DNA molecules.

  14. LPTAU, Quasi Random Sequence Generator

    International Nuclear Information System (INIS)

    Sobol, Ilya M.

    1993-01-01

    1 - Description of program or function: LPTAU generates quasi random sequences. These are uniformly distributed sets of L=M N points in the N-dimensional unit cube: I N =[0,1]x...x[0,1]. These sequences are used as nodes for multidimensional integration; as searching points in global optimization; as trial points in multi-criteria decision making; as quasi-random points for quasi Monte Carlo algorithms. 2 - Method of solution: Uses LP-TAU sequence generation (see references). 3 - Restrictions on the complexity of the problem: The number of points that can be generated is L 30 . The dimension of the space cannot exceed 51

  15. Preliminary study of pseudorandom binary sequence pulsing of ORELA

    International Nuclear Information System (INIS)

    Larson, N.M.; Olsen, D.K.

    1980-03-01

    It has been suggested that pseudorandom binary sequence (PRBS) pulsing might enhance the performance of the Oak Ridge Electron Linear Accelerator (ORELA) for neutron-induced, time-of-flight (TOF) cross-section measurements. In this technical memorandum, equations are developed for expected count rates, statistical variances, and backgrounds for a pulsing scheme in which a PRBS is superimposed on the periodic equalintensity ORELA bursts. Introduction of the PRBS modification permits neutrons of different energies originating from different bursts to reach the detector simultaneously, and the signal corresponding to a unique flight time to be extracted mathematically. Relative advantages and disadvantages of measurements from conventional and PRBS pulsing modes are discussed in terms of counting statistics and backgrounds. Computer models of TOF spectra are generated for both pulsing modes, using as examples a 20-meter 233 U fission-chamber measurement and a 155-meter 238 U sample-in transmission measurement. Detailed comparisons of PRBS vs conventional results are presented. This study indicates that although PRBS pulsing could enhance ORELA performance for selected measurements, for general ORELA operation the disadvantages from PRBS pulsing probably outweigh the advantages

  16. On the Roche constants for main-sequence binaries

    International Nuclear Information System (INIS)

    Giannuzzi, M.A.

    1979-01-01

    The ratios C 1 /C 2 of the constants defining the equipotential surfaces which describe the external forms of the components of a close binary system have been calculated on the basis of evolutionary models. Theoretical systems have been considered allowing for a wide range of input parameters (masses and separation) and taking into account the evolutionary effects on the radii of the stars during their Main-Sequence lifetime. The systems have not undergone any transfer of matter and are representative of detached binaries with Main-sequence components. The ratios of the constants are confined in limited intervals and, for the highest values of the mass-ratios, they are clustered around the unit. (Auth.)

  17. Composite Binary Sequences with a Large Ensemble and Zero Correlation Zone

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    S. S. Yudachev

    2015-01-01

    Full Text Available The article considers a proposed class of derived signals such as composite binary sequences for application in advanced spread spectrum radio systems of various purposes, using signals based on spectrum spreading by direct sequence method. Considered composite sequences, having a representative set of lengths and unique correlation properties, compares favorably with the widely used at present large ensembles formed on a single algorithmic basis. To evaluate the properties of the composite sequences generated on the basis of two components - the Barker code and Kerdock sequences, expressions of periodic and aperiodic correlation functions are given.An algorithm for generating practical ensembles of composite sequences is presented. On the basis of the algorithm and its software implementation in C #, the samples of the sequence ensembles of various lengths were obtained and their periodic and aperiodic correlation functions and statistical characteristics were studied in detail. As an illustration, some of the most typical correlation functions are presented. The most remarkable characteristics allowing a ssessing the feasibility of using this type of sequences in the design of specific types of radio systems are considered.On the basis of the proposed program and the performed calculations the conclusions can be drawn about the possibility of using the sequences of these classes, with the aim of reducing intra-system disturbance in the projected spread spectrum CDMA.

  18. Long period pseudo random number sequence generator

    Science.gov (United States)

    Wang, Charles C. (Inventor)

    1989-01-01

    A circuit for generating a sequence of pseudo random numbers, (A sub K). There is an exponentiator in GF(2 sup m) for the normal basis representation of elements in a finite field GF(2 sup m) each represented by m binary digits and having two inputs and an output from which the sequence (A sub K). Of pseudo random numbers is taken. One of the two inputs is connected to receive the outputs (E sub K) of maximal length shift register of n stages. There is a switch having a pair of inputs and an output. The switch outputs is connected to the other of the two inputs of the exponentiator. One of the switch inputs is connected for initially receiving a primitive element (A sub O) in GF(2 sup m). Finally, there is a delay circuit having an input and an output. The delay circuit output is connected to the other of the switch inputs and the delay circuit input is connected to the output of the exponentiator. Whereby after the exponentiator initially receives the primitive element (A sub O) in GF(2 sup m) through the switch, the switch can be switched to cause the exponentiator to receive as its input a delayed output A(K-1) from the exponentiator thereby generating (A sub K) continuously at the output of the exponentiator. The exponentiator in GF(2 sup m) is novel and comprises a cyclic-shift circuit; a Massey-Omura multiplier; and, a control logic circuit all operably connected together to perform the function U(sub i) = 92(sup i) (for n(sub i) = 1 or 1 (for n(subi) = 0).

  19. Spreading Sequences Generated Using Asymmetrical Integer-Number Maps

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    V. Sebesta

    2007-09-01

    Full Text Available Chaotic sequences produced by piecewise linear maps can be transformed to binary sequences. The binary sequences are optimal for the asynchronous DS/CDMA systems in case of certain shapes of the maps. This paper is devoted to the one-to-one integer-number maps derived from the suitable asymmetrical piecewise linear maps. Such maps give periodic integer-number sequences, which can be transformed to the binary sequences. The binary sequences produced via proposed modified integer-number maps are perfectly balanced and embody good autocorrelation and crosscorrelation properties. The number of different binary sequences is sizable. The sequences are suitable as spreading sequences in DS/CDMA systems.

  20. WHITE-DWARF-MAIN-SEQUENCE BINARIES IDENTIFIED FROM THE LAMOST PILOT SURVEY

    International Nuclear Information System (INIS)

    Ren Juanjuan; Luo Ali; Li Yinbi; Wei Peng; Zhao Jingkun; Zhao Yongheng; Song Yihan; Zhao Gang

    2013-01-01

    We present a set of white-dwarf-main-sequence (WDMS) binaries identified spectroscopically from the Large sky Area Multi-Object fiber Spectroscopic Telescope (LAMOST, also called the Guo Shou Jing Telescope) pilot survey. We develop a color selection criteria based on what is so far the largest and most complete Sloan Digital Sky Survey (SDSS) DR7 WDMS binary catalog and identify 28 WDMS binaries within the LAMOST pilot survey. The primaries in our binary sample are mostly DA white dwarfs except for one DB white dwarf. We derive the stellar atmospheric parameters, masses, and radii for the two components of 10 of our binaries. We also provide cooling ages for the white dwarf primaries as well as the spectral types for the companion stars of these 10 WDMS binaries. These binaries tend to contain hot white dwarfs and early-type companions. Through cross-identification, we note that nine binaries in our sample have been published in the SDSS DR7 WDMS binary catalog. Nineteen spectroscopic WDMS binaries identified by the LAMOST pilot survey are new. Using the 3σ radial velocity variation as a criterion, we find two post-common-envelope binary candidates from our WDMS binary sample

  1. Non-linear M -sequences Generation Method

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    Z. R. Garifullina

    2011-06-01

    Full Text Available The article deals with a new method for modeling a pseudorandom number generator based on R-blocks. The gist of the method is the replacement of a multi digit XOR element by a stochastic adder in a parallel binary linear feedback shift register scheme.

  2. Generation of two-dimensional binary mixtures in complex plasmas

    Science.gov (United States)

    Wieben, Frank; Block, Dietmar

    2016-10-01

    Complex plasmas are an excellent model system for strong coupling phenomena. Under certain conditions the dust particles immersed into the plasma form crystals which can be analyzed in terms of structure and dynamics. Previous experiments focussed mostly on monodisperse particle systems whereas dusty plasmas in nature and technology are polydisperse. Thus, a first and important step towards experiments in polydisperse systems are binary mixtures. Recent experiments on binary mixtures under microgravity conditions observed a phase separation of particle species with different radii even for small size disparities. This contradicts several numerical studies of 2D binary mixtures. Therefore, dedicated experiments are required to gain more insight into the physics of polydisperse systems. In this contribution first ground based experiments on two-dimensional binary mixtures are presented. Particular attention is paid to the requirements for the generation of such systems which involve the consideration of the temporal evolution of the particle properties. Furthermore, the structure of these two-component crystals is analyzed and compared to simulations. This work was supported by the Deutsche Forschungsgemeinschaft DFG in the framework of the SFB TR24 Greifswald Kiel, Project A3b.

  3. RANDNA: a random DNA sequence generator.

    Science.gov (United States)

    Piva, Francesco; Principato, Giovanni

    2006-01-01

    Monte Carlo simulations are useful to verify the significance of data. Genomic regularities, such as the nucleotide correlations or the not uniform distribution of the motifs throughout genomic or mature mRNA sequences, exist and their significance can be checked by means of the Monte Carlo test. The test needs good quality random sequences in order to work, moreover they should have the same nucleotide distribution as the sequences in which the regularities have been found. Random DNA sequences are also useful to estimate the background score of an alignment, that is a threshold below which the resulting score is merely due to chance. We have developed RANDNA, a free software which allows to produce random DNA or RNA sequences setting both their length and the percentage of nucleotide composition. Sequences having the same nucleotide distribution of exonic, intronic or intergenic sequences can be generated. Its graphic interface makes it possible to easily set the parameters that characterize the sequences being produced and saved in a text format file. The pseudo-random number generator function of Borland Delphi 6 is used, since it guarantees a good randomness, a long cycle length and a high speed. We have checked the quality of sequences generated by the software, by means of well-known tests, both by themselves and versus genuine random sequences. We show the good quality of the generated sequences. The software, complete with examples and documentation, is freely available to users from: http://www.introni.it/en/software.

  4. Polynomial sequences generated by infinite Hessenberg matrices

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    Verde-Star Luis

    2017-01-01

    Full Text Available We show that an infinite lower Hessenberg matrix generates polynomial sequences that correspond to the rows of infinite lower triangular invertible matrices. Orthogonal polynomial sequences are obtained when the Hessenberg matrix is tridiagonal. We study properties of the polynomial sequences and their corresponding matrices which are related to recurrence relations, companion matrices, matrix similarity, construction algorithms, and generating functions. When the Hessenberg matrix is also Toeplitz the polynomial sequences turn out to be of interpolatory type and we obtain additional results. For example, we show that every nonderogative finite square matrix is similar to a unique Toeplitz-Hessenberg matrix.

  5. "First generation" automated DNA sequencing technology.

    Science.gov (United States)

    Slatko, Barton E; Kieleczawa, Jan; Ju, Jingyue; Gardner, Andrew F; Hendrickson, Cynthia L; Ausubel, Frederick M

    2011-10-01

    Beginning in the 1980s, automation of DNA sequencing has greatly increased throughput, reduced costs, and enabled large projects to be completed more easily. The development of automation technology paralleled the development of other aspects of DNA sequencing: better enzymes and chemistry, separation and imaging technology, sequencing protocols, robotics, and computational advancements (including base-calling algorithms with quality scores, database developments, and sequence analysis programs). Despite the emergence of high-throughput sequencing platforms, automated Sanger sequencing technology remains useful for many applications. This unit provides background and a description of the "First-Generation" automated DNA sequencing technology. It also includes protocols for using the current Applied Biosystems (ABI) automated DNA sequencing machines. © 2011 by John Wiley & Sons, Inc.

  6. Galaxy LIMS for next-generation sequencing

    NARCIS (Netherlands)

    Scholtalbers, J.; Rossler, J.; Sorn, P.; Graaf, J. de; Boisguerin, V.; Castle, J.; Sahin, U.

    2013-01-01

    SUMMARY: We have developed a laboratory information management system (LIMS) for a next-generation sequencing (NGS) laboratory within the existing Galaxy platform. The system provides lab technicians standard and customizable sample information forms, barcoded submission forms, tracking of input

  7. Special Issue: Next Generation DNA Sequencing

    Directory of Open Access Journals (Sweden)

    Paul Richardson

    2010-10-01

    Full Text Available Next Generation Sequencing (NGS refers to technologies that do not rely on traditional dideoxy-nucleotide (Sanger sequencing where labeled DNA fragments are physically resolved by electrophoresis. These new technologies rely on different strategies, but essentially all of them make use of real-time data collection of a base level incorporation event across a massive number of reactions (on the order of millions versus 96 for capillary electrophoresis for instance. The major commercial NGS platforms available to researchers are the 454 Genome Sequencer (Roche, Illumina (formerly Solexa Genome analyzer, the SOLiD system (Applied Biosystems/Life Technologies and the Heliscope (Helicos Corporation. The techniques and different strategies utilized by these platforms are reviewed in a number of the papers in this special issue. These technologies are enabling new applications that take advantage of the massive data produced by this next generation of sequencing instruments. [...

  8. RAMBO-K: Rapid and Sensitive Removal of Background Sequences from Next Generation Sequencing Data.

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    Simon H Tausch

    Full Text Available The assembly of viral or endosymbiont genomes from Next Generation Sequencing (NGS data is often hampered by the predominant abundance of reads originating from the host organism. These reads increase the memory and CPU time usage of the assembler and can lead to misassemblies.We developed RAMBO-K (Read Assignment Method Based On K-mers, a tool which allows rapid and sensitive removal of unwanted host sequences from NGS datasets. Reaching a speed of 10 Megabases/s on 4 CPU cores and a standard hard drive, RAMBO-K is faster than any tool we tested, while showing a consistently high sensitivity and specificity across different datasets.RAMBO-K rapidly and reliably separates reads from different species without data preprocessing. It is suitable as a straightforward standard solution for workflows dealing with mixed datasets. Binaries and source code (java and python are available from http://sourceforge.net/projects/rambok/.

  9. PRIMITIVE MATRICES AND GENERATORS OF PSEUDO RANDOM SEQUENCES OF GALOIS

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    A. Beletsky

    2014-04-01

    Full Text Available In theory and practice of information cryptographic protection one of the key problems is the forming a binary pseudo-random sequences (PRS with a maximum length with acceptable statistical characteristics. PRS generators are usually implemented by linear shift register (LSR of maximum period with linear feedback [1]. In this paper we extend the concept of LSR, assuming that each of its rank (memory cell can be in one of the following condition. Let’s call such registers “generalized linear shift register.” The research goal is to develop algorithms for constructing Galois and Fibonacci generalized matrix of n-order over the field , which uniquely determined both the structure of corresponding generalized of n-order LSR maximal period, and formed on their basis Galois PRS generators of maximum length. Thus the article presents the questions of formation the primitive generalized Fibonacci and Galois arbitrary order matrix over the prime field . The synthesis of matrices is based on the use of irreducible polynomials of degree and primitive elements of the extended field generated by polynomial. The constructing methods of Galois and Fibonacci conjugated primitive matrices are suggested. The using possibilities of such matrices in solving the problem of constructing generalized generators of Galois pseudo-random sequences are discussed.

  10. Discovery and characterization of 3000+ main-sequence binaries from APOGEE spectra

    Science.gov (United States)

    El-Badry, Kareem; Ting, Yuan-Sen; Rix, Hans-Walter; Quataert, Eliot; Weisz, Daniel R.; Cargile, Phillip; Conroy, Charlie; Hogg, David W.; Bergemann, Maria; Liu, Chao

    2018-05-01

    We develop a data-driven spectral model for identifying and characterizing spatially unresolved multiple-star systems and apply it to APOGEE DR13 spectra of main-sequence stars. Binaries and triples are identified as targets whose spectra can be significantly better fit by a superposition of two or three model spectra, drawn from the same isochrone, than any single-star model. From an initial sample of ˜20 000 main-sequence targets, we identify ˜2500 binaries in which both the primary and secondary stars contribute detectably to the spectrum, simultaneously fitting for the velocities and stellar parameters of both components. We additionally identify and fit ˜200 triple systems, as well as ˜700 velocity-variable systems in which the secondary does not contribute detectably to the spectrum. Our model simplifies the process of simultaneously fitting single- or multi-epoch spectra with composite models and does not depend on a velocity offset between the two components of a binary, making it sensitive to traditionally undetectable systems with periods of hundreds or thousands of years. In agreement with conventional expectations, almost all the spectrally identified binaries with measured parallaxes fall above the main sequence in the colour-magnitude diagram. We find excellent agreement between spectrally and dynamically inferred mass ratios for the ˜600 binaries in which a dynamical mass ratio can be measured from multi-epoch radial velocities. We obtain full orbital solutions for 64 systems, including 14 close binaries within hierarchical triples. We make available catalogues of stellar parameters, abundances, mass ratios, and orbital parameters.

  11. White dwarf-main sequence binaries from LAMOST: the DR5 catalogue

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    Ren, J.-J.; Rebassa-Mansergas, A.; Parsons, S. G.; Liu, X.-W.; Luo, A.-L.; Kong, X.; Zhang, H.-T.

    2018-03-01

    We present the data release (DR) 5 catalogue of white dwarf-main sequence (WDMS) binaries from the Large Area Multi-Object fiber Spectroscopic Telescope (LAMOST). The catalogue contains 876 WDMS binaries, of which 757 are additions to our previous LAMOST DR1 sample and 357 are systems that have not been published before. We also describe a LAMOST-dedicated survey that aims at obtaining spectra of photometrically-selected WDMS binaries from the Sloan Digital Sky Survey (SDSS) that are expected to contain cool white dwarfs and/or early type M dwarf companions. This is a population under-represented in previous SDSS WDMS binary catalogues. We determine the stellar parameters (white dwarf effective temperatures, surface gravities and masses, and M dwarf spectral types) of the LAMOST DR5 WDMS binaries and make use of the parameter distributions to analyse the properties of the sample. We find that, despite our efforts, systems containing cool white dwarfs remain under-represented. Moreover, we make use of LAMOST DR5 and SDSS DR14 (when available) spectra to measure the Na I λλ 8183.27, 8194.81 absorption doublet and/or Hα emission radial velocities of our systems. This allows identifying 128 binaries displaying significant radial velocity variations, 76 of which are new. Finally, we cross-match our catalogue with the Catalina Surveys and identify 57 systems displaying light curve variations. These include 16 eclipsing systems, two of which are new, and nine binaries that are new eclipsing candidates. We calculate periodograms from the photometric data and measure (estimate) the orbital periods of 30 (15) WDMS binaries.

  12. Neural mechanisms of sequence generation in songbirds

    Science.gov (United States)

    Langford, Bruce

    Animal models in research are useful for studying more complex behavior. For example, motor sequence generation of actions requiring good muscle coordination such as writing with a pen, playing an instrument, or speaking, may involve the interaction of many areas in the brain, each a complex system in itself; thus it can be difficult to determine causal relationships between neural behavior and the behavior being studied. Birdsong, however, provides an excellent model behavior for motor sequence learning, memory, and generation. The song consists of learned sequences of notes that are spectrographically stereotyped over multiple renditions of the song, similar to syllables in human speech. The main areas of the songbird brain involve in singing are known, however, the mechanisms by which these systems store and produce song are not well understood. We used a custom built, head-mounted, miniature motorized microdrive to chronically record the neural firing patterns of identified neurons in HVC, a pre-motor cortical nucleus which has been shown to be important in song timing. These were done in Bengalese finch which generate a song made up of stereotyped notes but variable note sequences. We observed song related bursting in neurons projecting to Area X, a homologue to basal ganglia, and tonic firing in HVC interneurons. Interneuron had firing rate patterns that were consistent over multiple renditions of the same note sequence. We also designed and built a light-weight, low-powered wireless programmable neural stimulator using Bluetooth Low Energy Protocol. It was able to generate perturbations in the song when current pulses were administered to RA, which projects to the brainstem nucleus responsible for syringeal muscle control.

  13. Bioinformatics for Next Generation Sequencing Data

    Directory of Open Access Journals (Sweden)

    Alberto Magi

    2010-09-01

    Full Text Available The emergence of next-generation sequencing (NGS platforms imposes increasing demands on statistical methods and bioinformatic tools for the analysis and the management of the huge amounts of data generated by these technologies. Even at the early stages of their commercial availability, a large number of softwares already exist for analyzing NGS data. These tools can be fit into many general categories including alignment of sequence reads to a reference, base-calling and/or polymorphism detection, de novo assembly from paired or unpaired reads, structural variant detection and genome browsing. This manuscript aims to guide readers in the choice of the available computational tools that can be used to face the several steps of the data analysis workflow.

  14. A BiCMOS Binary Hysteresis Chaos Generator

    Science.gov (United States)

    Ahmadi, S.; Newcomb, R. W.

    A previous op-amp RC circuit which was proven to give chaotic signals is converted to a BiCMOS design more suitable to integrated circuit realization. The structure results from a degree two differential equation which includes binary hysteresis as its nonlinearity. The circuit is realized by differential (voltage to current) pairs feeding two capacitors, which carry the dynamics, with the key component being a (voltage to current) binary hysteresis circuit due to Linares.

  15. Sequence trajectory generation for garment handling systems

    OpenAIRE

    Liu, Honghai; Lin, Hua

    2008-01-01

    This paper presents a novel generic approach to the planning strategy of garment handling systems. An assumption is proposed to separate the components of such systems into a component for intelligent gripper techniques and a component for handling planning strategies. Researchers can concentrate on one of the two components first, then merge the two problems together. An algorithm is addressed to generate the trajectory position and a clothes handling sequence of clothes partitions, which ar...

  16. Least squares deconvolution for leak detection with a pseudo random binary sequence excitation

    Science.gov (United States)

    Nguyen, Si Tran Nguyen; Gong, Jinzhe; Lambert, Martin F.; Zecchin, Aaron C.; Simpson, Angus R.

    2018-01-01

    Leak detection and localisation is critical for water distribution system pipelines. This paper examines the use of the time-domain impulse response function (IRF) for leak detection and localisation in a pressurised water pipeline with a pseudo random binary sequence (PRBS) signal excitation. Compared to the conventional step wave generated using a single fast operation of a valve closure, a PRBS signal offers advantageous correlation properties, in that the signal has very low autocorrelation for lags different from zero and low cross correlation with other signals including noise and other interference. These properties result in a significant improvement in the IRF signal to noise ratio (SNR), leading to more accurate leak localisation. In this paper, the estimation of the system IRF is formulated as an optimisation problem in which the l2 norm of the IRF is minimised to suppress the impact of noise and interference sources. Both numerical and experimental data are used to verify the proposed technique. The resultant estimated IRF provides not only accurate leak location estimation, but also good sensitivity to small leak sizes due to the improved SNR.

  17. Formal definition of coherency and computation of minimal cut sequences for binary dynamic and repairable systems

    International Nuclear Information System (INIS)

    Chaux, Pierre-Yves

    2013-01-01

    Preventive risk assessment of a complex system rely on a dynamic models which describe the link between the system failure and the scenarios of failure and repair events from its components. The qualitative analyses of a binary dynamic and repairable system is aiming at computing and analyse the scenarios that lead to the system failure. Since such systems describe a large set of those, only the most representative ones, called Minimal Cut Sequences (MCS), are of interest for the safety engineer. The lack of a formal definition for the MCS has generated multiple definitions either specific to a given model (and thus not generic) or informal. This work proposes i) a formal framework and definition for the MCS while staying independent of the reliability model used, ii) the methodology to compute them using property extracted from their formal definition, iii) an extension of the formal framework for multi-states components in order to perform the qualitative analyses of Boolean logic Driven Markov Processes (BDMP) models. Under the hypothesis that the scenarios implicitly described by any reliability model can always be represented by a finite automaton, this work is defining the coherency for dynamic and repairable systems as the way to give a minimal representation of all scenarios that are leading to the system failure. (author)

  18. Generation of artificial FASTQ files to evaluate the performance of next-generation sequencing pipelines.

    Directory of Open Access Journals (Sweden)

    Matthew Frampton

    Full Text Available Pipelines for the analysis of Next-Generation Sequencing (NGS data are generally composed of a set of different publicly available software, configured together in order to map short reads of a genome and call variants. The fidelity of pipelines is variable. We have developed ArtificialFastqGenerator, which takes a reference genome sequence as input and outputs artificial paired-end FASTQ files containing Phred quality scores. Since these artificial FASTQs are derived from the reference genome, it provides a gold-standard for read-alignment and variant-calling, thereby enabling the performance of any NGS pipeline to be evaluated. The user can customise DNA template/read length, the modelling of coverage based on GC content, whether to use real Phred base quality scores taken from existing FASTQ files, and whether to simulate sequencing errors. Detailed coverage and error summary statistics are outputted. Here we describe ArtificialFastqGenerator and illustrate its implementation in evaluating a typical bespoke NGS analysis pipeline under different experimental conditions. ArtificialFastqGenerator was released in January 2012. Source code, example files and binaries are freely available under the terms of the GNU General Public License v3.0. from https://sourceforge.net/projects/artfastqgen/.

  19. Comparison of a newly developed binary typing with ribotyping and multilocus sequence typing methods for Clostridium difficile.

    Science.gov (United States)

    Li, Zhirong; Liu, Xiaolei; Zhao, Jianhong; Xu, Kaiyue; Tian, Tiantian; Yang, Jing; Qiang, Cuixin; Shi, Dongyan; Wei, Honglian; Sun, Suju; Cui, Qingqing; Li, Ruxin; Niu, Yanan; Huang, Bixing

    2018-04-01

    Clostridium difficile is the causative pathogen for antibiotic-related nosocomial diarrhea. For epidemiological study and identification of virulent clones, a new binary typing method was developed for C. difficile in this study. The usefulness of this newly developed optimized 10-loci binary typing method was compared with two widely used methods ribotyping and multilocus sequence typing (MLST) in 189 C. difficile samples. The binary typing, ribotyping and MLST typed the samples into 53 binary types (BTs), 26 ribotypes (RTs), and 33 MLST sequence types (STs), respectively. The typing ability of the binary method was better than that of either ribotyping or MLST expressed in Simpson Index (SI) at 0.937, 0.892 and 0.859, respectively. The ease of testing, portability and cost-effectiveness of the new binary typing would make it a useful typing alternative for outbreak investigations within healthcare facilities and epidemiological research. Copyright © 2018 Elsevier B.V. All rights reserved.

  20. AK Sco: a tidally induced atmospheric dynamo in a pre-main sequence binary?

    Science.gov (United States)

    Gómez de Castro, A. I.

    2009-02-01

    AK Sco is a unique source: a 10-30 Myrs old pre-main sequence spectroscopic binary composed by two nearly equal F5 stars that at periastron are separated by barely eleven stellar radii so, the stellar magnetospheres fill the Roche lobe at periastron. The orbit is not yet circularized (e = 0.47) and very strong tides are expected. This makes of AK Sco, the ideal laboratory to study the effect of gravitational tides in the stellar magnetic field building up during pre-main sequence evolution. Evidence of this effect is reported in this contribution.

  1. Statistical analysis of next generation sequencing data

    CERN Document Server

    Nettleton, Dan

    2014-01-01

    Next Generation Sequencing (NGS) is the latest high throughput technology to revolutionize genomic research. NGS generates massive genomic datasets that play a key role in the big data phenomenon that surrounds us today. To extract signals from high-dimensional NGS data and make valid statistical inferences and predictions, novel data analytic and statistical techniques are needed. This book contains 20 chapters written by prominent statisticians working with NGS data. The topics range from basic preprocessing and analysis with NGS data to more complex genomic applications such as copy number variation and isoform expression detection. Research statisticians who want to learn about this growing and exciting area will find this book useful. In addition, many chapters from this book could be included in graduate-level classes in statistical bioinformatics for training future biostatisticians who will be expected to deal with genomic data in basic biomedical research, genomic clinical trials and personalized med...

  2. Binary co-generative plants with height temperature SOFC fuel cells

    International Nuclear Information System (INIS)

    Tashevski, D; Dimitrov, K.; Armenski, S.

    2005-01-01

    In this paper, a field of binary co-generative plants with height temperature SOFC fuel cells is presented. Special attention of application of height temperature SOFC fuel cells and binary co-generative units has been given. These units made triple electricity and heat. Principle of combination of fuel cells with binary cycles has been presented. A model and computer programme for calculation of BKPFC, has been created. By using the program, all the important characteristic-results are calculated: power, efficiency, emission, dimension and economic analysis. On base of results, conclusions and recommendations has been given. (Author)

  3. Binary co-generative plants with height temperature SOFC fuel cells

    International Nuclear Information System (INIS)

    Tashevski, D; Dimitrov, K.; Armenski, S.

    2006-01-01

    In this paper, a field of binary co-generative plants with height temperature SOFC fuel cells is presented. Special attention of application of height temperature SOFC fuel cells and binary co-generative units has been given. These units made triple electricity and heat. Principle of combination of fuel cells with binary cycles has been presented. A model and computer programme for calculation of BKPFC, has been created. By using the program, all the important characteristic-results are calculated: power, efficiency, emission, dimension and economic analysis. On base of results, conclusions and recommendations has been given. (Author)

  4. Control of broadband optically generated ultrasound pulses using binary amplitude holograms.

    Science.gov (United States)

    Brown, Michael D; Jaros, Jiri; Cox, Ben T; Treeby, Bradley E

    2016-04-01

    In this work, the use of binary amplitude holography is investigated as a mechanism to focus broadband acoustic pulses generated by high peak-power pulsed lasers. Two algorithms are described for the calculation of the binary holograms; one using ray-tracing, and one using an optimization based on direct binary search. It is shown using numerical simulations that when a binary amplitude hologram is excited by a train of laser pulses at its design frequency, the acoustic field can be focused at a pre-determined distribution of points, including single and multiple focal points, and line and square foci. The numerical results are validated by acoustic field measurements from binary amplitude holograms, excited by a high peak-power laser.

  5. LETTER TO THE EDITOR: Exhaustive search for low-autocorrelation binary sequences

    Science.gov (United States)

    Mertens, S.

    1996-09-01

    Binary sequences with low autocorrelations are important in communication engineering and in statistical mechanics as ground states of the Bernasconi model. Computer searches are the main tool in the construction of such sequences. Owing to the exponential size 0305-4470/29/18/005/img1 of the configuration space, exhaustive searches are limited to short sequences. We discuss an exhaustive search algorithm with run-time characteristic 0305-4470/29/18/005/img2 and apply it to compile a table of exact ground states of the Bernasconi model up to N = 48. The data suggest F > 9 for the optimal merit factor in the limit 0305-4470/29/18/005/img3.

  6. Tablet—next generation sequence assembly visualization

    Science.gov (United States)

    Milne, Iain; Bayer, Micha; Cardle, Linda; Shaw, Paul; Stephen, Gordon; Wright, Frank; Marshall, David

    2010-01-01

    Summary: Tablet is a lightweight, high-performance graphical viewer for next-generation sequence assemblies and alignments. Supporting a range of input assembly formats, Tablet provides high-quality visualizations showing data in packed or stacked views, allowing instant access and navigation to any region of interest, and whole contig overviews and data summaries. Tablet is both multi-core aware and memory efficient, allowing it to handle assemblies containing millions of reads, even on a 32-bit desktop machine. Availability: Tablet is freely available for Microsoft Windows, Apple Mac OS X, Linux and Solaris. Fully bundled installers can be downloaded from http://bioinf.scri.ac.uk/tablet in 32- and 64-bit versions. Contact: tablet@scri.ac.uk PMID:19965881

  7. Fetal Kidney Anomalies: Next Generation Sequencing

    DEFF Research Database (Denmark)

    Rasmussen, Maria; Sunde, Lone; Nielsen, Marlene Louise

    Aim and Introduction Identification of abnormal kidneys in the fetus may lead to termination of the pregnancy and raises questions about the underlying cause and recurrence risk in future pregnancies. In this study, we investigate the effectiveness of targeted next generation sequencing in fetuses...... with prenatally detected kidney anomalies in order to uncover genetic explanations and assess recurrence risk. Also, we aim to study the relation between genetic findings and post mortem kidney histology. Methods The study comprises fetuses diagnosed prenatally with bilateral kidney anomalies that have undergone...... postmortem examination. The approximately 110 genes included in the targeted panel were chosen on the basis of their potential involvement in embryonic kidney development, cystic kidney disease, or the renin-angiotensin system. DNA was extracted from fetal tissue samples or cultured chorion villus cells...

  8. Binary pulsar PSR 1718-19 contains a stripped main-sequence turn-off star

    International Nuclear Information System (INIS)

    Zwitter, T.

    1993-05-01

    Lyne et al. (1993) have recently announced the discovery of a 1-second globular cluster pulsar, 1718-19, in a 6.2-hour binary system which is embedded in a cloud of material originating from the companion star. However the incident flux of the pulsar's radiation on the companion is too low to ablate it and a main sequence companion is too small to fill its Roche lobe. Here I argue that the companion is a stripped turn-off star of 0.2-0.4 solar masses (M sun ) and with approx. 0.1M sun helium core. It has approx. 1.8-times larger radius than a main sequence star of equal mass. Its position in the Hertzsprung-Russell diagram overlaps that of a ∼ 0.65M sun main-sequence star. The evolutionary state of the companion and the highly magnetized slowly rotating neutron star place the system on the verge of the low mass X-ray binary phase. (author). 19 refs, 2 figs

  9. Cardiorespiratory Kinetics Determined by Pseudo-Random Binary Sequences - Comparisons between Walking and Cycling.

    Science.gov (United States)

    Koschate, J; Drescher, U; Thieschäfer, L; Heine, O; Baum, K; Hoffmann, U

    2016-12-01

    This study aims to compare cardiorespiratory kinetics as a response to a standardised work rate protocol with pseudo-random binary sequences between cycling and walking in young healthy subjects. Muscular and pulmonary oxygen uptake (V̇O 2 ) kinetics as well as heart rate kinetics were expected to be similar for walking and cycling. Cardiac data and V̇O 2 of 23 healthy young subjects were measured in response to pseudo-random binary sequences. Kinetics were assessed applying time series analysis. Higher maxima of cross-correlation functions between work rate and the respective parameter indicate faster kinetics responses. Muscular V̇O 2 kinetics were estimated from heart rate and pulmonary V̇O 2 using a circulatory model. Muscular (walking vs. cycling [mean±SD in arbitrary units]: 0.40±0.08 vs. 0.41±0.08) and pulmonary V̇O 2 kinetics (0.35±0.06 vs. 0.35±0.06) were not different, although the time courses of the cross-correlation functions of pulmonary V̇O 2 showed unexpected biphasic responses. Heart rate kinetics (0.50±0.14 vs. 0.40±0.14; P=0.017) was faster for walking. Regarding the biphasic cross-correlation functions of pulmonary V̇O 2 during walking, the assessment of muscular V̇O 2 kinetics via pseudo-random binary sequences requires a circulatory model to account for cardio-dynamic distortions. Faster heart rate kinetics for walking should be considered by comparing results from cycle and treadmill ergometry. © Georg Thieme Verlag KG Stuttgart · New York.

  10. Generation of pseudo-random sequences for spread spectrum systems

    Science.gov (United States)

    Moser, R.; Stover, J.

    1985-05-01

    The characteristics of pseudo random radio signal sequences (PRS) are explored. The randomness of the PSR is a matter of artificially altering the sequence of binary digits broadcast. Autocorrelations of the two sequences shifted in time, if high, determine if the signals are the same and thus allow for position identification. Cross-correlation can also be calculated between sequences. Correlations closest to zero are obtained with large volume of prime numbers in the sequences. Techniques for selecting optimal and maximal lengths for the sequences are reviewed. If the correlations are near zero in the sequences, then signal channels can accommodate multiple users. Finally, Gold codes are discussed as a technique for maximizing the code lengths.

  11. Generation of binary holograms for deep scenes captured with a camera and a depth sensor

    Science.gov (United States)

    Leportier, Thibault; Park, Min-Chul

    2017-01-01

    This work presents binary hologram generation from images of a real object acquired from a Kinect sensor. Since hologram calculation from a point-cloud or polygon model presents a heavy computational burden, we adopted a depth-layer approach to generate the holograms. This method enables us to obtain holographic data of large scenes quickly. Our investigations focus on the performance of different methods, iterative and noniterative, to convert complex holograms into binary format. Comparisons were performed to examine the reconstruction of the binary holograms at different depths. We also propose to modify the direct binary search algorithm to take into account several reference image planes. Then, deep scenes featuring multiple planes of interest can be reconstructed with better efficiency.

  12. Sequence selection by dynamical symmetry breaking in an autocatalytic binary polymer model

    DEFF Research Database (Denmark)

    Fellermann, Harold; Tanaka, Shinpei; Rasmussen, Steen

    2017-01-01

    Template-directed replication of nucleic acids is at the essence of all living beings and a major milestone for any origin of life scenario. We present an idealized model of prebiotic sequence replication, where binary polymers act as templates for their autocatalytic replication, thereby serving...... as each others reactants and products in an intertwined molecular ecology. Our model demonstrates how autocatalysis alters the qualitative and quantitative system dynamics in counterintuitive ways. Most notably, numerical simulations reveal a very strong intrinsic selection mechanism that favors...... the appearance of a few population structures with highly ordered and repetitive sequence patterns when starting from a pool of monomers. We demonstrate both analytically and through simulation how this "selection of the dullest" is caused by continued symmetry breaking through random fluctuations...

  13. Next Generation Sequencing of Tubal Intraepithelial Carcinomas

    Science.gov (United States)

    McDaniel, Andrew S.; Stall, Jennifer N.; Hovelson, Daniel H.; Cani, Andi K.; Liu, Chia-Jen; Tomlins, Scott A.; Cho, Kathleen R.

    2016-01-01

    Importance High-grade serous carcinoma (HGSC) is the most prevalent and lethal form of ovarian cancer. HGSCs frequently arise in the distal fallopian tubes rather than the ovary, developing from small precursor lesions called serous tubal intraepithelial carcinomas (TICs or more specifically STICs). While STICs have been reported to harbor TP53 mutations, detailed molecular characterizations of these lesions are lacking. Observations We performed targeted next generation sequencing (NGS) on formalin-fixed, paraffin- embedded tissue from four women, two with HGSC and two with uterine endometrioid carcinoma (UEC) who were diagnosed with synchronous STICs. We detected concordant mutations in both HGSCs with synchronous STICs, including TP53 mutations as well as assumed germline BRCA1/2 alterations, confirming a clonal relationship between these lesions. NGS confirmed the presence of a STIC clonally unrelated to one case of UEC. NGS of the other tubal lesion diagnosed as a STIC unexpectedly supported the lesion as a micrometastasis from the associated UEC. Conclusions and Relevance We demonstrate that targeted NGS can identify genetic lesions in minute lesions such as TICs, and confirm TP53 mutations as early driving events for HGSC. NGS also demonstrated unexpected relationships between presumed STICs and synchronous carcinomas, suggesting potential diagnostic and translational research applications. PMID:26181193

  14. Modeling long correlation times using additive binary Markov chains: Applications to wind generation time series.

    Science.gov (United States)

    Weber, Juliane; Zachow, Christopher; Witthaut, Dirk

    2018-03-01

    Wind power generation exhibits a strong temporal variability, which is crucial for system integration in highly renewable power systems. Different methods exist to simulate wind power generation but they often cannot represent the crucial temporal fluctuations properly. We apply the concept of additive binary Markov chains to model a wind generation time series consisting of two states: periods of high and low wind generation. The only input parameter for this model is the empirical autocorrelation function. The two-state model is readily extended to stochastically reproduce the actual generation per period. To evaluate the additive binary Markov chain method, we introduce a coarse model of the electric power system to derive backup and storage needs. We find that the temporal correlations of wind power generation, the backup need as a function of the storage capacity, and the resting time distribution of high and low wind events for different shares of wind generation can be reconstructed.

  15. Modeling long correlation times using additive binary Markov chains: Applications to wind generation time series

    Science.gov (United States)

    Weber, Juliane; Zachow, Christopher; Witthaut, Dirk

    2018-03-01

    Wind power generation exhibits a strong temporal variability, which is crucial for system integration in highly renewable power systems. Different methods exist to simulate wind power generation but they often cannot represent the crucial temporal fluctuations properly. We apply the concept of additive binary Markov chains to model a wind generation time series consisting of two states: periods of high and low wind generation. The only input parameter for this model is the empirical autocorrelation function. The two-state model is readily extended to stochastically reproduce the actual generation per period. To evaluate the additive binary Markov chain method, we introduce a coarse model of the electric power system to derive backup and storage needs. We find that the temporal correlations of wind power generation, the backup need as a function of the storage capacity, and the resting time distribution of high and low wind events for different shares of wind generation can be reconstructed.

  16. Generation of Binary Off-axis Digital Fresnel Hologram with Enhanced Quality

    Directory of Open Access Journals (Sweden)

    Peter Wai Ming Tsang

    2015-06-01

    Full Text Available The emergence of high resolution printer and digital micromirror device (DMD has enabled real, off-axis holograms to be printed, or projected onto a screen. As most printers and DMD can only reproduce binary dots, the pixels in a hologram have to be truncated to 2 levels. However, direct binarizing a hologram will lead to severe degradation on its reconstructed image. In this paper, a method for generating binary off-axis digital Fresnel hologram is reported. A hologram generated with the proposed method is referred to as the "Enhanced Sampled Binary Hologram" (ESBH. The reconstructed image of the ESBH is superior in visual quality as compare with the one obtained with existing technique, and also resistant to noise contamination.

  17. BIG - a binary generator for testing digital devices

    International Nuclear Information System (INIS)

    Annuziata, M.; Sechi, G.

    1988-01-01

    This paper presents an unusual approach to the testing of digital devices. In order to test real time systems, we have designed, built, and used a digital generator able to send 2047 quadruples of 12-bit words, with a frequency range of up to 0.7 MHz. (orig.)

  18. Entropy of finite random binary sequences with weak long-range correlations.

    Science.gov (United States)

    Melnik, S S; Usatenko, O V

    2014-11-01

    We study the N-step binary stationary ergodic Markov chain and analyze its differential entropy. Supposing that the correlations are weak we express the conditional probability function of the chain through the pair correlation function and represent the entropy as a functional of the pair correlator. Since the model uses the two-point correlators instead of the block probability, it makes it possible to calculate the entropy of strings at much longer distances than using standard methods. A fluctuation contribution to the entropy due to finiteness of random chains is examined. This contribution can be of the same order as its regular part even at the relatively short lengths of subsequences. A self-similar structure of entropy with respect to the decimation transformations is revealed for some specific forms of the pair correlation function. Application of the theory to the DNA sequence of the R3 chromosome of Drosophila melanogaster is presented.

  19. On central ideals of finitely generated binary (-1,1)-algebras

    International Nuclear Information System (INIS)

    Pchelintsev, S V

    2002-01-01

    In 1975 the author proved that the centre of a free finitely generated (-1,1)-algebra contains a non-zero ideal of the whole algebra. Filippov proved that in a free alternative algebra of rank ≥4 there exists a trivial ideal contained in the associative centre. Il'tyakov established that the associative nucleus of a free alternative algebra of rank 3 coincides with the ideal of identities of the Cayley-Dickson algebra. In the present paper the above-mentioned theorem of the author is extended to free finitely generated binary (-1,1)-algebras. Theorem. The centre of a free finitely generated binary (-1,1)-algebra of rank ≥3 over a field of characteristic distinct from 2 and 3 contains a non-zero ideal of the whole algebra. As a by-product, we shall prove that the T-ideal generated by the function (z,x,(x,x,y)) in a free binary (-1,1)-algebra of finite rank is soluble. We deduce from this that the basis rank of the variety of binary (-1,1)-algebras is infinite

  20. The contribution of next generation sequencing to epilepsy genetics

    DEFF Research Database (Denmark)

    Møller, Rikke S.; Dahl, Hans A.; Helbig, Ingo

    2015-01-01

    During the last decade, next generation sequencing technologies such as targeted gene panels, whole exome sequencing and whole genome sequencing have led to an explosion of gene identifications in monogenic epilepsies including both familial epilepsies and severe epilepsies, often referred to as ...

  1. HLA typing: Conventional techniques v.next-generation sequencing

    African Journals Online (AJOL)

    The existing techniques have contributed significantly to our current knowledge of allelic diversity. At present, sequence-based typing (SBT) methods, in particular next-generation sequencing. (NGS), provide the highest possible resolution. NGS platforms were initially only used for genomic sequencing, but also showed.

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

    Science.gov (United States)

    Yang, Ye; Liu, Juan

    2015-01-01

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

  3. Automated Testing with Targeted Event Sequence Generation

    DEFF Research Database (Denmark)

    Jensen, Casper Svenning; Prasad, Mukul R.; Møller, Anders

    2013-01-01

    Automated software testing aims to detect errors by producing test inputs that cover as much of the application source code as possible. Applications for mobile devices are typically event-driven, which raises the challenge of automatically producing event sequences that result in high coverage...

  4. Image encryption using random sequence generated from generalized information domain

    International Nuclear Information System (INIS)

    Zhang Xia-Yan; Wu Jie-Hua; Zhang Guo-Ji; Li Xuan; Ren Ya-Zhou

    2016-01-01

    A novel image encryption method based on the random sequence generated from the generalized information domain and permutation–diffusion architecture is proposed. The random sequence is generated by reconstruction from the generalized information file and discrete trajectory extraction from the data stream. The trajectory address sequence is used to generate a P-box to shuffle the plain image while random sequences are treated as keystreams. A new factor called drift factor is employed to accelerate and enhance the performance of the random sequence generator. An initial value is introduced to make the encryption method an approximately one-time pad. Experimental results show that the random sequences pass the NIST statistical test with a high ratio and extensive analysis demonstrates that the new encryption scheme has superior security. (paper)

  5. Next-Generation Sequencing in the Mycology Lab.

    Science.gov (United States)

    Zoll, Jan; Snelders, Eveline; Verweij, Paul E; Melchers, Willem J G

    New state-of-the-art techniques in sequencing offer valuable tools in both detection of mycobiota and in understanding of the molecular mechanisms of resistance against antifungal compounds and virulence. Introduction of new sequencing platform with enhanced capacity and a reduction in costs for sequence analysis provides a potential powerful tool in mycological diagnosis and research. In this review, we summarize the applications of next-generation sequencing techniques in mycology.

  6. ABSOLUTE PROPERTIES OF THE PRE-MAIN-SEQUENCE ECLIPSING BINARY STAR NP PERSEI

    Energy Technology Data Exchange (ETDEWEB)

    Lacy, Claud H. Sandberg [Physics Department, University of Arkansas, Fayetteville, AR 72701 (United States); Fekel, Francis C.; Muterspaugh, Matthew W. [Center of Excellence in Information Systems, Tennessee State University, Nashville, TN 37209 (United States); Pavlovski, Krešimir [Department of Physics, Faculty of Science, University of Zagreb, Bijenička cesta 32, 10000 Zagreb (Croatia); Torres, Guillermo, E-mail: clacy@uark.edu, E-mail: fekel@evans.tsuniv.edu, E-mail: pavlovski@phy.hr, E-mail: gtorres@cfa.harvard.edu, E-mail: matthew1@coe.tsuniv.edu [Harvard-Smithsonian Center for Astrophysics, 60 Garden Street, Cambridge, MA 02138 (United States)

    2016-07-01

    NP Per is a well-detached, 2.2 day eclipsing binary whose components are both pre-main-sequence stars that are still contracting toward the main-sequence phase of evolution. We report extensive photometric and spectroscopic observations with which we have determined their properties accurately. Their surface temperatures are quite different: 6420 ± 90 K for the larger F5 primary star and 4540 ± 160 K for the smaller K5e star. Their masses and radii are 1.3207 ± 0.0087 solar masses and 1.372 ± 0.013 solar radii for the primary, and 1.0456 ± 0.0046 solar masses and 1.229 ± 0.013 solar radii for the secondary. The orbital period is variable over long periods of time. A comparison of the observations with current stellar evolution models from MESA indicates that the stars cannot be fit at a single age: the secondary appears significantly younger than the primary. If the stars are assumed to be coeval and to have the age of the primary (17 Myr), then the secondary is larger and cooler than predicted by current models. The H α spectral line of the secondary component is completely filled by, presumably, chromospheric emission due to a magnetic activity cycle.

  7. Droplet Digital™ PCR Next-Generation Sequencing Library QC Assay.

    Science.gov (United States)

    Heredia, Nicholas J

    2018-01-01

    Digital PCR is a valuable tool to quantify next-generation sequencing (NGS) libraries precisely and accurately. Accurately quantifying NGS libraries enable accurate loading of the libraries on to the sequencer and thus improve sequencing performance by reducing under and overloading error. Accurate quantification also benefits users by enabling uniform loading of indexed/barcoded libraries which in turn greatly improves sequencing uniformity of the indexed/barcoded samples. The advantages gained by employing the Droplet Digital PCR (ddPCR™) library QC assay includes the precise and accurate quantification in addition to size quality assessment, enabling users to QC their sequencing libraries with confidence.

  8. Comparison of next generation sequencing technologies for transcriptome characterization

    Directory of Open Access Journals (Sweden)

    Soltis Douglas E

    2009-08-01

    Full Text Available Abstract Background We have developed a simulation approach to help determine the optimal mixture of sequencing methods for most complete and cost effective transcriptome sequencing. We compared simulation results for traditional capillary sequencing with "Next Generation" (NG ultra high-throughput technologies. The simulation model was parameterized using mappings of 130,000 cDNA sequence reads to the Arabidopsis genome (NCBI Accession SRA008180.19. We also generated 454-GS20 sequences and de novo assemblies for the basal eudicot California poppy (Eschscholzia californica and the magnoliid avocado (Persea americana using a variety of methods for cDNA synthesis. Results The Arabidopsis reads tagged more than 15,000 genes, including new splice variants and extended UTR regions. Of the total 134,791 reads (13.8 MB, 119,518 (88.7% mapped exactly to known exons, while 1,117 (0.8% mapped to introns, 11,524 (8.6% spanned annotated intron/exon boundaries, and 3,066 (2.3% extended beyond the end of annotated UTRs. Sequence-based inference of relative gene expression levels correlated significantly with microarray data. As expected, NG sequencing of normalized libraries tagged more genes than non-normalized libraries, although non-normalized libraries yielded more full-length cDNA sequences. The Arabidopsis data were used to simulate additional rounds of NG and traditional EST sequencing, and various combinations of each. Our simulations suggest a combination of FLX and Solexa sequencing for optimal transcriptome coverage at modest cost. We have also developed ESTcalc http://fgp.huck.psu.edu/NG_Sims/ngsim.pl, an online webtool, which allows users to explore the results of this study by specifying individualized costs and sequencing characteristics. Conclusion NG sequencing technologies are a highly flexible set of platforms that can be scaled to suit different project goals. In terms of sequence coverage alone, the NG sequencing is a dramatic advance

  9. Zseq: An Approach for Preprocessing Next-Generation Sequencing Data.

    Science.gov (United States)

    Alkhateeb, Abedalrhman; Rueda, Luis

    2017-08-01

    Next-generation sequencing technology generates a huge number of reads (short sequences), which contain a vast amount of genomic data. The sequencing process, however, comes with artifacts. Preprocessing of sequences is mandatory for further downstream analysis. We present Zseq, a linear method that identifies the most informative genomic sequences and reduces the number of biased sequences, sequence duplications, and ambiguous nucleotides. Zseq finds the complexity of the sequences by counting the number of unique k-mers in each sequence as its corresponding score and also takes into the account other factors such as ambiguous nucleotides or high GC-content percentage in k-mers. Based on a z-score threshold, Zseq sweeps through the sequences again and filters those with a z-score less than the user-defined threshold. Zseq algorithm is able to provide a better mapping rate; it reduces the number of ambiguous bases significantly in comparison with other methods. Evaluation of the filtered reads has been conducted by aligning the reads and assembling the transcripts using the reference genome as well as de novo assembly. The assembled transcripts show a better discriminative ability to separate cancer and normal samples in comparison with another state-of-the-art method. Moreover, de novo assembled transcripts from the reads filtered by Zseq have longer genomic sequences than other tested methods. Estimating the threshold of the cutoff point is introduced using labeling rules with optimistic results.

  10. Next Generation Sequencing of Ancient DNA: Requirements, Strategies and Perspectives

    Directory of Open Access Journals (Sweden)

    Michael Knapp

    2010-07-01

    Full Text Available The invention of next-generation-sequencing has revolutionized almost all fields of genetics, but few have profited from it as much as the field of ancient DNA research. From its beginnings as an interesting but rather marginal discipline, ancient DNA research is now on its way into the centre of evolutionary biology. In less than a year from its invention next-generation-sequencing had increased the amount of DNA sequence data available from extinct organisms by several orders of magnitude. Ancient DNA  research is now not only adding a temporal aspect to evolutionary studies and allowing for the observation of evolution in real time, it also provides important data to help understand the origins of our own species. Here we review progress that has been made in next-generation-sequencing of ancient DNA over the past five years and evaluate sequencing strategies and future directions.

  11. Applying Next Generation Sequencing to Skeletal Development and Disease

    OpenAIRE

    Bowen, Margot Elizabeth

    2013-01-01

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

  12. SMITH: a LIMS for handling next-generation sequencing workflows

    OpenAIRE

    Venco, Francesco; Vaskin, Yuriy; Ceol, Arnaud; Muller, Heiko

    2014-01-01

    Background Life-science laboratories make increasing use of Next Generation Sequencing (NGS) for studying bio-macromolecules and their interactions. Array-based methods for measuring gene expression or protein-DNA interactions are being replaced by RNA-Seq and ChIP-Seq. Sequencing is generally performed by specialized facilities that have to keep track of sequencing requests, trace samples, ensure quality and make data available according to predefined privileges. An integrated tool helps to ...

  13. Chaotic generation of PN sequences : a VLSI implementation

    NARCIS (Netherlands)

    Dornbusch, A.; Pineda de Gyvez, J.

    1999-01-01

    Generation of repeatable pseudo-random sequences with chaotic analog electronics is not feasible using standard circuit topologies. Component variation caused by imperfect fabrication causes the same divergence of output sequences as does varying initial conditions. By quantizing the output of a

  14. Comparison of two Next Generation sequencing platforms for full genome sequencing of Classical Swine Fever Virus

    DEFF Research Database (Denmark)

    Fahnøe, Ulrik; Pedersen, Anders Gorm; Höper, Dirk

    2013-01-01

    to the consensus sequence. Additionally, we got an average sequence depth for the genome of 4000 for the Iontorrent PGM and 400 for the FLX platform making the mapping suitable for single nucleotide variant (SNV) detection. The analysis revealed a single non-silent SNV A10665G leading to the amino acid change D......Next Generation Sequencing (NGS) is becoming more adopted into viral research and will be the preferred technology in the years to come. We have recently sequenced several strains of Classical Swine Fever Virus (CSFV) by NGS on both Genome Sequencer FLX (GS FLX) and Iontorrent PGM platforms...

  15. Arbitrary digital pulse sequence generator with delay-loop timing

    Science.gov (United States)

    Hošák, Radim; Ježek, Miroslav

    2018-04-01

    We propose an idea of an electronic multi-channel arbitrary digital sequence generator with temporal granularity equal to two clock cycles. We implement the generator with 32 channels using a low-cost ARM microcontroller and demonstrate its capability to produce temporal delays ranging from tens of nanoseconds to hundreds of seconds, with 24 ns timing granularity and linear scaling of delay with respect to the number of delay loop iterations. The generator is optionally synchronized with an external clock source to provide 100 ps jitter and overall sequence repeatability within the whole temporal range. The generator is fully programmable and able to produce digital sequences of high complexity. The concept of the generator can be implemented using different microcontrollers and applied for controlling of various optical, atomic, and nuclear physics measurement setups.

  16. BetaBit: A fast generator of autocorrelated binary processes for geophysical research

    Science.gov (United States)

    Serinaldi, Francesco; Lombardo, Federico

    2017-05-01

    We introduce a fast and efficient non-iterative algorithm, called BetaBit, to simulate autocorrelated binary processes describing the occurrence of natural hazards, system failures, and other physical and geophysical phenomena characterized by persistence, temporal clustering, and low rate of occurrence. BetaBit overcomes the simulation constraints posed by the discrete nature of the marginal distributions of binary processes by using the link existing between the correlation coefficients of this process and those of the standard Gaussian processes. The performance of BetaBit is tested on binary signals with power-law and exponentially decaying autocorrelation functions (ACFs) corresponding to Hurst-Kolmogorov and Markov processes, respectively. An application to real-world sequences describing rainfall intermittency and the occurrence of strong positive phases of the North Atlantic Oscillation (NAO) index shows that BetaBit can also simulate surrogate data preserving the empirical ACF as well as signals with autoregressive moving average (ARMA) dependence structures. Extensions to cyclo-stationary processes accounting for seasonal fluctuations are also discussed.

  17. What can next generation sequencing do for you? Next generation sequencing as a valuable tool in plant research

    OpenAIRE

    Bräutigam, Andrea; Gowik, Udo

    2010-01-01

    Next generation sequencing (NGS) technologies have opened fascinating opportunities for the analysis of plants with and without a sequenced genome on a genomic scale. During the last few years, NGS methods have become widely available and cost effective. They can be applied to a wide variety of biological questions, from the sequencing of complete eukaryotic genomes and transcriptomes, to the genome-scale analysis of DNA-protein interactions. In this review, we focus on the use of NGS for pla...

  18. Generation of equal-intensity coherent optical beams by binary geometrical phase on metasurface

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Zheng-Han; Jiang, Shang-Chi; Xiong, Xiang; Peng, Ru-Wen, E-mail: rwpeng@nju.edu.cn, E-mail: muwang@nju.edu.cn; Wang, Mu, E-mail: rwpeng@nju.edu.cn, E-mail: muwang@nju.edu.cn [National Laboratory of Solid State Microstructures and School of Physics, and Collaborative Innovation Center of Advanced Microstructures, Nanjing University, Nanjing 210093 (China)

    2016-06-27

    We report here the design and realization of a broadband, equal-intensity optical beam splitter with a dispersion-free binary geometric phase on a metasurface with unit cell consisting of two mirror-symmetric elements. We demonstrate experimentally that two identical beams can be efficiently generated with incidence of any polarization. The efficiency of the device reaches 80% at 1120 nm and keeps larger than 70% in the range of 1000–1400 nm. We suggest that this approach for generating identical, coherent beams have wide applications in diffraction optics and in entangled photon light source for quantum communication.

  19. YSOVAR: SIX PRE-MAIN-SEQUENCE ECLIPSING BINARIES IN THE ORION NEBULA CLUSTER

    Energy Technology Data Exchange (ETDEWEB)

    Morales-Calderon, M.; Stauffer, J. R.; Rebull, L. M. [Spitzer Science Center, California Institute of Technology, 1200 E California Blvd., Pasadena, CA 91125 (United States); Stassun, K. G. [Physics and Astronomy Department, Vanderbilt University, 1807 Station B, Nashville, TN 37235 (United States); Vrba, F. J. [U. S. Naval Observatory, Flagstaff Station, 10391 W. Naval Observatory Road, Flagstaff, AZ 86001-8521 (United States); Prato, L. [Lowell Observatory, 1400 West Mars Hill Road, Flagstaff, AZ 86001 (United States); Hillenbrand, L. A.; Carpenter, J. M. [Astronomy Department, California Institute of Technology, 1200 E California Blvd., Pasadena, CA 91125 (United States); Terebey, S.; Angione, J. [Department of Physics and Astronomy, California State University at Los Angeles, Los Angeles, CA 90032 (United States); Covey, K. R. [Department of Astronomy, Cornell University, 226 Space Sciences Building, Ithaca, NY 14853 (United States); Terndrup, D. M. [Department of Astronomy, The Ohio State University, 140 West 18th Avenue, Columbus, OH 43210 (United States); Gutermuth, R. [Department of Astronomy, University of Massachusetts, Amherst, MA 01003 (United States); Song, I. [Physics and Astronomy Department, University of Georgia, Athens, GA 30602-2451 (United States); Plavchan, P. [NASA Exoplanet Science Institute, California Institute of Technology, Pasadena, CA 91125 (United States); Marchis, F. [SETI Institute, Carl Sagan Center, 189 N San Bernado Av, Mountain View, CA 94043 (United States); Garcia, E. V. [Department of Physics, Fisk University, 1000 17th Ave. N, Nashville, TN 37208 (United States); Margheim, S. [Gemini Observatory, Southern Operations Center, Casilla 603, La Serena (Chile); Luhman, K. L. [Department of Astronomy and Astrophysics, The Pennsylvania State University, University Park, PA 16802 (United States); Irwin, J. M., E-mail: mariamc@cab.inta-csic.es [Harvard-Smithsonian Center for Astrophysics, 60 Garden St., Cambridge, MA 02138 (United States)

    2012-07-10

    Eclipsing binaries (EBs) provide critical laboratories for empirically testing predictions of theoretical models of stellar structure and evolution. Pre-main-sequence (PMS) EBs are particularly valuable, both due to their rarity and the highly dynamic nature of PMS evolution, such that a dense grid of PMS EBs is required to properly calibrate theoretical PMS models. Analyzing multi-epoch, multi-color light curves for {approx}2400 candidate Orion Nebula Cluster (ONC) members from our Warm Spitzer Exploration Science Program YSOVAR, we have identified 12 stars whose light curves show eclipse features. Four of these 12 EBs are previously known. Supplementing our light curves with follow-up optical and near-infrared spectroscopy, we establish two of the candidates as likely field EBs lying behind the ONC. We confirm the remaining six candidate systems, however, as newly identified ONC PMS EBs. These systems increase the number of known PMS EBs by over 50% and include the highest mass ({theta}{sup 1} Ori E, for which we provide a complete set of well-determined parameters including component masses of 2.807 and 2.797 M{sub Sun }) and longest-period (ISOY J053505.71-052354.1, P {approx} 20 days) PMS EBs currently known. In two cases ({theta}{sup 1} Ori E and ISOY J053526.88-044730.7), enough photometric and spectroscopic data exist to attempt an orbit solution and derive the system parameters. For the remaining systems, we combine our data with literature information to provide a preliminary characterization sufficient to guide follow-up investigations of these rare, benchmark systems.

  20. YSOVAR: SIX PRE-MAIN-SEQUENCE ECLIPSING BINARIES IN THE ORION NEBULA CLUSTER

    International Nuclear Information System (INIS)

    Morales-Calderón, M.; Stauffer, J. R.; Rebull, L. M.; Stassun, K. G.; Vrba, F. J.; Prato, L.; Hillenbrand, L. A.; Carpenter, J. M.; Terebey, S.; Angione, J.; Covey, K. R.; Terndrup, D. M.; Gutermuth, R.; Song, I.; Plavchan, P.; Marchis, F.; García, E. V.; Margheim, S.; Luhman, K. L.; Irwin, J. M.

    2012-01-01

    Eclipsing binaries (EBs) provide critical laboratories for empirically testing predictions of theoretical models of stellar structure and evolution. Pre-main-sequence (PMS) EBs are particularly valuable, both due to their rarity and the highly dynamic nature of PMS evolution, such that a dense grid of PMS EBs is required to properly calibrate theoretical PMS models. Analyzing multi-epoch, multi-color light curves for ∼2400 candidate Orion Nebula Cluster (ONC) members from our Warm Spitzer Exploration Science Program YSOVAR, we have identified 12 stars whose light curves show eclipse features. Four of these 12 EBs are previously known. Supplementing our light curves with follow-up optical and near-infrared spectroscopy, we establish two of the candidates as likely field EBs lying behind the ONC. We confirm the remaining six candidate systems, however, as newly identified ONC PMS EBs. These systems increase the number of known PMS EBs by over 50% and include the highest mass (θ 1 Ori E, for which we provide a complete set of well-determined parameters including component masses of 2.807 and 2.797 M ☉ ) and longest-period (ISOY J053505.71–052354.1, P ∼ 20 days) PMS EBs currently known. In two cases (θ 1 Ori E and ISOY J053526.88–044730.7), enough photometric and spectroscopic data exist to attempt an orbit solution and derive the system parameters. For the remaining systems, we combine our data with literature information to provide a preliminary characterization sufficient to guide follow-up investigations of these rare, benchmark systems.

  1. Neural Sequence Generation Using Spatiotemporal Patterns of Inhibition.

    Directory of Open Access Journals (Sweden)

    Jonathan Cannon

    2015-11-01

    Full Text Available Stereotyped sequences of neural activity are thought to underlie reproducible behaviors and cognitive processes ranging from memory recall to arm movement. One of the most prominent theoretical models of neural sequence generation is the synfire chain, in which pulses of synchronized spiking activity propagate robustly along a chain of cells connected by highly redundant feedforward excitation. But recent experimental observations in the avian song production pathway during song generation have shown excitatory activity interacting strongly with the firing patterns of inhibitory neurons, suggesting a process of sequence generation more complex than feedforward excitation. Here we propose a model of sequence generation inspired by these observations in which a pulse travels along a spatially recurrent excitatory chain, passing repeatedly through zones of local feedback inhibition. In this model, synchrony and robust timing are maintained not through redundant excitatory connections, but rather through the interaction between the pulse and the spatiotemporal pattern of inhibition that it creates as it circulates the network. These results suggest that spatially and temporally structured inhibition may play a key role in sequence generation.

  2. Neural Sequence Generation Using Spatiotemporal Patterns of Inhibition.

    Science.gov (United States)

    Cannon, Jonathan; Kopell, Nancy; Gardner, Timothy; Markowitz, Jeffrey

    2015-11-01

    Stereotyped sequences of neural activity are thought to underlie reproducible behaviors and cognitive processes ranging from memory recall to arm movement. One of the most prominent theoretical models of neural sequence generation is the synfire chain, in which pulses of synchronized spiking activity propagate robustly along a chain of cells connected by highly redundant feedforward excitation. But recent experimental observations in the avian song production pathway during song generation have shown excitatory activity interacting strongly with the firing patterns of inhibitory neurons, suggesting a process of sequence generation more complex than feedforward excitation. Here we propose a model of sequence generation inspired by these observations in which a pulse travels along a spatially recurrent excitatory chain, passing repeatedly through zones of local feedback inhibition. In this model, synchrony and robust timing are maintained not through redundant excitatory connections, but rather through the interaction between the pulse and the spatiotemporal pattern of inhibition that it creates as it circulates the network. These results suggest that spatially and temporally structured inhibition may play a key role in sequence generation.

  3. A study of single and binary ion plasma expansion into laboratory-generated plasma wakes

    Science.gov (United States)

    Wright, Kenneth Herbert, Jr.

    1988-01-01

    Plasma expansion into the wake of a large rectangular plate immersed in a collisionless, supersonic plasma was investigated in laboratory experiments. The experimental conditions address both single ion and binary ion plasma flows for the case of a body whose size is large in comparison with the Debye length, when the potential difference between the body and the plasma is relatively small. A new plasma source was developed to generate equi-velocity, binary ion plasma flows, which allows access to new parameter space that have previously been unavailable for laboratory studies. Specifically, the new parameters are the ionic mass ratio and the ionic component density ratio. In a series of experiments, a krypton-neon plasma is employed where the ambient density ratio of neon to krypton is varied more than an order of magnitude. The expansion in both the single ion and binary ion plasma cases is limited to early times, i.e., a few ion plasma periods, by the combination of plasma density, plasma drift speed, and vacuum chamber size, which prevented detailed comparison with self-similar theory.

  4. Study of single and binary ion plasma expansion into laboratory-generated plasma wakes

    International Nuclear Information System (INIS)

    Wright, K.H. Jr.

    1988-02-01

    Plasma expansion into the wake of a large rectangular plate immersed in a collisionless, supersonic plasma was investigated in laboratory experiments. The experimental conditions address both single ion and binary ion plasma flows for the case of a body whose size is large in comparison with the Debye length, when the potential difference between the body and the plasma is relatively small. A new plasma source was developed to generate equi-velocity, binary ion plasma flows, which allows access to new parameter space that have previously been unavailable for laboratory studies. Specifically, the new parameters are the ionic mass ratio and the ionic component density ratio. In a series of experiments, a krypton-neon plasma is employed where the ambient density ratio of neon to krypton is varied more than an order of magnitude. The expansion in both the single ion and binary ion plasma cases is limited to early times, i.e., a few ion plasma periods, by the combination of plasma density, plasma drift speed, and vacuum chamber size, which prevented detailed comparison with self-similar theory

  5. Veldkamp-space aspects of a sequence of nested binary Segre varieties

    Czech Academy of Sciences Publication Activity Database

    Saniga, M.; Havlicek, H.; Holweck, F.; Planat, M.; Pracna, Petr

    2015-01-01

    Roč. 2, č. 3 (2015), s. 309-333 ISSN 2308-5827 Institutional support: RVO:61388955 Keywords : binary Segre varietes * Veldkamp spaces * hyperbolic quadrics Subject RIV: CF - Physical ; Theoretical Chemistry

  6. 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

  7. Application of Next-generation Sequencing in Clinical Molecular Diagnostics

    Directory of Open Access Journals (Sweden)

    Morteza Seifi

    2017-05-01

    Full Text Available ABSTRACT Next-generation sequencing (NGS is the catch all terms that used to explain several different modern sequencing technologies which let us to sequence nucleic acids much more rapidly and cheaply than the formerly used Sanger sequencing, and as such have revolutionized the study of molecular biology and genomics with excellent resolution and accuracy. Over the past years, many academic companies and institutions have continued technological advances to expand NGS applications from research to the clinic. In this review, the performance and technical features of current NGS platforms were described. Furthermore, advances in the applying of NGS technologies towards the progress of clinical molecular diagnostics were emphasized. General advantages and disadvantages of each sequencing system are summarized and compared to guide the selection of NGS platforms for specific research aims.

  8. Quantifying population genetic differentiation from next-generation sequencing data

    DEFF Research Database (Denmark)

    Fumagalli, Matteo; Garrett Vieira, Filipe Jorge; Korneliussen, Thorfinn Sand

    2013-01-01

    method for quantifying population genetic differentiation from next-generation sequencing data. In addition, we present a strategy to investigate population structure via Principal Components Analysis. Through extensive simulations, we compare the new method herein proposed to approaches based...... on genotype calling and demonstrate a marked improvement in estimation accuracy for a wide range of conditions. We apply the method to a large-scale genomic data set of domesticated and wild silkworms sequenced at low coverage. We find that we can infer the fine-scale genetic structure of the sampled......Over the last few years, new high-throughput DNA sequencing technologies have dramatically increased speed and reduced sequencing costs. However, the use of these sequencing technologies is often challenged by errors and biases associated with the bioinformatical methods used for analyzing the data...

  9. Next-Generation Sequencing: From Understanding Biology to Personalized Medicine

    Directory of Open Access Journals (Sweden)

    Benjamin Meder

    2013-03-01

    Full Text Available Within just a few years, the new methods for high-throughput next-generation sequencing have generated completely novel insights into the heritability and pathophysiology of human disease. In this review, we wish to highlight the benefits of the current state-of-the-art sequencing technologies for genetic and epigenetic research. We illustrate how these technologies help to constantly improve our understanding of genetic mechanisms in biological systems and summarize the progress made so far. This can be exemplified by the case of heritable heart muscle diseases, so-called cardiomyopathies. Here, next-generation sequencing is able to identify novel disease genes, and first clinical applications demonstrate the successful translation of this technology into personalized patient care.

  10. Automatic generation of randomized trial sequences for priming experiments.

    Science.gov (United States)

    Ihrke, Matthias; Behrendt, Jörg

    2011-01-01

    In most psychological experiments, a randomized presentation of successive displays is crucial for the validity of the results. For some paradigms, this is not a trivial issue because trials are interdependent, e.g., priming paradigms. We present a software that automatically generates optimized trial sequences for (negative-) priming experiments. Our implementation is based on an optimization heuristic known as genetic algorithms that allows for an intuitive interpretation due to its similarity to natural evolution. The program features a graphical user interface that allows the user to generate trial sequences and to interactively improve them. The software is based on freely available software and is released under the GNU General Public License.

  11. AN M DWARF COMPANION TO AN F-TYPE STAR IN A YOUNG MAIN-SEQUENCE BINARY

    Energy Technology Data Exchange (ETDEWEB)

    Eigmüller, Ph.; Csizmadia, Sz.; Erikson, A.; Fridlund, M.; Pasternacki, Th.; Rauer, H. [Institute of Planetary Research, German Aerospace Center Rutherfordstr. 2, D-12489 Berlin (Germany); Eislöffel, J.; Lehmann, H.; Hartmann, M.; Hatzes, A. [Thüringer Landessternwarte Tautenburg Sternwarte 5, D-07778 Tautenburg (Germany); Tkachenko, A. [Instituut voor Sterrenkunde, KU Leuven Celestijnenlaan 200D, 3001 Leuven (Belgium); Voss, H., E-mail: philipp.eigmueller@dlr.de [Universitat de Barcelona, Department of Astronomy and Meteorology Martí i Franquès, 1, E-08028 Barcelona (Spain)

    2016-03-15

    Only a few well characterized very low-mass M dwarfs are known today. Our understanding of M dwarfs is vital as these are the most common stars in our solar neighborhood. We aim to characterize the properties of a rare F+dM stellar system for a better understanding of the low-mass end of the Hertzsprung–Russel diagram. We used photometric light curves and radial velocity follow-up measurements to study the binary. Spectroscopic analysis was used in combination with isochrone fitting to characterize the primary star. The primary star is an early F-type main-sequence star with a mass of (1.493 ± 0.073) M{sub ⊙} and a radius of (1.474 ± 0.040) R{sub ⊙}. The companion is an M dwarf with a mass of (0.188 ± 0.014) M{sub ⊙} and a radius of (0.234 ± 0.009) R{sub ⊙}. The orbital period is (1.35121 ± 0.00001) days. The secondary star is among the lowest-mass M dwarfs known to date. The binary has not reached a 1:1 spin–orbit synchronization. This indicates a young main-sequence binary with an age below ∼250 Myr. The mass–radius relation of both components are in agreement with this finding.

  12. Empirical tests of pre-main-sequence stellar evolution models with eclipsing binaries

    Science.gov (United States)

    Stassun, Keivan G.; Feiden, Gregory A.; Torres, Guillermo

    2014-06-01

    We examine the performance of standard pre-main-sequence (PMS) stellar evolution models against the accurately measured properties of a benchmark sample of 26 PMS stars in 13 eclipsing binary (EB) systems having masses 0.04-4.0 M⊙ and nominal ages ≈1-20 Myr. We provide a definitive compilation of all fundamental properties for the EBs, with a careful and consistent reassessment of observational uncertainties. We also provide a definitive compilation of the various PMS model sets, including physical ingredients and limits of applicability. No set of model isochrones is able to successfully reproduce all of the measured properties of all of the EBs. In the H-R diagram, the masses inferred for the individual stars by the models are accurate to better than 10% at ≳1 M⊙, but below 1 M⊙ they are discrepant by 50-100%. Adjusting the observed radii and temperatures using empirical relations for the effects of magnetic activity helps to resolve the discrepancies in a few cases, but fails as a general solution. We find evidence that the failure of the models to match the data is linked to the triples in the EB sample; at least half of the EBs possess tertiary companions. Excluding the triples, the models reproduce the stellar masses to better than ∼10% in the H-R diagram, down to 0.5 M⊙, below which the current sample is fully contaminated by tertiaries. We consider several mechanisms by which a tertiary might cause changes in the EB properties and thus corrupt the agreement with stellar model predictions. We show that the energies of the tertiary orbits are comparable to that needed to potentially explain the scatter in the EB properties through injection of heat, perhaps involving tidal interaction. It seems from the evidence at hand that this mechanism, however it operates in detail, has more influence on the surface properties of the stars than on their internal structure, as the lithium abundances are broadly in good agreement with model predictions. The

  13. Standardization and quality management in next-generation sequencing.

    Science.gov (United States)

    Endrullat, Christoph; Glökler, Jörn; Franke, Philipp; Frohme, Marcus

    2016-09-01

    DNA sequencing continues to evolve quickly even after > 30 years. Many new platforms suddenly appeared and former established systems have vanished in almost the same manner. Since establishment of next-generation sequencing devices, this progress gains momentum due to the continually growing demand for higher throughput, lower costs and better quality of data. In consequence of this rapid development, standardized procedures and data formats as well as comprehensive quality management considerations are still scarce. Here, we listed and summarized current standardization efforts and quality management initiatives from companies, organizations and societies in form of published studies and ongoing projects. These comprise on the one hand quality documentation issues like technical notes, accreditation checklists and guidelines for validation of sequencing workflows. On the other hand, general standard proposals and quality metrics are developed and applied to the sequencing workflow steps with the main focus on upstream processes. Finally, certain standard developments for downstream pipeline data handling, processing and storage are discussed in brief. These standardization approaches represent a first basis for continuing work in order to prospectively implement next-generation sequencing in important areas such as clinical diagnostics, where reliable results and fast processing is crucial. Additionally, these efforts will exert a decisive influence on traceability and reproducibility of sequence data.

  14. Sequencing of BAC pools by different next generation sequencing platforms and strategies

    Directory of Open Access Journals (Sweden)

    Scholz Uwe

    2011-10-01

    Full Text Available Abstract Background Next generation sequencing of BACs is a viable option for deciphering the sequence of even large and highly repetitive genomes. In order to optimize this strategy, we examined the influence of read length on the quality of Roche/454 sequence assemblies, to what extent Illumina/Solexa mate pairs (MPs improve the assemblies by scaffolding and whether barcoding of BACs is dispensable. Results Sequencing four BACs with both FLX and Titanium technologies revealed similar sequencing accuracy, but showed that the longer Titanium reads produce considerably less misassemblies and gaps. The 454 assemblies of 96 barcoded BACs were improved by scaffolding 79% of the total contig length with MPs from a non-barcoded library. Assembly of the unmasked 454 sequences without separation by barcodes revealed chimeric contig formation to be a major problem, encompassing 47% of the total contig length. Masking the sequences reduced this fraction to 24%. Conclusion Optimal BAC pool sequencing should be based on the longest available reads, with barcoding essential for a comprehensive assessment of both repetitive and non-repetitive sequence information. When interest is restricted to non-repetitive regions and repeats are masked prior to assembly, barcoding is non-essential. In any case, the assemblies can be improved considerably by scaffolding with non-barcoded BAC pool MPs.

  15. Next-generation sequencing offers new insights into DNA degradation

    DEFF Research Database (Denmark)

    Overballe-Petersen, Søren; Orlando, Ludovic Antoine Alexandre; Willerslev, Eske

    2012-01-01

    The processes underlying DNA degradation are central to various disciplines, including cancer research, forensics and archaeology. The sequencing of ancient DNA molecules on next-generation sequencing platforms provides direct measurements of cytosine deamination, depurination and fragmentation...... rates that previously were obtained only from extrapolations of results from in vitro kinetic experiments performed over short timescales. For example, recent next-generation sequencing of ancient DNA reveals purine bases as one of the main targets of postmortem hydrolytic damage, through base...... elimination and strand breakage. It also shows substantially increased rates of DNA base-loss at guanosine. In this review, we argue that the latter results from an electron resonance structure unique to guanosine rather than adenosine having an extra resonance structure over guanosine as previously suggested....

  16. VOX POPULI: Automatic Generation of Biased Video Sequences

    NARCIS (Netherlands)

    S. Bocconi; F.-M. Nack (Frank)

    2004-01-01

    textabstractWe describe our experimental rhetoric engine Vox Populi that generates biased video-sequences from a repository of video interviews and other related audio-visual web sources. Users are thus able to explore their own opinions on controversial topics covered by the repository. The

  17. VOX POPULI: automatic generation of biased video sequences

    NARCIS (Netherlands)

    S. Bocconi; F.-M. Nack (Frank)

    2004-01-01

    textabstractWe describe our experimental rhetoric engine Vox Populi that generates biased video-sequences from a repository of video interviews and other related audio-visual web sources. Users are thus able to explore their own opinions on controversial topics covered by the repository. The

  18. HLA typing: Conventional techniques v. next-generation sequencing ...

    African Journals Online (AJOL)

    Background. The large number of population-specific polymorphisms present in the HLA complex in the South African (SA) population reduces the probability of finding an adequate HLA-matched donor for individuals in need of an unrelated haematopoietic stem cell transplantation (HSCT). Next-generation sequencing ...

  19. Digital chaotic sequence generator based on coupled chaotic systems

    International Nuclear Information System (INIS)

    Shu-Bo, Liu; Jing, Sun; Jin-Shuo, Liu; Zheng-Quan, Xu

    2009-01-01

    Chaotic systems perform well as a new rich source of cryptography and pseudo-random coding. Unfortunately their digital dynamical properties would degrade due to the finite computing precision. Proposed in this paper is a modified digital chaotic sequence generator based on chaotic logistic systems with a coupling structure where one chaotic subsystem generates perturbation signals to disturb the control parameter of the other one. The numerical simulations show that the length of chaotic orbits, the output distribution of chaotic system, and the security of chaotic sequences have been greatly improved. Moreover the chaotic sequence period can be extended at least by one order of magnitude longer than that of the uncoupled logistic system and the difficulty in decrypting increases 2 128 *2 128 times indicating that the dynamical degradation of digital chaos is effectively improved. A field programmable gate array (FPGA) implementation of an algorithm is given and the corresponding experiment shows that the output speed of the generated chaotic sequences can reach 571.4 Mbps indicating that the designed generator can be applied to the real-time video image encryption. (general)

  20. Next-generation sequencing approaches to understanding the oral microbiome

    NARCIS (Netherlands)

    Zaura, E.

    2012-01-01

    Until recently, the focus in dental research has been on studying a small fraction of the oral microbiome—so-called opportunistic pathogens. With the advent of next-generation sequencing (NGS) technologies, researchers now have the tools that allow for profiling of the microbiomes and metagenomes at

  1. Next generation sequencing reveals the hidden diversity of zooplankton assemblages.

    Directory of Open Access Journals (Sweden)

    Penelope K Lindeque

    Full Text Available BACKGROUND: Zooplankton play an important role in our oceans, in biogeochemical cycling and providing a food source for commercially important fish larvae. However, difficulties in correctly identifying zooplankton hinder our understanding of their roles in marine ecosystem functioning, and can prevent detection of long term changes in their community structure. The advent of massively parallel next generation sequencing technology allows DNA sequence data to be recovered directly from whole community samples. Here we assess the ability of such sequencing to quantify richness and diversity of a mixed zooplankton assemblage from a productive time series site in the Western English Channel. METHODOLOGY/PRINCIPLE FINDINGS: Plankton net hauls (200 µm were taken at the Western Channel Observatory station L4 in September 2010 and January 2011. These samples were analysed by microscopy and metagenetic analysis of the 18S nuclear small subunit ribosomal RNA gene using the 454 pyrosequencing platform. Following quality control a total of 419,041 sequences were obtained for all samples. The sequences clustered into 205 operational taxonomic units using a 97% similarity cut-off. Allocation of taxonomy by comparison with the National Centre for Biotechnology Information database identified 135 OTUs to species level, 11 to genus level and 1 to order, <2.5% of sequences were classified as unknowns. By comparison a skilled microscopic analyst was able to routinely enumerate only 58 taxonomic groups. CONCLUSIONS: Metagenetics reveals a previously hidden taxonomic richness, especially for Copepoda and hard-to-identify meroplankton such as Bivalvia, Gastropoda and Polychaeta. It also reveals rare species and parasites. We conclude that Next Generation Sequencing of 18S amplicons is a powerful tool for elucidating the true diversity and species richness of zooplankton communities. While this approach allows for broad diversity assessments of plankton it may

  2. BINARY PARTICLE SWARM OPTIMIZATION APPROACH FOR RANDOM GENERATION OUTAGE MAINTENANCE SCHEDULING

    Directory of Open Access Journals (Sweden)

    K. Suresh

    2013-01-01

    Full Text Available This paper presents a methodology for maintenance scheduling (MS of generators using binary particle swarm optimization (BPSO based probabilistic approach. The objective of this paper is to reduce the loss of load probability (LOLP for a power system. The capacity outage probability table (COPT is the initial step in creating maintenance schedule using the probabilistic levelized risk method. This paper proposes BPSO method which is used to construct the COPT. In order to mitigate the effects of probabilistic levelized risk method, BPSO based probabilistic levelized risk method is embarked on a MS problem. In order to validate the effectiveness of the proposed algorithm, case study results for simple five unit system can accomplish a significant levelization in the reliability indices that make possible to evaluate system generation system adequacy in the MS horizon of the power system. The proposed method shows better performance compared with other optimization methods and conventional method with improved search performance.

  3. Next-generation sequencing in schizophrenia and other neuropsychiatric disorders.

    Science.gov (United States)

    Schreiber, Matthew; Dorschner, Michael; Tsuang, Debby

    2013-10-01

    Schizophrenia is a debilitating lifelong illness that lacks a cure and poses a worldwide public health burden. The disease is characterized by a heterogeneous clinical and genetic presentation that complicates research efforts to identify causative genetic variations. This review examines the potential of current findings in schizophrenia and in other related neuropsychiatric disorders for application in next-generation technologies, particularly whole-exome sequencing (WES) and whole-genome sequencing (WGS). These approaches may lead to the discovery of underlying genetic factors for schizophrenia and may thereby identify and target novel therapeutic targets for this devastating disorder. © 2013 Wiley Periodicals, Inc.

  4. Diagnostics of Primary Immunodeficiencies through Next Generation Sequencing

    Directory of Open Access Journals (Sweden)

    Vera Gallo

    2016-11-01

    Full Text Available Background: Recently, a growing number of novel genetic defects underlying primary immunodeficiencies (PID have been identified, increasing the number of PID up to more than 250 well-defined forms. Next-generation sequencing (NGS technologies and proper filtering strategies greatly contributed to this rapid evolution, providing the possibility to rapidly and simultaneously analyze large numbers of genes or the whole exome. Objective: To evaluate the role of targeted next-generation sequencing and whole exome sequencing in the diagnosis of a case series, characterized by complex or atypical clinical features suggesting a PID, difficult to diagnose using the current diagnostic procedures.Methods: We retrospectively analyzed genetic variants identified through targeted next-generation sequencing or whole exome sequencing in 45 patients with complex PID of unknown etiology. Results: 40 variants were identified using targeted next-generation sequencing, while 5 were identified using whole exome sequencing. Newly identified genetic variants were classified into 4 groups: I variations associated with a well-defined PID; II variations associated with atypical features of a well-defined PID; III functionally relevant variations potentially involved in the immunological features; IV non-diagnostic genotype, in whom the link with phenotype is missing. We reached a conclusive genetic diagnosis in 7/45 patients (~16%. Among them, 4 patients presented with a typical well-defined PID. In the remaining 3 cases, mutations were associated with unexpected clinical features, expanding the phenotypic spectrum of typical PIDs. In addition, we identified 31 variants in 10 patients with complex phenotype, individually not causative per se of the disorder.Conclusion: NGS technologies represent a cost-effective and rapid first-line genetic approaches for the evaluation of complex PIDs. Whole exome sequencing, despite a moderate higher cost compared to targeted, is

  5. Programmable pulse sequence generator with multiple output lines

    Science.gov (United States)

    Drabczyk, Hubert

    2006-10-01

    This paper presents a novel concept of pulse sequence generator and its prototype as an electronic circuit testing laboratory tool. The generator has multiple output lines and is capable of using control data defining different pulse sequences to be given to the outputs. It is also possible to use different voltage levels in output signal and switch output lines for reading data from driven system. The pulse sequence generator can be used for runtime environment simulation, as hardware tester or auxiliary tool in new designs. Important design factors were to keep cost of the tool low and allow integration with other projects by using flexible architecture. The prototype was based on universal programmer with adjustable power supply, '51 microcontroller and Altera Cyclone chip. The generator communicates witch PC computer via RS232 port. Dedicated software was developed in the course of this project, to control the tool and data transmission. The prototype confirmed the possibility to create an inexpensive multipurpose laboratory tool for programming, testing and simulation of digital devices.

  6. Graph-based clustering and characterization of repetitive sequences in next-generation sequencing data

    Czech Academy of Sciences Publication Activity Database

    Novák, Petr; Neumann, Pavel; Macas, Jiří

    2010-01-01

    Roč. 11, č. 1 (2010), s. 378-389 ISSN 1471-2105 R&D Projects: GA MŠk(CZ) OC10037; GA MŠk(CZ) LC06004 Institutional research plan: CEZ:AV0Z50510513 Keywords : repetitive DNA * plant genome * next generation sequencing Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.028, year: 2010

  7. Implementation of Targeted Next Generation Sequencing in Clinical Diagnostics

    DEFF Research Database (Denmark)

    Larsen, Martin Jakob; Burton, Mark; Thomassen, Mads

    Accurate mutation detection is essential in clinical genetic diagnostics of monogenic hereditary diseases. Targeted next generation sequencing (NGS) provides a promising and cost-effective alternative to Sanger sequencing and MLPA analysis currently used in most diagnostic laboratories. One...... of mutation positive controls previously characterized by Sanger/MLPA analysis. Agilent SureSelect Target-Enrichment kits were used for capturing a set of genes associated with hereditary breast and ovarian cancer syndrome and a compilation of genes involved in multiple rare single gene disorders......, respectively. For diagnostics, the sequencing coverage is essential, wherefore a minimum coverage of 30x per nucleotide in the coding regions was used as our primary quality criterion. For the majority of the included genes, we obtained adequate gene coverage, in which we were able to detect 100% of the known...

  8. Machine-Checked Sequencer for Critical Embedded Code Generator

    Science.gov (United States)

    Izerrouken, Nassima; Pantel, Marc; Thirioux, Xavier

    This paper presents the development of a correct-by-construction block sequencer for GeneAuto a qualifiable (according to DO178B/ED12B recommendation) automatic code generator. It transforms Simulink models to MISRA C code for safety critical systems. Our approach which combines classical development process and formal specification and verification using proof-assistants, led to preliminary fruitful exchanges with certification authorities. We present parts of the classical user and tools requirements and derived formal specifications, implementation and verification for the correctness and termination of the block sequencer. This sequencer has been successfully applied to real-size industrial use cases from various transportation domain partners and led to requirement errors detection and a correct-by-construction implementation.

  9. Transcriptome sequencing of the Microarray Quality Control (MAQC RNA reference samples using next generation sequencing

    Directory of Open Access Journals (Sweden)

    Thierry-Mieg Danielle

    2009-06-01

    Full Text Available Abstract Background Transcriptome sequencing using next-generation sequencing platforms will soon be competing with DNA microarray technologies for global gene expression analysis. As a preliminary evaluation of these promising technologies, we performed deep sequencing of cDNA synthesized from the Microarray Quality Control (MAQC reference RNA samples using Roche's 454 Genome Sequencer FLX. Results We generated more that 3.6 million sequence reads of average length 250 bp for the MAQC A and B samples and introduced a data analysis pipeline for translating cDNA read counts into gene expression levels. Using BLAST, 90% of the reads mapped to the human genome and 64% of the reads mapped to the RefSeq database of well annotated genes with e-values ≤ 10-20. We measured gene expression levels in the A and B samples by counting the numbers of reads that mapped to individual RefSeq genes in multiple sequencing runs to evaluate the MAQC quality metrics for reproducibility, sensitivity, specificity, and accuracy and compared the results with DNA microarrays and Quantitative RT-PCR (QRTPCR from the MAQC studies. In addition, 88% of the reads were successfully aligned directly to the human genome using the AceView alignment programs with an average 90% sequence similarity to identify 137,899 unique exon junctions, including 22,193 new exon junctions not yet contained in the RefSeq database. Conclusion Using the MAQC metrics for evaluating the performance of gene expression platforms, the ExpressSeq results for gene expression levels showed excellent reproducibility, sensitivity, and specificity that improved systematically with increasing shotgun sequencing depth, and quantitative accuracy that was comparable to DNA microarrays and QRTPCR. In addition, a careful mapping of the reads to the genome using the AceView alignment programs shed new light on the complexity of the human transcriptome including the discovery of thousands of new splice variants.

  10. Second generation sequencing of the mesothelioma tumor genome.

    Directory of Open Access Journals (Sweden)

    Raphael Bueno

    2010-05-01

    Full Text Available The current paradigm for elucidating the molecular etiology of cancers relies on the interrogation of small numbers of genes, which limits the scope of investigation. Emerging second-generation massively parallel DNA sequencing technologies have enabled more precise definition of the cancer genome on a global scale. We examined the genome of a human primary malignant pleural mesothelioma (MPM tumor and matched normal tissue by using a combination of sequencing-by-synthesis and pyrosequencing methodologies to a 9.6X depth of coverage. Read density analysis uncovered significant aneuploidy and numerous rearrangements. Method-dependent informatics rules, which combined the results of different sequencing platforms, were developed to identify and validate candidate mutations of multiple types. Many more tumor-specific rearrangements than point mutations were uncovered at this depth of sequencing, resulting in novel, large-scale, inter- and intra-chromosomal deletions, inversions, and translocations. Nearly all candidate point mutations appeared to be previously unknown SNPs. Thirty tumor-specific fusions/translocations were independently validated with PCR and Sanger sequencing. Of these, 15 represented disrupted gene-encoding regions, including kinases, transcription factors, and growth factors. One large deletion in DPP10 resulted in altered transcription and expression of DPP10 transcripts in a set of 53 additional MPM tumors correlated with survival. Additionally, three point mutations were observed in the coding regions of NKX6-2, a transcription regulator, and NFRKB, a DNA-binding protein involved in modulating NFKB1. Several regions containing genes such as PCBD2 and DHFR, which are involved in growth factor signaling and nucleotide synthesis, respectively, were selectively amplified in the tumor. Second-generation sequencing uncovered all types of mutations in this MPM tumor, with DNA rearrangements representing the dominant type.

  11. Next-Generation Sequencing of Antibody Display Repertoires

    Directory of Open Access Journals (Sweden)

    Romain Rouet

    2018-02-01

    Full Text Available In vitro selection technology has transformed the development of therapeutic monoclonal antibodies. Using methods such as phage, ribosome, and yeast display, high affinity binders can be selected from diverse repertoires. Here, we review strategies for the next-generation sequencing (NGS of phage- and other antibody-display libraries, as well as NGS platforms and analysis tools. Moreover, we discuss recent examples relating to the use of NGS to assess library diversity, clonal enrichment, and affinity maturation.

  12. Statistical Approaches for Next-Generation Sequencing Data

    OpenAIRE

    Qiao, Dandi

    2012-01-01

    During the last two decades, genotyping technology has advanced rapidly, which enabled the tremendous success of genome-wide association studies (GWAS) in the search of disease susceptibility loci (DSLs). However, only a small fraction of the overall predicted heritability can be explained by the DSLs discovered. One possible explanation for this ”missing heritability” phenomenon is that many causal variants are rare. The recent development of high-throughput next-generation sequencing (NGS) ...

  13. Next Generation Sequencing Methods for Diagnosis of Epilepsy Syndromes

    Directory of Open Access Journals (Sweden)

    Paul Dunn

    2018-02-01

    Full Text Available Epilepsy is a neurological disorder characterized by an increased predisposition for seizures. Although this definition suggests that it is a single disorder, epilepsy encompasses a group of disorders with diverse aetiologies and outcomes. A genetic basis for epilepsy syndromes has been postulated for several decades, with several mutations in specific genes identified that have increased our understanding of the genetic influence on epilepsies. With 70-80% of epilepsy cases identified to have a genetic cause, there are now hundreds of genes identified to be associated with epilepsy syndromes which can be analyzed using next generation sequencing (NGS techniques such as targeted gene panels, whole exome sequencing (WES and whole genome sequencing (WGS. For effective use of these methodologies, diagnostic laboratories and clinicians require information on the relevant workflows including analysis and sequencing depth to understand the specific clinical application and diagnostic capabilities of these gene sequencing techniques. As epilepsy is a complex disorder, the differences associated with each technique influence the ability to form a diagnosis along with an accurate detection of the genetic etiology of the disorder. In addition, for diagnostic testing, an important parameter is the cost-effectiveness and the specific diagnostic outcome of each technique. Here, we review these commonly used NGS techniques to determine their suitability for application to epilepsy genetic diagnostic testing.

  14. Generation of control sequences for a pilot-disassembly system

    Science.gov (United States)

    Seliger, Guenther; Kim, Hyung-Ju; Keil, Thomas

    2002-02-01

    Closing the product and material cycles has emerged as a paradigm for industry in the 21st century. Disassembly plays a key role in a life cycle economy since it enables the recovery of resources. A partly automated disassembly system should adapt to a large variety of products and different degrees of devaluation. Also the amounts of products to be disassembled can vary strongly. To cope with these demands an approach to generate on-line disassembly control sequences will be presented. In order to react on these demands the technological feasibility is considered within a procedure for the generation of disassembly control sequences. Procedures are designed to find available and technologically feasible disassembly processes. The control system is formed by modularised and parameterised control units in the cell level within the entire control architecture. In the first development stage product and process analyses at the sample product washing machine were executed. Furthermore a generalized disassembly process was defined. Afterwards these processes were structured in primary and secondary functions. In the second stage the disassembly control at the technological level was investigated. Factors were the availability of the disassembly tools and the technological feasibility of the disassembly processes within the disassembly system. Technical alternative disassembly processes are determined as a result of availability of the tools and technological feasibility of processes. The fourth phase was the concept for the generation of the disassembly control sequences. The approach will be proved in a prototypical disassembly system.

  15. Random Sequence for Optimal Low-Power Laser Generated Ultrasound

    Science.gov (United States)

    Vangi, D.; Virga, A.; Gulino, M. S.

    2017-08-01

    Low-power laser generated ultrasounds are lately gaining importance in the research world, thanks to the possibility of investigating a mechanical component structural integrity through a non-contact and Non-Destructive Testing (NDT) procedure. The ultrasounds are, however, very low in amplitude, making it necessary to use pre-processing and post-processing operations on the signals to detect them. The cross-correlation technique is used in this work, meaning that a random signal must be used as laser input. For this purpose, a highly random and simple-to-create code called T sequence, capable of enhancing the ultrasound detectability, is introduced (not previously available at the state of the art). Several important parameters which characterize the T sequence can influence the process: the number of pulses Npulses , the pulse duration δ and the distance between pulses dpulses . A Finite Element FE model of a 3 mm steel disk has been initially developed to analytically study the longitudinal ultrasound generation mechanism and the obtainable outputs. Later, experimental tests have shown that the T sequence is highly flexible for ultrasound detection purposes, making it optimal to use high Npulses and δ but low dpulses . In the end, apart from describing all phenomena that arise in the low-power laser generation process, the results of this study are also important for setting up an effective NDT procedure using this technology.

  16. Next-Generation Sequencing of Tubal Intraepithelial Carcinomas.

    Science.gov (United States)

    McDaniel, Andrew S; Stall, Jennifer N; Hovelson, Daniel H; Cani, Andi K; Liu, Chia-Jen; Tomlins, Scott A; Cho, Kathleen R

    2015-11-01

    High-grade serous carcinoma (HGSC) is the most prevalent and lethal form of ovarian cancer. HGSCs frequently arise in the distal fallopian tubes rather than the ovary, developing from small precursor lesions called serous tubal intraepithelial carcinomas (TICs, or more specifically, STICs). While STICs have been reported to harbor TP53 mutations, detailed molecular characterizations of these lesions are lacking. We performed targeted next-generation sequencing (NGS) on formalin-fixed, paraffin-embedded tissue from 4 women, 2 with HGSC and 2 with uterine endometrioid carcinoma (UEC) who were diagnosed as having synchronous STICs. We detected concordant mutations in both HGSCs with synchronous STICs, including TP53 mutations as well as assumed germline BRCA1/2 alterations, confirming a clonal association between these lesions. Next-generation sequencing confirmed the presence of a STIC clonally unrelated to 1 case of UEC, and NGS of the other tubal lesion diagnosed as a STIC unexpectedly supported the lesion as a micrometastasis from the associated UEC. We demonstrate that targeted NGS can identify genetic alterations in minute lesions, such as TICs, and confirm TP53 mutations as early driving events for HGSC. Next-generation sequencing also demonstrated unexpected associations between presumed STICs and synchronous carcinomas, providing evidence that some TICs are actually metastases rather than HGSC precursors.

  17. Prediction and generation of binary Markov processes: Can a finite-state fox catch a Markov mouse?

    Science.gov (United States)

    Ruebeck, Joshua B.; James, Ryan G.; Mahoney, John R.; Crutchfield, James P.

    2018-01-01

    Understanding the generative mechanism of a natural system is a vital component of the scientific method. Here, we investigate one of the fundamental steps toward this goal by presenting the minimal generator of an arbitrary binary Markov process. This is a class of processes whose predictive model is well known. Surprisingly, the generative model requires three distinct topologies for different regions of parameter space. We show that a previously proposed generator for a particular set of binary Markov processes is, in fact, not minimal. Our results shed the first quantitative light on the relative (minimal) costs of prediction and generation. We find, for instance, that the difference between prediction and generation is maximized when the process is approximately independently, identically distributed.

  18. Next-Generation Sequencing and Genome Editing in Plant Virology

    Directory of Open Access Journals (Sweden)

    Ahmed Hadidi

    2016-08-01

    Full Text Available Next-generation sequencing (NGS has been applied to plant virology since 2009. NGS provides highly efficient, rapid, low cost DNA or RNA high-throughput sequencing of the genomes of plant viruses and viroids and of the specific small RNAs generated during the infection process. These small RNAs, which cover frequently the whole genome of the infectious agent, are 21-24 nt long and are known as vsRNAs for viruses and vd-sRNAs for viroids. NGS has been used in a number of studies in plant virology including, but not limited to, discovery of novel viruses and viroids as well as detection and identification of those pathogens already known, analysis of genome diversity and evolution, and study of pathogen epidemiology. The genome engineering editing method, clustered regularly interspaced short palindromic repeats (CRISPR-Cas9 system has been successfully used recently to engineer resistance to DNA geminiviruses (family, Geminiviridae by targeting different viral genome sequences in infected Nicotiana benthamiana or Arabidopsis plants. The DNA viruses targeted include tomato yellow leaf curl virus and merremia mosaic virus (begomovirus; beet curly top virus and beet severe curly top virus (curtovirus; and bean yellow dwarf virus (mastrevirus. The technique has also been used against the RNA viruses zucchini yellow mosaic virus, papaya ringspot virus and turnip mosaic virus (potyvirus and cucumber vein yellowing virus (ipomovirus, family, Potyviridae by targeting the translation initiation genes eIF4E in cucumber or Arabidopsis plants. From these recent advances of major importance, it is expected that NGS and CRISPR-Cas technologies will play a significant role in the very near future in advancing the field of plant virology and connecting it with other related fields of biology.Keywords: Next-generation sequencing, NGS, plant virology, plant viruses, viroids, resistance to plant viruses by CRISPR-Cas9

  19. Next generation sequencing and its applications in forensic genetics.

    Science.gov (United States)

    Børsting, Claus; Morling, Niels

    2015-09-01

    It has been almost a decade since the first next generation sequencing (NGS) technologies emerged and quickly changed the way genetic research is conducted. Today, full genomes are mapped and published almost weekly and with ever increasing speed and decreasing costs. NGS methods and platforms have matured during the last 10 years, and the quality of the sequences has reached a level where NGS is used in clinical diagnostics of humans. Forensic genetic laboratories have also explored NGS technologies and especially in the last year, there has been a small explosion in the number of scientific articles and presentations at conferences with forensic aspects of NGS. These contributions have demonstrated that NGS offers new possibilities for forensic genetic case work. More information may be obtained from unique samples in a single experiment by analyzing combinations of markers (STRs, SNPs, insertion/deletions, mRNA) that cannot be analyzed simultaneously with the standard PCR-CE methods used today. The true variation in core forensic STR loci has been uncovered, and previously unknown STR alleles have been discovered. The detailed sequence information may aid mixture interpretation and will increase the statistical weight of the evidence. In this review, we will give an introduction to NGS and single-molecule sequencing, and we will discuss the possible applications of NGS in forensic genetics. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  20. Molecular Characterization of Transgenic Events Using Next Generation Sequencing Approach.

    Science.gov (United States)

    Guttikonda, Satish K; Marri, Pradeep; Mammadov, Jafar; Ye, Liang; Soe, Khaing; Richey, Kimberly; Cruse, James; Zhuang, Meibao; Gao, Zhifang; Evans, Clive; Rounsley, Steve; Kumpatla, Siva P

    2016-01-01

    Demand for the commercial use of genetically modified (GM) crops has been increasing in light of the projected growth of world population to nine billion by 2050. A prerequisite of paramount importance for regulatory submissions is the rigorous safety assessment of GM crops. One of the components of safety assessment is molecular characterization at DNA level which helps to determine the copy number, integrity and stability of a transgene; characterize the integration site within a host genome; and confirm the absence of vector DNA. Historically, molecular characterization has been carried out using Southern blot analysis coupled with Sanger sequencing. While this is a robust approach to characterize the transgenic crops, it is both time- and resource-consuming. The emergence of next-generation sequencing (NGS) technologies has provided highly sensitive and cost- and labor-effective alternative for molecular characterization compared to traditional Southern blot analysis. Herein, we have demonstrated the successful application of both whole genome sequencing and target capture sequencing approaches for the characterization of single and stacked transgenic events and compared the results and inferences with traditional method with respect to key criteria required for regulatory submissions.

  1. Molecular Characterization of Transgenic Events Using Next Generation Sequencing Approach.

    Directory of Open Access Journals (Sweden)

    Satish K Guttikonda

    Full Text Available Demand for the commercial use of genetically modified (GM crops has been increasing in light of the projected growth of world population to nine billion by 2050. A prerequisite of paramount importance for regulatory submissions is the rigorous safety assessment of GM crops. One of the components of safety assessment is molecular characterization at DNA level which helps to determine the copy number, integrity and stability of a transgene; characterize the integration site within a host genome; and confirm the absence of vector DNA. Historically, molecular characterization has been carried out using Southern blot analysis coupled with Sanger sequencing. While this is a robust approach to characterize the transgenic crops, it is both time- and resource-consuming. The emergence of next-generation sequencing (NGS technologies has provided highly sensitive and cost- and labor-effective alternative for molecular characterization compared to traditional Southern blot analysis. Herein, we have demonstrated the successful application of both whole genome sequencing and target capture sequencing approaches for the characterization of single and stacked transgenic events and compared the results and inferences with traditional method with respect to key criteria required for regulatory submissions.

  2. Next generation sequencing and its applications in forensic genetics

    DEFF Research Database (Denmark)

    Børsting, Claus; Morling, Niels

    2015-01-01

    articles and presentations at conferences with forensic aspects of NGS. These contributions have demonstrated that NGS offers new possibilities for forensic genetic case work. More information may be obtained from unique samples in a single experiment by analyzing combinations of markers (STRs, SNPs......It has been almost a decade since the first next generation sequencing (NGS) technologies emerged and quickly changed the way genetic research is conducted. Today, full genomes are mapped and published almost weekly and with ever increasing speed and decreasing costs. NGS methods and platforms have...... matured during the last 10 years, and the quality of the sequences has reached a level where NGS is used in clinical diagnostics of humans. Forensic genetic laboratories have also explored NGS technologies and especially in the last year, there has been a small explosion in the number of scientific...

  3. Perspectives of Integrative Cancer Genomics in Next Generation Sequencing Era

    Directory of Open Access Journals (Sweden)

    So Mee Kwon

    2012-06-01

    Full Text Available The explosive development of genomics technologies including microarrays and next generation sequencing (NGS has provided comprehensive maps of cancer genomes, including the expression of mRNAs and microRNAs, DNA copy numbers, sequence variations, and epigenetic changes. These genome-wide profiles of the genetic aberrations could reveal the candidates for diagnostic and/or prognostic biomarkers as well as mechanistic insights into tumor development and progression. Recent efforts to establish the huge cancer genome compendium and integrative omics analyses, so-called "integromics", have extended our understanding on the cancer genome, showing its daunting complexity and heterogeneity. However, the challenges of the structured integration, sharing, and interpretation of the big omics data still remain to be resolved. Here, we review several issues raised in cancer omics data analysis, including NGS, focusing particularly on the study design and analysis strategies. This might be helpful to understand the current trends and strategies of the rapidly evolving cancer genomics research.

  4. Understanding Cancer Genome and Its Evolution by Next Generation Sequencing

    DEFF Research Database (Denmark)

    Hou, Yong

    Cancer will cause 13 million deaths by the year of 2030, ranking the second leading cause of death worldwide. Previous studies indicate that most of the cancers originate from cells that acquired somatic mutations and evolved as Darwin Theory. Ten biological insights of cancer have been summarized...... recently. Cutting-age technologies like next generation sequencing (NGS) enable exploring cancer genome and evolution much more efficiently. However, integrated cancer genome sequencing studies showed great inter-/intra-tumoral heterogeneity (ITH) and complex evolution patterns beyond the cancer biological...... knowledge we previously know. There is very limited knowledge of East Asia lung cancer genome except enrichment of EGFR mutations and lack of KRAS mutations. We carried out integrated genomic, transcriptomic and methylomic analysis of 335 primary Chinese lung adenocarcinomas (LUAD) and 35 corresponding...

  5. Fast precalculated triangular mesh algorithm for 3D binary computer-generated holograms.

    Science.gov (United States)

    Yang, Fan; Kaczorowski, Andrzej; Wilkinson, Tim D

    2014-12-10

    A new method for constructing computer-generated holograms using a precalculated triangular mesh is presented. The speed of calculation can be increased dramatically by exploiting both the precalculated base triangle and GPU parallel computing. Unlike algorithms using point-based sources, this method can reconstruct a more vivid 3D object instead of a "hollow image." In addition, there is no need to do a fast Fourier transform for each 3D element every time. A ferroelectric liquid crystal spatial light modulator is used to display the binary hologram within our experiment and the hologram of a base right triangle is produced by utilizing just a one-step Fourier transform in the 2D case, which can be expanded to the 3D case by multiplying by a suitable Fresnel phase plane. All 3D holograms generated in this paper are based on Fresnel propagation; thus, the Fresnel plane is treated as a vital element in producing the hologram. A GeForce GTX 770 graphics card with 2 GB memory is used to achieve parallel computing.

  6. Targeted next generation sequencing for molecular diagnosis of Usher syndrome.

    Science.gov (United States)

    Aparisi, María J; Aller, Elena; Fuster-García, Carla; García-García, Gema; Rodrigo, Regina; Vázquez-Manrique, Rafael P; Blanco-Kelly, Fiona; Ayuso, Carmen; Roux, Anne-Françoise; Jaijo, Teresa; Millán, José M

    2014-11-18

    Usher syndrome is an autosomal recessive disease that associates sensorineural hearing loss, retinitis pigmentosa and, in some cases, vestibular dysfunction. It is clinically and genetically heterogeneous. To date, 10 genes have been associated with the disease, making its molecular diagnosis based on Sanger sequencing, expensive and time-consuming. Consequently, the aim of the present study was to develop a molecular diagnostics method for Usher syndrome, based on targeted next generation sequencing. A custom HaloPlex panel for Illumina platforms was designed to capture all exons of the 10 known causative Usher syndrome genes (MYO7A, USH1C, CDH23, PCDH15, USH1G, CIB2, USH2A, GPR98, DFNB31 and CLRN1), the two Usher syndrome-related genes (HARS and PDZD7) and the two candidate genes VEZT and MYO15A. A cohort of 44 patients suffering from Usher syndrome was selected for this study. This cohort was divided into two groups: a test group of 11 patients with known mutations and another group of 33 patients with unknown mutations. Forty USH patients were successfully sequenced, 8 USH patients from the test group and 32 patients from the group composed of USH patients without genetic diagnosis. We were able to detect biallelic mutations in one USH gene in 22 out of 32 USH patients (68.75%) and to identify 79.7% of the expected mutated alleles. Fifty-three different mutations were detected. These mutations included 21 missense, 8 nonsense, 9 frameshifts, 9 intronic mutations and 6 large rearrangements. Targeted next generation sequencing allowed us to detect both point mutations and large rearrangements in a single experiment, minimizing the economic cost of the study, increasing the detection ratio of the genetic cause of the disease and improving the genetic diagnosis of Usher syndrome patients.

  7. Using next generation transcriptome sequencing to predict an ectomycorrhizal metabolome

    Directory of Open Access Journals (Sweden)

    Cseke Leland J

    2011-05-01

    Full Text Available Abstract Background Mycorrhizae, symbiotic interactions between soil fungi and tree roots, are ubiquitous in terrestrial ecosystems. The fungi contribute phosphorous, nitrogen and mobilized nutrients from organic matter in the soil and in return the fungus receives photosynthetically-derived carbohydrates. This union of plant and fungal metabolisms is the mycorrhizal metabolome. Understanding this symbiotic relationship at a molecular level provides important contributions to the understanding of forest ecosystems and global carbon cycling. Results We generated next generation short-read transcriptomic sequencing data from fully-formed ectomycorrhizae between Laccaria bicolor and aspen (Populus tremuloides roots. The transcriptomic data was used to identify statistically significantly expressed gene models using a bootstrap-style approach, and these expressed genes were mapped to specific metabolic pathways. Integration of expressed genes that code for metabolic enzymes and the set of expressed membrane transporters generates a predictive model of the ectomycorrhizal metabolome. The generated model of mycorrhizal metabolome predicts that the specific compounds glycine, glutamate, and allantoin are synthesized by L. bicolor and that these compounds or their metabolites may be used for the benefit of aspen in exchange for the photosynthetically-derived sugars fructose and glucose. Conclusions The analysis illustrates an approach to generate testable biological hypotheses to investigate the complex molecular interactions that drive ectomycorrhizal symbiosis. These models are consistent with experimental environmental data and provide insight into the molecular exchange processes for organisms in this complex ecosystem. The method used here for predicting metabolomic models of mycorrhizal systems from deep RNA sequencing data can be generalized and is broadly applicable to transcriptomic data derived from complex systems.

  8. Use of next-generation sequencing in oral cavity cancer

    DEFF Research Database (Denmark)

    Tabatabaeifar, Siavosh; Kruse, Torben A; Thomassen, Mads

    Background: Oral cavity cancer is a subgroup of head and neck cancer which is the world’s 6th most common cancer form. Oral squamous cell carcinomas (OSCC) constitute almost all oral cavity cancers, and OSCC are primarily attributed by excessive alcohol consumption and tobacco exposure...... of tumour cells exists. Conclusions: Use of next generation sequencing in oral cavity cancer can give valuable insight into the biology of the disease. By investigating intra tumour heterogeneity we see that the different tumour specimens in each patient are quite homogenous, but evidence of heterogeneous...

  9. AK SCO, FIRST DETECTION OF A HIGHLY DISTURBED ATMOSPHERE IN A PRE-MAIN-SEQUENCE CLOSE BINARY

    International Nuclear Information System (INIS)

    Gomez de Castro, Ana I.

    2009-01-01

    AK Sco is a unique source: a ∼10 Myr old pre-main-sequence (PMS) spectroscopic binary composed of two nearly equal F5 stars that at periastron are separated by barely 11 stellar radii, so the stellar magnetospheres fill the Roche lobe at periastron. The orbit is not yet circularized (e = 0.47) and very strong tides are expected. This makes AK Sco the ideal laboratory to study the effect of gravitational tides in the stellar magnetic field building up during PMS evolution. In this Letter, the detection of a highly disturbed (σ ≅ 100 km s -1 ) and very dense atmosphere (n e = 1.6 x 10 10 cm -3 ) is reported. Significant line broadening blurs any signs of ion belts or bow shocks in the spectrum of the atmospheric plasma. The radiative losses cannot be accounted for solely by the dissipation of energy from the tidal wave propagating in the stellar atmosphere or by the accreting material. The release of internal energy from the star seems to be the most likely source of the plasma heating. This is the first clear indication of a highly disturbed atmosphere surrounding a PMS close binary.

  10. AK Sco, First Detection of a Highly Disturbed Atmosphere in a Pre-Main-Sequence Close Binary

    Science.gov (United States)

    Gómez de Castro, Ana I.

    2009-06-01

    AK Sco is a unique source: a ~10 Myr old pre-main-sequence (PMS) spectroscopic binary composed of two nearly equal F5 stars that at periastron are separated by barely 11 stellar radii, so the stellar magnetospheres fill the Roche lobe at periastron. The orbit is not yet circularized (e = 0.47) and very strong tides are expected. This makes AK Sco the ideal laboratory to study the effect of gravitational tides in the stellar magnetic field building up during PMS evolution. In this Letter, the detection of a highly disturbed (σ sime 100 km s-1) and very dense atmosphere (n e = 1.6 × 1010 cm-3) is reported. Significant line broadening blurs any signs of ion belts or bow shocks in the spectrum of the atmospheric plasma. The radiative losses cannot be accounted for solely by the dissipation of energy from the tidal wave propagating in the stellar atmosphere or by the accreting material. The release of internal energy from the star seems to be the most likely source of the plasma heating. This is the first clear indication of a highly disturbed atmosphere surrounding a PMS close binary.

  11. Coding Local and Global Binary Visual Features Extracted From Video Sequences

    Science.gov (United States)

    Baroffio, Luca; Canclini, Antonio; Cesana, Matteo; Redondi, Alessandro; Tagliasacchi, Marco; Tubaro, Stefano

    2015-11-01

    Binary local features represent an effective alternative to real-valued descriptors, leading to comparable results for many visual analysis tasks, while being characterized by significantly lower computational complexity and memory requirements. When dealing with large collections, a more compact representation based on global features is often preferred, which can be obtained from local features by means of, e.g., the Bag-of-Visual-Word (BoVW) model. Several applications, including for example visual sensor networks and mobile augmented reality, require visual features to be transmitted over a bandwidth-limited network, thus calling for coding techniques that aim at reducing the required bit budget, while attaining a target level of efficiency. In this paper we investigate a coding scheme tailored to both local and global binary features, which aims at exploiting both spatial and temporal redundancy by means of intra- and inter-frame coding. In this respect, the proposed coding scheme can be conveniently adopted to support the Analyze-Then-Compress (ATC) paradigm. That is, visual features are extracted from the acquired content, encoded at remote nodes, and finally transmitted to a central controller that performs visual analysis. This is in contrast with the traditional approach, in which visual content is acquired at a node, compressed and then sent to a central unit for further processing, according to the Compress-Then-Analyze (CTA) paradigm. In this paper we experimentally compare ATC and CTA by means of rate-efficiency curves in the context of two different visual analysis tasks: homography estimation and content-based retrieval. Our results show that the novel ATC paradigm based on the proposed coding primitives can be competitive with CTA, especially in bandwidth limited scenarios.

  12. Coding Local and Global Binary Visual Features Extracted From Video Sequences.

    Science.gov (United States)

    Baroffio, Luca; Canclini, Antonio; Cesana, Matteo; Redondi, Alessandro; Tagliasacchi, Marco; Tubaro, Stefano

    2015-11-01

    Binary local features represent an effective alternative to real-valued descriptors, leading to comparable results for many visual analysis tasks while being characterized by significantly lower computational complexity and memory requirements. When dealing with large collections, a more compact representation based on global features is often preferred, which can be obtained from local features by means of, e.g., the bag-of-visual word model. Several applications, including, for example, visual sensor networks and mobile augmented reality, require visual features to be transmitted over a bandwidth-limited network, thus calling for coding techniques that aim at reducing the required bit budget while attaining a target level of efficiency. In this paper, we investigate a coding scheme tailored to both local and global binary features, which aims at exploiting both spatial and temporal redundancy by means of intra- and inter-frame coding. In this respect, the proposed coding scheme can conveniently be adopted to support the analyze-then-compress (ATC) paradigm. That is, visual features are extracted from the acquired content, encoded at remote nodes, and finally transmitted to a central controller that performs the visual analysis. This is in contrast with the traditional approach, in which visual content is acquired at a node, compressed and then sent to a central unit for further processing, according to the compress-then-analyze (CTA) paradigm. In this paper, we experimentally compare the ATC and the CTA by means of rate-efficiency curves in the context of two different visual analysis tasks: 1) homography estimation and 2) content-based retrieval. Our results show that the novel ATC paradigm based on the proposed coding primitives can be competitive with the CTA, especially in bandwidth limited scenarios.

  13. Investigation of a steam generator tube rupture sequence using VICTORIA

    International Nuclear Information System (INIS)

    Bixler, N.E.; Erickson, C.M.; Schaperow, J.H.

    1995-01-01

    VICTORIA-92 is a mechanistic computer code for analyzing fission product behavior within the reactor coolant system (RCS) during a severe reactor accident. It provides detailed predictions of the release of radionuclides and nonradioactive materials from the core and transport of these materials within the RCS. The modeling accounts for the chemical and aerosol processes that affect radionuclide behavior. Coupling of detailed chemistry and aerosol packages is a unique feature of VICTORIA; it allows exploration of phenomena involving deposition, revaporization, and re-entrainment that cannot be resolved with other codes. The purpose of this work is to determine the attenuation of fission products in the RCS and on the secondary side of the steam generator in an accident initiated by a steam generator tube rupture (SGTR). As a class, bypass sequences have been identified in NUREG-1150 as being risk dominant for the Surry and Sequoyah pressurized water reactor (PWR) plants

  14. Facial Expression Recognition from Video Sequences Based on Spatial-Temporal Motion Local Binary Pattern and Gabor Multiorientation Fusion Histogram

    Directory of Open Access Journals (Sweden)

    Lei Zhao

    2017-01-01

    Full Text Available This paper proposes novel framework for facial expressions analysis using dynamic and static information in video sequences. First, based on incremental formulation, discriminative deformable face alignment method is adapted to locate facial points to correct in-plane head rotation and break up facial region from background. Then, spatial-temporal motion local binary pattern (LBP feature is extracted and integrated with Gabor multiorientation fusion histogram to give descriptors, which reflect static and dynamic texture information of facial expressions. Finally, a one-versus-one strategy based multiclass support vector machine (SVM classifier is applied to classify facial expressions. Experiments on Cohn-Kanade (CK + facial expression dataset illustrate that integrated framework outperforms methods using single descriptors. Compared with other state-of-the-art methods on CK+, MMI, and Oulu-CASIA VIS datasets, our proposed framework performs better.

  15. Comparison of DNA Quantification Methods for Next Generation Sequencing.

    Science.gov (United States)

    Robin, Jérôme D; Ludlow, Andrew T; LaRanger, Ryan; Wright, Woodring E; Shay, Jerry W

    2016-04-06

    Next Generation Sequencing (NGS) is a powerful tool that depends on loading a precise amount of DNA onto a flowcell. NGS strategies have expanded our ability to investigate genomic phenomena by referencing mutations in cancer and diseases through large-scale genotyping, developing methods to map rare chromatin interactions (4C; 5C and Hi-C) and identifying chromatin features associated with regulatory elements (ChIP-seq, Bis-Seq, ChiA-PET). While many methods are available for DNA library quantification, there is no unambiguous gold standard. Most techniques use PCR to amplify DNA libraries to obtain sufficient quantities for optical density measurement. However, increased PCR cycles can distort the library's heterogeneity and prevent the detection of rare variants. In this analysis, we compared new digital PCR technologies (droplet digital PCR; ddPCR, ddPCR-Tail) with standard methods for the titration of NGS libraries. DdPCR-Tail is comparable to qPCR and fluorometry (QuBit) and allows sensitive quantification by analysis of barcode repartition after sequencing of multiplexed samples. This study provides a direct comparison between quantification methods throughout a complete sequencing experiment and provides the impetus to use ddPCR-based quantification for improvement of NGS quality.

  16. SMITH: a LIMS for handling next-generation sequencing workflows.

    Science.gov (United States)

    Venco, Francesco; Vaskin, Yuriy; Ceol, Arnaud; Muller, Heiko

    2014-01-01

    Life-science laboratories make increasing use of Next Generation Sequencing (NGS) for studying bio-macromolecules and their interactions. Array-based methods for measuring gene expression or protein-DNA interactions are being replaced by RNA-Seq and ChIP-Seq. Sequencing is generally performed by specialized facilities that have to keep track of sequencing requests, trace samples, ensure quality and make data available according to predefined privileges. An integrated tool helps to troubleshoot problems, to maintain a high quality standard, to reduce time and costs. Commercial and non-commercial tools called LIMS (Laboratory Information Management Systems) are available for this purpose. However, they often come at prohibitive cost and/or lack the flexibility and scalability needed to adjust seamlessly to the frequently changing protocols employed. In order to manage the flow of sequencing data produced at the Genomic Unit of the Italian Institute of Technology (IIT), we developed SMITH (Sequencing Machine Information Tracking and Handling). SMITH is a web application with a MySQL server at the backend. Wet-lab scientists of the Centre for Genomic Science and database experts from the Politecnico of Milan in the context of a Genomic Data Model Project developed SMITH. The data base schema stores all the information of an NGS experiment, including the descriptions of all protocols and algorithms used in the process. Notably, an attribute-value table allows associating an unconstrained textual description to each sample and all the data produced afterwards. This method permits the creation of metadata that can be used to search the database for specific files as well as for statistical analyses. SMITH runs automatically and limits direct human interaction mainly to administrative tasks. SMITH data-delivery procedures were standardized making it easier for biologists and analysts to navigate the data. Automation also helps saving time. The workflows are available

  17. SMITH: a LIMS for handling next-generation sequencing workflows

    Science.gov (United States)

    2014-01-01

    Background Life-science laboratories make increasing use of Next Generation Sequencing (NGS) for studying bio-macromolecules and their interactions. Array-based methods for measuring gene expression or protein-DNA interactions are being replaced by RNA-Seq and ChIP-Seq. Sequencing is generally performed by specialized facilities that have to keep track of sequencing requests, trace samples, ensure quality and make data available according to predefined privileges. An integrated tool helps to troubleshoot problems, to maintain a high quality standard, to reduce time and costs. Commercial and non-commercial tools called LIMS (Laboratory Information Management Systems) are available for this purpose. However, they often come at prohibitive cost and/or lack the flexibility and scalability needed to adjust seamlessly to the frequently changing protocols employed. In order to manage the flow of sequencing data produced at the Genomic Unit of the Italian Institute of Technology (IIT), we developed SMITH (Sequencing Machine Information Tracking and Handling). Methods SMITH is a web application with a MySQL server at the backend. Wet-lab scientists of the Centre for Genomic Science and database experts from the Politecnico of Milan in the context of a Genomic Data Model Project developed SMITH. The data base schema stores all the information of an NGS experiment, including the descriptions of all protocols and algorithms used in the process. Notably, an attribute-value table allows associating an unconstrained textual description to each sample and all the data produced afterwards. This method permits the creation of metadata that can be used to search the database for specific files as well as for statistical analyses. Results SMITH runs automatically and limits direct human interaction mainly to administrative tasks. SMITH data-delivery procedures were standardized making it easier for biologists and analysts to navigate the data. Automation also helps saving time. The

  18. THE CONTRIBUTIONS OF INTERACTIVE BINARY STARS TO DOUBLE MAIN-SEQUENCE TURNOFFS AND DUAL RED CLUMP OF INTERMEDIATE-AGE STAR CLUSTERS

    International Nuclear Information System (INIS)

    Yang Wuming; Bi Shaolan; Tian Zhijia; Li Tanda; Liu Kang; Meng Xiangcun

    2011-01-01

    Double or extended main-sequence turnoffs (DMSTOs) and dual red clump (RC) were observed in intermediate-age clusters, such as in NGC 1846 and 419. The DMSTOs are interpreted as that the cluster has two distinct stellar populations with differences in age of about 200-300 Myr but with the same metallicity. The dual RC is interpreted as a result of a prolonged star formation. Using a stellar population-synthesis method, we calculated the evolution of a binary-star stellar population. We found that binary interactions and merging can reproduce the dual RC in the color-magnitude diagrams of an intermediate-age cluster, whereas in actuality only a single population exists. Moreover, the binary interactions can lead to an extended main-sequence turnoff (MSTO) rather than DMSTOs. However, the rest of the main sequence, subgiant branch, and first giant branch are hardly spread by the binary interactions. Part of the observed dual RC and extended MSTO may be the results of binary interactions and mergers.

  19. Generation of binary holograms with a Kinect sensor for a high speed color holographic display

    Science.gov (United States)

    Leportier, Thibault; Park, Min-Chul; Yano, Sumio; Son, Jung-Young

    2017-05-01

    The Kinect sensor is a device that enables to capture a real scene with a camera and a depth sensor. A virtual model of the scene can then be obtained with a point cloud representation. A complex hologram can then be computed. However, complex data cannot be used directly because display devices cannot handle amplitude and phase modulation at the same time. Binary holograms are commonly used since they present several advantages. Among the methods that were proposed to convert holograms into a binary format, the direct-binary search (DBS) not only gives the best performance, it also offers the possibility to choose the display parameters of the binary hologram differently than the original complex hologram. Since wavelength and reconstruction distance can be modified, compensation of chromatic aberrations can be handled. In this study, we examine the potential of DBS for RGB holographic display.

  20. Products of prime powers in binary recurrence sequences : part II. The elliptic case, with an application to a mixed quadratic-exponential equation

    NARCIS (Netherlands)

    Weger, de B.M.M.

    1986-01-01

    In Part I the diophantine equation [formula] was studied, where [formula] is a linear binary recurrence sequence with positive discriminant. In this second part we extend this to negative discriminants. We use the p-adic and complex Gelfond-Baker theory to find explicit upper bounds for the

  1. Products of prime powers in binary recurrence sequences : part I. The hyperbolic case, with an application to the generalized Ramanujan-Nagell equation

    NARCIS (Netherlands)

    Pethö, A.; Weger, de B.M.M.

    1986-01-01

    We show how the Gelfond-Baker theory and diophantine approximation techniques can be applied to solve explicitly the diophantine equation [formula] (where [formula] is a binary recurrence sequence with positive discriminant), for arbitrary values of the parameters. We apply this to the equation

  2. Next Generation Sequencing and ALS: known genes, different phenotyphes.

    Science.gov (United States)

    Campopiano, Rosa; Ryskalin, Larisa; Giardina, Emiliano; Zampatti, Stefania; Busceti, Carla L; Biagioni, Francesca; Ferese, Rosangela; Storto, Marianna; Gambardella, Stefano; Fornai, Francesco

    2017-12-01

    Amyotrophic lateral sclerosis (ALS) is fatal neurodegenerative disease clinically characterized by upper and lower motor neuron dysfunction resulting in rapidly progressive paralysis and death from respiratory failure. Most cases appear to be sporadic, but 5-10 % of cases have a family history of the disease, and over the last decade, identification of mutations in about 20 genes predisposing to these disorders has provided the means to better understand their pathogenesis. Next Generation sequencing (NGS) is an advanced high-throughput DNA sequencing technology which have rapidly contributed to an acceleration in the discovery of genetic risk factors for both familial and sporadic neurological and neurodegenerative diseases. These strategies allowed to rapidly identify disease-associated variants and genetic risk factors for both familial (fALS) and sporadic ALS (sALS), strongly contributing to the knowledge of the genetic architecture of ALS. Moreover, as the number of ALS genes grows, many of the proteins they encode are in intracellular processes shared with other known diseases, suggesting an overlapping of clinical and phatological features between different diseases. To emphasize this concept, the review focuses on genes coding for Valosin-containing protein (VPC) and two Heterogeneous nuclear RNA-binding proteins (HNRNPA1 and hnRNPA2B1), recently idefied through NGS, where different mutations have been associated in both ALS and other neurological and neurodegenerative diseases.

  3. Next-generation sequence analysis of cancer xenograft models.

    Directory of Open Access Journals (Sweden)

    Fernando J Rossello

    Full Text Available Next-generation sequencing (NGS studies in cancer are limited by the amount, quality and purity of tissue samples. In this situation, primary xenografts have proven useful preclinical models. However, the presence of mouse-derived stromal cells represents a technical challenge to their use in NGS studies. We examined this problem in an established primary xenograft model of small cell lung cancer (SCLC, a malignancy often diagnosed from small biopsy or needle aspirate samples. Using an in silico strategy that assign reads according to species-of-origin, we prospectively compared NGS data from primary xenograft models with matched cell lines and with published datasets. We show here that low-coverage whole-genome analysis demonstrated remarkable concordance between published genome data and internal controls, despite the presence of mouse genomic DNA. Exome capture sequencing revealed that this enrichment procedure was highly species-specific, with less than 4% of reads aligning to the mouse genome. Human-specific expression profiling with RNA-Seq replicated array-based gene expression experiments, whereas mouse-specific transcript profiles correlated with published datasets from human cancer stroma. We conclude that primary xenografts represent a useful platform for complex NGS analysis in cancer research for tumours with limited sample resources, or those with prominent stromal cell populations.

  4. Next generation sequencing of pancreatic ductal adenocarcinoma: right or wrong?

    Science.gov (United States)

    Connor, Ashton A; Gallinger, Steven

    2017-07-01

    Pancreatic ductal adenocarcinoma (PDAC) has the highest mortality rate of all epithelial malignancies and a paradoxically rising incidence rate. Clinical translation of next generation sequencing (NGS) of tumour and germline samples may ameliorate outcomes by identifying prognostic and predictive genomic and transcriptomic features in appreciable fractions of patients, facilitating enrolment in biomarker-matched trials. Areas covered: The literature on precision oncology is reviewed. It is found that outcomes may be improved across various malignancies, and it is suggested that current issues of adequate tissue acquisition, turnaround times, analytic expertise and clinical trial accessibility may lessen as experience accrues. Also reviewed are PDAC genomic and transcriptomic NGS studies, emphasizing discoveries of promising biomarkers, though these require validation, and the fraction of patients that will benefit from these outside of the research setting is currently unknown. Expert commentary: Clinical use of NGS with PDAC should be used in investigational contexts in centers with multidisciplinary expertise in cancer sequencing and pancreatic cancer management. Biomarker directed studies will improve our understanding of actionable genomic variation in PDAC, and improve outcomes for this challenging disease.

  5. Targeted next-generation sequencing in monogenic dyslipidemias.

    Science.gov (United States)

    Hegele, Robert A; Ban, Matthew R; Cao, Henian; McIntyre, Adam D; Robinson, John F; Wang, Jian

    2015-04-01

    To evaluate the potential clinical translation of high-throughput next-generation sequencing (NGS) methods in diagnosis and management of dyslipidemia. Recent NGS experiments indicate that most causative genes for monogenic dyslipidemias are already known. Thus, monogenic dyslipidemias can now be diagnosed using targeted NGS. Targeting of dyslipidemia genes can be achieved by either: designing custom reagents for a dyslipidemia-specific NGS panel; or performing genome-wide NGS and focusing on genes of interest. Advantages of the former approach are lower cost and limited potential to detect incidental pathogenic variants unrelated to dyslipidemia. However, the latter approach is more flexible because masking criteria can be altered as knowledge advances, with no need for re-design of reagents or follow-up sequencing runs. Also, the cost of genome-wide analysis is decreasing and ethical concerns can likely be mitigated. DNA-based diagnosis is already part of the clinical diagnostic algorithms for familial hypercholesterolemia. Furthermore, DNA-based diagnosis is supplanting traditional biochemical methods to diagnose chylomicronemia caused by deficiency of lipoprotein lipase or its co-factors. The increasing availability and decreasing cost of clinical NGS for dyslipidemia means that its potential benefits can now be evaluated on a larger scale.

  6. Binary classifiers and latent sequence models for emotion detection in suicide notes.

    Science.gov (United States)

    Cherry, Colin; Mohammad, Saif M; de Bruijn, Berry

    2012-01-01

    This paper describes the National Research Council of Canada's submission to the 2011 i2b2 NLP challenge on the detection of emotions in suicide notes. In this task, each sentence of a suicide note is annotated with zero or more emotions, making it a multi-label sentence classification task. We employ two distinct large-margin models capable of handling multiple labels. The first uses one classifier per emotion, and is built to simplify label balance issues and to allow extremely fast development. This approach is very effective, scoring an F-measure of 55.22 and placing fourth in the competition, making it the best system that does not use web-derived statistics or re-annotated training data. Second, we present a latent sequence model, which learns to segment the sentence into a number of emotion regions. This model is intended to gracefully handle sentences that convey multiple thoughts and emotions. Preliminary work with the latent sequence model shows promise, resulting in comparable performance using fewer features.

  7. Next Generation DNA Sequencing and the Future of Genomic Medicine

    OpenAIRE

    Anderson, Matthew W.; Schrijver, Iris

    2010-01-01

    In the years since the first complete human genome sequence was reported, there has been a rapid development of technologies to facilitate high-throughput sequence analysis of DNA (termed “next-generation” sequencing). These novel approaches to DNA sequencing offer the promise of complete genomic analysis at a cost feasible for routine clinical diagnostics. However, the ability to more thoroughly interrogate genomic sequence raises a number of important issues with regard to result interpreta...

  8. Visual programming for next-generation sequencing data analytics.

    Science.gov (United States)

    Milicchio, Franco; Rose, Rebecca; Bian, Jiang; Min, Jae; Prosperi, Mattia

    2016-01-01

    High-throughput or next-generation sequencing (NGS) technologies have become an established and affordable experimental framework in biological and medical sciences for all basic and translational research. Processing and analyzing NGS data is challenging. NGS data are big, heterogeneous, sparse, and error prone. Although a plethora of tools for NGS data analysis has emerged in the past decade, (i) software development is still lagging behind data generation capabilities, and (ii) there is a 'cultural' gap between the end user and the developer. Generic software template libraries specifically developed for NGS can help in dealing with the former problem, whilst coupling template libraries with visual programming may help with the latter. Here we scrutinize the state-of-the-art low-level software libraries implemented specifically for NGS and graphical tools for NGS analytics. An ideal developing environment for NGS should be modular (with a native library interface), scalable in computational methods (i.e. serial, multithread, distributed), transparent (platform-independent), interoperable (with external software interface), and usable (via an intuitive graphical user interface). These characteristics should facilitate both the run of standardized NGS pipelines and the development of new workflows based on technological advancements or users' needs. We discuss in detail the potential of a computational framework blending generic template programming and visual programming that addresses all of the current limitations. In the long term, a proper, well-developed (although not necessarily unique) software framework will bridge the current gap between data generation and hypothesis testing. This will eventually facilitate the development of novel diagnostic tools embedded in routine healthcare.

  9. Chronic Meningitis Investigated via Metagenomic Next-Generation Sequencing

    Science.gov (United States)

    O’Donovan, Brian D.; Gelfand, Jeffrey M.; Sample, Hannah A.; Chow, Felicia C.; Betjemann, John P.; Shah, Maulik P.; Richie, Megan B.; Gorman, Mark P.; Hajj-Ali, Rula A.; Calabrese, Leonard H.; Zorn, Kelsey C.; Chow, Eric D.; Greenlee, John E.; Blum, Jonathan H.; Green, Gary; Khan, Lillian M.; Banerji, Debarko; Langelier, Charles; Bryson-Cahn, Chloe; Harrington, Whitney; Lingappa, Jairam R.; Shanbhag, Niraj M.; Green, Ari J.; Brew, Bruce J.; Soldatos, Ariane; Strnad, Luke; Doernberg, Sarah B.; Jay, Cheryl A.; Douglas, Vanja; Josephson, S. Andrew; DeRisi, Joseph L.

    2018-01-01

    Importance Identifying infectious causes of subacute or chronic meningitis can be challenging. Enhanced, unbiased diagnostic approaches are needed. Objective To present a case series of patients with diagnostically challenging subacute or chronic meningitis using metagenomic next-generation sequencing (mNGS) of cerebrospinal fluid (CSF) supported by a statistical framework generated from mNGS of control samples from the environment and from patients who were noninfectious. Design, Setting, and Participants In this case series, mNGS data obtained from the CSF of 94 patients with noninfectious neuroinflammatory disorders and from 24 water and reagent control samples were used to develop and implement a weighted scoring metric based on z scores at the species and genus levels for both nucleotide and protein alignments to prioritize and rank the mNGS results. Total RNA was extracted for mNGS from the CSF of 7 participants with subacute or chronic meningitis who were recruited between September 2013 and March 2017 as part of a multicenter study of mNGS pathogen discovery among patients with suspected neuroinflammatory conditions. The neurologic infections identified by mNGS in these 7 participants represented a diverse array of pathogens. The patients were referred from the University of California, San Francisco Medical Center (n = 2), Zuckerberg San Francisco General Hospital and Trauma Center (n = 2), Cleveland Clinic (n = 1), University of Washington (n = 1), and Kaiser Permanente (n = 1). A weighted z score was used to filter out environmental contaminants and facilitate efficient data triage and analysis. Main Outcomes and Measures Pathogens identified by mNGS and the ability of a statistical model to prioritize, rank, and simplify mNGS results. Results The 7 participants ranged in age from 10 to 55 years, and 3 (43%) were female. A parasitic worm (Taenia solium, in 2 participants), a virus (HIV-1), and 4 fungi (Cryptococcus neoformans

  10. Authentication of Herbal Supplements Using Next-Generation Sequencing.

    Directory of Open Access Journals (Sweden)

    Natalia V Ivanova

    Full Text Available DNA-based testing has been gaining acceptance as a tool for authentication of a wide range of food products; however, its applicability for testing of herbal supplements remains contentious.We utilized Sanger and Next-Generation Sequencing (NGS for taxonomic authentication of fifteen herbal supplements representing three different producers from five medicinal plants: Echinacea purpurea, Valeriana officinalis, Ginkgo biloba, Hypericum perforatum and Trigonella foenum-graecum. Experimental design included three modifications of DNA extraction, two lysate dilutions, Internal Amplification Control, and multiple negative controls to exclude background contamination. Ginkgo supplements were also analyzed using HPLC-MS for the presence of active medicinal components.All supplements yielded DNA from multiple species, rendering Sanger sequencing results for rbcL and ITS2 regions either uninterpretable or non-reproducible between the experimental replicates. Overall, DNA from the manufacturer-listed medicinal plants was successfully detected in seven out of eight dry herb form supplements; however, low or poor DNA recovery due to degradation was observed in most plant extracts (none detected by Sanger; three out of seven-by NGS. NGS also revealed a diverse community of fungi, known to be associated with live plant material and/or the fermentation process used in the production of plant extracts. HPLC-MS testing demonstrated that Ginkgo supplements with degraded DNA contained ten key medicinal components.Quality control of herbal supplements should utilize a synergetic approach targeting both DNA and bioactive components, especially for standardized extracts with degraded DNA. The NGS workflow developed in this study enables reliable detection of plant and fungal DNA and can be utilized by manufacturers for quality assurance of raw plant materials, contamination control during the production process, and the final product. Interpretation of results should

  11. [Diagnosis of mitochondrial disorders in children with next generation sequencing].

    Science.gov (United States)

    Liu, Zhimei; Fang, Fang; Ding, Changhong; Zhang, Weihua; Li, Jiuwei; Yang, Xinying; Wang, Xiaohui; Wu, Yun; Wang, Hongmei; Liu, Liying; Han, Tongli; Wang, Xu; Chen, Chunhong; Lyu, Junlan; Wu, Husheng

    2015-10-01

    To explore the application value of next generation sequencing (NGS) in the diagnosis of mitochondrial disorders. According to mitochondrial disease criteria, genomic DNA was extracted using standard procedure from peripheral venous blood of patients with suspected mitochondrial disease collected from neurological department of Beijing Children's Hospital Affiliated to Capital Medical University between October 2012 and February 2014. Targeted NGS to capture and sequence the entire mtDNA and exons of the 1 000 nuclear genes related to mitochondrial structure and function. Clinical data were collected from patients diagnosed at a molecular level, then clinical features and the relationship between genotype and phenotype were analyzed. Mutation was detected in 21 of 70 patients with suspected mitochondrial disease, in whom 10 harbored mtDNA mutation, while 11 nuclear DNA (nDNA) mutation. In 21 patients, 1 was diagnosed congenital myasthenic syndrome with episodic apnea due to CHAT gene p.I187T homozygous mutation, and 20 were diagnosed mitochondrial disease, in which 10 were Leigh syndrome, 4 were mitochondrial encephalomyopathy with lactic acidosis and stroke like episodes syndrome, 3 were Leber hereditary optic neuropathy (LHON) and LHON plus, 2 were mitochondrial DNA depletion syndrome and 1 was unknown. All the mtDNA mutations were point mutations, which contained A3243G, G3460A, G11778A, T14484C, T14502C and T14487C. Ten mitochondrial disease patients harbored homozygous or compound heterozygous mutations in 5 genes previously shown to cause disease: SURF1, PDHA1, NDUFV1, SUCLA2 and SUCLG1, which had 14 mutations, and 7 of the 14 mutations have not been reported. NGS has a certain application value in the diagnosis of mitochondrial diseases, especially in Leigh syndrome atypical mitochondrial syndrome and rare mitochondrial disorders.

  12. Authentication of Herbal Supplements Using Next-Generation Sequencing.

    Science.gov (United States)

    Ivanova, Natalia V; Kuzmina, Maria L; Braukmann, Thomas W A; Borisenko, Alex V; Zakharov, Evgeny V

    2016-01-01

    DNA-based testing has been gaining acceptance as a tool for authentication of a wide range of food products; however, its applicability for testing of herbal supplements remains contentious. We utilized Sanger and Next-Generation Sequencing (NGS) for taxonomic authentication of fifteen herbal supplements representing three different producers from five medicinal plants: Echinacea purpurea, Valeriana officinalis, Ginkgo biloba, Hypericum perforatum and Trigonella foenum-graecum. Experimental design included three modifications of DNA extraction, two lysate dilutions, Internal Amplification Control, and multiple negative controls to exclude background contamination. Ginkgo supplements were also analyzed using HPLC-MS for the presence of active medicinal components. All supplements yielded DNA from multiple species, rendering Sanger sequencing results for rbcL and ITS2 regions either uninterpretable or non-reproducible between the experimental replicates. Overall, DNA from the manufacturer-listed medicinal plants was successfully detected in seven out of eight dry herb form supplements; however, low or poor DNA recovery due to degradation was observed in most plant extracts (none detected by Sanger; three out of seven-by NGS). NGS also revealed a diverse community of fungi, known to be associated with live plant material and/or the fermentation process used in the production of plant extracts. HPLC-MS testing demonstrated that Ginkgo supplements with degraded DNA contained ten key medicinal components. Quality control of herbal supplements should utilize a synergetic approach targeting both DNA and bioactive components, especially for standardized extracts with degraded DNA. The NGS workflow developed in this study enables reliable detection of plant and fungal DNA and can be utilized by manufacturers for quality assurance of raw plant materials, contamination control during the production process, and the final product. Interpretation of results should involve an

  13. DNA Qualification Workflow for Next Generation Sequencing of Histopathological Samples

    Science.gov (United States)

    Simbolo, Michele; Gottardi, Marisa; Corbo, Vincenzo; Fassan, Matteo; Mafficini, Andrea; Malpeli, Giorgio; Lawlor, Rita T.; Scarpa, Aldo

    2013-01-01

    Histopathological samples are a treasure-trove of DNA for clinical research. However, the quality of DNA can vary depending on the source or extraction method applied. Thus a standardized and cost-effective workflow for the qualification of DNA preparations is essential to guarantee interlaboratory reproducible results. The qualification process consists of the quantification of double strand DNA (dsDNA) and the assessment of its suitability for downstream applications, such as high-throughput next-generation sequencing. We tested the two most frequently used instrumentations to define their role in this process: NanoDrop, based on UV spectroscopy, and Qubit 2.0, which uses fluorochromes specifically binding dsDNA. Quantitative PCR (qPCR) was used as the reference technique as it simultaneously assesses DNA concentration and suitability for PCR amplification. We used 17 genomic DNAs from 6 fresh-frozen (FF) tissues, 6 formalin-fixed paraffin-embedded (FFPE) tissues, 3 cell lines, and 2 commercial preparations. Intra- and inter-operator variability was negligible, and intra-methodology variability was minimal, while consistent inter-methodology divergences were observed. In fact, NanoDrop measured DNA concentrations higher than Qubit and its consistency with dsDNA quantification by qPCR was limited to high molecular weight DNA from FF samples and cell lines, where total DNA and dsDNA quantity virtually coincide. In partially degraded DNA from FFPE samples, only Qubit proved highly reproducible and consistent with qPCR measurements. Multiplex PCR amplifying 191 regions of 46 cancer-related genes was designated the downstream application, using 40 ng dsDNA from FFPE samples calculated by Qubit. All but one sample produced amplicon libraries suitable for next-generation sequencing. NanoDrop UV-spectrum verified contamination of the unsuccessful sample. In conclusion, as qPCR has high costs and is labor intensive, an alternative effective standard workflow for

  14. DNA qualification workflow for next generation sequencing of histopathological samples.

    Directory of Open Access Journals (Sweden)

    Michele Simbolo

    Full Text Available Histopathological samples are a treasure-trove of DNA for clinical research. However, the quality of DNA can vary depending on the source or extraction method applied. Thus a standardized and cost-effective workflow for the qualification of DNA preparations is essential to guarantee interlaboratory reproducible results. The qualification process consists of the quantification of double strand DNA (dsDNA and the assessment of its suitability for downstream applications, such as high-throughput next-generation sequencing. We tested the two most frequently used instrumentations to define their role in this process: NanoDrop, based on UV spectroscopy, and Qubit 2.0, which uses fluorochromes specifically binding dsDNA. Quantitative PCR (qPCR was used as the reference technique as it simultaneously assesses DNA concentration and suitability for PCR amplification. We used 17 genomic DNAs from 6 fresh-frozen (FF tissues, 6 formalin-fixed paraffin-embedded (FFPE tissues, 3 cell lines, and 2 commercial preparations. Intra- and inter-operator variability was negligible, and intra-methodology variability was minimal, while consistent inter-methodology divergences were observed. In fact, NanoDrop measured DNA concentrations higher than Qubit and its consistency with dsDNA quantification by qPCR was limited to high molecular weight DNA from FF samples and cell lines, where total DNA and dsDNA quantity virtually coincide. In partially degraded DNA from FFPE samples, only Qubit proved highly reproducible and consistent with qPCR measurements. Multiplex PCR amplifying 191 regions of 46 cancer-related genes was designated the downstream application, using 40 ng dsDNA from FFPE samples calculated by Qubit. All but one sample produced amplicon libraries suitable for next-generation sequencing. NanoDrop UV-spectrum verified contamination of the unsuccessful sample. In conclusion, as qPCR has high costs and is labor intensive, an alternative effective standard

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

    DEFF Research Database (Denmark)

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

    2014-01-01

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

  16. Analysis of heart rate and oxygen uptake kinetics studied by two different pseudo-random binary sequence work rate amplitudes.

    Science.gov (United States)

    Drescher, U; Koschate, J; Schiffer, T; Schneider, S; Hoffmann, U

    2017-06-01

    The aim of the study was to compare the kinetics responses of heart rate (HR), pulmonary (V˙O 2 pulm) and predicted muscular (V˙O 2 musc) oxygen uptake between two different pseudo-random binary sequence (PRBS) work rate (WR) amplitudes both below anaerobic threshold. Eight healthy individuals performed two PRBS WR protocols implying changes between 30W and 80W and between 30W and 110W. HR and V˙O 2 pulm were measured beat-to-beat and breath-by-breath, respectively. V˙O 2 musc was estimated applying the approach of Hoffmann et al. (Eur J Appl Physiol 113: 1745-1754, 2013) considering a circulatory model for venous return and cross-correlation functions (CCF) for the kinetics analysis. HR and V˙O 2 musc kinetics seem to be independent of WR intensity (p>0.05). V˙O 2 pulm kinetics show prominent differences in the lag of the CCF maximum (39±9s; 31±4s; p<0.05). A mean difference of 14W between the PRBS WR amplitudes impacts venous return significantly, while HR and V˙O 2 musc kinetics remain unchanged. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Observation of a sequence of wetting transitions in the binary water+ethylene glycol monobutyl ether mixture

    Science.gov (United States)

    Wu, Chih-Kang; Chen, Li-Jen

    2005-08-01

    A homemade pendant drop/bubble tensiometer was assembled and applied to perform the surface-interfacial tension measurements for the binary water+ethylene glycol monobutyl ether (C4E1) mixture over the temperature range from 50to128°C at 10bar. The symbol CiEj is the abbreviation of a nonionic polyoxyethylene alcohol CiH2i+1(OCH2CH2)jOH. The wetting behavior of the C4E1-rich phase at the interface separating the gas and the aqueous phases was systematically examined according to the wetting coefficient calculated from the experimental results of surface/interfacial tensions. It was found that the C4E1-rich phase exhibits a sequence of wetting transitions, nonwetting→partial wetting→complete wetting, at the gas-water interface in the water+C4E1 system along with increasing the temperature, consistent with the conjecture of Kahlweit and Busse [J. Chem. Phys. 91, 1339 (1989)]. In addition, the relationship of the mutual solubility and the interfacial tension of the interface separating the C4E1-rich phase and the aqueous phase is discussed.

  18. Accelerating next generation sequencing data analysis with system level optimizations.

    Science.gov (United States)

    Kathiresan, Nagarajan; Temanni, Ramzi; Almabrazi, Hakeem; Syed, Najeeb; Jithesh, Puthen V; Al-Ali, Rashid

    2017-08-22

    Next generation sequencing (NGS) data analysis is highly compute intensive. In-memory computing, vectorization, bulk data transfer, CPU frequency scaling are some of the hardware features in the modern computing architectures. To get the best execution time and utilize these hardware features, it is necessary to tune the system level parameters before running the application. We studied the GATK-HaplotypeCaller which is part of common NGS workflows, that consume more than 43% of the total execution time. Multiple GATK 3.x versions were benchmarked and the execution time of HaplotypeCaller was optimized by various system level parameters which included: (i) tuning the parallel garbage collection and kernel shared memory to simulate in-memory computing, (ii) architecture-specific tuning in the PairHMM library for vectorization, (iii) including Java 1.8 features through GATK source code compilation and building a runtime environment for parallel sorting and bulk data transfer (iv) the default 'on-demand' mode of CPU frequency is over-clocked by using 'performance-mode' to accelerate the Java multi-threads. As a result, the HaplotypeCaller execution time was reduced by 82.66% in GATK 3.3 and 42.61% in GATK 3.7. Overall, the execution time of NGS pipeline was reduced to 70.60% and 34.14% for GATK 3.3 and GATK 3.7 respectively.

  19. RANDOMNUMBERS, Random Number Sequence Generated from Gas Ionisation Chamber Data

    International Nuclear Information System (INIS)

    Frigerio, N.A.; Sanathanan, L.P.; Morley, M.; Tyler, S.A.; Clark, N.A.; Wang, J.

    1989-01-01

    1 - Description of problem or function: RANDOM NUMBERS is a data collection of almost 2.7 million 31-bit random numbers generated by using a high resolution gas ionization detector chamber in conjunction with a 4096-channel multichannel analyzer to record the radioactive decay of alpha particles from a U-235 source. The signals from the decaying alpha particles were fed to the 4096-channel analyzer, and for each channel the frequency of signals registered in a 20,000-microsecond interval was recorded. The parity bits of these frequency counts, 0 for an even count and 1 for and odd count, were then assembled in sequence to form 31-bit random numbers and transcribed onto magnetic tape. This cycle was repeated to obtain the random numbers. 2 - Method of solution: The frequency distribution of counts from the device conforms to the Brockwell-Moyal distribution which takes into account the dead time of the counter. The count data were analyzed and tests for randomness on a sample indicate that the device is a highly reliable source of truly random numbers. 3 - Restrictions on the complexity of the problem: The RANDOM NUMBERS tape contains 2,669,568 31-bit numbers

  20. Somatic mutations in histiocytic sarcoma identified by next generation sequencing.

    Science.gov (United States)

    Liu, Qingqing; Tomaszewicz, Keith; Hutchinson, Lloyd; Hornick, Jason L; Woda, Bruce; Yu, Hongbo

    2016-08-01

    Histiocytic sarcoma is a rare malignant neoplasm of presumed hematopoietic origin showing morphologic and immunophenotypic evidence of histiocytic differentiation. Somatic mutation importance in the pathogenesis or disease progression of histiocytic sarcoma was largely unknown. To identify somatic mutations in histiocytic sarcoma, we studied 5 histiocytic sarcomas [3 female and 2 male patients; mean age 54.8 (20-72), anatomic sites include lymph node, uterus, and pleura] and matched normal tissues from each patient as germ line controls. Somatic mutations in 50 "Hotspot" oncogenes and tumor suppressor genes were examined using next generation sequencing. Three (out of five) histiocytic sarcoma cases carried somatic mutations in BRAF. Among them, G464V [variant frequency (VF) of 43.6 %] and G466R (VF of 29.6 %) located at the P loop potentially interfere with the hydrophobic interaction between P and activating loops and ultimately activation of BRAF. Also detected was BRAF somatic mutation N581S (VF of 7.4 %), which was located at the catalytic loop of BRAF kinase domain: its role in modifying kinase activity was unclear. A similar mutational analysis was also performed on nine acute monocytic/monoblastic leukemia cases, which did not identify any BRAF somatic mutations. Our study detected several BRAF mutations in histiocytic sarcomas, which may be important in understanding the tumorigenesis of this rare neoplasm and providing mechanisms for potential therapeutical opportunities.

  1. Next generation sequencing and comparative analyses of Xenopus mitogenomes

    Directory of Open Access Journals (Sweden)

    Lloyd Rhiannon E

    2012-09-01

    -coding genes were shown to be under strong negative (purifying selection, with genes under the strongest pressure (Complex 4 also being the most highly expressed, highlighting their potentially crucial functions in the mitochondrial respiratory chain. Conclusions Next generation sequencing of long-PCR amplicons using single taxon or multi-taxon approaches enabled two new species of Xenopus mtDNA to be fully characterized. We anticipate our complete mitochondrial genome amplification methods to be applicable to other amphibians, helpful for identifying the most appropriate markers for differentiating species, populations and resolving phylogenies, a pressing need since amphibians are undergoing drastic global decline. Our mtDNAs also provide templates for conserved primer design and the assembly of RNA and DNA reads following high throughput “omic” techniques such as RNA- and ChIP-seq. These could help us better understand how processes such mitochondrial replication and gene expression influence xenopus growth and development, as well as how they evolved and are regulated.

  2. Localization of binary neutron star mergers with second and third generation gravitational-wave detectors

    Science.gov (United States)

    Mills, Cameron; Tiwari, Vaibhav; Fairhurst, Stephen

    2018-05-01

    The observation of gravitational wave signals from binary black hole and binary neutron star mergers has established the field of gravitational wave astronomy. It is expected that future networks of gravitational wave detectors will possess great potential in probing various aspects of astronomy. An important consideration for successive improvement of current detectors or establishment on new sites is knowledge of the minimum number of detectors required to perform precision astronomy. We attempt to answer this question by assessing the ability of future detector networks to detect and localize binary neutron stars mergers on the sky. Good localization ability is crucial for many of the scientific goals of gravitational wave astronomy, such as electromagnetic follow-up, measuring the properties of compact binaries throughout cosmic history, and cosmology. We find that although two detectors at improved sensitivity are sufficient to get a substantial increase in the number of observed signals, at least three detectors of comparable sensitivity are required to localize majority of the signals, typically to within around 10 deg2 —adequate for follow-up with most wide field of view optical telescopes.

  3. Automated Generation of Phase Diagrams for Binary Systems with Azeotropic Behavior

    DEFF Research Database (Denmark)

    Cismondi, Martin; Michelsen, Michael Locht; Zabaloy, Marcelo S.

    2008-01-01

    In this work, we propose a computational strategy and methods for the automated calculation of complete loci of homogeneous azeotropy of binary mixtures and the related Pxy and Txy diagrams for models of the equation-of-state (EOS) type. The strategy consists of first finding the system...

  4. KNIME4NGS: a comprehensive toolbox for next generation sequencing analysis.

    Science.gov (United States)

    Hastreiter, Maximilian; Jeske, Tim; Hoser, Jonathan; Kluge, Michael; Ahomaa, Kaarin; Friedl, Marie-Sophie; Kopetzky, Sebastian J; Quell, Jan-Dominik; Mewes, H Werner; Küffner, Robert

    2017-05-15

    Analysis of Next Generation Sequencing (NGS) data requires the processing of large datasets by chaining various tools with complex input and output formats. In order to automate data analysis, we propose to standardize NGS tasks into modular workflows. This simplifies reliable handling and processing of NGS data, and corresponding solutions become substantially more reproducible and easier to maintain. Here, we present a documented, linux-based, toolbox of 42 processing modules that are combined to construct workflows facilitating a variety of tasks such as DNAseq and RNAseq analysis. We also describe important technical extensions. The high throughput executor (HTE) helps to increase the reliability and to reduce manual interventions when processing complex datasets. We also provide a dedicated binary manager that assists users in obtaining the modules' executables and keeping them up to date. As basis for this actively developed toolbox we use the workflow management software KNIME. See http://ibisngs.github.io/knime4ngs for nodes and user manual (GPLv3 license). robert.kueffner@helmholtz-muenchen.de. Supplementary data are available at Bioinformatics online.

  5. Next generation sequencing (NGS)technologies and applications

    Energy Technology Data Exchange (ETDEWEB)

    Vuyisich, Momchilo [Los Alamos National Laboratory

    2012-09-11

    NGS technology overview: (1) NGS library preparation - Nucleic acids extraction, Sample quality control, RNA conversion to cDNA, Addition of sequencing adapters, Quality control of library; (2) Sequencing - Clonal amplification of library fragments, (except PacBio), Sequencing by synthesis, Data output (reads and quality); and (3) Data analysis - Read mapping, Genome assembly, Gene expression, Operon structure, sRNA discovery, and Epigenetic analyses.

  6. Applications of Next-Generation Sequencing Technologies to Diagnostic Virology

    Directory of Open Access Journals (Sweden)

    Giorgio Palù

    2011-11-01

    Full Text Available Novel DNA sequencing techniques, referred to as “next-generation” sequencing (NGS, provide high speed and throughput that can produce an enormous volume of sequences with many possible applications in research and diagnostic settings. In this article, we provide an overview of the many applications of NGS in diagnostic virology. NGS techniques have been used for high-throughput whole viral genome sequencing, such as sequencing of new influenza viruses, for detection of viral genome variability and evolution within the host, such as investigation of human immunodeficiency virus and human hepatitis C virus quasispecies, and monitoring of low-abundance antiviral drug-resistance mutations. NGS techniques have been applied to metagenomics-based strategies for the detection of unexpected disease-associated viruses and for the discovery of novel human viruses, including cancer-related viruses. Finally, the human virome in healthy and disease conditions has been described by NGS-based metagenomics.

  7. Sufficient condition for generation of multiple solidification front in one-dimensional solidification of binary alloys

    International Nuclear Information System (INIS)

    Bobula, E.; Kalicka, Z.

    1981-10-01

    In the paper we consider the one-dimensional solidification of binary alloys in the finite system. The authors present the sufficient condition for solidification in the liquid in front of the moving solid-liquid interface. The effect may produce a fluctuating concentration distributin in the solid. The convection in the liquid and supercooling required for homogeneous nucleation are omitted. A local-equilibrium approximation at the liquid-solid interface is supposed. (author)

  8. Biomolecule Sequencer: Next-Generation DNA Sequencing Technology for In-Flight Environmental Monitoring, Research, and Beyond

    Science.gov (United States)

    Smith, David J.; Burton, Aaron; Castro-Wallace, Sarah; John, Kristen; Stahl, Sarah E.; Dworkin, Jason Peter; Lupisella, Mark L.

    2016-01-01

    On the International Space Station (ISS), technologies capable of rapid microbial identification and disease diagnostics are not currently available. NASA still relies upon sample return for comprehensive, molecular-based sample characterization. Next-generation DNA sequencing is a powerful approach for identifying microorganisms in air, water, and surfaces onboard spacecraft. The Biomolecule Sequencer payload, manifested to SpaceX-9 and scheduled on the Increment 4748 research plan (June 2016), will assess the functionality of a commercially-available next-generation DNA sequencer in the microgravity environment of ISS. The MinION device from Oxford Nanopore Technologies (Oxford, UK) measures picoamp changes in electrical current dependent on nucleotide sequences of the DNA strand migrating through nanopores in the system. The hardware is exceptionally small (9.5 x 3.2 x 1.6 cm), lightweight (120 grams), and powered only by a USB connection. For the ISS technology demonstration, the Biomolecule Sequencer will be powered by a Microsoft Surface Pro3. Ground-prepared samples containing lambda bacteriophage, Escherichia coli, and mouse genomic DNA, will be launched and stored frozen on the ISS until experiment initiation. Immediately prior to sequencing, a crew member will collect and thaw frozen DNA samples, connect the sequencer to the Surface Pro3, inject thawed samples into a MinION flow cell, and initiate sequencing. At the completion of the sequencing run, data will be downlinked for ground analysis. Identical, synchronous ground controls will be used for data comparisons to determine sequencer functionality, run-time sequence, current dynamics, and overall accuracy. We will present our latest results from the ISS flight experiment the first time DNA has ever been sequenced in space and discuss the many potential applications of the Biomolecule Sequencer for environmental monitoring, medical diagnostics, higher fidelity and more adaptable Space Biology Human

  9. How Next-Generation Sequencing Has Aided Our Understanding of the Sequence Composition and Origin of B Chromosomes

    Directory of Open Access Journals (Sweden)

    Alevtina Ruban

    2017-10-01

    Full Text Available Accessory, supernumerary, or—most simply—B chromosomes, are found in many eukaryotic karyotypes. These small chromosomes do not follow the usual pattern of segregation, but rather are transmitted in a higher than expected frequency. As increasingly being demonstrated by next-generation sequencing (NGS, their structure comprises fragments of standard (A chromosomes, although in some plant species, their sequence also includes contributions from organellar genomes. Transcriptomic analyses of various animal and plant species have revealed that, contrary to what used to be the common belief, some of the B chromosome DNA is protein-encoding. This review summarizes the progress in understanding B chromosome biology enabled by the application of next-generation sequencing technology and state-of-the-art bioinformatics. In particular, a contrast is drawn between a direct sequencing approach and a strategy based on a comparative genomics as alternative routes that can be taken towards the identification of B chromosome sequences.

  10. Study and realisation of a programmable generator of pulse sequences, for nuclear magnetic resonance

    International Nuclear Information System (INIS)

    Lambert, Daniel

    1974-01-01

    After having recalled the operation of pulse-based nuclear magnetic resonance and the use of pulse sequences in NMR-based measurements, and outlined the need for a pulse sequence generator, the author reports the design and realisation of such a device. He describes its general organisation with its base sequence, base clock, sequence start, duration, displays, data transfers, data processing, and signal distribution. He presents the chosen technology (ECL logics), the sequence base set, time bases, multiplexers, comparison sets, the distribution set, the sequence programming, the sampling and output set. He reports tests and the use of the so-designed generator [fr

  11. Enrichment of target sequences for next-generation sequencing applications in research and diagnostics.

    Science.gov (United States)

    Altmüller, Janine; Budde, Birgit S; Nürnberg, Peter

    2014-02-01

    Abstract Targeted re-sequencing such as gene panel sequencing (GPS) has become very popular in medical genetics, both for research projects and in diagnostic settings. The technical principles of the different enrichment methods have been reviewed several times before; however, new enrichment products are constantly entering the market, and researchers are often puzzled about the requirement to take decisions about long-term commitments, both for the enrichment product and the sequencing technology. This review summarizes important considerations for the experimental design and provides helpful recommendations in choosing the best sequencing strategy for various research projects and diagnostic applications.

  12. Generation of Polymer Nanocomposites through Shear-Driven Aggregation of Binary Colloids

    Directory of Open Access Journals (Sweden)

    Xinxin Sheng

    2017-11-01

    Full Text Available Design of polymer nanocomposites has been an intense research topic in recent decades because hybrid nanomaterials are widely used in many fields. Throughout their development, there has often been a challenging issue how one can uniformly distribute nanoparticles (NPs in a polymer matrix, avoiding their agglomeration. In this short review, we first introduce the theory of colloidal aggregation/gelation purely based on intense shear forces. Then, we illustrate a methodology for preparing polymer nanocomposites where the NPs (as fillers are uniformly and randomly distributed inside a matrix of polymer NPs, based on intense shear-driven aggregation of binary colloids, without using any additives. Its feasibility has been demonstrated using two stable binary colloids composed of (1 poly-methyl methacrylate fillers and polystyrene NPs, and (2 graphene oxide sheets (fillers and poly-vinylidene fluoride NPs. The mechanism leading to capturing and distribution of the fillers inside the polymer NP matrix has been illustrated, and the advantages of the proposed methodology compared with the other common methods are also discussed.

  13. Applications and Case Studies of the Next-Generation Sequencing Technologies in Food, Nutrition and Agriculture.

    Science.gov (United States)

    Next-generation sequencing technologies are able to produce high-throughput short sequence reads in a cost-effective fashion. The emergence of these technologies has not only facilitated genome sequencing but also changed the landscape of life sciences. Here I survey their major applications ranging...

  14. Reprint of "Application of next generation sequencing in clinical microbiology and infection prevention"

    NARCIS (Netherlands)

    Deurenberg, Ruud H.; Bathoorn, Erik; Chlebowicz, Monika A.; Monge Gomes do Couto, Natacha; Ferdous, Mithila; Garcia-Cobos, Silvia; Kooistra-Smid, Anna M. D.; Raangs, Erwin C.; Rosema, Sigrid; Veloo, Alida C. M.; Zhou, Kai; Friedrich, Alexander W.; Rossen, John W. A.

    2017-01-01

    Current molecular diagnostics of human pathogens provide limited information that is often not sufficient for outbreak and transmission investigation. Next generation sequencing (NGS) determines the DNA sequence of a complete bacterial genome in a single sequence run, and from these data,

  15. Application of next generation sequencing in clinical microbiology and infection prevention

    NARCIS (Netherlands)

    Deurenberg, Ruud H.; Bathoorn, Erik; Chlebowicz, Monika A.; Couto, Natacha; Ferdous, Mithila; Garcia-Cobos, Silvia; Kooistra-Smid, Anna M. D.; Raangs, Erwin C.; Rosema, Sigrid; Veloo, Alida C. M.; Zhou, Kai; Friedrich, Alexander W.; Rossen, John W. A.

    2017-01-01

    Current molecular diagnostics of human pathogens provide limited information that is often not sufficient for outbreak and transmission investigation. Next generation sequencing (NGS) determines the DNA sequence of a complete bacterial genome in a single sequence run, and from these data,

  16. Generation and analysis of expressed sequence tags from Botrytis cinerea

    Directory of Open Access Journals (Sweden)

    EVELYN SILVA

    2006-01-01

    Full Text Available Botrytis cinerea is a filamentous plant pathogen of a wide range of plant species, and its infection may cause enormous damage both during plant growth and in the post-harvest phase. We have constructed a cDNA library from an isolate of B. cinerea and have sequenced 11,482 expressed sequence tags that were assembled into 1,003 contigs sequences and 3,032 singletons. Approximately 81% of the unigenes showed significant similarity to genes coding for proteins with known functions: more than 50% of the sequences code for genes involved in cellular metabolism, 12% for transport of metabolites, and approximately 10% for cellular organization. Other functional categories include responses to biotic and abiotic stimuli, cell communication, cell homeostasis, and cell development. We carried out pair-wise comparisons with fungal databases to determine the B. cinerea unisequence set with relevant similarity to genes in other fungal pathogenic counterparts. Among the 4,035 non-redundant B. cinerea unigenes, 1,338 (23% have significant homology with Fusarium verticillioides unigenes. Similar values were obtained for Saccharomyces cerevisiae and Aspergillus nidulans (22% and 24%, respectively. The lower percentages of homology were with Magnaporthe grisae and Neurospora crassa (13% and 19%, respectively. Several genes involved in putative and known fungal virulence and general pathogenicity were identified. The results provide important information for future research on this fungal pathogen

  17. Next-generation sequencing library preparation method for identification of RNA viruses on the Ion Torrent Sequencing Platform.

    Science.gov (United States)

    Chen, Guiqian; Qiu, Yuan; Zhuang, Qingye; Wang, Suchun; Wang, Tong; Chen, Jiming; Wang, Kaicheng

    2018-05-09

    Next generation sequencing (NGS) is a powerful tool for the characterization, discovery, and molecular identification of RNA viruses. There were multiple NGS library preparation methods published for strand-specific RNA-seq, but some methods are not suitable for identifying and characterizing RNA viruses. In this study, we report a NGS library preparation method to identify RNA viruses using the Ion Torrent PGM platform. The NGS sequencing adapters were directly inserted into the sequencing library through reverse transcription and polymerase chain reaction, without fragmentation and ligation of nucleic acids. The results show that this method is simple to perform, able to identify multiple species of RNA viruses in clinical samples.

  18. Autonomously generating operations sequences for a Mars Rover using AI-based planning

    Science.gov (United States)

    Sherwood, Rob; Mishkin, Andrew; Estlin, Tara; Chien, Steve; Backes, Paul; Cooper, Brian; Maxwell, Scott; Rabideau, Gregg

    2001-01-01

    This paper discusses a proof-of-concept prototype for ground-based automatic generation of validated rover command sequences from highlevel science and engineering activities. This prototype is based on ASPEN, the Automated Scheduling and Planning Environment. This Artificial Intelligence (AI) based planning and scheduling system will automatically generate a command sequence that will execute within resource constraints and satisfy flight rules.

  19. Identification of optimum sequencing depth especially for de novo genome assembly of small genomes using next generation sequencing data.

    Science.gov (United States)

    Desai, Aarti; Marwah, Veer Singh; Yadav, Akshay; Jha, Vineet; Dhaygude, Kishor; Bangar, Ujwala; Kulkarni, Vivek; Jere, Abhay

    2013-01-01

    Next Generation Sequencing (NGS) is a disruptive technology that has found widespread acceptance in the life sciences research community. The high throughput and low cost of sequencing has encouraged researchers to undertake ambitious genomic projects, especially in de novo genome sequencing. Currently, NGS systems generate sequence data as short reads and de novo genome assembly using these short reads is computationally very intensive. Due to lower cost of sequencing and higher throughput, NGS systems now provide the ability to sequence genomes at high depth. However, currently no report is available highlighting the impact of high sequence depth on genome assembly using real data sets and multiple assembly algorithms. Recently, some studies have evaluated the impact of sequence coverage, error rate and average read length on genome assembly using multiple assembly algorithms, however, these evaluations were performed using simulated datasets. One limitation of using simulated datasets is that variables such as error rates, read length and coverage which are known to impact genome assembly are carefully controlled. Hence, this study was undertaken to identify the minimum depth of sequencing required for de novo assembly for different sized genomes using graph based assembly algorithms and real datasets. Illumina reads for E.coli (4.6 MB) S.kudriavzevii (11.18 MB) and C.elegans (100 MB) were assembled using SOAPdenovo, Velvet, ABySS, Meraculous and IDBA-UD. Our analysis shows that 50X is the optimum read depth for assembling these genomes using all assemblers except Meraculous which requires 100X read depth. Moreover, our analysis shows that de novo assembly from 50X read data requires only 6-40 GB RAM depending on the genome size and assembly algorithm used. We believe that this information can be extremely valuable for researchers in designing experiments and multiplexing which will enable optimum utilization of sequencing as well as analysis resources.

  20. Detection of a divergent variant of grapevine virus F by next-generation sequencing.

    Science.gov (United States)

    Molenaar, Nicholas; Burger, Johan T; Maree, Hans J

    2015-08-01

    The complete genome sequence of a South African isolate of grapevine virus F (GVF) is presented. It was first detected by metagenomic next-generation sequencing of field samples and validated through direct Sanger sequencing. The genome sequence of GVF isolate V5 consists of 7539 nucleotides and contains a poly(A) tail. It has a typical vitivirus genome arrangement that comprises five open reading frames (ORFs), which share only 88.96 % nucleotide sequence identity with the existing complete GVF genome sequence (JX105428).

  1. A tale of three next generation sequencing platforms: comparison of Ion Torrent, Pacific Biosciences and Illumina MiSeq sequencers

    Directory of Open Access Journals (Sweden)

    Quail Michael A

    2012-07-01

    Full Text Available Abstract Background Next generation sequencing (NGS technology has revolutionized genomic and genetic research. The pace of change in this area is rapid with three major new sequencing platforms having been released in 2011: Ion Torrent’s PGM, Pacific Biosciences’ RS and the Illumina MiSeq. Here we compare the results obtained with those platforms to the performance of the Illumina HiSeq, the current market leader. In order to compare these platforms, and get sufficient coverage depth to allow meaningful analysis, we have sequenced a set of 4 microbial genomes with mean GC content ranging from 19.3 to 67.7%. Together, these represent a comprehensive range of genome content. Here we report our analysis of that sequence data in terms of coverage distribution, bias, GC distribution, variant detection and accuracy. Results Sequence generated by Ion Torrent, MiSeq and Pacific Biosciences technologies displays near perfect coverage behaviour on GC-rich, neutral and moderately AT-rich genomes, but a profound bias was observed upon sequencing the extremely AT-rich genome of Plasmodium falciparum on the PGM, resulting in no coverage for approximately 30% of the genome. We analysed the ability to call variants from each platform and found that we could call slightly more variants from Ion Torrent data compared to MiSeq data, but at the expense of a higher false positive rate. Variant calling from Pacific Biosciences data was possible but higher coverage depth was required. Context specific errors were observed in both PGM and MiSeq data, but not in that from the Pacific Biosciences platform. Conclusions All three fast turnaround sequencers evaluated here were able to generate usable sequence. However there are key differences between the quality of that data and the applications it will support.

  2. Consed: a graphical editor for next-generation sequencing

    OpenAIRE

    Gordon, David; Green, Phil

    2013-01-01

    Summary: The rapid growth of DNA sequencing throughput in recent years implies that graphical interfaces for viewing and correcting errors must now handle large numbers of reads, efficiently pinpoint regions of interest and automate as many tasks as possible. We have adapted consed to reflect this. To allow full-feature editing of large datasets while keeping memory requirements low, we developed a viewer, bamScape, that reads billion-read BAM files, identifies and displays problem areas for ...

  3. Supermassive Black Hole Binaries: Multi-Messenger Astrophysics and Long Baselines with the Next-Generation Very Large Array

    Science.gov (United States)

    Burke-Spolaor, Sarah; Lazio, Joseph; Nyland, Kristina; Blecha, Laura; Bogdanovic, Tamara; Comerford, Julie; Liu, Xin; Taylor, Gregory; Shen, Yue; Maccarone, T. J.; Chomiuk, Laura; Reines, Amy

    2018-01-01

    Dual ( physical processes that drive both the remnant's dynamics and the inspiral of the black hole pair. A systematic census of the dual supermassive black hole population will also directly constrain the strength and distribution of objects emitting gravitational waves that will be detected by pulsar timing arrays and future space-based laser interferometers. Although the population of dual supermassive black holes in galaxy merger products is central to these topics and others, few have yet been discovered.A suite of radio, visible-infrared, and X-ray telescopes have just begun to reveal the population of kiloparsec-separation dual active nuclei. This poster will present the unique capability of radio observations to explore the dual and binary population of supermassive black hole binaries, and will highlight the observational techniques and discoveries expected for the Next-Generation Very Large Array.Part of this research was carried out at the Jet Propulsion Laboratory, California Institute of Technology, under a contract with the National Aeronautics and Space Administration. The NANOGrav project receives support from NSF Physics Frontier Center award number 1430284.

  4. Theoretical extension and experimental demonstration of spectral compression in second-harmonic generation by Fresnel-inspired binary phase shaping

    Science.gov (United States)

    Li, Baihong; Dong, Ruifang; Zhou, Conghua; Xiang, Xiao; Li, Yongfang; Zhang, Shougang

    2018-05-01

    Selective two-photon microscopy and high-precision nonlinear spectroscopy rely on efficient spectral compression at the desired frequency. Previously, a Fresnel-inspired binary phase shaping (FIBPS) method was theoretically proposed for spectral compression of two-photon absorption and second-harmonic generation (SHG) with a square-chirped pulse. Here, we theoretically show that the FIBPS can introduce a negative quadratic frequency phase (negative chirp) by analogy with the spatial-domain phase function of Fresnel zone plate. Thus, the previous theoretical model can be extended to the case where the pulse can be transformed limited and in any symmetrical spectral shape. As an example, we experimentally demonstrate spectral compression in SHG by FIBPS for a Gaussian transform-limited pulse and show good agreement with the theory. Given the fundamental pulse bandwidth, a narrower SHG bandwidth with relatively high intensity can be obtained by simply increasing the number of binary phases. The experimental results also verify that our method is superior to that proposed in [Phys. Rev. A 46, 2749 (1992), 10.1103/PhysRevA.46.2749]. This method will significantly facilitate the applications of selective two-photon microscopy and spectroscopy. Moreover, as it can introduce negative dispersion, hence it can also be generalized to other applications in the field of dispersion compensation.

  5. A genome-wide analysis of lentivector integration sites using targeted sequence capture and next generation sequencing technology.

    Science.gov (United States)

    Ustek, Duran; Sirma, Sema; Gumus, Ergun; Arikan, Muzaffer; Cakiris, Aris; Abaci, Neslihan; Mathew, Jaicy; Emrence, Zeliha; Azakli, Hulya; Cosan, Fulya; Cakar, Atilla; Parlak, Mahmut; Kursun, Olcay

    2012-10-01

    One application of next-generation sequencing (NGS) is the targeted resequencing of interested genes which has not been used in viral integration site analysis of gene therapy applications. Here, we combined targeted sequence capture array and next generation sequencing to address the whole genome profiling of viral integration sites. Human 293T and K562 cells were transduced with a HIV-1 derived vector. A custom made DNA probe sets targeted pLVTHM vector used to capture lentiviral vector/human genome junctions. The captured DNA was sequenced using GS FLX platform. Seven thousand four hundred and eighty four human genome sequences flanking the long terminal repeats (LTR) of pLVTHM fragment sequences matched with an identity of at least 98% and minimum 50 bp criteria in both cells. In total, 203 unique integration sites were identified. The integrations in both cell lines were totally distant from the CpG islands and from the transcription start sites and preferentially located in introns. A comparison between the two cell lines showed that the lentiviral-transduced DNA does not have the same preferred regions in the two different cell lines. Copyright © 2012 Elsevier B.V. All rights reserved.

  6. Exact distribution of a pattern in a set of random sequences generated by a Markov source: applications to biological data

    Directory of Open Access Journals (Sweden)

    Regad Leslie

    2010-01-01

    Full Text Available Abstract Background In bioinformatics it is common to search for a pattern of interest in a potentially large set of rather short sequences (upstream gene regions, proteins, exons, etc.. Although many methodological approaches allow practitioners to compute the distribution of a pattern count in a random sequence generated by a Markov source, no specific developments have taken into account the counting of occurrences in a set of independent sequences. We aim to address this problem by deriving efficient approaches and algorithms to perform these computations both for low and high complexity patterns in the framework of homogeneous or heterogeneous Markov models. Results The latest advances in the field allowed us to use a technique of optimal Markov chain embedding based on deterministic finite automata to introduce three innovative algorithms. Algorithm 1 is the only one able to deal with heterogeneous models. It also permits to avoid any product of convolution of the pattern distribution in individual sequences. When working with homogeneous models, Algorithm 2 yields a dramatic reduction in the complexity by taking advantage of previous computations to obtain moment generating functions efficiently. In the particular case of low or moderate complexity patterns, Algorithm 3 exploits power computation and binary decomposition to further reduce the time complexity to a logarithmic scale. All these algorithms and their relative interest in comparison with existing ones were then tested and discussed on a toy-example and three biological data sets: structural patterns in protein loop structures, PROSITE signatures in a bacterial proteome, and transcription factors in upstream gene regions. On these data sets, we also compared our exact approaches to the tempting approximation that consists in concatenating the sequences in the data set into a single sequence. Conclusions Our algorithms prove to be effective and able to handle real data sets with

  7. Exact distribution of a pattern in a set of random sequences generated by a Markov source: applications to biological data.

    Science.gov (United States)

    Nuel, Gregory; Regad, Leslie; Martin, Juliette; Camproux, Anne-Claude

    2010-01-26

    In bioinformatics it is common to search for a pattern of interest in a potentially large set of rather short sequences (upstream gene regions, proteins, exons, etc.). Although many methodological approaches allow practitioners to compute the distribution of a pattern count in a random sequence generated by a Markov source, no specific developments have taken into account the counting of occurrences in a set of independent sequences. We aim to address this problem by deriving efficient approaches and algorithms to perform these computations both for low and high complexity patterns in the framework of homogeneous or heterogeneous Markov models. The latest advances in the field allowed us to use a technique of optimal Markov chain embedding based on deterministic finite automata to introduce three innovative algorithms. Algorithm 1 is the only one able to deal with heterogeneous models. It also permits to avoid any product of convolution of the pattern distribution in individual sequences. When working with homogeneous models, Algorithm 2 yields a dramatic reduction in the complexity by taking advantage of previous computations to obtain moment generating functions efficiently. In the particular case of low or moderate complexity patterns, Algorithm 3 exploits power computation and binary decomposition to further reduce the time complexity to a logarithmic scale. All these algorithms and their relative interest in comparison with existing ones were then tested and discussed on a toy-example and three biological data sets: structural patterns in protein loop structures, PROSITE signatures in a bacterial proteome, and transcription factors in upstream gene regions. On these data sets, we also compared our exact approaches to the tempting approximation that consists in concatenating the sequences in the data set into a single sequence. Our algorithms prove to be effective and able to handle real data sets with multiple sequences, as well as biological patterns of

  8. Consed: a graphical editor for next-generation sequencing.

    Science.gov (United States)

    Gordon, David; Green, Phil

    2013-11-15

    The rapid growth of DNA sequencing throughput in recent years implies that graphical interfaces for viewing and correcting errors must now handle large numbers of reads, efficiently pinpoint regions of interest and automate as many tasks as possible. We have adapted consed to reflect this. To allow full-feature editing of large datasets while keeping memory requirements low, we developed a viewer, bamScape, that reads billion-read BAM files, identifies and displays problem areas for user review and launches the consed graphical editor on user-selected regions, allowing, in addition to longstanding consed capabilities such as assembly editing, a variety of new features including direct editing of the reference sequence, variant and error detection, display of annotation tracks and the ability to simultaneously process a group of reads. Many batch processing capabilities have been added. The consed package is free to academic, government and non-profit users, and licensed to others for a fee by the University of Washington. The current version (26.0) is available for linux, macosx and solaris systems or as C++ source code. It includes a user's manual (with exercises) and example datasets. http://www.phrap.org/consed/consed.html dgordon@uw.edu .

  9. Application of Next Generation Sequencing on Genetic Testing

    DEFF Research Database (Denmark)

    Li, Jian

    The discovery of genetic factors behind increasing number of human diseases and the growth of education of genetic knowledge to the public make demands for genetic testing increase rapidly. However, traditional genetic testing methods cannot meet all kinds of the requirements. Next generation seq...

  10. Next-generation sequencing can reveal in vitro-generated PCR crossover products: some artifactual sequences correspond to HLA alleles in the IMGT/HLA database.

    Science.gov (United States)

    Holcomb, C L; Rastrou, M; Williams, T C; Goodridge, D; Lazaro, A M; Tilanus, M; Erlich, H A

    2014-01-01

    The high-resolution human leukocyte antigen (HLA) genotyping assay that we developed using 454 sequencing and Conexio software uses generic polymerase chain reaction (PCR) primers for DRB exon 2. Occasionally, we observed low abundance DRB amplicon sequences that resulted from in vitro PCR 'crossing over' between DRB1 and DRB3/4/5. These hybrid sequences, revealed by the clonal sequencing property of the 454 system, were generally observed at a read depth of 5%-10% of the true alleles. They usually contained at least one mismatch with the IMGT/HLA database, and consequently, were easily recognizable and did not cause a problem for HLA genotyping. Sometimes, however, these artifactual sequences matched a rare allele and the automatic genotype assignment was incorrect. These observations raised two issues: (1) could PCR conditions be modified to reduce such artifacts? and (2) could some of the rare alleles listed in the IMGT/HLA database be artifacts rather than true alleles? Because PCR crossing over occurs during late cycles of PCR, we compared DRB genotypes resulting from 28 and (our standard) 35 cycles of PCR. For all 21 cell line DNAs amplified for 35 cycles, crossover products were detected. In 33% of the cases, these hybrid sequences corresponded to named alleles. With amplification for only 28 cycles, these artifactual sequences were not detectable. To investigate whether some rare alleles in the IMGT/HLA database might be due to PCR artifacts, we analyzed four samples obtained from the investigators who submitted the sequences. In three cases, the sequences were generated from true alleles. In one case, our 454 sequencing revealed an error in the previously submitted sequence. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  11. Assessment of metagenomic assembly using simulated next generation sequencing data

    DEFF Research Database (Denmark)

    Mende, Daniel R; Waller, Alison S; Sunagawa, Shinichi

    2012-01-01

    with platform-specific (Sanger, pyrosequencing, Illumina) base-error models, and simulated metagenomes of differing community complexities. We first evaluated the effect of rigorous quality control on Illumina data. Although quality filtering removed a large proportion of the data, it greatly improved...... the accuracy and contig lengths of resulting assemblies. We then compared the quality-trimmed Illumina assemblies to those from Sanger and pyrosequencing. For the simple community (10 genomes) all sequencing technologies assembled a similar amount and accurately represented the expected functional composition...... the Sanger reads still represented the overall functional composition reasonably well. We further examined the effect of scaffolding of contigs using paired-end Illumina reads. It dramatically increased contig lengths of the simple community and yielded minor improvements to the more complex communities...

  12. Next generation sequencing of DNA-launched Chikungunya vaccine virus

    Energy Technology Data Exchange (ETDEWEB)

    Hidajat, Rachmat; Nickols, Brian [Medigen, Inc., 8420 Gas House Pike, Suite S, Frederick, MD 21701 (United States); Forrester, Naomi [Institute for Human Infections and Immunity, Sealy Center for Vaccine Development and Department of Pathology, University of Texas Medical Branch, GNL, 301 University Blvd., Galveston, TX 77555 (United States); Tretyakova, Irina [Medigen, Inc., 8420 Gas House Pike, Suite S, Frederick, MD 21701 (United States); Weaver, Scott [Institute for Human Infections and Immunity, Sealy Center for Vaccine Development and Department of Pathology, University of Texas Medical Branch, GNL, 301 University Blvd., Galveston, TX 77555 (United States); Pushko, Peter, E-mail: ppushko@medigen-usa.com [Medigen, Inc., 8420 Gas House Pike, Suite S, Frederick, MD 21701 (United States)

    2016-03-15

    Chikungunya virus (CHIKV) represents a pandemic threat with no approved vaccine available. Recently, we described a novel vaccination strategy based on iDNA® infectious clone designed to launch a live-attenuated CHIKV vaccine from plasmid DNA in vitro or in vivo. As a proof of concept, we prepared iDNA plasmid pCHIKV-7 encoding the full-length cDNA of the 181/25 vaccine. The DNA-launched CHIKV-7 virus was prepared and compared to the 181/25 virus. Illumina HiSeq2000 sequencing revealed that with the exception of the 3′ untranslated region, CHIKV-7 viral RNA consistently showed a lower frequency of single-nucleotide polymorphisms than the 181/25 RNA including at the E2-12 and E2-82 residues previously identified as attenuating mutations. In the CHIKV-7, frequencies of reversions at E2-12 and E2-82 were 0.064% and 0.086%, while in the 181/25, frequencies were 0.179% and 0.133%, respectively. We conclude that the DNA-launched virus has a reduced probability of reversion mutations, thereby enhancing vaccine safety. - Highlights: • Chikungunya virus (CHIKV) is an emerging pandemic threat. • In vivo DNA-launched attenuated CHIKV is a novel vaccine technology. • DNA-launched virus was sequenced using HiSeq2000 and compared to the 181/25 virus. • DNA-launched virus has lower frequency of SNPs at E2-12 and E2-82 attenuation loci.

  13. Next generation sequencing of DNA-launched Chikungunya vaccine virus

    International Nuclear Information System (INIS)

    Hidajat, Rachmat; Nickols, Brian; Forrester, Naomi; Tretyakova, Irina; Weaver, Scott; Pushko, Peter

    2016-01-01

    Chikungunya virus (CHIKV) represents a pandemic threat with no approved vaccine available. Recently, we described a novel vaccination strategy based on iDNA® infectious clone designed to launch a live-attenuated CHIKV vaccine from plasmid DNA in vitro or in vivo. As a proof of concept, we prepared iDNA plasmid pCHIKV-7 encoding the full-length cDNA of the 181/25 vaccine. The DNA-launched CHIKV-7 virus was prepared and compared to the 181/25 virus. Illumina HiSeq2000 sequencing revealed that with the exception of the 3′ untranslated region, CHIKV-7 viral RNA consistently showed a lower frequency of single-nucleotide polymorphisms than the 181/25 RNA including at the E2-12 and E2-82 residues previously identified as attenuating mutations. In the CHIKV-7, frequencies of reversions at E2-12 and E2-82 were 0.064% and 0.086%, while in the 181/25, frequencies were 0.179% and 0.133%, respectively. We conclude that the DNA-launched virus has a reduced probability of reversion mutations, thereby enhancing vaccine safety. - Highlights: • Chikungunya virus (CHIKV) is an emerging pandemic threat. • In vivo DNA-launched attenuated CHIKV is a novel vaccine technology. • DNA-launched virus was sequenced using HiSeq2000 and compared to the 181/25 virus. • DNA-launched virus has lower frequency of SNPs at E2-12 and E2-82 attenuation loci.

  14. From Conventional to Next Generation Sequencing of Epstein-Barr Virus Genomes.

    Science.gov (United States)

    Kwok, Hin; Chiang, Alan Kwok Shing

    2016-02-24

    Genomic sequences of Epstein-Barr virus (EBV) have been of interest because the virus is associated with cancers, such as nasopharyngeal carcinoma, and conditions such as infectious mononucleosis. The progress of whole-genome EBV sequencing has been limited by the inefficiency and cost of the first-generation sequencing technology. With the advancement of next-generation sequencing (NGS) and target enrichment strategies, increasing number of EBV genomes has been published. These genomes were sequenced using different approaches, either with or without EBV DNA enrichment. This review provides an overview of the EBV genomes published to date, and a description of the sequencing technology and bioinformatic analyses employed in generating these sequences. We further explored ways through which the quality of sequencing data can be improved, such as using DNA oligos for capture hybridization, and longer insert size and read length in the sequencing runs. These advances will enable large-scale genomic sequencing of EBV which will facilitate a better understanding of the genetic variations of EBV in different geographic regions and discovery of potentially pathogenic variants in specific diseases.

  15. MiSeq: A Next Generation Sequencing Platform for Genomic Analysis.

    Science.gov (United States)

    Ravi, Rupesh Kanchi; Walton, Kendra; Khosroheidari, Mahdieh

    2018-01-01

    MiSeq, Illumina's integrated next generation sequencing instrument, uses reversible-terminator sequencing-by-synthesis technology to provide end-to-end sequencing solutions. The MiSeq instrument is one of the smallest benchtop sequencers that can perform onboard cluster generation, amplification, genomic DNA sequencing, and data analysis, including base calling, alignment and variant calling, in a single run. It performs both single- and paired-end runs with adjustable read lengths from 1 × 36 base pairs to 2 × 300 base pairs. A single run can produce output data of up to 15 Gb in as little as 4 h of runtime and can output up to 25 M single reads and 50 M paired-end reads. Thus, MiSeq provides an ideal platform for rapid turnaround time. MiSeq is also a cost-effective tool for various analyses focused on targeted gene sequencing (amplicon sequencing and target enrichment), metagenomics, and gene expression studies. For these reasons, MiSeq has become one of the most widely used next generation sequencing platforms. Here, we provide a protocol to prepare libraries for sequencing using the MiSeq instrument and basic guidelines for analysis of output data from the MiSeq sequencing run.

  16. From Conventional to Next Generation Sequencing of Epstein-Barr Virus Genomes

    Directory of Open Access Journals (Sweden)

    Hin Kwok

    2016-02-01

    Full Text Available Genomic sequences of Epstein–Barr virus (EBV have been of interest because the virus is associated with cancers, such as nasopharyngeal carcinoma, and conditions such as infectious mononucleosis. The progress of whole-genome EBV sequencing has been limited by the inefficiency and cost of the first-generation sequencing technology. With the advancement of next-generation sequencing (NGS and target enrichment strategies, increasing number of EBV genomes has been published. These genomes were sequenced using different approaches, either with or without EBV DNA enrichment. This review provides an overview of the EBV genomes published to date, and a description of the sequencing technology and bioinformatic analyses employed in generating these sequences. We further explored ways through which the quality of sequencing data can be improved, such as using DNA oligos for capture hybridization, and longer insert size and read length in the sequencing runs. These advances will enable large-scale genomic sequencing of EBV which will facilitate a better understanding of the genetic variations of EBV in different geographic regions and discovery of potentially pathogenic variants in specific diseases.

  17. Now And Next Generation Sequencing Techniques: Future of Sequence Analysis using Cloud Computing

    Directory of Open Access Journals (Sweden)

    Radhe Shyam Thakur

    2012-12-01

    Full Text Available Advancements in the field of sequencing techniques resulted in the huge sequenced data to be produced at a very faster rate. It is going cumbersome for the datacenter to maintain the databases. Data mining and sequence analysis approaches needs to analyze the databases several times to reach any efficient conclusion. To cope with such overburden on computer resources and to reach efficient and effective conclusions quickly, the virtualization of the resources and computation on pay as you go concept was introduced and termed as cloud computing. The datacenter’s hardware and software is collectively known as cloud which when available publicly is termed as public cloud. The datacenter’s resources are provided in a virtual mode to the clients via a service provider like Amazon, Google and Joyent which charges on pay as you go manner. The workload is shifted to the provider which is maintained by the required hardware and software upgradation. The service provider manages it by upgrading the requirements in the virtual mode. Basically a virtual environment is created according to the need of the user by taking permission from datacenter via internet, the task is performed and the environment is deleted after the task is over. In this discussion, we are focusing on the basics of cloud computing, the prerequisites and overall working of clouds. Furthermore, briefly the applications of cloud computing in biological systems, especially in comparative genomics, genome informatics and SNP detection with reference to traditional workflow are discussed.

  18. Now and next-generation sequencing techniques: future of sequence analysis using cloud computing.

    Science.gov (United States)

    Thakur, Radhe Shyam; Bandopadhyay, Rajib; Chaudhary, Bratati; Chatterjee, Sourav

    2012-01-01

    Advances in the field of sequencing techniques have resulted in the greatly accelerated production of huge sequence datasets. This presents immediate challenges in database maintenance at datacenters. It provides additional computational challenges in data mining and sequence analysis. Together these represent a significant overburden on traditional stand-alone computer resources, and to reach effective conclusions quickly and efficiently, the virtualization of the resources and computation on a pay-as-you-go concept (together termed "cloud computing") has recently appeared. The collective resources of the datacenter, including both hardware and software, can be available publicly, being then termed a public cloud, the resources being provided in a virtual mode to the clients who pay according to the resources they employ. Examples of public companies providing these resources include Amazon, Google, and Joyent. The computational workload is shifted to the provider, which also implements required hardware and software upgrades over time. A virtual environment is created in the cloud corresponding to the computational and data storage needs of the user via the internet. The task is then performed, the results transmitted to the user, and the environment finally deleted after all tasks are completed. In this discussion, we focus on the basics of cloud computing, and go on to analyze the prerequisites and overall working of clouds. Finally, the applications of cloud computing in biological systems, particularly in comparative genomics, genome informatics, and SNP detection are discussed with reference to traditional workflows.

  19. Next-generation phylogeography: a targeted approach for multilocus sequencing of non-model organisms.

    Directory of Open Access Journals (Sweden)

    Jonathan B Puritz

    Full Text Available The field of phylogeography has long since realized the need and utility of incorporating nuclear DNA (nDNA sequences into analyses. However, the use of nDNA sequence data, at the population level, has been hindered by technical laboratory difficulty, sequencing costs, and problematic analytical methods dealing with genotypic sequence data, especially in non-model organisms. Here, we present a method utilizing the 454 GS-FLX Titanium pyrosequencing platform with the capacity to simultaneously sequence two species of sea star (Meridiastra calcar and Parvulastra exigua at five different nDNA loci across 16 different populations of 20 individuals each per species. We compare results from 3 populations with traditional Sanger sequencing based methods, and demonstrate that this next-generation sequencing platform is more time and cost effective and more sensitive to rare variants than Sanger based sequencing. A crucial advantage is that the high coverage of clonally amplified sequences simplifies haplotype determination, even in highly polymorphic species. This targeted next-generation approach can greatly increase the use of nDNA sequence loci in phylogeographic and population genetic studies by mitigating many of the time, cost, and analytical issues associated with highly polymorphic, diploid sequence markers.

  20. Combining next-generation sequencing and online databases for microsatellite development in non-model organisms.

    Science.gov (United States)

    Rico, Ciro; Normandeau, Eric; Dion-Côté, Anne-Marie; Rico, María Inés; Côté, Guillaume; Bernatchez, Louis

    2013-12-03

    Next-generation sequencing (NGS) is revolutionising marker development and the rapidly increasing amount of transcriptomes published across a wide variety of taxa is providing valuable sequence databases for the identification of genetic markers without the need to generate new sequences. Microsatellites are still the most important source of polymorphic markers in ecology and evolution. Motivated by our long-term interest in the adaptive radiation of a non-model species complex of whitefishes (Coregonus spp.), in this study, we focus on microsatellite characterisation and multiplex optimisation using transcriptome sequences generated by Illumina® and Roche-454, as well as online databases of Expressed Sequence Tags (EST) for the study of whitefish evolution and demographic history. We identified and optimised 40 polymorphic loci in multiplex PCR reactions and validated the robustness of our analyses by testing several population genetics and phylogeographic predictions using 494 fish from five lakes and 2 distinct ecotypes.

  1. Applications of nanotechnology, next generation sequencing and microarrays in biomedical research.

    Science.gov (United States)

    Elingaramil, Sauli; Li, Xiaolong; He, Nongyue

    2013-07-01

    Next-generation sequencing technologies, microarrays and advances in bio nanotechnology have had an enormous impact on research within a short time frame. This impact appears certain to increase further as many biomedical institutions are now acquiring these prevailing new technologies. Beyond conventional sampling of genome content, wide-ranging applications are rapidly evolving for next-generation sequencing, microarrays and nanotechnology. To date, these technologies have been applied in a variety of contexts, including whole-genome sequencing, targeted re sequencing and discovery of transcription factor binding sites, noncoding RNA expression profiling and molecular diagnostics. This paper thus discusses current applications of nanotechnology, next-generation sequencing technologies and microarrays in biomedical research and highlights the transforming potential these technologies offer.

  2. 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.

  3. The Quest for Rare Variants: Pooled Multiplexed Next Generation Sequencing in Plants

    OpenAIRE

    Fabio eMarroni; Sara ePinosio; Sara ePinosio; Michele eMorgante

    2012-01-01

    Next generation sequencing (NGS) instruments produce an unprecedented amount of sequence data at contained costs. This gives researchers the possibility of designing studies with adequate power to identify rare variants at a fraction of the economic and labor resources required by individual Sanger sequencing. As of today, only three research groups working in plant sciences have exploited this potentiality. They showed that pooled NGS can provide results in excellent agreement with those obt...

  4. The Quest for Rare Variants: Pooled Multiplexed Next Generation Sequencing in Plants

    OpenAIRE

    Marroni, Fabio; Pinosio, Sara; Morgante, Michele

    2012-01-01

    Next generation sequencing (NGS) instruments produce an unprecedented amount of sequence data at contained costs. This gives researchers the possibility of designing studies with adequate power to identify rare variants at a fraction of the economic and labor resources required by individual Sanger sequencing. As of today, few research groups working in plant sciences have exploited this potentiality, showing that pooled NGS provides results in excellent agreement with those obtained by indiv...

  5. Uncovering of Classical Swine Fever Virus adaptive response to vaccination by Next Generation Sequencing

    DEFF Research Database (Denmark)

    Fahnøe, Ulrik; Orton, Richard; Höper, Dirk

    Next Generation Sequencing (NGS) has rapidly become the preferred technology in nucleotide sequencing, and can be applied to unravel molecular adaptation of RNA viruses such as Classical Swine Fever Virus (CSFV). However, the detection of low frequency variants within viral populations by NGS...... is affected by errors introduced during sample preparation and sequencing, and so far no definitive solution to this problem has been presented....

  6. NGSUtils: a software suite for analyzing and manipulating next-generation sequencing datasets

    OpenAIRE

    Breese, Marcus R.; Liu, Yunlong

    2013-01-01

    Summary: NGSUtils is a suite of software tools for manipulating data common to next-generation sequencing experiments, such as FASTQ, BED and BAM format files. These tools provide a stable and modular platform for data management and analysis.

  7. Clinical utility of a 377 gene custom next-generation sequencing ...

    Indian Academy of Sciences (India)

    JEN BEVILACQUA

    2017-07-26

    Jul 26, 2017 ... Clinical utility of a 377 gene custom next-generation sequencing epilepsy panel ... number of genes, making it a very attractive option for a condition as .... clinical value of various test offerings to guide decision making.

  8. Exploring the potential of second-generation sequencing in diverse biological contexts

    DEFF Research Database (Denmark)

    Fordyce, Sarah Louise

    Second generation sequencing (SGS) has revolutionized the study of DNA, allowing massive parallel sequencing of nucleic acids with unprecedented depths of coverage. The research undertaken in this thesis occurred in parallel with the increased accessibility of SGS platforms for routine genetic...

  9. Generating Correlated Gamma Sequences for Sea-Clutter Simulation

    Science.gov (United States)

    2012-03-01

    generation of correlated Gamma random fields via SIRP theory is examined in [Conte et al. 1991, Armstrong & Griffiths 1991]. In these papers , the Gamma...2 〉2 + |〈x[n]x∗[n+ k]〉|2 . (4) Because 〈 |x|2 〉2 = z̄2 and |〈x[n]x∗[n+ k]〉|2 ≥ 0, this results in 〈z[n]z[n+ k]〉 ≥ z̄2 if the real- isation of z[n] is...linear map- ping. In a practical situation, a process with a given auto-covariance function would be specified. It is shown that by using an

  10. A Window Into Clinical Next-Generation Sequencing-Based Oncology Testing Practices.

    Science.gov (United States)

    Nagarajan, Rakesh; Bartley, Angela N; Bridge, Julia A; Jennings, Lawrence J; Kamel-Reid, Suzanne; Kim, Annette; Lazar, Alexander J; Lindeman, Neal I; Moncur, Joel; Rai, Alex J; Routbort, Mark J; Vasalos, Patricia; Merker, Jason D

    2017-12-01

    - Detection of acquired variants in cancer is a paradigm of precision medicine, yet little has been reported about clinical laboratory practices across a broad range of laboratories. - To use College of American Pathologists proficiency testing survey results to report on the results from surveys on next-generation sequencing-based oncology testing practices. - College of American Pathologists proficiency testing survey results from more than 250 laboratories currently performing molecular oncology testing were used to determine laboratory trends in next-generation sequencing-based oncology testing. - These presented data provide key information about the number of laboratories that currently offer or are planning to offer next-generation sequencing-based oncology testing. Furthermore, we present data from 60 laboratories performing next-generation sequencing-based oncology testing regarding specimen requirements and assay characteristics. The findings indicate that most laboratories are performing tumor-only targeted sequencing to detect single-nucleotide variants and small insertions and deletions, using desktop sequencers and predesigned commercial kits. Despite these trends, a diversity of approaches to testing exists. - This information should be useful to further inform a variety of topics, including national discussions involving clinical laboratory quality systems, regulation and oversight of next-generation sequencing-based oncology testing, and precision oncology efforts in a data-driven manner.

  11. GenRGenS: Software for Generating Random Genomic Sequences and Structures

    OpenAIRE

    Ponty , Yann; Termier , Michel; Denise , Alain

    2006-01-01

    International audience; GenRGenS is a software tool dedicated to randomly generating genomic sequences and structures. It handles several classes of models useful for sequence analysis, such as Markov chains, hidden Markov models, weighted context-free grammars, regular expressions and PROSITE expressions. GenRGenS is the only program that can handle weighted context-free grammars, thus allowing the user to model and to generate structured objects (such as RNA secondary structures) of any giv...

  12. Next-Generation Sequencing in Neuropathologic Diagnosis of Infections of the Nervous System (Open Access)

    Science.gov (United States)

    2016-06-13

    nervous system ABSTRACT Objective: To determine the feasibility of next-generation sequencing (NGS) microbiome ap- proaches in the diagnosis of infectious...V, van Doorn HR, Nghia HD, et al. Identification of a new cyclovirus in cerebrospinal fluid of patients with acute central nervous system infections...Kumar, et al. system Next-generation sequencing in neuropathologic diagnosis of infections of the nervous This information is current as of June 13

  13. General simulation algorithm for autocorrelated binary processes.

    Science.gov (United States)

    Serinaldi, Francesco; Lombardo, Federico

    2017-02-01

    The apparent ubiquity of binary random processes in physics and many other fields has attracted considerable attention from the modeling community. However, generation of binary sequences with prescribed autocorrelation is a challenging task owing to the discrete nature of the marginal distributions, which makes the application of classical spectral techniques problematic. We show that such methods can effectively be used if we focus on the parent continuous process of beta distributed transition probabilities rather than on the target binary process. This change of paradigm results in a simulation procedure effectively embedding a spectrum-based iterative amplitude-adjusted Fourier transform method devised for continuous processes. The proposed algorithm is fully general, requires minimal assumptions, and can easily simulate binary signals with power-law and exponentially decaying autocorrelation functions corresponding, for instance, to Hurst-Kolmogorov and Markov processes. An application to rainfall intermittency shows that the proposed algorithm can also simulate surrogate data preserving the empirical autocorrelation.

  14. General simulation algorithm for autocorrelated binary processes

    Science.gov (United States)

    Serinaldi, Francesco; Lombardo, Federico

    2017-02-01

    The apparent ubiquity of binary random processes in physics and many other fields has attracted considerable attention from the modeling community. However, generation of binary sequences with prescribed autocorrelation is a challenging task owing to the discrete nature of the marginal distributions, which makes the application of classical spectral techniques problematic. We show that such methods can effectively be used if we focus on the parent continuous process of beta distributed transition probabilities rather than on the target binary process. This change of paradigm results in a simulation procedure effectively embedding a spectrum-based iterative amplitude-adjusted Fourier transform method devised for continuous processes. The proposed algorithm is fully general, requires minimal assumptions, and can easily simulate binary signals with power-law and exponentially decaying autocorrelation functions corresponding, for instance, to Hurst-Kolmogorov and Markov processes. An application to rainfall intermittency shows that the proposed algorithm can also simulate surrogate data preserving the empirical autocorrelation.

  15. Next generation sequencing in clinical medicine: Challenges and lessons for pathology and biomedical informatics

    Directory of Open Access Journals (Sweden)

    Rama R Gullapalli

    2012-01-01

    Full Text Available The Human Genome Project (HGP provided the initial draft of mankind′s DNA sequence in 2001. The HGP was produced by 23 collaborating laboratories using Sanger sequencing of mapped regions as well as shotgun sequencing techniques in a process that occupied 13 years at a cost of ~$3 billion. Today, Next Generation Sequencing (NGS techniques represent the next phase in the evolution of DNA sequencing technology at dramatically reduced cost compared to traditional Sanger sequencing. A single laboratory today can sequence the entire human genome in a few days for a few thousand dollars in reagents and staff time. Routine whole exome or even whole genome sequencing of clinical patients is well within the realm of affordability for many academic institutions across the country. This paper reviews current sequencing technology methods and upcoming advancements in sequencing technology as well as challenges associated with data generation, data manipulation and data storage. Implementation of routine NGS data in cancer genomics is discussed along with potential pitfalls in the interpretation of the NGS data. The overarching importance of bioinformatics in the clinical implementation of NGS is emphasized. [7] We also review the issue of physician education which also is an important consideration for the successful implementation of NGS in the clinical workplace. NGS technologies represent a golden opportunity for the next generation of pathologists to be at the leading edge of the personalized medicine approaches coming our way. Often under-emphasized issues of data access and control as well as potential ethical implications of whole genome NGS sequencing are also discussed. Despite some challenges, it′s hard not to be optimistic about the future of personalized genome sequencing and its potential impact on patient care and the advancement of knowledge of human biology and disease in the near future.

  16. Next-Generation Sequencing Workflow for NSCLC Critical Samples Using a Targeted Sequencing Approach by Ion Torrent PGM™ Platform.

    Science.gov (United States)

    Vanni, Irene; Coco, Simona; Truini, Anna; Rusmini, Marta; Dal Bello, Maria Giovanna; Alama, Angela; Banelli, Barbara; Mora, Marco; Rijavec, Erika; Barletta, Giulia; Genova, Carlo; Biello, Federica; Maggioni, Claudia; Grossi, Francesco

    2015-12-03

    Next-generation sequencing (NGS) is a cost-effective technology capable of screening several genes simultaneously; however, its application in a clinical context requires an established workflow to acquire reliable sequencing results. Here, we report an optimized NGS workflow analyzing 22 lung cancer-related genes to sequence critical samples such as DNA from formalin-fixed paraffin-embedded (FFPE) blocks and circulating free DNA (cfDNA). Snap frozen and matched FFPE gDNA from 12 non-small cell lung cancer (NSCLC) patients, whose gDNA fragmentation status was previously evaluated using a multiplex PCR-based quality control, were successfully sequenced with Ion Torrent PGM™. The robust bioinformatic pipeline allowed us to correctly call both Single Nucleotide Variants (SNVs) and indels with a detection limit of 5%, achieving 100% specificity and 96% sensitivity. This workflow was also validated in 13 FFPE NSCLC biopsies. Furthermore, a specific protocol for low input gDNA capable of producing good sequencing data with high coverage, high uniformity, and a low error rate was also optimized. In conclusion, we demonstrate the feasibility of obtaining gDNA from FFPE samples suitable for NGS by performing appropriate quality controls. The optimized workflow, capable of screening low input gDNA, highlights NGS as a potential tool in the detection, disease monitoring, and treatment of NSCLC.

  17. Mining and Development of Novel SSR Markers Using Next Generation Sequencing (NGS Data in Plants

    Directory of Open Access Journals (Sweden)

    Sima Taheri

    2018-02-01

    Full Text Available Microsatellites, or simple sequence repeats (SSRs, are one of the most informative and multi-purpose genetic markers exploited in plant functional genomics. However, the discovery of SSRs and development using traditional methods are laborious, time-consuming, and costly. Recently, the availability of high-throughput sequencing technologies has enabled researchers to identify a substantial number of microsatellites at less cost and effort than traditional approaches. Illumina is a noteworthy transcriptome sequencing technology that is currently used in SSR marker development. Although 454 pyrosequencing datasets can be used for SSR development, this type of sequencing is no longer supported. This review aims to present an overview of the next generation sequencing, with a focus on the efficient use of de novo transcriptome sequencing (RNA-Seq and related tools for mining and development of microsatellites in plants.

  18. Method for Generating Pseudorandom Sequences with the Assured Period Based on R-blocks

    Directory of Open Access Journals (Sweden)

    M. A. Ivanov

    2011-03-01

    Full Text Available The article describes the characteristics of a new class of fast-acting pseudorandom number generators, based on the use of stochastic adders or R-blocks. A new method for generating pseudorandom sequences with the assured length of period is offered.

  19. Security problems for a pseudorandom sequence generator based on the Chen chaotic system

    Science.gov (United States)

    Özkaynak, Fatih; Yavuz, Sırma

    2013-09-01

    Recently, a novel pseudorandom number generator scheme based on the Chen chaotic system was proposed. In this study, we analyze the security weaknesses of the proposed generator. By applying a brute force attack on a reduced key space, we show that 66% of the generated pseudorandom number sequences can be revealed. Executable C# code is given for the proposed attack. The computational complexity of this attack is O(n), where n is the sequence length. Both mathematical proofs and experimental results are presented to support the proposed attack.

  20. Exome-wide DNA capture and next generation sequencing in domestic and wild species

    Directory of Open Access Journals (Sweden)

    Ng Sarah B

    2011-07-01

    Full Text Available Abstract Background Gene-targeted and genome-wide markers are crucial to advance evolutionary biology, agriculture, and biodiversity conservation by improving our understanding of genetic processes underlying adaptation and speciation. Unfortunately, for eukaryotic species with large genomes it remains costly to obtain genome sequences and to develop genome resources such as genome-wide SNPs. A method is needed to allow gene-targeted, next-generation sequencing that is flexible enough to include any gene or number of genes, unlike transcriptome sequencing. Such a method would allow sequencing of many individuals, avoiding ascertainment bias in subsequent population genetic analyses. We demonstrate the usefulness of a recent technology, exon capture, for genome-wide, gene-targeted marker discovery in species with no genome resources. We use coding gene sequences from the domestic cow genome sequence (Bos taurus to capture (enrich for, and subsequently sequence, thousands of exons of B. taurus, B. indicus, and Bison bison (wild bison. Our capture array has probes for 16,131 exons in 2,570 genes, including 203 candidate genes with known function and of interest for their association with disease and other fitness traits. Results We successfully sequenced and mapped exon sequences from across the 29 autosomes and X chromosome in the B. taurus genome sequence. Exon capture and high-throughput sequencing identified thousands of putative SNPs spread evenly across all reference chromosomes, in all three individuals, including hundreds of SNPs in our targeted candidate genes. Conclusions This study shows exon capture can be customized for SNP discovery in many individuals and for non-model species without genomic resources. Our captured exome subset was small enough for affordable next-generation sequencing, and successfully captured exons from a divergent wild species using the domestic cow genome as reference.

  1. Exome-wide DNA capture and next generation sequencing in domestic and wild species.

    Science.gov (United States)

    Cosart, Ted; Beja-Pereira, Albano; Chen, Shanyuan; Ng, Sarah B; Shendure, Jay; Luikart, Gordon

    2011-07-05

    Gene-targeted and genome-wide markers are crucial to advance evolutionary biology, agriculture, and biodiversity conservation by improving our understanding of genetic processes underlying adaptation and speciation. Unfortunately, for eukaryotic species with large genomes it remains costly to obtain genome sequences and to develop genome resources such as genome-wide SNPs. A method is needed to allow gene-targeted, next-generation sequencing that is flexible enough to include any gene or number of genes, unlike transcriptome sequencing. Such a method would allow sequencing of many individuals, avoiding ascertainment bias in subsequent population genetic analyses.We demonstrate the usefulness of a recent technology, exon capture, for genome-wide, gene-targeted marker discovery in species with no genome resources. We use coding gene sequences from the domestic cow genome sequence (Bos taurus) to capture (enrich for), and subsequently sequence, thousands of exons of B. taurus, B. indicus, and Bison bison (wild bison). Our capture array has probes for 16,131 exons in 2,570 genes, including 203 candidate genes with known function and of interest for their association with disease and other fitness traits. We successfully sequenced and mapped exon sequences from across the 29 autosomes and X chromosome in the B. taurus genome sequence. Exon capture and high-throughput sequencing identified thousands of putative SNPs spread evenly across all reference chromosomes, in all three individuals, including hundreds of SNPs in our targeted candidate genes. This study shows exon capture can be customized for SNP discovery in many individuals and for non-model species without genomic resources. Our captured exome subset was small enough for affordable next-generation sequencing, and successfully captured exons from a divergent wild species using the domestic cow genome as reference.

  2. Humans can consciously generate random number sequences: a possible test for artificial intelligence.

    Science.gov (United States)

    Persaud, Navindra

    2005-01-01

    Computer algorithms can only produce seemingly random or pseudorandom numbers whereas certain natural phenomena, such as the decay of radioactive particles, can be utilized to produce truly random numbers. In this study, the ability of humans to generate random numbers was tested in healthy adults. Subjects were simply asked to generate and dictate random numbers. Generated numbers were tested for uniformity, independence and information density. The results suggest that humans can generate random numbers that are uniformly distributed, independent of one another and unpredictable. If humans can generate sequences of random numbers then neural networks or forms of artificial intelligence, which are purported to function in ways essentially the same as the human brain, should also be able to generate sequences of random numbers. Elucidating the precise mechanism by which humans generate random number sequences and the underlying neural substrates may have implications in the cognitive science of decision-making. It is possible that humans use their random-generating neural machinery to make difficult decisions in which all expected outcomes are similar. It is also possible that certain people, perhaps those with neurological or psychiatric impairments, are less able or unable to generate random numbers. If the random-generating neural machinery is employed in decision making its impairment would have profound implications in matters of agency and free will.

  3. Efficiency to Discovery Transgenic Loci in GM Rice Using Next Generation Sequencing Whole Genome Re-sequencing

    Directory of Open Access Journals (Sweden)

    Doori Park

    2015-09-01

    Full Text Available Molecular characterization technology in genetically modified organisms, in addition to how transgenic biotechnologies are developed now require full transparency to assess the risk to living modified and non-modified organisms. Next generation sequencing (NGS methodology is suggested as an effective means in genome characterization and detection of transgenic insertion locations. In the present study, we applied NGS to insert transgenic loci, specifically the epidermal growth factor (EGF in genetically modified rice cells. A total of 29.3 Gb (~72× coverage was sequenced with a 2 × 150 bp paired end method by Illumina HiSeq2500, which was consecutively mapped to the rice genome and T-vector sequence. The compatible pairs of reads were successfully mapped to 10 loci on the rice chromosome and vector sequences were validated to the insertion location by polymerase chain reaction (PCR amplification. The EGF transgenic site was confirmed only on chromosome 4 by PCR. Results of this study demonstrated the success of NGS data to characterize the rice genome. Bioinformatics analyses must be developed in association with NGS data to identify highly accurate transgenic sites.

  4. NeSSM: a Next-generation Sequencing Simulator for Metagenomics.

    Directory of Open Access Journals (Sweden)

    Ben Jia

    Full Text Available BACKGROUND: Metagenomics can reveal the vast majority of microbes that have been missed by traditional cultivation-based methods. Due to its extremely wide range of application areas, fast metagenome sequencing simulation systems with high fidelity are in great demand to facilitate the development and comparison of metagenomics analysis tools. RESULTS: We present here a customizable metagenome simulation system: NeSSM (Next-generation Sequencing Simulator for Metagenomics. Combining complete genomes currently available, a community composition table, and sequencing parameters, it can simulate metagenome sequencing better than existing systems. Sequencing error models based on the explicit distribution of errors at each base and sequencing coverage bias are incorporated in the simulation. In order to improve the fidelity of simulation, tools are provided by NeSSM to estimate the sequencing error models, sequencing coverage bias and the community composition directly from existing metagenome sequencing data. Currently, NeSSM supports single-end and pair-end sequencing for both 454 and Illumina platforms. In addition, a GPU (graphics processing units version of NeSSM is also developed to accelerate the simulation. By comparing the simulated sequencing data from NeSSM with experimental metagenome sequencing data, we have demonstrated that NeSSM performs better in many aspects than existing popular metagenome simulators, such as MetaSim, GemSIM and Grinder. The GPU version of NeSSM is more than one-order of magnitude faster than MetaSim. CONCLUSIONS: NeSSM is a fast simulation system for high-throughput metagenome sequencing. It can be helpful to develop tools and evaluate strategies for metagenomics analysis and it's freely available for academic users at http://cbb.sjtu.edu.cn/~ccwei/pub/software/NeSSM.php.

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

    Science.gov (United States)

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

    2012-06-25

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

  6. Real-time UAV trajectory generation using feature points matching between video image sequences

    Science.gov (United States)

    Byun, Younggi; Song, Jeongheon; Han, Dongyeob

    2017-09-01

    Unmanned aerial vehicles (UAVs), equipped with navigation systems and video capability, are currently being deployed for intelligence, reconnaissance and surveillance mission. In this paper, we present a systematic approach for the generation of UAV trajectory using a video image matching system based on SURF (Speeded up Robust Feature) and Preemptive RANSAC (Random Sample Consensus). Video image matching to find matching points is one of the most important steps for the accurate generation of UAV trajectory (sequence of poses in 3D space). We used the SURF algorithm to find the matching points between video image sequences, and removed mismatching by using the Preemptive RANSAC which divides all matching points to outliers and inliers. The inliers are only used to determine the epipolar geometry for estimating the relative pose (rotation and translation) between image sequences. Experimental results from simulated video image sequences showed that our approach has a good potential to be applied to the automatic geo-localization of the UAVs system

  7. “Shovel-ready” Sequences as a Stimulus for the Next Generation of Life Scientists

    Science.gov (United States)

    Boyle, Michael D.

    2010-01-01

    Genomics and bioinformatics are dynamic fields well-suited for capturing the imagination of undergraduates in both research laboratories and classrooms. Currently, raw nucleotide sequence is being provided, as part of several genomics research initiatives, for undergraduate research and teaching. These initiatives could be easily extended and much more effective if the source of the sequenced material and the subsequent focus of the data analysis were aligned with the research interests of individual faculty at undergraduate institutions. By judicious use of surplus capacity in existing nucleotide sequencing cores, raw sequence data could be generated to support ongoing research efforts involving undergraduates. This would allow these students to participate actively in discovery research, with a goal of making novel contributions to their field through original research while nurturing the next generation of talented research scientists. PMID:23653696

  8. "Shovel-ready" Sequences as a Stimulus for the Next Generation of Life Scientists.

    Science.gov (United States)

    Boyle, Michael D

    2010-01-01

    Genomics and bioinformatics are dynamic fields well-suited for capturing the imagination of undergraduates in both research laboratories and classrooms. Currently, raw nucleotide sequence is being provided, as part of several genomics research initiatives, for undergraduate research and teaching. These initiatives could be easily extended and much more effective if the source of the sequenced material and the subsequent focus of the data analysis were aligned with the research interests of individual faculty at undergraduate institutions. By judicious use of surplus capacity in existing nucleotide sequencing cores, raw sequence data could be generated to support ongoing research efforts involving undergraduates. This would allow these students to participate actively in discovery research, with a goal of making novel contributions to their field through original research while nurturing the next generation of talented research scientists.

  9. “Shovel-ready” Sequences as a Stimulus for the Next Generation of Life Scientists

    Directory of Open Access Journals (Sweden)

    Michael D. Boyle

    2010-04-01

    Full Text Available Genomics and bioinformatics are dynamic fields well-suited for capturing the imagination of undergraduates in both research laboratories and classrooms. Currently, raw nucleotide sequence is being provided, as part of several genomics research initiatives, for undergraduate research and teaching. These initiatives could be easily extended and much more effective if the source of the sequenced material and the subsequent focus of the data analysis were aligned with the research interests of individual faculty at undergraduate institutions. By judicious use of surplus capacity in existing nucleotide sequencing cores, raw sequence data could be generated to support ongoing research efforts involving undergraduates. This would allow these students to participate actively in discovery research, with a goal of making novel contributions to their field through original research while nurturing the next generation of talented research scientists.

  10. Genome survey sequencing and genetic background characterization of Gracilariopsis lemaneiformis (Rhodophyta) based on next-generation sequencing.

    Science.gov (United States)

    Zhou, Wei; Hu, Yiyi; Sui, Zhenghong; Fu, Feng; Wang, Jinguo; Chang, Lianpeng; Guo, Weihua; Li, Binbin

    2013-01-01

    Gracilariopsis lemaneiformis has a high economic value and is one of the most important aquaculture species in China. Despite it is economic importance, it has remained largely unstudied at the genomic level. In this study, we conducted a genome survey of Gp. lemaneiformis using next-generation sequencing (NGS) technologies. In total, 18.70 Gb of high-quality sequence data with an estimated genome size of 97 Mb were obtained by HiSeq 2000 sequencing for Gp. lemaneiformis. These reads were assembled into 160,390 contigs with a N50 length of 3.64 kb, which were further assembled into 125,685 scaffolds with a total length of 81.17 Mb. Genome analysis predicted 3490 genes and a GC% content of 48%. The identified genes have an average transcript length of 1,429 bp, an average coding sequence size of 1,369 bp, 1.36 exons per gene, exon length of 1,008 bp, and intron length of 191 bp. From the initial assembled scaffold, transposable elements constituted 54.64% (44.35 Mb) of the genome, and 7737 simple sequence repeats (SSRs) were identified. Among these SSRs, the trinucleotide repeat type was the most abundant (up to 73.20% of total SSRs), followed by the di- (17.41%), tetra- (5.49%), hexa- (2.90%), and penta- (1.00%) nucleotide repeat type. These characteristics suggest that Gp. lemaneiformis is a model organism for genetic study. This is the first report of genome-wide characterization within this taxon.

  11. Genome Survey Sequencing and Genetic Background Characterization of Gracilariopsis lemaneiformis (Rhodophyta) Based on Next-Generation Sequencing

    Science.gov (United States)

    Sui, Zhenghong; Fu, Feng; Wang, Jinguo; Chang, Lianpeng; Guo, Weihua; Li, Binbin

    2013-01-01

    Gracilariopsis lemaneiformis has a high economic value and is one of the most important aquaculture species in China. Despite it is economic importance, it has remained largely unstudied at the genomic level. In this study, we conducted a genome survey of Gp. lemaneiformis using next-generation sequencing (NGS) technologies. In total, 18.70 Gb of high-quality sequence data with an estimated genome size of 97 Mb were obtained by HiSeq 2000 sequencing for Gp. lemaneiformis. These reads were assembled into 160,390 contigs with a N50 length of 3.64 kb, which were further assembled into 125,685 scaffolds with a total length of 81.17 Mb. Genome analysis predicted 3490 genes and a GC% content of 48%. The identified genes have an average transcript length of 1,429 bp, an average coding sequence size of 1,369 bp, 1.36 exons per gene, exon length of 1,008 bp, and intron length of 191 bp. From the initial assembled scaffold, transposable elements constituted 54.64% (44.35 Mb) of the genome, and 7737 simple sequence repeats (SSRs) were identified. Among these SSRs, the trinucleotide repeat type was the most abundant (up to 73.20% of total SSRs), followed by the di- (17.41%), tetra- (5.49%), hexa- (2.90%), and penta- (1.00%) nucleotide repeat type. These characteristics suggest that Gp. lemaneiformis is a model organism for genetic study. This is the first report of genome-wide characterization within this taxon. PMID:23875008

  12. An integrated SNP mining and utilization (ISMU) pipeline for next generation sequencing data.

    Science.gov (United States)

    Azam, Sarwar; Rathore, Abhishek; Shah, Trushar M; Telluri, Mohan; Amindala, BhanuPrakash; Ruperao, Pradeep; Katta, Mohan A V S K; Varshney, Rajeev K

    2014-01-01

    Open source single nucleotide polymorphism (SNP) discovery pipelines for next generation sequencing data commonly requires working knowledge of command line interface, massive computational resources and expertise which is a daunting task for biologists. Further, the SNP information generated may not be readily used for downstream processes such as genotyping. Hence, a comprehensive pipeline has been developed by integrating several open source next generation sequencing (NGS) tools along with a graphical user interface called Integrated SNP Mining and Utilization (ISMU) for SNP discovery and their utilization by developing genotyping assays. The pipeline features functionalities such as pre-processing of raw data, integration of open source alignment tools (Bowtie2, BWA, Maq, NovoAlign and SOAP2), SNP prediction (SAMtools/SOAPsnp/CNS2snp and CbCC) methods and interfaces for developing genotyping assays. The pipeline outputs a list of high quality SNPs between all pairwise combinations of genotypes analyzed, in addition to the reference genome/sequence. Visualization tools (Tablet and Flapjack) integrated into the pipeline enable inspection of the alignment and errors, if any. The pipeline also provides a confidence score or polymorphism information content value with flanking sequences for identified SNPs in standard format required for developing marker genotyping (KASP and Golden Gate) assays. The pipeline enables users to process a range of NGS datasets such as whole genome re-sequencing, restriction site associated DNA sequencing and transcriptome sequencing data at a fast speed. The pipeline is very useful for plant genetics and breeding community with no computational expertise in order to discover SNPs and utilize in genomics, genetics and breeding studies. The pipeline has been parallelized to process huge datasets of next generation sequencing. It has been developed in Java language and is available at http://hpc.icrisat.cgiar.org/ISMU as a standalone

  13. Transcriptome analysis of carnation (Dianthus caryophyllus L.) based on next-generation sequencing technology.

    Science.gov (United States)

    Tanase, Koji; Nishitani, Chikako; Hirakawa, Hideki; Isobe, Sachiko; Tabata, Satoshi; Ohmiya, Akemi; Onozaki, Takashi

    2012-07-02

    Carnation (Dianthus caryophyllus L.), in the family Caryophyllaceae, can be found in a wide range of colors and is a model system for studies of flower senescence. In addition, it is one of the most important flowers in the global floriculture industry. However, few genomics resources, such as sequences and markers are available for carnation or other members of the Caryophyllaceae. To increase our understanding of the genetic control of important characters in carnation, we generated an expressed sequence tag (EST) database for a carnation cultivar important in horticulture by high-throughput sequencing using 454 pyrosequencing technology. We constructed a normalized cDNA library and a 3'-UTR library of carnation, obtaining a total of 1,162,126 high-quality reads. These reads were assembled into 300,740 unigenes consisting of 37,844 contigs and 262,896 singlets. The contigs were searched against an Arabidopsis sequence database, and 61.8% (23,380) of them had at least one BLASTX hit. These contigs were also annotated with Gene Ontology (GO) and were found to cover a broad range of GO categories. Furthermore, we identified 17,362 potential simple sequence repeats (SSRs) in 14,291 of the unigenes. We focused on gene discovery in the areas of flower color and ethylene biosynthesis. Transcripts were identified for almost every gene involved in flower chlorophyll and carotenoid metabolism and in anthocyanin biosynthesis. Transcripts were also identified for every step in the ethylene biosynthesis pathway. We present the first large-scale sequence data set for carnation, generated using next-generation sequencing technology. The large EST database generated from these sequences is an informative resource for identifying genes involved in various biological processes in carnation and provides an EST resource for understanding the genetic diversity of this plant.

  14. Transcriptome analysis of carnation (Dianthus caryophyllus L. based on next-generation sequencing technology

    Directory of Open Access Journals (Sweden)

    Tanase Koji

    2012-07-01

    Full Text Available Abstract Background Carnation (Dianthus caryophyllus L., in the family Caryophyllaceae, can be found in a wide range of colors and is a model system for studies of flower senescence. In addition, it is one of the most important flowers in the global floriculture industry. However, few genomics resources, such as sequences and markers are available for carnation or other members of the Caryophyllaceae. To increase our understanding of the genetic control of important characters in carnation, we generated an expressed sequence tag (EST database for a carnation cultivar important in horticulture by high-throughput sequencing using 454 pyrosequencing technology. Results We constructed a normalized cDNA library and a 3’-UTR library of carnation, obtaining a total of 1,162,126 high-quality reads. These reads were assembled into 300,740 unigenes consisting of 37,844 contigs and 262,896 singlets. The contigs were searched against an Arabidopsis sequence database, and 61.8% (23,380 of them had at least one BLASTX hit. These contigs were also annotated with Gene Ontology (GO and were found to cover a broad range of GO categories. Furthermore, we identified 17,362 potential simple sequence repeats (SSRs in 14,291 of the unigenes. We focused on gene discovery in the areas of flower color and ethylene biosynthesis. Transcripts were identified for almost every gene involved in flower chlorophyll and carotenoid metabolism and in anthocyanin biosynthesis. Transcripts were also identified for every step in the ethylene biosynthesis pathway. Conclusions We present the first large-scale sequence data set for carnation, generated using next-generation sequencing technology. The large EST database generated from these sequences is an informative resource for identifying genes involved in various biological processes in carnation and provides an EST resource for understanding the genetic diversity of this plant.

  15. Screening for SNPs with Allele-Specific Methylation based on Next-Generation Sequencing Data

    OpenAIRE

    Hu, Bo; Ji, Yuan; Xu, Yaomin; Ting, Angela H

    2013-01-01

    Allele-specific methylation (ASM) has long been studied but mainly documented in the context of genomic imprinting and X chromosome inactivation. Taking advantage of the next-generation sequencing technology, we conduct a high-throughput sequencing experiment with four prostate cell lines to survey the whole genome and identify single nucleotide polymorphisms (SNPs) with ASM. A Bayesian approach is proposed to model the counts of short reads for each SNP conditional on its genotypes of multip...

  16. Next-Generation Sequencing for Typing and Detection of ESBL and MBL E. coli causing UTI

    OpenAIRE

    Nabakishore Nayak; Mahesh Chanda Sahu

    2017-01-01

    Next-generation sequencing (NGS) has the potential to provide typing results and detect resistance genes in a single assay, thus guiding timely treatment decisions and allowing rapid tracking of transmission of resistant clones. We can be evaluated the performance of a new NGS assay during an outbreak of sequence type 131 (ST131) Escherichia coli infections in a teaching hospital. The assay will be performed on 100 extended-spectrum- beta-lactamase (ESBL) E. coli isolates collected from UTI d...

  17. Generation of synthetic sequences of electricity demand: Application in South Australia

    International Nuclear Information System (INIS)

    Magnano, L.; Boland, J.W.

    2007-01-01

    We have developed a model to generate synthetic sequences of half-hourly electricity demand. The generated sequences represent possible realisations of electricity load that could have occurred. Each of the components included in the model has a physical interpretation. These components are yearly and daily seasonality which were modelled using Fourier series, weekly seasonality modelled with dummy variables, and the relationship with current temperature described by polynomial functions of temperature. Finally the stochastic component was modelled with autoregressive moving average (ARMA) processes. These synthetic sequences were developed for two purposes. The first one is to use them as input data in market simulation software. The second one is to build probability distributions of the outputs to calculate probabilistic forecasts. As an application several summers of half-hourly electricity demand were generated and from them the value of demand that is not expected to be exceeded more than once in 10 years was calculated

  18. Identifying Corneal Infections in Formalin-Fixed Specimens Using Next Generation Sequencing.

    Science.gov (United States)

    Li, Zhigang; Breitwieser, Florian P; Lu, Jennifer; Jun, Albert S; Asnaghi, Laura; Salzberg, Steven L; Eberhart, Charles G

    2018-01-01

    We test the ability of next-generation sequencing, combined with computational analysis, to identify a range of organisms causing infectious keratitis. This retrospective study evaluated 16 cases of infectious keratitis and four control corneas in formalin-fixed tissues from the pathology laboratory. Infectious cases also were analyzed in the microbiology laboratory using culture, polymerase chain reaction, and direct staining. Classified sequence reads were analyzed with two different metagenomics classification engines, Kraken and Centrifuge, and visualized using the Pavian software tool. Sequencing generated 20 to 46 million reads per sample. On average, 96% of the reads were classified as human, 0.3% corresponded to known vectors or contaminant sequences, 1.7% represented microbial sequences, and 2.4% could not be classified. The two computational strategies successfully identified the fungal, bacterial, and amoebal pathogens in most patients, including all four bacterial and mycobacterial cases, five of six fungal cases, three of three Acanthamoeba cases, and one of three herpetic keratitis cases. In several cases, additional potential pathogens also were identified. In one case with cytomegalovirus identified by Kraken and Centrifuge, the virus was confirmed by direct testing, while two where Staphylococcus aureus or cytomegalovirus were identified by Centrifuge but not Kraken could not be confirmed. Confirmation was not attempted for an additional three potential pathogens identified by Kraken and 11 identified by Centrifuge. Next generation sequencing combined with computational analysis can identify a wide range of pathogens in formalin-fixed corneal specimens, with potential applications in clinical diagnostics and research.

  19. Analysis of quality raw data of second generation sequencers with Quality Assessment Software.

    Science.gov (United States)

    Ramos, Rommel Tj; Carneiro, Adriana R; Baumbach, Jan; Azevedo, Vasco; Schneider, Maria Pc; Silva, Artur

    2011-04-18

    Second generation technologies have advantages over Sanger; however, they have resulted in new challenges for the genome construction process, especially because of the small size of the reads, despite the high degree of coverage. Independent of the program chosen for the construction process, DNA sequences are superimposed, based on identity, to extend the reads, generating contigs; mismatches indicate a lack of homology and are not included. This process improves our confidence in the sequences that are generated. We developed Quality Assessment Software, with which one can review graphs showing the distribution of quality values from the sequencing reads. This software allow us to adopt more stringent quality standards for sequence data, based on quality-graph analysis and estimated coverage after applying the quality filter, providing acceptable sequence coverage for genome construction from short reads. Quality filtering is a fundamental step in the process of constructing genomes, as it reduces the frequency of incorrect alignments that are caused by measuring errors, which can occur during the construction process due to the size of the reads, provoking misassemblies. Application of quality filters to sequence data, using the software Quality Assessment, along with graphing analyses, provided greater precision in the definition of cutoff parameters, which increased the accuracy of genome construction.

  20. Generation of novel motor sequences: the neural correlates of musical improvisation.

    Science.gov (United States)

    Berkowitz, Aaron L; Ansari, Daniel

    2008-06-01

    While some motor behavior is instinctive and stereotyped or learned and re-executed, much action is a spontaneous response to a novel set of environmental conditions. The neural correlates of both pre-learned and cued motor sequences have been previously studied, but novel motor behavior has thus far not been examined through brain imaging. In this paper, we report a study of musical improvisation in trained pianists with functional magnetic resonance imaging (fMRI), using improvisation as a case study of novel action generation. We demonstrate that both rhythmic (temporal) and melodic (ordinal) motor sequence creation modulate activity in a network of brain regions comprised of the dorsal premotor cortex, the rostral cingulate zone of the anterior cingulate cortex, and the inferior frontal gyrus. These findings are consistent with a role for the dorsal premotor cortex in movement coordination, the rostral cingulate zone in voluntary selection, and the inferior frontal gyrus in sequence generation. Thus, the invention of novel motor sequences in musical improvisation recruits a network of brain regions coordinated to generate possible sequences, select among them, and execute the decided-upon sequence.

  1. Analyzing Plasmodium falciparum erythrocyte membrane protein 1 gene expression by a next generation sequencing based method

    DEFF Research Database (Denmark)

    Jespersen, Jakob S.; Petersen, Bent; Seguin-Orlando, Andaine

    2013-01-01

    at identifying PfEMP1 features associated with high virulence. Here we present the first effective method for sequence analysis of var genes expressed in field samples: a sequential PCR and next generation sequencing based technique applied on expressed var sequence tags and subsequently on long range PCR......, encoded by ~60 highly variable 'var' genes per haploid genome. PfEMP1 is exported to the surface of infected erythrocytes and is thought to be fundamental to immune evasion by adhesion to host and parasite factors. The highly variable nature has constituted a roadblock in var expression studies aimed...

  2. Genome wide SNP discovery in flax through next generation sequencing of reduced representation libraries

    Directory of Open Access Journals (Sweden)

    Kumar Santosh

    2012-12-01

    Full Text Available Abstract Background Flax (Linum usitatissimum L. is a significant fibre and oilseed crop. Current flax molecular markers, including isozymes, RAPDs, AFLPs and SSRs are of limited use in the construction of high density linkage maps and for association mapping applications due to factors such as low reproducibility, intense labour requirements and/or limited numbers. We report here on the use of a reduced representation library strategy combined with next generation Illumina sequencing for rapid and large scale discovery of SNPs in eight flax genotypes. SNP discovery was performed through in silico analysis of the sequencing data against the whole genome shotgun sequence assembly of flax genotype CDC Bethune. Genotyping-by-sequencing of an F6-derived recombinant inbred line population provided validation of the SNPs. Results Reduced representation libraries of eight flax genotypes were sequenced on the Illumina sequencing platform resulting in sequence coverage ranging from 4.33 to 15.64X (genome equivalents. Depending on the relatedness of the genotypes and the number and length of the reads, between 78% and 93% of the reads mapped onto the CDC Bethune whole genome shotgun sequence assembly. A total of 55,465 SNPs were discovered with the largest number of SNPs belonging to the genotypes with the highest mapping coverage percentage. Approximately 84% of the SNPs discovered were identified in a single genotype, 13% were shared between any two genotypes and the remaining 3% in three or more. Nearly a quarter of the SNPs were found in genic regions. A total of 4,706 out of 4,863 SNPs discovered in Macbeth were validated using genotyping-by-sequencing of 96 F6 individuals from a recombinant inbred line population derived from a cross between CDC Bethune and Macbeth, corresponding to a validation rate of 96.8%. Conclusions Next generation sequencing of reduced representation libraries was successfully implemented for genome-wide SNP discovery from

  3. The history and advances of reversible terminators used in new generations of sequencing technology.

    Science.gov (United States)

    Chen, Fei; Dong, Mengxing; Ge, Meng; Zhu, Lingxiang; Ren, Lufeng; Liu, Guocheng; Mu, Rong

    2013-02-01

    DNA sequencing using reversible terminators, as one sequencing by synthesis strategy, has garnered a great deal of interest due to its popular application in the second-generation high-throughput DNA sequencing technology. In this review, we provided its history of development, classification, and working mechanism of this technology. We also outlined the screening strategies for DNA polymerases to accommodate the reversible terminators as substrates during polymerization; particularly, we introduced the "REAP" method developed by us. At the end of this review, we discussed current limitations of this approach and provided potential solutions to extend its application. Copyright © 2013. Production and hosting by Elsevier Ltd.

  4. Generation and Analysis of Full-length cDNA Sequences from Elephant Shark (Callorhinchus milii)

    KAUST Repository

    Kodzius, Rimantas

    2009-03-17

    Cartilaginous fishes are the oldest living group of jawed vertebrates and therefore is an important group for understanding the evolution of vertebrate genomes including the human genome. Our laboratory has proposed elephant shark (C. milii) as a model cartilaginous fish genome because of its relatively small genome size (910 Mb). The whole genome of C. milii is being sequenced (first cartilaginous fish genome to be sequenced completely). To characterize the transcriptome of C. milii and to assist in annotating exon-intron boundaries, transcriptional start sites and alternatively spliced transcripts, we are generating full-length cDNA sequences from C. milii.

  5. NGS QC Toolkit: a toolkit for quality control of next generation sequencing data.

    Directory of Open Access Journals (Sweden)

    Ravi K Patel

    Full Text Available Next generation sequencing (NGS technologies provide a high-throughput means to generate large amount of sequence data. However, quality control (QC of sequence data generated from these technologies is extremely important for meaningful downstream analysis. Further, highly efficient and fast processing tools are required to handle the large volume of datasets. Here, we have developed an application, NGS QC Toolkit, for quality check and filtering of high-quality data. This toolkit is a standalone and open source application freely available at http://www.nipgr.res.in/ngsqctoolkit.html. All the tools in the application have been implemented in Perl programming language. The toolkit is comprised of user-friendly tools for QC of sequencing data generated using Roche 454 and Illumina platforms, and additional tools to aid QC (sequence format converter and trimming tools and analysis (statistics tools. A variety of options have been provided to facilitate the QC at user-defined parameters. The toolkit is expected to be very useful for the QC of NGS data to facilitate better downstream analysis.

  6. Maturity onset diabetes of youth (MODY) in Turkish children: sequence analysis of 11 causative genes by next generation sequencing.

    Science.gov (United States)

    Ağladıoğlu, Sebahat Yılmaz; Aycan, Zehra; Çetinkaya, Semra; Baş, Veysel Nijat; Önder, Aşan; Peltek Kendirci, Havva Nur; Doğan, Haldun; Ceylaner, Serdar

    2016-04-01

    Maturity-onset diabetes of the youth (MODY), is a genetically and clinically heterogeneous group of diseasesand is often misdiagnosed as type 1 or type 2 diabetes. The aim of this study is to investigate both novel and proven mutations of 11 MODY genes in Turkish children by using targeted next generation sequencing. A panel of 11 MODY genes were screened in 43 children with MODY diagnosed by clinical criterias. Studies of index cases was done with MISEQ-ILLUMINA, and family screenings and confirmation studies of mutations was done by Sanger sequencing. We identified 28 (65%) point mutations among 43 patients. Eighteen patients have GCK mutations, four have HNF1A, one has HNF4A, one has HNF1B, two have NEUROD1, one has PDX1 gene variations and one patient has both HNF1A and HNF4A heterozygote mutations. This is the first study including molecular studies of 11 MODY genes in Turkish children. GCK is the most frequent type of MODY in our study population. Very high frequency of novel mutations (42%) in our study population, supports that in heterogenous disorders like MODY sequence analysis provides rapid, cost effective and accurate genetic diagnosis.

  7. A P-N Sequence Generator Using LFSR with Dual Edge Trigger Technique

    Directory of Open Access Journals (Sweden)

    Naghwal Nitin Kumar

    2016-01-01

    Full Text Available This paper represents the design and implementation of a low power 4-bit LFSR using Dual edge triggered flip flop. A linear feedback shift register (LFSR is assembled by N number of flip flops connected in series and a combinational logic generally xor gate. An LFSR can generate random number sequence which acts as cipher in cryptography. A known text encrypted over long PN sequence, in order to improve security sequence made longer ie 128 bit; require long chain of flip flop leads to more power consumption. In this paper a novel circuit of random sequence generator using dual edge triggered flip flop has been proposed. Data has been generated on every edge of flip flop instead of single edge. A DETFF-LFSR can generate random number require with less number of clock cycle, it minimizes the number of flip flop result in power saving. In this paper we concentrates on the designing of power competent Test Pattern Generator (TPG using four dual edge triggered flip-flops as the basic building block, overall there is reduction of power around 25% by using these techniques.

  8. Combined DECS Analysis and Next-Generation Sequencing Enable Efficient Detection of Novel Plant RNA Viruses

    Directory of Open Access Journals (Sweden)

    Hironobu Yanagisawa

    2016-03-01

    Full Text Available The presence of high molecular weight double-stranded RNA (dsRNA within plant cells is an indicator of infection with RNA viruses as these possess genomic or replicative dsRNA. DECS (dsRNA isolation, exhaustive amplification, cloning, and sequencing analysis has been shown to be capable of detecting unknown viruses. We postulated that a combination of DECS analysis and next-generation sequencing (NGS would improve detection efficiency and usability of the technique. Here, we describe a model case in which we efficiently detected the presumed genome sequence of Blueberry shoestring virus (BSSV, a member of the genus Sobemovirus, which has not so far been reported. dsRNAs were isolated from BSSV-infected blueberry plants using the dsRNA-binding protein, reverse-transcribed, amplified, and sequenced using NGS. A contig of 4,020 nucleotides (nt that shared similarities with sequences from other Sobemovirus species was obtained as a candidate of the BSSV genomic sequence. Reverse transcription (RT-PCR primer sets based on sequences from this contig enabled the detection of BSSV in all BSSV-infected plants tested but not in healthy controls. A recombinant protein encoded by the putative coat protein gene was bound by the BSSV-antibody, indicating that the candidate sequence was that of BSSV itself. Our results suggest that a combination of DECS analysis and NGS, designated here as “DECS-C,” is a powerful method for detecting novel plant viruses.

  9. Statistical inference of the generation probability of T-cell receptors from sequence repertoires.

    Science.gov (United States)

    Murugan, Anand; Mora, Thierry; Walczak, Aleksandra M; Callan, Curtis G

    2012-10-02

    Stochastic rearrangement of germline V-, D-, and J-genes to create variable coding sequence for certain cell surface receptors is at the origin of immune system diversity. This process, known as "VDJ recombination", is implemented via a series of stochastic molecular events involving gene choices and random nucleotide insertions between, and deletions from, genes. We use large sequence repertoires of the variable CDR3 region of human CD4+ T-cell receptor beta chains to infer the statistical properties of these basic biochemical events. Because any given CDR3 sequence can be produced in multiple ways, the probability distribution of hidden recombination events cannot be inferred directly from the observed sequences; we therefore develop a maximum likelihood inference method to achieve this end. To separate the properties of the molecular rearrangement mechanism from the effects of selection, we focus on nonproductive CDR3 sequences in T-cell DNA. We infer the joint distribution of the various generative events that occur when a new T-cell receptor gene is created. We find a rich picture of correlation (and absence thereof), providing insight into the molecular mechanisms involved. The generative event statistics are consistent between individuals, suggesting a universal biochemical process. Our probabilistic model predicts the generation probability of any specific CDR3 sequence by the primitive recombination process, allowing us to quantify the potential diversity of the T-cell repertoire and to understand why some sequences are shared between individuals. We argue that the use of formal statistical inference methods, of the kind presented in this paper, will be essential for quantitative understanding of the generation and evolution of diversity in the adaptive immune system.

  10. Interacting binaries

    International Nuclear Information System (INIS)

    Eggleton, P.P.; Pringle, J.E.

    1985-01-01

    This volume contains 15 review articles in the field of binary stars. The subjects reviewed span considerably, from the shortest period of interacting binaries to the longest, symbiotic stars. Also included are articles on Algols, X-ray binaries and Wolf-Rayet stars (single and binary). Contents: Preface. List of Participants. Activity of Contact Binary Systems. Wolf-Rayet Stars and Binarity. Symbiotic Stars. Massive X-ray Binaries. Stars that go Hump in the Night: The SU UMa Stars. Interacting Binaries - Summing Up

  11. 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.

  12. Partial sequence homogenization in the 5S multigene families may generate sequence chimeras and spurious results in phylogenetic reconstructions.

    Science.gov (United States)

    Galián, José A; Rosato, Marcela; Rosselló, Josep A

    2014-03-01

    Multigene families have provided opportunities for evolutionary biologists to assess molecular evolution processes and phylogenetic reconstructions at deep and shallow systematic levels. However, the use of these markers is not free of technical and analytical challenges. Many evolutionary studies that used the nuclear 5S rDNA gene family rarely used contiguous 5S coding sequences due to the routine use of head-to-tail polymerase chain reaction primers that are anchored to the coding region. Moreover, the 5S coding sequences have been concatenated with independent, adjacent gene units in many studies, creating simulated chimeric genes as the raw data for evolutionary analysis. This practice is based on the tacitly assumed, but rarely tested, hypothesis that strict intra-locus concerted evolution processes are operating in 5S rDNA genes, without any empirical evidence as to whether it holds for the recovered data. The potential pitfalls of analysing the patterns of molecular evolution and reconstructing phylogenies based on these chimeric genes have not been assessed to date. Here, we compared the sequence integrity and phylogenetic behavior of entire versus concatenated 5S coding regions from a real data set obtained from closely related plant species (Medicago, Fabaceae). Our results suggest that within arrays sequence homogenization is partially operating in the 5S coding region, which is traditionally assumed to be highly conserved. Consequently, concatenating 5S genes increases haplotype diversity, generating novel chimeric genotypes that most likely do not exist within the genome. In addition, the patterns of gene evolution are distorted, leading to incorrect haplotype relationships in some evolutionary reconstructions.

  13. Development and evaluation of a panel of filovirus sequence capture probes for pathogen detection by next-generation sequencing.

    Directory of Open Access Journals (Sweden)

    Jeffrey W Koehler

    Full Text Available A detailed understanding of the circulating pathogens in a particular geographic location aids in effectively utilizing targeted, rapid diagnostic assays, thus allowing for appropriate therapeutic and containment procedures. This is especially important in regions prevalent for highly pathogenic viruses co-circulating with other endemic pathogens such as the malaria parasite. The importance of biosurveillance is highlighted by the ongoing Ebola virus disease outbreak in West Africa. For example, a more comprehensive assessment of the regional pathogens could have identified the risk of a filovirus disease outbreak earlier and led to an improved diagnostic and response capacity in the region. In this context, being able to rapidly screen a single sample for multiple pathogens in a single tube reaction could improve both diagnostics as well as pathogen surveillance. Here, probes were designed to capture identifying filovirus sequence for the ebolaviruses Sudan, Ebola, Reston, Taï Forest, and Bundibugyo and the Marburg virus variants Musoke, Ci67, and Angola. These probes were combined into a single probe panel, and the captured filovirus sequence was successfully identified using the MiSeq next-generation sequencing platform. This panel was then used to identify the specific filovirus from nonhuman primates experimentally infected with Ebola virus as well as Bundibugyo virus in human sera samples from the Democratic Republic of the Congo, thus demonstrating the utility for pathogen detection using clinical samples. While not as sensitive and rapid as real-time PCR, this panel, along with incorporating additional sequence capture probe panels, could be used for broad pathogen screening and biosurveillance.

  14. Generation and analysis of expressed sequence tags from the ciliate protozoan parasite Ichthyophthirius multifiliis

    Directory of Open Access Journals (Sweden)

    Arias Covadonga

    2007-06-01

    Full Text Available Abstract Background The ciliate protozoan Ichthyophthirius multifiliis (Ich is an important parasite of freshwater fish that causes 'white spot disease' leading to significant losses. A genomic resource for large-scale studies of this parasite has been lacking. To study gene expression involved in Ich pathogenesis and virulence, our goal was to generate expressed sequence tags (ESTs for the development of a powerful microarray platform for the analysis of global gene expression in this species. Here, we initiated a project to sequence and analyze over 10,000 ESTs. Results We sequenced 10,368 EST clones using a normalized cDNA library made from pooled samples of the trophont, tomont, and theront life-cycle stages, and generated 9,769 sequences (94.2% success rate. Post-sequencing processing led to 8,432 high quality sequences. Clustering analysis of these ESTs allowed identification of 4,706 unique sequences containing 976 contigs and 3,730 singletons. These unique sequences represent over two million base pairs (~10% of Plasmodium falciparum genome, a phylogenetically related protozoan. BLASTX searches produced 2,518 significant (E-value -5 hits and further Gene Ontology (GO analysis annotated 1,008 of these genes. The ESTs were analyzed comparatively against the genomes of the related protozoa Tetrahymena thermophila and P. falciparum, allowing putative identification of additional genes. All the EST sequences were deposited by dbEST in GenBank (GenBank: EG957858–EG966289. Gene discovery and annotations are presented and discussed. Conclusion This set of ESTs represents a significant proportion of the Ich transcriptome, and provides a material basis for the development of microarrays useful for gene expression studies concerning Ich development, pathogenesis, and virulence.

  15. Some properties of spectral binary stars

    International Nuclear Information System (INIS)

    Krajcheva, Z.T.; Popova, E.I.; Tutukov, A.V.; Yungel'son, L.R.; AN SSSR, Moscow. Astronomicheskij Sovet)

    1978-01-01

    Statistical investigations of spectra binary stars are carried out. Binary systems consisting of main sequence stars are considered. For 826 binary stars masses of components, ratios of component masses, semiaxes of orbits and orbital angular momenta are calculated. The distributions of these parameters and their correlations are analyzed. The dependences of statistical properties of spectral binary stars on their origin and evolution are discussed

  16. The age-metallicity relation in the solar neighbourhood from a pilot sample of white dwarf-main sequence binaries

    OpenAIRE

    Rebassa-Mansergas, A.; Anguiano, B.; García-Berro, E.; Freeman, K. C.; Cojocaru, R.; Manser, C. J.; Pala, A. F.; Gänsicke, B. T.; Liu, X. -W.

    2016-01-01

    The age–metallicity relation (AMR) is a fundamental observational constraint for understanding how the Galactic disc formed and evolved chemically in time. However, there is not yet an agreement on the observational properties of the AMR for the solar neighbourhood, primarily due to the difficulty in obtaining accurate stellar ages for individual field stars. We have started an observational campaign for providing the much needed observational input by using wide white-dwarf–main-sequence (WD...

  17. Recurrence Relations and Generating Functions of the Sequence of Sums of Corresponding Factorials and Triangular Numbers

    Directory of Open Access Journals (Sweden)

    Romer C. Castillo

    2015-11-01

    Full Text Available This study established some recurrence relations and exponential generating functions of the sequence of factoriangular numbers. A factoriangular number is defined as a sum of corresponding factorial and triangular number. The proofs utilize algebraic manipulations with some known results from calculus, particularly on power series and Maclaurin’s series. The recurrence relations were found by manipulating the formula defining a factoringular number while the ascertained exponential generating functions were in the closed form.

  18. [Molecular and prenatal diagnosis of a family with Fanconi anemia by next generation sequencing].

    Science.gov (United States)

    Gong, Zhuwen; Yu, Yongguo; Zhang, Qigang; Gu, Xuefan

    2015-04-01

    To provide prenatal diagnosis for a pregnant woman who had given birth to a child with Fanconi anemia with combined next-generation sequencing (NGS) and Sanger sequencing. For the affected child, potential mutations of the FANCA gene were analyzed with NGS. Suspected mutation was verified with Sanger sequencing. For prenatal diagnosis, genomic DNA was extracted from cultured fetal amniotic fluid cells and subjected to analysis of the same mutations. A low-frequency frameshifting mutation c.989_995del7 (p.H330LfsX2, inherited from his father) and a truncating mutation c.3971C>T (p.P1324L, inherited from his mother) have been identified in the affected child and considered to be pathogenic. The two mutations were subsequently verified by Sanger sequencing. Upon prenatal diagnosis, the fetus was found to carry two mutations. The combined next-generation sequencing and Sanger sequencing can reduce the time for diagnosis and identify subtypes of Fanconi anemia and the mutational sites, which has enabled reliable prenatal diagnosis of this disease.

  19. Targeted Next-generation Sequencing and Bioinformatics Pipeline to Evaluate Genetic Determinants of Constitutional Disease.

    Science.gov (United States)

    Dilliott, Allison A; Farhan, Sali M K; Ghani, Mahdi; Sato, Christine; Liang, Eric; Zhang, Ming; McIntyre, Adam D; Cao, Henian; Racacho, Lemuel; Robinson, John F; Strong, Michael J; Masellis, Mario; Bulman, Dennis E; Rogaeva, Ekaterina; Lang, Anthony; Tartaglia, Carmela; Finger, Elizabeth; Zinman, Lorne; Turnbull, John; Freedman, Morris; Swartz, Rick; Black, Sandra E; Hegele, Robert A

    2018-04-04

    Next-generation sequencing (NGS) is quickly revolutionizing how research into the genetic determinants of constitutional disease is performed. The technique is highly efficient with millions of sequencing reads being produced in a short time span and at relatively low cost. Specifically, targeted NGS is able to focus investigations to genomic regions of particular interest based on the disease of study. Not only does this further reduce costs and increase the speed of the process, but it lessens the computational burden that often accompanies NGS. Although targeted NGS is restricted to certain regions of the genome, preventing identification of potential novel loci of interest, it can be an excellent technique when faced with a phenotypically and genetically heterogeneous disease, for which there are previously known genetic associations. Because of the complex nature of the sequencing technique, it is important to closely adhere to protocols and methodologies in order to achieve sequencing reads of high coverage and quality. Further, once sequencing reads are obtained, a sophisticated bioinformatics workflow is utilized to accurately map reads to a reference genome, to call variants, and to ensure the variants pass quality metrics. Variants must also be annotated and curated based on their clinical significance, which can be standardized by applying the American College of Medical Genetics and Genomics Pathogenicity Guidelines. The methods presented herein will display the steps involved in generating and analyzing NGS data from a targeted sequencing panel, using the ONDRISeq neurodegenerative disease panel as a model, to identify variants that may be of clinical significance.

  20. Challenges and opportunities in estimating viral genetic diversity from next-generation sequencing data

    Directory of Open Access Journals (Sweden)

    Niko eBeerenwinkel

    2012-09-01

    Full Text Available Many viruses, including the clinically relevant RNA viruses HIV and HCV, exist in large populations and display high genetic heterogeneity within and between infected hosts. Assessing intra-patient viral genetic diversity is essential for understanding the evolutionary dynamics of viruses, for designing effective vaccines, and for the success of antiviral therapy. Next-generation sequencing technologies allow the rapid and cost-effective acquisition of thousands to millions of short DNA sequences from a single sample. However, this approach entails several challenges in experimental design and computational data analysis. Here, we review the entire process of inferring viral diversity from sample collection to computing measures of genetic diversity. We discuss sample preparation, including reverse transcription and amplification, and the effect of experimental conditions on diversity estimates due to in vitro base substitutions, insertions, deletions, and recombination. The use of different next-generation sequencing platforms and their sequencing error profiles are compared in the context of various applications of diversity estimation, ranging from the detection of single nucleotide variants to the reconstruction of whole-genome haplotypes. We describe the statistical and computational challenges arising from these technical artifacts, and we review existing approaches, including available software, for their solution. Finally, we discuss open problems, and highlight successful biomedical applications and potential future clinical use of next-generation sequencing to estimate viral diversity.

  1. ReQON: a Bioconductor package for recalibrating quality scores from next-generation sequencing data

    Directory of Open Access Journals (Sweden)

    Cabanski Christopher R

    2012-09-01

    Full Text Available Abstract Background Next-generation sequencing technologies have become important tools for genome-wide studies. However, the quality scores that are assigned to each base have been shown to be inaccurate. If the quality scores are used in downstream analyses, these inaccuracies can have a significant impact on the results. Results Here we present ReQON, a tool that recalibrates the base quality scores from an input BAM file of aligned sequencing data using logistic regression. ReQON also generates diagnostic plots showing the effectiveness of the recalibration. We show that ReQON produces quality scores that are both more accurate, in the sense that they more closely correspond to the probability of a sequencing error, and do a better job of discriminating between sequencing errors and non-errors than the original quality scores. We also compare ReQON to other available recalibration tools and show that ReQON is less biased and performs favorably in terms of quality score accuracy. Conclusion ReQON is an open source software package, written in R and available through Bioconductor, for recalibrating base quality scores for next-generation sequencing data. ReQON produces a new BAM file with more accurate quality scores, which can improve the results of downstream analysis, and produces several diagnostic plots showing the effectiveness of the recalibration.

  2. Navigating the tip of the genomic iceberg: Next-generation sequencing for plant systematics.

    Science.gov (United States)

    Straub, Shannon C K; Parks, Matthew; Weitemier, Kevin; Fishbein, Mark; Cronn, Richard C; Liston, Aaron

    2012-02-01

    Just as Sanger sequencing did more than 20 years ago, next-generation sequencing (NGS) is poised to revolutionize plant systematics. By combining multiplexing approaches with NGS throughput, systematists may no longer need to choose between more taxa or more characters. Here we describe a genome skimming (shallow sequencing) approach for plant systematics. Through simulations, we evaluated optimal sequencing depth and performance of single-end and paired-end short read sequences for assembly of nuclear ribosomal DNA (rDNA) and plastomes and addressed the effect of divergence on reference-guided plastome assembly. We also used simulations to identify potential phylogenetic markers from low-copy nuclear loci at different sequencing depths. We demonstrated the utility of genome skimming through phylogenetic analysis of the Sonoran Desert clade (SDC) of Asclepias (Apocynaceae). Paired-end reads performed better than single-end reads. Minimum sequencing depths for high quality rDNA and plastome assemblies were 40× and 30×, respectively. Divergence from the reference significantly affected plastome assembly, but relatively similar references are available for most seed plants. Deeper rDNA sequencing is necessary to characterize intragenomic polymorphism. The low-copy fraction of the nuclear genome was readily surveyed, even at low sequencing depths. Nearly 160000 bp of sequence from three organelles provided evidence of phylogenetic incongruence in the SDC. Adoption of NGS will facilitate progress in plant systematics, as whole plastome and rDNA cistrons, partial mitochondrial genomes, and low-copy nuclear markers can now be efficiently obtained for molecular phylogenetics studies.

  3. Quantitative miRNA expression analysis: comparing microarrays with next-generation sequencing

    DEFF Research Database (Denmark)

    Willenbrock, Hanni; Salomon, Jesper; Søkilde, Rolf

    2009-01-01

    Recently, next-generation sequencing has been introduced as a promising, new platform for assessing the copy number of transcripts, while the existing microarray technology is considered less reliable for absolute, quantitative expression measurements. Nonetheless, so far, results from the two...... technologies have only been compared based on biological data, leading to the conclusion that, although they are somewhat correlated, expression values differ significantly. Here, we use synthetic RNA samples, resembling human microRNA samples, to find that microarray expression measures actually correlate...... better with sample RNA content than expression measures obtained from sequencing data. In addition, microarrays appear highly sensitive and perform equivalently to next-generation sequencing in terms of reproducibility and relative ratio quantification....

  4. Counting of oligomers in sequences generated by markov chains for DNA motif discovery.

    Science.gov (United States)

    Shan, Gao; Zheng, Wei-Mou

    2009-02-01

    By means of the technique of the imbedded Markov chain, an efficient algorithm is proposed to exactly calculate first, second moments of word counts and the probability for a word to occur at least once in random texts generated by a Markov chain. A generating function is introduced directly from the imbedded Markov chain to derive asymptotic approximations for the problem. Two Z-scores, one based on the number of sequences with hits and the other on the total number of word hits in a set of sequences, are examined for discovery of motifs on a set of promoter sequences extracted from A. thaliana genome. Source code is available at http://www.itp.ac.cn/zheng/oligo.c.

  5. Karect: accurate correction of substitution, insertion and deletion errors for next-generation sequencing data

    KAUST Repository

    Allam, Amin

    2015-07-14

    Motivation: Next-generation sequencing generates large amounts of data affected by errors in the form of substitutions, insertions or deletions of bases. Error correction based on the high-coverage information, typically improves de novo assembly. Most existing tools can correct substitution errors only; some support insertions and deletions, but accuracy in many cases is low. Results: We present Karect, a novel error correction technique based on multiple alignment. Our approach supports substitution, insertion and deletion errors. It can handle non-uniform coverage as well as moderately covered areas of the sequenced genome. Experiments with data from Illumina, 454 FLX and Ion Torrent sequencing machines demonstrate that Karect is more accurate than previous methods, both in terms of correcting individual-bases errors (up to 10% increase in accuracy gain) and post de novo assembly quality (up to 10% increase in NGA50). We also introduce an improved framework for evaluating the quality of error correction.

  6. Am I my Family's Keeper? : Disclosure Dilemmas in Next Generation Sequencing

    NARCIS (Netherlands)

    Wouters, Roel H P; Bijlsma, Rhodé M; Ausems, Margreet G E M; van Delden, Johannes J M; Voest, Emile E; Bredenoord, Annelien L

    2016-01-01

    Ever since genetic testing is possible for specific mutations, ethical debate has sparked on the question of whether professionals have a duty to warn not only patients but also their relatives that might be at risk for hereditary diseases. As next generation sequencing swiftly finds its way into

  7. Unifying and generating of space vector modulation sequences for multilevel converter

    DEFF Research Database (Denmark)

    Ma, Ke; Blaabjerg, Frede

    2014-01-01

    Space Vector Modulation (SVM) is a powerful method which enables some freedom to generate the modulation sequences and modify the performances of converter. However, in the multi-level converter structures, the number of switching state redundancies significantly increases, and the determination...

  8. Mutation Detection in Patients with Retinal Dystrophies Using Targeted Next Generation Sequencing

    DEFF Research Database (Denmark)

    Weisschuh, Nicole; Mayer, Anja K; Strom, Tim M

    2016-01-01

    Retinal dystrophies (RD) constitute a group of blinding diseases that are characterized by clinical variability and pronounced genetic heterogeneity. The different nonsyndromic and syndromic forms of RD can be attributed to mutations in more than 200 genes. Consequently, next generation sequencing...

  9. Next-generation sequencing in NSCLC and melanoma patients : A cost and budget impact analysis

    NARCIS (Netherlands)

    Van Amerongen, Rosa A.; Retèl, Valesca P.; Coupé, Veerle M.H.; Nederlof, Petra M.; Vogel, Maartje J.; Van Harten, Wim H.

    2016-01-01

    Next-generation sequencing (NGS) has reached the molecular diagnostic laboratories. Although the NGS technology aims to improve the effectiveness of therapies by selecting the most promising therapy, concerns are that NGS testing is expensive and that the 'benefits' are not yet in relation to these

  10. A generative Bezier curve model for surf-zone tracking in coastal image sequences

    CSIR Research Space (South Africa)

    Burke, Michael G

    2017-09-01

    Full Text Available This work introduces a generative Bezier curve model suitable for surf-zone curve tracking in coastal image sequences. The model combines an adaptive curve parametrised by control points governed by local random walks with a global sinusoidal motion...

  11. Bringing Next-Generation Sequencing into the Classroom through a Comparison of Molecular Biology Techniques

    Science.gov (United States)

    Bowling, Bethany; Zimmer, Erin; Pyatt, Robert E.

    2014-01-01

    Although the development of next-generation (NextGen) sequencing technologies has revolutionized genomic research and medicine, the incorporation of these topics into the classroom is challenging, given an implied high degree of technical complexity. We developed an easy-to-implement, interactive classroom activity investigating the similarities…

  12. Estimation of allele frequency and association mapping using next-generation sequencing data

    DEFF Research Database (Denmark)

    Kim, Su Yeon; Lohmueller, Kirk E; Albrechtsen, Anders

    2011-01-01

    Estimation of allele frequency is of fundamental importance in population genetic analyses and in association mapping. In most studies using next-generation sequencing, a cost effective approach is to use medium or low-coverage data (e.g., frequency estimation...

  13. Next-generation sequencing for endocrine cancers: Recent advances and challenges.

    Science.gov (United States)

    Suresh, Padmanaban S; Venkatesh, Thejaswini; Tsutsumi, Rie; Shetty, Abhishek

    2017-05-01

    Contemporary molecular biology research tools have enriched numerous areas of biomedical research that address challenging diseases, including endocrine cancers (pituitary, thyroid, parathyroid, adrenal, testicular, ovarian, and neuroendocrine cancers). These tools have placed several intriguing clues before the scientific community. Endocrine cancers pose a major challenge in health care and research despite considerable attempts by researchers to understand their etiology. Microarray analyses have provided gene signatures from many cells, tissues, and organs that can differentiate healthy states from diseased ones, and even show patterns that correlate with stages of a disease. Microarray data can also elucidate the responses of endocrine tumors to therapeutic treatments. The rapid progress in next-generation sequencing methods has overcome many of the initial challenges of these technologies, and their advantages over microarray techniques have enabled them to emerge as valuable aids for clinical research applications (prognosis, identification of drug targets, etc.). A comprehensive review describing the recent advances in next-generation sequencing methods and their application in the evaluation of endocrine and endocrine-related cancers is lacking. The main purpose of this review is to illustrate the concepts that collectively constitute our current view of the possibilities offered by next-generation sequencing technological platforms, challenges to relevant applications, and perspectives on the future of clinical genetic testing of patients with endocrine tumors. We focus on recent discoveries in the use of next-generation sequencing methods for clinical diagnosis of endocrine tumors in patients and conclude with a discussion on persisting challenges and future objectives.

  14. Body fluid identification of blood, saliva and semen using second generation sequencing of micro-RNA

    DEFF Research Database (Denmark)

    Petersen, Christel H.; Hjort, Benjamin Benn; Tvedebrink, Torben

    2013-01-01

    We report a new second generation sequencing method for identification micro-RNA (miRNA) that can be used to identify body fluids and tissues. Principal component analysis of 10 miRNAs with high expression in 16 samples of blood, saliva and semen showed clear differences in the expression of mi...

  15. PheoSeq : A Targeted Next-Generation Sequencing Assay for Pheochromocytoma and Paraganglioma Diagnostics

    NARCIS (Netherlands)

    Currás-Freixes, Maria; Piñeiro-Yañez, Elena; Montero-Conde, Cristina; Apellániz-Ruiz, María; Calsina, Bruna; Mancikova, Veronika; Remacha, Laura; Richter, Susan; Ercolino, Tonino; Rogowski-Lehmann, Natalie; Deutschbein, Timo; Calatayud, María; Guadalix, Sonsoles; Álvarez-Escolá, Cristina; Lamas, Cristina; Aller, Javier; Sastre-Marcos, Julia; Lázaro, Conxi; Galofré, Juan C.; Patiño-García, Ana; Meoro-Avilés, Amparo; Balmaña-Gelpi, Judith; De Miguel-Novoa, Paz; Balbín, Milagros; Matías-Guiu, Xavier; Letón, Rocío; Inglada-Pérez, Lucía; Torres-Pérez, Rafael; Roldán-Romero, Juan M.; Rodríguez-Antona, Cristina; Fliedner, Stephanie M J; Opocher, Giuseppe; Pacak, Karel; Korpershoek, Esther; de Krijger, Ronald R.; Vroonen, Laurent; Mannelli, Massimo; Fassnacht, Martin; Beuschlein, Felix; Eisenhofer, Graeme; Cascón, Alberto; Al-Shahrour, Fátima; Robledo, Mercedes

    2017-01-01

    Genetic diagnosis is recommended for all pheochromocytoma and paraganglioma (PPGL) cases, as driver mutations are identified in approximately 80% of the cases. As the list of related genes expands, genetic diagnosis becomes more time-consuming, and targeted next-generation sequencing (NGS) has

  16. The role of next generation sequencing for the development and testing of veterinary biologics

    Science.gov (United States)

    Next generation sequencing technology has become widely available and it offers many new opportunities in vaccine technology. Both human and veterinary medicine has numerous examples of adventitious agents being found in live vaccines. In veterinary medicine a continuing trend is the use of viral ...

  17. Unveiling distribution patterns of freshwater phytoplankton by a next generation sequencing based approach

    Czech Academy of Sciences Publication Activity Database

    Eiler, A.; Drakare, S.; Bertilsson, S.; Pernthaler, J.; Peura, S.; Rofner, C.; Šimek, Karel; Yang, Y.; Znachor, Petr; Lindström, E.S.

    2013-01-01

    Roč. 8, č. 1 (2013), e53516 E-ISSN 1932-6203 R&D Projects: GA ČR(CZ) GA206/08/0015 Institutional support: RVO:60077344 Keywords : phytoplankton * next generation sequencing * diversity Subject RIV: EE - Microbiology, Virology Impact factor: 3.534, year: 2013

  18. DNA fingerprinting, DNA barcoding, and next generation sequencing technology in plants.

    Science.gov (United States)

    Sucher, Nikolaus J; Hennell, James R; Carles, Maria C

    2012-01-01

    DNA fingerprinting of plants has become an invaluable tool in forensic, scientific, and industrial laboratories all over the world. PCR has become part of virtually every variation of the plethora of approaches used for DNA fingerprinting today. DNA sequencing is increasingly used either in combination with or as a replacement for traditional DNA fingerprinting techniques. A prime example is the use of short, standardized regions of the genome as taxon barcodes for biological identification of plants. Rapid advances in "next generation sequencing" (NGS) technology are driving down the cost of sequencing and bringing large-scale sequencing projects into the reach of individual investigators. We present an overview of recent publications that demonstrate the use of "NGS" technology for DNA fingerprinting and DNA barcoding applications.

  19. New tool to assemble repetitive regions using next-generation sequencing data

    Science.gov (United States)

    Kuśmirek, Wiktor; Nowak, Robert M.; Neumann, Łukasz

    2017-08-01

    The next generation sequencing techniques produce a large amount of sequencing data. Some part of the genome are composed of repetitive DNA sequences, which are very problematic for the existing genome assemblers. We propose a modification of the algorithm for a DNA assembly, which uses the relative frequency of reads to properly reconstruct repetitive sequences. The new approach was implemented and tested, as a demonstration of the capability of our software we present some results for model organisms. The new implementation, using a three-layer software architecture was selected, where the presentation layer, data processing layer, and data storage layer were kept separate. Source code as well as demo application with web interface and the additional data are available at project web-page: http://dnaasm.sourceforge.net.

  20. Illuminating the evolution of equids and rodents with next-generation sequencing of ancient specimens

    DEFF Research Database (Denmark)

    Mouatt, Julia Thidamarth Vilstrup

    enrichment methods and the massive throughput and latest advances within DNA sequencing, the field of ancient DNA has flourished in later years. Those advances have even enabled the sequencing of complete genomes from the past, moving the field into genomic sciences. In this thesis we have used these latest......The sequencing of ancient DNA provides perspectives on the genetic history of past populations and extinct species. However, ancient DNA research presents specific limitations mostly due to DNA survival, damage and contamination. Yet with stringent laboratory procedures, the sensitivity of target...... developments within ancient DNA research, including target enrichment capture and Next-Generation Sequencing, to address a range of evolutionary questions related to two major mammalian groups, equids and rodents. In particular we have resolved phylogenetic relationships within equids using complete mitochond...

  1. Evaluating multiplexed next-generation sequencing as a method in palynology for mixed pollen samples.

    Science.gov (United States)

    Keller, A; Danner, N; Grimmer, G; Ankenbrand, M; von der Ohe, K; von der Ohe, W; Rost, S; Härtel, S; Steffan-Dewenter, I

    2015-03-01

    The identification of pollen plays an important role in ecology, palaeo-climatology, honey quality control and other areas. Currently, expert knowledge and reference collections are essential to identify pollen origin through light microscopy. Pollen identification through molecular sequencing and DNA barcoding has been proposed as an alternative approach, but the assessment of mixed pollen samples originating from multiple plant species is still a tedious and error-prone task. Next-generation sequencing has been proposed to avoid this hindrance. In this study we assessed mixed pollen probes through next-generation sequencing of amplicons from the highly variable, species-specific internal transcribed spacer 2 region of nuclear ribosomal DNA. Further, we developed a bioinformatic workflow to analyse these high-throughput data with a newly created reference database. To evaluate the feasibility, we compared results from classical identification based on light microscopy from the same samples with our sequencing results. We assessed in total 16 mixed pollen samples, 14 originated from honeybee colonies and two from solitary bee nests. The sequencing technique resulted in higher taxon richness (deeper assignments and more identified taxa) compared to light microscopy. Abundance estimations from sequencing data were significantly correlated with counted abundances through light microscopy. Simulation analyses of taxon specificity and sensitivity indicate that 96% of taxa present in the database are correctly identifiable at the genus level and 70% at the species level. Next-generation sequencing thus presents a useful and efficient workflow to identify pollen at the genus and species level without requiring specialised palynological expert knowledge. © 2014 German Botanical Society and The Royal Botanical Society of the Netherlands.

  2. The simultaneous use of several pseudo-random binary sequences in the identification of linear multivariable dynamic systems

    International Nuclear Information System (INIS)

    Cummins, J.D.

    1965-02-01

    With several white noise sources the various transmission paths of a linear multivariable system may be determined simultaneously. This memorandum considers the restrictions on pseudo-random two state sequences to effect simultaneous identification of several transmission paths and the consequential rejection of cross-coupled signals in linear multivariable systems. The conditions for simultaneous identification are established by an example, which shows that the integration time required is large i.e. tends to infinity, as it does when white noise sources are used. (author)

  3. BINARY VECTOR CONSTRUCTION OF KAPPA(κ-CARRAGEENASE GENE AND TRANSFORMATION TO Agrobacterium tumefaciens AS MEDIATOR FOR SEAWEED TRANSGENIC GENERATION

    Directory of Open Access Journals (Sweden)

    Muh Alias L. Rajamuddin

    2016-11-01

    Full Text Available Increasing of kappa (κ-carrageenan content in Kappaphycus alvarezii seaweed is potentially be achieved by applying transgenesis technology. This study was performed to obtain a construction of  κ-Carrageenase gene and Agrobacterium tumefaciens to carry those construction genes.  The κ-Carrageenase (κ-Car gene was involved in κ-carrageenan biosynthesis. The κ-Car gene sequence was ligated between the 35S CaMV promoter and tNos terminator sequences to generate pMSH/κ-Car expression vector. Transformation of pMSH/κ-Car plasmid to Escherichia coli was performed by heat-shock method, and to Agrobacterium tumefaciens by tri-parental mating method. The results showed that several colonies of E. coli and A. tumefaciens grew in the selective culture mediums containing antibiotic. PCR analysis using primers 35S-Forward and tNos-Reverse with DNA template from those bacterial colonies resulted DNA fragment of about 2,000 bp, the same as the total length of 35S CaMV promoter, κ-Car gene and tNos terminator sequences. Therefore, the construction of pMSH/κ-Car gene was succeeded and a colony of A. tumefaciens transformant carrying pMSH/κ-Car plasmid was successfully produced.                                                                                   Keywords:  Agrobacterium tumefaciens, kappa(κ-Carrageenase gene, transgenesis, vector

  4. SIS: a program to generate draft genome sequence scaffolds for prokaryotes

    Directory of Open Access Journals (Sweden)

    Dias Zanoni

    2012-05-01

    Full Text Available Abstract Background Decreasing costs of DNA sequencing have made prokaryotic draft genome sequences increasingly common. A contig scaffold is an ordering of contigs in the correct orientation. A scaffold can help genome comparisons and guide gap closure efforts. One popular technique for obtaining contig scaffolds is to map contigs onto a reference genome. However, rearrangements that may exist between the query and reference genomes may result in incorrect scaffolds, if these rearrangements are not taken into account. Large-scale inversions are common rearrangement events in prokaryotic genomes. Even in draft genomes it is possible to detect the presence of inversions given sufficient sequencing coverage and a sufficiently close reference genome. Results We present a linear-time algorithm that can generate a set of contig scaffolds for a draft genome sequence represented in contigs given a reference genome. The algorithm is aimed at prokaryotic genomes and relies on the presence of matching sequence patterns between the query and reference genomes that can be interpreted as the result of large-scale inversions; we call these patterns inversion signatures. Our algorithm is capable of correctly generating a scaffold if at least one member of every inversion signature pair is present in contigs and no inversion signatures have been overwritten in evolution. The algorithm is also capable of generating scaffolds in the presence of any kind of inversion, even though in this general case there is no guarantee that all scaffolds in the scaffold set will be correct. We compare the performance of sis, the program that implements the algorithm, to seven other scaffold-generating programs. The results of our tests show that sis has overall better performance. Conclusions sis is a new easy-to-use tool to generate contig scaffolds, available both as stand-alone and as a web server. The good performance of sis in our tests adds evidence that large

  5. Localization accuracy of compact binary coalescences detected by the third-generation gravitational-wave detectors and implication for cosmology

    Science.gov (United States)

    Zhao, Wen; Wen, Linqing

    2018-03-01

    We use the Fisher information matrix to investigate the angular resolution and luminosity distance uncertainty for coalescing binary neutron stars (BNSs) and neutron star-black hole binaries (NSBHs) detected by the third-generation (3G) gravitational-wave (GW) detectors. Our study focuses on an individual 3G detector and a network of up to four 3G detectors at different locations including the United States, Europe, China, and Australia for the proposed Einstein Telescope (ET) and Cosmic Explorer (CE) detectors. In particular, we examine the effect of the Earth's rotation, as GW signals from BNS and low-mass NSBH systems could be hours long for 3G detectors. In this case, an individual detector can be effectively treated as a detector network with long baselines formed by the trajectory of the detector as it rotates with the Earth. Therefore, a single detector or two-detector networks could also be used to localize the GW sources effectively. We find that a time-dependent antenna beam-pattern function can help better localize BNS and NSBH sources, especially edge-on ones. The medium angular resolution for one ET-D detector is around 150 deg2 for BNSs at a redshift of z =0.1 , which improves rapidly with a decreasing low-frequency cutoff flow in sensitivity. The medium angular resolution for a network of two CE detectors in the United States and Europe, respectively, is around 20 deg2 at z =0.2 for the simulated BNS and NSBH samples. While for a network of two ET-D detectors, the similar angular resolution can be achieved at a much higher redshift of z =0.5 . The angular resolution of a network of three detectors is mainly determined by the baselines between detectors regardless of the CE or ET detector type. The medium angular resolution of BNS for a network of three detectors of the ET-D or CE type in the United States, Europe, and Australia is around 10 deg2 at z =2 . We discuss the implications of our results for multimessenger astronomy and, in particular, for

  6. Generation of expressed sequence tags for discovery of genes responsible for floral traits of Chrysanthemum morifolium by next-generation sequencing technology.

    Science.gov (United States)

    Sasaki, Katsutomo; Mitsuda, Nobutaka; Nashima, Kenji; Kishimoto, Kyutaro; Katayose, Yuichi; Kanamori, Hiroyuki; Ohmiya, Akemi

    2017-09-04

    Chrysanthemum morifolium is one of the most economically valuable ornamental plants worldwide. Chrysanthemum is an allohexaploid plant with a large genome that is commercially propagated by vegetative reproduction. New cultivars with different floral traits, such as color, morphology, and scent, have been generated mainly by classical cross-breeding and mutation breeding. However, only limited genetic resources and their genome information are available for the generation of new floral traits. To obtain useful information about molecular bases for floral traits of chrysanthemums, we read expressed sequence tags (ESTs) of chrysanthemums by high-throughput sequencing using the 454 pyrosequencing technology. We constructed normalized cDNA libraries, consisting of full-length, 3'-UTR, and 5'-UTR cDNAs derived from various tissues of chrysanthemums. These libraries produced a total number of 3,772,677 high-quality reads, which were assembled into 213,204 contigs. By comparing the data obtained with those of full genome-sequenced species, we confirmed that our chrysanthemum contig set contained the majority of all expressed genes, which was sufficient for further molecular analysis in chrysanthemums. We confirmed that our chrysanthemum EST set (contigs) contained a number of contigs that encoded transcription factors and enzymes involved in pigment and aroma compound metabolism that was comparable to that of other species. This information can serve as an informative resource for identifying genes involved in various biological processes in chrysanthemums. Moreover, the findings of our study will contribute to a better understanding of the floral characteristics of chrysanthemums including the myriad cultivars at the molecular level.

  7. Next-generation sequencing reveals phylogeographic structure and a species tree for recent bird divergences

    DEFF Research Database (Denmark)

    McCormack, John E.; Maley, James M.; Hird, Sarah M.

    2012-01-01

    divergence in four phylogenetically diverse avian systems using a method for quick and cost-effective generation of primary DNA sequence data using pyrosequencing. NGS data were processed using an analytical pipeline that reduces many reads into two called alleles per locus per individual. Using single...... throughout the genome. Using eight loci found in Zonotrichia and Junco lineages, we were also able to generate a species tree of these sparrow sister genera, demonstrating the potential of this method for generating data amenable to coalescent-based analysis. We discuss improvements that should enhance...

  8. Fiscal 1995 survey of promotion of the geothermal development. Report on a usage feasibility test of a small scale geothermal binary cycle power generation system; 1995 nendo chinetsu kaihatsu sokushin chosa. Chusho chinetsu binary hatsuden system jissho shiken hokokusho

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1997-03-01

    In this survey, studies for popularization and practical utilization of small and medium size geothermal binary cycle power systems which assesses low and medium temperature geothermal resources were conducted, and studies for development of the system to be introduced for practical use and for promotion of the popularization were made. A study was carried out of preconditions and various conditions of a demonstrative test plant (100kW class, 500kW class) in view of the initial cost of the actual plant, and an analysis was made of the power generation cost. Acceptability of the demonstrative test plant (100kW class) was examined to analyze problems on the introduction. A thermodynamic analysis was made of the output of geothermal binary cycle power generation. Analysis/evaluation of the results of the 100kW demonstrative test plant were carried out in view of the operation results of the plant of the same kind, and checks/reviews were conducted of performance and reliability of the system, equipment simplification, etc. Inspection of the system was made in the stage of design/manufacture of the 500kW demonstrative test plant. Concerning the spread/expansion of the system, studied were multiple stage geothermal utilization and PR promotion method. 14 refs., 62 figs., 55 tabs.

  9. Iterative normalization technique for reference sequence generation for zero-tail discrete fourier transform spread orthogonal frequency division multiplexing

    DEFF Research Database (Denmark)

    2017-01-01

    Systems, methods, apparatuses, and computer program products for generating sequences for zero-tail discrete fourier transform (DFT)-spread-orthogonal frequency division multiplexing (OFDM) (ZT DFT-s-OFDM) reference signals. One method includes adding a zero vector to an input sequence...... of each of the elements, converting the sequence to time domain, generating a zero-padded sequence by forcing a zero head and tail of the sequence, and repeating the steps until a final sequence with zero-tail and flat frequency response is obtained....

  10. Rapid evaluation and quality control of next generation sequencing data with FaQCs.

    Science.gov (United States)

    Lo, Chien-Chi; Chain, Patrick S G

    2014-11-19

    Next generation sequencing (NGS) technologies that parallelize the sequencing process and produce thousands to millions, or even hundreds of millions of sequences in a single sequencing run, have revolutionized genomic and genetic research. Because of the vagaries of any platform's sequencing chemistry, the experimental processing, machine failure, and so on, the quality of sequencing reads is never perfect, and often declines as the read is extended. These errors invariably affect downstream analysis/application and should therefore be identified early on to mitigate any unforeseen effects. Here we present a novel FastQ Quality Control Software (FaQCs) that can rapidly process large volumes of data, and which improves upon previous solutions to monitor the quality and remove poor quality data from sequencing runs. Both the speed of processing and the memory footprint of storing all required information have been optimized via algorithmic and parallel processing solutions. The trimmed output compared side-by-side with the original data is part of the automated PDF output. We show how this tool can help data analysis by providing a few examples, including an increased percentage of reads recruited to references, improved single nucleotide polymorphism identification as well as de novo sequence assembly metrics. FaQCs combines several features of currently available applications into a single, user-friendly process, and includes additional unique capabilities such as filtering the PhiX control sequences, conversion of FASTQ formats, and multi-threading. The original data and trimmed summaries are reported within a variety of graphics and reports, providing a simple way to do data quality control and assurance.

  11. Model-based quality assessment and base-calling for second-generation sequencing data.

    Science.gov (United States)

    Bravo, Héctor Corrada; Irizarry, Rafael A

    2010-09-01

    Second-generation sequencing (sec-gen) technology can sequence millions of short fragments of DNA in parallel, making it capable of assembling complex genomes for a small fraction of the price and time of previous technologies. In fact, a recently formed international consortium, the 1000 Genomes Project, plans to fully sequence the genomes of approximately 1200 people. The prospect of comparative analysis at the sequence level of a large number of samples across multiple populations may be achieved within the next five years. These data present unprecedented challenges in statistical analysis. For instance, analysis operates on millions of short nucleotide sequences, or reads-strings of A,C,G, or T's, between 30 and 100 characters long-which are the result of complex processing of noisy continuous fluorescence intensity measurements known as base-calling. The complexity of the base-calling discretization process results in reads of widely varying quality within and across sequence samples. This variation in processing quality results in infrequent but systematic errors that we have found to mislead downstream analysis of the discretized sequence read data. For instance, a central goal of the 1000 Genomes Project is to quantify across-sample variation at the single nucleotide level. At this resolution, small error rates in sequencing prove significant, especially for rare variants. Sec-gen sequencing is a relatively new technology for which potential biases and sources of obscuring variation are not yet fully understood. Therefore, modeling and quantifying the uncertainty inherent in the generation of sequence reads is of utmost importance. In this article, we present a simple model to capture uncertainty arising in the base-calling procedure of the Illumina/Solexa GA platform. Model parameters have a straightforward interpretation in terms of the chemistry of base-calling allowing for informative and easily interpretable metrics that capture the variability in

  12. Masking as an effective quality control method for next-generation sequencing data analysis.

    Science.gov (United States)

    Yun, Sajung; Yun, Sijung

    2014-12-13

    Next generation sequencing produces base calls with low quality scores that can affect the accuracy of identifying simple nucleotide variation calls, including single nucleotide polymorphisms and small insertions and deletions. Here we compare the effectiveness of two data preprocessing methods, masking and trimming, and the accuracy of simple nucleotide variation calls on whole-genome sequence data from Caenorhabditis elegans. Masking substitutes low quality base calls with 'N's (undetermined bases), whereas trimming removes low quality bases that results in a shorter read lengths. We demonstrate that masking is more effective than trimming in reducing the false-positive rate in single nucleotide polymorphism (SNP) calling. However, both of the preprocessing methods did not affect the false-negative rate in SNP calling with statistical significance compared to the data analysis without preprocessing. False-positive rate and false-negative rate for small insertions and deletions did not show differences between masking and trimming. We recommend masking over trimming as a more effective preprocessing method for next generation sequencing data analysis since masking reduces the false-positive rate in SNP calling without sacrificing the false-negative rate although trimming is more commonly used currently in the field. The perl script for masking is available at http://code.google.com/p/subn/. The sequencing data used in the study were deposited in the Sequence Read Archive (SRX450968 and SRX451773).

  13. The Quest for Rare Variants: Pooled Multiplexed Next Generation Sequencing in Plants

    Directory of Open Access Journals (Sweden)

    Fabio eMarroni

    2012-06-01

    Full Text Available Next generation sequencing (NGS instruments produce an unprecedented amount of sequence data at contained costs. This gives researchers the possibility of designing studies with adequate power to identify rare variants at a fraction of the economic and labor resources required by individual Sanger sequencing. As of today, only three research groups working in plant sciences have exploited this potentiality. They showed that pooled NGS can provide results in excellent agreement with those obtained by individual Sanger sequencing. Aim of this review is to convey to the reader the general ideas underlying the use of pooled NGS for the identification of rare variants. To facilitate a thorough understanding of the possibilities of the method we will explain in detail the variations in study design and discuss their advantages and disadvantages. We will show that information on allele frequency obtained by pooled next generation sequencing can be used to accurately compute basic population genetics indexes such as allele frequency, nucleotide diversity and Tajima’s D. Finally we will discuss applications and future perspectives of the multiplexed NGS approach.

  14. Clinical Use of Next-Generation Sequencing in the Diagnosis of Wilson’s Disease

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    Dániel Németh

    2016-01-01

    Full Text Available Objective. Wilson’s disease is a disorder of copper metabolism which is fatal without treatment. The great number of disease-causing ATP7B gene mutations and the variable clinical presentation of WD may cause a real diagnostic challenge. The emergence of next-generation sequencing provides a time-saving, cost-effective method for full sequencing of the whole ATP7B gene compared to the traditional Sanger sequencing. This is the first report on the clinical use of NGS to examine ATP7B gene. Materials and Methods. We used Ion Torrent Personal Genome Machine in four heterozygous patients for the identification of the other mutations and also in two patients with no known mutation. One patient with acute on chronic liver failure was a candidate for acute liver transplantation. The results were validated by Sanger sequencing. Results. In each case, the diagnosis of Wilson’s disease was confirmed by identifying the mutations in both alleles within 48 hours. One novel mutation (p.Ala1270Ile was found beyond the eight other known ones. The rapid detection of the mutations made possible the prompt diagnosis of WD in a patient with acute liver failure. Conclusions. According to our results we found next-generation sequencing a very useful, reliable, time-saving, and cost-effective method for diagnosing Wilson’s disease in selected cases.

  15. Internally generated hippocampal sequences as a vantage point to probe future-oriented cognition.

    Science.gov (United States)

    Pezzulo, Giovanni; Kemere, Caleb; van der Meer, Matthijs A A

    2017-05-01

    Information processing in the rodent hippocampus is fundamentally shaped by internally generated sequences (IGSs), expressed during two different network states: theta sequences, which repeat and reset at the ∼8 Hz theta rhythm associated with active behavior, and punctate sharp wave-ripple (SWR) sequences associated with wakeful rest or slow-wave sleep. A potpourri of diverse functional roles has been proposed for these IGSs, resulting in a fragmented conceptual landscape. Here, we advance a unitary view of IGSs, proposing that they reflect an inferential process that samples a policy from the animal's generative model, supported by hippocampus-specific priors. The same inference affords different cognitive functions when the animal is in distinct dynamical modes, associated with specific functional networks. Theta sequences arise when inference is coupled to the animal's action-perception cycle, supporting online spatial decisions, predictive processing, and episode encoding. SWR sequences arise when the animal is decoupled from the action-perception cycle and may support offline cognitive processing, such as memory consolidation, the prospective simulation of spatial trajectories, and imagination. We discuss the empirical bases of this proposal in relation to rodent studies and highlight how the proposed computational principles can shed light on the mechanisms of future-oriented cognition in humans. © 2017 New York Academy of Sciences.

  16. Autonomously Generating Operations Sequences for a Mars Rover Using Artificial Intelligence-Based Planning

    Science.gov (United States)

    Sherwood, R.; Mutz, D.; Estlin, T.; Chien, S.; Backes, P.; Norris, J.; Tran, D.; Cooper, B.; Rabideau, G.; Mishkin, A.; Maxwell, S.

    2001-07-01

    This article discusses a proof-of-concept prototype for ground-based automatic generation of validated rover command sequences from high-level science and engineering activities. This prototype is based on ASPEN, the Automated Scheduling and Planning Environment. This artificial intelligence (AI)-based planning and scheduling system will automatically generate a command sequence that will execute within resource constraints and satisfy flight rules. An automated planning and scheduling system encodes rover design knowledge and uses search and reasoning techniques to automatically generate low-level command sequences while respecting rover operability constraints, science and engineering preferences, environmental predictions, and also adhering to hard temporal constraints. This prototype planning system has been field-tested using the Rocky 7 rover at JPL and will be field-tested on more complex rovers to prove its effectiveness before transferring the technology to flight operations for an upcoming NASA mission. Enabling goal-driven commanding of planetary rovers greatly reduces the requirements for highly skilled rover engineering personnel. This in turn greatly reduces mission operations costs. In addition, goal-driven commanding permits a faster response to changes in rover state (e.g., faults) or science discoveries by removing the time-consuming manual sequence validation process, allowing rapid "what-if" analyses, and thus reducing overall cycle times.

  17. Modeling the Process of Event Sequence Data Generated for Working Condition Diagnosis

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    Jianwei Ding

    2015-01-01

    Full Text Available Condition monitoring systems are widely used to monitor the working condition of equipment, generating a vast amount and variety of telemetry data in the process. The main task of surveillance focuses on analyzing these routinely collected telemetry data to help analyze the working condition in the equipment. However, with the rapid increase in the volume of telemetry data, it is a nontrivial task to analyze all the telemetry data to understand the working condition of the equipment without any a priori knowledge. In this paper, we proposed a probabilistic generative model called working condition model (WCM, which is capable of simulating the process of event sequence data generated and depicting the working condition of equipment at runtime. With the help of WCM, we are able to analyze how the event sequence data behave in different working modes and meanwhile to detect the working mode of an event sequence (working condition diagnosis. Furthermore, we have applied WCM to illustrative applications like automated detection of an anomalous event sequence for the runtime of equipment. Our experimental results on the real data sets demonstrate the effectiveness of the model.

  18. 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.

  19. Generating quantitative models describing the sequence specificity of biological processes with the stabilized matrix method

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    Sette Alessandro

    2005-05-01

    Full Text Available Abstract Background Many processes in molecular biology involve the recognition of short sequences of nucleic-or amino acids, such as the binding of immunogenic peptides to major histocompatibility complex (MHC molecules. From experimental data, a model of the sequence specificity of these processes can be constructed, such as a sequence motif, a scoring matrix or an artificial neural network. The purpose of these models is two-fold. First, they can provide a summary of experimental results, allowing for a deeper understanding of the mechanisms involved in sequence recognition. Second, such models can be used to predict the experimental outcome for yet untested sequences. In the past we reported the development of a method to generate such models called the Stabilized Matrix Method (SMM. This method has been successfully applied to predicting peptide binding to MHC molecules, peptide transport by the transporter associated with antigen presentation (TAP and proteasomal cleavage of protein sequences. Results Herein we report the implementation of the SMM algorithm as a publicly available software package. Specific features determining the type of problems the method is most appropriate for are discussed. Advantageous features of the package are: (1 the output generated is easy to interpret, (2 input and output are both quantitative, (3 specific computational strategies to handle experimental noise are built in, (4 the algorithm is designed to effectively handle bounded experimental data, (5 experimental data from randomized peptide libraries and conventional peptides can easily be combined, and (6 it is possible to incorporate pair interactions between positions of a sequence. Conclusion Making the SMM method publicly available enables bioinformaticians and experimental biologists to easily access it, to compare its performance to other prediction methods, and to extend it to other applications.

  20. Next-generation Sequencing-based genomic profiling: Fostering innovation in cancer care?

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    Gustavo S. Fernandes

    Full Text Available OBJECTIVES: With the development of next-generation sequencing (NGS technologies, DNA sequencing has been increasingly utilized in clinical practice. Our goal was to investigate the impact of genomic evaluation on treatment decisions for heavily pretreated patients with metastatic cancer. METHODS: We analyzed metastatic cancer patients from a single institution whose cancers had progressed after all available standard-of-care therapies and whose tumors underwent next-generation sequencing analysis. We determined the percentage of patients who received any therapy directed by the test, and its efficacy. RESULTS: From July 2013 to December 2015, 185 consecutive patients were tested using a commercially available next-generation sequencing-based test, and 157 patients were eligible. Sixty-six patients (42.0% were female, and 91 (58.0% were male. The mean age at diagnosis was 52.2 years, and the mean number of pre-test lines of systemic treatment was 2.7. One hundred and seventy-seven patients (95.6% had at least one identified gene alteration. Twenty-four patients (15.2% underwent systemic treatment directed by the test result. Of these, one patient had a complete response, four (16.7% had partial responses, two (8.3% had stable disease, and 17 (70.8% had disease progression as the best result. The median progression-free survival time with matched therapy was 1.6 months, and the median overall survival was 10 months. CONCLUSION: We identified a high prevalence of gene alterations using an next-generation sequencing test. Although some benefit was associated with the matched therapy, most of the patients had disease progression as the best response, indicating the limited biological potential and unclear clinical relevance of this practice.

  1. Aptaligner: automated software for aligning pseudorandom DNA X-aptamers from next-generation sequencing data.

    Science.gov (United States)

    Lu, Emily; Elizondo-Riojas, Miguel-Angel; Chang, Jeffrey T; Volk, David E

    2014-06-10

    Next-generation sequencing results from bead-based aptamer libraries have demonstrated that traditional DNA/RNA alignment software is insufficient. This is particularly true for X-aptamers containing specialty bases (W, X, Y, Z, ...) that are identified by special encoding. Thus, we sought an automated program that uses the inherent design scheme of bead-based X-aptamers to create a hypothetical reference library and Markov modeling techniques to provide improved alignments. Aptaligner provides this feature as well as length error and noise level cutoff features, is parallelized to run on multiple central processing units (cores), and sorts sequences from a single chip into projects and subprojects.

  2. Evaluating next-generation sequencing for direct clinical diagnostics in diarrhoeal disease

    DEFF Research Database (Denmark)

    Joensen, Katrine Grimstrup; Engsbro, A L Ø; Lukjancenko, Oksana

    2017-01-01

    The accurate microbiological diagnosis of diarrhoea involves numerous laboratory tests and, often, the pathogen is not identified in time to guide clinical management. With next-generation sequencing (NGS) becoming cheaper, it has huge potential in routine diagnostics. The aim of this study...... was to evaluate the potential of NGS-based diagnostics through direct sequencing of faecal samples. Fifty-eight clinical faecal samples were obtained from patients with diarrhoea as part of the routine diagnostics at Hvidovre University Hospital, Denmark. Ten samples from healthy individuals were also included...

  3. Yleaf: Software for Human Y-Chromosomal Haplogroup Inference from Next-Generation Sequencing Data.

    Science.gov (United States)

    Ralf, Arwin; Montiel González, Diego; Zhong, Kaiyin; Kayser, Manfred

    2018-05-01

    Next-generation sequencing (NGS) technologies offer immense possibilities given the large genomic data they simultaneously deliver. The human Y-chromosome serves as good example how NGS benefits various applications in evolution, anthropology, genealogy, and forensics. Prior to NGS, the Y-chromosome phylogenetic tree consisted of a few hundred branches, based on NGS data, it now contains many thousands. The complexity of both, Y tree and NGS data provide challenges for haplogroup assignment. For effective analysis and interpretation of Y-chromosome NGS data, we present Yleaf, a publically available, automated, user-friendly software for high-resolution Y-chromosome haplogroup inference independently of library and sequencing methods.

  4. Generation of sequence signatures from DNA amplification fingerprints with mini-hairpin and microsatellite primers.

    Science.gov (United States)

    Caetano-Anollés, G; Gresshoff, P M

    1996-06-01

    DNA amplification fingerprinting (DAF) with mini-hairpins harboring arbitrary "core" sequences at their 3' termini were used to fingerprint a variety of templates, including PCR products and whole genomes, to establish genetic relationships between plant tax at the interspecific and intraspecific level, and to identify closely related fungal isolates and plant accessions. No correlation was observed between the sequence of the arbitrary core, the stability of the mini-hairpin structure and DAF efficiency. Mini-hairpin primers with short arbitrary cores and primers complementary to simple sequence repeats present in microsatellites were also used to generate arbitrary signatures from amplification profiles (ASAP). The ASAP strategy is a dual-step amplification procedure that uses at least one primer in each fingerprinting stage. ASAP was able to reproducibly amplify DAF products (representing about 10-15 kb of sequence) following careful optimization of amplification parameters such as primer and template concentration. Avoidance of primer sequences partially complementary to DAF product termini was necessary in order to produce distinct fingerprints. This allowed the combinatorial use of oligomers in nucleic acid screening, with numerous ASAP fingerprinting reactions based on a limited number of primer sequences. Mini-hairpin primers and ASAP analysis significantly increased detection of polymorphic DNA, separating closely related bermudagrass (Cynodon) cultivars and detecting putatively linked markers in bulked segregant analysis of the soybean (Glycine max) supernodulation (nitrate-tolerant symbiosis) locus.

  5. An integrative variant analysis suite for whole exome next-generation sequencing data

    Directory of Open Access Journals (Sweden)

    Challis Danny

    2012-01-01

    Full Text Available Abstract Background Whole exome capture sequencing allows researchers to cost-effectively sequence the coding regions of the genome. Although the exome capture sequencing methods have become routine and well established, there is currently a lack of tools specialized for variant calling in this type of data. Results Using statistical models trained on validated whole-exome capture sequencing data, the Atlas2 Suite is an integrative variant analysis pipeline optimized for variant discovery on all three of the widely used next generation sequencing platforms (SOLiD, Illumina, and Roche 454. The suite employs logistic regression models in conjunction with user-adjustable cutoffs to accurately separate true SNPs and INDELs from sequencing and mapping errors with high sensitivity (96.7%. Conclusion We have implemented the Atlas2 Suite and applied it to 92 whole exome samples from the 1000 Genomes Project. The Atlas2 Suite is available for download at http://sourceforge.net/projects/atlas2/. In addition to a command line version, the suite has been integrated into the Genboree Workbench, allowing biomedical scientists with minimal informatics expertise to remotely call, view, and further analyze variants through a simple web interface. The existing genomic databases displayed via the Genboree browser also streamline the process from variant discovery to functional genomics analysis, resulting in an off-the-shelf toolkit for the broader community.

  6. Evaluation of second-generation sequencing of 19 dilated cardiomyopathy genes for clinical applications.

    Science.gov (United States)

    Gowrisankar, Sivakumar; Lerner-Ellis, Jordan P; Cox, Stephanie; White, Emily T; Manion, Megan; LeVan, Kevin; Liu, Jonathan; Farwell, Lisa M; Iartchouk, Oleg; Rehm, Heidi L; Funke, Birgit H

    2010-11-01

    Medical sequencing for diseases with locus and allelic heterogeneities has been limited by the high cost and low throughput of traditional sequencing technologies. "Second-generation" sequencing (SGS) technologies allow the parallel processing of a large number of genes and, therefore, offer great promise for medical sequencing; however, their use in clinical laboratories is still in its infancy. Our laboratory offers clinical resequencing for dilated cardiomyopathy (DCM) using an array-based platform that interrogates 19 of more than 30 genes known to cause DCM. We explored both the feasibility and cost effectiveness of using PCR amplification followed by SGS technology for sequencing these 19 genes in a set of five samples enriched for known sequence alterations (109 unique substitutions and 27 insertions and deletions). While the analytical sensitivity for substitutions was comparable to that of the DCM array (98%), SGS technology performed better than the DCM array for insertions and deletions (90.6% versus 58%). Overall, SGS performed substantially better than did the current array-based testing platform; however, the operational cost and projected turnaround time do not meet our current standards. Therefore, efficient capture methods and/or sample pooling strategies that shorten the turnaround time and decrease reagent and labor costs are needed before implementing this platform into routine clinical applications.

  7. Histoimmunogenetics Markup Language 1.0: Reporting next generation sequencing-based HLA and KIR genotyping.

    Science.gov (United States)

    Milius, Robert P; Heuer, Michael; Valiga, Daniel; Doroschak, Kathryn J; Kennedy, Caleb J; Bolon, Yung-Tsi; Schneider, Joel; Pollack, Jane; Kim, Hwa Ran; Cereb, Nezih; Hollenbach, Jill A; Mack, Steven J; Maiers, Martin

    2015-12-01

    We present an electronic format for exchanging data for HLA and KIR genotyping with extensions for next-generation sequencing (NGS). This format addresses NGS data exchange by refining the Histoimmunogenetics Markup Language (HML) to conform to the proposed Minimum Information for Reporting Immunogenomic NGS Genotyping (MIRING) reporting guidelines (miring.immunogenomics.org). Our refinements of HML include two major additions. First, NGS is supported by new XML structures to capture additional NGS data and metadata required to produce a genotyping result, including analysis-dependent (dynamic) and method-dependent (static) components. A full genotype, consensus sequence, and the surrounding metadata are included directly, while the raw sequence reads and platform documentation are externally referenced. Second, genotype ambiguity is fully represented by integrating Genotype List Strings, which use a hierarchical set of delimiters to represent allele and genotype ambiguity in a complete and accurate fashion. HML also continues to enable the transmission of legacy methods (e.g. site-specific oligonucleotide, sequence-specific priming, and Sequence Based Typing (SBT)), adding features such as allowing multiple group-specific sequencing primers, and fully leveraging techniques that combine multiple methods to obtain a single result, such as SBT integrated with NGS. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  8. Using Next Generation RAD Sequencing to Isolate Multispecies Microsatellites for Pilosocereus (Cactaceae.

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    Isabel A S Bonatelli

    Full Text Available Microsatellite markers (also known as SSRs, Simple Sequence Repeats are widely used in plant science and are among the most informative molecular markers for population genetic investigations, but the development of such markers presents substantial challenges. In this report, we discuss how next generation sequencing can replace the cloning, Sanger sequencing, identification of polymorphic loci, and testing cross-amplification that were previously required to develop microsatellites. We report the development of a large set of microsatellite markers for five species of the Neotropical cactus genus Pilosocereus using a restriction-site-associated DNA sequencing (RAD-seq on a Roche 454 platform. We identified an average of 165 microsatellites per individual, with the absolute numbers across individuals proportional to the sequence reads obtained per individual. Frequency distribution of the repeat units was similar in the five species, with shorter motifs such as di- and trinucleotide being the most abundant repeats. In addition, we provide 72 microsatellites that could be potentially amplified in the sampled species and 22 polymorphic microsatellites validated in two populations of the species Pilosocereus machrisii. Although low coverage sequencing among individuals was observed for most of the loci, which we suggest to be more related to the nature of the microsatellite markers and the possible bias inserted by the restriction enzymes than to the genome size, our work demonstrates that an NGS approach is an efficient method to isolate multispecies microsatellites even in non-model organisms.

  9. Using Next Generation RAD Sequencing to Isolate Multispecies Microsatellites for Pilosocereus (Cactaceae).

    Science.gov (United States)

    Bonatelli, Isabel A S; Carstens, Bryan C; Moraes, Evandro M

    2015-01-01

    Microsatellite markers (also known as SSRs, Simple Sequence Repeats) are widely used in plant science and are among the most informative molecular markers for population genetic investigations, but the development of such markers presents substantial challenges. In this report, we discuss how next generation sequencing can replace the cloning, Sanger sequencing, identification of polymorphic loci, and testing cross-amplification that were previously required to develop microsatellites. We report the development of a large set of microsatellite markers for five species of the Neotropical cactus genus Pilosocereus using a restriction-site-associated DNA sequencing (RAD-seq) on a Roche 454 platform. We identified an average of 165 microsatellites per individual, with the absolute numbers across individuals proportional to the sequence reads obtained per individual. Frequency distribution of the repeat units was similar in the five species, with shorter motifs such as di- and trinucleotide being the most abundant repeats. In addition, we provide 72 microsatellites that could be potentially amplified in the sampled species and 22 polymorphic microsatellites validated in two populations of the species Pilosocereus machrisii. Although low coverage sequencing among individuals was observed for most of the loci, which we suggest to be more related to the nature of the microsatellite markers and the possible bias inserted by the restriction enzymes than to the genome size, our work demonstrates that an NGS approach is an efficient method to isolate multispecies microsatellites even in non-model organisms.

  10. Stepwise threshold clustering: a new method for genotyping MHC loci using next-generation sequencing technology.

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    William E Stutz

    Full Text Available Genes of the vertebrate major histocompatibility complex (MHC are of great interest to biologists because of their important role in immunity and disease, and their extremely high levels of genetic diversity. Next generation sequencing (NGS technologies are quickly becoming the method of choice for high-throughput genotyping of multi-locus templates like MHC in non-model organisms. Previous approaches to genotyping MHC genes using NGS technologies suffer from two problems:1 a "gray zone" where low frequency alleles and high frequency artifacts can be difficult to disentangle and 2 a similar sequence problem, where very similar alleles can be difficult to distinguish as two distinct alleles. Here were present a new method for genotyping MHC loci--Stepwise Threshold Clustering (STC--that addresses these problems by taking full advantage of the increase in sequence data provided by NGS technologies. Unlike previous approaches for genotyping MHC with NGS data that attempt to classify individual sequences as alleles or artifacts, STC uses a quasi-Dirichlet clustering algorithm to cluster similar sequences at increasing levels of sequence similarity. By applying frequency and similarity based criteria to clusters rather than individual sequences, STC is able to successfully identify clusters of sequences that correspond to individual or similar alleles present in the genomes of individual samples. Furthermore, STC does not require duplicate runs of all samples, increasing the number of samples that can be genotyped in a given project. We show how the STC method works using a single sample library. We then apply STC to 295 threespine stickleback (Gasterosteus aculeatus samples from four populations and show that neighboring populations differ significantly in MHC allele pools. We show that STC is a reliable, accurate, efficient, and flexible method for genotyping MHC that will be of use to biologists interested in a variety of downstream applications.

  11. A next generation semiconductor based sequencing approach for the identification of meat species in DNA mixtures.

    Directory of Open Access Journals (Sweden)

    Francesca Bertolini

    Full Text Available The identification of the species of origin of meat and meat products is an important issue to prevent and detect frauds that might have economic, ethical and health implications. In this paper we evaluated the potential of the next generation semiconductor based sequencing technology (Ion Torrent Personal Genome Machine for the identification of DNA from meat species (pig, horse, cattle, sheep, rabbit, chicken, turkey, pheasant, duck, goose and pigeon as well as from human and rat in DNA mixtures through the sequencing of PCR products obtained from different couples of universal primers that amplify 12S and 16S rRNA mitochondrial DNA genes. Six libraries were produced including PCR products obtained separately from 13 species or from DNA mixtures containing DNA from all species or only avian or only mammalian species at equimolar concentration or at 1:10 or 1:50 ratios for pig and horse DNA. Sequencing obtained a total of 33,294,511 called nucleotides of which 29,109,688 with Q20 (87.43% in a total of 215,944 reads. Different alignment algorithms were used to assign the species based on sequence data. Error rate calculated after confirmation of the obtained sequences by Sanger sequencing ranged from 0.0003 to 0.02 for the different species. Correlation about the number of reads per species between different libraries was high for mammalian species (0.97 and lower for avian species (0.70. PCR competition limited the efficiency of amplification and sequencing for avian species for some primer pairs. Detection of low level of pig and horse DNA was possible with reads obtained from different primer pairs. The sequencing of the products obtained from different universal PCR primers could be a useful strategy to overcome potential problems of amplification. Based on these results, the Ion Torrent technology can be applied for the identification of meat species in DNA mixtures.

  12. Molecular diagnosis of Usher syndrome: application of two different next generation sequencing-based procedures.

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    Danilo Licastro

    Full Text Available Usher syndrome (USH is a clinically and genetically heterogeneous disorder characterized by visual and hearing impairments. Clinically, it is subdivided into three subclasses with nine genes identified so far. In the present study, we investigated whether the currently available Next Generation Sequencing (NGS technologies are already suitable for molecular diagnostics of USH. We analyzed a total of 12 patients, most of which were negative for previously described mutations in known USH genes upon primer extension-based microarray genotyping. We enriched the NGS template either by whole exome capture or by Long-PCR of the known USH genes. The main NGS sequencing platforms were used: SOLiD for whole exome sequencing, Illumina (Genome Analyzer II and Roche 454 (GS FLX for the Long-PCR sequencing. Long-PCR targeting was more efficient with up to 94% of USH gene regions displaying an overall coverage higher than 25×, whereas whole exome sequencing yielded a similar coverage for only 50% of those regions. Overall this integrated analysis led to the identification of 11 novel sequence variations in USH genes (2 homozygous and 9 heterozygous out of 18 detected. However, at least two cases were not genetically solved. Our result highlights the current limitations in the diagnostic use of NGS for USH patients. The limit for whole exome sequencing is linked to the need of a strong coverage and to the correct interpretation of sequence variations with a non obvious, pathogenic role, whereas the targeted approach suffers from the high genetic heterogeneity of USH that may be also caused by the presence of additional causative genes yet to be identified.

  13. Molecular Diagnosis of Usher Syndrome: Application of Two Different Next Generation Sequencing-Based Procedures

    Science.gov (United States)

    Licastro, Danilo; Mutarelli, Margherita; Peluso, Ivana; Neveling, Kornelia; Wieskamp, Nienke; Rispoli, Rossella; Vozzi, Diego; Athanasakis, Emmanouil; D'Eustacchio, Angela; Pizzo, Mariateresa; D'Amico, Francesca; Ziviello, Carmela; Simonelli, Francesca; Fabretto, Antonella; Scheffer, Hans; Gasparini, Paolo; Banfi, Sandro; Nigro, Vincenzo

    2012-01-01

    Usher syndrome (USH) is a clinically and genetically heterogeneous disorder characterized by visual and hearing impairments. Clinically, it is subdivided into three subclasses with nine genes identified so far. In the present study, we investigated whether the currently available Next Generation Sequencing (NGS) technologies are already suitable for molecular diagnostics of USH. We analyzed a total of 12 patients, most of which were negative for previously described mutations in known USH genes upon primer extension-based microarray genotyping. We enriched the NGS template either by whole exome capture or by Long-PCR of the known USH genes. The main NGS sequencing platforms were used: SOLiD for whole exome sequencing, Illumina (Genome Analyzer II) and Roche 454 (GS FLX) for the Long-PCR sequencing. Long-PCR targeting was more efficient with up to 94% of USH gene regions displaying an overall coverage higher than 25×, whereas whole exome sequencing yielded a similar coverage for only 50% of those regions. Overall this integrated analysis led to the identification of 11 novel sequence variations in USH genes (2 homozygous and 9 heterozygous) out of 18 detected. However, at least two cases were not genetically solved. Our result highlights the current limitations in the diagnostic use of NGS for USH patients. The limit for whole exome sequencing is linked to the need of a strong coverage and to the correct interpretation of sequence variations with a non obvious, pathogenic role, whereas the targeted approach suffers from the high genetic heterogeneity of USH that may be also caused by the presence of additional causative genes yet to be identified. PMID:22952768

  14. Association testing for next-generation sequencing data using score statistics

    DEFF Research Database (Denmark)

    Skotte, Line; Korneliussen, Thorfinn Sand; Albrechtsen, Anders

    2012-01-01

    computationally feasible due to the use of score statistics. As part of the joint likelihood, we model the distribution of the phenotypes using a generalized linear model framework, which works for both quantitative and discrete phenotypes. Thus, the method presented here is applicable to case-control studies...... of genotype calls into account have been proposed; most require numerical optimization which for large-scale data is not always computationally feasible. We show that using a score statistic for the joint likelihood of observed phenotypes and observed sequencing data provides an attractive approach...... to association testing for next-generation sequencing data. The joint model accounts for the genotype classification uncertainty via the posterior probabilities of the genotypes given the observed sequencing data, which gives the approach higher power than methods based on called genotypes. This strategy remains...

  15. [Diagnosis of a case with oculocutaneous albinism type Ⅲ with next generation exome capture sequencing].

    Science.gov (United States)

    Lyu, Yuqiang; Huang, Jing; Zhang, Kaihui; Liu, Guohua; Gao, Min; Gai, Zhongtao; Liu, Yi

    2017-02-10

    To explore the clinical and genetic features of a Chinese boy with oculocutaneous albinism. The clinical features of the patient were analyzed. The DNA of the patient and his parents was extracted and sequenced by next generation exome capture sequencing. The nature and impact of detected mutation were predicted and validated. The child has displayed strabismus, poor vision, nystagmus and brown hair. DNA sequencing showed that the patient has carried compound heterozygous mutations of the TYRP1 gene, namely c.1214C>A (p.T405N) and c.1333dupG, which were inherited from his mother and father, respectively. Neither mutation was reported previously. The child has suffered from oculocutaneous albinism type Ⅲ caused by mutations of the TYRP1 gene.

  16. Next generation sequencing yields the complete mitochondrial genome of the largescale mullet, Liza macrolepis (Teleostei: Mugilidae).

    Science.gov (United States)

    Shen, Kang-Ning; Tsai, Shiou-Yi; Chen, Ching-Hung; Hsiao, Chung-Der; Durand, Jean-Dominique

    2016-11-01

    In this study, the complete mitogenome sequence of largescale mullet (Teleostei: Mugilidae) has been sequenced by the next-generation sequencing method. The assembled mitogenome, consisting of 16,832 bp, had the typical vertebrate mitochondrial gene arrangement, including 13 protein-coding genes, 22 transfer RNAs, two ribosomal RNAs genes, and a non-coding control region of D-loop. D-loop which has a length of 1094 bp is located between tRNA-Pro and tRNA-Phe. The overall base composition of largescale mullet is 27.8% for A, 30.1% for C, 16.2% for G, and 25.9% for T. The complete mitogenome may provide essential and important DNA molecular data for further phylogenetic and evolutionary analysis for Mugilidae.

  17. Next generation sequencing yields the complete mitochondrial genome of the Hornlip mullet Plicomugil labiosus (Teleostei: Mugilidae).

    Science.gov (United States)

    Shen, Kang-Ning; Chen, Ching-Hung; Hsiao, Chung-Der

    2016-05-01

    In this study, the complete mitogenome sequence of hornlip mullet Plicomugil labiosus (Teleostei: Mugilidae) has been sequenced by next-generation sequencing method. The assembled mitogenome, consisting of 16,829 bp, had the typical vertebrate mitochondrial gene arrangement, including 13 protein coding genes, 22 transfer RNAs, 2 ribosomal RNAs genes and a non-coding control region of D-loop. D-loop contains 1057 bp length is located between tRNA-Pro and tRNA-Phe. The overall base composition of P. labiosus is 28.0% for A, 29.3% for C, 15.5% for G and 27.2% for T. The complete mitogenome may provide essential and important DNA molecular data for further population, phylogenetic and evolutionary analysis for Mugilidae.

  18. Cracking the Code of Human Diseases Using Next-Generation Sequencing: Applications, Challenges, and Perspectives

    Directory of Open Access Journals (Sweden)

    Vincenza Precone

    2015-01-01

    Full Text Available Next-generation sequencing (NGS technologies have greatly impacted on every field of molecular research mainly because they reduce costs and increase throughput of DNA sequencing. These features, together with the technology’s flexibility, have opened the way to a variety of applications including the study of the molecular basis of human diseases. Several analytical approaches have been developed to selectively enrich regions of interest from the whole genome in order to identify germinal and/or somatic sequence variants and to study DNA methylation. These approaches are now widely used in research, and they are already being used in routine molecular diagnostics. However, some issues are still controversial, namely, standardization of methods, data analysis and storage, and ethical aspects. Besides providing an overview of the NGS-based approaches most frequently used to study the molecular basis of human diseases at DNA level, we discuss the principal challenges and applications of NGS in the field of human genomics.

  19. Rapid and Easy Protocol for Quantification of Next-Generation Sequencing Libraries.

    Science.gov (United States)

    Hawkins, Steve F C; Guest, Paul C

    2018-01-01

    The emergence of next-generation sequencing (NGS) over the last 10 years has increased the efficiency of DNA sequencing in terms of speed, ease, and price. However, the exact quantification of a NGS library is crucial in order to obtain good data on sequencing platforms developed by the current market leader Illumina. Different approaches for DNA quantification are available currently and the most commonly used are based on analysis of the physical properties of the DNA through spectrophotometric or fluorometric methods. Although these methods are technically simple, they do not allow exact quantification as can be achieved using a real-time quantitative PCR (qPCR) approach. A qPCR protocol for DNA quantification with applications in NGS library preparation studies is presented here. This can be applied in various fields of study such as medical disorders resulting from nutritional programming disturbances.

  20. Generation and analysis of expressed sequence tags in the extreme large genomes Lilium and Tulipa

    Directory of Open Access Journals (Sweden)

    Shahin Arwa

    2012-11-01

    Full Text Available Abstract Background Bulbous flowers such as lily and tulip (Liliaceae family are monocot perennial herbs that are economically very important ornamental plants worldwide. However, there are hardly any genetic studies performed and genomic resources are lacking. To build genomic resources and develop tools to speed up the breeding in both crops, next generation sequencing was implemented. We sequenced and assembled transcriptomes of four lily and five tulip genotypes using 454 pyro-sequencing technology. Results Successfully, we developed the first set of 81,791 contigs with an average length of 514 bp for tulip, and enriched the very limited number of 3,329 available ESTs (Expressed Sequence Tags for lily with 52,172 contigs with an average length of 555 bp. The contigs together with singletons covered on average 37% of lily and 39% of tulip estimated transcriptome. Mining lily and tulip sequence data for SSRs (Simple Sequence Repeats showed that di-nucleotide repeats were twice more abundant in UTRs (UnTranslated Regions compared to coding regions, while tri-nucleotide repeats were equally spread over coding and UTR regions. Two sets of single nucleotide polymorphism (SNP markers suitable for high throughput genotyping were developed. In the first set, no SNPs flanking the target SNP (50 bp on either side were allowed. In the second set, one SNP in the flanking regions was allowed, which resulted in a 2 to 3 fold increase in SNP marker numbers compared with the first set. Orthologous groups between the two flower bulbs: lily and tulip (12,017 groups and among the three monocot species: lily, tulip, and rice (6,900 groups were determined using OrthoMCL. Orthologous groups were screened for common SNP markers and EST-SSRs to study synteny between lily and tulip, which resulted in 113 common SNP markers and 292 common EST-SSR. Lily and tulip contigs generated were annotated and described according to Gene Ontology terminology. Conclusions

  1. Generation and analysis of expressed sequence tags in the extreme large genomes Lilium and Tulipa.

    Science.gov (United States)

    Shahin, Arwa; van Kaauwen, Martijn; Esselink, Danny; Bargsten, Joachim W; van Tuyl, Jaap M; Visser, Richard G F; Arens, Paul

    2012-11-20

    Bulbous flowers such as lily and tulip (Liliaceae family) are monocot perennial herbs that are economically very important ornamental plants worldwide. However, there are hardly any genetic studies performed and genomic resources are lacking. To build genomic resources and develop tools to speed up the breeding in both crops, next generation sequencing was implemented. We sequenced and assembled transcriptomes of four lily and five tulip genotypes using 454 pyro-sequencing technology. Successfully, we developed the first set of 81,791 contigs with an average length of 514 bp for tulip, and enriched the very limited number of 3,329 available ESTs (Expressed Sequence Tags) for lily with 52,172 contigs with an average length of 555 bp. The contigs together with singletons covered on average 37% of lily and 39% of tulip estimated transcriptome. Mining lily and tulip sequence data for SSRs (Simple Sequence Repeats) showed that di-nucleotide repeats were twice more abundant in UTRs (UnTranslated Regions) compared to coding regions, while tri-nucleotide repeats were equally spread over coding and UTR regions. Two sets of single nucleotide polymorphism (SNP) markers suitable for high throughput genotyping were developed. In the first set, no SNPs flanking the target SNP (50 bp on either side) were allowed. In the second set, one SNP in the flanking regions was allowed, which resulted in a 2 to 3 fold increase in SNP marker numbers compared with the first set. Orthologous groups between the two flower bulbs: lily and tulip (12,017 groups) and among the three monocot species: lily, tulip, and rice (6,900 groups) were determined using OrthoMCL. Orthologous groups were screened for common SNP markers and EST-SSRs to study synteny between lily and tulip, which resulted in 113 common SNP markers and 292 common EST-SSR. Lily and tulip contigs generated were annotated and described according to Gene Ontology terminology. Two transcriptome sets were built that are valuable

  2. Next-generation sequencing for diagnosis of rare diseases in the neonatal intensive care unit

    Science.gov (United States)

    Daoud, Hussein; Luco, Stephanie M.; Li, Rui; Bareke, Eric; Beaulieu, Chandree; Jarinova, Olga; Carson, Nancy; Nikkel, Sarah M.; Graham, Gail E.; Richer, Julie; Armour, Christine; Bulman, Dennis E.; Chakraborty, Pranesh; Geraghty, Michael; Lines, Matthew A.; Lacaze-Masmonteil, Thierry; Majewski, Jacek; Boycott, Kym M.; Dyment, David A.

    2016-01-01

    Background: Rare diseases often present in the first days and weeks of life and may require complex management in the setting of a neonatal intensive care unit (NICU). Exhaustive consultations and traditional genetic or metabolic investigations are costly and often fail to arrive at a final diagnosis when no recognizable syndrome is suspected. For this pilot project, we assessed the feasibility of next-generation sequencing as a tool to improve the diagnosis of rare diseases in newborns in the NICU. Methods: We retrospectively identified and prospectively recruited newborns and infants admitted to the NICU of the Children’s Hospital of Eastern Ontario and the Ottawa Hospital, General Campus, who had been referred to the medical genetics or metabolics inpatient consult service and had features suggesting an underlying genetic or metabolic condition. DNA from the newborns and parents was enriched for a panel of clinically relevant genes and sequenced on a MiSeq sequencing platform (Illumina Inc.). The data were interpreted with a standard informatics pipeline and reported to care providers, who assessed the importance of genotype–phenotype correlations. Results: Of 20 newborns studied, 8 received a diagnosis on the basis of next-generation sequencing (diagnostic rate 40%). The diagnoses were renal tubular dysgenesis, SCN1A-related encephalopathy syndrome, myotubular myopathy, FTO deficiency syndrome, cranioectodermal dysplasia, congenital myasthenic syndrome, autosomal dominant intellectual disability syndrome type 7 and Denys–Drash syndrome. Interpretation: This pilot study highlighted the potential of next-generation sequencing to deliver molecular diagnoses rapidly with a high success rate. With broader use, this approach has the potential to alter health care delivery in the NICU. PMID:27241786

  3. Next-generation sequencing for diagnosis of rare diseases in the neonatal intensive care unit.

    Science.gov (United States)

    Daoud, Hussein; Luco, Stephanie M; Li, Rui; Bareke, Eric; Beaulieu, Chandree; Jarinova, Olga; Carson, Nancy; Nikkel, Sarah M; Graham, Gail E; Richer, Julie; Armour, Christine; Bulman, Dennis E; Chakraborty, Pranesh; Geraghty, Michael; Lines, Matthew A; Lacaze-Masmonteil, Thierry; Majewski, Jacek; Boycott, Kym M; Dyment, David A

    2016-08-09

    Rare diseases often present in the first days and weeks of life and may require complex management in the setting of a neonatal intensive care unit (NICU). Exhaustive consultations and traditional genetic or metabolic investigations are costly and often fail to arrive at a final diagnosis when no recognizable syndrome is suspected. For this pilot project, we assessed the feasibility of next-generation sequencing as a tool to improve the diagnosis of rare diseases in newborns in the NICU. We retrospectively identified and prospectively recruited newborns and infants admitted to the NICU of the Children's Hospital of Eastern Ontario and the Ottawa Hospital, General Campus, who had been referred to the medical genetics or metabolics inpatient consult service and had features suggesting an underlying genetic or metabolic condition. DNA from the newborns and parents was enriched for a panel of clinically relevant genes and sequenced on a MiSeq sequencing platform (Illumina Inc.). The data were interpreted with a standard informatics pipeline and reported to care providers, who assessed the importance of genotype-phenotype correlations. Of 20 newborns studied, 8 received a diagnosis on the basis of next-generation sequencing (diagnostic rate 40%). The diagnoses were renal tubular dysgenesis, SCN1A-related encephalopathy syndrome, myotubular myopathy, FTO deficiency syndrome, cranioectodermal dysplasia, congenital myasthenic syndrome, autosomal dominant intellectual disability syndrome type 7 and Denys-Drash syndrome. This pilot study highlighted the potential of next-generation sequencing to deliver molecular diagnoses rapidly with a high success rate. With broader use, this approach has the potential to alter health care delivery in the NICU. © 2016 Canadian Medical Association or its licensors.

  4. The synthesis of the correlation function of pseudorandom binary numbers at the output shift register

    Science.gov (United States)

    Galustov, G. G.; Voronin, V. V.

    2017-05-01

    The sequence generator generates a sequence of pseudorandom binary numbers using a linear-feedback shift register (LFSR). This block implements LFSR using a simple shift register generator (SSRG, or Fibonacci) configuration. In this article we introduce the concept of probabilistic binary element provides requirements, which ensure compliance with the criterion of "uniformity" in the implementation of the basic physical generators uniformly distributed random number sequences. Based on these studies, we obtained an analytic relation between the parameters of the binary sequence and parameters of a numerical sequence with the shift register output. The received analytical dependencies can help in evaluating the statistical characteristics of the processes in solving problems of statistical modeling. It is supposed that the formation of the binary sequence output from the binary probabilistic element is produced using a physical noise process. It is shown that the observed errors in statistical modeling using pseudo-random numbers do not occur if the model examines linear systems with constant parameters, but in case models of nonlinear systems, higher order moments can have a Gaussian distribution.

  5. Frontal dynamic aphasia in progressive supranuclear palsy: Distinguishing between generation and fluent sequencing of novel thoughts.

    Science.gov (United States)

    Robinson, Gail A; Spooner, Donna; Harrison, William J

    2015-10-01

    Frontal dynamic aphasia is characterised by a profound reduction in spontaneous speech despite well-preserved naming, repetition and comprehension. Since Luria (1966, 1970) designated this term, two main forms of dynamic aphasia have been identified: one, a language-specific selection deficit at the level of word/sentence generation, associated with left inferior frontal lesions; and two, a domain-general impairment in generating multiple responses or connected speech, associated with more extensive bilateral frontal and/or frontostriatal damage. Both forms of dynamic aphasia have been interpreted as arising due to disturbances in early prelinguistic conceptual preparation mechanisms that are critical for language production. We investigate language-specific and domain-general accounts of dynamic aphasia and address two issues: one, whether deficits in multiple conceptual preparation mechanisms can co-occur; and two, the contribution of broader cognitive processes such as energization, the ability to initiate and sustain response generation over time, to language generation failure. Thus, we report patient WAL who presented with frontal dynamic aphasia in the context of progressive supranuclear palsy (PSP). WAL was given a series of experimental tests that showed that his dynamic aphasia was not underpinned by a language-specific deficit in selection or in microplanning. By contrast, WAL presented with a domain-general deficit in fluent sequencing of novel thoughts. The latter replicated the pattern documented in a previous PSP patient (Robinson, et al., 2006); however, unique to WAL, generating novel thoughts was impaired but there was no evidence of a sequencing deficit because perseveration was absent. Thus, WAL is the first unequivocal case to show a distinction between novel thought generation and subsequent fluent sequencing. Moreover, WAL's generation deficit encompassed verbal and non-verbal responses, showing a similar (but more profoundly reduced) pattern

  6. Next Generation Sequencing As an Aid to Diagnosis and Treatment of an Unusual Pediatric Brain Cancer

    Directory of Open Access Journals (Sweden)

    John Glod

    2014-07-01

    Full Text Available Classification of pediatric brain tumors with unusual histologic and clinical features may be a diagnostic challenge to the pathologist. We present a case of a 12-year-old girl with a primary intracranial tumor. The tumor classification was not certain initially, and the site of origin and clinical behavior were unusual. Genomic characterization of the tumor using a Clinical Laboratory Improvement Amendment (CLIA-certified next-generation sequencing assay assisted in the diagnosis and translated into patient benefit, albeit transient. Our case argues that next generation sequencing may play a role in the pathological classification of pediatric brain cancers and guiding targeted therapy, supporting additional studies of genetically targeted therapeutics.

  7. Implementation of Cloud based next generation sequencing data analysis in a clinical laboratory.

    Science.gov (United States)

    Onsongo, Getiria; Erdmann, Jesse; Spears, Michael D; Chilton, John; Beckman, Kenneth B; Hauge, Adam; Yohe, Sophia; Schomaker, Matthew; Bower, Matthew; Silverstein, Kevin A T; Thyagarajan, Bharat

    2014-05-23

    The introduction of next generation sequencing (NGS) has revolutionized molecular diagnostics, though several challenges remain limiting the widespread adoption of NGS testing into clinical practice. One such difficulty includes the development of a robust bioinformatics pipeline that can handle the volume of data generated by high-throughput sequencing in a cost-effective manner. Analysis of sequencing data typically requires a substantial level of computing power that is often cost-prohibitive to most clinical diagnostics laboratories. To address this challenge, our institution has developed a Galaxy-based data analysis pipeline which relies on a web-based, cloud-computing infrastructure to process NGS data and identify genetic variants. It provides additional flexibility, needed to control storage costs, resulting in a pipeline that is cost-effective on a per-sample basis. It does not require the usage of EBS disk to run a sample. We demonstrate the validation and feasibility of implementing this bioinformatics pipeline in a molecular diagnostics laboratory. Four samples were analyzed in duplicate pairs and showed 100% concordance in mutations identified. This pipeline is currently being used in the clinic and all identified pathogenic variants confirmed using Sanger sequencing further validating the software.

  8. SNP calling, genotype calling, and sample allele frequency estimation from new-generation sequencing data

    DEFF Research Database (Denmark)

    Nielsen, Rasmus; Korneliussen, Thorfinn Sand; Albrechtsen, Anders

    2012-01-01

    We present a statistical framework for estimation and application of sample allele frequency spectra from New-Generation Sequencing (NGS) data. In this method, we first estimate the allele frequency spectrum using maximum likelihood. In contrast to previous methods, the likelihood function is cal...... be extended to various other cases including cases with deviations from Hardy-Weinberg equilibrium. We evaluate the statistical properties of the methods using simulations and by application to a real data set....

  9. INTEGRATED APPROACH TO GENERATION OF PRECEDENCE RELATIONS AND PRECEDENCE GRAPHS FOR ASSEMBLY SEQUENCE PLANNING

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    An integrated approach to generation of precedence relations and precedence graphs for assembly sequence planning is presented, which contains more assembly flexibility. The approach involves two stages. Based on the assembly model, the components in the assembly can be divided into partially constrained components and completely constrained components in the first stage, and then geometric precedence relation for every component is generated automatically. According to the result of the first stage, the second stage determines and constructs all precedence graphs. The algorithms of these two stages proposed are verified by two assembly examples.

  10. Next-Generation Sequencing in Clinical Molecular Diagnostics of Cancer: Advantages and Challenges

    Directory of Open Access Journals (Sweden)

    Rajyalakshmi Luthra

    2015-10-01

    Full Text Available The application of next-generation sequencing (NGS to characterize cancer genomes has resulted in the discovery of numerous genetic markers. Consequently, the number of markers that warrant routine screening in molecular diagnostic laboratories, often from limited tumor material, has increased. This increased demand has been difficult to manage by traditional low- and/or medium-throughput sequencing platforms. Massively parallel sequencing capabilities of NGS provide a much-needed alternative for mutation screening in multiple genes with a single low investment of DNA. However, implementation of NGS technologies, most of which are for research use only (RUO, in a diagnostic laboratory, needs extensive validation in order to establish Clinical Laboratory Improvement Amendments (CLIA and College of American Pathologists (CAP-compliant performance characteristics. Here, we have reviewed approaches for validation of NGS technology for routine screening of tumors. We discuss the criteria for selecting gene markers to include in the NGS panel and the deciding factors for selecting target capture approaches and sequencing platforms. We also discuss challenges in result reporting, storage and retrieval of the voluminous sequencing data and the future potential of clinical NGS.

  11. A microfluidic DNA library preparation platform for next-generation sequencing.

    Directory of Open Access Journals (Sweden)

    Hanyoup Kim

    Full Text Available Next-generation sequencing (NGS is emerging as a powerful tool for elucidating genetic information for a wide range of applications. Unfortunately, the surging popularity of NGS has not yet been accompanied by an improvement in automated techniques for preparing formatted sequencing libraries. To address this challenge, we have developed a prototype microfluidic system for preparing sequencer-ready DNA libraries for analysis by Illumina sequencing. Our system combines droplet-based digital microfluidic (DMF sample handling with peripheral modules to create a fully-integrated, sample-in library-out platform. In this report, we use our automated system to prepare NGS libraries from samples of human and bacterial genomic DNA. E. coli libraries prepared on-device from 5 ng of total DNA yielded excellent sequence coverage over the entire bacterial genome, with >99% alignment to the reference genome, even genome coverage, and good quality scores. Furthermore, we produced a de novo assembly on a previously unsequenced multi-drug resistant Klebsiella pneumoniae strain BAA-2146 (KpnNDM. The new method described here is fast, robust, scalable, and automated. Our device for library preparation will assist in the integration of NGS technology into a wide variety of laboratories, including small research laboratories and clinical laboratories.

  12. A microfluidic DNA library preparation platform for next-generation sequencing.

    Science.gov (United States)

    Kim, Hanyoup; Jebrail, Mais J; Sinha, Anupama; Bent, Zachary W; Solberg, Owen D; Williams, Kelly P; Langevin, Stanley A; Renzi, Ronald F; Van De Vreugde, James L; Meagher, Robert J; Schoeniger, Joseph S; Lane, Todd W; Branda, Steven S; Bartsch, Michael S; Patel, Kamlesh D

    2013-01-01

    Next-generation sequencing (NGS) is emerging as a powerful tool for elucidating genetic information for a wide range of applications. Unfortunately, the surging popularity of NGS has not yet been accompanied by an improvement in automated techniques for preparing formatted sequencing libraries. To address this challenge, we have developed a prototype microfluidic system for preparing sequencer-ready DNA libraries for analysis by Illumina sequencing. Our system combines droplet-based digital microfluidic (DMF) sample handling with peripheral modules to create a fully-integrated, sample-in library-out platform. In this report, we use our automated system to prepare NGS libraries from samples of human and bacterial genomic DNA. E. coli libraries prepared on-device from 5 ng of total DNA yielded excellent sequence coverage over the entire bacterial genome, with >99% alignment to the reference genome, even genome coverage, and good quality scores. Furthermore, we produced a de novo assembly on a previously unsequenced multi-drug resistant Klebsiella pneumoniae strain BAA-2146 (KpnNDM). The new method described here is fast, robust, scalable, and automated. Our device for library preparation will assist in the integration of NGS technology into a wide variety of laboratories, including small research laboratories and clinical laboratories.

  13. Next Generation Sequencing of Actinobacteria for the Discovery of Novel Natural Products

    Science.gov (United States)

    Gomez-Escribano, Juan Pablo; Alt, Silke; Bibb, Mervyn J.

    2016-01-01

    Like many fields of the biosciences, actinomycete natural products research has been revolutionised by next-generation DNA sequencing (NGS). Hundreds of new genome sequences from actinobacteria are made public every year, many of them as a result of projects aimed at identifying new natural products and their biosynthetic pathways through genome mining. Advances in these technologies in the last five years have meant not only a reduction in the cost of whole genome sequencing, but also a substantial increase in the quality of the data, having moved from obtaining a draft genome sequence comprised of several hundred short contigs, sometimes of doubtful reliability, to the possibility of obtaining an almost complete and accurate chromosome sequence in a single contig, allowing a detailed study of gene clusters and the design of strategies for refactoring and full gene cluster synthesis. The impact that these technologies are having in the discovery and study of natural products from actinobacteria, including those from the marine environment, is only starting to be realised. In this review we provide a historical perspective of the field, analyse the strengths and limitations of the most relevant technologies, and share the insights acquired during our genome mining projects. PMID:27089350

  14. 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.

  15. BG7: A New Approach for Bacterial Genome Annotation Designed for Next Generation Sequencing Data

    Science.gov (United States)

    Pareja-Tobes, Pablo; Manrique, Marina; Pareja-Tobes, Eduardo; Pareja, Eduardo; Tobes, Raquel

    2012-01-01

    BG7 is a new system for de novo bacterial, archaeal and viral genome annotation based on a new approach specifically designed for annotating genomes sequenced with next generation sequencing technologies. The system is versatile and able to annotate genes even in the step of preliminary assembly of the genome. It is especially efficient detecting unexpected genes horizontally acquired from bacterial or archaeal distant genomes, phages, plasmids, and mobile elements. From the initial phases of the gene annotation process, BG7 exploits the massive availability of annotated protein sequences in databases. BG7 predicts ORFs and infers their function based on protein similarity with a wide set of reference proteins, integrating ORF prediction and functional annotation phases in just one step. BG7 is especially tolerant to sequencing errors in start and stop codons, to frameshifts, and to assembly or scaffolding errors. The system is also tolerant to the high level of gene fragmentation which is frequently found in not fully assembled genomes. BG7 current version – which is developed in Java, takes advantage of Amazon Web Services (AWS) cloud computing features, but it can also be run locally in any operating system. BG7 is a fast, automated and scalable system that can cope with the challenge of analyzing the huge amount of genomes that are being sequenced with NGS technologies. Its capabilities and efficiency were demonstrated in the 2011 EHEC Germany outbreak in which BG7 was used to get the first annotations right the next day after the first entero-hemorrhagic E. coli genome sequences were made publicly available. The suitability of BG7 for genome annotation has been proved for Illumina, 454, Ion Torrent, and PacBio sequencing technologies. Besides, thanks to its plasticity, our system could be very easily adapted to work with new technologies in the future. PMID:23185310

  16. Simultaneous genomic identification and profiling of a single cell using semiconductor-based next generation sequencing

    Directory of Open Access Journals (Sweden)

    Manabu Watanabe

    2014-09-01

    Full Text Available Combining single-cell methods and next-generation sequencing should provide a powerful means to understand single-cell biology and obviate the effects of sample heterogeneity. Here we report a single-cell identification method and seamless cancer gene profiling using semiconductor-based massively parallel sequencing. A549 cells (adenocarcinomic human alveolar basal epithelial cell line were used as a model. Single-cell capture was performed using laser capture microdissection (LCM with an Arcturus® XT system, and a captured single cell and a bulk population of A549 cells (≈106 cells were subjected to whole genome amplification (WGA. For cell identification, a multiplex PCR method (AmpliSeq™ SNP HID panel was used to enrich 136 highly discriminatory SNPs with a genotype concordance probability of 1031–35. For cancer gene profiling, we used mutation profiling that was performed in parallel using a hotspot panel for 50 cancer-related genes. Sequencing was performed using a semiconductor-based bench top sequencer. The distribution of sequence reads for both HID and Cancer panel amplicons was consistent across these samples. For the bulk population of cells, the percentages of sequence covered at coverage of more than 100× were 99.04% for the HID panel and 98.83% for the Cancer panel, while for the single cell percentages of sequence covered at coverage of more than 100× were 55.93% for the HID panel and 65.96% for the Cancer panel. Partial amplification failure or randomly distributed non-amplified regions across samples from single cells during the WGA procedures or random allele drop out probably caused these differences. However, comparative analyses showed that this method successfully discriminated a single A549 cancer cell from a bulk population of A549 cells. Thus, our approach provides a powerful means to overcome tumor sample heterogeneity when searching for somatic mutations.

  17. BG7: a new approach for bacterial genome annotation designed for next generation sequencing data.

    Directory of Open Access Journals (Sweden)

    Pablo Pareja-Tobes

    Full Text Available BG7 is a new system for de novo bacterial, archaeal and viral genome annotation based on a new approach specifically designed for annotating genomes sequenced with next generation sequencing technologies. The system is versatile and able to annotate genes even in the step of preliminary assembly of the genome. It is especially efficient detecting unexpected genes horizontally acquired from bacterial or archaeal distant genomes, phages, plasmids, and mobile elements. From the initial phases of the gene annotation process, BG7 exploits the massive availability of annotated protein sequences in databases. BG7 predicts ORFs and infers their function based on protein similarity with a wide set of reference proteins, integrating ORF prediction and functional annotation phases in just one step. BG7 is especially tolerant to sequencing errors in start and stop codons, to frameshifts, and to assembly or scaffolding errors. The system is also tolerant to the high level of gene fragmentation which is frequently found in not fully assembled genomes. BG7 current version - which is developed in Java, takes advantage of Amazon Web Services (AWS cloud computing features, but it can also be run locally in any operating system. BG7 is a fast, automated and scalable system that can cope with the challenge of analyzing the huge amount of genomes that are being sequenced with NGS technologies. Its capabilities and efficiency were demonstrated in the 2011 EHEC Germany outbreak in which BG7 was used to get the first annotations right the next day after the first entero-hemorrhagic E. coli genome sequences were made publicly available. The suitability of BG7 for genome annotation has been proved for Illumina, 454, Ion Torrent, and PacBio sequencing technologies. Besides, thanks to its plasticity, our system could be very easily adapted to work with new technologies in the future.

  18. WIND-ACCRETION DISKS IN WIDE BINARIES, SECOND-GENERATION PROTOPLANETARY DISKS, AND ACCRETION ONTO WHITE DWARFS

    Energy Technology Data Exchange (ETDEWEB)

    Perets, Hagai B. [Technion-Israel Institute of Technology, Haifa (Israel); Kenyon, Scott J., E-mail: hperets@physics.technion.ac.il [Harvard-Smithsonian Center for Astrophysics, 60 Garden Street, Cambridge, MA 02138 (United States)

    2013-02-20

    Mass transfer from an evolved donor star to its binary companion is a standard feature of stellar evolution in binaries. In wide binaries, the companion star captures some of the mass ejected in a wind by the primary star. The captured material forms an accretion disk. Here, we study the evolution of wind-accretion disks, using a numerical approach which allows us to follow the long-term evolution. For a broad range of initial conditions, we derive the radial density and temperature profiles of the disk. In most cases, wind accretion leads to long-lived stable disks over the lifetime of the asymptotic giant branch donor star. The disks have masses of a few times 10{sup -5}-10{sup -3} M {sub Sun }, with surface density and temperature profiles that follow broken power laws. The total mass in the disk scales approximately linearly with the viscosity parameter used. Roughly, 50%-80% of the mass falling into the disk accretes onto the central star; the rest flows out through the outer edge of the disk into the stellar wind of the primary. For systems with large accretion rates, the secondary accretes as much as 0.1 M {sub Sun }. When the secondary is a white dwarf, accretion naturally leads to nova and supernova eruptions. For all types of secondary star, the surface density and temperature profiles of massive disks resemble structures observed in protoplanetary disks, suggesting that coordinated observational programs might improve our understanding of uncertain disk physics.

  19. Generation of a double binary transgenic zebrafish model to study myeloid gene regulation in response to oncogene activation in melanocytes.

    Science.gov (United States)

    Kenyon, Amy; Gavriouchkina, Daria; Zorman, Jernej; Chong-Morrison, Vanessa; Napolitani, Giorgio; Cerundolo, Vincenzo; Sauka-Spengler, Tatjana

    2018-04-06

    A complex network of inflammatory genes is closely linked to somatic cell transformation and malignant disease. Immune cells and their associated molecules are responsible for detecting and eliminating cancer cells as they establish themselves as the precursors of a tumour. By the time a patient has a detectable solid tumour, cancer cells have escaped the initial immune response mechanisms. Here, we describe the development of a double binary zebrafish model that enables regulatory programming of the myeloid cells as they respond to oncogene-activated melanocytes to be explored, focussing on the initial phase when cells become the precursors of cancer. A hormone-inducible binary system allows for temporal control of expression of different Ras oncogenes ( NRas Q61K , HRas G12V and KRas G12V ) in melanocytes, leading to proliferation and changes in morphology of the melanocytes. This model was coupled to binary cell-specific biotagging models allowing in vivo biotinylation and subsequent isolation of macrophage or neutrophil nuclei for regulatory profiling of their active transcriptomes. Nuclear transcriptional profiling of neutrophils, performed as they respond to the earliest precursors of melanoma in vivo , revealed an intricate landscape of regulatory factors that may promote progression to melanoma, including Serpinb1l4, Fgf1, Fgf6, Cathepsin H, Galectin 1 and Galectin 3. The model presented here provides a powerful platform to study the myeloid response to the earliest precursors of melanoma. © 2018. Published by The Company of Biologists Ltd.

  20. WIND-ACCRETION DISKS IN WIDE BINARIES, SECOND-GENERATION PROTOPLANETARY DISKS, AND ACCRETION ONTO WHITE DWARFS

    International Nuclear Information System (INIS)

    Perets, Hagai B.; Kenyon, Scott J.

    2013-01-01

    Mass transfer from an evolved donor star to its binary companion is a standard feature of stellar evolution in binaries. In wide binaries, the companion star captures some of the mass ejected in a wind by the primary star. The captured material forms an accretion disk. Here, we study the evolution of wind-accretion disks, using a numerical approach which allows us to follow the long-term evolution. For a broad range of initial conditions, we derive the radial density and temperature profiles of the disk. In most cases, wind accretion leads to long-lived stable disks over the lifetime of the asymptotic giant branch donor star. The disks have masses of a few times 10 –5 -10 –3 M ☉ , with surface density and temperature profiles that follow broken power laws. The total mass in the disk scales approximately linearly with the viscosity parameter used. Roughly, 50%-80% of the mass falling into the disk accretes onto the central star; the rest flows out through the outer edge of the disk into the stellar wind of the primary. For systems with large accretion rates, the secondary accretes as much as 0.1 M ☉ . When the secondary is a white dwarf, accretion naturally leads to nova and supernova eruptions. For all types of secondary star, the surface density and temperature profiles of massive disks resemble structures observed in protoplanetary disks, suggesting that coordinated observational programs might improve our understanding of uncertain disk physics.

  1. Calculation of Tajima's D and other neutrality test statistics from low depth next-generation sequencing data

    DEFF Research Database (Denmark)

    Korneliussen, Thorfinn Sand; Moltke, Ida; Albrechtsen, Anders

    2013-01-01

    A number of different statistics are used for detecting natural selection using DNA sequencing data, including statistics that are summaries of the frequency spectrum, such as Tajima's D. These statistics are now often being applied in the analysis of Next Generation Sequencing (NGS) data. Howeve......, estimates of frequency spectra from NGS data are strongly affected by low sequencing coverage; the inherent technology dependent variation in sequencing depth causes systematic differences in the value of the statistic among genomic regions....

  2. 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.

  3. WiseScaffolder: an algorithm for the semi-automatic scaffolding of Next Generation Sequencing data.

    Science.gov (United States)

    Farrant, Gregory K; Hoebeke, Mark; Partensky, Frédéric; Andres, Gwendoline; Corre, Erwan; Garczarek, Laurence

    2015-09-03

    The sequencing depth provided by high-throughput sequencing technologies has allowed a rise in the number of de novo sequenced genomes that could potentially be closed without further sequencing. However, genome scaffolding and closure require costly human supervision that often results in genomes being published as drafts. A number of automatic scaffolders were recently released, which improved the global quality of genomes published in the last few years. Yet, none of them reach the efficiency of manual scaffolding. Here, we present an innovative semi-automatic scaffolder that additionally helps with chimerae resolution and generates valuable contig maps and outputs for manual improvement of the automatic scaffolding. This software was tested on the newly sequenced marine cyanobacterium Synechococcus sp. WH8103 as well as two reference datasets used in previous studies, Rhodobacter sphaeroides and Homo sapiens chromosome 14 (http://gage.cbcb.umd.edu/). The quality of resulting scaffolds was compared to that of three other stand-alone scaffolders: SSPACE, SOPRA and SCARPA. For all three model organisms, WiseScaffolder produced better results than other scaffolders in terms of contiguity statistics (number of genome fragments, N50, LG50, etc.) and, in the case of WH8103, the reliability of the scaffolds was confirmed by whole genome alignment against a closely related reference genome. We also propose an efficient computer-assisted strategy for manual improvement of the scaffolding, using outputs generated by WiseScaffolder, as well as for genome finishing that in our hands led to the circularization of the WH8103 genome. Altogether, WiseScaffolder proved more efficient than three other scaffolders for both prokaryotic and eukaryotic genomes and is thus likely applicable to most genome projects. The scaffolding pipeline described here should be of particular interest to biologists wishing to take advantage of the high added value of complete genomes.

  4. Analysis of selected genes associated with cardiomyopathy by next-generation sequencing.

    Science.gov (United States)

    Szabadosova, Viktoria; Boronova, Iveta; Ferenc, Peter; Tothova, Iveta; Bernasovska, Jarmila; Zigova, Michaela; Kmec, Jan; Bernasovsky, Ivan

    2018-02-01

    As the leading cause of congestive heart failure, cardiomyopathy represents a heterogenous group of heart muscle disorders. Despite considerable progress being made in the genetic diagnosis of cardiomyopathy by detection of the mutations in the most prevalent cardiomyopathy genes, the cause remains unsolved in many patients. High-throughput mutation screening in the disease genes for cardiomyopathy is now possible because of using target enrichment followed by next-generation sequencing. The aim of the study was to analyze a panel of genes associated with dilated or hypertrophic cardiomyopathy based on previously published results in order to identify the subjects at risk. The method of next-generation sequencing by IlluminaHiSeq 2500 platform was used to detect sequence variants in 16 individuals diagnosed with dilated or hypertrophic cardiomyopathy. Detected variants were filtered and the functional impact of amino acid changes was predicted by computational programs. DNA samples of the 16 patients were analyzed by whole exome sequencing. We identified six nonsynonymous variants that were shown to be pathogenic in all used prediction softwares: rs3744998 (EPG5), rs11551768 (MGME1), rs148374985 (MURC), rs78461695 (PLEC), rs17158558 (RET) and rs2295190 (SYNE1). Two of the analyzed sequence variants had minor allele frequency (MAF)MURC), rs34580776 (MYBPC3). Our data support the potential role of the detected variants in pathogenesis of dilated or hypertrophic cardiomyopathy; however, the possibility that these variants might not be true disease-causing variants but are susceptibility alleles that require additional mutations or injury to cause the clinical phenotype of disease must be considered. © 2017 Wiley Periodicals, Inc.

  5. Investigation of next-generation sequencing data of Klebsiella pneumoniae using web-based tools.

    Science.gov (United States)

    Brhelova, Eva; Antonova, Mariya; Pardy, Filip; Kocmanova, Iva; Mayer, Jiri; Racil, Zdenek; Lengerova, Martina

    2017-11-01

    Rapid identification and characterization of multidrug-resistant Klebsiella pneumoniae strains is necessary due to the increasing frequency of severe infections in patients. The decreasing cost of next-generation sequencing enables us to obtain a comprehensive overview of genetic information in one step. The aim of this study is to demonstrate and evaluate the utility and scope of the application of web-based databases to next-generation sequenced (NGS) data. The whole genomes of 11 clinical Klebsiella pneumoniae isolates were sequenced using Illumina MiSeq. Selected web-based tools were used to identify a variety of genetic characteristics, such as acquired antimicrobial resistance genes, multilocus sequence types, plasmid replicons, and identify virulence factors, such as virulence genes, cps clusters, urease-nickel clusters and efflux systems. Using web-based tools hosted by the Center for Genomic Epidemiology, we detected resistance to 8 main antimicrobial groups with at least 11 acquired resistance genes. The isolates were divided into eight sequence types (ST11, 23, 37, 323, 433, 495 and 562, and a new one, ST1646). All of the isolates carried replicons of large plasmids. Capsular types, virulence factors and genes coding AcrAB and OqxAB efflux pumps were detected using BIGSdb-Kp, whereas the selected virulence genes, identified in almost all of the isolates, were detected using CLC Genomic Workbench software. Applying appropriate web-based online tools to NGS data enables the rapid extraction of comprehensive information that can be used for more efficient diagnosis and treatment of patients, while data processing is free of charge, easy and time-efficient.

  6. SNP discovery in the transcriptome of white Pacific shrimp Litopenaeus vannamei by next generation sequencing.

    Directory of Open Access Journals (Sweden)

    Yang Yu

    Full Text Available The application of next generation sequencing technology has greatly facilitated high throughput single nucleotide polymorphism (SNP discovery and genotyping in genetic research. In the present study, SNPs were discovered based on two transcriptomes of Litopenaeus vannamei (L. vannamei generated from Illumina sequencing platform HiSeq 2000. One transcriptome of L. vannamei was obtained through sequencing on the RNA from larvae at mysis stage and its reference sequence was de novo assembled. The data from another transcriptome were downloaded from NCBI and the reads of the two transcriptomes were mapped separately to the assembled reference by BWA. SNP calling was performed using SAMtools. A total of 58,717 and 36,277 SNPs with high quality were predicted from the two transcriptomes, respectively. SNP calling was also performed using the reads of two transcriptomes together, and a total of 96,040 SNPs with high quality were predicted. Among these 96,040 SNPs, 5,242 and 29,129 were predicted as non-synonymous and synonymous SNPs respectively. Characterization analysis of the predicted SNPs in L. vannamei showed that the estimated SNP frequency was 0.21% (one SNP per 476 bp and the estimated ratio for transition to transversion was 2.0. Fifty SNPs were randomly selected for validation by Sanger sequencing after PCR amplification and 76% of SNPs were confirmed, which indicated that the SNPs predicted in this study were reliable. These SNPs will be very useful for genetic study in L. vannamei, especially for the high density linkage map construction and genome-wide association studies.

  7. Analysis of Litopenaeus vannamei transcriptome using the next-generation DNA sequencing technique.

    Directory of Open Access Journals (Sweden)

    Chaozheng Li

    Full Text Available BACKGROUND: Pacific white shrimp (Litopenaeus vannamei, the major species of farmed shrimps in the world, has been attracting extensive studies, which require more and more genome background knowledge. The now available transcriptome data of L. vannamei are insufficient for research requirements, and have not been adequately assembled and annotated. METHODOLOGY/PRINCIPAL FINDINGS: This is the first study that used a next-generation high-throughput DNA sequencing technique, the Solexa/Illumina GA II method, to analyze the transcriptome from whole bodies of L. vannamei larvae. More than 2.4 Gb of raw data were generated, and 109,169 unigenes with a mean length of 396 bp were assembled using the SOAP denovo software. 73,505 unigenes (>200 bp with good quality sequences were selected and subjected to annotation analysis, among which 37.80% can be matched in NCBI Nr database, 37.3% matched in Swissprot, and 44.1% matched in TrEMBL. Using BLAST and BLAST2Go softwares, 11,153 unigenes were classified into 25 Clusters of Orthologous Groups of proteins (COG categories, 8171 unigenes were assigned into 51 Gene ontology (GO functional groups, and 18,154 unigenes were divided into 220 Kyoto Encyclopedia of Genes and Genomes (KEGG pathways. To primarily verify part of the results of assembly and annotations, 12 assembled unigenes that are homologous to many embryo development-related genes were chosen and subjected to RT-PCR for electrophoresis and Sanger sequencing analyses, and to real-time PCR for expression profile analyses during embryo development. CONCLUSIONS/SIGNIFICANCE: The L. vannamei transcriptome analyzed using the next-generation sequencing technique enriches the information of L. vannamei genes, which will facilitate our understanding of the genome background of crustaceans, and promote the studies on L. vannamei.

  8. Next-generation sequencing for genetic testing of familial colorectal cancer syndromes.

    Science.gov (United States)

    Simbolo, Michele; Mafficini, Andrea; Agostini, Marco; Pedrazzani, Corrado; Bedin, Chiara; Urso, Emanuele D; Nitti, Donato; Turri, Giona; Scardoni, Maria; Fassan, Matteo; Scarpa, Aldo

    2015-01-01

    Genetic screening in families with high risk to develop colorectal cancer (CRC) prevents incurable disease and permits personalized therapeutic and follow-up strategies. The advancement of next-generation sequencing (NGS) technologies has revolutionized the throughput of DNA sequencing. A series of 16 probands for either familial adenomatous polyposis (FAP; 8 cases) or hereditary nonpolyposis colorectal cancer (HNPCC; 8 cases) were investigated for intragenic mutations in five CRC familial syndromes-associated genes (APC, MUTYH, MLH1, MSH2, MSH6) applying both a custom multigene Ion AmpliSeq NGS panel and conventional Sanger sequencing. Fourteen pathogenic variants were detected in 13/16 FAP/HNPCC probands (81.3 %); one FAP proband presented two co-existing pathogenic variants, one in APC and one in MUTYH. Thirteen of these 14 pathogenic variants were detected by both NGS and Sanger, while one MSH2 mutation (L280FfsX3) was identified only by Sanger sequencing. This is due to a limitation of the NGS approach in resolving sequences close or within homopolymeric stretches of DNA. To evaluate the performance of our NGS custom panel we assessed its capability to resolve the DNA sequences corresponding to 2225 pathogenic variants reported in the COSMIC database for APC, MUTYH, MLH1, MSH2, MSH6. Our NGS custom panel resolves the sequences where 2108 (94.7 %) of these variants occur. The remaining 117 mutations reside inside or in close proximity to homopolymer stretches; of these 27 (1.2 %) are imprecisely identified by the software but can be resolved by visual inspection of the region, while the remaining 90 variants (4.0 %) are blind spots. In summary, our custom panel would miss 4 % (90/2225) of pathogenic variants that would need a small set of Sanger sequencing reactions to be solved. The multiplex NGS approach has the advantage of analyzing multiple genes in multiple samples simultaneously, requiring only a reduced number of Sanger sequences to resolve

  9. Single nucleotide polymorphism analysis of Korean native chickens using next generation sequencing data.

    Science.gov (United States)

    Seo, Dong-Won; Oh, Jae-Don; Jin, Shil; Song, Ki-Duk; Park, Hee-Bok; Heo, Kang-Nyeong; Shin, Younhee; Jung, Myunghee; Park, Junhyung; Jo, Cheorun; Lee, Hak-Kyo; Lee, Jun-Heon

    2015-02-01

    There are five native chicken lines in Korea, which are mainly classified by plumage colors (black, white, red, yellow, gray). These five lines are very important genetic resources in the Korean poultry industry. Based on a next generation sequencing technology, whole genome sequence and reference assemblies were performed using Gallus_gallus_4.0 (NCBI) with whole genome sequences from these lines to identify common and novel single nucleotide polymorphisms (SNPs). We obtained 36,660,731,136 ± 1,257,159,120 bp of raw sequence and average 26.6-fold of 25-29 billion reference assembly sequences representing 97.288 % coverage. Also, 4,006,068 ± 97,534 SNPs were observed from 29 autosomes and the Z chromosome and, of these, 752,309 SNPs are the common SNPs across lines. Among the identified SNPs, the number of novel- and known-location assigned SNPs was 1,047,951 ± 14,956 and 2,948,648 ± 81,414, respectively. The number of unassigned known SNPs was 1,181 ± 150 and unassigned novel SNPs was 8,238 ± 1,019. Synonymous SNPs, non-synonymous SNPs, and SNPs having character changes were 26,266 ± 1,456, 11,467 ± 604, 8,180 ± 458, respectively. Overall, 443,048 ± 26,389 SNPs in each bird were identified by comparing with dbSNP in NCBI. The presently obtained genome sequence and SNP information in Korean native chickens have wide applications for further genome studies such as genetic diversity studies to detect causative mutations for economic and disease related traits.

  10. Use of four next-generation sequencing platforms to determine HIV-1 coreceptor tropism.

    Science.gov (United States)

    Archer, John; Weber, Jan; Henry, Kenneth; Winner, Dane; Gibson, Richard; Lee, Lawrence; Paxinos, Ellen; Arts, Eric J; Robertson, David L; Mimms, Larry; Quiñones-Mateu, Miguel E

    2012-01-01

    HIV-1 coreceptor tropism assays are required to rule out the presence of CXCR4-tropic (non-R5) viruses prior treatment with CCR5 antagonists. Phenotypic (e.g., Trofile™, Monogram Biosciences) and genotypic (e.g., population sequencing linked to bioinformatic algorithms) assays are the most widely used. Although several next-generation sequencing (NGS) platforms are available, to date all published deep sequencing HIV-1 tropism studies have used the 454™ Life Sciences/Roche platform. In this study, HIV-1 co-receptor usage was predicted for twelve patients scheduled to start a maraviroc-based antiretroviral regimen. The V3 region of the HIV-1 env gene was sequenced using four NGS platforms: 454™, PacBio® RS (Pacific Biosciences), Illumina®, and Ion Torrent™ (Life Technologies). Cross-platform variation was evaluated, including number of reads, read length and error rates. HIV-1 tropism was inferred using Geno2Pheno, Web PSSM, and the 11/24/25 rule and compared with Trofile™ and virologic response to antiretroviral therapy. Error rates related to insertions/deletions (indels) and nucleotide substitutions introduced by the four NGS platforms were low compared to the actual HIV-1 sequence variation. Each platform detected all major virus variants within the HIV-1 population with similar frequencies. Identification of non-R5 viruses was comparable among the four platforms, with minor differences attributable to the algorithms used to infer HIV-1 tropism. All NGS platforms showed similar concordance with virologic response to the maraviroc-based regimen (75% to 80% range depending on the algorithm used), compared to Trofile (80%) and population sequencing (70%). In conclusion, all four NGS platforms were able to detect minority non-R5 variants at comparable levels suggesting that any NGS-based method can be used to predict HIV-1 coreceptor usage.

  11. Use of four next-generation sequencing platforms to determine HIV-1 coreceptor tropism.

    Directory of Open Access Journals (Sweden)

    John Archer

    Full Text Available HIV-1 coreceptor tropism assays are required to rule out the presence of CXCR4-tropic (non-R5 viruses prior treatment with CCR5 antagonists. Phenotypic (e.g., Trofile™, Monogram Biosciences and genotypic (e.g., population sequencing linked to bioinformatic algorithms assays are the most widely used. Although several next-generation sequencing (NGS platforms are available, to date all published deep sequencing HIV-1 tropism studies have used the 454™ Life Sciences/Roche platform. In this study, HIV-1 co-receptor usage was predicted for twelve patients scheduled to start a maraviroc-based antiretroviral regimen. The V3 region of the HIV-1 env gene was sequenced using four NGS platforms: 454™, PacBio® RS (Pacific Biosciences, Illumina®, and Ion Torrent™ (Life Technologies. Cross-platform variation was evaluated, including number of reads, read length and error rates. HIV-1 tropism was inferred using Geno2Pheno, Web PSSM, and the 11/24/25 rule and compared with Trofile™ and virologic response to antiretroviral therapy. Error rates related to insertions/deletions (indels and nucleotide substitutions introduced by the four NGS platforms were low compared to the actual HIV-1 sequence variation. Each platform detected all major virus variants within the HIV-1 population with similar frequencies. Identification of non-R5 viruses was comparable among the four platforms, with minor differences attributable to the algorithms used to infer HIV-1 tropism. All NGS platforms showed similar concordance with virologic response to the maraviroc-based regimen (75% to 80% range depending on the algorithm used, compared to Trofile (80% and population sequencing (70%. In conclusion, all four NGS platforms were able to detect minority non-R5 variants at comparable levels suggesting that any NGS-based method can be used to predict HIV-1 coreceptor usage.

  12. Combining information from linkage and association mapping for next-generation sequencing longitudinal family data.

    Science.gov (United States)

    Balliu, Brunilda; Uh, Hae-Won; Tsonaka, Roula; Boehringer, Stefan; Helmer, Quinta; Houwing-Duistermaat, Jeanine J

    2014-01-01

    In this analysis, we investigate the contributions that linkage-based methods, such as identical-by-descent mapping, can make to association mapping to identify rare variants in next-generation sequencing data. First, we identify regions in which cases share more segments identical-by-descent around a putative causal variant than do controls. Second, we use a two-stage mixed-effect model approach to summarize the single-nucleotide polymorphism data within each region and include them as covariates in the model for the phenotype. We assess the impact of linkage disequilibrium in determining identical-by-descent states between individuals by using markers with and without linkage disequilibrium for the first part and the impact of imputation in testing for association by using imputed genome-wide association studies or raw sequence markers for the second part. We apply the method to next-generation sequencing longitudinal family data from Genetic Association Workshop 18 and identify a significant region at chromosome 3: 40249244-41025167 (p-value = 2.3 × 10(-3)).

  13. Mapping RNA Structure In Vitro with SHAPE Chemistry and Next-Generation Sequencing (SHAPE-Seq).

    Science.gov (United States)

    Watters, Kyle E; Lucks, Julius B

    2016-01-01

    Mapping RNA structure with selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) chemistry has proven to be a versatile method for characterizing RNA structure in a variety of contexts. SHAPE reagents covalently modify RNAs in a structure-dependent manner to create adducts at the 2'-OH group of the ribose backbone at nucleotides that are structurally flexible. The positions of these adducts are detected using reverse transcriptase (RT) primer extension, which stops one nucleotide before the modification, to create a pool of cDNAs whose lengths reflect the location of SHAPE modification. Quantification of the cDNA pools is used to estimate the "reactivity" of each nucleotide in an RNA molecule to the SHAPE reagent. High reactivities indicate nucleotides that are structurally flexible, while low reactivities indicate nucleotides that are inflexible. These SHAPE reactivities can then be used to infer RNA structures by restraining RNA structure prediction algorithms. Here, we provide a state-of-the-art protocol describing how to perform in vitro RNA structure probing with SHAPE chemistry using next-generation sequencing to quantify cDNA pools and estimate reactivities (SHAPE-Seq). The use of next-generation sequencing allows for higher throughput, more consistent data analysis, and multiplexing capabilities. The technique described herein, SHAPE-Seq v2.0, uses a universal reverse transcription priming site that is ligated to the RNA after SHAPE modification. The introduced priming site allows for the structural analysis of an RNA independent of its sequence.

  14. Somatic mosaicism of a CDKL5 mutation identified by next-generation sequencing.

    Science.gov (United States)

    Kato, Takeshi; Morisada, Naoya; Nagase, Hiroaki; Nishiyama, Masahiro; Toyoshima, Daisaku; Nakagawa, Taku; Maruyama, Azusa; Fu, Xue Jun; Nozu, Kandai; Wada, Hiroko; Takada, Satoshi; Iijima, Kazumoto

    2015-10-01

    CDKL5-related encephalopathy is an X-linked dominantly inherited disorder that is characterized by early infantile epileptic encephalopathy or atypical Rett syndrome. We describe a 5-year-old Japanese boy with intractable epilepsy, severe developmental delay, and Rett syndrome-like features. Onset was at 2 months, when his electroencephalogram showed sporadic single poly spikes and diffuse irregular poly spikes. We conducted a genetic analysis using an Illumina® TruSight™ One sequencing panel on a next-generation sequencer. We identified two epilepsy-associated single nucleotide variants in our case: CDKL5 p.Ala40Val and KCNQ2 p.Glu515Asp. CDKL5 p.Ala40Val has been previously reported to be responsible for early infantile epileptic encephalopathy. In our case, the CDKL5 heterozygous mutation showed somatic mosaicism because the boy's karyotype was 46,XY. The KCNQ2 variant p.Glu515Asp is known to cause benign familial neonatal seizures-1, and this variant showed paternal inheritance. Although we believe that the somatic mosaic CDKL5 mutation is mainly responsible for the neurological phenotype in the patient, the KCNQ2 variant might have some neurological effect. Genetic analysis by next-generation sequencing is capable of identifying multiple variants in a patient. Copyright © 2015 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.

  15. Molecular typing of lung adenocarcinoma on cytological samples using a multigene next generation sequencing panel.

    Directory of Open Access Journals (Sweden)

    Aldo Scarpa

    Full Text Available Identification of driver mutations in lung adenocarcinoma has led to development of targeted agents that are already approved for clinical use or are in clinical trials. Therefore, the number of biomarkers that will be needed to assess is expected to rapidly increase. This calls for the implementation of methods probing the mutational status of multiple genes for inoperable cases, for which limited cytological or bioptic material is available. Cytology specimens from 38 lung adenocarcinomas were subjected to the simultaneous assessment of 504 mutational hotspots of 22 lung cancer-associated genes using 10 nanograms of DNA and Ion Torrent PGM next-generation sequencing. Thirty-six cases were successfully sequenced (95%. In 24/36 cases (67% at least one mutated gene was observed, including EGFR, KRAS, PIK3CA, BRAF, TP53, PTEN, MET, SMAD4, FGFR3, STK11, MAP2K1. EGFR and KRAS mutations, respectively found in 6/36 (16% and 10/36 (28% cases, were mutually exclusive. Nine samples (25% showed concurrent alterations in different genes. The next-generation sequencing test used is superior to current standard methodologies, as it interrogates multiple genes and requires limited amounts of DNA. Its applicability to routine cytology samples might allow a significant increase in the fraction of lung cancer patients eligible for personalized therapy.

  16. Identification of Five Novel Variants in Chinese Oculocutaneous Albinism by Targeted Next-Generation Sequencing.

    Science.gov (United States)

    Qiu, Biyuan; Ma, Tao; Peng, Chunyan; Zheng, Xiaoqin; Yang, Jiyun

    2018-04-01

    The diagnosis of oculocutaneous albinism (OCA) is established using clinical signs and symptoms. OCA is, however, a highly genetically heterogeneous disease with mutations identified in at least nineteen unique genes, many of which produce overlapping phenotypic traits. Thus, differentiating genetic OCA subtypes for diagnoses and genetic counseling is challenging, based on clinical presentation alone, and would benefit from a comprehensive molecular diagnostic. To develop and validate a more comprehensive, targeted, next-generation-sequencing-based diagnostic for the identification of OCA-causing variants. The genomic DNA samples from 28 OCA probands were analyzed by targeted next-generation sequencing (NGS), and the candidate variants were confirmed through Sanger sequencing. We observed mutations in the TYR, OCA2, and SLC45A2 genes in 25/28 (89%) patients with OCA. We identified 38 pathogenic variants among these three genes, including 5 novel variants: c.1970G>T (p.Gly657Val), c.1669A>C (p.Thr557Pro), c.2339-2A>C, and c.1349C>G (p.Thr450Arg) in OCA2; c.459_470delTTTTGCTGCCGA (p.Ala155_Phe158del) in SLC45A2. Our findings expand the mutational spectrum of OCA in the Chinese population, and the assay we developed should be broadly useful as a molecular diagnostic, and as an aid for genetic counseling for OCA patients.

  17. Read length and repeat resolution: exploring prokaryote genomes using next-generation sequencing technologies.

    Directory of Open Access Journals (Sweden)

    Matt J Cahill

    Full Text Available BACKGROUND: There are a growing number of next-generation sequencing technologies. At present, the most cost-effective options also produce the shortest reads. However, even for prokaryotes, there is uncertainty concerning the utility of these technologies for the de novo assembly of complete genomes. This reflects an expectation that short reads will be unable to resolve small, but presumably abundant, repeats. METHODOLOGY/PRINCIPAL FINDINGS: Using a simple model of repeat assembly, we develop and test a technique that, for any read length, can estimate the occurrence of unresolvable repeats in a genome, and thus predict the number of gaps that would need to be closed to produce a complete sequence. We apply this technique to 818 prokaryote genome sequences. This provides a quantitative assessment of the relative performance of various lengths. Notably, unpaired reads of only 150nt can reconstruct approximately 50% of the analysed genomes with fewer than 96 repeat-induced gaps. Nonetheless, there is considerable variation amongst prokaryotes. Some genomes can be assembled to near contiguity using very short reads while others require much longer reads. CONCLUSIONS: Given the diversity of prokaryote genomes, a sequencing strategy should be tailored to the organism under study. Our results will provide researchers with a practical resource to guide the selection of the appropriate read length.

  18. The utility of Next Generation Sequencing for molecular diagnostics in Rett syndrome.

    Science.gov (United States)

    Vidal, Silvia; Brandi, Núria; Pacheco, Paola; Gerotina, Edgar; Blasco, Laura; Trotta, Jean-Rémi; Derdak, Sophia; Del Mar O'Callaghan, Maria; Garcia-Cazorla, Àngels; Pineda, Mercè; Armstrong, Judith

    2017-09-25

    Rett syndrome (RTT) is an early-onset neurodevelopmental disorder that almost exclusively affects girls and is totally disabling. Three genes have been identified that cause RTT: MECP2, CDKL5 and FOXG1. However, the etiology of some of RTT patients still remains unknown. Recently, next generation sequencing (NGS) has promoted genetic diagnoses because of the quickness and affordability of the method. To evaluate the usefulness of NGS in genetic diagnosis, we present the genetic study of RTT-like patients using different techniques based on this technology. We studied 1577 patients with RTT-like clinical diagnoses and reviewed patients who were previously studied and thought to have RTT genes by Sanger sequencing. Genetically, 477 of 1577 patients with a RTT-like suspicion have been diagnosed. Positive results were found in 30% by Sanger sequencing, 23% with a custom panel, 24% with a commercial panel and 32% with whole exome sequencing. A genetic study using NGS allows the study of a larger number of genes associated with RTT-like symptoms simultaneously, providing genetic study of a wider group of patients as well as significantly reducing the response time and cost of the study.

  19. Next generation sequencing and analysis of a conserved transcriptome of New Zealand's kiwi.

    Science.gov (United States)

    Subramanian, Sankar; Huynen, Leon; Millar, Craig D; Lambert, David M

    2010-12-15

    Kiwi is a highly distinctive, flightless and endangered ratite bird endemic to New Zealand. To understand the patterns of molecular evolution of the nuclear protein-coding genes in brown kiwi (Apteryx australis mantelli) and to determine the timescale of avian history we sequenced a transcriptome obtained from a kiwi embryo using next generation sequencing methods. We then assembled the conserved protein-coding regions using the chicken proteome as a scaffold. Using 1,543 conserved protein coding genes we estimated the neutral evolutionary divergence between the kiwi and chicken to be ~45%, which is approximately equal to the divergence computed for the human-mouse pair using the same set of genes. A large fraction of genes was found to be under high selective constraint, as most of the expressed genes appeared to be involved in developmental gene regulation. Our study suggests a significant relationship between gene expression levels and protein evolution. Using sequences from over 700 nuclear genes we estimated the divergence between the two basal avian groups, Palaeognathae and Neognathae to be 132 million years, which is consistent with previous studies using mitochondrial genes. The results of this investigation revealed patterns of mutation and purifying selection in conserved protein coding regions in birds. Furthermore this study suggests a relatively cost-effective way of obtaining a glimpse into the fundamental molecular evolutionary attributes of a genome, particularly when no closely related genomic sequence is available.

  20. Next generation sequencing and analysis of a conserved transcriptome of New Zealand's kiwi

    Directory of Open Access Journals (Sweden)

    Huynen Leon

    2010-12-01

    Full Text Available Abstract Background Kiwi is a highly distinctive, flightless and endangered ratite bird endemic to New Zealand. To understand the patterns of molecular evolution of the nuclear protein-coding genes in brown kiwi (Apteryx australis mantelli and to determine the timescale of avian history we sequenced a transcriptome obtained from a kiwi embryo using next generation sequencing methods. We then assembled the conserved protein-coding regions using the chicken proteome as a scaffold. Results Using 1,543 conserved protein coding genes we estimated the neutral evolutionary divergence between the kiwi and chicken to be ~45%, which is approximately equal to the divergence computed for the human-mouse pair using the same set of genes. A large fraction of genes was found to be under high selective constraint, as most of the expressed genes appeared to be involved in developmental gene regulation. Our study suggests a significant relationship between gene expression levels and protein evolution. Using sequences from over 700 nuclear genes we estimated the divergence between the two basal avian groups, Palaeognathae and Neognathae to be 132 million years, which is consistent with previous studies using mitochondrial genes. Conclusions The results of this investigation revealed patterns of mutation and purifying selection in conserved protein coding regions in birds. Furthermore this study suggests a relatively cost-effective way of obtaining a glimpse into the fundamental molecular evolutionary attributes of a genome, particularly when no closely related genomic sequence is available.

  1. Applications and challenges of next-generation sequencing in Brassica species.

    Science.gov (United States)

    Wei, Lijuan; Xiao, Meili; Hayward, Alice; Fu, Donghui

    2013-12-01

    Next-generation sequencing (NGS) produces numerous (often millions) short DNA sequence reads, typically varying between 25 and 400 bp in length, at a relatively low cost and in a short time. This revolutionary technology is being increasingly applied in whole-genome, transcriptome, epigenome and small RNA sequencing, molecular marker and gene discovery, comparative and evolutionary genomics, and association studies. The Brassica genus comprises some of the most agro-economically important crops, providing abundant vegetables, condiments, fodder, oil and medicinal products. Many Brassica species have undergone the process of polyploidization, which makes their genomes exceptionally complex and can create difficulties in genomics research. NGS injects new vigor into Brassica research, yet also faces specific challenges in the analysis of complex crop genomes and traits. In this article, we review the advantages and limitations of different NGS technologies and their applications and challenges, using Brassica as an advanced model system for agronomically important, polyploid crops. Specifically, we focus on the use of NGS for genome resequencing, transcriptome sequencing, development of single-nucleotide polymorphism markers, and identification of novel microRNAs and their targets. We present trends and advances in NGS technology in relation to Brassica crop improvement, with wide application for sophisticated genomics research into agronomically important polyploid crops.

  2. Read length and repeat resolution: Exploring prokaryote genomes using next-generation sequencing technologies

    KAUST Repository

    Cahill, Matt J.

    2010-07-12

    Background: There are a growing number of next-generation sequencing technologies. At present, the most cost-effective options also produce the shortest reads. However, even for prokaryotes, there is uncertainty concerning the utility of these technologies for the de novo assembly of complete genomes. This reflects an expectation that short reads will be unable to resolve small, but presumably abundant, repeats. Methodology/Principal Findings: Using a simple model of repeat assembly, we develop and test a technique that, for any read length, can estimate the occurrence of unresolvable repeats in a genome, and thus predict the number of gaps that would need to be closed to produce a complete sequence. We apply this technique to 818 prokaryote genome sequences. This provides a quantitative assessment of the relative performance of various lengths. Notably, unpaired reads of only 150nt can reconstruct approximately 50% of the analysed genomes with fewer than 96 repeat-induced gaps. Nonetheless, there is considerable variation amongst prokaryotes. Some genomes can be assembled to near contiguity using very short reads while others require much longer reads. Conclusions: Given the diversity of prokaryote genomes, a sequencing strategy should be tailored to the organism under study. Our results will provide researchers with a practical resource to guide the selection of the appropriate read length. 2010 Cahill et al.

  3. Clinical validation of targeted next-generation sequencing for inherited disorders.

    Science.gov (United States)

    Yohe, Sophia; Hauge, Adam; Bunjer, Kari; Kemmer, Teresa; Bower, Matthew; Schomaker, Matthew; Onsongo, Getiria; Wilson, Jon; Erdmann, Jesse; Zhou, Yi; Deshpande, Archana; Spears, Michael D; Beckman, Kenneth; Silverstein, Kevin A T; Thyagarajan, Bharat

    2015-02-01

    Although next-generation sequencing (NGS) can revolutionize molecular diagnostics, several hurdles remain in the implementation of this technology in clinical laboratories. To validate and implement an NGS panel for genetic diagnosis of more than 100 inherited diseases, such as neurologic conditions, congenital hearing loss and eye disorders, developmental disorders, nonmalignant diseases treated by hematopoietic cell transplantation, familial cancers, connective tissue disorders, metabolic disorders, disorders of sexual development, and cardiac disorders. The diagnostic gene panels ranged from 1 to 54 genes with most of panels containing 10 genes or fewer. We used a liquid hybridization-based, target-enrichment strategy to enrich 10 067 exons in 568 genes, followed by NGS with a HiSeq 2000 sequencing system (Illumina, San Diego, California). We successfully sequenced 97.6% (9825 of 10 067) of the targeted exons to obtain a minimum coverage of 20× at all bases. We demonstrated 100% concordance in detecting 19 pathogenic single-nucleotide variations and 11 pathogenic insertion-deletion mutations ranging in size from 1 to 18 base pairs across 18 samples that were previously characterized by Sanger sequencing. Using 4 pairs of blinded, duplicate samples, we demonstrated a high degree of concordance (>99%) among the blinded, duplicate pairs. We have successfully demonstrated the feasibility of using the NGS platform to multiplex genetic tests for several rare diseases and the use of cloud computing for bioinformatics analysis as a relatively low-cost solution for implementing NGS in clinical laboratories.

  4. DNA copy number, including telomeres and mitochondria, assayed using next-generation sequencing

    Directory of Open Access Journals (Sweden)

    Jackson Stuart

    2010-04-01

    Full Text Available Abstract Background DNA copy number variations occur within populations and aberrations can cause disease. We sought to develop an improved lab-automatable, cost-efficient, accurate platform to profile DNA copy number. Results We developed a sequencing-based assay of nuclear, mitochondrial, and telomeric DNA copy number that draws on the unbiased nature of next-generation sequencing and incorporates techniques developed for RNA expression profiling. To demonstrate this platform, we assayed UMC-11 cells using 5 million 33 nt reads and found tremendous copy number variation, including regions of single and homogeneous deletions and amplifications to 29 copies; 5 times more mitochondria and 4 times less telomeric sequence than a pool of non-diseased, blood-derived DNA; and that UMC-11 was derived from a male individual. Conclusion The described assay outputs absolute copy number, outputs an error estimate (p-value, and is more accurate than array-based platforms at high copy number. The platform enables profiling of mitochondrial levels and telomeric length. The assay is lab-automatable and has a genomic resolution and cost that are tunable based on the number of sequence reads.

  5. Validation of Metagenomic Next-Generation Sequencing Tests for Universal Pathogen Detection.

    Science.gov (United States)

    Schlaberg, Robert; Chiu, Charles Y; Miller, Steve; Procop, Gary W; Weinstock, George

    2017-06-01

    - Metagenomic sequencing can be used for detection of any pathogens using unbiased, shotgun next-generation sequencing (NGS), without the need for sequence-specific amplification. Proof-of-concept has been demonstrated in infectious disease outbreaks of unknown causes and in patients with suspected infections but negative results for conventional tests. Metagenomic NGS tests hold great promise to improve infectious disease diagnostics, especially in immunocompromised and critically ill patients. - To discuss challenges and provide example solutions for validating metagenomic pathogen detection tests in clinical laboratories. A summary of current regulatory requirements, largely based on prior guidance for NGS testing in constitutional genetics and oncology, is provided. - Examples from 2 separate validation studies are provided for steps from assay design, and validation of wet bench and bioinformatics protocols, to quality control and assurance. - Although laboratory and data analysis workflows are still complex, metagenomic NGS tests for infectious diseases are increasingly being validated in clinical laboratories. Many parallels exist to NGS tests in other fields. Nevertheless, specimen preparation, rapidly evolving data analysis algorithms, and incomplete reference sequence databases are idiosyncratic to the field of microbiology and often overlooked.

  6. Coval: improving alignment quality and variant calling accuracy for next-generation sequencing data.

    Directory of Open Access Journals (Sweden)

    Shunichi Kosugi

    Full Text Available Accurate identification of DNA polymorphisms using next-generation sequencing technology is challenging because of a high rate of sequencing error and incorrect mapping of reads to reference genomes. Currently available short read aligners and DNA variant callers suffer from these problems. We developed the Coval software to improve the quality of short read alignments. Coval is designed to minimize the incidence of spurious alignment of short reads, by filtering mismatched reads that remained in alignments after local realignment and error correction of mismatched reads. The error correction is executed based on the base quality and allele frequency at the non-reference positions for an individual or pooled sample. We demonstrated the utility of Coval by applying it to simulated genomes and experimentally obtained short-read data of rice, nematode, and mouse. Moreover, we found an unexpectedly large number of incorrectly mapped reads in 'targeted' alignments, where the whole genome sequencing reads had been aligned to a local genomic segment, and showed that Coval effectively eliminated such spurious alignments. We conclude that Coval significantly improves the quality of short-read sequence alignments, thereby increasing the calling accuracy of currently available tools for SNP and indel identification. Coval is available at http://sourceforge.net/projects/coval105/.

  7. The quest for rare variants: pooled multiplexed next generation sequencing in plants.

    Science.gov (United States)

    Marroni, Fabio; Pinosio, Sara; Morgante, Michele

    2012-01-01

    Next generation sequencing (NGS) instruments produce an unprecedented amount of sequence data at contained costs. This gives researchers the possibility of designing studies with adequate power to identify rare variants at a fraction of the economic and labor resources required by individual Sanger sequencing. As of today, few research groups working in plant sciences have exploited this potentiality, showing that pooled NGS provides results in excellent agreement with those obtained by individual Sanger sequencing. The aim of this review is to convey to the reader the general ideas underlying the use of pooled NGS for the identification of rare variants. To facilitate a thorough understanding of the possibilities of the method, we will explain in detail the possible experimental and analytical approaches and discuss their advantages and disadvantages. We will show that information on allele frequency obtained by pooled NGS can be used to accurately compute basic population genetics indexes such as allele frequency, nucleotide diversity, and Tajima's D. Finally, we will discuss applications and future perspectives of the multiplexed NGS approach.

  8. Read length and repeat resolution: Exploring prokaryote genomes using next-generation sequencing technologies

    KAUST Repository

    Cahill, Matt J.; Kö ser, Claudio U.; Ross, Nicholas E.; Archer, John A.C.

    2010-01-01

    Background: There are a growing number of next-generation sequencing technologies. At present, the most cost-effective options also produce the shortest reads. However, even for prokaryotes, there is uncertainty concerning the utility of these technologies for the de novo assembly of complete genomes. This reflects an expectation that short reads will be unable to resolve small, but presumably abundant, repeats. Methodology/Principal Findings: Using a simple model of repeat assembly, we develop and test a technique that, for any read length, can estimate the occurrence of unresolvable repeats in a genome, and thus predict the number of gaps that would need to be closed to produce a complete sequence. We apply this technique to 818 prokaryote genome sequences. This provides a quantitative assessment of the relative performance of various lengths. Notably, unpaired reads of only 150nt can reconstruct approximately 50% of the analysed genomes with fewer than 96 repeat-induced gaps. Nonetheless, there is considerable variation amongst prokaryotes. Some genomes can be assembled to near contiguity using very short reads while others require much longer reads. Conclusions: Given the diversity of prokaryote genomes, a sequencing strategy should be tailored to the organism under study. Our results will provide researchers with a practical resource to guide the selection of the appropriate read length. 2010 Cahill et al.

  9. Development of Genome-Wide SSR Markers from Angelica gigas Nakai Using Next Generation Sequencing.

    Science.gov (United States)

    Gil, Jinsu; Um, Yurry; Kim, Serim; Kim, Ok Tae; Koo, Sung Cheol; Reddy, Chinreddy Subramanyam; Kim, Seong-Cheol; Hong, Chang Pyo; Park, Sin-Gi; Kim, Ho Bang; Lee, Dong Hoon; Jeong, Byung-Hoon; Chung, Jong-Wook; Lee, Yi

    2017-09-21

    Angelica gigas Nakai is an important medicinal herb, widely utilized in Asian countries especially in Korea, Japan, and China. Although it is a vital medicinal herb, the lack of sequencing data and efficient molecular markers has limited the application of a genetic approach for horticultural improvements. Simple sequence repeats (SSRs) are universally accepted molecular markers for population structure study. In this study, we found over 130,000 SSRs, ranging from di- to deca-nucleotide motifs, using the genome sequence of Manchu variety (MV) of A. gigas, derived from next generation sequencing (NGS). From the putative SSR regions identified, a total of 16,496 primer sets were successfully designed. Among them, we selected 848 SSR markers that showed polymorphism from in silico analysis and contained tri- to hexa-nucleotide motifs. We tested 36 SSR primer sets for polymorphism in 16 A. gigas accessions. The average polymorphism information content (PIC) was 0.69; the average observed heterozygosity ( H O ) values, and the expected heterozygosity ( H E ) values were 0.53 and 0.73, respectively. These newly developed SSR markers would be useful tools for molecular genetics, genotype identification, genetic mapping, molecular breeding, and studying species relationships of the Angelica genus.

  10. Protection algorithm for a wind turbine generator based on positive- and negative-sequence fault components

    DEFF Research Database (Denmark)

    Zheng, Tai-Ying; Cha, Seung-Tae; Crossley, Peter A.

    2011-01-01

    A protection relay for a wind turbine generator (WTG) based on positive- and negative-sequence fault components is proposed in the paper. The relay uses the magnitude of the positive-sequence component in the fault current to detect a fault on a parallel WTG, connected to the same power collection...... feeder, or a fault on an adjacent feeder; but for these faults, the relay remains stable and inoperative. A fault on the power collection feeder or a fault on the collection bus, both of which require an instantaneous tripping response, are distinguished from an inter-tie fault or a grid fault, which...... in the fault current is used to decide on either instantaneous or delayed operation. The operating performance of the relay is then verified using various fault scenarios modelled using EMTP-RV. The scenarios involve changes in the position and type of fault, and the faulted phases. Results confirm...

  11. [Application of next-generation semiconductor sequencing technologies in genetic diagnosis of inherited cardiomyopathies].

    Science.gov (United States)

    Zhao, Yue; Zhang, Hong; Xia, Xue-shan

    2015-07-01

    Inherited cardiomyopathy is the most common hereditary cardiac disease. It also causes a significant proportion of sudden cardiac deaths in young adults and athletes. So far, approximately one hundred genes have been reported to be involved in cardiomyopathies through different mechanisms. Therefore, the identification of the genetic basis and disease mechanisms of cardiomyopathies are important for establishing a clinical diagnosis and genetic testing. Next-generation semiconductor sequencing (NGSS) technology platform is a high-throughput sequencer capable of analyzing clinically derived genomes with high productivity, sensitivity and specificity. It was launched in 2010 by Life Technologies of USA, and it is based on a high density semiconductor chip, which was covered with tens of thousands of wells. NGSS has been successfully used in candidate gene mutation screening to identify hereditary disease. In this review, we summarize these genetic variations, challenge and application of NGSS in inherited cardiomyopathy, and its value in disease diagnosis, prevention and treatment.

  12. Compression of computer generated phase-shifting hologram sequence using AVC and HEVC

    Science.gov (United States)

    Xing, Yafei; Pesquet-Popescu, Béatrice; Dufaux, Frederic

    2013-09-01

    With the capability of achieving twice the compression ratio of Advanced Video Coding (AVC) with similar reconstruction quality, High Efficiency Video Coding (HEVC) is expected to become the newleading technique of video coding. In order to reduce the storage and transmission burden of digital holograms, in this paper we propose to use HEVC for compressing the phase-shifting digital hologram sequences (PSDHS). By simulating phase-shifting digital holography (PSDH) interferometry, interference patterns between illuminated three dimensional( 3D) virtual objects and the stepwise phase changed reference wave are generated as digital holograms. The hologram sequences are obtained by the movement of the virtual objects and compressed by AVC and HEVC. The experimental results show that AVC and HEVC are efficient to compress PSDHS, with HEVC giving better performance. Good compression rate and reconstruction quality can be obtained with bitrate above 15000kbps.

  13. Next-generation sequencing: hype and hope for development of personalized radiation therapy?

    International Nuclear Information System (INIS)

    Tinhofer, Ingeborg; Niehr, Franziska; Konschak, Robert; Liebs, Sandra; Munz, Matthias; Stenzinger, Albrecht; Weichert, Wilko; Keilholz, Ulrich; Budach, Volker

    2015-01-01

    The introduction of next-generation sequencing (NGS) in the field of cancer research has boosted worldwide efforts of genome-wide personalized oncology aiming at identifying predictive biomarkers and novel actionable targets. Despite considerable progress in understanding the molecular biology of distinct cancer entities by the use of this revolutionary technology and despite contemporaneous innovations in drug development, translation of NGS findings into improved concepts for cancer treatment remains a challenge. The aim of this article is to describe shortly the NGS platforms for DNA sequencing and in more detail key achievements and unresolved hurdles. A special focus will be given on potential clinical applications of this innovative technique in the field of radiation oncology

  14. Screening for SNPs with Allele-Specific Methylation based on Next-Generation Sequencing Data.

    Science.gov (United States)

    Hu, Bo; Ji, Yuan; Xu, Yaomin; Ting, Angela H

    2013-05-01

    Allele-specific methylation (ASM) has long been studied but mainly documented in the context of genomic imprinting and X chromosome inactivation. Taking advantage of the next-generation sequencing technology, we conduct a high-throughput sequencing experiment with four prostate cell lines to survey the whole genome and identify single nucleotide polymorphisms (SNPs) with ASM. A Bayesian approach is proposed to model the counts of short reads for each SNP conditional on its genotypes of multiple subjects, leading to a posterior probability of ASM. We flag SNPs with high posterior probabilities of ASM by accounting for multiple comparisons based on posterior false discovery rates. Applying the Bayesian approach to the in-house prostate cell line data, we identify 269 SNPs as candidates of ASM. A simulation study is carried out to demonstrate the quantitative performance of the proposed approach.

  15. Analysis Of Segmental Duplications In The Pig Genome Based On Next-Generation Sequencing

    DEFF Research Database (Denmark)

    Fadista, João; Bendixen, Christian

    Segmental duplications are >1kb segments of duplicated DNA present in a genome with high sequence identity (>90%). They are associated with genomic rearrangements and provide a significant source of gene and genome evolution within mammalian genomes. Although segmental duplications have been...... extensively studied in other organisms, its analysis in pig has been hampered by the lack of a complete pig genome assembly. By measuring the depth of coverage of Illumina whole-genome shotgun sequencing reads of the Tabasco animal aligned to the latest pig genome assembly (Sus scrofa 10 – based also...... and their associated copy number alterations, focusing on the global organization of these segments and their possible functional significance in porcine phenotypes. This work provides insights into mammalian genome evolution and generates a valuable resource for porcine genomics research...

  16. Next-generation sequence detects ARAP3 as a novel oncogene in papillary thyroid carcinoma

    Directory of Open Access Journals (Sweden)

    Wang QX

    2016-11-01

    Full Text Available Qing-Xuan Wang, En-Dong Chen, Ye-Feng Cai, Yi-Li Zhou, Zhou-Ci Zheng, Ying-Hao Wang, Yi-Xiang Jin, Wen-Xu Jin, Xiao-Hua Zhang, Ou-Chen Wang Department of Oncology, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang Province, China Purpose: Thyroid cancer is the most frequent malignancies of the endocrine system, and it has became the fastest growing type of cancer worldwide. Much still remains unknown about the molecular mechanisms of thyroid cancer. Studies have found that some certain relationship between ARAP3 and human cancer. However, the role of ARAP3 in thyroid cancer has not been well explained. This study aimed to investigate the role of ARAP3 gene in papillary thyroid carcinoma. Methods: Whole exon sequence and whole genome sequence of primary papillary thyroid carcinoma (PTC samples and matched adjacent normal thyroid tissue samples were performed and then bioinformatics analysis was carried out. PTC cell lines (TPC1, BCPAP, and KTC-1 with transfection of small interfering RNA were used to investigate the functions of ARAP3 gene, including cell proliferation assay, colony formation assay, migration assay, and invasion assay. Results: Using next-generation sequence and bioinformatics analysis, we found ARAP3 genes may play an important role in thyroid cancer. Downregulation of ARAP3 significantly suppressed PTC cell lines (TPC1, BCPAP, and KTC-1, cell proliferation, colony formation, migration, and invasion. Conclusion: This study indicated that ARAP3 genes have important biological implications and may act as a potentially drugable target in PTC. Keywords: papillary thyroid carcinoma, next-generation sequence, ARAP3, oncogene

  17. Generation, analysis and functional annotation of expressed sequence tags from the ectoparasitic mite Psoroptes ovis

    Directory of Open Access Journals (Sweden)

    Kenyon Fiona

    2011-07-01

    Full Text Available Abstract Background Sheep scab is caused by Psoroptes ovis and is arguably the most important ectoparasitic disease affecting sheep in the UK. The disease is highly contagious and causes and considerable pruritis and irritation and is therefore a major welfare concern. Current methods of treatment are unsustainable and in order to elucidate novel methods of disease control a more comprehensive understanding of the parasite is required. To date, no full genomic DNA sequence or large scale transcript datasets are available and prior to this study only 484 P. ovis expressed sequence tags (ESTs were accessible in public databases. Results In order to further expand upon the transcriptomic coverage of P. ovis thus facilitating novel insights into the mite biology we undertook a larger scale EST approach, incorporating newly generated and previously described P. ovis transcript data and representing the largest collection of P. ovis ESTs to date. We sequenced 1,574 ESTs and assembled these along with 484 previously generated P. ovis ESTs, which resulted in the identification of 1,545 unique P. ovis sequences. BLASTX searches identified 961 ESTs with significant hits (E-value P. ovis ESTs. Gene Ontology (GO analysis allowed the functional annotation of 880 ESTs and included predictions of signal peptide and transmembrane domains; allowing the identification of potential P. ovis excreted/secreted factors, and mapping of metabolic pathways. Conclusions This dataset currently represents the largest collection of P. ovis ESTs, all of which are publicly available in the GenBank EST database (dbEST (accession numbers FR748230 - FR749648. Functional analysis of this dataset identified important homologues, including house dust mite allergens and tick salivary factors. These findings offer new insights into the underlying biology of P. ovis, facilitating further investigations into mite biology and the identification of novel methods of intervention.

  18. Short-term hydro generation scheduling of Xiluodu and Xiangjiaba cascade hydropower stations using improved binary-real coded bee colony optimization algorithm

    International Nuclear Information System (INIS)

    Lu, Peng; Zhou, Jianzhong; Wang, Chao; Qiao, Qi; Mo, Li

    2015-01-01

    Highlights: • STHGS problem is decomposed into two parallel sub-problems of UC and ELD. • Binary coded BCO is used to solve UC sub-problem with 0–1 discrete variables. • Real coded BCO is used to solve ELD sub-problem with continuous variables. • Some heuristic repairing strategies are designed to handle various constraints. • The STHGS of Xiluodu and Xiangjiaba cascade stations is solved by IB-RBCO. - Abstract: Short-term hydro generation scheduling (STHGS) of cascade hydropower stations is a typical nonlinear mixed integer optimization problem to minimize the total water consumption while simultaneously meeting the grid requirements and other hydraulic and electrical constraints. In this paper, STHGS problem is decomposed into two parallel sub-problems of unit commitment (UC) and economic load dispatch (ELD), and the methodology of improved binary-real coded bee colony optimization (IB-RBCO) algorithm is proposed to solve them. Firstly, the improved binary coded BCO is used to solve the UC sub-problem with 0–1 discrete variables, and the heuristic repairing strategy for unit state constrains is applied to generate the feasible unit commitment schedule. Then, the improved real coded BCO is used to solve the ELD sub-problem with continuous variables, and an effective method is introduced to handle various unit operation constraints. Especially, the new updating strategy of DE/best/2/bin method with dynamic parameter control mechanism is applied to real coded BCO to improve the search ability of IB-RBCO. Finally, to verify the feasibility and effectiveness of the proposed IB-RBCO method, it is applied to solve the STHGS problem of Xiluodu and Xiangjiaba cascaded hydropower stations, and the simulating results are compared with other intelligence algorithms. The simulation results demonstrate that the proposed IB-RBCO method can get higher-quality solutions with less water consumption and shorter calculating time when facing the complex STHGS problem

  19. BATCH-GE: Batch analysis of Next-Generation Sequencing data for genome editing assessment

    Science.gov (United States)

    Boel, Annekatrien; Steyaert, Woutert; De Rocker, Nina; Menten, Björn; Callewaert, Bert; De Paepe, Anne; Coucke, Paul; Willaert, Andy

    2016-01-01

    Targeted mutagenesis by the CRISPR/Cas9 system is currently revolutionizing genetics. The ease of this technique has enabled genome engineering in-vitro and in a range of model organisms and has pushed experimental dimensions to unprecedented proportions. Due to its tremendous progress in terms of speed, read length, throughput and cost, Next-Generation Sequencing (NGS) has been increasingly used for the analysis of CRISPR/Cas9 genome editing experiments. However, the current tools for genome editing assessment lack flexibility and fall short in the analysis of large amounts of NGS data. Therefore, we designed BATCH-GE, an easy-to-use bioinformatics tool for batch analysis of NGS-generated genome editing data, available from https://github.com/WouterSteyaert/BATCH-GE.git. BATCH-GE detects and reports indel mutations and other precise genome editing events and calculates the corresponding mutagenesis efficiencies for a large number of samples in parallel. Furthermore, this new tool provides flexibility by allowing the user to adapt a number of input variables. The performance of BATCH-GE was evaluated in two genome editing experiments, aiming to generate knock-out and knock-in zebrafish mutants. This tool will not only contribute to the evaluation of CRISPR/Cas9-based experiments, but will be of use in any genome editing experiment and has the ability to analyze data from every organism with a sequenced genome. PMID:27461955

  20. Characterization of the cutaneous mycobiota in healthy and allergic cats using next generation sequencing.

    Science.gov (United States)

    Meason-Smith, Courtney; Diesel, Alison; Patterson, Adam P; Older, Caitlin E; Johnson, Timothy J; Mansell, Joanne M; Suchodolski, Jan S; Rodrigues Hoffmann, Aline

    2017-02-01

    Next generation sequencing (NGS) studies have demonstrated a diverse skin-associated microbiota and microbial dysbiosis associated with atopic dermatitis in people and in dogs. The skin of cats has yet to be investigated using NGS techniques. We hypothesized that the fungal microbiota of healthy feline skin would be similar to that of dogs, with a predominance of environmental fungi, and that fungal dysbiosis would be present on the skin of allergic cats. Eleven healthy cats and nine cats diagnosed with one or more cutaneous hypersensitivity disorders, including flea bite, food-induced and nonflea nonfood-induced hypersensitivity. Healthy cats were sampled at twelve body sites and allergic cats at six sites. DNA was isolated and Illumina sequencing was performed targeting the internal transcribed spacer region of fungi. Sequences were processed using the bioinformatics software QIIME. The most abundant fungal sequences from the skin of all cats were classified as Cladosporium and Alternaria. The mucosal sites, including nostril, conjunctiva and reproductive tracts, had the fewest number of fungi, whereas the pre-aural space had the most. Allergic feline skin had significantly greater amounts of Agaricomycetes and Sordariomycetes, and significantly less Epicoccum compared to healthy feline skin. The skin of healthy cats appears to have a more diverse fungal microbiota compared to previous studies, and a fungal dysbiosis is noted in the skin of allergic cats. Future studies assessing the temporal stability of the skin microbiota in cats will be useful in determining whether the microbiota sequenced using NGS are colonizers or transient microbes. © 2016 ESVD and ACVD.

  1. Next-Generation Sequencing Analysis and Algorithms for PDX and CDX Models.

    Science.gov (United States)

    Khandelwal, Garima; Girotti, María Romina; Smowton, Christopher; Taylor, Sam; Wirth, Christopher; Dynowski, Marek; Frese, Kristopher K; Brady, Ged; Dive, Caroline; Marais, Richard; Miller, Crispin

    2017-08-01

    Patient-derived xenograft (PDX) and circulating tumor cell-derived explant (CDX) models are powerful methods for the study of human disease. In cancer research, these methods have been applied to multiple questions, including the study of metastatic progression, genetic evolution, and therapeutic drug responses. As PDX and CDX models can recapitulate the highly heterogeneous characteristics of a patient tumor, as well as their response to chemotherapy, there is considerable interest in combining them with next-generation sequencing to monitor the genomic, transcriptional, and epigenetic changes that accompany oncogenesis. When used for this purpose, their reliability is highly dependent on being able to accurately distinguish between sequencing reads that originate from the host, and those that arise from the xenograft itself. Here, we demonstrate that failure to correctly identify contaminating host reads when analyzing DNA- and RNA-sequencing (DNA-Seq and RNA-Seq) data from PDX and CDX models is a major confounding factor that can lead to incorrect mutation calls and a failure to identify canonical mutation signatures associated with tumorigenicity. In addition, a highly sensitive algorithm and open source software tool for identifying and removing contaminating host sequences is described. Importantly, when applied to PDX and CDX models of melanoma, these data demonstrate its utility as a sensitive and selective tool for the correction of PDX- and CDX-derived whole-exome and RNA-Seq data. Implications: This study describes a sensitive method to identify contaminating host reads in xenograft and explant DNA- and RNA-Seq data and is applicable to other forms of deep sequencing. Mol Cancer Res; 15(8); 1012-6. ©2017 AACR . ©2017 American Association for Cancer Research.

  2. Graph mining for next generation sequencing: leveraging the assembly graph for biological insights.

    Science.gov (United States)

    Warnke-Sommer, Julia; Ali, Hesham

    2016-05-06

    The assembly of Next Generation Sequencing (NGS) reads remains a challenging task. This is especially true for the assembly of metagenomics data that originate from environmental samples potentially containing hundreds to thousands of unique species. The principle objective of current assembly tools is to assemble NGS reads into contiguous stretches of sequence called contigs while maximizing for both accuracy and contig length. The end goal of this process is to produce longer contigs with the major focus being on assembly only. Sequence read assembly is an aggregative process, during which read overlap relationship information is lost as reads are merged into longer sequences or contigs. The assembly graph is information rich and capable of capturing the genomic architecture of an input read data set. We have developed a novel hybrid graph in which nodes represent sequence regions at different levels of granularity. This model, utilized in the assembly and analysis pipeline Focus, presents a concise yet feature rich view of a given input data set, allowing for the extraction of biologically relevant graph structures for graph mining purposes. Focus was used to create hybrid graphs to model metagenomics data sets obtained from the gut microbiomes of five individuals with Crohn's disease and eight healthy individuals. Repetitive and mobile genetic elements are found to be associated with hybrid graph structure. Using graph mining techniques, a comparative study of the Crohn's disease and healthy data sets was conducted with focus on antibiotics resistance genes associated with transposase genes. Results demonstrated significant differences in the phylogenetic distribution of categories of antibiotics resistance genes in the healthy and diseased patients. Focus was also evaluated as a pure assembly tool and produced excellent results when compared against the Meta-velvet, Omega, and UD-IDBA assemblers. Mining the hybrid graph can reveal biological phenomena captured

  3. A Review of Study Designs and Statistical Methods for Genomic Epidemiology Studies using Next Generation Sequencing

    Directory of Open Access Journals (Sweden)

    Qian eWang

    2015-04-01

    Full Text Available Results from numerous linkage and association studies have greatly deepened scientists’ understanding of the genetic basis of many human diseases, yet some important questions remain unanswered. For example, although a large number of disease-associated loci have been identified from genome-wide association studies (GWAS in the past 10 years, it is challenging to interpret these results as most disease-associated markers have no clear functional roles in disease etiology, and all the identified genomic factors only explain a small portion of disease heritability. With the help of next-generation sequencing (NGS, diverse types of genomic and epigenetic variations can be detected with high accuracy. More importantly, instead of using linkage disequilibrium to detect association signals based on a set of pre-set probes, NGS allows researchers to directly study all the variants in each individual, therefore promises opportunities for identifying functional variants and a more comprehensive dissection of disease heritability. Although the current scale of NGS studies is still limited due to the high cost, the success of several recent studies suggests the great potential for applying NGS in genomic epidemiology, especially as the cost of sequencing continues to drop. In this review, we discuss several pioneer applications of NGS, summarize scientific discoveries for rare and complex diseases, and compare various study designs including targeted sequencing and whole-genome sequencing using population-based and family-based cohorts. Finally, we highlight recent advancements in statistical methods proposed for sequencing analysis, including group-based association tests, meta-analysis techniques, and annotation tools for variant prioritization.

  4. Genomic Selection in the Era of Next Generation Sequencing for Complex Traits in Plant Breeding.

    Science.gov (United States)

    Bhat, Javaid A; Ali, Sajad; Salgotra, Romesh K; Mir, Zahoor A; Dutta, Sutapa; Jadon, Vasudha; Tyagi, Anshika; Mushtaq, Muntazir; Jain, Neelu; Singh, Pradeep K; Singh, Gyanendra P; Prabhu, K V

    2016-01-01

    Genomic selection (GS) is a promising approach exploiting molecular genetic markers to design novel breeding programs and to develop new markers-based models for genetic evaluation. In plant breeding, it provides opportunities to increase genetic gain of complex traits per unit time and cost. The cost-benefit balance was an important consideration for GS to work in crop plants. Availability of genome-wide high-throughput, cost-effective and flexible markers, having low ascertainment bias, suitable for large population size as well for both model and non-model crop species with or without the reference genome sequence was the most important factor for its successful and effective implementation in crop species. These factors were the major limitations to earlier marker systems viz., SSR and array-based, and was unimaginable before the availability of next-generation sequencing (NGS) technologies which have provided novel SNP genotyping platforms especially the genotyping by sequencing. These marker technologies have changed the entire scenario of marker applications and made the use of GS a routine work for crop improvement in both model and non-model crop species. The NGS-based genotyping have increased genomic-estimated breeding value prediction accuracies over other established marker platform in cereals and other crop species, and made the dream of GS true in crop breeding. But to harness the true benefits from GS, these marker technologies will be combined with high-throughput phenotyping for achieving the valuable genetic gain from complex traits. Moreover, the continuous decline in sequencing cost will make the WGS feasible and cost effective for GS in near future. Till that time matures the targeted sequencing seems to be more cost-effective option for large scale marker discovery and GS, particularly in case of large and un-decoded genomes.

  5. Third-Generation Sequencing and Analysis of Four Complete Pig Liver Esterase Gene Sequences in Clones Identified by Screening BAC Library.

    Science.gov (United States)

    Zhou, Qiongqiong; Sun, Wenjuan; Liu, Xiyan; Wang, Xiliang; Xiao, Yuncai; Bi, Dingren; Yin, Jingdong; Shi, Deshi

    2016-01-01

    Pig liver carboxylesterase (PLE) gene sequences in GenBank are incomplete, which has led to difficulties in studying the genetic structure and regulation mechanisms of gene expression of PLE family genes. The aim of this study was to obtain and analysis of complete gene sequences of PLE family by screening from a Rongchang pig BAC library and third-generation PacBio gene sequencing. After a number of existing incomplete PLE isoform gene sequences were analysed, primers were designed based on conserved regions in PLE exons, and the whole pig genome used as a template for Polymerase chain reaction (PCR) amplification. Specific primers were then selected based on the PCR amplification results. A three-step PCR screening method was used to identify PLE-positive clones by screening a Rongchang pig BAC library and PacBio third-generation sequencing was performed. BLAST comparisons and other bioinformatics methods were applied for sequence analysis. Five PLE-positive BAC clones, designated BAC-10, BAC-70, BAC-75, BAC-119 and BAC-206, were identified. Sequence analysis yielded the complete sequences of four PLE genes, PLE1, PLE-B9, PLE-C4, and PLE-G2. Complete PLE gene sequences were defined as those containing regulatory sequences, exons, and introns. It was found that, not only did the PLE exon sequences of the four genes show a high degree of homology, but also that the intron sequences were highly similar. Additionally, the regulatory region of the genes contained two 720bps reverse complement sequences that may have an important function in the regulation of PLE gene expression. This is the first report to confirm the complete sequences of four PLE genes. In addition, the study demonstrates that each PLE isoform is encoded by a single gene and that the various genes exhibit a high degree of sequence homology, suggesting that the PLE family evolved from a single ancestral gene. Obtaining the complete sequences of these PLE genes provides the necessary foundation for

  6. Cloning and Identification of Recombinant Argonaute-Bound Small RNAs Using Next-Generation Sequencing.

    Science.gov (United States)

    Gangras, Pooja; Dayeh, Daniel M; Mabin, Justin W; Nakanishi, Kotaro; Singh, Guramrit

    2018-01-01

    Argonaute proteins (AGOs) are loaded with small RNAs as guides to recognize target mRNAs. Since the target specificity heavily depends on the base complementarity between two strands, it is important to identify small guide and long target RNAs bound to AGOs. For this purpose, next-generation sequencing (NGS) technologies have extended our appreciation truly to the nucleotide level. However, the identification of RNAs via NGS from scarce RNA samples remains a challenge. Further, most commercial and published methods are compatible with either small RNAs or long RNAs, but are not equally applicable to both. Therefore, a single method that yields quantitative, bias-free NGS libraries to identify small and long RNAs from low levels of input will be of wide interest. Here, we introduce such a procedure that is based on several modifications of two published protocols and allows robust, sensitive, and reproducible cloning and sequencing of small amounts of RNAs of variable lengths. The method was applied to the identification of small RNAs bound to a purified eukaryotic AGO. Following ligation of a DNA adapter to RNA 3'-end, the key feature of this method is to use the adapter for priming reverse transcription (RT) wherein biotinylated deoxyribonucleotides specifically incorporated into the extended complementary DNA. Such RT products are enriched on streptavidin beads, circularized while immobilized on beads and directly used for PCR amplification. We provide a stepwise guide to generate RNA-Seq libraries, their purification, quantification, validation, and preparation for next-generation sequencing. We also provide basic steps in post-NGS data analyses using Galaxy, an open-source, web-based platform.

  7. Pseudo-Random Sequences Generated by a Class of One-Dimensional Smooth Map

    Science.gov (United States)

    Wang, Xing-Yuan; Qin, Xue; Xie, Yi-Xin

    2011-08-01

    We extend a class of a one-dimensional smooth map. We make sure that for each desired interval of the parameter the map's Lyapunov exponent is positive. Then we propose a novel parameter perturbation method based on the good property of the extended one-dimensional smooth map. We perturb the parameter r in each iteration by the real number xi generated by the iteration. The auto-correlation function and NIST statistical test suite are taken to illustrate the method's randomness finally. We provide an application of this method in image encryption. Experiments show that the pseudo-random sequences are suitable for this application.

  8. Pseudo-Random Sequences Generated by a Class of One-Dimensional Smooth Map

    International Nuclear Information System (INIS)

    Wang Xing-Yuan; Qin Xue; Xie Yi-Xin

    2011-01-01

    We extend a class of a one-dimensional smooth map. We make sure that for each desired interval of the parameter the map's Lyapunov exponent is positive. Then we propose a novel parameter perturbation method based on the good property of the extended one-dimensional smooth map. We perturb the parameter r in each iteration by the real number x i generated by the iteration. The auto-correlation function and NIST statistical test suite are taken to illustrate the method's randomness finally. We provide an application of this method in image encryption. Experiments show that the pseudo-random sequences are suitable for this application. (general)

  9. Analysis of breast cancer metastasis candidate genes from next generation-sequencing via systematic functional genomics

    DEFF Research Database (Denmark)

    Blomstrøm, Monica Marie

    2016-01-01

    several growth modulators and invasion modulators were identified and independently validated. These candidates revealed a group of genes with metastasis-related functions in vitro that are involved in RNA-related processes, such as RNA-processing. Moreover, a general feature was that proliferation......) and non-CSCs. The main goal of this project was to functionally characterize a set of candidate genes recovered from next-generation sequencing analysis for their role in breast cancer metastasis formation. The starting gene set comprised 104 gene variants; i.e. 57 wildtype and 47 mutated variants. During...

  10. Next-generation DNA sequencing of HEXA: a step in the right direction for carrier screening

    OpenAIRE

    Hoffman, Jodi D; Greger, Valerie; Strovel, Erin T; Blitzer, Miriam G; Umbarger, Mark A; Kennedy, Caleb; Bishop, Brian; Saunders, Patrick; Porreca, Gregory J; Schienda, Jaclyn; Davie, Jocelyn; Hallam, Stephanie; Towne, Charles

    2013-01-01

    Tay-Sachs disease (TSD) is the prototype for ethnic-based carrier screening, with a carrier rate of ∼1/27 in Ashkenazi Jews and French Canadians. HexA enzyme analysis is the current gold standard for TSD carrier screening (detection rate ∼98%), but has technical limitations. We compared DNA analysis by next-generation DNA sequencing (NGS) plus an assay for the 7.6 kb deletion to enzyme analysis for TSD carrier screening using 74 samples collected from participants at a TSD family conference. ...

  11. Using next generation sequencing to tackle non-typhoidal Salmonella infections

    DEFF Research Database (Denmark)

    Wain, John; Keddy, Karen H.; Hendriksen, Rene S.

    2013-01-01

    The publication of studies using next generation sequencing to analyse large numbers of bacterial isolates from global epidemics is transforming microbiology, epidemiology and public health. The emergence of multidrug resistant Salmonella Typhimurium ST313 is one example. While the epidemiology...... in Africa appears to be human-to-human spread and the association with invasive disease almost absolute, more needs to be done to exclude the possibility of animal reservoirs and to transfer the ability to track all Salmonella infections to the laboratories in the front line. In this mini-review we...

  12. Computational Approach to Annotating Variants of Unknown Significance in Clinical Next Generation Sequencing.

    Science.gov (United States)

    Schulz, Wade L; Tormey, Christopher A; Torres, Richard

    2015-01-01

    Next generation sequencing (NGS) has become a common technology in the clinical laboratory, particularly for the analysis of malignant neoplasms. However, most mutations identified by NGS are variants of unknown clinical significance (VOUS). Although the approach to define these variants differs by institution, software algorithms that predict variant effect on protein function may be used. However, these algorithms commonly generate conflicting results, potentially adding uncertainty to interpretation. In this review, we examine several computational tools used to predict whether a variant has clinical significance. In addition to describing the role of these tools in clinical diagnostics, we assess their efficacy in analyzing known pathogenic and benign variants in hematologic malignancies. Copyright© by the American Society for Clinical Pathology (ASCP).

  13. Viral Bacterial Artificial Chromosomes: Generation, Mutagenesis, and Removal of Mini-F Sequences

    Directory of Open Access Journals (Sweden)

    B. Karsten Tischer

    2012-01-01

    Full Text Available Maintenance and manipulation of large DNA and RNA virus genomes had presented an obstacle for virological research. BAC vectors provided a solution to both problems as they can harbor large DNA sequences and can efficiently be modified using well-established mutagenesis techniques in Escherichia coli. Numerous DNA virus genomes of herpesvirus and pox virus were cloned into mini-F vectors. In addition, several reverse genetic systems for RNA viruses such as members of Coronaviridae and Flaviviridae could be established based on BAC constructs. Transfection into susceptible eukaryotic cells of virus DNA cloned as a BAC allows reconstitution of recombinant viruses. In this paper, we provide an overview on the strategies that can be used for the generation of virus BAC vectors and also on systems that are currently available for various virus species. Furthermore, we address common mutagenesis techniques that allow modification of BACs from single-nucleotide substitutions to deletion of viral genes or insertion of foreign sequences. Finally, we review the reconstitution of viruses from BAC vectors and the removal of the bacterial sequences from the virus genome during this process.

  14. Quality control of next-generation sequencing library through an integrative digital microfluidic platform.

    Science.gov (United States)

    Thaitrong, Numrin; Kim, Hanyoup; Renzi, Ronald F; Bartsch, Michael S; Meagher, Robert J; Patel, Kamlesh D

    2012-12-01

    We have developed an automated quality control (QC) platform for next-generation sequencing (NGS) library characterization by integrating a droplet-based digital microfluidic (DMF) system with a capillary-based reagent delivery unit and a quantitative CE module. Using an in-plane capillary-DMF interface, a prepared sample droplet was actuated into position between the ground electrode and the inlet of the separation capillary to complete the circuit for an electrokinetic injection. Using a DNA ladder as an internal standard, the CE module with a compact LIF detector was capable of detecting dsDNA in the range of 5-100 pg/μL, suitable for the amount of DNA required by the Illumina Genome Analyzer sequencing platform. This DMF-CE platform consumes tenfold less sample volume than the current Agilent BioAnalyzer QC technique, preserving precious sample while providing necessary sensitivity and accuracy for optimal sequencing performance. The ability of this microfluidic system to validate NGS library preparation was demonstrated by examining the effects of limited-cycle PCR amplification on the size distribution and the yield of Illumina-compatible libraries, demonstrating that as few as ten cycles of PCR bias the size distribution of the library toward undesirable larger fragments. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. A new feedback image encryption scheme based on perturbation with dynamical compound chaotic sequence cipher generator

    Science.gov (United States)

    Tong, Xiaojun; Cui, Minggen; Wang, Zhu

    2009-07-01

    The design of the new compound two-dimensional chaotic function is presented by exploiting two one-dimensional chaotic functions which switch randomly, and the design is used as a chaotic sequence generator which is proved by Devaney's definition proof of chaos. The properties of compound chaotic functions are also proved rigorously. In order to improve the robustness against difference cryptanalysis and produce avalanche effect, a new feedback image encryption scheme is proposed using the new compound chaos by selecting one of the two one-dimensional chaotic functions randomly and a new image pixels method of permutation and substitution is designed in detail by array row and column random controlling based on the compound chaos. The results from entropy analysis, difference analysis, statistical analysis, sequence randomness analysis, cipher sensitivity analysis depending on key and plaintext have proven that the compound chaotic sequence cipher can resist cryptanalytic, statistical and brute-force attacks, and especially it accelerates encryption speed, and achieves higher level of security. By the dynamical compound chaos and perturbation technology, the paper solves the problem of computer low precision of one-dimensional chaotic function.

  16. Clinical pharmacogenomics testing in the era of next generation sequencing: challenges and opportunities for precision medicine.

    Science.gov (United States)

    Ji, Yuan; Si, Yue; McMillin, Gwendolyn A; Lyon, Elaine

    2018-04-23

    The rapid development and dramatic decrease in cost of sequencing techniques have ushered the implementation of genomic testing in patient care. Next generation DNA sequencing (NGS) techniques have been used increasingly in clinical laboratories to scan the whole or part of the human genome in order to facilitate diagnosis and/or prognostics of genetic disease. Despite many hurdles and debates, pharmacogenomics (PGx) is believed to be an area of genomic medicine where precision medicine could have immediate impact in the near future. Areas covered: This review focuses on lessons learned through early attempts of clinically implementing PGx testing; the challenges and opportunities that PGx testing brings to precision medicine in the era of NGS. Expert commentary: Replacing targeted analysis approach with NGS for PGx testing is neither technically feasible nor necessary currently due to several technical limitations and uncertainty involved in interpreting variants of uncertain significance for PGx variants. However, reporting PGx variants out of clinical whole exome or whole genome sequencing (WES/WGS) might represent additional benefits for patients who are tested by WES/WGS.

  17. Reprint of "Application of next generation sequencing in clinical microbiology and infection prevention".

    Science.gov (United States)

    Deurenberg, Ruud H; Bathoorn, Erik; Chlebowicz, Monika A; Couto, Natacha; Ferdous, Mithila; García-Cobos, Silvia; Kooistra-Smid, Anna M D; Raangs, Erwin C; Rosema, Sigrid; Veloo, Alida C M; Zhou, Kai; Friedrich, Alexander W; Rossen, John W A

    2017-05-20

    Current molecular diagnostics of human pathogens provide limited information that is often not sufficient for outbreak and transmission investigation. Next generation sequencing (NGS) determines the DNA sequence of a complete bacterial genome in a single sequence run, and from these data, information on resistance and virulence, as well as information for typing is obtained, useful for outbreak investigation. The obtained genome data can be further used for the development of an outbreak-specific screening test. In this review, a general introduction to NGS is presented, including the library preparation and the major characteristics of the most common NGS platforms, such as the MiSeq (Illumina) and the Ion PGM™ (ThermoFisher). An overview of the software used for NGS data analyses used at the medical microbiology diagnostic laboratory in the University Medical Center Groningen in The Netherlands is given. Furthermore, applications of NGS in the clinical setting are described, such as outbreak management, molecular case finding, characterization and surveillance of pathogens, rapid identification of bacteria using the 16S-23S rRNA region, taxonomy, metagenomics approaches on clinical samples, and the determination of the transmission of zoonotic micro-organisms from animals to humans. Finally, we share our vision on the use of NGS in personalised microbiology in the near future, pointing out specific requirements. Copyright © 2017. Published by Elsevier B.V.

  18. A practical comparison of de novo genome assembly software tools for next-generation sequencing technologies.

    Directory of Open Access Journals (Sweden)

    Wenyu Zhang

    Full Text Available The advent of next-generation sequencing technologies is accompanied with the development of many whole-genome sequence assembly methods and software, especially for de novo fragment assembly. Due to the poor knowledge about the applicability and performance of these software tools, choosing a befitting assembler becomes a tough task. Here, we provide the information of adaptivity for each program, then above all, compare the performance of eight distinct tools against eight groups of simulated datasets from Solexa sequencing platform. Considering the computational time, maximum random access memory (RAM occupancy, assembly accuracy and integrity, our study indicate that string-based assemblers, overlap-layout-consensus (OLC assemblers are well-suited for very short reads and longer reads of small genomes respectively. For large datasets of more than hundred millions of short reads, De Bruijn graph-based assemblers would be more appropriate. In terms of software implementation, string-based assemblers are superior to graph-based ones, of which SOAPdenovo is complex for the creation of configuration file. Our comparison study will assist researchers in selecting a well-suited assembler and offer essential information for the improvement of existing assemblers or the developing of novel assemblers.

  19. Human Y chromosome copy number variation in the next generation sequencing era and beyond.

    Science.gov (United States)

    Massaia, Andrea; Xue, Yali

    2017-05-01

    The human Y chromosome provides a fertile ground for structural rearrangements owing to its haploidy and high content of repeated sequences. The methodologies used for copy number variation (CNV) studies have developed over the years. Low-throughput techniques based on direct observation of rearrangements were developed early on, and are still used, often to complement array-based or sequencing approaches which have limited power in regions with high repeat content and specifically in the presence of long, identical repeats, such as those found in human sex chromosomes. Some specific rearrangements have been investigated for decades; because of their effects on fertility, or their outstanding evolutionary features, the interest in these has not diminished. However, following the flourishing of large-scale genomics, several studies have investigated CNVs across the whole chromosome. These studies sometimes employ data generated within large genomic projects such as the DDD study or the 1000 Genomes Project, and often survey large samples of healthy individuals without any prior selection. Novel technologies based on sequencing long molecules and combinations of technologies, promise to stimulate the study of Y-CNVs in the immediate future.

  20. Assessment of genetic variation for the LINE-1 retrotransposon from next generation sequence data

    Directory of Open Access Journals (Sweden)

    Ramos Kenneth

    2010-10-01

    Full Text Available Abstract Background In humans, copies of the Long Interspersed Nuclear Element 1 (LINE-1 retrotransposon comprise 21% of the reference genome, and have been shown to modulate expression and produce novel splice isoforms of transcripts from genes that span or neighbor the LINE-1 insertion site. Results In this work, newly released pilot data from the 1000 Genomes Project is analyzed to detect previously unreported full length insertions of the retrotransposon LINE-1. By direct analysis of the sequence data, we have identified 22 previously unreported LINE-1 insertion sites within the sequence data reported for a mother/father/daughter trio. Conclusions It is demonstrated here that next generation sequencing data, as well as emerging high quality datasets from individual genome projects allow us to assess the amount of heterogeneity with respect to the LINE-1 retrotransposon amongst humans, and provide us with a wealth of testable hypotheses as to the impact that this diversity may have on the health of individuals and populations.

  1. Application of next generation sequencing in clinical microbiology and infection prevention.

    Science.gov (United States)

    Deurenberg, Ruud H; Bathoorn, Erik; Chlebowicz, Monika A; Couto, Natacha; Ferdous, Mithila; García-Cobos, Silvia; Kooistra-Smid, Anna M D; Raangs, Erwin C; Rosema, Sigrid; Veloo, Alida C M; Zhou, Kai; Friedrich, Alexander W; Rossen, John W A

    2017-02-10

    Current molecular diagnostics of human pathogens provide limited information that is often not sufficient for outbreak and transmission investigation. Next generation sequencing (NGS) determines the DNA sequence of a complete bacterial genome in a single sequence run, and from these data, information on resistance and virulence, as well as information for typing is obtained, useful for outbreak investigation. The obtained genome data can be further used for the development of an outbreak-specific screening test. In this review, a general introduction to NGS is presented, including the library preparation and the major characteristics of the most common NGS platforms, such as the MiSeq (Illumina) and the Ion PGM™ (ThermoFisher). An overview of the software used for NGS data analyses used at the medical microbiology diagnostic laboratory in the University Medical Center Groningen in The Netherlands is given. Furthermore, applications of NGS in the clinical setting are described, such as outbreak management, molecular case finding, characterization and surveillance of pathogens, rapid identification of bacteria using the 16S-23S rRNA region, taxonomy, metagenomics approaches on clinical samples, and the determination of the transmission of zoonotic micro-organisms from animals to humans. Finally, we share our vision on the use of NGS in personalised microbiology in the near future, pointing out specific requirements. Copyright © 2016 The Author(s). Published by Elsevier B.V. All rights reserved.

  2. Next generation sequencing and molecular analysis of artichoke Italian latent virus.

    Science.gov (United States)

    Elbeaino, Toufic; Belghacem, Imen; Mascia, Tiziana; Gallitelli, Donato; Digiaro, Michele

    2017-06-01

    Next-generation sequencing (NGS) allowed the assembly of the complete RNA-1 and RNA-2 sequences of a grapevine isolate of artichoke Italian latent virus (AILV). RNA-1 and RNA-2 are 7,338 and 4,630 nucleotides in length excluding the 3' terminal poly(A) tail, and encode two putative polyproteins of 255.8 kDa (p1) and 149.6 kDa (p2), respectively. All conserved motifs and predicted cleavage sites, typical for nepovirus polyproteins, were found in p1 and p2. AILV p1 and p2 share high amino acid identity with their homologues in beet ringspot virus (p1, 81% and p2, 71%), tomato black ring virus (p1, 79% and p2, 63%), grapevine Anatolian ringspot virus (p1, 65% and p2, 63%), and grapevine chrome mosaic virus (p1, 60% and p2, 54%), and to a lesser extent with other grapevine nepoviruses of subgroup A and C. Phylogenetic and sequence analyses, all confirmed the strict relationship of AILV with members classified in subgroup B of genus Nepovirus.

  3. Identification of parasitic communities within European ticks using next-generation sequencing.

    Directory of Open Access Journals (Sweden)

    Sarah Bonnet

    2014-03-01

    Full Text Available Risk assessment of tick-borne and zoonotic disease emergence necessitates sound knowledge of the particular microorganisms circulating within the communities of these major vectors. Assessment of pathogens carried by wild ticks must be performed without a priori, to allow for the detection of new or unexpected agents.We evaluated the potential of Next-Generation Sequencing techniques (NGS to produce an inventory of parasites carried by questing ticks. Sequences corresponding to parasites from two distinct genera were recovered in Ixodes ricinus ticks collected in Eastern France: Babesia spp. and Theileria spp. Four Babesia species were identified, three of which were zoonotic: B. divergens, Babesia sp. EU1 and B. microti; and one which infects cattle, B. major. This is the first time that these last two species have been identified in France. This approach also identified new sequences corresponding to as-yet unknown organisms similar to tropical Theileria species.Our findings demonstrate the capability of NGS to produce an inventory of live tick-borne parasites, which could potentially be transmitted by the ticks, and uncovers unexpected parasites in Western Europe.

  4. Cloud-based bioinformatics workflow platform for large-scale next-generation sequencing analyses.

    Science.gov (United States)

    Liu, Bo; Madduri, Ravi K; Sotomayor, Borja; Chard, Kyle; Lacinski, Lukasz; Dave, Utpal J; Li, Jianqiang; Liu, Chunchen; Foster, Ian T

    2014-06-01

    Due to the upcoming data deluge of genome data, the need for storing and processing large-scale genome data, easy access to biomedical analyses tools, efficient data sharing and retrieval has presented significant challenges. The variability in data volume results in variable computing and storage requirements, therefore biomedical researchers are pursuing more reliable, dynamic and convenient methods for conducting sequencing analyses. This paper proposes a Cloud-based bioinformatics workflow platform for large-scale next-generation sequencing analyses, which enables reliable and highly scalable execution of sequencing analyses workflows in a fully automated manner. Our platform extends the existing Galaxy workflow system by adding data management capabilities for transferring large quantities of data efficiently and reliably (via Globus Transfer), domain-specific analyses tools preconfigured for immediate use by researchers (via user-specific tools integration), automatic deployment on Cloud for on-demand resource allocation and pay-as-you-go pricing (via Globus Provision), a Cloud provisioning tool for auto-scaling (via HTCondor scheduler), and the support for validating the correctness of workflows (via semantic verification tools). Two bioinformatics workflow use cases as well as performance evaluation are presented to validate the feasibility of the proposed approach. Copyright © 2014 Elsevier Inc. All rights reserved.

  5. Mutation Detection in Patients with Retinal Dystrophies Using Targeted Next Generation Sequencing.

    Directory of Open Access Journals (Sweden)

    Nicole Weisschuh

    Full Text Available Retinal dystrophies (RD constitute a group of blinding diseases that are characterized by clinical variability and pronounced genetic heterogeneity. The different nonsyndromic and syndromic forms of RD can be attributed to mutations in more than 200 genes. Consequently, next generation sequencing (NGS technologies are among the most promising approaches to identify mutations in RD. We screened a large cohort of patients comprising 89 independent cases and families with various subforms of RD applying different NGS platforms. While mutation screening in 50 cases was performed using a RD gene capture panel, 47 cases were analyzed using whole exome sequencing. One family was analyzed using whole genome sequencing. A detection rate of 61% was achieved including mutations in 34 known and two novel RD genes. A total of 69 distinct mutations were identified, including 39 novel mutations. Notably, genetic findings in several families were not consistent with the initial clinical diagnosis. Clinical reassessment resulted in refinement of the clinical diagnosis in some of these families and confirmed the broad clinical spectrum associated with mutations in RD genes.

  6. Interacting binaries

    CERN Document Server

    Shore, S N; van den Heuvel, EPJ

    1994-01-01

    This volume contains lecture notes presented at the 22nd Advanced Course of the Swiss Society for Astrophysics and Astronomy. The contributors deal with symbiotic stars, cataclysmic variables, massive binaries and X-ray binaries, in an attempt to provide a better understanding of stellar evolution.

  7. Generation of an infectious clone of a new Korean isolate of apple chlorotic leaf spot virus (ACLSV) driven by dual 35S and T7 promoters in a versatile binary vector

    Science.gov (United States)

    The full-length sequence of a new isolate of Apple chlorotic leaf spot virus (ACLSV) from Korea was divergent, but most closely related to the Japanese isolate A4, at 84% nucleotide identity. The full-length cDNA of the Korean isolate of ACLSV was cloned into a binary vector downstream of the bacter...

  8. 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/.

  9. Enhancing Next-Generation Sequencing-Guided Cancer Care Through Cognitive Computing.

    Science.gov (United States)

    Patel, Nirali M; Michelini, Vanessa V; Snell, Jeff M; Balu, Saianand; Hoyle, Alan P; Parker, Joel S; Hayward, Michele C; Eberhard, David A; Salazar, Ashley H; McNeillie, Patrick; Xu, Jia; Huettner, Claudia S; Koyama, Takahiko; Utro, Filippo; Rhrissorrakrai, Kahn; Norel, Raquel; Bilal, Erhan; Royyuru, Ajay; Parida, Laxmi; Earp, H Shelton; Grilley-Olson, Juneko E; Hayes, D Neil; Harvey, Stephen J; Sharpless, Norman E; Kim, William Y

    2018-02-01

    Using next-generation sequencing (NGS) to guide cancer therapy has created challenges in analyzing and reporting large volumes of genomic data to patients and caregivers. Specifically, providing current, accurate information on newly approved therapies and open clinical trials requires considerable manual curation performed mainly by human "molecular tumor boards" (MTBs). The purpose of this study was to determine the utility of cognitive computing as performed by Watson for Genomics (WfG) compared with a human MTB. One thousand eighteen patient cases that previously underwent targeted exon sequencing at the University of North Carolina (UNC) and subsequent analysis by the UNCseq informatics pipeline and the UNC MTB between November 7, 2011, and May 12, 2015, were analyzed with WfG, a cognitive computing technology for genomic analysis. Using a WfG-curated actionable gene list, we identified additional genomic events of potential significance (not discovered by traditional MTB curation) in 323 (32%) patients. The majority of these additional genomic events were considered actionable based upon their ability to qualify patients for biomarker-selected clinical trials. Indeed, the opening of a relevant clinical trial within 1 month prior to WfG analysis provided the rationale for identification of a new actionable event in nearly a quarter of the 323 patients. This automated analysis took potentially improve patient care by providing a rapid, comprehensive approach for data analysis and consideration of up-to-date availability of clinical trials. The results of this study demonstrate that the interpretation and actionability of somatic next-generation sequencing results are evolving too rapidly to rely solely on human curation. Molecular tumor boards empowered by cognitive computing can significantly improve patient care by providing a fast, cost-effective, and comprehensive approach for data analysis in the delivery of precision medicine. Patients and physicians who

  10. Accurate estimation of short read mapping quality for next-generation genome sequencing

    Science.gov (United States)

    Ruffalo, Matthew; Koyutürk, Mehmet; Ray, Soumya; LaFramboise, Thomas

    2012-01-01

    Motivation: Several software tools specialize in the alignment of short next-generation sequencing reads to a reference sequence. Some of these tools report a mapping quality score for each alignment—in principle, this quality score tells researchers the likelihood that the alignment is correct. However, the reported mapping quality often correlates weakly with actual accuracy and the qualities of many mappings are underestimated, encouraging the researchers to discard correct mappings. Further, these low-quality mappings tend to correlate with variations in the genome (both single nucleotide and structural), and such mappings are important in accurately identifying genomic variants. Approach: We develop a machine learning tool, LoQuM (LOgistic regression tool for calibrating the Quality of short read mappings, to assign reliable mapping quality scores to mappings of Illumina reads returned by any alignment tool. LoQuM uses statistics on the read (base quality scores reported by the sequencer) and the alignment (number of matches, mismatches and deletions, mapping quality score returned by the alignment tool, if available, and number of mappings) as features for classification and uses simulated reads to learn a logistic regression model that relates these features to actual mapping quality. Results: We test the predictions of LoQuM on an independent dataset generated by the ART short read simulation software and observe that LoQuM can ‘resurrect’ many mappings that are assigned zero quality scores by the alignment tools and are therefore likely to be discarded by researchers. We also observe that the recalibration of mapping quality scores greatly enhances the precision of called single nucleotide polymorphisms. Availability: LoQuM is available as open source at http://compbio.case.edu/loqum/. Contact: matthew.ruffalo@case.edu. PMID:22962451

  11. MicroRNA Profiling in Aqueous Humor of Individual Human Eyes by Next-Generation Sequencing.

    Science.gov (United States)

    Wecker, Thomas; Hoffmeier, Klaus; Plötner, Anne; Grüning, Björn Andreas; Horres, Ralf; Backofen, Rolf; Reinhard, Thomas; Schlunck, Günther

    2016-04-01

    Extracellular microRNAs (miRNAs) in aqueous humor were suggested to have a role in transcellular signaling and may serve as disease biomarkers. The authors adopted next-generation sequencing (NGS) techniques to further characterize the miRNA profile in single samples of 60 to 80 μL human aqueous humor. Samples were obtained at the outset of cataract surgery in nine independent, otherwise healthy eyes. Four samples were used to extract RNA and generate sequencing libraries, followed by an adapter-driven amplification step, electrophoretic size selection, sequencing, and data analysis. Five samples were used for quantitative PCR (qPCR) validation of NGS results. Published NGS data on circulating miRNAs in blood were analyzed in comparison. One hundred fifty-eight miRNAs were consistently detected by NGS in all four samples; an additional 59 miRNAs were present in at least three samples. The aqueous humor miRNA profile shows some overlap with published NGS-derived inventories of circulating miRNAs in blood plasma with high prevalence of human miR-451a, -21, and -16. In contrast to blood, miR-184, -4448, -30a, -29a, -29c, -19a, -30d, -205, -24, -22, and -3074 were detected among the 20 most prevalent miRNAs in aqueous humor. Relative expression patterns of miR-451a, -202, and -144 suggested by NGS were confirmed by qPCR. Our data illustrate the feasibility of miRNA analysis by NGS in small individual aqueous humor samples. Intraocular cells as well as blood plasma contribute to the extracellular aqueous humor miRNome. The data suggest possible roles of miRNA in intraocular cell adhesion and signaling by TGF-β and Wnt, which are important in intraocular pressure regulation and glaucoma.

  12. GenNon-h: Generating multiple sequence alignments on nonhomogeneous phylogenetic trees

    Directory of Open Access Journals (Sweden)

    Kedzierska Anna M

    2012-08-01

    Full Text Available Abstract Background A number of software packages are available to generate DNA multiple sequence alignments (MSAs evolved under continuous-time Markov processes on phylogenetic trees. On the other hand, methods of simulating the DNA MSA directly from the transition matrices do not exist. Moreover, existing software restricts to the time-reversible models and it is not optimized to generate nonhomogeneous data (i.e. placing distinct substitution rates at different lineages. Results We present the first package designed to generate MSAs evolving under discrete-time Markov processes on phylogenetic trees, directly from probability substitution matrices. Based on the input model and a phylogenetic tree in the Newick format (with branch lengths measured as the expected number of substitutions per site, the algorithm produces DNA alignments of desired length. GenNon-h is publicly available for download. Conclusion The software presented here is an efficient tool to generate DNA MSAs on a given phylogenetic tree. GenNon-h provides the user with the nonstationary or nonhomogeneous phylogenetic data that is well suited for testing complex biological hypotheses, exploring the limits of the reconstruction algorithms and their robustness to such models.

  13. Evaluation of next generation sequencing for the analysis of Eimeria communities in wildlife.

    Science.gov (United States)

    Vermeulen, Elke T; Lott, Matthew J; Eldridge, Mark D B; Power, Michelle L

    2016-05-01

    Next-generation sequencing (NGS) techniques are well-established for studying bacterial communities but not yet for microbial eukaryotes. Parasite communities remain poorly studied, due in part to the lack of reliable and accessible molecular methods to analyse eukaryotic communities. We aimed to develop and evaluate a methodology to analyse communities of the protozoan parasite Eimeria from populations of the Australian marsupial Petrogale penicillata (brush-tailed rock-wallaby) using NGS. An oocyst purification method for small sample sizes and polymerase chain reaction (PCR) protocol for the 18S rRNA locus targeting Eimeria was developed and optimised prior to sequencing on the Illumina MiSeq platform. A data analysis approach was developed by modifying methods from bacterial metagenomics and utilising existing Eimeria sequences in GenBank. Operational taxonomic unit (OTU) assignment at a high similarity threshold (97%) was more accurate at assigning Eimeria contigs into Eimeria OTUs but at a lower threshold (95%) there was greater resolution between OTU consensus sequences. The assessment of two amplification PCR methods prior to Illumina MiSeq, single and nested PCR, determined that single PCR was more sensitive to Eimeria as more Eimeria OTUs were detected in single amplicons. We have developed a simple and cost-effective approach to a data analysis pipeline for community analysis of eukaryotic organisms using Eimeria communities as a model. The pipeline provides a basis for evaluation using other eukaryotic organisms and potential for diverse community analysis studies. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. Organism-specific rRNA capture system for application in next-generation sequencing.

    Directory of Open Access Journals (Sweden)

    Sai-Kam Li

    Full Text Available RNA-sequencing is a powerful tool in studying RNomics. However, the highly abundance of ribosomal RNAs (rRNA and transfer RNA (tRNA have predominated in the sequencing reads, thereby hindering the study of lowly expressed genes. Therefore, rRNA depletion prior to sequencing is often performed in order to preserve the subtle alteration in gene expression especially those at relatively low expression levels. One of the commercially available methods is to use DNA or RNA probes to hybridize to the target RNAs. However, there is always a concern with the non-specific binding and unintended removal of messenger RNA (mRNA when the same set of probes is applied to different organisms. The degree of such unintended mRNA removal varies among organisms due to organism-specific genomic variation. We developed a computer-based method to design probes to deplete rRNA in an organism-specific manner. Based on the computation results, biotinylated-RNA-probes were produced by in vitro transcription and were used to perform rRNA depletion with subtractive hybridization. We demonstrated that the designed probes of 16S rRNAs and 23S rRNAs can efficiently remove rRNAs from Mycobacterium smegmatis. In comparison with a commercial subtractive hybridization-based rRNA removal kit, using organism-specific probes is better in preserving the RNA integrity and abundance. We believe the computer-based design approach can be used as a generic method in preparing RNA of any organisms for next-generation sequencing, particularly for the transcriptome analysis of microbes.

  15. Validation of a next-generation sequencing assay for clinical molecular oncology.

    Science.gov (United States)

    Cottrell, Catherine E; Al-Kateb, Hussam; Bredemeyer, Andrew J; Duncavage, Eric J; Spencer, David H; Abel, Haley J; Lockwood, Christina M; Hagemann, Ian S; O'Guin, Stephanie M; Burcea, Lauren C; Sawyer, Christopher S; Oschwald, Dayna M; Stratman, Jennifer L; Sher, Dorie A; Johnson, Mark R; Brown, Justin T; Cliften, Paul F; George, Bijoy; McIntosh, Leslie D; Shrivastava, Savita; Nguyen, Tudung T; Payton, Jacqueline E; Watson, Mark A; Crosby, Seth D; Head, Richard D; Mitra, Robi D; Nagarajan, Rakesh; Kulkarni, Shashikant; Seibert, Karen; Virgin, Herbert W; Milbrandt, Jeffrey; Pfeifer, John D

    2014-01-01

    Currently, oncology testing includes molecular studies and cytogenetic analysis to detect genetic aberrations of clinical significance. Next-generation sequencing (NGS) allows rapid analysis of multiple genes for clinically actionable somatic variants. The WUCaMP assay uses targeted capture for NGS analysis of 25 cancer-associated genes to detect mutations at actionable loci. We present clinical validation of the assay and a detailed framework for design and validation of similar clinical assays. Deep sequencing of 78 tumor specimens (≥ 1000× average unique coverage across the capture region) achieved high sensitivity for detecting somatic variants at low allele fraction (AF). Validation revealed sensitivities and specificities of 100% for detection of single-nucleotide variants (SNVs) within coding regions, compared with SNP array sequence data (95% CI = 83.4-100.0 for sensitivity and 94.2-100.0 for specificity) or whole-genome sequencing (95% CI = 89.1-100.0 for sensitivity and 99.9-100.0 for specificity) of HapMap samples. Sensitivity for detecting variants at an observed 10% AF was 100% (95% CI = 93.2-100.0) in HapMap mixes. Analysis of 15 masked specimens harboring clinically reported variants yielded concordant calls for 13/13 variants at AF of ≥ 15%. The WUCaMP assay is a robust and sensitive method to detect somatic variants of clinical significance in molecular oncology laboratories, with reduced time and cost of genetic analysis allowing for strategic patient management. Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

  16. Targeted next generation sequencing reveals a novel intragenic deletion of the TPO gene in a family with intellectual disability

    NARCIS (Netherlands)

    Iqbal, Z.; Neveling, K.; Razzaq, A.; Shahzad, M.; Zahoor, M.Y.; Qasim, M.; Gilissen, C.F.H.A.; Wieskamp, N.; Kwint, M.P.; Gijsen, S.; de Brouwer, A.P.; Veltman, J.A.; Riazuddin, S.; Bokhoven, J.H.L.M. van

    2012-01-01

    BACKGROUNDS AND AIMS: Next generation sequencing (NGS) approaches have revolutionized the identification of mutations underlying genetic disorders. This technology is particularly useful for the identification of mutations in known and new genes for conditions with extensive genetic heterogeneity.

  17. Models of Prime-Like Sequences Generated by Least Element Sieve Operations Like the Sieve of Eratosthenes

    OpenAIRE

    Baum, Leonard E.

    2017-01-01

    We suggest other models of sieve generated sequences like the Sieve of Eratosthenes to explain randomness properties of the prime numbers, like the twin prime conjecture, the lim sup conjecture, the Riemann conjecture, and the prime number theorem.

  18. Is the $1000 Genome as Near as We Think? A Cost Analysis of Next-Generation Sequencing

    NARCIS (Netherlands)

    Nimwegen, K.J.M. van; Soest, R.A.; Veltman, J.A.; Nelen, M.R.; Wilt, G.J. van der; Peart-Vissers, L.E.L.M.; Grutters, J.P.C.

    2016-01-01

    BACKGROUND: The substantial technological advancements in next-generation sequencing (NGS), combined with dropping costs, have allowed for a swift diffusion of NGS applications in clinical settings. Although several commercial parties report to have broken the $1000 barrier for sequencing an entire

  19. AQME: A forensic mitochondrial DNA analysis tool for next-generation sequencing data.

    Science.gov (United States)

    Sturk-Andreaggi, Kimberly; Peck, Michelle A; Boysen, Cecilie; Dekker, Patrick; McMahon, Timothy P; Marshall, Charla K

    2017-11-01

    The feasibility of generating mitochondrial DNA (mtDNA) data has expanded considerably with the advent of next-generation sequencing (NGS), specifically in the generation of entire mtDNA genome (mitogenome) sequences. However, the analysis of these data has emerged as the greatest challenge to implementation in forensics. To address this need, a custom toolkit for use in the CLC Genomics Workbench (QIAGEN, Hilden, Germany) was developed through a collaborative effort between the Armed Forces Medical Examiner System - Armed Forces DNA Identification Laboratory (AFMES-AFDIL) and QIAGEN Bioinformatics. The AFDIL-QIAGEN mtDNA Expert, or AQME, generates an editable mtDNA profile that employs forensic conventions and includes the interpretation range required for mtDNA data reporting. AQME also integrates an mtDNA haplogroup estimate into the analysis workflow, which provides the analyst with phylogenetic nomenclature guidance and a profile quality check without the use of an external tool. Supplemental AQME outputs such as nucleotide-per-position metrics, configurable export files, and an audit trail are produced to assist the analyst during review. AQME is applied to standard CLC outputs and thus can be incorporated into any mtDNA bioinformatics pipeline within CLC regardless of sample type, library preparation or NGS platform. An evaluation of AQME was performed to demonstrate its functionality and reliability for the analysis of mitogenome NGS data. The study analyzed Illumina mitogenome data from 21 samples (including associated controls) of varying quality and sample preparations with the AQME toolkit. A total of 211 tool edits were automatically applied to 130 of the 698 total variants reported in an effort to adhere to forensic nomenclature. Although additional manual edits were required for three samples, supplemental tools such as mtDNA haplogroup estimation assisted in identifying and guiding these necessary modifications to the AQME-generated profile. Along

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

    Directory of Open Access Journals (Sweden)

    Hideyuki Tamaki

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

  1. Generation of mast cells from mouse fetus: analysis of differentiation and functionality, and transcriptome profiling using next generation sequencer.

    Directory of Open Access Journals (Sweden)

    Nobuyuki Fukuishi

    Full Text Available While gene knockout technology can reveal the roles of proteins in cellular functions, including in mast cells, fetal death due to gene manipulation frequently interrupts experimental analysis. We generated mast cells from mouse fetal liver (FLMC, and compared the fundamental functions of FLMC with those of bone marrow-derived mouse mast cells (BMMC. Under electron microscopy, numerous small and electron-dense granules were observed in FLMC. In FLMC, the expression levels of a subunit of the FcεRI receptor and degranulation by IgE cross-linking were comparable with BMMC. By flow cytometry we observed surface expression of c-Kit prior to that of FcεRI on FLMC, although on BMMC the expression of c-Kit came after FcεRI. The surface expression levels of Sca-1 and c-Kit, a marker of putative mast cell precursors, were slightly different between bone marrow cells and fetal liver cells, suggesting that differentiation stage or cell type are not necessarily equivalent between both lineages. Moreover, this indicates that phenotypically similar mast cells may not have undergone an identical process of differentiation. By comprehensive analysis using the next generation sequencer, the same frequency of gene expression was observed for 98.6% of all transcripts in both cell types. These results indicate that FLMC could represent a new and useful tool for exploring mast cell differentiation, and may help to elucidate the roles of individual proteins in the function of mast cells where gene manipulation can induce embryonic lethality in the mid to late stages of pregnancy.

  2. Generation of mast cells from mouse fetus: analysis of differentiation and functionality, and transcriptome profiling using next generation sequencer.

    Science.gov (United States)

    Fukuishi, Nobuyuki; Igawa, Yuusuke; Kunimi, Tomoyo; Hamano, Hirofumi; Toyota, Masao; Takahashi, Hironobu; Kenmoku, Hiromichi; Yagi, Yasuyuki; Matsui, Nobuaki; Akagi, Masaaki

    2013-01-01

    While gene knockout technology can reveal the roles of proteins in cellular functions, including in mast cells, fetal death due to gene manipulation frequently interrupts experimental analysis. We generated mast cells from mouse fetal liver (FLMC), and compared the fundamental functions of FLMC with those of bone marrow-derived mouse mast cells (BMMC). Under electron microscopy, numerous small and electron-dense granules were observed in FLMC. In FLMC, the expression levels of a subunit of the FcεRI receptor and degranulation by IgE cross-linking were comparable with BMMC. By flow cytometry we observed surface expression of c-Kit prior to that of FcεRI on FLMC, although on BMMC the expression of c-Kit came after FcεRI. The surface expression levels of Sca-1 and c-Kit, a marker of putative mast cell precursors, were slightly different between bone marrow cells and fetal liver cells, suggesting that differentiation stage or cell type are not necessarily equivalent between both lineages. Moreover, this indicates that phenotypically similar mast cells may not have undergone an identical process of differentiation. By comprehensive analysis using the next generation sequencer, the same frequency of gene expression was observed for 98.6% of all transcripts in both cell types. These results indicate that FLMC could represent a new and useful tool for exploring mast cell differentiation, and may help to elucidate the roles of individual proteins in the function of mast cells where gene manipulation can induce embryonic lethality in the mid to late stages of pregnancy.

  3. Transcriptional profiling of endocrine cerebro-osteodysplasia using microarray and next-generation sequencing.

    Directory of Open Access Journals (Sweden)

    Piya Lahiry

    Full Text Available BACKGROUND: Transcriptome profiling of patterns of RNA expression is a powerful approach to identify networks of genes that play a role in disease. To date, most mRNA profiling of tissues has been accomplished using microarrays, but next-generation sequencing can offer a richer and more comprehensive picture. METHODOLOGY/PRINCIPAL FINDINGS: ECO is a rare multi-system developmental disorder caused by a homozygous mutation in ICK encoding intestinal cell kinase. We performed gene expression profiling using both cDNA microarrays and next-generation mRNA sequencing (mRNA-seq of skin fibroblasts from ECO-affected subjects. We then validated a subset of differentially expressed transcripts identified by each method using quantitative reverse transcription-polymerase chain reaction (qRT-PCR. Finally, we used gene ontology (GO to identify critical pathways and processes that were abnormal according to each technical platform. Methodologically, mRNA-seq identifies a much larger number of differentially expressed genes with much better correlation to qRT-PCR results than the microarray (r² = 0.794 and 0.137, respectively. Biologically, cDNA microarray identified functional pathways focused on anatomical structure and development, while the mRNA-seq platform identified a higher proportion of genes involved in cell division and DNA replication pathways. CONCLUSIONS/SIGNIFICANCE: Transcriptome profiling with mRNA-seq had greater sensitivity, range and accuracy than the microarray. The two platforms generated different but complementary hypotheses for further evaluation.

  4. XMM-NEWTON MONITORING OF THE CLOSE PRE-MAIN-SEQUENCE BINARY AK SCO. EVIDENCE OF TIDE-DRIVEN FILLING OF THE INNER GAP IN THE CIRCUMBINARY DISK

    Energy Technology Data Exchange (ETDEWEB)

    Gomez de Castro, Ana Ines [S. D. Astronomia y Geodesia and Instituto de Matematica Interdisciplinar, Fac. de CC Matematicas, Universidad Complutense, E-28040 Madrid (Spain); Lopez-Santiago, Javier [Departamento de Astrofisica, Fac de CC Fisicas, Universidad Complutense, E-28040 Madrid (Spain); Talavera, Antonio [European Space Astronomy Center, Villanueva de la Canada, E-28691, Madrid (Spain); Sytov, A. Yu.; Bisikalo, D. [Institute of Astronomy of the Russian Academy of Sciences, Pyatnitskaya St. 48, 109017 Moscow (Russian Federation)

    2013-03-20

    AK Sco stands out among pre-main-sequence binaries because of its prominent ultraviolet excess, the high eccentricity of its orbit, and the strong tides driven by it. AK Sco consists of two F5-type stars that get as close as 11 R{sub *} at periastron passage. The presence of a dense (n{sub e} {approx} 10{sup 11} cm{sup -3}) extended envelope has been unveiled recently. In this article, we report the results from an XMM-Newton-based monitoring of the system. We show that at periastron, X-ray and UV fluxes are enhanced by a factor of {approx}3 with respect to the apastron values. The X-ray radiation is produced in an optically thin plasma with T {approx} 6.4 Multiplication-Sign 10{sup 6} K and it is found that the N{sub H} column density rises from 0.35 Multiplication-Sign 10{sup 21} cm{sup -2} at periastron to 1.11 Multiplication-Sign 10{sup 21} cm{sup -2} at apastron, in good agreement with previous polarimetric observations. The UV emission detected in the Optical Monitor band seems to be caused by the reprocessing of the high-energy magnetospheric radiation on the circumstellar material. Further evidence of the strong magnetospheric disturbances is provided by the detection of line broadening of 278.7 km s{sup -1} in the N V line with Hubble Space Telescope/Space Telescope Imaging Spectrograph. Numerical simulations of the mass flow from the circumbinary disk to the components have been carried out. They provide a consistent scenario with which to interpret AK Sco observations. We show that the eccentric orbit acts like a gravitational piston. At apastron, matter is dragged efficiently from the inner disk border, filling the inner gap and producing accretion streams that end as ring-like structures around each component of the system. At periastron, the ring-like structures come into contact, leading to angular momentum loss, and thus producing an accretion outburst.

  5. Comparative analyses of two Geraniaceae transcriptomes using next-generation sequencing.

    Science.gov (United States)

    Zhang, Jin; Ruhlman, Tracey A; Mower, Jeffrey P; Jansen, Robert K

    2013-12-29

    Organelle genomes of Geraniaceae exhibit several unusual evolutionary phenomena compared to other angiosperm families including accelerated nucleotide substitution rates, widespread gene loss, reduced RNA editing, and extensive genomic rearrangements. Since most organelle-encoded proteins function in multi-subunit complexes that also contain nuclear-encoded proteins, it is likely that the atypical organellar phenomena affect the evolution of nuclear genes encoding organellar proteins. To begin to unravel the complex co-evolutionary interplay between organellar and nuclear genomes in this family, we sequenced nuclear transcriptomes of two species, Geranium maderense and Pelargonium x hortorum. Normalized cDNA libraries of G. maderense and P. x hortorum were used for transcriptome sequencing. Five assemblers (MIRA, Newbler, SOAPdenovo, SOAPdenovo-trans [SOAPtrans], Trinity) and two next-generation technologies (454 and Illumina) were compared to determine the optimal transcriptome sequencing approach. Trinity provided the highest quality assembly of Illumina data with the deepest transcriptome coverage. An analysis to determine the amount of sequencing needed for de novo assembly revealed diminishing returns of coverage and quality with data sets larger than sixty million Illumina paired end reads for both species. The G. maderense and P. x hortorum transcriptomes contained fewer transcripts encoding the PLS subclass of PPR proteins relative to other angiosperms, consistent with reduced mitochondrial RNA editing activity in Geraniaceae. In addition, transcripts for all six plastid targeted sigma factors were identified in both transcriptomes, suggesting that one of the highly divergent rpoA-like ORFs in the P. x hortorum plastid genome is functional. The findings support the use of the Illumina platform and assemblers optimized for transcriptome assembly, such as Trinity or SOAPtrans, to generate high-quality de novo transcriptomes with broad coverage. In addition

  6. Application of Next Generation Sequencing in Mammalian Embryogenomics: Lessons Learned from Endogenous Betaretroviruses of Sheep

    Science.gov (United States)

    Spencer, Thomas E.; Palmarini, Massimo

    2012-01-01

    Endogenous retroviruses (ERVs) are present in the genome of all vertebrates and are remnants of ancient exogenous retroviral infections of the host germline transmitted vertically from generation to generation. The sheep genome contains 27 JSRV-related endogenous betaretroviruses (enJSRVs) related to the pathogenic Jaagsiekte sheep retrovirus (JSRV) that have been integrating in the host genome for the last 5 to 7 million years. The exogenous JSRV is a causative agent of a transmissible lung cancer in sheep, and enJSRVs are able to protect the host against JSRV infection. In sheep, the enJSRVs are most abundantly expressed in the uterine epithelia as well as in the conceptus (embryo and associated extraembryonic membranes) trophectoderm. Sixteen of the 27 enJSRV loci contain an envelope (env) gene with an intact open reading frame, and in utero loss-of-function experiments found the enJSRVs Env to be essential for trophoblast outgrowth and conceptus elongation. Collectively, available evidence supports the ideas that genes captured from ancestral retroviruses were pivotal in the acquisition of new, important functions in mammalian evolution and were positively selected for biological roles in genome plasticity, protection of the host against infection of related pathogenic and exogenous retroviruses, and a convergent physiological role in placental morphogenesis and thus mammalian reproduction. The discovery of ERVs in mammals was initially based on molecular cloning discovery techniques and will be boosted forward by next generation sequencing technologies and in silico discovery techniques. PMID:22951118

  7. Assessing the 5S ribosomal RNA heterogeneity in Arabidopsis thaliana using short RNA next generation sequencing data.

    Science.gov (United States)

    Szymanski, Maciej; Karlowski, Wojciech M

    2016-01-01

    In eukaryotes, ribosomal 5S rRNAs are products of multigene families organized within clusters of tandemly repeated units. Accumulation of genomic data obtained from a variety of organisms demonstrated that the potential 5S rRNA coding sequences show a large number of variants, often incompatible with folding into a correct secondary structure. Here, we present results of an analysis of a large set of short RNA sequences generated by the next generation sequencing techniques, to address the problem of heterogeneity of the 5S rRNA transcripts in Arabidopsis and identification of potentially functional rRNA-derived fragments.

  8. Low-coverage MiSeq next generation sequencing reveals the mitochondrial genome of the Eastern Rock Lobster, Sagmariasus verreauxi.

    Science.gov (United States)

    Doyle, Stephen R; Griffith, Ian S; Murphy, Nick P; Strugnell, Jan M

    2015-01-01

    The complete mitochondrial genome of the Eastern Rock lobster, Sagmariasus verreauxi, is reported for the first time. Using low-coverage, long read MiSeq next generation sequencing, we constructed and determined the mtDNA genome organization of the 15,470 bp sequence from two isolates from Eastern Tasmania, Australia and Northern New Zealand, and identified 46 polymorphic nucleotides between the two sequences. This genome sequence and its genetic polymorphisms will likely be useful in understanding the distribution and population connectivity of the Eastern Rock Lobster, and in the fisheries management of this commercially important species.

  9. Next Generation Semiconductor Based Sequencing of the Donkey (Equus asinus) Genome Provided Comparative Sequence Data against the Horse Genome and a Few Millions of Single Nucleotide Polymorphisms

    Science.gov (United States)

    Bertolini, Francesca; Scimone, Concetta; Geraci, Claudia; Schiavo, Giuseppina; Utzeri, Valerio Joe; Chiofalo, Vincenzo; Fontanesi, Luca

    2015-01-01

    Few studies investigated the donkey (Equus asinus) at the whole genome level so far. Here, we sequenced the genome of two male donkeys using a next generation semiconductor based sequencing platform (the Ion Proton sequencer) and compared obtained sequence information with the available donkey draft genome (and its Illumina reads from which it was originated) and with the EquCab2.0 assembly of the horse genome. Moreover, the Ion Torrent Personal Genome Analyzer was used to sequence reduced representation libraries (RRL) obtained from a DNA pool including donkeys of different breeds (Grigio Siciliano, Ragusano and Martina Franca). The number of next generation sequencing reads aligned with the EquCab2.0 horse genome was larger than those aligned with the draft donkey genome. This was due to the larger N50 for contigs and scaffolds of the horse genome. Nucleotide divergence between E. caballus and E. asinus was estimated to be ~ 0.52-0.57%. Regions with low nucleotide divergence were identified in several autosomal chromosomes and in the whole chromosome X. These regions might be evolutionally important in equids. Comparing Y-chromosome regions we identified variants that could be useful to track donkey paternal lineages. Moreover, about 4.8 million of single nucleotide polymorphisms (SNPs) in the donkey genome were identified and annotated combining sequencing data from Ion Proton (whole genome sequencing) and Ion Torrent (RRL) runs with Illumina reads. A higher density of SNPs was present in regions homologous to horse chromosome 12, in which several studies reported a high frequency of copy number variants. The SNPs we identified constitute a first resource useful to describe variability at the population genomic level in E. asinus and to establish monitoring systems for the conservation of donkey genetic resources. PMID:26151450

  10. Next Generation Semiconductor Based Sequencing of the Donkey (Equus asinus Genome Provided Comparative Sequence Data against the Horse Genome and a Few Millions of Single Nucleotide Polymorphisms.

    Directory of Open Access Journals (Sweden)

    Francesca Bertolini

    Full Text Available Few studies investigated the donkey (Equus asinus at the whole genome level so far. Here, we sequenced the genome of two male donkeys using a next generation semiconductor based sequencing platform (the Ion Proton sequencer and compared obtained sequence information with the available donkey draft genome (and its Illumina reads from which it was originated and with the EquCab2.0 assembly of the horse genome. Moreover, the Ion Torrent Personal Genome Analyzer was used to sequence reduced representation libraries (RRL obtained from a DNA pool including donkeys of different breeds (Grigio Siciliano, Ragusano and Martina Franca. The number of next generation sequencing reads aligned with the EquCab2.0 horse genome was larger than those aligned with the draft donkey genome. This was due to the larger N50 for contigs and scaffolds of the horse genome. Nucleotide divergence between E. caballus and E. asinus was estimated to be ~ 0.52-0.57%. Regions with low nucleotide divergence were identified in several autosomal chromosomes and in the whole chromosome X. These regions might be evolutionally important in equids. Comparing Y-chromosome regions we identified variants that could be useful to track donkey paternal lineages. Moreover, about 4.8 million of single nucleotide polymorphisms (SNPs in the donkey genome were identified and annotated combining sequencing data from Ion Proton (whole genome sequencing and Ion Torrent (RRL runs with Illumina reads. A higher density of SNPs was present in regions homologous to horse chromosome 12, in which several studies reported a high frequency of copy number variants. The SNPs we identified constitute a first resource useful to describe variability at the population genomic level in E. asinus and to establish monitoring systems for the conservation of donkey genetic resources.

  11. Targeted next-generation sequencing analysis identifies novel mutations in families with severe familial exudative vitreoretinopathy

    Science.gov (United States)

    Huang, Xiao-Yan; Zhuang, Hong; Wu, Ji-Hong; Li, Jian-Kang; Hu, Fang-Yuan; Zheng, Yu; Tellier, Laurent Christian Asker M.; Zhang, Sheng-Hai; Gao, Feng-Juan; Zhang, Jian-Guo

    2017-01-01

    Purpose Familial exudative vitreoretinopathy (FEVR) is a genetically and clinically heterogeneous disease, characterized by failure of vascular development of the peripheral retina. The symptoms of FEVR vary widely among patients in the same family, and even between the two eyes of a given patient. This study was designed to identify the genetic defect in a patient cohort of ten Chinese families with a definitive diagnosis of FEVR. Methods To identify the causative gene, next-generation sequencing (NGS)-based target capture sequencing was performed. Segregation analysis of the candidate variant was performed in additional family members by using Sanger sequencing and quantitative real-time PCR (QPCR). Results Of the cohort of ten FEVR families, six pathogenic variants were identified, including four novel and two known heterozygous mutations. Of the variants identified, four were missense variants, and two were novel heterozygous deletion mutations [LRP5, c.4053 DelC (p.Ile1351IlefsX88); TSPAN12, EX8Del]. The two novel heterozygous deletion mutations were not observed in the control subjects and could give rise to a relatively severe FEVR phenotype, which could be explained by the protein function prediction. Conclusions We identified two novel heterozygous deletion mutations [LRP5, c.4053 DelC (p.Ile1351IlefsX88); TSPAN12, EX8Del] using targeted NGS as a causative mutation for FEVR. These genetic deletion variations exhibit a severe form of FEVR, with tractional retinal detachments compared with other known point mutations. The data further enrich the mutation spectrum of FEVR and enhance our understanding of genotype–phenotype correlations to provide useful information for disease diagnosis, prognosis, and effective genetic counseling. PMID:28867931

  12. Probabilistic Inference on Multiple Normalized Signal Profiles from Next Generation Sequencing: Transcription Factor Binding Sites

    KAUST Repository

    Wong, Ka-Chun; Peng, Chengbin; Li, Yue

    2015-01-01

    With the prevalence of chromatin immunoprecipitation (ChIP) with sequencing (ChIP-Seq) technology, massive ChIP-Seq data has been accumulated. The ChIP-Seq technology measures the genome-wide occupancy of DNA-binding proteins in vivo. It is well-known that different DNA-binding protein occupancies may result in a gene being regulated in different conditions (e.g. different cell types). To fully understand a gene's function, it is essential to develop probabilistic models on multiple ChIP-Seq profiles for deciphering the gene transcription causalities. In this work, we propose and describe two probabilistic models. Assuming the conditional independence of different DNA-binding proteins' occupancies, the first method (SignalRanker) is developed as an intuitive method for ChIP-Seq genome-wide signal profile inference. Unfortunately, such an assumption may not always hold in some gene regulation cases. Thus, we propose and describe another method (FullSignalRanker) which does not make the conditional independence assumption. The proposed methods are compared with other existing methods on ENCODE ChIP-Seq datasets, demonstrating its regression and classification ability. The results suggest that FullSignalRanker is the best-performing method for recovering the signal ranks on the promoter and enhancer regions. In addition, FullSignalRanker is also the best-performing method for peak sequence classification. We envision that SignalRanker and FullSignalRanker will become important in the era of next generation sequencing. FullSignalRanker program is available on the following website: http://www.cs.toronto.edu/∼wkc/FullSignalRanker/ © 2015 IEEE.

  13. An artificial functional family filter in homolog searching in next-generation sequencing metagenomics.

    Directory of Open Access Journals (Sweden)

    Ruofei Du

    Full Text Available In functional metagenomics, BLAST homology search is a common method to classify metagenomic reads into protein/domain sequence families such as Clusters of Orthologous Groups of proteins (COGs in order to quantify the abundance of each COG in the community. The resulting functional profile of the community is then used in downstream analysis to correlate the change in abundance to environmental perturbation, clinical variation, and so on. However, the short read length coupled with next-generation sequencing technologies poses a barrier in this approach, essentially because similarity significance cannot be discerned by searching with short reads. Consequently, artificial functional families are produced, in which those with a large number of reads assigned decreases the accuracy of functional profile dramatically. There is no method available to address this problem. We intended to fill this gap in this paper. We revealed that BLAST similarity scores of homologues for short reads from COG protein members coding sequences are distributed differently from the scores of those derived elsewhere. We showed that, by choosing an appropriate score cut-off, we are able to filter out most artificial families and simultaneously to preserve sufficient information in order to build the functional profile. We also showed that, by incorporated application of BLAST and RPS-BLAST, some artificial families with large read counts can be further identified after the score cutoff filtration. Evaluated on three experimental metagenomic datasets with different coverages, we found that the proposed method is robust against read coverage and consistently outperforms the other E-value cutoff methods currently used in literatures.

  14. Probabilistic Inference on Multiple Normalized Signal Profiles from Next Generation Sequencing: Transcription Factor Binding Sites

    KAUST Repository

    Wong, Ka-Chun

    2015-04-20

    With the prevalence of chromatin immunoprecipitation (ChIP) with sequencing (ChIP-Seq) technology, massive ChIP-Seq data has been accumulated. The ChIP-Seq technology measures the genome-wide occupancy of DNA-binding proteins in vivo. It is well-known that different DNA-binding protein occupancies may result in a gene being regulated in different conditions (e.g. different cell types). To fully understand a gene\\'s function, it is essential to develop probabilistic models on multiple ChIP-Seq profiles for deciphering the gene transcription causalities. In this work, we propose and describe two probabilistic models. Assuming the conditional independence of different DNA-binding proteins\\' occupancies, the first method (SignalRanker) is developed as an intuitive method for ChIP-Seq genome-wide signal profile inference. Unfortunately, such an assumption may not always hold in some gene regulation cases. Thus, we propose and describe another method (FullSignalRanker) which does not make the conditional independence assumption. The proposed methods are compared with other existing methods on ENCODE ChIP-Seq datasets, demonstrating its regression and classification ability. The results suggest that FullSignalRanker is the best-performing method for recovering the signal ranks on the promoter and enhancer regions. In addition, FullSignalRanker is also the best-performing method for peak sequence classification. We envision that SignalRanker and FullSignalRanker will become important in the era of next generation sequencing. FullSignalRanker program is available on the following website: http://www.cs.toronto.edu/∼wkc/FullSignalRanker/ © 2015 IEEE.

  15. Assuring the Quality of Next-Generation Sequencing in Clinical Microbiology and Public Health Laboratories.

    Science.gov (United States)

    Gargis, Amy S; Kalman, Lisa; Lubin, Ira M

    2016-12-01

    Clinical microbiology and public health laboratories are beginning to utilize next-generation sequencing (NGS) for a range of applications. This technology has the potential to transform the field by providing approaches that will complement, or even replace, many conventional laboratory tests. While the benefits of NGS are significant, the complexities of these assays require an evolving set of standards to ensure testing quality. Regulatory and accreditation requirements, professional guidelines, and best practices that help ensure the quality of NGS-based tests are emerging. This review highlights currently available standards and guidelines for the implementation of NGS in the clinical and public health laboratory setting, and it includes considerations for NGS test validation, quality control procedures, proficiency testing, and reference materials. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  16. A Next-Generation Sequencing Approach Uncovers Viral Transcripts Incorporated in Poxvirus Virions

    Directory of Open Access Journals (Sweden)

    Marica Grossegesse

    2017-10-01

    Full Text Available Transcripts are known to be incorporated in particles of DNA viruses belonging to the families of Herpesviridae and Mimiviridae, but the presence of transcripts in other DNA viruses, such as poxviruses, has not been analyzed yet. Therefore, we first established a next-generation-sequencing (NGS-based protocol, enabling the unbiased identification of transcripts in virus particles. Subsequently, we applied our protocol to analyze RNA in an emerging zoonotic member of the Poxviridae family, namely Cowpox virus. Our results revealed the incorporation of 19 viral transcripts, while host identifications were restricted to ribosomal and mitochondrial RNA. Most viral transcripts had an unknown and immunomodulatory function, suggesting that transcript incorporation may be beneficial for poxvirus immune evasion. Notably, the most abundant transcript originated from the D5L/I1R gene that encodes a viral inhibitor of the host cytoplasmic DNA sensing machinery.

  17. Comparing microarrays and next-generation sequencing technologies for microbial ecology research.

    Science.gov (United States)

    Roh, Seong Woon; Abell, Guy C J; Kim, Kyoung-Ho; Nam, Young-Do; Bae, Jin-Woo

    2010-06-01

    Recent advances in molecular biology have resulted in the application of DNA microarrays and next-generation sequencing (NGS) technologies to the field of microbial ecology. This review aims to examine the strengths and weaknesses of each of the methodologies, including depth and ease of analysis, throughput and cost-effectiveness. It also intends to highlight the optimal application of each of the individual technologies toward the study of a particular environment and identify potential synergies between the two main technologies, whereby both sample number and coverage can be maximized. We suggest that the efficient use of microarray and NGS technologies will allow researchers to advance the field of microbial ecology, and importantly, improve our understanding of the role of microorganisms in their various environments.

  18. Genetic counselors’ (GC) knowledge, awareness, and understanding of clinical next-generation sequencing (NGS) genomic testing

    Science.gov (United States)

    Boland, PM; Ruth, K; Matro, JM; Rainey, KL; Fang, CY; Wong, YN; Daly, MB; Hall, MJ

    2014-01-01

    Genomic tests are increasingly complex, less expensive, and more widely available with the advent of next-generation sequencing (NGS). We assessed knowledge and perceptions among genetic counselors pertaining to NGS genomic testing via an online survey. Associations between selected characteristics and perceptions were examined. Recent education on NGS testing was common, but practical experience limited. Perceived understanding of clinical NGS was modest, specifically concerning tumor testing. Greater perceived understanding of clinical NGS testing correlated with more time spent in cancer-related counseling, exposure to NGS testing, and NGS-focused education. Substantial disagreement about the role of counseling for tumor-based testing was seen. Finally, a majority of counselors agreed with the need for more education about clinical NGS testing, supporting this approach to optimizing implementation. PMID:25523111

  19. Next generation sequencing identifies abnormal Y chromosome and candidate causal variants in premature ovarian failure patients.

    Science.gov (United States)

    Lee, Yujung; Kim, Changshin; Park, YoungJoon; Pyun, Jung-A; Kwack, KyuBum

    2016-12-01

    Premature ovarian failure (POF) is characterized by heterogeneous genetic causes such as chromosomal abnormalities and variants in causal genes. Recently, development of techniques made next generation sequencing (NGS) possible to detect genome wide variants including chromosomal abnormalities. Among 37 Korean POF patients, XY karyotype with distal part deletions of Y chromosome, Yp11.32-31 and Yp12 end part, was observed in two patients through NGS. Six deleterious variants in POF genes were also detected which might explain the pathogenesis of POF with abnormalities in the sex chromosomes. Additionally, the two POF patients had no mutation in SRY but three non-synonymous variants were detected in genes regarding sex reversal. These findings suggest candidate causes of POF and sex reversal and show the propriety of NGS to approach the heterogeneous pathogenesis of POF. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. Next Generation Sequencing approach to molecular diagnosis of Duchenne muscular dystrophy; identification of a novel mutation.

    Science.gov (United States)

    Ebrahimzadeh-Vesal, Reza; Teymoori, Atieh; Azimi-Nezhad, Mohsen; Hosseini, Forough Sadat

    2018-02-20

    Duchenne Muscular Dystrophy (DMD; MIM 310200) is one of the most common and severe type of hereditary muscular dystrophies. The disease is caused by mutations in the dystrophin gene. The dystrophin gene is associated with X-linked recessive Duchenne and Becker muscular dystrophy. This disease occurs almost exclusively in males. The clinical symptoms of muscle weakness usually begin at childhood. The main symptoms of this disorder are gradually muscular weakness. The affected patients have inability to standing up and walking. Death is usually due to respiratory infection or cardiomyopathy. In this article, we have reported the discovery of a new nonsense mutation that creates abnormal stop codon in the dystrophin gene. This mutation was detected using Next Generation Sequencing (NGS) technique. The subject was a 17-year-old male with muscular dystrophy that who was suspected of having DMD. He was referred to Hakim medical genetics center of Neyshabur, IRAN. Copyright © 2017. Published by Elsevier B.V.

  1. How next-generation sequencing and multiscale data analysis will transform infectious disease management.

    Science.gov (United States)

    Pak, Theodore R; Kasarskis, Andrew

    2015-12-01

    Recent reviews have examined the extent to which routine next-generation sequencing (NGS) on clinical specimens will improve the capabilities of clinical microbiology laboratories in the short term, but do not explore integrating NGS with clinical data from electronic medical records (EMRs), immune profiling data, and other rich datasets to create multiscale predictive models. This review introduces a range of "omics" and patient data sources relevant to managing infections and proposes 3 potentially disruptive applications for these data in the clinical workflow. The combined threats of healthcare-associated infections and multidrug-resistant organisms may be addressed by multiscale analysis of NGS and EMR data that is ideally updated and refined over time within each healthcare organization. Such data and analysis should form the cornerstone of future learning health systems for infectious disease. © The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America.

  2. No more non-model species: the promise of next generation sequencing for comparative immunology.

    Science.gov (United States)

    Dheilly, Nolwenn M; Adema, Coen; Raftos, David A; Gourbal, Benjamin; Grunau, Christoph; Du Pasquier, Louis

    2014-07-01

    Next generation sequencing (NGS) allows for the rapid, comprehensive and cost effective analysis of entire genomes and transcriptomes. NGS provides approaches for immune response gene discovery, profiling gene expression over the course of parasitosis, studying mechanisms of diversification of immune receptors and investigating the role of epigenetic mechanisms in regulating immune gene expression and/or diversification. NGS will allow meaningful comparisons to be made between organisms from different taxa in an effort to understand the selection of diverse strategies for host defence under different environmental pathogen pressures. At the same time, it will reveal the shared and unique components of the immunological toolkit and basic functional aspects that are essential for immune defence throughout the living world. In this review, we argue that NGS will revolutionize our understanding of immune responses throughout the animal kingdom because the depth of information it provides will circumvent the need to concentrate on a few "model" species. Copyright © 2014 Elsevier Ltd. All rights reserved.

  3. Development of microsatellite markers using next-generation sequencing for the columnar cactus Echinopsis chiloensis (Cactaceae).

    Science.gov (United States)

    Ossa, Carmen G; Larridon, Isabel; Peralta, Gioconda; Asselman, Pieter; Pérez, Fernanda

    2016-12-01

    The aim of this study was to develop microsatellite markers as a tool to study population structure, genetic diversity and effective population size of Echinopsis chiloensis, an endemic cactus from arid and semiarid regions of Central Chile. We developed 12 polymorphic microsatellite markers for E. chiloensis using next-generation sequencing and tested them in 60 individuals from six sites, covering all the latitudinal range of this species. The number of alleles per locus ranged from 3 to 8, while the observed (Ho) and expected (He) heterozygosity ranged from 0.0 to 0.80 and from 0.10 to 0.76, respectively. We also detected significant differences between sites, with F ST values ranging from 0.05 to 0.29. Microsatellite markers will enable us to estimate genetic diversity and population structure of E. chiloensis in future ecological and phylogeographic studies.

  4. Genetic counselors' (GC) knowledge, awareness, understanding of clinical next-generation sequencing (NGS) genomic testing.

    Science.gov (United States)

    Boland, P M; Ruth, K; Matro, J M; Rainey, K L; Fang, C Y; Wong, Y N; Daly, M B; Hall, M J

    2015-12-01

    Genomic tests are increasingly complex, less expensive, and more widely available with the advent of next-generation sequencing (NGS). We assessed knowledge and perceptions among genetic counselors pertaining to NGS genomic testing via an online survey. Associations between selected characteristics and perceptions were examined. Recent education on NGS testing was common, but practical experience limited. Perceived understanding of clinical NGS was modest, specifically concerning tumor testing. Greater perceived understanding of clinical NGS testing correlated with more time spent in cancer-related counseling, exposure to NGS testing, and NGS-focused education. Substantial disagreement about the role of counseling for tumor-based testing was seen. Finally, a majority of counselors agreed with the need for more education about clinical NGS testing, supporting this approach to optimizing implementation. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  5. Trojan Binaries

    Science.gov (United States)

    Noll, K. S.

    2017-12-01

    The Jupiter Trojans, in the context of giant planet migration models, can be thought of as an extension of the small body populations found beyond Neptune in the Kuiper Belt. Binaries are a distinctive feature of small body populations in the Kuiper Belt with an especially high fraction apparent among the brightest Cold Classicals. The binary fraction, relative sizes, and separations in the dynamically excited populations (Scattered, Resonant) reflects processes that may have eroded a more abundant initial population. This trend continues in the Centaurs and Trojans where few binaries have been found. We review new evidence including a third resolved Trojan binary and lightcurve studies to understand how the Trojans are related to the small body populations that originated in the outer protoplanetary disk.

  6. Applying Ancestry and Sex Computation as a Quality Control Tool in Targeted Next-Generation Sequencing.

    Science.gov (United States)

    Mathias, Patrick C; Turner, Emily H; Scroggins, Sheena M; Salipante, Stephen J; Hoffman, Noah G; Pritchard, Colin C; Shirts, Brian H

    2016-03-01

    To apply techniques for ancestry and sex computation from next-generation sequencing (NGS) data as an approach to confirm sample identity and detect sample processing errors. We combined a principal component analysis method with k-nearest neighbors classification to compute the ancestry of patients undergoing NGS testing. By combining this calculation with X chromosome copy number data, we determined the sex and ancestry of patients for comparison with self-report. We also modeled the sensitivity of this technique in detecting sample processing errors. We applied this technique to 859 patient samples with reliable self-report data. Our k-nearest neighbors ancestry screen had an accuracy of 98.7% for patients reporting a single ancestry. Visual inspection of principal component plots was consistent with self-report in 99.6% of single-ancestry and mixed-ancestry patients. Our model demonstrates that approximately two-thirds of potential sample swaps could be detected in our patient population using this technique. Patient ancestry can be estimated from NGS data incidentally sequenced in targeted panels, enabling an inexpensive quality control method when coupled with patient self-report. © American Society for Clinical Pathology, 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  7. Quantifying low-frequency revertants in oral poliovirus vaccine using next generation sequencing.

    Science.gov (United States)

    Sarcey, Eric; Serres, Aurélie; Tindy, Fabrice; Chareyre, Audrey; Ng, Siemon; Nicolas, Marine; Vetter, Emmanuelle; Bonnevay, Thierry; Abachin, Eric; Mallet, Laurent

    2017-08-01

    Spontaneous reversion to neurovirulence of live attenuated oral poliovirus vaccine (OPV) serotype 3 (chiefly involving the n.472U>C mutation), must be monitored during production to ensure vaccine safety and consistency. Mutant analysis by polymerase chain reaction and restriction enzyme cleavage (MAPREC) has long been endorsed by the World Health Organization as the preferred in vitro test for this purpose; however, it requires radiolabeling, which is no longer supported by many laboratories. We evaluated the performance and suitability of next generation sequencing (NGS) as an alternative to MAPREC. The linearity of NGS was demonstrated at revertant concentrations equivalent to the study range of 0.25%-1.5%. NGS repeatability and intermediate precision were comparable across all tested samples, and NGS was highly reproducible, irrespective of sequencing platform or analysis software used. NGS was performed on OPV serotype 3 working seed lots and monovalent bulks (n=21) that were previously tested using MAPREC, and which covered the representative range of vaccine production. Percentages of 472-C revertants identified by NGS and MAPREC were comparable and highly correlated (r≥0.80), with a Pearson correlation coefficient of 0.95585 (p<0.0001). NGS demonstrated statistically equivalent performance to that of MAPREC for quantifying low-frequency OPV serotype 3 revertants, and offers a valid alternative to MAPREC. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  8. Sequencing and generation of an infectious clone of the pathogenic goose parvovirus strain LH.

    Science.gov (United States)

    Wang, Jianye; Duan, Jinkun; Zhu, Liqian; Jiang, Zhiwei; Zhu, Guoqiang

    2015-03-01

    In this study, the complete genome of the virulent strain LH of goose parvovirus (GPV) was sequenced and cloned into the pBluescript II (SK) plasmid vector. Sequence alignments of the inverted terminal repeats (ITR) of GPV strains revealed a common 14-nt-pair deletion in the stem of the palindromic structure in the LH strain and three other strains isolated after 1982 when compared to three GPV strains isolated earlier than that time. Transfection of 11-day-old embryonated goose eggs with the plasmid pLH, which contains the entire genome of strain LH, resulted in successful rescue of the infectious virus. Death of embryos after transfection via the chorioallantoic membrane infiltration route occurred earlier than when transfection was done via the allantoic cavity inoculation route. The rescued virus exhibited virulence similar to that of its parental virus, as evaluated by the mortality rate in goslings. Generation of the pathogenic infectious clone provides us with a powerful tool to elucidate the molecular pathogenesis of GPV in the future.

  9. QuickNGS elevates Next-Generation Sequencing data analysis to a new level of automation.

    Science.gov (United States)

    Wagle, Prerana; Nikolić, Miloš; Frommolt, Peter

    2015-07-01

    Next-Generation Sequencing (NGS) has emerged as a widely used tool in molecular biology. While time and cost for the sequencing itself are decreasing, the analysis of the massive amounts of data remains challenging. Since multiple algorithmic approaches for the basic data analysis have been developed, there is now an increasing need to efficiently use these tools to obtain results in reasonable time. We have developed QuickNGS, a new workflow system for laboratories with the need to analyze data from multiple NGS projects at a time. QuickNGS takes advantage of parallel computing resources, a comprehensive back-end database, and a careful selection of previously published algorithmic approaches to build fully automated data analysis workflows. We demonstrate the efficiency of our new software by a comprehensive analysis of 10 RNA-Seq samples which we can finish in only a few minutes of hands-on time. The approach we have taken is suitable to process even much larger numbers of samples and multiple projects at a time. Our approach considerably reduces the barriers that still limit the usability of the powerful NGS technology and finally decreases the time to be spent before proceeding to further downstream analysis and interpretation of the data.

  10. Next-Generation Sequencing for Typing and Detection of ESBL and MBL E. coli causing UTI

    Directory of Open Access Journals (Sweden)

    Nabakishore Nayak

    2017-10-01

    Full Text Available Next-generation sequencing (NGS has the potential to provide typing results and detect resistance genes in a single assay, thus guiding timely treatment decisions and allowing rapid tracking of transmission of resistant clones. We can be evaluated the performance of a new NGS assay during an outbreak of sequence type 131 (ST131 Escherichia coli infections in a teaching hospital. The assay will be performed on 100 extended-spectrum- beta-lactamase (ESBL E. coli isolates collected from UTI during last 5 years. Typing results will be compared to those of amplified fragment length polymorphism (AFLP, whereby we will be visually assessed the agreement of the Bio-Detection phylogenetic tree with clusters defined by AFLP. A microarray will be considered the gold standard for detection of resistance genes. AFLP will be identified a large cluster of different indistinguishable isolates on adjacent departments, indicating clonal spread. The BioDetection phylogenetic tree will be showed that all isolates of this outbreak cluster will be strongly related, while the further arrangement of the tree also largely agreed with other clusters defined by AFLP. With these experiments we will detect the ESBL and MBL strains and the patient can be prescribed the antibiotics accordingly.

  11. Statistical framework for detection of genetically modified organisms based on Next Generation Sequencing.

    Science.gov (United States)

    Willems, Sander; Fraiture, Marie-Alice; Deforce, Dieter; De Keersmaecker, Sigrid C J; De Loose, Marc; Ruttink, Tom; Herman, Philippe; Van Nieuwerburgh, Filip; Roosens, Nancy

    2016-02-01

    Because the number and diversity of genetically modified (GM) crops has significantly increased, their analysis based on real-time PCR (qPCR) methods is becoming increasingly complex and laborious. While several pioneers already investigated Next Generation Sequencing (NGS) as an alternative to qPCR, its practical use has not been assessed for routine analysis. In this study a statistical framework was developed to predict the number of NGS reads needed to detect transgene sequences, to prove their integration into the host genome and to identify the specific transgene event in a sample with known composition. This framework was validated by applying it to experimental data from food matrices composed of pure GM rice, processed GM rice (noodles) or a 10% GM/non-GM rice mixture, revealing some influential factors. Finally, feasibility of NGS for routine analysis of GM crops was investigated by applying the framework to samples commonly encountered in routine analysis of GM crops. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  12. Detecting Positive Selection of Korean Native Goat Populations Using Next-Generation Sequencing

    Science.gov (United States)

    Lee, Wonseok; Ahn, Sojin; Taye, Mengistie; Sung, Samsun; Lee, Hyun-Jeong; Cho, Seoae; Kim, Heebal

    2016-01-01

    Goats (Capra hircus) are one of the oldest species of domesticated animals. Native Korean goats are a particularly interesting group, as they are indigenous to the area and were raised in the Korean peninsula almost 2,000 years ago. Although they have a small body size and produce low volumes of milk and meat, they are quite resistant to lumbar paralysis. Our study aimed to reveal the distinct genetic features and patterns of selection in native Korean goats by comparing the genomes of native Korean goat and crossbred goat populations. We sequenced the whole genome of 15 native Korean goats and 11 crossbred goats using next-generation sequencing (Illumina platform) to compare the genomes of the two populations. We found decreased nucleotide diversity in the native Korean goats compared to the crossbred goats. Genetic structural analysis demonstrated that the native Korean goat and crossbred goat populations shared a common ancestry, but were clearly distinct. Finally, to reveal the native Korean goat’s selective sweep region, selective sweep signals were identified in the native Korean goat genome using cross-population extended haplotype homozygosity (XP-EHH) and a cross-population composite likelihood ratio test (XP-CLR). As a result, we were able to identify candidate genes for recent selection, such as the CCR3 gene, which is related to lumbar paralysis resistance. Combined with future studies and recent goat genome information, this study will contribute to a thorough understanding of the native Korean goat genome. PMID:27989103

  13. Development of next generation sequencing panel for UMOD and association with kidney disease.

    Directory of Open Access Journals (Sweden)

    Caitlin Bailie

    Full Text Available Chronic kidney disease (CKD has a prevalence of approximately 10% in adult populations. CKD can progress to end-stage renal disease (ESRD and this is usually fatal unless some form of renal replacement therapy (chronic dialysis or renal transplantation is provided. There is an inherited predisposition to CKD with several genetic risk markers now identified. The UMOD gene has been associated with CKD of varying aetiologies. An AmpliSeq next generation sequencing panel was developed to facilitate comprehensive sequencing of the UMOD gene, covering exonic and regulatory regions. SNPs and CpG sites in the genomic region encompassing UMOD were evaluated for association with CKD in two studies; the UK Wellcome Trust Case-Control 3 Renal Transplant Dysfunction Study (n = 1088 and UK-ROI GENIE GWAS (n = 1726. A technological comparison of two Ion Torrent machines revealed 100% allele call concordance between S5 XL™ and PGM™ machines. One SNP (rs183962941, located in a non-coding region of UMOD, was nominally associated with ESRD (p = 0.008. No association was identified between UMOD variants and estimated glomerular filtration rate. Analysis of methylation data for over 480,000 CpG sites revealed differential methylation patterns within UMOD, the most significant of these was cg03140788 p = 3.7 x 10-10.

  14. Detecting Positive Selection of Korean Native Goat Populations Using Next-Generation Sequencing.

    Science.gov (United States)

    Lee, Wonseok; Ahn, Sojin; Taye, Mengistie; Sung, Samsun; Lee, Hyun-Jeong; Cho, Seoae; Kim, Heebal

    2016-12-01

    Goats ( Capra hircus ) are one of the oldest species of domesticated animals. Native Korean goats are a particularly interesting group, as they are indigenous to the area and were raised in the Korean peninsula almost 2,000 years ago. Although they have a small body size and produce low volumes of milk and meat, they are quite resistant to lumbar paralysis. Our study aimed to reveal the distinct genetic features and patterns of selection in native Korean goats by comparing the genomes of native Korean goat and crossbred goat populations. We sequenced the whole genome of 15 native Korean goats and 11 crossbred goats using next-generation sequencing (Illumina platform) to compare the genomes of the two populations. We found decreased nucleotide diversity in the native Korean goats compared to the crossbred goats. Genetic structural analysis demonstrated that the native Korean goat and crossbred goat populations shared a common ancestry, but were clearly distinct. Finally, to reveal the native Korean goat's selective sweep region, selective sweep signals were identified in the native Korean goat genome using cross-population extended haplotype homozygosity (XP-EHH) and a cross-population composite likelihood ratio test (XP-CLR). As a result, we were able to identify candidate genes for recent selection, such as the CCR3 gene, which is related to lumbar paralysis resistance. Combined with future studies and recent goat genome information, this study will contribute to a thorough understanding of the native Korean goat genome.

  15. nQuire: a statistical framework for ploidy estimation using next generation sequencing.

    Science.gov (United States)

    Weiß, Clemens L; Pais, Marina; Cano, Liliana M; Kamoun, Sophien; Burbano, Hernán A

    2018-04-04

    Intraspecific variation in ploidy occurs in a wide range of species including pathogenic and nonpathogenic eukaryotes such as yeasts and oomycetes. Ploidy can be inferred indirectly - without measuring DNA content - from experiments using next-generation sequencing (NGS). We present nQuire, a statistical framework that distinguishes between diploids, triploids and tetraploids using NGS. The command-line tool models the distribution of base frequencies at variable sites using a Gaussian Mixture Model, and uses maximum likelihood to select the most plausible ploidy model. nQuire handles large genomes at high coverage efficiently and uses standard input file formats. We demonstrate the utility of nQuire analyzing individual samples of the pathogenic oomycete Phytophthora infestans and the Baker's yeast Saccharomyces cerevisiae. Using these organisms we show the dependence between reliability of the ploidy assignment and sequencing depth. Additionally, we employ normalized maximized log- likelihoods generated by nQuire to ascertain ploidy level in a population of samples with ploidy heterogeneity. Using these normalized values we cluster samples in three dimensions using multivariate Gaussian mixtures. The cluster assignments retrieved from a S. cerevisiae population recovered the true ploidy level in over 96% of samples. Finally, we show that nQuire can be used regionally to identify chromosomal aneuploidies. nQuire provides a statistical framework to study organisms with intraspecific variation in ploidy. nQuire is likely to be useful in epidemiological studies of pathogens, artificial selection experiments, and for historical or ancient samples where intact nuclei are not preserved. It is implemented as a stand-alone Linux command line tool in the C programming language and is available at https://github.com/clwgg/nQuire under the MIT license.

  16. CFSAN SNP Pipeline: an automated method for constructing SNP matrices from next-generation sequence data

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    Steve Davis

    2015-08-01

    Full Text Available The analysis of next-generation sequence (NGS data is often a fragmented step-wise process. For example, multiple pieces of software are typically needed to map NGS reads, extract variant sites, and construct a DNA sequence matrix containing only single nucleotide polymorphisms (i.e., a SNP matrix for a set of individuals. The management and chaining of these software pieces and their outputs can often be a cumbersome and difficult task. Here, we present CFSAN SNP Pipeline, which combines into a single package the mapping of NGS reads to a reference genome with Bowtie2, processing of those mapping (BAM files using SAMtools, identification of variant sites using VarScan, and production of a SNP matrix using custom Python scripts. We also introduce a Python package (CFSAN SNP Mutator that when given a reference genome will generate variants of known position against which we validate our pipeline. We created 1,000 simulated Salmonella enterica sp. enterica Serovar Agona genomes at 100× and 20× coverage, each containing 500 SNPs, 20 single-base insertions and 20 single-base deletions. For the 100× dataset, the CFSAN SNP Pipeline recovered 98.9% of the introduced SNPs and had a false positive rate of 1.04 × 10−6; for the 20× dataset 98.8% of SNPs were recovered and the false positive rate was 8.34 × 10−7. Based on these results, CFSAN SNP Pipeline is a robust and accurate tool that it is among the first to combine into a single executable the myriad steps required to produce a SNP matrix from NGS data. Such a tool is useful to those working in an applied setting (e.g., food safety traceback investigations as well as for those interested in evolutionary questions.

  17. Using next-generation sequencing to develop molecular diagnostics for Pseudoperonospora cubensis, the cucurbit downy mildew pathogen

    Science.gov (United States)

    Advances in Next Generation Sequencing (NGS) allow for rapid development of genomics resources needed to generate molecular diagnostics assays for infectious agents. NGS approaches are particularly helpful for organisms that cannot be cultured, such as the downy mildew pathogens, a group of biotrop...

  18. The Use of Next Generation Sequencing and Junction Sequence Analysis Bioinformatics to Achieve Molecular Characterization of Crops Improved Through Modern Biotechnology

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    David Kovalic

    2012-11-01

    Full Text Available The assessment of genetically modified (GM crops for regulatory approval currently requires a detailed molecular characterization of the DNA sequence and integrity of the transgene locus. In addition, molecular characterization is a critical component of event selection and advancement during product development. Typically, molecular characterization has relied on Southern blot analysis to establish locus and copy number along with targeted sequencing of polymerase chain reaction products spanning any inserted DNA to complete the characterization process. Here we describe the use of next generation (NexGen sequencing and junction sequence analysis bioinformatics in a new method for achieving full molecular characterization of a GM event without the need for Southern blot analysis. In this study, we examine a typical GM soybean [ (L. Merr.] line and demonstrate that this new method provides molecular characterization equivalent to the current Southern blot-based method. We also examine an event containing in vivo DNA rearrangement of multiple transfer DNA inserts to demonstrate that the new method is effective at identifying complex cases. Next generation sequencing and bioinformatics offers certain advantages over current approaches, most notably the simplicity, efficiency, and consistency of the method, and provides a viable alternative for efficiently and robustly achieving molecular characterization of GM crops.

  19. Identification of SNP and SSR Markers in Finger Millet Using Next Generation Sequencing Technologies.

    Science.gov (United States)

    Gimode, Davis; Odeny, Damaris A; de Villiers, Etienne P; Wanyonyi, Solomon; Dida, Mathews M; Mneney, Emmarold E; Muchugi, Alice; Machuka, Jesse; de Villiers, Santie M

    2016-01-01

    Finger millet is an important cereal crop in eastern Africa and southern India with excellent grain storage quality and unique ability to thrive in extreme environmental conditions. Since negligible attention has been paid to improving this crop to date, the current study used Next Generation Sequencing (NGS) technologies to develop both Simple Sequence Repeat (SSR) and Single Nucleotide Polymorphism (SNP) markers. Genomic DNA from cultivated finger millet genotypes KNE755 and KNE796 was sequenced using both Roche 454 and Illumina technologies. Non-organelle sequencing reads were assembled into 207 Mbp representing approximately 13% of the finger millet genome. We identified 10,327 SSRs and 23,285 non-homeologous SNPs and tested 101 of each for polymorphism across a diverse set of wild and cultivated finger millet germplasm. For the 49 polymorphic SSRs, the mean polymorphism information content (PIC) was 0.42, ranging from 0.16 to 0.77. We also validated 92 SNP markers, 80 of which were polymorphic with a mean PIC of 0.29 across 30 wild and 59 cultivated accessions. Seventy-six of the 80 SNPs were polymorphic across 30 wild germplasm with a mean PIC of 0.30 while only 22 of the SNP markers showed polymorphism among the 59 cultivated accessions with an average PIC value of 0.15. Genetic diversity analysis using the polymorphic SNP markers revealed two major clusters; one of wild and another of cultivated accessions. Detailed STRUCTURE analysis confirmed this grouping pattern and further revealed 2 sub-populations within wild E. coracana subsp. africana. Both STRUCTURE and genetic diversity analysis assisted with the correct identification of the new germplasm collections. These polymorphic SSR and SNP markers are a significant addition to the existing 82 published SSRs, especially with regard to the previously reported low polymorphism levels in finger millet. Our results also reveal an unexploited finger millet genetic resource that can be included in the regional

  20. Identification of SNP and SSR Markers in Finger Millet Using Next Generation Sequencing Technologies.

    Directory of Open Access Journals (Sweden)

    Davis Gimode

    Full Text Available Finger millet is an important cereal crop in eastern Africa and southern India with excellent grain storage quality and unique ability to thrive in extreme environmental conditions. Since negligible attention has been paid to improving this crop to date, the current study used Next Generation Sequencing (NGS technologies to develop both Simple Sequence Repeat (SSR and Single Nucleotide Polymorphism (SNP markers. Genomic DNA from cultivated finger millet genotypes KNE755 and KNE796 was sequenced using both Roche 454 and Illumina technologies. Non-organelle sequencing reads were assembled into 207 Mbp representing approximately 13% of the finger millet genome. We identified 10,327 SSRs and 23,285 non-homeologous SNPs and tested 101 of each for polymorphism across a diverse set of wild and cultivated finger millet germplasm. For the 49 polymorphic SSRs, the mean polymorphism information content (PIC was 0.42, ranging from 0.16 to 0.77. We also validated 92 SNP markers, 80 of which were polymorphic with a mean PIC of 0.29 across 30 wild and 59 cultivated accessions. Seventy-six of the 80 SNPs were polymorphic across 30 wild germplasm with a mean PIC of 0.30 while only 22 of the SNP markers showed polymorphism among the 59 cultivated accessions with an average PIC value of 0.15. Genetic diversity analysis using the polymorphic SNP markers revealed two major clusters; one of wild and another of cultivated accessions. Detailed STRUCTURE analysis confirmed this grouping pattern and further revealed 2 sub-populations within wild E. coracana subsp. africana. Both STRUCTURE and genetic diversity analysis assisted with the correct identification of the new germplasm collections. These polymorphic SSR and SNP markers are a significant addition to the existing 82 published SSRs, especially with regard to the previously reported low polymorphism levels in finger millet. Our results also reveal an unexploited finger millet genetic resource that can be included

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

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    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

  2. Integration of microbiological, epidemiological and next generation sequencing technologies data for the managing of nosocomial infections

    Directory of Open Access Journals (Sweden)

    Matteo Brilli

    2018-02-01

    Full Text Available At its core, the work of clinical microbiologists consists in the retrieving of a few bytes of information (species identification; metabolic capacities; staining and antigenic properties; antibiotic resistance profiles, etc. from pathogenic agents. The development of next generation sequencing technologies (NGS, and the possibility to determine the entire genome for bacterial pathogens, fungi and protozoans will likely introduce a breakthrough in the amount of information generated by clinical microbiology laboratories: from bytes to Megabytes of information, for a single isolate. In parallel, the development of novel informatics tools, designed for the management and analysis of the so-called Big Data, offers the possibility to search for patterns in databases collecting genomic and microbiological information on the pathogens, as well as epidemiological data and information on the clinical parameters of the patients. Nosocomial infections and antibiotic resistance will likely represent major challenges for clinical microbiologists, in the next decades. In this paper, we describe how bacterial genomics based on NGS, integrated with novel informatic tools, could contribute to the control of hospital infections and multi-drug resistant pathogens.

  3. ChronQC: a quality control monitoring system for clinical next generation sequencing.

    Science.gov (United States)

    Tawari, Nilesh R; Seow, Justine Jia Wen; Perumal, Dharuman; Ow, Jack L; Ang, Shimin; Devasia, Arun George; Ng, Pauline C

    2018-05-15

    ChronQC is a quality control (QC) tracking system for clinical implementation of next-generation sequencing (NGS). ChronQC generates time series plots for various QC metrics to allow comparison of current runs to historical runs. ChronQC has multiple features for tracking QC data including Westgard rules for clinical validity, laboratory-defined thresholds and historical observations within a specified time period. Users can record their notes and corrective actions directly onto the plots for long-term recordkeeping. ChronQC facilitates regular monitoring of clinical NGS to enable adherence to high quality clinical standards. ChronQC is freely available on GitHub (https://github.com/nilesh-tawari/ChronQC), Docker (https://hub.docker.com/r/nileshtawari/chronqc/) and the Python Package Index. ChronQC is implemented in Python and runs on all common operating systems (Windows, Linux and Mac OS X). tawari.nilesh@gmail.com or pauline.c.ng@gmail.com. Supplementary data are available at Bioinformatics online.

  4. Structural variation discovery in the cancer genome using next generation sequencing: Computational solutions and perspectives

    Science.gov (United States)

    Liu, Biao; Conroy, Jeffrey M.; Morrison, Carl D.; Odunsi, Adekunle O.; Qin, Maochun; Wei, Lei; Trump, Donald L.; Johnson, Candace S.; Liu, Song; Wang, Jianmin

    2015-01-01

    Somatic Structural Variations (SVs) are a complex collection of chromosomal mutations that could directly contribute to carcinogenesis. Next Generation Sequencing (NGS) technology has emerged as the primary means of interrogating the SVs of the cancer genome in recent investigations. Sophisticated computational methods are required to accurately identify the SV events and delineate their breakpoints from the massive amounts of reads generated by a NGS experiment. In this review, we provide an overview of current analytic tools used for SV detection in NGS-based cancer studies. We summarize the features of common SV groups and the primary types of NGS signatures that can be used in SV detection methods. We discuss the principles and key similarities and differences of existing computational programs and comment on unresolved issues related to this research field. The aim of this article is to provide a practical guide of relevant concepts, computational methods, software tools and important factors for analyzing and interpreting NGS data for the detection of SVs in the cancer genome. PMID:25849937

  5. repDNA: a Python package to generate various modes of feature vectors for DNA sequences by incorporating user-defined physicochemical properties and sequence-order effects.

    Science.gov (United States)

    Liu, Bin; Liu, Fule; Fang, Longyun; Wang, Xiaolong; Chou, Kuo-Chen

    2015-04-15

    In order to develop powerful computational predictors for identifying the biological features or attributes of DNAs, one of the most challenging problems is to find a suitable approach to effectively represent the DNA sequences. To facilitate the studies of DNAs and nucleotides, we developed a Python package called representations of DNAs (repDNA) for generating the widely used features reflecting the physicochemical properties and sequence-order effects of DNAs and nucleotides. There are three feature groups composed of 15 features. The first group calculates three nucleic acid composition features describing the local sequence information by means of kmers; the second group calculates six autocorrelation features describing the level of correlation between two oligonucleotides along a DNA sequence in terms of their specific physicochemical properties; the third group calculates six pseudo nucleotide composition features, which can be used to represent a DNA sequence with a discrete model or vector yet still keep considerable sequence-order information via the physicochemical properties of its constituent oligonucleotides. In addition, these features can be easily calculated based on both the built-in and user-defined properties via using repDNA. The repDNA Python package is freely accessible to the public at http://bioinformatics.hitsz.edu.cn/repDNA/. bliu@insun.hit.edu.cn or kcchou@gordonlifescience.org Supplementary data are available at Bioinformatics online. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  6. Design and optimization of a high-efficiency array generator in the mid-IR with binary subwavelength grooves.

    Science.gov (United States)

    Bloom, Guillaume; Larat, Christian; Lallier, Eric; Lee-Bouhours, Mane-Si Laure; Loiseaux, Brigitte; Huignard, Jean-Pierre

    2011-02-10

    We have designed a high-efficiency array generator composed of subwavelength grooves etched in a GaAs substrate for operation at 4.5 μm. The method used combines rigorous coupled wave analysis with an optimization algorithm. The optimized beam splitter has both a high efficiency (∼96%) and a good intensity uniformity (∼0.2%). The fabrication error tolerances are numerically calculated, and it is shown that this subwavelength array generator could be fabricated with current electron beam writers and inductively coupled plasma etching. Finally, we studied the effect of a simple and realistic antireflection coating on the performance of the beam splitter.

  7. The origin of the RS CVn binaries

    International Nuclear Information System (INIS)

    Biermann, P.

    1976-01-01

    Six possible origins for the RS CVn binaries are considered based on the following possibilities. RS CVn binaries might now be either pre-main-sequence or post-main-sequence. A pre-main-sequence binary might not always have been a binary but might have resulted from fission of a rapidly rotating single pre-main-sequence star. The main-sequence counterparts might be either single stars or binaries. To decide which of the six origins is possible, the following observed data for the RS CVn binaries are considered: total mass, total angular momentum, lack of observed connection with regions of star formation, large space density, kinematical age, and the visual companion of WW Dra. In addition lifetimes and space densities of single stars and other types of binaries are considered. The only origin possible is that the RS CVn binaries are in a thermal phase following fission of a main-sequence single star. In this explanation the single star had a rapidly rotating core which became unstable due to the core contraction which made it begin to evolve off the main sequence. The present Be stars might be examples of such parent single stars. (Auth.)

  8. Molecular Diagnostics of Gliomas Using Next Generation Sequencing of a Glioma-Tailored Gene Panel.

    Science.gov (United States)

    Zacher, Angela; Kaulich, Kerstin; Stepanow, Stefanie; Wolter, Marietta; Köhrer, Karl; Felsberg, Jörg; Malzkorn, Bastian; Reifenberger, Guido

    2017-03-01

    Current classification of gliomas is based on histological criteria according to the World Health Organization (WHO) classification of tumors of the central nervous system. Over the past years, characteristic genetic profiles have been identified in various glioma types. These can refine tumor diagnostics and provide important prognostic and predictive information. We report on the establishment and validation of gene panel next generation sequencing (NGS) for the molecular diagnostics of gliomas. We designed a glioma-tailored gene panel covering 660 amplicons derived from 20 genes frequently aberrant in different glioma types. Sensitivity and specificity of glioma gene panel NGS for detection of DNA sequence variants and copy number changes were validated by single gene analyses. NGS-based mutation detection was optimized for application on formalin-fixed paraffin-embedded tissue specimens including small stereotactic biopsy samples. NGS data obtained in a retrospective analysis of 121 gliomas allowed for their molecular classification into distinct biological groups, including (i) isocitrate dehydrogenase gene (IDH) 1 or 2 mutant astrocytic gliomas with frequent α-thalassemia/mental retardation syndrome X-linked (ATRX) and tumor protein p53 (TP53) gene mutations, (ii) IDH mutant oligodendroglial tumors with 1p/19q codeletion, telomerase reverse transcriptase (TERT) promoter mutation and frequent Drosophila homolog of capicua (CIC) gene mutation, as well as (iii) IDH wildtype glioblastomas with frequent TERT promoter mutation, phosphatase and tensin homolog (PTEN) mutation and/or epidermal growth factor receptor (EGFR) amplification. Oligoastrocytic gliomas were genetically assigned to either of these groups. Our findings implicate gene panel NGS as a promising diagnostic technique that may facilitate integrated histological and molecular glioma classification. © 2016 International Society of Neuropathology.

  9. Molecular diagnostics for congenital hearing loss including 15 deafness genes using a next generation sequencing platform

    Directory of Open Access Journals (Sweden)

    De Keulenaer Sarah

    2012-05-01

    Full Text Available Abstract Background Hereditary hearing loss (HL can originate from mutations in one of many genes involved in the complex process of hearing. Identification of the genetic defects in patients is currently labor intensive and expensive. While screening with Sanger sequencing for GJB2 mutations is common, this is not the case for the other known deafness genes (> 60. Next generation sequencing technology (NGS has the potential to be much more cost efficient. Published methods mainly use hybridization based target enrichment procedures that are time saving and efficient, but lead to loss in sensitivity. In this study we used a semi-automated PCR amplification and NGS in order to combine high sensitivity, speed and cost efficiency. Results In this proof of concept study, we screened 15 autosomal recessive deafness genes in 5 patients with congenital genetic deafness. 646 specific primer pairs for all exons and most of the UTR of the 15 selected genes were designed using primerXL. Using patient specific identifiers, all amplicons were pooled and analyzed using the Roche 454 NGS technology. Three of these patients are members of families in which a region of interest has previously been characterized by linkage studies. In these, we were able to identify two new mutations in CDH23 and OTOF. For another patient, the etiology of deafness was unclear, and no causal mutation was found. In a fifth patient, included as a positive control, we could confirm a known mutation in TMC1. Conclusions We have developed an assay that holds great promise as a tool for screening patients with familial autosomal recessive nonsyndromal hearing loss (ARNSHL. For the first time, an efficient, reliable and cost effective genetic test, based on PCR enrichment, for newborns with undiagnosed deafness is available.

  10. Ligation bias in illumina next-generation DNA libraries: implications for sequencing ancient genomes.

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    Andaine Seguin-Orlando

    Full Text Available Ancient DNA extracts consist of a mixture of endogenous molecules and contaminant DNA templates, often originating from environmental microbes. These two populations of templates exhibit different chemical characteristics, with the former showing depurination and cytosine deamination by-products, resulting from post-mortem DNA damage. Such chemical modifications can interfere with the molecular tools used for building second-generation DNA libraries, and limit our ability to fully characterize the true complexity of ancient DNA extracts. In this study, we first use fresh DNA extracts to demonstrate that library preparation based on adapter ligation at AT-overhangs are biased against DNA templates starting with thymine residues, contrarily to blunt-end adapter ligation. We observe the same bias on fresh DNA extracts sheared on Bioruptor, Covaris and nebulizers. This contradicts previous reports suggesting that this bias could originate from the methods used for shearing DNA. This also suggests that AT-overhang adapter ligation efficiency is affected in a sequence-dependent manner and results in an uneven representation of different genomic contexts. We then show how this bias could affect the base composition of ancient DNA libraries prepared following AT-overhang ligation, mainly by limiting the ability to ligate DNA templates starting with thymines and therefore deaminated cytosines. This results in particular nucleotide misincorporation damage patterns, deviating from the signature generally expected for authenticating ancient sequence data. Consequently, we show that models adequate for estimating post-mortem DNA damage levels must be robust to the molecular tools used for building ancient DNA libraries.

  11. Next-generation sequencing in NSCLC and melanoma patients: a cost and budget impact analysis

    Science.gov (United States)

    van Amerongen, Rosa A; Retèl, Valesca P; Coupé, Veerle MH; Nederlof, Petra M; Vogel, Maartje J; van Harten, Wim H

    2016-01-01

    Next-generation sequencing (NGS) has reached the molecular diagnostic laboratories. Although the NGS technology aims to improve the effectiveness of therapies by selecting the most promising therapy, concerns are that NGS testing is expensive and that the ‘benefits’ are not yet in relation to these costs. In this study, we give an estimation of the costs and an institutional and national budget impact of various types of NGS tests in non-small-cell lung cancer (NSCLC) and melanoma patients within The Netherlands. First, an activity-based costing (ABC) analysis has been conducted on the costs of two examples of NGS panels (small- and medium-targeted gene panel (TGP)) based on data of The Netherlands Cancer Institute (NKI). Second, we performed a budget impact analysis (BIA) to estimate the current (2015) and future (2020) budget impact of NGS on molecular diagnostics for NSCLC and melanoma patients in The Netherlands. Literature, expert opinions, and a data set of patients within the NKI (n = 172) have been included in the BIA. Based on our analysis, we expect that the NGS test cost concerns will be limited. In the current situation, NGS can indeed result in higher diagnostic test costs, which is mainly related to required additional tests besides the small TGP. However, in the future, we expect that the use of whole-genome sequencing (WGS) will increase, for which it is expected that additional tests can be (partly) avoided. Although the current clinical benefits are expected to be limited, the research potentials of NGS are already an important advantage. PMID:27899957

  12. A Next-Generation Sequencing Strategy for Evaluating the Most Common Genetic Abnormalities in Multiple Myeloma.

    Science.gov (United States)

    Jiménez, Cristina; Jara-Acevedo, María; Corchete, Luis A; Castillo, David; Ordóñez, Gonzalo R; Sarasquete, María E; Puig, Noemí; Martínez-López, Joaquín; Prieto-Conde, María I; García-Álvarez, María; Chillón, María C; Balanzategui, Ana; Alcoceba, Miguel; Oriol, Albert; Rosiñol, Laura; Palomera, Luis; Teruel, Ana I; Lahuerta, Juan J; Bladé, Joan; Mateos, María V; Orfão, Alberto; San Miguel, Jesús F; González, Marcos; Gutiérrez, Norma C; García-Sanz, Ramón

    2017-01-01

    Identification and characterization of genetic alterations are essential for diagnosis of multiple myeloma and may guide therapeutic decisions. Currently, genomic analysis of myeloma to cover the diverse range of alterations with prognostic impact requires fluorescence in situ hybridization (FISH), single nucleotide polymorphism arrays, and sequencing techniques, which are costly and labor intensive and require large numbers of plasma cells. To overcome these limitations, we designed a targeted-capture next-generation sequencing approach for one-step identification of IGH translocations, V(D)J clonal rearrangements, the IgH isotype, and somatic mutations to rapidly identify risk groups and specific targetable molecular lesions. Forty-eight newly diagnosed myeloma patients were tested with the panel, which included IGH and six genes that are recurrently mutated in myeloma: NRAS, KRAS, HRAS, TP53, MYC, and BRAF. We identified 14 of 17 IGH translocations previously detected by FISH and three confirmed translocations not detected by FISH, with the additional advantage of breakpoint identification, which can be used as a target for evaluating minimal residual disease. IgH subclass and V(D)J rearrangements were identified in 77% and 65% of patients, respectively. Mutation analysis revealed the presence of missense protein-coding alterations in at least one of the evaluating genes in 16 of 48 patients (33%). This method may represent a time- and cost-effective diagnostic method for the molecular characterization of multiple myeloma. Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

  13. Estimating Exceptionally Rare Germline and Somatic Mutation Frequencies via Next Generation Sequencing.

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    Jordan Eboreime

    Full Text Available We used targeted next generation deep-sequencing (Safe Sequencing System to measure ultra-rare de novo mutation frequencies in the human male germline by attaching a unique identifier code to each target DNA molecule. Segments from three different human genes (FGFR3, MECP2 and PTPN11 were studied. Regardless of the gene segment, the particular testis donor or the 73 different testis pieces used, the frequencies for any one of the six different mutation types were consistent. Averaging over the C>T/G>A and G>T/C>A mutation types the background mutation frequency was 2.6x10-5 per base pair, while for the four other mutation types the average background frequency was lower at 1.5x10-6 per base pair. These rates far exceed the well documented human genome average frequency per base pair (~10-8 suggesting a non-biological explanation for our data. By computational modeling and a new experimental procedure to distinguish between pre-mutagenic lesion base mismatches and a fully mutated base pair in the original DNA molecule, we argue that most of the base-dependent variation in background frequency is due to a mixture of deamination and oxidation during the first two PCR cycles. Finally, we looked at a previously studied disease mutation in the PTPN11 gene and could easily distinguish true mutations from the SSS background. We also discuss the limits and possibilities of this and other methods to measure exceptionally rare mutation frequencies, and we present calculations for other scientists seeking to design their own such experiments.

  14. Identification of HIV Mutation as Diagnostic Biomarker through Next Generation Sequencing.

    Science.gov (United States)

    Shaw, Wen Hui; Lin, Qianqian; Muhammad, Zikry Zhiwei Bin Roslee; Lee, Jia Jun; Khong, Wei Xin; Ng, Oon Tek; Tan, Eng Lee; Li, Peng

    2016-07-01

    Current clinical detection of Human immunodeficiency virus 1 (HIV-1) is used to target viral genes and proteins. However, the immunoassay, such as viral culture or Polymerase Chain Reaction (PCR), lacks accuracy in the diagnosis, as these conventional assays rely on the stable genome and HIV-1 is a highly-mutated virus. Next generation sequencing (NGS) promises to be transformative for the practice of infectious disease, and the rapidly reducing cost and processing time mean that this will become a feasible technology in diagnostic and research laboratories in the near future. The technology offers the superior sensitivity to detect the pathogenic viruses, including unknown and unexpected strains. To leverage the NGS technology in order to improve current HIV-1 diagnosis and genotyping methods. Ten blood samples were collected from HIV-1 infected patients which were diagnosed by RT PCR at Singapore Communicable Disease Centre, Tan Tock Seng Hospital from October 2014 to March 2015. Viral RNAs were extracted from blood plasma and reversed into cDNA. The HIV-1 cDNA samples were cleaned up using a PCR purification kit and the sequencing library was prepared and identified through MiSeq. Two common mutations were observed in all ten samples. The common mutations were identified at genome locations 1908 and 2104 as missense and silent mutations respectively, conferring S37N and S3S found on aspartic protease and reverse transcriptase subunits. The common mutations identified in this study were not previously reported, therefore suggesting the potential for them to be used for identification of viral infection, disease transmission and drug resistance. This was especially the case for, missense mutation S37N which could cause an amino acid change in viral proteases thus reducing the binding affinity of some protease inhibitors. Thus, the unique common mutations identified in this study could be used as diagnostic biomarkers to indicate the origin of infection as being

  15. Killer Immunoglobulin-Like Receptor Allele Determination Using Next-Generation Sequencing Technology

    Directory of Open Access Journals (Sweden)

    Bercelin Maniangou

    2017-05-01

    Full Text Available The impact of natural killer (NK cell alloreactivity on hematopoietic stem cell transplantation (HSCT outcome is still debated due to the complexity of graft parameters, HLA class I environment, the nature of killer cell immunoglobulin-like receptor (KIR/KIR ligand genetic combinations studied, and KIR+ NK cell repertoire size. KIR genes are known to be polymorphic in terms of gene content, copy number variation, and number of alleles. These allelic polymorphisms may impact both the phenotype and function of KIR+ NK cells. We, therefore, speculate that polymorphisms may alter donor KIR+ NK cell phenotype/function thus modulating post-HSCT KIR+ NK cell alloreactivity. To investigate KIR allele polymorphisms of all KIR genes, we developed a next-generation sequencing (NGS technology on a MiSeq platform. To ensure the reliability and specificity of our method, genomic DNA from well-characterized cell lines were used; high-resolution KIR typing results obtained were then compared to those previously reported. Two different bioinformatic pipelines were used allowing the attribution of sequencing reads to specific KIR genes and the assignment of KIR alleles for each KIR gene. Our results demonstrated successful long-range KIR gene amplifications of all reference samples using intergenic KIR primers. The alignment of reads to the human genome reference (hg19 using BiRD pipeline or visualization of data using Profiler software demonstrated that all KIR genes were completely sequenced with a sufficient read depth (mean 317× for all loci and a high percentage of mapping (mean 93% for all loci. Comparison of high-resolution KIR typing obtained to those published data using exome capture resulted in a reported concordance rate of 95% for centromeric and telomeric KIR genes. Overall, our results suggest that NGS can be used to investigate the broad KIR allelic polymorphism. Hence, these data improve our knowledge, not only on KIR+ NK cell alloreactivity in

  16. Next-generation sequencing (NGS) for assessment of microbial water quality: current progress, challenges, and future opportunities

    OpenAIRE

    BoonFei eTan; Charmaine Marie Ng; Jean Pierre Nshimyimana; Jean Pierre Nshimyimana; Lay-Leng eLoh; Lay-Leng eLoh; Karina Yew-Hoong Gin; Janelle Renee Thompson; Janelle Renee Thompson

    2015-01-01

    Water quality is an emergent property of a complex system comprised of interacting microbial populations and introduced microbial and chemical contaminants. Studies leveraging next-generation sequencing (NGS) technologies are providing new insights into the ecology of microbially mediated processes that influence fresh water quality such as algal blooms, contaminant biodegradation, and pathogen dissemination. In addition, sequencing methods targeting small subunit (SSU) rRNA hypervariable reg...

  17. Depletion of Human DNA in Spiked Clinical Specimens for Improvement of Sensitivity of Pathogen Detection by Next-Generation Sequencing

    OpenAIRE

    Hasan, Mohammad R.; Rawat, Arun; Tang, Patrick; Jithesh, Puthen V.; Thomas, Eva; Tan, Rusung; Tilley, Peter

    2016-01-01

    Next-generation sequencing (NGS) technology has shown promise for the detection of human pathogens from clinical samples. However, one of the major obstacles to the use of NGS in diagnostic microbiology is the low ratio of pathogen DNA to human DNA in most clinical specimens. In this study, we aimed to develop a specimen-processing protocol to remove human DNA and enrich specimens for bacterial and viral DNA for shotgun metagenomic sequencing. Cerebrospinal fluid (CSF) and nasopharyngeal aspi...

  18. Highly conserved intragenic HSV-2 sequences: Results from next-generation sequencing of HSV-2 UL and US regions from genital swabs collected from 3 continents.

    Science.gov (United States)

    Johnston, Christine; Magaret, Amalia; Roychoudhury, Pavitra; Greninger, Alexander L; Cheng, Anqi; Diem, Kurt; Fitzgibbon, Matthew P; Huang, Meei-Li; Selke, Stacy; Lingappa, Jairam R; Celum, Connie; Jerome, Keith R; Wald, Anna; Koelle, David M

    2017-10-01

    Understanding the variability in circulating herpes simplex virus type 2 (HSV-2) genomic sequences is critical to the development of HSV-2 vaccines. Genital lesion swabs containing ≥ 10 7 log 10 copies HSV DNA collected from Africa, the USA, and South America underwent next-generation sequencing, followed by K-mer based filtering and de novo genomic assembly. Sites of heterogeneity within coding regions in unique long and unique short (U L _U S ) regions were identified. Phylogenetic trees were created using maximum likelihood reconstruction. Among 46 samples from 38 persons, 1468 intragenic base-pair substitutions were identified. The maximum nucleotide distance between strains for concatenated U L_ U S segments was 0.4%. Phylogeny did not reveal geographic clustering. The most variable proteins had non-synonymous mutations in < 3% of amino acids. Unenriched HSV-2 DNA can undergo next-generation sequencing to identify intragenic variability. The use of clinical swabs for sequencing expands the information that can be gathered directly from these specimens. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Discovery and validation of Barrett's esophagus microRNA transcriptome by next generation sequencing.

    Directory of Open Access Journals (Sweden)

    Ajay Bansal

    Full Text Available Barrett's esophagus (BE is transition from squamous to columnar mucosa as a result of gastroesophageal reflux disease (GERD. The role of microRNA during this transition has not been systematically studied.For initial screening, total RNA from 5 GERD and 6 BE patients was size fractionated. RNA <70 nucleotides was subjected to SOLiD 3 library preparation and next generation sequencing (NGS. Bioinformatics analysis was performed using R package "DEseq". A p value<0.05 adjusted for a false discovery rate of 5% was considered significant. NGS-identified miRNA were validated using qRT-PCR in an independent group of 40 GERD and 27 BE patients. MicroRNA expression of human BE tissues was also compared with three BE cell lines.NGS detected 19.6 million raw reads per sample. 53.1% of filtered reads mapped to miRBase version 18. NGS analysis followed by qRT-PCR validation found 10 differentially expressed miRNA; several are novel (-708-5p, -944, -224-5p and -3065-5p. Up- or down- regulation predicted by NGS was matched by qRT-PCR in every case. Human BE tissues and BE cell lines showed a high degree of concordance (70-80% in miRNA expression. Prediction analysis identified targets that mapped to developmental signaling pathways such as TGFβ and Notch and inflammatory pathways such as toll-like receptor signaling and TGFβ. Cluster analysis found similarly regulated (up or down miRNA to share common targets suggesting coordination between miRNA.Using highly sensitive next-generation sequencing, we have performed a comprehensive genome wide analysis of microRNA in BE and GERD patients. Differentially expressed miRNA between BE and GERD have been further validated. Expression of miRNA between BE human tissues and BE cell lines are highly correlated. These miRNA should be studied in biological models to further understand BE development.

  20. The de novo assembly of mitochondrial genomes of the extinct passenger pigeon (Ectopistes migratorius with next generation sequencing.

    Directory of Open Access Journals (Sweden)

    Chih-Ming Hung

    Full Text Available The information from ancient DNA (aDNA provides an unparalleled opportunity to infer phylogenetic relationships and population history of extinct species and to investigate genetic evolution directly. However, the degraded and fragmented nature of aDNA has posed technical challenges for studies based on conventional PCR amplification. In this study, we present an approach based on next generation sequencing to efficiently sequence the complete mitochondrial genome (mitogenome of two extinct passenger pigeons (Ectopistes migratorius using de novo assembly of massive short (90 bp, paired-end or single-end reads. Although varying levels of human contamination and low levels of postmortem nucleotide lesion were observed, they did not impact sequencing accuracy. Our results demonstrated that the de novo assembly of shotgun sequence reads could be a potent approach to sequence mitogenomes, and offered an efficient way to infer evolutionary history of extinct species.

  1. The De Novo Assembly of Mitochondrial Genomes of the Extinct Passenger Pigeon (Ectopistes migratorius) with Next Generation Sequencing

    Science.gov (United States)

    Hung, Chih-Ming; Lin, Rong-Chien; Chu, Jui-Hua; Yeh, Chia-Fen; Yao, Chiou-Ju; Li, Shou-Hsien

    2013-01-01

    The information from ancient DNA (aDNA) provides an unparalleled opportunity to infer phylogenetic relationships and population history of extinct species and to investigate genetic evolution directly. However, the degraded and fragmented nature of aDNA has posed technical challenges for studies based on conventional PCR amplification. In this study, we present an approach based on next generation sequencing to efficiently sequence the complete mitochondrial genome (mitogenome) of two extinct passenger pigeons (Ectopistes migratorius) using de novo assembly of massive short (90 bp), paired-end or single-end reads. Although varying levels of human contamination and low levels of postmortem nucleotide lesion were observed, they did not impact sequencing accuracy. Our results demonstrated that the de novo assembly of shotgun sequence reads could be a potent approach to sequence mitogenomes, and offered an efficient way to infer evolutionary history of extinct species. PMID:23437111

  2. Long-PCR based next generation sequencing of the whole mitochondrial genome of the peacock skate Pavoraja nitida (Elasmobranchii: Arhynchobatidae).

    Science.gov (United States)

    Yang, Lei; Naylor, Gavin J P

    2016-01-01

    We determined the complete mitochondrial genome sequence (16,760 bp) of the peacock skate Pavoraja nitida using a long-PCR based next generation sequencing method. It has 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and 1 control region in the typical vertebrate arrangement. Primers, protocols, and procedures used to obtain this mitogenome are provided. We anticipate that this approach will facilitate rapid collection of mitogenome sequences for studies on phylogenetic relationships, population genetics, and conservation of cartilaginous fishes.

  3. Translational Bioinformatics for Diagnostic and Prognostic Prediction of Prostate Cancer in the Next-Generation Sequencing Era

    Directory of Open Access Journals (Sweden)

    Jiajia Chen

    2013-01-01

    Full Text Available The discovery of prostate cancer biomarkers has been boosted by the advent of next-generation sequencing (NGS technologies. Nevertheless, many challenges still exist in exploiting the flood of sequence data and translating them into routine diagnostics and prognosis of prostate cancer. Here we review the recent developments in prostate cancer biomarkers by high throughput sequencing technologies. We highlight some fundamental issues of translational bioinformatics and the potential use of cloud computing in NGS data processing for the improvement of prostate cancer treatment.

  4. Pseudo-random Trees: Multiple Independent Sequence Generators for Parallel and Branching Computations

    Science.gov (United States)

    Halton, John H.

    1989-09-01

    A class of families of linear congruential pseudo-random sequences is defined, for which it is possible to branch at any event without changing the sequence of random numbers used in the original random walk and for which the sequences in different branches show properties analogous to mutual statistical independence. This is a hitherto unavailable, and computationally desirable, tool.

  5. miRanalyzer: a microRNA detection and analysis tool for next-generation sequencing experiments.

    Science.gov (United States)

    Hackenberg, Michael; Sturm, Martin; Langenberger, David; Falcón-Pérez, Juan Manuel; Aransay, Ana M

    2009-07-01

    Next-generation sequencing allows now the sequencing of small RNA molecules and the estimation of their expression levels. Consequently, there will be a high demand of bioinformatics tools to cope with the several gigabytes of sequence data generated in each single deep-sequencing experiment. Given this scene, we developed miRanalyzer, a web server tool for the analysis of deep-sequencing experiments for small RNAs. The web server tool requires a simple input file containing a list of unique reads and its copy numbers (expression levels). Using these data, miRanalyzer (i) detects all known microRNA sequences annotated in miRBase, (ii) finds all perfect matches against other libraries of transcribed sequences and (iii) predicts new microRNAs. The prediction of new microRNAs is an especially important point as there are many species with very few known microRNAs. Therefore, we implemented a highly accurate machine learning algorithm for the prediction of new microRNAs that reaches AUC values of 97.9% and recall values of up to 75% on unseen data. The web tool summarizes all the described steps in a single output page, which provides a comprehensive overview of the analysis, adding links to more detailed output pages for each analysis module. miRanalyzer is available at http://web.bioinformatics.cicbiogune.es/microRNA/.

  6. A white dwarf companion to the main-sequence star 4 Omicron(1) Orionis and the binary hypothesis for the origin of peculiar red giants

    Science.gov (United States)

    Ake, Thomas B.; Johnson, Hollis R.

    1988-01-01

    Ultraviolet spectra of the peculiar red giants (PRGs) called MS stars are investigated, and the discovery of a white dwarf (WD) companion to the MS star 4 Omicron(1) Orionis is reported. The observations and data analysis are discussed and compared with those for field WDs in order to derive parameters for the WD and the luminosity of the primary. Detection limits for the other MS stars investigated are derived, and the binary hypothesis for PRGs is reviewed.

  7. Human papillomavirus genome integration in squamous carcinogenesis: what have next-generation sequencing studies taught us?

    Science.gov (United States)

    Groves, Ian J; Coleman, Nicholas

    2018-05-01

    Human papillomavirus (HPV) infection is associated with ∼5% of all human cancers, including a range of squamous cell carcinomas. Persistent infection by high-risk HPVs (HRHPVs) is associated with the integration of virus genomes (which are usually stably maintained as extrachromosomal episomes) into host chromosomes. Although HRHPV integration rates differ across human sites of infection, this process appears to be an important event in HPV-associated neoplastic progression, leading to deregulation of virus oncogene expression, host gene expression modulation, and further genomic instability. However, the mechanisms by which HRHPV integration occur and by which the subsequent gene expression changes take place are incompletely understood. The advent of next-generation sequencing (NGS) of both RNA and DNA has allowed powerful interrogation of the association of HRHPVs with human disease, including precise determination of the sites of integration and the genomic rearrangements at integration loci. In turn, these data have indicated that integration occurs through two main mechanisms: looping integration and direct insertion. Improved understanding of integration sites is allowing further investigation of the factors that provide a competitive advantage to some integrants during disease progression. Furthermore, advanced approaches to the generation of genome-wide samples have given novel insights into the three-dimensional interactions within the nucleus, which could act as another layer of epigenetic control of both virus and host transcription. It is hoped that further advances in NGS techniques and analysis will not only allow the examination of further unanswered questions regarding HPV infection, but also direct new approaches to treating HPV-associated human disease. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John

  8. Population clustering based on copy number variations detected from next generation sequencing data.

    Science.gov (United States)

    Duan, Junbo; Zhang, Ji-Gang; Wan, Mingxi; Deng, Hong-Wen; Wang, Yu-Ping

    2014-08-01

    Copy number variations (CNVs) can be used as significant bio-markers and next generation sequencing (NGS) provides a high resolution detection of these CNVs. But how to extract features from CNVs and further apply them to genomic studies such as population clustering have become a big challenge. In this paper, we propose a novel method for population clustering based on CNVs from NGS. First, CNVs are extracted from each sample to form a feature matrix. Then, this feature matrix is decomposed into the source matrix and weight matrix with non-negative matrix factorization (NMF). The source matrix consists of common CNVs that are shared by all the samples from the same group, and the weight matrix indicates the corresponding level of CNVs from each sample. Therefore, using NMF of CNVs one can differentiate samples from different ethnic groups, i.e. population clustering. To validate the approach, we applied it to the analysis of both simulation data and two real data set from the 1000 Genomes Project. The results on simulation data demonstrate that the proposed method can recover the true common CNVs with high quality. The results on the first real data analysis show that the proposed method can cluster two family trio with different ancestries into two ethnic groups and the results on the second real data analysis show that the proposed method can be applied to the whole-genome with large sample size consisting of multiple groups. Both results demonstrate the potential of the proposed method for population clustering.

  9. Targeted next generation sequencing in Italian patients with Usher syndrome: phenotype-genotype correlations.

    Science.gov (United States)

    Eandi, Chiara M; Dallorto, Laura; Spinetta, Roberta; Micieli, Maria Pia; Vanzetti, Mario; Mariottini, Alessandro; Passerini, Ilaria; Torricelli, Francesca; Alovisi, Camilla; Marchese, Cristiana

    2017-11-15

    We report results of DNA analysis with next generation sequencing