WorldWideScience

Sample records for quantitative deep sequencing

  1. Quantitative phenotyping via deep barcode sequencing.

    Science.gov (United States)

    Smith, Andrew M; Heisler, Lawrence E; Mellor, Joseph; Kaper, Fiona; Thompson, Michael J; Chee, Mark; Roth, Frederick P; Giaever, Guri; Nislow, Corey

    2009-10-01

    Next-generation DNA sequencing technologies have revolutionized diverse genomics applications, including de novo genome sequencing, SNP detection, chromatin immunoprecipitation, and transcriptome analysis. Here we apply deep sequencing to genome-scale fitness profiling to evaluate yeast strain collections in parallel. This method, Barcode analysis by Sequencing, or "Bar-seq," outperforms the current benchmark barcode microarray assay in terms of both dynamic range and throughput. When applied to a complex chemogenomic assay, Bar-seq quantitatively identifies drug targets, with performance superior to the benchmark microarray assay. We also show that Bar-seq is well-suited for a multiplex format. We completely re-sequenced and re-annotated the yeast deletion collection using deep sequencing, found that approximately 20% of the barcodes and common priming sequences varied from expectation, and used this revised list of barcode sequences to improve data quality. Together, this new assay and analysis routine provide a deep-sequencing-based toolkit for identifying gene-environment interactions on a genome-wide scale.

  2. DNA Replication Profiling Using Deep Sequencing.

    Science.gov (United States)

    Saayman, Xanita; Ramos-Pérez, Cristina; Brown, Grant W

    2018-01-01

    Profiling of DNA replication during progression through S phase allows a quantitative snap-shot of replication origin usage and DNA replication fork progression. We present a method for using deep sequencing data to profile DNA replication in S. cerevisiae.

  3. DeepSimulator: a deep simulator for Nanopore sequencing

    KAUST Repository

    Li, Yu; Han, Renmin; Bi, Chongwei; Li, Mo; Wang, Sheng; Gao, Xin

    2017-01-01

    or assembled contigs, we simulate the electrical current signals by a context-dependent deep learning model, followed by a base-calling procedure to yield simulated reads. This workflow mimics the sequencing procedure more naturally. The thorough experiments

  4. A quantitative lubricant test for deep drawing

    DEFF Research Database (Denmark)

    Olsson, David Dam; Bay, Niels; Andreasen, Jan L.

    2010-01-01

    A tribological test for deep drawing has been developed by which the performance of lubricants may be evaluated quantitatively measuring the maximum backstroke force on the punch owing to friction between tool and workpiece surface. The forming force is found not to give useful information...

  5. DeepSimulator: a deep simulator for Nanopore sequencing

    KAUST Repository

    Li, Yu

    2017-12-23

    Motivation: Oxford Nanopore sequencing is a rapidly developed sequencing technology in recent years. To keep pace with the explosion of the downstream data analytical tools, a versatile Nanopore sequencing simulator is needed to complement the experimental data as well as to benchmark those newly developed tools. However, all the currently available simulators are based on simple statistics of the produced reads, which have difficulty in capturing the complex nature of the Nanopore sequencing procedure, the main task of which is the generation of raw electrical current signals. Results: Here we propose a deep learning based simulator, DeepSimulator, to mimic the entire pipeline of Nanopore sequencing. Starting from a given reference genome or assembled contigs, we simulate the electrical current signals by a context-dependent deep learning model, followed by a base-calling procedure to yield simulated reads. This workflow mimics the sequencing procedure more naturally. The thorough experiments performed across four species show that the signals generated by our context-dependent model are more similar to the experimentally obtained signals than the ones generated by the official context-independent pore model. In terms of the simulated reads, we provide a parameter interface to users so that they can obtain the reads with different accuracies ranging from 83% to 97%. The reads generated by the default parameter have almost the same properties as the real data. Two case studies demonstrate the application of DeepSimulator to benefit the development of tools in de novo assembly and in low coverage SNP detection. Availability: The software can be accessed freely at: https://github.com/lykaust15/DeepSimulator.

  6. Quantitative phase microscopy using deep neural networks

    Science.gov (United States)

    Li, Shuai; Sinha, Ayan; Lee, Justin; Barbastathis, George

    2018-02-01

    Deep learning has been proven to achieve ground-breaking accuracy in various tasks. In this paper, we implemented a deep neural network (DNN) to achieve phase retrieval in a wide-field microscope. Our DNN utilized the residual neural network (ResNet) architecture and was trained using the data generated by a phase SLM. The results showed that our DNN was able to reconstruct the profile of the phase target qualitatively. In the meantime, large error still existed, which indicated that our approach still need to be improved.

  7. Predicting effects of noncoding variants with deep learning-based sequence model.

    Science.gov (United States)

    Zhou, Jian; Troyanskaya, Olga G

    2015-10-01

    Identifying functional effects of noncoding variants is a major challenge in human genetics. To predict the noncoding-variant effects de novo from sequence, we developed a deep learning-based algorithmic framework, DeepSEA (http://deepsea.princeton.edu/), that directly learns a regulatory sequence code from large-scale chromatin-profiling data, enabling prediction of chromatin effects of sequence alterations with single-nucleotide sensitivity. We further used this capability to improve prioritization of functional variants including expression quantitative trait loci (eQTLs) and disease-associated variants.

  8. Development of a quantitative lubricant test for deep drawing

    DEFF Research Database (Denmark)

    Olsson, David Dam; Bay, Niels; Andreasen, Jan Lasson

    2004-01-01

    A tribological test for deep drawing has been developed by which the performance of lubricants may be evaluated quantitatively measuring the maximum backstroke force on the punch due to sliding friction between tool and work piece surface. The forming force is found not to give useful information...

  9. Geoseq: a tool for dissecting deep-sequencing datasets

    Directory of Open Access Journals (Sweden)

    Homann Robert

    2010-10-01

    Full Text Available Abstract Background Datasets generated on deep-sequencing platforms have been deposited in various public repositories such as the Gene Expression Omnibus (GEO, Sequence Read Archive (SRA hosted by the NCBI, or the DNA Data Bank of Japan (ddbj. Despite being rich data sources, they have not been used much due to the difficulty in locating and analyzing datasets of interest. Results Geoseq http://geoseq.mssm.edu provides a new method of analyzing short reads from deep sequencing experiments. Instead of mapping the reads to reference genomes or sequences, Geoseq maps a reference sequence against the sequencing data. It is web-based, and holds pre-computed data from public libraries. The analysis reduces the input sequence to tiles and measures the coverage of each tile in a sequence library through the use of suffix arrays. The user can upload custom target sequences or use gene/miRNA names for the search and get back results as plots and spreadsheet files. Geoseq organizes the public sequencing data using a controlled vocabulary, allowing identification of relevant libraries by organism, tissue and type of experiment. Conclusions Analysis of small sets of sequences against deep-sequencing datasets, as well as identification of public datasets of interest, is simplified by Geoseq. We applied Geoseq to, a identify differential isoform expression in mRNA-seq datasets, b identify miRNAs (microRNAs in libraries, and identify mature and star sequences in miRNAS and c to identify potentially mis-annotated miRNAs. The ease of using Geoseq for these analyses suggests its utility and uniqueness as an analysis tool.

  10. Quantiprot - a Python package for quantitative analysis of protein sequences.

    Science.gov (United States)

    Konopka, Bogumił M; Marciniak, Marta; Dyrka, Witold

    2017-07-17

    The field of protein sequence analysis is dominated by tools rooted in substitution matrices and alignments. A complementary approach is provided by methods of quantitative characterization. A major advantage of the approach is that quantitative properties defines a multidimensional solution space, where sequences can be related to each other and differences can be meaningfully interpreted. Quantiprot is a software package in Python, which provides a simple and consistent interface to multiple methods for quantitative characterization of protein sequences. The package can be used to calculate dozens of characteristics directly from sequences or using physico-chemical properties of amino acids. Besides basic measures, Quantiprot performs quantitative analysis of recurrence and determinism in the sequence, calculates distribution of n-grams and computes the Zipf's law coefficient. We propose three main fields of application of the Quantiprot package. First, quantitative characteristics can be used in alignment-free similarity searches, and in clustering of large and/or divergent sequence sets. Second, a feature space defined by quantitative properties can be used in comparative studies of protein families and organisms. Third, the feature space can be used for evaluating generative models, where large number of sequences generated by the model can be compared to actually observed sequences.

  11. Deep-Sea, Deep-Sequencing: Metabarcoding Extracellular DNA from Sediments of Marine Canyons.

    Directory of Open Access Journals (Sweden)

    Magdalena Guardiola

    Full Text Available Marine sediments are home to one of the richest species pools on Earth, but logistics and a dearth of taxonomic work-force hinders the knowledge of their biodiversity. We characterized α- and β-diversity of deep-sea assemblages from submarine canyons in the western Mediterranean using an environmental DNA metabarcoding. We used a new primer set targeting a short eukaryotic 18S sequence (ca. 110 bp. We applied a protocol designed to obtain extractions enriched in extracellular DNA from replicated sediment corers. With this strategy we captured information from DNA (local or deposited from the water column that persists adsorbed to inorganic particles and buffered short-term spatial and temporal heterogeneity. We analysed replicated samples from 20 localities including 2 deep-sea canyons, 1 shallower canal, and two open slopes (depth range 100-2,250 m. We identified 1,629 MOTUs, among which the dominant groups were Metazoa (with representatives of 19 phyla, Alveolata, Stramenopiles, and Rhizaria. There was a marked small-scale heterogeneity as shown by differences in replicates within corers and within localities. The spatial variability between canyons was significant, as was the depth component in one of the canyons where it was tested. Likewise, the composition of the first layer (1 cm of sediment was significantly different from deeper layers. We found that qualitative (presence-absence and quantitative (relative number of reads data showed consistent trends of differentiation between samples and geographic areas. The subset of exclusively benthic MOTUs showed similar patterns of β-diversity and community structure as the whole dataset. Separate analyses of the main metazoan phyla (in number of MOTUs showed some differences in distribution attributable to different lifestyles. Our results highlight the differentiation that can be found even between geographically close assemblages, and sets the ground for future monitoring and conservation

  12. Detection of Emerging Vaccine-Related Polioviruses by Deep Sequencing.

    Science.gov (United States)

    Sahoo, Malaya K; Holubar, Marisa; Huang, ChunHong; Mohamed-Hadley, Alisha; Liu, Yuanyuan; Waggoner, Jesse J; Troy, Stephanie B; Garcia-Garcia, Lourdes; Ferreyra-Reyes, Leticia; Maldonado, Yvonne; Pinsky, Benjamin A

    2017-07-01

    Oral poliovirus vaccine can mutate to regain neurovirulence. To date, evaluation of these mutations has been performed primarily on culture-enriched isolates by using conventional Sanger sequencing. We therefore developed a culture-independent, deep-sequencing method targeting the 5' untranslated region (UTR) and P1 genomic region to characterize vaccine-related poliovirus variants. Error analysis of the deep-sequencing method demonstrated reliable detection of poliovirus mutations at levels of vaccinated, asymptomatic children and their close contacts collected during a prospective cohort study in Veracruz, Mexico, revealed no vaccine-derived polioviruses. This was expected given that the longest duration between sequenced sample collection and the end of the most recent national immunization week was 66 days. However, we identified many low-level variants (Sabin serotypes, as well as vaccine-related viruses with multiple canonical mutations associated with phenotypic reversion present at high levels (>90%). These results suggest that monitoring emerging vaccine-related poliovirus variants by deep sequencing may aid in the poliovirus endgame and efforts to ensure global polio eradication. Copyright © 2017 Sahoo et al.

  13. Unified Deep Learning Architecture for Modeling Biology Sequence.

    Science.gov (United States)

    Wu, Hongjie; Cao, Chengyuan; Xia, Xiaoyan; Lu, Qiang

    2017-10-09

    Prediction of the spatial structure or function of biological macromolecules based on their sequence remains an important challenge in bioinformatics. When modeling biological sequences using traditional sequencing models, characteristics, such as long-range interactions between basic units, the complicated and variable output of labeled structures, and the variable length of biological sequences, usually lead to different solutions on a case-by-case basis. This study proposed the use of bidirectional recurrent neural networks based on long short-term memory or a gated recurrent unit to capture long-range interactions by designing the optional reshape operator to adapt to the diversity of the output labels and implementing a training algorithm to support the training of sequence models capable of processing variable-length sequences. Additionally, the merge and pooling operators enhanced the ability to capture short-range interactions between basic units of biological sequences. The proposed deep-learning model and its training algorithm might be capable of solving currently known biological sequence-modeling problems through the use of a unified framework. We validated our model on one of the most difficult biological sequence-modeling problems currently known, with our results indicating the ability of the model to obtain predictions of protein residue interactions that exceeded the accuracy of current popular approaches by 10% based on multiple benchmarks.

  14. DSAP: deep-sequencing small RNA analysis pipeline.

    Science.gov (United States)

    Huang, Po-Jung; Liu, Yi-Chung; Lee, Chi-Ching; Lin, Wei-Chen; Gan, Richie Ruei-Chi; Lyu, Ping-Chiang; Tang, Petrus

    2010-07-01

    DSAP is an automated multiple-task web service designed to provide a total solution to analyzing deep-sequencing small RNA datasets generated by next-generation sequencing technology. DSAP uses a tab-delimited file as an input format, which holds the unique sequence reads (tags) and their corresponding number of copies generated by the Solexa sequencing platform. The input data will go through four analysis steps in DSAP: (i) cleanup: removal of adaptors and poly-A/T/C/G/N nucleotides; (ii) clustering: grouping of cleaned sequence tags into unique sequence clusters; (iii) non-coding RNA (ncRNA) matching: sequence homology mapping against a transcribed sequence library from the ncRNA database Rfam (http://rfam.sanger.ac.uk/); and (iv) known miRNA matching: detection of known miRNAs in miRBase (http://www.mirbase.org/) based on sequence homology. The expression levels corresponding to matched ncRNAs and miRNAs are summarized in multi-color clickable bar charts linked to external databases. DSAP is also capable of displaying miRNA expression levels from different jobs using a log(2)-scaled color matrix. Furthermore, a cross-species comparative function is also provided to show the distribution of identified miRNAs in different species as deposited in miRBase. DSAP is available at http://dsap.cgu.edu.tw.

  15. Transcriptome sequences resolve deep relationships of the grape family.

    Science.gov (United States)

    Wen, Jun; Xiong, Zhiqiang; Nie, Ze-Long; Mao, Likai; Zhu, Yabing; Kan, Xian-Zhao; Ickert-Bond, Stefanie M; Gerrath, Jean; Zimmer, Elizabeth A; Fang, Xiao-Dong

    2013-01-01

    Previous phylogenetic studies of the grape family (Vitaceae) yielded poorly resolved deep relationships, thus impeding our understanding of the evolution of the family. Next-generation sequencing now offers access to protein coding sequences very easily, quickly and cost-effectively. To improve upon earlier work, we extracted 417 orthologous single-copy nuclear genes from the transcriptomes of 15 species of the Vitaceae, covering its phylogenetic diversity. The resulting transcriptome phylogeny provides robust support for the deep relationships, showing the phylogenetic utility of transcriptome data for plants over a time scale at least since the mid-Cretaceous. The pros and cons of transcriptome data for phylogenetic inference in plants are also evaluated.

  16. Transcriptome sequences resolve deep relationships of the grape family.

    Directory of Open Access Journals (Sweden)

    Jun Wen

    Full Text Available Previous phylogenetic studies of the grape family (Vitaceae yielded poorly resolved deep relationships, thus impeding our understanding of the evolution of the family. Next-generation sequencing now offers access to protein coding sequences very easily, quickly and cost-effectively. To improve upon earlier work, we extracted 417 orthologous single-copy nuclear genes from the transcriptomes of 15 species of the Vitaceae, covering its phylogenetic diversity. The resulting transcriptome phylogeny provides robust support for the deep relationships, showing the phylogenetic utility of transcriptome data for plants over a time scale at least since the mid-Cretaceous. The pros and cons of transcriptome data for phylogenetic inference in plants are also evaluated.

  17. Deep-sequencing protocols influence the results obtained in small-RNA sequencing.

    Directory of Open Access Journals (Sweden)

    Joern Toedling

    Full Text Available Second-generation sequencing is a powerful method for identifying and quantifying small-RNA components of cells. However, little attention has been paid to the effects of the choice of sequencing platform and library preparation protocol on the results obtained. We present a thorough comparison of small-RNA sequencing libraries generated from the same embryonic stem cell lines, using different sequencing platforms, which represent the three major second-generation sequencing technologies, and protocols. We have analysed and compared the expression of microRNAs, as well as populations of small RNAs derived from repetitive elements. Despite the fact that different libraries display a good correlation between sequencing platforms, qualitative and quantitative variations in the results were found, depending on the protocol used. Thus, when comparing libraries from different biological samples, it is strongly recommended to use the same sequencing platform and protocol in order to ensure the biological relevance of the comparisons.

  18. Deep whole-genome sequencing of 90 Han Chinese genomes.

    Science.gov (United States)

    Lan, Tianming; Lin, Haoxiang; Zhu, Wenjuan; Laurent, Tellier Christian Asker Melchior; Yang, Mengcheng; Liu, Xin; Wang, Jun; Wang, Jian; Yang, Huanming; Xu, Xun; Guo, Xiaosen

    2017-09-01

    Next-generation sequencing provides a high-resolution insight into human genetic information. However, the focus of previous studies has primarily been on low-coverage data due to the high cost of sequencing. Although the 1000 Genomes Project and the Haplotype Reference Consortium have both provided powerful reference panels for imputation, low-frequency and novel variants remain difficult to discover and call with accuracy on the basis of low-coverage data. Deep sequencing provides an optimal solution for the problem of these low-frequency and novel variants. Although whole-exome sequencing is also a viable choice for exome regions, it cannot account for noncoding regions, sometimes resulting in the absence of important, causal variants. For Han Chinese populations, the majority of variants have been discovered based upon low-coverage data from the 1000 Genomes Project. However, high-coverage, whole-genome sequencing data are limited for any population, and a large amount of low-frequency, population-specific variants remain uncharacterized. We have performed whole-genome sequencing at a high depth (∼×80) of 90 unrelated individuals of Chinese ancestry, collected from the 1000 Genomes Project samples, including 45 Northern Han Chinese and 45 Southern Han Chinese samples. Eighty-three of these 90 have been sequenced by the 1000 Genomes Project. We have identified 12 568 804 single nucleotide polymorphisms, 2 074 210 short InDels, and 26 142 structural variations from these 90 samples. Compared to the Han Chinese data from the 1000 Genomes Project, we have found 7 000 629 novel variants with low frequency (defined as minor allele frequency genome. Compared to the 1000 Genomes Project, these Han Chinese deep sequencing data enhance the characterization of a large number of low-frequency, novel variants. This will be a valuable resource for promoting Chinese genetics research and medical development. Additionally, it will provide a valuable supplement to the 1000

  19. deepTools2: a next generation web server for deep-sequencing data analysis.

    Science.gov (United States)

    Ramírez, Fidel; Ryan, Devon P; Grüning, Björn; Bhardwaj, Vivek; Kilpert, Fabian; Richter, Andreas S; Heyne, Steffen; Dündar, Friederike; Manke, Thomas

    2016-07-08

    We present an update to our Galaxy-based web server for processing and visualizing deeply sequenced data. Its core tool set, deepTools, allows users to perform complete bioinformatic workflows ranging from quality controls and normalizations of aligned reads to integrative analyses, including clustering and visualization approaches. Since we first described our deepTools Galaxy server in 2014, we have implemented new solutions for many requests from the community and our users. Here, we introduce significant enhancements and new tools to further improve data visualization and interpretation. deepTools continue to be open to all users and freely available as a web service at deeptools.ie-freiburg.mpg.de The new deepTools2 suite can be easily deployed within any Galaxy framework via the toolshed repository, and we also provide source code for command line usage under Linux and Mac OS X. A public and documented API for access to deepTools functionality is also available. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  20. Deep sequencing methods for protein engineering and design.

    Science.gov (United States)

    Wrenbeck, Emily E; Faber, Matthew S; Whitehead, Timothy A

    2017-08-01

    The advent of next-generation sequencing (NGS) has revolutionized protein science, and the development of complementary methods enabling NGS-driven protein engineering have followed. In general, these experiments address the functional consequences of thousands of protein variants in a massively parallel manner using genotype-phenotype linked high-throughput functional screens followed by DNA counting via deep sequencing. We highlight the use of information rich datasets to engineer protein molecular recognition. Examples include the creation of multiple dual-affinity Fabs targeting structurally dissimilar epitopes and engineering of a broad germline-targeted anti-HIV-1 immunogen. Additionally, we highlight the generation of enzyme fitness landscapes for conducting fundamental studies of protein behavior and evolution. We conclude with discussion of technological advances. Copyright © 2016 Elsevier Ltd. All rights reserved.

  1. Error Analysis of Deep Sequencing of Phage Libraries: Peptides Censored in Sequencing

    Directory of Open Access Journals (Sweden)

    Wadim L. Matochko

    2013-01-01

    Full Text Available Next-generation sequencing techniques empower selection of ligands from phage-display libraries because they can detect low abundant clones and quantify changes in the copy numbers of clones without excessive selection rounds. Identification of errors in deep sequencing data is the most critical step in this process because these techniques have error rates >1%. Mechanisms that yield errors in Illumina and other techniques have been proposed, but no reports to date describe error analysis in phage libraries. Our paper focuses on error analysis of 7-mer peptide libraries sequenced by Illumina method. Low theoretical complexity of this phage library, as compared to complexity of long genetic reads and genomes, allowed us to describe this library using convenient linear vector and operator framework. We describe a phage library as N×1 frequency vector n=ni, where ni is the copy number of the ith sequence and N is the theoretical diversity, that is, the total number of all possible sequences. Any manipulation to the library is an operator acting on n. Selection, amplification, or sequencing could be described as a product of a N×N matrix and a stochastic sampling operator (Sa. The latter is a random diagonal matrix that describes sampling of a library. In this paper, we focus on the properties of Sa and use them to define the sequencing operator (Seq. Sequencing without any bias and errors is Seq=Sa IN, where IN is a N×N unity matrix. Any bias in sequencing changes IN to a nonunity matrix. We identified a diagonal censorship matrix (CEN, which describes elimination or statistically significant downsampling, of specific reads during the sequencing process.

  2. Protein model discrimination using mutational sensitivity derived from deep sequencing.

    Science.gov (United States)

    Adkar, Bharat V; Tripathi, Arti; Sahoo, Anusmita; Bajaj, Kanika; Goswami, Devrishi; Chakrabarti, Purbani; Swarnkar, Mohit K; Gokhale, Rajesh S; Varadarajan, Raghavan

    2012-02-08

    A major bottleneck in protein structure prediction is the selection of correct models from a pool of decoys. Relative activities of ∼1,200 individual single-site mutants in a saturation library of the bacterial toxin CcdB were estimated by determining their relative populations using deep sequencing. This phenotypic information was used to define an empirical score for each residue (RankScore), which correlated with the residue depth, and identify active-site residues. Using these correlations, ∼98% of correct models of CcdB (RMSD ≤ 4Å) were identified from a large set of decoys. The model-discrimination methodology was further validated on eleven different monomeric proteins using simulated RankScore values. The methodology is also a rapid, accurate way to obtain relative activities of each mutant in a large pool and derive sequence-structure-function relationships without protein isolation or characterization. It can be applied to any system in which mutational effects can be monitored by a phenotypic readout. Copyright © 2012 Elsevier Ltd. All rights reserved.

  3. Sequencing Infrastructure Investments under Deep Uncertainty Using Real Options Analysis

    Directory of Open Access Journals (Sweden)

    Nishtha Manocha

    2018-02-01

    Full Text Available The adaptation tipping point and adaptation pathway approach developed to make decisions under deep uncertainty do not shed light on which among the multiple available pathways should be chosen as the preferred pathway. This creates the need to extend these approaches by means of suitable tools that can help sequence actions and subsequently enable the outlining of relevant policies. This paper presents two sequencing approaches, namely, the “Build to Target” and “Build Up” approach, to aid in sub-selecting a set of preferred pathways. Both approaches differ in the levels of flexibility they offer. They are exemplified by means of two case studies wherein the Net Present Valuation and the Real Options Analysis are employed as selection criterions. The results demonstrate the benefit of these two approaches when used in conjunction with the adaptation pathways and show how the pathways selected by means of a Build to Target approach generally have a value greater than, or at least the same as, the pathways selected by the Build Up approach. Further, this paper also demonstrates the capacity of Real Options to quantify and capture the economic value of flexibility, which cannot be done by traditional valuation approaches such as Net Present Valuation.

  4. DeepBase: annotation and discovery of microRNAs and other noncoding RNAs from deep-sequencing data.

    Science.gov (United States)

    Yang, Jian-Hua; Qu, Liang-Hu

    2012-01-01

    Recent advances in high-throughput deep-sequencing technology have produced large numbers of short and long RNA sequences and enabled the detection and profiling of known and novel microRNAs (miRNAs) and other noncoding RNAs (ncRNAs) at unprecedented sensitivity and depth. In this chapter, we describe the use of deepBase, a database that we have developed to integrate all public deep-sequencing data and to facilitate the comprehensive annotation and discovery of miRNAs and other ncRNAs from these data. deepBase provides an integrative, interactive, and versatile web graphical interface to evaluate miRBase-annotated miRNA genes and other known ncRNAs, explores the expression patterns of miRNAs and other ncRNAs, and discovers novel miRNAs and other ncRNAs from deep-sequencing data. deepBase also provides a deepView genome browser to comparatively analyze these data at multiple levels. deepBase is available at http://deepbase.sysu.edu.cn/.

  5. Key roles for freshwater Actinobacteria revealed by deep metagenomic sequencing.

    Science.gov (United States)

    Ghai, Rohit; Mizuno, Carolina Megumi; Picazo, Antonio; Camacho, Antonio; Rodriguez-Valera, Francisco

    2014-12-01

    Freshwater ecosystems are critical but fragile environments directly affecting society and its welfare. However, our understanding of genuinely freshwater microbial communities, constrained by our capacity to manipulate its prokaryotic participants in axenic cultures, remains very rudimentary. Even the most abundant components, freshwater Actinobacteria, remain largely unknown. Here, applying deep metagenomic sequencing to the microbial community of a freshwater reservoir, we were able to circumvent this traditional bottleneck and reconstruct de novo seven distinct streamlined actinobacterial genomes. These genomes represent three new groups of photoheterotrophic, planktonic Actinobacteria. We describe for the first time genomes of two novel clades, acMicro (Micrococcineae, related to Luna2,) and acAMD (Actinomycetales, related to acTH1). Besides, an aggregate of contigs belonged to a new branch of the Acidimicrobiales. All are estimated to have small genomes (approximately 1.2 Mb), and their GC content varied from 40 to 61%. One of the Micrococcineae genomes encodes a proteorhodopsin, a rhodopsin type reported for the first time in Actinobacteria. The remarkable potential capacity of some of these genomes to transform recalcitrant plant detrital material, particularly lignin-derived compounds, suggests close linkages between the terrestrial and aquatic realms. Moreover, abundances of Actinobacteria correlate inversely to those of Cyanobacteria that are responsible for prolonged and frequently irretrievable damage to freshwater ecosystems. This suggests that they might serve as sentinels of impending ecological catastrophes. © 2014 John Wiley & Sons Ltd.

  6. Evaluation of MRI sequences for quantitative T1 brain mapping

    Science.gov (United States)

    Tsialios, P.; Thrippleton, M.; Glatz, A.; Pernet, C.

    2017-11-01

    T1 mapping constitutes a quantitative MRI technique finding significant application in brain imaging. It allows evaluation of contrast uptake, blood perfusion, volume, providing a more specific biomarker of disease progression compared to conventional T1-weighted images. While there are many techniques for T1-mapping there is a wide range of reported T1-values in tissues, raising the issue of protocols reproducibility and standardization. The gold standard for obtaining T1-maps is based on acquiring IR-SE sequence. Widely used alternative sequences are IR-SE-EPI, VFA (DESPOT), DESPOT-HIFI and MP2RAGE that speed up scanning and fitting procedures. A custom MRI phantom was used to assess the reproducibility and accuracy of the different methods. All scans were performed using a 3T Siemens Prisma scanner. The acquired data processed using two different codes. The main difference was observed for VFA (DESPOT) which grossly overestimated T1 relaxation time by 214 ms [126 270] compared to the IR-SE sequence. MP2RAGE and DESPOT-HIFI sequences gave slightly shorter time than IR-SE (~20 to 30ms) and can be considered as alternative and time-efficient methods for acquiring accurate T1 maps of the human brain, while IR-SE-EPI gave identical result, at a cost of a lower image quality.

  7. Quantitative assessment of deep gas migration in Fennoscandian sites

    International Nuclear Information System (INIS)

    Delos, Anne; Trinchero, Paolo; Richard, Laurent; Molinero, Jorge; Dentz, Marco; Pitkaenen, Petteri

    2010-11-01

    turns out that its results cannot be reliably used to estimate gas fluxes. They can rather provide an estimate of the effective in situ gas production (i.e. radiogenic production) averaged over the model domain. Such effective in situ gas productions have been computed and discussed for the 3 sites. Helium profiles have been modelled using two different approaches: calibrating the residence time or estimating the release fraction (i.e. the rate of Helium production actually released to the water). Methane and Hydrogen in situ productions have been then determined either setting time equal to the age of formation of the rock or using the value of residence time obtained from the analysis of the Helium profiles. A new analytical solution that can take into account not only the radiogenic production but also the flux from a deep source of helium has been developed. This solution in companion with further field characterization (e.g. isotopic measurements of Helium) provides a powerful tool that allows accounting for the coupled effect of a (limited in space) in situ production and a source occurring at a large depth in the crust or mantle. It is thought that this new analytical solution could be used for future quantitative modelling of gas migration when more data were available

  8. Quantitative assessment of deep gas migration in Fennoscandian sites

    Energy Technology Data Exchange (ETDEWEB)

    Delos, Anne; Trinchero, Paolo; Richard, Laurent; Molinero, Jorge (Amphos 21 Consulting S.L., Barcelona (Spain)); Dentz, Marco (IDAEA-CSIC Instituto de Diagnostico Ambiental y Estudios del Agua, Barcelona (Spain)); Pitkaenen, Petteri (Posiva Oy, Olkiluoto, Eurajoki (Finland))

    2010-11-15

    (which, strictly speaking, is infinite). It turns out that its results cannot be reliably used to estimate gas fluxes. They can rather provide an estimate of the effective in situ gas production (i.e. radiogenic production) averaged over the model domain. Such effective in situ gas productions have been computed and discussed for the 3 sites. Helium profiles have been modelled using two different approaches: calibrating the residence time or estimating the release fraction (i.e. the rate of Helium production actually released to the water). Methane and Hydrogen in situ productions have been then determined either setting time equal to the age of formation of the rock or using the value of residence time obtained from the analysis of the Helium profiles. A new analytical solution that can take into account not only the radiogenic production but also the flux from a deep source of helium has been developed. This solution in companion with further field characterization (e.g. isotopic measurements of Helium) provides a powerful tool that allows accounting for the coupled effect of a (limited in space) in situ production and a source occurring at a large depth in the crust or mantle. It is thought that this new analytical solution could be used for future quantitative modelling of gas migration when more data were available

  9. DeepProbe: Information Directed Sequence Understanding and Chatbot Design via Recurrent Neural Networks

    OpenAIRE

    Yin, Zi; Chang, Keng-hao; Zhang, Ruofei

    2017-01-01

    Information extraction and user intention identification are central topics in modern query understanding and recommendation systems. In this paper, we propose DeepProbe, a generic information-directed interaction framework which is built around an attention-based sequence to sequence (seq2seq) recurrent neural network. DeepProbe can rephrase, evaluate, and even actively ask questions, leveraging the generative ability and likelihood estimation made possible by seq2seq models. DeepProbe makes...

  10. Deep sequencing of cardiac microRNA-mRNA interactomes in clinical and experimental cardiomyopathy.

    Science.gov (United States)

    Matkovich, Scot J; Dorn, Gerald W

    2015-01-01

    MicroRNAs are a family of short (~21 nucleotide) noncoding RNAs that serve key roles in cellular growth and differentiation and the response of the heart to stress stimuli. As the sequence-specific recognition element of RNA-induced silencing complexes (RISCs), microRNAs bind mRNAs and prevent their translation via mechanisms that may include transcript degradation and/or prevention of ribosome binding. Short microRNA sequences and the ability of microRNAs to bind to mRNA sites having only partial/imperfect sequence complementarity complicate purely computational analyses of microRNA-mRNA interactomes. Furthermore, computational microRNA target prediction programs typically ignore biological context, and therefore the principal determinants of microRNA-mRNA binding: the presence and quantity of each. To address these deficiencies we describe an empirical method, developed via studies of stressed and failing hearts, to determine disease-induced changes in microRNAs, mRNAs, and the mRNAs targeted to the RISC, without cross-linking mRNAs to RISC proteins. Deep sequencing methods are used to determine RNA abundances, delivering unbiased, quantitative RNA data limited only by their annotation in the genome of interest. We describe the laboratory bench steps required to perform these experiments, experimental design strategies to achieve an appropriate number of sequencing reads per biological replicate, and computer-based processing tools and procedures to convert large raw sequencing data files into gene expression measures useful for differential expression analyses.

  11. Discovery radiomics via evolutionary deep radiomic sequencer discovery for pathologically proven lung cancer detection.

    Science.gov (United States)

    Shafiee, Mohammad Javad; Chung, Audrey G; Khalvati, Farzad; Haider, Masoom A; Wong, Alexander

    2017-10-01

    While lung cancer is the second most diagnosed form of cancer in men and women, a sufficiently early diagnosis can be pivotal in patient survival rates. Imaging-based, or radiomics-driven, detection methods have been developed to aid diagnosticians, but largely rely on hand-crafted features that may not fully encapsulate the differences between cancerous and healthy tissue. Recently, the concept of discovery radiomics was introduced, where custom abstract features are discovered from readily available imaging data. We propose an evolutionary deep radiomic sequencer discovery approach based on evolutionary deep intelligence. Motivated by patient privacy concerns and the idea of operational artificial intelligence, the evolutionary deep radiomic sequencer discovery approach organically evolves increasingly more efficient deep radiomic sequencers that produce significantly more compact yet similarly descriptive radiomic sequences over multiple generations. As a result, this framework improves operational efficiency and enables diagnosis to be run locally at the radiologist's computer while maintaining detection accuracy. We evaluated the evolved deep radiomic sequencer (EDRS) discovered via the proposed evolutionary deep radiomic sequencer discovery framework against state-of-the-art radiomics-driven and discovery radiomics methods using clinical lung CT data with pathologically proven diagnostic data from the LIDC-IDRI dataset. The EDRS shows improved sensitivity (93.42%), specificity (82.39%), and diagnostic accuracy (88.78%) relative to previous radiomics approaches.

  12. microRNA expression profiling in fetal single ventricle malformation identified by deep sequencing.

    Science.gov (United States)

    Yu, Zhang-Bin; Han, Shu-Ping; Bai, Yun-Fei; Zhu, Chun; Pan, Ya; Guo, Xi-Rong

    2012-01-01

    microRNAs (miRNAs) have emerged as key regulators in many biological processes, particularly cardiac growth and development, although the specific miRNA expression profile associated with this process remains to be elucidated. This study aimed to characterize the cellular microRNA profile involved in the development of congenital heart malformation, through the investigation of single ventricle (SV) defects. Comprehensive miRNA profiling in human fetal SV cardiac tissue was performed by deep sequencing. Differential expression of 48 miRNAs was revealed by sequencing by oligonucleotide ligation and detection (SOLiD) analysis. Of these, 38 were down-regulated and 10 were up-regulated in differentiated SV cardiac tissue, compared to control cardiac tissue. This was confirmed by real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis. Predicted target genes of the 48 differentially expressed miRNAs were analyzed by gene ontology and categorized according to cellular process, regulation of biological process and metabolic process. Pathway-Express analysis identified the WNT and mTOR signaling pathways as the most significant processes putatively affected by the differential expression of these miRNAs. The candidate genes involved in cardiac development were identified as potential targets for these differentially expressed microRNAs and the collaborative network of microRNAs and cardiac development related-mRNAs was constructed. These data provide the basis for future investigation of the mechanism of the occurrence and development of fetal SV malformations.

  13. Accurate identification of RNA editing sites from primitive sequence with deep neural networks.

    Science.gov (United States)

    Ouyang, Zhangyi; Liu, Feng; Zhao, Chenghui; Ren, Chao; An, Gaole; Mei, Chuan; Bo, Xiaochen; Shu, Wenjie

    2018-04-16

    RNA editing is a post-transcriptional RNA sequence alteration. Current methods have identified editing sites and facilitated research but require sufficient genomic annotations and prior-knowledge-based filtering steps, resulting in a cumbersome, time-consuming identification process. Moreover, these methods have limited generalizability and applicability in species with insufficient genomic annotations or in conditions of limited prior knowledge. We developed DeepRed, a deep learning-based method that identifies RNA editing from primitive RNA sequences without prior-knowledge-based filtering steps or genomic annotations. DeepRed achieved 98.1% and 97.9% area under the curve (AUC) in training and test sets, respectively. We further validated DeepRed using experimentally verified U87 cell RNA-seq data, achieving 97.9% positive predictive value (PPV). We demonstrated that DeepRed offers better prediction accuracy and computational efficiency than current methods with large-scale, mass RNA-seq data. We used DeepRed to assess the impact of multiple factors on editing identification with RNA-seq data from the Association of Biomolecular Resource Facilities and Sequencing Quality Control projects. We explored developmental RNA editing pattern changes during human early embryogenesis and evolutionary patterns in Drosophila species and the primate lineage using DeepRed. Our work illustrates DeepRed's state-of-the-art performance; it may decipher the hidden principles behind RNA editing, making editing detection convenient and effective.

  14. Deep sequencing as a method of typing bluetongue virus isolates.

    Science.gov (United States)

    Rao, Pavuluri Panduranga; Reddy, Yella Narasimha; Ganesh, Kapila; Nair, Shreeja G; Niranjan, Vidya; Hegde, Nagendra R

    2013-11-01

    Bluetongue (BT) is an economically important endemic disease of livestock in tropics and subtropics. In addition, its recent spread to temperate regions like North America and Northern Europe is of serious concern. Rapid serotyping and characterization of BT virus (BTV) is an essential step in the identification of origin of the virus and for controlling the disease. Serotyping of BTV is typically performed by serum neutralization, and of late by nucleotide sequencing. This report describes the near complete genome sequencing and typing of two isolates of BTV using Illumina next generation sequencing platform. Two of the BTV RNAs were multiplexed with ten other unknown samples. Viral RNA was isolated and fragmented, reverse transcribed, the cDNA ends were repaired and ligated with a multiplex oligo. The genome library was amplified using primers complementary to the ligated oligo and subjected to single and paired end sequencing. The raw reads were assembled using a de novo method and reference-based assembly was performed based on the contig data. Near complete sequences of all segments of BTV were obtained with more than 20× coverage, and single read sequencing method was sufficient to identify the genotype and serotype of the virus. The two viruses used in this study were typed as BTV-1 and BTV-9E. Copyright © 2013 Elsevier B.V. All rights reserved.

  15. Deep amplicon sequencing reveals mixed phytoplasma infection within single grapevine plants

    DEFF Research Database (Denmark)

    Nicolaisen, Mogens; Contaldo, Nicoletta; Makarova, Olga

    2011-01-01

    The diversity of phytoplasmas within single plants has not yet been fully investigated. In this project, deep amplicon sequencing was used to generate 50,926 phytoplasma sequences from 11 phytoplasma-infected grapevine samples from a PCR amplicon in the 5' end of the 16S region. After clustering ...

  16. Deep sequencing of foot-and-mouth disease virus reveals RNA sequences involved in genome packaging.

    Science.gov (United States)

    Logan, Grace; Newman, Joseph; Wright, Caroline F; Lasecka-Dykes, Lidia; Haydon, Daniel T; Cottam, Eleanor M; Tuthill, Tobias J

    2017-10-18

    Non-enveloped viruses protect their genomes by packaging them into an outer shell or capsid of virus-encoded proteins. Packaging and capsid assembly in RNA viruses can involve interactions between capsid proteins and secondary structures in the viral genome as exemplified by the RNA bacteriophage MS2 and as proposed for other RNA viruses of plants, animals and human. In the picornavirus family of non-enveloped RNA viruses, the requirements for genome packaging remain poorly understood. Here we show a novel and simple approach to identify predicted RNA secondary structures involved in genome packaging in the picornavirus foot-and-mouth disease virus (FMDV). By interrogating deep sequencing data generated from both packaged and unpackaged populations of RNA we have determined multiple regions of the genome with constrained variation in the packaged population. Predicted secondary structures of these regions revealed stem loops with conservation of structure and a common motif at the loop. Disruption of these features resulted in attenuation of virus growth in cell culture due to a reduction in assembly of mature virions. This study provides evidence for the involvement of predicted RNA structures in picornavirus packaging and offers a readily transferable methodology for identifying packaging requirements in many other viruses. Importance In order to transmit their genetic material to a new host, non-enveloped viruses must protect their genomes by packaging them into an outer shell or capsid of virus-encoded proteins. For many non-enveloped RNA viruses the requirements for this critical part of the viral life cycle remain poorly understood. We have identified RNA sequences involved in genome packaging of the picornavirus foot-and-mouth disease virus. This virus causes an economically devastating disease of livestock affecting both the developed and developing world. The experimental methods developed to carry out this work are novel, simple and transferable to the

  17. A simple method for the parallel deep sequencing of full influenza A genomes

    DEFF Research Database (Denmark)

    Kampmann, Marie-Louise; Fordyce, Sarah Louise; Avila Arcos, Maria del Carmen

    2011-01-01

    Given the major threat of influenza A to human and animal health, and its ability to evolve rapidly through mutation and reassortment, tools that enable its timely characterization are necessary to help monitor its evolution and spread. For this purpose, deep sequencing can be a very valuable tool....... This study reports a comprehensive method that enables deep sequencing of the complete genomes of influenza A subtypes using the Illumina Genome Analyzer IIx (GAIIx). By using this method, the complete genomes of nine viruses were sequenced in parallel, representing the 2009 pandemic H1N1 virus, H5N1 virus...

  18. LookSeq: A browser-based viewer for deep sequencing data

    OpenAIRE

    Manske, Heinrich Magnus; Kwiatkowski, Dominic P.

    2009-01-01

    Sequencing a genome to great depth can be highly informative about heterogeneity within an individual or a population. Here we address the problem of how to visualize the multiple layers of information contained in deep sequencing data. We propose an interactive AJAX-based web viewer for browsing large data sets of aligned sequence reads. By enabling seamless browsing and fast zooming, the LookSeq program assists the user to assimilate information at different levels of resolution, from an ov...

  19. Short-range quantitative precipitation forecasting using Deep Learning approaches

    Science.gov (United States)

    Akbari Asanjan, A.; Yang, T.; Gao, X.; Hsu, K. L.; Sorooshian, S.

    2017-12-01

    Predicting short-range quantitative precipitation is very important for flood forecasting, early flood warning and other hydrometeorological purposes. This study aims to improve the precipitation forecasting skills using a recently developed and advanced machine learning technique named Long Short-Term Memory (LSTM). The proposed LSTM learns the changing patterns of clouds from Cloud-Top Brightness Temperature (CTBT) images, retrieved from the infrared channel of Geostationary Operational Environmental Satellite (GOES), using a sophisticated and effective learning method. After learning the dynamics of clouds, the LSTM model predicts the upcoming rainy CTBT events. The proposed model is then merged with a precipitation estimation algorithm termed Precipitation Estimation from Remotely Sensed Information using Artificial Neural Networks (PERSIANN) to provide precipitation forecasts. The results of merged LSTM with PERSIANN are compared to the results of an Elman-type Recurrent Neural Network (RNN) merged with PERSIANN and Final Analysis of Global Forecast System model over the states of Oklahoma, Florida and Oregon. The performance of each model is investigated during 3 storm events each located over one of the study regions. The results indicate the outperformance of merged LSTM forecasts comparing to the numerical and statistical baselines in terms of Probability of Detection (POD), False Alarm Ratio (FAR), Critical Success Index (CSI), RMSE and correlation coefficient especially in convective systems. The proposed method shows superior capabilities in short-term forecasting over compared methods.

  20. Uniform, optimal signal processing of mapped deep-sequencing data.

    Science.gov (United States)

    Kumar, Vibhor; Muratani, Masafumi; Rayan, Nirmala Arul; Kraus, Petra; Lufkin, Thomas; Ng, Huck Hui; Prabhakar, Shyam

    2013-07-01

    Despite their apparent diversity, many problems in the analysis of high-throughput sequencing data are merely special cases of two general problems, signal detection and signal estimation. Here we adapt formally optimal solutions from signal processing theory to analyze signals of DNA sequence reads mapped to a genome. We describe DFilter, a detection algorithm that identifies regulatory features in ChIP-seq, DNase-seq and FAIRE-seq data more accurately than assay-specific algorithms. We also describe EFilter, an estimation algorithm that accurately predicts mRNA levels from as few as 1-2 histone profiles (R ∼0.9). Notably, the presence of regulatory motifs in promoters correlates more with histone modifications than with mRNA levels, suggesting that histone profiles are more predictive of cis-regulatory mechanisms. We show by applying DFilter and EFilter to embryonic forebrain ChIP-seq data that regulatory protein identification and functional annotation are feasible despite tissue heterogeneity. The mathematical formalism underlying our tools facilitates integrative analysis of data from virtually any sequencing-based functional profile.

  1. Protein sequences bound to mineral surfaces persist into deep time

    DEFF Research Database (Denmark)

    Demarchi, Beatrice; Hall, Shaun; Roncal-Herrero, Teresa

    2016-01-01

    of Laetoli (3.8 Ma) and Olduvai Gorge (1.3 Ma) in Tanzania. By tracking protein diagenesis back in time we find consistent patterns of preservation, demonstrating authenticity of the surviving sequences. Molecular dynamics simulations of struthiocalcin-1 and -2, the dominant proteins within the eggshell......, reveal that distinct domains bind to the mineral surface. It is the domain with the strongest calculated binding energy to the calcite surface that is selectively preserved. Thermal age calculations demonstrate that the Laetoli and Olduvai peptides are 50 times older than any previously authenticated...

  2. DeepGO: predicting protein functions from sequence and interactions using a deep ontology-aware classifier

    KAUST Repository

    Kulmanov, Maxat

    2017-09-27

    Motivation A large number of protein sequences are becoming available through the application of novel high-throughput sequencing technologies. Experimental functional characterization of these proteins is time-consuming and expensive, and is often only done rigorously for few selected model organisms. Computational function prediction approaches have been suggested to fill this gap. The functions of proteins are classified using the Gene Ontology (GO), which contains over 40 000 classes. Additionally, proteins have multiple functions, making function prediction a large-scale, multi-class, multi-label problem. Results We have developed a novel method to predict protein function from sequence. We use deep learning to learn features from protein sequences as well as a cross-species protein–protein interaction network. Our approach specifically outputs information in the structure of the GO and utilizes the dependencies between GO classes as background information to construct a deep learning model. We evaluate our method using the standards established by the Computational Assessment of Function Annotation (CAFA) and demonstrate a significant improvement over baseline methods such as BLAST, in particular for predicting cellular locations.

  3. DeepGO: predicting protein functions from sequence and interactions using a deep ontology-aware classifier.

    Science.gov (United States)

    Kulmanov, Maxat; Khan, Mohammed Asif; Hoehndorf, Robert; Wren, Jonathan

    2018-02-15

    A large number of protein sequences are becoming available through the application of novel high-throughput sequencing technologies. Experimental functional characterization of these proteins is time-consuming and expensive, and is often only done rigorously for few selected model organisms. Computational function prediction approaches have been suggested to fill this gap. The functions of proteins are classified using the Gene Ontology (GO), which contains over 40 000 classes. Additionally, proteins have multiple functions, making function prediction a large-scale, multi-class, multi-label problem. We have developed a novel method to predict protein function from sequence. We use deep learning to learn features from protein sequences as well as a cross-species protein-protein interaction network. Our approach specifically outputs information in the structure of the GO and utilizes the dependencies between GO classes as background information to construct a deep learning model. We evaluate our method using the standards established by the Computational Assessment of Function Annotation (CAFA) and demonstrate a significant improvement over baseline methods such as BLAST, in particular for predicting cellular locations. Web server: http://deepgo.bio2vec.net, Source code: https://github.com/bio-ontology-research-group/deepgo. robert.hoehndorf@kaust.edu.sa. Supplementary data are available at Bioinformatics online. © The Author(s) 2017. Published by Oxford University Press.

  4. Determining mutant spectra of three RNA viral samples using ultra-deep sequencing

    Energy Technology Data Exchange (ETDEWEB)

    Chen, H

    2012-06-06

    RNA viruses have extremely high mutation rates that enable the virus to adapt to new host environments and even jump from one species to another. As part of a viral transmission study, three viral samples collected from naturally infected animals were sequenced using Illumina paired-end technology at ultra-deep coverage. In order to determine the mutant spectra within the viral quasispecies, it is critical to understand the sequencing error rates and control for false positive calls of viral variants (point mutantations). I will estimate the sequencing error rate from two control sequences and characterize the mutant spectra in the natural samples with this error rate.

  5. Deep Sequencing of Urinary RNAs for Bladder Cancer Molecular Diagnostics.

    Science.gov (United States)

    Sin, Mandy L Y; Mach, Kathleen E; Sinha, Rahul; Wu, Fan; Trivedi, Dharati R; Altobelli, Emanuela; Jensen, Kristin C; Sahoo, Debashis; Lu, Ying; Liao, Joseph C

    2017-07-15

    Purpose: The majority of bladder cancer patients present with localized disease and are managed by transurethral resection. However, the high rate of recurrence necessitates lifetime cystoscopic surveillance. Developing a sensitive and specific urine-based test would significantly improve bladder cancer screening, detection, and surveillance. Experimental Design: RNA-seq was used for biomarker discovery to directly assess the gene expression profile of exfoliated urothelial cells in urine derived from bladder cancer patients ( n = 13) and controls ( n = 10). Eight bladder cancer specific and 3 reference genes identified by RNA-seq were quantitated by qPCR in a training cohort of 102 urine samples. A diagnostic model based on the training cohort was constructed using multiple logistic regression. The model was further validated in an independent cohort of 101 urines. Results: A total of 418 genes were found to be differentially expressed between bladder cancer and controls. Validation of a subset of these genes was used to construct an equation for computing a probability of bladder cancer score (P BC ) based on expression of three markers ( ROBO1, WNT5A , and CDC42BPB ). Setting P BC = 0.45 as the cutoff for a positive test, urine testing using the three-marker panel had overall 88% sensitivity and 92% specificity in the training cohort. The accuracy of the three-marker panel in the independent validation cohort yielded an AUC of 0.87 and overall 83% sensitivity and 89% specificity. Conclusions: Urine-based molecular diagnostics using this three-marker signature could provide a valuable adjunct to cystoscopy and may lead to a reduction of unnecessary procedures for bladder cancer diagnosis. Clin Cancer Res; 23(14); 3700-10. ©2017 AACR . ©2017 American Association for Cancer Research.

  6. Workup of Human Blood Samples for Deep Sequencing of HIV-1 Genomes

    NARCIS (Netherlands)

    Cornelissen, Marion; Gall, Astrid; van der Kuyl, Antoinette; Wymant, Chris; Blanquart, François; Fraser, Christophe; Berkhout, Ben

    2018-01-01

    We describe a detailed protocol for the manual workup of blood (plasma/serum) samples from individuals infected with the human immunodeficiency virus type 1 (HIV-1) for deep sequence analysis of the viral genome. The study optimizing the assay was performed in the context of the BEEHIVE (Bridging

  7. Efficient forward propagation of time-sequences in convolutional neural networks using Deep Shifting

    NARCIS (Netherlands)

    K.L. Groenland (Koen); S.M. Bohte (Sander)

    2016-01-01

    textabstractWhen a Convolutional Neural Network is used for on-the-fly evaluation of continuously updating time-sequences, many redundant convolution operations are performed. We propose the method of Deep Shifting, which remembers previously calculated results of convolution operations in order

  8. Deep RNA Sequencing of the Skeletal Muscle Transcriptome in Swimming Fish

    NARCIS (Netherlands)

    Palstra, A.P.; Beltran, S.; Burgerhout, E.; Brittijn, S.A.; Magnoni, L.J.; Henkel, C.V.; Jansen, A.; Thillart, G.E.E.J.M.; Spaink, H.P.; Planas, J.V.

    2013-01-01

    Deep RNA sequencing (RNA-seq) was performed to provide an in-depth view of the transcriptome of red and white skeletal muscle of exercised and non-exercised rainbow trout (Oncorhynchus mykiss) with the specific objective to identify expressed genes and quantify the transcriptomic effects of

  9. Reduced deep regional cerebral venous oxygen saturation in hemodialysis patients using quantitative susceptibility mapping.

    Science.gov (United States)

    Chai, Chao; Liu, Saifeng; Fan, Linlin; Liu, Lei; Li, Jinping; Zuo, Chao; Qian, Tianyi; Haacke, E Mark; Shen, Wen; Xia, Shuang

    2018-02-01

    Cerebral venous oxygen saturation (SvO 2 ) is an important indicator of brain function. There was debate about lower cerebral oxygen metabolism in hemodialysis patients and there were no reports about the changes of deep regional cerebral SvO 2 in hemodialysis patients. In this study, we aim to explore the deep regional cerebral SvO 2 from straight sinus using quantitative susceptibility mapping (QSM) and the correlation with clinical risk factors and neuropsychiatric testing . 52 hemodialysis patients and 54 age-and gender-matched healthy controls were enrolled. QSM reconstructed from original phase data of 3.0 T susceptibility-weighted imaging was used to measure the susceptibility of straight sinus. The susceptibility was used to calculate the deep regional cerebral SvO 2 and compare with healthy individuals. Correlation analysis was performed to investigate the correlation between deep regional cerebral SvO 2 , clinical risk factors and neuropsychiatric testing. The deep regional cerebral SvO 2 of hemodialysis patients (72.5 ± 3.7%) was significantly lower than healthy controls (76.0 ± 2.1%) (P deep regional cerebral SvO 2 in patients. The Mini-Mental State Examination (MMSE) scores of hemodialysis patients were significantly lower than healthy controls (P deep regional cerebral SvO 2 did not correlate with MMSE scores (P = 0.630). In summary, the decreased deep regional cerebral SvO 2 occurred in hemodialysis patients and dialysis duration, parathyroid hormone, hematocrit, hemoglobin and red blood cell may be clinical risk factors.

  10. AUC-Maximized Deep Convolutional Neural Fields for Protein Sequence Labeling.

    Science.gov (United States)

    Wang, Sheng; Sun, Siqi; Xu, Jinbo

    2016-09-01

    Deep Convolutional Neural Networks (DCNN) has shown excellent performance in a variety of machine learning tasks. This paper presents Deep Convolutional Neural Fields (DeepCNF), an integration of DCNN with Conditional Random Field (CRF), for sequence labeling with an imbalanced label distribution. The widely-used training methods, such as maximum-likelihood and maximum labelwise accuracy, do not work well on imbalanced data. To handle this, we present a new training algorithm called maximum-AUC for DeepCNF. That is, we train DeepCNF by directly maximizing the empirical Area Under the ROC Curve (AUC), which is an unbiased measurement for imbalanced data. To fulfill this, we formulate AUC in a pairwise ranking framework, approximate it by a polynomial function and then apply a gradient-based procedure to optimize it. Our experimental results confirm that maximum-AUC greatly outperforms the other two training methods on 8-state secondary structure prediction and disorder prediction since their label distributions are highly imbalanced and also has similar performance as the other two training methods on solvent accessibility prediction, which has three equally-distributed labels. Furthermore, our experimental results show that our AUC-trained DeepCNF models greatly outperform existing popular predictors of these three tasks. The data and software related to this paper are available at https://github.com/realbigws/DeepCNF_AUC.

  11. Shallow Investigations of the Deep Seafloor: Quantitative Morphology in the Levant Basin, Eastern Mediterranean

    Science.gov (United States)

    Kanari, M.; Ketter, T.; Tibor, G.; Schattner, U.

    2017-12-01

    We aim to characterize the seafloor morphology and its shallow sub-surface structures and deformations in the deep part of the Levant basin (eastern Mediterranean) using recently acquired high-resolution shallow seismic reflection data and multibeam bathymetry, which allow quantitative analysis of morphology and structure. The Levant basin at the eastern Mediterranean is considered a passive continental margin, where most of the recent geological processes were related in literature to salt tectonics rooted at the Messinian deposits from 6Ma. We analyzed two sets of recently acquired high-resolution data from multibeam bathymetry and 3.5 kHz Chirp sub-bottom seismic reflection in the deep basin of the continental shelf offshore Israel (water depths up to 2100 m). Semi-automatic mapping of seafloor features and seismic data interpretation resulted in quantitative morphological analysis of the seafloor and its underlying sediment with penetration depth up to 60 m. The quantitative analysis and its interpretation are still in progress. Preliminary results reveal distinct morphologies of four major elements: channels, faults, folds and sediment waves, validated by seismic data. From the spatial distribution and orientation analyses of these phenomena, we identify two primary process types which dominate the formation of the seafloor in the Levant basin: structural and sedimentary. Characterization of the geological and geomorphological processes forming the seafloor helps to better understand the transport mechanisms and the relations between sediment transport and deposition in deep water and the shallower parts of the shelf and slope.

  12. Enhanced arbovirus surveillance with deep sequencing: Identification of novel rhabdoviruses and bunyaviruses in Australian mosquitoes.

    Science.gov (United States)

    Coffey, Lark L; Page, Brady L; Greninger, Alexander L; Herring, Belinda L; Russell, Richard C; Doggett, Stephen L; Haniotis, John; Wang, Chunlin; Deng, Xutao; Delwart, Eric L

    2014-01-05

    Viral metagenomics characterizes known and identifies unknown viruses based on sequence similarities to any previously sequenced viral genomes. A metagenomics approach was used to identify virus sequences in Australian mosquitoes causing cytopathic effects in inoculated mammalian cell cultures. Sequence comparisons revealed strains of Liao Ning virus (Reovirus, Seadornavirus), previously detected only in China, livestock-infecting Stretch Lagoon virus (Reovirus, Orbivirus), two novel dimarhabdoviruses, named Beaumont and North Creek viruses, and two novel orthobunyaviruses, named Murrumbidgee and Salt Ash viruses. The novel virus proteomes diverged by ≥ 50% relative to their closest previously genetically characterized viral relatives. Deep sequencing also generated genomes of Warrego and Wallal viruses, orbiviruses linked to kangaroo blindness, whose genomes had not been fully characterized. This study highlights viral metagenomics in concert with traditional arbovirus surveillance to characterize known and new arboviruses in field-collected mosquitoes. Follow-up epidemiological studies are required to determine whether the novel viruses infect humans. © 2013 Elsevier Inc. All rights reserved.

  13. Simultaneous identification of DNA and RNA viruses present in pig faeces using process-controlled deep sequencing.

    Directory of Open Access Journals (Sweden)

    Jana Sachsenröder

    Full Text Available BACKGROUND: Animal faeces comprise a community of many different microorganisms including bacteria and viruses. Only scarce information is available about the diversity of viruses present in the faeces of pigs. Here we describe a protocol, which was optimized for the purification of the total fraction of viral particles from pig faeces. The genomes of the purified DNA and RNA viruses were simultaneously amplified by PCR and subjected to deep sequencing followed by bioinformatic analyses. The efficiency of the method was monitored using a process control consisting of three bacteriophages (T4, M13 and MS2 with different morphology and genome types. Defined amounts of the bacteriophages were added to the sample and their abundance was assessed by quantitative PCR during the preparation procedure. RESULTS: The procedure was applied to a pooled faecal sample of five pigs. From this sample, 69,613 sequence reads were generated. All of the added bacteriophages were identified by sequence analysis of the reads. In total, 7.7% of the reads showed significant sequence identities with published viral sequences. They mainly originated from bacteriophages (73.9% and mammalian viruses (23.9%; 0.8% of the sequences showed identities to plant viruses. The most abundant detected porcine viruses were kobuvirus, rotavirus C, astrovirus, enterovirus B, sapovirus and picobirnavirus. In addition, sequences with identities to the chimpanzee stool-associated circular ssDNA virus were identified. Whole genome analysis indicates that this virus, tentatively designated as pig stool-associated circular ssDNA virus (PigSCV, represents a novel pig virus. CONCLUSION: The established protocol enables the simultaneous detection of DNA and RNA viruses in pig faeces including the identification of so far unknown viruses. It may be applied in studies investigating aetiology, epidemiology and ecology of diseases. The implemented process control serves as quality control, ensures

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

    Science.gov (United States)

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

    2016-10-01

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

  15. MicroRNA discovery and analysis of pinewood nematode Bursaphelenchus xylophilus by deep sequencing.

    Directory of Open Access Journals (Sweden)

    Qi-Xing Huang

    Full Text Available BACKGROUND: MicroRNAs (miRNAs are considered to be very important in regulating the growth, development, behavior and stress response in animals and plants in post-transcriptional gene regulation. Pinewood nematode, Bursaphelenchus xylophilus, is an important invasive plant parasitic nematode in Asia. To have a comprehensive knowledge about miRNAs of the nematode is necessary for further in-depth study on roles of miRNAs in the ecological adaptation of the invasive species. METHODS AND FINDINGS: Five small RNA libraries were constructed and sequenced by Illumina/Solexa deep-sequencing technology. A total of 810 miRNA candidates (49 conserved and 761 novel were predicted by a computational pipeline, of which 57 miRNAs (20 conserved and 37 novel encoded by 53 miRNA precursors were identified by experimental methods. Ten novel miRNAs were considered to be species-specific miRNAs of B. xylophilus. Comparison of expression profiles of miRNAs in the five small RNA libraries showed that many miRNAs exhibited obviously different expression levels in the third-stage dispersal juvenile and at a cold-stressed status. Most of the miRNAs exhibited obviously down-regulated expression in the dispersal stage. But differences among the three geographic libraries were not prominent. A total of 979 genes were predicted to be targets of these authentic miRNAs. Among them, seven heat shock protein genes were targeted by 14 miRNAs, and six FMRFamide-like neuropeptides genes were targeted by 17 miRNAs. A real-time quantitative polymerase chain reaction was used to quantify the mRNA expression levels of target genes. CONCLUSIONS: Basing on the fact that a negative correlation existed between the expression profiles of miRNAs and the mRNA expression profiles of their target genes (hsp, flp by comparing those of the nematodes at a cold stressed status and a normal status, we suggested that miRNAs might participate in ecological adaptation and behavior regulation of the

  16. Ultra-deep sequencing of intra-host rabies virus populations during cross-species transmission.

    Directory of Open Access Journals (Sweden)

    Monica K Borucki

    2013-11-01

    Full Text Available One of the hurdles to understanding the role of viral quasispecies in RNA virus cross-species transmission (CST events is the need to analyze a densely sampled outbreak using deep sequencing in order to measure the amount of mutation occurring on a small time scale. In 2009, the California Department of Public Health reported a dramatic increase (350 in the number of gray foxes infected with a rabies virus variant for which striped skunks serve as a reservoir host in Humboldt County. To better understand the evolution of rabies, deep-sequencing was applied to 40 unpassaged rabies virus samples from the Humboldt outbreak. For each sample, approximately 11 kb of the 12 kb genome was amplified and sequenced using the Illumina platform. Average coverage was 17,448 and this allowed characterization of the rabies virus population present in each sample at unprecedented depths. Phylogenetic analysis of the consensus sequence data demonstrated that samples clustered according to date (1995 vs. 2009 and geographic location (northern vs. southern. A single amino acid change in the G protein distinguished a subset of northern foxes from a haplotype present in both foxes and skunks, suggesting this mutation may have played a role in the observed increased transmission among foxes in this region. Deep-sequencing data indicated that many genetic changes associated with the CST event occurred prior to 2009 since several nonsynonymous mutations that were present in the consensus sequences of skunk and fox rabies samples obtained from 20032010 were present at the sub-consensus level (as rare variants in the viral population in skunk and fox samples from 1995. These results suggest that analysis of rare variants within a viral population may yield clues to ancestral genomes and identify rare variants that have the potential to be selected for if environment conditions change.

  17. Chiron: translating nanopore raw signal directly into nucleotide sequence using deep learning

    KAUST Repository

    Teng, Haotian; Cao, Minh Duc; Hall, Michael B; Duarte, Tania; Wang, Sheng; Coin, Lachlan J M

    2018-01-01

    Sequencing by translocating DNA fragments through an array of nanopores is a rapidly maturing technology that offers faster and cheaper sequencing than other approaches. However, accurately deciphering the DNA sequence from the noisy and complex electrical signal is challenging. Here, we report Chiron, the first deep learning model to achieve end-to-end basecalling and directly translate the raw signal to DNA sequence without the error-prone segmentation step. Trained with only a small set of 4,000 reads, we show that our model provides state-of-the-art basecalling accuracy, even on previously unseen species. Chiron achieves basecalling speeds of more than 2,000 bases per second using desktop computer graphics processing units.

  18. Chiron: translating nanopore raw signal directly into nucleotide sequence using deep learning

    KAUST Repository

    Teng, Haotian

    2018-04-10

    Sequencing by translocating DNA fragments through an array of nanopores is a rapidly maturing technology that offers faster and cheaper sequencing than other approaches. However, accurately deciphering the DNA sequence from the noisy and complex electrical signal is challenging. Here, we report Chiron, the first deep learning model to achieve end-to-end basecalling and directly translate the raw signal to DNA sequence without the error-prone segmentation step. Trained with only a small set of 4,000 reads, we show that our model provides state-of-the-art basecalling accuracy, even on previously unseen species. Chiron achieves basecalling speeds of more than 2,000 bases per second using desktop computer graphics processing units.

  19. LookSeq: a browser-based viewer for deep sequencing data.

    Science.gov (United States)

    Manske, Heinrich Magnus; Kwiatkowski, Dominic P

    2009-11-01

    Sequencing a genome to great depth can be highly informative about heterogeneity within an individual or a population. Here we address the problem of how to visualize the multiple layers of information contained in deep sequencing data. We propose an interactive AJAX-based web viewer for browsing large data sets of aligned sequence reads. By enabling seamless browsing and fast zooming, the LookSeq program assists the user to assimilate information at different levels of resolution, from an overview of a genomic region to fine details such as heterogeneity within the sample. A specific problem, particularly if the sample is heterogeneous, is how to depict information about structural variation. LookSeq provides a simple graphical representation of paired sequence reads that is more revealing about potential insertions and deletions than are conventional methods.

  20. A Quantitative Accident Sequence Analysis for a VHTR

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Jintae; Lee, Joeun; Jae, Moosung [Hanyang University, Seoul (Korea, Republic of)

    2016-05-15

    In Korea, the basic design features of VHTR are currently discussed in the various design concepts. Probabilistic risk assessment (PRA) offers a logical and structured method to assess risks of a large and complex engineered system, such as a nuclear power plant. It will be introduced at an early stage in the design, and will be upgraded at various design and licensing stages as the design matures and the design details are defined. Risk insights to be developed from the PRA are viewed as essential to developing a design that is optimized in meeting safety objectives and in interpreting the applicability of the existing demands to the safety design approach of the VHTR. In this study, initiating events which may occur in VHTRs were selected through MLD method. The initiating events were then grouped into four categories for the accident sequence analysis. Initiating events frequency and safety systems failure rate were calculated by using reliability data obtained from the available sources and fault tree analysis. After quantification, uncertainty analysis was conducted. The SR and LR frequency are calculated respectively 7.52E- 10/RY and 7.91E-16/RY, which are relatively less than the core damage frequency of LWRs.

  1. Prognostic value of deep sequencing method for minimal residual disease detection in multiple myeloma

    Science.gov (United States)

    Lahuerta, Juan J.; Pepin, François; González, Marcos; Barrio, Santiago; Ayala, Rosa; Puig, Noemí; Montalban, María A.; Paiva, Bruno; Weng, Li; Jiménez, Cristina; Sopena, María; Moorhead, Martin; Cedena, Teresa; Rapado, Immaculada; Mateos, María Victoria; Rosiñol, Laura; Oriol, Albert; Blanchard, María J.; Martínez, Rafael; Bladé, Joan; San Miguel, Jesús; Faham, Malek; García-Sanz, Ramón

    2014-01-01

    We assessed the prognostic value of minimal residual disease (MRD) detection in multiple myeloma (MM) patients using a sequencing-based platform in bone marrow samples from 133 MM patients in at least very good partial response (VGPR) after front-line therapy. Deep sequencing was carried out in patients in whom a high-frequency myeloma clone was identified and MRD was assessed using the IGH-VDJH, IGH-DJH, and IGK assays. The results were contrasted with those of multiparametric flow cytometry (MFC) and allele-specific oligonucleotide polymerase chain reaction (ASO-PCR). The applicability of deep sequencing was 91%. Concordance between sequencing and MFC and ASO-PCR was 83% and 85%, respectively. Patients who were MRD– by sequencing had a significantly longer time to tumor progression (TTP) (median 80 vs 31 months; P < .0001) and overall survival (median not reached vs 81 months; P = .02), compared with patients who were MRD+. When stratifying patients by different levels of MRD, the respective TTP medians were: MRD ≥10−3 27 months, MRD 10−3 to 10−5 48 months, and MRD <10−5 80 months (P = .003 to .0001). Ninety-two percent of VGPR patients were MRD+. In complete response patients, the TTP remained significantly longer for MRD– compared with MRD+ patients (131 vs 35 months; P = .0009). PMID:24646471

  2. miRBase: integrating microRNA annotation and deep-sequencing data.

    Science.gov (United States)

    Kozomara, Ana; Griffiths-Jones, Sam

    2011-01-01

    miRBase is the primary online repository for all microRNA sequences and annotation. The current release (miRBase 16) contains over 15,000 microRNA gene loci in over 140 species, and over 17,000 distinct mature microRNA sequences. Deep-sequencing technologies have delivered a sharp rise in the rate of novel microRNA discovery. We have mapped reads from short RNA deep-sequencing experiments to microRNAs in miRBase and developed web interfaces to view these mappings. The user can view all read data associated with a given microRNA annotation, filter reads by experiment and count, and search for microRNAs by tissue- and stage-specific expression. These data can be used as a proxy for relative expression levels of microRNA sequences, provide detailed evidence for microRNA annotations and alternative isoforms of mature microRNAs, and allow us to revisit previous annotations. miRBase is available online at: http://www.mirbase.org/.

  3. Analysis of Plasmodium falciparum diversity in natural infections by deep sequencing

    OpenAIRE

    Manske, Magnus; Miotto, Olivo; Campino, Susana; Auburn, Sarah; Almagro-Garcia, Jacob; Maslen, Gareth; O?Brien, Jack; Djimde, Abdoulaye; Doumbo, Ogobara; Zongo, Issaka; Ouedraogo, Jean-Bosco; Michon, Pascal; Mueller, Ivo; Siba, Peter; Nzila, Alexis

    2012-01-01

    : Malaria elimination strategies require surveillance of the parasite population for genetic changes that demand a public health response, such as new forms of drug resistance. Here we describe methods for the large-scale analysis of genetic variation in Plasmodium falciparum by deep sequencing of parasite DNA obtained from the blood of patients with malaria, either directly or after short-term culture. Analysis of 86,158 exonic single nucleotide polymorphisms that passed genotyping quality c...

  4. A novel wavelet sequence based on deep bidirectional LSTM network model for ECG signal classification.

    Science.gov (United States)

    Yildirim, Özal

    2018-05-01

    Long-short term memory networks (LSTMs), which have recently emerged in sequential data analysis, are the most widely used type of recurrent neural networks (RNNs) architecture. Progress on the topic of deep learning includes successful adaptations of deep versions of these architectures. In this study, a new model for deep bidirectional LSTM network-based wavelet sequences called DBLSTM-WS was proposed for classifying electrocardiogram (ECG) signals. For this purpose, a new wavelet-based layer is implemented to generate ECG signal sequences. The ECG signals were decomposed into frequency sub-bands at different scales in this layer. These sub-bands are used as sequences for the input of LSTM networks. New network models that include unidirectional (ULSTM) and bidirectional (BLSTM) structures are designed for performance comparisons. Experimental studies have been performed for five different types of heartbeats obtained from the MIT-BIH arrhythmia database. These five types are Normal Sinus Rhythm (NSR), Ventricular Premature Contraction (VPC), Paced Beat (PB), Left Bundle Branch Block (LBBB), and Right Bundle Branch Block (RBBB). The results show that the DBLSTM-WS model gives a high recognition performance of 99.39%. It has been observed that the wavelet-based layer proposed in the study significantly improves the recognition performance of conventional networks. This proposed network structure is an important approach that can be applied to similar signal processing problems. Copyright © 2018 Elsevier Ltd. All rights reserved.

  5. Continuous Distributed Representation of Biological Sequences for Deep Proteomics and Genomics.

    Directory of Open Access Journals (Sweden)

    Ehsaneddin Asgari

    Full Text Available We introduce a new representation and feature extraction method for biological sequences. Named bio-vectors (BioVec to refer to biological sequences in general with protein-vectors (ProtVec for proteins (amino-acid sequences and gene-vectors (GeneVec for gene sequences, this representation can be widely used in applications of deep learning in proteomics and genomics. In the present paper, we focus on protein-vectors that can be utilized in a wide array of bioinformatics investigations such as family classification, protein visualization, structure prediction, disordered protein identification, and protein-protein interaction prediction. In this method, we adopt artificial neural network approaches and represent a protein sequence with a single dense n-dimensional vector. To evaluate this method, we apply it in classification of 324,018 protein sequences obtained from Swiss-Prot belonging to 7,027 protein families, where an average family classification accuracy of 93%±0.06% is obtained, outperforming existing family classification methods. In addition, we use ProtVec representation to predict disordered proteins from structured proteins. Two databases of disordered sequences are used: the DisProt database as well as a database featuring the disordered regions of nucleoporins rich with phenylalanine-glycine repeats (FG-Nups. Using support vector machine classifiers, FG-Nup sequences are distinguished from structured protein sequences found in Protein Data Bank (PDB with a 99.8% accuracy, and unstructured DisProt sequences are differentiated from structured DisProt sequences with 100.0% accuracy. These results indicate that by only providing sequence data for various proteins into this model, accurate information about protein structure can be determined. Importantly, this model needs to be trained only once and can then be applied to extract a comprehensive set of information regarding proteins of interest. Moreover, this representation can be

  6. CPSS: a computational platform for the analysis of small RNA deep sequencing data.

    Science.gov (United States)

    Zhang, Yuanwei; Xu, Bo; Yang, Yifan; Ban, Rongjun; Zhang, Huan; Jiang, Xiaohua; Cooke, Howard J; Xue, Yu; Shi, Qinghua

    2012-07-15

    Next generation sequencing (NGS) techniques have been widely used to document the small ribonucleic acids (RNAs) implicated in a variety of biological, physiological and pathological processes. An integrated computational tool is needed for handling and analysing the enormous datasets from small RNA deep sequencing approach. Herein, we present a novel web server, CPSS (a computational platform for the analysis of small RNA deep sequencing data), designed to completely annotate and functionally analyse microRNAs (miRNAs) from NGS data on one platform with a single data submission. Small RNA NGS data can be submitted to this server with analysis results being returned in two parts: (i) annotation analysis, which provides the most comprehensive analysis for small RNA transcriptome, including length distribution and genome mapping of sequencing reads, small RNA quantification, prediction of novel miRNAs, identification of differentially expressed miRNAs, piwi-interacting RNAs and other non-coding small RNAs between paired samples and detection of miRNA editing and modifications and (ii) functional analysis, including prediction of miRNA targeted genes by multiple tools, enrichment of gene ontology terms, signalling pathway involvement and protein-protein interaction analysis for the predicted genes. CPSS, a ready-to-use web server that integrates most functions of currently available bioinformatics tools, provides all the information wanted by the majority of users from small RNA deep sequencing datasets. CPSS is implemented in PHP/PERL+MySQL+R and can be freely accessed at http://mcg.ustc.edu.cn/db/cpss/index.html or http://mcg.ustc.edu.cn/sdap1/cpss/index.html.

  7. Deep sequencing reveals double mutations in cis of MPL exon 10 in myeloproliferative neoplasms.

    Science.gov (United States)

    Pietra, Daniela; Brisci, Angela; Rumi, Elisa; Boggi, Sabrina; Elena, Chiara; Pietrelli, Alessandro; Bordoni, Roberta; Ferrari, Maurizio; Passamonti, Francesco; De Bellis, Gianluca; Cremonesi, Laura; Cazzola, Mario

    2011-04-01

    Somatic mutations of MPL exon 10, mainly involving a W515 substitution, have been described in JAK2 (V617F)-negative patients with essential thrombocythemia and primary myelofibrosis. We used direct sequencing and high-resolution melt analysis to identify mutations of MPL exon 10 in 570 patients with myeloproliferative neoplasms, and allele specific PCR and deep sequencing to further characterize a subset of mutated patients. Somatic mutations were detected in 33 of 221 patients (15%) with JAK2 (V617F)-negative essential thrombocythemia or primary myelofibrosis. Only one patient with essential thrombocythemia carried both JAK2 (V617F) and MPL (W515L). High-resolution melt analysis identified abnormal patterns in all the MPL mutated cases, while direct sequencing did not detect the mutant MPL in one fifth of them. In 3 cases carrying double MPL mutations, deep sequencing analysis showed identical load and location in cis of the paired lesions, indicating their simultaneous occurrence on the same chromosome.

  8. Deep sequencing analysis of HBV genotype shift and correlation with antiviral efficiency during adefovir dipivoxil therapy.

    Directory of Open Access Journals (Sweden)

    Yuwei Wang

    Full Text Available Viral genotype shift in chronic hepatitis B (CHB patients during antiviral therapy has been reported, but the underlying mechanism remains elusive.38 CHB patients treated with ADV for one year were selected for studying genotype shift by both deep sequencing and Sanger sequencing method.Sanger sequencing method found that 7.9% patients showed mixed genotype before ADV therapy. In contrast, all 38 patients showed mixed genotype before ADV treatment by deep sequencing. 95.5% mixed genotype rate was also obtained from additional 200 treatment-naïve CHB patients. Of the 13 patients with genotype shift, the fraction of the minor genotype in 5 patients (38% increased gradually during the course of ADV treatment. Furthermore, responses to ADV and HBeAg seroconversion were associated with the high rate of genotype shift, suggesting drug and immune pressure may be key factors to induce genotype shift. Interestingly, patients with genotype C had a significantly higher rate of genotype shift than genotype B. In genotype shift group, ADV treatment induced a marked enhancement of genotype B ratio accompanied by a reduction of genotype C ratio, suggesting genotype C may be more sensitive to ADV than genotype B. Moreover, patients with dominant genotype C may have a better therapeutic effect. Finally, genotype shifts was correlated with clinical improvement in terms of ALT.Our findings provided a rational explanation for genotype shift among ADV-treated CHB patients. The genotype and genotype shift might be associated with antiviral efficiency.

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

    Science.gov (United States)

    Kozomara, Ana; Griffiths-Jones, Sam

    2014-01-01

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

  10. Exploring the Mechanisms of Gastrointestinal Cancer Development Using Deep Sequencing Analysis

    International Nuclear Information System (INIS)

    Matsumoto, Tomonori; Shimizu, Takahiro; Takai, Atsushi; Marusawa, Hiroyuki

    2015-01-01

    Next-generation sequencing (NGS) technologies have revolutionized cancer genomics due to their high throughput sequencing capacity. Reports of the gene mutation profiles of various cancers by many researchers, including international cancer genome research consortia, have increased over recent years. In addition to detecting somatic mutations in tumor cells, NGS technologies enable us to approach the subject of carcinogenic mechanisms from new perspectives. Deep sequencing, a method of optimizing the high throughput capacity of NGS technologies, allows for the detection of genetic aberrations in small subsets of premalignant and/or tumor cells in noncancerous chronically inflamed tissues. Genome-wide NGS data also make it possible to clarify the mutational signatures of each cancer tissue by identifying the precise pattern of nucleotide alterations in the cancer genome, providing new information regarding the mechanisms of tumorigenesis. In this review, we highlight these new methods taking advantage of NGS technologies, and discuss our current understanding of carcinogenic mechanisms elucidated from such approaches

  11. Exploring the Mechanisms of Gastrointestinal Cancer Development Using Deep Sequencing Analysis

    Energy Technology Data Exchange (ETDEWEB)

    Matsumoto, Tomonori; Shimizu, Takahiro; Takai, Atsushi; Marusawa, Hiroyuki, E-mail: maru@kuhp.kyoto-u.ac.jp [Department of Gastroenterology and Hepatology, Graduate School of Medicine, Kyoto University, 54 Shogoin-Kawahara-cho, Sakyo-ku, Kyoto 606-8507 (Japan)

    2015-06-15

    Next-generation sequencing (NGS) technologies have revolutionized cancer genomics due to their high throughput sequencing capacity. Reports of the gene mutation profiles of various cancers by many researchers, including international cancer genome research consortia, have increased over recent years. In addition to detecting somatic mutations in tumor cells, NGS technologies enable us to approach the subject of carcinogenic mechanisms from new perspectives. Deep sequencing, a method of optimizing the high throughput capacity of NGS technologies, allows for the detection of genetic aberrations in small subsets of premalignant and/or tumor cells in noncancerous chronically inflamed tissues. Genome-wide NGS data also make it possible to clarify the mutational signatures of each cancer tissue by identifying the precise pattern of nucleotide alterations in the cancer genome, providing new information regarding the mechanisms of tumorigenesis. In this review, we highlight these new methods taking advantage of NGS technologies, and discuss our current understanding of carcinogenic mechanisms elucidated from such approaches.

  12. Quantitative methods for evaluating the efficacy of thalamic deep brain stimulation in patients with essential tremor.

    Science.gov (United States)

    Wastensson, Gunilla; Holmberg, Björn; Johnels, Bo; Barregard, Lars

    2013-01-01

    Deep brain stimulation (DBS) of the thalamus is a safe and efficient method for treatment of disabling tremor in patient with essential tremor (ET). However, successful tremor suppression after surgery requires careful selection of stimulus parameters. Our aim was to examine the possible use of certain quantitative methods for evaluating the efficacy of thalamic DBS in ET patients in clinical practice, and to compare these methods with traditional clinical tests. We examined 22 patients using the Essential Tremor Rating Scale (ETRS) and quantitative assessment of tremor with the stimulator both activated and deactivated. We used an accelerometer (CATSYS tremor Pen) for quantitative measurement of postural tremor, and a eurythmokinesimeter (EKM) to evaluate kinetic tremor in a rapid pointing task. The efficacy of DBS on tremor suppression was prominent irrespective of the method used. The agreement between clinical rating of postural tremor and tremor intensity as measured by the CATSYS tremor pen was relatively high (rs = 0.74). The agreement between kinetic tremor as assessed by the ETRS and the main outcome variable from the EKM test was low (rs = 0.34). The lack of agreement indicates that the EKM test is not comparable with the clinical test. Quantitative methods, such as the CATSYS tremor pen, could be a useful complement to clinical tremor assessment in evaluating the efficacy of DBS in clinical practice. Future studies should evaluate the precision of these methods and long-term impact on tremor suppression, activities of daily living (ADL) function and quality of life.

  13. Quantitative trait locus mapping of deep rooting by linkage and association analysis in rice.

    Science.gov (United States)

    Lou, Qiaojun; Chen, Liang; Mei, Hanwei; Wei, Haibin; Feng, Fangjun; Wang, Pei; Xia, Hui; Li, Tiemei; Luo, Lijun

    2015-08-01

    Deep rooting is a very important trait for plants' drought avoidance, and it is usually represented by the ratio of deep rooting (RDR). Three sets of rice populations were used to determine the genetic base for RDR. A linkage mapping population with 180 recombinant inbred lines and an association mapping population containing 237 rice varieties were used to identify genes linked to RDR. Six quantitative trait loci (QTLs) of RDR were identified as being located on chromosomes 1, 2, 4, 7, and 10. Using 1 019 883 single-nucleotide polymorphisms (SNPs), a genome-wide association study of the RDR was performed. Forty-eight significant SNPs of the RDR were identified and formed a clear peak on the short arm of chromosome 1 in a Manhattan plot. Compared with the shallow-rooting group and the whole collection, the deep-rooting group had selective sweep regions on chromosomes 1 and 2, especially in the major QTL region on chromosome 2. Seven of the nine candidate SNPs identified by association mapping were verified in two RDR extreme groups. The findings from this study will be beneficial to rice drought-resistance research and breeding. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  14. Deep Learning Automates the Quantitative Analysis of Individual Cells in Live-Cell Imaging Experiments.

    Science.gov (United States)

    Van Valen, David A; Kudo, Takamasa; Lane, Keara M; Macklin, Derek N; Quach, Nicolas T; DeFelice, Mialy M; Maayan, Inbal; Tanouchi, Yu; Ashley, Euan A; Covert, Markus W

    2016-11-01

    Live-cell imaging has opened an exciting window into the role cellular heterogeneity plays in dynamic, living systems. A major critical challenge for this class of experiments is the problem of image segmentation, or determining which parts of a microscope image correspond to which individual cells. Current approaches require many hours of manual curation and depend on approaches that are difficult to share between labs. They are also unable to robustly segment the cytoplasms of mammalian cells. Here, we show that deep convolutional neural networks, a supervised machine learning method, can solve this challenge for multiple cell types across the domains of life. We demonstrate that this approach can robustly segment fluorescent images of cell nuclei as well as phase images of the cytoplasms of individual bacterial and mammalian cells from phase contrast images without the need for a fluorescent cytoplasmic marker. These networks also enable the simultaneous segmentation and identification of different mammalian cell types grown in co-culture. A quantitative comparison with prior methods demonstrates that convolutional neural networks have improved accuracy and lead to a significant reduction in curation time. We relay our experience in designing and optimizing deep convolutional neural networks for this task and outline several design rules that we found led to robust performance. We conclude that deep convolutional neural networks are an accurate method that require less curation time, are generalizable to a multiplicity of cell types, from bacteria to mammalian cells, and expand live-cell imaging capabilities to include multi-cell type systems.

  15. Sequence-based prediction of protein protein interaction using a deep-learning algorithm.

    Science.gov (United States)

    Sun, Tanlin; Zhou, Bo; Lai, Luhua; Pei, Jianfeng

    2017-05-25

    Protein-protein interactions (PPIs) are critical for many biological processes. It is therefore important to develop accurate high-throughput methods for identifying PPI to better understand protein function, disease occurrence, and therapy design. Though various computational methods for predicting PPI have been developed, their robustness for prediction with external datasets is unknown. Deep-learning algorithms have achieved successful results in diverse areas, but their effectiveness for PPI prediction has not been tested. We used a stacked autoencoder, a type of deep-learning algorithm, to study the sequence-based PPI prediction. The best model achieved an average accuracy of 97.19% with 10-fold cross-validation. The prediction accuracies for various external datasets ranged from 87.99% to 99.21%, which are superior to those achieved with previous methods. To our knowledge, this research is the first to apply a deep-learning algorithm to sequence-based PPI prediction, and the results demonstrate its potential in this field.

  16. Deep sequence characterisation of a divergent HPIV-4a from an adult with prolonged influenza-like illness

    Directory of Open Access Journals (Sweden)

    Katherine E. Arden

    2015-12-01

    Deep sequencing allowed identification and genomic characterisation of a possible pathogen from an ILI as well as being an important tool to aid future understanding of the linkages between viral genetic variation, transmission and disease prognosis.

  17. An introduction to deep learning on biological sequence data: examples and solutions.

    Science.gov (United States)

    Jurtz, Vanessa Isabell; Johansen, Alexander Rosenberg; Nielsen, Morten; Almagro Armenteros, Jose Juan; Nielsen, Henrik; Sønderby, Casper Kaae; Winther, Ole; Sønderby, Søren Kaae

    2017-11-15

    Deep neural network architectures such as convolutional and long short-term memory networks have become increasingly popular as machine learning tools during the recent years. The availability of greater computational resources, more data, new algorithms for training deep models and easy to use libraries for implementation and training of neural networks are the drivers of this development. The use of deep learning has been especially successful in image recognition; and the development of tools, applications and code examples are in most cases centered within this field rather than within biology. Here, we aim to further the development of deep learning methods within biology by providing application examples and ready to apply and adapt code templates. Given such examples, we illustrate how architectures consisting of convolutional and long short-term memory neural networks can relatively easily be designed and trained to state-of-the-art performance on three biological sequence problems: prediction of subcellular localization, protein secondary structure and the binding of peptides to MHC Class II molecules. All implementations and datasets are available online to the scientific community at https://github.com/vanessajurtz/lasagne4bio. skaaesonderby@gmail.com. Supplementary data are available at Bioinformatics online. © The Author (2017). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

  18. Quantitative comparison between a multiecho sequence and a single-echo sequence for susceptibility-weighted phase imaging.

    Science.gov (United States)

    Gilbert, Guillaume; Savard, Geneviève; Bard, Céline; Beaudoin, Gilles

    2012-06-01

    The aim of this study was to investigate the benefits arising from the use of a multiecho sequence for susceptibility-weighted phase imaging using a quantitative comparison with a standard single-echo acquisition. Four healthy adult volunteers were imaged on a clinical 3-T system using a protocol comprising two different three-dimensional susceptibility-weighted gradient-echo sequences: a standard single-echo sequence and a multiecho sequence. Both sequences were repeated twice in order to evaluate the local noise contribution by a subtraction of the two acquisitions. For the multiecho sequence, the phase information from each echo was independently unwrapped, and the background field contribution was removed using either homodyne filtering or the projection onto dipole fields method. The phase information from all echoes was then combined using a weighted linear regression. R2 maps were also calculated from the multiecho acquisitions. The noise standard deviation in the reconstructed phase images was evaluated for six manually segmented regions of interest (frontal white matter, posterior white matter, globus pallidus, putamen, caudate nucleus and lateral ventricle). The use of the multiecho sequence for susceptibility-weighted phase imaging led to a reduction of the noise standard deviation for all subjects and all regions of interest investigated in comparison to the reference single-echo acquisition. On average, the noise reduction ranged from 18.4% for the globus pallidus to 47.9% for the lateral ventricle. In addition, the amount of noise reduction was found to be strongly inversely correlated to the estimated R2 value (R=-0.92). In conclusion, the use of a multiecho sequence is an effective way to decrease the noise contribution in susceptibility-weighted phase images, while preserving both contrast and acquisition time. The proposed approach additionally permits the calculation of R2 maps. Copyright © 2012 Elsevier Inc. All rights reserved.

  19. Deep sequencing discovery of novel and conserved microRNAs in trifoliate orange (Citrus trifoliata

    Directory of Open Access Journals (Sweden)

    Yu Huaping

    2010-07-01

    Full Text Available Abstract Background MicroRNAs (miRNAs play a critical role in post-transcriptional gene regulation and have been shown to control many genes involved in various biological and metabolic processes. There have been extensive studies to discover miRNAs and analyze their functions in model plant species, such as Arabidopsis and rice. Deep sequencing technologies have facilitated identification of species-specific or lowly expressed as well as conserved or highly expressed miRNAs in plants. Results In this research, we used Solexa sequencing to discover new microRNAs in trifoliate orange (Citrus trifoliata which is an important rootstock of citrus. A total of 13,106,753 reads representing 4,876,395 distinct sequences were obtained from a short RNA library generated from small RNA extracted from C. trifoliata flower and fruit tissues. Based on sequence similarity and hairpin structure prediction, we found that 156,639 reads representing 63 sequences from 42 highly conserved miRNA families, have perfect matches to known miRNAs. We also identified 10 novel miRNA candidates whose precursors were all potentially generated from citrus ESTs. In addition, five miRNA* sequences were also sequenced. These sequences had not been earlier described in other plant species and accumulation of the 10 novel miRNAs were confirmed by qRT-PCR analysis. Potential target genes were predicted for most conserved and novel miRNAs. Moreover, four target genes including one encoding IRX12 copper ion binding/oxidoreductase and three genes encoding NB-LRR disease resistance protein have been experimentally verified by detection of the miRNA-mediated mRNA cleavage in C. trifoliata. Conclusion Deep sequencing of short RNAs from C. trifoliata flowers and fruits identified 10 new potential miRNAs and 42 highly conserved miRNA families, indicating that specific miRNAs exist in C. trifoliata. These results show that regulatory miRNAs exist in agronomically important trifoliate orange

  20. Quantitative analysis of patients with celiac disease by video capsule endoscopy: A deep learning method.

    Science.gov (United States)

    Zhou, Teng; Han, Guoqiang; Li, Bing Nan; Lin, Zhizhe; Ciaccio, Edward J; Green, Peter H; Qin, Jing

    2017-06-01

    Celiac disease is one of the most common diseases in the world. Capsule endoscopy is an alternative way to visualize the entire small intestine without invasiveness to the patient. It is useful to characterize celiac disease, but hours are need to manually analyze the retrospective data of a single patient. Computer-aided quantitative analysis by a deep learning method helps in alleviating the workload during analysis of the retrospective videos. Capsule endoscopy clips from 6 celiac disease patients and 5 controls were preprocessed for training. The frames with a large field of opaque extraluminal fluid or air bubbles were removed automatically by using a pre-selection algorithm. Then the frames were cropped and the intensity was corrected prior to frame rotation in the proposed new method. The GoogLeNet is trained with these frames. Then, the clips of capsule endoscopy from 5 additional celiac disease patients and 5 additional control patients are used for testing. The trained GoogLeNet was able to distinguish the frames from capsule endoscopy clips of celiac disease patients vs controls. Quantitative measurement with evaluation of the confidence was developed to assess the severity level of pathology in the subjects. Relying on the evaluation confidence, the GoogLeNet achieved 100% sensitivity and specificity for the testing set. The t-test confirmed the evaluation confidence is significant to distinguish celiac disease patients from controls. Furthermore, it is found that the evaluation confidence may also relate to the severity level of small bowel mucosal lesions. A deep convolutional neural network was established for quantitative measurement of the existence and degree of pathology throughout the small intestine, which may improve computer-aided clinical techniques to assess mucosal atrophy and other etiologies in real-time with videocapsule endoscopy. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. Development of genic-SSR markers by deep transcriptome sequencing in pigeonpea [Cajanus cajan (L.) Millspaugh

    Science.gov (United States)

    2011-01-01

    Background Pigeonpea [Cajanus cajan (L.) Millspaugh], one of the most important food legumes of semi-arid tropical and subtropical regions, has limited genomic resources, particularly expressed sequence based (genic) markers. We report a comprehensive set of validated genic simple sequence repeat (SSR) markers using deep transcriptome sequencing, and its application in genetic diversity analysis and mapping. Results In this study, 43,324 transcriptome shotgun assembly unigene contigs were assembled from 1.696 million 454 GS-FLX sequence reads of separate pooled cDNA libraries prepared from leaf, root, stem and immature seed of two pigeonpea varieties, Asha and UPAS 120. A total of 3,771 genic-SSR loci, excluding homopolymeric and compound repeats, were identified; of which 2,877 PCR primer pairs were designed for marker development. Dinucleotide was the most common repeat motif with a frequency of 60.41%, followed by tri- (34.52%), hexa- (2.62%), tetra- (1.67%) and pentanucleotide (0.76%) repeat motifs. Primers were synthesized and tested for 772 of these loci with repeat lengths of ≥18 bp. Of these, 550 markers were validated for consistent amplification in eight diverse pigeonpea varieties; 71 were found to be polymorphic on agarose gel electrophoresis. Genetic diversity analysis was done on 22 pigeonpea varieties and eight wild species using 20 highly polymorphic genic-SSR markers. The number of alleles at these loci ranged from 4-10 and the polymorphism information content values ranged from 0.46 to 0.72. Neighbor-joining dendrogram showed distinct separation of the different groups of pigeonpea cultivars and wild species. Deep transcriptome sequencing of the two parental lines helped in silico identification of polymorphic genic-SSR loci to facilitate the rapid development of an intra-species reference genetic map, a subset of which was validated for expected allelic segregation in the reference mapping population. Conclusion We developed 550 validated genic

  2. 3' terminal diversity of MRP RNA and other human noncoding RNAs revealed by deep sequencing.

    Science.gov (United States)

    Goldfarb, Katherine C; Cech, Thomas R

    2013-09-21

    Post-transcriptional 3' end processing is a key component of RNA regulation. The abundant and essential RNA subunit of RNase MRP has been proposed to function in three distinct cellular compartments and therefore may utilize this mode of regulation. Here we employ 3' RACE coupled with high-throughput sequencing to characterize the 3' terminal sequences of human MRP RNA and other noncoding RNAs that form RNP complexes. The 3' terminal sequence of MRP RNA from HEK293T cells has a distinctive distribution of genomically encoded termini (including an assortment of U residues) with a portion of these selectively tagged by oligo(A) tails. This profile contrasts with the relatively homogenous 3' terminus of an in vitro transcribed MRP RNA control and the differing 3' terminal profiles of U3 snoRNA, RNase P RNA, and telomerase RNA (hTR). 3' RACE coupled with deep sequencing provides a valuable framework for the functional characterization of 3' terminal sequences of noncoding RNAs.

  3. 3′ terminal diversity of MRP RNA and other human noncoding RNAs revealed by deep sequencing

    Science.gov (United States)

    2013-01-01

    Background Post-transcriptional 3′ end processing is a key component of RNA regulation. The abundant and essential RNA subunit of RNase MRP has been proposed to function in three distinct cellular compartments and therefore may utilize this mode of regulation. Here we employ 3′ RACE coupled with high-throughput sequencing to characterize the 3′ terminal sequences of human MRP RNA and other noncoding RNAs that form RNP complexes. Results The 3′ terminal sequence of MRP RNA from HEK293T cells has a distinctive distribution of genomically encoded termini (including an assortment of U residues) with a portion of these selectively tagged by oligo(A) tails. This profile contrasts with the relatively homogenous 3′ terminus of an in vitro transcribed MRP RNA control and the differing 3′ terminal profiles of U3 snoRNA, RNase P RNA, and telomerase RNA (hTR). Conclusions 3′ RACE coupled with deep sequencing provides a valuable framework for the functional characterization of 3′ terminal sequences of noncoding RNAs. PMID:24053768

  4. Hybridization-based reconstruction of small non-coding RNA transcripts from deep sequencing data.

    Science.gov (United States)

    Ragan, Chikako; Mowry, Bryan J; Bauer, Denis C

    2012-09-01

    Recent advances in RNA sequencing technology (RNA-Seq) enables comprehensive profiling of RNAs by producing millions of short sequence reads from size-fractionated RNA libraries. Although conventional tools for detecting and distinguishing non-coding RNAs (ncRNAs) from reference-genome data can be applied to sequence data, ncRNA detection can be improved by harnessing the full information content provided by this new technology. Here we present NorahDesk, the first unbiased and universally applicable method for small ncRNAs detection from RNA-Seq data. NorahDesk utilizes the coverage-distribution of small RNA sequence data as well as thermodynamic assessments of secondary structure to reliably predict and annotate ncRNA classes. Using publicly available mouse sequence data from brain, skeletal muscle, testis and ovary, we evaluated our method with an emphasis on the performance for microRNAs (miRNAs) and piwi-interacting small RNA (piRNA). We compared our method with Dario and mirDeep2 and found that NorahDesk produces longer transcripts with higher read coverage. This feature makes it the first method particularly suitable for the prediction of both known and novel piRNAs.

  5. In vivo quantitative NMR imaging of fruit tissues during growth using Spoiled Gradient Echo sequence

    DEFF Research Database (Denmark)

    Kenouche, S.; Perrier, M.; Bertin, N.

    2014-01-01

    of this study was to design a robust and accurate quantitative measurement method based on NMR imaging combined with contrast agent (CA) for mapping and quantifying water transport in growing cherry tomato fruits. A multiple flip-angle Spoiled Gradient Echo (SGE) imaging sequence was used to evaluate...

  6. Quantitative diffusion characteristics of the human brain depend on MRI sequence parameters

    Energy Technology Data Exchange (ETDEWEB)

    Wilson, M.; Blumhardt, L.D. [University of Nottingham, Department of Neurology, Royal Preston Hospital, Preston (United Kingdom); Morgan, P.S. [Division of Academic Radiology, Queens Medical Centre, Nottingham (United Kingdom)

    2002-07-01

    Quantitative diffusion-weighted MRI has been applied to the study of neurological diseases, including multiple sclerosis, where the molecular self-diffusion coefficient D has been measured in both lesions and normal-appearing white matter. Histograms of D have been used as a novel measure of the ''lesion load'', with potential applications that include the monitoring of efficacy in new treatment trials. However different ways of measuring D may affect its value, making comparison between different centres and research groups impossible. We aimed to assess the effect, if any, of using two different MRI sequences on the value of D. We studied 13 healthy volunteers, using two different quantitative diffusion sequences (including different b{sub max} values and gradient applications). Maps of D were analysed using both regions of interest (ROI) in white matter and ''whole brain'' histograms, and compared between the two sequences. In addition, we studied three standardised test liquids (with known values of D) using both sequences. Histograms from the two sequences had different distributions, with a greater spread and higher peak position from the sequence with lower b{sub max}. This greater spread of D was also evident in the white matter and test liquid ROI. ''Limits of agreement'' analysis demonstrated that the differences could be clinically relevant, despite significant correlations between the sequences obtained using simple rank methods. We conclude that different quantitative diffusion sequences are unlikely to produce directly comparable values of D, particularly if different b{sub max} values are used. In addition, the use of inappropriate statistical tests may give false impressions of close agreement. Standardisation of methods for the measurement of D are required if these techniques are to become useful tools, for example in monitoring changes in the disease burden of multiple sclerosis. (orig.)

  7. Quantitative diffusion characteristics of the human brain depend on MRI sequence parameters

    International Nuclear Information System (INIS)

    Wilson, M.; Blumhardt, L.D.; Morgan, P.S.

    2002-01-01

    Quantitative diffusion-weighted MRI has been applied to the study of neurological diseases, including multiple sclerosis, where the molecular self-diffusion coefficient D has been measured in both lesions and normal-appearing white matter. Histograms of D have been used as a novel measure of the ''lesion load'', with potential applications that include the monitoring of efficacy in new treatment trials. However different ways of measuring D may affect its value, making comparison between different centres and research groups impossible. We aimed to assess the effect, if any, of using two different MRI sequences on the value of D. We studied 13 healthy volunteers, using two different quantitative diffusion sequences (including different b max values and gradient applications). Maps of D were analysed using both regions of interest (ROI) in white matter and ''whole brain'' histograms, and compared between the two sequences. In addition, we studied three standardised test liquids (with known values of D) using both sequences. Histograms from the two sequences had different distributions, with a greater spread and higher peak position from the sequence with lower b max . This greater spread of D was also evident in the white matter and test liquid ROI. ''Limits of agreement'' analysis demonstrated that the differences could be clinically relevant, despite significant correlations between the sequences obtained using simple rank methods. We conclude that different quantitative diffusion sequences are unlikely to produce directly comparable values of D, particularly if different b max values are used. In addition, the use of inappropriate statistical tests may give false impressions of close agreement. Standardisation of methods for the measurement of D are required if these techniques are to become useful tools, for example in monitoring changes in the disease burden of multiple sclerosis. (orig.)

  8. Deep sequencing reveals persistence of cell-associated mumps vaccine virus in chronic encephalitis.

    Science.gov (United States)

    Morfopoulou, Sofia; Mee, Edward T; Connaughton, Sarah M; Brown, Julianne R; Gilmour, Kimberly; Chong, W K 'Kling'; Duprex, W Paul; Ferguson, Deborah; Hubank, Mike; Hutchinson, Ciaran; Kaliakatsos, Marios; McQuaid, Stephen; Paine, Simon; Plagnol, Vincent; Ruis, Christopher; Virasami, Alex; Zhan, Hong; Jacques, Thomas S; Schepelmann, Silke; Qasim, Waseem; Breuer, Judith

    2017-01-01

    Routine childhood vaccination against measles, mumps and rubella has virtually abolished virus-related morbidity and mortality. Notwithstanding this, we describe here devastating neurological complications associated with the detection of live-attenuated mumps virus Jeryl Lynn (MuV JL5 ) in the brain of a child who had undergone successful allogeneic transplantation for severe combined immunodeficiency (SCID). This is the first confirmed report of MuV JL5 associated with chronic encephalitis and highlights the need to exclude immunodeficient individuals from immunisation with live-attenuated vaccines. The diagnosis was only possible by deep sequencing of the brain biopsy. Sequence comparison of the vaccine batch to the MuV JL5 isolated from brain identified biased hypermutation, particularly in the matrix gene, similar to those found in measles from cases of SSPE. The findings provide unique insights into the pathogenesis of paramyxovirus brain infections.

  9. Identification of miRNAs and their target genes in developing soybean seeds by deep sequencing

    Directory of Open Access Journals (Sweden)

    Chen Shou-Yi

    2011-01-01

    Full Text Available Abstract Background MicroRNAs (miRNAs regulate gene expression by mediating gene silencing at transcriptional and post-transcriptional levels in higher plants. miRNAs and related target genes have been widely studied in model plants such as Arabidopsis and rice; however, the number of identified miRNAs in soybean (Glycine max is limited, and global identification of the related miRNA targets has not been reported in previous research. Results In our study, a small RNA library and a degradome library were constructed from developing soybean seeds for deep sequencing. We identified 26 new miRNAs in soybean by bioinformatic analysis and further confirmed their expression by stem-loop RT-PCR. The miRNA star sequences of 38 known miRNAs and 8 new miRNAs were also discovered, providing additional evidence for the existence of miRNAs. Through degradome sequencing, 145 and 25 genes were identified as targets of annotated miRNAs and new miRNAs, respectively. GO analysis indicated that many of the identified miRNA targets may function in soybean seed development. Additionally, a soybean homolog of Arabidopsis SUPPRESSOR OF GENE SLIENCING 3 (AtSGS3 was detected as a target of the newly identified miRNA Soy_25, suggesting the presence of feedback control of miRNA biogenesis. Conclusions We have identified large numbers of miRNAs and their related target genes through deep sequencing of a small RNA library and a degradome library. Our study provides more information about the regulatory network of miRNAs in soybean and advances our understanding of miRNA functions during seed development.

  10. Evaluation of Nine Somatic Variant Callers for Detection of Somatic Mutations in Exome and Targeted Deep Sequencing Data

    DEFF Research Database (Denmark)

    Krøigård, Anne Bruun; Thomassen, Mads; Lænkholm, Anne Vibeke

    2016-01-01

    a comprehensive evaluation using exome sequencing and targeted deep sequencing data of paired tumor-normal samples from five breast cancer patients to evaluate the performance of nine publicly available somatic variant callers: EBCall, Mutect, Seurat, Shimmer, Indelocator, Somatic Sniper, Strelka, VarScan 2...

  11. Poly(A)-tag deep sequencing data processing to extract poly(A) sites.

    Science.gov (United States)

    Wu, Xiaohui; Ji, Guoli; Li, Qingshun Quinn

    2015-01-01

    Polyadenylation [poly(A)] is an essential posttranscriptional processing step in the maturation of eukaryotic mRNA. The advent of next-generation sequencing (NGS) technology has offered feasible means to generate large-scale data and new opportunities for intensive study of polyadenylation, particularly deep sequencing of the transcriptome targeting the junction of 3'-UTR and the poly(A) tail of the transcript. To take advantage of this unprecedented amount of data, we present an automated workflow to identify polyadenylation sites by integrating NGS data cleaning, processing, mapping, normalizing, and clustering. In this pipeline, a series of Perl scripts are seamlessly integrated to iteratively map the single- or paired-end sequences to the reference genome. After mapping, the poly(A) tags (PATs) at the same genome coordinate are grouped into one cleavage site, and the internal priming artifacts removed. Then the ambiguous region is introduced to parse the genome annotation for cleavage site clustering. Finally, cleavage sites within a close range of 24 nucleotides and from different samples can be clustered into poly(A) clusters. This procedure could be used to identify thousands of reliable poly(A) clusters from millions of NGS sequences in different tissues or treatments.

  12. Deep sequencing analysis of the developing mouse brain reveals a novel microRNA

    Directory of Open Access Journals (Sweden)

    Piltz Sandra

    2011-04-01

    Full Text Available Abstract Background MicroRNAs (miRNAs are small non-coding RNAs that can exert multilevel inhibition/repression at a post-transcriptional or protein synthesis level during disease or development. Characterisation of miRNAs in adult mammalian brains by deep sequencing has been reported previously. However, to date, no small RNA profiling of the developing brain has been undertaken using this method. We have performed deep sequencing and small RNA analysis of a developing (E15.5 mouse brain. Results We identified the expression of 294 known miRNAs in the E15.5 developing mouse brain, which were mostly represented by let-7 family and other brain-specific miRNAs such as miR-9 and miR-124. We also discovered 4 putative 22-23 nt miRNAs: mm_br_e15_1181, mm_br_e15_279920, mm_br_e15_96719 and mm_br_e15_294354 each with a 70-76 nt predicted pre-miRNA. We validated the 4 putative miRNAs and further characterised one of them, mm_br_e15_1181, throughout embryogenesis. Mm_br_e15_1181 biogenesis was Dicer1-dependent and was expressed in E3.5 blastocysts and E7 whole embryos. Embryo-wide expression patterns were observed at E9.5 and E11.5 followed by a near complete loss of expression by E13.5, with expression restricted to a specialised layer of cells within the developing and early postnatal brain. Mm_br_e15_1181 was upregulated during neurodifferentiation of P19 teratocarcinoma cells. This novel miRNA has been identified as miR-3099. Conclusions We have generated and analysed the first deep sequencing dataset of small RNA sequences of the developing mouse brain. The analysis revealed a novel miRNA, miR-3099, with potential regulatory effects on early embryogenesis, and involvement in neuronal cell differentiation/function in the brain during late embryonic and early neonatal development.

  13. Mapping vaccinia virus DNA replication origins at nucleotide level by deep sequencing.

    Science.gov (United States)

    Senkevich, Tatiana G; Bruno, Daniel; Martens, Craig; Porcella, Stephen F; Wolf, Yuri I; Moss, Bernard

    2015-09-01

    Poxviruses reproduce in the host cytoplasm and encode most or all of the enzymes and factors needed for expression and synthesis of their double-stranded DNA genomes. Nevertheless, the mode of poxvirus DNA replication and the nature and location of the replication origins remain unknown. A current but unsubstantiated model posits only leading strand synthesis starting at a nick near one covalently closed end of the genome and continuing around the other end to generate a concatemer that is subsequently resolved into unit genomes. The existence of specific origins has been questioned because any plasmid can replicate in cells infected by vaccinia virus (VACV), the prototype poxvirus. We applied directional deep sequencing of short single-stranded DNA fragments enriched for RNA-primed nascent strands isolated from the cytoplasm of VACV-infected cells to pinpoint replication origins. The origins were identified as the switching points of the fragment directions, which correspond to the transition from continuous to discontinuous DNA synthesis. Origins containing a prominent initiation point mapped to a sequence within the hairpin loop at one end of the VACV genome and to the same sequence within the concatemeric junction of replication intermediates. These findings support a model for poxvirus genome replication that involves leading and lagging strand synthesis and is consistent with the requirements for primase and ligase activities as well as earlier electron microscopic and biochemical studies implicating a replication origin at the end of the VACV genome.

  14. Deep sequencing of the Camellia sinensis transcriptome revealed candidate genes for major metabolic pathways of tea-specific compounds

    Energy Technology Data Exchange (ETDEWEB)

    Shi, CY; Yang, H; Wei, CL; Yu, O; Zhang, ZZ; Sun, J; Wan, XC

    2011-01-01

    Tea is one of the most popular non-alcoholic beverages worldwide. However, the tea plant, Camellia sinensis, is difficult to culture in vitro, to transform, and has a large genome, rendering little genomic information available. Recent advances in large-scale RNA sequencing (RNA-seq) provide a fast, cost-effective, and reliable approach to generate large expression datasets for functional genomic analysis, which is especially suitable for non-model species with un-sequenced genomes. Using high-throughput Illumina RNA-seq, the transcriptome from poly (A){sup +} RNA of C. sinensis was analyzed at an unprecedented depth (2.59 gigabase pairs). Approximate 34.5 million reads were obtained, trimmed, and assembled into 127,094 unigenes, with an average length of 355 bp and an N50 of 506 bp, which consisted of 788 contig clusters and 126,306 singletons. This number of unigenes was 10-fold higher than existing C. sinensis sequences deposited in GenBank (as of August 2010). Sequence similarity analyses against six public databases (Uniprot, NR and COGs at NCBI, Pfam, InterPro and KEGG) found 55,088 unigenes that could be annotated with gene descriptions, conserved protein domains, or gene ontology terms. Some of the unigenes were assigned to putative metabolic pathways. Targeted searches using these annotations identified the majority of genes associated with several primary metabolic pathways and natural product pathways that are important to tea quality, such as flavonoid, theanine and caffeine biosynthesis pathways. Novel candidate genes of these secondary pathways were discovered. Comparisons with four previously prepared cDNA libraries revealed that this transcriptome dataset has both a high degree of consistency with previous EST data and an approximate 20 times increase in coverage. Thirteen unigenes related to theanine and flavonoid synthesis were validated. Their expression patterns in different organs of the tea plant were analyzed by RT-PCR and quantitative real

  15. Deep sequencing of the Camellia sinensis transcriptome revealed candidate genes for major metabolic pathways of tea-specific compounds

    Directory of Open Access Journals (Sweden)

    Chen Qi

    2011-02-01

    analyzed by RT-PCR and quantitative real time PCR (qRT-PCR. Conclusions An extensive transcriptome dataset has been obtained from the deep sequencing of tea plant. The coverage of the transcriptome is comprehensive enough to discover all known genes of several major metabolic pathways. This transcriptome dataset can serve as an important public information platform for gene expression, genomics, and functional genomic studies in C. sinensis.

  16. High-throughput sequencing and analysis of the gill tissue transcriptome from the deep-sea hydrothermal vent mussel Bathymodiolus azoricus

    Directory of Open Access Journals (Sweden)

    Gomes Paula

    2010-10-01

    Full Text Available Abstract Background Bathymodiolus azoricus is a deep-sea hydrothermal vent mussel found in association with large faunal communities living in chemosynthetic environments at the bottom of the sea floor near the Azores Islands. Investigation of the exceptional physiological reactions that vent mussels have adopted in their habitat, including responses to environmental microbes, remains a difficult challenge for deep-sea biologists. In an attempt to reveal genes potentially involved in the deep-sea mussel innate immunity we carried out a high-throughput sequence analysis of freshly collected B. azoricus transcriptome using gills tissues as the primary source of immune transcripts given its strategic role in filtering the surrounding waterborne potentially infectious microorganisms. Additionally, a substantial EST data set was produced and from which a comprehensive collection of genes coding for putative proteins was organized in a dedicated database, "DeepSeaVent" the first deep-sea vent animal transcriptome database based on the 454 pyrosequencing technology. Results A normalized cDNA library from gills tissue was sequenced in a full 454 GS-FLX run, producing 778,996 sequencing reads. Assembly of the high quality reads resulted in 75,407 contigs of which 3,071 were singletons. A total of 39,425 transcripts were conceptually translated into amino-sequences of which 22,023 matched known proteins in the NCBI non-redundant protein database, 15,839 revealed conserved protein domains through InterPro functional classification and 9,584 were assigned with Gene Ontology terms. Queries conducted within the database enabled the identification of genes putatively involved in immune and inflammatory reactions which had not been previously evidenced in the vent mussel. Their physical counterpart was confirmed by semi-quantitative quantitative Reverse-Transcription-Polymerase Chain Reactions (RT-PCR and their RNA transcription level by quantitative PCR (q

  17. Advancing Eucalyptus genomics: identification and sequencing of lignin biosynthesis genes from deep-coverage BAC libraries

    Directory of Open Access Journals (Sweden)

    Kudrna David

    2011-03-01

    Full Text Available Abstract Background Eucalyptus species are among the most planted hardwoods in the world because of their rapid growth, adaptability and valuable wood properties. The development and integration of genomic resources into breeding practice will be increasingly important in the decades to come. Bacterial artificial chromosome (BAC libraries are key genomic tools that enable positional cloning of important traits, synteny evaluation, and the development of genome framework physical maps for genetic linkage and genome sequencing. Results We describe the construction and characterization of two deep-coverage BAC libraries EG_Ba and EG_Bb obtained from nuclear DNA fragments of E. grandis (clone BRASUZ1 digested with HindIII and BstYI, respectively. Genome coverages of 17 and 15 haploid genome equivalents were estimated for EG_Ba and EG_Bb, respectively. Both libraries contained large inserts, with average sizes ranging from 135 Kb (Eg_Bb to 157 Kb (Eg_Ba, very low extra-nuclear genome contamination providing a probability of finding a single copy gene ≥ 99.99%. Libraries were screened for the presence of several genes of interest via hybridizations to high-density BAC filters followed by PCR validation. Five selected BAC clones were sequenced and assembled using the Roche GS FLX technology providing the whole sequence of the E. grandis chloroplast genome, and complete genomic sequences of important lignin biosynthesis genes. Conclusions The two E. grandis BAC libraries described in this study represent an important milestone for the advancement of Eucalyptus genomics and forest tree research. These BAC resources have a highly redundant genome coverage (> 15×, contain large average inserts and have a very low percentage of clones with organellar DNA or empty vectors. These publicly available BAC libraries are thus suitable for a broad range of applications in genetic and genomic research in Eucalyptus and possibly in related species of Myrtaceae

  18. Analysis of Plasmodium falciparum diversity in natural infections by deep sequencing

    Science.gov (United States)

    Manske, Magnus; Miotto, Olivo; Campino, Susana; Auburn, Sarah; Almagro-Garcia, Jacob; Maslen, Gareth; O’Brien, Jack; Djimde, Abdoulaye; Doumbo, Ogobara; Zongo, Issaka; Ouedraogo, Jean-Bosco; Michon, Pascal; Mueller, Ivo; Siba, Peter; Nzila, Alexis; Borrmann, Steffen; Kiara, Steven M.; Marsh, Kevin; Jiang, Hongying; Su, Xin-Zhuan; Amaratunga, Chanaki; Fairhurst, Rick; Socheat, Duong; Nosten, Francois; Imwong, Mallika; White, Nicholas J.; Sanders, Mandy; Anastasi, Elisa; Alcock, Dan; Drury, Eleanor; Oyola, Samuel; Quail, Michael A.; Turner, Daniel J.; Rubio, Valentin Ruano; Jyothi, Dushyanth; Amenga-Etego, Lucas; Hubbart, Christina; Jeffreys, Anna; Rowlands, Kate; Sutherland, Colin; Roper, Cally; Mangano, Valentina; Modiano, David; Tan, John C.; Ferdig, Michael T.; Amambua-Ngwa, Alfred; Conway, David J.; Takala-Harrison, Shannon; Plowe, Christopher V.; Rayner, Julian C.; Rockett, Kirk A.; Clark, Taane G.; Newbold, Chris I.; Berriman, Matthew; MacInnis, Bronwyn; Kwiatkowski, Dominic P.

    2013-01-01

    Malaria elimination strategies require surveillance of the parasite population for genetic changes that demand a public health response, such as new forms of drug resistance. 1,2 Here we describe methods for large-scale analysis of genetic variation in Plasmodium falciparum by deep sequencing of parasite DNA obtained from the blood of patients with malaria, either directly or after short term culture. Analysis of 86,158 exonic SNPs that passed genotyping quality control in 227 samples from Africa, Asia and Oceania provides genome-wide estimates of allele frequency distribution, population structure and linkage disequilibrium. By comparing the genetic diversity of individual infections with that of the local parasite population, we derive a metric of within-host diversity that is related to the level of inbreeding in the population. An open-access web application has been established for exploration of regional differences in allele frequency and of highly differentiated loci in the P. falciparum genome. PMID:22722859

  19. Draft Genome Sequence of Deep-Sea Alteromonas sp. Strain V450 Isolated from the Marine Sponge Leiodermatium sp.

    Science.gov (United States)

    Wang, Guojun; Barrett, Nolan H; McCarthy, Peter J

    2017-02-02

    The proteobacterium Alteromonas sp. strain V450 was isolated from the Atlantic deep-sea sponge Leiodermatium sp. Here, we report the draft genome sequence of this strain, with a genome size of approx. 4.39 Mb and a G+C content of 44.01%. The results will aid deep-sea microbial ecology, evolution, and sponge-microbe association studies. Copyright © 2017 Wang et al.

  20. Deep RNA sequencing of the skeletal muscle transcriptome in swimming fish.

    Directory of Open Access Journals (Sweden)

    Arjan P Palstra

    Full Text Available Deep RNA sequencing (RNA-seq was performed to provide an in-depth view of the transcriptome of red and white skeletal muscle of exercised and non-exercised rainbow trout (Oncorhynchus mykiss with the specific objective to identify expressed genes and quantify the transcriptomic effects of swimming-induced exercise. Pubertal autumn-spawning seawater-raised female rainbow trout were rested (n = 10 or swum (n = 10 for 1176 km at 0.75 body-lengths per second in a 6,000-L swim-flume under reproductive conditions for 40 days. Red and white muscle RNA of exercised and non-exercised fish (4 lanes was sequenced and resulted in 15-17 million reads per lane that, after de novo assembly, yielded 149,159 red and 118,572 white muscle contigs. Most contigs were annotated using an iterative homology search strategy against salmonid ESTs, the zebrafish Danio rerio genome and general Metazoan genes. When selecting for large contigs (>500 nucleotides, a number of novel rainbow trout gene sequences were identified in this study: 1,085 and 1,228 novel gene sequences for red and white muscle, respectively, which included a number of important molecules for skeletal muscle function. Transcriptomic analysis revealed that sustained swimming increased transcriptional activity in skeletal muscle and specifically an up-regulation of genes involved in muscle growth and developmental processes in white muscle. The unique collection of transcripts will contribute to our understanding of red and white muscle physiology, specifically during the long-term reproductive migration of salmonids.

  1. Deep sequencing-based transcriptome analysis of Plutella xylostella larvae parasitized by Diadegma semiclausum

    Science.gov (United States)

    2011-01-01

    Background Parasitoid insects manipulate their hosts' physiology by injecting various factors into their host upon parasitization. Transcriptomic approaches provide a powerful approach to study insect host-parasitoid interactions at the molecular level. In order to investigate the effects of parasitization by an ichneumonid wasp (Diadegma semiclausum) on the host (Plutella xylostella), the larval transcriptome profile was analyzed using a short-read deep sequencing method (Illumina). Symbiotic polydnaviruses (PDVs) associated with ichneumonid parasitoids, known as ichnoviruses, play significant roles in host immune suppression and developmental regulation. In the current study, D. semiclausum ichnovirus (DsIV) genes expressed in P. xylostella were identified and their sequences compared with other reported PDVs. Five of these genes encode proteins of unknown identity, that have not previously been reported. Results De novo assembly of cDNA sequence data generated 172,660 contigs between 100 and 10000 bp in length; with 35% of > 200 bp in length. Parasitization had significant impacts on expression levels of 928 identified insect host transcripts. Gene ontology data illustrated that the majority of the differentially expressed genes are involved in binding, catalytic activity, and metabolic and cellular processes. In addition, the results show that transcription levels of antimicrobial peptides, such as gloverin, cecropin E and lysozyme, were up-regulated after parasitism. Expression of ichnovirus genes were detected in parasitized larvae with 19 unique sequences identified from five PDV gene families including vankyrin, viral innexin, repeat elements, a cysteine-rich motif, and polar residue rich protein. Vankyrin 1 and repeat element 1 genes showed the highest transcription levels among the DsIV genes. Conclusion This study provides detailed information on differential expression of P. xylostella larval genes following parasitization, DsIV genes expressed in the

  2. Use of deep whole-genome sequencing data to identify structure risk variants in breast cancer susceptibility genes.

    Science.gov (United States)

    Guo, Xingyi; Shi, Jiajun; Cai, Qiuyin; Shu, Xiao-Ou; He, Jing; Wen, Wanqing; Allen, Jamie; Pharoah, Paul; Dunning, Alison; Hunter, David J; Kraft, Peter; Easton, Douglas F; Zheng, Wei; Long, Jirong

    2018-03-01

    Functional disruptions of susceptibility genes by large genomic structure variant (SV) deletions in germlines are known to be associated with cancer risk. However, few studies have been conducted to systematically search for SV deletions in breast cancer susceptibility genes. We analysed deep (> 30x) whole-genome sequencing (WGS) data generated in blood samples from 128 breast cancer patients of Asian and European descent with either a strong family history of breast cancer or early cancer onset disease. To identify SV deletions in known or suspected breast cancer susceptibility genes, we used multiple SV calling tools including Genome STRiP, Delly, Manta, BreakDancer and Pindel. SV deletions were detected by at least three of these bioinformatics tools in five genes. Specifically, we identified heterozygous deletions covering a fraction of the coding regions of BRCA1 (with approximately 80kb in two patients), and TP53 genes (with ∼1.6 kb in two patients), and of intronic regions (∼1 kb) of the PALB2 (one patient), PTEN (three patients) and RAD51C genes (one patient). We confirmed the presence of these deletions using real-time quantitative PCR (qPCR). Our study identified novel SV deletions in breast cancer susceptibility genes and the identification of such SV deletions may improve clinical testing.

  3. A method to prioritize quantitative traits and individuals for sequencing in family-based studies.

    Directory of Open Access Journals (Sweden)

    Kaanan P Shah

    Full Text Available Owing to recent advances in DNA sequencing, it is now technically feasible to evaluate the contribution of rare variation to complex traits and diseases. However, it is still cost prohibitive to sequence the whole genome (or exome of all individuals in each study. For quantitative traits, one strategy to reduce cost is to sequence individuals in the tails of the trait distribution. However, the next challenge becomes how to prioritize traits and individuals for sequencing since individuals are often characterized for dozens of medically relevant traits. In this article, we describe a new method, the Rare Variant Kinship Test (RVKT, which leverages relationship information in family-based studies to identify quantitative traits that are likely influenced by rare variants. Conditional on nuclear families and extended pedigrees, we evaluate the power of the RVKT via simulation. Not unexpectedly, the power of our method depends strongly on effect size, and to a lesser extent, on the frequency of the rare variant and the number and type of relationships in the sample. As an illustration, we also apply our method to data from two genetic studies in the Old Order Amish, a founder population with extensive genealogical records. Remarkably, we implicate the presence of a rare variant that lowers fasting triglyceride levels in the Heredity and Phenotype Intervention (HAPI Heart study (p = 0.044, consistent with the presence of a previously identified null mutation in the APOC3 gene that lowers fasting triglyceride levels in HAPI Heart study participants.

  4. 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....

  5. DeepGO: predicting protein functions from sequence and interactions using a deep ontology-aware classifier

    KAUST Repository

    Kulmanov, Maxat; Khan, Mohammed Asif; Hoehndorf, Robert

    2017-01-01

    A large number of protein sequences are becoming available through the application of novel high-throughput sequencing technologies. Experimental functional characterization of these proteins is time-consuming and expensive, and is often

  6. Evaluation of Nine Somatic Variant Callers for Detection of Somatic Mutations in Exome and Targeted Deep Sequencing Data.

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    Anne Bruun Krøigård

    Full Text Available Next generation sequencing is extensively applied to catalogue somatic mutations in cancer, in research settings and increasingly in clinical settings for molecular diagnostics, guiding therapy decisions. Somatic variant callers perform paired comparisons of sequencing data from cancer tissue and matched normal tissue in order to detect somatic mutations. The advent of many new somatic variant callers creates a need for comparison and validation of the tools, as no de facto standard for detection of somatic mutations exists and only limited comparisons have been reported. We have performed a comprehensive evaluation using exome sequencing and targeted deep sequencing data of paired tumor-normal samples from five breast cancer patients to evaluate the performance of nine publicly available somatic variant callers: EBCall, Mutect, Seurat, Shimmer, Indelocator, Somatic Sniper, Strelka, VarScan 2 and Virmid for the detection of single nucleotide mutations and small deletions and insertions. We report a large variation in the number of calls from the nine somatic variant callers on the same sequencing data and highly variable agreement. Sequencing depth had markedly diverse impact on individual callers, as for some callers, increased sequencing depth highly improved sensitivity. For SNV calling, we report EBCall, Mutect, Virmid and Strelka to be the most reliable somatic variant callers for both exome sequencing and targeted deep sequencing. For indel calling, EBCall is superior due to high sensitivity and robustness to changes in sequencing depths.

  7. Evaluation of Nine Somatic Variant Callers for Detection of Somatic Mutations in Exome and Targeted Deep Sequencing Data.

    Science.gov (United States)

    Krøigård, Anne Bruun; Thomassen, Mads; Lænkholm, Anne-Vibeke; Kruse, Torben A; Larsen, Martin Jakob

    2016-01-01

    Next generation sequencing is extensively applied to catalogue somatic mutations in cancer, in research settings and increasingly in clinical settings for molecular diagnostics, guiding therapy decisions. Somatic variant callers perform paired comparisons of sequencing data from cancer tissue and matched normal tissue in order to detect somatic mutations. The advent of many new somatic variant callers creates a need for comparison and validation of the tools, as no de facto standard for detection of somatic mutations exists and only limited comparisons have been reported. We have performed a comprehensive evaluation using exome sequencing and targeted deep sequencing data of paired tumor-normal samples from five breast cancer patients to evaluate the performance of nine publicly available somatic variant callers: EBCall, Mutect, Seurat, Shimmer, Indelocator, Somatic Sniper, Strelka, VarScan 2 and Virmid for the detection of single nucleotide mutations and small deletions and insertions. We report a large variation in the number of calls from the nine somatic variant callers on the same sequencing data and highly variable agreement. Sequencing depth had markedly diverse impact on individual callers, as for some callers, increased sequencing depth highly improved sensitivity. For SNV calling, we report EBCall, Mutect, Virmid and Strelka to be the most reliable somatic variant callers for both exome sequencing and targeted deep sequencing. For indel calling, EBCall is superior due to high sensitivity and robustness to changes in sequencing depths.

  8. Quantitative methods for structural characterization of proteins based on deep UV resonance Raman spectroscopy.

    Science.gov (United States)

    Shashilov, Victor A; Sikirzhytski, Vitali; Popova, Ludmila A; Lednev, Igor K

    2010-09-01

    Here we report on novel quantitative approaches for protein structural characterization using deep UV resonance Raman (DUVRR) spectroscopy. Specifically, we propose a new method combining hydrogen-deuterium (HD) exchange and Bayesian source separation for extracting the DUVRR signatures of various structural elements of aggregated proteins including the cross-beta core and unordered parts of amyloid fibrils. The proposed method is demonstrated using the set of DUVRR spectra of hen egg white lysozyme acquired at various stages of HD exchange. Prior information about the concentration matrix and the spectral features of the individual components was incorporated into the Bayesian equation to eliminate the ill-conditioning of the problem caused by 100% correlation of the concentration profiles of protonated and deuterated species. Secondary structure fractions obtained by partial least squares (PLS) and least squares support vector machines (LS-SVMs) were used as the initial guess for the Bayessian source separation. Advantages of the PLS and LS-SVMs methods over the classical least squares calibration (CLSC) are discussed and illustrated using the DUVRR data of the prion protein in its native and aggregated forms. Copyright (c) 2010 Elsevier Inc. All rights reserved.

  9. Deep neural nets as a method for quantitative structure-activity relationships.

    Science.gov (United States)

    Ma, Junshui; Sheridan, Robert P; Liaw, Andy; Dahl, George E; Svetnik, Vladimir

    2015-02-23

    Neural networks were widely used for quantitative structure-activity relationships (QSAR) in the 1990s. Because of various practical issues (e.g., slow on large problems, difficult to train, prone to overfitting, etc.), they were superseded by more robust methods like support vector machine (SVM) and random forest (RF), which arose in the early 2000s. The last 10 years has witnessed a revival of neural networks in the machine learning community thanks to new methods for preventing overfitting, more efficient training algorithms, and advancements in computer hardware. In particular, deep neural nets (DNNs), i.e. neural nets with more than one hidden layer, have found great successes in many applications, such as computer vision and natural language processing. Here we show that DNNs can routinely make better prospective predictions than RF on a set of large diverse QSAR data sets that are taken from Merck's drug discovery effort. The number of adjustable parameters needed for DNNs is fairly large, but our results show that it is not necessary to optimize them for individual data sets, and a single set of recommended parameters can achieve better performance than RF for most of the data sets we studied. The usefulness of the parameters is demonstrated on additional data sets not used in the calibration. Although training DNNs is still computationally intensive, using graphical processing units (GPUs) can make this issue manageable.

  10. MicroRNA repertoire for functional genome research in tilapia identified by deep sequencing.

    Science.gov (United States)

    Yan, Biao; Wang, Zhen-Hua; Zhu, Chang-Dong; Guo, Jin-Tao; Zhao, Jin-Liang

    2014-08-01

    The Nile tilapia (Oreochromis niloticus; Cichlidae) is an economically important species in aquaculture and occupies a prominent position in the aquaculture industry. MicroRNAs (miRNAs) are a class of noncoding RNAs that post-transcriptionally regulate gene expression involved in diverse biological and metabolic processes. To increase the repertoire of miRNAs characterized in tilapia, we used the Illumina/Solexa sequencing technology to sequence a small RNA library using pooled RNA sample isolated from the different developmental stages of tilapia. Bioinformatic analyses suggest that 197 conserved and 27 novel miRNAs are expressed in tilapia. Sequence alignments indicate that all tested miRNAs and miRNAs* are highly conserved across many species. In addition, we characterized the tissue expression patterns of five miRNAs using real-time quantitative PCR. We found that miR-1/206, miR-7/9, and miR-122 is abundantly expressed in muscle, brain, and liver, respectively, implying a potential role in the regulation of tissue differentiation or the maintenance of tissue identity. Overall, our results expand the number of tilapia miRNAs, and the discovery of miRNAs in tilapia genome contributes to a better understanding the role of miRNAs in regulating diverse biological processes.

  11. Ultra Deep Sequencing of a Baculovirus Population Reveals Widespread Genomic Variations

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    Aurélien Chateigner

    2015-07-01

    Full Text Available Viruses rely on widespread genetic variation and large population size for adaptation. Large DNA virus populations are thought to harbor little variation though natural populations may be polymorphic. To measure the genetic variation present in a dsDNA virus population, we deep sequenced a natural strain of the baculovirus Autographa californica multiple nucleopolyhedrovirus. With 124,221X average genome coverage of our 133,926 bp long consensus, we could detect low frequency mutations (0.025%. K-means clustering was used to classify the mutations in four categories according to their frequency in the population. We found 60 high frequency non-synonymous mutations under balancing selection distributed in all functional classes. These mutants could alter viral adaptation dynamics, either through competitive or synergistic processes. Lastly, we developed a technique for the delimitation of large deletions in next generation sequencing data. We found that large deletions occur along the entire viral genome, with hotspots located in homologous repeat regions (hrs. Present in 25.4% of the genomes, these deletion mutants presumably require functional complementation to complete their infection cycle. They might thus have a large impact on the fitness of the baculovirus population. Altogether, we found a wide breadth of genomic variation in the baculovirus population, suggesting it has high adaptive potential.

  12. Incorporation of unique molecular identifiers in TruSeq adapters improves the accuracy of quantitative sequencing.

    Science.gov (United States)

    Hong, Jungeui; Gresham, David

    2017-11-01

    Quantitative analysis of next-generation sequencing (NGS) data requires discriminating duplicate reads generated by PCR from identical molecules that are of unique origin. Typically, PCR duplicates are identified as sequence reads that align to the same genomic coordinates using reference-based alignment. However, identical molecules can be independently generated during library preparation. Misidentification of these molecules as PCR duplicates can introduce unforeseen biases during analyses. Here, we developed a cost-effective sequencing adapter design by modifying Illumina TruSeq adapters to incorporate a unique molecular identifier (UMI) while maintaining the capacity to undertake multiplexed, single-index sequencing. Incorporation of UMIs into TruSeq adapters (TrUMIseq adapters) enables identification of bona fide PCR duplicates as identically mapped reads with identical UMIs. Using TrUMIseq adapters, we show that accurate removal of PCR duplicates results in improved accuracy of both allele frequency (AF) estimation in heterogeneous populations using DNA sequencing and gene expression quantification using RNA-Seq.

  13. Recurrent chimeric RNAs enriched in human prostate cancer identified by deep sequencing

    Science.gov (United States)

    Kannan, Kalpana; Wang, Liguo; Wang, Jianghua; Ittmann, Michael M.; Li, Wei; Yen, Laising

    2011-01-01

    Transcription-induced chimeric RNAs, possessing sequences from different genes, are expected to increase the proteomic diversity through chimeric proteins or altered regulation. Despite their importance, few studies have focused on chimeric RNAs especially regarding their presence/roles in human cancers. By deep sequencing the transcriptome of 20 human prostate cancer and 10 matched benign prostate tissues, we obtained 1.3 billion sequence reads, which led to the identification of 2,369 chimeric RNA candidates. Chimeric RNAs occurred in significantly higher frequency in cancer than in matched benign samples. Experimental investigation of a selected 46 set led to the confirmation of 32 chimeric RNAs, of which 27 were highly recurrent and previously undescribed in prostate cancer. Importantly, a subset of these chimeras was present in prostate cancer cell lines, but not detectable in primary human prostate epithelium cells, implying their associations with cancer. These chimeras contain discernable 5′ and 3′ splice sites at the RNA junction, indicating that their formation is mediated by splicing. Their presence is also largely independent of the expression of parental genes, suggesting that other factors are involved in their production and regulation. One chimera, TMEM79-SMG5, is highly differentially expressed in human cancer samples and therefore a potential biomarker. The prevalence of chimeric RNAs may allow the limited number of human genes to encode a substantially larger number of RNAs and proteins, forming an additional layer of cellular complexity. Together, our results suggest that chimeric RNAs are widespread, and increased chimeric RNA events could represent a unique class of molecular alteration in cancer. PMID:21571633

  14. Ultra-deep sequencing of mouse mitochondrial DNA: mutational patterns and their origins.

    Directory of Open Access Journals (Sweden)

    Adam Ameur

    2011-03-01

    Full Text Available Somatic mutations of mtDNA are implicated in the aging process, but there is no universally accepted method for their accurate quantification. We have used ultra-deep sequencing to study genome-wide mtDNA mutation load in the liver of normally- and prematurely-aging mice. Mice that are homozygous for an allele expressing a proof-reading-deficient mtDNA polymerase (mtDNA mutator mice have 10-times-higher point mutation loads than their wildtype siblings. In addition, the mtDNA mutator mice have increased levels of a truncated linear mtDNA molecule, resulting in decreased sequence coverage in the deleted region. In contrast, circular mtDNA molecules with large deletions occur at extremely low frequencies in mtDNA mutator mice and can therefore not drive the premature aging phenotype. Sequence analysis shows that the main proportion of the mutation load in heterozygous mtDNA mutator mice and their wildtype siblings is inherited from their heterozygous mothers consistent with germline transmission. We found no increase in levels of point mutations or deletions in wildtype C57Bl/6N mice with increasing age, thus questioning the causative role of these changes in aging. In addition, there was no increased frequency of transversion mutations with time in any of the studied genotypes, arguing against oxidative damage as a major cause of mtDNA mutations. Our results from studies of mice thus indicate that most somatic mtDNA mutations occur as replication errors during development and do not result from damage accumulation in adult life.

  15. 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.

  16. Quantitative and phylogenetic study of the Deep Sea Archaeal Group in sediments of the arctic mid-ocean spreading ridge

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    Steffen Leth eJørgensen

    2013-10-01

    Full Text Available In marine sediments archaea often constitute a considerable part of the microbial community, of which the Deep Sea Archaeal Group (DSAG is one of the most predominant. Despite their high abundance no members from this archaeal group have so far been characterized and thus their metabolism is unknown. Here we show that the relative abundance of DSAG marker genes can be correlated with geochemical parameters, allowing prediction of both the potential electron donors and acceptors of these organisms. We estimated the abundance of 16S rRNA genes from Archaea, Bacteria and DSAG in 52 sediment horizons from two cores collected at the slow-spreading Arctic Mid-Ocean Ridge, using qPCR. The results indicate that members of the DSAG make up the entire archaeal population in certain horizons and constitute up to ~ 50% of the total microbial community. The quantitative data were correlated to 30 different geophysical and geochemical parameters obtained from the same sediment horizons. We observed a significant correlation between the relative abundance of DSAG 16S rRNA genes and the content of organic carbon (p < 0.0001. Further, significant co-variation with iron oxide, and dissolved iron and manganese (all p < 0.0000, indicated a direct or indirect link to iron and manganese cycling. Neither of these parameters correlated with the relative abundance of archaeal or bacterial 16S rRNA genes, nor did any other major electron donor or acceptor measured. Phylogenetic analysis of DSAG 16S rRNA gene sequences reveals three monophyletic lineages with no apparent habitat-specific distribution. In this study we support the hypothesis that members of the DSAG are tightly linked to the content of organic carbon and directly or indirectly involved in the cycling of iron and/or manganese compounds. Further, we provide a molecular tool to assess their abundance in environmental samples and enrichment cultures.

  17. Profile of microbial communities on carbonate stones of the medieval church of San Leonardo di Siponto (Italy) by Illumina-based deep sequencing.

    Science.gov (United States)

    Chimienti, Guglielmina; Piredda, Roberta; Pepe, Gabriella; van der Werf, Inez Dorothé; Sabbatini, Luigia; Crecchio, Carmine; Ricciuti, Patrizia; D'Erchia, Anna Maria; Manzari, Caterina; Pesole, Graziano

    2016-10-01

    Comprehensive studies of the biodiversity of the microbial epilithic community on monuments may provide critical insights for clarifying factors involved in the colonization processes. We carried out a high-throughput investigation of the communities colonizing the medieval church of San Leonardo di Siponto (Italy) by Illumina-based deep sequencing. The metagenomic analysis of sequences revealed the presence of Archaea, Bacteria, and Eukarya. Bacteria were Actinobacteria, Proteobacteria, Bacteroidetes, Cyanobacteria, Chloroflexi, Firmicutes and Candidatus Saccharibacteria. The predominant phylum was Actinobacteria, with the orders Actynomycetales and Rubrobacteriales, represented by the genera Pseudokineococcus, Sporichthya, Blastococcus, Arthrobacter, Geodermatophilus, Friedmanniella, Modestobacter, and Rubrobacter, respectively. Cyanobacteria sequences showing strong similarity with an uncultured bacterium sequence were identified. The presence of the green algae Oocystaceae and Trebuxiaceae was revealed. The microbial diversity was explored at qualitative and quantitative levels, evaluating the richness (the number of operational taxonomic units (OTUs)) and the abundance of reads associated with each OTU. The rarefaction curves approached saturation, suggesting that the majority of OTUs were recovered. The results highlighted a structured community, showing low diversity, made up of extremophile organisms adapted to desiccation and UV radiation. Notably, the microbiome appeared to be composed not only of microorganisms possibly involved in biodeterioration but also of carbonatogenic bacteria, such as those belonging to the genus Arthrobacter, which could be useful in bioconservation. Our investigation demonstrated that molecular tools, and in particular the easy-to-run next-generation sequencing, are powerful to perform a microbiological diagnosis in order to plan restoration and protection strategies.

  18. Identification of ribonucleotide reductase mutation causing temperature-sensitivity of herpes simplex virus isolates from whitlow by deep sequencing.

    Science.gov (United States)

    Daikoku, Tohru; Oyama, Yukari; Yajima, Misako; Sekizuka, Tsuyoshi; Kuroda, Makoto; Shimada, Yuka; Takehara, Kazuhiko; Miwa, Naoko; Okuda, Tomoko; Sata, Tetsutaro; Shiraki, Kimiyasu

    2015-06-01

    Herpes simplex virus 2 caused a genital ulcer, and a secondary herpetic whitlow appeared during acyclovir therapy. The secondary and recurrent whitlow isolates were acyclovir-resistant and temperature-sensitive in contrast to a genital isolate. We identified the ribonucleotide reductase mutation responsible for temperature-sensitivity by deep-sequencing analysis.

  19. Genotypic tropism testing by massively parallel sequencing: qualitative and quantitative analysis

    Directory of Open Access Journals (Sweden)

    Thiele Bernhard

    2011-05-01

    Full Text Available Abstract Background Inferring viral tropism from genotype is a fast and inexpensive alternative to phenotypic testing. While being highly predictive when performed on clonal samples, sensitivity of predicting CXCR4-using (X4 variants drops substantially in clinical isolates. This is mainly attributed to minor variants not detected by standard bulk-sequencing. Massively parallel sequencing (MPS detects single clones thereby being much more sensitive. Using this technology we wanted to improve genotypic prediction of coreceptor usage. Methods Plasma samples from 55 antiretroviral-treated patients tested for coreceptor usage with the Monogram Trofile Assay were sequenced with standard population-based approaches. Fourteen of these samples were selected for further analysis with MPS. Tropism was predicted from each sequence with geno2pheno[coreceptor]. Results Prediction based on bulk-sequencing yielded 59.1% sensitivity and 90.9% specificity compared to the trofile assay. With MPS, 7600 reads were generated on average per isolate. Minorities of sequences with high confidence in CXCR4-usage were found in all samples, irrespective of phenotype. When using the default false-positive-rate of geno2pheno[coreceptor] (10%, and defining a minority cutoff of 5%, the results were concordant in all but one isolate. Conclusions The combination of MPS and coreceptor usage prediction results in a fast and accurate alternative to phenotypic assays. The detection of X4-viruses in all isolates suggests that coreceptor usage as well as fitness of minorities is important for therapy outcome. The high sensitivity of this technology in combination with a quantitative description of the viral population may allow implementing meaningful cutoffs for predicting response to CCR5-antagonists in the presence of X4-minorities.

  20. Genotypic tropism testing by massively parallel sequencing: qualitative and quantitative analysis.

    Science.gov (United States)

    Däumer, Martin; Kaiser, Rolf; Klein, Rolf; Lengauer, Thomas; Thiele, Bernhard; Thielen, Alexander

    2011-05-13

    Inferring viral tropism from genotype is a fast and inexpensive alternative to phenotypic testing. While being highly predictive when performed on clonal samples, sensitivity of predicting CXCR4-using (X4) variants drops substantially in clinical isolates. This is mainly attributed to minor variants not detected by standard bulk-sequencing. Massively parallel sequencing (MPS) detects single clones thereby being much more sensitive. Using this technology we wanted to improve genotypic prediction of coreceptor usage. Plasma samples from 55 antiretroviral-treated patients tested for coreceptor usage with the Monogram Trofile Assay were sequenced with standard population-based approaches. Fourteen of these samples were selected for further analysis with MPS. Tropism was predicted from each sequence with geno2pheno[coreceptor]. Prediction based on bulk-sequencing yielded 59.1% sensitivity and 90.9% specificity compared to the trofile assay. With MPS, 7600 reads were generated on average per isolate. Minorities of sequences with high confidence in CXCR4-usage were found in all samples, irrespective of phenotype. When using the default false-positive-rate of geno2pheno[coreceptor] (10%), and defining a minority cutoff of 5%, the results were concordant in all but one isolate. The combination of MPS and coreceptor usage prediction results in a fast and accurate alternative to phenotypic assays. The detection of X4-viruses in all isolates suggests that coreceptor usage as well as fitness of minorities is important for therapy outcome. The high sensitivity of this technology in combination with a quantitative description of the viral population may allow implementing meaningful cutoffs for predicting response to CCR5-antagonists in the presence of X4-minorities.

  1. Deep sequencing-based analysis of the anaerobic stimulon in Neisseria gonorrhoeae

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    Clark Virginia L

    2011-01-01

    Full Text Available Abstract Background Maintenance of an anaerobic denitrification system in the obligate human pathogen, Neisseria gonorrhoeae, suggests that an anaerobic lifestyle may be important during the course of infection. Furthermore, mounting evidence suggests that reduction of host-produced nitric oxide has several immunomodulary effects on the host. However, at this point there have been no studies analyzing the complete gonococcal transcriptome response to anaerobiosis. Here we performed deep sequencing to compare the gonococcal transcriptomes of aerobically and anaerobically grown cells. Using the information derived from this sequencing, we discuss the implications of the robust transcriptional response to anaerobic growth. Results We determined that 198 chromosomal genes were differentially expressed (~10% of the genome in response to anaerobic conditions. We also observed a large induction of genes encoded within the cryptic plasmid, pJD1. Validation of RNA-seq data using translational-lacZ fusions or RT-PCR demonstrated the RNA-seq results to be very reproducible. Surprisingly, many genes of prophage origin were induced anaerobically, as well as several transcriptional regulators previously unknown to be involved in anaerobic growth. We also confirmed expression and regulation of a small RNA, likely a functional equivalent of fnrS in the Enterobacteriaceae family. We also determined that many genes found to be responsive to anaerobiosis have also been shown to be responsive to iron and/or oxidative stress. Conclusions Gonococci will be subject to many forms of environmental stress, including oxygen-limitation, during the course of infection. Here we determined that the anaerobic stimulon in gonococci was larger than previous studies would suggest. Many new targets for future research have been uncovered, and the results derived from this study may have helped to elucidate factors or mechanisms of virulence that may have otherwise been overlooked.

  2. A Bioinformatic Pipeline for Monitoring of the Mutational Stability of Viral Drug Targets with Deep-Sequencing Technology.

    Science.gov (United States)

    Kravatsky, Yuri; Chechetkin, Vladimir; Fedoseeva, Daria; Gorbacheva, Maria; Kravatskaya, Galina; Kretova, Olga; Tchurikov, Nickolai

    2017-11-23

    The efficient development of antiviral drugs, including efficient antiviral small interfering RNAs (siRNAs), requires continuous monitoring of the strict correspondence between a drug and the related highly variable viral DNA/RNA target(s). Deep sequencing is able to provide an assessment of both the general target conservation and the frequency of particular mutations in the different target sites. The aim of this study was to develop a reliable bioinformatic pipeline for the analysis of millions of short, deep sequencing reads corresponding to selected highly variable viral sequences that are drug target(s). The suggested bioinformatic pipeline combines the available programs and the ad hoc scripts based on an original algorithm of the search for the conserved targets in the deep sequencing data. We also present the statistical criteria for the threshold of reliable mutation detection and for the assessment of variations between corresponding data sets. These criteria are robust against the possible sequencing errors in the reads. As an example, the bioinformatic pipeline is applied to the study of the conservation of RNA interference (RNAi) targets in human immunodeficiency virus 1 (HIV-1) subtype A. The developed pipeline is freely available to download at the website http://virmut.eimb.ru/. Brief comments and comparisons between VirMut and other pipelines are also presented.

  3. A Bioinformatic Pipeline for Monitoring of the Mutational Stability of Viral Drug Targets with Deep-Sequencing Technology

    Directory of Open Access Journals (Sweden)

    Yuri Kravatsky

    2017-11-01

    Full Text Available The efficient development of antiviral drugs, including efficient antiviral small interfering RNAs (siRNAs, requires continuous monitoring of the strict correspondence between a drug and the related highly variable viral DNA/RNA target(s. Deep sequencing is able to provide an assessment of both the general target conservation and the frequency of particular mutations in the different target sites. The aim of this study was to develop a reliable bioinformatic pipeline for the analysis of millions of short, deep sequencing reads corresponding to selected highly variable viral sequences that are drug target(s. The suggested bioinformatic pipeline combines the available programs and the ad hoc scripts based on an original algorithm of the search for the conserved targets in the deep sequencing data. We also present the statistical criteria for the threshold of reliable mutation detection and for the assessment of variations between corresponding data sets. These criteria are robust against the possible sequencing errors in the reads. As an example, the bioinformatic pipeline is applied to the study of the conservation of RNA interference (RNAi targets in human immunodeficiency virus 1 (HIV-1 subtype A. The developed pipeline is freely available to download at the website http://virmut.eimb.ru/. Brief comments and comparisons between VirMut and other pipelines are also presented.

  4. Deep Ion Torrent sequencing identifies soil fungal community shifts after frequent prescribed fires in a southeastern US forest ecosystem.

    Science.gov (United States)

    Brown, Shawn P; Callaham, Mac A; Oliver, Alena K; Jumpponen, Ari

    2013-12-01

    Prescribed burning is a common management tool to control fuel loads, ground vegetation, and facilitate desirable game species. We evaluated soil fungal community responses to long-term prescribed fire treatments in a loblolly pine forest on the Piedmont of Georgia and utilized deep Internal Transcribed Spacer Region 1 (ITS1) amplicon sequencing afforded by the recent Ion Torrent Personal Genome Machine (PGM). These deep sequence data (19,000 + reads per sample after subsampling) indicate that frequent fires (3-year fire interval) shift soil fungus communities, whereas infrequent fires (6-year fire interval) permit system resetting to a state similar to that without prescribed fire. Furthermore, in nonmetric multidimensional scaling analyses, primarily ectomycorrhizal taxa were correlated with axes associated with long fire intervals, whereas soil saprobes tended to be correlated with the frequent fire recurrence. We conclude that (1) multiplexed Ion Torrent PGM analyses allow deep cost effective sequencing of fungal communities but may suffer from short read lengths and inconsistent sequence quality adjacent to the sequencing adaptor; (2) frequent prescribed fires elicit a shift in soil fungal communities; and (3) such shifts do not occur when fire intervals are longer. Our results emphasize the general responsiveness of these forests to management, and the importance of fire return intervals in meeting management objectives. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  5. Application of Tandem Two-Dimensional Mass Spectrometry for Top-Down Deep Sequencing of Calmodulin.

    Science.gov (United States)

    Floris, Federico; Chiron, Lionel; Lynch, Alice M; Barrow, Mark P; Delsuc, Marc-André; O'Connor, Peter B

    2018-06-04

    Two-dimensional mass spectrometry (2DMS) involves simultaneous acquisition of the fragmentation patterns of all the analytes in a mixture by correlating their precursor and fragment ions by modulating precursor ions systematically through a fragmentation zone. Tandem two-dimensional mass spectrometry (MS/2DMS) unites the ultra-high accuracy of Fourier transform ion cyclotron resonance (FT-ICR) MS/MS and the simultaneous data-independent fragmentation of 2DMS to achieve extensive inter-residue fragmentation of entire proteins. 2DMS was recently developed for top-down proteomics (TDP), and applied to the analysis of calmodulin (CaM), reporting a cleavage coverage of about ~23% using infrared multiphoton dissociation (IRMPD) as fragmentation technique. The goal of this work is to expand the utility of top-down protein analysis using MS/2DMS in order to extend the cleavage coverage in top-down proteomics further into the interior regions of the protein. In this case, using MS/2DMS, the cleavage coverage of CaM increased from ~23% to ~42%. Graphical Abstract Two-dimensional mass spectrometry, when applied to primary fragment ions from the source, allows deep-sequencing of the protein calmodulin.

  6. Genomic region operation kit for flexible processing of deep sequencing data.

    Science.gov (United States)

    Ovaska, Kristian; Lyly, Lauri; Sahu, Biswajyoti; Jänne, Olli A; Hautaniemi, Sampsa

    2013-01-01

    Computational analysis of data produced in deep sequencing (DS) experiments is challenging due to large data volumes and requirements for flexible analysis approaches. Here, we present a mathematical formalism based on set algebra for frequently performed operations in DS data analysis to facilitate translation of biomedical research questions to language amenable for computational analysis. With the help of this formalism, we implemented the Genomic Region Operation Kit (GROK), which supports various DS-related operations such as preprocessing, filtering, file conversion, and sample comparison. GROK provides high-level interfaces for R, Python, Lua, and command line, as well as an extension C++ API. It supports major genomic file formats and allows storing custom genomic regions in efficient data structures such as red-black trees and SQL databases. To demonstrate the utility of GROK, we have characterized the roles of two major transcription factors (TFs) in prostate cancer using data from 10 DS experiments. GROK is freely available with a user guide from >http://csbi.ltdk.helsinki.fi/grok/.

  7. Deep Sequence Analysis of AgoshRNA Processing Reveals 3' A Addition and Trimming.

    Science.gov (United States)

    Harwig, Alex; Herrera-Carrillo, Elena; Jongejan, Aldo; van Kampen, Antonius Hubertus; Berkhout, Ben

    2015-07-14

    The RNA interference (RNAi) pathway, in which microprocessor and Dicer collaborate to process microRNAs (miRNA), was recently expanded by the description of alternative processing routes. In one of these noncanonical pathways, Dicer action is replaced by the Argonaute2 (Ago2) slicer function. It was recently shown that the stem-length of precursor-miRNA or short hairpin RNA (shRNA) molecules is a major determinant for Dicer versus Ago2 processing. Here we present the results of a deep sequence study on the processing of shRNAs with different stem length and a top G·U wobble base pair (bp). This analysis revealed some unexpected properties of these so-called AgoshRNA molecules that are processed by Ago2 instead of Dicer. First, we confirmed the gradual shift from Dicer to Ago2 processing upon shortening of the hairpin length. Second, hairpins with a stem larger than 19 base pair are inefficiently cleaved by Ago2 and we noticed a shift in the cleavage site. Third, the introduction of a top G·U bp in a regular shRNA can promote Ago2-cleavage, which coincides with a loss of Ago2-loading of the Dicer-cleaved 3' strand. Fourth, the Ago2-processed AgoshRNAs acquire a short 3' tail of 1-3 A-nucleotides (nt) and we present evidence that this product is subsequently trimmed by the poly(A)-specific ribonuclease (PARN).

  8. High-Quality Draft Single-Cell Genome Sequence Belonging to the Archaeal Candidate Division SA1, Isolated from Nereus Deep in the Red Sea

    KAUST Repository

    Ngugi, David

    2018-05-09

    Candidate division SA1 encompasses a phylogenetically coherent archaeal group ubiquitous in deep hypersaline anoxic brines around the globe. Recently, the genome sequences of two cultivated representatives from hypersaline soda lake sediments were published. Here, we present a single-cell genome sequence from Nereus Deep in the Red Sea that represents a putatively novel family within SA1.

  9. High-Quality Draft Single-Cell Genome Sequence Belonging to the Archaeal Candidate Division SA1, Isolated from Nereus Deep in the Red Sea

    KAUST Repository

    Ngugi, David; Stingl, Ulrich

    2018-01-01

    Candidate division SA1 encompasses a phylogenetically coherent archaeal group ubiquitous in deep hypersaline anoxic brines around the globe. Recently, the genome sequences of two cultivated representatives from hypersaline soda lake sediments were published. Here, we present a single-cell genome sequence from Nereus Deep in the Red Sea that represents a putatively novel family within SA1.

  10. Transcriptome analysis of the model protozoan, Tetrahymena thermophila, using Deep RNA sequencing.

    Directory of Open Access Journals (Sweden)

    Jie Xiong

    Full Text Available BACKGROUND: The ciliated protozoan Tetrahymena thermophila is a well-studied single-celled eukaryote model organism for cellular and molecular biology. However, the lack of extensive T. thermophila cDNA libraries or a large expressed sequence tag (EST database limited the quality of the original genome annotation. METHODOLOGY/PRINCIPAL FINDINGS: This RNA-seq study describes the first deep sequencing analysis of the T. thermophila transcriptome during the three major stages of the life cycle: growth, starvation and conjugation. Uniquely mapped reads covered more than 96% of the 24,725 predicted gene models in the somatic genome. More than 1,000 new transcribed regions were identified. The great dynamic range of RNA-seq allowed detection of a nearly six order-of-magnitude range of measurable gene expression orchestrated by this cell. RNA-seq also allowed the first prediction of transcript untranslated regions (UTRs and an updated (larger size estimate of the T. thermophila transcriptome: 57 Mb, or about 55% of the somatic genome. Our study identified nearly 1,500 alternative splicing (AS events distributed over 5.2% of T. thermophila genes. This percentage represents a two order-of-magnitude increase over previous EST-based estimates in Tetrahymena. Evidence of stage-specific regulation of alternative splicing was also obtained. Finally, our study allowed us to completely confirm about 26.8% of the genes originally predicted by the gene finder, to correct coding sequence boundaries and intron-exon junctions for about a third, and to reassign microarray probes and correct earlier microarray data. CONCLUSIONS/SIGNIFICANCE: RNA-seq data significantly improve the genome annotation and provide a fully comprehensive view of the global transcriptome of T. thermophila. To our knowledge, 5.2% of T. thermophila genes with AS is the highest percentage of genes showing AS reported in a unicellular eukaryote. Tetrahymena thus becomes an excellent unicellular

  11. Detection and quantification of Plasmodium falciparum in blood samples using quantitative nucleic acid sequence-based amplification

    NARCIS (Netherlands)

    Schoone, G. J.; Oskam, L.; Kroon, N. C.; Schallig, H. D.; Omar, S. A.

    2000-01-01

    A quantitative nucleic acid sequence-based amplification (QT-NASBA) assay for the detection of Plasmodium parasites has been developed. Primers and probes were selected on the basis of the sequence of the small-subunit rRNA gene. Quantification was achieved by coamplification of the RNA in the

  12. Feasibility of a RARE-based sequence for quantitative diffusion-weighted MRI of the spine

    International Nuclear Information System (INIS)

    Raya, J.G.; Dietrich, O.; Sommer, J.; Reiser, M.F.; Baur-Melnyk, A.; Birkenmaier, C.

    2007-01-01

    The feasibility of a diffusion-weighted single-shot fast-spin-echo sequence for the diagnostic work-up of bone marrow diseases was assessed. Twenty healthy controls and 16 patients with various bone marrow pathologies of the spine (bone marrow edema, tumor and inflammation) were examined with a diffusion-weighted single-shot sequence based on a modified rapid acquisition with relaxation enhancement (mRARE) technique; four diffusion weightings (b-values: 50, 250, 500 and 750 s/mm 2 ) in three orthogonal orientations were applied. Apparent diffusion coefficients (ADCs) were determined in the bone marrow and in the intervertebral discs of healthy volunteers and in diseased bone marrow. Ten of the 20 volunteers were repeatedly scanned within 30 min to examine short-time reproducibility. Spatial reproducibility was assessed by measuring ADCs in two different slices including the same lesion in 12 patients. The ADCs of the lesions exhibited significantly higher values, (1.27 ± 0.32) x 10 -3 mm 2 /s, compared with healthy bone marrow, (0.21 ± 0.10) x 10 -3 mm 2 /s. Short-time and spatial reproducibility had a mean coefficient of variation of 2.1% and 6.4%, respectively. The diffusion-weighted mRARE sequence provides a reliable tool for determining quantitative ADCs in vertebral bone marrow with adequate image quality. (orig.)

  13. Deep Sequencing Reveals the Complete Genome and Evidence for Transcriptional Activity of the First Virus-Like Sequences Identified in Aristotelia chilensis (Maqui Berry

    Directory of Open Access Journals (Sweden)

    Javier Villacreses

    2015-04-01

    Full Text Available Here, we report the genome sequence and evidence for transcriptional activity of a virus-like element in the native Chilean berry tree Aristotelia chilensis. We propose to name the endogenous sequence as Aristotelia chilensis Virus 1 (AcV1. High-throughput sequencing of the genome of this tree uncovered an endogenous viral element, with a size of 7122 bp, corresponding to the complete genome of AcV1. Its sequence contains three open reading frames (ORFs: ORFs 1 and 2 shares 66%–73% amino acid similarity with members of the Caulimoviridae virus family, especially the Petunia vein clearing virus (PVCV, Petuvirus genus. ORF1 encodes a movement protein (MP; ORF2 a Reverse Transcriptase (RT and a Ribonuclease H (RNase H domain; and ORF3 showed no amino acid sequence similarity with any other known virus proteins. Analogous to other known endogenous pararetrovirus sequences (EPRVs, AcV1 is integrated in the genome of Maqui Berry and showed low viral transcriptional activity, which was detected by deep sequencing technology (DNA and RNA-seq. Phylogenetic analysis of AcV1 and other pararetroviruses revealed a closer resemblance with Petuvirus. Overall, our data suggests that AcV1 could be a new member of Caulimoviridae family, genus Petuvirus, and the first evidence of this kind of virus in a fruit plant.

  14. Microbial Dark Matter: Unusual intervening sequences in 16S rRNA genes of candidate phyla from the deep subsurface

    Energy Technology Data Exchange (ETDEWEB)

    Jarett, Jessica; Stepanauskas, Ramunas; Kieft, Thomas; Onstott, Tullis; Woyke, Tanja

    2014-03-17

    The Microbial Dark Matter project has sequenced genomes from over 200 single cells from candidate phyla, greatly expanding our knowledge of the ecology, inferred metabolism, and evolution of these widely distributed, yet poorly understood lineages. The second phase of this project aims to sequence an additional 800 single cells from known as well as potentially novel candidate phyla derived from a variety of environments. In order to identify whole genome amplified single cells, screening based on phylogenetic placement of 16S rRNA gene sequences is being conducted. Briefly, derived 16S rRNA gene sequences are aligned to a custom version of the Greengenes reference database and added to a reference tree in ARB using parsimony. In multiple samples from deep subsurface habitats but not from other habitats, a large number of sequences proved difficult to align and therefore to place in the tree. Based on comparisons to reference sequences and structural alignments using SSU-ALIGN, many of these ?difficult? sequences appear to originate from candidate phyla, and contain intervening sequences (IVSs) within the 16S rRNA genes. These IVSs are short (39 - 79 nt) and do not appear to be self-splicing or to contain open reading frames. IVSs were found in the loop regions of stem-loop structures in several different taxonomic groups. Phylogenetic placement of sequences is strongly affected by IVSs; two out of three groups investigated were classified as different phyla after their removal. Based on data from samples screened in this project, IVSs appear to be more common in microbes occurring in deep subsurface habitats, although the reasons for this remain elusive.

  15. Deep Learning for Magnetic Resonance Fingerprinting: A New Approach for Predicting Quantitative Parameter Values from Time Series.

    Science.gov (United States)

    Hoppe, Elisabeth; Körzdörfer, Gregor; Würfl, Tobias; Wetzl, Jens; Lugauer, Felix; Pfeuffer, Josef; Maier, Andreas

    2017-01-01

    The purpose of this work is to evaluate methods from deep learning for application to Magnetic Resonance Fingerprinting (MRF). MRF is a recently proposed measurement technique for generating quantitative parameter maps. In MRF a non-steady state signal is generated by a pseudo-random excitation pattern. A comparison of the measured signal in each voxel with the physical model yields quantitative parameter maps. Currently, the comparison is done by matching a dictionary of simulated signals to the acquired signals. To accelerate the computation of quantitative maps we train a Convolutional Neural Network (CNN) on simulated dictionary data. As a proof of principle we show that the neural network implicitly encodes the dictionary and can replace the matching process.

  16. Deep sequencing shows that oocytes are not prone to accumulate mtDNA heteroplasmic mutations during ovarian ageing.

    Science.gov (United States)

    Boucret, L; Bris, C; Seegers, V; Goudenège, D; Desquiret-Dumas, V; Domin-Bernhard, M; Ferré-L'Hotellier, V; Bouet, P E; Descamps, P; Reynier, P; Procaccio, V; May-Panloup, P

    2017-10-01

    Does ovarian ageing increase the number of heteroplasmic mitochondrial DNA (mtDNA) point mutations in oocytes? Our results suggest that oocytes are not subject to the accumulation of mtDNA point mutations during ovarian ageing. Ageing is associated with the alteration of mtDNA integrity in various tissues. Primary oocytes, present in the ovary since embryonic life, may accumulate mtDNA mutations during the process of ovarian ageing. This was an observational study of 53 immature oocyte-cumulus complexes retrieved from 35 women undergoing IVF at the University Hospital of Angers, France, from March 2013 to March 2014. The women were classified in two groups, one including 19 women showing signs of ovarian ageing objectified by a diminished ovarian reserve (DOR), and the other, including 16 women with a normal ovarian reserve (NOR), which served as a control group. mtDNA was extracted from isolated oocytes, and from their corresponding cumulus cells (CCs) considered as a somatic cell compartment. The average mtDNA content of each sample was assessed by using a quantitative real-time PCR technique. Deep sequencing was performed using the Ion Torrent Proton for Next-Generation Sequencing. Signal processing and base calling were done by the embedded pre-processing pipeline and the variants were analyzed using an in-house workflow. The distribution of the different variants between DOR and NOR patients, on one hand, and oocyte and CCs, on the other, was analyzed with the generalized mixed linear model to take into account the cluster of cells belonging to a given mother. There were no significant differences between the numbers of mtDNA variants between the DOR and the NOR patients, either in the oocytes (P = 0.867) or in the surrounding CCs (P = 0.154). There were also no differences in terms of variants with potential functional consequences. De-novo mtDNA variants were found in 28% of the oocytes and in 66% of the CCs with the mean number of variants being

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

    Directory of Open Access Journals (Sweden)

    Federico M Ibarbalz

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

  18. Develop a quantitative understanding of rockmass behaviour near excavations in deep mines, part 1

    CSIR Research Space (South Africa)

    Napier, JAL

    1995-12-01

    Full Text Available Control of the rock mass deformation near deep level stopes and the avoidance of damaging incidents of violent rock failure require a fundamental understanding of rock failure mechanisms. Research work to gain this understanding has been undertaken...

  19. Deep Sequencing of 71 Candidate Genes to Characterize Variation Associated with Alcohol Dependence.

    Science.gov (United States)

    Clark, Shaunna L; McClay, Joseph L; Adkins, Daniel E; Kumar, Gaurav; Aberg, Karolina A; Nerella, Srilaxmi; Xie, Linying; Collins, Ann L; Crowley, James J; Quackenbush, Corey R; Hilliard, Christopher E; Shabalin, Andrey A; Vrieze, Scott I; Peterson, Roseann E; Copeland, William E; Silberg, Judy L; McGue, Matt; Maes, Hermine; Iacono, William G; Sullivan, Patrick F; Costello, Elizabeth J; van den Oord, Edwin J

    2017-04-01

    Previous genomewide association studies (GWASs) have identified a number of putative risk loci for alcohol dependence (AD). However, only a few loci have replicated and these replicated variants only explain a small proportion of AD risk. Using an innovative approach, the goal of this study was to generate hypotheses about potentially causal variants for AD that can be explored further through functional studies. We employed targeted capture of 71 candidate loci and flanking regions followed by next-generation deep sequencing (mean coverage 78X) in 806 European Americans. Regions included in our targeted capture library were genes identified through published GWAS of alcohol, all human alcohol and aldehyde dehydrogenases, reward system genes including dopaminergic and opioid receptors, prioritized candidate genes based on previous associations, and genes involved in the absorption, distribution, metabolism, and excretion of drugs. We performed single-locus tests to determine if any single variant was associated with AD symptom count. Sets of variants that overlapped with biologically meaningful annotations were tested for association in aggregate. No single, common variant was significantly associated with AD in our study. We did, however, find evidence for association with several variant sets. Two variant sets were significant at the q-value <0.10 level: a genic enhancer for ADHFE1 (p = 1.47 × 10 -5 ; q = 0.019), an alcohol dehydrogenase, and ADORA1 (p = 5.29 × 10 -5 ; q = 0.035), an adenosine receptor that belongs to a G-protein-coupled receptor gene family. To our knowledge, this is the first sequencing study of AD to examine variants in entire genes, including flanking and regulatory regions. We found that in addition to protein coding variant sets, regulatory variant sets may play a role in AD. From these findings, we have generated initial functional hypotheses about how these sets may influence AD. Copyright © 2017 by the Research Society on

  20. The Ebola virus VP35 protein binds viral immunostimulatory and host RNAs identified through deep sequencing.

    Directory of Open Access Journals (Sweden)

    Kari A Dilley

    Full Text Available Ebola virus and Marburg virus are members of the Filovirdae family and causative agents of hemorrhagic fever with high fatality rates in humans. Filovirus virulence is partially attributed to the VP35 protein, a well-characterized inhibitor of the RIG-I-like receptor pathway that triggers the antiviral interferon (IFN response. Prior work demonstrates the ability of VP35 to block potent RIG-I activators, such as Sendai virus (SeV, and this IFN-antagonist activity is directly correlated with its ability to bind RNA. Several structural studies demonstrate that VP35 binds short synthetic dsRNAs; yet, there are no data that identify viral immunostimulatory RNAs (isRNA or host RNAs bound to VP35 in cells. Utilizing a SeV infection model, we demonstrate that both viral isRNA and host RNAs are bound to Ebola and Marburg VP35s in cells. By deep sequencing the purified VP35-bound RNA, we identified the SeV copy-back defective interfering (DI RNA, previously identified as a robust RIG-I activator, as the isRNA bound by multiple filovirus VP35 proteins, including the VP35 protein from the West African outbreak strain (Makona EBOV. Moreover, RNAs isolated from a VP35 RNA-binding mutant were not immunostimulatory and did not include the SeV DI RNA. Strikingly, an analysis of host RNAs bound by wild-type, but not mutant, VP35 revealed that select host RNAs are preferentially bound by VP35 in cell culture. Taken together, these data support a model in which VP35 sequesters isRNA in virus-infected cells to avert RIG-I like receptor (RLR activation.

  1. Deep sequencing-based identification of small regulatory RNAs in Synechocystis sp. PCC 6803.

    Directory of Open Access Journals (Sweden)

    Wen Xu

    Full Text Available Synechocystis sp. PCC 6803 is a genetically tractable model organism for photosynthesis research. The genome of Synechocystis sp. PCC 6803 consists of a circular chromosome and seven plasmids. The importance of small regulatory RNAs (sRNAs as mediators of a number of cellular processes in bacteria has begun to be recognized. However, little is known regarding sRNAs in Synechocystis sp. PCC 6803. To provide a comprehensive overview of sRNAs in this model organism, the sRNAs of Synechocystis sp. PCC 6803 were analyzed using deep sequencing, and 7,951,189 reads were obtained. High quality mapping reads (6,127,890 were mapped onto the genome and assembled into 16,192 transcribed regions (clusters based on read overlap. A total number of 5211 putative sRNAs were revealed from the genome and the 4 megaplasmids, and 27 of these molecules, including four from plasmids, were confirmed by RT-PCR. In addition, possible target genes regulated by all of the putative sRNAs identified in this study were predicted by IntaRNA and analyzed for functional categorization and biological pathways, which provided evidence that sRNAs are indeed involved in many different metabolic pathways, including basic metabolic pathways, such as glycolysis/gluconeogenesis, the citrate cycle, fatty acid metabolism and adaptations to environmentally stress-induced changes. The information from this study provides a valuable reservoir for understanding the sRNA-mediated regulation of the complex physiology and metabolic processes of cyanobacteria.

  2. Deep sequencing reveals a novel closterovirus associated with wild rose leaf rosette disease.

    Science.gov (United States)

    He, Yan; Yang, Zuokun; Hong, Ni; Wang, Guoping; Ning, Guogui; Xu, Wenxing

    2015-06-01

    A bizarre virus-like symptom of a leaf rosette formed by dense small leaves on branches of wild roses (Rosa multiflora Thunb.), designated as 'wild rose leaf rosette disease' (WRLRD), was observed in China. To investigate the presumed causal virus, a wild rose sample affected by WRLRD was subjected to deep sequencing of small interfering RNAs (siRNAs) for a complete survey of the infecting viruses and viroids. The assembly of siRNAs led to the reconstruction of the complete genomes of three known viruses, namely Apple stem grooving virus (ASGV), Blackberry chlorotic ringspot virus (BCRV) and Prunus necrotic ringspot virus (PNRSV), and of a novel virus provisionally named 'rose leaf rosette-associated virus' (RLRaV). Phylogenetic analysis clearly placed RLRaV alongside members of the genus Closterovirus, family Closteroviridae. Genome organization of RLRaV RNA (17,653 nucleotides) showed 13 open reading frames (ORFs), except ORF1 and the quintuple gene block, most of which showed no significant similarities with known viral proteins, but, instead, had detectable identities to fungal or bacterial proteins. Additional novel molecular features indicated that RLRaV seems to be the most complex virus among the known genus members. To our knowledge, this is the first report of WRLRD and its associated closterovirus, as well as two ilarviruses and one capilovirus, infecting wild roses. Our findings present novel information about the closterovirus and the aetiology of this rose disease which should facilitate its control. More importantly, the novel features of RLRaV help to clarify the molecular and evolutionary features of the closterovirus. © 2014 BSPP AND JOHN WILEY & SONS LTD.

  3. The Ebola virus VP35 protein binds viral immunostimulatory and host RNAs identified through deep sequencing.

    Science.gov (United States)

    Dilley, Kari A; Voorhies, Alexander A; Luthra, Priya; Puri, Vinita; Stockwell, Timothy B; Lorenzi, Hernan; Basler, Christopher F; Shabman, Reed S

    2017-01-01

    Ebola virus and Marburg virus are members of the Filovirdae family and causative agents of hemorrhagic fever with high fatality rates in humans. Filovirus virulence is partially attributed to the VP35 protein, a well-characterized inhibitor of the RIG-I-like receptor pathway that triggers the antiviral interferon (IFN) response. Prior work demonstrates the ability of VP35 to block potent RIG-I activators, such as Sendai virus (SeV), and this IFN-antagonist activity is directly correlated with its ability to bind RNA. Several structural studies demonstrate that VP35 binds short synthetic dsRNAs; yet, there are no data that identify viral immunostimulatory RNAs (isRNA) or host RNAs bound to VP35 in cells. Utilizing a SeV infection model, we demonstrate that both viral isRNA and host RNAs are bound to Ebola and Marburg VP35s in cells. By deep sequencing the purified VP35-bound RNA, we identified the SeV copy-back defective interfering (DI) RNA, previously identified as a robust RIG-I activator, as the isRNA bound by multiple filovirus VP35 proteins, including the VP35 protein from the West African outbreak strain (Makona EBOV). Moreover, RNAs isolated from a VP35 RNA-binding mutant were not immunostimulatory and did not include the SeV DI RNA. Strikingly, an analysis of host RNAs bound by wild-type, but not mutant, VP35 revealed that select host RNAs are preferentially bound by VP35 in cell culture. Taken together, these data support a model in which VP35 sequesters isRNA in virus-infected cells to avert RIG-I like receptor (RLR) activation.

  4. Differential genomic arrangements in Caryophyllales through deep transcriptome sequencing of A. hypochondriacus.

    Directory of Open Access Journals (Sweden)

    Meeta Sunil

    Full Text Available Genome duplication event in edible dicots under the orders Rosid and Asterid, common during the oligocene period, is missing for species under the order Caryophyllales. Despite this, grain amaranths not only survived this period but display many desirable traits missing in species under rosids and asterids. For example, grain amaranths display traits like C4 photosynthesis, high-lysine seeds, high-yield, drought resistance, tolerance to infection and resilience to stress. It is, therefore, of interest to look for minor genome rearrangements with potential functional implications that are unique to grain amaranths. Here, by deep sequencing and assembly of 16 transcriptomes (86.8 billion bases we have interrogated differential genome rearrangement unique to Amaranthus hypochondriacus with potential links to these phenotypes. We have predicted 125,581 non-redundant transcripts including 44,529 protein coding transcripts identified based on homology to known proteins and 13,529 predicted as novel/amaranth specific coding transcripts. Of the protein coding de novo assembled transcripts, we have identified 1810 chimeric transcripts. More than 30% and 19% of the gene pairs within the chimeric transcripts are found within the same loci in the genomes of A. hypochondriacus and Beta vulgaris respectively and are considered real positives. Interestingly, one of the chimeric transcripts comprises two important genes, namely DHDPS1, a key enzyme implicated in the biosynthesis of lysine, and alpha-glucosidase, an enzyme involved in sucrose catabolism, in close proximity to each other separated by a distance of 612 bases in the genome of A. hypochondriacus in a convergent configuration. We have experimentally validated that transcripts of these two genes are also overlapping in the 3' UTR with their expression negatively correlated from bud to mature seed, suggesting a potential link between the high seed lysine trait and unique genome organization.

  5. Deep Sequence Analysis of AgoshRNA Processing Reveals 3’ A Addition and Trimming

    Directory of Open Access Journals (Sweden)

    Alex Harwig

    2015-01-01

    Full Text Available The RNA interference (RNAi pathway, in which microprocessor and Dicer collaborate to process microRNAs (miRNA, was recently expanded by the description of alternative processing routes. In one of these noncanonical pathways, Dicer action is replaced by the Argonaute2 (Ago2 slicer function. It was recently shown that the stem-length of precursor-miRNA or short hairpin RNA (shRNA molecules is a major determinant for Dicer versus Ago2 processing. Here we present the results of a deep sequence study on the processing of shRNAs with different stem length and a top G·U wobble base pair (bp. This analysis revealed some unexpected properties of these so-called AgoshRNA molecules that are processed by Ago2 instead of Dicer. First, we confirmed the gradual shift from Dicer to Ago2 processing upon shortening of the hairpin length. Second, hairpins with a stem larger than 19 base pair are inefficiently cleaved by Ago2 and we noticed a shift in the cleavage site. Third, the introduction of a top G·U bp in a regular shRNA can promote Ago2-cleavage, which coincides with a loss of Ago2-loading of the Dicer-cleaved 3’ strand. Fourth, the Ago2-processed AgoshRNAs acquire a short 3’ tail of 1–3 A-nucleotides (nt and we present evidence that this product is subsequently trimmed by the poly(A-specific ribonuclease (PARN.

  6. Deep sequencing of the mitochondrial genome reveals common heteroplasmic sites in NADH dehydrogenase genes.

    Science.gov (United States)

    Liu, Chunyu; Fetterman, Jessica L; Liu, Poching; Luo, Yan; Larson, Martin G; Vasan, Ramachandran S; Zhu, Jun; Levy, Daniel

    2018-03-01

    Increasing evidence implicates mitochondrial dysfunction in aging and age-related conditions. But little is known about the molecular basis for this connection. A possible cause may be mutations in the mitochondrial DNA (mtDNA), which are often heteroplasmic-the joint presence of different alleles at a single locus in the same individual. However, the involvement of mtDNA heteroplasmy in aging and age-related conditions has not been investigated thoroughly. We deep-sequenced the complete mtDNA genomes of 356 Framingham Heart Study participants (52% women, mean age 43, mean coverage 4570-fold), identified 2880 unique mutations and comprehensively annotated them by MITOMAP and PolyPhen-2. We discovered 11 heteroplasmic "hot" spots [NADH dehydrogenase (ND) subunit 1, 4, 5 and 6 genes, n = 7; cytochrome c oxidase I (COI), n = 2; 16S rRNA, n = 1; D-loop, n = 1] for which the alternative-to-reference allele ratios significantly increased with advancing age (Bonferroni correction p < 0.001). Four of these heteroplasmic mutations in ND and COI genes were predicted to be deleterious nonsynonymous mutations which may have direct impact on ATP production. We confirmed previous findings that healthy individuals carry many low-frequency heteroplasmy mutations with potentially deleterious effects. We hypothesize that the effect of a single deleterious heteroplasmy may be minimal due to a low mutant-to-wildtype allele ratio, whereas the aggregate effects of many deleterious mutations may cause changes in mitochondrial function and contribute to age-related diseases. The identification of age-related mtDNA mutations is an important step to understand the genetic architecture of age-related diseases and may uncover novel therapeutic targets for such diseases.

  7. Analysis of microRNA profile of Anopheles sinensis by deep sequencing and bioinformatic approaches.

    Science.gov (United States)

    Feng, Xinyu; Zhou, Xiaojian; Zhou, Shuisen; Wang, Jingwen; Hu, Wei

    2018-03-12

    microRNAs (miRNAs) are small non-coding RNAs widely identified in many mosquitoes. They are reported to play important roles in development, differentiation and innate immunity. However, miRNAs in Anopheles sinensis, one of the Chinese malaria mosquitoes, remain largely unknown. We investigated the global miRNA expression profile of An. sinensis using Illumina Hiseq 2000 sequencing. Meanwhile, we applied a bioinformatic approach to identify potential miRNAs in An. sinensis. The identified miRNA profiles were compared and analyzed by two approaches. The selected miRNAs from the sequencing result and the bioinformatic approach were confirmed with qRT-PCR. Moreover, target prediction, GO annotation and pathway analysis were carried out to understand the role of miRNAs in An. sinensis. We identified 49 conserved miRNAs and 12 novel miRNAs by next-generation high-throughput sequencing technology. In contrast, 43 miRNAs were predicted by the bioinformatic approach, of which two were assigned as novel. Comparative analysis of miRNA profiles by two approaches showed that 21 miRNAs were shared between them. Twelve novel miRNAs did not match any known miRNAs of any organism, indicating that they are possibly species-specific. Forty miRNAs were found in many mosquito species, indicating that these miRNAs are evolutionally conserved and may have critical roles in the process of life. Both the selected known and novel miRNAs (asi-miR-281, asi-miR-184, asi-miR-14, asi-miR-nov5, asi-miR-nov4, asi-miR-9383, and asi-miR-2a) could be detected by quantitative real-time PCR (qRT-PCR) in the sequenced sample, and the expression patterns of these miRNAs measured by qRT-PCR were in concordance with the original miRNA sequencing data. The predicted targets for the known and the novel miRNAs covered many important biological roles and pathways indicating the diversity of miRNA functions. We also found 21 conserved miRNAs and eight counterparts of target immune pathway genes in An. sinensis

  8. Deep sequencing of the Camellia chekiangoleosa transcriptome revealed candidate genes for anthocyanin biosynthesis.

    Science.gov (United States)

    Wang, Zhong-Wei; Jiang, Cong; Wen, Qiang; Wang, Na; Tao, Yuan-Yuan; Xu, Li-An

    2014-03-15

    Camellia chekiangoleosa is an important species of genus Camellia. It provides high-quality edible oil and has great ornamental value. The flowers are big and red which bloom between February and March. Flower pigmentation is closely related to the accumulation of anthocyanin. Although anthocyanin biosynthesis has been studied extensively in herbaceous plants, little molecular information on the anthocyanin biosynthesis pathway of C. chekiangoleosa is yet known. In the present study, a cDNA library was constructed to obtain detailed and general data from the flowers of C. chekiangoleosa. To explore the transcriptome of C. chekiangoleosa and investigate genes involved in anthocyanin biosynthesis, a 454 GS FLX Titanium platform was used to generate an EST dataset. About 46,279 sequences were obtained, and 24,593 (53.1%) were annotated. Using Blast search against the AGRIS, 1740 unigenes were found homologous to 599 Arabidopsis transcription factor genes. Based on the transcriptome dataset, nine anthocyanin biosynthesis pathway genes (PAL, CHS1, CHS2, CHS3, CHI, F3H, DFR, ANS, and UFGT) were identified and cloned. The spatio-temporal expression patterns of these genes were also analyzed using quantitative real-time polymerase chain reaction. The study results not only enrich the gene resource but also provide valuable information for further studies concerning anthocyanin biosynthesis. Copyright © 2014 Elsevier B.V. All rights reserved.

  9. Deep Sequencing of Porphyromonas gingivalis and comparative transcriptome analysis of a LuxS mutant

    Directory of Open Access Journals (Sweden)

    Takanoi eHirano

    2012-06-01

    Full Text Available Porphyromonas gingivalis is a major etiological agent and chronic and aggressive forms of periodontal disease. The organism is an assacharolytic anaerobe and is a constituent of mixed species biofilms in a variety of microenvironments in the oral cavity. P. gingivalis expresses a range of virulence factors over which it exerts tight control. High-throughput sequencing technologies provide the opportunity to relate functional genomics to basic biology. In this study we report qualitative and quantitative RNA-Seq analysis of the transcriptome of P. gingivalis. We have also applied RNA-Seq to the transcriptome of a ΔluxS mutant of P. gingivalis deficient in AI-2-mediated bacterial communication. The transcriptome analysis confirmed the expression of all predicted ORFs for strain ATCC 33277, including 854 hypothetical proteins, and allowed the identification of hitherto unknown transcriptional units. Twelve noncoding RNAs were identified, including 11 small RNAs and one cobalamine riboswitch. Fifty seven genes were differentially regulated in the LuxS mutant. Addition of exogenous synthetic 4,5-dihydroxy-2,3-pentanedione (DPD, AI-2 precursor to the ΔluxS mutant culture complemented expression of a subset of genes, indicating that LuxS is involved in both AI-2 signaling and non-signaling dependent systems in P. gingivalis. This work provides an important dataset for future study of P. gingivalis pathophysiology and further defines the LuxS regulon in this oral pathogen.

  10. Quantitative trait loci markers derived from whole genome sequence data increases the reliability of genomic prediction

    DEFF Research Database (Denmark)

    Brøndum, Rasmus Froberg; Su, Guosheng; Janss, Luc

    2015-01-01

    This study investigated the effect on the reliability of genomic prediction when a small number of significant variants from single marker analysis based on whole genome sequence data were added to the regular 54k single nucleotide polymorphism (SNP) array data. The extra markers were selected...... with the aim of augmenting the custom low-density Illumina BovineLD SNP chip (San Diego, CA) used in the Nordic countries. The single-marker analysis was done breed-wise on all 16 index traits included in the breeding goals for Nordic Holstein, Danish Jersey, and Nordic Red cattle plus the total merit index...... itself. Depending on the trait’s economic weight, 15, 10, or 5 quantitative trait loci (QTL) were selected per trait per breed and 3 to 5 markers were selected to tag each QTL. After removing duplicate markers (same marker selected for more than one trait or breed) and filtering for high pairwise linkage...

  11. The subclonal structure and genomic evolution of oral squamous cell carcinoma revealed by ultra-deep sequencing

    DEFF Research Database (Denmark)

    Tabatabaeifar, Siavosh; Thomassen, Mads; Larsen, Martin J

    2017-01-01

    Recent studies suggest that head and neck squamous cell carcinomas are very heterogeneous between patients; however the subclonal structure remains unexplored mainly due to studies using only a single biopsy per patient. To deconvolutethe clonal structure and describe the genomic cancer evolution......, we applied whole-exome sequencing combined with ultra-deep targeted sequencing on oral squamous cell carcinomas (OSCC). From each patient, a set of biopsies was sampled from distinct geographical sites in primary tumor and lymph node metastasis.We demonstrate that the included OSCCs show a high...

  12. Rapid and Deep Proteomes by Faster Sequencing on a Benchtop Quadrupole Ultra-High-Field Orbitrap Mass Spectrometer

    DEFF Research Database (Denmark)

    Kelstrup, Christian D; Jersie-Christensen, Rosa R; Batth, Tanveer Singh

    2014-01-01

    per second or up to 600 new peptides sequenced per gradient minute. We identify 4400 proteins from one microgram of HeLa digest using a one hour gradient, which is an approximately 30% improvement compared to previous instrumentation. In addition, we show very deep proteome coverage can be achieved...... in less than 24 hours of analysis time by offline high pH reversed-phase peptide fractionation from which we identify more than 140,000 unique peptide sequences. This is comparable to state-of-the-art multi-day, multi-enzyme efforts. Finally the acquisition methods are evaluated for single...

  13. Identifications of Putative PKA Substrates with Quantitative Phosphoproteomics and Primary-Sequence-Based Scoring.

    Science.gov (United States)

    Imamura, Haruna; Wagih, Omar; Niinae, Tomoya; Sugiyama, Naoyuki; Beltrao, Pedro; Ishihama, Yasushi

    2017-04-07

    Protein kinase A (PKA or cAMP-dependent protein kinase) is a serine/threonine kinase that plays essential roles in the regulation of proliferation, differentiation, and apoptosis. To better understand the functions of PKA, it is necessary to elucidate the direct interplay between PKA and their substrates in living human cells. To identify kinase target substrates in a high-throughput manner, we first quantified the change of phosphoproteome in the cells of which PKA activity was perturbed by drug stimulations. LC-MS/MS analyses identified 2755 and 3191 phosphopeptides from experiments with activator or inhibitor of PKA. To exclude potential indirect targets of PKA, we built a computational model to characterize the kinase sequence specificity toward the substrate target site based on known kinase-substrate relationships. Finally, by combining the sequence recognition model with the quantitative changes in phosphorylation measured in the two drug perturbation experiments, we identified 29 reliable candidates of PKA targeting residues in living cells including 8 previously known substrates. Moreover, 18 of these sites were confirmed to be site-specifically phosphorylated in vitro. Altogether this study proposed a confident list of PKA substrate candidates, expanding our knowledge of PKA signaling network.

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

    Science.gov (United States)

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

    2017-02-01

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

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

    Science.gov (United States)

    2012-01-01

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

  16. Identification and Characterization of Liver MicroRNAs of the Chinese Tree Shrew via Deep Sequencing.

    Science.gov (United States)

    Feng, Yue; Feng, Yue-Mei; Feng, Yang; Lu, Caixia; Liu, Li; Sun, Xiaomei; Dai, Jiejie; Xia, Xueshan

    2015-10-01

    Chinese tree shrew (Tupaia belangeri chinensis) is a small animal that possess many features, which are valuable in biomedical research, as experimental models. Currently, there are numerous attempts to utilize tree shrews as models for hepatitis C virus (HCV) infection. This study aimed to construct a liver microRNA (miRNA) data of the tree shrew. Three second filial generation tree shrews were used in this study. Total RNA was extracted from each liver of the tree shrew and equal quality mixed, then reverse-transcribed to complementary DNA (cDNA). The cDNAs were amplified by polymerase chain reaction and subjected to high-throughput sequencing. A total of 2060 conserved miRNAs were identified through alignment with the mature miRNAs in miRBase 20.0 database. The gene ontology and Kyoto encyclopedia of genes and genomes analyses of the target genes of the miRNAs revealed several candidate miRNAs, genes and pathways that may involve in the process of HCV infection. The abundance of miR-122 and Let-7 families and their other characteristics provided us more evidences for the utilization of this animal, as a potential model for HCV infection and other related biomedical research. Moreover, 80 novel microRNAs were predicted using the software Mireap. The top 3 abundant miRNAs were validated in other tree samples, based on stem-loop quantitative reverse transcription-polymerase chain reaction. According to the liver microRNA data of Chinese tree shrew, characteristics of the miR-122 and Let-7 families further highlight the suitability of tree shrew as the animal model in HCV research.

  17. An introduction to Deep learning on biological sequence data - Examples and solutions

    DEFF Research Database (Denmark)

    Jurtz, Vanessa Isabell; Johansen, Alexander Rosenberg; Nielsen, Morten

    2017-01-01

    Deep neural network architectures such as convolutional and long short-term memory networks have become increasingly popular as machine learning tools during the recent years. The availability of greater computational resources, more data, new algorithms for training deep models and easy to use....... Here, we aim to further the development of deep learning methods within biology by providing application examples and ready to apply and adapt code templates. Given such examples, we illustrate how architectures consisting of convolutional and long short-term memory neural networks can relatively...

  18. Deep Sequencing Analysis of miRNA Expression in Breast Muscle of Fast-Growing and Slow-Growing Broilers

    Directory of Open Access Journals (Sweden)

    Hongjia Ouyang

    2015-07-01

    Full Text Available Growth performance is an important economic trait in chicken. MicroRNAs (miRNAs have been shown to play important roles in various biological processes, but their functions in chicken growth are not yet clear. To investigate the function of miRNAs in chicken growth, breast muscle tissues of the two-tail samples (highest and lowest body weight from Recessive White Rock (WRR and Xinghua Chickens (XH were performed on high throughput small RNA deep sequencing. In this study, a total of 921 miRNAs were identified, including 733 known mature miRNAs and 188 novel miRNAs. There were 200, 279, 257 and 297 differentially expressed miRNAs in the comparisons of WRRh vs. WRRl, WRRh vs. XHh, WRRl vs. XHl, and XHh vs. XHl group, respectively. A total of 22 highly differentially expressed miRNAs (fold change > 2 or < 0.5; p-value < 0.05; q-value < 0.01, which also have abundant expression (read counts > 1000 were found in our comparisons. As far as two analyses (WRRh vs. WRRl, and XHh vs. XHl are concerned, we found 80 common differentially expressed miRNAs, while 110 miRNAs were found in WRRh vs. XHh and WRRl vs. XHl. Furthermore, 26 common miRNAs were identified among all four comparisons. Four differentially expressed miRNAs (miR-223, miR-16, miR-205a and miR-222b-5p were validated by quantitative real-time RT-PCR (qRT-PCR. Regulatory networks of interactions among miRNAs and their targets were constructed using integrative miRNA target-prediction and network-analysis. Growth hormone receptor (GHR was confirmed as a target of miR-146b-3p by dual-luciferase assay and qPCR, indicating that miR-34c, miR-223, miR-146b-3p, miR-21 and miR-205a are key growth-related target genes in the network. These miRNAs are proposed as candidate miRNAs for future studies concerning miRNA-target function on regulation of chicken growth.

  19. SHARAKU: an algorithm for aligning and clustering read mapping profiles of deep sequencing in non-coding RNA processing.

    Science.gov (United States)

    Tsuchiya, Mariko; Amano, Kojiro; Abe, Masaya; Seki, Misato; Hase, Sumitaka; Sato, Kengo; Sakakibara, Yasubumi

    2016-06-15

    Deep sequencing of the transcripts of regulatory non-coding RNA generates footprints of post-transcriptional processes. After obtaining sequence reads, the short reads are mapped to a reference genome, and specific mapping patterns can be detected called read mapping profiles, which are distinct from random non-functional degradation patterns. These patterns reflect the maturation processes that lead to the production of shorter RNA sequences. Recent next-generation sequencing studies have revealed not only the typical maturation process of miRNAs but also the various processing mechanisms of small RNAs derived from tRNAs and snoRNAs. We developed an algorithm termed SHARAKU to align two read mapping profiles of next-generation sequencing outputs for non-coding RNAs. In contrast with previous work, SHARAKU incorporates the primary and secondary sequence structures into an alignment of read mapping profiles to allow for the detection of common processing patterns. Using a benchmark simulated dataset, SHARAKU exhibited superior performance to previous methods for correctly clustering the read mapping profiles with respect to 5'-end processing and 3'-end processing from degradation patterns and in detecting similar processing patterns in deriving the shorter RNAs. Further, using experimental data of small RNA sequencing for the common marmoset brain, SHARAKU succeeded in identifying the significant clusters of read mapping profiles for similar processing patterns of small derived RNA families expressed in the brain. The source code of our program SHARAKU is available at http://www.dna.bio.keio.ac.jp/sharaku/, and the simulated dataset used in this work is available at the same link. Accession code: The sequence data from the whole RNA transcripts in the hippocampus of the left brain used in this work is available from the DNA DataBank of Japan (DDBJ) Sequence Read Archive (DRA) under the accession number DRA004502. yasu@bio.keio.ac.jp Supplementary data are available

  20. Optimizing a massive parallel sequencing workflow for quantitative miRNA expression analysis.

    Directory of Open Access Journals (Sweden)

    Francesca Cordero

    Full Text Available BACKGROUND: Massive Parallel Sequencing methods (MPS can extend and improve the knowledge obtained by conventional microarray technology, both for mRNAs and short non-coding RNAs, e.g. miRNAs. The processing methods used to extract and interpret the information are an important aspect of dealing with the vast amounts of data generated from short read sequencing. Although the number of computational tools for MPS data analysis is constantly growing, their strengths and weaknesses as part of a complex analytical pipe-line have not yet been well investigated. PRIMARY FINDINGS: A benchmark MPS miRNA dataset, resembling a situation in which miRNAs are spiked in biological replication experiments was assembled by merging a publicly available MPS spike-in miRNAs data set with MPS data derived from healthy donor peripheral blood mononuclear cells. Using this data set we observed that short reads counts estimation is strongly under estimated in case of duplicates miRNAs, if whole genome is used as reference. Furthermore, the sensitivity of miRNAs detection is strongly dependent by the primary tool used in the analysis. Within the six aligners tested, specifically devoted to miRNA detection, SHRiMP and MicroRazerS show the highest sensitivity. Differential expression estimation is quite efficient. Within the five tools investigated, two of them (DESseq, baySeq show a very good specificity and sensitivity in the detection of differential expression. CONCLUSIONS: The results provided by our analysis allow the definition of a clear and simple analytical optimized workflow for miRNAs digital quantitative analysis.

  1. Optimizing a massive parallel sequencing workflow for quantitative miRNA expression analysis.

    Science.gov (United States)

    Cordero, Francesca; Beccuti, Marco; Arigoni, Maddalena; Donatelli, Susanna; Calogero, Raffaele A

    2012-01-01

    Massive Parallel Sequencing methods (MPS) can extend and improve the knowledge obtained by conventional microarray technology, both for mRNAs and short non-coding RNAs, e.g. miRNAs. The processing methods used to extract and interpret the information are an important aspect of dealing with the vast amounts of data generated from short read sequencing. Although the number of computational tools for MPS data analysis is constantly growing, their strengths and weaknesses as part of a complex analytical pipe-line have not yet been well investigated. A benchmark MPS miRNA dataset, resembling a situation in which miRNAs are spiked in biological replication experiments was assembled by merging a publicly available MPS spike-in miRNAs data set with MPS data derived from healthy donor peripheral blood mononuclear cells. Using this data set we observed that short reads counts estimation is strongly under estimated in case of duplicates miRNAs, if whole genome is used as reference. Furthermore, the sensitivity of miRNAs detection is strongly dependent by the primary tool used in the analysis. Within the six aligners tested, specifically devoted to miRNA detection, SHRiMP and MicroRazerS show the highest sensitivity. Differential expression estimation is quite efficient. Within the five tools investigated, two of them (DESseq, baySeq) show a very good specificity and sensitivity in the detection of differential expression. The results provided by our analysis allow the definition of a clear and simple analytical optimized workflow for miRNAs digital quantitative analysis.

  2. Eigenspectra optoacoustic tomography achieves quantitative blood oxygenation imaging deep in tissues

    Science.gov (United States)

    Tzoumas, Stratis; Nunes, Antonio; Olefir, Ivan; Stangl, Stefan; Symvoulidis, Panagiotis; Glasl, Sarah; Bayer, Christine; Multhoff, Gabriele; Ntziachristos, Vasilis

    2016-06-01

    Light propagating in tissue attains a spectrum that varies with location due to wavelength-dependent fluence attenuation, an effect that causes spectral corruption. Spectral corruption has limited the quantification accuracy of optical and optoacoustic spectroscopic methods, and impeded the goal of imaging blood oxygen saturation (sO2) deep in tissues; a critical goal for the assessment of oxygenation in physiological processes and disease. Here we describe light fluence in the spectral domain and introduce eigenspectra multispectral optoacoustic tomography (eMSOT) to account for wavelength-dependent light attenuation, and estimate blood sO2 within deep tissue. We validate eMSOT in simulations, phantoms and animal measurements and spatially resolve sO2 in muscle and tumours, validating our measurements with histology data. eMSOT shows substantial sO2 accuracy enhancement over previous optoacoustic methods, potentially serving as a valuable tool for imaging tissue pathophysiology.

  3. Eigenspectra optoacoustic tomography achieves quantitative blood oxygenation imaging deep in tissues.

    Science.gov (United States)

    Tzoumas, Stratis; Nunes, Antonio; Olefir, Ivan; Stangl, Stefan; Symvoulidis, Panagiotis; Glasl, Sarah; Bayer, Christine; Multhoff, Gabriele; Ntziachristos, Vasilis

    2016-06-30

    Light propagating in tissue attains a spectrum that varies with location due to wavelength-dependent fluence attenuation, an effect that causes spectral corruption. Spectral corruption has limited the quantification accuracy of optical and optoacoustic spectroscopic methods, and impeded the goal of imaging blood oxygen saturation (sO2) deep in tissues; a critical goal for the assessment of oxygenation in physiological processes and disease. Here we describe light fluence in the spectral domain and introduce eigenspectra multispectral optoacoustic tomography (eMSOT) to account for wavelength-dependent light attenuation, and estimate blood sO2 within deep tissue. We validate eMSOT in simulations, phantoms and animal measurements and spatially resolve sO2 in muscle and tumours, validating our measurements with histology data. eMSOT shows substantial sO2 accuracy enhancement over previous optoacoustic methods, potentially serving as a valuable tool for imaging tissue pathophysiology.

  4. Identification and Removal of Contaminant Sequences From Ribosomal Gene Databases: Lessons From the Census of Deep Life.

    Science.gov (United States)

    Sheik, Cody S; Reese, Brandi Kiel; Twing, Katrina I; Sylvan, Jason B; Grim, Sharon L; Schrenk, Matthew O; Sogin, Mitchell L; Colwell, Frederick S

    2018-01-01

    Earth's subsurface environment is one of the largest, yet least studied, biomes on Earth, and many questions remain regarding what microorganisms are indigenous to the subsurface. Through the activity of the Census of Deep Life (CoDL) and the Deep Carbon Observatory, an open access 16S ribosomal RNA gene sequence database from diverse subsurface environments has been compiled. However, due to low quantities of biomass in the deep subsurface, the potential for incorporation of contaminants from reagents used during sample collection, processing, and/or sequencing is high. Thus, to understand the ecology of subsurface microorganisms (i.e., the distribution, richness, or survival), it is necessary to minimize, identify, and remove contaminant sequences that will skew the relative abundances of all taxa in the sample. In this meta-analysis, we identify putative contaminants associated with the CoDL dataset, recommend best practices for removing contaminants from samples, and propose a series of best practices for subsurface microbiology sampling. The most abundant putative contaminant genera observed, independent of evenness across samples, were Propionibacterium , Aquabacterium , Ralstonia , and Acinetobacter . While the top five most frequently observed genera were Pseudomonas , Propionibacterium , Acinetobacter , Ralstonia , and Sphingomonas . The majority of the most frequently observed genera (high evenness) were associated with reagent or potential human contamination. Additionally, in DNA extraction blanks, we observed potential archaeal contaminants, including methanogens, which have not been discussed in previous contamination studies. Such contaminants would directly affect the interpretation of subsurface molecular studies, as methanogenesis is an important subsurface biogeochemical process. Utilizing previously identified contaminant genera, we found that ∼27% of the total dataset were identified as contaminant sequences that likely originate from DNA

  5. Quantitative Determination of Plasmodium vivax Gametocytes by Real-Time Quantitative Nucleic Acid Sequence-Based Amplification in Clinical Samples

    NARCIS (Netherlands)

    Beurskens, Martijn; Mens, Pètra; Schallig, Henk; Syafruddin, Din; Asih, Puji Budi Setia; Hermsen, Rob; Sauerwein, Robert

    2009-01-01

    Microscopic detection of Plasmodium vivax gametocytes, the sexual life stage of this malaria parasite, is insensitive because P vivax parasitaemia is low. To detect and quantity gametocytes a more sensitive, quantitative realtime Pvs25-QT-NASBA based oil Pvs25 mRNA was developed and tested in two

  6. Comparison of illumina and 454 deep sequencing in participants failing raltegravir-based antiretroviral therapy.

    Directory of Open Access Journals (Sweden)

    Jonathan Z Li

    Full Text Available The impact of raltegravir-resistant HIV-1 minority variants (MVs on raltegravir treatment failure is unknown. Illumina sequencing offers greater throughput than 454, but sequence analysis tools for viral sequencing are needed. We evaluated Illumina and 454 for the detection of HIV-1 raltegravir-resistant MVs.A5262 was a single-arm study of raltegravir and darunavir/ritonavir in treatment-naïve patients. Pre-treatment plasma was obtained from 5 participants with raltegravir resistance at the time of virologic failure. A control library was created by pooling integrase clones at predefined proportions. Multiplexed sequencing was performed with Illumina and 454 platforms at comparable costs. Illumina sequence analysis was performed with the novel snp-assess tool and 454 sequencing was analyzed with V-Phaser.Illumina sequencing resulted in significantly higher sequence coverage and a 0.095% limit of detection. Illumina accurately detected all MVs in the control library at ≥0.5% and 7/10 MVs expected at 0.1%. 454 sequencing failed to detect any MVs at 0.1% with 5 false positive calls. For MVs detected in the patient samples by both 454 and Illumina, the correlation in the detected variant frequencies was high (R2 = 0.92, P<0.001. Illumina sequencing detected 2.4-fold greater nucleotide MVs and 2.9-fold greater amino acid MVs compared to 454. The only raltegravir-resistant MV detected was an E138K mutation in one participant by Illumina sequencing, but not by 454.In participants of A5262 with raltegravir resistance at virologic failure, baseline raltegravir-resistant MVs were rarely detected. At comparable costs to 454 sequencing, Illumina demonstrated greater depth of coverage, increased sensitivity for detecting HIV MVs, and fewer false positive variant calls.

  7. High-resolution deep sequencing reveals biodiversity, population structure, and persistence of HIV-1 quasispecies within host ecosystems

    Directory of Open Access Journals (Sweden)

    Yin Li

    2012-12-01

    Full Text Available Abstract Background Deep sequencing provides the basis for analysis of biodiversity of taxonomically similar organisms in an environment. While extensively applied to microbiome studies, population genetics studies of viruses are limited. To define the scope of HIV-1 population biodiversity within infected individuals, a suite of phylogenetic and population genetic algorithms was applied to HIV-1 envelope hypervariable domain 3 (Env V3 within peripheral blood mononuclear cells from a group of perinatally HIV-1 subtype B infected, therapy-naïve children. Results Biodiversity of HIV-1 Env V3 quasispecies ranged from about 70 to 270 unique sequence clusters across individuals. Viral population structure was organized into a limited number of clusters that included the dominant variants combined with multiple clusters of low frequency variants. Next generation viral quasispecies evolved from low frequency variants at earlier time points through multiple non-synonymous changes in lineages within the evolutionary landscape. Minor V3 variants detected as long as four years after infection co-localized in phylogenetic reconstructions with early transmitting viruses or with subsequent plasma virus circulating two years later. Conclusions Deep sequencing defines HIV-1 population complexity and structure, reveals the ebb and flow of dominant and rare viral variants in the host ecosystem, and identifies an evolutionary record of low-frequency cell-associated viral V3 variants that persist for years. Bioinformatics pipeline developed for HIV-1 can be applied for biodiversity studies of virome populations in human, animal, or plant ecosystems.

  8. Foundations of Sequence-to-Sequence Modeling for Time Series

    OpenAIRE

    Kuznetsov, Vitaly; Mariet, Zelda

    2018-01-01

    The availability of large amounts of time series data, paired with the performance of deep-learning algorithms on a broad class of problems, has recently led to significant interest in the use of sequence-to-sequence models for time series forecasting. We provide the first theoretical analysis of this time series forecasting framework. We include a comparison of sequence-to-sequence modeling to classical time series models, and as such our theory can serve as a quantitative guide for practiti...

  9. Synthetic internal control sequences to increase negative call veracity in multiplexed, quantitative PCR assays for Phakopsora pachyrhizi

    Science.gov (United States)

    Quantitative PCR (Q-PCR) utilizing specific primer sequences and a fluorogenic, 5’-exonuclease linear hydrolysis probe is well established as a detection and identification method for Phakopsora pachyrhizi, the soybean rust pathogen. Because of the extreme sensitivity of Q-PCR, the DNA of a single u...

  10. Genome-wide analyses of long noncoding RNA expression profiles correlated with radioresistance in nasopharyngeal carcinoma via next-generation deep sequencing.

    Science.gov (United States)

    Li, Guo; Liu, Yong; Liu, Chao; Su, Zhongwu; Ren, Shuling; Wang, Yunyun; Deng, Tengbo; Huang, Donghai; Tian, Yongquan; Qiu, Yuanzheng

    2016-09-06

    Radioresistance is one of the major factors limiting the therapeutic efficacy and prognosis of patients with nasopharyngeal carcinoma (NPC). Accumulating evidence has suggested that aberrant expression of long noncoding RNAs (lncRNAs) contributes to cancer progression. Therefore, here we identified lncRNAs associated with radioresistance in NPC. The differential expression profiles of lncRNAs associated with NPC radioresistance were constructed by next-generation deep sequencing by comparing radioresistant NPC cells with their parental cells. LncRNA-related mRNAs were predicted and analyzed using bioinformatics algorithms compared with the mRNA profiles related to radioresistance obtained in our previous study. Several lncRNAs and associated mRNAs were validated in established NPC radioresistant cell models and NPC tissues. By comparison between radioresistant CNE-2-Rs and parental CNE-2 cells by next-generation deep sequencing, a total of 781 known lncRNAs and 2054 novel lncRNAs were annotated. The top five upregulated and downregulated known/novel lncRNAs were detected using quantitative real-time reverse transcription-polymerase chain reaction, and 7/10 known lncRNAs and 3/10 novel lncRNAs were demonstrated to have significant differential expression trends that were the same as those predicted by deep sequencing. From the prediction process, 13 pairs of lncRNAs and their associated genes were acquired, and the prediction trends of three pairs were validated in both radioresistant CNE-2-Rs and 6-10B-Rs cell lines, including lncRNA n373932 and SLITRK5, n409627 and PRSS12, and n386034 and RIMKLB. LncRNA n373932 and its related SLITRK5 showed dramatic expression changes in post-irradiation radioresistant cells and a negative expression correlation in NPC tissues (R = -0.595, p < 0.05). Our study provides an overview of the expression profiles of radioresistant lncRNAs and potentially related mRNAs, which will facilitate future investigations into the

  11. Quantitative comparison of cortical and deep grey matter in pathological subtypes of unilateral cerebral palsy.

    Science.gov (United States)

    Scheck, Simon M; Pannek, Kerstin; Fiori, Simona; Boyd, Roslyn N; Rose, Stephen E

    2014-10-01

    The aim of this study was to quantify grey matter changes in children with unilateral cerebral palsy (UCP), differentiating between cortical or deep grey matter (CDGM) lesions, periventricular white matter (PWM) lesions, and unilateral and bilateral lesions. In a cross-sectional study we obtained high resolution structural magnetic resonance images from 72 children (41 males, 31 females, mean age 10y 9mo [SD 3y 1mo], range 5y 1mo-17y 1mo) with UCP (33 left, 39 right hemiplegia; Manual Ability Classification System level I n=29, II n=43; Gross Motor Function Classification System level I n=46, II n=26), and 19 children with typical development (CTD; eight males, 11 females, mean age 11y 2mo [SD 2y 7mo], range 7y 8mo-16y 4mo). Images were classified by lesion type and analyzed using voxel-based morphometry (VBM) and subcortical volumetric analysis. Deep grey matter volumes were not significantly different between children with CDGM and PWM lesions, with the thalamus, putamen, and globus pallidus being reduced unilaterally in both groups compared with CTD (p≤0.001). Children with CDGM lesions additionally showed widespread cortical changes involving all lobes using VBM (p<0.01). Children with bilateral lesions had reduced thalamus and putamen volumes bilaterally (p<0.001). The thalamic volume was reduced bilaterally in children with unilateral lesions (p=0.004). Lesions to the PWM cause secondary changes to the deep grey matter structures similar to primary changes seen in CDGM lesions. Despite having a unilateral phenotype, grey matter changes are observed bilaterally, even in children with unilateral lesions. © 2014 Mac Keith Press.

  12. Quantitative analysis of the anti-noise performance of an m-sequence in an electromagnetic method

    Science.gov (United States)

    Yuan, Zhe; Zhang, Yiming; Zheng, Qijia

    2018-02-01

    An electromagnetic method with a transmitted waveform coded by an m-sequence achieved better anti-noise performance compared to the conventional manner with a square-wave. The anti-noise performance of the m-sequence varied with multiple coding parameters; hence, a quantitative analysis of the anti-noise performance for m-sequences with different coding parameters was required to optimize them. This paper proposes the concept of an identification system, with the identified Earth impulse response obtained by measuring the system output with the input of the voltage response. A quantitative analysis of the anti-noise performance of the m-sequence was achieved by analyzing the amplitude-frequency response of the corresponding identification system. The effects of the coding parameters on the anti-noise performance are summarized by numerical simulation, and their optimization is further discussed in our conclusions; the validity of the conclusions is further verified by field experiment. The quantitative analysis method proposed in this paper provides a new insight into the anti-noise mechanism of the m-sequence, and could be used to evaluate the anti-noise performance of artificial sources in other time-domain exploration methods, such as the seismic method.

  13. Sequence of structures in fine-grained turbidites: Comparison of recent deep-sea and ancient flysch sediments

    Science.gov (United States)

    Stow, Dorrik A. V.; Shanmugam, Ganapathy

    1980-01-01

    A comparative study of the sequence of sedimentary structures in ancient and modern fine-grained turbidites is made in three contrasting areas. They are (1) Holocene and Pleistocene deep-sea muds of the Nova Scotian Slope and Rise, (2) Middle Ordovician Sevier Shale of the Valley and Ridge Province of the Southern Appalachians, and (3) Cambro-Ordovician Halifax Slate of the Meguma Group in Nova Scotia. A standard sequence of structures is proposed for fine-grained turbidites. The complete sequence has nine sub-divisions that are here termed T 0 to T 8. "The lower subdivision (T 0) comprises a silt lamina which has a sharp, scoured and load-cast base, internal parallel-lamination and cross-lamination, and a sharp current-lineated or wavy surface with 'fading-ripples' (= Type C etc. …)." (= Type C ripple-drift cross-lamination, Jopling and Walker, 1968). The overlying sequence shows textural and compositional grading through alternating silt and mud laminae. A convolute-laminated sub-division (T 1) is overlain by low-amplitude climbing ripples (T 2), thin regular laminae (T 3), thin indistinct laminae (T 4), and thin wipsy or convolute laminae (T 5). The topmost three divisions, graded mud (T 6), ungraded mud (T 7) and bioturbated mud (T 8), do not have silt laminae but rare patchy silt lenses and silt pseudonodules and a thin zone of micro-burrowing near the upper surface. The proposed sequence is analogous to the Bouma (1962) structural scheme for sandy turbidites and is approximately equivalent to Bouma's (C)DE divisions. The repetition of partial sequences characterizes different parts of the slope/base-of-slope/basin plain environment, and represents deposition from different stages of evolution of a large, muddy, turbidity flow. Microstructural detail and sequence are well preserved in ancient and even slightly metamorphosed sediments. Their recognition is important for determining depositional processes and for palaeoenvironmental interpretation.

  14. A quantitative assessment of the Hadoop framework for analyzing massively parallel DNA sequencing data.

    Science.gov (United States)

    Siretskiy, Alexey; Sundqvist, Tore; Voznesenskiy, Mikhail; Spjuth, Ola

    2015-01-01

    New high-throughput technologies, such as massively parallel sequencing, have transformed the life sciences into a data-intensive field. The most common e-infrastructure for analyzing this data consists of batch systems that are based on high-performance computing resources; however, the bioinformatics software that is built on this platform does not scale well in the general case. Recently, the Hadoop platform has emerged as an interesting option to address the challenges of increasingly large datasets with distributed storage, distributed processing, built-in data locality, fault tolerance, and an appealing programming methodology. In this work we introduce metrics and report on a quantitative comparison between Hadoop and a single node of conventional high-performance computing resources for the tasks of short read mapping and variant calling. We calculate efficiency as a function of data size and observe that the Hadoop platform is more efficient for biologically relevant data sizes in terms of computing hours for both split and un-split data files. We also quantify the advantages of the data locality provided by Hadoop for NGS problems, and show that a classical architecture with network-attached storage will not scale when computing resources increase in numbers. Measurements were performed using ten datasets of different sizes, up to 100 gigabases, using the pipeline implemented in Crossbow. To make a fair comparison, we implemented an improved preprocessor for Hadoop with better performance for splittable data files. For improved usability, we implemented a graphical user interface for Crossbow in a private cloud environment using the CloudGene platform. All of the code and data in this study are freely available as open source in public repositories. From our experiments we can conclude that the improved Hadoop pipeline scales better than the same pipeline on high-performance computing resources, we also conclude that Hadoop is an economically viable

  15. Benchmarking of the Oxford Nanopore MinION sequencing for quantitative and qualitative assessment of cDNA populations.

    Science.gov (United States)

    Oikonomopoulos, Spyros; Wang, Yu Chang; Djambazian, Haig; Badescu, Dunarel; Ragoussis, Jiannis

    2016-08-24

    To assess the performance of the Oxford Nanopore Technologies MinION sequencing platform, cDNAs from the External RNA Controls Consortium (ERCC) RNA Spike-In mix were sequenced. This mix mimics mammalian mRNA species and consists of 92 polyadenylated transcripts with known concentration. cDNA libraries were generated using a template switching protocol to facilitate the direct comparison between different sequencing platforms. The MinION performance was assessed for its ability to sequence the cDNAs directly with good accuracy in terms of abundance and full length. The abundance of the ERCC cDNA molecules sequenced by MinION agreed with their expected concentration. No length or GC content bias was observed. The majority of cDNAs were sequenced as full length. Additionally, a complex cDNA population derived from a human HEK-293 cell line was sequenced on an Illumina HiSeq 2500, PacBio RS II and ONT MinION platforms. We observed that there was a good agreement in the measured cDNA abundance between PacBio RS II and ONT MinION (rpearson = 0.82, isoforms with length more than 700bp) and between Illumina HiSeq 2500 and ONT MinION (rpearson = 0.75). This indicates that the ONT MinION can sequence quantitatively both long and short full length cDNA molecules.

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

    Science.gov (United States)

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

    2017-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Wycliff M. Kinoti

    2017-06-01

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

  18. Characterization and Development of EST-SSRs by Deep Transcriptome Sequencing in Chinese Cabbage (Brassica rapa L. ssp. pekinensis

    Directory of Open Access Journals (Sweden)

    Qian Ding

    2015-01-01

    Full Text Available Simple sequence repeats (SSRs are among the most important markers for population analysis and have been widely used in plant genetic mapping and molecular breeding. Expressed sequence tag-SSR (EST-SSR markers, located in the coding regions, are potentially more efficient for QTL mapping, gene targeting, and marker-assisted breeding. In this study, we investigated 51,694 nonredundant unigenes, assembled from clean reads from deep transcriptome sequencing with a Solexa/Illumina platform, for identification and development of EST-SSRs in Chinese cabbage. In total, 10,420 EST-SSRs with over 12 bp were identified and characterized, among which 2744 EST-SSRs are new and 2317 are known ones showing polymorphism with previously reported SSRs. A total of 7877 PCR primer pairs for 1561 EST-SSR loci were designed, and primer pairs for twenty-four EST-SSRs were selected for primer evaluation. In nineteen EST-SSR loci (79.2%, amplicons were successfully generated with high quality. Seventeen (89.5% showed polymorphism in twenty-four cultivars of Chinese cabbage. The polymorphic alleles of each polymorphic locus were sequenced, and the results showed that most polymorphisms were due to variations of SSR repeat motifs. The EST-SSRs identified and characterized in this study have important implications for developing new tools for genetics and molecular breeding in Chinese cabbage.

  19. Geochemical features and effects on deep-seated fluids during the May-June 2012 southern Po Valley seismic sequence

    Directory of Open Access Journals (Sweden)

    Francesco Italiano

    2012-10-01

    Full Text Available A periodic sampling of the groundwaters and dissolved and free gases in selected deep wells located in the area affected by the May-June 2012 southern Po Valley seismic sequence has provided insight into seismogenic-induced changes of the local aquifer systems. The results obtained show progressive changes in the fluid geochemistry, allowing it to be established that deep-seated fluids were mobilized during the seismic sequence and reached surface layers along faults and fractures, which generated significant geochemical anomalies. The May-June 2012 seismic swarm (mainshock on May 29, 2012, M 5.8; 7 shocks M >5, about 200 events 3 > M > 5 induced several modifications in the circulating fluids. This study reports the preliminary results obtained for the geochemical features of the waters and gases collected over the epicentral area from boreholes drilled at different depths, thus intercepting water and gases with different origins and circulation. The aim of the investigations was to improve our knowledge of the fluids circulating over the seismic area (e.g. origin, provenance, interactions, mixing of different components, temporal changes. This was achieved by collecting samples from both shallow and deep-drilled boreholes, and then, after the selection of the relevant sites, we looked for temporal changes with mid-to-long-term monitoring activity following a constant sampling rate. This allowed us to gain better insight into the relationships between the fluid circulation and the faulting activity. The sampling sites are listed in Table 1, along with the analytical results of the gas phase. […

  20. Discovery and profiling of novel and conserved microRNAs during flower development in Carya cathayensis via deep sequencing.

    Science.gov (United States)

    Wang, Zheng Jia; Huang, Jian Qin; Huang, You Jun; Li, Zheng; Zheng, Bing Song

    2012-08-01

    Hickory (Carya cathayensis Sarg.) is an economically important woody plant in China, but its long juvenile phase delays yield. MicroRNAs (miRNAs) are critical regulators of genes and important for normal plant development and physiology, including flower development. We used Solexa technology to sequence two small RNA libraries from two floral differentiation stages in hickory to identify miRNAs related to flower development. We identified 39 conserved miRNA sequences from 114 loci belonging to 23 families as well as two novel and ten potential novel miRNAs belonging to nine families. Moreover, 35 conserved miRNA*s and two novel miRNA*s were detected. Twenty miRNA sequences from 49 loci belonging to 11 families were differentially expressed; all were up-regulated at the later stage of flower development in hickory. Quantitative real-time PCR of 12 conserved miRNA sequences, five novel miRNA families, and two novel miRNA*s validated that all were expressed during hickory flower development, and the expression patterns were similar to those detected with Solexa sequencing. Finally, a total of 146 targets of the novel and conserved miRNAs were predicted. This study identified a diverse set of miRNAs that were closely related to hickory flower development and that could help in plant floral induction.

  1. Mitochondrial genome sequences reveal deep divergences among Anopheles punctulatus sibling species in Papua New Guinea

    Directory of Open Access Journals (Sweden)

    Logue Kyle

    2013-02-01

    Full Text Available Abstract Background Members of the Anopheles punctulatus group (AP group are the primary vectors of human malaria in Papua New Guinea. The AP group includes 13 sibling species, most of them morphologically indistinguishable. Understanding why only certain species are able to transmit malaria requires a better comprehension of their evolutionary history. In particular, understanding relationships and divergence times among Anopheles species may enable assessing how malaria-related traits (e.g. blood feeding behaviours, vector competence have evolved. Methods DNA sequences of 14 mitochondrial (mt genomes from five AP sibling species and two species of the Anopheles dirus complex of Southeast Asia were sequenced. DNA sequences from all concatenated protein coding genes (10,770 bp were then analysed using a Bayesian approach to reconstruct phylogenetic relationships and date the divergence of the AP sibling species. Results Phylogenetic reconstruction using the concatenated DNA sequence of all mitochondrial protein coding genes indicates that the ancestors of the AP group arrived in Papua New Guinea 25 to 54 million years ago and rapidly diverged to form the current sibling species. Conclusion Through evaluation of newly described mt genome sequences, this study has revealed a divergence among members of the AP group in Papua New Guinea that would significantly predate the arrival of humans in this region, 50 thousand years ago. The divergence observed among the mtDNA sequences studied here may have resulted from reproductive isolation during historical changes in sea-level through glacial minima and maxima. This leads to a hypothesis that the AP sibling species have evolved independently for potentially thousands of generations. This suggests that the evolution of many phenotypes, such as insecticide resistance will arise independently in each of the AP sibling species studied here.

  2. Deep Proteomics of Mouse Skeletal Muscle Enables Quantitation of Protein Isoforms, Metabolic Pathways, and Transcription Factors*

    Science.gov (United States)

    Deshmukh, Atul S.; Murgia, Marta; Nagaraj, Nagarjuna; Treebak, Jonas T.; Cox, Jürgen; Mann, Matthias

    2015-01-01

    Skeletal muscle constitutes 40% of individual body mass and plays vital roles in locomotion and whole-body metabolism. Proteomics of skeletal muscle is challenging because of highly abundant contractile proteins that interfere with detection of regulatory proteins. Using a state-of-the art MS workflow and a strategy to map identifications from the C2C12 cell line model to tissues, we identified a total of 10,218 proteins, including skeletal muscle specific transcription factors like myod1 and myogenin and circadian clock proteins. We obtain absolute abundances for proteins expressed in a muscle cell line and skeletal muscle, which should serve as a valuable resource. Quantitation of protein isoforms of glucose uptake signaling pathways and in glucose and lipid metabolic pathways provides a detailed metabolic map of the cell line compared with tissue. This revealed unexpectedly complex regulation of AMP-activated protein kinase and insulin signaling in muscle tissue at the level of enzyme isoforms. PMID:25616865

  3. Improved detection of CXCR4-using HIV by V3 genotyping: application of population-based and "deep" sequencing to plasma RNA and proviral DNA.

    Science.gov (United States)

    Swenson, Luke C; Moores, Andrew; Low, Andrew J; Thielen, Alexander; Dong, Winnie; Woods, Conan; Jensen, Mark A; Wynhoven, Brian; Chan, Dennison; Glascock, Christopher; Harrigan, P Richard

    2010-08-01

    Tropism testing should rule out CXCR4-using HIV before treatment with CCR5 antagonists. Currently, the recombinant phenotypic Trofile assay (Monogram) is most widely utilized; however, genotypic tests may represent alternative methods. Independent triplicate amplifications of the HIV gp120 V3 region were made from either plasma HIV RNA or proviral DNA. These underwent standard, population-based sequencing with an ABI3730 (RNA n = 63; DNA n = 40), or "deep" sequencing with a Roche/454 Genome Sequencer-FLX (RNA n = 12; DNA n = 12). Position-specific scoring matrices (PSSMX4/R5) (-6.96 cutoff) and geno2pheno[coreceptor] (5% false-positive rate) inferred tropism from V3 sequence. These methods were then independently validated with a separate, blinded dataset (n = 278) of screening samples from the maraviroc MOTIVATE trials. Standard sequencing of HIV RNA with PSSM yielded 69% sensitivity and 91% specificity, relative to Trofile. The validation dataset gave 75% sensitivity and 83% specificity. Proviral DNA plus PSSM gave 77% sensitivity and 71% specificity. "Deep" sequencing of HIV RNA detected >2% inferred-CXCR4-using virus in 8/8 samples called non-R5 by Trofile, and <2% in 4/4 samples called R5. Triplicate analyses of V3 standard sequence data detect greater proportions of CXCR4-using samples than previously achieved. Sequencing proviral DNA and "deep" V3 sequencing may also be useful tools for assessing tropism.

  4. Ultra-deep sequencing reveals the subclonal structure and genomic evolution of oral squamous cell carcinoma

    DEFF Research Database (Denmark)

    Tabatabaeifar, Siavosh; Thomassen, Mads; Larsen, Martin Jakob

    Background: Oral squamous cell carcinoma (OSCC), a subgroup of head and neck squamous cell carcinoma (HNSCC), is primarily caused by alcohol consumption and tobacco use. Recent DNA sequencing studies suggests that HNSCC are very heterogeneous between patients; however the intra-patient subclonal...

  5. MicroRNA identity and abundance in porcine skeletal muscles determined by deep sequencing

    DEFF Research Database (Denmark)

    Nielsen, M; Hansen, J H; Hedegaard, J

    2010-01-01

    levels of 212 annotated miRNA genes, thereby providing a thorough account of the miRNA transcriptome in porcine muscle tissue. The expression levels displayed a very large range, as reflected by the number of sequence reads, which varied from single counts for rare miRNAs to several million reads...

  6. High diversity of picornaviruses in rats from different continents revealed by deep sequencing

    DEFF Research Database (Denmark)

    Arn Hansen, Thomas; Mollerup, Sarah; Nguyen, Nam-Phuong

    2016-01-01

    Outbreaks of zoonotic diseases in humans and livestock are not uncommon, and an important component in containment of such emerging viral diseases is rapid and reliable diagnostics. Such methods are often PCR-based and hence require the availability of sequence data from the pathogen. Rattus norv...

  7. High diversity of picornaviruses in rats from different continents revealed by deep sequencing

    DEFF Research Database (Denmark)

    Arn Hansen, Thomas; Mollerup, Sarah; Nguyen, Nam-Phuong

    2016-01-01

    Outbreaks of zoonotic diseases in humans and livestock are not uncommon, and an important component in containment of such emerging viral diseases is rapid and reliable diagnostics. Such methods are often PCR-based and hence require the availability of sequence data from the pathogen. Rattus...

  8. Deep-sequencing revealed Citrus bark cracking viroid (CBCVd) as a highly aggressive pathogen on hop

    Czech Academy of Sciences Publication Activity Database

    Jakše, J.; Radišek, S.; Pokorn, T.; Matoušek, Jaroslav; Javornik, B.

    2015-01-01

    Roč. 64, č. 4 (2015), s. 831-842 ISSN 0032-0862 R&D Projects: GA MŠk(CZ) LH14255 Institutional support: RVO:60077344 Keywords : Bioinformatic * Citrus bark cracking viroid * Hop * Next-generation sequencing Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.383, year: 2015

  9. Using small RNA (sRNA) deep sequencing to understand global virus distribution in plants

    Science.gov (United States)

    Small RNAs (sRNAs), a class of regulatory RNAs, have been used to serve as the specificity determinants of suppressing gene expression in plants and animals. Next generation sequencing (NGS) uncovered the sRNA landscape in most organisms including their associated microbes. In the current study, w...

  10. Deep sequencing of uveal melanoma identifies a recurrent mutation in PLCB4

    DEFF Research Database (Denmark)

    Johansson, Peter; Aoude, Lauren G; Wadt, Karin

    2016-01-01

    Next generation sequencing of uveal melanoma (UM) samples has identified a number of recurrent oncogenic or loss-of-function mutations in key driver genes including: GNAQ, GNA11, EIF1AX, SF3B1 and BAP1. To search for additional driver mutations in this tumor type we carried out whole......, instead, a BRCA mutation signature predominated. In addition to mutations in the known UM driver genes, we found a recurrent mutation in PLCB4 (c.G1888T, p.D630Y, NM_000933), which was validated using Sanger sequencing. The identical mutation was also found in published UM sequence data (1 of 56 tumors......-genome or whole-exome sequencing of 28 tumors or primary cell lines. These samples have a low mutation burden, with a mean of 10.6 protein changing mutations per sample (range 0 to 53). As expected for these sun-shielded melanomas the mutation spectrum was not consistent with an ultraviolet radiation signature...

  11. Deep proteomics of mouse skeletal muscle enables quantitation of protein isoforms, metabolic pathways, and transcription factors.

    Science.gov (United States)

    Deshmukh, Atul S; Murgia, Marta; Nagaraj, Nagarjuna; Treebak, Jonas T; Cox, Jürgen; Mann, Matthias

    2015-04-01

    Skeletal muscle constitutes 40% of individual body mass and plays vital roles in locomotion and whole-body metabolism. Proteomics of skeletal muscle is challenging because of highly abundant contractile proteins that interfere with detection of regulatory proteins. Using a state-of-the art MS workflow and a strategy to map identifications from the C2C12 cell line model to tissues, we identified a total of 10,218 proteins, including skeletal muscle specific transcription factors like myod1 and myogenin and circadian clock proteins. We obtain absolute abundances for proteins expressed in a muscle cell line and skeletal muscle, which should serve as a valuable resource. Quantitation of protein isoforms of glucose uptake signaling pathways and in glucose and lipid metabolic pathways provides a detailed metabolic map of the cell line compared with tissue. This revealed unexpectedly complex regulation of AMP-activated protein kinase and insulin signaling in muscle tissue at the level of enzyme isoforms. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. Quantitative, multiplexed workflow for deep analysis of human blood plasma and biomarker discovery by mass spectrometry.

    Science.gov (United States)

    Keshishian, Hasmik; Burgess, Michael W; Specht, Harrison; Wallace, Luke; Clauser, Karl R; Gillette, Michael A; Carr, Steven A

    2017-08-01

    Proteomic characterization of blood plasma is of central importance to clinical proteomics and particularly to biomarker discovery studies. The vast dynamic range and high complexity of the plasma proteome have, however, proven to be serious challenges and have often led to unacceptable tradeoffs between depth of coverage and sample throughput. We present an optimized sample-processing pipeline for analysis of the human plasma proteome that provides greatly increased depth of detection, improved quantitative precision and much higher sample analysis throughput as compared with prior methods. The process includes abundant protein depletion, isobaric labeling at the peptide level for multiplexed relative quantification and ultra-high-performance liquid chromatography coupled to accurate-mass, high-resolution tandem mass spectrometry analysis of peptides fractionated off-line by basic pH reversed-phase (bRP) chromatography. The overall reproducibility of the process, including immunoaffinity depletion, is high, with a process replicate coefficient of variation (CV) of 4,500 proteins are detected and quantified per patient sample on average, with two or more peptides per protein and starting from as little as 200 μl of plasma. The approach can be multiplexed up to 10-plex using tandem mass tags (TMT) reagents, further increasing throughput, albeit with some decrease in the number of proteins quantified. In addition, we provide a rapid protocol for analysis of nonfractionated depleted plasma samples analyzed in 10-plex. This provides ∼600 quantified proteins for each of the ten samples in ∼5 h of instrument time.

  13. Surface studies of water isotopes in Antarctica for quantitative interpretation of deep ice core data

    Science.gov (United States)

    Landais, Amaelle; Casado, Mathieu; Prié, Frédéric; Magand, Olivier; Arnaud, Laurent; Ekaykin, Alexey; Petit, Jean-Robert; Picard, Ghislain; Fily, Michel; Minster, Bénédicte; Touzeau, Alexandra; Goursaud, Sentia; Masson-Delmotte, Valérie; Jouzel, Jean; Orsi, Anaïs

    2017-07-01

    Polar ice cores are unique climate archives. Indeed, most of them have a continuous stratigraphy and present high temporal resolution of many climate variables in a single archive. While water isotopic records (δD or δ18O) in ice cores are often taken as references for past atmospheric temperature variations, their relationship to temperature is associated with a large uncertainty. Several reasons are invoked to explain the limitation of such an approach; in particular, post-deposition effects are important in East Antarctica because of the low accumulation rates. The strong influence of post-deposition processes highlights the need for surface polar research programs in addition to deep drilling programs. We present here new results on water isotopes from several recent surface programs, mostly over East Antarctica. Together with previously published data, the new data presented in this study have several implications for the climatic reconstructions based on ice core isotopic data: (1) The spatial relationship between surface mean temperature and mean snow isotopic composition over the first meters in depth can be explained quite straightforwardly using simple isotopic models tuned to d-excess vs. δ18O evolution in transects on the East Antarctic sector. The observed spatial slopes are significantly higher (∼ 0.7-0.8‰·°C-1 for δ18O vs. temperature) than seasonal slopes inferred from precipitation data at Vostok and Dome C (0.35 to 0.46‰·°C-1). We explain these differences by changes in condensation versus surface temperature between summer and winter in the central East Antarctic plateau, where the inversion layer vanishes in summer. (2) Post-deposition effects linked to exchanges between the snow surface and the atmospheric water vapor lead to an evolution of δ18O in the surface snow, even in the absence of any precipitation event. This evolution preserves the positive correlation between the δ18O of snow and surface temperature, but is

  14. A Retrospective Examination of Feline Leukemia Subgroup Characterization: Viral Interference Assays to Deep Sequencing

    Directory of Open Access Journals (Sweden)

    Elliott S. Chiu

    2018-01-01

    Full Text Available Feline leukemia virus (FeLV was the first feline retrovirus discovered, and is associated with multiple fatal disease syndromes in cats, including lymphoma. The original research conducted on FeLV employed classical virological techniques. As methods have evolved to allow FeLV genetic characterization, investigators have continued to unravel the molecular pathology associated with this fascinating agent. In this review, we discuss how FeLV classification, transmission, and disease-inducing potential have been defined sequentially by viral interference assays, Sanger sequencing, PCR, and next-generation sequencing. In particular, we highlight the influences of endogenous FeLV and host genetics that represent FeLV research opportunities on the near horizon.

  15. A Retrospective Examination of Feline Leukemia Subgroup Characterization: Viral Interference Assays to Deep Sequencing.

    Science.gov (United States)

    Chiu, Elliott S; Hoover, Edward A; VandeWoude, Sue

    2018-01-10

    Feline leukemia virus (FeLV) was the first feline retrovirus discovered, and is associated with multiple fatal disease syndromes in cats, including lymphoma. The original research conducted on FeLV employed classical virological techniques. As methods have evolved to allow FeLV genetic characterization, investigators have continued to unravel the molecular pathology associated with this fascinating agent. In this review, we discuss how FeLV classification, transmission, and disease-inducing potential have been defined sequentially by viral interference assays, Sanger sequencing, PCR, and next-generation sequencing. In particular, we highlight the influences of endogenous FeLV and host genetics that represent FeLV research opportunities on the near horizon.

  16. Deep sequencing of the oral microbiome reveals signatures of periodontal disease.

    Directory of Open Access Journals (Sweden)

    Bo Liu

    Full Text Available The oral microbiome, the complex ecosystem of microbes inhabiting the human mouth, harbors several thousands of bacterial types. The proliferation of pathogenic bacteria within the mouth gives rise to periodontitis, an inflammatory disease known to also constitute a risk factor for cardiovascular disease. While much is known about individual species associated with pathogenesis, the system-level mechanisms underlying the transition from health to disease are still poorly understood. Through the sequencing of the 16S rRNA gene and of whole community DNA we provide a glimpse at the global genetic, metabolic, and ecological changes associated with periodontitis in 15 subgingival plaque samples, four from each of two periodontitis patients, and the remaining samples from three healthy individuals. We also demonstrate the power of whole-metagenome sequencing approaches in characterizing the genomes of key players in the oral microbiome, including an unculturable TM7 organism. We reveal the disease microbiome to be enriched in virulence factors, and adapted to a parasitic lifestyle that takes advantage of the disrupted host homeostasis. Furthermore, diseased samples share a common structure that was not found in completely healthy samples, suggesting that the disease state may occupy a narrow region within the space of possible configurations of the oral microbiome. Our pilot study demonstrates the power of high-throughput sequencing as a tool for understanding the role of the oral microbiome in periodontal disease. Despite a modest level of sequencing (~2 lanes Illumina 76 bp PE and high human DNA contamination (up to ~90% we were able to partially reconstruct several oral microbes and to preliminarily characterize some systems-level differences between the healthy and diseased oral microbiomes.

  17. High diversity of picornaviruses in rats from different continents revealed by deep sequencing.

    Science.gov (United States)

    Hansen, Thomas Arn; Mollerup, Sarah; Nguyen, Nam-Phuong; White, Nicole E; Coghlan, Megan; Alquezar-Planas, David E; Joshi, Tejal; Jensen, Randi Holm; Fridholm, Helena; Kjartansdóttir, Kristín Rós; Mourier, Tobias; Warnow, Tandy; Belsham, Graham J; Bunce, Michael; Willerslev, Eske; Nielsen, Lars Peter; Vinner, Lasse; Hansen, Anders Johannes

    2016-08-17

    Outbreaks of zoonotic diseases in humans and livestock are not uncommon, and an important component in containment of such emerging viral diseases is rapid and reliable diagnostics. Such methods are often PCR-based and hence require the availability of sequence data from the pathogen. Rattus norvegicus (R. norvegicus) is a known reservoir for important zoonotic pathogens. Transmission may be direct via contact with the animal, for example, through exposure to its faecal matter, or indirectly mediated by arthropod vectors. Here we investigated the viral content in rat faecal matter (n=29) collected from two continents by analyzing 2.2 billion next-generation sequencing reads derived from both DNA and RNA. Among other virus families, we found sequences from members of the Picornaviridae to be abundant in the microbiome of all the samples. Here we describe the diversity of the picornavirus-like contigs including near-full-length genomes closely related to the Boone cardiovirus and Theiler's encephalomyelitis virus. From this study, we conclude that picornaviruses within R. norvegicus are more diverse than previously recognized. The virome of R. norvegicus should be investigated further to assess the full potential for zoonotic virus transmission.

  18. Deep transcriptome sequencing provides new insights into the structural and functional organization of the wheat genome.

    Science.gov (United States)

    Pingault, Lise; Choulet, Frédéric; Alberti, Adriana; Glover, Natasha; Wincker, Patrick; Feuillet, Catherine; Paux, Etienne

    2015-02-10

    Because of its size, allohexaploid nature, and high repeat content, the bread wheat genome is a good model to study the impact of the genome structure on gene organization, function, and regulation. However, because of the lack of a reference genome sequence, such studies have long been hampered and our knowledge of the wheat gene space is still limited. The access to the reference sequence of the wheat chromosome 3B provided us with an opportunity to study the wheat transcriptome and its relationships to genome and gene structure at a level that has never been reached before. By combining this sequence with RNA-seq data, we construct a fine transcriptome map of the chromosome 3B. More than 8,800 transcription sites are identified, that are distributed throughout the entire chromosome. Expression level, expression breadth, alternative splicing as well as several structural features of genes, including transcript length, number of exons, and cumulative intron length are investigated. Our analysis reveals a non-monotonic relationship between gene expression and structure and leads to the hypothesis that gene structure is determined by its function, whereas gene expression is subject to energetic cost. Moreover, we observe a recombination-based partitioning at the gene structure and function level. Our analysis provides new insights into the relationships between gene and genome structure and function. It reveals mechanisms conserved with other plant species as well as superimposed evolutionary forces that shaped the wheat gene space, likely participating in wheat adaptation.

  19. Single nucleotide polymorphism discovery in rainbow trout by deep sequencing of a reduced representation library

    Directory of Open Access Journals (Sweden)

    Salem Mohamed

    2009-11-01

    Full Text Available Abstract Background To enhance capabilities for genomic analyses in rainbow trout, such as genomic selection, a large suite of polymorphic markers that are amenable to high-throughput genotyping protocols must be identified. Expressed Sequence Tags (ESTs have been used for single nucleotide polymorphism (SNP discovery in salmonids. In those strategies, the salmonid semi-tetraploid genomes often led to assemblies of paralogous sequences and therefore resulted in a high rate of false positive SNP identification. Sequencing genomic DNA using primers identified from ESTs proved to be an effective but time consuming methodology of SNP identification in rainbow trout, therefore not suitable for high throughput SNP discovery. In this study, we employed a high-throughput strategy that used pyrosequencing technology to generate data from a reduced representation library constructed with genomic DNA pooled from 96 unrelated rainbow trout that represent the National Center for Cool and Cold Water Aquaculture (NCCCWA broodstock population. Results The reduced representation library consisted of 440 bp fragments resulting from complete digestion with the restriction enzyme HaeIII; sequencing produced 2,000,000 reads providing an average 6 fold coverage of the estimated 150,000 unique genomic restriction fragments (300,000 fragment ends. Three independent data analyses identified 22,022 to 47,128 putative SNPs on 13,140 to 24,627 independent contigs. A set of 384 putative SNPs, randomly selected from the sets produced by the three analyses were genotyped on individual fish to determine the validation rate of putative SNPs among analyses, distinguish apparent SNPs that actually represent paralogous loci in the tetraploid genome, examine Mendelian segregation, and place the validated SNPs on the rainbow trout linkage map. Approximately 48% (183 of the putative SNPs were validated; 167 markers were successfully incorporated into the rainbow trout linkage map. In

  20. Single nucleotide polymorphism discovery in rainbow trout by deep sequencing of a reduced representation library.

    Science.gov (United States)

    Sánchez, Cecilia Castaño; Smith, Timothy P L; Wiedmann, Ralph T; Vallejo, Roger L; Salem, Mohamed; Yao, Jianbo; Rexroad, Caird E

    2009-11-25

    To enhance capabilities for genomic analyses in rainbow trout, such as genomic selection, a large suite of polymorphic markers that are amenable to high-throughput genotyping protocols must be identified. Expressed Sequence Tags (ESTs) have been used for single nucleotide polymorphism (SNP) discovery in salmonids. In those strategies, the salmonid semi-tetraploid genomes often led to assemblies of paralogous sequences and therefore resulted in a high rate of false positive SNP identification. Sequencing genomic DNA using primers identified from ESTs proved to be an effective but time consuming methodology of SNP identification in rainbow trout, therefore not suitable for high throughput SNP discovery. In this study, we employed a high-throughput strategy that used pyrosequencing technology to generate data from a reduced representation library constructed with genomic DNA pooled from 96 unrelated rainbow trout that represent the National Center for Cool and Cold Water Aquaculture (NCCCWA) broodstock population. The reduced representation library consisted of 440 bp fragments resulting from complete digestion with the restriction enzyme HaeIII; sequencing produced 2,000,000 reads providing an average 6 fold coverage of the estimated 150,000 unique genomic restriction fragments (300,000 fragment ends). Three independent data analyses identified 22,022 to 47,128 putative SNPs on 13,140 to 24,627 independent contigs. A set of 384 putative SNPs, randomly selected from the sets produced by the three analyses were genotyped on individual fish to determine the validation rate of putative SNPs among analyses, distinguish apparent SNPs that actually represent paralogous loci in the tetraploid genome, examine Mendelian segregation, and place the validated SNPs on the rainbow trout linkage map. Approximately 48% (183) of the putative SNPs were validated; 167 markers were successfully incorporated into the rainbow trout linkage map. In addition, 2% of the sequences from the

  1. Genetic control of environmental variation of two quantitative traits of Drosophila melanogaster revealed by whole-genome sequencing

    DEFF Research Database (Denmark)

    Sørensen, Peter; de los Campos, Gustavo; Morgante, Fabio

    2015-01-01

    and others more volatile performance. Understanding the mechanisms responsible for environmental variability not only informs medical questions but is relevant in evolution and in agricultural science. In this work fully sequenced inbred lines of Drosophila melanogaster were analyzed to study the nature...... of genetic control of environmental variance for two quantitative traits: starvation resistance (SR) and startle response (SL). The evidence for genetic control of environmental variance is compelling for both traits. Sequence information is incorporated in random regression models to study the underlying...... genetic signals, which are shown to be different in the two traits. Genomic variance in sexual dimorphism was found for SR but not for SL. Indeed, the proportion of variance captured by sequence information and the contribution to this variance from four chromosome segments differ between sexes in SR...

  2. Metagenomes obtained by "deep sequencing" - what do they tell about the EBPR communities

    DEFF Research Database (Denmark)

    Albertsen, Mads; Saunders, Aaron Marc; Nielsen, Kåre Lehmann

    on phylogenetic and functional level (Fig. 1). Even though the samples were taken at different times of the year (August vs. December) and from different EBPR plants, they cluster tightly, which may be attributed to the wide range of selection pressures acting on the EBPR communities. These results confirm...... the findings of a core microbial community using quantitative fluorescence in situ hybridization (qFISH) and other techniques (Nielsen et al., 2010; 2011). Through the use of qFISH probes we investigated the micro-diversity of the key PAO Accumulibacter (clade I and II) in the two EBPR plants. In Aalborg East...

  3. Identification of microRNAs Involved in the Host Response to Enterovirus 71 Infection by a Deep Sequencing Approach

    Directory of Open Access Journals (Sweden)

    Lunbiao Cui

    2010-01-01

    Full Text Available Role of microRNA (miRNA has been highlighted in pathogen-host interactions recently. To identify cellular miRNAs involved in the host response to enterovirus 71 (EV71 infection, we performed a comprehensive miRNA profiling in EV71-infected Hep2 cells through deep sequencing. 64 miRNAs were found whose expression levels changed for more than 2-fold in response to EV71 infection. Gene ontology analysis revealed that many of these mRNAs play roles in neurological process, immune response, and cell death pathways, which are known to be associated with the extreme virulence of EV71. To our knowledge, this is the first study on host miRNAs expression alteration response to EV71 infection. Our findings supported the hypothesis that certain miRNAs might be essential in the host-pathogen interactions.

  4. Laser Capture and Deep Sequencing Reveals the Transcriptomic Programmes Regulating the Onset of Pancreas and Liver Differentiation in Human Embryos

    Directory of Open Access Journals (Sweden)

    Rachel E. Jennings

    2017-11-01

    Full Text Available To interrogate the alternative fates of pancreas and liver in the earliest stages of human organogenesis, we developed laser capture, RNA amplification, and computational analysis of deep sequencing. Pancreas-enriched gene expression was less conserved between human and mouse than for liver. The dorsal pancreatic bud was enriched for components of Notch, Wnt, BMP, and FGF signaling, almost all genes known to cause pancreatic agenesis or hypoplasia, and over 30 unexplored transcription factors. SOX9 and RORA were imputed as key regulators in pancreas compared with EP300, HNF4A, and FOXA family members in liver. Analyses implied that current in vitro human stem cell differentiation follows a dorsal rather than a ventral pancreatic program and pointed to additional factors for hepatic differentiation. In summary, we provide the transcriptional codes regulating the start of human liver and pancreas development to facilitate stem cell research and clinical interpretation without inter-species extrapolation.

  5. Insights into the genetic structure and diversity of 38 South Asian Indians from deep whole-genome sequencing.

    Directory of Open Access Journals (Sweden)

    Lai-Ping Wong

    2014-05-01

    Full Text Available South Asia possesses a significant amount of genetic diversity due to considerable intergroup differences in culture and language. There have been numerous reports on the genetic structure of Asian Indians, although these have mostly relied on genotyping microarrays or targeted sequencing of the mitochondria and Y chromosomes. Asian Indians in Singapore are primarily descendants of immigrants from Dravidian-language-speaking states in south India, and 38 individuals from the general population underwent deep whole-genome sequencing with a target coverage of 30X as part of the Singapore Sequencing Indian Project (SSIP. The genetic structure and diversity of these samples were compared against samples from the Singapore Sequencing Malay Project and populations in Phase 1 of the 1,000 Genomes Project (1 KGP. SSIP samples exhibited greater intra-population genetic diversity and possessed higher heterozygous-to-homozygous genotype ratio than other Asian populations. When compared against a panel of well-defined Asian Indians, the genetic makeup of the SSIP samples was closely related to South Indians. However, even though the SSIP samples clustered distinctly from the Europeans in the global population structure analysis with autosomal SNPs, eight samples were assigned to mitochondrial haplogroups that were predominantly present in Europeans and possessed higher European admixture than the remaining samples. An analysis of the relative relatedness between SSIP with two archaic hominins (Denisovan, Neanderthal identified higher ancient admixture in East Asian populations than in SSIP. The data resource for these samples is publicly available and is expected to serve as a valuable complement to the South Asian samples in Phase 3 of 1 KGP.

  6. Insights into the genetic structure and diversity of 38 South Asian Indians from deep whole-genome sequencing.

    Science.gov (United States)

    Wong, Lai-Ping; Lai, Jason Kuan-Han; Saw, Woei-Yuh; Ong, Rick Twee-Hee; Cheng, Anthony Youzhi; Pillai, Nisha Esakimuthu; Liu, Xuanyao; Xu, Wenting; Chen, Peng; Foo, Jia-Nee; Tan, Linda Wei-Lin; Koo, Seok-Hwee; Soong, Richie; Wenk, Markus Rene; Lim, Wei-Yen; Khor, Chiea-Chuen; Little, Peter; Chia, Kee-Seng; Teo, Yik-Ying

    2014-05-01

    South Asia possesses a significant amount of genetic diversity due to considerable intergroup differences in culture and language. There have been numerous reports on the genetic structure of Asian Indians, although these have mostly relied on genotyping microarrays or targeted sequencing of the mitochondria and Y chromosomes. Asian Indians in Singapore are primarily descendants of immigrants from Dravidian-language-speaking states in south India, and 38 individuals from the general population underwent deep whole-genome sequencing with a target coverage of 30X as part of the Singapore Sequencing Indian Project (SSIP). The genetic structure and diversity of these samples were compared against samples from the Singapore Sequencing Malay Project and populations in Phase 1 of the 1,000 Genomes Project (1 KGP). SSIP samples exhibited greater intra-population genetic diversity and possessed higher heterozygous-to-homozygous genotype ratio than other Asian populations. When compared against a panel of well-defined Asian Indians, the genetic makeup of the SSIP samples was closely related to South Indians. However, even though the SSIP samples clustered distinctly from the Europeans in the global population structure analysis with autosomal SNPs, eight samples were assigned to mitochondrial haplogroups that were predominantly present in Europeans and possessed higher European admixture than the remaining samples. An analysis of the relative relatedness between SSIP with two archaic hominins (Denisovan, Neanderthal) identified higher ancient admixture in East Asian populations than in SSIP. The data resource for these samples is publicly available and is expected to serve as a valuable complement to the South Asian samples in Phase 3 of 1 KGP.

  7. Deep Sequencing of Plant and Animal DNA Contained within Traditional Chinese Medicines Reveals Legality Issues and Health Safety Concerns

    Science.gov (United States)

    Coghlan, Megan L.; Haile, James; Houston, Jayne; Murray, Dáithí C.; White, Nicole E.; Moolhuijzen, Paula; Bellgard, Matthew I.; Bunce, Michael

    2012-01-01

    Traditional Chinese medicine (TCM) has been practiced for thousands of years, but only within the last few decades has its use become more widespread outside of Asia. Concerns continue to be raised about the efficacy, legality, and safety of many popular complementary alternative medicines, including TCMs. Ingredients of some TCMs are known to include derivatives of endangered, trade-restricted species of plants and animals, and therefore contravene the Convention on International Trade in Endangered Species (CITES) legislation. Chromatographic studies have detected the presence of heavy metals and plant toxins within some TCMs, and there are numerous cases of adverse reactions. It is in the interests of both biodiversity conservation and public safety that techniques are developed to screen medicinals like TCMs. Targeting both the p-loop region of the plastid trnL gene and the mitochondrial 16S ribosomal RNA gene, over 49,000 amplicon sequence reads were generated from 15 TCM samples presented in the form of powders, tablets, capsules, bile flakes, and herbal teas. Here we show that second-generation, high-throughput sequencing (HTS) of DNA represents an effective means to genetically audit organic ingredients within complex TCMs. Comparison of DNA sequence data to reference databases revealed the presence of 68 different plant families and included genera, such as Ephedra and Asarum, that are potentially toxic. Similarly, animal families were identified that include genera that are classified as vulnerable, endangered, or critically endangered, including Asiatic black bear (Ursus thibetanus) and Saiga antelope (Saiga tatarica). Bovidae, Cervidae, and Bufonidae DNA were also detected in many of the TCM samples and were rarely declared on the product packaging. This study demonstrates that deep sequencing via HTS is an efficient and cost-effective way to audit highly processed TCM products and will assist in monitoring their legality and safety especially when

  8. Population-genomic variation within RNA viruses of the Western honey bee, Apis mellifera, inferred from deep sequencing.

    Science.gov (United States)

    Cornman, Robert Scott; Boncristiani, Humberto; Dainat, Benjamin; Chen, Yanping; vanEngelsdorp, Dennis; Weaver, Daniel; Evans, Jay D

    2013-03-07

    Deep sequencing of viruses isolated from infected hosts is an efficient way to measure population-genetic variation and can reveal patterns of dispersal and natural selection. In this study, we mined existing Illumina sequence reads to investigate single-nucleotide polymorphisms (SNPs) within two RNA viruses of the Western honey bee (Apis mellifera), deformed wing virus (DWV) and Israel acute paralysis virus (IAPV). All viral RNA was extracted from North American samples of honey bees or, in one case, the ectoparasitic mite Varroa destructor. Coverage depth was generally lower for IAPV than DWV, and marked gaps in coverage occurred in several narrow regions (selection. The Kakugo strain of DWV fell outside of all other DWV sequences at 100% bootstrap support. IAPV consensus sequences supported the existence of multiple clades as had been previously reported, and Fu and Li's D was closer to neutral expectation overall, although a sliding-window analysis identified a significantly positive D within the protease region, suggesting selection maintains diversity in that region. Within-sample mean diversity was comparable between the two viruses on average, although for both viruses there was substantial variation among samples in mean diversity at third codon positions and in the number of high-diversity sites. FST values were bimodal for DWV, likely reflecting neutral divergence in two low-diversity populations, whereas IAPV had several sites that were strong outliers with very low FST. This initial survey of genetic variation within honey bee RNA viruses suggests future directions for studies examining the underlying causes of population-genetic structure in these economically important pathogens.

  9. Deep sequencing-based analysis of the Cymbidium ensifolium floral transcriptome.

    Directory of Open Access Journals (Sweden)

    Xiaobai Li

    Full Text Available Cymbidium ensifolium is a Chinese Cymbidium with an elegant shape, beautiful appearance, and a fragrant aroma. C. ensifolium has a long history of cultivation in China and it has excellent commercial value as a potted plant and cut flower. The development of C. ensifolium genomic resources has been delayed because of its large genome size. Taking advantage of technical and cost improvement of RNA-Seq, we extracted total mRNA from flower buds and mature flowers and obtained a total of 9.52 Gb of filtered nucleotides comprising 98,819,349 filtered reads. The filtered reads were assembled into 101,423 isotigs, representing 51,696 genes. Of the 101,423 isotigs, 41,873 were putative homologs of annotated sequences in the public databases, of which 158 were associated with floral development and 119 were associated with flowering. The isotigs were categorized according to their putative functions. In total, 10,212 of the isotigs were assigned into 25 eukaryotic orthologous groups (KOGs, 41,690 into 58 gene ontology (GO terms, and 9,830 into 126 Arabidopsis Kyoto Encyclopedia of Genes and Genomes (KEGG pathways, and 9,539 isotigs into 123 rice pathways. Comparison of the isotigs with those of the two related orchid species P. equestris and C. sinense showed that 17,906 isotigs are unique to C. ensifolium. In addition, a total of 7,936 SSRs and 16,676 putative SNPs were identified. To our knowledge, this transcriptome database is the first major genomic resource for C. ensifolium and the most comprehensive transcriptomic resource for genus Cymbidium. These sequences provide valuable information for understanding the molecular mechanisms of floral development and flowering. Sequences predicted to be unique to C. ensifolium would provide more insights into C. ensifolium gene diversity. The numerous SNPs and SSRs identified in the present study will contribute to marker development for C. ensifolium.

  10. Congruent Deep Relationships in the Grape Family (Vitaceae) Based on Sequences of Chloroplast Genomes and Mitochondrial Genes via Genome Skimming.

    Science.gov (United States)

    Zhang, Ning; Wen, Jun; Zimmer, Elizabeth A

    2015-01-01

    Vitaceae is well-known for having one of the most economically important fruits, i.e., the grape (Vitis vinifera). The deep phylogeny of the grape family was not resolved until a recent phylogenomic analysis of 417 nuclear genes from transcriptome data. However, it has been reported extensively that topologies based on nuclear and organellar genes may be incongruent due to differences in their evolutionary histories. Therefore, it is important to reconstruct a backbone phylogeny of the grape family using plastomes and mitochondrial genes. In this study,next-generation sequencing data sets of 27 species were obtained using genome skimming with total DNAs from silica-gel preserved tissue samples on an Illumina NextSeq 500 instrument [corrected]. Plastomes were assembled using the combination of de novo and reference genome (of V. vinifera) methods. Sixteen mitochondrial genes were also obtained via genome skimming using the reference genome of V. vinifera. Extensive phylogenetic analyses were performed using maximum likelihood and Bayesian methods. The topology based on either plastome data or mitochondrial genes is congruent with the one using hundreds of nuclear genes, indicating that the grape family did not exhibit significant reticulation at the deep level. The results showcase the power of genome skimming in capturing extensive phylogenetic data: especially from chloroplast and mitochondrial DNAs.

  11. Congruent Deep Relationships in the Grape Family (Vitaceae Based on Sequences of Chloroplast Genomes and Mitochondrial Genes via Genome Skimming.

    Directory of Open Access Journals (Sweden)

    Ning Zhang

    Full Text Available Vitaceae is well-known for having one of the most economically important fruits, i.e., the grape (Vitis vinifera. The deep phylogeny of the grape family was not resolved until a recent phylogenomic analysis of 417 nuclear genes from transcriptome data. However, it has been reported extensively that topologies based on nuclear and organellar genes may be incongruent due to differences in their evolutionary histories. Therefore, it is important to reconstruct a backbone phylogeny of the grape family using plastomes and mitochondrial genes. In this study,next-generation sequencing data sets of 27 species were obtained using genome skimming with total DNAs from silica-gel preserved tissue samples on an Illumina NextSeq 500 instrument [corrected]. Plastomes were assembled using the combination of de novo and reference genome (of V. vinifera methods. Sixteen mitochondrial genes were also obtained via genome skimming using the reference genome of V. vinifera. Extensive phylogenetic analyses were performed using maximum likelihood and Bayesian methods. The topology based on either plastome data or mitochondrial genes is congruent with the one using hundreds of nuclear genes, indicating that the grape family did not exhibit significant reticulation at the deep level. The results showcase the power of genome skimming in capturing extensive phylogenetic data: especially from chloroplast and mitochondrial DNAs.

  12. Predicting backbone Cα angles and dihedrals from protein sequences by stacked sparse auto-encoder deep neural network.

    Science.gov (United States)

    Lyons, James; Dehzangi, Abdollah; Heffernan, Rhys; Sharma, Alok; Paliwal, Kuldip; Sattar, Abdul; Zhou, Yaoqi; Yang, Yuedong

    2014-10-30

    Because a nearly constant distance between two neighbouring Cα atoms, local backbone structure of proteins can be represented accurately by the angle between C(αi-1)-C(αi)-C(αi+1) (θ) and a dihedral angle rotated about the C(αi)-C(αi+1) bond (τ). θ and τ angles, as the representative of structural properties of three to four amino-acid residues, offer a description of backbone conformations that is complementary to φ and ψ angles (single residue) and secondary structures (>3 residues). Here, we report the first machine-learning technique for sequence-based prediction of θ and τ angles. Predicted angles based on an independent test have a mean absolute error of 9° for θ and 34° for τ with a distribution on the θ-τ plane close to that of native values. The average root-mean-square distance of 10-residue fragment structures constructed from predicted θ and τ angles is only 1.9Å from their corresponding native structures. Predicted θ and τ angles are expected to be complementary to predicted ϕ and ψ angles and secondary structures for using in model validation and template-based as well as template-free structure prediction. The deep neural network learning technique is available as an on-line server called Structural Property prediction with Integrated DEep neuRal network (SPIDER) at http://sparks-lab.org. Copyright © 2014 Wiley Periodicals, Inc.

  13. Metagenomes obtained by "deep sequencing" - what do they tell about the EBPR communities?

    DEFF Research Database (Denmark)

    Albertsen, Mads; Saunders, Aaron Marc; Nielsen, Kåre Lehmann

    2013-01-01

    Metagenomics enables studies of the genomic potential of complex microbial communities by sequencing bulk genomic DNA directly from the environment. Knowledge of the genetic potential of a community can be used to formulate and test ecological hypotheses about stability and performance...... demonstrate that metagenomics can be used as a powerful tool for system wide characterization of the EBPR community as well as for a deeper understanding of the function of specific community members. Furthermore, we discuss and illustrate some of the general pitfalls in metagenomics and stress the need...

  14. Evolution of simeprevir-resistant variants over time by ultra-deep sequencing in HCV genotype 1b.

    Science.gov (United States)

    Akuta, Norio; Suzuki, Fumitaka; Sezaki, Hitomi; Suzuki, Yoshiyuki; Hosaka, Tetsuya; Kobayashi, Masahiro; Kobayashi, Mariko; Saitoh, Satoshi; Ikeda, Kenji; Kumada, Hiromitsu

    2014-08-01

    Using ultra-deep sequencing technology, the present study was designed to investigate the evolution of simeprevir-resistant variants (amino acid substitutions of aa80, aa155, aa156, and aa168 positions in HCV NS3 region) over time. In Toranomon Hospital, 18 Japanese patients infected with HCV genotype 1b, received triple therapy of simeprevir/PEG-IFN/ribavirin (DRAGON or CONCERT study). Sustained virological response rate was 67%, and that was significantly higher in patients with IL28B rs8099917 TT than in those with non-TT. Six patients, who did not achieve sustained virological response, were tested for resistant variants by ultra-deep sequencing, at the baseline, at the time of re-elevation of viral loads, and at 96 weeks after the completion of treatment. Twelve of 18 resistant variants, detected at re-elevation of viral load, were de novo resistant variants. Ten of 12 de novo resistant variants become undetectable over time, and that five of seven resistant variants, detected at baseline, persisted over time. In one patient, variants of Q80R at baseline (0.3%) increased at 96-week after the cessation of treatment (10.2%), and de novo resistant variants of D168E (0.3%) also increased at 96-week after the cessation of treatment (9.7%). In conclusion, the present study indicates that the emergence of simeprevir-resistant variants after the start of treatment could not be predicted at baseline, and the majority of de novo resistant variants become undetectable over time. Further large-scale prospective studies should be performed to investigate the clinical utility in detecting simeprevir-resistant variants. © 2014 Wiley Periodicals, Inc.

  15. DEEPre: sequence-based enzyme EC number prediction by deep learning

    KAUST Repository

    Li, Yu

    2017-10-20

    Annotation of enzyme function has a broad range of applications, such as metagenomics, industrial biotechnology, and diagnosis of enzyme deficiency-caused diseases. However, the time and resource required make it prohibitively expensive to experimentally determine the function of every enzyme. Therefore, computational enzyme function prediction has become increasingly important. In this paper, we develop such an approach, determining the enzyme function by predicting the Enzyme Commission number.We propose an end-to-end feature selection and classification model training approach, as well as an automatic and robust feature dimensionality uniformization method, DEEPre, in the field of enzyme function prediction. Instead of extracting manuallycrafted features from enzyme sequences, our model takes the raw sequence encoding as inputs, extracting convolutional and sequential features from the raw encoding based on the classification result to directly improve the prediction performance. The thorough cross-fold validation experiments conducted on two large-scale datasets show that DEEPre improves the prediction performance over the previous state-of-the-art methods. In addition, our server outperforms five other servers in determining the main class of enzymes on a separate low-homology dataset. Two case studies demonstrate DEEPre\\'s ability to capture the functional difference of enzyme isoforms.The server could be accessed freely at http://www.cbrc.kaust.edu.sa/DEEPre.

  16. Deep sequencing reveals distinct patterns of DNA methylation in prostate cancer.

    Science.gov (United States)

    Kim, Jung H; Dhanasekaran, Saravana M; Prensner, John R; Cao, Xuhong; Robinson, Daniel; Kalyana-Sundaram, Shanker; Huang, Christina; Shankar, Sunita; Jing, Xiaojun; Iyer, Matthew; Hu, Ming; Sam, Lee; Grasso, Catherine; Maher, Christopher A; Palanisamy, Nallasivam; Mehra, Rohit; Kominsky, Hal D; Siddiqui, Javed; Yu, Jindan; Qin, Zhaohui S; Chinnaiyan, Arul M

    2011-07-01

    Beginning with precursor lesions, aberrant DNA methylation marks the entire spectrum of prostate cancer progression. We mapped the global DNA methylation patterns in select prostate tissues and cell lines using MethylPlex-next-generation sequencing (M-NGS). Hidden Markov model-based next-generation sequence analysis identified ∼68,000 methylated regions per sample. While global CpG island (CGI) methylation was not differential between benign adjacent and cancer samples, overall promoter CGI methylation significantly increased from ~12.6% in benign samples to 19.3% and 21.8% in localized and metastatic cancer tissues, respectively (P-value prostate tissues, 2481 differentially methylated regions (DMRs) are cancer-specific, including numerous novel DMRs. A novel cancer-specific DMR in the WFDC2 promoter showed frequent methylation in cancer (17/22 tissues, 6/6 cell lines), but not in the benign tissues (0/10) and normal PrEC cells. Integration of LNCaP DNA methylation and H3K4me3 data suggested an epigenetic mechanism for alternate transcription start site utilization, and these modifications segregated into distinct regions when present on the same promoter. Finally, we observed differences in repeat element methylation, particularly LINE-1, between ERG gene fusion-positive and -negative cancers, and we confirmed this observation using pyrosequencing on a tissue panel. This comprehensive methylome map will further our understanding of epigenetic regulation in prostate cancer progression.

  17. Deep sequencing reveals exceptional diversity and modes of transmission for bacterial sponge symbionts.

    Science.gov (United States)

    Webster, Nicole S; Taylor, Michael W; Behnam, Faris; Lücker, Sebastian; Rattei, Thomas; Whalan, Stephen; Horn, Matthias; Wagner, Michael

    2010-08-01

    Marine sponges contain complex bacterial communities of considerable ecological and biotechnological importance, with many of these organisms postulated to be specific to sponge hosts. Testing this hypothesis in light of the recent discovery of the rare microbial biosphere, we investigated three Australian sponges by massively parallel 16S rRNA gene tag pyrosequencing. Here we show bacterial diversity that is unparalleled in an invertebrate host, with more than 250,000 sponge-derived sequence tags being assigned to 23 bacterial phyla and revealing up to 2996 operational taxonomic units (95% sequence similarity) per sponge species. Of the 33 previously described 'sponge-specific' clusters that were detected in this study, 48% were found exclusively in adults and larvae - implying vertical transmission of these groups. The remaining taxa, including 'Poribacteria', were also found at very low abundance among the 135,000 tags retrieved from surrounding seawater. Thus, members of the rare seawater biosphere may serve as seed organisms for widely occurring symbiont populations in sponges and their host association might have evolved much more recently than previously thought. © 2009 Society for Applied Microbiology and Blackwell Publishing Ltd.

  18. Deep Sequencing Insights in Therapeutic shRNA Processing and siRNA Target Cleavage Precision.

    Science.gov (United States)

    Denise, Hubert; Moschos, Sterghios A; Sidders, Benjamin; Burden, Frances; Perkins, Hannah; Carter, Nikki; Stroud, Tim; Kennedy, Michael; Fancy, Sally-Ann; Lapthorn, Cris; Lavender, Helen; Kinloch, Ross; Suhy, David; Corbau, Romu

    2014-02-04

    TT-034 (PF-05095808) is a recombinant adeno-associated virus serotype 8 (AAV8) agent expressing three short hairpin RNA (shRNA) pro-drugs that target the hepatitis C virus (HCV) RNA genome. The cytosolic enzyme Dicer cleaves each shRNA into multiple, potentially active small interfering RNA (siRNA) drugs. Using next-generation sequencing (NGS) to identify and characterize active shRNAs maturation products, we observed that each TT-034-encoded shRNA could be processed into as many as 95 separate siRNA strands. Few of these appeared active as determined by Sanger 5' RNA Ligase-Mediated Rapid Amplification of cDNA Ends (5-RACE) and through synthetic shRNA and siRNA analogue studies. Moreover, NGS scrutiny applied on 5-RACE products (RACE-seq) suggested that synthetic siRNAs could direct cleavage in not one, but up to five separate positions on targeted RNA, in a sequence-dependent manner. These data support an on-target mechanism of action for TT-034 without cytotoxicity and question the accepted precision of substrate processing by the key RNA interference (RNAi) enzymes Dicer and siRNA-induced silencing complex (siRISC).Molecular Therapy-Nucleic Acids (2014) 3, e145; doi:10.1038/mtna.2013.73; published online 4 February 2014.

  19. DEEPre: sequence-based enzyme EC number prediction by deep learning

    KAUST Repository

    Li, Yu; Wang, Sheng; Umarov, Ramzan; Xie, Bingqing; Fan, Ming; Li, Lihua; Gao, Xin

    2017-01-01

    Annotation of enzyme function has a broad range of applications, such as metagenomics, industrial biotechnology, and diagnosis of enzyme deficiency-caused diseases. However, the time and resource required make it prohibitively expensive to experimentally determine the function of every enzyme. Therefore, computational enzyme function prediction has become increasingly important. In this paper, we develop such an approach, determining the enzyme function by predicting the Enzyme Commission number.We propose an end-to-end feature selection and classification model training approach, as well as an automatic and robust feature dimensionality uniformization method, DEEPre, in the field of enzyme function prediction. Instead of extracting manuallycrafted features from enzyme sequences, our model takes the raw sequence encoding as inputs, extracting convolutional and sequential features from the raw encoding based on the classification result to directly improve the prediction performance. The thorough cross-fold validation experiments conducted on two large-scale datasets show that DEEPre improves the prediction performance over the previous state-of-the-art methods. In addition, our server outperforms five other servers in determining the main class of enzymes on a separate low-homology dataset. Two case studies demonstrate DEEPre's ability to capture the functional difference of enzyme isoforms.The server could be accessed freely at http://www.cbrc.kaust.edu.sa/DEEPre.

  20. Evolutionary Relations of Hexanchiformes Deep-Sea Sharks Elucidated by Whole Mitochondrial Genome Sequences

    Science.gov (United States)

    Tanaka, Keiko; Tomita, Taketeru; Suzuki, Shingo; Hosomichi, Kazuyoshi; Sano, Kazumi; Doi, Hiroyuki; Kono, Azumi; Inoko, Hidetoshi; Kulski, Jerzy K.; Tanaka, Sho

    2013-01-01

    Hexanchiformes is regarded as a monophyletic taxon, but the morphological and genetic relationships between the five extant species within the order are still uncertain. In this study, we determined the whole mitochondrial DNA (mtDNA) sequences of seven sharks including representatives of the five Hexanchiformes, one squaliform, and one carcharhiniform and inferred the phylogenetic relationships among those species and 12 other Chondrichthyes (cartilaginous fishes) species for which the complete mitogenome is available. The monophyly of Hexanchiformes and its close relation with all other Squaliformes sharks were strongly supported by likelihood and Bayesian phylogenetic analysis of 13,749 aligned nucleotides of 13 protein coding genes and two rRNA genes that were derived from the whole mDNA sequences of the 19 species. The phylogeny suggested that Hexanchiformes is in the superorder Squalomorphi, Chlamydoselachus anguineus (frilled shark) is the sister species to all other Hexanchiformes, and the relations within Hexanchiformes are well resolved as Chlamydoselachus, (Notorynchus, (Heptranchias, (Hexanchus griseus, H. nakamurai))). Based on our phylogeny, we discussed evolutionary scenarios of the jaw suspension mechanism and gill slit numbers that are significant features in the sharks. PMID:24089661

  1. Deep sequencing of subseafloor eukaryotic rRNA reveals active Fungi across marine subsurface provinces.

    Directory of Open Access Journals (Sweden)

    William Orsi

    Full Text Available The deep marine subsurface is a vast habitat for microbial life where cells may live on geologic timescales. Because DNA in sediments may be preserved on long timescales, ribosomal RNA (rRNA is suggested to be a proxy for the active fraction of a microbial community in the subsurface. During an investigation of eukaryotic 18S rRNA by amplicon pyrosequencing, unique profiles of Fungi were found across a range of marine subsurface provinces including ridge flanks, continental margins, and abyssal plains. Subseafloor fungal populations exhibit statistically significant correlations with total organic carbon (TOC, nitrate, sulfide, and dissolved inorganic carbon (DIC. These correlations are supported by terminal restriction length polymorphism (TRFLP analyses of fungal rRNA. Geochemical correlations with fungal pyrosequencing and TRFLP data from this geographically broad sample set suggests environmental selection of active Fungi in the marine subsurface. Within the same dataset, ancient rRNA signatures were recovered from plants and diatoms in marine sediments ranging from 0.03 to 2.7 million years old, suggesting that rRNA from some eukaryotic taxa may be much more stable than previously considered in the marine subsurface.

  2. Genome-wide analysis of SRSF10-regulated alternative splicing by deep sequencing of chicken transcriptome

    Directory of Open Access Journals (Sweden)

    Xuexia Zhou

    2014-12-01

    Full Text Available Splicing factor SRSF10 is known to function as a sequence-specific splicing activator that is capable of regulating alternative splicing both in vitro and in vivo. We recently used an RNA-seq approach coupled with bioinformatics analysis to identify the extensive splicing network regulated by SRSF10 in chicken cells. We found that SRSF10 promoted both exon inclusion and exclusion. Functionally, many of the SRSF10-verified alternative exons are linked to pathways of response to external stimulus. Here we describe in detail the experimental design, bioinformatics analysis and GO/pathway enrichment analysis of SRSF10-regulated genes to correspond with our data in the Gene Expression Omnibus with accession number GSE53354. Our data thus provide a resource for studying regulation of alternative splicing in vivo that underlines biological functions of splicing regulatory proteins in cells.

  3. Deep Sequencing Reveals Uncharted Isoform Heterogeneity of the Protein-Coding Transcriptome in Cerebral Ischemia.

    Science.gov (United States)

    Bhattarai, Sunil; Aly, Ahmed; Garcia, Kristy; Ruiz, Diandra; Pontarelli, Fabrizio; Dharap, Ashutosh

    2018-06-03

    Gene expression in cerebral ischemia has been a subject of intense investigations for several years. Studies utilizing probe-based high-throughput methodologies such as microarrays have contributed significantly to our existing knowledge but lacked the capacity to dissect the transcriptome in detail. Genome-wide RNA-sequencing (RNA-seq) enables comprehensive examinations of transcriptomes for attributes such as strandedness, alternative splicing, alternative transcription start/stop sites, and sequence composition, thus providing a very detailed account of gene expression. Leveraging this capability, we conducted an in-depth, genome-wide evaluation of the protein-coding transcriptome of the adult mouse cortex after transient focal ischemia at 6, 12, or 24 h of reperfusion using RNA-seq. We identified a total of 1007 transcripts at 6 h, 1878 transcripts at 12 h, and 1618 transcripts at 24 h of reperfusion that were significantly altered as compared to sham controls. With isoform-level resolution, we identified 23 splice variants arising from 23 genes that were novel mRNA isoforms. For a subset of genes, we detected reperfusion time-point-dependent splice isoform switching, indicating an expression and/or functional switch for these genes. Finally, for 286 genes across all three reperfusion time-points, we discovered multiple, distinct, simultaneously expressed and differentially altered isoforms per gene that were generated via alternative transcription start/stop sites. Of these, 165 isoforms derived from 109 genes were novel mRNAs. Together, our data unravel the protein-coding transcriptome of the cerebral cortex at an unprecedented depth to provide several new insights into the flexibility and complexity of stroke-related gene transcription and transcript organization.

  4. Transcriptional responses of Acropora hyacinthus embryo under the benzo(a)pyrene stress by deep sequencing.

    Science.gov (United States)

    Xiao, Rong; Zhou, Hailong; Chen, Chien-Min; Cheng, Huamin; Li, Hongwu; Xie, Jia; Zhao, Hongwei; Han, Qian; Diao, Xiaoping

    2018-04-24

    Coral embryos are a critical and sensitive period for the early growth and development of coral. Benzo(a)pyrene (BaP) is widely distributed in the ocean and has strong toxicity, but there is little information on the toxic effects to coral embryos exposed to this widespread environmental contaminant. Thus, in this study, we utilized the Illumina Hiseq™ 4000 platform to explore the gene response of Acropora hyacinthus embryos under the BaP stress. A total of 130,042 Unigenes were obtained and analyzed, and approximately 37.67% of those matched with sequences from four different species. In total, 2606 Unigenes were up-regulated, and 3872 Unigenes were down-regulated. After Gene Ontology (GO) annotation, the results show that the "cellular process" and "metabolic process" were leading in the category of biological processes, which the "binding" and "catalytic activity" were the most abundant subcategories in molecular function. Based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, the most differentially expressed genes (DEGs) were enriched, as well as down-regulated in the pathways of oxidative phosphorylation, metabolism of xenobiotics, immune-related genes, apoptosis and human disease genes. At the same time, 388,197 of Single-nucleotide Polymorphisms (SNPs) and 6164 of Simple Sequence Repeats (SSRs) were obtained, which can be served as the richer and more valuable SSRs molecular markers in the future. The results of this study can help to better understand the toxicological mechanism of coral embryo exposed to BaP, and it is also essential for the protection and restoration of coral reef ecosystem in the future. Copyright © 2018 Elsevier Ltd. All rights reserved.

  5. Identification and characterization of novel serum microRNA candidates from deep sequencing in cervical cancer patients.

    Science.gov (United States)

    Juan, Li; Tong, Hong-li; Zhang, Pengjun; Guo, Guanghong; Wang, Zi; Wen, Xinyu; Dong, Zhennan; Tian, Ya-ping

    2014-09-03

    Small non-coding microRNAs (miRNAs) are involved in cancer development and progression, and serum profiles of cervical cancer patients may be useful for identifying novel miRNAs. We performed deep sequencing on serum pools of cervical cancer patients and healthy controls with 3 replicates and constructed a small RNA library. We used MIREAP to predict novel miRNAs and identified 2 putative novel miRNAs between serum pools of cervical cancer patients and healthy controls after filtering out pseudo-pre-miRNAs using Triplet-SVM analysis. The 2 putative novel miRNAs were validated by real time PCR and were significantly decreased in cervical cancer patients compared with healthy controls. One novel miRNA had an area under curve (AUC) of 0.921 (95% CI: 0.883, 0.959) with a sensitivity of 85.7% and a specificity of 88.2% when discriminating between cervical cancer patients and healthy controls. Our results suggest that characterizing serum profiles of cervical cancers by Solexa sequencing may be a good method for identifying novel miRNAs and that the validated novel miRNAs described here may be cervical cancer-associated biomarkers.

  6. Deciphering KRAS and NRAS mutated clone dynamics in MLL-AF4 paediatric leukaemia by ultra deep sequencing analysis.

    Science.gov (United States)

    Trentin, Luca; Bresolin, Silvia; Giarin, Emanuela; Bardini, Michela; Serafin, Valentina; Accordi, Benedetta; Fais, Franco; Tenca, Claudya; De Lorenzo, Paola; Valsecchi, Maria Grazia; Cazzaniga, Giovanni; Kronnie, Geertruy Te; Basso, Giuseppe

    2016-10-04

    To induce and sustain the leukaemogenic process, MLL-AF4+ leukaemia seems to require very few genetic alterations in addition to the fusion gene itself. Studies of infant and paediatric patients with MLL-AF4+ B cell precursor acute lymphoblastic leukaemia (BCP-ALL) have reported mutations in KRAS and NRAS with incidences ranging from 25 to 50%. Whereas previous studies employed Sanger sequencing, here we used next generation amplicon deep sequencing for in depth evaluation of RAS mutations in 36 paediatric patients at diagnosis of MLL-AF4+ leukaemia. RAS mutations including those in small sub-clones were detected in 63.9% of patients. Furthermore, the mutational analysis of 17 paired samples at diagnosis and relapse revealed complex RAS clone dynamics and showed that the mutated clones present at relapse were almost all originated from clones that were already detectable at diagnosis and survived to the initial therapy. Finally, we showed that mutated patients were indeed characterized by a RAS related signature at both transcriptional and protein levels and that the targeting of the RAS pathway could be of beneficial for treatment of MLL-AF4+ BCP-ALL clones carrying somatic RAS mutations.

  7. Identifying genomic changes associated with insecticide resistance in the dengue mosquito Aedes aegypti by deep targeted sequencing

    Science.gov (United States)

    Faucon, Frederic; Dusfour, Isabelle; Gaude, Thierry; Navratil, Vincent; Boyer, Frederic; Chandre, Fabrice; Sirisopa, Patcharawan; Thanispong, Kanutcharee; Juntarajumnong, Waraporn; Poupardin, Rodolphe; Chareonviriyaphap, Theeraphap; Girod, Romain; Corbel, Vincent; Reynaud, Stephane; David, Jean-Philippe

    2015-01-01

    The capacity of mosquitoes to resist insecticides threatens the control of diseases such as dengue and malaria. Until alternative control tools are implemented, characterizing resistance mechanisms is crucial for managing resistance in natural populations. Insecticide biodegradation by detoxification enzymes is a common resistance mechanism; however, the genomic changes underlying this mechanism have rarely been identified, precluding individual resistance genotyping. In particular, the role of copy number variations (CNVs) and polymorphisms of detoxification enzymes have never been investigated at the genome level, although they can represent robust markers of metabolic resistance. In this context, we combined target enrichment with high-throughput sequencing for conducting the first comprehensive screening of gene amplifications and polymorphisms associated with insecticide resistance in mosquitoes. More than 760 candidate genes were captured and deep sequenced in several populations of the dengue mosquito Ae. aegypti displaying distinct genetic backgrounds and contrasted resistance levels to the insecticide deltamethrin. CNV analysis identified 41 gene amplifications associated with resistance, most affecting cytochrome P450s overtranscribed in resistant populations. Polymorphism analysis detected more than 30,000 variants and strong selection footprints in specific genomic regions. Combining Bayesian and allele frequency filtering approaches identified 55 nonsynonymous variants strongly associated with resistance. Both CNVs and polymorphisms were conserved within regions but differed across continents, confirming that genomic changes underlying metabolic resistance to insecticides are not universal. By identifying novel DNA markers of insecticide resistance, this study opens the way for tracking down metabolic changes developed by mosquitoes to resist insecticides within and among populations. PMID:26206155

  8. Real-Time Human Detection for Aerial Captured Video Sequences via Deep Models

    Directory of Open Access Journals (Sweden)

    Nouar AlDahoul

    2018-01-01

    Full Text Available Human detection in videos plays an important role in various real life applications. Most of traditional approaches depend on utilizing handcrafted features which are problem-dependent and optimal for specific tasks. Moreover, they are highly susceptible to dynamical events such as illumination changes, camera jitter, and variations in object sizes. On the other hand, the proposed feature learning approaches are cheaper and easier because highly abstract and discriminative features can be produced automatically without the need of expert knowledge. In this paper, we utilize automatic feature learning methods which combine optical flow and three different deep models (i.e., supervised convolutional neural network (S-CNN, pretrained CNN feature extractor, and hierarchical extreme learning machine for human detection in videos captured using a nonstatic camera on an aerial platform with varying altitudes. The models are trained and tested on the publicly available and highly challenging UCF-ARG aerial dataset. The comparison between these models in terms of training, testing accuracy, and learning speed is analyzed. The performance evaluation considers five human actions (digging, waving, throwing, walking, and running. Experimental results demonstrated that the proposed methods are successful for human detection task. Pretrained CNN produces an average accuracy of 98.09%. S-CNN produces an average accuracy of 95.6% with soft-max and 91.7% with Support Vector Machines (SVM. H-ELM has an average accuracy of 95.9%. Using a normal Central Processing Unit (CPU, H-ELM’s training time takes 445 seconds. Learning in S-CNN takes 770 seconds with a high performance Graphical Processing Unit (GPU.

  9. Small RNA and transcriptome deep sequencing proffers insight into floral gene regulation in Rosa cultivars

    Directory of Open Access Journals (Sweden)

    Kim Jungeun

    2012-11-01

    Full Text Available Abstract Background Roses (Rosa sp., which belong to the family Rosaceae, are the most economically important ornamental plants—making up 30% of the floriculture market. However, given high demand for roses, rose breeding programs are limited in molecular resources which can greatly enhance and speed breeding efforts. A better understanding of important genes that contribute to important floral development and desired phenotypes will lead to improved rose cultivars. For this study, we analyzed rose miRNAs and the rose flower transcriptome in order to generate a database to expound upon current knowledge regarding regulation of important floral characteristics. A rose genetic database will enable comprehensive analysis of gene expression and regulation via miRNA among different Rosa cultivars. Results We produced more than 0.5 million reads from expressed sequences, totalling more than 110 million bp. From these, we generated 35,657, 31,434, 34,725, and 39,722 flower unigenes from Rosa hybrid: ‘Vital’, ‘Maroussia’, and ‘Sympathy’ and Rosa rugosa Thunb. , respectively. The unigenes were assigned functional annotations, domains, metabolic pathways, Gene Ontology (GO terms, Plant Ontology (PO terms, and MIPS Functional Catalogue (FunCat terms. Rose flower transcripts were compared with genes from whole genome sequences of Rosaceae members (apple, strawberry, and peach and grape. We also produced approximately 40 million small RNA reads from flower tissue for Rosa, representing 267 unique miRNA tags. Among identified miRNAs, 25 of them were novel and 242 of them were conserved miRNAs. Statistical analyses of miRNA profiles revealed both shared and species-specific miRNAs, which presumably effect flower development and phenotypes. Conclusions In this study, we constructed a Rose miRNA and transcriptome database, and we analyzed the miRNAs and transcriptome generated from the flower tissues of four Rosa cultivars. The database provides a

  10. Analysis of hepatitis C NS5A resistance associated polymorphisms using ultra deep single molecule real time (SMRT) sequencing.

    Science.gov (United States)

    Bergfors, Assar; Leenheer, Daniël; Bergqvist, Anders; Ameur, Adam; Lennerstrand, Johan

    2016-02-01

    Development of Hepatitis C virus (HCV) resistance against direct-acting antivirals (DAAs), including NS5A inhibitors, is an obstacle to successful treatment of HCV when DAAs are used in sub-optimal combinations. Furthermore, it has been shown that baseline (pre-existing) resistance against DAAs is present in treatment naïve-patients and this will potentially complicate future treatment strategies in different HCV genotypes (GTs). Thus the aim was to detect low levels of NS5A resistant associated variants (RAVs) in a limited sample set of treatment-naïve patients of HCV GT1a and 3a, since such polymorphisms can display in vitro resistance as high as 60000 fold. Ultra-deep single molecule real time (SMRT) sequencing with the Pacific Biosciences (PacBio) RSII instrument was used to detect these RAVs. The SMRT sequencing was conducted on ten samples; three of them positive with Sanger sequencing (GT1a Q30H and Y93N, and GT3a Y93H), five GT1a samples, and two GT3a non-positive samples. The same methods were applied to the HCV GT1a H77-plasmid in a dilution series, in order to determine the error rates of replication, which in turn was used to determine the limit of detection (LOD), as defined by mean + 3SD, of minority variants down to 0.24%. We found important baseline NS5A RAVs at levels between 0.24 and 0.5%, which could potentially have clinical relevance. This new method with low level detection of baseline RAVs could be useful in predicting the most cost-efficient combination of DAA treatment, and reduce the treatment duration for an HCV infected individual. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. The 2007 Nazko, British Columbia, earthquake sequence: Injection of magma deep in the crust beneath the Anahim volcanic belt

    Science.gov (United States)

    Cassidy, J.F.; Balfour, N.; Hickson, C.; Kao, H.; White, Rickie; Caplan-Auerbach, J.; Mazzotti, S.; Rogers, Gary C.; Al-Khoubbi, I.; Bird, A.L.; Esteban, L.; Kelman, M.; Hutchinson, J.; McCormack, D.

    2011-01-01

    On 9 October 2007, an unusual sequence of earthquakes began in central British Columbia about 20 km west of the Nazko cone, the most recent (circa 7200 yr) volcanic center in the Anahim volcanic belt. Within 25 hr, eight earthquakes of magnitude 2.3-2.9 occurred in a region where no earthquakes had previously been recorded. During the next three weeks, more than 800 microearthquakes were located (and many more detected), most at a depth of 25-31 km and within a radius of about 5 km. After about two months, almost all activity ceased. The clear P- and S-wave arrivals indicated that these were high-frequency (volcanic-tectonic) earthquakes and the b value of 1.9 that we calculated is anomalous for crustal earthquakes but consistent with volcanic-related events. Analysis of receiver functions at a station immediately above the seismicity indicated a Moho near 30 km depth. Precise relocation of the seismicity using a double-difference method suggested a horizontal migration at the rate of about 0:5 km=d, with almost all events within the lowermost crust. Neither harmonic tremor nor long-period events were observed; however, some spasmodic bursts were recorded and determined to be colocated with the earthquake hypocenters. These observations are all very similar to a deep earthquake sequence recorded beneath Lake Tahoe, California, in 2003-2004. Based on these remarkable similarities, we interpret the Nazko sequence as an indication of an injection of magma into the lower crust beneath the Anahim volcanic belt. This magma injection fractures rock, producing high-frequency, volcanic-tectonic earthquakes and spasmodic bursts.

  12. Genomic DNA sequences from mastodon and woolly mammoth reveal deep speciation of forest and savanna elephants.

    Directory of Open Access Journals (Sweden)

    Nadin Rohland

    2010-12-01

    Full Text Available To elucidate the history of living and extinct elephantids, we generated 39,763 bp of aligned nuclear DNA sequence across 375 loci for African savanna elephant, African forest elephant, Asian elephant, the extinct American mastodon, and the woolly mammoth. Our data establish that the Asian elephant is the closest living relative of the extinct mammoth in the nuclear genome, extending previous findings from mitochondrial DNA analyses. We also find that savanna and forest elephants, which some have argued are the same species, are as or more divergent in the nuclear genome as mammoths and Asian elephants, which are considered to be distinct genera, thus resolving a long-standing debate about the appropriate taxonomic classification of the African elephants. Finally, we document a much larger effective population size in forest elephants compared with the other elephantid taxa, likely reflecting species differences in ancient geographic structure and range and differences in life history traits such as variance in male reproductive success.

  13. Revealing stable processing products from ribosome-associated small RNAs by deep-sequencing data analysis.

    Science.gov (United States)

    Zywicki, Marek; Bakowska-Zywicka, Kamilla; Polacek, Norbert

    2012-05-01

    The exploration of the non-protein-coding RNA (ncRNA) transcriptome is currently focused on profiling of microRNA expression and detection of novel ncRNA transcription units. However, recent studies suggest that RNA processing can be a multi-layer process leading to the generation of ncRNAs of diverse functions from a single primary transcript. Up to date no methodology has been presented to distinguish stable functional RNA species from rapidly degraded side products of nucleases. Thus the correct assessment of widespread RNA processing events is one of the major obstacles in transcriptome research. Here, we present a novel automated computational pipeline, named APART, providing a complete workflow for the reliable detection of RNA processing products from next-generation-sequencing data. The major features include efficient handling of non-unique reads, detection of novel stable ncRNA transcripts and processing products and annotation of known transcripts based on multiple sources of information. To disclose the potential of APART, we have analyzed a cDNA library derived from small ribosome-associated RNAs in Saccharomyces cerevisiae. By employing the APART pipeline, we were able to detect and confirm by independent experimental methods multiple novel stable RNA molecules differentially processed from well known ncRNAs, like rRNAs, tRNAs or snoRNAs, in a stress-dependent manner.

  14. Deep sequencing and ecological characterization of gut microbial communities of diverse bumble bee species.

    Directory of Open Access Journals (Sweden)

    Haw Chuan Lim

    Full Text Available Gut bacterial communities of bumble bees are correlated with defense against pathogens. Further understanding this host-microbe association is vitally important as bumble bees are currently experiencing global population declines, potentially due in part to emergent diseases. In this study, we used pyrosequencing and community fingerprinting (ARISA to characterize the gut microbial communities of nine bumble species from across the Bombus phylogeny. Overall, we delimited 74 bacterial taxa (operational taxonomic units or OTUs belonging to Betaproteobacteria, Gammaproteobacteria, Bacilli, Actinobacteria, Flavobacteria and Alphaproteobacteria. Each bacterial community was taxonomically simple, containing an average of 1.9 common (relative abundance per sample > 5% bacterial OTUs. The most abundant and prevalent (occurring in 92% of the samples bacterial OTU, based on 16S rRNA sequences, closely matched that of the previously described Betaproteobacteria species Snodgrassella alvi. Bacteria that were first described in bee-related external environments dominated a number of gut bacterial communities, suggesting that they are not strictly dependent on the internal gut environment. The ARISA data showed a correlation between bacterial community structures and the geographic locations where the bees were sampled, suggesting that at least a subset of the bacterial species may be transmitted environmentally. Using light and fluorescent microscopy, we demonstrated that the gut bacteria form a biofilm on the internal epithelial surface of the ileum, corroborating results obtained from Apis mellifera.

  15. Quantitative assessment of hematopoietic chimerism by quantitative real-time polymerase chain reaction of sequence polymorphism systems after hematopoietic stem cell transplantation.

    Science.gov (United States)

    Qin, Xiao-ying; Li, Guo-xuan; Qin, Ya-zhen; Wang, Yu; Wang, Feng-rong; Liu, Dai-hong; Xu, Lan-ping; Chen, Huan; Han, Wei; Wang, Jing-zhi; Zhang, Xiao-hui; Li, Jin-lan; Li, Ling-di; Liu, Kai-yan; Huang, Xiao-jun

    2011-08-01

    Analysis of changes in recipient and donor hematopoietic cell origin is extremely useful to monitor the effect of hematopoietic stem cell transplantation (HSCT) and sequential adoptive immunotherapy by donor lymphocyte infusions. We developed a sensitive, reliable and rapid real-time PCR method based on sequence polymorphism systems to quantitatively assess the hematopoietic chimerism after HSCT. A panel of 29 selected sequence polymorphism (SP) markers was screened by real-time PCR in 101 HSCT patients with leukemia and other hematological diseases. The chimerism kinetics of bone marrow samples of 8 HSCT patients in remission and relapse situations were followed longitudinally. Recipient genotype discrimination was possible in 97.0% (98 of 101) with a mean number of 2.5 (1-7) informative markers per recipient/donor pair. Using serial dilutions of plasmids containing specific SP markers, the linear correlation (r) of 0.99, the slope between -3.2 and -3.7 and the sensitivity of 0.1% were proved reproducible. By this method, it was possible to very accurately detect autologous signals in the range from 0.1% to 30%. The accuracy of the method in the very important range of autologous signals below 5% was extraordinarily high (standard deviation real-time PCR method over short tandem repeat PCR chimerism assays is the absence of PCR competition and plateau biases, with demonstrated greater sensitivity and linearity. Finally, we prospectively analyzed bone marrow samples of 8 patients who received allografts and presented the chimerism kinetics of remission and relapse situations that illustrated the sensitivity level and the promising clinical application of this method. This SP-based real-time PCR assay provides a rapid, sensitive, and accurate quantitative assessment of mixed chimerism that can be useful in predicting graft rejection and early relapse.

  16. Deep sequencing whole transcriptome exploration of the σE regulon in Neisseria meningitidis.

    Directory of Open Access Journals (Sweden)

    Robert Antonius Gerhardus Huis in 't Veld

    Full Text Available Bacteria live in an ever-changing environment and must alter protein expression promptly to adapt to these changes and survive. Specific response genes that are regulated by a subset of alternative σ(70-like transcription factors have evolved in order to respond to this changing environment. Recently, we have described the existence of a σ(E regulon including the anti-σ-factor MseR in the obligate human bacterial pathogen Neisseria meningitidis. To unravel the complete σ(E regulon in N. meningitidis, we sequenced total RNA transcriptional content of wild type meningococci and compared it with that of mseR mutant cells (ΔmseR in which σ(E is highly expressed. Eleven coding genes and one non-coding gene were found to be differentially expressed between H44/76 wildtype and H44/76ΔmseR cells. Five of the 6 genes of the σ(E operon, msrA/msrB, and the gene encoding a pepSY-associated TM helix family protein showed enhanced transcription, whilst aniA encoding a nitrite reductase and nspA encoding the vaccine candidate Neisserial surface protein A showed decreased transcription. Analysis of differential expression in IGRs showed enhanced transcription of a non-coding RNA molecule, identifying a σ(E dependent small non-coding RNA. Together this constitutes the first complete exploration of an alternative σ-factor regulon in N. meningitidis. The results direct to a relatively small regulon indicative for a strictly defined response consistent with a relatively stable niche, the human throat, where N. meningitidis resides.

  17. Draft Genome Sequence of Pseudoalteromonas sp. Strain XI10 Isolated from the Brine-Seawater Interface of Erba Deep in the Red Sea

    KAUST Repository

    Zhang, Guishan; Haroon, Mohamed; Zhang, Ruifu; Hikmawan, Tyas I.; Stingl, Ulrich

    2016-01-01

    Pseudoalteromonas sp. strain XI10 was isolated from the brine-seawater interface of Erba Deep in the Red Sea, Saudi Arabia. Here, we present the draft genome sequence of strain XI10, a gammaproteobacterium that synthesizes polysaccharides for biofilm formation when grown in liquid culture.

  18. Draft Genome Sequences of TwoThiomicrospiraStrains Isolated from the Brine-Seawater Interface of Kebrit Deep in the Red Sea

    KAUST Repository

    Zhang, Guishan

    2016-03-11

    Two Thiomicrospira strains, WB1 and XS5, were isolated from the Kebrit Deep brine-seawater interface in the Red Sea, Saudi Arabia. Here, we present the draft genome sequences of these gammaproteobacteria, which both produce sulfuric acid from thiosulfate in culture.

  19. Draft Genome Sequences of TwoThiomicrospiraStrains Isolated from the Brine-Seawater Interface of Kebrit Deep in the Red Sea

    KAUST Repository

    Zhang, Guishan; Haroon, Mohamed; Zhang, Ruifu; Hikmawan, Tyas I.; Stingl, Ulrich

    2016-01-01

    Two Thiomicrospira strains, WB1 and XS5, were isolated from the Kebrit Deep brine-seawater interface in the Red Sea, Saudi Arabia. Here, we present the draft genome sequences of these gammaproteobacteria, which both produce sulfuric acid from thiosulfate in culture.

  20. Draft Genome Sequence of Pseudoalteromonas sp. Strain XI10 Isolated from the Brine-Seawater Interface of Erba Deep in the Red Sea

    KAUST Repository

    Zhang, Guishan

    2016-03-10

    Pseudoalteromonas sp. strain XI10 was isolated from the brine-seawater interface of Erba Deep in the Red Sea, Saudi Arabia. Here, we present the draft genome sequence of strain XI10, a gammaproteobacterium that synthesizes polysaccharides for biofilm formation when grown in liquid culture.

  1. A tapping device for recording and quantitative characterization of rhythmic/auditory sequences.

    Science.gov (United States)

    Piazza, Caterina; Cesareo, Ambra; Caccia, Martina; Reni, Gianluigi; Lorusso, Maria L

    2017-07-01

    The processing of auditory stimuli is essential for the correct perception of language and deficits in this ability are often related to the presence or development of language disorders. The motor imitation (e.g. tapping or beating) of rhythmic sequences can be a very sensitive correlate of deficits in auditory processing. Thus, the study of the tapping performance, with the investigation of both temporal and intensity information, might be very useful. The present work is aimed at the development and preliminary testing of a tapping device to be used for the imitation and/or the production of rhythmic sequences, allowing the recording of both tapping duration and intensity. The device is essentially made up of a Force Sensing Resistor and an Arduino UNO board. It was validated using different sampling frequencies (f s ) in a group of 10 young healthy adults investigating its efficacy in terms of touch and intensity detection by means of two testing procedures. Results demonstrated a good performance of the device when programmed with fs equal to 50 and 100Hz. Moreover, both temporal and intensity parameters were extracted, thus supporting the potential use of the device for the analysis of the imitation or production of rhythmic sequences. This work represents a first step for the development of a useful, low cost tool to support the diagnosis, training and rehabilitation of language disorders.

  2. Analyses of Tissue Culture Adaptation of Human Herpesvirus-6A by Whole Genome Deep Sequencing Redefines the Reference Sequence and Identifies Virus Entry Complex Changes.

    Science.gov (United States)

    Tweedy, Joshua G; Escriva, Eric; Topf, Maya; Gompels, Ursula A

    2017-12-31

    Tissue-culture adaptation of viruses can modulate infection. Laboratory passage and bacterial artificial chromosome (BAC)mid cloning of human cytomegalovirus, HCMV, resulted in genomic deletions and rearrangements altering genes encoding the virus entry complex, which affected cellular tropism, virulence, and vaccine development. Here, we analyse these effects on the reference genome for related betaherpesviruses, Roseolovirus, human herpesvirus 6A (HHV-6A) strain U1102. This virus is also naturally "cloned" by germline subtelomeric chromosomal-integration in approximately 1% of human populations, and accurate references are key to understanding pathological relationships between exogenous and endogenous virus. Using whole genome next-generation deep-sequencing Illumina-based methods, we compared the original isolate to tissue-culture passaged and the BACmid-cloned virus. This re-defined the reference genome showing 32 corrections and 5 polymorphisms. Furthermore, minor variant analyses of passaged and BACmid virus identified emerging populations of a further 32 single nucleotide polymorphisms (SNPs) in 10 loci, half non-synonymous indicating cell-culture selection. Analyses of the BAC-virus genome showed deletion of the BAC cassette via loxP recombination removing green fluorescent protein (GFP)-based selection. As shown for HCMV culture effects, select HHV-6A SNPs mapped to genes encoding mediators of virus cellular entry, including virus envelope glycoprotein genes gB and the gH/gL complex. Comparative models suggest stabilisation of the post-fusion conformation. These SNPs are essential to consider in vaccine-design, antimicrobial-resistance, and pathogenesis.

  3. InFusion: Advancing Discovery of Fusion Genes and Chimeric Transcripts from Deep RNA-Sequencing Data.

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    Konstantin Okonechnikov

    Full Text Available Analysis of fusion transcripts has become increasingly important due to their link with cancer development. Since high-throughput sequencing approaches survey fusion events exhaustively, several computational methods for the detection of gene fusions from RNA-seq data have been developed. This kind of analysis, however, is complicated by native trans-splicing events, the splicing-induced complexity of the transcriptome and biases and artefacts introduced in experiments and data analysis. There are a number of tools available for the detection of fusions from RNA-seq data; however, certain differences in specificity and sensitivity between commonly used approaches have been found. The ability to detect gene fusions of different types, including isoform fusions and fusions involving non-coding regions, has not been thoroughly studied yet. Here, we propose a novel computational toolkit called InFusion for fusion gene detection from RNA-seq data. InFusion introduces several unique features, such as discovery of fusions involving intergenic regions, and detection of anti-sense transcription in chimeric RNAs based on strand-specificity. Our approach demonstrates superior detection accuracy on simulated data and several public RNA-seq datasets. This improved performance was also evident when evaluating data from RNA deep-sequencing of two well-established prostate cancer cell lines. InFusion identified 26 novel fusion events that were validated in vitro, including alternatively spliced gene fusion isoforms and chimeric transcripts that include intergenic regions. The toolkit is freely available to download from http:/bitbucket.org/kokonech/infusion.

  4. Deep developmental transcriptome sequencing uncovers numerous new genes and enhances gene annotation in the sponge Amphimedon queenslandica.

    Science.gov (United States)

    Fernandez-Valverde, Selene L; Calcino, Andrew D; Degnan, Bernard M

    2015-05-15

    The demosponge Amphimedon queenslandica is amongst the few early-branching metazoans with an assembled and annotated draft genome, making it an important species in the study of the origin and early evolution of animals. Current gene models in this species are largely based on in silico predictions and low coverage expressed sequence tag (EST) evidence. Amphimedon queenslandica protein-coding gene models are improved using deep RNA-Seq data from four developmental stages and CEL-Seq data from 82 developmental samples. Over 86% of previously predicted genes are retained in the new gene models, although 24% have additional exons; there is also a marked increase in the total number of annotated 3' and 5' untranslated regions (UTRs). Importantly, these new developmental transcriptome data reveal numerous previously unannotated protein-coding genes in the Amphimedon genome, increasing the total gene number by 25%, from 30,060 to 40,122. In general, Amphimedon genes have introns that are markedly smaller than those in other animals and most of the alternatively spliced genes in Amphimedon undergo intron-retention; exon-skipping is the least common mode of alternative splicing. Finally, in addition to canonical polyadenylation signal sequences, Amphimedon genes are enriched in a number of unique AT-rich motifs in their 3' UTRs. The inclusion of developmental transcriptome data has substantially improved the structure and composition of protein-coding gene models in Amphimedon queenslandica, providing a more accurate and comprehensive set of genes for functional and comparative studies. These improvements reveal the Amphimedon genome is comprised of a remarkably high number of tightly packed genes. These genes have small introns and there is pervasive intron retention amongst alternatively spliced transcripts. These aspects of the sponge genome are more similar unicellular opisthokont genomes than to other animal genomes.

  5. Deep RNA sequencing reveals dynamic regulation of myocardial noncoding RNAs in failing human heart and remodeling with mechanical circulatory support.

    Science.gov (United States)

    Yang, Kai-Chien; Yamada, Kathryn A; Patel, Akshar Y; Topkara, Veli K; George, Isaac; Cheema, Faisal H; Ewald, Gregory A; Mann, Douglas L; Nerbonne, Jeanne M

    2014-03-04

    Microarrays have been used extensively to profile transcriptome remodeling in failing human heart, although the genomic coverage provided is limited and fails to provide a detailed picture of the myocardial transcriptome landscape. Here, we describe sequencing-based transcriptome profiling, providing comprehensive analysis of myocardial mRNA, microRNA (miRNA), and long noncoding RNA (lncRNA) expression in failing human heart before and after mechanical support with a left ventricular (LV) assist device (LVAD). Deep sequencing of RNA isolated from paired nonischemic (NICM; n=8) and ischemic (ICM; n=8) human failing LV samples collected before and after LVAD and from nonfailing human LV (n=8) was conducted. These analyses revealed high abundance of mRNA (37%) and lncRNA (71%) of mitochondrial origin. miRNASeq revealed 160 and 147 differentially expressed miRNAs in ICM and NICM, respectively, compared with nonfailing LV. Among these, only 2 (ICM) and 5 (NICM) miRNAs are normalized with LVAD. RNASeq detected 18 480, including 113 novel, lncRNAs in human LV. Among the 679 (ICM) and 570 (NICM) lncRNAs differentially expressed with heart failure, ≈10% are improved or normalized with LVAD. In addition, the expression signature of lncRNAs, but not miRNAs or mRNAs, distinguishes ICM from NICM. Further analysis suggests that cis-gene regulation represents a major mechanism of action of human cardiac lncRNAs. The myocardial transcriptome is dynamically regulated in advanced heart failure and after LVAD support. The expression profiles of lncRNAs, but not mRNAs or miRNAs, can discriminate failing hearts of different pathologies and are markedly altered in response to LVAD support. These results suggest an important role for lncRNAs in the pathogenesis of heart failure and in reverse remodeling observed with mechanical support.

  6. Comparative sequence analyses of the major quantitative trait locus phosphorus uptake 1 (Pup1) reveal a complex genetic structure.

    Science.gov (United States)

    Heuer, Sigrid; Lu, Xiaochun; Chin, Joong Hyoun; Tanaka, Juan Pariasca; Kanamori, Hiroyuki; Matsumoto, Takashi; De Leon, Teresa; Ulat, Victor Jun; Ismail, Abdelbagi M; Yano, Masahiro; Wissuwa, Matthias

    2009-06-01

    The phosphorus uptake 1 (Pup1) locus was identified as a major quantitative trait locus (QTL) for tolerance of phosphorus deficiency in rice. Near-isogenic lines with the Pup1 region from tolerant donor parent Kasalath typically show threefold higher phosphorus uptake and grain yield in phosphorus-deficient field trials than the intolerant parent Nipponbare. In this study, we report the fine mapping of the Pup1 locus to the long arm of chromosome 12 (15.31-15.47 Mb). Genes in the region were initially identified on the basis of the Nipponbare reference genome, but did not reveal any obvious candidate genes related to phosphorus uptake. Kasalath BAC clones were therefore sequenced and revealed a 278-kbp sequence significantly different from the syntenic regions in Nipponbare (145 kb) and in the indica reference genome of 93-11 (742 kbp). Size differences are caused by large insertions or deletions (INDELs), and an exceptionally large number of retrotransposon and transposon-related elements (TEs) present in all three sequences (45%-54%). About 46 kb of the Kasalath sequence did not align with the entire Nipponbare genome, and only three Nipponbare genes (fatty acid alpha-dioxygenase, dirigent protein and aspartic proteinase) are highly conserved in Kasalath. Two Nipponbare genes (expressed proteins) might have evolved by at least three TE integrations in an ancestor gene that is still present in Kasalath. Several predicted Kasalath genes are novel or unknown genes that are mainly located within INDEL regions. Our results highlight the importance of sequencing QTL regions in the respective donor parent, as important genes might not be present in the current reference genomes.

  7. Deep Sea Coral voucher sequence dataset - Identification of deep-sea corals collected during the 2009 - 2014 West Coast Groundfish Bottom Trawl Survey

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — Data for this project resides in the West Coast Groundfish Bottom Trawl Survey Database. Deep-sea corals are often components of trawling bycatch, though their...

  8. Deconstructing the genetic basis of spent sulphite liquor tolerance using deep sequencing of genome-shuffled yeast.

    Science.gov (United States)

    Pinel, Dominic; Colatriano, David; Jiang, Heng; Lee, Hung; Martin, Vincent Jj

    2015-01-01

    Identifying the genetic basis of complex microbial phenotypes is currently a major barrier to our understanding of multigenic traits and our ability to rationally design biocatalysts with highly specific attributes for the biotechnology industry. Here, we demonstrate that strain evolution by meiotic recombination-based genome shuffling coupled with deep sequencing can be used to deconstruct complex phenotypes and explore the nature of multigenic traits, while providing concrete targets for strain development. We determined genomic variations found within Saccharomyces cerevisiae previously evolved in our laboratory by genome shuffling for tolerance to spent sulphite liquor. The representation of these variations was backtracked through parental mutant pools and cross-referenced with RNA-seq gene expression analysis to elucidate the importance of single mutations and key biological processes that play a role in our trait of interest. Our findings pinpoint novel genes and biological determinants of lignocellulosic hydrolysate inhibitor tolerance in yeast. These include the following: protein homeostasis constituents, including Ubp7p and Art5p, related to ubiquitin-mediated proteolysis; stress response transcriptional repressor, Nrg1p; and NADPH-dependent glutamate dehydrogenase, Gdh1p. Reverse engineering a prominent mutation in ubiquitin-specific protease gene UBP7 in a laboratory S. cerevisiae strain effectively increased spent sulphite liquor tolerance. This study advances understanding of yeast tolerance mechanisms to inhibitory substrates and biocatalyst design for a biomass-to-biofuel/biochemical industry, while providing insights into the process of mutation accumulation that occurs during genome shuffling.

  9. Quantitative analysis of polycomb response elements (PREs at identical genomic locations distinguishes contributions of PRE sequence and genomic environment

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    Okulski Helena

    2011-03-01

    Full Text Available Abstract Background Polycomb/Trithorax response elements (PREs are cis-regulatory elements essential for the regulation of several hundred developmentally important genes. However, the precise sequence requirements for PRE function are not fully understood, and it is also unclear whether these elements all function in a similar manner. Drosophila PRE reporter assays typically rely on random integration by P-element insertion, but PREs are extremely sensitive to genomic position. Results We adapted the ΦC31 site-specific integration tool to enable systematic quantitative comparison of PREs and sequence variants at identical genomic locations. In this adaptation, a miniwhite (mw reporter in combination with eye-pigment analysis gives a quantitative readout of PRE function. We compared the Hox PRE Frontabdominal-7 (Fab-7 with a PRE from the vestigial (vg gene at four landing sites. The analysis revealed that the Fab-7 and vg PREs have fundamentally different properties, both in terms of their interaction with the genomic environment at each site and their inherent silencing abilities. Furthermore, we used the ΦC31 tool to examine the effect of deletions and mutations in the vg PRE, identifying a 106 bp region containing a previously predicted motif (GTGT that is essential for silencing. Conclusions This analysis showed that different PREs have quantifiably different properties, and that changes in as few as four base pairs have profound effects on PRE function, thus illustrating the power and sensitivity of ΦC31 site-specific integration as a tool for the rapid and quantitative dissection of elements of PRE design.

  10. Using whole-genome sequence data to predict quantitative trait phenotypes in Drosophila melanogaster.

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    Ulrike Ober

    Full Text Available Predicting organismal phenotypes from genotype data is important for plant and animal breeding, medicine, and evolutionary biology. Genomic-based phenotype prediction has been applied for single-nucleotide polymorphism (SNP genotyping platforms, but not using complete genome sequences. Here, we report genomic prediction for starvation stress resistance and startle response in Drosophila melanogaster, using ∼2.5 million SNPs determined by sequencing the Drosophila Genetic Reference Panel population of inbred lines. We constructed a genomic relationship matrix from the SNP data and used it in a genomic best linear unbiased prediction (GBLUP model. We assessed predictive ability as the correlation between predicted genetic values and observed phenotypes by cross-validation, and found a predictive ability of 0.239±0.008 (0.230±0.012 for starvation resistance (startle response. The predictive ability of BayesB, a Bayesian method with internal SNP selection, was not greater than GBLUP. Selection of the 5% SNPs with either the highest absolute effect or variance explained did not improve predictive ability. Predictive ability decreased only when fewer than 150,000 SNPs were used to construct the genomic relationship matrix. We hypothesize that predictive power in this population stems from the SNP-based modeling of the subtle relationship structure caused by long-range linkage disequilibrium and not from population structure or SNPs in linkage disequilibrium with causal variants. We discuss the implications of these results for genomic prediction in other organisms.

  11. Transmission Bottleneck Size Estimation from Pathogen Deep-Sequencing Data, with an Application to Human Influenza A Virus.

    Science.gov (United States)

    Sobel Leonard, Ashley; Weissman, Daniel B; Greenbaum, Benjamin; Ghedin, Elodie; Koelle, Katia

    2017-07-15

    The bottleneck governing infectious disease transmission describes the size of the pathogen population transferred from the donor to the recipient host. Accurate quantification of the bottleneck size is particularly important for rapidly evolving pathogens such as influenza virus, as narrow bottlenecks reduce the amount of transferred viral genetic diversity and, thus, may decrease the rate of viral adaptation. Previous studies have estimated bottleneck sizes governing viral transmission by using statistical analyses of variants identified in pathogen sequencing data. These analyses, however, did not account for variant calling thresholds and stochastic viral replication dynamics within recipient hosts. Because these factors can skew bottleneck size estimates, we introduce a new method for inferring bottleneck sizes that accounts for these factors. Through the use of a simulated data set, we first show that our method, based on beta-binomial sampling, accurately recovers transmission bottleneck sizes, whereas other methods fail to do so. We then apply our method to a data set of influenza A virus (IAV) infections for which viral deep-sequencing data from transmission pairs are available. We find that the IAV transmission bottleneck size estimates in this study are highly variable across transmission pairs, while the mean bottleneck size of 196 virions is consistent with a previous estimate for this data set. Furthermore, regression analysis shows a positive association between estimated bottleneck size and donor infection severity, as measured by temperature. These results support findings from experimental transmission studies showing that bottleneck sizes across transmission events can be variable and influenced in part by epidemiological factors. IMPORTANCE The transmission bottleneck size describes the size of the pathogen population transferred from the donor to the recipient host and may affect the rate of pathogen adaptation within host populations. Recent

  12. Ultrasensitive quantitation of human papillomavirus type 16 E6 oncogene sequences by nested real time PCR

    Directory of Open Access Journals (Sweden)

    López-Revilla Rubén

    2010-05-01

    Full Text Available Abstract Background We have developed an ultrasensitive method based on conventional PCR preamplification followed by nested amplification through real time PCR (qPCR in the presence of the DNA intercalating agent EvaGreen. Results Amplification mixtures calibrated with a known number of pHV101 copies carrying a 645 base pair (bp-long insert of the human papillomavirus type 16 (HPV16 E6 oncogene were used to generate the E6-1 amplicon of 645 bp by conventional PCR and then the E6-2 amplicon of 237 bp by nested qPCR. Direct and nested qPCR mixtures for E6-2 amplification corresponding to 2.5 × 102-2.5 × 106 initial pHV101 copies had threshold cycle (Ct values in the ranges of 18.7-29.0 and 10.0-25.0, respectively. The Ct of qPCR mixtures prepared with 1/50 volumes of preamplified mixtures containing 50 ng of DNA of the SiHa cell line (derived from an invasive cervical cancer with one HPV16 genome per cell was 19.9. Thermal fluorescence extinction profiles of E6-2 amplicons generated from pHV101 and SiHa DNA were identical, with a peak at 85.5°C. Conclusions Our method based on conventional preamplification for 15 cycles increased 10,750 times the sensitivity of nested qPCR for the quantitation of the E6 viral oncogene and confirmed that the SiHa cell line contains one E6-HPV16 copy per cell.

  13. High-throughput deep sequencing reveals that microRNAs play important roles in salt tolerance of euhalophyte Salicornia europaea.

    Science.gov (United States)

    Feng, Juanjuan; Wang, Jinhui; Fan, Pengxiang; Jia, Weitao; Nie, Lingling; Jiang, Ping; Chen, Xianyang; Lv, Sulian; Wan, Lichuan; Chang, Sandra; Li, Shizhong; Li, Yinxin

    2015-02-26

    microRNAs (miRNAs) are implicated in plant development processes and play pivotal roles in plant adaptation to environmental stresses. Salicornia europaea, a salt mash euhalophyte, is a suitable model plant to study salt adaptation mechanisms. S. europaea is also a vegetable, forage, and oilseed that can be used for saline land reclamation and biofuel precursor production on marginal lands. Despite its importance, no miRNA has been identified from S. europaea thus far. Deep sequencing was performed to investigate small RNA transcriptome of S. europaea. Two hundred and ten conserved miRNAs comprising 51 families and 31 novel miRNAs (including seven miRNA star sequences) belonging to 30 families were identified. About half (13 out of 31) of the novel miRNAs were only detected in salt-treated samples. The expression of 43 conserved and 13 novel miRNAs significantly changed in response to salinity. In addition, 53 conserved and 13 novel miRNAs were differentially expressed between the shoots and roots. Furthermore, 306 and 195 S. europaea unigenes were predicted to be targets of 41 conserved and 29 novel miRNA families, respectively. These targets encoded a wide range of proteins, and genes involved in transcription regulation constituted the largest category. Four of these genes encoding laccase, F-box family protein, SAC3/GANP family protein, and NADPH cytochrome P-450 reductase were validated using 5'-RACE. Our results indicate that specific miRNAs are tightly regulated by salinity in the shoots and/or roots of S. europaea, which may play important roles in salt tolerance of this euhalophyte. The S. europaea salt-responsive miRNAs and miRNAs that target transcription factors, nucleotide binding site-leucine-rich repeat proteins and enzymes involved in lignin biosynthesis as well as carbon and nitrogen metabolism may be applied in genetic engineering of crops with high stress tolerance, and genetic modification of biofuel crops with high biomass and regulatable

  14. RNA deep sequencing reveals novel candidate genes and polymorphisms in boar testis and liver tissues with divergent androstenone levels.

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    Asep Gunawan

    Full Text Available Boar taint is an unpleasant smell and taste of pork meat derived from some entire male pigs. The main causes of boar taint are the two compounds androstenone (5α-androst-16-en-3-one and skatole (3-methylindole. It is crucial to understand the genetic mechanism of boar taint to select pigs for lower androstenone levels and thus reduce boar taint. The aim of the present study was to investigate transcriptome differences in boar testis and liver tissues with divergent androstenone levels using RNA deep sequencing (RNA-Seq. The total number of reads produced for each testis and liver sample ranged from 13,221,550 to 33,206,723 and 12,755,487 to 46,050,468, respectively. In testis samples 46 genes were differentially regulated whereas 25 genes showed differential expression in the liver. The fold change values ranged from -4.68 to 2.90 in testis samples and -2.86 to 3.89 in liver samples. Differentially regulated genes in high androstenone testis and liver samples were enriched in metabolic processes such as lipid metabolism, small molecule biochemistry and molecular transport. This study provides evidence for transcriptome profile and gene polymorphisms of boars with divergent androstenone level using RNA-Seq technology. Digital gene expression analysis identified candidate genes in flavin monooxygenease family, cytochrome P450 family and hydroxysteroid dehydrogenase family. Moreover, polymorphism and association analysis revealed mutation in IRG6, MX1, IFIT2, CYP7A1, FMO5 and KRT18 genes could be potential candidate markers for androstenone levels in boars. Further studies are required for proving the role of candidate genes to be used in genomic selection against boar taint in pig breeding programs.

  15. Combining real-time PCR and next-generation DNA sequencing to provide quantitative comparisons of fungal aerosol populations

    Science.gov (United States)

    Dannemiller, Karen C.; Lang-Yona, Naama; Yamamoto, Naomichi; Rudich, Yinon; Peccia, Jordan

    2014-02-01

    We examined fungal communities associated with the PM10 mass of Rehovot, Israel outdoor air samples collected in the spring and fall seasons. Fungal communities were described by 454 pyrosequencing of the internal transcribed spacer (ITS) region of the fungal ribosomal RNA encoding gene. To allow for a more quantitative comparison of fungal exposure in humans, the relative abundance values of specific taxa were transformed to absolute concentrations through multiplying these values by the sample's total fungal spore concentration (derived from universal fungal qPCR). Next, the sequencing-based absolute concentrations for Alternaria alternata, Cladosporium cladosporioides, Epicoccum nigrum, and Penicillium/Aspergillus spp. were compared to taxon-specific qPCR concentrations for A. alternata, C. cladosporioides, E. nigrum, and Penicillium/Aspergillus spp. derived from the same spring and fall aerosol samples. Results of these comparisons showed that the absolute concentration values generated from pyrosequencing were strongly associated with the concentration values derived from taxon-specific qPCR (for all four species, p 0.70). The correlation coefficients were greater for species present in higher concentrations. Our microbial aerosol population analyses demonstrated that fungal diversity (number of fungal operational taxonomic units) was higher in the spring compared to the fall (p = 0.02), and principal coordinate analysis showed distinct seasonal differences in taxa distribution (ANOSIM p = 0.004). Among genera containing allergenic and/or pathogenic species, the absolute concentrations of Alternaria, Aspergillus, Fusarium, and Cladosporium were greater in the fall, while Cryptococcus, Penicillium, and Ulocladium concentrations were greater in the spring. The transformation of pyrosequencing fungal population relative abundance data to absolute concentrations can improve next-generation DNA sequencing-based quantitative aerosol exposure assessment.

  16. Quantitative trait loci mapping of heat tolerance in broccoli (Brassica oleracea var. italica) using genotyping-by-sequencing.

    Science.gov (United States)

    Branham, Sandra E; Stansell, Zachary J; Couillard, David M; Farnham, Mark W

    2017-03-01

    Five quantitative trait loci and one epistatic interaction were associated with heat tolerance in a doubled haploid population of broccoli evaluated in three summer field trials. Predicted rising global temperatures due to climate change have generated a demand for crops that are resistant to yield and quality losses from heat stress. Broccoli (Brassica oleracea var. italica) is a cool weather crop with high temperatures during production decreasing both head quality and yield. Breeding for heat tolerance in broccoli has potential to both expand viable production areas and extend the growing season but breeding efficiency is constrained by limited genetic information. A doubled haploid (DH) broccoli population segregating for heat tolerance was evaluated for head quality in three summer fields in Charleston, SC, USA. Multiple quantitative trait loci (QTL) mapping of 1,423 single nucleotide polymorphisms developed through genotyping-by-sequencing identified five QTL and one positive epistatic interaction that explained 62.1% of variation in heat tolerance. The QTL identified here can be used to develop markers for marker-assisted selection and to increase our understanding of the molecular mechanisms underlying plant response to heat stress.

  17. Quantitative and qualitative differences in celiac disease epitopes among durum wheat varieties identified through deep RNA-amplican sequencing

    NARCIS (Netherlands)

    Salentijn, E.M.J.; Esselink, D.G.; Goryunova, S.V.; Meer, van der I.M.; Gilissen, L.J.W.J.; Smulders, M.J.M.

    2013-01-01

    Background - Wheat gluten is important for the industrial quality of bread wheat (Triticum aestivum L.) and durum wheat (T. turgidum L.). Gluten proteins are also the source of immunogenic peptides that can trigger a T cell reaction in celiac disease (CD) patients, leading to inflammatory responses

  18. Discovery of MicroRNAs associated with myogenesis by deep sequencing of serial developmental skeletal muscles in pigs.

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    Xinhua Hou

    Full Text Available MicroRNAs (miRNAs are short, single-stranded non-coding RNAs that repress their target genes by binding their 3' UTRs. These RNAs play critical roles in myogenesis. To gain knowledge about miRNAs involved in the regulation of myogenesis, porcine longissimus muscles were collected from 18 developmental stages (33-, 40-, 45-, 50-, 55-, 60-, 65-, 70-, 75-, 80-, 85-, 90-, 95-, 100- and 105-day post-gestation fetuses, 0 and 10-day postnatal piglets and adult pigs to identify miRNAs using Solexa sequencing technology. We detected 197 known miRNAs and 78 novel miRNAs according to comparison with known miRNAs in the miRBase (release 17.0 database. Moreover, variations in sequence length and single nucleotide polymorphisms were also observed in 110 known miRNAs. Expression analysis of the 11 most abundant miRNAs were conducted using quantitative PCR (qPCR in eleven tissues (longissimus muscles, leg muscles, heart, liver, spleen, lung, kidney, stomach, small intestine and colon, and the results revealed that ssc-miR-378, ssc-miR-1 and ssc-miR-206 were abundantly expressed in skeletal muscles. During skeletal muscle development, the expression level of ssc-miR-378 was low at 33 days post-coitus (dpc, increased at 65 and 90 dpc, peaked at postnatal day 0, and finally declined and maintained a comparatively stable level. This expression profile suggested that ssc-miR-378 was a new candidate miRNA for myogenesis and participated in skeletal muscle development in pigs. Target prediction and KEGG pathway analysis suggested that bone morphogenetic protein 2 (BMP2 and mitogen-activated protein kinase 1 (MAPK1, both of which were relevant to proliferation and differentiation, might be the potential targets of miR-378. Luciferase activities of report vectors containing the 3'UTR of porcine BMP2 or MAPK1 were downregulated by miR-378, which suggested that miR-378 probably regulated myogenesis though the regulation of these two genes.

  19. Genome-wide identification of soybean microRNA responsive to soybean cyst nematodes infection by deep sequencing.

    Science.gov (United States)

    Tian, Bin; Wang, Shichen; Todd, Timothy C; Johnson, Charles D; Tang, Guiliang; Trick, Harold N

    2017-08-02

    The soybean cyst nematode (SCN), Heterodera glycines, is one of the most devastating diseases limiting soybean production worldwide. It is known that small RNAs, including microRNAs (miRNAs) and small interfering RNAs (siRNAs), play important roles in regulating plant growth and development, defense against pathogens, and responses to environmental changes. In order to understand the role of soybean miRNAs during SCN infection, we analyzed 24 small RNA libraries including three biological replicates from two soybean cultivars (SCN susceptible KS4607, and SCN HG Type 7 resistant KS4313N) that were grown under SCN-infested and -noninfested soil at two different time points (SCN feeding establishment and egg production). In total, 537 known and 70 putative novel miRNAs in soybean were identified from a total of 0.3 billion reads (average about 13.5 million reads for each sample) with the programs of Bowtie and miRDeep2 mapper. Differential expression analyses were carried out using edgeR to identify miRNAs involved in the soybean-SCN interaction. Comparative analysis of miRNA profiling indicated a total of 60 miRNAs belonging to 25 families that might be specifically related to cultivar responses to SCN. Quantitative RT-PCR validated similar miRNA interaction patterns as sequencing results. These findings suggest that miRNAs are likely to play key roles in soybean response to SCN. The present work could provide a framework for miRNA functional identification and the development of novel approaches for improving soybean SCN resistance in future studies.

  20. Deep Sequencing of Three Loci Implicated in Large-Scale Genome-Wide Association Study Smoking Meta-Analyses.

    Science.gov (United States)

    Clark, Shaunna L; McClay, Joseph L; Adkins, Daniel E; Aberg, Karolina A; Kumar, Gaurav; Nerella, Sri; Xie, Linying; Collins, Ann L; Crowley, James J; Quakenbush, Corey R; Hillard, Christopher E; Gao, Guimin; Shabalin, Andrey A; Peterson, Roseann E; Copeland, William E; Silberg, Judy L; Maes, Hermine; Sullivan, Patrick F; Costello, Elizabeth J; van den Oord, Edwin J

    2016-05-01

    Genome-wide association study meta-analyses have robustly implicated three loci that affect susceptibility for smoking: CHRNA5\\CHRNA3\\CHRNB4, CHRNB3\\CHRNA6 and EGLN2\\CYP2A6. Functional follow-up studies of these loci are needed to provide insight into biological mechanisms. However, these efforts have been hampered by a lack of knowledge about the specific causal variant(s) involved. In this study, we prioritized variants in terms of the likelihood they account for the reported associations. We employed targeted capture of the CHRNA5\\CHRNA3\\CHRNB4, CHRNB3\\CHRNA6, and EGLN2\\CYP2A6 loci and flanking regions followed by next-generation deep sequencing (mean coverage 78×) to capture genomic variation in 363 individuals. We performed single locus tests to determine if any single variant accounts for the association, and examined if sets of (rare) variants that overlapped with biologically meaningful annotations account for the associations. In total, we investigated 963 variants, of which 71.1% were rare (minor allele frequency < 0.01), 6.02% were insertion/deletions, and 51.7% were catalogued in dbSNP141. The single variant results showed that no variant fully accounts for the association in any region. In the variant set results, CHRNB4 accounts for most of the signal with significant sets consisting of directly damaging variants. CHRNA6 explains most of the signal in the CHRNB3\\CHRNA6 locus with significant sets indicating a regulatory role for CHRNA6. Significant sets in CYP2A6 involved directly damaging variants while the significant variant sets suggested a regulatory role for EGLN2. We found that multiple variants implicating multiple processes explain the signal. Some variants can be prioritized for functional follow-up. © The Author 2015. Published by Oxford University Press on behalf of the Society for Research on Nicotine and Tobacco. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  1. Phylogenetic and genome-wide deep-sequencing analyses of canine parvovirus reveal co-infection with field variants and emergence of a recent recombinant strain.

    Directory of Open Access Journals (Sweden)

    Ruben Pérez

    Full Text Available Canine parvovirus (CPV, a fast-evolving single-stranded DNA virus, comprises three antigenic variants (2a, 2b, and 2c with different frequencies and genetic variability among countries. The contribution of co-infection and recombination to the genetic variability of CPV is far from being fully elucidated. Here we took advantage of a natural CPV population, recently formed by the convergence of divergent CPV-2c and CPV-2a strains, to study co-infection and recombination. Complete sequences of the viral coding region of CPV-2a and CPV-2c strains from 40 samples were generated and analyzed using phylogenetic tools. Two samples showed co-infection and were further analyzed by deep sequencing. The sequence profile of one of the samples revealed the presence of CPV-2c and CPV-2a strains that differed at 29 nucleotides. The other sample included a minor CPV-2a strain (13.3% of the viral population and a major recombinant strain (86.7%. The recombinant strain arose from inter-genotypic recombination between CPV-2c and CPV-2a strains within the VP1/VP2 gene boundary. Our findings highlight the importance of deep-sequencing analysis to provide a better understanding of CPV molecular diversity.

  2. Phylogenetic and Genome-Wide Deep-Sequencing Analyses of Canine Parvovirus Reveal Co-Infection with Field Variants and Emergence of a Recent Recombinant Strain

    Science.gov (United States)

    Pérez, Ruben; Calleros, Lucía; Marandino, Ana; Sarute, Nicolás; Iraola, Gregorio; Grecco, Sofia; Blanc, Hervé; Vignuzzi, Marco; Isakov, Ofer; Shomron, Noam; Carrau, Lucía; Hernández, Martín; Francia, Lourdes; Sosa, Katia; Tomás, Gonzalo; Panzera, Yanina

    2014-01-01

    Canine parvovirus (CPV), a fast-evolving single-stranded DNA virus, comprises three antigenic variants (2a, 2b, and 2c) with different frequencies and genetic variability among countries. The contribution of co-infection and recombination to the genetic variability of CPV is far from being fully elucidated. Here we took advantage of a natural CPV population, recently formed by the convergence of divergent CPV-2c and CPV-2a strains, to study co-infection and recombination. Complete sequences of the viral coding region of CPV-2a and CPV-2c strains from 40 samples were generated and analyzed using phylogenetic tools. Two samples showed co-infection and were further analyzed by deep sequencing. The sequence profile of one of the samples revealed the presence of CPV-2c and CPV-2a strains that differed at 29 nucleotides. The other sample included a minor CPV-2a strain (13.3% of the viral population) and a major recombinant strain (86.7%). The recombinant strain arose from inter-genotypic recombination between CPV-2c and CPV-2a strains within the VP1/VP2 gene boundary. Our findings highlight the importance of deep-sequencing analysis to provide a better understanding of CPV molecular diversity. PMID:25365348

  3. Deep-sequencing to resolve complex diversity of apicomplexan parasites in platypuses and echidnas: Proof of principle for wildlife disease investigation.

    Science.gov (United States)

    Šlapeta, Jan; Saverimuttu, Stefan; Vogelnest, Larry; Sangster, Cheryl; Hulst, Frances; Rose, Karrie; Thompson, Paul; Whittington, Richard

    2017-11-01

    The short-beaked echidna (Tachyglossus aculeatus) and the platypus (Ornithorhynchus anatinus) are iconic egg-laying monotremes (Mammalia: Monotremata) from Australasia. The aim of this study was to demonstrate the utility of diversity profiles in disease investigations of monotremes. Using small subunit (18S) rDNA amplicon deep-sequencing we demonstrated the presence of apicomplexan parasites and confirmed by direct and cloned amplicon gene sequencing Theileria ornithorhynchi, Theileria tachyglossi, Eimeria echidnae and Cryptosporidium fayeri. Using a combination of samples from healthy and diseased animals, we show a close evolutionary relationship between species of coccidia (Eimeria) and piroplasms (Theileria) from the echidna and platypus. The presence of E. echidnae was demonstrated in faeces and tissues affected by disseminated coccidiosis. Moreover, the presence of E. echidnae DNA in the blood of echidnas was associated with atoxoplasma-like stages in white blood cells, suggesting Hepatozoon tachyglossi blood stages are disseminated E. echidnae stages. These next-generation DNA sequencing technologies are suited to material and organisms that have not been previously characterised and for which the material is scarce. The deep sequencing approach supports traditional diagnostic methods, including microscopy, clinical pathology and histopathology, to better define the status quo. This approach is particularly suitable for wildlife disease investigation. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Genomic variation in macrophage-cultured European porcine reproductive and respiratory syndrome virus Olot/91 revealed using ultra-deep next generation sequencing.

    Science.gov (United States)

    Lu, Zen H; Brown, Alexander; Wilson, Alison D; Calvert, Jay G; Balasch, Monica; Fuentes-Utrilla, Pablo; Loecherbach, Julia; Turner, Frances; Talbot, Richard; Archibald, Alan L; Ait-Ali, Tahar

    2014-03-04

    Porcine Reproductive and Respiratory Syndrome (PRRS) is a disease of major economic impact worldwide. The etiologic agent of this disease is the PRRS virus (PRRSV). Increasing evidence suggest that microevolution within a coexisting quasispecies population can give rise to high sequence heterogeneity in PRRSV. We developed a pipeline based on the ultra-deep next generation sequencing approach to first construct the complete genome of a European PRRSV, strain Olot/9, cultured on macrophages and then capture the rare variants representative of the mixed quasispecies population. Olot/91 differs from the reference Lelystad strain by about 5% and a total of 88 variants, with frequencies as low as 1%, were detected in the mixed population. These variants included 16 non-synonymous variants concentrated in the genes encoding structural and nonstructural proteins; including Glycoprotein 2a and 5. Using an ultra-deep sequencing methodology, the complete genome of Olot/91 was constructed without any prior knowledge of the sequence. Rare variants that constitute minor fractions of the heterogeneous PRRSV population could successfully be detected to allow further exploration of microevolutionary events.

  5. deepBase v2.0: identification, expression, evolution and function of small RNAs, LncRNAs and circular RNAs from deep-sequencing data.

    Science.gov (United States)

    Zheng, Ling-Ling; Li, Jun-Hao; Wu, Jie; Sun, Wen-Ju; Liu, Shun; Wang, Ze-Lin; Zhou, Hui; Yang, Jian-Hua; Qu, Liang-Hu

    2016-01-04

    Small non-coding RNAs (e.g. miRNAs) and long non-coding RNAs (e.g. lincRNAs and circRNAs) are emerging as key regulators of various cellular processes. However, only a very small fraction of these enigmatic RNAs have been well functionally characterized. In this study, we describe deepBase v2.0 (http://biocenter.sysu.edu.cn/deepBase/), an updated platform, to decode evolution, expression patterns and functions of diverse ncRNAs across 19 species. deepBase v2.0 has been updated to provide the most comprehensive collection of ncRNA-derived small RNAs generated from 588 sRNA-Seq datasets. Moreover, we developed a pipeline named lncSeeker to identify 176 680 high-confidence lncRNAs from 14 species. Temporal and spatial expression patterns of various ncRNAs were profiled. We identified approximately 24 280 primate-specific, 5193 rodent-specific lncRNAs, and 55 highly conserved lncRNA orthologs between human and zebrafish. We annotated 14 867 human circRNAs, 1260 of which are orthologous to mouse circRNAs. By combining expression profiles and functional genomic annotations, we developed lncFunction web-server to predict the function of lncRNAs based on protein-lncRNA co-expression networks. This study is expected to provide considerable resources to facilitate future experimental studies and to uncover ncRNA functions. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  6. Complete genome sequence of the aerobic, heterotroph Marinithermus hydrothermalis type strain (T1T) from a deep-sea hydrothermal vent chimney

    Energy Technology Data Exchange (ETDEWEB)

    Copeland, A [U.S. Department of Energy, Joint Genome Institute; Gu, Wei [U.S. Department of Energy, Joint Genome Institute; Yasawong, Montri [HZI - Helmholtz Centre for Infection Research, Braunschweig, Germany; Lapidus, Alla L. [U.S. Department of Energy, Joint Genome Institute; Lucas, Susan [U.S. Department of Energy, Joint Genome Institute; Deshpande, Shweta [U.S. Department of Energy, Joint Genome Institute; Pagani, Ioanna [U.S. Department of Energy, Joint Genome Institute; Tapia, Roxanne [Los Alamos National Laboratory (LANL); Cheng, Jan-Fang [U.S. Department of Energy, Joint Genome Institute; Goodwin, Lynne A. [Los Alamos National Laboratory (LANL); Pitluck, Sam [U.S. Department of Energy, Joint Genome Institute; Liolios, Konstantinos [U.S. Department of Energy, Joint Genome Institute; Ivanova, N [U.S. Department of Energy, Joint Genome Institute; Mavromatis, K [U.S. Department of Energy, Joint Genome Institute; Mikhailova, Natalia [U.S. Department of Energy, Joint Genome Institute; Pati, Amrita [U.S. Department of Energy, Joint Genome Institute; Chen, Amy [U.S. Department of Energy, Joint Genome Institute; Palaniappan, Krishna [U.S. Department of Energy, Joint Genome Institute; Land, Miriam L [ORNL; Pan, Chongle [ORNL; Brambilla, Evelyne-Marie [DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany; Rohde, Manfred [HZI - Helmholtz Centre for Infection Research, Braunschweig, Germany; Tindall, Brian [DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany; Sikorski, Johannes [DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany; Goker, Markus [DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany; Detter, J. Chris [U.S. Department of Energy, Joint Genome Institute; Bristow, James [U.S. Department of Energy, Joint Genome Institute; Eisen, Jonathan [U.S. Department of Energy, Joint Genome Institute; Markowitz, Victor [U.S. Department of Energy, Joint Genome Institute; Hugenholtz, Philip [U.S. Department of Energy, Joint Genome Institute; Kyrpides, Nikos C [U.S. Department of Energy, Joint Genome Institute; Klenk, Hans-Peter [DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany; Woyke, Tanja [U.S. Department of Energy, Joint Genome Institute

    2012-01-01

    Marinithermus hydrothermalis Sako et al. 2003 is the type species of the monotypic genus Marinithermus. M. hydrothermalis T1 T was the first isolate within the phylum ThermusDeinococcus to exhibit optimal growth under a salinity equivalent to that of sea water and to have an absolute requirement for NaCl for growth. M. hydrothermalis T1 T is of interest because it may provide a new insight into the ecological significance of the aerobic, thermophilic decomposers in the circulation of organic compounds in deep-sea hydrothermal vent ecosystems. This is the first completed genome sequence of a member of the genus Marinithermus and the seventh sequence from the family Thermaceae. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 2,269,167 bp long genome with its 2,251 protein-coding and 59 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.

  7. Complete genome sequence of the aerobic, heterotroph Marinithermus hydrothermalis type strain (T1(T)) from a deep-sea hydrothermal vent chimney.

    Science.gov (United States)

    Copeland, Alex; Gu, Wei; Yasawong, Montri; Lapidus, Alla; Lucas, Susan; Deshpande, Shweta; Pagani, Ioanna; Tapia, Roxanne; Cheng, Jan-Fang; Goodwin, Lynne A; Pitluck, Sam; Liolios, Konstantinos; Ivanova, Natalia; Mavromatis, Konstantinos; Mikhailova, Natalia; Pati, Amrita; Chen, Amy; Palaniappan, Krishna; Land, Miriam; Pan, Chongle; Brambilla, Evelyne-Marie; Rohde, Manfred; Tindall, Brian J; Sikorski, Johannes; Göker, Markus; Detter, John C; Bristow, James; Eisen, Jonathan A; Markowitz, Victor; Hugenholtz, Philip; Kyrpides, Nikos C; Klenk, Hans-Peter; Woyke, Tanja

    2012-03-19

    Marinithermus hydrothermalis Sako et al. 2003 is the type species of the monotypic genus Marinithermus. M. hydrothermalis T1(T) was the first isolate within the phylum "Thermus-Deinococcus" to exhibit optimal growth under a salinity equivalent to that of sea water and to have an absolute requirement for NaCl for growth. M. hydrothermalis T1(T) is of interest because it may provide a new insight into the ecological significance of the aerobic, thermophilic decomposers in the circulation of organic compounds in deep-sea hydrothermal vent ecosystems. This is the first completed genome sequence of a member of the genus Marinithermus and the seventh sequence from the family Thermaceae. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 2,269,167 bp long genome with its 2,251 protein-coding and 59 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.

  8. An improved method for quantitatively measuring the sequences of total organic carbon and black carbon in marine sediment cores

    Science.gov (United States)

    Xu, Xiaoming; Zhu, Qing; Zhou, Qianzhi; Liu, Jinzhong; Yuan, Jianping; Wang, Jianghai

    2018-01-01

    Understanding global carbon cycle is critical to uncover the mechanisms of global warming and remediate its adverse effects on human activities. Organic carbon in marine sediments is an indispensable part of the global carbon reservoir in global carbon cycling. Evaluating such a reservoir calls for quantitative studies of marine carbon burial, which closely depend on quantifying total organic carbon and black carbon in marine sediment cores and subsequently on obtaining their high-resolution temporal sequences. However, the conventional methods for detecting the contents of total organic carbon or black carbon cannot resolve the following specific difficulties, i.e., (1) a very limited amount of each subsample versus the diverse analytical items, (2) a low and fluctuating recovery rate of total organic carbon or black carbon versus the reproducibility of carbon data, and (3) a large number of subsamples versus the rapid batch measurements. In this work, (i) adopting the customized disposable ceramic crucibles with the microporecontrolled ability, (ii) developing self-made or customized facilities for the procedures of acidification and chemothermal oxidization, and (iii) optimizing procedures and carbon-sulfur analyzer, we have built a novel Wang-Xu-Yuan method (the WXY method) for measuring the contents of total organic carbon or black carbon in marine sediment cores, which includes the procedures of pretreatment, weighing, acidification, chemothermal oxidation and quantification; and can fully meet the requirements of establishing their highresolution temporal sequences, whatever in the recovery, experimental efficiency, accuracy and reliability of the measurements, and homogeneity of samples. In particular, the usage of disposable ceramic crucibles leads to evidently simplify the experimental scenario, which further results in the very high recovery rates for total organic carbon and black carbon. This new technique may provide a significant support for

  9. Quantitative Single-letter Sequencing: a method for simultaneously monitoring numerous known allelic variants in single DNA samples

    Directory of Open Access Journals (Sweden)

    Duborjal Hervé

    2008-02-01

    Full Text Available Abstract Background Pathogens such as fungi, bacteria and especially viruses, are highly variable even within an individual host, intensifying the difficulty of distinguishing and accurately quantifying numerous allelic variants co-existing in a single nucleic acid sample. The majority of currently available techniques are based on real-time PCR or primer extension and often require multiplexing adjustments that impose a practical limitation of the number of alleles that can be monitored simultaneously at a single locus. Results Here, we describe a novel method that allows the simultaneous quantification of numerous allelic variants in a single reaction tube and without multiplexing. Quantitative Single-letter Sequencing (QSS begins with a single PCR amplification step using a pair of primers flanking the polymorphic region of interest. Next, PCR products are submitted to single-letter sequencing with a fluorescently-labelled primer located upstream of the polymorphic region. The resulting monochromatic electropherogram shows numerous specific diagnostic peaks, attributable to specific variants, signifying their presence/absence in the DNA sample. Moreover, peak fluorescence can be quantified and used to estimate the frequency of the corresponding variant in the DNA population. Using engineered allelic markers in the genome of Cauliflower mosaic virus, we reliably monitored six different viral genotypes in DNA extracted from infected plants. Evaluation of the intrinsic variance of this method, as applied to both artificial plasmid DNA mixes and viral genome populations, demonstrates that QSS is a robust and reliable method of detection and quantification for variants with a relative frequency of between 0.05 and 1. Conclusion This simple method is easily transferable to many other biological systems and questions, including those involving high throughput analysis, and can be performed in any laboratory since it does not require specialized

  10. Universal and blocking primer mismatches limit the use of high-throughput DNA sequencing for the quantitative metabarcoding of arthropods.

    Science.gov (United States)

    Piñol, J; Mir, G; Gomez-Polo, P; Agustí, N

    2015-07-01

    The quantification of the biological diversity in environmental samples using high-throughput DNA sequencing is hindered by the PCR bias caused by variable primer-template mismatches of the individual species. In some dietary studies, there is the added problem that samples are enriched with predator DNA, so often a predator-specific blocking oligonucleotide is used to alleviate the problem. However, specific blocking oligonucleotides could coblock nontarget species to some degree. Here, we accurately estimate the extent of the PCR biases induced by universal and blocking primers on a mock community prepared with DNA of twelve species of terrestrial arthropods. We also compare universal and blocking primer biases with those induced by variable annealing temperature and number of PCR cycles. The results show that reads of all species were recovered after PCR enrichment at our control conditions (no blocking oligonucleotide, 45 °C annealing temperature and 40 cycles) and high-throughput sequencing. They also show that the four factors considered biased the final proportions of the species to some degree. Among these factors, the number of primer-template mismatches of each species had a disproportionate effect (up to five orders of magnitude) on the amplification efficiency. In particular, the number of primer-template mismatches explained most of the variation (~3/4) in the amplification efficiency of the species. The effect of blocking oligonucleotide concentration on nontarget species relative abundance was also significant, but less important (below one order of magnitude). Considering the results reported here, the quantitative potential of the technique is limited, and only qualitative results (the species list) are reliable, at least when targeting the barcoding COI region. © 2014 John Wiley & Sons Ltd.

  11. Arthropod phylogenetics in light of three novel millipede (myriapoda: diplopoda) mitochondrial genomes with comments on the appropriateness of mitochondrial genome sequence data for inferring deep level relationships.

    Science.gov (United States)

    Brewer, Michael S; Swafford, Lynn; Spruill, Chad L; Bond, Jason E

    2013-01-01

    Arthropods are the most diverse group of eukaryotic organisms, but their phylogenetic relationships are poorly understood. Herein, we describe three mitochondrial genomes representing orders of millipedes for which complete genomes had not been characterized. Newly sequenced genomes are combined with existing data to characterize the protein coding regions of myriapods and to attempt to reconstruct the evolutionary relationships within the Myriapoda and Arthropoda. The newly sequenced genomes are similar to previously characterized millipede sequences in terms of synteny and length. Unique translocations occurred within the newly sequenced taxa, including one half of the Appalachioria falcifera genome, which is inverted with respect to other millipede genomes. Across myriapods, amino acid conservation levels are highly dependent on the gene region. Additionally, individual loci varied in the level of amino acid conservation. Overall, most gene regions showed low levels of conservation at many sites. Attempts to reconstruct the evolutionary relationships suffered from questionable relationships and low support values. Analyses of phylogenetic informativeness show the lack of signal deep in the trees (i.e., genes evolve too quickly). As a result, the myriapod tree resembles previously published results but lacks convincing support, and, within the arthropod tree, well established groups were recovered as polyphyletic. The novel genome sequences described herein provide useful genomic information concerning millipede groups that had not been investigated. Taken together with existing sequences, the variety of compositions and evolution of myriapod mitochondrial genomes are shown to be more complex than previously thought. Unfortunately, the use of mitochondrial protein-coding regions in deep arthropod phylogenetics appears problematic, a result consistent with previously published studies. Lack of phylogenetic signal renders the resulting tree topologies as suspect

  12. Arthropod phylogenetics in light of three novel millipede (myriapoda: diplopoda mitochondrial genomes with comments on the appropriateness of mitochondrial genome sequence data for inferring deep level relationships.

    Directory of Open Access Journals (Sweden)

    Michael S Brewer

    Full Text Available BACKGROUND: Arthropods are the most diverse group of eukaryotic organisms, but their phylogenetic relationships are poorly understood. Herein, we describe three mitochondrial genomes representing orders of millipedes for which complete genomes had not been characterized. Newly sequenced genomes are combined with existing data to characterize the protein coding regions of myriapods and to attempt to reconstruct the evolutionary relationships within the Myriapoda and Arthropoda. RESULTS: The newly sequenced genomes are similar to previously characterized millipede sequences in terms of synteny and length. Unique translocations occurred within the newly sequenced taxa, including one half of the Appalachioria falcifera genome, which is inverted with respect to other millipede genomes. Across myriapods, amino acid conservation levels are highly dependent on the gene region. Additionally, individual loci varied in the level of amino acid conservation. Overall, most gene regions showed low levels of conservation at many sites. Attempts to reconstruct the evolutionary relationships suffered from questionable relationships and low support values. Analyses of phylogenetic informativeness show the lack of signal deep in the trees (i.e., genes evolve too quickly. As a result, the myriapod tree resembles previously published results but lacks convincing support, and, within the arthropod tree, well established groups were recovered as polyphyletic. CONCLUSIONS: The novel genome sequences described herein provide useful genomic information concerning millipede groups that had not been investigated. Taken together with existing sequences, the variety of compositions and evolution of myriapod mitochondrial genomes are shown to be more complex than previously thought. Unfortunately, the use of mitochondrial protein-coding regions in deep arthropod phylogenetics appears problematic, a result consistent with previously published studies. Lack of phylogenetic

  13. Transcriptional Slippage and RNA Editing Increase the Diversity of Transcripts in Chloroplasts: Insight from Deep Sequencing of Vigna radiata Genome and Transcriptome.

    Directory of Open Access Journals (Sweden)

    Ching-Ping Lin

    Full Text Available We performed deep sequencing of the nuclear and organellar genomes of three mungbean genotypes: Vigna radiata ssp. sublobata TC1966, V. radiata var. radiata NM92 and the recombinant inbred line RIL59 derived from a cross between TC1966 and NM92. Moreover, we performed deep sequencing of the RIL59 transcriptome to investigate transcript variability. The mungbean chloroplast genome has a quadripartite structure including a pair of inverted repeats separated by two single copy regions. A total of 213 simple sequence repeats were identified in the chloroplast genomes of NM92 and RIL59; 78 single nucleotide variants and nine indels were discovered in comparing the chloroplast genomes of TC1966 and NM92. Analysis of the mungbean chloroplast transcriptome revealed mRNAs that were affected by transcriptional slippage and RNA editing. Transcriptional slippage frequency was positively correlated with the length of simple sequence repeats of the mungbean chloroplast genome (R2=0.9911. In total, 41 C-to-U editing sites were found in 23 chloroplast genes and in one intergenic spacer. No editing site that swapped U to C was found. A combination of bioinformatics and experimental methods revealed that the plastid-encoded RNA polymerase-transcribed genes psbF and ndhA are affected by transcriptional slippage in mungbean and in main lineages of land plants, including three dicots (Glycine max, Brassica rapa, and Nicotiana tabacum, two monocots (Oryza sativa and Zea mays, two gymnosperms (Pinus taeda and Ginkgo biloba and one moss (Physcomitrella patens. Transcript analysis of the rps2 gene showed that transcriptional slippage could affect transcripts at single sequence repeat regions with poly-A runs. It showed that transcriptional slippage together with incomplete RNA editing may cause sequence diversity of transcripts in chloroplasts of land plants.

  14. Toxic Cyanobacterial Bloom Triggers in Missisquoi Bay, Lake Champlain, as Determined by Next-Generation Sequencing and Quantitative PCR

    Directory of Open Access Journals (Sweden)

    Nathalie Fortin

    2015-05-01

    Full Text Available Missisquoi Bay (MB is a temperate eutrophic freshwater lake that frequently experiences toxic Microcystis-dominated cyanobacterial blooms. Non-point sources are responsible for the high concentrations of phosphorus and nitrogen in the bay. This study combined data from environmental parameters, E. coli counts, high-throughput sequencing of 16S rRNA gene amplicons, quantitative PCR (16S rRNA and mcyD genes and toxin analyses to identify the main bloom-promoting factors. In 2009, nutrient concentrations correlated with E. coli counts, abundance of total cyanobacterial cells, Microcystis 16S rRNA and mcyD genes and intracellular microcystin. Total and dissolved phosphorus also correlated significantly with rainfall. The major cyanobacterial taxa were members of the orders Chroococcales and Nostocales. The genus Microcystis was the main mcyD-carrier and main microcystin producer. Our results suggested that increasing nutrient concentrations and total nitrogen:total phosphorus (TN:TP ratios approaching 11:1, coupled with an increase in temperature, promoted Microcystis-dominated toxic blooms. Although the importance of nutrient ratios and absolute concentrations on cyanobacterial and Microcystis dynamics have been documented in other laboratories, an optimum TN:TP ratio for Microcystis dominance has not been previously observed in situ. This observation provides further support that nutrient ratios are an important determinant of species composition in natural phytoplankton assemblages.

  15. High-fidelity target sequencing of individual molecules identified using barcode sequences: de novo detection and absolute quantitation of mutations in plasma cell-free DNA from cancer patients.

    Science.gov (United States)

    Kukita, Yoji; Matoba, Ryo; Uchida, Junji; Hamakawa, Takuya; Doki, Yuichiro; Imamura, Fumio; Kato, Kikuya

    2015-08-01

    Circulating tumour DNA (ctDNA) is an emerging field of cancer research. However, current ctDNA analysis is usually restricted to one or a few mutation sites due to technical limitations. In the case of massively parallel DNA sequencers, the number of false positives caused by a high read error rate is a major problem. In addition, the final sequence reads do not represent the original DNA population due to the global amplification step during the template preparation. We established a high-fidelity target sequencing system of individual molecules identified in plasma cell-free DNA using barcode sequences; this system consists of the following two steps. (i) A novel target sequencing method that adds barcode sequences by adaptor ligation. This method uses linear amplification to eliminate the errors introduced during the early cycles of polymerase chain reaction. (ii) The monitoring and removal of erroneous barcode tags. This process involves the identification of individual molecules that have been sequenced and for which the number of mutations have been absolute quantitated. Using plasma cell-free DNA from patients with gastric or lung cancer, we demonstrated that the system achieved near complete elimination of false positives and enabled de novo detection and absolute quantitation of mutations in plasma cell-free DNA. © The Author 2015. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

  16. Prevalence of Hepatitis C Virus Subgenotypes 1a and 1b in Japanese Patients: Ultra-Deep Sequencing Analysis of HCV NS5B Genotype-Specific Region

    Science.gov (United States)

    Wu, Shuang; Kanda, Tatsuo; Nakamoto, Shingo; Jiang, Xia; Miyamura, Tatsuo; Nakatani, Sueli M.; Ono, Suzane Kioko; Takahashi-Nakaguchi, Azusa; Gonoi, Tohru; Yokosuka, Osamu

    2013-01-01

    Background Hepatitis C virus (HCV) subgenotypes 1a and 1b have different impacts on the treatment response to peginterferon plus ribavirin with direct-acting antivirals (DAAs) against patients infected with HCV genotype 1, as the emergence rates of resistance mutations are different between these two subgenotypes. In Japan, almost all of HCV genotype 1 belongs to subgenotype 1b. Methods and Findings To determine HCV subgenotype 1a or 1b in Japanese patients infected with HCV genotype 1, real-time PCR-based method and Sanger method were used for the HCV NS5B region. HCV subgenotypes were determined in 90% by real-time PCR-based method. We also analyzed the specific probe regions for HCV subgenotypes 1a and 1b using ultra-deep sequencing, and uncovered mutations that could not be revealed using direct-sequencing by Sanger method. We estimated the prevalence of HCV subgenotype 1a as 1.2-2.5% of HCV genotype 1 patients in Japan. Conclusions Although real-time PCR-based HCV subgenotyping method seems fair for differentiating HCV subgenotypes 1a and 1b, it may not be sufficient for clinical practice. Ultra-deep sequencing is useful for revealing the resistant strain(s) of HCV before DAA treatment as well as mixed infection with different genotypes or subgenotypes of HCV. PMID:24069214

  17. NNAlign: A Web-Based Prediction Method Allowing Non-Expert End-User Discovery of Sequence Motifs in Quantitative Peptide Data

    DEFF Research Database (Denmark)

    Andreatta, Massimo; Schafer-Nielsen, Claus; Lund, Ole

    2011-01-01

    Recent advances in high-throughput technologies have made it possible to generate both gene and protein sequence data at an unprecedented rate and scale thereby enabling entirely new "omics"-based approaches towards the analysis of complex biological processes. However, the amount and complexity...... to interpret large data sets. We have recently developed a method, NNAlign, which is generally applicable to any biological problem where quantitative peptide data is available. This method efficiently identifies underlying sequence patterns by simultaneously aligning peptide sequences and identifying motifs...... associated with quantitative readouts. Here, we provide a web-based implementation of NNAlign allowing non-expert end-users to submit their data (optionally adjusting method parameters), and in return receive a trained method (including a visual representation of the identified motif) that subsequently can...

  18. Quantitative controls on location and architecture of carbonate depositional sequences: upper miocene, cabo de gata region, se Spain

    Science.gov (United States)

    Franseen, E.K.; Goldstein, R.H.; Farr, M.R.

    1998-01-01

    Sequence stratigraphy, pinning-point relative sea-level curves, and magnetostratigraphy provide the quantitative data necessary to understand how rates of sea-level change and different substrate paleoslopes are dominant controls on accumulation rate, carbonate depositional sequence location, and internal architecture. Five third-order (1-10 my) and fourth-order (0.1-1.0 my) upper Miocene carbonate depositional sequences (DS1A, DS1B, DS2, DS3, TCC) formed with superimposed higher-frequency sea-level cycles in an archipelago setting in SE Spain. Overall, our study indicates when areas of high substrate slope (> 15??) are in shallow water, independent of climate, the location and internal architecture of carbonate deposits are not directly linked to sea-level position but, instead, are controlled by location of gently sloping substrates and processes of bypass. In contrast, if carbonate sediments are generated where substrates of low slope ( 15.6 cm/ky to ??? 2 cm/ky and overall relative sea level rose at rates of 17-21.4 cm/ky. Higher frequency sea-level rates were about 111 to more than 260 cm/ky, producing onlapping, fining- (deepening-) upward cycles. Decreasing accumulation rates resulted from decreasing surface area for shallow-water sediment production, drowning of shallow-water substrates, and complex sediment dispersal related to the archipelago setting. Typical systems tract and parasequence development should not be expected in "bypass ramp" settings; facies of onlapping strata do not track base level and are likely to be significantly different compared to onlapping strata associated with coastal onlap. Basal and upper DS2 reef megabreccias (indicating the transition from cool to warmer climatic conditions) were eroded from steep upslope positions and redeposited downslope onto areas of gentle substrate during rapid sea-level falls (> 22.7 cm/ky) of short duration. Such rapid sea-level falls and presence of steep slopes are not conducive to formation of

  19. Deep sequencing of ESTs from nacreous and prismatic layer producing tissues and a screen for novel shell formation-related genes in the pearl oyster.

    Directory of Open Access Journals (Sweden)

    Shigeharu Kinoshita

    Full Text Available BACKGROUND: Despite its economic importance, we have a limited understanding of the molecular mechanisms underlying shell formation in pearl oysters, wherein the calcium carbonate crystals, nacre and prism, are formed in a highly controlled manner. We constructed comprehensive expressed gene profiles in the shell-forming tissues of the pearl oyster Pinctada fucata and identified novel shell formation-related genes candidates. PRINCIPAL FINDINGS: We employed the GS FLX 454 system and constructed transcriptome data sets from pallial mantle and pearl sac, which form the nacreous layer, and from the mantle edge, which forms the prismatic layer in P. fucata. We sequenced 260477 reads and obtained 29682 unique sequences. We also screened novel nacreous and prismatic gene candidates by a combined analysis of sequence and expression data sets, and identified various genes encoding lectin, protease, protease inhibitors, lysine-rich matrix protein, and secreting calcium-binding proteins. We also examined the expression of known nacreous and prismatic genes in our EST library and identified novel isoforms with tissue-specific expressions. CONCLUSIONS: We constructed EST data sets from the nacre- and prism-producing tissues in P. fucata and found 29682 unique sequences containing novel gene candidates for nacreous and prismatic layer formation. This is the first report of deep sequencing of ESTs in the shell-forming tissues of P. fucata and our data provide a powerful tool for a comprehensive understanding of the molecular mechanisms of molluscan biomineralization.

  20. Deep sequencing reveals different compositions of mRNA transcribed from the F8 gene in a panel of FVIII-producing CHO cell lines

    DEFF Research Database (Denmark)

    Kaas, Christian Schrøder; Bolt, Gert; Hansen, Jens J

    2015-01-01

    orders of magnitude lower than for antibodies. In the present study we investigated CHO DXB11 cells transfected with a plasmid encoding human coagulation factor VIII. Single cell clones were isolated from the pool of transfectants and a panel of 14 clones representing a dynamic range of FVIII...... FVIII productivity. It was found that three MTX resistant, nonproducing clones had different truncations of the F8 transcripts. We find that by using deep sequencing, in contrast to microarray technology, for determining the transcriptome from CHO transfectants, we are able to accurately deduce...

  1. Genome sequence of Halorhabdus tiamatea, the first archaeon isolated from a deep-sea anoxic brine lake.

    KAUST Repository

    Antunes, Andre

    2011-09-01

    We present the draft genome of Halorhabdus tiamatea, the first member of the Archaea ever isolated from a deep-sea anoxic brine. Genome comparison with Halorhabdus utahensis revealed some striking differences, including a marked increase in genes associated with transmembrane transport and putative genes for a trehalose synthase and a lactate dehydrogenase.

  2. Genome sequence of Haloplasma contractile, an unusual contractile bacterium from a deep-sea anoxic brine lake.

    KAUST Repository

    Antunes, Andre; Alam, Intikhab; El Dorry, Hamza; Siam, Rania; Robertson, Anthony J.; Bajic, Vladimir B.; Stingl, Ulrich

    2011-01-01

    We present the draft genome of Haloplasma contractile, isolated from a deep-sea brine and representing a new order between Firmicutes and Mollicutes. Its complex morphology with contractile protrusions might be strongly influenced by the presence of seven MreB/Mbl homologs, which appears to be the highest copy number ever reported.

  3. Genome Sequence of Aeribacillus pallidus Strain GS3372, an Endospore-Forming Bacterium Isolated in a Deep Geothermal Reservoir

    OpenAIRE

    Sevasti Filippidou; Marion Jaussi; Thomas Junier; Tina Wunderlin; Nicole Jeanneret; Simona Regenspurg; Po-E Li; Chien-Chi Lo; Shannon Johnson; Kim McMurry; Cheryl D. Gleasner; Momchilo Vuyisich; Patrick S. Chain; Pilar Junier

    2015-01-01

    The genome of strain GS3372 is the first publicly available strain of Aeribacillus pallidus. This endospore-forming thermophilic strain was isolated from a deep geothermal reservoir. The availability of this genome can contribute to the clarification of the taxonomy of the closely related Anoxybacillus, Geobacillus, and Aeribacillus genera.

  4. Genome Sequence of Aeribacillus pallidus Strain GS3372, an Endospore-Forming Bacterium Isolated in a Deep Geothermal Reservoir.

    Science.gov (United States)

    Filippidou, Sevasti; Jaussi, Marion; Junier, Thomas; Wunderlin, Tina; Jeanneret, Nicole; Regenspurg, Simona; Li, Po-E; Lo, Chien-Chi; Johnson, Shannon; McMurry, Kim; Gleasner, Cheryl D; Vuyisich, Momchilo; Chain, Patrick S; Junier, Pilar

    2015-08-27

    The genome of strain GS3372 is the first publicly available strain of Aeribacillus pallidus. This endospore-forming thermophilic strain was isolated from a deep geothermal reservoir. The availability of this genome can contribute to the clarification of the taxonomy of the closely related Anoxybacillus, Geobacillus, and Aeribacillus genera. Copyright © 2015 Filippidou et al.

  5. Genome sequence of Haloplasma contractile, an unusual contractile bacterium from a deep-sea anoxic brine lake.

    KAUST Repository

    Antunes, Andre

    2011-09-01

    We present the draft genome of Haloplasma contractile, isolated from a deep-sea brine and representing a new order between Firmicutes and Mollicutes. Its complex morphology with contractile protrusions might be strongly influenced by the presence of seven MreB/Mbl homologs, which appears to be the highest copy number ever reported.

  6. Genome sequence of Halorhabdus tiamatea, the first archaeon isolated from a deep-sea anoxic brine lake.

    KAUST Repository

    Antunes, Andre; Alam, Intikhab; Bajic, Vladimir B.; Stingl, Ulrich

    2011-01-01

    We present the draft genome of Halorhabdus tiamatea, the first member of the Archaea ever isolated from a deep-sea anoxic brine. Genome comparison with Halorhabdus utahensis revealed some striking differences, including a marked increase in genes associated with transmembrane transport and putative genes for a trehalose synthase and a lactate dehydrogenase.

  7. Deep sequencing of the Trypanosoma cruzi GP63 surface proteases reveals diversity and diversifying selection among chronic and congenital Chagas disease patients.

    Science.gov (United States)

    Llewellyn, Martin S; Messenger, Louisa A; Luquetti, Alejandro O; Garcia, Lineth; Torrico, Faustino; Tavares, Suelene B N; Cheaib, Bachar; Derome, Nicolas; Delepine, Marc; Baulard, Céline; Deleuze, Jean-Francois; Sauer, Sascha; Miles, Michael A

    2015-04-01

    Chagas disease results from infection with the diploid protozoan parasite Trypanosoma cruzi. T. cruzi is highly genetically diverse, and multiclonal infections in individual hosts are common, but little studied. In this study, we explore T. cruzi infection multiclonality in the context of age, sex and clinical profile among a cohort of chronic patients, as well as paired congenital cases from Cochabamba, Bolivia and Goias, Brazil using amplicon deep sequencing technology. A 450bp fragment of the trypomastigote TcGP63I surface protease gene was amplified and sequenced across 70 chronic and 22 congenital cases on the Illumina MiSeq platform. In addition, a second, mitochondrial target--ND5--was sequenced across the same cohort of cases. Several million reads were generated, and sequencing read depths were normalized within patient cohorts (Goias chronic, n = 43, Goias congenital n = 2, Bolivia chronic, n = 27; Bolivia congenital, n = 20), Among chronic cases, analyses of variance indicated no clear correlation between intra-host sequence diversity and age, sex or symptoms, while principal coordinate analyses showed no clustering by symptoms between patients. Between congenital pairs, we found evidence for the transmission of multiple sequence types from mother to infant, as well as widespread instances of novel genotypes in infants. Finally, non-synonymous to synonymous (dn:ds) nucleotide substitution ratios among sequences of TcGP63Ia and TcGP63Ib subfamilies within each cohort provided powerful evidence of strong diversifying selection at this locus. Our results shed light on the diversity of parasite DTUs within each patient, as well as the extent to which parasite strains pass between mother and foetus in congenital cases. Although we were unable to find any evidence that parasite diversity accumulates with age in our study cohorts, putative diversifying selection within members of the TcGP63I gene family suggests a link between genetic diversity within this gene

  8. Discovery of Bovine Digital Dermatitis-Associated Treponema spp. in the Dairy Herd Environment by a Targeted Deep-Sequencing Approach

    DEFF Research Database (Denmark)

    Schou, Kirstine Klitgaard; Weiss Nielsen, Martin; Ingerslev, Hans-Christian

    2014-01-01

    The bacteria associated with the infectious claw disease bovine digital dermatitis (DD) are spirochetes of the genus Treponema; however, their environmental reservoir remains unknown. To our knowledge, the current study is the first report of the discovery and phylogenetic characterization of r...... of this disease among cows within a herd as well as between herds. To address the issue of DD infection reservoirs, we searched for evidence of DD-associated treponemes in fresh feces, in slurry, and in hoof lesions by deep sequencing of the V3 and V4 hypervariable regions of the 16S rRNA gene coupled...... with identification at the operational-taxonomic-unit level. Using treponeme-specific primers in this high-throughput approach, we identified small amounts of DNA (on average 0.6% of the total amount of sequence reads) from DD-associated treponemes in 43 of 64 samples from slurry and cow feces collected from six...

  9. Deep learning

    CERN Document Server

    Goodfellow, Ian; Courville, Aaron

    2016-01-01

    Deep learning is a form of machine learning that enables computers to learn from experience and understand the world in terms of a hierarchy of concepts. Because the computer gathers knowledge from experience, there is no need for a human computer operator to formally specify all the knowledge that the computer needs. The hierarchy of concepts allows the computer to learn complicated concepts by building them out of simpler ones; a graph of these hierarchies would be many layers deep. This book introduces a broad range of topics in deep learning. The text offers mathematical and conceptual background, covering relevant concepts in linear algebra, probability theory and information theory, numerical computation, and machine learning. It describes deep learning techniques used by practitioners in industry, including deep feedforward networks, regularization, optimization algorithms, convolutional networks, sequence modeling, and practical methodology; and it surveys such applications as natural language proces...

  10. Identification of microRNAs from Amur grape (Vitis amurensis Rupr.) by deep sequencing and analysis of microRNA variations with bioinformatics.

    Science.gov (United States)

    Wang, Chen; Han, Jian; Liu, Chonghuai; Kibet, Korir Nicholas; Kayesh, Emrul; Shangguan, Lingfei; Li, Xiaoying; Fang, Jinggui

    2012-03-29

    MicroRNA (miRNA) is a class of functional non-coding small RNA with 19-25 nucleotides in length while Amur grape (Vitis amurensis Rupr.) is an important wild fruit crop with the strongest cold resistance among the Vitis species, is used as an excellent breeding parent for grapevine, and has elicited growing interest in wine production. To date, there is a relatively large number of grapevine miRNAs (vv-miRNAs) from cultivated grapevine varieties such as Vitis vinifera L. and hybrids of V. vinifera and V. labrusca, but there is no report on miRNAs from Vitis amurensis Rupr, a wild grapevine species. A small RNA library from Amur grape was constructed and Solexa technology used to perform deep sequencing of the library followed by subsequent bioinformatics analysis to identify new miRNAs. In total, 126 conserved miRNAs belonging to 27 miRNA families were identified, and 34 known but non-conserved miRNAs were also found. Significantly, 72 new potential Amur grape-specific miRNAs were discovered. The sequences of these new potential va-miRNAs were further validated through miR-RACE, and accumulation of 18 new va-miRNAs in seven tissues of grapevines confirmed by real time RT-PCR (qRT-PCR) analysis. The expression levels of va-miRNAs in flowers and berries were found to be basically consistent in identity to those from deep sequenced sRNAs libraries of combined corresponding tissues. We also describe the conservation and variation of va-miRNAs using miR-SNPs and miR-LDs during plant evolution based on comparison of orthologous sequences, and further reveal that the number and sites of miR-SNP in diverse miRNA families exhibit distinct divergence. Finally, 346 target genes for the new miRNAs were predicted and they include a number of Amur grape stress tolerance genes and many genes regulating anthocyanin synthesis and sugar metabolism. Deep sequencing of short RNAs from Amur grape flowers and berries identified 72 new potential miRNAs and 34 known but non-conserved mi

  11. Identification of microRNAs from Amur grape (vitis amurensis Rupr. by deep sequencing and analysis of microRNA variations with bioinformatics

    Directory of Open Access Journals (Sweden)

    Wang Chen

    2012-03-01

    Full Text Available Abstract Background MicroRNA (miRNA is a class of functional non-coding small RNA with 19-25 nucleotides in length while Amur grape (Vitis amurensis Rupr. is an important wild fruit crop with the strongest cold resistance among the Vitis species, is used as an excellent breeding parent for grapevine, and has elicited growing interest in wine production. To date, there is a relatively large number of grapevine miRNAs (vv-miRNAs from cultivated grapevine varieties such as Vitis vinifera L. and hybrids of V. vinifera and V. labrusca, but there is no report on miRNAs from Vitis amurensis Rupr, a wild grapevine species. Results A small RNA library from Amur grape was constructed and Solexa technology used to perform deep sequencing of the library followed by subsequent bioinformatics analysis to identify new miRNAs. In total, 126 conserved miRNAs belonging to 27 miRNA families were identified, and 34 known but non-conserved miRNAs were also found. Significantly, 72 new potential Amur grape-specific miRNAs were discovered. The sequences of these new potential va-miRNAs were further validated through miR-RACE, and accumulation of 18 new va-miRNAs in seven tissues of grapevines confirmed by real time RT-PCR (qRT-PCR analysis. The expression levels of va-miRNAs in flowers and berries were found to be basically consistent in identity to those from deep sequenced sRNAs libraries of combined corresponding tissues. We also describe the conservation and variation of va-miRNAs using miR-SNPs and miR-LDs during plant evolution based on comparison of orthologous sequences, and further reveal that the number and sites of miR-SNP in diverse miRNA families exhibit distinct divergence. Finally, 346 target genes for the new miRNAs were predicted and they include a number of Amur grape stress tolerance genes and many genes regulating anthocyanin synthesis and sugar metabolism. Conclusions Deep sequencing of short RNAs from Amur grape flowers and berries identified 72

  12. Ultra-deep sequencing reveals high prevalence and broad structural diversity of hepatitis B surface antigen mutations in a global population.

    Science.gov (United States)

    Gencay, Mikael; Hübner, Kirsten; Gohl, Peter; Seffner, Anja; Weizenegger, Michael; Neofytos, Dionysios; Batrla, Richard; Woeste, Andreas; Kim, Hyon-Suk; Westergaard, Gaston; Reinsch, Christine; Brill, Eva; Thu Thuy, Pham Thi; Hoang, Bui Huu; Sonderup, Mark; Spearman, C Wendy; Pabinger, Stephan; Gautier, Jérémie; Brancaccio, Giuseppina; Fasano, Massimo; Santantonio, Teresa; Gaeta, Giovanni B; Nauck, Markus; Kaminski, Wolfgang E

    2017-01-01

    The diversity of the hepatitis B surface antigen (HBsAg) has a significant impact on the performance of diagnostic screening tests and the clinical outcome of hepatitis B infection. Neutralizing or diagnostic antibodies against the HBsAg are directed towards its highly conserved major hydrophilic region (MHR), in particular towards its "a" determinant subdomain. Here, we explored, on a global scale, the genetic diversity of the HBsAg MHR in a large, multi-ethnic cohort of randomly selected subjects with HBV infection from four continents. A total of 1553 HBsAg positive blood samples of subjects originating from 20 different countries across Africa, America, Asia and central Europe were characterized for amino acid variation in the MHR. Using highly sensitive ultra-deep sequencing, we found 72.8% of the successfully sequenced subjects (n = 1391) demonstrated amino acid sequence variation in the HBsAg MHR. This indicates that the global variation frequency in the HBsAg MHR is threefold higher than previously reported. The majority of the amino acid mutations were found in the HBV genotypes B (28.9%) and C (25.4%). Collectively, we identified 345 distinct amino acid mutations in the MHR. Among these, we report 62 previously unknown mutations, which extends the worldwide pool of currently known HBsAg MHR mutations by 22%. Importantly, topological analysis identified the "a" determinant upstream flanking region as the structurally most diverse subdomain of the HBsAg MHR. The highest prevalence of "a" determinant region mutations was observed in subjects from Asia, followed by the African, American and European cohorts, respectively. Finally, we found that more than half (59.3%) of all HBV subjects investigated carried multiple MHR mutations. Together, this worldwide ultra-deep sequencing based genotyping study reveals that the global prevalence and structural complexity of variation in the hepatitis B surface antigen have, to date, been significantly underappreciated.

  13. Multiple viral infections in Agaricus bisporus - Characterisation of 18 unique RNA viruses and 8 ORFans identified by deep sequencing

    OpenAIRE

    Deakin, Gregory; Dobbs, Edward; Bennett, Julie M.; Jones, Ian M.; Grogan, Helen M.; Burton, Kerry S.

    2017-01-01

    Thirty unique non-host RNAs were sequenced in the cultivated fungus, Agaricus bisporus, comprising 18 viruses each encoding an RdRp domain with an additional 8 ORFans (non-host RNAs with no similarity to known sequences). Two viruses were multipartite with component RNAs showing correlative abundances and common 3′ motifs. The viruses, all positive sense single-stranded, were classified into diverse orders/families. Multiple infections of Agaricus may represent a diverse, dynamic and interact...

  14. Trace Fossils as Indicators of Depositional Sequence Boundaries in Lower Carboniferous Deep-Sea Fan Environment Moravice Formation, Czech Republic

    Czech Academy of Sciences Publication Activity Database

    Lehotský, T.; Bábek, O.; Mikuláš, Radek; Zapletal, J.

    2002-01-01

    Roč. 14, - (2002), s. 59-60 ISSN 1210-9606. [Áelazno 2002. Meeting of the Czech Tectonic Studies Group /7./. Áelazno, 09.05.2002-12.05.2002] R&D Projects: GA ČR GA205/00/0118 Keywords : trace fossils * Carboniferous * Deep- Sea Environment Subject RIV: DB - Geology ; Mineralogy http://geolines.gli.cas.cz/fileadmin/volumes/volume14/G14-059.pdf

  15. Time and space resolved deep metagenomics to investigate selection pressures on low abundant species in complex environments

    DEFF Research Database (Denmark)

    Albertsen, Mads; Saunders, Aaron Marc; Nielsen, Kåre Lehmann

    and between EBPR plants we sequenced a total of 10 samples from 3 different plants over a 3 year period at a depth of 25 Gb each. In addition, one time point was selected for deep sequencing, generating 200 Gb of sequence divided between replicates. Quantitative FISH analysis using >30 oligonucleotide probes...

  16. Inspecting Targeted Deep Sequencing of Whole Genome Amplified DNA Versus Fresh DNA for Somatic Mutation Detection: A Genetic Study in Myelodysplastic Syndrome Patients.

    Science.gov (United States)

    Palomo, Laura; Fuster-Tormo, Francisco; Alvira, Daniel; Ademà, Vera; Armengol, María Pilar; Gómez-Marzo, Paula; de Haro, Nuri; Mallo, Mar; Xicoy, Blanca; Zamora, Lurdes; Solé, Francesc

    2017-08-01

    Whole genome amplification (WGA) has become an invaluable method for preserving limited samples of precious stock material and has been used during the past years as an alternative tool to increase the amount of DNA before library preparation for next-generation sequencing. Myelodysplastic syndromes (MDS) are a group of clonal hematopoietic stem cell disorders characterized by presenting somatic mutations in several myeloid-related genes. In this work, targeted deep sequencing has been performed on four paired fresh DNA and WGA DNA samples from bone marrow of MDS patients, to assess the feasibility of using WGA DNA for detecting somatic mutations. The results of this study highlighted that, in general, the sequencing and alignment statistics of fresh DNA and WGA DNA samples were similar. However, after variant calling and when considering variants detected at all frequencies, there was a high level of discordance between fresh DNA and WGA DNA (overall, a higher number of variants was detected in WGA DNA). After proper filtering, a total of three somatic mutations were detected in the cohort. All somatic mutations detected in fresh DNA were also identified in WGA DNA and validated by whole exome sequencing.

  17. Quantitative statistical analysis of cis-regulatory sequences in ABA/VP1- and CBF/DREB1-regulated genes of Arabidopsis.

    Science.gov (United States)

    Suzuki, Masaharu; Ketterling, Matthew G; McCarty, Donald R

    2005-09-01

    We have developed a simple quantitative computational approach for objective analysis of cis-regulatory sequences in promoters of coregulated genes. The program, designated MotifFinder, identifies oligo sequences that are overrepresented in promoters of coregulated genes. We used this approach to analyze promoter sequences of Viviparous1 (VP1)/abscisic acid (ABA)-regulated genes and cold-regulated genes, respectively, of Arabidopsis (Arabidopsis thaliana). We detected significantly enriched sequences in up-regulated genes but not in down-regulated genes. This result suggests that gene activation but not repression is mediated by specific and common sequence elements in promoters. The enriched motifs include several known cis-regulatory sequences as well as previously unidentified motifs. With respect to known cis-elements, we dissected the flanking nucleotides of the core sequences of Sph element, ABA response elements (ABREs), and the C repeat/dehydration-responsive element. This analysis identified the motif variants that may correlate with qualitative and quantitative differences in gene expression. While both VP1 and cold responses are mediated in part by ABA signaling via ABREs, these responses correlate with unique ABRE variants distinguished by nucleotides flanking the ACGT core. ABRE and Sph motifs are tightly associated uniquely in the coregulated set of genes showing a strict dependence on VP1 and ABA signaling. Finally, analysis of distribution of the enriched sequences revealed a striking concentration of enriched motifs in a proximal 200-base region of VP1/ABA and cold-regulated promoters. Overall, each class of coregulated genes possesses a discrete set of the enriched motifs with unique distributions in their promoters that may account for the specificity of gene regulation.

  18. Deep sequencing of Salmonella RNA associated with heterologous Hfq proteins in vivo reveals small RNAs as a major target class and identifies RNA processing phenotypes.

    Science.gov (United States)

    Sittka, Alexandra; Sharma, Cynthia M; Rolle, Katarzyna; Vogel, Jörg

    2009-01-01

    The bacterial Sm-like protein, Hfq, is a key factor for the stability and function of small non-coding RNAs (sRNAs) in Escherichia coli. Homologues of this protein have been predicted in many distantly related organisms yet their functional conservation as sRNA-binding proteins has not entirely been clear. To address this, we expressed in Salmonella the Hfq proteins of two eubacteria (Neisseria meningitides, Aquifex aeolicus) and an archaeon (Methanocaldococcus jannaschii), and analyzed the associated RNA by deep sequencing. This in vivo approach identified endogenous Salmonella sRNAs as a major target of the foreign Hfq proteins. New Salmonella sRNA species were also identified, and some of these accumulated specifically in the presence of a foreign Hfq protein. In addition, we observed specific RNA processing defects, e.g., suppression of precursor processing of SraH sRNA by Methanocaldococcus Hfq, or aberrant accumulation of extracytoplasmic target mRNAs of the Salmonella GcvB, MicA or RybB sRNAs. Taken together, our study provides evidence of a conserved inherent sRNA-binding property of Hfq, which may facilitate the lateral transmission of regulatory sRNAs among distantly related species. It also suggests that the expression of heterologous RNA-binding proteins combined with deep sequencing analysis of RNA ligands can be used as a molecular tool to dissect individual steps of RNA metabolism in vivo.

  19. Deep Sequencing of T-cell Receptor DNA as a Biomarker of Clonally Expanded TILs in Breast Cancer after Immunotherapy.

    Science.gov (United States)

    Page, David B; Yuan, Jianda; Redmond, David; Wen, Y Hanna; Durack, Jeremy C; Emerson, Ryan; Solomon, Stephen; Dong, Zhiwan; Wong, Phillip; Comstock, Christopher; Diab, Adi; Sung, Janice; Maybody, Majid; Morris, Elizabeth; Brogi, Edi; Morrow, Monica; Sacchini, Virgilio; Elemento, Olivier; Robins, Harlan; Patil, Sujata; Allison, James P; Wolchok, Jedd D; Hudis, Clifford; Norton, Larry; McArthur, Heather L

    2016-10-01

    In early-stage breast cancer, the degree of tumor-infiltrating lymphocytes (TIL) predicts response to chemotherapy and overall survival. Combination immunotherapy with immune checkpoint antibody plus tumor cryoablation can induce lymphocytic infiltrates and improve survival in mice. We used T-cell receptor (TCR) DNA sequencing to evaluate both the effect of cryoimmunotherapy in humans and the feasibility of TCR sequencing in early-stage breast cancer. In a pilot clinical trial, 18 women with early-stage breast cancer were treated preoperatively with cryoablation, single-dose anti-CTLA-4 (ipilimumab), or cryoablation + ipilimumab. TCRs within serially collected peripheral blood and tumor tissue were sequenced. In baseline tumor tissues, T-cell density as measured by TCR sequencing correlated with TIL scores obtained by hematoxylin and eosin (H&E) staining. However, tumors with little or no lymphocytes by H&E contained up to 3.6 × 10 6 TCR DNA sequences, highlighting the sensitivity of the ImmunoSEQ platform. In this dataset, ipilimumab increased intratumoral T-cell density over time, whereas cryoablation ± ipilimumab diversified and remodeled the intratumoral T-cell clonal repertoire. Compared with monotherapy, cryoablation plus ipilimumab was associated with numerically greater numbers of peripheral blood and intratumoral T-cell clones expanding robustly following therapy. In conclusion, TCR sequencing correlates with H&E lymphocyte scoring and provides additional information on clonal diversity. These findings support further study of the use of TCR sequencing as a biomarker for T-cell responses to therapy and for the study of cryoimmunotherapy in early-stage breast cancer. Cancer Immunol Res; 4(10); 835-44. ©2016 AACR. ©2016 American Association for Cancer Research.

  20. Genotyping-by-sequencing markers facilitate the identification of quantitative trait loci controlling resistance to Penicillium expansum in Malus sieversii.

    Directory of Open Access Journals (Sweden)

    John L Norelli

    Full Text Available Blue mold caused by Penicillium expansum is the most important postharvest disease of apple worldwide and results in significant financial losses. There are no defined sources of resistance to blue mold in domesticated apple. However, resistance has been described in wild Malus sieversii accessions, including plant introduction (PI613981. The objective of the present study was to identify the genetic loci controlling resistance to blue mold in this accession. We describe the first quantitative trait loci (QTL reported in the Rosaceae tribe Maleae conditioning resistance to P. expansum on genetic linkage group 3 (qM-Pe3.1 and linkage group 10 (qM-Pe10.1. These loci were identified in a M.× domestica 'Royal Gala' X M. sieversii PI613981 family (GMAL4593 based on blue mold lesion diameter seven days post-inoculation in mature, wounded apple fruit inoculated with P. expansum. Phenotypic analyses were conducted in 169 progeny over a four year period. PI613981 was the source of the resistance allele for qM-Pe3.1, a QTL with a major effect on blue mold resistance, accounting for 27.5% of the experimental variability. The QTL mapped from 67.3 to 74 cM on linkage group 3 of the GMAL4593 genetic linkage map. qM-Pe10.1 mapped from 73.6 to 81.8 cM on linkage group 10. It had less of an effect on resistance, accounting for 14% of the experimental variation. 'Royal Gala' was the primary contributor to the resistance effect of this QTL. However, resistance-associated alleles in both parents appeared to contribute to the least square mean blue mold lesion diameter in an additive manner at qM-Pe10.1. A GMAL4593 genetic linkage map composed of simple sequence repeats and 'Golden Delicious' single nucleotide polymorphism markers was able to detect qM-Pe10.1, but failed to detect qM-Pe3.1. The subsequent addition of genotyping-by-sequencing markers to the linkage map provided better coverage of the PI613981 genome on linkage group 3 and facilitated discovery of q

  1. Quantitation of next generation sequencing library preparation protocol efficiencies using droplet digital PCR assays - a systematic comparison of DNA library preparation kits for Illumina sequencing.

    Science.gov (United States)

    Aigrain, Louise; Gu, Yong; Quail, Michael A

    2016-06-13

    The emergence of next-generation sequencing (NGS) technologies in the past decade has allowed the democratization of DNA sequencing both in terms of price per sequenced bases and ease to produce DNA libraries. When it comes to preparing DNA sequencing libraries for Illumina, the current market leader, a plethora of kits are available and it can be difficult for the users to determine which kit is the most appropriate and efficient for their applications; the main concerns being not only cost but also minimal bias, yield and time efficiency. We compared 9 commercially available library preparation kits in a systematic manner using the same DNA sample by probing the amount of DNA remaining after each protocol steps using a new droplet digital PCR (ddPCR) assay. This method allows the precise quantification of fragments bearing either adaptors or P5/P7 sequences on both ends just after ligation or PCR enrichment. We also investigated the potential influence of DNA input and DNA fragment size on the final library preparation efficiency. The overall library preparations efficiencies of the libraries show important variations between the different kits with the ones combining several steps into a single one exhibiting some final yields 4 to 7 times higher than the other kits. Detailed ddPCR data also reveal that the adaptor ligation yield itself varies by more than a factor of 10 between kits, certain ligation efficiencies being so low that it could impair the original library complexity and impoverish the sequencing results. When a PCR enrichment step is necessary, lower adaptor-ligated DNA inputs leads to greater amplification yields, hiding the latent disparity between kits. We describe a ddPCR assay that allows us to probe the efficiency of the most critical step in the library preparation, ligation, and to draw conclusion on which kits is more likely to preserve the sample heterogeneity and reduce the need of amplification.

  2. Qualitative and quantitative comparison of contrast-enhanced fluid-attenuated inversion recovery, magnetization transfer spin echo, and fat-saturation T1-weighted sequences in infectious meningitis

    International Nuclear Information System (INIS)

    Azad, Rajiv; Tayal, Mohit; Azad, Sheenam; Sharma, Garima; Srivastava, Rajendra Kumar

    2017-01-01

    To compare the contrast-enhanced fluid-attenuated inversion recovery (CE-FLAIR), the CE T1-weighted (CE-T1W) sequence with fat suppression (FS) and magnetization transfer (MT) for early detection and characterization of infectious meningitis. Fifty patients and 10 control subjects were evaluated with the CE-FLAIR and the CE-T1W sequences with FS and MT. Qualitative assessment was done by two observers for presence and grading of abnormal leptomeningeal enhancement. Quantitative assessment included computation of net meningeal enhancement, using single pixel signal intensity software. A newly devised FLAIR based scoring system, based on certain imaging features including ventricular dilatation, ependymal enhancement, infarcts and subdural effusions was used to indicate the etiology. Data were analysed using the Student's t test, Cohen's Kappa coefficient, Pearson's correlation coefficient, the intraclass correlation coefficient, one way analysis of variance, and Fisher's exact test with Bonferroni correction as the post hoc test. The CE-FLAIR sequence demonstrated a better sensitivity (100%), diagnostic accuracy (95%), and a stronger correlation with the cerebrospinal fluid, total leukocyte count (r = 0.75), protein (r = 0.77), adenosine deaminase (r = 0.81) and blood glucose (r = -0.6) values compared to the CE-T1W sequences. Qualitative grades and quantitative meningeal enhancement on the CE-FLAIR sequence were also significantly greater than those on the other sequences. The FLAIR based scoring system yielded a diagnostic accuracy of 91.6% and a sensitivity of 96%. A strong inverse Pearson's correlation (r = -0.95) was found between the assigned score and patient's Glasgow Coma Scale at the time of admission. The CE-FLAIR sequence is better suited for evaluating infectious meningitis and could be included as a part of the routine MR imaging protocol

  3. Qualitative and quantitative comparison of contrast-enhanced fluid-attenuated inversion recovery, magnetization transfer spin echo, and fat-saturation T1-weighted sequences in infectious meningitis

    Energy Technology Data Exchange (ETDEWEB)

    Azad, Rajiv; Tayal, Mohit; Azad, Sheenam; Sharma, Garima; Srivastava, Rajendra Kumar [SGRR Institute of Medical and Health Sciences, Patel Nagar, Dehradun (India)

    2017-11-15

    To compare the contrast-enhanced fluid-attenuated inversion recovery (CE-FLAIR), the CE T1-weighted (CE-T1W) sequence with fat suppression (FS) and magnetization transfer (MT) for early detection and characterization of infectious meningitis. Fifty patients and 10 control subjects were evaluated with the CE-FLAIR and the CE-T1W sequences with FS and MT. Qualitative assessment was done by two observers for presence and grading of abnormal leptomeningeal enhancement. Quantitative assessment included computation of net meningeal enhancement, using single pixel signal intensity software. A newly devised FLAIR based scoring system, based on certain imaging features including ventricular dilatation, ependymal enhancement, infarcts and subdural effusions was used to indicate the etiology. Data were analysed using the Student's t test, Cohen's Kappa coefficient, Pearson's correlation coefficient, the intraclass correlation coefficient, one way analysis of variance, and Fisher's exact test with Bonferroni correction as the post hoc test. The CE-FLAIR sequence demonstrated a better sensitivity (100%), diagnostic accuracy (95%), and a stronger correlation with the cerebrospinal fluid, total leukocyte count (r = 0.75), protein (r = 0.77), adenosine deaminase (r = 0.81) and blood glucose (r = -0.6) values compared to the CE-T1W sequences. Qualitative grades and quantitative meningeal enhancement on the CE-FLAIR sequence were also significantly greater than those on the other sequences. The FLAIR based scoring system yielded a diagnostic accuracy of 91.6% and a sensitivity of 96%. A strong inverse Pearson's correlation (r = -0.95) was found between the assigned score and patient's Glasgow Coma Scale at the time of admission. The CE-FLAIR sequence is better suited for evaluating infectious meningitis and could be included as a part of the routine MR imaging protocol.

  4. The Subclonal Structure and Genomic Evolution of Oral Squamous Cell Carcinoma Revealed by Ultra-deep Sequencing

    DEFF Research Database (Denmark)

    Tabatabaeifar, Siavosh; Thomassen, Mads; Larsen, Martin Jakob

    Background: Oral squamous cell carcinoma (OSCC), a subgroup of head and neck squamous cell carcinoma (HNSCC), is primarily caused by alcohol consumption and tobacco use. Recent DNA sequencing studies suggests that HNSCC are very heterogeneous between patients; however the intra-patient subclonal...

  5. Deep Illumina-based shotgun sequencing reveals dietary effects on the structure and function of the fecal microbiome of growing kittens.

    Directory of Open Access Journals (Sweden)

    Oliver Deusch

    Full Text Available Previously, we demonstrated that dietary protein:carbohydrate ratio dramatically affects the fecal microbial taxonomic structure of kittens using targeted 16S gene sequencing. The present study, using the same fecal samples, applied deep Illumina shotgun sequencing to identify the diet-associated functional potential and analyze taxonomic changes of the feline fecal microbiome.Fecal samples from kittens fed one of two diets differing in protein and carbohydrate content (high-protein, low-carbohydrate, HPLC; and moderate-protein, moderate-carbohydrate, MPMC were collected at 8, 12 and 16 weeks of age (n = 6 per group. A total of 345.3 gigabases of sequence were generated from 36 samples, with 99.75% of annotated sequences identified as bacterial. At the genus level, 26% and 39% of reads were annotated for HPLC- and MPMC-fed kittens, with HPLC-fed cats showing greater species richness and microbial diversity. Two phyla, ten families and fifteen genera were responsible for more than 80% of the sequences at each taxonomic level for both diet groups, consistent with the previous taxonomic study. Significantly different abundances between diet groups were observed for 324 genera (56% of all genera identified demonstrating widespread diet-induced changes in microbial taxonomic structure. Diversity was not affected over time. Functional analysis identified 2,013 putative enzyme function groups were different (p<0.000007 between the two dietary groups and were associated to 194 pathways, which formed five discrete clusters based on average relative abundance. Of those, ten contained more (p<0.022 enzyme functions with significant diet effects than expected by chance. Six pathways were related to amino acid biosynthesis and metabolism linking changes in dietary protein with functional differences of the gut microbiome.These data indicate that feline feces-derived microbiomes have large structural and functional differences relating to the dietary

  6. OCT structure, COB location and magmatic type of the S Angolan & SE Brazilian margins from integrated quantitative analysis of deep seismic reflection and gravity anomaly data

    Science.gov (United States)

    Cowie, Leanne; Kusznir, Nick; Horn, Brian

    2014-05-01

    Integrated quantitative analysis using deep seismic reflection data and gravity inversion have been applied to the S Angolan and SE Brazilian margins to determine OCT structure, COB location and magmatic type. Knowledge of these margin parameters are of critical importance for understanding rifted continental margin formation processes and in evaluating petroleum systems in deep-water frontier oil and gas exploration. The OCT structure, COB location and magmatic type of the S Angolan and SE Brazilian rifted continental margins are much debated; exhumed and serpentinised mantle have been reported at these margins. Gravity anomaly inversion, incorporating a lithosphere thermal gravity anomaly correction, has been used to determine Moho depth, crustal basement thickness and continental lithosphere thinning. Residual Depth Anomaly (RDA) analysis has been used to investigate OCT bathymetric anomalies with respect to expected oceanic bathymetries and subsidence analysis has been used to determine the distribution of continental lithosphere thinning. These techniques have been validated for profiles Lusigal 12 and ISE-01 on the Iberian margin. In addition a joint inversion technique using deep seismic reflection and gravity anomaly data has been applied to the ION-GXT BS1-575 SE Brazil and ION-GXT CS1-2400 S Angola deep seismic reflection lines. The joint inversion method solves for coincident seismic and gravity Moho in the time domain and calculates the lateral variations in crustal basement densities and velocities along the seismic profiles. Gravity inversion, RDA and subsidence analysis along the ION-GXT BS1-575 profile, which crosses the Sao Paulo Plateau and Florianopolis Ridge of the SE Brazilian margin, predict the COB to be located SE of the Florianopolis Ridge. Integrated quantitative analysis shows no evidence for exhumed mantle on this margin profile. The joint inversion technique predicts oceanic crustal thicknesses of between 7 and 8 km thickness with

  7. MicroRNAs in Amoebozoa: deep sequencing of the small RNA population in the social amoeba Dictyostelium discoideum reveals developmentally regulated microRNAs.

    Science.gov (United States)

    Avesson, Lotta; Reimegård, Johan; Wagner, E Gerhart H; Söderbom, Fredrik

    2012-10-01

    The RNA interference machinery has served as a guardian of eukaryotic genomes since the divergence from prokaryotes. Although the basic components have a shared origin, silencing pathways directed by small RNAs have evolved in diverse directions in different eukaryotic lineages. Micro (mi)RNAs regulate protein-coding genes and play vital roles in plants and animals, but less is known about their functions in other organisms. Here, we report, for the first time, deep sequencing of small RNAs from the social amoeba Dictyostelium discoideum. RNA from growing single-cell amoebae as well as from two multicellular developmental stages was sequenced. Computational analyses combined with experimental data reveal the expression of miRNAs, several of them exhibiting distinct expression patterns during development. To our knowledge, this is the first report of miRNAs in the Amoebozoa supergroup. We also show that overexpressed miRNA precursors generate miRNAs and, in most cases, miRNA* sequences, whose biogenesis is dependent on the Dicer-like protein DrnB, further supporting the presence of miRNAs in D. discoideum. In addition, we find miRNAs processed from hairpin structures originating from an intron as well as from a class of repetitive elements. We believe that these repetitive elements are sources for newly evolved miRNAs.

  8. Genome-wide discovery and differential regulation of conserved and novel microRNAs in chickpea via deep sequencing.

    Science.gov (United States)

    Jain, Mukesh; Chevala, V V S Narayana; Garg, Rohini

    2014-11-01

    MicroRNAs (miRNAs) are essential components of complex gene regulatory networks that orchestrate plant development. Although several genomic resources have been developed for the legume crop chickpea, miRNAs have not been discovered until now. For genome-wide discovery of miRNAs in chickpea (Cicer arietinum), we sequenced the small RNA content from seven major tissues/organs employing Illumina technology. About 154 million reads were generated, which represented more than 20 million distinct small RNA sequences. We identified a total of 440 conserved miRNAs in chickpea based on sequence similarity with known miRNAs in other plants. In addition, 178 novel miRNAs were identified using a miRDeep pipeline with plant-specific scoring. Some of the conserved and novel miRNAs with significant sequence similarity were grouped into families. The chickpea miRNAs targeted a wide range of mRNAs involved in diverse cellular processes, including transcriptional regulation (transcription factors), protein modification and turnover, signal transduction, and metabolism. Our analysis revealed several miRNAs with differential spatial expression. Many of the chickpea miRNAs were expressed in a tissue-specific manner. The conserved and differential expression of members of the same miRNA family in different tissues was also observed. Some of the same family members were predicted to target different chickpea mRNAs, which suggested the specificity and complexity of miRNA-mediated developmental regulation. This study, for the first time, reveals a comprehensive set of conserved and novel miRNAs along with their expression patterns and putative targets in chickpea, and provides a framework for understanding regulation of developmental processes in legumes. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  9. Genome re-sequencing of semi-wild soybean reveals a complex Soja population structure and deep introgression.

    Directory of Open Access Journals (Sweden)

    Jie Qiu

    Full Text Available Semi-wild soybean is a unique type of soybean that retains both wild and domesticated characteristics, which provides an important intermediate type for understanding the evolution of the subgenus Soja population in the Glycine genus. In this study, a semi-wild soybean line (Maliaodou and a wild line (Lanxi 1 collected from the lower Yangtze regions were deeply sequenced while nine other semi-wild lines were sequenced to a 3-fold genome coverage. Sequence analysis revealed that (1 no independent phylogenetic branch covering all 10 semi-wild lines was observed in the Soja phylogenetic tree; (2 besides two distinct subpopulations of wild and cultivated soybean in the Soja population structure, all semi-wild lines were mixed with some wild lines into a subpopulation rather than an independent one or an intermediate transition type of soybean domestication; (3 high heterozygous rates (0.19-0.49 were observed in several semi-wild lines; and (4 over 100 putative selective regions were identified by selective sweep analysis, including those related to the development of seed size. Our results suggested a hybridization origin for the semi-wild soybean, which makes a complex Soja population structure.

  10. Detection of low frequency FGFR3 mutations in the urine of bladder cancer patients using next-generation deep sequencing

    Directory of Open Access Journals (Sweden)

    Millholl

    2012-06-01

    Full Text Available John M Millholland, Shuqiang Li, Cecilia A Fernandez, Anthony P ShuberPredictive Biosciences Inc, Lexington, MA, USAAbstract: Biological fluid-based noninvasive biomarker assays for monitoring and diagnosing disease are clinically powerful. A major technical hurdle for developing these assays is the requirement of high analytical sensitivity so that biomarkers present at very low levels can be consistently detected. In the case of biological fluid-based cancer diagnostic assays, sensitivities similar to those of tissue-based assays are difficult to achieve with DNA markers due to the high abundance of normal DNA background present in the sample. Here we describe a new urine-based assay that uses ultradeep sequencing technology to detect single mutant molecules of fibroblast growth factor receptor 3 (FGFR3 DNA that are indicative of bladder cancer. Detection of FGFR3 mutations in urine would provide clinicians with a noninvasive means of diagnosing early-stage bladder cancer. The single-molecule assay detects FGFR3 mutant DNA when present at as low as 0.02% of total urine DNA and results in 91% concordance with the frequency that FGFR3 mutations are detected in bladder cancer tumors, significantly improving diagnostic performance. To our knowledge, this is the first practical application of next-generation sequencing technology for noninvasive cancer diagnostics.Keywords: FGFR3, mutation, urine, single molecule, sequencing, bladder cancer

  11. In-vivo quantitative evaluation of perfusion zones and perfusion gradient in the deep inferior epigastric artery perforator flap

    Science.gov (United States)

    Saint-Cyr, Michel; Lakhiani, Chrisovalantis; Cheng, Angela; Mangum, Michael; Liang, Jinyang; Teotia, Sumeet; Livingston, Edward H.; Zuzak, Karel J.

    2013-03-01

    The selection of well-vascularized tissue during DIEP flap harvest remains controversial. While several studies have elucidated cross-midline perfusion, further characterization of perfusion to the ipsilateral hemiabdomen is necessary for minimizing rates of fat necrosis or partial fat necrosis in bilateral DIEP flaps. Eighteen patients (29 flaps) underwent DIEP flap harvest using a prospectively designed protocol. Perforators were marked and imaged with a novel system for quantitatively measuring tissue oxygenation, the Digital Light Hyperspectral Imager. Images were then analyzed to determine if perforator selection influenced ipsilateral flap perfusion. Flaps based on a single lateral row perforator (SLRP) were found to have a higher level of hemoglobin oxygenation in Zone I (mean %HbO2 = 76.1) compared to single medial row perforator (SMRP) flaps (%HbO2 = 71.6). Perfusion of Zone III relative to Zone I was similar between SLRP and SMRP flaps (97.4% vs. 97.9%, respectively). These differences were not statistically significant (p>0.05). Perfusion to the lateral edge of the flap was slightly greater for SLRP flaps compared SMRP flaps (92.1% vs. 89.5%, respectively). SMRP flaps had superior perfusion travelling inferiorly compared to SLRP flaps (88.8% vs. 83.9%, respectively). Overall, it was observed that flaps were better perfused in the lateral direction than inferiorly. Significant differences in perfusion gradients directed inferiorly or laterally were observed, and perforator selection influenced perfusion in the most distal or inferior aspects of the flap. This suggests broader clinical implications for flap design that merit further investigation.

  12. Virus pathotype and deep sequencing of the HA gene of a low pathogenicity H7N1 avian influenza virus causing mortality in Turkeys.

    Directory of Open Access Journals (Sweden)

    Munir Iqbal

    Full Text Available Low pathogenicity avian influenza (LPAI viruses of the H7 subtype generally cause mild disease in poultry. However the evolution of a LPAI virus into highly pathogenic avian influenza (HPAI virus results in the generation of a virus that can cause severe disease and death. The classification of these two pathotypes is based, in part, on disease signs and death in chickens, as assessed in an intravenous pathogenicity test, but the effect of LPAI viruses in turkeys is less well understood. During an investigation of LPAI virus infection of turkeys, groups of three-week-old birds inoculated with A/chicken/Italy/1279/99 (H7N1 showed severe disease signs and died or were euthanised within seven days of infection. Virus was detected in many internal tissues and organs from culled birds. To examine the possible evolution of the infecting virus to a highly pathogenic form in these turkeys, sequence analysis of the haemagglutinin (HA gene cleavage site was carried out by analysing multiple cDNA amplicons made from swabs and tissue sample extracts employing Sanger and Next Generation Sequencing. In addition, a RT-PCR assay to detect HPAI virus was developed. There was no evidence of the presence of HPAI virus in either the virus used as inoculum or from swabs taken from infected birds. However, a small proportion (<0.5% of virus carried in individual tracheal or liver samples did contain a molecular signature typical of a HPAI virus at the HA cleavage site. All the signature sequences were identical and were similar to HPAI viruses collected during the Italian epizootic in 1999/2000. We assume that the detection of HPAI virus in tissue samples following infection with A/chicken/Italy/1279/99 reflected amplification of a virus present at very low levels within the mixed inoculum but, strikingly, we observed no new HPAI virus signatures in the amplified DNA analysed by deep-sequencing.

  13. The induced earthquake sequence related to the St. Gallen deep geothermal project (Switzerland): Fault reactivation and fluid interactions imaged by microseismicity

    Science.gov (United States)

    Diehl, T.; Kraft, T.; Kissling, E.; Wiemer, S.

    2017-09-01

    In July 2013, a sequence of more than 340 earthquakes was induced by reservoir stimulations and well-control procedures following a gas kick at a deep geothermal drilling project close to the city of St. Gallen, Switzerland. The sequence culminated in an ML 3.5 earthquake, which was felt within 10-15 km from the epicenter. High-quality earthquake locations and 3-D reflection seismic data acquired in the St. Gallen project provide a unique data set, which allows high-resolution studies of earthquake triggering related to the injection of fluids into macroscopic fault zones. In this study, we present a high-precision earthquake catalog of the induced sequence. Absolute locations are constrained by a coupled hypocenter-velocity inversion, and subsequent double-difference relocations image the geometry of the ML 3.5 rupture and resolve the spatiotemporal evolution of seismicity. A joint interpretation of earthquake and seismic data shows that the majority of the seismicity occurred in the pre-Mesozoic basement, hundreds of meters below the borehole and the targeted Mesozoic sequence. We propose a hydraulic connectivity between the reactivated fault and the borehole, likely through faults mapped by seismic data. Despite the excellent quality of the seismic data, the association of seismicity with mapped faults remains ambiguous. In summary, our results document that the actual hydraulic properties of a fault system and hydraulic connections between its fault segments are complex and may not be predictable upfront. Incomplete knowledge of fault structures and stress heterogeneities within highly complex fault systems additionally challenge the degree of predictability of induced seismicity related to underground fluid injections.

  14. Appearances can be deceptive: revealing a hidden viral infection with deep sequencing in a plant quarantine context.

    Science.gov (United States)

    Candresse, Thierry; Filloux, Denis; Muhire, Brejnev; Julian, Charlotte; Galzi, Serge; Fort, Guillaume; Bernardo, Pauline; Daugrois, Jean-Heindrich; Fernandez, Emmanuel; Martin, Darren P; Varsani, Arvind; Roumagnac, Philippe

    2014-01-01

    Comprehensive inventories of plant viral diversity are essential for effective quarantine and sanitation efforts. The safety of regulated plant material exchanges presently relies heavily on techniques such as PCR or nucleic acid hybridisation, which are only suited to the detection and characterisation of specific, well characterised pathogens. Here, we demonstrate the utility of sequence-independent next generation sequencing (NGS) of both virus-derived small interfering RNAs (siRNAs) and virion-associated nucleic acids (VANA) for the detailed identification and characterisation of viruses infecting two quarantined sugarcane plants. Both plants originated from Egypt and were known to be infected with Sugarcane streak Egypt Virus (SSEV; Genus Mastrevirus, Family Geminiviridae), but were revealed by the NGS approaches to also be infected by a second highly divergent mastrevirus, here named Sugarcane white streak Virus (SWSV). This novel virus had escaped detection by all routine quarantine detection assays and was found to also be present in sugarcane plants originating from Sudan. Complete SWSV genomes were cloned and sequenced from six plants and all were found to share >91% genome-wide identity. With the exception of two SWSV variants, which potentially express unusually large RepA proteins, the SWSV isolates display genome characteristics very typical to those of all other previously described mastreviruses. An analysis of virus-derived siRNAs for SWSV and SSEV showed them to be strongly influenced by secondary structures within both genomic single stranded DNA and mRNA transcripts. In addition, the distribution of siRNA size frequencies indicates that these mastreviruses are likely subject to both transcriptional and post-transcriptional gene silencing. Our study stresses the potential advantages of NGS-based virus metagenomic screening in a plant quarantine setting and indicates that such techniques could dramatically reduce the numbers of non

  15. Appearances can be deceptive: revealing a hidden viral infection with deep sequencing in a plant quarantine context.

    Directory of Open Access Journals (Sweden)

    Thierry Candresse

    Full Text Available Comprehensive inventories of plant viral diversity are essential for effective quarantine and sanitation efforts. The safety of regulated plant material exchanges presently relies heavily on techniques such as PCR or nucleic acid hybridisation, which are only suited to the detection and characterisation of specific, well characterised pathogens. Here, we demonstrate the utility of sequence-independent next generation sequencing (NGS of both virus-derived small interfering RNAs (siRNAs and virion-associated nucleic acids (VANA for the detailed identification and characterisation of viruses infecting two quarantined sugarcane plants. Both plants originated from Egypt and were known to be infected with Sugarcane streak Egypt Virus (SSEV; Genus Mastrevirus, Family Geminiviridae, but were revealed by the NGS approaches to also be infected by a second highly divergent mastrevirus, here named Sugarcane white streak Virus (SWSV. This novel virus had escaped detection by all routine quarantine detection assays and was found to also be present in sugarcane plants originating from Sudan. Complete SWSV genomes were cloned and sequenced from six plants and all were found to share >91% genome-wide identity. With the exception of two SWSV variants, which potentially express unusually large RepA proteins, the SWSV isolates display genome characteristics very typical to those of all other previously described mastreviruses. An analysis of virus-derived siRNAs for SWSV and SSEV showed them to be strongly influenced by secondary structures within both genomic single stranded DNA and mRNA transcripts. In addition, the distribution of siRNA size frequencies indicates that these mastreviruses are likely subject to both transcriptional and post-transcriptional gene silencing. Our study stresses the potential advantages of NGS-based virus metagenomic screening in a plant quarantine setting and indicates that such techniques could dramatically reduce the numbers of non

  16. Detection of Inter-lineage Natural Recombination in Avian Paramyxovirus Serotype 1 using Simplified Deep Sequencing Platform

    Directory of Open Access Journals (Sweden)

    Dilan Amila Satharasinghe

    2016-11-01

    Full Text Available Newcastle disease virus (NDV is a prototype member of avian paramyxovirus serotype 1 (APMV-1, which causes severe and contagious disease in the commercial poultry and wild birds. Despite extensive vaccination programs and other control measures, the disease remains endemic around the globe especially in Asia, Africa, and the Middle East. Being a single serotype, genotype II based vaccines remained most acceptable means of immunization. However, the evidence is emerging on failures of vaccines mainly due to evolving nature of the virus and higher genetic gaps between vaccine and field strains of APMV-1. Most of the epidemiological and genetic characterizations of APMVs are based on conventional methods, which are prone to mask the diverse population of viruses in complex samples. In this study, we report the application of a simple, robust, and less resource-demanding methodology for the whole genome sequencing of NDV, using next-generation sequencing on the Illumina MiSeq platform. Using this platform, we sequenced full genomes of five virulent Malaysian NDV strains collected during 2004-2013. All isolates clustered within highly prevalent lineage 5 (specifically in lineage 5a; however, a significantly greater genetic divergence was observed in isolates collected from 2004 to 2011. Interestingly, genetic characterization of one isolate collected in 2013 (IBS025/13 shown natural recombination between lineage 2 and lineage 5. In the event of recombination, the isolate (IBS025/13 carried nucleocapsid protein consist of 55-1801 nucleotides (nts and near-complete phosphoprotein (1804-3254 nts genes of lineage 2 whereas surface glycoproteins (fusion, hemagglutinin-neuraminidase and large polymerase of lineage 5. Additionally, the recombinant virus has a genome size of 15,186 nts which is characteristics for the old genotypes I to IV isolated from 1930 to 1960. Taken together, we report the occurrence of a natural recombination in circulating strains

  17. Integrative analysis of deep sequencing data identifies estrogen receptor early response genes and links ATAD3B to poor survival in breast cancer.

    Directory of Open Access Journals (Sweden)

    Kristian Ovaska

    Full Text Available Identification of responsive genes to an extra-cellular cue enables characterization of pathophysiologically crucial biological processes. Deep sequencing technologies provide a powerful means to identify responsive genes, which creates a need for computational methods able to analyze dynamic and multi-level deep sequencing data. To answer this need we introduce here a data-driven algorithm, SPINLONG, which is designed to search for genes that match the user-defined hypotheses or models. SPINLONG is applicable to various experimental setups measuring several molecular markers in parallel. To demonstrate the SPINLONG approach, we analyzed ChIP-seq data reporting PolII, estrogen receptor α (ERα, H3K4me3 and H2A.Z occupancy at five time points in the MCF-7 breast cancer cell line after estradiol stimulus. We obtained 777 ERa early responsive genes and compared the biological functions of the genes having ERα binding within 20 kb of the transcription start site (TSS to genes without such binding site. Our results show that the non-genomic action of ERα via the MAPK pathway, instead of direct ERa binding, may be responsible for early cell responses to ERα activation. Our results also indicate that the ERα responsive genes triggered by the genomic pathway are transcribed faster than those without ERα binding sites. The survival analysis of the 777 ERα responsive genes with 150 primary breast cancer tumors and in two independent validation cohorts indicated the ATAD3B gene, which does not have ERα binding site within 20 kb of its TSS, to be significantly associated with poor patient survival.

  18. Small RNA Deep Sequencing and the Effects of microRNA408 on Root Gravitropic Bending in Arabidopsis

    Science.gov (United States)

    Li, Huasheng; Lu, Jinying; Sun, Qiao; Chen, Yu; He, Dacheng; Liu, Min

    2015-11-01

    MicroRNA (miRNA) is a non-coding small RNA composed of 20 to 24 nucleotides that influences plant root development. This study analyzed the miRNA expression in Arabidopsis root tip cells using Illumina sequencing and real-time PCR before (sample 0) and 15 min after (sample 15) a 3-D clinostat rotational treatment was administered. After stimulation was performed, the expression levels of seven miRNA genes, including Arabidopsis miR160, miR161, miR394, miR402, miR403, miR408, and miR823, were significantly upregulated. Illumina sequencing results also revealed two novel miRNAsthat have not been previously reported, The target genes of these miRNAs included pentatricopeptide repeat-containing protein and diadenosine tetraphosphate hydrolase. An overexpression vector of Arabidopsis miR408 was constructed and transferred to Arabidopsis plant. The roots of plants over expressing miR408 exhibited a slower reorientation upon gravistimulation in comparison with those of wild-type. This result indicate that miR408 could play a role in root gravitropic response.

  19. DanQ: a hybrid convolutional and recurrent deep neural network for quantifying the function of DNA sequences.

    Science.gov (United States)

    Quang, Daniel; Xie, Xiaohui

    2016-06-20

    Modeling the properties and functions of DNA sequences is an important, but challenging task in the broad field of genomics. This task is particularly difficult for non-coding DNA, the vast majority of which is still poorly understood in terms of function. A powerful predictive model for the function of non-coding DNA can have enormous benefit for both basic science and translational research because over 98% of the human genome is non-coding and 93% of disease-associated variants lie in these regions. To address this need, we propose DanQ, a novel hybrid convolutional and bi-directional long short-term memory recurrent neural network framework for predicting non-coding function de novo from sequence. In the DanQ model, the convolution layer captures regulatory motifs, while the recurrent layer captures long-term dependencies between the motifs in order to learn a regulatory 'grammar' to improve predictions. DanQ improves considerably upon other models across several metrics. For some regulatory markers, DanQ can achieve over a 50% relative improvement in the area under the precision-recall curve metric compared to related models. We have made the source code available at the github repository http://github.com/uci-cbcl/DanQ. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  20. HIV-1 transmission patterns in antiretroviral therapy-naive, HIV-infected North Americans based on phylogenetic analysis by population level and ultra-deep DNA sequencing.

    Directory of Open Access Journals (Sweden)

    Lisa L Ross

    Full Text Available Factors that contribute to the transmission of human immunodeficiency virus type 1 (HIV-1, especially drug-resistant HIV-1 variants remain a significant public health concern. In-depth phylogenetic analyses of viral sequences obtained in the screening phase from antiretroviral-naïve HIV-infected patients seeking enrollment in EPZ108859, a large open-label study in the USA, Canada and Puerto Rico (ClinicalTrials.gov NCT00440947 were examined for insights into the roles of drug resistance and epidemiological factors that could impact disease dissemination. Viral transmission clusters (VTCs were initially predicted from a phylogenetic analysis of population level HIV-1 pol sequences obtained from 690 antiretroviral-naïve subjects in 2007. Subsequently, the predicted VTCs were tested for robustness by ultra deep sequencing (UDS using pyrosequencing technology and further phylogenetic analyses. The demographic characteristics of clustered and non-clustered subjects were then compared. From 690 subjects, 69 were assigned to 1 of 30 VTCs, each containing 2 to 5 subjects. Race composition of VTCs were significantly more likely to be white (72% vs. 60%; p = 0.04. VTCs had fewer reverse transcriptase and major PI resistance mutations (9% vs. 24%; p = 0.002 than non-clustered sequences. Both men-who-have-sex-with-men (MSM (68% vs. 48%; p = 0.001 and Canadians (29% vs. 14%; p = 0.03 were significantly more frequent in VTCs than non-clustered sequences. Of the 515 subjects who initiated antiretroviral therapy, 33 experienced confirmed virologic failure through 144 weeks while only 3/33 were from VTCs. Fewer VTCs subjects (as compared to those with non-clustering virus had HIV-1 with resistance-associated mutations or experienced virologic failure during the course of the study. Our analysis shows specific geographical and drug resistance trends that correlate well with transmission clusters defined by HIV sequences of similarity

  1. Quantitative assessment of hepatic function: modified look-locker inversion recovery (MOLLI) sequence for T1 mapping on Gd-EOB-DTPA-enhanced liver MR imaging

    Energy Technology Data Exchange (ETDEWEB)

    Yoon, Jeong Hee [Seoul National University Hospital, Department of Radiology, Seoul (Korea, Republic of); Lee, Jeong Min; Han, Joon Koo; Choi, Byung Ihn [Seoul National University Hospital, Department of Radiology, Seoul (Korea, Republic of); Seoul National University College of Medicine, Institute of Radiation Medicine, Jongno-gu, Seoul (Korea, Republic of); Paek, Munyoung [Siemens Healthcare, Seoul (Korea, Republic of)

    2016-06-15

    To determine whether multislice T1 mapping of the liver using a modified look-locker inversion recovery (MOLLI) sequence on gadoxetic acid-enhanced magnetic resonance imaging (MRI) can be used as a quantitative tool to estimate liver function and predict the presence of oesophageal or gastric varices. Phantoms filled with gadoxetic acid were scanned three times using MOLLI sequence to test repeatability. Patients with chronic liver disease or liver cirrhosis who underwent gadoxetic acid-enhanced liver MRI including MOLLI sequence at 3 T were included (n = 343). Pre- and postcontrast T1 relaxation times of the liver (T1liver), changes between pre- and postcontrast T1liver (ΔT1liver), and adjusted postcontrast T1liver (postcontrast T1liver-T1spleen/T1spleen) were compared among Child-Pugh classes. In 62 patients who underwent endoscopy, all T1 parameters and spleen sizes were correlated with varices. Phantom study showed excellent repeatability of MOLLI sequence. As Child-Pugh scores increased, pre- and postcontrast T1liver were significantly prolonged (P < 0.001), and ΔT1liver and adjusted postcontrast T1liver decreased (P< 0.001). Adjusted postcontrast T1liver and spleen size were independently associated with varices (R{sup 2} = 0.29, P < 0.001). T1 mapping of the liver using MOLLI sequence on gadoxetic acid-enhanced MRI demonstrated potential in quantitatively estimating liver function, and adjusted postcontrast T1liver was significantly associated with varices. (orig.)

  2. Detection of Inter-Lineage Natural Recombination in Avian Paramyxovirus Serotype 1 Using Simplified Deep Sequencing Platform.

    Science.gov (United States)

    Satharasinghe, Dilan A; Murulitharan, Kavitha; Tan, Sheau W; Yeap, Swee K; Munir, Muhammad; Ideris, Aini; Omar, Abdul R

    2016-01-01

    Newcastle disease virus (NDV) is a prototype member of avian paramyxovirus serotype 1 (APMV-1), which causes severe and contagious disease in the commercial poultry and wild birds. Despite extensive vaccination programs and other control measures, the disease remains endemic around the globe especially in Asia, Africa, and the Middle East. Being a single serotype, genotype II based vaccines remained most acceptable means of immunization. However, the evidence is emerging on failures of vaccines mainly due to evolving nature of the virus and higher genetic gaps between vaccine and field strains of APMV-1. Most of the epidemiological and genetic characterizations of APMVs are based on conventional methods, which are prone to mask the diverse population of viruses in complex samples. In this study, we report the application of a simple, robust, and less resource-demanding methodology for the whole genome sequencing of NDV, using next-generation sequencing (NGS) on the Illumina MiSeq platform. Using this platform, we sequenced full genomes of five virulent Malaysian NDV strains collected during 2004-2013. All isolates clustered within highly prevalent lineage 5 (specifically in lineage 5a); however, a significantly greater genetic divergence was observed in isolates collected from 2004 to 2011. Interestingly, genetic characterization of one isolate collected in 2013 (IBS025/13) shown natural recombination between lineage 2 and lineage 5. In the event of recombination, the isolate (IBS025/13) carried nucleocapsid protein consist of 55-1801 nucleotides (nts) and near-complete phosphoprotein (1804-3254 nts) genes of lineage 2 whereas surface glycoproteins (fusion, hemagglutinin-neuraminidase) and large polymerase of lineage 5. Additionally, the recombinant virus has a genome size of 15,186 nts which is characteristics for the old genotypes I-IV isolated from 1930 to 1960. Taken together, we report the occurrence of a natural recombination in circulating strains of NDV in

  3. Targeted deep sequencing of mucinous ovarian tumors reveals multiple overlapping RAS-pathway activating mutations in borderline and cancerous neoplasms

    International Nuclear Information System (INIS)

    Mackenzie, Robertson; Kommoss, Stefan; Winterhoff, Boris J.; Kipp, Benjamin R.; Garcia, Joaquin J.; Voss, Jesse; Halling, Kevin; Karnezis, Anthony; Senz, Janine; Yang, Winnie; Prigge, Elena-Sophie; Reuschenbach, Miriam; Doeberitz, Magnus Von Knebel; Gilks, Blake C.; Huntsman, David G.; Bakkum-Gamez, Jamie; McAlpine, Jessica N.; Anglesio, Michael S.

    2015-01-01

    Mucinous ovarian tumors represent a distinct histotype of epithelial ovarian cancer. The rarest (2-4 % of ovarian carcinomas) of the five major histotypes, their genomic landscape remains poorly described. We undertook hotspot sequencing of 50 genes commonly mutated in human cancer across 69 mucinous ovarian tumors. Our goals were to establish the overall frequency of cancer-hotspot mutations across a large cohort, especially those tumors previously thought to be “RAS-pathway alteration negative”, using highly-sensitive next-generation sequencing as well as further explore a small number of cases with apparent heterogeneity in RAS-pathway activating alterations. Using the Ion Torrent PGM platform, we performed next generation sequencing analysis using the v2 Cancer Hotspot Panel. Regions of disparate ERBB2-amplification status were sequenced independently for two mucinous carcinoma (MC) cases, previously established as showing ERBB2 amplification/overexpression heterogeneity, to assess the hypothesis of subclonal populations containing either KRAS mutation or ERBB2 amplification independently or simultaneously. We detected mutations in KRAS, TP53, CDKN2A, PIK3CA, PTEN, BRAF, FGFR2, STK11, CTNNB1, SRC, SMAD4, GNA11 and ERBB2. KRAS mutations remain the most frequently observed alteration among MC (64.9 %) and mucinous borderline tumors (MBOT) (92.3 %). TP53 mutation occurred more frequently in carcinomas than borderline tumors (56.8 % and 11.5 %, respectively), and combined IHC and mutation data suggest alterations occur in approximately 68 % of MC and as many as 20 % of MBOT. Proven and potential RAS-pathway activating changes were observed in all but one MC. Concurrent ERBB2 amplification and KRAS mutation were observed in a substantial number of cases (7/63 total), as was co-occurrence of KRAS and BRAF mutations (one case). Microdissection of ERBB2-amplified regions of tumors harboring KRAS mutation suggests these alterations are occurring in the same cell

  4. Revisiting bovine pyometra-New insights into the disease using a culture-independent deep sequencing approach

    DEFF Research Database (Denmark)

    Knudsen, Lif Rødtness Vesterby; Karstrup, Cecilia Christensen; Pedersen, Hanne Gervi

    2015-01-01

    -independent studies have demonstrated that the bacterial diversity in most environments is underestimated in culture-based studies. Consequently, fastidious pyometra-associated pathogens may have been overlooked. Therefore, the primary purpose of this study was to investigate the diversity of bacteria in the uterus......The bacteria present in the uterus during pyometra have previously been studied using bacteriological culturing. These studies identified Fusobacterium necrophorum and Trueperella pyogenes as the major contributors to the pathogenesis of pyometra. However, an increasing number of culture...... of cows with pyometra by using culture-independent 16S rRNA PCR combined with next generation sequencing. We investigated the microbial composition in the uterus of 21 cows with pyometra, which were obtained from a Danish slaughterhouse. Similar to the observations from the culture studies...

  5. Event-specific qualitative and quantitative PCR detection of the GMO carnation (Dianthus caryophyllus) variety Moonlite based upon the 5'-transgene integration sequence.

    Science.gov (United States)

    Li, P; Jia, J W; Jiang, L X; Zhu, H; Bai, L; Wang, J B; Tang, X M; Pan, A H

    2012-04-27

    To ensure the implementation of genetically modified organism (GMO)-labeling regulations, an event-specific detection method was developed based on the junction sequence of an exogenous integrant in the transgenic carnation variety Moonlite. The 5'-transgene integration sequence was isolated by thermal asymmetric interlaced PCR. Based upon the 5'-transgene integration sequence, the event-specific primers and TaqMan probe were designed to amplify the fragments, which spanned the exogenous DNA and carnation genomic DNA. Qualitative and quantitative PCR assays were developed employing the designed primers and probe. The detection limit of the qualitative PCR assay was 0.05% for Moonlite in 100 ng total carnation genomic DNA, corresponding to about 79 copies of the carnation haploid genome; the limit of detection and quantification of the quantitative PCR assay were estimated to be 38 and 190 copies of haploid carnation genomic DNA, respectively. Carnation samples with different contents of genetically modified components were quantified and the bias between the observed and true values of three samples were lower than the acceptance criterion (GMO detection method. These results indicated that these event-specific methods would be useful for the identification and quantification of the GMO carnation Moonlite.

  6. Deep sequencing reveals transcriptome re-programming of Taxus × media cells to the elicitation with methyl jasmonate.

    Science.gov (United States)

    Sun, Guiling; Yang, Yanfang; Xie, Fuliang; Wen, Jian-Fan; Wu, Jianqiang; Wilson, Iain W; Tang, Qi; Liu, Hongwei; Qiu, Deyou

    2013-01-01

    Plant cell culture represents an alternative source for producing high-value secondary metabolites including paclitaxel (Taxol®), which is mainly produced in Taxus and has been widely used in cancer chemotherapy. The phytohormone methyl jasmonate (MeJA) can significantly increase the production of paclitaxel, which is induced in plants as a secondary metabolite possibly in defense against herbivores and pathogens. In cell culture, MeJA also elicits the accumulation of paclitaxel; however, the mechanism is still largely unknown. To obtain insight into the global regulation mechanism of MeJA in the steady state of paclitaxel production (7 days after MeJA addition), especially on paclitaxel biosynthesis, we sequenced the transcriptomes of MeJA-treated and untreated Taxus × media cells and obtained ∼ 32.5 M high quality reads, from which 40,348 unique sequences were obtained by de novo assembly. Expression level analysis indicated that a large number of genes were associated with transcriptional regulation, DNA and histone modification, and MeJA signaling network. All the 29 known genes involved in the biosynthesis of terpenoid backbone and paclitaxel were found with 18 genes showing increased transcript abundance following elicitation of MeJA. The significantly up-regulated changes of 9 genes in paclitaxel biosynthesis were validated by qRT-PCR assays. According to the expression changes and the previously proposed enzyme functions, multiple candidates for the unknown steps in paclitaxel biosynthesis were identified. We also found some genes putatively involved in the transport and degradation of paclitaxel. Potential target prediction of miRNAs indicated that miRNAs may play an important role in the gene expression regulation following the elicitation of MeJA. Our results shed new light on the global regulation mechanism by which MeJA regulates the physiology of Taxus cells and is helpful to understand how MeJA elicits other plant species besides Taxus.

  7. Compositional Bias in Naïve and Chemically-modified Phage-Displayed Libraries uncovered by Paired-end Deep Sequencing.

    Science.gov (United States)

    He, Bifang; Tjhung, Katrina F; Bennett, Nicholas J; Chou, Ying; Rau, Andrea; Huang, Jian; Derda, Ratmir

    2018-01-19

    Understanding the composition of a genetically-encoded (GE) library is instrumental to the success of ligand discovery. In this manuscript, we investigate the bias in GE-libraries of linear, macrocyclic and chemically post-translationally modified (cPTM) tetrapeptides displayed on the M13KE platform, which are produced via trinucleotide cassette synthesis (19 codons) and NNK-randomized codon. Differential enrichment of synthetic DNA {S}, ligated vector {L} (extension and ligation of synthetic DNA into the vector), naïve libraries {N} (transformation of the ligated vector into the bacteria followed by expression of the library for 4.5 hours to yield a "naïve" library), and libraries chemically modified by aldehyde ligation and cysteine macrocyclization {M} characterized by paired-end deep sequencing, detected a significant drop in diversity in {L} → {N}, but only a minor compositional difference in {S} → {L} and {N} → {M}. Libraries expressed at the N-terminus of phage protein pIII censored positively charged amino acids Arg and Lys; libraries expressed between pIII domains N1 and N2 overcame Arg/Lys-censorship but introduced new bias towards Gly and Ser. Interrogation of biases arising from cPTM by aldehyde ligation and cysteine macrocyclization unveiled censorship of sequences with Ser/Phe. Analogous analysis can be used to explore library diversity in new display platforms and optimize cPTM of these libraries.

  8. Use of whole genome deep sequencing to define emerging minority variants in virus envelope genes in herpesvirus treated with novel antimicrobial K21.

    Science.gov (United States)

    Tweedy, Joshua G; Prusty, Bhupesh K; Gompels, Ursula A

    2017-10-01

    New antivirals are required to prevent rising antimicrobial resistance from replication inhibitors. The aim of this study was to analyse the range of emerging mutations in herpesvirus by whole genome deep sequencing. We tested human herpesvirus 6 treatment with novel antiviral K21, where evidence indicated distinct effects on virus envelope proteins. We treated BACmid cloned virus in order to analyse mechanisms and candidate targets for resistance. Illumina based next generation sequencing technology enabled analyses of mutations in 85 genes to depths of 10,000 per base detecting low prevalent minority variants (<1%). After four passages in tissue culture the untreated virus accumulated mutations in infected cells giving an emerging mixed population (45-73%) of non-synonymous SNPs in six genes including two envelope glycoproteins. Strikingly, treatment with K21 did not accumulate the passage mutations; instead a high frequency mutation was selected in envelope protein gQ2, part of the gH/gL complex essential for herpesvirus infection. This introduced a stop codon encoding a truncation mutation previously observed in increased virion production. There was reduced detection of the glycoprotein complex in infected cells. This supports a novel pathway for K21 targeting virion envelopes distinct from replication inhibition. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  9. A Quantitative Tool to Distinguish Isobaric Leucine and Isoleucine Residues for Mass Spectrometry-Based De Novo Monoclonal Antibody Sequencing

    Science.gov (United States)

    Poston, Chloe N.; Higgs, Richard E.; You, Jinsam; Gelfanova, Valentina; Hale, John E.; Knierman, Michael D.; Siegel, Robert; Gutierrez, Jesus A.

    2014-07-01

    De novo sequencing by mass spectrometry (MS) allows for the determination of the complete amino acid (AA) sequence of a given protein based on the mass difference of detected ions from MS/MS fragmentation spectra. The technique relies on obtaining specific masses that can be attributed to characteristic theoretical masses of AAs. A major limitation of de novo sequencing by MS is the inability to distinguish between the isobaric residues leucine (Leu) and isoleucine (Ile). Incorrect identification of Ile as Leu or vice versa often results in loss of activity in recombinant antibodies. This functional ambiguity is commonly resolved with costly and time-consuming AA mutation and peptide sequencing experiments. Here, we describe a set of orthogonal biochemical protocols, which experimentally determine the identity of Ile or Leu residues in monoclonal antibodies (mAb) based on the selectivity that leucine aminopeptidase shows for n-terminal Leu residues and the cleavage preference for Leu by chymotrypsin. The resulting observations are combined with germline frequencies and incorporated into a logistic regression model, called Predictor for Xle Sites (PXleS) to provide a statistical likelihood for the identity of Leu at an ambiguous site. We demonstrate that PXleS can generate a probability for an Xle site in mAbs with 96% accuracy. The implementation of PXleS precludes the expression of several possible sequences and, therefore, reduces the overall time and resources required to go from spectra generation to a biologically active sequence for a mAb when an Ile or Leu residue is in question.

  10. Three-dimensional fluid-attenuated inversion recovery sequence for visualisation of subthalamic nucleus for deep brain stimulation in Parkinson's disease

    Energy Technology Data Exchange (ETDEWEB)

    Heo, Young Jin [University of Ulsan College of Medicine, Asan Medical Center, Department of Radiology, Research Institute of Radiology, Seoul (Korea, Republic of); Inje University, Department of Radiology, Busan Paik Hospital, Busan (Korea, Republic of); Kim, Sang Joon; Kim, Ho Sung; Choi, Choong Gon; Jung, Seung Chai [University of Ulsan College of Medicine, Asan Medical Center, Department of Radiology, Research Institute of Radiology, Seoul (Korea, Republic of); Lee, Jung Kyo [University of Ulsan College of Medicine, Asan Medical Center, Department of Neurosurgery, Seoul (Korea, Republic of); Lee, Chong Sik; Chung, Sun J. [University of Ulsan College of Medicine, Asan Medical Center, Department of Neurology, Seoul (Korea, Republic of); Cho, So Hyun [Department of Radiology, Busan (Korea, Republic of); Lee, Gyoung Ro [Philips HealthCare Korea, Seoul (Korea, Republic of)

    2015-09-15

    Deep brain stimulation (DBS) of the subthalamic nucleus (STN) is an accepted treatment for advanced Parkinson's disease (PD). However, targeting the STN is difficult due to its relatively small size and variable location. The purpose of this study was to assess which of the following sequences obtained with the 3.0 T MR system can accurately delineate the STN: coronal 3D fluid-attenuated inversion recovery (FLAIR), 2D T2*-weighted fast-field echo (T2*-FFE) and 2D T2-weighted turbo spin-echo (TSE) sequences. We included 20 consecutive patients with PD who underwent 3.0 T MR for DBS targeting. 3D FLAIR, 2D T2*-FFE and T2-TSE images were obtained for all study patients. Image quality and demarcation of the STN were analysed using 4-point scales, and contrast ratio (CR) of the STN and normal white matter was calculated. The Friedman test was used to compare the three sequences. In qualitative analysis, the 2D T2*-FFE image showed more artefacts than 3D FLAIR or 2D T2-TSE, but the difference did not reach statistical significance. 3D FLAIR images showed significantly superior demarcation of the STN compared with 2D T2*-FFE and T2-TSE images (P < 0.001, respectively). The CR of 3D FLAIR was significantly higher than that of 2D T2*-FFE or T2-TSE images in multiple comparison correction (P < 0.001), but there was no significant difference in the CR between 2D T2*-FFE and T2-TSE images. Coronal 3D FLAIR images showed the most accurate demarcation of the STN for DBS targeting among coronal 3D FLAIR, 2D T2*-FFE and T2-TSE images. (orig.)

  11. Integrated analysis of gene expression, CpG island methylation, and gene copy number in breast cancer cells by deep sequencing.

    Directory of Open Access Journals (Sweden)

    Zhifu Sun

    Full Text Available We used deep sequencing technology to profile the transcriptome, gene copy number, and CpG island methylation status simultaneously in eight commonly used breast cell lines to develop a model for how these genomic features are integrated in estrogen receptor positive (ER+ and negative breast cancer. Total mRNA sequence, gene copy number, and genomic CpG island methylation were carried out using the Illumina Genome Analyzer. Sequences were mapped to the human genome to obtain digitized gene expression data, DNA copy number in reference to the non-tumor cell line (MCF10A, and methylation status of 21,570 CpG islands to identify differentially expressed genes that were correlated with methylation or copy number changes. These were evaluated in a dataset from 129 primary breast tumors. Gene expression in cell lines was dominated by ER-associated genes. ER+ and ER- cell lines formed two distinct, stable clusters, and 1,873 genes were differentially expressed in the two groups. Part of chromosome 8 was deleted in all ER- cells and part of chromosome 17 amplified in all ER+ cells. These loci encoded 30 genes that were overexpressed in ER+ cells; 9 of these genes were overexpressed in ER+ tumors. We identified 149 differentially expressed genes that exhibited differential methylation of one or more CpG islands within 5 kb of the 5' end of the gene and for which mRNA abundance was inversely correlated with CpG island methylation status. In primary tumors we identified 84 genes that appear to be robust components of the methylation signature that we identified in ER+ cell lines. Our analyses reveal a global pattern of differential CpG island methylation that contributes to the transcriptome landscape of ER+ and ER- breast cancer cells and tumors. The role of gene amplification/deletion appears to more modest, although several potentially significant genes appear to be regulated by copy number aberrations.

  12. Three-dimensional fluid-attenuated inversion recovery sequence for visualisation of subthalamic nucleus for deep brain stimulation in Parkinson's disease

    International Nuclear Information System (INIS)

    Heo, Young Jin; Kim, Sang Joon; Kim, Ho Sung; Choi, Choong Gon; Jung, Seung Chai; Lee, Jung Kyo; Lee, Chong Sik; Chung, Sun J.; Cho, So Hyun; Lee, Gyoung Ro

    2015-01-01

    Deep brain stimulation (DBS) of the subthalamic nucleus (STN) is an accepted treatment for advanced Parkinson's disease (PD). However, targeting the STN is difficult due to its relatively small size and variable location. The purpose of this study was to assess which of the following sequences obtained with the 3.0 T MR system can accurately delineate the STN: coronal 3D fluid-attenuated inversion recovery (FLAIR), 2D T2*-weighted fast-field echo (T2*-FFE) and 2D T2-weighted turbo spin-echo (TSE) sequences. We included 20 consecutive patients with PD who underwent 3.0 T MR for DBS targeting. 3D FLAIR, 2D T2*-FFE and T2-TSE images were obtained for all study patients. Image quality and demarcation of the STN were analysed using 4-point scales, and contrast ratio (CR) of the STN and normal white matter was calculated. The Friedman test was used to compare the three sequences. In qualitative analysis, the 2D T2*-FFE image showed more artefacts than 3D FLAIR or 2D T2-TSE, but the difference did not reach statistical significance. 3D FLAIR images showed significantly superior demarcation of the STN compared with 2D T2*-FFE and T2-TSE images (P < 0.001, respectively). The CR of 3D FLAIR was significantly higher than that of 2D T2*-FFE or T2-TSE images in multiple comparison correction (P < 0.001), but there was no significant difference in the CR between 2D T2*-FFE and T2-TSE images. Coronal 3D FLAIR images showed the most accurate demarcation of the STN for DBS targeting among coronal 3D FLAIR, 2D T2*-FFE and T2-TSE images. (orig.)

  13. Deep sequencing-based transcriptome profiling analysis of bacteria-challenged Lateolabrax japonicus reveals insight into the immune-relevant genes in marine fish

    Directory of Open Access Journals (Sweden)

    Xiang Li-xin

    2010-08-01

    Full Text Available Abstract Background Systematic research on fish immunogenetics is indispensable in understanding the origin and evolution of immune systems. This has long been a challenging task because of the limited number of deep sequencing technologies and genome backgrounds of non-model fish available. The newly developed Solexa/Illumina RNA-seq and Digital gene expression (DGE are high-throughput sequencing approaches and are powerful tools for genomic studies at the transcriptome level. This study reports the transcriptome profiling analysis of bacteria-challenged Lateolabrax japonicus using RNA-seq and DGE in an attempt to gain insights into the immunogenetics of marine fish. Results RNA-seq analysis generated 169,950 non-redundant consensus sequences, among which 48,987 functional transcripts with complete or various length encoding regions were identified. More than 52% of these transcripts are possibly involved in approximately 219 known metabolic or signalling pathways, while 2,673 transcripts were associated with immune-relevant genes. In addition, approximately 8% of the transcripts appeared to be fish-specific genes that have never been described before. DGE analysis revealed that the host transcriptome profile of Vibrio harveyi-challenged L. japonicus is considerably altered, as indicated by the significant up- or down-regulation of 1,224 strong infection-responsive transcripts. Results indicated an overall conservation of the components and transcriptome alterations underlying innate and adaptive immunity in fish and other vertebrate models. Analysis suggested the acquisition of numerous fish-specific immune system components during early vertebrate evolution. Conclusion This study provided a global survey of host defence gene activities against bacterial challenge in a non-model marine fish. Results can contribute to the in-depth study of candidate genes in marine fish immunity, and help improve current understanding of host

  14. Quantitative profiling of selective Sox/POU pairing on hundreds of sequences in parallel by Coop-seq.

    Science.gov (United States)

    Chang, Yiming K; Srivastava, Yogesh; Hu, Caizhen; Joyce, Adam; Yang, Xiaoxiao; Zuo, Zheng; Havranek, James J; Stormo, Gary D; Jauch, Ralf

    2017-01-25

    Cooperative binding of transcription factors is known to be important in the regulation of gene expression programs conferring cellular identities. However, current methods to measure cooperativity parameters have been laborious and therefore limited to studying only a few sequence variants at a time. We developed Coop-seq (cooperativity by sequencing) that is capable of efficiently and accurately determining the cooperativity parameters for hundreds of different DNA sequences in a single experiment. We apply Coop-seq to 12 dimer pairs from the Sox and POU families of transcription factors using 324 unique sequences with changed half-site orientation, altered spacing and discrete randomization within the binding elements. The study reveals specific dimerization profiles of different Sox factors with Oct4. By contrast, Oct4 and the three neural class III POU factors Brn2, Brn4 and Oct6 assemble with Sox2 in a surprisingly indistinguishable manner. Two novel half-site configurations can support functional Sox/Oct dimerization in addition to known composite motifs. Moreover, Coop-seq uncovers a nucleotide switch within the POU half-site when spacing is altered, which is mirrored in genomic loci bound by Sox2/Oct4 complexes. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  15. Minimal Residual Disease Detection and Evolved IGH Clones Analysis in Acute B Lymphoblastic Leukemia Using IGH Deep Sequencing.

    Science.gov (United States)

    Wu, Jinghua; Jia, Shan; Wang, Changxi; Zhang, Wei; Liu, Sixi; Zeng, Xiaojing; Mai, Huirong; Yuan, Xiuli; Du, Yuanping; Wang, Xiaodong; Hong, Xueyu; Li, Xuemei; Wen, Feiqiu; Xu, Xun; Pan, Jianhua; Li, Changgang; Liu, Xiao

    2016-01-01

    Acute B lymphoblastic leukemia (B-ALL) is one of the most common types of childhood cancer worldwide and chemotherapy is the main treatment approach. Despite good response rates to chemotherapy regiments, many patients eventually relapse and minimal residual disease (MRD) is the leading risk factor for relapse. The evolution of leukemic clones during disease development and treatment may have clinical significance. In this study, we performed immunoglobulin heavy chain ( IGH ) repertoire high throughput sequencing (HTS) on the diagnostic and post-treatment samples of 51 pediatric B-ALL patients. We identified leukemic IGH clones in 92.2% of the diagnostic samples and nearly half of the patients were polyclonal. About one-third of the leukemic clones have correct open reading frame in the complementarity determining region 3 (CDR3) of IGH , which demonstrates that the leukemic B cells were in the early developmental stage. We also demonstrated the higher sensitivity of HTS in MRD detection and investigated the clinical value of using peripheral blood in MRD detection and monitoring the clonal IGH evolution. In addition, we found leukemic clones were extensively undergoing continuous clonal IGH evolution by variable gene replacement. Dynamic frequency change and newly emerged evolved IGH clones were identified upon the pressure of chemotherapy. In summary, we confirmed the high sensitivity and universal applicability of HTS in MRD detection. We also reported the ubiquitous evolved IGH clones in B-ALL samples and their response to chemotherapy during treatment.

  16. Deep RNA sequencing reveals hidden features and dynamics of early gene transcription in Paramecium bursaria chlorella virus 1.

    Directory of Open Access Journals (Sweden)

    Guillaume Blanc

    Full Text Available Paramecium bursaria chlorella virus 1 (PBCV-1 is the prototype of the genus Chlorovirus (family Phycodnaviridae that infects the unicellular, eukaryotic green alga Chlorella variabilis NC64A. The 331-kb PBCV-1 genome contains 416 major open reading frames. A mRNA-seq approach was used to analyze PBCV-1 transcriptomes at 6 progressive times during the first hour of infection. The alignment of 17 million reads to the PBCV-1 genome allowed the construction of single-base transcriptome maps. Significant transcription was detected for a subset of 50 viral genes as soon as 7 min after infection. By 20 min post infection (p.i., transcripts were detected for most PBCV-1 genes and transcript levels continued to increase globally up to 60 min p.i., at which time 41% or the poly (A+-containing RNAs in the infected cells mapped to the PBCV-1 genome. For some viral genes, the number of transcripts in the latter time points (20 to 60 min p.i. was much higher than that of the most highly expressed host genes. RNA-seq data revealed putative polyadenylation signal sequences in PBCV-1 genes that were identical to the polyadenylation signal AAUAAA of green algae. Several transcripts have an RNA fragment excised. However, the frequency of excision and the resulting putative shortened protein products suggest that most of these excision events have no functional role but are probably the result of the activity of misled splicesomes.

  17. Deep sequencing-based transcriptome analysis of chicken spleen in response to avian pathogenic Escherichia coli (APEC infection.

    Directory of Open Access Journals (Sweden)

    Qinghua Nie

    Full Text Available Avian pathogenic Escherichia coli (APEC leads to economic losses in poultry production and is also a threat to human health. The goal of this study was to characterize the chicken spleen transcriptome and to identify candidate genes for response and resistance to APEC infection using Solexa sequencing. We obtained 14422935, 14104324, and 14954692 Solexa read pairs for non-challenged (NC, challenged-mild pathology (MD, and challenged-severe pathology (SV, respectively. A total of 148197 contigs and 98461 unigenes were assembled, of which 134949 contigs and 91890 unigenes match the chicken genome. In total, 12272 annotated unigenes take part in biological processes (11664, cellular components (11927, and molecular functions (11963. Summing three specific contrasts, 13650 significantly differentially expressed unigenes were found in NC Vs. MD (6844, NC Vs. SV (7764, and MD Vs. SV (2320. Some unigenes (e.g. CD148, CD45 and LCK were involved in crucial pathways, such as the T cell receptor (TCR signaling pathway and microbial metabolism in diverse environments. This study facilitates understanding of the genetic architecture of the chicken spleen transcriptome, and has identified candidate genes for host response to APEC infection.

  18. Deep sequencing of the Mexican avocado transcriptome, an ancient angiosperm with a high content of fatty acids.

    Science.gov (United States)

    Ibarra-Laclette, Enrique; Méndez-Bravo, Alfonso; Pérez-Torres, Claudia Anahí; Albert, Victor A; Mockaitis, Keithanne; Kilaru, Aruna; López-Gómez, Rodolfo; Cervantes-Luevano, Jacob Israel; Herrera-Estrella, Luis

    2015-08-13

    Avocado (Persea americana) is an economically important tropical fruit considered to be a good source of fatty acids. Despite its importance, the molecular and cellular characterization of biochemical and developmental processes in avocado is limited due to the lack of transcriptome and genomic information. The transcriptomes of seeds, roots, stems, leaves, aerial buds and flowers were determined using different sequencing platforms. Additionally, the transcriptomes of three different stages of fruit ripening (pre-climacteric, climacteric and post-climacteric) were also analyzed. The analysis of the RNAseqatlas presented here reveals strong differences in gene expression patterns between different organs, especially between root and flower, but also reveals similarities among the gene expression patterns in other organs, such as stem, leaves and aerial buds (vegetative organs) or seed and fruit (storage organs). Important regulators, functional categories, and differentially expressed genes involved in avocado fruit ripening were identified. Additionally, to demonstrate the utility of the avocado gene expression atlas, we investigated the expression patterns of genes implicated in fatty acid metabolism and fruit ripening. A description of transcriptomic changes occurring during fruit ripening was obtained in Mexican avocado, contributing to a dynamic view of the expression patterns of genes involved in fatty acid biosynthesis and the fruit ripening process.

  19. OCT structure, COB location and magmatic type of the SE Brazilian & S Angolan margins from integrated quantitative analysis of deep seismic reflection and gravity anomaly data

    Science.gov (United States)

    Cowie, L.; Kusznir, N. J.; Horn, B.

    2013-12-01

    Knowledge of ocean-continent transition (OCT) structure, continent-ocean boundary (COB) location and magmatic type are of critical importance for understanding rifted continental margin formation processes and in evaluating petroleum systems in deep-water frontier oil and gas exploration. The OCT structure, COB location and magmatic type of the SE Brazilian and S Angolan rifted continental margins are much debated; exhumed and serpentinised mantle have been reported at these margins. Integrated quantitative analysis using deep seismic reflection data and gravity inversion have been used to determine OCT structure, COB location and magmatic type for the SE Brazilian and S Angolan margins. Gravity inversion has been used to determine Moho depth, crustal basement thickness and continental lithosphere thinning. Residual Depth Anomaly (RDA) analysis has been used to investigate OCT bathymetric anomalies with respect to expected oceanic bathymetries and subsidence analysis has been used to determine the distribution of continental lithosphere thinning. These techniques have been validated on the Iberian margin for profiles IAM9 and ISE-01. In addition a joint inversion technique using deep seismic reflection and gravity anomaly data has been applied to the ION-GXT BS1-575 SE Brazil and ION-GXT CS1-2400 S Angola. The joint inversion method solves for coincident seismic and gravity Moho in the time domain and calculates the lateral variations in crustal basement densities and velocities along profile. Gravity inversion, RDA and subsidence analysis along the S Angolan ION-GXT CS1-2400 profile has been used to determine OCT structure and COB location. Analysis suggests that exhumed mantle, corresponding to a magma poor margin, is absent beneath the allochthonous salt. The thickness of earliest oceanic crust, derived from gravity and deep seismic reflection data is approximately 7km. The joint inversion predicts crustal basement densities and seismic velocities which are

  20. MR imaging of hyaline cartilage at 0.5 T: a quantitative and qualitative in vitro evaluation of three types of sequences

    International Nuclear Information System (INIS)

    Linden, E. van der; Kroon, H.M.; Doornbos, J.; Bloem, J.L.; Hermans, J.

    1998-01-01

    Objective. To identify an optimal pulse sequence for in vitro imaging of hyaline cartilage at 0.5 T. Materials and methods. Twelve holes of varying diameter and depth were drilled in cartilage of two pig knees. These were submerged in saline and scanned with a 0.5-T MR system. Sixteen T1-weighted gradient echo (GE), two T2-weighted GE, and 16 fast spin echo sequences were used, by varying repetition time (TR), echo time (TE), flip angle (FA), echo train length, profile order, and by use of fat saturation. Contrast-to-noise ratios (CNR) of cartilage versus saline solution and cartilage versus subchondral bone were measured. Cartilaginous lesions were evaluated separately by three independent observers. Interobserver variability and correlation between the quantitative and qualitative analyses were calculated. Results. The mean CNRs of two specimens of cartilage versus saline solution ranged from 6.3 (±2.1) to 27.7 (±2.5), and those of cartilage versus subchondral bone from 0.3 (±0.2) to 22.5 (±1.4). The highest CNR was obtained with a T1-weighted spoiled 3D-GE technique (TR 65 ms, TE 11.5 ms, FA 45 ). The number of lesions observed per sequence varied from 35 to 69. Observer agreement was fair to good. The T1-weighted spoiled GE sequences with a TR of 65 ms, TE of 11.5 ms and FA of 30 and 45 were significantly superior to the other 34 sequences in the qualitative analysis. Conclusion. T1-weighted spoiled 3D-GE sequences with a TR of 65 ms, a TE of 11.5 ms, and a FA of 30-45 were found to be optimal for in vitro imaging of cartilage at 0.5 T. (orig.)

  1. MR imaging of hyaline cartilage at 0.5 T: a quantitative and qualitative in vitro evaluation of three types of sequences

    Energy Technology Data Exchange (ETDEWEB)

    Linden, E. van der; Kroon, H.M.; Doornbos, J.; Bloem, J.L. [Department of Radiology C2-S, Albinusdreef 2, Leiden University Medical Center, Postbus 9600, NL-2300 RC Leiden (Netherlands); Hermans, J. [Department of Medical Statistics, Leiden University Medical Center, Leiden (Netherlands)

    1998-06-01

    Objective. To identify an optimal pulse sequence for in vitro imaging of hyaline cartilage at 0.5 T. Materials and methods. Twelve holes of varying diameter and depth were drilled in cartilage of two pig knees. These were submerged in saline and scanned with a 0.5-T MR system. Sixteen T1-weighted gradient echo (GE), two T2-weighted GE, and 16 fast spin echo sequences were used, by varying repetition time (TR), echo time (TE), flip angle (FA), echo train length, profile order, and by use of fat saturation. Contrast-to-noise ratios (CNR) of cartilage versus saline solution and cartilage versus subchondral bone were measured. Cartilaginous lesions were evaluated separately by three independent observers. Interobserver variability and correlation between the quantitative and qualitative analyses were calculated. Results. The mean CNRs of two specimens of cartilage versus saline solution ranged from 6.3 ({+-}2.1) to 27.7 ({+-}2.5), and those of cartilage versus subchondral bone from 0.3 ({+-}0.2) to 22.5 ({+-}1.4). The highest CNR was obtained with a T1-weighted spoiled 3D-GE technique (TR 65 ms, TE 11.5 ms, FA 45 ). The number of lesions observed per sequence varied from 35 to 69. Observer agreement was fair to good. The T1-weighted spoiled GE sequences with a TR of 65 ms, TE of 11.5 ms and FA of 30 and 45 were significantly superior to the other 34 sequences in the qualitative analysis. Conclusion. T1-weighted spoiled 3D-GE sequences with a TR of 65 ms, a TE of 11.5 ms, and a FA of 30-45 were found to be optimal for in vitro imaging of cartilage at 0.5 T. (orig.) With 8 figs., 1 tab., 31 refs.

  2. RNA deep sequencing reveals differential microRNA expression during development of sea urchin and sea star.

    Directory of Open Access Journals (Sweden)

    Sabah Kadri

    Full Text Available microRNAs (miRNAs are small (20-23 nt, non-coding single stranded RNA molecules that act as post-transcriptional regulators of mRNA gene expression. They have been implicated in regulation of developmental processes in diverse organisms. The echinoderms, Strongylocentrotus purpuratus (sea urchin and Patiria miniata (sea star are excellent model organisms for studying development with well-characterized transcriptional networks. However, to date, nothing is known about the role of miRNAs during development in these organisms, except that the genes that are involved in the miRNA biogenesis pathway are expressed during their developmental stages. In this paper, we used Illumina Genome Analyzer (Illumina, Inc. to sequence small RNA libraries in mixed stage population of embryos from one to three days after fertilization of sea urchin and sea star (total of 22,670,000 reads. Analysis of these data revealed the miRNA populations in these two species. We found that 47 and 38 known miRNAs are expressed in sea urchin and sea star, respectively, during early development (32 in common. We also found 13 potentially novel miRNAs in the sea urchin embryonic library. miRNA expression is generally conserved between the two species during development, but 7 miRNAs are highly expressed in only one species. We expect that our two datasets will be a valuable resource for everyone working in the field of developmental biology and the regulatory networks that affect it. The computational pipeline to analyze Illumina reads is available at http://www.benoslab.pitt.edu/services.html.

  3. RNA Deep Sequencing Reveals Differential MicroRNA Expression during Development of Sea Urchin and Sea Star

    Science.gov (United States)

    Kadri, Sabah; Hinman, Veronica F.; Benos, Panayiotis V.

    2011-01-01

    microRNAs (miRNAs) are small (20–23 nt), non-coding single stranded RNA molecules that act as post-transcriptional regulators of mRNA gene expression. They have been implicated in regulation of developmental processes in diverse organisms. The echinoderms, Strongylocentrotus purpuratus (sea urchin) and Patiria miniata (sea star) are excellent model organisms for studying development with well-characterized transcriptional networks. However, to date, nothing is known about the role of miRNAs during development in these organisms, except that the genes that are involved in the miRNA biogenesis pathway are expressed during their developmental stages. In this paper, we used Illumina Genome Analyzer (Illumina, Inc.) to sequence small RNA libraries in mixed stage population of embryos from one to three days after fertilization of sea urchin and sea star (total of 22,670,000 reads). Analysis of these data revealed the miRNA populations in these two species. We found that 47 and 38 known miRNAs are expressed in sea urchin and sea star, respectively, during early development (32 in common). We also found 13 potentially novel miRNAs in the sea urchin embryonic library. miRNA expression is generally conserved between the two species during development, but 7 miRNAs are highly expressed in only one species. We expect that our two datasets will be a valuable resource for everyone working in the field of developmental biology and the regulatory networks that affect it. The computational pipeline to analyze Illumina reads is available at http://www.benoslab.pitt.edu/services.html. PMID:22216218

  4. Deep sequencing of natural and experimental populations of Drosophila melanogaster reveals biases in the spectrum of new mutations.

    Science.gov (United States)

    Assaf, Zoe June; Tilk, Susanne; Park, Jane; Siegal, Mark L; Petrov, Dmitri A

    2017-12-01

    Mutations provide the raw material of evolution, and thus our ability to study evolution depends fundamentally on having precise measurements of mutational rates and patterns. We generate a data set for this purpose using (1) de novo mutations from mutation accumulation experiments and (2) extremely rare polymorphisms from natural populations. The first, mutation accumulation (MA) lines are the product of maintaining flies in tiny populations for many generations, therefore rendering natural selection ineffective and allowing new mutations to accrue in the genome. The second, rare genetic variation from natural populations allows the study of mutation because extremely rare polymorphisms are relatively unaffected by the filter of natural selection. We use both methods in Drosophila melanogaster , first generating our own novel data set of sequenced MA lines and performing a meta-analysis of all published MA mutations (∼2000 events) and then identifying a high quality set of ∼70,000 extremely rare (≤0.1%) polymorphisms that are fully validated with resequencing. We use these data sets to precisely measure mutational rates and patterns. Highlights of our results include: a high rate of multinucleotide mutation events at both short (∼5 bp) and long (∼1 kb) genomic distances, showing that mutation drives GC content lower in already GC-poor regions, and using our precise context-dependent mutation rates to predict long-term evolutionary patterns at synonymous sites. We also show that de novo mutations from independent MA experiments display similar patterns of single nucleotide mutation and well match the patterns of mutation found in natural populations. © 2017 Assaf et al.; Published by Cold Spring Harbor Laboratory Press.

  5. Identification of novel and conserved microRNAs related to drought stress in potato by deep sequencing.

    Science.gov (United States)

    Zhang, Ning; Yang, Jiangwei; Wang, Zemin; Wen, Yikai; Wang, Jie; He, Wenhui; Liu, Bailin; Si, Huaijun; Wang, Di

    2014-01-01

    MicroRNAs (miRNAs) are a group of small, non-coding RNAs that play important roles in plant growth, development and stress response. There have been an increasing number of investigations aimed at discovering miRNAs and analyzing their functions in model plants (such as Arabidopsis thaliana and rice). In this research, we constructed small RNA libraries from both polyethylene glycol (PEG 6,000) treated and control potato samples, and a large number of known and novel miRNAs were identified. Differential expression analysis showed that 100 of the known miRNAs were down-regulated and 99 were up-regulated as a result of PEG stress, while 119 of the novel miRNAs were up-regulated and 151 were down-regulated. Based on target prediction, annotation and expression analysis of the miRNAs and their putative target genes, 4 miRNAs were identified as regulating drought-related genes (miR811, miR814, miR835, miR4398). Their target genes were MYB transcription factor (CV431094), hydroxyproline-rich glycoprotein (TC225721), quaporin (TC223412) and WRKY transcription factor (TC199112), respectively. Relative expression trends of those miRNAs were the same as that predicted by Solexa sequencing and they showed a negative correlation with the expression of the target genes. The results provide molecular evidence for the possible involvement of miRNAs in the process of drought response and/or tolerance in the potato plant.

  6. Transcriptome profiling and digital gene expression by deep sequencing in early somatic embryogenesis of endangered medicinal Eleutherococcus senticosus Maxim.

    Science.gov (United States)

    Tao, Lei; Zhao, Yue; Wu, Ying; Wang, Qiuyu; Yuan, Hongmei; Zhao, Lijuan; Guo, Wendong; You, Xiangling

    2016-03-01

    Somatic embryogenesis (SE) has been studied as a model system to understand molecular events in physiology, biochemistry, and cytology during plant embryo development. In particular, it is exceedingly difficult to access the morphological and early regulatory events in zygotic embryos. To understand the molecular mechanisms regulating early SE in Eleutherococcus senticosus Maxim., we used high-throughput RNA-Seq technology to investigate its transcriptome. We obtained 58,327,688 reads, which were assembled into 75,803 unique unigenes. To better understand their functions, the unigenes were annotated using the Clusters of Orthologous Groups, Gene Ontology, and Kyoto Encyclopedia of Genes and Genomes databases. Digital gene expression libraries revealed differences in gene expression profiles at different developmental stages (embryogenic callus, yellow embryogenic callus, global embryo). We obtained a sequencing depth of >5.6 million tags per sample and identified many differentially expressed genes at various stages of SE. The initiation of SE affected gene expression in many KEGG pathways, but predominantly that in metabolic pathways, biosynthesis of secondary metabolites, and plant hormone signal transduction. This information on the changes in the multiple pathways related to SE induction in E. senticosus Maxim. embryogenic tissue will contribute to a more comprehensive understanding of the mechanisms involved in early SE. Additionally, the differentially expressed genes may act as molecular markers and could play very important roles in the early stage of SE. The results are a comprehensive molecular biology resource for investigating SE of E. senticosus Maxim. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. Deep sequencing of Brachypodium small RNAs at the global genome level identifies microRNAs involved in cold stress response

    Directory of Open Access Journals (Sweden)

    Chong Kang

    2009-09-01

    Full Text Available Abstract Background MicroRNAs (miRNAs are endogenous small RNAs having large-scale regulatory effects on plant development and stress responses. Extensive studies of miRNAs have only been performed in a few model plants. Although miRNAs are proved to be involved in plant cold stress responses, little is known for winter-habit monocots. Brachypodium distachyon, with close evolutionary relationship to cool-season cereals, has recently emerged as a novel model plant. There are few reports of Brachypodium miRNAs. Results High-throughput sequencing and whole-genome-wide data mining led to the identification of 27 conserved miRNAs, as well as 129 predicted miRNAs in Brachypodium. For multiple-member conserved miRNA families, their sizes in Brachypodium were much smaller than those in rice and Populus. The genome organization of miR395 family in Brachypodium was quite different from that in rice. The expression of 3 conserved miRNAs and 25 predicted miRNAs showed significant changes in response to cold stress. Among these miRNAs, some were cold-induced and some were cold-suppressed, but all the conserved miRNAs were up-regulated under cold stress condition. Conclusion Our results suggest that Brachypodium miRNAs are composed of a set of conserved miRNAs and a large proportion of non-conserved miRNAs with low expression levels. Both kinds of miRNAs were involved in cold stress response, but all the conserved miRNAs were up-regulated, implying an important role for cold-induced miRNAs. The different size and genome organization of miRNA families in Brachypodium and rice suggest that the frequency of duplication events or the selection pressure on duplicated miRNAs are different between these two closely related plant species.

  8. A computational approach to distinguish somatic vs. germline origin of genomic alterations from deep sequencing of cancer specimens without a matched normal.

    Directory of Open Access Journals (Sweden)

    James X Sun

    2018-02-01

    Full Text Available A key constraint in genomic testing in oncology is that matched normal specimens are not commonly obtained in clinical practice. Thus, while well-characterized genomic alterations do not require normal tissue for interpretation, a significant number of alterations will be unknown in whether they are germline or somatic, in the absence of a matched normal control. We introduce SGZ (somatic-germline-zygosity, a computational method for predicting somatic vs. germline origin and homozygous vs. heterozygous or sub-clonal state of variants identified from deep massively parallel sequencing (MPS of cancer specimens. The method does not require a patient matched normal control, enabling broad application in clinical research. SGZ predicts the somatic vs. germline status of each alteration identified by modeling the alteration's allele frequency (AF, taking into account the tumor content, tumor ploidy, and the local copy number. Accuracy of the prediction depends on the depth of sequencing and copy number model fit, which are achieved in our clinical assay by sequencing to high depth (>500x using MPS, covering 394 cancer-related genes and over 3,500 genome-wide single nucleotide polymorphisms (SNPs. Calls are made using a statistic based on read depth and local variability of SNP AF. To validate the method, we first evaluated performance on samples from 30 lung and colon cancer patients, where we sequenced tumors and matched normal tissue. We examined predictions for 17 somatic hotspot mutations and 20 common germline SNPs in 20,182 clinical cancer specimens. To assess the impact of stromal admixture, we examined three cell lines, which were titrated with their matched normal to six levels (10-75%. Overall, predictions were made in 85% of cases, with 95-99% of variants predicted correctly, a significantly superior performance compared to a basic approach based on AF alone. We then applied the SGZ method to the COSMIC database of known somatic variants

  9. Quantitative risk trends deriving from PSA-based event analyses. Analysis of results from U.S.NRC's accident sequence precursor program

    International Nuclear Information System (INIS)

    Watanabe, Norio

    2004-01-01

    The United States Nuclear Regulatory Commission (U.S.NRC) has been carrying out the Accident Sequence Precursor (ASP) Program to identify and categorize precursors to potential severe core damage accident sequences using the probabilistic safety assessment (PSA) technique. The ASP Program has identified a lot of risk significant events as precursors that occurred at U.S. nuclear power plants. Although the results from the ASP Program include valuable information that could be useful for obtaining and characterizing risk significant insights and for monitoring risk trends in nuclear power industry, there are only a few attempts to determine and develop the trends using the ASP results. The present study examines and discusses quantitative risk trends for the industry level, using two indicators, that is, the occurrence frequency of precursors and the annual core damage probability, deriving from the results of the ASP analysis. It is shown that the core damage risk at U.S. nuclear power plants has been lowered and the likelihood of risk significant events has been remarkably decreasing. As well, the present study demonstrates that two risk indicators used here can provide quantitative information useful for examining and monitoring the risk trends and/or risk characteristics in nuclear power industry. (author)

  10. Single-nucleotide variant in multiple copies of a deleted in azoospermia (DAZ) sequence - a human Y chromosome quantitative polymorphism.

    Science.gov (United States)

    Szmulewicz, Martin N; Ruiz, Luis M; Reategui, Erika P; Hussini, Saeed; Herrera, Rene J

    2002-01-01

    The evolution of the deleted in azoospermia (DAZ) gene family supports prevalent theories on the origin and development of sex chromosomes and sexual dimorphism. The ancestral DAZL gene in human chromosome 3 is known to be involved in germline development of both males and females. The available phylogenetic data suggest that some time after the divergence of the New World and Old World monkey lineages, the DAZL gene, which is found in all mammals, was copied to the Y chromosome of an ancestor to the Old World monkeys, but not New World monkeys. In modern man, the Y-linked DAZ gene complex is located on the distal part of the q arm. It is thought that after being copied to the Y chromosome, and after the divergence of the human and great ape lineages, the DAZ gene in the former underwent internal rearrangements. This included tandem duplications as well as a T > C transition altering an MboI restriction enzyme site in a duplicated sequence. In this study, we report on the ratios of MboI-/MboI+ variant sequences in individuals from seven worldwide human populations (Basque, Benin, Egypt, Formosa, Kungurtug, Oman and Rwanda) in the DAZ complex. The ratio of PCR MboI- and MboI+ amplicons can be used to characterize individuals and populations. Our results show a nonrandom distribution of MboI-/MboI+ sequence ratios in all populations examined, as well as significant differences in ratios between populations when compared pairwise. The multiple ratios imply that there have been more than one recent reorganization events at this locus. Considering the dynamic nature of this locus and its involvement in male fertility, we investigated the extent and distribution of this polymorphism. Copyright 2002 S. Karger AG, Basel

  11. Chimeric proteins for detection and quantitation of DNA mutations, DNA sequence variations, DNA damage and DNA mismatches

    Science.gov (United States)

    McCutchen-Maloney, Sandra L.

    2002-01-01

    Chimeric proteins having both DNA mutation binding activity and nuclease activity are synthesized by recombinant technology. The proteins are of the general formula A-L-B and B-L-A where A is a peptide having DNA mutation binding activity, L is a linker and B is a peptide having nuclease activity. The chimeric proteins are useful for detection and identification of DNA sequence variations including DNA mutations (including DNA damage and mismatches) by binding to the DNA mutation and cutting the DNA once the DNA mutation is detected.

  12. Identification and characterization of lipid metabolism-related microRNAs in the liver of genetically improved farmed tilapia (GIFT, Oreochromis niloticus) by deep sequencing.

    Science.gov (United States)

    Tao, Yi-Fan; Qiang, Jun; Yin, Guo-Jun; Xu, Pao; Shi, Qiong; Bao, Jing-Wen

    2017-10-01

    MicroRNAs (miRNAs) play vital roles in modulating diverse metabolic processes in the liver, including lipid metabolism. Genetically improved farmed tilapia (GIFT, Oreochromis niloticus), an important aquaculture species in China, is susceptible to hepatic steatosis when reared in intensive culture systems. To investigate the miRNAs involved in GIFT lipid metabolism, two hepatic small RNA libraries from high-fat diet-fed and normal-fat diet-fed GIFT were constructed and sequenced using high-throughput sequencing technology. A total of 204 known and 56 novel miRNAs were identified by aligning the sequencing data with known Danio rerio miRNAs listed in miRBase 21.0. Six known miRNAs (miR-30a-5p, miR-34a, miR-145-5p, miR-29a, miR-205-5p, and miR-23a-3p) that were differentially expressed between the high-fat diet and normal-fat diet groups were validated by quantitative real-time PCR. Bioinformatics tools were used to predict the potential target genes of these differentially expressed miRNAs, and Gene Ontology enrichment analysis indicated that these miRNAs may play important roles in diet-induced hepatic steatosis in GIFT. Our results provide a foundation for further studies of the role of miRNAs in tilapia lipid homeostasis regulation, and may help to identify novel targets for therapeutic interventions to reduce the occurrence of fatty liver disease in farmed tilapia. Copyright © 2017. Published by Elsevier Ltd.

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

    Science.gov (United States)

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

    2017-07-01

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

  14. Carbon transformations in deep granitic groundwater by attached bacterial populations characterized with 16S-rRNA gene sequencing technique and scanning electron microscopy

    International Nuclear Information System (INIS)

    Ekendahl, S.; Arlinger, J.; Staahl, F.; Pedersen, K.

    1993-10-01

    This report presents molecular characterization of attached bacterial populations growing in slowly flowing (1-3 mm s -1 ) artesian groundwater from deep crystalline bed-rock of the Stripa research mine, south central Sweden. The assimilation rate of CO 2 and lactate, and the lactate respiration rates were also determined. The bacteria studied grew in anoxic, high pH, 9-10, and low redox artesian groundwater flowing up through tubings from two levels of a borehole designated V2, 812-820 m and 970-1240 m below ground. The major groups of bacteria were found. Signature bases placed them in the appropriate systematic groups. All belonged to the Proteobacterial groups beta and gamma. One group was found only at the 812-820 m level, where it constituted 63% of the sequenced clones, whereas the second group existed almost exclusively and constituted 85% of the sequenced clones at the 970-1240 m level. The third group was equally distributed between the levels. A few other bacteria were also found. None of the 16S-rRNA genes from the dominating bacteria resembled any of the other by more than 90% similarity, and none of them resembled anything in the database by more than 96%. Temperature did not seem to have any effect on species composition at the deeper level. SEM images showed rods appearing in microcolonies. The difference in population diversity between the two levels studied presumably reflect the different environments. The earlier proposed presence of sulphate reducing bacteria could no be confirmed

  15. Prevalence and evolution of low frequency HIV drug resistance mutations detected by ultra deep sequencing in patients experiencing first line antiretroviral therapy failure.

    Science.gov (United States)

    Vandenhende, Marie-Anne; Bellecave, Pantxika; Recordon-Pinson, Patricia; Reigadas, Sandrine; Bidet, Yannick; Bruyand, Mathias; Bonnet, Fabrice; Lazaro, Estibaliz; Neau, Didier; Fleury, Hervé; Dabis, François; Morlat, Philippe; Masquelier, Bernard

    2014-01-01

    Clinical relevance of low-frequency HIV-1 variants carrying drug resistance associated mutations (DRMs) is still unclear. We aimed to study the prevalence of low-frequency DRMs, detected by Ultra-Deep Sequencing (UDS) before antiretroviral therapy (ART) and at virological failure (VF), in HIV-1 infected patients experiencing VF on first-line ART. Twenty-nine ART-naive patients followed up in the ANRS-CO3 Aquitaine Cohort, having initiated ART between 2000 and 2009 and experiencing VF (2 plasma viral loads (VL) >500 copies/ml or one VL >1000 copies/ml) were included. Reverse transcriptase and protease DRMs were identified using Sanger sequencing (SS) and UDS at baseline (before ART initiation) and VF. Additional low-frequency variants with PI-, NNRTI- and NRTI-DRMs were found by UDS at baseline and VF, significantly increasing the number of detected DRMs by 1.35 fold (plow-frequency DRMs modified ARV susceptibility predictions to the prescribed treatment for 1 patient at baseline, in whom low-frequency DRM was found at high frequency at VF, and 6 patients at VF. DRMs found at VF were rarely detected as low-frequency DRMs prior to treatment. The rare low-frequency NNRTI- and NRTI-DRMs detected at baseline that correlated with the prescribed treatment were most often found at high-frequency at VF. Low frequency DRMs detected before ART initiation and at VF in patients experiencing VF on first-line ART can increase the overall burden of resistance to PI, NRTI and NNRTI.

  16. Deep sequencing analysis of HIV-1 reverse transcriptase at baseline and time of failure in patients receiving rilpivirine in the phase III studies ECHO and THRIVE.

    Science.gov (United States)

    Van Eygen, Veerle; Thys, Kim; Van Hove, Carl; Rimsky, Laurence T; De Meyer, Sandra; Aerssens, Jeroen; Picchio, Gaston; Vingerhoets, Johan

    2016-05-01

    Minority variants (1.0-25.0%) were evaluated by deep sequencing (DS) at baseline and virological failure (VF) in a selection of antiretroviral treatment-naïve, HIV-1-infected patients from the rilpivirine ECHO/THRIVE phase III studies. Linkage between frequently emerging resistance-associated mutations (RAMs) was determined. DS (llIumina®) and population sequencing (PS) results were available at baseline for 47 VFs and time of failure for 48 VFs; and at baseline for 49 responders matched for baseline characteristics. Minority mutations were accurately detected at frequencies down to 1.2% of the HIV-1 quasispecies. No baseline minority rilpivirine RAMs were detected in VFs; one responder carried 1.9% F227C. Baseline minority mutations associated with resistance to other non-nucleoside reverse transcriptase inhibitors (NNRTIs) were detected in 8/47 VFs (17.0%) and 7/49 responders (14.3%). Baseline minority nucleoside/nucleotide reverse transcriptase inhibitor (NRTI) RAMs M184V and L210W were each detected in one VF (none in responders). At failure, two patients without NNRTI RAMs by PS carried minority rilpivirine RAMs K101E and/or E138K; and five additional patients carried other minority NNRTI RAMs V90I, V106I, V179I, V189I, and Y188H. Overall at failure, minority NNRTI RAMs and NRTI RAMs were found in 29/48 (60.4%) and 16/48 VFs (33.3%), respectively. Linkage analysis showed that E138K and K101E were usually not observed on the same viral genome. In conclusion, baseline minority rilpivirine RAMs and other NNRTI/NRTI RAMs were uncommon in the rilpivirine arm of the ECHO and THRIVE studies. DS at failure showed emerging NNRTI resistant minority variants in seven rilpivirine VFs who had no detectable NNRTI RAMs by PS. © 2015 Wiley Periodicals, Inc.

  17. Visualization of the internal globus pallidus: sequence and orientation for deep brain stimulation using a standard installation protocol at 3.0 Tesla.

    Science.gov (United States)

    Nölte, Ingo S; Gerigk, Lars; Al-Zghloul, Mansour; Groden, Christoph; Kerl, Hans U

    2012-03-01

    Deep-brain stimulation (DBS) of the internal globus pallidus (GPi) has shown remarkable therapeutic benefits for treatment-resistant neurological disorders including dystonia and Parkinson's disease (PD). The success of the DBS is critically dependent on the reliable visualization of the GPi. The aim of the study was to evaluate promising 3.0 Tesla magnetic resonance imaging (MRI) methods for pre-stereotactic visualization of the GPi using a standard installation protocol. MRI at 3.0 T of nine healthy individuals and of one patient with PD was acquired (FLAIR, T1-MPRAGE, T2-SPACE, T2*-FLASH2D, susceptibility-weighted imaging mapping (SWI)). Image quality and visualization of the GPi for each sequence were assessed by two neuroradiologists independently using a 6-point scale. Axial, coronal, and sagittal planes of the T2*-FLASH2D images were compared. Inter-rater reliability, contrast-to-noise ratios (CNR) and signal-to-noise ratios (SNR) for the GPi were determined. For illustration, axial T2*-FLASH2D images were fused with a section schema of the Schaltenbrand-Wahren stereotactic atlas. The GPi was best and reliably visualized in axial and to a lesser degree on coronal T2*-FLASH2D images. No major artifacts in the GPi were observed in any of the sequences. SWI offered a significantly higher CNR for the GPi compared to standard T2-weighted imaging using the standard parameters. The fusion of the axial T2*-FLASH2D images and the atlas projected the GPi clearly in the boundaries of the section schema. Using a standard installation protocol at 3.0 T T2*-FLASH2D imaging (particularly axial view) provides optimal and reliable delineation of the GPi.

  18. Development on quantitative safety analysis method of accident scenario. The automatic scenario generator development for event sequence construction of accident

    International Nuclear Information System (INIS)

    Kojima, Shigeo; Onoue, Akira; Kawai, Katsunori

    1998-01-01

    This study intends to develop a more sophisticated tool that will advance the current event tree method used in all PSA, and to focus on non-catastrophic events, specifically a non-core melt sequence scenario not included in an ordinary PSA. In the non-catastrophic event PSA, it is necessary to consider various end states and failure combinations for the purpose of multiple scenario construction. Therefore it is anticipated that an analysis work should be reduced and automated method and tool is required. A scenario generator that can automatically handle scenario construction logic and generate the enormous size of sequences logically identified by state-of-the-art methodology was developed. To fulfill the scenario generation as a technical tool, a simulation model associated with AI technique and graphical interface, was introduced. The AI simulation model in this study was verified for the feasibility of its capability to evaluate actual systems. In this feasibility study, a spurious SI signal was selected to test the model's applicability. As a result, the basic capability of the scenario generator could be demonstrated and important scenarios were generated. The human interface with a system and its operation, as well as time dependent factors and their quantification in scenario modeling, was added utilizing human scenario generator concept. Then the feasibility of an improved scenario generator was tested for actual use. Automatic scenario generation with a certain level of credibility, was achieved by this study. (author)

  19. [Cloning and sequence analysis of the DHBV genome of the brown ducks in Guilin region and establishment of the quantitative method for detecting DHBV].

    Science.gov (United States)

    Su, He-Ling; Huang, Ri-Dong; He, Song-Qing; Xu, Qing; Zhu, Hua; Mo, Zhi-Jing; Liu, Qing-Bo; Liu, Yong-Ming

    2013-03-01

    Brown ducks carrying DHBV were widely used as hepatitis B animal model in the research of the activity and toxicity of anti-HBV dugs. Studies showed that the ratio of DHBV carriers in the brown ducks in Guilin region was relatively high. Nevertheless, the characters of the DHBV genome of Guilin brown duck remain unknown. Here we report the cloning of the genome of Guilin brown duck DHBV and the sequence analysis of the genome. The full length of the DHBV genome of Guilin brown duck was 3 027bp. Analysis using ORF finder found that there was an ORF for an unknown peptide other than S-ORF, PORF and C-ORF in the genome of the DHBV. Vector NTI 8. 0 analysis revealed that the unknown peptide contained a motif which binded to HLA * 0201. Aligning with the DHBV sequences from different countries and regions indicated that there were no obvious differences of regional distribution among the sequences. A fluorescence quantitative PCR for detecting DHBV was establishment based on the recombinant plasmid pGEM-DHBV-S constructed. This study laid the groundwork for using Guilin brown duck as a hepatitis B animal model.

  20. Comparison of the live attenuated yellow fever vaccine 17D-204 strain to its virulent parental strain Asibi by deep sequencing.

    Science.gov (United States)

    Beck, Andrew; Tesh, Robert B; Wood, Thomas G; Widen, Steven G; Ryman, Kate D; Barrett, Alan D T

    2014-02-01

    The first comparison of a live RNA viral vaccine strain to its wild-type parental strain by deep sequencing is presented using as a model the yellow fever virus (YFV) live vaccine strain 17D-204 and its wild-type parental strain, Asibi. The YFV 17D-204 vaccine genome was compared to that of the parental strain Asibi by massively parallel methods. Variability was compared on multiple scales of the viral genomes. A modeled exploration of small-frequency variants was performed to reconstruct plausible regions of mutational plasticity. Overt quasispecies diversity is a feature of the parental strain, whereas the live vaccine strain lacks diversity according to multiple independent measurements. A lack of attenuating mutations in the Asibi population relative to that of 17D-204 was observed, demonstrating that the vaccine strain was derived by discrete mutation of Asibi and not by selection of genomes in the wild-type population. Relative quasispecies structure is a plausible correlate of attenuation for live viral vaccines. Analyses such as these of attenuated viruses improve our understanding of the molecular basis of vaccine attenuation and provide critical information on the stability of live vaccines and the risk of reversion to virulence.

  1. Expression profiles of mRNA and long noncoding RNA in the ovaries of letrozole-induced polycystic ovary syndrome rat model through deep sequencing.

    Science.gov (United States)

    Fu, Lu-Lu; Xu, Ying; Li, Dan-Dan; Dai, Xiao-Wei; Xu, Xin; Zhang, Jing-Shun; Ming, Hao; Zhang, Xue-Ying; Zhang, Guo-Qing; Ma, Ya-Lan; Zheng, Lian-Wen

    2018-05-30

    Polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders in reproductive-aged women. However, the exact pathophysiology of PCOS remains largely unclear. We performed deep sequencing to investigate the mRNA and long noncoding RNA (lncRNA) expression profiles in the ovarian tissues of letrozole-induced PCOS rat model and control rats. A total of 2147 mRNAs and 158 lncRNAs were differentially expressed between the PCOS models and control. Gene ontology analysis indicated that differentially expressed mRNAs were associated with biological adhesion, reproduction, and metabolic process. Pathway analysis results indicated that these aberrantly expressed mRNAs were related to several specific signaling pathways, including insulin resistance, steroid hormone biosynthesis, PPAR signaling pathway, cell adhesion molecules, autoimmune thyroid disease, and AMPK signaling pathway. The relative expression levels of mRNAs and lncRNAs were validated through qRT-PCR. LncRNA-miRNA-mRNA network was constructed to explore ceRNAs involved in the PCOS model and were also verified by qRTPCR experiment. These findings may provide insight into the pathogenesis of PCOS and clues to find key diagnostic and therapeutic roles of lncRNA in PCOS. Copyright © 2018 Elsevier B.V. All rights reserved.

  2. Quantitative evaluation of deep and shallow tissue layers' contribution to fNIRS signal using multi-distance optodes and independent component analysis.

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    Funane, Tsukasa; Atsumori, Hirokazu; Katura, Takusige; Obata, Akiko N; Sato, Hiroki; Tanikawa, Yukari; Okada, Eiji; Kiguchi, Masashi

    2014-01-15

    To quantify the effect of absorption changes in the deep tissue (cerebral) and shallow tissue (scalp, skin) layers on functional near-infrared spectroscopy (fNIRS) signals, a method using multi-distance (MD) optodes and independent component analysis (ICA), referred to as the MD-ICA method, is proposed. In previous studies, when the signal from the shallow tissue layer (shallow signal) needs to be eliminated, it was often assumed that the shallow signal had no correlation with the signal from the deep tissue layer (deep signal). In this study, no relationship between the waveforms of deep and shallow signals is assumed, and instead, it is assumed that both signals are linear combinations of multiple signal sources, which allows the inclusion of a "shared component" (such as systemic signals) that is contained in both layers. The method also assumes that the partial optical path length of the shallow layer does not change, whereas that of the deep layer linearly increases along with the increase of the source-detector (S-D) distance. Deep- and shallow-layer contribution ratios of each independent component (IC) are calculated using the dependence of the weight of each IC on the S-D distance. Reconstruction of deep- and shallow-layer signals are performed by the sum of ICs weighted by the deep and shallow contribution ratio. Experimental validation of the principle of this technique was conducted using a dynamic phantom with two absorbing layers. Results showed that our method is effective for evaluating deep-layer contributions even if there are high correlations between deep and shallow signals. Next, we applied the method to fNIRS signals obtained on a human head with 5-, 15-, and 30-mm S-D distances during a verbal fluency task, a verbal working memory task (prefrontal area), a finger tapping task (motor area), and a tetrametric visual checker-board task (occipital area) and then estimated the deep-layer contribution ratio. To evaluate the signal separation

  3. Consistency and reproducibility of next-generation sequencing and other multigene mutational assays: A worldwide ring trial study on quantitative cytological molecular reference specimens.

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    Malapelle, Umberto; Mayo-de-Las-Casas, Clara; Molina-Vila, Miguel A; Rosell, Rafael; Savic, Spasenija; Bihl, Michel; Bubendorf, Lukas; Salto-Tellez, Manuel; de Biase, Dario; Tallini, Giovanni; Hwang, David H; Sholl, Lynette M; Luthra, Rajyalakshmi; Weynand, Birgit; Vander Borght, Sara; Missiaglia, Edoardo; Bongiovanni, Massimo; Stieber, Daniel; Vielh, Philippe; Schmitt, Fernando; Rappa, Alessandra; Barberis, Massimo; Pepe, Francesco; Pisapia, Pasquale; Serra, Nicola; Vigliar, Elena; Bellevicine, Claudio; Fassan, Matteo; Rugge, Massimo; de Andrea, Carlos E; Lozano, Maria D; Basolo, Fulvio; Fontanini, Gabriella; Nikiforov, Yuri E; Kamel-Reid, Suzanne; da Cunha Santos, Gilda; Nikiforova, Marina N; Roy-Chowdhuri, Sinchita; Troncone, Giancarlo

    2017-08-01

    Molecular testing of cytological lung cancer specimens includes, beyond epidermal growth factor receptor (EGFR), emerging predictive/prognostic genomic biomarkers such as Kirsten rat sarcoma viral oncogene homolog (KRAS), neuroblastoma RAS viral [v-ras] oncogene homolog (NRAS), B-Raf proto-oncogene, serine/threonine kinase (BRAF), and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α (PIK3CA). Next-generation sequencing (NGS) and other multigene mutational assays are suitable for cytological specimens, including smears. However, the current literature reflects single-institution studies rather than multicenter experiences. Quantitative cytological molecular reference slides were produced with cell lines designed to harbor concurrent mutations in the EGFR, KRAS, NRAS, BRAF, and PIK3CA genes at various allelic ratios, including low allele frequencies (AFs; 1%). This interlaboratory ring trial study included 14 institutions across the world that performed multigene mutational assays, from tissue extraction to data analysis, on these reference slides, with each laboratory using its own mutation analysis platform and methodology. All laboratories using NGS (n = 11) successfully detected the study's set of mutations with minimal variations in the means and standard errors of variant fractions at dilution points of 10% (P = .171) and 5% (P = .063) despite the use of different sequencing platforms (Illumina, Ion Torrent/Proton, and Roche). However, when mutations at a low AF of 1% were analyzed, the concordance of the NGS results was low, and this reflected the use of different thresholds for variant calling among the institutions. In contrast, laboratories using matrix-assisted laser desorption/ionization-time of flight (n = 2) showed lower concordance in terms of mutation detection and mutant AF quantification. Quantitative molecular reference slides are a useful tool for monitoring the performance of different multigene mutational

  4. Deep sequencing of small RNA libraries from human prostate epithelial and stromal cells reveal distinct pattern of microRNAs primarily predicted to target growth factors.

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    Singh, Savita; Zheng, Yun; Jagadeeswaran, Guru; Ebron, Jey Sabith; Sikand, Kavleen; Gupta, Sanjay; Sunker, Ramanjulu; Shukla, Girish C

    2016-02-28

    Complex epithelial and stromal cell interactions are required during the development and progression of prostate cancer. Regulatory small non-coding microRNAs (miRNAs) participate in the spatiotemporal regulation of messenger RNA (mRNA) and regulation of translation affecting a large number of genes involved in prostate carcinogenesis. In this study, through deep-sequencing of size fractionated small RNA libraries we profiled the miRNAs of prostate epithelial (PrEC) and stromal (PrSC) cells. Over 50 million reads were obtained for PrEC in which 860,468 were unique sequences. Similarly, nearly 76 million reads for PrSC were obtained in which over 1 million were unique reads. Expression of many miRNAs of broadly conserved and poorly conserved miRNA families were identified. Sixteen highly expressed miRNAs with significant change in expression in PrSC than PrEC were further analyzed in silico. ConsensusPathDB showed the target genes of these miRNAs were significantly involved in adherence junction, cell adhesion, EGRF, TGF-β and androgen signaling. Let-7 family of tumor-suppressor miRNAs expression was highly pervasive in both, PrEC and PrSC cells. In addition, we have also identified several miRNAs that are unique to PrEC or PrSC cells and their predicted putative targets are a group of transcription factors. This study provides perspective on the miRNA expression in PrEC and PrSC, and reveals a global trend in miRNA interactome. We conclude that the most abundant miRNAs are potential regulators of development and differentiation of the prostate gland by targeting a set of growth factors. Additionally, high level expression of the most members of let-7 family miRNAs suggests their role in the fine tuning of the growth and proliferation of prostate epithelial and stromal cells. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  5. A translational study of resistance emergence using sequential direct-acting antiviral agents for hepatitis C using ultra-deep sequencing.

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    Abe, Hiromi; Hayes, C Nelson; Hiraga, Nobuhiko; Imamura, Michio; Tsuge, Masataka; Miki, Daiki; Takahashi, Shoichi; Ochi, Hidenori; Chayama, Kazuaki

    2013-09-01

    Direct-acting antiviral agents (DAAs) against hepatitis C virus (HCV) have recently been developed and are ultimately hoped to replace interferon-based therapy. However, DAA monotherapy results in rapid emergence of resistant strains and DAAs must be used in combinations that present a high genetic barrier to resistance, although viral kinetics of multidrug-resistant strains remain poorly characterized. The aim of this study is to track the emergence and fitness of resistance using combinations of telaprevir and NS5A or NS5B inhibitors with genotype 1b clones. HCV-infected chimeric mice were treated with DAAs, and resistance was monitored using direct and ultra-deep sequencing. Combination therapy with telaprevir and BMS-788329 (NS5A inhibitor) reduced serum HCV RNA to undetectable levels. The presence of an NS3-V36A telaprevir resistance mutation resulted in poor response to telaprevir monotherapy but showed significant HCV reduction when telaprevir was combined with BMS-788329. However, a BMS-788329-resistant strain emerged at low frequency. Infection with a BMS-788329-resistant NS5A-L31V mutation rapidly resulted in gain of an additional NS5A-Y93A mutation that conferred telaprevir resistance during combination therapy. Infection with dual NS5AL31V/NS5AY93H mutations resulted in poor response to combination therapy and development of telaprevir resistance. Although HCV RNA became undetectable soon after the beginning of combination therapy with BMS-788329 and BMS-821095 (NS5B inhibitor), rebound with emergence of resistance against all three drugs occurred. Triple resistance also occurred following infection with the NS3V36A/NS5AL31V/NS5AY93H triple mutation. Resistant strains easily develop from cloned virus strains. Sequential use of DAAs should be avoided to prevent emergence of multidrug-resistant strains.

  6. SRY mutation analysis by next generation (deep sequencing in a cohort of chromosomal Disorders of Sex Development (DSD patients with a mosaic karyotype

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    Hersmus Remko

    2012-11-01

    Full Text Available Abstract Background The presence of the Y-chromosome or Y chromosome-derived material is seen in 4-60% of Turner syndrome patients (Chromosomal Disorders of Sex Development (DSD. DSD patients with specific Y-chromosomal material in their karyotype, the GonadoBlastoma on the Y-chromosome (GBY region, have an increased risk of developing type II germ cell tumors/cancer (GCC, most likely related to TSPY. The Sex determining Region on the Y gene (SRY is located on the short arm of the Y-chromosome and is the crucial switch that initiates testis determination and subsequent male development. Mutations in this gene are responsible for sex reversal in approximately 10-15% of 46,XY pure gonadal dysgenesis (46,XY DSD cases. The majority of the mutations described are located in the central HMG domain, which is involved in the binding and bending of the DNA and harbors two nuclear localization signals. SRY mutations have also been found in a small number of patients with a 45,X/46,XY karyotype and might play a role in the maldevelopment of the gonads. Methods To thoroughly investigate the presence of possible SRY gene mutations in mosaic DSD patients, we performed next generation (deep sequencing on the genomic DNA of fourteen independent patients (twelve 45,X/46,XY, one 45,X/46,XX/46,XY, and one 46,XX/46,XY. Results and conclusions The results demonstrate that aberrations in SRY are rare in mosaic DSD patients and therefore do not play a significant role in the etiology of the disease.

  7. Mutations Related to Antiretroviral Resistance Identified by Ultra-Deep Sequencing in HIV-1 Infected Children under Structured Interruptions of HAART.

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    Jose Manuel Vazquez-Guillen

    Full Text Available Although Structured Treatment Interruptions (STI are currently not considered an alternative strategy for antiretroviral treatment, their true benefits and limitations have not been fully established. Some studies suggest the possibility of improving the quality of life of patients with this strategy; however, the information that has been obtained corresponds mostly to studies conducted in adults, with a lack of knowledge about its impact on children. Furthermore, mutations associated with antiretroviral resistance could be selected due to sub-therapeutic levels of HAART at each interruption period. Genotyping methods to determine the resistance profiles of the infecting viruses have become increasingly important for the management of patients under STI, thus low-abundance antiretroviral drug-resistant mutations (DRM's at levels under limit of detection of conventional genotyping (<20% of quasispecies could increase the risk of virologic failure. In this work, we analyzed the protease and reverse transcriptase regions of the pol gene by ultra-deep sequencing in pediatric patients under STI with the aim of determining the presence of high- and low-abundance DRM's in the viral rebounds generated by the STI. High-abundance mutations in protease and high- and low-abundance mutations in reverse transcriptase were detected but no one of these are directly associated with resistance to antiretroviral drugs. The results could suggest that the evaluated STI program is virologically safe, but strict and carefully planned studies, with greater numbers of patients and interruption/restart cycles, are still needed to evaluate the selection of DRM's during STI.

  8. B1 mapping for bias-correction in quantitative T1 imaging of the brain at 3T using standard pulse sequences.

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    Boudreau, Mathieu; Tardif, Christine L; Stikov, Nikola; Sled, John G; Lee, Wayne; Pike, G Bruce

    2017-12-01

    B 1 mapping is important for many quantitative imaging protocols, particularly those that include whole-brain T 1 mapping using the variable flip angle (VFA) technique. However, B 1 mapping sequences are not typically available on many magnetic resonance imaging (MRI) scanners. The aim of this work was to demonstrate that B 1 mapping implemented using standard scanner product pulse sequences can produce B 1 (and VFA T 1 ) maps comparable in quality and acquisition time to advanced techniques. Six healthy subjects were scanned at 3.0T. An interleaved multislice spin-echo echo planar imaging double-angle (EPI-DA) B 1 mapping protocol, using a standard product pulse sequence, was compared to two alternative methods (actual flip angle imaging, AFI, and Bloch-Siegert shift, BS). Single-slice spin-echo DA B 1 maps were used as a reference for comparison (Ref. DA). VFA flip angles were scaled using each B 1 map prior to fitting T 1 ; the nominal flip angle case was also compared. The pooled-subject voxelwise correlation (ρ) for B 1 maps (BS/AFI/EPI-DA) relative to the reference B 1 scan (Ref. DA) were ρ = 0.92/0.95/0.98. VFA T 1 correlations using these maps were ρ = 0.86/0.88/0.96, much better than without B 1 correction (ρ = 0.53). The relative error for each B 1 map (BS/AFI/EPI-DA/Nominal) had 95 th percentiles of 5/4/3/13%. Our findings show that B 1 mapping implemented using product pulse sequences can provide excellent quality B 1 (and VFA T 1 ) maps, comparable to other custom techniques. This fast whole-brain measurement (∼2 min) can serve as an excellent alternative for researchers without access to advanced B 1 pulse sequences. 1 Technical Efficacy: Stage 1 J. Magn. Reson. Imaging 2017;46:1673-1682. © 2017 International Society for Magnetic Resonance in Medicine.

  9. Deep sequencing and flow cytometric characterization of expanded effector memory CD8+CD57+ T cells frequently reveals T-cell receptor Vβ oligoclonality and CDR3 homology in acquired aplastic anemia.

    Science.gov (United States)

    Giudice, Valentina; Feng, Xingmin; Lin, Zenghua; Hu, Wei; Zhang, Fanmao; Qiao, Wangmin; Ibanez, Maria Del Pilar Fernandez; Rios, Olga; Young, Neal S

    2018-05-01

    Oligoclonal expansion of CD8 + CD28 - lymphocytes has been considered indirect evidence for a pathogenic immune response in acquired aplastic anemia. A subset of CD8 + CD28 - cells with CD57 expression, termed effector memory cells, is expanded in several immune-mediated diseases and may have a role in immune surveillance. We hypothesized that effector memory CD8 + CD28 - CD57 + cells may drive aberrant oligoclonal expansion in aplastic anemia. We found CD8 + CD57 + cells frequently expanded in the blood of aplastic anemia patients, with oligoclonal characteristics by flow cytometric Vβ usage analysis: skewing in 1-5 Vβ families and frequencies of immunodominant clones ranging from 1.98% to 66.5%. Oligoclonal characteristics were also observed in total CD8 + cells from aplastic anemia patients with CD8 + CD57 + cell expansion by T-cell receptor deep sequencing, as well as the presence of 1-3 immunodominant clones. Oligoclonality was confirmed by T-cell receptor repertoire deep sequencing of enriched CD8 + CD57 + cells, which also showed decreased diversity compared to total CD4 + and CD8 + cell pools. From analysis of complementarity-determining region 3 sequences in the CD8 + cell pool, a total of 29 sequences were shared between patients and controls, but these sequences were highly expressed in aplastic anemia subjects and also present in their immunodominant clones. In summary, expansion of effector memory CD8 + T cells is frequent in aplastic anemia and mirrors Vβ oligoclonal expansion. Flow cytometric Vβ usage analysis combined with deep sequencing technologies allows high resolution characterization of the T-cell receptor repertoire, and might represent a useful tool in the diagnosis and periodic evaluation of aplastic anemia patients. (Registered at clinicaltrials.gov identifiers: 00001620, 01623167, 00001397, 00071045, 00081523, 00961064 ). Copyright © 2018 Ferrata Storti Foundation.

  10. Deep sequencing of the viral phoH gene reveals temporal variation, depth-specific composition, and persistent dominance of the same viral phoH genes in the Sargasso Sea

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    Dawn B. Goldsmith

    2015-06-01

    Full Text Available Deep sequencing of the viral phoH gene, a host-derived auxiliary metabolic gene, was used to track viral diversity throughout the water column at the Bermuda Atlantic Time-series Study (BATS site in the summer (September and winter (March of three years. Viral phoH sequences reveal differences in the viral communities throughout a depth profile and between seasons in the same year. Variation was also detected between the same seasons in subsequent years, though these differences were not as great as the summer/winter distinctions. Over 3,600 phoH operational taxonomic units (OTUs; 97% sequence identity were identified. Despite high richness, most phoH sequences belong to a few large, common OTUs whereas the majority of the OTUs are small and rare. While many OTUs make sporadic appearances at just a few times or depths, a small number of OTUs dominate the community throughout the seasons, depths, and years.

  11. Personalized mapping of the deep brain with a white matter attenuated inversion recovery (WAIR) sequence at 1.5-tesla: Experience based on a series of 156 patients.

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    Zerroug, A; Gabrillargues, J; Coll, G; Vassal, F; Jean, B; Chabert, E; Claise, B; Khalil, T; Sakka, L; Feschet, F; Durif, F; Boyer, L; Coste, J; Lemaire, J-J

    2016-08-01

    Deep brain mapping has been proposed for direct targeting in stereotactic functional surgery, aiming to personalize electrode implantation according to individual MRI anatomy without atlas or statistical template. We report our clinical experience of direct targeting in a series of 156 patients operated on using a dedicated Inversion Recovery Turbo Spin Echo sequence at 1.5-tesla, called White Matter Attenuated Inversion Recovery (WAIR). After manual contouring of all pertinent structures and 3D planning of trajectories, 312 DBS electrodes were implanted. Detailed anatomy of close neighbouring structures, whether gray nuclei or white matter regions, was identified during each planning procedure. We gathered the experience of these 312 deep brain mappings and elaborated consistent procedures of anatomical MRI mapping for pallidal, subthalamic and ventral thalamic regions. We studied the number of times the central track anatomically optimized was selected for implantation of definitive electrodes. WAIR sequence provided high-quality images of most common functional targets, successfully used for pure direct stereotactic targeting: the central track corresponding to the optimized primary anatomical trajectory was chosen for implantation of definitive electrodes in 90.38%. WAIR sequence is anatomically reliable, enabling precise deep brain mapping and direct stereotactic targeting under routine clinical conditions. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  12. Large-Scale Genotyping-by-Sequencing Indicates High Levels of Gene Flow in the Deep-Sea Octocoral Swiftia simplex (Nutting 1909 on the West Coast of the United States.

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    Meredith V Everett

    Full Text Available Deep-sea corals are a critical component of habitat in the deep-sea, existing as regional hotspots for biodiversity, and are associated with increased assemblages of fish, including commercially important species. Because sampling these species is so difficult, little is known about the connectivity and life history of deep-sea octocoral populations. This study evaluates the genetic connectivity among 23 individuals of the deep-sea octocoral Swiftia simplex collected from Eastern Pacific waters along the west coast of the United States. We utilized high-throughput restriction-site associated DNA (RAD-tag sequencing to develop the first molecular genetic resource for the deep-sea octocoral, Swiftia simplex. Using this technique we discovered thousands of putative genome-wide SNPs in this species, and after quality control, successfully genotyped 1,145 SNPs across individuals sampled from California to Washington. These SNPs were used to assess putative population structure across the region. A STRUCTURE analysis as well as a principal coordinates analysis both failed to detect any population differentiation across all geographic areas in these collections. Additionally, after assigning individuals to putative population groups geographically, no significant FST values could be detected (FST for the full data set 0.0056, and no significant isolation by distance could be detected (p = 0.999. Taken together, these results indicate a high degree of connectivity and potential panmixia in S. simplex along this portion of the continental shelf.

  13. Directional RNA deep sequencing sheds new light on the transcriptional response of Anabaena sp. strain PCC 7120 to combined-nitrogen deprivation

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    Head Steven R

    2011-06-01

    Full Text Available Abstract Background Cyanobacteria are potential sources of renewable chemicals and biofuels and serve as model organisms for bacterial photosynthesis, nitrogen fixation, and responses to environmental changes. Anabaena (Nostoc sp. strain PCC 7120 (hereafter Anabaena is a multicellular filamentous cyanobacterium that can "fix" atmospheric nitrogen into ammonia when grown in the absence of a source of combined nitrogen. Because the nitrogenase enzyme is oxygen sensitive, Anabaena forms specialized cells called heterocysts that create a microoxic environment for nitrogen fixation. We have employed directional RNA-seq to map the Anabaena transcriptome during vegetative cell growth and in response to combined-nitrogen deprivation, which induces filaments to undergo heterocyst development. Our data provide an unprecedented view of transcriptional changes in Anabaena filaments during the induction of heterocyst development and transition to diazotrophic growth. Results Using the Illumina short read platform and a directional RNA-seq protocol, we obtained deep sequencing data for RNA extracted from filaments at 0, 6, 12, and 21 hours after the removal of combined nitrogen. The RNA-seq data provided information on transcript abundance and boundaries for the entire transcriptome. From these data, we detected novel antisense transcripts within the UTRs (untranslated regions and coding regions of key genes involved in heterocyst development, suggesting that antisense RNAs may be important regulators of the nitrogen response. In addition, many 5' UTRs were longer than anticipated, sometimes extending into upstream open reading frames (ORFs, and operons often showed complex structure and regulation. Finally, many genes that had not been previously identified as being involved in heterocyst development showed regulation, providing new candidates for future studies in this model organism. Conclusions Directional RNA-seq data were obtained that provide

  14. Deep sequencing uncovers commonality in small RNA profiles between transgene-induced and naturally occurring RNA silencing of chalcone synthase-A gene in petunia.

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    Kasai, Megumi; Matsumura, Hideo; Yoshida, Kentaro; Terauchi, Ryohei; Taneda, Akito; Kanazawa, Akira

    2013-01-30

    Introduction of a transgene that transcribes RNA homologous to an endogenous gene in the plant genome can induce silencing of both genes, a phenomenon termed cosuppression. Cosuppression was first discovered in transgenic petunia plants transformed with the CHS-A gene encoding chalcone synthase, in which nonpigmented sectors in flowers or completely white flowers are produced. Some of the flower-color patterns observed in transgenic petunias having CHS-A cosuppression resemble those in existing nontransgenic varieties. Although the mechanism by which white sectors are generated in nontransgenic petunia is known to be due to RNA silencing of the CHS-A gene as in cosuppression, whether the same trigger(s) and/or pattern of RNA degradation are involved in these phenomena has not been known. Here, we addressed this question using deep-sequencing and bioinformatic analyses of small RNAs. We analyzed short interfering RNAs (siRNAs) produced in nonpigmented sectors of petal tissues in transgenic petunia plants that have CHS-A cosuppression and a nontransgenic petunia variety Red Star, that has naturally occurring CHS-A RNA silencing. In both silencing systems, 21-nt and 22-nt siRNAs were the most and the second-most abundant size classes, respectively. CHS-A siRNA production was confined to exon 2, indicating that RNA degradation through the RNA silencing pathway occurred in this exon. Common siRNAs were detected in cosuppression and naturally occurring RNA silencing, and their ranks based on the number of siRNAs in these plants were correlated with each other. Noticeably, highly abundant siRNAs were common in these systems. Phased siRNAs were detected in multiple phases at multiple sites, and some of the ends of the regions that produced phased siRNAs were conserved. The features of siRNA production found to be common to cosuppression and naturally occurring silencing of the CHS-A gene indicate mechanistic similarities between these silencing systems especially in the

  15. Quantitative T1 and T2* carotid atherosclerotic plaque imaging using a three-dimensional multi-echo phase-sensitive inversion recovery sequence: a feasibility study.

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    Fujiwara, Yasuhiro; Maruyama, Hirotoshi; Toyomaru, Kanako; Nishizaka, Yuri; Fukamatsu, Masahiro

    2018-06-01

    Magnetic resonance imaging (MRI) is widely used to detect carotid atherosclerotic plaques. Although it is important to evaluate vulnerable carotid plaques containing lipids and intra-plaque hemorrhages (IPHs) using T 1 -weighted images, the image contrast changes depending on the imaging settings. Moreover, to distinguish between a thrombus and a hemorrhage, it is useful to evaluate the iron content of the plaque using both T 1 -weighted and T 2 *-weighted images. Therefore, a quantitative evaluation of carotid atherosclerotic plaques using T 1 and T 2 * values may be necessary for the accurate evaluation of plaque components. The purpose of this study was to determine whether the multi-echo phase-sensitive inversion recovery (mPSIR) sequence can improve T 1 contrast while simultaneously providing accurate T 1 and T 2 * values of an IPH. T 1 and T 2 * values measured using mPSIR were compared to values from conventional methods in phantom and in vivo studies. In the phantom study, the T 1 and T 2 * values estimated using mPSIR were linearly correlated with those of conventional methods. In the in vivo study, mPSIR demonstrated higher T 1 contrast between the IPH phantom and sternocleidomastoid muscle than the conventional method. Moreover, the T 1 and T 2 * values of the blood vessel wall and sternocleidomastoid muscle estimated using mPSIR were correlated with values measured by conventional methods and with values reported previously. The mPSIR sequence improved T 1 contrast while simultaneously providing accurate T 1 and T 2 * values of the neck region. Although further study is required to evaluate the clinical utility, mPSIR may improve carotid atherosclerotic plaque detection and provide detailed information about plaque components.

  16. Fine mapping and candidate gene search of quantitative trait loci for growth and obesity using mouse intersubspecific subcongenic intercrosses and exome sequencing.

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    Akira Ishikawa

    Full Text Available Although growth and body composition traits are quantitative traits of medical and agricultural importance, the genetic and molecular basis of those traits remains elusive. Our previous genome-wide quantitative trait locus (QTL analyses in an intersubspecific backcross population between C57BL/6JJcl (B6 and wild Mus musculus castaneus mice revealed a major growth QTL (named Pbwg1 on a proximal region of mouse chromosome 2. Using the B6.Cg-Pbwg1 intersubspecific congenic strain created, we revealed 12 closely linked QTLs for body weight and body composition traits on an approximately 44.1-Mb wild-derived congenic region. In this study, we narrowed down genomic regions harboring three (Pbwg1.12, Pbwg1.3 and Pbwg1.5 of the 12 linked QTLs and searched for possible candidate genes for the QTLs. By phenotypic analyses of F2 intercross populations between B6 and each of four B6.Cg-Pbwg1 subcongenic strains with overlapping and non-overlapping introgressed regions, we physically defined Pbwg1.12 affecting body weight to a 3.8-Mb interval (61.5-65.3 Mb on chromosome 2. We fine-mapped Pbwg1.3 for body length to an 8.0-Mb interval (57.3-65.3 and Pbwg1.5 for abdominal white fat weight to a 2.1-Mb interval (59.4-61.5. The wild-derived allele at Pbwg1.12 and Pbwg1.3 uniquely increased body weight and length despite the fact that the wild mouse has a smaller body size than that of B6, whereas it decreased fat weight at Pbwg1.5. Exome sequencing and candidate gene prioritization suggested that Gcg and Grb14 are putative candidate genes for Pbwg1.12 and that Ly75 and Itgb6 are putative candidate genes for Pbwg1.5. These genes had nonsynonymous SNPs, but the SNPs were predicted to be not harmful to protein functions. These results provide information helpful to identify wild-derived quantitative trait genes causing enhanced growth and resistance to obesity.

  17. Quantitative analysis by next generation sequencing of hematopoietic stem and progenitor cells (LSK and of splenic B cells transcriptomes from wild-type and Usp3-knockout mice

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    Cesare Lancini

    2016-03-01

    Full Text Available The data described here provide genome-wide expression profiles of murine primitive hematopoietic stem and progenitor cells (LSK and of B cell populations, obtained by high throughput sequencing. Cells are derived from wild-type mice and from mice deficient for the ubiquitin-specific protease 3 (USP3; Usp3Δ/Δ. Modification of histone proteins by ubiquitin plays a crucial role in the cellular response to DNA damage (DDR (Jackson and Durocher, 2013 [1]. USP3 is a histone H2A deubiquitinating enzyme (DUB that regulates ubiquitin-dependent DDR in response to DNA double-strand breaks (Nicassio et al., 2007; Doil et al., 2008 [2,3]. Deletion of USP3 in mice increases the incidence of spontaneous tumors and affects hematopoiesis [4]. In particular, Usp3-knockout mice show progressive loss of B and T cells and decreased functional potential of hematopoietic stem cells (HSCs during aging. USP3-deficient cells, including HSCs, display enhanced histone ubiquitination, accumulate spontaneous DNA damage and are hypersensitive to ionizing radiation (Lancini et al., 2014 [4]. To address whether USP3 loss leads to deregulation of specific molecular pathways relevant to HSC homeostasis and/or B cell development, we have employed the RNA-sequencing technology and investigated transcriptional differences between wild-type and Usp3Δ/Δ LSK, naïve B cells or in vitro activated B cells. The data relate to the research article “Tight regulation of ubiquitin-mediated DNA damage response by USP3 preserves the functional integrity of hematopoietic stem cells” (Lancini et al., 2014 [4]. The RNA-sequencing and analysis data sets have been deposited in NCBI׳s Gene Expression Omnibus (Edgar et al., 2002 [5] and are accessible through GEO Series accession number GSE58495 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE58495. With this article, we present validation of the RNA-seq data set through quantitative real-time PCR and comparative analysis. Keywords: B

  18. Quantitative analysis by next generation sequencing of hematopoietic stem and progenitor cells (LSK) and of splenic B cells transcriptomes from wild-type and Usp3-knockout mice.

    Science.gov (United States)

    Lancini, Cesare; Gargiulo, Gaetano; van den Berk, Paul C M; Citterio, Elisabetta

    2016-03-01

    The data described here provide genome-wide expression profiles of murine primitive hematopoietic stem and progenitor cells (LSK) and of B cell populations, obtained by high throughput sequencing. Cells are derived from wild-type mice and from mice deficient for the ubiquitin-specific protease 3 (USP3; Usp3Δ/Δ). Modification of histone proteins by ubiquitin plays a crucial role in the cellular response to DNA damage (DDR) (Jackson and Durocher, 2013) [1]. USP3 is a histone H2A deubiquitinating enzyme (DUB) that regulates ubiquitin-dependent DDR in response to DNA double-strand breaks (Nicassio et al., 2007; Doil et al., 2008) [2], [3]. Deletion of USP3 in mice increases the incidence of spontaneous tumors and affects hematopoiesis [4]. In particular, Usp3-knockout mice show progressive loss of B and T cells and decreased functional potential of hematopoietic stem cells (HSCs) during aging. USP3-deficient cells, including HSCs, display enhanced histone ubiquitination, accumulate spontaneous DNA damage and are hypersensitive to ionizing radiation (Lancini et al., 2014) [4]. To address whether USP3 loss leads to deregulation of specific molecular pathways relevant to HSC homeostasis and/or B cell development, we have employed the RNA-sequencing technology and investigated transcriptional differences between wild-type and Usp3Δ/Δ LSK, naïve B cells or in vitro activated B cells. The data relate to the research article "Tight regulation of ubiquitin-mediated DNA damage response by USP3 preserves the functional integrity of hematopoietic stem cells" (Lancini et al., 2014) [4]. The RNA-sequencing and analysis data sets have been deposited in NCBI׳s Gene Expression Omnibus (Edgar et al., 2002) [5] and are accessible through GEO Series accession number GSE58495 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE58495). With this article, we present validation of the RNA-seq data set through quantitative real-time PCR and comparative analysis.

  19. Quantitative assessment of rest and action tremor and the effect of cueing in Parkinson’s disease patients treated with deep brain stimulation

    NARCIS (Netherlands)

    Heida, Tjitske; Wentink, E.C.; Marani, Enrico

    2011-01-01

    Introduction: In Parkinson’s disease rest and action tremor may occur. High frequency deep brain stimulation in basal ganglia nuclei has proved to be effective in the suppression of tremor. In addition, rhythmic auditory cues have shown to result in improved performance of repetitive movements,

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

    Science.gov (United States)

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

    2006-08-01

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

  1. Usefulness of R2* maps generated by iterative decomposition of water and fat with echo asymmetry and least-squares estimation quantitation sequence for cerebral artery dissection

    Energy Technology Data Exchange (ETDEWEB)

    Kato, Ayumi; Shinohara, Yuki; Fujii, Shinya; Miyoshi, Fuminori; Kuya, Keita; Ogawa, Toshihide [Tottori University, Division of Radiology, Department of Pathophysiological, and Therapeutic Science, Faculty of Medicine, Yonago (Japan); Yamashita, Eijiro [Tottori University Hospital, Division of Clinical Radiology, Yonago (Japan)

    2015-09-15

    Acute intramural hematoma resulting from cerebral artery dissection is usually visualized as a region of intermediate signal intensity on T1-weighted images (WI). This often causes problems with distinguishing acute atheromatous lesions from surrounding parenchyma and dissection. The present study aimed to determine whether or not R2* maps generated by the iterative decomposition of water and fat with echo asymmetry and least-squares estimation quantitation sequence (IDEAL IQ) can distinguish cerebral artery dissection more effectively than three-dimensional variable refocusing flip angle TSE T1WI (T1-CUBE) and T2*WI. We reviewed data from nine patients with arterial dissection who were assessed by MR images including R2* maps, T2*WI, T1-CUBE, and 3D time-of-flight (TOF)-MRA. We visually assessed intramural hematomas in each patient as positive (clearly visible susceptibility effect reflecting intramural hematoma as hyperintensity on R2* map and hypointensity on T2*WI), negative (absent intramural hematoma), equivocal (difficult to distinguish between intramural hematoma and other paramagnetic substances such as veins, vessel wall calcification, or hemorrhage) and not evaluable (difficult to determine intramural hematoma due to susceptibility artifacts arising from skull base). Eight of nine patients were assessed during the acute phase. Lesions in all eight patients were positive for intramural hematoma corresponding to dissection sites on R2* maps, while two lesions were positive on T2*WI and three lesions showed high-intensity on T1-CUBE reflected intramural hematoma during the acute phase. R2* maps generated using IDEAL IQ can detect acute intramural hematoma associated with cerebral artery dissection more effectively than T2*WI and earlier than T1-CUBE. (orig.)

  2. Sequence of deep-focus earthquakes beneath the Bonin Islands identified by the NIED nationwide dense seismic networks Hi-net and F-net

    Science.gov (United States)

    Takemura, Shunsuke; Saito, Tatsuhiko; Shiomi, Katsuhiko

    2017-03-01

    An M 6.8 ( Mw 6.5) deep-focus earthquake occurred beneath the Bonin Islands at 21:18 (JST) on June 23, 2015. Observed high-frequency (>1 Hz) seismograms across Japan, which contain several sets of P- and S-wave arrivals for the 10 min after the origin time, indicate that moderate-to-large earthquakes occurred sequentially around Japan. Snapshots of the seismic energy propagation illustrate that after one deep-focus earthquake occurred beneath the Sea of Japan, two deep-focus earthquakes occurred sequentially after the first ( Mw 6.5) event beneath the Bonin Islands in the next 4 min. The United States Geological Survey catalog includes three Bonin deep-focus earthquakes with similar hypocenter locations, but their estimated magnitudes are inconsistent with seismograms from across Japan. The maximum-amplitude patterns of the latter two earthquakes were similar to that of the first Bonin earthquake, which indicates similar locations and mechanisms. Furthermore, based on the ratios of the S-wave amplitudes to that of the first event, the magnitudes of the latter events are estimated as M 6.5 ± 0.02 and M 5.8 ± 0.02, respectively. Three magnitude-6-class earthquakes occurred sequentially within 4 min in the Pacific slab at 480 km depth, where complex heterogeneities exist within the slab.[Figure not available: see fulltext.

  3. Complete genome sequence of the highly Mn(II) tolerant Staphylococcus sp. AntiMn-1 isolated from deep-sea sediment in the Clarion-Clipperton Zone.

    Science.gov (United States)

    Wang, Xing; Lin, Danqiu; Jing, Xiaohuan; Zhu, Sidong; Yang, Jifang; Chen, Jigang

    2018-01-20

    Staphylococcus sp. AntiMn-1 is a deep-sea bacterium inhabiting seafloor sediment in the Clarion-Clipperton Zone (CCZ) that is highly tolerant to Mn(II) and displays efficient Mn(II) oxidation. Herein, we present the assembly and annotation of its genome. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Deep Sequencing-Identified Kanamycin-Resistant Paenibacillus sp. Strain KS1 Isolated from Epiphyte Tillandsia usneoides (Spanish Moss) in Central Florida, USA.

    Science.gov (United States)

    Lata, Pushpa; Govindarajan, Subramaniam S; Qi, Feng; Li, Jian-Liang; Sahoo, Malaya K

    2017-02-02

    Paenibacillus sp. strain KS1 was isolated from an epiphyte, Tillandsia usneoides (Spanish moss), in central Florida, USA. Here, we report a draft genome sequence of this strain, which consists of a total of 398 contigs spanning 6,508,195 bp, with a G+C content of 46.5% and comprising 5,401 predicted coding sequences. Copyright © 2017 Lata et al.

  5. Testing the Role of Microbial Ecology, Redox-Mediated Deep Water Production and Hypersalinity on TEX86: Lipids and 16s Sequences from Archaea and Bacteria in the Water Column and Sediments of Orca Basin

    Science.gov (United States)

    Warren, C.; Romero, I.; Ellis, G.; Goddard, E.; Krishnan, S.; Nigro, L. M.; Super, J. R.; Zhang, Y.; Zhuang, G.; Hollander, D. J.; Pagani, M.

    2014-12-01

    Mesophilic marine archaea and bacteria are known to substantially contribute to the oceanic microbial biomass and play critical roles in global carbon, nitrogen and nutrient cycles. The Orca Basin, a 2400 meter deep bathymetric depression on the continental slope of the north-central Gulf of Mexico, is an ideal environment to examine how redox-dependent biochemical processes control the input and cycling of bacterial and archaea-derived lipid compounds from formation in near-surface water, through secondary recycling processes operating at the redox-transition in the water column, to sedimentary diagenetic processes operating in oxic to anoxic zones within the basin. The lowermost 180 meters of the Orca Basin is characterized by an anoxic, hypersaline brine that is separated from the overlying oxic seawater by a well-defined redox sequence associated with a systematic increasing in salinity from 35 - 250‰. While surface water conditions are viewed as normal marine with a seasonally productive water column, the sub-oxic to anoxic transition zones within the deep-water column and the sediment spans over 200 m allowing the unique opportunity for discrete sampling of resident organisms and lipids. Here we present 16s rRNA sequence data of Bacteria and Archaea collected parallel to GDGT lipid profiles and in situ environmental measurements from the sediment and overlying water column in the intermediate zone of the basin, where movements of chemical transition zones are preserved. We evaluated GDGTs and corresponding taxa across the surface water, chlorophyll maximum, thermocline, and the deep redox boundary, including oxygenation, denitrification, manganese, iron and sulfate reduction zones, to determine if GDGTs are being produced under these conditions and how surface-derived GDGT lipids and the TEX86 signal may be altered. The results have implications for the application of the TEX86 paleotemperature proxy.

  6. Deep nirS amplicon sequencing of San Francisco Bay sediments enables prediction of geography and environmental conditions from denitrifying community composition.

    Science.gov (United States)

    Lee, Jessica A; Francis, Christopher A

    2017-12-01

    Denitrification is a dominant nitrogen loss process in the sediments of San Francisco Bay. In this study, we sought to understand the ecology of denitrifying bacteria by using next-generation sequencing (NGS) to survey the diversity of a denitrification functional gene, nirS (encoding cytchrome-cd 1 nitrite reductase), along the salinity gradient of San Francisco Bay over the course of a year. We compared our dataset to a library of nirS sequences obtained previously from the same samples by standard PCR cloning and Sanger sequencing, and showed that both methods similarly demonstrated geography, salinity and, to a lesser extent, nitrogen, to be strong determinants of community composition. Furthermore, the depth afforded by NGS enabled novel techniques for measuring the association between environment and community composition. We used Random Forests modelling to demonstrate that the site and salinity of a sample could be predicted from its nirS sequences, and to identify indicator taxa associated with those environmental characteristics. This work contributes significantly to our understanding of the distribution and dynamics of denitrifying communities in San Francisco Bay, and provides valuable tools for the further study of this key N-cycling guild in all estuarine systems. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

  7. Anterior foregut microbiota of the glassy-winged sharpshooter explored using deep 16S rRNA gene sequencing from individual insects.

    Directory of Open Access Journals (Sweden)

    Elizabeth E Rogers

    Full Text Available The glassy-winged sharpshooter (GWSS is an invasive insect species that transmits Xylella fastidiosa, the bacterium causing Pierce's disease of grapevine and other leaf scorch diseases. X. fastidiosa has been shown to colonize the anterior foregut (cibarium and precibarium of sharpshooters, where it may interact with other naturally-occurring bacterial species. To evaluate such interactions, a comprehensive list of bacterial species associated with the sharpshooter cibarium and precibarium is needed. Here, a survey of microbiota associated with the GWSS anterior foregut was conducted. Ninety-six individual GWSS, 24 from each of 4 locations (Bakersfield, CA; Ojai, CA; Quincy, FL; and a laboratory colony, were characterized for bacteria in dissected sharpshooter cibaria and precibaria by amplification and sequencing of a portion of the 16S rRNA gene using Illumina MiSeq technology. An average of approximately 150,000 sequence reads were obtained per insect. The most common genus detected was Wolbachia; sequencing of the Wolbachia ftsZ gene placed this strain in supergroup B, one of two Wolbachia supergroups most commonly associated with arthropods. X. fastidiosa was detected in all 96 individuals examined. By multilocus sequence typing, both X. fastidiosa subspecies fastidiosa and subspecies sandyi were present in GWSS from California and the colony; only subspecies fastidiosa was detected in GWSS from Florida. In addition to Wolbachia and X. fastidiosa, 23 other bacterial genera were detected at or above an average incidence of 0.1%; these included plant-associated microbes (Methylobacterium, Sphingomonas, Agrobacterium, and Ralstonia and soil- or water-associated microbes (Anoxybacillus, Novosphingobium, Caulobacter, and Luteimonas. Sequences belonging to species of the family Enterobacteriaceae also were detected but it was not possible to assign these to individual genera. Many of these species likely interact with X. fastidiosa in the

  8. miRDis: a Web tool for endogenous and exogenous microRNA discovery based on deep-sequencing data analysis.

    Science.gov (United States)

    Zhang, Hanyuan; Vieira Resende E Silva, Bruno; Cui, Juan

    2018-05-01

    Small RNA sequencing is the most widely used tool for microRNA (miRNA) discovery, and shows great potential for the efficient study of miRNA cross-species transport, i.e., by detecting the presence of exogenous miRNA sequences in the host species. Because of the increased appreciation of dietary miRNAs and their far-reaching implication in human health, research interests are currently growing with regard to exogenous miRNAs bioavailability, mechanisms of cross-species transport and miRNA function in cellular biological processes. In this article, we present microRNA Discovery (miRDis), a new small RNA sequencing data analysis pipeline for both endogenous and exogenous miRNA detection. Specifically, we developed and deployed a Web service that supports the annotation and expression profiling data of known host miRNAs and the detection of novel miRNAs, other noncoding RNAs, and the exogenous miRNAs from dietary species. As a proof-of-concept, we analyzed a set of human plasma sequencing data from a milk-feeding study where 225 human miRNAs were detected in the plasma samples and 44 show elevated expression after milk intake. By examining the bovine-specific sequences, data indicate that three bovine miRNAs (bta-miR-378, -181* and -150) are present in human plasma possibly because of the dietary uptake. Further evaluation based on different sets of public data demonstrates that miRDis outperforms other state-of-the-art tools in both detection and quantification of miRNA from either animal or plant sources. The miRDis Web server is available at: http://sbbi.unl.edu/miRDis/index.php.

  9. Quantitative assessment of irradiated lung volume and lung mass in breast cancer patients treated with tangential fields in combination with deep inspiration breath hold (DIBH)

    International Nuclear Information System (INIS)

    Kapp, Karin Sigrid; Zurl, Brigitte; Stranzl, Heidi; Winkler, Peter

    2010-01-01

    Purpose: Comparison of the amount of irradiated lung tissue volume and mass in patients with breast cancer treated with an optimized tangential-field technique with and without a deep inspiration breath-hold (DIBH) technique and its impact on the normal-tissue complication probability (NTCP). Material and Methods: Computed tomography datasets of 60 patients in normal breathing (NB) and subsequently in DIBH were compared. With a Real-Time Position Management Respiratory Gating System (RPM), anteroposterior movement of the chest wall was monitored and a lower and upper threshold were defined. Ipsilateral lung and a restricted tangential region of the lung were delineated and the mean and maximum doses calculated. Irradiated lung tissue mass was computed based on density values. NTCP for lung was calculated using a modified Lyman-Kutcher-Burman (LKB) model. Results: Mean dose to the ipsilateral lung in DIBH versus NB was significantly reduced by 15%. Mean lung mass calculation in the restricted area receiving ≤ 20 Gy (M 20 ) was reduced by 17% in DIBH but associated with an increase in volume. NTCP showed an improvement in DIBH of 20%. The correlation of individual breathing amplitude with NTCP proved to be independent. Conclusion: The delineation of a restricted area provides the lung mass calculation in patients treated with tangential fields. DIBH reduces ipsilateral lung dose by inflation so that less tissue remains in the irradiated region and its efficiency is supported by a decrease of NTCP. (orig.)

  10. Comparison between quantitative nucleic acid sequence-based amplification, real-time reverse transcriptase PCR, and real-time PCR for quantification of Leishmania parasites

    NARCIS (Netherlands)

    van der Meide, Wendy; Guerra, Jorge; Schoone, Gerard; Farenhorst, Marit; Coelho, Leila; Faber, William; Peekel, Inge; Schallig, Henk

    2008-01-01

    DNA or RNA amplification methods for detection of Leishmania parasites have advantages regarding sensitivity and potential quantitative characteristics in comparison with conventional diagnostic methods but are often still not routinely applied. However, the use and application of molecular assays

  11. Complete genome sequence of the thermophilic sulfur-reducer Desulfurobacterium thermolithotrophum type strain (BSAT) from a deep-sea hydrothermal vent

    Energy Technology Data Exchange (ETDEWEB)

    Goker, Markus [DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany; Daligault, Hajnalka E. [Los Alamos National Laboratory (LANL); Mwirichia, Romano [Jomo Kenyatta University of Agriculture and Technology, Nairobi, Kenya; Lapidus, Alla L. [U.S. Department of Energy, Joint Genome Institute; Deshpande, Shweta [U.S. Department of Energy, Joint Genome Institute; Pagani, Ioanna [U.S. Department of Energy, Joint Genome Institute; Tapia, Roxanne [Los Alamos National Laboratory (LANL); Cheng, Jan-Fang [U.S. Department of Energy, Joint Genome Institute; Goodwin, Lynne A. [Los Alamos National Laboratory (LANL); Pitluck, Sam [U.S. Department of Energy, Joint Genome Institute; Liolios, Konstantinos [U.S. Department of Energy, Joint Genome Institute; Ivanova, N [U.S. Department of Energy, Joint Genome Institute; Mavromatis, K [U.S. Department of Energy, Joint Genome Institute; Mikhailova, Natalia [U.S. Department of Energy, Joint Genome Institute; Pati, Amrita [U.S. Department of Energy, Joint Genome Institute; Chen, Amy [U.S. Department of Energy, Joint Genome Institute; Palaniappan, Krishna [U.S. Department of Energy, Joint Genome Institute; Han, Cliff [Los Alamos National Laboratory (LANL); Land, Miriam L [ORNL; Hauser, Loren John [ORNL; Pan, Chongle [ORNL; Brambilla, Evelyne-Marie [DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany; Rohde, Manfred [HZI - Helmholtz Centre for Infection Research, Braunschweig, Germany; Spring, Stefan [DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany; Sikorski, Johannes [DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany; Wirth, Reinhard [Universitat Regensburg, Regensburg, Germany; Detter, J. Chris [U.S. Department of Energy, Joint Genome Institute; Woyke, Tanja [U.S. Department of Energy, Joint Genome Institute; Bristow, James [U.S. Department of Energy, Joint Genome Institute; Eisen, Jonathan [U.S. Department of Energy, Joint Genome Institute; Markowitz, Victor [U.S. Department of Energy, Joint Genome Institute; Hugenholtz, Philip [U.S. Department of Energy, Joint Genome Institute; Kyrpides, Nikos C [U.S. Department of Energy, Joint Genome Institute; Klenk, Hans-Peter [DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany

    2011-01-01

    Desulfurobacterium thermolithotrophum L'Haridon et al. 1998 is the type species of the ge- nus Desulfurobacterium which belongs to the family Desulfurobacteriaceae. The species is of interest because it represents the first thermophilic bacterium that can act as a primary pro- ducer in the temperature range of 45-75 C (optimum 70 C) and is incapable of growing un- der microaerophilic conditions. Strain BSAT preferentially synthesizes high-melting-point fatty acids (C18 and C20) which is hypothesized to be a strategy to ensure the functionality of the membrane at high growth temperatures. This is the second completed genome sequence of a member of the family Desulfurobacteriaceae and the first sequence from the genus Desulfu- robacterium. The 1,541,968 bp long genome harbors 1,543 protein-coding and 51 RNA genes and is a part of the Genomic Encyclopedia of Bacteria and Archaea project.

  12. Quantitative Visualization of Dynamic Tracer Transportation in the Extracellular Space of Deep Brain Regions Using Tracer-Based Magnetic Resonance Imaging

    Science.gov (United States)

    Hou, Jin; Wang, Wei; Quan, Xianyue; Liang, Wen; Li, Zhiming; Han, Hongbin; Chen, Deji

    2017-01-01

    Background This study assessed an innovative tracer-based magnetic resonance imaging (MRI) system to visualize the dynamic transportation of tracers in regions of deep brain extracellular space (ECS) and to measure transportation ability and ECS structure. Material/Methods Gadolinium-diethylene triamine pentaacetic acid (Gd-DTPA) was the chosen tracer and was injected into the caudate nucleus and thalamus. Real-time dynamic transportation of Gd-DTPA in ECS was observed and the results were verified by laser scanning confocal microscopy. Using Transwell assay across the blood-brain barrier, a modified diffusion equation was further simplified. Effective diffusion coefficient D* and tortuosity λ were calculated. Immunohistochemical staining and Western blot analysis were used to investigate the extracellular matrix contributing to ECS structure. Results Tracers injected into the caudate nucleus were transported to the ipsilateral frontal and temporal cortices away from the injection points, while both of them injected into the thalamus were only distributed on site. Although the caudate nucleus was closely adjacent to the thalamus, tracer transportation between partitions was not observed. In addition, D* and the λ showed statistically significant differences between partitions. ECS was shown to be a physiologically partitioned system, and its division is characterized by the unique distribution territory and transportation ability of substances located in it. Versican and Tenascin R are possible contributors to the tortuosity of ECS. Conclusions Tracer-based MRI will improve our understanding of the brain microenvironment, improve the techniques for local delivery of drugs, and highlight brain tissue engineering fields in the future. PMID:28866708

  13. Quantitative Visualization of Dynamic Tracer Transportation in the Extracellular Space of Deep Brain Regions Using Tracer-Based Magnetic Resonance Imaging.

    Science.gov (United States)

    Hou, Jin; Wang, Wei; Quan, Xianyue; Liang, Wen; Li, Zhiming; Chen, Deji; Han, Hongbin

    2017-09-03

    BACKGROUND This study assessed an innovative tracer-based magnetic resonance imaging (MRI) system to visualize the dynamic transportation of tracers in regions of deep brain extracellular space (ECS) and to measure transportation ability and ECS structure. MATERIAL AND METHODS Gadolinium-diethylene triamine pentaacetic acid (Gd-DTPA) was the chosen tracer and was injected into the caudate nucleus and thalamus. Real-time dynamic transportation of Gd-DTPA in ECS was observed and the results were verified by laser scanning confocal microscopy. Using Transwell assay across the blood-brain barrier, a modified diffusion equation was further simplified. Effective diffusion coefficient D* and tortuosity λ were calculated. Immunohistochemical staining and Western blot analysis were used to investigate the extracellular matrix contributing to ECS structure. RESULTS Tracers injected into the caudate nucleus were transported to the ipsilateral frontal and temporal cortices away from the injection points, while both of them injected into the thalamus were only distributed on site. Although the caudate nucleus was closely adjacent to the thalamus, tracer transportation between partitions was not observed. In addition, D* and the λ showed statistically significant differences between partitions. ECS was shown to be a physiologically partitioned system, and its division is characterized by the unique distribution territory and transportation ability of substances located in it. Versican and Tenascin R are possible contributors to the tortuosity of ECS. CONCLUSIONS Tracer-based MRI will improve our understanding of the brain microenvironment, improve the techniques for local delivery of drugs, and highlight brain tissue engineering fields in the future.

  14. Deep mRNA sequencing of the Tritonia diomedea brain transcriptome provides access to gene homologues for neuronal excitability, synaptic transmission and peptidergic signalling.

    Directory of Open Access Journals (Sweden)

    Adriano Senatore

    Full Text Available The sea slug Tritonia diomedea (Mollusca, Gastropoda, Nudibranchia, has a simple and highly accessible nervous system, making it useful for studying neuronal and synaptic mechanisms underlying behavior. Although many important contributions have been made using Tritonia, until now, a lack of genetic information has impeded exploration at the molecular level.We performed Illumina sequencing of central nervous system mRNAs from Tritonia, generating 133.1 million 100 base pair, paired-end reads. De novo reconstruction of the RNA-Seq data yielded a total of 185,546 contigs, which partitioned into 123,154 non-redundant gene clusters (unigenes. BLAST comparison with RefSeq and Swiss-Prot protein databases, as well as mRNA data from other invertebrates (gastropod molluscs: Aplysia californica, Lymnaea stagnalis and Biomphalaria glabrata; cnidarian: Nematostella vectensis revealed that up to 76,292 unigenes in the Tritonia transcriptome have putative homologues in other databases, 18,246 of which are below a more stringent E-value cut-off of 1x10-6. In silico prediction of secreted proteins from the Tritonia transcriptome shotgun assembly (TSA produced a database of 579 unique sequences of secreted proteins, which also exhibited markedly higher expression levels compared to other genes in the TSA.Our efforts greatly expand the availability of gene sequences available for Tritonia diomedea. We were able to extract full length protein sequences for most queried genes, including those involved in electrical excitability, synaptic vesicle release and neurotransmission, thus confirming that the transcriptome will serve as a useful tool for probing the molecular correlates of behavior in this species. We also generated a neurosecretome database that will serve as a useful tool for probing peptidergic signalling systems in the Tritonia brain.

  15. Deep Sequencing Data and Infectivity Assays Indicate that Chickpea Chlorotic Dwarf Virus is the Etiological Agent of the “Hard Fruit Syndrome” of Watermelon

    Directory of Open Access Journals (Sweden)

    Takoua Zaagueri

    2017-10-01

    Full Text Available Chickpea chlorotic dwarf virus (CpCDV, a polyphagous mastrevirus, family Geminiviridae, has been recently linked to the onset of the “hard fruit syndrome” of watermelon, first described in Tunisia, that makes fruits unmarketable due to the presence of white hard portions in the flesh, chlorotic mottling on the rind, and an unpleasant taste. To investigate the etiological agent of this disease, total RNA extracted from symptomatic watermelon fruits was subjected to small RNA sequencing through next generation sequencing (NGS techniques. Data obtained showed the presence of CpCDV and two other viral species. However, following validation through polymerase chain reaction (PCR, CpCDV was the only viral species consistently detected in all samples. Watermelon seedlings were then challenged by an agroinfectious CpCDV clone; several plants proved to be CpCDV-infected, and were able to produce fruits. CpCDV infected and replicated in watermelon fruits and leaves, leading to abnormality in fruits and in seed production, similar to those described in field. These results indicate that CpCDV is the etiological agent of the “hard fruit syndrome” of watermelon.

  16. Deep Sequencing of Myxilla (Ectyomyxilla) methanophila, an Epibiotic Sponge on Cold-Seep Tubeworms, Reveals Methylotrophic, Thiotrophic, and Putative Hydrocarbon-Degrading Microbial Associations

    KAUST Repository

    Arellano, Shawn M.

    2012-10-11

    The encrusting sponge Myxilla (Ectyomyxilla) methanophila (Poecilosclerida: Myxillidae) is an epibiont on vestimentiferan tubeworms at hydrocarbon seeps on the upper Louisiana slope of the Gulf of Mexico. It has long been suggested that this sponge harbors methylotrophic bacteria due to its low δ13C value and high methanol dehydrogenase activity, yet the full community of microbial associations in M. methanophila remained uncharacterized. In this study, we sequenced 16S rRNA genes representing the microbial community in M. methanophila collected from two hydrocarbon-seep sites (GC234 and Bush Hill) using both Sanger sequencing and next-generation 454 pyrosequencing technologies. Additionally, we compared the microbial community in M. methanophila to that of the biofilm collected from the associated tubeworm. Our results revealed that the microbial diversity in the sponges from both sites was low but the community structure was largely similar, showing a high proportion of methylotrophic bacteria of the genus Methylohalomonas and polycyclic aromatic hydrocarbon (PAH)-degrading bacteria of the genera Cycloclasticus and Neptunomonas. Furthermore, the sponge microbial clone library revealed the dominance of thioautotrophic gammaproteobacterial symbionts in M. methanophila. In contrast, the biofilm communities on the tubeworms were more diverse and dominated by the chemoorganotrophic Moritella at GC234 and methylotrophic Methylomonas and Methylohalomonas at Bush Hill. Overall, our study provides evidence to support previous suggestion that M. methanophila harbors methylotrophic symbionts and also reveals the association of PAH-degrading and thioautotrophic microbes in the sponge. © 2012 Springer Science+Business Media New York.

  17. Deep sequencing of Myxilla (Ectyomyxilla) methanophila, an epibiotic sponge on cold-seep tubeworms, reveals methylotrophic, thiotrophic, and putative hydrocarbon-degrading microbial associations.

    Science.gov (United States)

    Arellano, Shawn M; Lee, On On; Lafi, Feras F; Yang, Jiangke; Wang, Yong; Young, Craig M; Qian, Pei-Yuan

    2013-02-01

    The encrusting sponge Myxilla (Ectyomyxilla) methanophila (Poecilosclerida: Myxillidae) is an epibiont on vestimentiferan tubeworms at hydrocarbon seeps on the upper Louisiana slope of the Gulf of Mexico. It has long been suggested that this sponge harbors methylotrophic bacteria due to its low δ(13)C value and high methanol dehydrogenase activity, yet the full community of microbial associations in M. methanophila remained uncharacterized. In this study, we sequenced 16S rRNA genes representing the microbial community in M. methanophila collected from two hydrocarbon-seep sites (GC234 and Bush Hill) using both Sanger sequencing and next-generation 454 pyrosequencing technologies. Additionally, we compared the microbial community in M. methanophila to that of the biofilm collected from the associated tubeworm. Our results revealed that the microbial diversity in the sponges from both sites was low but the community structure was largely similar, showing a high proportion of methylotrophic bacteria of the genus Methylohalomonas and polycyclic aromatic hydrocarbon (PAH)-degrading bacteria of the genera Cycloclasticus and Neptunomonas. Furthermore, the sponge microbial clone library revealed the dominance of thioautotrophic gammaproteobacterial symbionts in M. methanophila. In contrast, the biofilm communities on the tubeworms were more diverse and dominated by the chemoorganotrophic Moritella at GC234 and methylotrophic Methylomonas and Methylohalomonas at Bush Hill. Overall, our study provides evidence to support previous suggestion that M. methanophila harbors methylotrophic symbionts and also reveals the association of PAH-degrading and thioautotrophic microbes in the sponge.

  18. Redox front formation in an uplifting sedimentary rock sequence: An analogue for redox-controlling processes in the geosphere around deep geological repositories for radioactive waste

    International Nuclear Information System (INIS)

    Yoshida, H.; Metcalfe, R.; Yamamoto, K.; Murakami, Y.; Hoshii, D.; Kanekiyo, A.; Naganuma, T.; Hayashi, T.

    2008-01-01

    Subsurface redox fronts control the mobilization and fixation of many trace elements, including potential pollutants such as certain radionuclides. Any safety assessment for a deep geological repository for radioactive wastes needs to take into account adequately the long-term redox processes in the geosphere surrounding the repository. To build confidence in understanding these processes, a redox front in a reduced siliceous sedimentary rock distributed in an uplifting area in Japan has been studied in detail. Geochemical analyses show increased concentrations of Fe and trace elements, including rare earth elements (REEs), at the redox front, even though concentrations of reduced rock matrix constituents show little change. Detailed SEM observations revealed that fossilized microorganisms composed of amorphous granules made exclusively of Fe and Si occur in the rock's pore space. Microbial 16S rDNA analysis suggests that there is presently a zonation of different bacterial groups within the redox band, and bacterial zonation played an important role in the concentration of Fe-oxyhydroxides at the redox front. These water-rock-microbe interactions can be considered analogous to the processes occurring in the redox fronts that would develop around geological repositories for radioactive waste. Once formed, the Fe-oxyhydroxides within such a front would be preserved even after reducing conditions resume following repository closure

  19. Redox front formation in an uplifting sedimentary rock sequence: An analogue for redox-controlling processes in the geosphere around deep geological repositories for radioactive waste

    Energy Technology Data Exchange (ETDEWEB)

    Yoshida, H. [Nagoya University Museum, Material Research Section, Furocho, Nagoya 464-8602 (Japan)], E-mail: dora@num.nagoya-u.ac.jp; Metcalfe, R. [Quintessa Japan, Queen' s Tower A7-707, Minatomirai, Yokohama 220-6007 (Japan); Yamamoto, K. [Nagoya University Museum, Material Research Section, Furocho, Nagoya 464-8602 (Japan); Murakami, Y. [Japan Atomic Energy Agency (JAEA), Tono Geoscience Centre (Japan); Hoshii, D.; Kanekiyo, A.; Naganuma, T. [Hiroshima University, Higashi Hiroshima, Kagamiyama 1-4-4 (Japan); Hayashi, T. [Asahi University, Department of Dental Pharmacology, Hozumi, Gifu (Japan)

    2008-08-15

    Subsurface redox fronts control the mobilization and fixation of many trace elements, including potential pollutants such as certain radionuclides. Any safety assessment for a deep geological repository for radioactive wastes needs to take into account adequately the long-term redox processes in the geosphere surrounding the repository. To build confidence in understanding these processes, a redox front in a reduced siliceous sedimentary rock distributed in an uplifting area in Japan has been studied in detail. Geochemical analyses show increased concentrations of Fe and trace elements, including rare earth elements (REEs), at the redox front, even though concentrations of reduced rock matrix constituents show little change. Detailed SEM observations revealed that fossilized microorganisms composed of amorphous granules made exclusively of Fe and Si occur in the rock's pore space. Microbial 16S rDNA analysis suggests that there is presently a zonation of different bacterial groups within the redox band, and bacterial zonation played an important role in the concentration of Fe-oxyhydroxides at the redox front. These water-rock-microbe interactions can be considered analogous to the processes occurring in the redox fronts that would develop around geological repositories for radioactive waste. Once formed, the Fe-oxyhydroxides within such a front would be preserved even after reducing conditions resume following repository closure.

  20. High throughput deep degradome sequencing reveals microRNAs and their targets in response to drought stress in mulberry (Morus alba).

    Science.gov (United States)

    Li, Ruixue; Chen, Dandan; Wang, Taichu; Wan, Yizhen; Li, Rongfang; Fang, Rongjun; Wang, Yuting; Hu, Fei; Zhou, Hong; Li, Long; Zhao, Weiguo

    2017-01-01

    MicroRNAs (miRNAs) play important regulatory roles by targeting mRNAs for cleavage or translational repression. Identification of miRNA targets is essential to better understanding the roles of miRNAs. miRNA targets have not been well characterized in mulberry (Morus alba). To anatomize miRNA guided gene regulation under drought stress, transcriptome-wide high throughput degradome sequencing was used in this study to directly detect drought stress responsive miRNA targets in mulberry. A drought library (DL) and a contrast library (CL) were constructed to capture the cleaved mRNAs for sequencing. In CL, 409 target genes of 30 conserved miRNA families and 990 target genes of 199 novel miRNAs were identified. In DL, 373 target genes of 30 conserved miRNA families and 950 target genes of 195 novel miRNAs were identified. Of the conserved miRNA families in DL, mno-miR156, mno-miR172, and mno-miR396 had the highest number of targets with 54, 52 and 41 transcripts, respectively, indicating that these three miRNA families and their target genes might play important functions in response to drought stress in mulberry. Additionally, we found that many of the target genes were transcription factors. By analyzing the miRNA-target molecular network, we found that the DL independent networks consisted of 838 miRNA-mRNA pairs (63.34%). The expression patterns of 11 target genes and 12 correspondent miRNAs were detected using qRT-PCR. Six miRNA targets were further verified by RNA ligase-mediated 5' rapid amplification of cDNA ends (RLM-5' RACE). Gene Ontology (GO) annotations and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that these target transcripts were implicated in a broad range of biological processes and various metabolic pathways. This is the first study to comprehensively characterize target genes and their associated miRNAs in response to drought stress by degradome sequencing in mulberry. This study provides a framework for understanding

  1. Strategy for Extracting DNA from Clay Soil and Detecting a Specific Target Sequence via Selective Enrichment and Real-Time (Quantitative) PCR Amplification ▿

    Science.gov (United States)

    Yankson, Kweku K.; Steck, Todd R.

    2009-01-01

    We present a simple strategy for isolating and accurately enumerating target DNA from high-clay-content soils: desorption with buffers, an optional magnetic capture hybridization step, and quantitation via real-time PCR. With the developed technique, μg quantities of DNA were extracted from mg samples of pure kaolinite and a field clay soil. PMID:19633108

  2. Deep sequencing of atrial fibrillation patients with mitral valve regurgitation shows no evidence of mosaicism but reveals novel rare germline variants

    DEFF Research Database (Denmark)

    Gregers, Emilie; Ahlberg, Gustav; Christensen, Thea

    2017-01-01

    the HaloPlex Target Enrichment System. MuTect software was used for identification of somatic point variants. We functionally characterized selected variants using electrophysiologic techniques. RESULTS: No somatic variants were identified in the cardiac tissue. Thirty-three patients (75%) had a rare...... patient population undergoing surgery for mitral valve regurgitation (MVR) to determine whether these patients are genetically predisposed to AF. METHODS: DNA was extracted from blood and left atrial tissue from 44 AF patients with MVR. Using next-generation sequencing, we investigated 110 genes using...... germline variation in ≥1 candidate genes. Fourteen variants were novel. Fifteen variants were predicted damaging or likely damaging in ≥6 in silico predictions. We identified rare variants in genes never directly associated with AF: KCNE4, SCN4B, NEURL1, and CAND2. Interestingly, 7 patients (16%) had...

  3. Identification and characterization of novel and differentially expressed microRNAs in peripheral blood from healthy and mastitis Holstein cattle by deep sequencing.

    Science.gov (United States)

    Li, Zhixiong; Wang, Hongliang; Chen, Ling; Wang, Lijun; Liu, Xiaolin; Ru, Caixia; Song, Ailong

    2014-02-01

    MicroRNA (miRNA) mediates post-transcriptional gene regulation and plays an important role in regulating the development of immune cells and in modulating innate and adaptive immune responses in mammals, including cattle. In the present study, we identified novel and differentially expressed miRNAs in peripheral blood from healthy and mastitis Holstein cattle by Solexa sequencing and bioinformatics. In total, 608 precursor hairpins (pre-miRNAs) encoding for 753 mature miRNAs were detected. Statistically, 173 unique miRNAs (of 753, 22.98%) were identified that had significant differential expression between healthy and mastitis Holstein cattle (P mastitis Holstein cattle, which provide important information on mastitis in miRNAs expression. Diverse miRNAs may play an important role in the treatment of mastitis in Holstein cattle. © 2013 Stichting International Foundation for Animal Genetics.

  4. Deep sequencing shows low-level oncogenic hepatitis B virus variants persists post-liver transplant despite potent anti-HBV prophylaxis.

    Science.gov (United States)

    Lau, K C K; Osiowy, C; Giles, E; Lusina, B; van Marle, G; Burak, K W; Coffin, C S

    2018-01-06

    Recent studies suggest that withdrawal of hepatitis B immune globulin (HBIG) and nucleos(t)ide analogues (NA) prophylaxis may be considered in HBV surface antigen (HBsAg)-negative liver transplant (LT) recipients with a low risk of disease recurrence. However, the frequency of occult HBV infection (OBI) and HBV variants after LT in the current era of potent NA therapy is unknown. Twelve LT recipients on prophylaxis were tested in matched plasma and peripheral blood mononuclear cells (PBMCs) for HBV quasispecies by in-house nested PCR and next-generation sequencing of amplicons. HBV covalently closed circular DNA (cccDNA) was detected in Hirt DNA isolated from PBMCs with cccDNA-specific primers and confirmed by nucleic acid hybridization and Sanger sequencing. HBV mRNA in PBMC was detected with reverse-transcriptase nested PCR. In LT recipients on immunosuppressive therapy (10/12 male; median age 57.5 [IQR: 39.8-66.5]; median follow-up post-LT 60 months; 6 pre-LT hepatocellular carcinoma [HCC]), 9 were HBsAg-. HBV DNA was detected in all plasma and PBMC tested; cccDNA and/or mRNA was detected in the PBMC of 10/12 patients. Significant HBV quasispecies diversity (ie 143-2212 nonredundant HBV species) was noted in both sites, and single nucleotide polymorphisms associated with cirrhosis and HCC were detected at varying frequencies. In conclusion, OBI and HBV variants associated with severe liver disease persist in LT recipients on prophylaxis. Although HBV control and cccDNA transcriptional silencing may occur despite immunosuppression, complete virological eradication does not occur in LT recipients with a history of HBV-related end-stage liver disease. © 2018 John Wiley & Sons Ltd.

  5. Deep sequencing of H7N8 avian influenza viruses from surveillance zone supports H7N8 high pathogenicity avian influenza was limited to a single outbreak farm in Indiana during 2016.

    Science.gov (United States)

    Lee, Dong-Hun; Torchetti, Mia Kim; Killian, Mary Lea; Swayne, David E

    2017-07-01

    In mid-January 2016, an outbreak of H7N8 high-pathogenicity avian influenza virus (HPAIV) in commercial turkeys occurred in Indiana. Surveillance within the 10km control zone identified H7N8 low-pathogenicity avian influenza virus (LPAIV) in nine surrounding turkey flocks but no other HPAIV-affected premises. We sequenced four of the H7N8 HPAIV isolated from the single farm and nine LPAIV identified during control zone surveillance. Evaluation included phylogenetic network analysis indicating close relatedness across the HPAIV and LPAIV, and that the progenitor H7N8 LPAIV spread among the affected turkey farms in Indiana, followed by spontaneous mutation to HPAIV on a single premise through acquisition of three basic amino acids at the hemagglutinin cleavage site. Deep sequencing of the available viruses failed to identify subpopulations in either the HPAIV or LPAIV suggesting mutation to HPAIV likely occurred on a single farm and the HPAIV did not spread to epidemiologically linked LPAIV-affected farms. Published by Elsevier Inc.

  6. Deep sequencing shows microRNA involvement in bovine mammary gland adaptation to diets supplemented with linseed oil or safflower oil.

    Science.gov (United States)

    Li, Ran; Beaudoin, Frédéric; Ammah, Adolf A; Bissonnette, Nathalie; Benchaar, Chaouki; Zhao, Xin; Lei, Chuzhao; Ibeagha-Awemu, Eveline M

    2015-10-30

    Bovine milk fat composition is responsive to dietary manipulation providing an avenue to modify the content of fatty acids and especially some specific unsaturated fatty acid (USFA) isomers of benefit to human health. MicroRNAs (miRNAs) regulate gene expression but their specific roles in bovine mammary gland lipogenesis are unclear. The objective of this study was to determine the expression pattern of miRNAs following mammary gland adaptation to dietary supplementation with 5 % linseed or safflower oil using next generation RNA-sequencing. Twenty-four Canadian Holstein dairy cows (twelve per treatment) in mid lactation were fed a control diet (total mixed ration of corn:grass silages) for 28 days followed by a treatment period (control diet supplemented with 5 % linseed or safflower oil) of 28 days. Milk samples were collected weekly for fat and individual fatty acid determination. RNA from mammary gland biopsies harvested on day-14 (control period) and on days +7 and +28 (treatment period) from six randomly selected cows per treatment was subjected to small RNA sequencing. Milk fat percentage decreased significantly (P safflower oil treatments, respectively. Seven miRNAs including six up-regulated (bta-miR-199c, miR-199a-3p, miR-98, miR-378, miR-148b and miR-21-5p) and one down-regulated (bta-miR-200a) were found to be regulated (P < 0.05) by both treatments, and thus considered core differentially expressed (DE) miRNAs. The gene targets of core DE miRNAs have functions related to gene expression and general cellular metabolism (P < 0.05) and are enriched in four pathways of lipid metabolism (3-phosphoinositide biosynthesis, 3-phosphoinositide degradation, D-myo-inisitol-5-phosphate metabolism and the superpathway of inositol phosphate compounds). Our results suggest that DE miRNAs in this study might be important regulators of bovine mammary lipogenesis and metabolism. The novel miRNAs identified in this study will further enrich the bovine miRNome repertoire

  7. A deep x-ray survey of the Pleiades cluster and the B6-A3 main sequence stars in Orion

    Science.gov (United States)

    Caillault, Jean-Pierre

    1993-01-01

    We have obtained deep ROSAT images of three regions within the Pleiades open cluster. We have detected 317 X-ray sources in these ROSAT PSPC images, 171 of which we associate with certain probable members of the Pleiades cluster. We detect nearly all Pleiades members with spectral types later than G0 and within 25 arcminutes of our three field centers where our sensitivity is highest. This has allowed us to derive for the first time the luminosity function for the G, K, and M dwarfs of an open cluster without the need to use statistical techniques to account for the presence of upper limits in the data sample. Because of our high X-ray detection frequency down to the faint limit of the optical catalog, we suspect that some of our unidentified X-ray sources are previously unknown, very low-mass members of the Pleiades. A large fraction of the Pleiades members detected with ROSAT have published rotational velocities. Plots of L(sub x)/L(sub bol) versus spectroscopic rotational velocity show tightly correlated 'saturation' type relations for stars with (B - V)(sub O) greater than 0.60. For each of several color ranges, X-ray luminosities rise rapidly with increasing rotation rate until v sin i approximately equals 15 km/s, and then remain essentially flat for rotation rates up to at least v sin i approximately equal to 100 km/s. The dispersion in rotation among low-mass stars in the Pleiades is by far the dominant contributor to the dispersion in L(subx) at a given mass. Only about 35 percent of the B.A. and early F stars in the Pleiades are detected as X-ray sources in our survey. There is no correlation between X-ray flux and rotation for these stars. The X-ray luminosity function for the early-type Pleiades stars appears to be bimodal, with only a few exceptions. We either detect these stars at fluxes in the range found for low-mass stars or we derive X-ray limits below the level found for most Pleiades dwarfs. The X-ray spectra for the early-type Pleiades stars

  8. Deep sequencing of small RNAs identifies canonical and non-canonical miRNA and endogenous siRNAs in mammalian somatic tissues.

    Science.gov (United States)

    Castellano, Leandro; Stebbing, Justin

    2013-03-01

    MicroRNAs (miRNAs) are small RNA molecules that regulate gene expression. They are characterized by specific maturation processes defined by canonical and non-canonical biogenic pathways. Analysis of ∼0.5 billion sequences from mouse data sets derived from different tissues, developmental stages and cell types, partly characterized by either ablation or mutation of the main proteins belonging to miRNA processor complexes, reveals 66 high-confidence new genomic loci coding for miRNAs that could be processed in a canonical or non-canonical manner. A proportion of the newly discovered miRNAs comprises mirtrons, for which we define a new sub-class. Notably, some of these newly discovered miRNAs are generated from untranslated and open reading frames of coding genes, and we experimentally validate these. We also show that many annotated miRNAs do not present miRNA-like features, as they are neither processed by known processing complexes nor loaded on AGO2; this indicates that the current miRNA miRBase database list should be refined and re-defined. Accordingly, a group of them map on ribosomal RNA molecules, whereas others cannot undergo genuine miRNA biogenesis. Notably, a group of annotated miRNAs are Dgcr8 independent and DICER dependent endogenous small interfering RNAs that derive from a unique hairpin formed from a short interspersed nuclear element.

  9. Seismic-sequence stratigraphy and geologic structure of the Floridan aquifer system near "Boulder Zone" deep wells in Miami-Dade County, Florida

    Science.gov (United States)

    Cunningham, Kevin J.

    2015-01-01

    The U.S. Geological Survey, in cooperation with the Miami-Dade Water and Sewer Department, acquired, processed, and interpreted seismic-reflection data near the North and South District “Boulder Zone” Well Fields to determine if geologic factors may contribute to the upward migration of injected effluent into that upper part of the Floridan aquifer system designated by the U.S. Environmental Protection Agency as an underground source of drinking water. The depth of the Boulder Zone at the North and South District “Boulder Zone” Well Fields ranges from about 2,750 to 3,300 feet below land surface (ft bls), whereas overlying permeable zones used as alternative drinking water supply range in depth from about 825 to 1,580 ft bls at the North and South District “Boulder Zone” Well Fields. Seismic-sequence stratigraphy and geologic structures imaged on seismic-reflection profiles created for the study describe the part of the Floridan aquifer system overlying and within the Boulder Zone. Features of the Floridan aquifer system underlying the Boulder Zone were not studied because seismic-reflection profiles acquired near the North and South District “Boulder Zone” Well Fields lacked adequate resolution at such depths.

  10. Transcriptome profiling of the cancer, adjacent non-tumor and distant normal tissues from a colorectal cancer patient by deep sequencing.

    Directory of Open Access Journals (Sweden)

    Yan'an Wu

    Full Text Available Colorectal cancer (CRC is one of the most commonly diagnosed cancers in the world. A genome-wide screening of transcriptome dysregulation between cancer and normal tissue would provide insight into the molecular basis of CRC initiation and progression. Compared with microarray technology, which is commonly used to identify transcriptional changes, the recently developed RNA-seq technique has the ability to detect other abnormal regulations in the cancer transcriptome, such as alternative splicing, novel transcripts or gene fusion. In this study, we performed high-throughput transcriptome sequencing at ~50× coverage on CRC, adjacent non-tumor and distant normal tissue. The results revealed cancer-specific, differentially expressed genes and differential alternative splicing, suggesting that the extracellular matrix and metabolic pathways are activated and the genes related to cell homeostasis are suppressed in CRC. In addition, one tumor-restricted gene fusion, PRTEN-NOTCH2, was also detected and experimentally confirmed. This study reveals some common features in tumor invasion and provides a comprehensive survey of the CRC transcriptome, which provides better insight into the complexity of regulatory changes during tumorigenesis.

  11. In Silico Derivation of HLA-Specific Alloreactivity Potential from Whole Exome Sequencing of Stem Cell Transplant Donors and Recipients: Understanding the Quantitative Immunobiology of Allogeneic Transplantation

    Directory of Open Access Journals (Sweden)

    Max eJameson-Lee

    2014-11-01

    Full Text Available Donor T cell mediated graft versus host effects (GVH may result from the aggregate alloreactivity to minor histocompatibility antigens (mHA presented by the HLA molecules in each donor-recipient pair undergoing stem cell transplantation (SCT. Whole exome sequencing has previously demonstrated a large number of nonsynonymous single nucleotide polymorphisms (SNP present in HLA-matched recipients of SCT donors (GVH direction. The nucleotide sequence flanking each of these SNPs was obtained and the amino acid sequence determined. All the possible nonameric-peptides incorporating the variant amino acid resulting from these SNPs were interrogated in-silico for their likelihood to be presented by the HLA class I molecules using the Immune Epitope Database stabilized matrix method (SMM and NetMHCpan algorithms. The SMM algorithm predicted that a median of 18,396 peptides weakly bound HLA class I molecules in individual SCT recipients, and 2,254 peptides displayed strong binding. A similar library of presented peptides was identified when the data was interrogated using the NetMHCpan algorithm. The bioinformatic algorithm presented here demonstrates that there may be a high level of mHA variation in HLA-matched individuals, constituting an HLA-specific alloreactivity potential.

  12. Identification and comparative profiling of miRNAs in an early flowering mutant of trifoliate orange and its wild type by genome-wide deep sequencing.

    Directory of Open Access Journals (Sweden)

    Lei-Ming Sun

    Full Text Available MicroRNAs (miRNAs are a new class of small, endogenous RNAs that play a regulatory role in various biological and metabolic processes by negatively affecting gene expression at the post-transcriptional level. While the number of known Arabidopsis and rice miRNAs is continuously increasing, information regarding miRNAs from woody plants such as citrus remains limited. Solexa sequencing was performed at different developmental stages on both an early flowering mutant of trifoliate orange (precocious trifoliate orange, Poncirus trifoliata L. Raf. and its wild-type in this study, resulting in the obtainment of 141 known miRNAs belonging to 99 families and 75 novel miRNAs in four libraries. A total of 317 potential target genes were predicted based on the 51 novel miRNAs families, GO and KEGG annotation revealed that high ranked miRNA-target genes are those implicated in diverse cellular processes in plants, including development, transcription, protein degradation and cross adaptation. To characterize those miRNAs expressed at the juvenile and adult development stages of the mutant and its wild-type, further analysis on the expression profiles of several miRNAs through real-time PCR was performed. The results revealed that most miRNAs were down-regulated at adult stage compared with juvenile stage for both the mutant and its wild-type. These results indicate that both conserved and novel miRNAs may play important roles in citrus growth and development, stress responses and other physiological processes.

  13. Analysis of resistance-associated substitutions in acute hepatitis C virus infection by deep sequencing across six genotypes and three continents.

    Science.gov (United States)

    Eltahla, A A; Rodrigo, C; Betz-Stablein, B; Grebely, J; Applegate, T; Luciani, F; Schinkel, J; Dore, G J; Page, K; Bruneau, J; Morris, M D; Cox, A L; Kim, A Y; Shoukry, N H; Lauer, G M; Maher, L; Hellard, M; Prins, M; Lloyd, A R; Bull, R A

    2017-01-01

    Several direct-acting antivirals (DAAs) have been approved for the treatment of chronic hepatitis C virus (HCV) infections, opening the door to highly effective interferon-free treatment regimens. Resistance-associated substitutions (RASs) have been reported both in treatment-naïve patients and following treatment with protease (NS3), phosphoprotein (NS5A) and polymerase (NS5B) inhibitors. The prevalence of naturally occurring RASs in untreated HCV-infected individuals has mostly been analysed in those infected with genotype 1 (GT1), in the late phase of infection, and only within limited regions of the genome. Furthermore, the geographic distribution of RASs remains poorly characterized. In this study, we used next-generation sequencing to analyse full-length HCV genomes for the prevalence of RASs in acute HCV infections identified in nine international prospective cohorts. RASs were analysed in 179 participants infected with all six major HCV genotypes (GT1-GT6), and the geographic distribution of RASs was assessed in 107 GT1a and GT3a samples. While RASs were detected at varied frequencies across the three genomic regions, and between genotypes, RASs relevant to multiple DAAs in the leading IFN-free regimens were rarely detected in combination. Low-frequency RASs (<10% of the viral population) were also shown to have a GT-specific distribution. The main RASs with geographic associations were NS3 Q80K in GT1a samples and NS5B N142T in GT3a. These data provide the backdrop for prospective surveillance of RASs during DAA treatment scale-up. © 2016 John Wiley & Sons Ltd.

  14. Bacterial community dynamics in a cooling tower with emphasis on pathogenic bacteria and Legionella species using universal and genus-specific deep sequencing.

    Science.gov (United States)

    Pereira, Rui P A; Peplies, Jörg; Höfle, Manfred G; Brettar, Ingrid

    2017-10-01

    Cooling towers are the major source of outbreaks of legionellosis in Europe and worldwide. These outbreaks are mostly associated with Legionella species, primarily L. pneumophila, and its surveillance in cooling tower environments is of high relevance to public health. In this study, a combined NGS-based approach was used to study the whole bacterial community, specific waterborne and water-based bacterial pathogens, especially Legionella species, targeting the 16S rRNA gene. This approach was applied to water from a cooling tower obtained by monthly sampling during two years. The studied cooling tower was an open circuit cooling tower with lamellar cooling situated in Braunschweig, Germany. A highly diverse bacterial community was observed with 808 genera including 25 potentially pathogenic taxa using universal 16S rRNA primers. Sphingomonas and Legionella were the most abundant pathogenic genera. By applying genus-specific primers for Legionella, a diverse community with 85 phylotypes, and a representative core community with substantial temporal heterogeneity was observed. A high percentage of sequences (65%) could not be affiliated to an acknowledged species. L. pneumophila was part of the core community and the most abundant Legionella species reinforcing the importance of cooling towers as its environmental reservoir. Major temperature shifts (>10 °C) were the key environmental factor triggering the reduction or dominance of the Legionella species in the Legionella community dynamics. In addition, interventions by chlorine dioxide had a strong impact on the Legionella community composition but not on the whole bacterial community. Overall, the presented results demonstrated the value of a combined NGS approach for the molecular monitoring and surveillance of health related pathogens in man-made freshwater systems. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. Iron deposition of the deep grey matter in patients with multiple sclerosis and neuromyelitis optica: A control quantitative study by 3D-enhanced susceptibility-weighted angiography (ESWAN)

    International Nuclear Information System (INIS)

    Chen Xuan; Zeng Chun; Luo Tianyou; Ouyang Yu; Lv Fajin; Rumzan, Reshiana; Wang Zhongping; Li Qi; Wang Jingjie; Hou Huanxin

    2012-01-01

    Purpose: Previous studies have detected abnormal iron deposition in the deep grey matter (DGM) of multiple sclerosis (MS). The regional specificity of the DGM iron deposition in neuromyelitis optica (NMO) is still unclear. We compared the differences in the DGM iron concentration between MS and NMO patients. Methods: We enrolled 42 relapsing–remitting MS (RRMS) patients, 42 NMO patients and 42 healthy controls undergoing brain conventional MRI and three-dimensional (3D)-enhanced T 2 *-weighted angiography (ESWAN) sequences. We obtained the mean phase values (MPVs) for ESWAN-filtered phase images. An analysis of covariance (ANCOVA) was used to compare MPVs among three groups. The correlations of MPVs changes with disease duration and expanded disability status scale (EDSS) were analyzed. Results: The RRMS patients had higher DGM iron concentration than did the NMO and control groups, but only the bilateral substantia nigra (SN) showed a significant statistical difference among three groups (p 0.05). Furthermore, no correlations were found between the DGM iron concentration and EDSS (p > 0.05). Conclusions: We confirm the iron concentration in the DGM iron content of MS patients is more than NMO patients and healthy controls in the same age range. Furthermore, the disease duration was found to be a significant contributor to patients with MS.

  16. Characterisation of the human uterine microbiome in non-pregnant women through deep sequencing of the V1-2 region of the 16S rRNA gene

    Directory of Open Access Journals (Sweden)

    Hans Verstraelen

    2016-01-01

    Full Text Available Background. It is widely assumed that the uterine cavity in non-pregnant women is physiologically sterile, also as a premise to the long-held view that human infants develop in a sterile uterine environment, though likely reflecting under-appraisal of the extent of the human bacterial metacommunity. In an exploratory study, we aimed to investigate the putative presence of a uterine microbiome in a selected series of non-pregnant women through deep sequencing of the V1-2 hypervariable region of the 16S ribosomal RNA (rRNA gene.Methods. Nineteen women with various reproductive conditions, including subfertility, scheduled for hysteroscopy and not showing uterine anomalies were recruited. Subjects were highly diverse with regard to demographic and medical history and included nulliparous and parous women. Endometrial tissue and mucus harvesting was performed by use of a transcervical device designed to obtain endometrial biopsy, while avoiding cervicovaginal contamination. Bacteria were targeted by use of a barcoded Illumina MiSeq paired-end sequencing method targeting the 16S rRNA gene V1-2 region, yielding an average of 41,194 reads per sample after quality filtering. Taxonomic annotation was pursued by comparison with sequences available through the Ribosomal Database Project and the NCBI database.Results. Out of 183 unique 16S rRNA gene amplicon sequences, 15 phylotypes were present in all samples. In some 90% of the women included, community architecture was fairly similar inasmuch B. xylanisolvens, B. thetaiotaomicron, B. fragilis and an undetermined Pelomonas taxon constituted over one third of the endometrial bacterial community. On the singular phylotype level, six women showed predominance of L. crispatus or L. iners in the presence of the Bacteroides core. Two endometrial communities were highly dissimilar, largely lacking the Bacteroides core, one dominated by L. crispatus and another consisting of a highly diverse community, including

  17. Comparison of 454 Ultra-Deep Sequencing and Allele-Specific Real-Time PCR with Regard to the Detection of Emerging Drug-Resistant Minor HIV-1 Variants after Antiretroviral Prophylaxis for Vertical Transmission.

    Directory of Open Access Journals (Sweden)

    Andrea Hauser

    Full Text Available Pregnant HIV-infected women were screened for the development of HIV-1 drug resistance after implementation of a triple-antiretroviral transmission prophylaxis as recommended by the WHO in 2006. The study offered the opportunity to compare amplicon-based 454 ultra-deep sequencing (UDS and allele-specific real-time PCR (ASPCR for the detection of drug-resistant minor variants in the HIV-1 reverse transcriptase (RT.Plasma samples from 34 Tanzanian women were previously analysed by ASPCR for key resistance mutations in the viral RT selected by AZT, 3TC, and NVP (K70R, K103N, Y181C, M184V, T215Y/F. In this study, the RT region of the same samples was investigated by amplicon-based UDS for resistance mutations using the 454 GS FLX System.Drug-resistant HIV-variants were identified in 69% (20/29 of women by UDS and in 45% (13/29 by ASPCR. The absolute number of resistance mutations identified by UDS was twice that identified by ASPCR (45 vs 24. By UDS 14 of 24 ASPCR-detected resistance mutations were identified at the same position. The overall concordance between UDS and ASPCR was 61.0% (25/41. The proportions of variants quantified by UDS were approximately 2-3 times lower than by ASPCR. Amplicon generation from samples with viral loads below 20,000 copies/ml failed more frequently by UDS compared to ASPCR (limit of detection = 650 copies/ml, resulting in missing or insufficient sequence coverage.Both methods can provide useful information about drug-resistant minor HIV-1 variants. ASPCR has a higher sensitivity than UDS, but is restricted to single resistance mutations. In contrast, UDS is limited by its requirement for high viral loads to achieve sufficient sequence coverage, but the sequence information reveals the complete resistance patterns within the genomic region analysed. Improvements to the UDS limit of detection are in progress, and UDS could then facilitate monitoring of drug-resistant minor variants in the HIV-1 quasispecies.

  18. Deep frying

    NARCIS (Netherlands)

    Koerten, van K.N.

    2016-01-01

    Deep frying is one of the most used methods in the food processing industry. Though practically any food can be fried, French fries are probably the most well-known deep fried products. The popularity of French fries stems from their unique taste and texture, a crispy outside with a mealy soft

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

    Science.gov (United States)

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

    2017-07-28

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

  20. NetMHCpan, a method for quantitative predictions of peptide binding to any HLA-A and -B locus protein of known sequence

    DEFF Research Database (Denmark)

    Nielsen, Morten; Lundegaard, Claus; Blicher, Thomas

    2007-01-01

    BACKGROUND: Binding of peptides to Major Histocompatibility Complex (MHC) molecules is the single most selective step in the recognition of pathogens by the cellular immune system. The human MHC class I system (HLA-I) is extremely polymorphic. The number of registered HLA-I molecules has now surp...... to provide new basic insights into HLA structure-function relationships. The method is available at http://www.cbs.dtu.dk/services/NetMHCpan....... surpassed 1500. Characterizing the specificity of each separately would be a major undertaking. PRINCIPAL FINDINGS: Here, we have drawn on a large database of known peptide-HLA-I interactions to develop a bioinformatics method, which takes both peptide and HLA sequence information into account...... successfully validate this method. We further demonstrate that the method can be applied to perform a clustering analysis of MHC specificities and suggest using this clustering to select particularly informative novel MHC molecules for future biochemical and functional analysis. CONCLUSIONS: Encompassing all...

  1. 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.

  2. Learning from Past Classification Errors: Exploring Methods for Improving the Performance of a Deep Learning-based Building Extraction Model through Quantitative Analysis of Commission Errors for Optimal Sample Selection

    Science.gov (United States)

    Swan, B.; Laverdiere, M.; Yang, L.

    2017-12-01

    In the past five years, deep Convolutional Neural Networks (CNN) have been increasingly favored for computer vision applications due to their high accuracy and ability to generalize well in very complex problems; however, details of how they function and in turn how they may be optimized are still imperfectly understood. In particular, their complex and highly nonlinear network architecture, including many hidden layers and self-learned parameters, as well as their mathematical implications, presents open questions about how to effectively select training data. Without knowledge of the exact ways the model processes and transforms its inputs, intuition alone may fail as a guide to selecting highly relevant training samples. Working in the context of improving a CNN-based building extraction model used for the LandScan USA gridded population dataset, we have approached this problem by developing a semi-supervised, highly-scalable approach to select training samples from a dataset of identified commission errors. Due to the large scope this project, tens of thousands of potential samples could be derived from identified commission errors. To efficiently trim those samples down to a manageable and effective set for creating additional training sample, we statistically summarized the spectral characteristics of areas with rates of commission errors at the image tile level and grouped these tiles using affinity propagation. Highly representative members of each commission error cluster were then used to select sites for training sample creation. The model will be incrementally re-trained with the new training data to allow for an assessment of how the addition of different types of samples affects the model performance, such as precision and recall rates. By using quantitative analysis and data clustering techniques to select highly relevant training samples, we hope to improve model performance in a manner that is resource efficient, both in terms of training process

  3. In chronic myeloid leukemia patients on second-line tyrosine kinase inhibitor therapy, deep sequencing of BCR-ABL1 at the time of warning may allow sensitive detection of emerging drug-resistant mutants.

    Science.gov (United States)

    Soverini, Simona; De Benedittis, Caterina; Castagnetti, Fausto; Gugliotta, Gabriele; Mancini, Manuela; Bavaro, Luana; Machova Polakova, Katerina; Linhartova, Jana; Iurlo, Alessandra; Russo, Domenico; Pane, Fabrizio; Saglio, Giuseppe; Rosti, Gianantonio; Cavo, Michele; Baccarani, Michele; Martinelli, Giovanni

    2016-08-02

    Imatinib-resistant chronic myeloid leukemia (CML) patients receiving second-line tyrosine kinase inhibitor (TKI) therapy with dasatinib or nilotinib have a higher risk of disease relapse and progression and not infrequently BCR-ABL1 kinase domain (KD) mutations are implicated in therapeutic failure. In this setting, earlier detection of emerging BCR-ABL1 KD mutations would offer greater chances of efficacy for subsequent salvage therapy and limit the biological consequences of full BCR-ABL1 kinase reactivation. Taking advantage of an already set up and validated next-generation deep amplicon sequencing (DS) assay, we aimed to assess whether DS may allow a larger window of detection of emerging BCR-ABL1 KD mutants predicting for an impending relapse. a total of 125 longitudinal samples from 51 CML patients who had acquired dasatinib- or nilotinib-resistant mutations during second-line therapy were analyzed by DS from the time of failure and mutation detection by conventional sequencing backwards. BCR-ABL1/ABL1%(IS) transcript levels were used to define whether the patient had 'optimal response', 'warning' or 'failure' at the time of first mutation detection by DS. DS was able to backtrack dasatinib- or nilotinib-resistant mutations to the previous sample(s) in 23/51 (45 %) pts. Median mutation burden at the time of first detection by DS was 5.5 % (range, 1.5-17.5 %); median interval between detection by DS and detection by conventional sequencing was 3 months (range, 1-9 months). In 5 cases, the mutations were detectable at baseline. In the remaining cases, response level at the time mutations were first detected by DS could be defined as 'Warning' (according to the 2013 ELN definitions of response to 2nd-line therapy) in 13 cases, as 'Optimal response' in one case, as 'Failure' in 4 cases. No dasatinib- or nilotinib-resistant mutations were detected by DS in 15 randomly selected patients with 'warning' at various timepoints, that later turned into optimal

  4. Viroid quasispecies revealed by deep sequencing

    Czech Academy of Sciences Publication Activity Database

    Brass, J.R.J.; Owens, R.A.; Matoušek, Jaroslav; Steger, G.

    2017-01-01

    Roč. 14, č. 3 (2017), s. 317-325 ISSN 1547-6286 Institutional support: RVO:60077344 Keywords : hepatitis-delta-virus * small rnas * biolistic inoculation * tomato plants Subject RIV: EB - Gene tics ; Molecular Biology OBOR OECD: Biochemistry and molecular biology Impact factor: 3.900, year: 2016

  5. Genome-Wide Mapping of Growth-Related Quantitative Trait Loci in Orange-Spotted Grouper (Epinephelus coioides) Using Double Digest Restriction-Site Associated DNA Sequencing (ddRADseq).

    Science.gov (United States)

    Yu, Hui; You, Xinxin; Li, Jia; Liu, Hankui; Meng, Zining; Xiao, Ling; Zhang, Haifa; Lin, Hao-Ran; Zhang, Yong; Shi, Qiong

    2016-04-06

    Mapping of quantitative trait loci (QTL) is essential for the discovery of genetic structures that related to complex quantitative traits. In this study, we identified 264,072 raw SNPs (single-nucleotide polymorphisms) by double digest restriction site associated DNA sequencing (ddRADseq), and utilized 3029 of these SNPs to construct a genetic linkage map in orange-spotted grouper (Epinephelus coioides) using a regression mapping algorithm. The genetic map contained 24 linkage groups (LGs) spanning a total genetic distance of 1231.98 cM. Twenty-seven significant growth-related QTLs were identified. Furthermore, we identified 17 genes (fez2, alg3, ece2, arvcf, sla27a4, sgk223, camk2, prrc2b, mchr1, sardh, pappa, syk, tert, wdrcp91, ftz-f1, mate1 and notch1) including three (tert, ftz-f1 and notch1) that have been reported to be involved in fish growth. To summarize, we mapped growth-related QTLs in the orange-spotted grouper. These QTLs will be useful in marker-assisted selection (MAS) efforts to improve growth-related traits in this economically important fish.

  6. DeepRT: deep learning for peptide retention time prediction in proteomics

    OpenAIRE

    Ma, Chunwei; Zhu, Zhiyong; Ye, Jun; Yang, Jiarui; Pei, Jianguo; Xu, Shaohang; Zhou, Ruo; Yu, Chang; Mo, Fan; Wen, Bo; Liu, Siqi

    2017-01-01

    Accurate predictions of peptide retention times (RT) in liquid chromatography have many applications in mass spectrometry-based proteomics. Herein, we present DeepRT, a deep learning based software for peptide retention time prediction. DeepRT automatically learns features directly from the peptide sequences using the deep convolutional Neural Network (CNN) and Recurrent Neural Network (RNN) model, which eliminates the need to use hand-crafted features or rules. After the feature learning, pr...

  7. Characterization of the collagen component of cartilage repair tissue of the talus with quantitative MRI: comparison of T2 relaxation time measurements with a diffusion-weighted double-echo steady-state sequence (dwDESS)

    International Nuclear Information System (INIS)

    Kretzschmar, M.; Hainc, N.; Studler, U.; Bieri, O.; Miska, M.; Wiewiorski, M.; Valderrabano, V.

    2015-01-01

    The purpose of this study was to characterize the collagen component of repair tissue (RT) of the talus after autologous matrix-induced chondrogenesis (AMIC) using quantitative T2 and diffusion-weighted imaging. Mean T2 values and diffusion coefficients of AMIC-RT and normal cartilage of the talus of 25 patients with posttraumatic osteochondral lesions and AMIC repair were compared in a cross-sectional design using partially spoiled steady-state free precession (pSSFP) for T2 quantification, and diffusion-weighted double-echo steady-state (dwDESS) for diffusion measurement. RT and cartilage were graded with modified Noyes and MOCART scores on morphological sequences. An association between follow-up interval and quantitative MRI measures was assessed using multivariate regression, after stratifying the cohort according to time interval between surgery and MRI. Mean T2 of the AMIC-RT and cartilage were 43.1 ms and 39.1 ms, respectively (p = 0.26). Mean diffusivity of the RT (1.76 μm 2 /ms) was significantly higher compared to normal cartilage (1.46 μm 2 /ms) (p = 0.0092). No correlation was found between morphological and quantitative parameters. RT diffusivity was lowest in the subgroup with follow-up >28 months (p = 0.027). Compared to T2-mapping, dwDESS demonstrated greater sensitivity in detecting differences in the collagen matrix between AMIC-RT and cartilage. Decreased diffusivity in patients with longer follow-up times may indicate an increased matrix organization of RT. (orig.)

  8. Characterization of the collagen component of cartilage repair tissue of the talus with quantitative MRI: comparison of T2 relaxation time measurements with a diffusion-weighted double-echo steady-state sequence (dwDESS)

    Energy Technology Data Exchange (ETDEWEB)

    Kretzschmar, M.; Hainc, N.; Studler, U. [University Hospital Basel, Department of Radiology, Basel (Switzerland); Bieri, O. [University Hospital Basel, Division of Radiological Physics, Basel (Switzerland); Miska, M. [University Hospital, Department of Orthopedics, Heidelberg (Germany); Wiewiorski, M.; Valderrabano, V. [University Hospital Basel, Department of Orthopedic Surgery, Basel (Switzerland)

    2015-04-01

    The purpose of this study was to characterize the collagen component of repair tissue (RT) of the talus after autologous matrix-induced chondrogenesis (AMIC) using quantitative T2 and diffusion-weighted imaging. Mean T2 values and diffusion coefficients of AMIC-RT and normal cartilage of the talus of 25 patients with posttraumatic osteochondral lesions and AMIC repair were compared in a cross-sectional design using partially spoiled steady-state free precession (pSSFP) for T2 quantification, and diffusion-weighted double-echo steady-state (dwDESS) for diffusion measurement. RT and cartilage were graded with modified Noyes and MOCART scores on morphological sequences. An association between follow-up interval and quantitative MRI measures was assessed using multivariate regression, after stratifying the cohort according to time interval between surgery and MRI. Mean T2 of the AMIC-RT and cartilage were 43.1 ms and 39.1 ms, respectively (p = 0.26). Mean diffusivity of the RT (1.76 μm{sup 2}/ms) was significantly higher compared to normal cartilage (1.46 μm{sup 2}/ms) (p = 0.0092). No correlation was found between morphological and quantitative parameters. RT diffusivity was lowest in the subgroup with follow-up >28 months (p = 0.027). Compared to T2-mapping, dwDESS demonstrated greater sensitivity in detecting differences in the collagen matrix between AMIC-RT and cartilage. Decreased diffusivity in patients with longer follow-up times may indicate an increased matrix organization of RT. (orig.)

  9. Unveiling the Biodiversity of Deep-Sea Nematodes through Metabarcoding: Are We Ready to Bypass the Classical Taxonomy?

    Science.gov (United States)

    Dell'Anno, Antonio; Carugati, Laura; Corinaldesi, Cinzia; Riccioni, Giulia; Danovaro, Roberto

    2015-01-01

    Nematodes inhabiting benthic deep-sea ecosystems account for >90% of the total metazoan abundances and they have been hypothesised to be hyper-diverse, but their biodiversity is still largely unknown. Metabarcoding could facilitate the census of biodiversity, especially for those tiny metazoans for which morphological identification is difficult. We compared, for the first time, different DNA extraction procedures based on the use of two commercial kits and a previously published laboratory protocol and tested their suitability for sequencing analyses of 18S rDNA of marine nematodes. We also investigated the reliability of Roche 454 sequencing analyses for assessing the biodiversity of deep-sea nematode assemblages previously morphologically identified. Finally, intra-genomic variation in 18S rRNA gene repeats was investigated by Illumina MiSeq in different deep-sea nematode morphospecies to assess the influence of polymorphisms on nematode biodiversity estimates. Our results indicate that the two commercial kits should be preferred for the molecular analysis of biodiversity of deep-sea nematodes since they consistently provide amplifiable DNA suitable for sequencing. We report that the morphological identification of deep-sea nematodes matches the results obtained by metabarcoding analysis only at the order-family level and that a large portion of Operational Clustered Taxonomic Units (OCTUs) was not assigned. We also show that independently from the cut-off criteria and bioinformatic pipelines used, the number of OCTUs largely exceeds the number of individuals and that 18S rRNA gene of different morpho-species of nematodes displayed intra-genomic polymorphisms. Our results indicate that metabarcoding is an important tool to explore the diversity of deep-sea nematodes, but still fails in identifying most of the species due to limited number of sequences deposited in the public databases, and in providing quantitative data on the species encountered. These aspects

  10. Differential evolution of a CXCR4-using HIV-1 strain in CCR5wt/wt and CCR5∆32/∆32 hosts revealed by longitudinal deep sequencing and phylogenetic reconstruction.

    Science.gov (United States)

    Le, Anh Q; Taylor, Jeremy; Dong, Winnie; McCloskey, Rosemary; Woods, Conan; Danroth, Ryan; Hayashi, Kanna; Milloy, M-J; Poon, Art F Y; Brumme, Zabrina L

    2015-12-03

    Rare individuals homozygous for a naturally-occurring 32 base pair deletion in the CCR5 gene (CCR5∆32/∆32) are resistant to infection by CCR5-using ("R5") HIV-1 strains but remain susceptible to less common CXCR4-using ("X4") strains. The evolutionary dynamics of X4 infections however, remain incompletely understood. We identified two individuals, one CCR5wt/wt and one CCR5∆32/∆32, within the Vancouver Injection Drug Users Study who were infected with a genetically similar X4 HIV-1 strain. While early-stage plasma viral loads were comparable in the two individuals (~4.5-5 log10 HIV-1 RNA copies/ml), CD4 counts in the CCR5wt/wt individual reached a nadir of 250 cells/mm(3) in the CCR5∆32/∆32 individual. Ancestral phylogenetic reconstructions using longitudinal envelope-V3 deep sequences suggested that both individuals were infected by a single transmitted/founder (T/F) X4 virus that differed at only one V3 site (codon 24). While substantial within-host HIV-1 V3 diversification was observed in plasma and PBMC in both individuals, the CCR5wt/wt individual's HIV-1 population gradually reverted from 100% X4 to ~60% R5 over ~4 years whereas the CCR5∆32/∆32 individual's remained consistently X4. Our observations illuminate early dynamics of X4 HIV-1 infections and underscore the influence of CCR5 genotype on HIV-1 V3 evolution.

  11. Sequences in language and text

    CERN Document Server

    Mikros, George K

    2015-01-01

    The aim of this volume is to present the diverse but highly interesting area of the quantitative analysis of the sequence of various linguistic structures. The collected articles present a wide spectrum of quantitative analyses of linguistic syntagmatic structures and explore novel sequential linguistic entities. This volume will be interesting to all researchers studying linguistics using quantitative methods.

  12. Whole Body MRI at 3T with Quantitative Diffusion Weighted Imaging and Contrast-Enhanced Sequences for the Characterization of Peripheral Lesions in Patients with Neurofibromatosis Type 2 and Schwannomatosis

    International Nuclear Information System (INIS)

    Fayad, Laura M.; Blakeley, Jaishri; Plotkin, Scott; Widemann, Brigitte; Jacobs, Michael A.

    2013-01-01

    Purpose. WB-MRI is mainly used for tumor detection and surveillance. The purpose of this study is to establish the feasibility of WB-MRI at 3T for lesion characterization, with DWI/ADC-mapping and contrast-enhanced sequences, in patients with neurofibromatosis type 2 (NF-2) and schwannomatosis. Materials and Methods. At 3T, WB-MRI was performed in 11 subjects (10 NF-2 and 1 schwannomatosis) with STIR, T1, contrast-enhanced T1, and DWI/ADC mapping (b = 50, 400, 800 s/mm 2 ). Two readers reviewed imaging for the presence and character of peripheral lesions. Lesion size and features (signal intensity, heterogeneity, enhancement characteristics, and ADC values) were recorded. Descriptive statistics were reported. Results. Twenty-three lesions were identified, with average size of 4.6 ± 2.8 cm. Lesions were characterized as tumors (21/23) or cysts (2/23) by contrast-enhancement properties (enhancement in tumors, no enhancement in cysts). On T1, tumors were homogeneously isointense (5/21) or hypointense (16/21); on STIR, tumors were hyperintense and homogeneous (10/21) or heterogeneous (11/21); on postcontrast T1, tumors enhanced homogeneously (14/21) or heterogeneously (7/21); on DWI, tumor ADC values were variable (range 0.8–2.7), suggesting variability in intrinsic tumor properties. Conclusion. WB-MRI with quantitative DWI and contrast-enhanced sequences at 3T is feasible and advances the utility of WB-MRI not only to include detection, but also to provide additional metrics for lesion characterization

  13. Deep Learning

    DEFF Research Database (Denmark)

    Jensen, Morten Bornø; Bahnsen, Chris Holmberg; Nasrollahi, Kamal

    2018-01-01

    I løbet af de sidste 10 år er kunstige neurale netværk gået fra at være en støvet, udstødt tekno-logi til at spille en hovedrolle i udviklingen af kunstig intelligens. Dette fænomen kaldes deep learning og er inspireret af hjernens opbygning.......I løbet af de sidste 10 år er kunstige neurale netværk gået fra at være en støvet, udstødt tekno-logi til at spille en hovedrolle i udviklingen af kunstig intelligens. Dette fænomen kaldes deep learning og er inspireret af hjernens opbygning....

  14. Deep geothermics

    International Nuclear Information System (INIS)

    Anon.

    1995-01-01

    The hot-dry-rocks located at 3-4 km of depth correspond to low permeable rocks carrying a large amount of heat. The extraction of this heat usually requires artificial hydraulic fracturing of the rock to increase its permeability before water injection. Hot-dry-rocks geothermics or deep geothermics is not today a commercial channel but only a scientific and technological research field. The Soultz-sous-Forets site (Northern Alsace, France) is characterized by a 6 degrees per meter geothermal gradient and is used as a natural laboratory for deep geothermal and geological studies in the framework of a European research program. Two boreholes have been drilled up to 3600 m of depth in the highly-fractured granite massif beneath the site. The aim is to create a deep heat exchanger using only the natural fracturing for water transfer. A consortium of german, french and italian industrial companies (Pfalzwerke, Badenwerk, EdF and Enel) has been created for a more active participation to the pilot phase. (J.S.). 1 fig., 2 photos

  15. Invited talk: Deep Learning Meets Physics

    CERN Multimedia

    CERN. Geneva

    2018-01-01

    Deep Learning has emerged as one of the most successful fields of machine learning and artificial intelligence with overwhelming success in industrial speech, text and vision benchmarks. Consequently it evolved into the central field of research for IT giants like Google, facebook, Microsoft, Baidu, and Amazon. Deep Learning is founded on novel neural network techniques, the recent availability of very fast computers, and massive data sets. In its core, Deep Learning discovers multiple levels of abstract representations of the input. The main obstacle to learning deep neural networks is the vanishing gradient problem. The vanishing gradient impedes credit assignment to the first layers of a deep network or to early elements of a sequence, therefore limits model selection. Major advances in Deep Learning can be related to avoiding the vanishing gradient like stacking, ReLUs, residual networks, highway networks, and LSTM. For Deep Learning, we suggested self-normalizing neural networks (SNNs) which automatica...

  16. Deep learning for computational chemistry.

    Science.gov (United States)

    Goh, Garrett B; Hodas, Nathan O; Vishnu, Abhinav

    2017-06-15

    The rise and fall of artificial neural networks is well documented in the scientific literature of both computer science and computational chemistry. Yet almost two decades later, we are now seeing a resurgence of interest in deep learning, a machine learning algorithm based on multilayer neural networks. Within the last few years, we have seen the transformative impact of deep learning in many domains, particularly in speech recognition and computer vision, to the extent that the majority of expert practitioners in those field are now regularly eschewing prior established models in favor of deep learning models. In this review, we provide an introductory overview into the theory of deep neural networks and their unique properties that distinguish them from traditional machine learning algorithms used in cheminformatics. By providing an overview of the variety of emerging applications of deep neural networks, we highlight its ubiquity and broad applicability to a wide range of challenges in the field, including quantitative structure activity relationship, virtual screening, protein structure prediction, quantum chemistry, materials design, and property prediction. In reviewing the performance of deep neural networks, we observed a consistent outperformance against non-neural networks state-of-the-art models across disparate research topics, and deep neural network-based models often exceeded the "glass ceiling" expectations of their respective tasks. Coupled with the maturity of GPU-accelerated computing for training deep neural networks and the exponential growth of chemical data on which to train these networks on, we anticipate that deep learning algorithms will be a valuable tool for computational chemistry. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  17. The objective of this program is to develop innovative DNA detection technologies to achieve fast microbial community assessment. The specific approaches are (1) to develop inexpensive and reliable sequence-proof hybridization DNA detection technology (2) to develop quantitative DNA hybridization technology for microbial community assessment and (3) to study the microbes which have demonstrated the potential to have nuclear waste bioremediation

    International Nuclear Information System (INIS)

    Chen, Chung H.

    2004-01-01

    The objective of this program is to develop innovative DNA detection technologies to achieve fast microbial community assessment. The specific approaches are (1) to develop inexpensive and reliable sequence-proof hybridization DNA detection technology (2) to develop quantitative DNA hybridization technology for microbial community assessment and (3) to study the microbes which have demonstrated the potential to have nuclear waste bioremediation

  18. Deep smarts.

    Science.gov (United States)

    Leonard, Dorothy; Swap, Walter

    2004-09-01

    When a person sizes up a complex situation and rapidly comes to a decision that proves to be not just good but brilliant, you think, "That was smart." After you watch him do this a few times, you realize you're in the presence of something special. It's not raw brainpower, though that helps. It's not emotional intelligence, either, though that, too, is often involved. It's deep smarts. Deep smarts are not philosophical--they're not"wisdom" in that sense, but they're as close to wisdom as business gets. You see them in the manager who understands when and how to move into a new international market, in the executive who knows just what kind of talk to give when her organization is in crisis, in the technician who can track a product failure back to an interaction between independently produced elements. These are people whose knowledge would be hard to purchase on the open market. Their insight is based on know-how more than on know-what; it comprises a system view as well as expertise in individual areas. Because deep smarts are experienced based and often context specific, they can't be produced overnight or readily imported into an organization. It takes years for an individual to develop them--and no time at all for an organization to lose them when a valued veteran walks out the door. They can be taught, however, with the right techniques. Drawing on their forthcoming book Deep Smarts, Dorothy Leonard and Walter Swap say the best way to transfer such expertise to novices--and, on a larger scale, to make individual knowledge institutional--isn't through PowerPoint slides, a Web site of best practices, online training, project reports, or lectures. Rather, the sage needs to teach the neophyte individually how to draw wisdom from experience. Companies have to be willing to dedicate time and effort to such extensive training, but the investment more than pays for itself.

  19. Deep Recurrent Convolutional Neural Network: Improving Performance For Speech Recognition

    OpenAIRE

    Zhang, Zewang; Sun, Zheng; Liu, Jiaqi; Chen, Jingwen; Huo, Zhao; Zhang, Xiao

    2016-01-01

    A deep learning approach has been widely applied in sequence modeling problems. In terms of automatic speech recognition (ASR), its performance has significantly been improved by increasing large speech corpus and deeper neural network. Especially, recurrent neural network and deep convolutional neural network have been applied in ASR successfully. Given the arising problem of training speed, we build a novel deep recurrent convolutional network for acoustic modeling and then apply deep resid...

  20. Quantitative Susceptibility Mapping in Parkinson's Disease.

    Science.gov (United States)

    Langkammer, Christian; Pirpamer, Lukas; Seiler, Stephan; Deistung, Andreas; Schweser, Ferdinand; Franthal, Sebastian; Homayoon, Nina; Katschnig-Winter, Petra; Koegl-Wallner, Mariella; Pendl, Tamara; Stoegerer, Eva Maria; Wenzel, Karoline; Fazekas, Franz; Ropele, Stefan; Reichenbach, Jürgen Rainer; Schmidt, Reinhold; Schwingenschuh, Petra

    2016-01-01

    Quantitative susceptibility mapping (QSM) and R2* relaxation rate mapping have demonstrated increased iron deposition in the substantia nigra of patients with idiopathic Parkinson's disease (PD). However, the findings in other subcortical deep gray matter nuclei are converse and the sensitivity of QSM and R2* for morphological changes and their relation to clinical measures of disease severity has so far been investigated only sparsely. The local ethics committee approved this study and all subjects gave written informed consent. 66 patients with idiopathic Parkinson's disease and 58 control subjects underwent quantitative MRI at 3T. Susceptibility and R2* maps were reconstructed from a spoiled multi-echo 3D gradient echo sequence. Mean susceptibilities and R2* rates were measured in subcortical deep gray matter nuclei and compared between patients with PD and controls as well as related to clinical variables. Compared to control subjects, patients with PD had increased R2* values in the substantia nigra. QSM also showed higher susceptibilities in patients with PD in substantia nigra, in the nucleus ruber, thalamus, and globus pallidus. Magnetic susceptibility of several of these structures was correlated with the levodopa-equivalent daily dose (LEDD) and clinical markers of motor and non-motor disease severity (total MDS-UPDRS, MDS-UPDRS-I and II). Disease severity as assessed by the Hoehn & Yahr scale was correlated with magnetic susceptibility in the substantia nigra. The established finding of higher R2* rates in the substantia nigra was extended by QSM showing superior sensitivity for PD-related tissue changes in nigrostriatal dopaminergic pathways. QSM additionally reflected the levodopa-dosage and disease severity. These results suggest a more widespread pathologic involvement and QSM as a novel means for its investigation, more sensitive than current MRI techniques.

  1. Using high-throughput DNA sequencing, genetic fingerprinting, and quantitative PCR as tools for monitoring bloom-forming and toxigenic cyanobacteria in Upper Klamath Lake, Oregon, 2013 and 2014

    Science.gov (United States)

    Caldwell Eldridge, Sara L.; Driscoll, Conner; Dreher, Theo W.

    2017-06-05

    Monitoring the community structure and metabolic activities of cyanobacterial blooms in Upper Klamath Lake, Oregon, is critical to lake management because these blooms degrade water quality and produce toxic microcystins that are harmful to humans, domestic animals, and wildlife. Genetic tools, such as DNA fingerprinting by terminal restriction fragment length polymorphism (T-RFLP) analysis, high-throughput DNA sequencing (HTS), and real-time, quantitative polymerase chain reaction (qPCR), provide more sensitive and rapid assessments of bloom ecology than traditional techniques. The objectives of this study were (1) to characterize the microbial community at one site in Upper Klamath Lake and determine changes in the cyanobacterial community through time using T-RFLP and HTS in comparison with traditional light microscopy; (2) to determine relative abundances and changes in abundance over time of toxigenic Microcystis using qPCR; and (3) to determine relative abundances and changes in abundance over time of Aphanizomenon, Microcystis, and total cyanobacteria using qPCR. T-RFLP analysis of total cyanobacteria showed a dominance of only one or two distinct genotypes in samples from 2013, but results of HTS in 2013 and 2014 showed more variations in the bloom cycle that fit with the previous understanding of bloom dynamics in Upper Klamath Lake and indicated that potentially toxigenic Microcystis was more prevalent in 2014 than in years prior. The qPCR-estimated copy numbers of all target genes were higher in 2014 than in 2013, when microcystin concentrations also were higher. Total Microcystis density was shown with qPCR to be a better predictor of late-season increases in microcystin concentrations than the relative proportions of potentially toxigenic cells. In addition, qPCR targeting Aphanizomenon at one site in Upper Klamath Lake indicated a moderate bloom of this species (corresponding to chlorophyll a concentrations between approximately 75 and 200 micrograms

  2. DeepPy: Pythonic deep learning

    DEFF Research Database (Denmark)

    Larsen, Anders Boesen Lindbo

    This technical report introduces DeepPy – a deep learning framework built on top of NumPy with GPU acceleration. DeepPy bridges the gap between highperformance neural networks and the ease of development from Python/NumPy. Users with a background in scientific computing in Python will quickly...... be able to understand and change the DeepPy codebase as it is mainly implemented using high-level NumPy primitives. Moreover, DeepPy supports complex network architectures by letting the user compose mathematical expressions as directed graphs. The latest version is available at http...

  3. Quantitative research.

    Science.gov (United States)

    Watson, Roger

    2015-04-01

    This article describes the basic tenets of quantitative research. The concepts of dependent and independent variables are addressed and the concept of measurement and its associated issues, such as error, reliability and validity, are explored. Experiments and surveys – the principal research designs in quantitative research – are described and key features explained. The importance of the double-blind randomised controlled trial is emphasised, alongside the importance of longitudinal surveys, as opposed to cross-sectional surveys. Essential features of data storage are covered, with an emphasis on safe, anonymous storage. Finally, the article explores the analysis of quantitative data, considering what may be analysed and the main uses of statistics in analysis.

  4. Deep Learning and Its Applications in Biomedicine.

    Science.gov (United States)

    Cao, Chensi; Liu, Feng; Tan, Hai; Song, Deshou; Shu, Wenjie; Li, Weizhong; Zhou, Yiming; Bo, Xiaochen; Xie, Zhi

    2018-02-01

    Advances in biological and medical technologies have been providing us explosive volumes of biological and physiological data, such as medical images, electroencephalography, genomic and protein sequences. Learning from these data facilitates the understanding of human health and disease. Developed from artificial neural networks, deep learning-based algorithms show great promise in extracting features and learning patterns from complex data. The aim of this paper is to provide an overview of deep learning techniques and some of the state-of-the-art applications in the biomedical field. We first introduce the development of artificial neural network and deep learning. We then describe two main components of deep learning, i.e., deep learning architectures and model optimization. Subsequently, some examples are demonstrated for deep learning applications, including medical image classification, genomic sequence analysis, as well as protein structure classification and prediction. Finally, we offer our perspectives for the future directions in the field of deep learning. Copyright © 2018. Production and hosting by Elsevier B.V.

  5. Genome-wide detection and analysis of hippocampus core promoters using DeepCAGE

    DEFF Research Database (Denmark)

    Valen, Eivind; Pascarella, Giovanni; Chalk, Alistair

    2009-01-01

    in a given tissue. Here, we present a new method for high-throughput sequencing of 5' cDNA tags-DeepCAGE: merging the Cap Analysis of Gene Expression method with ultra-high-throughput sequence technology. We apply DeepCAGE to characterize 1.4 million sequenced TSS from mouse hippocampus and reveal a wealth...

  6. Quantitative habitability.

    Science.gov (United States)

    Shock, Everett L; Holland, Melanie E

    2007-12-01

    A framework is proposed for a quantitative approach to studying habitability. Considerations of environmental supply and organismal demand of energy lead to the conclusions that power units are most appropriate and that the units for habitability become watts per organism. Extreme and plush environments are revealed to be on a habitability continuum, and extreme environments can be quantified as those where power supply only barely exceeds demand. Strategies for laboratory and field experiments are outlined that would quantify power supplies, power demands, and habitability. An example involving a comparison of various metabolisms pursued by halophiles is shown to be well on the way to a quantitative habitability analysis.

  7. Greedy Deep Dictionary Learning

    OpenAIRE

    Tariyal, Snigdha; Majumdar, Angshul; Singh, Richa; Vatsa, Mayank

    2016-01-01

    In this work we propose a new deep learning tool called deep dictionary learning. Multi-level dictionaries are learnt in a greedy fashion, one layer at a time. This requires solving a simple (shallow) dictionary learning problem, the solution to this is well known. We apply the proposed technique on some benchmark deep learning datasets. We compare our results with other deep learning tools like stacked autoencoder and deep belief network; and state of the art supervised dictionary learning t...

  8. Assessment of deep tissue hyperalgesia in the groin – a method comparison of electrical vs. pressure stimulation

    DEFF Research Database (Denmark)

    Aasvang, E K; Werner, M U; Kehlet, H

    2014-01-01

    BACKGROUND: Deep pain complaints are more frequent than cutaneous in post-surgical patients, and a prevalent finding in quantitative sensory testing studies. However, the preferred assessment method - pressure algometry - is indirect and tissue unspecific, hindering advances in treatment and prev......BACKGROUND: Deep pain complaints are more frequent than cutaneous in post-surgical patients, and a prevalent finding in quantitative sensory testing studies. However, the preferred assessment method - pressure algometry - is indirect and tissue unspecific, hindering advances in treatment...... thresholds to pressure algometry, by performing identical test-retest sequences 10 days apart, in deep tissues in the groin region. Electrical stimulation was performed by five up-and-down staircase series of single impulses of 0.04 ms duration, starting from 0 mA in increments of 0.2 mA until a threshold......: The presented tissue-specific direct deep tissue electrical stimulation technique has equal or superior reliability compared with the indirect tissue-unspecific stimulation by pressure algometry. This method may facilitate advances in mechanism based preventive and treatment strategies in acute and chronic post...

  9. Sequence assembly

    DEFF Research Database (Denmark)

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

    2009-01-01

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