The Performance of a PN Spread Spectrum Receiver Preceded by an Adaptive Interference Suppression Filter.

DTIC Science & Technology

1982-12-01

Sequence dj Estimate of the Desired Signal DEL Sampling Time Interval DS Direct Sequence c Sufficient Statistic E/T Signal Power Erfc Complimentary Error...Namely, a white Gaussian noise (WGN) generator was added. Also, a statistical subroutine was added in order to assess performance improvement at the...reference code and then passed through a correlation detector whose output is the sufficient 1 statistic , e . Using a threshold device and the sufficient

Isotopically enhanced triple-quantum-dot qubit

PubMed Central

Eng, Kevin; Ladd, Thaddeus D.; Smith, Aaron; Borselli, Matthew G.; Kiselev, Andrey A.; Fong, Bryan H.; Holabird, Kevin S.; Hazard, Thomas M.; Huang, Biqin; Deelman, Peter W.; Milosavljevic, Ivan; Schmitz, Adele E.; Ross, Richard S.; Gyure, Mark F.; Hunter, Andrew T.

2015-01-01

Like modern microprocessors today, future processors of quantum information may be implemented using all-electrical control of silicon-based devices. A semiconductor spin qubit may be controlled without the use of magnetic fields by using three electrons in three tunnel-coupled quantum dots. Triple dots have previously been implemented in GaAs, but this material suffers from intrinsic nuclear magnetic noise. Reduction of this noise is possible by fabricating devices using isotopically purified silicon. We demonstrate universal coherent control of a triple-quantum-dot qubit implemented in an isotopically enhanced Si/SiGe heterostructure. Composite pulses are used to implement spin-echo type sequences, and differential charge sensing enables single-shot state readout. These experiments demonstrate sufficient control with sufficiently low noise to enable the long pulse sequences required for exchange-only two-qubit logic and randomized benchmarking. PMID:26601186

BiRen: predicting enhancers with a deep-learning-based model using the DNA sequence alone.

PubMed

Yang, Bite; Liu, Feng; Ren, Chao; Ouyang, Zhangyi; Xie, Ziwei; Bo, Xiaochen; Shu, Wenjie

2017-07-01

Enhancer elements are noncoding stretches of DNA that play key roles in controlling gene expression programmes. Despite major efforts to develop accurate enhancer prediction methods, identifying enhancer sequences continues to be a challenge in the annotation of mammalian genomes. One of the major issues is the lack of large, sufficiently comprehensive and experimentally validated enhancers for humans or other species. Thus, the development of computational methods based on limited experimentally validated enhancers and deciphering the transcriptional regulatory code encoded in the enhancer sequences is urgent. We present a deep-learning-based hybrid architecture, BiRen, which predicts enhancers using the DNA sequence alone. Our results demonstrate that BiRen can learn common enhancer patterns directly from the DNA sequence and exhibits superior accuracy, robustness and generalizability in enhancer prediction relative to other state-of-the-art enhancer predictors based on sequence characteristics. Our BiRen will enable researchers to acquire a deeper understanding of the regulatory code of enhancer sequences. Our BiRen method can be freely accessed at https://github.com/wenjiegroup/BiRen . shuwj@bmi.ac.cn or boxc@bmi.ac.cn. Supplementary data are available at Bioinformatics online. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

Tertiary alphabet for the observable protein structural universe

PubMed Central

Mackenzie, Craig O.; Zhou, Jianfu; Grigoryan, Gevorg

2016-01-01

Here, we systematically decompose the known protein structural universe into its basic elements, which we dub tertiary structural motifs (TERMs). A TERM is a compact backbone fragment that captures the secondary, tertiary, and quaternary environments around a given residue, comprising one or more disjoint segments (three on average). We seek the set of universal TERMs that capture all structure in the Protein Data Bank (PDB), finding remarkable degeneracy. Only ∼600 TERMs are sufficient to describe 50% of the PDB at sub-Angstrom resolution. However, more rare geometries also exist, and the overall structural coverage grows logarithmically with the number of TERMs. We go on to show that universal TERMs provide an effective mapping between sequence and structure. We demonstrate that TERM-based statistics alone are sufficient to recapitulate close-to-native sequences given either NMR or X-ray backbones. Furthermore, sequence variability predicted from TERM data agrees closely with evolutionary variation. Finally, locations of TERMs in protein chains can be predicted from sequence alone based on sequence signatures emergent from TERM instances in the PDB. For multisegment motifs, this method identifies spatially adjacent fragments that are not contiguous in sequence—a major bottleneck in structure prediction. Although all TERMs recur in diverse proteins, some appear specialized for certain functions, such as interface formation, metal coordination, or even water binding. Structural biology has benefited greatly from previously observed degeneracies in structure. The decomposition of the known structural universe into a finite set of compact TERMs offers exciting opportunities toward better understanding, design, and prediction of protein structure. PMID:27810958

Impact of sequencing depth in ChIP-seq experiments

PubMed Central

Jung, Youngsook L.; Luquette, Lovelace J.; Ho, Joshua W.K.; Ferrari, Francesco; Tolstorukov, Michael; Minoda, Aki; Issner, Robbyn; Epstein, Charles B.; Karpen, Gary H.; Kuroda, Mitzi I.; Park, Peter J.

2014-01-01

In a chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) experiment, an important consideration in experimental design is the minimum number of sequenced reads required to obtain statistically significant results. We present an extensive evaluation of the impact of sequencing depth on identification of enriched regions for key histone modifications (H3K4me3, H3K36me3, H3K27me3 and H3K9me2/me3) using deep-sequenced datasets in human and fly. We propose to define sufficient sequencing depth as the number of reads at which detected enrichment regions increase <1% for an additional million reads. Although the required depth depends on the nature of the mark and the state of the cell in each experiment, we observe that sufficient depth is often reached at <20 million reads for fly. For human, there are no clear saturation points for the examined datasets, but our analysis suggests 40–50 million reads as a practical minimum for most marks. We also devise a mathematical model to estimate the sufficient depth and total genomic coverage of a mark. Lastly, we find that the five algorithms tested do not agree well for broad enrichment profiles, especially at lower depths. Our findings suggest that sufficient sequencing depth and an appropriate peak-calling algorithm are essential for ensuring robustness of conclusions derived from ChIP-seq data. PMID:24598259

Amplicon Sequencing of the slpH Locus Permits Culture-Independent Strain Typing of Lactobacillus helveticus in Dairy Products

PubMed Central

Moser, Aline; Wüthrich, Daniel; Bruggmann, Rémy; Eugster-Meier, Elisabeth; Meile, Leo; Irmler, Stefan

2017-01-01

The advent of massive parallel sequencing technologies has opened up possibilities for the study of the bacterial diversity of ecosystems without the need for enrichment or single strain isolation. By exploiting 78 genome data-sets from Lactobacillus helveticus strains, we found that the slpH locus that encodes a putative surface layer protein displays sufficient genetic heterogeneity to be a suitable target for strain typing. Based on high-throughput slpH gene sequencing and the detection of single-base DNA sequence variations, we established a culture-independent method to assess the biodiversity of the L. helveticus strains present in fermented dairy food. When we applied the method to study the L. helveticus strain composition in 15 natural whey cultures (NWCs) that were collected at different Gruyère, a protected designation of origin (PDO) production facilities, we detected a total of 10 sequence types (STs). In addition, we monitored the development of a three-strain mix in raclette cheese for 17 weeks. PMID:28775722

ECB deacylase mutants

DOEpatents

Arnold, Frances H.; Shao, Zhixin; Zhao, Huimin; Giver, Lorraine J.

2002-01-01

A method for in vitro mutagenesis and recombination of polynucleotide sequences based on polymerase-catalyzed extension of primer oligonucleotides is disclosed. The method involves priming template polynucleotide(s) with random-sequences or defined-sequence primers to generate a pool of short DNA fragments with a low level of point mutations. The DNA fragments are subjected to denaturization followed by annealing and further enzyme-catalyzed DNA polymerization. This procedure is repeated a sufficient number of times to produce full-length genes which comprise mutants of the original template polynucleotides. These genes can be further amplified by the polymerase chain reaction and cloned into a vector for expression of the encoded proteins.

Fast selection of miRNA candidates based on large-scale pre-computed MFE sets of randomized sequences.

PubMed

Warris, Sven; Boymans, Sander; Muiser, Iwe; Noback, Michiel; Krijnen, Wim; Nap, Jan-Peter

2014-01-13

Small RNAs are important regulators of genome function, yet their prediction in genomes is still a major computational challenge. Statistical analyses of pre-miRNA sequences indicated that their 2D structure tends to have a minimal free energy (MFE) significantly lower than MFE values of equivalently randomized sequences with the same nucleotide composition, in contrast to other classes of non-coding RNA. The computation of many MFEs is, however, too intensive to allow for genome-wide screenings. Using a local grid infrastructure, MFE distributions of random sequences were pre-calculated on a large scale. These distributions follow a normal distribution and can be used to determine the MFE distribution for any given sequence composition by interpolation. It allows on-the-fly calculation of the normal distribution for any candidate sequence composition. The speedup achieved makes genome-wide screening with this characteristic of a pre-miRNA sequence practical. Although this particular property alone will not be able to distinguish miRNAs from other sequences sufficiently discriminative, the MFE-based P-value should be added to the parameters of choice to be included in the selection of potential miRNA candidates for experimental verification.

Sequencing Needs for Viral Diagnostics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gardner, S N; Lam, M; Mulakken, N J

2004-01-26

We built a system to guide decisions regarding the amount of genomic sequencing required to develop diagnostic DNA signatures, which are short sequences that are sufficient to uniquely identify a viral species. We used our existing DNA diagnostic signature prediction pipeline, which selects regions of a target species genome that are conserved among strains of the target (for reliability, to prevent false negatives) and unique relative to other species (for specificity, to avoid false positives). We performed simulations, based on existing sequence data, to assess the number of genome sequences of a target species and of close phylogenetic relatives (''nearmore » neighbors'') that are required to predict diagnostic signature regions that are conserved among strains of the target species and unique relative to other bacterial and viral species. For DNA viruses such as variola (smallpox), three target genomes provide sufficient guidance for selecting species-wide signatures. Three near neighbor genomes are critical for species specificity. In contrast, most RNA viruses require four target genomes and no near neighbor genomes, since lack of conservation among strains is more limiting than uniqueness. SARS and Ebola Zaire are exceptional, as additional target genomes currently do not improve predictions, but near neighbor sequences are urgently needed. Our results also indicate that double stranded DNA viruses are more conserved among strains than are RNA viruses, since in most cases there was at least one conserved signature candidate for the DNA viruses and zero conserved signature candidates for the RNA viruses.« less

A Simple Method for the Extraction, PCR-amplification, Cloning, and Sequencing of Pasteuria 16S rDNA from Small Numbers of Endospores.

PubMed

Atibalentja, N; Noel, G R; Ciancio, A

2004-03-01

For many years the taxonomy of the genus Pasteuria has been marred with confusion because the bacterium could not be cultured in vitro and, therefore, descriptions were based solely on morphological, developmental, and pathological characteristics. The current study sought to devise a simple method for PCR-amplification, cloning, and sequencing of Pasteuria 16S rDNA from small numbers of endospores, with no need for prior DNA purification. Results show that DNA extracts from plain glass bead-beating of crude suspensions containing 10,000 endospores at 0.2 x 10 endospores ml(-1) were sufficient for PCR-amplification of Pasteuria 16S rDNA, when used in conjunction with specific primers. These results imply that for P. penetrans and P. nishizawae only one parasitized female of Meloidogyne spp. and Heterodera glycines, respectively, should be sufficient, and as few as eight cadavers of Belonolaimus longicaudatus with an average number of 1,250 endospores of "Candidatus Pasteuria usgae" are needed for PCR-amplification of Pasteuria 16S rDNA. The method described in this paper should facilitate the sequencing of the 16S rDNA of the many Pasteuria isolates that have been reported on nematodes and, consequently, expedite the classification of those isolates through comparative sequence analysis.

HYBRIDIZATION PROPERTIES OF DNA SEQUENCES DIRECTING THE SYNTHESIS OF MESSENGER RNA AND HETEROGENEOUS NUCLEAR RNA

PubMed Central

Greenberg, Jay R.; Perry, Robert P.

1971-01-01

The relationship of the DNA sequences from which polyribosomal messenger RNA (mRNA) and heterogeneous nuclear RNA (NRNA) of mouse L cells are transcribed was investigated by means of hybridization kinetics and thermal denaturation of the hybrids. Hybridization was performed in formamide solutions at DNA excess. Under these conditions most of the hybridizing mRNA and NRNA react at values of Dot (DNA concentration multiplied by time) expected for RNA transcribed from the nonrepeated or rarely repeated fraction of the genome. However, a fraction of both mRNA and NRNA hybridize at values of Dot about 10,000 times lower, and therefore must be transcribed from highly redundant DNA sequences. The fraction of NRNA hybridizing to highly repeated sequences is about 1.7 times greater than the corresponding fraction of mRNA. The hybrids formed by the rapidly reacting fractions of both NRNA and mRNA melt over a narrow temperature range with a midpoint about 11°C below that of native L cell DNA. This indicates that these hybrids consist of partially complementary sequences with approximately 11% mismatching of bases. Hybrids formed by the slowly reacting fraction of NRNA melt within 4°–6°C of native DNA, indicating very little, if any, mismatching of bases. Hybrids of the slowly reacting components of mRNA, formed under conditions of sufficiently low RNA input, have a high thermal stability, similar to that observed for hybrids of the slowly reacting NRNA component. However, when higher inputs of mRNA are used, hybrids are formed which have a strikingly lower thermal stability. This observation can be explained by assuming that there is sufficient similarity among the relatively rare DNA sequences coding for mRNA so that under hybridization conditions, in which these DNA sequences are not truly in excess, reversible hybrids exhibiting a considerable amount of mispairing are formed. The fact that a comparable phenomenon has not been observed for NRNA may mean that there is less similarity among the relatively rare DNA sequences coding for NRNA than there is among the rare sequences coding for mRNA. PMID:4999767

Discriminatory usefulness of pulsed-field gel electrophoresis and sequence-based typing in Legionella outbreaks.

PubMed

Quero, Sara; García-Núñez, Marian; Párraga-Niño, Noemí; Barrabeig, Irene; Pedro-Botet, Maria L; de Simon, Mercè; Sopena, Nieves; Sabrià, Miquel

2016-06-01

To compare the discriminatory power of pulsed-field gel electrophoresis (PFGE) and sequence-based typing (SBT) in Legionella outbreaks for determining the infection source. Twenty-five investigations of Legionnaires' disease were analyzed by PFGE, SBT and Dresden monoclonal antibody. The results suggested that monoclonal antibody could reduce the number of Legionella isolates to be characterized by molecular methods. The epidemiological concordance PFGE-SBT was 100%, while the molecular concordance was 64%. Adjusted Wallace index (AW) showed that PFGE has better discriminatory power than SBT (AWSBT→PFGE = 0.767; AWPFGE→SBT = 1). The discrepancies appeared mostly in sequence type (ST) 1, a worldwide distributed ST for which PFGE discriminated different profiles. SBT discriminatory power was not sufficient verifying the infection source, especially in worldwide distributed STs, which were classified into different PFGE patterns.

Comparison of methods for calculating conditional expectations of sufficient statistics for continuous time Markov chains.

PubMed

Tataru, Paula; Hobolth, Asger

2011-12-05

Continuous time Markov chains (CTMCs) is a widely used model for describing the evolution of DNA sequences on the nucleotide, amino acid or codon level. The sufficient statistics for CTMCs are the time spent in a state and the number of changes between any two states. In applications past evolutionary events (exact times and types of changes) are unaccessible and the past must be inferred from DNA sequence data observed in the present. We describe and implement three algorithms for computing linear combinations of expected values of the sufficient statistics, conditioned on the end-points of the chain, and compare their performance with respect to accuracy and running time. The first algorithm is based on an eigenvalue decomposition of the rate matrix (EVD), the second on uniformization (UNI), and the third on integrals of matrix exponentials (EXPM). The implementation in R of the algorithms is available at http://www.birc.au.dk/~paula/. We use two different models to analyze the accuracy and eight experiments to investigate the speed of the three algorithms. We find that they have similar accuracy and that EXPM is the slowest method. Furthermore we find that UNI is usually faster than EVD.

Direct repeat sequences in the Streptomyces chitinase-63 promoter direct both glucose repression and chitin induction

PubMed Central

Ni, Xiangyang; Westpheling, Janet

1997-01-01

The chi63 promoter directs glucose-sensitive, chitin-dependent transcription of a gene involved in the utilization of chitin as carbon source. Analysis of 5′ and 3′ deletions of the promoter region revealed that a 350-bp segment is sufficient for wild-type levels of expression and regulation. The analysis of single base changes throughout the promoter region, introduced by random and site-directed mutagenesis, identified several sequences to be important for activity and regulation. Single base changes at −10, −12, −32, −33, −35, and −37 upstream of the transcription start site resulted in loss of activity from the promoter, suggesting that bases in these positions are important for RNA polymerase interaction. The sequences centered around −10 (TATTCT) and −35 (TTGACC) in this promoter are, in fact, prototypical of eubacterial promoters. Overlapping the RNA polymerase binding site is a perfect 12-bp direct repeat sequence. Some base changes within this direct repeat resulted in constitutive expression, suggesting that this sequence is an operator for negative regulation. Other base changes resulted in loss of glucose repression while retaining the requirement for chitin induction, suggesting that this sequence is also involved in glucose repression. The fact that cis-acting mutations resulted in glucose resistance but not inducer independence rules out the possibility that glucose repression acts exclusively by inducer exclusion. The fact that mutations that affect glucose repression and chitin induction fall within the same direct repeat sequence module suggests that the direct repeat sequence facilitates both chitin induction and glucose repression. PMID:9371809

Exploring the sequence-structure protein landscape in the glycosyltransferase family

PubMed Central

Zhang, Ziding; Kochhar, Sunil; Grigorov, Martin

2003-01-01

To understand the molecular basis of glycosyltransferases’ (GTFs) catalytic mechanism, extensive structural information is required. Here, fold recognition methods were employed to assign 3D protein shapes (folds) to the currently known GTF sequences, available in public databases such as GenBank and Swissprot. First, GTF sequences were retrieved and classified into clusters, based on sequence similarity only. Intracluster sequence similarity was chosen sufficiently high to ensure that the same fold is found within a given cluster. Then, a representative sequence from each cluster was selected to compose a subset of GTF sequences. The members of this reduced set were processed by three different fold recognition methods: 3D-PSSM, FUGUE, and GeneFold. Finally, the results from different fold recognition methods were analyzed and compared to sequence-similarity search methods (i.e., BLAST and PSI-BLAST). It was established that the folds of about 70% of all currently known GTF sequences can be confidently assigned by fold recognition methods, a value which is higher than the fold identification rate based on sequence comparison alone (48% for BLAST and 64% for PSI-BLAST). The identified folds were submitted to 3D clustering, and we found that most of the GTF sequences adopt the typical GTF A or GTF B folds. Our results indicate a lack of evidence that new GTF folds (i.e., folds other than GTF A and B) exist. Based on cases where fold identification was not possible, we suggest several sequences as the most promising targets for a structural genomics initiative focused on the GTF protein family. PMID:14500887

Supporting Teachers' Use of Research-Based Instructional Sequences

ERIC Educational Resources Information Center

Cobb, Paul; Jackson, Kara

2015-01-01

In this paper, we frame the dissemination of the products of classroom design studies as a process of supporting the learning of large numbers of teachers. We argue that high-quality pull-out professional development is essential but not sufficient, and go on to consider teacher collaboration and one-on-one coaching in the classroom as additional…

Fast selection of miRNA candidates based on large-scale pre-computed MFE sets of randomized sequences

PubMed Central

2014-01-01

Background Small RNAs are important regulators of genome function, yet their prediction in genomes is still a major computational challenge. Statistical analyses of pre-miRNA sequences indicated that their 2D structure tends to have a minimal free energy (MFE) significantly lower than MFE values of equivalently randomized sequences with the same nucleotide composition, in contrast to other classes of non-coding RNA. The computation of many MFEs is, however, too intensive to allow for genome-wide screenings. Results Using a local grid infrastructure, MFE distributions of random sequences were pre-calculated on a large scale. These distributions follow a normal distribution and can be used to determine the MFE distribution for any given sequence composition by interpolation. It allows on-the-fly calculation of the normal distribution for any candidate sequence composition. Conclusion The speedup achieved makes genome-wide screening with this characteristic of a pre-miRNA sequence practical. Although this particular property alone will not be able to distinguish miRNAs from other sequences sufficiently discriminative, the MFE-based P-value should be added to the parameters of choice to be included in the selection of potential miRNA candidates for experimental verification. PMID:24418292

Phylogenetic Placement of Exact Amplicon Sequences Improves Associations with Clinical Information

PubMed Central

McDonald, Daniel; Gonzalez, Antonio; Navas-Molina, Jose A.; Jiang, Lingjing; Xu, Zhenjiang Zech; Winker, Kevin; Kado, Deborah M.; Orwoll, Eric; Manary, Mark; Mirarab, Siavash

2018-01-01

ABSTRACT Recent algorithmic advances in amplicon-based microbiome studies enable the inference of exact amplicon sequence fragments. These new methods enable the investigation of sub-operational taxonomic units (sOTU) by removing erroneous sequences. However, short (e.g., 150-nucleotide [nt]) DNA sequence fragments do not contain sufficient phylogenetic signal to reproduce a reasonable tree, introducing a barrier in the utilization of critical phylogenetically aware metrics such as Faith’s PD or UniFrac. Although fragment insertion methods do exist, those methods have not been tested for sOTUs from high-throughput amplicon studies in insertions against a broad reference phylogeny. We benchmarked the SATé-enabled phylogenetic placement (SEPP) technique explicitly against 16S V4 sequence fragments and showed that it outperforms the conceptually problematic but often-used practice of reconstructing de novo phylogenies. In addition, we provide a BSD-licensed QIIME2 plugin (https://github.com/biocore/q2-fragment-insertion) for SEPP and integration into the microbial study management platform QIITA. IMPORTANCE The move from OTU-based to sOTU-based analysis, while providing additional resolution, also introduces computational challenges. We demonstrate that one popular method of dealing with sOTUs (building a de novo tree from the short sequences) can provide incorrect results in human gut metagenomic studies and show that phylogenetic placement of the new sequences with SEPP resolves this problem while also yielding other benefits over existing methods. PMID:29719869

The hormone response element mimic sequence of GAS5 lncRNA is sufficient to induce apoptosis in breast cancer cells

PubMed Central

Pickard, Mark R.; Williams, Gwyn T.

2016-01-01

Growth arrest-specific 5 (GAS5) lncRNA promotes apoptosis, and its expression is down-regulated in breast cancer. GAS5 lncRNA is a decoy of glucocorticoid/related receptors; a stem-loop sequence constitutes the GAS5 hormone response element mimic (HREM), which is essential for the regulation of breast cancer cell apoptosis. This preclinical study aimed to determine if the GAS5 HREM sequence alone promotes the apoptosis of breast cancer cells. Nucleofection of hormone-sensitive and –insensitive breast cancer cell lines with a GAS5 HREM DNA oligonucleotide increased both basal and ultraviolet-C-induced apoptosis, and decreased culture viability and clonogenic growth, similar to GAS5 lncRNA. The HREM oligonucleotide demonstrated similar sequence specificity to the native HREM for its functional activity and had no effect on endogenous GAS5 lncRNA levels. Certain chemically modified HREM oligonucleotides, notably DNA and RNA phosphorothioates, retained pro-apoptotic. activity. Crucially the HREM oligonucleotide could overcome apoptosis resistance secondary to deficient endogenous GAS5 lncRNA levels. Thus, the GAS5 lncRNA HREM sequence alone is sufficient to induce apoptosis in breast cancer cells, including triple-negative breast cancer cells. These findings further suggest that emerging knowledge of structure/function relationships in the field of lncRNA biology can be exploited for the development of entirely novel, oligonucleotide mimic-based, cancer therapies. PMID:26862727

Influence of gag and RRE Sequences on HIV-1 RNA Packaging Signal Structure and Function.

PubMed

Kharytonchyk, Siarhei; Brown, Joshua D; Stilger, Krista; Yasin, Saif; Iyer, Aishwarya S; Collins, John; Summers, Michael F; Telesnitsky, Alice

2018-07-06

The packaging signal (Ψ) and Rev-responsive element (RRE) enable unspliced HIV-1 RNAs' export from the nucleus and packaging into virions. For some retroviruses, engrafting Ψ onto a heterologous RNA is sufficient to direct encapsidation. In contrast, HIV-1 RNA packaging requires 5' leader Ψ elements plus poorly defined additional features. We previously defined minimal 5' leader sequences competitive with intact Ψ for HIV-1 packaging, and here examined the potential roles of additional downstream elements. The findings confirmed that together, HIV-1 5' leader Ψ sequences plus a nuclear export element are sufficient to specify packaging. However, RNAs trafficked using a heterologous export element did not compete well with RNAs using HIV-1's RRE. Furthermore, some RNA additions to well-packaged minimal vectors rendered them packaging-defective. These defects were rescued by extending gag sequences in their native context. To understand these packaging defects' causes, in vitro dimerization properties of RNAs containing minimal packaging elements were compared to RNAs with sequence extensions that were or were not compatible with packaging. In vitro dimerization was found to correlate with packaging phenotypes, suggesting that HIV-1 evolved to prevent 5' leader residues' base pairing with downstream residues and misfolding of the packaging signal. Our findings explain why gag sequences have been implicated in packaging and show that RRE's packaging contributions appear more specific than nuclear export alone. Paired with recent work showing that sequences upstream of Ψ can dictate RNA folds, the current work explains how genetic context of minimal packaging elements contributes to HIV-1 RNA fate determination. Copyright © 2018 Elsevier Ltd. All rights reserved.

Monitoring Arthrobacter protophormiae RKJ100 in a 'tag and chase' method during p-nitrophenol bio-remediation in soil microcosms.

PubMed

Pandey, Gunjan; Pandey, Janmejay; Jain, Rakesh K

2006-05-01

Monitoring of micro-organisms released deliberately into the environment is essential to assess their movement during the bio-remediation process. During the last few years, DNA-based genetic methods have emerged as the preferred method for such monitoring; however, their use is restricted in cases where organisms used for bio-remediation are not well characterized or where the public domain databases do not provide sufficient information regarding their sequence. For monitoring of such micro-organisms, alternate approaches have to be undertaken. In this study, we have specifically monitored a p-nitrophenol (PNP)-degrading organism, Arthrobacter protophormiae RKJ100, using molecular methods during PNP degradation in soil microcosm. Cells were tagged with a transposon-based foreign DNA sequence prior to their introduction into PNP-contaminated microcosms. Later, this artificially introduced DNA sequence was PCR-amplified to distinguish the bio-augmented organism from the indigenous microflora during PNP bio-remediation.

Distance-dependent duplex DNA destabilization proximal to G-quadruplex/i-motif sequences

PubMed Central

König, Sebastian L. B.; Huppert, Julian L.; Sigel, Roland K. O.; Evans, Amanda C.

2013-01-01

G-quadruplexes and i-motifs are complementary examples of non-canonical nucleic acid substructure conformations. G-quadruplex thermodynamic stability has been extensively studied for a variety of base sequences, but the degree of duplex destabilization that adjacent quadruplex structure formation can cause has yet to be fully addressed. Stable in vivo formation of these alternative nucleic acid structures is likely to be highly dependent on whether sufficient spacing exists between neighbouring duplex- and quadruplex-/i-motif-forming regions to accommodate quadruplexes or i-motifs without disrupting duplex stability. Prediction of putative G-quadruplex-forming regions is likely to be assisted by further understanding of what distance (number of base pairs) is required for duplexes to remain stable as quadruplexes or i-motifs form. Using oligonucleotide constructs derived from precedented G-quadruplexes and i-motif-forming bcl-2 P1 promoter region, initial biophysical stability studies indicate that the formation of G-quadruplex and i-motif conformations do destabilize proximal duplex regions. The undermining effect that quadruplex formation can have on duplex stability is mitigated with increased distance from the duplex region: a spacing of five base pairs or more is sufficient to maintain duplex stability proximal to predicted quadruplex/i-motif-forming regions. PMID:23771141

The fuzzy polynucleotide space: basic properties.

PubMed

Torres, Angela; Nieto, Juan J

2003-03-22

Any triplet codon may be regarded as a 12-dimensional fuzzy code. Sufficient information about a particular sequence may not be available in certain situations. The investigator will be confronted with imprecise sequences, yet want to make comparisons of sequences. Fuzzy polynucleotides can be compared by using geometrical interpretation of fuzzy sets as points in a hypercube. We introduce the space of fuzzy polynucleotides and a means of measuring dissimilitudes between them. We establish mathematical principles to measure dissimilarities between fuzzy polynucleotides and present several examples in this metric space. We calculate the frequencies of the nucleotides at the three base sites of a codon in the coding sequences of Escherichia coli K-12 and Mycobacterium tuberculosis H37Rv, and consider them as points in that fuzzy space. We compute the distance between the genomes of E.coli and M.tuberculosis.

Simultaneous electroencephalography-functional MRI at 3 T: an analysis of safety risks imposed by performing anatomical reference scans with the EEG equipment in place.

PubMed

Nöth, Ulrike; Laufs, Helmut; Stoermer, Robert; Deichmann, Ralf

2012-03-01

To describe heating effects to be expected in simultaneous electroencephalography (EEG) and magnetic resonance imaging (MRI) when deviating from the EEG manufacturer's instructions; to test which anatomical MRI sequences have a sufficiently low specific absorption rate (SAR) to be performed with the EEG equipment in place; and to suggest precautions to reduce the risk of heating. Heating was determined in vivo below eight EEG electrodes, using both head and body coil transmission and sequences covering the whole range of SAR values. Head transmit coil: temperature increases were below 2.2°C for low SAR sequences, but reached 4.6°C (one subject, clavicle) for high SAR sequences; the equilibrium temperature T(eq) remained below 39°C. Body transmit coil: temperature increases were higher and more frequent over subjects and electrodes, with values below 2.6°C for low SAR sequences, reaching 6.9°C for high SAR sequences (T8 electrode) with T(eq) exceeding a critical level of 40°C. Anatomical imaging should be based on T1-weighted sequences (FLASH, MPRAGE, MDEFT) with an SAR below values for functional MRI sequences based on gradient echo planar imaging. Anatomical sequences with a high SAR can pose a significant risk, which is reduced by using head coil transmission. Copyright © 2011 Wiley-Liss, Inc.

Nucleic and Amino Acid Sequences Support Structure-Based Viral Classification.

PubMed

Sinclair, Robert M; Ravantti, Janne J; Bamford, Dennis H

2017-04-15

Viral capsids ensure viral genome integrity by protecting the enclosed nucleic acids. Interactions between the genome and capsid and between individual capsid proteins (i.e., capsid architecture) are intimate and are expected to be characterized by strong evolutionary conservation. For this reason, a capsid structure-based viral classification has been proposed as a way to bring order to the viral universe. The seeming lack of sufficient sequence similarity to reproduce this classification has made it difficult to reject structural convergence as the basis for the classification. We reinvestigate whether the structure-based classification for viral coat proteins making icosahedral virus capsids is in fact supported by previously undetected sequence similarity. Since codon choices can influence nascent protein folding cotranslationally, we searched for both amino acid and nucleotide sequence similarity. To demonstrate the sensitivity of the approach, we identify a candidate gene for the pandoravirus capsid protein. We show that the structure-based classification is strongly supported by amino acid and also nucleotide sequence similarities, suggesting that the similarities are due to common descent. The correspondence between structure-based and sequence-based analyses of the same proteins shown here allow them to be used in future analyses of the relationship between linear sequence information and macromolecular function, as well as between linear sequence and protein folds. IMPORTANCE Viral capsids protect nucleic acid genomes, which in turn encode capsid proteins. This tight coupling of protein shell and nucleic acids, together with strong functional constraints on capsid protein folding and architecture, leads to the hypothesis that capsid protein-coding nucleotide sequences may retain signatures of ancient viral evolution. We have been able to show that this is indeed the case, using the major capsid proteins of viruses forming icosahedral capsids. Importantly, we detected similarity at the nucleotide level between capsid protein-coding regions from viruses infecting cells belonging to all three domains of life, reproducing a previously established structure-based classification of icosahedral viral capsids. Copyright © 2017 Sinclair et al.

Nucleic and Amino Acid Sequences Support Structure-Based Viral Classification

PubMed Central

Sinclair, Robert M.; Ravantti, Janne J.

2017-01-01

ABSTRACT Viral capsids ensure viral genome integrity by protecting the enclosed nucleic acids. Interactions between the genome and capsid and between individual capsid proteins (i.e., capsid architecture) are intimate and are expected to be characterized by strong evolutionary conservation. For this reason, a capsid structure-based viral classification has been proposed as a way to bring order to the viral universe. The seeming lack of sufficient sequence similarity to reproduce this classification has made it difficult to reject structural convergence as the basis for the classification. We reinvestigate whether the structure-based classification for viral coat proteins making icosahedral virus capsids is in fact supported by previously undetected sequence similarity. Since codon choices can influence nascent protein folding cotranslationally, we searched for both amino acid and nucleotide sequence similarity. To demonstrate the sensitivity of the approach, we identify a candidate gene for the pandoravirus capsid protein. We show that the structure-based classification is strongly supported by amino acid and also nucleotide sequence similarities, suggesting that the similarities are due to common descent. The correspondence between structure-based and sequence-based analyses of the same proteins shown here allow them to be used in future analyses of the relationship between linear sequence information and macromolecular function, as well as between linear sequence and protein folds. IMPORTANCE Viral capsids protect nucleic acid genomes, which in turn encode capsid proteins. This tight coupling of protein shell and nucleic acids, together with strong functional constraints on capsid protein folding and architecture, leads to the hypothesis that capsid protein-coding nucleotide sequences may retain signatures of ancient viral evolution. We have been able to show that this is indeed the case, using the major capsid proteins of viruses forming icosahedral capsids. Importantly, we detected similarity at the nucleotide level between capsid protein-coding regions from viruses infecting cells belonging to all three domains of life, reproducing a previously established structure-based classification of icosahedral viral capsids. PMID:28122979

Tempo and mode of genomic mutations unveil human evolutionary history.

PubMed

Hara, Yuichiro

2015-01-01

Mutations that have occurred in human genomes provide insight into various aspects of evolutionary history such as speciation events and degrees of natural selection. Comparing genome sequences between human and great apes or among humans is a feasible approach for inferring human evolutionary history. Recent advances in high-throughput or so-called 'next-generation' DNA sequencing technologies have enabled the sequencing of thousands of individual human genomes, as well as a variety of reference genomes of hominids, many of which are publicly available. These sequence data can help to unveil the detailed demographic history of the lineage leading to humans as well as the explosion of modern human population size in the last several thousand years. In addition, high-throughput sequencing illustrates the tempo and mode of de novo mutations, which are producing human genetic variation at this moment. Pedigree-based human genome sequencing has shown that mutation rates vary significantly across the human genome. These studies have also provided an improved timescale of human evolution, because the mutation rate estimated from pedigree analysis is half that estimated from traditional analyses based on molecular phylogeny. Because of the dramatic reduction in sequencing cost, sequencing on-demand samples designed for specific studies is now also becoming popular. To produce data of sufficient quality to meet the requirements of the study, it is necessary to set an explicit sequencing plan that includes the choice of sample collection methods, sequencing platforms, and number of sequence reads.

How close is close: 16S rRNA sequence identity may not be sufficient to guarantee species identity

NASA Technical Reports Server (NTRS)

Fox, G. E.; Wisotzkey, J. D.; Jurtshuk, P. Jr

1992-01-01

16S rRNA (genes coding for rRNA) sequence comparisons were conducted with the following three psychrophilic strains: Bacillus globisporus W25T (T = type strain) and Bacillus psychrophilus W16AT, and W5. These strains exhibited more than 99.5% sequence identity and within experimental uncertainty could be regarded as identical. Their close taxonomic relationship was further documented by phenotypic similarities. In contrast, previously published DNA-DNA hybridization results have convincingly established that these strains do not belong to the same species if current standards are used. These results emphasize the important point that effective identity of 16S rRNA sequences is not necessarily a sufficient criterion to guarantee species identity. Thus, although 16S rRNA sequences can be used routinely to distinguish and establish relationships between genera and well-resolved species, very recently diverged species may not be recognizable.

Limitations and potentials of current motif discovery algorithms

PubMed Central

Hu, Jianjun; Li, Bin; Kihara, Daisuke

2005-01-01

Computational methods for de novo identification of gene regulation elements, such as transcription factor binding sites, have proved to be useful for deciphering genetic regulatory networks. However, despite the availability of a large number of algorithms, their strengths and weaknesses are not sufficiently understood. Here, we designed a comprehensive set of performance measures and benchmarked five modern sequence-based motif discovery algorithms using large datasets generated from Escherichia coli RegulonDB. Factors that affect the prediction accuracy, scalability and reliability are characterized. It is revealed that the nucleotide and the binding site level accuracy are very low, while the motif level accuracy is relatively high, which indicates that the algorithms can usually capture at least one correct motif in an input sequence. To exploit diverse predictions from multiple runs of one or more algorithms, a consensus ensemble algorithm has been developed, which achieved 6–45% improvement over the base algorithms by increasing both the sensitivity and specificity. Our study illustrates limitations and potentials of existing sequence-based motif discovery algorithms. Taking advantage of the revealed potentials, several promising directions for further improvements are discussed. Since the sequence-based algorithms are the baseline of most of the modern motif discovery algorithms, this paper suggests substantial improvements would be possible for them. PMID:16284194

Protein Structure Determination using Metagenome sequence data

PubMed Central

Ovchinnikov, Sergey; Park, Hahnbeom; Varghese, Neha; Huang, Po-Ssu; Pavlopoulos, Georgios A.; Kim, David E.; Kamisetty, Hetunandan; Kyrpides, Nikos C.; Baker, David

2017-01-01

Despite decades of work by structural biologists, there are still ~5200 protein families with unknown structure outside the range of comparative modeling. We show that Rosetta structure prediction guided by residue-residue contacts inferred from evolutionary information can accurately model proteins that belong to large families, and that metagenome sequence data more than triples the number of protein families with sufficient sequences for accurate modeling. We then integrate metagenome data, contact based structure matching and Rosetta structure calculations to generate models for 614 protein families with currently unknown structures; 206 are membrane proteins and 137 have folds not represented in the PDB. This approach provides the representative models for large protein families originally envisioned as the goal of the protein structure initiative at a fraction of the cost. PMID:28104891

Linear reduction method for predictive and informative tag SNP selection.

PubMed

He, Jingwu; Westbrooks, Kelly; Zelikovsky, Alexander

2005-01-01

Constructing a complete human haplotype map is helpful when associating complex diseases with their related SNPs. Unfortunately, the number of SNPs is very large and it is costly to sequence many individuals. Therefore, it is desirable to reduce the number of SNPs that should be sequenced to a small number of informative representatives called tag SNPs. In this paper, we propose a new linear algebra-based method for selecting and using tag SNPs. We measure the quality of our tag SNP selection algorithm by comparing actual SNPs with SNPs predicted from selected linearly independent tag SNPs. Our experiments show that for sufficiently long haplotypes, knowing only 0.4% of all SNPs the proposed linear reduction method predicts an unknown haplotype with the error rate below 2% based on 10% of the population.

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

PubMed

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.

Mumps virus F gene and HN gene sequencing as a molecular tool to study mumps virus transmission.

PubMed

Gouma, Sigrid; Cremer, Jeroen; Parkkali, Saara; Veldhuijzen, Irene; van Binnendijk, Rob S; Koopmans, Marion P G

2016-11-01

Various mumps outbreaks have occurred in the Netherlands since 2004, particularly among persons who had received 2 doses of measles, mumps, and rubella (MMR) vaccination. Genomic typing of pathogens can be used to track outbreaks, but the established genotyping of mumps virus based on the small hydrophobic (SH) gene sequences did not provide sufficient resolution. Therefore, we expanded the sequencing to include fusion (F) gene and haemagglutinin-neuraminidase (HN) gene sequences in addition to the SH gene sequences from 109 mumps virus genotype G strains obtained between 2004 and mid 2015 in the Netherlands. When the molecular information from these 3 genes was combined, we were able to identify separate mumps virus clusters and track mumps virus transmission. The analyses suggested that multiple mumps virus introductions occurred in the Netherlands between 2004 and 2015 resulting in several mumps outbreaks throughout this period, whereas during some local outbreaks the molecular data pointed towards endemic circulation. Combined analysis of epidemiological data and sequence data collected in 2015 showed good support for the phylogenetic clustering. Copyright © 2016 Elsevier B.V. All rights reserved.

Draft versus finished sequence data for DNA and protein diagnostic signature development

PubMed Central

Gardner, Shea N.; Lam, Marisa W.; Smith, Jason R.; Torres, Clinton L.; Slezak, Tom R.

2005-01-01

Sequencing pathogen genomes is costly, demanding careful allocation of limited sequencing resources. We built a computational Sequencing Analysis Pipeline (SAP) to guide decisions regarding the amount of genomic sequencing necessary to develop high-quality diagnostic DNA and protein signatures. SAP uses simulations to estimate the number of target genomes and close phylogenetic relatives (near neighbors or NNs) to sequence. We use SAP to assess whether draft data are sufficient or finished sequencing is required using Marburg and variola virus sequences. Simulations indicate that intermediate to high-quality draft with error rates of 10−3–10−5 (∼8× coverage) of target organisms is suitable for DNA signature prediction. Low-quality draft with error rates of ∼1% (3× to 6× coverage) of target isolates is inadequate for DNA signature prediction, although low-quality draft of NNs is sufficient, as long as the target genomes are of high quality. For protein signature prediction, sequencing errors in target genomes substantially reduce the detection of amino acid sequence conservation, even if the draft is of high quality. In summary, high-quality draft of target and low-quality draft of NNs appears to be a cost-effective investment for DNA signature prediction, but may lead to underestimation of predicted protein signatures. PMID:16243783

Multiplexed microsatellite recovery using massively parallel sequencing

USGS Publications Warehouse

Jennings, T.N.; Knaus, B.J.; Mullins, T.D.; Haig, S.M.; Cronn, R.C.

2011-01-01

Conservation and management of natural populations requires accurate and inexpensive genotyping methods. Traditional microsatellite, or simple sequence repeat (SSR), marker analysis remains a popular genotyping method because of the comparatively low cost of marker development, ease of analysis and high power of genotype discrimination. With the availability of massively parallel sequencing (MPS), it is now possible to sequence microsatellite-enriched genomic libraries in multiplex pools. To test this approach, we prepared seven microsatellite-enriched, barcoded genomic libraries from diverse taxa (two conifer trees, five birds) and sequenced these on one lane of the Illumina Genome Analyzer using paired-end 80-bp reads. In this experiment, we screened 6.1 million sequences and identified 356958 unique microreads that contained di- or trinucleotide microsatellites. Examination of four species shows that our conversion rate from raw sequences to polymorphic markers compares favourably to Sanger- and 454-based methods. The advantage of multiplexed MPS is that the staggering capacity of modern microread sequencing is spread across many libraries; this reduces sample preparation and sequencing costs to less than $400 (USD) per species. This price is sufficiently low that microsatellite libraries could be prepared and sequenced for all 1373 organisms listed as 'threatened' and 'endangered' in the United States for under $0.5M (USD).

Rapid isolation of microsatellite DNAs and identification of polymorphic mitochondrial DNA regions in the fish rotan (Perccottus glenii) invading European Russia

USGS Publications Warehouse

King, Timothy L.; Eackles, Michael S.; Reshetnikov, Andrey N.

2015-01-01

Human-mediated translocations and subsequent large-scale colonization by the invasive fish rotan (Perccottus glenii Dybowski, 1877; Perciformes, Odontobutidae), also known as Amur or Chinese sleeper, has resulted in dramatic transformations of small lentic ecosystems. However, no detailed genetic information exists on population structure, levels of effective movement, or relatedness among geographic populations of P. glenii within the European part of the range. We used massively parallel genomic DNA shotgun sequencing on the semiconductor-based Ion Torrent Personal Genome Machine (PGM) sequencing platform to identify nuclear microsatellite and mitochondrial DNA sequences in P. glenii from European Russia. Here we describe the characterization of nine nuclear microsatellite loci, ascertain levels of allelic diversity, heterozygosity, and demographic status of P. glenii collected from Ilev, Russia, one of several initial introduction points in European Russia. In addition, we mapped sequence reads to the complete P. glenii mitochondrial DNA sequence to identify polymorphic regions. Nuclear microsatellite markers developed for P. glenii yielded sufficient genetic diversity to: (1) produce unique multilocus genotypes; (2) elucidate structure among geographic populations; and (3) provide unique perspectives for analysis of population sizes and historical demographics. Among 4.9 million filtered P. glenii Ion Torrent PGM sequence reads, 11,304 mapped to the mitochondrial genome (NC_020350). This resulted in 100 % coverage of this genome to a mean coverage depth of 102X. A total of 130 variable sites were observed between the publicly available genome from China and the studied composite mitochondrial genome. Among these, 82 were diagnostic and monomorphic between the mitochondrial genomes and distributed among 15 genome regions. The polymorphic sites (N = 48) were distributed among 11 mitochondrial genome regions. Our results also indicate that sequence reads generated from two three-hour runs on the Ion Torrent PGM can generate a sufficient number of nuclear and mitochondrial markers to improve understanding of the evolutionary and ecological dynamics of non-model and in particular, invasive species.

OSG-GEM: Gene Expression Matrix Construction Using the Open Science Grid.

PubMed

Poehlman, William L; Rynge, Mats; Branton, Chris; Balamurugan, D; Feltus, Frank A

2016-01-01

High-throughput DNA sequencing technology has revolutionized the study of gene expression while introducing significant computational challenges for biologists. These computational challenges include access to sufficient computer hardware and functional data processing workflows. Both these challenges are addressed with our scalable, open-source Pegasus workflow for processing high-throughput DNA sequence datasets into a gene expression matrix (GEM) using computational resources available to U.S.-based researchers on the Open Science Grid (OSG). We describe the usage of the workflow (OSG-GEM), discuss workflow design, inspect performance data, and assess accuracy in mapping paired-end sequencing reads to a reference genome. A target OSG-GEM user is proficient with the Linux command line and possesses basic bioinformatics experience. The user may run this workflow directly on the OSG or adapt it to novel computing environments.

OSG-GEM: Gene Expression Matrix Construction Using the Open Science Grid

PubMed Central

Poehlman, William L.; Rynge, Mats; Branton, Chris; Balamurugan, D.; Feltus, Frank A.

2016-01-01

High-throughput DNA sequencing technology has revolutionized the study of gene expression while introducing significant computational challenges for biologists. These computational challenges include access to sufficient computer hardware and functional data processing workflows. Both these challenges are addressed with our scalable, open-source Pegasus workflow for processing high-throughput DNA sequence datasets into a gene expression matrix (GEM) using computational resources available to U.S.-based researchers on the Open Science Grid (OSG). We describe the usage of the workflow (OSG-GEM), discuss workflow design, inspect performance data, and assess accuracy in mapping paired-end sequencing reads to a reference genome. A target OSG-GEM user is proficient with the Linux command line and possesses basic bioinformatics experience. The user may run this workflow directly on the OSG or adapt it to novel computing environments. PMID:27499617

Recombinant pinoresinol/lariciresinol reductase, recombinant dirigent protein, and methods of use

DOEpatents

Lewis, Norman G.; Davin, Laurence B.; Dinkova-Kostova, Albena T.; Fujita, Masayuki; Gang, David R.; Sarkanen, Simo; Ford, Joshua D.

2001-04-03

Dirigent proteins and pinoresinol/lariciresinol reductases have been isolated, together with cDNAs encoding dirigent proteins and pinoresinol/lariciresinol reductases. Accordingly, isolated DNA sequences are provided which code for the expression of dirigent proteins and pinoresinol/lariciresinol reductases. In other aspects, replicable recombinant cloning vehicles are provided which code for dirigent proteins or pinoresinol/lariciresinol reductases or for a base sequence sufficiently complementary to at least a portion of dirigent protein or pinoresinol/lariciresinol reductase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding dirigent protein or pinoresinol/lariciresinol reductase. Thus, systems and methods are provided for the recombinant expression of dirigent proteins and/or pinoresinol/lariciresinol reductases.

A novel alignment-free method for detection of lateral genetic transfer based on TF-IDF.

PubMed

Cong, Yingnan; Chan, Yao-Ban; Ragan, Mark A

2016-07-25

Lateral genetic transfer (LGT) plays an important role in the evolution of microbes. Existing computational methods for detecting genomic regions of putative lateral origin scale poorly to large data. Here, we propose a novel method based on TF-IDF (Term Frequency-Inverse Document Frequency) statistics to detect not only regions of lateral origin, but also their origin and direction of transfer, in sets of hierarchically structured nucleotide or protein sequences. This approach is based on the frequency distributions of k-mers in the sequences. If a set of contiguous k-mers appears sufficiently more frequently in another phyletic group than in its own, we infer that they have been transferred from the first group to the second. We performed rigorous tests of TF-IDF using simulated and empirical datasets. With the simulated data, we tested our method under different parameter settings for sequence length, substitution rate between and within groups and post-LGT, deletion rate, length of transferred region and k size, and found that we can detect LGT events with high precision and recall. Our method performs better than an established method, ALFY, which has high recall but low precision. Our method is efficient, with runtime increasing approximately linearly with sequence length.

The tolerance to exchanges of the Watson–Crick base pair in the hammerhead ribozyme core is determined by surrounding elements

PubMed Central

Przybilski, Rita; Hammann, Christian

2007-01-01

Tertiary interacting elements are important features of functional RNA molecules, for example, in all small nucleolytic ribozymes. The recent crystal structure of a tertiary stabilized type I hammerhead ribozyme revealed a conventional Watson–Crick base pair in the catalytic core, formed between nucleotides C3 and G8. We show that any Watson–Crick base pair between these positions retains cleavage competence in two type III ribozymes. In the Arabidopsis thaliana sequence, only moderate differences in cleavage rates are observed for the different base pairs, while the peach latent mosaic viroid (PLMVd) ribozyme exhibits a preference for a pyrimidine at position 3 and a purine at position 8. To understand these differences, we created a series of chimeric ribozymes in which we swapped sequence elements that surround the catalytic core. The kinetic characterization of the resulting ribozymes revealed that the tertiary interacting loop sequences of the PLMVd ribozyme are sufficient to induce the preference for Y3–R8 base pairs in the A. thaliana hammerhead ribozyme. In contrast to this, only when the entire stem–loops I and II of the A. thaliana sequences are grafted on the PLMVd ribozyme is any Watson–Crick base pair similarly tolerated. The data provide evidence for a complex interplay of secondary and tertiary structure elements that lead, mediated by long-range effects, to an individual modulation of the local structure in the catalytic core of different hammerhead ribozymes. PMID:17666711

Molecular characterization of Calocedrus rupestris Aver., H.T. Nguyen & L.K. Phan, 2008 (Cupressaceae) based on ITS1 partial sequence.

PubMed

Long, P K; Trang, N T P; Averyanov, L V; Loc, P K

2011-11-21

Calocedrus rupestris Aver., H.T. Nguyen & L.K. Phan was described in 2008 based on some morphological characters that were not sufficiently significant to discriminate it as a species distinct from C. macrolepis Kurz. We applied a new approach to resolve these conflicting views by using sequence data from DNA (ITS) to elucidate phylogenetic relationships between the two species. Analyses of a partial ITS1 sequence in 5 individuals of 2 subpopulations of C. macrolepis and 18 individuals of 8 subpopulations of C. rupestris collected in Vietnam were done. Molecular characterization of the two species showed its low divergence with the lack of autapomorphic characters. In addition, the ITS1 partial sequences of some C. rupestris individuals were identical with C. macrolepis. Due to the less distinctive morphology between C. rupestris and C. macrolepis, the divergence between them does not exceed the interspecific levels, and therefore, C. rupestris could not be regarded as an independent species in relation to C. macrolepis but only as one of its varieties, C. macrolepis var. rupestris (Aver., H.T. Nguyen & L.K. Phan) L.K. Phan, Long K. Phan & Aver.

A method for release and multiple strand amplification of small quantities of DNA from endospores of the fastidious bacterium Pasteuria penetrans.

PubMed

Mauchline, T H; Mohan, S; Davies, K G; Schaff, J E; Opperman, C H; Kerry, B R; Hirsch, P R

2010-05-01

To establish a reliable protocol to extract DNA from Pasteuria penetrans endospores for use as template in multiple strand amplification, thus providing sufficient material for genetic analyses. To develop a highly sensitive PCR-based diagnostic tool for P. penetrans. An optimized method to decontaminate endospores, release and purify DNA enabled multiple strand amplification. DNA purity was assessed by cloning and sequencing gyrB and 16S rRNA gene fragments obtained from PCR using generic primers. Samples indicated to be 100%P. penetrans by the gyrB assay were estimated at 46% using the 16S rRNA gene. No bias was detected on cloning and sequencing 12 housekeeping and sporulation gene fragments from amplified DNA. The detection limit by PCR with Pasteuria-specific 16S rRNA gene primers following multiple strand amplification of DNA extracted using the method was a single endospore. Generation of large quantities DNA will facilitate genomic sequencing of P. penetrans. Apparent differences in sample purity are explained by variations in 16S rRNA gene copy number in Eubacteria leading to exaggerated estimations of sample contamination. Detection of single endospores will facilitate investigations of P. penetrans molecular ecology. These methods will advance studies on P. penetrans and facilitate research on other obligate and fastidious micro-organisms where it is currently impractical to obtain DNA in sufficient quantity and quality.

Precise assignment of the heavy-strand promoter of mouse mitochondrial DNA: cognate start sites are not required for transcriptional initiation.

PubMed Central

Chang, D D; Clayton, D A

1986-01-01

Transcription of the heavy strand of mouse mitochondrial DNA starts from two closely spaced, distinct sites located in the displacement loop region of the genome. We report here an analysis of regulatory sequences required for faithful transcription from these two sites. Data obtained from in vitro assays demonstrated that a 51-base-pair region, encompassing nucleotides -40 to +11 of the downstream start site, contains sufficient information for accurate transcription from both start sites. Deletion of the 3' flanking sequences, including one or both start sites to -17, resulted in the initiation of transcription by the mitochondrial RNA polymerase from alternative sites within vector DNA sequences. This feature places the mouse heavy-strand promoter uniquely among other known mitochondrial promoters, all of which absolutely require cognate start sites for transcription. Comparison of the heavy-strand promoter with those of other vertebrate mitochondrial DNAs revealed a remarkably high rate of sequence divergence among species. Images PMID:3785226

AFLP fragment isolation technique as a method to produce random sequences for single nucleotide polymorphism discovery in the green turtle, Chelonia mydas.

PubMed

Roden, Suzanne E; Dutton, Peter H; Morin, Phillip A

2009-01-01

The green sea turtle, Chelonia mydas, was used as a case study for single nucleotide polymorphism (SNP) discovery in a species that has little genetic sequence information available. As green turtles have a complex population structure, additional nuclear markers other than microsatellites could add to our understanding of their complex life history. Amplified fragment length polymorphism technique was used to generate sets of random fragments of genomic DNA, which were then electrophoretically separated with precast gels, stained with SYBR green, excised, and directly sequenced. It was possible to perform this method without the use of polyacrylamide gels, radioactive or fluorescent labeled primers, or hybridization methods, reducing the time, expense, and safety hazards of SNP discovery. Within 13 loci, 2547 base pairs were screened, resulting in the discovery of 35 SNPs. Using this method, it was possible to yield a sufficient number of loci to screen for SNP markers without the availability of prior sequence information.

cWINNOWER algorithm for finding fuzzy dna motifs

NASA Technical Reports Server (NTRS)

Liang, S.; Samanta, M. P.; Biegel, B. A.

2004-01-01

The cWINNOWER algorithm detects fuzzy motifs in DNA sequences rich in protein-binding signals. A signal is defined as any short nucleotide pattern having up to d mutations differing from a motif of length l. The algorithm finds such motifs if a clique consisting of a sufficiently large number of mutated copies of the motif (i.e., the signals) is present in the DNA sequence. The cWINNOWER algorithm substantially improves the sensitivity of the winnower method of Pevzner and Sze by imposing a consensus constraint, enabling it to detect much weaker signals. We studied the minimum detectable clique size qc as a function of sequence length N for random sequences. We found that qc increases linearly with N for a fast version of the algorithm based on counting three-member sub-cliques. Imposing consensus constraints reduces qc by a factor of three in this case, which makes the algorithm dramatically more sensitive. Our most sensitive algorithm, which counts four-member sub-cliques, needs a minimum of only 13 signals to detect motifs in a sequence of length N = 12,000 for (l, d) = (15, 4). Copyright Imperial College Press.

Method for performing site-specific affinity fractionation for use in DNA sequencing

DOEpatents

Mirzabekov, Andrei Darievich; Lysov, Yuri Petrovich; Dubley, Svetlana A.

1999-01-01

A method for fractionating and sequencing DNA via affinity interaction is provided comprising contacting cleaved DNA to a first array of oligonucleotide molecules to facilitate hybridization between said cleaved DNA and the molecules; extracting the hybridized DNA from the molecules; contacting said extracted hybridized DNA with a second array of oligonucleotide molecules, wherein the oligonucleotide molecules in the second array have specified base sequences that are complementary to said extracted hybridized DNA; and attaching labeled DNA to the second array of oligonucleotide molecules, wherein the labeled re-hybridized DNA have sequences that are complementary to the oligomers. The invention further provides a method for performing multi-step conversions of the chemical structure of compounds comprising supplying an array of polyacrylamide vessels separated by hydrophobic surfaces; immobilizing a plurality of reactants, such as enzymes, in the vessels so that each vessel contains one reactant; contacting the compounds to each of the vessels in a predetermined sequence and for a sufficient time to convert the compounds to a desired state; and isolating the converted compounds from said array.

cWINNOWER Algorithm for Finding Fuzzy DNA Motifs

NASA Technical Reports Server (NTRS)

Liang, Shoudan

2003-01-01

The cWINNOWER algorithm detects fuzzy motifs in DNA sequences rich in protein-binding signals. A signal is defined as any short nucleotide pattern having up to d mutations differing from a motif of length l. The algorithm finds such motifs if multiple mutated copies of the motif (i.e., the signals) are present in the DNA sequence in sufficient abundance. The cWINNOWER algorithm substantially improves the sensitivity of the winnower method of Pevzner and Sze by imposing a consensus constraint, enabling it to detect much weaker signals. We studied the minimum number of detectable motifs qc as a function of sequence length N for random sequences. We found that qc increases linearly with N for a fast version of the algorithm based on counting three-member sub-cliques. Imposing consensus constraints reduces qc, by a factor of three in this case, which makes the algorithm dramatically more sensitive. Our most sensitive algorithm, which counts four-member sub-cliques, needs a minimum of only 13 signals to detect motifs in a sequence of length N = 12000 for (l,d) = (15,4).

Miniaturized reaction vessel system, method for performing site-specific biochemical reactions and affinity fractionation for use in DNA sequencing

DOEpatents

Mirzabekov, Andrei Darievich; Lysov, Yuri Petrovich; Dubley, Svetlana A.

2000-01-01

A method for fractionating and sequencing DNA via affinity interaction is provided comprising contacting cleaved DNA to a first array of oligonucleotide molecules to facilitate hybridization between said cleaved DNA and the molecules; extracting the hybridized DNA from the molecules; contacting said extracted hybridized DNA with a second array of oligonucleotide molecules, wherein the oligonucleotide molecules in the second array have specified base sequences that are complementary to said extracted hybridized DNA; and attaching labeled DNA to the second array of oligonucleotide molecules, wherein the labeled re-hybridized DNA have sequences that are complementary to the oligomers. The invention further provides a method for performing multi-step conversions of the chemical structure of compounds comprising supplying an array of polyacrylamide vessels separated by hydrophobic surfaces; immobilizing a plurality of reactants, such as enzymes, in the vessels so that each vessel contains one reactant; contacting the compounds to each of the vessels in a predetermined sequence and for a sufficient time to convert the compounds to a desired state; and isolating the converted compounds from said array.

Method for performing site-specific affinity fractionation for use in DNA sequencing

DOEpatents

Mirzabekov, A.D.; Lysov, Y.P.; Dubley, S.A.

1999-05-18

A method for fractionating and sequencing DNA via affinity interaction is provided comprising contacting cleaved DNA to a first array of oligonucleotide molecules to facilitate hybridization between the cleaved DNA and the molecules; extracting the hybridized DNA from the molecules; contacting the extracted hybridized DNA with a second array of oligonucleotide molecules, wherein the oligonucleotide molecules in the second array have specified base sequences that are complementary to the extracted hybridized DNA; and attaching labeled DNA to the second array of oligonucleotide molecules, wherein the labeled re-hybridized DNA have sequences that are complementary to the oligomers. The invention further provides a method for performing multi-step conversions of the chemical structure of compounds comprising supplying an array of polyacrylamide vessels separated by hydrophobic surfaces; immobilizing a plurality of reactants, such as enzymes, in the vessels so that each vessel contains one reactant; contacting the compounds to each of the vessels in a predetermined sequence and for a sufficient time to convert the compounds to a desired state; and isolating the converted compounds from the array. 14 figs.

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

PubMed Central

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

2015-01-01

For 150 years or more, specimens were routinely collected and deposited in natural history collections without preserving fresh tissue samples for genetic analysis. In the case of most herpetological specimens (i.e. amphibians and reptiles), attempts to extract and sequence DNA from formalin-fixed, ethanol-preserved specimens—particularly for use in phylogenetic analyses—has been laborious and largely ineffective due to the highly fragmented nature of the DNA. As a result, tens of thousands of specimens in herpetological collections have not been available for sequence-based phylogenetic studies. Massively parallel High-Throughput Sequencing methods and the associated bioinformatics, however, are particularly suited to recovering meaningful genetic markers from severely degraded/fragmented DNA sequences such as DNA damaged by formalin-fixation. In this study, we compared previously published DNA extraction methods on three tissue types subsampled from formalin-fixed specimens of Anolis carolinensis, followed by sequencing. Sufficient quality DNA was recovered from liver tissue, making this technique minimally destructive to museum specimens. Sequencing was only successful for the more recently collected specimen (collected ~30 ybp). We suspect this could be due either to the conditions of preservation and/or the amount of tissue used for extraction purposes. For the successfully sequenced sample, we found a high rate of base misincorporation. After rigorous trimming, we successfully mapped 27.93% of the cleaned reads to the reference genome, were able to reconstruct the complete mitochondrial genome, and recovered an accurate phylogenetic placement for our specimen. We conclude that the amount of DNA available, which can vary depending on specimen age and preservation conditions, will determine if sequencing will be successful. The technique described here will greatly improve the value of museum collections by making many formalin-fixed specimens available for genetic analysis. PMID:26505622

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

PubMed

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

2015-01-01

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

SAM: String-based sequence search algorithm for mitochondrial DNA database queries

PubMed Central

Röck, Alexander; Irwin, Jodi; Dür, Arne; Parsons, Thomas; Parson, Walther

2011-01-01

The analysis of the haploid mitochondrial (mt) genome has numerous applications in forensic and population genetics, as well as in disease studies. Although mtDNA haplotypes are usually determined by sequencing, they are rarely reported as a nucleotide string. Traditionally they are presented in a difference-coded position-based format relative to the corrected version of the first sequenced mtDNA. This convention requires recommendations for standardized sequence alignment that is known to vary between scientific disciplines, even between laboratories. As a consequence, database searches that are vital for the interpretation of mtDNA data can suffer from biased results when query and database haplotypes are annotated differently. In the forensic context that would usually lead to underestimation of the absolute and relative frequencies. To address this issue we introduce SAM, a string-based search algorithm that converts query and database sequences to position-free nucleotide strings and thus eliminates the possibility that identical sequences will be missed in a database query. The mere application of a BLAST algorithm would not be a sufficient remedy as it uses a heuristic approach and does not address properties specific to mtDNA, such as phylogenetically stable but also rapidly evolving insertion and deletion events. The software presented here provides additional flexibility to incorporate phylogenetic data, site-specific mutation rates, and other biologically relevant information that would refine the interpretation of mitochondrial DNA data. The manuscript is accompanied by freeware and example data sets that can be used to evaluate the new software (http://stringvalidation.org). PMID:21056022

Using genic sequence capture in combination with a syntenic pseudo genome to map a deletion mutant in a wheat species.

PubMed

Gardiner, Laura-Jayne; Gawroński, Piotr; Olohan, Lisa; Schnurbusch, Thorsten; Hall, Neil; Hall, Anthony

2014-12-01

Mapping-by-sequencing analyses have largely required a complete reference sequence and employed whole genome re-sequencing. In species such as wheat, no finished genome reference sequence is available. Additionally, because of its large genome size (17 Gb), re-sequencing at sufficient depth of coverage is not practical. Here, we extend the utility of mapping by sequencing, developing a bespoke pipeline and algorithm to map an early-flowering locus in einkorn wheat (Triticum monococcum L.) that is closely related to the bread wheat genome A progenitor. We have developed a genomic enrichment approach using the gene-rich regions of hexaploid bread wheat to design a 110-Mbp NimbleGen SeqCap EZ in solution capture probe set, representing the majority of genes in wheat. Here, we use the capture probe set to enrich and sequence an F2 mapping population of the mutant. The mutant locus was identified in T. monococcum, which lacks a complete genome reference sequence, by mapping the enriched data set onto pseudo-chromosomes derived from the capture probe target sequence, with a long-range order of genes based on synteny of wheat with Brachypodium distachyon. Using this approach we are able to map the region and identify a set of deleted genes within the interval. © 2014 The Authors.The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.

Rapid and Accurate Sequencing of Enterovirus Genomes Using MinION Nanopore Sequencer.

PubMed

Wang, Ji; Ke, Yue Hua; Zhang, Yong; Huang, Ke Qiang; Wang, Lei; Shen, Xin Xin; Dong, Xiao Ping; Xu, Wen Bo; Ma, Xue Jun

2017-10-01

Knowledge of an enterovirus genome sequence is very important in epidemiological investigation to identify transmission patterns and ascertain the extent of an outbreak. The MinION sequencer is increasingly used to sequence various viral pathogens in many clinical situations because of its long reads, portability, real-time accessibility of sequenced data, and very low initial costs. However, information is lacking on MinION sequencing of enterovirus genomes. In this proof-of-concept study using Enterovirus 71 (EV71) and Coxsackievirus A16 (CA16) strains as examples, we established an amplicon-based whole genome sequencing method using MinION. We explored the accuracy, minimum sequencing time, discrimination and high-throughput sequencing ability of MinION, and compared its performance with Sanger sequencing. Within the first minute (min) of sequencing, the accuracy of MinION was 98.5% for the single EV71 strain and 94.12%-97.33% for 10 genetically-related CA16 strains. In as little as 14 min, 99% identity was reached for the single EV71 strain, and in 17 min (on average), 99% identity was achieved for 10 CA16 strains in a single run. MinION is suitable for whole genome sequencing of enteroviruses with sufficient accuracy and fine discrimination and has the potential as a fast, reliable and convenient method for routine use. Copyright © 2017 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.

Validation of Genotyping-By-Sequencing Analysis in Populations of Tetraploid Alfalfa by 454 Sequencing

PubMed Central

Rocher, Solen; Jean, Martine; Castonguay, Yves; Belzile, François

2015-01-01

Genotyping-by-sequencing (GBS) is a relatively low-cost high throughput genotyping technology based on next generation sequencing and is applicable to orphan species with no reference genome. A combination of genome complexity reduction and multiplexing with DNA barcoding provides a simple and affordable way to resolve allelic variation between plant samples or populations. GBS was performed on ApeKI libraries using DNA from 48 genotypes each of two heterogeneous populations of tetraploid alfalfa (Medicago sativa spp. sativa): the synthetic cultivar Apica (ATF0) and a derived population (ATF5) obtained after five cycles of recurrent selection for superior tolerance to freezing (TF). Nearly 400 million reads were obtained from two lanes of an Illumina HiSeq 2000 sequencer and analyzed with the Universal Network-Enabled Analysis Kit (UNEAK) pipeline designed for species with no reference genome. Following the application of whole dataset-level filters, 11,694 single nucleotide polymorphism (SNP) loci were obtained. About 60% had a significant match on the Medicago truncatula syntenic genome. The accuracy of allelic ratios and genotype calls based on GBS data was directly assessed using 454 sequencing on a subset of SNP loci scored in eight plant samples. Sequencing depth in this study was not sufficient for accurate tetraploid allelic dosage, but reliable genotype calls based on diploid allelic dosage were obtained when using additional quality filtering. Principal Component Analysis of SNP loci in plant samples revealed that a small proportion (<5%) of the genetic variability assessed by GBS is able to differentiate ATF0 and ATF5. Our results confirm that analysis of GBS data using UNEAK is a reliable approach for genome-wide discovery of SNP loci in outcrossed polyploids. PMID:26115486

A few nucleotide polymorphisms are sufficient to recruit nuclear factors differentially to the intron 1 of HPV-16 intratypic variants.

PubMed

López-Urrutia, Eduardo; Valdés, Jesús; Bonilla-Moreno, Raúl; Martínez-Salazar, Martha; Martínez-Garcia, Martha; Berumen, Jaime; Villegas-Sepúlveda, Nicolás

2012-06-01

The HPV-16 E6/E7 genes, which contain intron 1, are processed by alternative splicing and its transcripts are detected with a heterogeneous profile in tumours cells. Frequently, the HPV-16 positive carcinoma cells bear viral variants that contain single nucleotide polymorphisms into its DNA sequence. We were interested in analysing the contribution of this polymorphism to the heterogeneity in the pattern of the E6/E7 spliced transcripts. Using the E6/E7 sequences from three closely related HPV-16 variants, we have shown that a few nucleotide changes are sufficient to produce heterogeneity in the splicing profile. Furthermore, using mutants that contained a single SNP, we also showed that one nucleotide change was sufficient to reproduce the heterogeneous splicing profile. Additionally, a difference of two or three SNPs among these viral sequences was sufficient to recruit differentially several splicing factors to the polymorphic E6/E7 transcripts. Moreover, only one SNP was sufficient to alter the binding site of at least one splicing factor, changing the ability of splicing factors to bind the transcript. Finally, the factors that were differentially bound to the short form of intron 1 of one of these E6/E7 variants were identified as TIA1 and/or TIAR and U1-70k, while U2AF65, U5-52k and PTB were preferentially bound to the transcript of the other variants. Copyright © 2012 Elsevier B.V. All rights reserved.

Recominant Pinoresino-Lariciresinol Reductase, Recombinant Dirigent Protein And Methods Of Use

DOEpatents

Lewis, Norman G.; Davin, Laurence B.; Dinkova-Kostova, Albena T.; Fujita, Masayuki , Gang; David R. , Sarkanen; Simo , Ford; Joshua D.

2003-10-21

Dirigent proteins and pinoresinol/lariciresinol reductases have been isolated, together with cDNAs encoding dirigent proteins and pinoresinol/lariciresinol reductases. Accordingly, isolated DNA sequences are provided from source species Forsythia intermedia, Thuja plicata, Tsuga heterophylla, Eucommia ulmoides, Linum usitatissimum, and Schisandra chinensis, which code for the expression of dirigent proteins and pinoresinol/lariciresinol reductases. In other aspects, replicable recombinant cloning vehicles are provided which code for dirigent proteins or pinoresinol/lariciresinol reductases or for a base sequence sufficiently complementary to at least a portion of dirigent protein or pinoresinol/lariciresinol reductase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding dirigent protein or pinoresinol/lariciresinol reductase. Thus, systems and methods are provided for the recombinant expression of dirigent proteins and/or pinoresinol/lariciresinol reductases.

A Method for Preparing DNA Sequencing Templates Using a DNA-Binding Microplate

PubMed Central

Yang, Yu; Hebron, Haroun R.; Hang, Jun

2009-01-01

A DNA-binding matrix was immobilized on the surface of a 96-well microplate and used for plasmid DNA preparation for DNA sequencing. The same DNA-binding plate was used for bacterial growth, cell lysis, DNA purification, and storage. In a single step using one buffer, bacterial cells were lysed by enzymes, and released DNA was captured on the plate simultaneously. After two wash steps, DNA was eluted and stored in the same plate. Inclusion of phosphates in the culture medium was found to enhance the yield of plasmid significantly. Purified DNA samples were used successfully in DNA sequencing with high consistency and reproducibility. Eleven vectors and nine libraries were tested using this method. In 10 μl sequencing reactions using 3 μl sample and 0.25 μl BigDye Terminator v3.1, the results from a 3730xl sequencer gave a success rate of 90–95% and read-lengths of 700 bases or more. The method is fully automatable and convenient for manual operation as well. It enables reproducible, high-throughput, rapid production of DNA with purity and yields sufficient for high-quality DNA sequencing at a substantially reduced cost. PMID:19568455

Analysis of the cytochrome c oxidase subunit II (COX2) gene in giant panda, Ailuropoda melanoleuca.

PubMed

Ling, S S; Zhu, Y; Lan, D; Li, D S; Pang, H Z; Wang, Y; Li, D Y; Wei, R P; Zhang, H M; Wang, C D; Hu, Y D

2017-01-23

The giant panda, Ailuropoda melanoleuca (Ursidae), has a unique bamboo-based diet; however, this low-energy intake has been sufficient to maintain the metabolic processes of this species since the fourth ice age. As mitochondria are the main sites for energy metabolism in animals, the protein-coding genes involved in mitochondrial respiratory chains, particularly cytochrome c oxidase subunit II (COX2), which is the rate-limiting enzyme in electron transfer, could play an important role in giant panda metabolism. Therefore, the present study aimed to isolate, sequence, and analyze the COX2 DNA from individuals kept at the Giant Panda Protection and Research Center, China, and compare these sequences with those of the other Ursidae family members. Multiple sequence alignment showed that the COX2 gene had three point mutations that defined three haplotypes, with 60% of the sequences corresponding to haplotype I. The neutrality tests revealed that the COX2 gene was conserved throughout evolution, and the maximum likelihood phylogenetic analysis, using homologous sequences from other Ursidae species, showed clustering of the COX2 sequences of giant pandas, suggesting that this gene evolved differently in them.

Is sequence awareness mandatory for perceptual sequence learning: An assessment using a pure perceptual sequence learning design.

PubMed

Deroost, Natacha; Coomans, Daphné

2018-02-01

We examined the role of sequence awareness in a pure perceptual sequence learning design. Participants had to react to the target's colour that changed according to a perceptual sequence. By varying the mapping of the target's colour onto the response keys, motor responses changed randomly. The effect of sequence awareness on perceptual sequence learning was determined by manipulating the learning instructions (explicit versus implicit) and assessing the amount of sequence awareness after the experiment. In the explicit instruction condition (n = 15), participants were instructed to intentionally search for the colour sequence, whereas in the implicit instruction condition (n = 15), they were left uninformed about the sequenced nature of the task. Sequence awareness after the sequence learning task was tested by means of a questionnaire and the process-dissociation-procedure. The results showed that the instruction manipulation had no effect on the amount of perceptual sequence learning. Based on their report to have actively applied their sequence knowledge during the experiment, participants were subsequently regrouped in a sequence strategy group (n = 14, of which 4 participants from the implicit instruction condition and 10 participants from the explicit instruction condition) and a no-sequence strategy group (n = 16, of which 11 participants from the implicit instruction condition and 5 participants from the explicit instruction condition). Only participants of the sequence strategy group showed reliable perceptual sequence learning and sequence awareness. These results indicate that perceptual sequence learning depends upon the continuous employment of strategic cognitive control processes on sequence knowledge. Sequence awareness is suggested to be a necessary but not sufficient condition for perceptual learning to take place. Copyright © 2018 Elsevier B.V. All rights reserved.

A three-nucleotide helix I is sufficient for full activity of a hammerhead ribozyme: advantages of an asymmetric design.

PubMed Central

Tabler, M; Homann, M; Tzortzakaki, S; Sczakiel, G

1994-01-01

Trans-cleaving hammerhead ribozymes with long target-specific antisense sequences flanking the catalytic domain share some features with conventional antisense RNA and are therefore termed 'catalytic antisense RNAs'. Sequences 5' to the catalytic domain form helix I and sequences 3' to it form helix III when complexed with the target RNA. A catalytic antisense RNA of more than 400 nucleotides, and specific for the human immunodeficiency virus type 1 (HIV-1), was systematically truncated within the arm that constituted originally a helix I of 128 base pairs. The resulting ribozymes formed helices I of 13, 8, 5, 3, 2, 1 and 0 nucleotides, respectively, and a helix III of about 280 nucleotides. When their in vitro cleavage activity was compared with the original catalytic antisense RNA, it was found that a helix I of as little as three nucleotides was sufficient for full endonucleolytic activity. The catalytically active constructs inhibited HIV-1 replication about four-fold more effectively than the inactive ones when tested in human cells. A conventional hammerhead ribozyme having helices of just 8 nucleotides on either side failed to cleave the target RNA in vitro when tested under the conditions for catalytic antisense RNA. Cleavage activity could only be detected after heat-treatment of the ribozyme substrate mixture which indicates that hammerhead ribozymes with short arms do not associate as efficiently to the target RNA as catalytic antisense RNA. The requirement of just a three-nucleotide helix I allows simple PCR-based generation strategies for asymmetric hammerhead ribozymes. Advantages of an asymmetric design will be discussed. Images PMID:7937118

Robustness of Next Generation Sequencing on Older Formalin-Fixed Paraffin-Embedded Tissue

PubMed Central

Carrick, Danielle Mercatante; Mehaffey, Michele G.; Sachs, Michael C.; Altekruse, Sean; Camalier, Corinne; Chuaqui, Rodrigo; Cozen, Wendy; Das, Biswajit; Hernandez, Brenda Y.; Lih, Chih-Jian; Lynch, Charles F.; Makhlouf, Hala; McGregor, Paul; McShane, Lisa M.; Phillips Rohan, JoyAnn; Walsh, William D.; Williams, Paul M.; Gillanders, Elizabeth M.; Mechanic, Leah E.; Schully, Sheri D.

2015-01-01

Next Generation Sequencing (NGS) technologies are used to detect somatic mutations in tumors and study germ line variation. Most NGS studies use DNA isolated from whole blood or fresh frozen tissue. However, formalin-fixed paraffin-embedded (FFPE) tissues are one of the most widely available clinical specimens. Their potential utility as a source of DNA for NGS would greatly enhance population-based cancer studies. While preliminary studies suggest FFPE tissue may be used for NGS, the feasibility of using archived FFPE specimens in population based studies and the effect of storage time on these specimens needs to be determined. We conducted a study to determine whether DNA in archived FFPE high-grade ovarian serous adenocarcinomas from Surveillance, Epidemiology and End Results (SEER) registries Residual Tissue Repositories (RTR) was present in sufficient quantity and quality for NGS assays. Fifty-nine FFPE tissues, stored from 3 to 32 years, were obtained from three SEER RTR sites. DNA was extracted, quantified, quality assessed, and subjected to whole exome sequencing (WES). Following DNA extraction, 58 of 59 specimens (98%) yielded DNA and moved on to the library generation step followed by WES. Specimens stored for longer periods of time had significantly lower coverage of the target region (6% lower per 10 years, 95% CI: 3-10%) and lower average read depth (40x lower per 10 years, 95% CI: 18-60), although sufficient quality and quantity of WES data was obtained for data mining. Overall, 90% (53/59) of specimens provided usable NGS data regardless of storage time. This feasibility study demonstrates FFPE specimens acquired from SEER registries after varying lengths of storage time and under varying storage conditions are a promising source of DNA for NGS. PMID:26222067

Controllability of switched singular mix-valued logical control networks with constraints

NASA Astrophysics Data System (ADS)

Deng, Lei; Gong, Mengmeng; Zhu, Peiyong

2018-03-01

The present paper investigates the controllability problem of switched singular mix-valued logical control networks (SSMLCNs) with constraints on states and controls. First, using the semi-tenser product (STP) of matrices, the SSMLCN is expressed in an algebraic form, based on which a necessary and sufficient condition is given for the uniqueness of solution of SSMLCNs. Second, a necessary and sufficient criteria is derived for the controllability of constrained SSMLCNs, by converting a constrained SSMLCN into a parallel constrained switched mix-valued logical control network. Third, an algorithm is presented to design a proper switching sequence and a control scheme which force a state to a reachable state. Finally, a numerical example is given to demonstrate the efficiency of the results obtained in this paper.

Delimitation of the Thoracosphaeraceae (Dinophyceae), including the calcareous dinoflagellates, based on large amounts of ribosomal RNA sequence data.

PubMed

Gottschling, Marc; Soehner, Sylvia; Zinssmeister, Carmen; John, Uwe; Plötner, Jörg; Schweikert, Michael; Aligizaki, Katerina; Elbrächter, Malte

2012-01-01

The phylogenetic relationships of the Dinophyceae (Alveolata) are not sufficiently resolved at present. The Thoracosphaeraceae (Peridiniales) are the only group of the Alveolata that include members with calcareous coccoid stages; this trait is considered apomorphic. Although the coccoid stage apparently is not calcareous, Bysmatrum has been assigned to the Thoracosphaeraceae based on thecal morphology. We tested the monophyly of the Thoracosphaeraceae using large sets of ribosomal RNA sequence data of the Alveolata including the Dinophyceae. Phylogenetic analyses were performed using Maximum Likelihood and Bayesian approaches. The Thoracosphaeraceae were monophyletic, but included also a number of non-calcareous dinophytes (such as Pentapharsodinium and Pfiesteria) and even parasites (such as Duboscquodinium and Tintinnophagus). Bysmatrum had an isolated and uncertain phylogenetic position outside the Thoracosphaeraceae. The phylogenetic relationships among calcareous dinophytes appear complex, and the assumption of the single origin of the potential to produce calcareous structures is challenged. The application of concatenated ribosomal RNA sequence data may prove promising for phylogenetic reconstructions of the Dinophyceae in future. Copyright © 2011 Elsevier GmbH. All rights reserved.

Report on the Human Genome Initiative for the Office of Health and Environmental Research

DOE R&D Accomplishments Database

Tinoco, I.; Cahill, G.; Cantor, C.; Caskey, T.; Dulbecco, R.; Engelhardt, D. L.; Hood, L.; Lerman, L. S.; Mendelsohn, M. L.; Sinsheimer, R. L.; Smith, T.; Soll, D.; Stormo, G.; White, R. L.

1987-04-01

The report urges DOE and the Nation to commit to a large, multi-year, multidisciplinary, technological undertaking to order and sequence the human genome. This effort will first require significant innovation in general capability to manipulate DNA, major new analytical methods for ordering and sequencing, theoretical developments in computer science and mathematical biology, and great expansions in our ability to store and manipulate the information and to interface it with other large and diverse genetic databases. The actual ordering and sequencing involves the coordinated processing of some 3 billion bases from a reference human genome. Science is poised on the rudimentary edge of being able to read and understand human genes. A concerted, broadly based, scientific effort to provide new methods of sufficient power and scale should transform this activity from an inefficient one-gene-at-a-time, single laboratory effort into a coordinated, worldwide, comprehensive reading of "the book of man". The effort will be extraordinary in scope and magnitude, but so will be the benefit to biological understanding, new technology and the diagnosis and treatment of human disease.

Neptune: a bioinformatics tool for rapid discovery of genomic variation in bacterial populations

PubMed Central

Marinier, Eric; Zaheer, Rahat; Berry, Chrystal; Weedmark, Kelly A.; Domaratzki, Michael; Mabon, Philip; Knox, Natalie C.; Reimer, Aleisha R.; Graham, Morag R.; Chui, Linda; Patterson-Fortin, Laura; Zhang, Jian; Pagotto, Franco; Farber, Jeff; Mahony, Jim; Seyer, Karine; Bekal, Sadjia; Tremblay, Cécile; Isaac-Renton, Judy; Prystajecky, Natalie; Chen, Jessica; Slade, Peter

2017-01-01

Abstract The ready availability of vast amounts of genomic sequence data has created the need to rethink comparative genomics algorithms using ‘big data’ approaches. Neptune is an efficient system for rapidly locating differentially abundant genomic content in bacterial populations using an exact k-mer matching strategy, while accommodating k-mer mismatches. Neptune’s loci discovery process identifies sequences that are sufficiently common to a group of target sequences and sufficiently absent from non-targets using probabilistic models. Neptune uses parallel computing to efficiently identify and extract these loci from draft genome assemblies without requiring multiple sequence alignments or other computationally expensive comparative sequence analyses. Tests on simulated and real datasets showed that Neptune rapidly identifies regions that are both sensitive and specific. We demonstrate that this system can identify trait-specific loci from different bacterial lineages. Neptune is broadly applicable for comparative bacterial analyses, yet will particularly benefit pathogenomic applications, owing to efficient and sensitive discovery of differentially abundant genomic loci. The software is available for download at: http://github.com/phac-nml/neptune. PMID:29048594

Identifying functional cancer-specific miRNA-mRNA interactions in testicular germ cell tumor.

PubMed

Sedaghat, Nafiseh; Fathy, Mahmood; Modarressi, Mohammad Hossein; Shojaie, Ali

2016-09-07

Testicular cancer is the most common cancer in men aged between 15 and 35 and more than 90% of testicular neoplasms are originated at germ cells. Recent research has shown the impact of microRNAs (miRNAs) in different types of cancer, including testicular germ cell tumor (TGCT). MicroRNAs are small non-coding RNAs which affect the development and progression of cancer cells by binding to mRNAs and regulating their expressions. The identification of functional miRNA-mRNA interactions in cancers, i.e. those that alter the expression of genes in cancer cells, can help delineate post-regulatory mechanisms and may lead to new treatments to control the progression of cancer. A number of sequence-based methods have been developed to predict miRNA-mRNA interactions based on the complementarity of sequences. While necessary, sequence complementarity is, however, not sufficient for presence of functional interactions. Alternative methods have thus been developed to refine the sequence-based interactions using concurrent expression profiles of miRNAs and mRNAs. This study aims to find functional cancer-specific miRNA-mRNA interactions in TGCT. To this end, the sequence-based predicted interactions are first refined using an ensemble learning method, based on two well-known methods of learning miRNA-mRNA interactions, namely, TaLasso and GenMiR++. Additional functional analyses were then used to identify a subset of interactions to be most likely functional and specific to TGCT. The final list of 13 miRNA-mRNA interactions can be potential targets for identifying TGCT-specific interactions and future laboratory experiments to develop new therapies. Copyright © 2016 Elsevier Ltd. All rights reserved.

Galerkin approximation for inverse problems for nonautonomous nonlinear distributed systems

NASA Technical Reports Server (NTRS)

Banks, H. T.; Reich, Simeon; Rosen, I. G.

1988-01-01

An abstract framework and convergence theory is developed for Galerkin approximation for inverse problems involving the identification of nonautonomous nonlinear distributed parameter systems. A set of relatively easily verified conditions is provided which are sufficient to guarantee the existence of optimal solutions and their approximation by a sequence of solutions to a sequence of approximating finite dimensional identification problems. The approach is based on the theory of monotone operators in Banach spaces and is applicable to a reasonably broad class of nonlinear distributed systems. Operator theoretic and variational techniques are used to establish a fundamental convergence result. An example involving evolution systems with dynamics described by nonstationary quasilinear elliptic operators along with some applications are presented and discussed.

Improving the prospects of cleavage-based nanopore sequencing engines

NASA Astrophysics Data System (ADS)

Brady, Kyle T.; Reiner, Joseph E.

2015-08-01

Recently proposed methods for DNA sequencing involve the use of cleavage-based enzymes attached to the opening of a nanopore. The idea is that DNA interacting with either an exonuclease or polymerase protein will lead to a small molecule being cleaved near the mouth of the nanopore, and subsequent entry into the pore will yield information about the DNA sequence. The prospects for this approach seem promising, but it has been shown that diffusion related effects impose a limit on the capture probability of molecules by the pore, which limits the efficacy of the technique. Here, we revisit the problem with the goal of optimizing the capture probability via a step decrease in the nucleotide diffusion coefficient between the pore and bulk solutions. It is shown through random walk simulations and a simplified analytical model that decreasing the molecule's diffusion coefficient in the bulk relative to its value in the pore increases the nucleotide capture probability. Specifically, we show that at sufficiently high applied transmembrane potentials (≥100 mV), increasing the potential by a factor f is equivalent to decreasing the diffusion coefficient ratio Dbulk/Dpore by the same factor f. This suggests a promising route toward implementation of cleavage-based sequencing protocols. We also discuss the feasibility of forming a step function in the diffusion coefficient across the pore-bulk interface.

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

PubMed

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.

A Pan-HIV Strategy for Complete Genome Sequencing

PubMed Central

Yamaguchi, Julie; Alessandri-Gradt, Elodie; Tell, Robert W.; Brennan, Catherine A.

2015-01-01

Molecular surveillance is essential to monitor HIV diversity and track emerging strains. We have developed a universal library preparation method (HIV-SMART [i.e., switching mechanism at 5′ end of RNA transcript]) for next-generation sequencing that harnesses the specificity of HIV-directed priming to enable full genome characterization of all HIV-1 groups (M, N, O, and P) and HIV-2. Broad application of the HIV-SMART approach was demonstrated using a panel of diverse cell-cultured virus isolates. HIV-1 non-subtype B-infected clinical specimens from Cameroon were then used to optimize the protocol to sequence directly from plasma. When multiplexing 8 or more libraries per MiSeq run, full genome coverage at a median ∼2,000× depth was routinely obtained for either sample type. The method reproducibly generated the same consensus sequence, consistently identified viral sequence heterogeneity present in specimens, and at viral loads of ≤4.5 log copies/ml yielded sufficient coverage to permit strain classification. HIV-SMART provides an unparalleled opportunity to identify diverse HIV strains in patient specimens and to determine phylogenetic classification based on the entire viral genome. Easily adapted to sequence any RNA virus, this technology illustrates the utility of next-generation sequencing (NGS) for viral characterization and surveillance. PMID:26699702

Impact of sequencing depth and read length on single cell RNA sequencing data of T cells.

PubMed

Rizzetto, Simone; Eltahla, Auda A; Lin, Peijie; Bull, Rowena; Lloyd, Andrew R; Ho, Joshua W K; Venturi, Vanessa; Luciani, Fabio

2017-10-06

Single cell RNA sequencing (scRNA-seq) provides great potential in measuring the gene expression profiles of heterogeneous cell populations. In immunology, scRNA-seq allowed the characterisation of transcript sequence diversity of functionally relevant T cell subsets, and the identification of the full length T cell receptor (TCRαβ), which defines the specificity against cognate antigens. Several factors, e.g. RNA library capture, cell quality, and sequencing output affect the quality of scRNA-seq data. We studied the effects of read length and sequencing depth on the quality of gene expression profiles, cell type identification, and TCRαβ reconstruction, utilising 1,305 single cells from 8 publically available scRNA-seq datasets, and simulation-based analyses. Gene expression was characterised by an increased number of unique genes identified with short read lengths (<50 bp), but these featured higher technical variability compared to profiles from longer reads. Successful TCRαβ reconstruction was achieved for 6 datasets (81% - 100%) with at least 0.25 millions (PE) reads of length >50 bp, while it failed for datasets with <30 bp reads. Sufficient read length and sequencing depth can control technical noise to enable accurate identification of TCRαβ and gene expression profiles from scRNA-seq data of T cells.

Systematic and fully automated identification of protein sequence patterns.

PubMed

Hart, R K; Royyuru, A K; Stolovitzky, G; Califano, A

2000-01-01

We present an efficient algorithm to systematically and automatically identify patterns in protein sequence families. The procedure is based on the Splash deterministic pattern discovery algorithm and on a framework to assess the statistical significance of patterns. We demonstrate its application to the fully automated discovery of patterns in 974 PROSITE families (the complete subset of PROSITE families which are defined by patterns and contain DR records). Splash generates patterns with better specificity and undiminished sensitivity, or vice versa, in 28% of the families; identical statistics were obtained in 48% of the families, worse statistics in 15%, and mixed behavior in the remaining 9%. In about 75% of the cases, Splash patterns identify sequence sites that overlap more than 50% with the corresponding PROSITE pattern. The procedure is sufficiently rapid to enable its use for daily curation of existing motif and profile databases. Third, our results show that the statistical significance of discovered patterns correlates well with their biological significance. The trypsin subfamily of serine proteases is used to illustrate this method's ability to exhaustively discover all motifs in a family that are statistically and biologically significant. Finally, we discuss applications of sequence patterns to multiple sequence alignment and the training of more sensitive score-based motif models, akin to the procedure used by PSI-BLAST. All results are available at httpl//www.research.ibm.com/spat/.

Definition of Eight Mulberry Species in the Genus Morus by Internal Transcribed Spacer-Based Phylogeny

PubMed Central

Zeng, Qiwei; Chen, Hongyu; Zhang, Chao; Han, Minjing; Li, Tian; Qi, Xiwu; Xiang, Zhonghuai; He, Ningjia

2015-01-01

Mulberry, belonging to the order Rosales, family Moraceae, and genus Morus, has received attention because of both its economic and medicinal value, as well as for its important ecological function. The genus Morus has a worldwide distribution, however, its taxonomy remains complex and disputed. Many studies have attempted to classify Morus species, resulting in varied numbers of designated Morus spp. To address this issue, we used information from internal transcribed spacer (ITS) genetic sequences to study the taxonomy of all the members of generally accepted genus Morus. We found that intraspecific 5.8S rRNA sequences were identical but that interspecific 5.8S sequences were diverse. M. alba and M. notabilis showed the shortest (215 bp) and the longest (233 bp) ITS1 sequence length, respectively. With the completion of the mulberry genome, we could identify single nucleotide polymorphisms within the ITS locus in the M. notabilis genome. From reconstruction of a phylogenetic tree based on the complete ITS data, we propose that the Morus genus should be classified into eight species, including M. alba, M. nigra, M. notabilis, M. serrata, M. celtidifolia, M. insignis, M. rubra, and M. mesozygia. Furthermore, the classification of the ITS sequences of known interspecific hybrid clones into both paternal and maternal clades indicated that ITS variation was sufficient to distinguish interspecific hybrids in the genus Morus. PMID:26266951

Phylogenetic Analysis of Prevalent Tuberculosis and Non-Tuberculosis Mycobacteria in Isfahan, Iran, Based on a 360 bp Sequence of the rpoB Gene

PubMed Central

Nasr Esfahani, Bahram; Moghim, Sharareh; Ghasemian Safaei, Hajieh; Moghoofei, Mohsen; Sedighi, Mansour; Hadifar, Shima

2016-01-01

Background Taxonomic and phylogenetic studies of Mycobacterium species have been based around the 16sRNA gene for many years. However, due to the high strain similarity between species in the Mycobacterium genus (94.3% - 100%), defining a valid phylogenetic tree is difficult; consequently, its use in estimating the boundaries between species is limited. The sequence of the rpoB gene makes it an appropriate gene for phylogenetic analysis, especially in bacteria with limited variation. Objectives In the present study, a 360bp sequence of rpoB was used for precise classification of Mycobacterium strains isolated in Isfahan, Iran. Materials and Methods From February to October 2013, 57 clinical and environmental isolates were collected, subcultured, and identified by phenotypic methods. After DNA extraction, a 360bp fragment was PCR-amplified and sequenced. The phylogenetic tree was constructed based on consensus sequence data, using MEGA5 software. Results Slow and fast-growing groups of the Mycobacterium strains were clearly differentiated based on the constructed tree of 56 common Mycobacterium isolates. Each species with a unique title in the tree was identified; in total, 13 nods with a bootstrap value of over 50% were supported. Among the slow-growing group was Mycobacterium kansasii, with M. tuberculosis in a cluster with a bootstrap value of 98% and M. gordonae in another cluster with a bootstrap value of 90%. In the fast-growing group, one cluster with a bootstrap value of 89% was defined, including all fast-growing members present in this study. Conclusions The results suggest that only the application of the rpoB gene sequence is sufficient for taxonomic categorization and definition of a new Mycobacterium species, due to its high resolution power and proper variation in its sequence (85% - 100%); the resulting tree has high validity. PMID:27284397

A Compressed Sensing Based Ultra-Wideband Communication System

DTIC Science & Technology

2009-06-01

principle, most of the processing at the receiver can be moved to the transmitter—where energy consumption and computation are sufficient for many advanced...extended to continuous time signals. We use ∗ to denote the convolution process in a linear time-invariant (LTI) system. Assume that there is an analog...Filter Channel Low Rate A/D Processing Sparse Bit Sequence UWB Pulse Generator α̂ Waves)(RadioGHz 5 MHz125 θ Ψ Φ y θ̂ 1 ˆ arg min s.t. yθ

Genomic insights into the taxonomic status of the Bacillus cereus group

PubMed Central

Liu, Yang; Lai, Qiliang; Göker, Markus; Meier-Kolthoff, Jan P.; Wang, Meng; Sun, Yamin; Wang, Lei; Shao, Zongze

2015-01-01

The identification and phylogenetic relationships of bacteria within the Bacillus cereus group are controversial. This study aimed at determining the taxonomic affiliations of these strains using the whole-genome sequence-based Genome BLAST Distance Phylogeny (GBDP) approach. The GBDP analysis clearly separated 224 strains into 30 clusters, representing eleven known, partially merged species and accordingly 19–20 putative novel species. Additionally, 16S rRNA gene analysis, a novel variant of multi-locus sequence analysis (nMLSA) and screening of virulence genes were performed. The 16S rRNA gene sequence was not sufficient to differentiate the bacteria within this group due to its high conservation. The nMLSA results were consistent with GBDP. Moreover, a fast typing method was proposed using the pycA gene, and where necessary, the ccpA gene. The pXO plasmids and cry genes were widely distributed, suggesting little correlation with the phylogenetic positions of the host bacteria. This might explain why classifications based on virulence characteristics proved unsatisfactory in the past. In summary, this is the first large-scale and systematic study of the taxonomic status of the bacteria within the B. cereus group using whole-genome sequences, and is likely to contribute to further insights into their pathogenicity, phylogeny and adaptation to diverse environments. PMID:26373441

Camps 2.0: exploring the sequence and structure space of prokaryotic, eukaryotic, and viral membrane proteins.

PubMed

Neumann, Sindy; Hartmann, Holger; Martin-Galiano, Antonio J; Fuchs, Angelika; Frishman, Dmitrij

2012-03-01

Structural bioinformatics of membrane proteins is still in its infancy, and the picture of their fold space is only beginning to emerge. Because only a handful of three-dimensional structures are available, sequence comparison and structure prediction remain the main tools for investigating sequence-structure relationships in membrane protein families. Here we present a comprehensive analysis of the structural families corresponding to α-helical membrane proteins with at least three transmembrane helices. The new version of our CAMPS database (CAMPS 2.0) covers nearly 1300 eukaryotic, prokaryotic, and viral genomes. Using an advanced classification procedure, which is based on high-order hidden Markov models and considers both sequence similarity as well as the number of transmembrane helices and loop lengths, we identified 1353 structurally homogeneous clusters roughly corresponding to membrane protein folds. Only 53 clusters are associated with experimentally determined three-dimensional structures, and for these clusters CAMPS is in reasonable agreement with structure-based classification approaches such as SCOP and CATH. We therefore estimate that ∼1300 structures would need to be determined to provide a sufficient structural coverage of polytopic membrane proteins. CAMPS 2.0 is available at http://webclu.bio.wzw.tum.de/CAMPS2.0/. Copyright © 2011 Wiley Periodicals, Inc.

A machine learning model to determine the accuracy of variant calls in capture-based next generation sequencing.

PubMed

van den Akker, Jeroen; Mishne, Gilad; Zimmer, Anjali D; Zhou, Alicia Y

2018-04-17

Next generation sequencing (NGS) has become a common technology for clinical genetic tests. The quality of NGS calls varies widely and is influenced by features like reference sequence characteristics, read depth, and mapping accuracy. With recent advances in NGS technology and software tools, the majority of variants called using NGS alone are in fact accurate and reliable. However, a small subset of difficult-to-call variants that still do require orthogonal confirmation exist. For this reason, many clinical laboratories confirm NGS results using orthogonal technologies such as Sanger sequencing. Here, we report the development of a deterministic machine-learning-based model to differentiate between these two types of variant calls: those that do not require confirmation using an orthogonal technology (high confidence), and those that require additional quality testing (low confidence). This approach allows reliable NGS-based calling in a clinical setting by identifying the few important variant calls that require orthogonal confirmation. We developed and tested the model using a set of 7179 variants identified by a targeted NGS panel and re-tested by Sanger sequencing. The model incorporated several signals of sequence characteristics and call quality to determine if a variant was identified at high or low confidence. The model was tuned to eliminate false positives, defined as variants that were called by NGS but not confirmed by Sanger sequencing. The model achieved very high accuracy: 99.4% (95% confidence interval: +/- 0.03%). It categorized 92.2% (6622/7179) of the variants as high confidence, and 100% of these were confirmed to be present by Sanger sequencing. Among the variants that were categorized as low confidence, defined as NGS calls of low quality that are likely to be artifacts, 92.1% (513/557) were found to be not present by Sanger sequencing. This work shows that NGS data contains sufficient characteristics for a machine-learning-based model to differentiate low from high confidence variants. Additionally, it reveals the importance of incorporating site-specific features as well as variant call features in such a model.

Quasispecies Analyses of the HIV-1 Near-full-length Genome With Illumina MiSeq

PubMed Central

Ode, Hirotaka; Matsuda, Masakazu; Matsuoka, Kazuhiro; Hachiya, Atsuko; Hattori, Junko; Kito, Yumiko; Yokomaku, Yoshiyuki; Iwatani, Yasumasa; Sugiura, Wataru

2015-01-01

Human immunodeficiency virus type-1 (HIV-1) exhibits high between-host genetic diversity and within-host heterogeneity, recognized as quasispecies. Because HIV-1 quasispecies fluctuate in terms of multiple factors, such as antiretroviral exposure and host immunity, analyzing the HIV-1 genome is critical for selecting effective antiretroviral therapy and understanding within-host viral coevolution mechanisms. Here, to obtain HIV-1 genome sequence information that includes minority variants, we sought to develop a method for evaluating quasispecies throughout the HIV-1 near-full-length genome using the Illumina MiSeq benchtop deep sequencer. To ensure the reliability of minority mutation detection, we applied an analysis method of sequence read mapping onto a consensus sequence derived from de novo assembly followed by iterative mapping and subsequent unique error correction. Deep sequencing analyses of aHIV-1 clone showed that the analysis method reduced erroneous base prevalence below 1% in each sequence position and discarded only < 1% of all collected nucleotides, maximizing the usage of the collected genome sequences. Further, we designed primer sets to amplify the HIV-1 near-full-length genome from clinical plasma samples. Deep sequencing of 92 samples in combination with the primer sets and our analysis method provided sufficient coverage to identify >1%-frequency sequences throughout the genome. When we evaluated sequences of pol genes from 18 treatment-naïve patients' samples, the deep sequencing results were in agreement with Sanger sequencing and identified numerous additional minority mutations. The results suggest that our deep sequencing method would be suitable for identifying within-host viral population dynamics throughout the genome. PMID:26617593

BiPPred: Combined sequence- and structure-based prediction of peptide binding to the Hsp70 chaperone BiP.

PubMed

Schneider, Markus; Rosam, Mathias; Glaser, Manuel; Patronov, Atanas; Shah, Harpreet; Back, Katrin Christiane; Daake, Marina Angelika; Buchner, Johannes; Antes, Iris

2016-10-01

Substrate binding to Hsp70 chaperones is involved in many biological processes, and the identification of potential substrates is important for a comprehensive understanding of these events. We present a multi-scale pipeline for an accurate, yet efficient prediction of peptides binding to the Hsp70 chaperone BiP by combining sequence-based prediction with molecular docking and MMPBSA calculations. First, we measured the binding of 15mer peptides from known substrate proteins of BiP by peptide array (PA) experiments and performed an accuracy assessment of the PA data by fluorescence anisotropy studies. Several sequence-based prediction models were fitted using this and other peptide binding data. A structure-based position-specific scoring matrix (SB-PSSM) derived solely from structural modeling data forms the core of all models. The matrix elements are based on a combination of binding energy estimations, molecular dynamics simulations, and analysis of the BiP binding site, which led to new insights into the peptide binding specificities of the chaperone. Using this SB-PSSM, peptide binders could be predicted with high selectivity even without training of the model on experimental data. Additional training further increased the prediction accuracies. Subsequent molecular docking (DynaDock) and MMGBSA/MMPBSA-based binding affinity estimations for predicted binders allowed the identification of the correct binding mode of the peptides as well as the calculation of nearly quantitative binding affinities. The general concept behind the developed multi-scale pipeline can readily be applied to other protein-peptide complexes with linearly bound peptides, for which sufficient experimental binding data for the training of classical sequence-based prediction models is not available. Proteins 2016; 84:1390-1407. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Species composition of the genus Saprolegnia in fin fish aquaculture environments, as determined by nucleotide sequence analysis of the nuclear rDNA ITS regions.

PubMed

de la Bastide, Paul Y; Leung, Wai Lam; Hintz, William E

2015-01-01

The ITS region of the rDNA gene was compared for Saprolegnia spp. in order to improve our understanding of nucleotide sequence variability within and between species of this genus, determine species composition in Canadian fin fish aquaculture facilities, and to assess the utility of ITS sequence variability in genetic marker development. From a collection of more than 400 field isolates, ITS region nucleotide sequences were studied and it was determined that there was sufficient consistent inter-specific variation to support the designation of species identity based on ITS sequence data. This non-subjective approach to species identification does not rely upon transient morphological features. Phylogenetic analyses comparing our ITS sequences and species designations with data from previous studies generally supported the clade scheme of Diéguez-Uribeondo et al. (2007) and found agreement with the molecular taxonomic cluster system of Sandoval-Sierra et al. (2014). Our Canadian ITS sequence collection will thus contribute to the public database and assist the clarification of Saprolegnia spp. taxonomy. The analysis of ITS region sequence variability facilitated genus- and species-level identification of unknown samples from aquaculture facilities and provided useful information on species composition. A unique ITS-RFLP for the identification of S. parasitica was also described. Copyright © 2014 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Exploiting rice-sorghum synteny for targeted development of EST-SSRs to enrich the sorghum genetic linkage map.

PubMed

Ramu, P; Kassahun, B; Senthilvel, S; Ashok Kumar, C; Jayashree, B; Folkertsma, R T; Reddy, L Ananda; Kuruvinashetti, M S; Haussmann, B I G; Hash, C T

2009-11-01

The sequencing and detailed comparative functional analysis of genomes of a number of select botanical models open new doors into comparative genomics among the angiosperms, with potential benefits for improvement of many orphan crops that feed large populations. In this study, a set of simple sequence repeat (SSR) markers was developed by mining the expressed sequence tag (EST) database of sorghum. Among the SSR-containing sequences, only those sharing considerable homology with rice genomic sequences across the lengths of the 12 rice chromosomes were selected. Thus, 600 SSR-containing sorghum EST sequences (50 homologous sequences on each of the 12 rice chromosomes) were selected, with the intention of providing coverage for corresponding homologous regions of the sorghum genome. Primer pairs were designed and polymorphism detection ability was assessed using parental pairs of two existing sorghum mapping populations. About 28% of these new markers detected polymorphism in this 4-entry panel. A subset of 55 polymorphic EST-derived SSR markers were mapped onto the existing skeleton map of a recombinant inbred population derived from cross N13 x E 36-1, which is segregating for Striga resistance and the stay-green component of terminal drought tolerance. These new EST-derived SSR markers mapped across all 10 sorghum linkage groups, mostly to regions expected based on prior knowledge of rice-sorghum synteny. The ESTs from which these markers were derived were then mapped in silico onto the aligned sorghum genome sequence, and 88% of the best hits corresponded to linkage-based positions. This study demonstrates the utility of comparative genomic information in targeted development of markers to fill gaps in linkage maps of related crop species for which sufficient genomic tools are not available.

Computational Redesign of Thioredoxin Is Hypersensitive toward Minor Conformational Changes in the Backbone Template

PubMed Central

Christensen, Signe; Horowitz, Scott; Bardwell, James C.A.; Olsen, Johan G.; Willemoës, Martin; Lindorff-Larsen, Kresten; Ferkinghoff-Borg, Jesper; Hamelryck, Thomas; Winther, Jakob R.

2017-01-01

Despite the development of powerful computational tools, the full-sequence design of proteins still remains a challenging task. To investigate the limits and capabilities of computational tools, we conducted a study of the ability of the program Rosetta to predict sequences that recreate the authentic fold of thioredoxin. Focusing on the influence of conformational details in the template structures, we based our study on 8 experimentally determined template structures and generated 120 designs from each. For experimental evaluation, we chose six sequences from each of the eight templates by objective criteria. The 48 selected sequences were evaluated based on their progressive ability to (1) produce soluble protein in Escherichia coli and (2) yield stable monomeric protein, and (3) on the ability of the stable, soluble proteins to adopt the target fold. Of the 48 designs, we were able to synthesize 32, 20 of which resulted in soluble protein. Of these, only two were sufficiently stable to be purified. An X-ray crystal structure was solved for one of the designs, revealing a close resemblance to the target structure. We found a significant difference among the eight template structures to realize the above three criteria despite their high structural similarity. Thus, in order to improve the success rate of computational full-sequence design methods, we recommend that multiple template structures are used. Furthermore, this study shows that special care should be taken when optimizing the geometry of a structure prior to computational design when using a method that is based on rigid conformations. PMID:27659562

Computational Redesign of Thioredoxin Is Hypersensitive toward Minor Conformational Changes in the Backbone Template.

PubMed

Johansson, Kristoffer E; Tidemand Johansen, Nicolai; Christensen, Signe; Horowitz, Scott; Bardwell, James C A; Olsen, Johan G; Willemoës, Martin; Lindorff-Larsen, Kresten; Ferkinghoff-Borg, Jesper; Hamelryck, Thomas; Winther, Jakob R

2016-10-23

Despite the development of powerful computational tools, the full-sequence design of proteins still remains a challenging task. To investigate the limits and capabilities of computational tools, we conducted a study of the ability of the program Rosetta to predict sequences that recreate the authentic fold of thioredoxin. Focusing on the influence of conformational details in the template structures, we based our study on 8 experimentally determined template structures and generated 120 designs from each. For experimental evaluation, we chose six sequences from each of the eight templates by objective criteria. The 48 selected sequences were evaluated based on their progressive ability to (1) produce soluble protein in Escherichia coli and (2) yield stable monomeric protein, and (3) on the ability of the stable, soluble proteins to adopt the target fold. Of the 48 designs, we were able to synthesize 32, 20 of which resulted in soluble protein. Of these, only two were sufficiently stable to be purified. An X-ray crystal structure was solved for one of the designs, revealing a close resemblance to the target structure. We found a significant difference among the eight template structures to realize the above three criteria despite their high structural similarity. Thus, in order to improve the success rate of computational full-sequence design methods, we recommend that multiple template structures are used. Furthermore, this study shows that special care should be taken when optimizing the geometry of a structure prior to computational design when using a method that is based on rigid conformations. Copyright © 2016 Elsevier Ltd. All rights reserved.

Robustness of Massively Parallel Sequencing Platforms

PubMed Central

Kavak, Pınar; Yüksel, Bayram; Aksu, Soner; Kulekci, M. Oguzhan; Güngör, Tunga; Hach, Faraz; Şahinalp, S. Cenk; Alkan, Can; Sağıroğlu, Mahmut Şamil

2015-01-01

The improvements in high throughput sequencing technologies (HTS) made clinical sequencing projects such as ClinSeq and Genomics England feasible. Although there are significant improvements in accuracy and reproducibility of HTS based analyses, the usability of these types of data for diagnostic and prognostic applications necessitates a near perfect data generation. To assess the usability of a widely used HTS platform for accurate and reproducible clinical applications in terms of robustness, we generated whole genome shotgun (WGS) sequence data from the genomes of two human individuals in two different genome sequencing centers. After analyzing the data to characterize SNPs and indels using the same tools (BWA, SAMtools, and GATK), we observed significant number of discrepancies in the call sets. As expected, the most of the disagreements between the call sets were found within genomic regions containing common repeats and segmental duplications, albeit only a small fraction of the discordant variants were within the exons and other functionally relevant regions such as promoters. We conclude that although HTS platforms are sufficiently powerful for providing data for first-pass clinical tests, the variant predictions still need to be confirmed using orthogonal methods before using in clinical applications. PMID:26382624

SELMAP - SELEX affinity landscape MAPping of transcription factor binding sites using integrated microfluidics

PubMed Central

Chen, Dana; Orenstein, Yaron; Golodnitsky, Rada; Pellach, Michal; Avrahami, Dorit; Wachtel, Chaim; Ovadia-Shochat, Avital; Shir-Shapira, Hila; Kedmi, Adi; Juven-Gershon, Tamar; Shamir, Ron; Gerber, Doron

2016-01-01

Transcription factors (TFs) alter gene expression in response to changes in the environment through sequence-specific interactions with the DNA. These interactions are best portrayed as a landscape of TF binding affinities. Current methods to study sequence-specific binding preferences suffer from limited dynamic range, sequence bias, lack of specificity and limited throughput. We have developed a microfluidic-based device for SELEX Affinity Landscape MAPping (SELMAP) of TF binding, which allows high-throughput measurement of 16 proteins in parallel. We used it to measure the relative affinities of Pho4, AtERF2 and Btd full-length proteins to millions of different DNA binding sites, and detected both high and low-affinity interactions in equilibrium conditions, generating a comprehensive landscape of the relative TF affinities to all possible DNA 6-mers, and even DNA10-mers with increased sequencing depth. Low quantities of both the TFs and DNA oligomers were sufficient for obtaining high-quality results, significantly reducing experimental costs. SELMAP allows in-depth screening of hundreds of TFs, and provides a means for better understanding of the regulatory processes that govern gene expression. PMID:27628341

Evaluating information content of SNPs for sample-tagging in re-sequencing projects.

PubMed

Hu, Hao; Liu, Xiang; Jin, Wenfei; Hilger Ropers, H; Wienker, Thomas F

2015-05-15

Sample-tagging is designed for identification of accidental sample mix-up, which is a major issue in re-sequencing studies. In this work, we develop a model to measure the information content of SNPs, so that we can optimize a panel of SNPs that approach the maximal information for discrimination. The analysis shows that as low as 60 optimized SNPs can differentiate the individuals in a population as large as the present world, and only 30 optimized SNPs are in practice sufficient in labeling up to 100 thousand individuals. In the simulated populations of 100 thousand individuals, the average Hamming distances, generated by the optimized set of 30 SNPs are larger than 18, and the duality frequency, is lower than 1 in 10 thousand. This strategy of sample discrimination is proved robust in large sample size and different datasets. The optimized sets of SNPs are designed for Whole Exome Sequencing, and a program is provided for SNP selection, allowing for customized SNP numbers and interested genes. The sample-tagging plan based on this framework will improve re-sequencing projects in terms of reliability and cost-effectiveness.

Magnetic resonance imaging of the sacroiliac joints in the early detection of spondyloarthritis: no added value of gadolinium compared with short tau inversion recovery sequence.

PubMed

de Hooge, Manouk; van den Berg, Rosaline; Navarro-Compán, Victoria; van Gaalen, Floris; van der Heijde, Désirée; Huizinga, Tom; Reijnierse, Monique

2013-07-01

To investigate the additional value of T1 fat-saturated after gadolinium (T1/Gd) compared with T1 and short tau inversion recovery (STIR) sequence in detecting active lesions of the SI joints typical of axial SpA (axSpA) in a prospective cohort study, the SpondyloArthritis Caught Early (SPACE) cohort, and to assess its influence on final MRI diagnosis of the SI joint (MRI-SIJ) based on the Assessment of Spondyloarthritis International Society (ASAS) definition of active sacroiliitis. Patients in the SPACE cohort received baseline and 3-month follow-up MRI-SIJ with coronal oblique T1, STIR and T1/Gd sequences. Bone marrow oedema (BME), capsulitis/enthesitis and synovitis and active sacroiliitis according to the ASAS definition were evaluated by three blinded readers. A total of 127 patients received an MRI-SIJ at baseline and 67 patients also received an MRI-SIJ at 3 months follow-up since the Gd protocol was added some months after the start of the SPACE project. Twenty-five of the 127 patients (19.7%) with a baseline MRI-SIJ and 14 of 67 patients (20.6%) with a follow-up MRI-SIJ presented BME on the STIR sequence sufficient to fulfill the ASAS definition for a positive MRI-SIJ. In eight patients, additional synovitis and/or capsulitis/enthesitis was observed; however, no additional BME was visualized on T1/Gd. One patient, without clinical diagnosis of axSpA, showed synovitis as an isolated finding. Synovitis and capsulitis/enthesitis are detectable with the administration of Gd. However, they are always observed in the presence of BME. Therefore T1 and STIR sequence alone are sufficient in the MRI assessment that, among others, is used for diagnosing patients with early axSpA.

Genomic Sequencing of Bordetella pertussis for Epidemiology and Global Surveillance of Whooping Cough.

PubMed

Bouchez, Valérie; Guglielmini, Julien; Dazas, Mélody; Landier, Annie; Toubiana, Julie; Guillot, Sophie; Criscuolo, Alexis; Brisse, Sylvain

2018-06-01

Bordetella pertussis causes whooping cough, a highly contagious respiratory disease that is reemerging in many world regions. The spread of antigen-deficient strains may threaten acellular vaccine efficacy. Dynamics of strain transmission are poorly defined because of shortcomings in current strain genotyping methods. Our objective was to develop a whole-genome genotyping strategy with sufficient resolution for local epidemiologic questions and sufficient reproducibility to enable international comparisons of clinical isolates. We defined a core genome multilocus sequence typing scheme comprising 2,038 loci and demonstrated its congruence with whole-genome single-nucleotide polymorphism variation. Most cases of intrafamilial groups of isolates or of multiple isolates recovered from the same patient were distinguished from temporally and geographically cocirculating isolates. However, epidemiologically unrelated isolates were sometimes nearly undistinguishable. We set up a publicly accessible core genome multilocus sequence typing database to enable global comparisons of B. pertussis isolates, opening the way for internationally coordinated surveillance.

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

PubMed

Schwessinger, Benjamin; Rathjen, John P

2017-01-01

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

The Neandertal genome and ancient DNA authenticity

PubMed Central

Green, Richard E; Briggs, Adrian W; Krause, Johannes; Prüfer, Kay; Burbano, Hernán A; Siebauer, Michael; Lachmann, Michael; Pääbo, Svante

2009-01-01

Recent advances in high-thoughput DNA sequencing have made genome-scale analyses of genomes of extinct organisms possible. With these new opportunities come new difficulties in assessing the authenticity of the DNA sequences retrieved. We discuss how these difficulties can be addressed, particularly with regard to analyses of the Neandertal genome. We argue that only direct assays of DNA sequence positions in which Neandertals differ from all contemporary humans can serve as a reliable means to estimate human contamination. Indirect measures, such as the extent of DNA fragmentation, nucleotide misincorporations, or comparison of derived allele frequencies in different fragment size classes, are unreliable. Fortunately, interim approaches based on mtDNA differences between Neandertals and current humans, detection of male contamination through Y chromosomal sequences, and repeated sequencing from the same fossil to detect autosomal contamination allow initial large-scale sequencing of Neandertal genomes. This will result in the discovery of fixed differences in the nuclear genome between Neandertals and current humans that can serve as future direct assays for contamination. For analyses of other fossil hominins, which may become possible in the future, we suggest a similar ‘boot-strap' approach in which interim approaches are applied until sufficient data for more definitive direct assays are acquired. PMID:19661919

Genetic differentiation of Artyfechinostomum malayanum and A. sufrartyfex (Trematoda: Echinostomatidae) based on internal transcribed spacer sequences.

PubMed

Tantrawatpan, Chairat; Saijuntha, Weerachai; Sithithaworn, Paiboon; Andrews, Ross H; Petney, Trevor N

2013-01-01

Genetic differentiation between two synonymous echinostomes species, Artyfechinostomum malayanum and Artyfechinostomum sufrartyfex was determined by using the first and second internal transcribed spacers (ITS1 and ITS2), the non-coding region of rDNA as genetic makers. Of the 699 bp of combined ITS1 and ITS2 sequences examined, 18 variable nucleotide positions (2.58 %) were observed. Of these, 17 positions could be used as diagnostic position between these two sibling species, whereas the other one variation was intraspecific variation of A. malayanum. A clade of A. malayanum was closely aligned with A. sufrartyfex and clearly distance from the cluster of other echinostomes. Our results may sufficiently suggest that the current synonymy of these species is not valid.

Not all transmembrane helices are born equal: Towards the extension of the sequence homology concept to membrane proteins

PubMed Central

2011-01-01

Background Sequence homology considerations widely used to transfer functional annotation to uncharacterized protein sequences require special precautions in the case of non-globular sequence segments including membrane-spanning stretches composed of non-polar residues. Simple, quantitative criteria are desirable for identifying transmembrane helices (TMs) that must be included into or should be excluded from start sequence segments in similarity searches aimed at finding distant homologues. Results We found that there are two types of TMs in membrane-associated proteins. On the one hand, there are so-called simple TMs with elevated hydrophobicity, low sequence complexity and extraordinary enrichment in long aliphatic residues. They merely serve as membrane-anchoring device. In contrast, so-called complex TMs have lower hydrophobicity, higher sequence complexity and some functional residues. These TMs have additional roles besides membrane anchoring such as intra-membrane complex formation, ligand binding or a catalytic role. Simple and complex TMs can occur both in single- and multi-membrane-spanning proteins essentially in any type of topology. Whereas simple TMs have the potential to confuse searches for sequence homologues and to generate unrelated hits with seemingly convincing statistical significance, complex TMs contain essential evolutionary information. Conclusion For extending the homology concept onto membrane proteins, we provide a necessary quantitative criterion to distinguish simple TMs (and a sufficient criterion for complex TMs) in query sequences prior to their usage in homology searches based on assessment of hydrophobicity and sequence complexity of the TM sequence segments. Reviewers This article was reviewed by Shamil Sunyaev, L. Aravind and Arcady Mushegian. PMID:22024092

Whole-Genome Sequences of Thirteen Isolates of Borrelia burgdorferi

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schutzer S. E.; Dunn J.; Fraser-Liggett, C. M.

2011-02-01

Borrelia burgdorferi is a causative agent of Lyme disease in North America and Eurasia. The first complete genome sequence of B. burgdorferi strain 31, available for more than a decade, has assisted research on the pathogenesis of Lyme disease. Because a single genome sequence is not sufficient to understand the relationship between genotypic and geographic variation and disease phenotype, we determined the whole-genome sequences of 13 additional B. burgdorferi isolates that span the range of natural variation. These sequences should allow improved understanding of pathogenesis and provide a foundation for novel detection, diagnosis, and prevention strategies.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Belinsky, Steven A; Palmisano, William A

A molecular marker-based method for monitoring and detecting cancer in humans. Aberrant methylation of gene promoters is a marker for cancer risk in humans. A two-stage, or "nested" polymerase chain reaction method is disclosed for detecting methylated DNA sequences at sufficiently high levels of sensitivity to permit cancer screening in biological fluid samples, such as sputum, obtained non-invasively. The method is for detecting the aberrant methylation of the p16 gene, O 6-methylguanine-DNA methyltransferase gene, Death-associated protein kinase gene, RAS-associated family 1 gene, or other gene promoters. The method offers a potentially powerful approach to population-based screening for the detection ofmore » lung and other cancers.« less

Real-Time PCR Typing of Escherichia coli Based on Multiple Single Nucleotide Polymorphisms--a Convenient and Rapid Method.

PubMed

Lager, Malin; Mernelius, Sara; Löfgren, Sture; Söderman, Jan

2016-01-01

Healthcare-associated infections caused by Escherichia coli and antibiotic resistance due to extended-spectrum beta-lactamase (ESBL) production constitute a threat against patient safety. To identify, track, and control outbreaks and to detect emerging virulent clones, typing tools of sufficient discriminatory power that generate reproducible and unambiguous data are needed. A probe based real-time PCR method targeting multiple single nucleotide polymorphisms (SNP) was developed. The method was based on the multi locus sequence typing scheme of Institute Pasteur and by adaptation of previously described typing assays. An 8 SNP-panel that reached a Simpson's diversity index of 0.95 was established, based on analysis of sporadic E. coli cases (ESBL n = 27 and non-ESBL n = 53). This multi-SNP assay was used to identify the sequence type 131 (ST131) complex according to the Achtman's multi locus sequence typing scheme. However, it did not fully discriminate within the complex but provided a diagnostic signature that outperformed a previously described detection assay. Pulsed-field gel electrophoresis typing of isolates from a presumed outbreak (n = 22) identified two outbreaks (ST127 and ST131) and three different non-outbreak-related isolates. Multi-SNP typing generated congruent data except for one non-outbreak-related ST131 isolate. We consider multi-SNP real-time PCR typing an accessible primary generic E. coli typing tool for rapid and uniform type identification.

DNA Extraction Protocols for Whole-Genome Sequencing in Marine Organisms.

PubMed

Panova, Marina; Aronsson, Henrik; Cameron, R Andrew; Dahl, Peter; Godhe, Anna; Lind, Ulrika; Ortega-Martinez, Olga; Pereyra, Ricardo; Tesson, Sylvie V M; Wrange, Anna-Lisa; Blomberg, Anders; Johannesson, Kerstin

2016-01-01

The marine environment harbors a large proportion of the total biodiversity on this planet, including the majority of the earths' different phyla and classes. Studying the genomes of marine organisms can bring interesting insights into genome evolution. Today, almost all marine organismal groups are understudied with respect to their genomes. One potential reason is that extraction of high-quality DNA in sufficient amounts is challenging for many marine species. This is due to high polysaccharide content, polyphenols and other secondary metabolites that will inhibit downstream DNA library preparations. Consequently, protocols developed for vertebrates and plants do not always perform well for invertebrates and algae. In addition, many marine species have large population sizes and, as a consequence, highly variable genomes. Thus, to facilitate the sequence read assembly process during genome sequencing, it is desirable to obtain enough DNA from a single individual, which is a challenge in many species of invertebrates and algae. Here, we present DNA extraction protocols for seven marine species (four invertebrates, two algae, and a marine yeast), optimized to provide sufficient DNA quality and yield for de novo genome sequencing projects.

Late Quaternary climate and environmental reconstruction based on leaf wax analyses in the loess sequence of Möhlin, Switzerland

NASA Astrophysics Data System (ADS)

Wüthrich, Lorenz; Bliedtner, Marcel; Kathrin Schäfer, Imke; Zech, Jana; Shajari, Fatemeh; Gaar, Dorian; Preusser, Frank; Salazar, Gary; Szidat, Sönke; Zech, Roland

2017-12-01

We present the results of leaf wax analyses (long-chain n-alkanes) from the 6.8 m deep loess sequence of Möhlin, Switzerland, spanning the last ˜ 70 kyr. Leaf waxes are well preserved and occur in sufficient amounts only down to 0.4 m and below 1.8 m depth, so no paleoenvironmental reconstructions can be done for marine isotope stage (MIS) 2. Compound-specific δ2Hwax analyses yielded similar values for late MIS 3 compared to the uppermost samples, indicating that various effects (e.g., more negative values due to lower temperatures, more positive values due to an enriched moisture source) cancel each other out. A pronounced ˜ 30 ‰ shift towards more negative values probably reflects more humid conditions before ˜ 32 ka. Radiocarbon dating of the n-alkanes corroborates the stratigraphic integrity of leaf waxes and their potential for dating loess-paleosol sequences (LPS) back to ˜ 30 ka.

Sequence-Based Prediction of RNA-Binding Proteins Using Random Forest with Minimum Redundancy Maximum Relevance Feature Selection.

PubMed

Ma, Xin; Guo, Jing; Sun, Xiao

2015-01-01

The prediction of RNA-binding proteins is one of the most challenging problems in computation biology. Although some studies have investigated this problem, the accuracy of prediction is still not sufficient. In this study, a highly accurate method was developed to predict RNA-binding proteins from amino acid sequences using random forests with the minimum redundancy maximum relevance (mRMR) method, followed by incremental feature selection (IFS). We incorporated features of conjoint triad features and three novel features: binding propensity (BP), nonbinding propensity (NBP), and evolutionary information combined with physicochemical properties (EIPP). The results showed that these novel features have important roles in improving the performance of the predictor. Using the mRMR-IFS method, our predictor achieved the best performance (86.62% accuracy and 0.737 Matthews correlation coefficient). High prediction accuracy and successful prediction performance suggested that our method can be a useful approach to identify RNA-binding proteins from sequence information.

The study of human Y chromosome variation through ancient DNA.

PubMed

Kivisild, Toomas

2017-05-01

High throughput sequencing methods have completely transformed the study of human Y chromosome variation by offering a genome-scale view on genetic variation retrieved from ancient human remains in context of a growing number of high coverage whole Y chromosome sequence data from living populations from across the world. The ancient Y chromosome sequences are providing us the first exciting glimpses into the past variation of male-specific compartment of the genome and the opportunity to evaluate models based on previously made inferences from patterns of genetic variation in living populations. Analyses of the ancient Y chromosome sequences are challenging not only because of issues generally related to ancient DNA work, such as DNA damage-induced mutations and low content of endogenous DNA in most human remains, but also because of specific properties of the Y chromosome, such as its highly repetitive nature and high homology with the X chromosome. Shotgun sequencing of uniquely mapping regions of the Y chromosomes to sufficiently high coverage is still challenging and costly in poorly preserved samples. To increase the coverage of specific target SNPs capture-based methods have been developed and used in recent years to generate Y chromosome sequence data from hundreds of prehistoric skeletal remains. Besides the prospects of testing directly as how much genetic change in a given time period has accompanied changes in material culture the sequencing of ancient Y chromosomes allows us also to better understand the rate at which mutations accumulate and get fixed over time. This review considers genome-scale evidence on ancient Y chromosome diversity that has recently started to accumulate in geographic areas favourable to DNA preservation. More specifically the review focuses on examples of regional continuity and change of the Y chromosome haplogroups in North Eurasia and in the New World.

BAUM: improving genome assembly by adaptive unique mapping and local overlap-layout-consensus approach.

PubMed

Wang, Anqi; Wang, Zhanyu; Li, Zheng; Li, Lei M

2018-06-15

It is highly desirable to assemble genomes of high continuity and consistency at low cost. The current bottleneck of draft genome continuity using the second generation sequencing (SGS) reads is primarily caused by uncertainty among repetitive sequences. Even though the single-molecule real-time sequencing technology is very promising to overcome the uncertainty issue, its relatively high cost and error rate add burden on budget or computation. Many long-read assemblers take the overlap-layout-consensus (OLC) paradigm, which is less sensitive to sequencing errors, heterozygosity and variability of coverage. However, current assemblers of SGS data do not sufficiently take advantage of the OLC approach. Aiming at minimizing uncertainty, the proposed method BAUM, breaks the whole genome into regions by adaptive unique mapping; then the local OLC is used to assemble each region in parallel. BAUM can (i) perform reference-assisted assembly based on the genome of a close species (ii) or improve the results of existing assemblies that are obtained based on short or long sequencing reads. The tests on two eukaryote genomes, a wild rice Oryza longistaminata and a parrot Melopsittacus undulatus, show that BAUM achieved substantial improvement on genome size and continuity. Besides, BAUM reconstructed a considerable amount of repetitive regions that failed to be assembled by existing short read assemblers. We also propose statistical approaches to control the uncertainty in different steps of BAUM. http://www.zhanyuwang.xin/wordpress/index.php/2017/07/21/baum. Supplementary data are available at Bioinformatics online.

Convolutional neural network architectures for predicting DNA–protein binding

PubMed Central

Zeng, Haoyang; Edwards, Matthew D.; Liu, Ge; Gifford, David K.

2016-01-01

Motivation: Convolutional neural networks (CNN) have outperformed conventional methods in modeling the sequence specificity of DNA–protein binding. Yet inappropriate CNN architectures can yield poorer performance than simpler models. Thus an in-depth understanding of how to match CNN architecture to a given task is needed to fully harness the power of CNNs for computational biology applications. Results: We present a systematic exploration of CNN architectures for predicting DNA sequence binding using a large compendium of transcription factor datasets. We identify the best-performing architectures by varying CNN width, depth and pooling designs. We find that adding convolutional kernels to a network is important for motif-based tasks. We show the benefits of CNNs in learning rich higher-order sequence features, such as secondary motifs and local sequence context, by comparing network performance on multiple modeling tasks ranging in difficulty. We also demonstrate how careful construction of sequence benchmark datasets, using approaches that control potentially confounding effects like positional or motif strength bias, is critical in making fair comparisons between competing methods. We explore how to establish the sufficiency of training data for these learning tasks, and we have created a flexible cloud-based framework that permits the rapid exploration of alternative neural network architectures for problems in computational biology. Availability and Implementation: All the models analyzed are available at http://cnn.csail.mit.edu. Contact: gifford@mit.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:27307608

The C-terminal portion of the cleaved HT motif is necessary and sufficient to mediate export of proteins from the malaria parasite into its host cell

PubMed Central

Tarr, Sarah J; Cryar, Adam; Thalassinos, Konstantinos; Haldar, Kasturi; Osborne, Andrew R

2013-01-01

The malaria parasite exports proteins across its plasma membrane and a surrounding parasitophorous vacuole membrane, into its host erythrocyte. Most exported proteins contain a Host Targeting motif (HT motif) that targets them for export. In the parasite secretory pathway, the HT motif is cleaved by the protease plasmepsin V, but the role of the newly generated N-terminal sequence in protein export is unclear. Using a model protein that is cleaved by an exogenous viral protease, we show that the new N-terminal sequence, normally generated by plasmepsin V cleavage, is sufficient to target a protein for export, and that cleavage by plasmepsin V is not coupled directly to the transfer of a protein to the next component in the export pathway. Mutation of the fourth and fifth positions of the HT motif, as well as amino acids further downstream, block or affect the efficiency of protein export indicating that this region is necessary for efficient export. We also show that the fifth position of the HT motif is important for plasmepsin V cleavage. Our results indicate that plasmepsin V cleavage is required to generate a new N-terminal sequence that is necessary and sufficient to mediate protein export by the malaria parasite. PMID:23279267

Sequencing degraded DNA from non-destructively sampled museum specimens for RAD-tagging and low-coverage shotgun phylogenetics.

PubMed

Tin, Mandy Man-Ying; Economo, Evan Philip; Mikheyev, Alexander Sergeyevich

2014-01-01

Ancient and archival DNA samples are valuable resources for the study of diverse historical processes. In particular, museum specimens provide access to biotas distant in time and space, and can provide insights into ecological and evolutionary changes over time. However, archival specimens are difficult to handle; they are often fragile and irreplaceable, and typically contain only short segments of denatured DNA. Here we present a set of tools for processing such samples for state-of-the-art genetic analysis. First, we report a protocol for minimally destructive DNA extraction of insect museum specimens, which produced sequenceable DNA from all of the samples assayed. The 11 specimens analyzed had fragmented DNA, rarely exceeding 100 bp in length, and could not be amplified by conventional PCR targeting the mitochondrial cytochrome oxidase I gene. Our approach made these samples amenable to analysis with commonly used next-generation sequencing-based molecular analytic tools, including RAD-tagging and shotgun genome re-sequencing. First, we used museum ant specimens from three species, each with its own reference genome, for RAD-tag mapping. Were able to use the degraded DNA sequences, which were sequenced in full, to identify duplicate reads and filter them prior to base calling. Second, we re-sequenced six Hawaiian Drosophila species, with millions of years of divergence, but with only a single available reference genome. Despite a shallow coverage of 0.37 ± 0.42 per base, we could recover a sufficient number of overlapping SNPs to fully resolve the species tree, which was consistent with earlier karyotypic studies, and previous molecular studies, at least in the regions of the tree that these studies could resolve. Although developed for use with degraded DNA, all of these techniques are readily applicable to more recent tissue, and are suitable for liquid handling automation.

Bartonella dromedarii sp. nov. isolated from domesticated camels (Camelus dromedarius) in Israel.

PubMed

Rasis, Michal; Rudoler, Nir; Schwartz, David; Giladi, Michael

2014-11-01

Bartonella spp. are fastidious, Gram-negative bacilli that cause a wide spectrum of diseases in humans. Most Bartonella spp. have adapted to a specific host, generally a domestic or wild mammal. Dromedary camels (Camelus dromedarius) have become a focus of growing public-health interest because they have been identified as a reservoir host for the Middle East respiratory syndrome coronavirus. Nevertheless, data on camel zoonoses are limited. We aimed to study the occurrence of Bartonella bacteremia among dromedaries in Israel. Nine of 51 (17.6%) camels were found to be bacteremic with Bartonella spp.; bacteremia levels ranged from five to >1000 colony-forming units/mL. Phylogenetic reconstruction based on the concatenated sequences of gltA and rpoB genes demonstrated that the dromedary Bartonella isolates are closely related to other ruminant-derived Bartonella spp., with B. bovis being the nearest relative. Using electron microscopy, the novel isolates were shown to be flagellated, whereas B. bovis is nonflagellated. Sequence comparisons analysis of the housekeeping genes ftsZ, ribC, and groEL showed the highest homology to B. chomelii, B. capreoli, and B. birtlesii, respectively. Sequence analysis of the gltA and rpoB revealed ∼96% identity to B. bovis, a previously suggested cutoff value for sequence-based differentiation of Bartonella spp., suggesting that this approach does not have sufficient discriminatory power for differentiating ruminant-related Bartonella spp. A comprehensive multilocus sequence typing (MLST) analysis based on nine genetic loci (gltA, rpoB, ftsZ, internal transcribed spacer (ITS), 16S rRNA, ribC, groEL, nuoG, and SsrA) identified seven sequence types of the new dromedary isolates. This is the first description of a Bartonella sp. from camelids. On the basis of a distinct reservoir and ecological niche, sequence analyses, and expression of flagella, we designate these isolates as a novel Bartonella sp. named Bartonella dromedarii sp. nov. Further studies are required to explore its zoonotic potential.

Impact of Therapy Sequence with Alkylating Agents and MGMT Status in Patients with Advanced Neuroendocrine Tumors.

PubMed

Krug, Sebastian; Boch, Michael; Rexin, Peter; Gress, Thomas M; Michl, Patrick; Rinke, Anja

2017-05-01

Alkylating chemotherapeutics with either a streptozotocin-(STZ) or temozolomide-(TEM) backbone are routinely used in patients with progressive and unresectable pancreatic neuroendocrine tumors (PNET). In addition, dacarbazine (DTIC) was described as an alternative alkylating therapy option for PNETs. The optimal treatment sequence with alkylating compounds and a potential use of O6-methylguanine-DNA methyltransferase (MGMT) level as predictive biomarker have not yet been sufficiently elucidated. The aim of our study was the evaluation of therapy sequence with either STZ-based treatment followed by DTIC (group A) or the inverse schedule with upfront DTIC (group B) and to correlate MGMT status with clinicopathological characteristics and response to therapy. We retrospectively analyzed 28 patients with neuroendocrine tumors (NET) who were treated with STZ-based therapy and DTIC. Additionally, in a second group MGMT immunohistochemistry was performed from primary and metastatic tumor sites. For statistical evaluation Kaplan-Meier analysis, Cox regression methods and Fisher's exact test were used. There was no difference of objective response and disease control between either STZ-based therapy followed by DTIC treatment (group A) after progression or the reverse sequence (group B). Median time to progression (TTP) was estimated to be 21 months in both arms. First-line STZ-based chemotherapy was not superior to first-line DTIC treatment (16 vs. 13 months; p=0.8). MGMT status did not correlate with clinicopathological characteristics or response to therapy with these alkylating agents. Upfront chemotherapy with either STZ-based treatment or DTIC monotherapy showed similar efficacy and median TTP rates. In this study, MGMT protein expression assessed by immunohistochemistry did not play an important role as a predictive marker for alkylating agents. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Identification of simple objects in image sequences

NASA Astrophysics Data System (ADS)

Geiselmann, Christoph; Hahn, Michael

1994-08-01

We present an investigation in the identification and location of simple objects in color image sequences. As an example the identification of traffic signs is discussed. Three aspects are of special interest. First regions have to be detected which may contain the object. The separation of those regions from the background can be based on color, motion, and contours. In the experiments all three possibilities are investigated. The second aspect focuses on the extraction of suitable features for the identification of the objects. For that purpose the border line of the region of interest is used. For planar objects a sufficient approximation of perspective projection is affine mapping. In consequence, it is near at hand to extract affine-invariant features from the border line. The investigation includes invariant features based on Fourier descriptors and moments. Finally, the object is identified by maximum likelihood classification. In the experiments all three basic object types are correctly identified. The probabilities for misclassification have been found to be below 1%

Versatile single-chip event sequencer for atomic physics experiments

NASA Astrophysics Data System (ADS)

Eyler, Edward

2010-03-01

A very inexpensive dsPIC microcontroller with internal 32-bit counters is used to produce a flexible timing signal generator with up to 16 TTL-compatible digital outputs, with a time resolution and accuracy of 50 ns. This time resolution is easily sufficient for event sequencing in typical experiments involving cold atoms or laser spectroscopy. This single-chip device is capable of triggered operation and can also function as a sweeping delay generator. With one additional chip it can also concurrently produce accurately timed analog ramps, and another one-chip addition allows real-time control from an external computer. Compared to an FPGA-based digital pattern generator, this design is slower but simpler and more flexible, and it can be reprogrammed using ordinary `C' code without special knowledge. I will also describe the use of the same microcontroller with additional hardware to implement a digital lock-in amplifier and PID controller for laser locking, including a simple graphics-based control unit. This work is supported in part by the NSF.

Scene-based nonuniformity correction with video sequences and registration.

PubMed

Hardie, R C; Hayat, M M; Armstrong, E; Yasuda, B

2000-03-10

We describe a new, to our knowledge, scene-based nonuniformity correction algorithm for array detectors. The algorithm relies on the ability to register a sequence of observed frames in the presence of the fixed-pattern noise caused by pixel-to-pixel nonuniformity. In low-to-moderate levels of nonuniformity, sufficiently accurate registration may be possible with standard scene-based registration techniques. If the registration is accurate, and motion exists between the frames, then groups of independent detectors can be identified that observe the same irradiance (or true scene value). These detector outputs are averaged to generate estimates of the true scene values. With these scene estimates, and the corresponding observed values through a given detector, a curve-fitting procedure is used to estimate the individual detector response parameters. These can then be used to correct for detector nonuniformity. The strength of the algorithm lies in its simplicity and low computational complexity. Experimental results, to illustrate the performance of the algorithm, include the use of visible-range imagery with simulated nonuniformity and infrared imagery with real nonuniformity.

Lazy evaluation of FP programs: A data-flow approach

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wei, Y.H.; Gaudiot, J.L.

1988-12-31

This paper presents a lazy evaluation system for the list-based functional language, Backus` FP in data-driven environment. A superset language of FP, called DFP (Demand-driven FP), is introduced. FP eager programs are transformed into DFP lazy programs which contain the notions of demands. The data-driven execution of DFP programs has the same effects of lazy evaluation. DFP lazy programs have the property of always evaluating a sufficient and necessary result. The infinite sequence generator is used to demonstrate the eager-lazy program transformation and the execution of the lazy programs.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Merkley, Eric D.; Sego, Landon H.; Lin, Andy

Adaptive processes in bacterial species can occur rapidly in laboratory culture, leading to genetic divergence between naturally occurring and laboratory-adapted strains. Differentiating wild and closely-related laboratory strains is clearly important for biodefense and bioforensics; however, DNA sequence data alone has thus far not provided a clear signature, perhaps due to lack of understanding of how diverse genome changes lead to adapted phenotypes. Protein abundance profiles from mass spectrometry-based proteomics analyses are a molecular measure of phenotype. Proteomics data contains sufficient information that powerful statistical methods can uncover signatures that distinguish wild strains of Yersinia pestis from laboratory-adapted strains.

Successful isolation and PCR amplification of DNA from National Institute of Standards and Technology herbal dietary supplement standard reference material powders and extracts.

PubMed

Cimino, Matthew T

2010-03-01

Twenty-four herbal dietary supplement powder and extract reference standards provided by the National Institute of Standards and Technology (NIST) were investigated using three different commercially available DNA extraction kits to evaluate DNA availability for downstream nucleotide-based applications. The material included samples of Camellia, Citrus, Ephedra, Ginkgo, Hypericum, Serenoa, And Vaccinium. Protocols from Qiagen, MoBio, and Phytopure were used to isolate and purify DNA from the NIST standards. The resulting DNA concentration was quantified using SYBR Green fluorometry. Each of the 24 samples yielded DNA, though the concentration of DNA from each approach was notably different. The Phytopure method consistently yielded more DNA. The average yield ratio was 22 : 3 : 1 (ng/microL; Phytopure : Qiagen : MoBio). Amplification of the internal transcribed spacer II region using PCR was ultimately successful in 22 of the 24 samples. Direct sequencing chromatograms of the amplified material suggested that most of the samples were comprised of mixtures. However, the sequencing chromatograms of 12 of the 24 samples were sufficient to confirm the identity of the target material. The successful extraction, amplification, and sequencing of DNA from these herbal dietary supplement extracts and powders supports a continued effort to explore nucleotide sequence-based tools for the authentication and identification of plants in dietary supplements. (c) Georg Thieme Verlag KG Stuttgart . New York.

Sonication-based isolation and enrichment of Chlorella protothecoides chloroplasts for illumina genome sequencing

DOE Office of Scientific and Technical Information (OSTI.GOV)

Angelova, Angelina; Park, Sang-Hycuk; Kyndt, John

2013-09-01

With the increasing world demand for biofuel, a number of oleaginous algal species are being considered as renewable sources of oil. Chlorella protothecoides Krüger synthesizes triacylglycerols (TAGs) as storage compounds that can be converted into renewable fuel utilizing an anabolic pathway that is poorly understood. The paucity of algal chloroplast genome sequences has been an important constraint to chloroplast transformation and for studying gene expression in TAGs pathways. In this study, the intact chloroplasts were released from algal cells using sonication followed by sucrose gradient centrifugation, resulting in a 2.36-fold enrichment of chloroplasts from C. protothecoides, based on qPCR analysis.more » The C. protothecoides chloroplast genome (cpDNA) was determined using the Illumina HiSeq 2000 sequencing platform and found to be 84,576 Kb in size (8.57 Kb) in size, with a GC content of 30.8 %. This is the first report of an optimized protocol that uses a sonication step, followed by sucrose gradient centrifugation, to release and enrich intact chloroplasts from a microalga (C. prototheocoides) of sufficient quality to permit chloroplast genome sequencing with high coverage, while minimizing nuclear genome contamination. The approach is expected to guide chloroplast isolation from other oleaginous algal species for a variety of uses that benefit from enrichment of chloroplasts, ranging from biochemical analysis to genomics studies.« less

How Good Are Statistical Models at Approximating Complex Fitness Landscapes?

PubMed Central

du Plessis, Louis; Leventhal, Gabriel E.; Bonhoeffer, Sebastian

2016-01-01

Fitness landscapes determine the course of adaptation by constraining and shaping evolutionary trajectories. Knowledge of the structure of a fitness landscape can thus predict evolutionary outcomes. Empirical fitness landscapes, however, have so far only offered limited insight into real-world questions, as the high dimensionality of sequence spaces makes it impossible to exhaustively measure the fitness of all variants of biologically meaningful sequences. We must therefore revert to statistical descriptions of fitness landscapes that are based on a sparse sample of fitness measurements. It remains unclear, however, how much data are required for such statistical descriptions to be useful. Here, we assess the ability of regression models accounting for single and pairwise mutations to correctly approximate a complex quasi-empirical fitness landscape. We compare approximations based on various sampling regimes of an RNA landscape and find that the sampling regime strongly influences the quality of the regression. On the one hand it is generally impossible to generate sufficient samples to achieve a good approximation of the complete fitness landscape, and on the other hand systematic sampling schemes can only provide a good description of the immediate neighborhood of a sequence of interest. Nevertheless, we obtain a remarkably good and unbiased fit to the local landscape when using sequences from a population that has evolved under strong selection. Thus, current statistical methods can provide a good approximation to the landscape of naturally evolving populations. PMID:27189564

Functional region prediction with a set of appropriate homologous sequences-an index for sequence selection by integrating structure and sequence information with spatial statistics

PubMed Central

2012-01-01

Background The detection of conserved residue clusters on a protein structure is one of the effective strategies for the prediction of functional protein regions. Various methods, such as Evolutionary Trace, have been developed based on this strategy. In such approaches, the conserved residues are identified through comparisons of homologous amino acid sequences. Therefore, the selection of homologous sequences is a critical step. It is empirically known that a certain degree of sequence divergence in the set of homologous sequences is required for the identification of conserved residues. However, the development of a method to select homologous sequences appropriate for the identification of conserved residues has not been sufficiently addressed. An objective and general method to select appropriate homologous sequences is desired for the efficient prediction of functional regions. Results We have developed a novel index to select the sequences appropriate for the identification of conserved residues, and implemented the index within our method to predict the functional regions of a protein. The implementation of the index improved the performance of the functional region prediction. The index represents the degree of conserved residue clustering on the tertiary structure of the protein. For this purpose, the structure and sequence information were integrated within the index by the application of spatial statistics. Spatial statistics is a field of statistics in which not only the attributes but also the geometrical coordinates of the data are considered simultaneously. Higher degrees of clustering generate larger index scores. We adopted the set of homologous sequences with the highest index score, under the assumption that the best prediction accuracy is obtained when the degree of clustering is the maximum. The set of sequences selected by the index led to higher functional region prediction performance than the sets of sequences selected by other sequence-based methods. Conclusions Appropriate homologous sequences are selected automatically and objectively by the index. Such sequence selection improved the performance of functional region prediction. As far as we know, this is the first approach in which spatial statistics have been applied to protein analyses. Such integration of structure and sequence information would be useful for other bioinformatics problems. PMID:22643026

Diversity analysis in Cannabis sativa based on large-scale development of expressed sequence tag-derived simple sequence repeat markers.

PubMed

Gao, Chunsheng; Xin, Pengfei; Cheng, Chaohua; Tang, Qing; Chen, Ping; Wang, Changbiao; Zang, Gonggu; Zhao, Lining

2014-01-01

Cannabis sativa L. is an important economic plant for the production of food, fiber, oils, and intoxicants. However, lack of sufficient simple sequence repeat (SSR) markers has limited the development of cannabis genetic research. Here, large-scale development of expressed sequence tag simple sequence repeat (EST-SSR) markers was performed to obtain more informative genetic markers, and to assess genetic diversity in cannabis (Cannabis sativa L.). Based on the cannabis transcriptome, 4,577 SSRs were identified from 3,624 ESTs. From there, a total of 3,442 complementary primer pairs were designed as SSR markers. Among these markers, trinucleotide repeat motifs (50.99%) were the most abundant, followed by hexanucleotide (25.13%), dinucleotide (16.34%), tetranucloetide (3.8%), and pentanucleotide (3.74%) repeat motifs, respectively. The AAG/CTT trinucleotide repeat (17.96%) was the most abundant motif detected in the SSRs. One hundred and seventeen EST-SSR markers were randomly selected to evaluate primer quality in 24 cannabis varieties. Among these 117 markers, 108 (92.31%) were successfully amplified and 87 (74.36%) were polymorphic. Forty-five polymorphic primer pairs were selected to evaluate genetic diversity and relatedness among the 115 cannabis genotypes. The results showed that 115 varieties could be divided into 4 groups primarily based on geography: Northern China, Europe, Central China, and Southern China. Moreover, the coefficient of similarity when comparing cannabis from Northern China with the European group cannabis was higher than that when comparing with cannabis from the other two groups, owing to a similar climate. This study outlines the first large-scale development of SSR markers for cannabis. These data may serve as a foundation for the development of genetic linkage, quantitative trait loci mapping, and marker-assisted breeding of cannabis.

Diversity Analysis in Cannabis sativa Based on Large-Scale Development of Expressed Sequence Tag-Derived Simple Sequence Repeat Markers

PubMed Central

Cheng, Chaohua; Tang, Qing; Chen, Ping; Wang, Changbiao; Zang, Gonggu; Zhao, Lining

2014-01-01

Cannabis sativa L. is an important economic plant for the production of food, fiber, oils, and intoxicants. However, lack of sufficient simple sequence repeat (SSR) markers has limited the development of cannabis genetic research. Here, large-scale development of expressed sequence tag simple sequence repeat (EST-SSR) markers was performed to obtain more informative genetic markers, and to assess genetic diversity in cannabis (Cannabis sativa L.). Based on the cannabis transcriptome, 4,577 SSRs were identified from 3,624 ESTs. From there, a total of 3,442 complementary primer pairs were designed as SSR markers. Among these markers, trinucleotide repeat motifs (50.99%) were the most abundant, followed by hexanucleotide (25.13%), dinucleotide (16.34%), tetranucloetide (3.8%), and pentanucleotide (3.74%) repeat motifs, respectively. The AAG/CTT trinucleotide repeat (17.96%) was the most abundant motif detected in the SSRs. One hundred and seventeen EST-SSR markers were randomly selected to evaluate primer quality in 24 cannabis varieties. Among these 117 markers, 108 (92.31%) were successfully amplified and 87 (74.36%) were polymorphic. Forty-five polymorphic primer pairs were selected to evaluate genetic diversity and relatedness among the 115 cannabis genotypes. The results showed that 115 varieties could be divided into 4 groups primarily based on geography: Northern China, Europe, Central China, and Southern China. Moreover, the coefficient of similarity when comparing cannabis from Northern China with the European group cannabis was higher than that when comparing with cannabis from the other two groups, owing to a similar climate. This study outlines the first large-scale development of SSR markers for cannabis. These data may serve as a foundation for the development of genetic linkage, quantitative trait loci mapping, and marker-assisted breeding of cannabis. PMID:25329551

Matrix Transformations between Certain Sequence Spaces over the Non-Newtonian Complex Field

PubMed Central

Efe, Hakan

2014-01-01

In some cases, the most general linear operator between two sequence spaces is given by an infinite matrix. So the theory of matrix transformations has always been of great interest in the study of sequence spaces. In the present paper, we introduce the matrix transformations in sequence spaces over the field ℂ* and characterize some classes of infinite matrices with respect to the non-Newtonian calculus. Also we give the necessary and sufficient conditions on an infinite matrix transforming one of the classical sets over ℂ* to another one. Furthermore, the concept for sequence-to-sequence and series-to-series methods of summability is given with some illustrated examples. PMID:25110740

Use of Formulaic Sequences in Monologues of Chinese EFL Learners

ERIC Educational Resources Information Center

Qi, Yan; Ding, Yanren

2011-01-01

The literature on formulaic language lacks sufficient research on how L2 learners make progress in native-like formulaicity of their target language. This study analyzed the use of formulaic sequences (FSs) by 56 Chinese university English majors in their prepared monologues at the beginning and end of a three-year period and compared the student…

Mitochondrial sequencing reveals five separate origins of 'black' Apis mellifera (Hymenoptera: Apidae) in eastern Australian commercial colonies.

PubMed

Oxley, P R; Oldroyd, B P

2009-04-01

Establishment of a closed population honey bee, Apis mellifera L. (Hymenoptera: Apidae), breeding program based on 'black' strains has been proposed for eastern Australia. Long-term success of such a program requires a high level of genetic variance. To determine the likely extent of genetic variation available, 50 colonies from 11 different commercial apiaries were sequenced in the mitochondrial cytochrome oxidase I and II intergenic region. Five distinct and novel mitotypes were identified. No colonies were found with the A. mellifera mellifera mitotype, which is often associated with undesirable feral strains. One group of mitotypes was consistent with a caucasica origin, two with carnica, and two with ligustica. The results suggest that there is sufficient genetic diversity to support a breeding program provided all these five sources were pooled.

Constructing and Modifying Sequence Statistics for relevent Using informR in 𝖱

PubMed Central

Marcum, Christopher Steven; Butts, Carter T.

2015-01-01

The informR package greatly simplifies the analysis of complex event histories in 𝖱 by providing user friendly tools to build sufficient statistics for the relevent package. Historically, building sufficient statistics to model event sequences (of the form a→b) using the egocentric generalization of Butts’ (2008) relational event framework for modeling social action has been cumbersome. The informR package simplifies the construction of the complex list of arrays needed by the rem() model fitting for a variety of cases involving egocentric event data, multiple event types, and/or support constraints. This paper introduces these tools using examples from real data extracted from the American Time Use Survey. PMID:26185488

Nucleic acid molecules encoding isopentenyl monophosphate kinase, and methods of use

DOEpatents

Croteau, Rodney B.; Lange, Bernd M.

2001-01-01

A cDNA encoding isopentenyl monophosphate kinase (IPK) from peppermint (Mentha x piperita) has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Accordingly, an isolated DNA sequence (SEQ ID NO:1) is provided which codes for the expression of isopentenyl monophosphate kinase (SEQ ID NO:2), from peppermint (Mentha x piperita). In other aspects, replicable recombinant cloning vehicles are provided which code for isopentenyl monophosphate kinase, or for a base sequence sufficiently complementary to at least a portion of isopentenyl monophosphate kinase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding isopentenyl monophosphate kinase. Thus, systems and methods are provided for the recombinant expression of the aforementioned recombinant isopentenyl monophosphate kinase that may be used to facilitate its production, isolation and purification in significant amounts. Recombinant isopentenyl monophosphate kinase may be used to obtain expression or enhanced expression of isopentenyl monophosphate kinase in plants in order to enhance the production of isopentenyl monophosphate kinase, or isoprenoids derived therefrom, or may be otherwise employed for the regulation or expression of isopentenyl monophosphate kinase, or the production of its products.

Deblurring sequential ocular images from multi-spectral imaging (MSI) via mutual information.

PubMed

Lian, Jian; Zheng, Yuanjie; Jiao, Wanzhen; Yan, Fang; Zhao, Bojun

2018-06-01

Multi-spectral imaging (MSI) produces a sequence of spectral images to capture the inner structure of different species, which was recently introduced into ocular disease diagnosis. However, the quality of MSI images can be significantly degraded by motion blur caused by the inevitable saccades and exposure time required for maintaining a sufficiently high signal-to-noise ratio. This degradation may confuse an ophthalmologist, reduce the examination quality, or defeat various image analysis algorithms. We propose an early work specially on deblurring sequential MSI images, which is distinguished from many of the current image deblurring techniques by resolving the blur kernel simultaneously for all the images in an MSI sequence. It is accomplished by incorporating several a priori constraints including the sharpness of the latent clear image, the spatial and temporal smoothness of the blur kernel and the similarity between temporally-neighboring images in MSI sequence. Specifically, we model the similarity between MSI images with mutual information considering the different wavelengths used for capturing different images in MSI sequence. The optimization of the proposed approach is based on a multi-scale framework and stepwise optimization strategy. Experimental results from 22 MSI sequences validate that our approach outperforms several state-of-the-art techniques in natural image deblurring.

Sequences spanning the leader-repeat junction mediate CRISPR adaptation to phage in Streptococcus thermophilus

PubMed Central

Wei, Yunzhou; Chesne, Megan T.; Terns, Rebecca M.; Terns, Michael P.

2015-01-01

CRISPR-Cas systems are RNA-based immune systems that protect prokaryotes from invaders such as phages and plasmids. In adaptation, the initial phase of the immune response, short foreign DNA fragments are captured and integrated into host CRISPR loci to provide heritable defense against encountered foreign nucleic acids. Each CRISPR contains a ∼100–500 bp leader element that typically includes a transcription promoter, followed by an array of captured ∼35 bp sequences (spacers) sandwiched between copies of an identical ∼35 bp direct repeat sequence. New spacers are added immediately downstream of the leader. Here, we have analyzed adaptation to phage infection in Streptococcus thermophilus at the CRISPR1 locus to identify cis-acting elements essential for the process. We show that the leader and a single repeat of the CRISPR locus are sufficient for adaptation in this system. Moreover, we identified a leader sequence element capable of stimulating adaptation at a dormant repeat. We found that sequences within 10 bp of the site of integration, in both the leader and repeat of the CRISPR, are required for the process. Our results indicate that information at the CRISPR leader-repeat junction is critical for adaptation in this Type II-A system and likely other CRISPR-Cas systems. PMID:25589547

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

PubMed

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

2012-09-11

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

A survey and evaluations of histogram-based statistics in alignment-free sequence comparison.

PubMed

Luczak, Brian B; James, Benjamin T; Girgis, Hani Z

2017-12-06

Since the dawn of the bioinformatics field, sequence alignment scores have been the main method for comparing sequences. However, alignment algorithms are quadratic, requiring long execution time. As alternatives, scientists have developed tens of alignment-free statistics for measuring the similarity between two sequences. We surveyed tens of alignment-free k-mer statistics. Additionally, we evaluated 33 statistics and multiplicative combinations between the statistics and/or their squares. These statistics are calculated on two k-mer histograms representing two sequences. Our evaluations using global alignment scores revealed that the majority of the statistics are sensitive and capable of finding similar sequences to a query sequence. Therefore, any of these statistics can filter out dissimilar sequences quickly. Further, we observed that multiplicative combinations of the statistics are highly correlated with the identity score. Furthermore, combinations involving sequence length difference or Earth Mover's distance, which takes the length difference into account, are always among the highest correlated paired statistics with identity scores. Similarly, paired statistics including length difference or Earth Mover's distance are among the best performers in finding the K-closest sequences. Interestingly, similar performance can be obtained using histograms of shorter words, resulting in reducing the memory requirement and increasing the speed remarkably. Moreover, we found that simple single statistics are sufficient for processing next-generation sequencing reads and for applications relying on local alignment. Finally, we measured the time requirement of each statistic. The survey and the evaluations will help scientists with identifying efficient alternatives to the costly alignment algorithm, saving thousands of computational hours. The source code of the benchmarking tool is available as Supplementary Materials. © The Author 2017. Published by Oxford University Press.

Analyzing the relationship between sequence divergence and nodal support using Bayesian phylogenetic analyses.

PubMed

Makowsky, Robert; Cox, Christian L; Roelke, Corey; Chippindale, Paul T

2010-11-01

Determining the appropriate gene for phylogeny reconstruction can be a difficult process. Rapidly evolving genes tend to resolve recent relationships, but suffer from alignment issues and increased homoplasy among distantly related species. Conversely, slowly evolving genes generally perform best for deeper relationships, but lack sufficient variation to resolve recent relationships. We determine the relationship between sequence divergence and Bayesian phylogenetic reconstruction ability using both natural and simulated datasets. The natural data are based on 28 well-supported relationships within the subphylum Vertebrata. Sequences of 12 genes were acquired and Bayesian analyses were used to determine phylogenetic support for correct relationships. Simulated datasets were designed to determine whether an optimal range of sequence divergence exists across extreme phylogenetic conditions. Across all genes we found that an optimal range of divergence for resolving the correct relationships does exist, although this level of divergence expectedly depends on the distance metric. Simulated datasets show that an optimal range of sequence divergence exists across diverse topologies and models of evolution. We determine that a simple to measure property of genetic sequences (genetic distance) is related to phylogenic reconstruction ability in Bayesian analyses. This information should be useful for selecting the most informative gene to resolve any relationships, especially those that are difficult to resolve, as well as minimizing both cost and confounding information during project design. Copyright © 2010. Published by Elsevier Inc.

Automated use of mutagenesis data in structure prediction.

PubMed

Nanda, Vikas; DeGrado, William F

2005-05-15

In the absence of experimental structural determination, numerous methods are available to indirectly predict or probe the structure of a target molecule. Genetic modification of a protein sequence is a powerful tool for identifying key residues involved in binding reactions or protein stability. Mutagenesis data is usually incorporated into the modeling process either through manual inspection of model compatibility with empirical data, or through the generation of geometric constraints linking sensitive residues to a binding interface. We present an approach derived from statistical studies of lattice models for introducing mutation information directly into the fitness score. The approach takes into account the phenotype of mutation (neutral or disruptive) and calculates the energy for a given structure over an ensemble of sequences. The structure prediction procedure searches for the optimal conformation where neutral sequences either have no impact or improve stability and disruptive sequences reduce stability relative to wild type. We examine three types of sequence ensembles: information from saturation mutagenesis, scanning mutagenesis, and homologous proteins. Incorporating multiple sequences into a statistical ensemble serves to energetically separate the native state and misfolded structures. As a result, the prediction of structure with a poor force field is sufficiently enhanced by mutational information to improve accuracy. Furthermore, by separating misfolded conformations from the target score, the ensemble energy serves to speed up conformational search algorithms such as Monte Carlo-based methods. Copyright 2005 Wiley-Liss, Inc.

A novel multi-locus sequence typing (MLST) protocol for Leuconostoc lactis isolates from traditional dairy products in China and Mongolia.

PubMed

Dan, Tong; Liu, Wenjun; Sun, Zhihong; Lv, Qiang; Xu, Haiyan; Song, Yuqin; Zhang, Heping

2014-06-09

Economically, Leuconostoc lactis is one of the most important species in the genus Leuconostoc. It plays an important role in the food industry including the production of dextrans and bacteriocins. Currently, traditional molecular typing approaches for characterisation of this species at the isolate level are either unavailable or are not sufficiently reliable for practical use. Multilocus sequence typing (MLST) is a robust and reliable method for characterising bacterial and fungal species at the molecular level. In this study, a novel MLST protocol was developed for 50 L. lactis isolates from Mongolia and China. Sequences from eight targeted genes (groEL, carB, recA, pheS, murC, pyrG, rpoB and uvrC) were obtained. Sequence analysis indicated 20 different sequence types (STs), with 13 of them being represented by a single isolate. Phylogenetic analysis based on the sequences of eight MLST loci indicated that the isolates belonged to two major groups, A (34 isolates) and B (16 isolates). Linkage disequilibrium analyses indicated that recombination occurred at a low frequency in L. lactis, indicating a clonal population structure. Split-decomposition analysis indicated that intraspecies recombination played a role in generating genotypic diversity amongst isolates. Our results indicated that MLST is a valuable tool for typing L. lactis isolates that can be used for further monitoring of evolutionary changes and population genetics.

Structurally detailed coarse-grained model for Sec-facilitated co-translational protein translocation and membrane integration

PubMed Central

Miller, Thomas F.

2017-01-01

We present a coarse-grained simulation model that is capable of simulating the minute-timescale dynamics of protein translocation and membrane integration via the Sec translocon, while retaining sufficient chemical and structural detail to capture many of the sequence-specific interactions that drive these processes. The model includes accurate geometric representations of the ribosome and Sec translocon, obtained directly from experimental structures, and interactions parameterized from nearly 200 μs of residue-based coarse-grained molecular dynamics simulations. A protocol for mapping amino-acid sequences to coarse-grained beads enables the direct simulation of trajectories for the co-translational insertion of arbitrary polypeptide sequences into the Sec translocon. The model reproduces experimentally observed features of membrane protein integration, including the efficiency with which polypeptide domains integrate into the membrane, the variation in integration efficiency upon single amino-acid mutations, and the orientation of transmembrane domains. The central advantage of the model is that it connects sequence-level protein features to biological observables and timescales, enabling direct simulation for the mechanistic analysis of co-translational integration and for the engineering of membrane proteins with enhanced membrane integration efficiency. PMID:28328943

Nested methylation-specific polymerase chain reaction cancer detection method

DOEpatents

Belinsky, Steven A [Albuquerque, NM; Palmisano, William A [Edgewood, NM

2007-05-08

A molecular marker-based method for monitoring and detecting cancer in humans. Aberrant methylation of gene promoters is a marker for cancer risk in humans. A two-stage, or "nested" polymerase chain reaction method is disclosed for detecting methylated DNA sequences at sufficiently high levels of sensitivity to permit cancer screening in biological fluid samples, such as sputum, obtained non-invasively. The method is for detecting the aberrant methylation of the p16 gene, O 6-methylguanine-DNA methyltransferase gene, Death-associated protein kinase gene, RAS-associated family 1 gene, or other gene promoters. The method offers a potentially powerful approach to population-based screening for the detection of lung and other cancers.

Polymerase ribozyme efficiency increased by G/T-rich DNA oligonucleotides

PubMed Central

Yao, Chengguo; Müller, Ulrich F.

2011-01-01

The RNA world hypothesis states that the early evolution of life went through a stage where RNA served as genome and as catalyst. The replication of RNA world organisms would have been facilitated by ribozymes that catalyze RNA polymerization. To recapitulate an RNA world in the laboratory, a series of RNA polymerase ribozymes was developed previously. However, these ribozymes have a polymerization efficiency that is too low for self-replication, and the most efficient ribozymes prefer one specific template sequence. The limiting factor for polymerization efficiency is the weak sequence-independent binding to its primer/template substrate. Most of the known polymerase ribozymes bind an RNA heptanucleotide to form the P2 duplex on the ribozyme. By modifying this heptanucleotide, we were able to significantly increase polymerization efficiency. Truncations at the 3′-terminus of this heptanucleotide increased full-length primer extension by 10-fold, on a specific template sequence. In contrast, polymerization on several different template sequences was improved dramatically by replacing the RNA heptanucleotide with DNA oligomers containing randomized sequences of 15 nt. The presence of G and T in the random sequences was sufficient for this effect, with an optimal composition of 60% G and 40% T. Our results indicate that these DNA sequences function by establishing many weak and nonspecific base-pairing interactions to the single-stranded portion of the template. Such low-specificity interactions could have had important functions in an RNA world. PMID:21622900

On the necessity of dissecting sequence similarity scores into segment-specific contributions for inferring protein homology, function prediction and annotation

PubMed Central

2014-01-01

Background Protein sequence similarities to any types of non-globular segments (coiled coils, low complexity regions, transmembrane regions, long loops, etc. where either positional sequence conservation is the result of a very simple, physically induced pattern or rather integral sequence properties are critical) are pertinent sources for mistaken homologies. Regretfully, these considerations regularly escape attention in large-scale annotation studies since, often, there is no substitute to manual handling of these cases. Quantitative criteria are required to suppress events of function annotation transfer as a result of false homology assignments. Results The sequence homology concept is based on the similarity comparison between the structural elements, the basic building blocks for conferring the overall fold of a protein. We propose to dissect the total similarity score into fold-critical and other, remaining contributions and suggest that, for a valid homology statement, the fold-relevant score contribution should at least be significant on its own. As part of the article, we provide the DissectHMMER software program for dissecting HMMER2/3 scores into segment-specific contributions. We show that DissectHMMER reproduces HMMER2/3 scores with sufficient accuracy and that it is useful in automated decisions about homology for instructive sequence examples. To generalize the dissection concept for cases without 3D structural information, we find that a dissection based on alignment quality is an appropriate surrogate. The approach was applied to a large-scale study of SMART and PFAM domains in the space of seed sequences and in the space of UniProt/SwissProt. Conclusions Sequence similarity core dissection with regard to fold-critical and other contributions systematically suppresses false hits and, additionally, recovers previously obscured homology relationships such as the one between aquaporins and formate/nitrite transporters that, so far, was only supported by structure comparison. PMID:24890864

Brownian dynamics simulations of sequence-dependent duplex denaturation in dynamically superhelical DNA

NASA Astrophysics Data System (ADS)

Mielke, Steven P.; Grønbech-Jensen, Niels; Krishnan, V. V.; Fink, William H.; Benham, Craig J.

2005-09-01

The topological state of DNA in vivo is dynamically regulated by a number of processes that involve interactions with bound proteins. In one such process, the tracking of RNA polymerase along the double helix during transcription, restriction of rotational motion of the polymerase and associated structures, generates waves of overtwist downstream and undertwist upstream from the site of transcription. The resulting superhelical stress is often sufficient to drive double-stranded DNA into a denatured state at locations such as promoters and origins of replication, where sequence-specific duplex opening is a prerequisite for biological function. In this way, transcription and other events that actively supercoil the DNA provide a mechanism for dynamically coupling genetic activity with regulatory and other cellular processes. Although computer modeling has provided insight into the equilibrium dynamics of DNA supercoiling, to date no model has appeared for simulating sequence-dependent DNA strand separation under the nonequilibrium conditions imposed by the dynamic introduction of torsional stress. Here, we introduce such a model and present results from an initial set of computer simulations in which the sequences of dynamically superhelical, 147 base pair DNA circles were systematically altered in order to probe the accuracy with which the model can predict location, extent, and time of stress-induced duplex denaturation. The results agree both with well-tested statistical mechanical calculations and with available experimental information. Additionally, we find that sites susceptible to denaturation show a propensity for localizing to supercoil apices, suggesting that base sequence determines locations of strand separation not only through the energetics of interstrand interactions, but also by influencing the geometry of supercoiling.

Brownian dynamics simulations of sequence-dependent duplex denaturation in dynamically superhelical DNA.

PubMed

Mielke, Steven P; Grønbech-Jensen, Niels; Krishnan, V V; Fink, William H; Benham, Craig J

2005-09-22

The topological state of DNA in vivo is dynamically regulated by a number of processes that involve interactions with bound proteins. In one such process, the tracking of RNA polymerase along the double helix during transcription, restriction of rotational motion of the polymerase and associated structures, generates waves of overtwist downstream and undertwist upstream from the site of transcription. The resulting superhelical stress is often sufficient to drive double-stranded DNA into a denatured state at locations such as promoters and origins of replication, where sequence-specific duplex opening is a prerequisite for biological function. In this way, transcription and other events that actively supercoil the DNA provide a mechanism for dynamically coupling genetic activity with regulatory and other cellular processes. Although computer modeling has provided insight into the equilibrium dynamics of DNA supercoiling, to date no model has appeared for simulating sequence-dependent DNA strand separation under the nonequilibrium conditions imposed by the dynamic introduction of torsional stress. Here, we introduce such a model and present results from an initial set of computer simulations in which the sequences of dynamically superhelical, 147 base pair DNA circles were systematically altered in order to probe the accuracy with which the model can predict location, extent, and time of stress-induced duplex denaturation. The results agree both with well-tested statistical mechanical calculations and with available experimental information. Additionally, we find that sites susceptible to denaturation show a propensity for localizing to supercoil apices, suggesting that base sequence determines locations of strand separation not only through the energetics of interstrand interactions, but also by influencing the geometry of supercoiling.

Swellix: a computational tool to explore RNA conformational space.

PubMed

Sloat, Nathan; Liu, Jui-Wen; Schroeder, Susan J

2017-11-21

The sequence of nucleotides in an RNA determines the possible base pairs for an RNA fold and thus also determines the overall shape and function of an RNA. The Swellix program presented here combines a helix abstraction with a combinatorial approach to the RNA folding problem in order to compute all possible non-pseudoknotted RNA structures for RNA sequences. The Swellix program builds on the Crumple program and can include experimental constraints on global RNA structures such as the minimum number and lengths of helices from crystallography, cryoelectron microscopy, or in vivo crosslinking and chemical probing methods. The conceptual advance in Swellix is to count helices and generate all possible combinations of helices rather than counting and combining base pairs. Swellix bundles similar helices and includes improvements in memory use and efficient parallelization. Biological applications of Swellix are demonstrated by computing the reduction in conformational space and entropy due to naturally modified nucleotides in tRNA sequences and by motif searches in Human Endogenous Retroviral (HERV) RNA sequences. The Swellix motif search reveals occurrences of protein and drug binding motifs in the HERV RNA ensemble that do not occur in minimum free energy or centroid predicted structures. Swellix presents significant improvements over Crumple in terms of efficiency and memory use. The efficient parallelization of Swellix enables the computation of sequences as long as 418 nucleotides with sufficient experimental constraints. Thus, Swellix provides a practical alternative to free energy minimization tools when multiple structures, kinetically determined structures, or complex RNA-RNA and RNA-protein interactions are present in an RNA folding problem.

Towards a Logical Distinction Between Swarms and Aftershock Sequences

NASA Astrophysics Data System (ADS)

Gardine, M.; Burris, L.; McNutt, S.

2007-12-01

The distinction between swarms and aftershock sequences has, up to this point, been fairly arbitrary and non- uniform. Typically 0.5 to 1 order of magnitude difference between the mainshock and largest aftershock has been a traditional choice, but there are many exceptions. Seismologists have generally assumed that the mainshock carries most of the energy, but this is only true if it is sufficiently large compared to the size and numbers of aftershocks. Here we present a systematic division based on energy of the aftershock sequence compared to the energy of the largest event of the sequence. It is possible to calculate the amount of aftershock energy assumed to be in the sequence using the b-value of the frequency-magnitude relation with a fixed choice of magnitude separation (M-mainshock minus M-largest aftershock). Assuming that the energy of an aftershock sequence is less than the energy of the mainshock, the b-value at which the aftershock energy exceeds that of the mainshock energy determines the boundary between aftershock sequences and swarms. The amount of energy for various choices of b-value is also calculated using different values of magnitude separation. When the minimum b-value at which the sequence energy exceeds that of the largest event/mainshock is plotted against the magnitude separation, a linear trend emerges. Values plotting above this line represent swarms and values plotting below it represent aftershock sequences. This scheme has the advantage that it represents a physical quantity - energy - rather than only statistical features of earthquake distributions. As such it may be useful to help distinguish swarms from mainshock/aftershock sequences and to better determine the underlying causes of earthquake swarms.

Evolution of biological sequences implies an extreme value distribution of type I for both global and local pairwise alignment scores.

PubMed

Bastien, Olivier; Maréchal, Eric

2008-08-07

Confidence in pairwise alignments of biological sequences, obtained by various methods such as Blast or Smith-Waterman, is critical for automatic analyses of genomic data. Two statistical models have been proposed. In the asymptotic limit of long sequences, the Karlin-Altschul model is based on the computation of a P-value, assuming that the number of high scoring matching regions above a threshold is Poisson distributed. Alternatively, the Lipman-Pearson model is based on the computation of a Z-value from a random score distribution obtained by a Monte-Carlo simulation. Z-values allow the deduction of an upper bound of the P-value (1/Z-value2) following the TULIP theorem. Simulations of Z-value distribution is known to fit with a Gumbel law. This remarkable property was not demonstrated and had no obvious biological support. We built a model of evolution of sequences based on aging, as meant in Reliability Theory, using the fact that the amount of information shared between an initial sequence and the sequences in its lineage (i.e., mutual information in Information Theory) is a decreasing function of time. This quantity is simply measured by a sequence alignment score. In systems aging, the failure rate is related to the systems longevity. The system can be a machine with structured components, or a living entity or population. "Reliability" refers to the ability to operate properly according to a standard. Here, the "reliability" of a sequence refers to the ability to conserve a sufficient functional level at the folded and maturated protein level (positive selection pressure). Homologous sequences were considered as systems 1) having a high redundancy of information reflected by the magnitude of their alignment scores, 2) which components are the amino acids that can independently be damaged by random DNA mutations. From these assumptions, we deduced that information shared at each amino acid position evolved with a constant rate, corresponding to the information hazard rate, and that pairwise sequence alignment scores should follow a Gumbel distribution, which parameters could find some theoretical rationale. In particular, one parameter corresponds to the information hazard rate. Extreme value distribution of alignment scores, assessed from high scoring segments pairs following the Karlin-Altschul model, can also be deduced from the Reliability Theory applied to molecular sequences. It reflects the redundancy of information between homologous sequences, under functional conservative pressure. This model also provides a link between concepts of biological sequence analysis and of systems biology.

Complete genome sequence of a potyvirus infecting yam beans (Pachyrhizus spp.) in Peru.

PubMed

Fuentes, Segundo; Heider, Bettina; Tasso, Ruby Carolina; Romero, Elisa; Zum Felde, Thomas; Kreuze, Jan Frederik

2012-04-01

In 2010, yam beans in a field trial in Peru showed viral disease symptoms. Graft-transmission and positive ELISA results using potyvirus-specific antibodies suggested that the symptoms could be the result of a potyviral infection. Small interfering RNA (siRNA) were extracted from one of the samples and sent for high-throughput sequencing. The full genome of a new potyvirus could be assembled from the resulting siRNA sequences, and it was sufficiently different from other sequences to be considered a member of a new species, which we have designated Yam bean mosaic virus (YBMV). Sequence similarity suggests that YBMV has also been detected in yam beans in Indonesia.

Terminator oligo blocking efficiently eliminates rRNA from Drosophila small RNA sequencing libraries.

PubMed

Wickersheim, Michelle L; Blumenstiel, Justin P

2013-11-01

A large number of methods are available to deplete ribosomal RNA reads from high-throughput RNA sequencing experiments. Such methods are critical for sequencing Drosophila small RNAs between 20 and 30 nucleotides because size selection is not typically sufficient to exclude the highly abundant class of 30 nucleotide 2S rRNA. Here we demonstrate that pre-annealing terminator oligos complimentary to Drosophila 2S rRNA prior to 5' adapter ligation and reverse transcription efficiently depletes 2S rRNA sequences from the sequencing reaction in a simple and inexpensive way. This depletion is highly specific and is achieved with minimal perturbation of miRNA and piRNA profiles.

High quality methylome-wide investigations through next-generation sequencing of DNA from a single archived dry blood spot

PubMed Central

Aberg, Karolina A.; Xie, Lin Y.; Nerella, Srilaxmi; Copeland, William E.; Costello, E. Jane; van den Oord, Edwin J.C.G.

2013-01-01

The potential importance of DNA methylation in the etiology of complex diseases has led to interest in the development of methylome-wide association studies (MWAS) aimed at interrogating all methylation sites in the human genome. When using blood as biomaterial for a MWAS the DNA is typically extracted directly from fresh or frozen whole blood that was collected via venous puncture. However, DNA extracted from dry blood spots may also be an alternative starting material. In the present study, we apply a methyl-CpG binding domain (MBD) protein enrichment-based technique in combination with next generation sequencing (MBD-seq) to assess the methylation status of the ~27 million CpGs in the human autosomal reference genome. We investigate eight methylomes using DNA from blood spots. This data are compared with 1,500 methylomes previously assayed with the same MBD-seq approach using DNA from whole blood. When investigating the sequence quality and the enrichment profile across biological features, we find that DNA extracted from blood spots gives comparable results with DNA extracted from whole blood. Only if the amount of starting material is ≤ 0.5µg DNA we observe a slight decrease in the assay performance. In conclusion, we show that high quality methylome-wide investigations using MBD-seq can be conducted in DNA extracted from archived dry blood spots without sacrificing quality and without bias in enrichment profile as long as the amount of starting material is sufficient. In general, the amount of DNA extracted from a single blood spot is sufficient for methylome-wide investigations with the MBD-seq approach. PMID:23644822

High quality methylome-wide investigations through next-generation sequencing of DNA from a single archived dry blood spot.

PubMed

Aberg, Karolina A; Xie, Lin Y; Nerella, Srilaxmi; Copeland, William E; Costello, E Jane; van den Oord, Edwin J C G

2013-05-01

The potential importance of DNA methylation in the etiology of complex diseases has led to interest in the development of methylome-wide association studies (MWAS) aimed at interrogating all methylation sites in the human genome. When using blood as biomaterial for a MWAS the DNA is typically extracted directly from fresh or frozen whole blood that was collected via venous puncture. However, DNA extracted from dry blood spots may also be an alternative starting material. In the present study, we apply a methyl-CpG binding domain (MBD) protein enrichment-based technique in combination with next generation sequencing (MBD-seq) to assess the methylation status of the ~27 million CpGs in the human autosomal reference genome. We investigate eight methylomes using DNA from blood spots. This data are compared with 1,500 methylomes previously assayed with the same MBD-seq approach using DNA from whole blood. When investigating the sequence quality and the enrichment profile across biological features, we find that DNA extracted from blood spots gives comparable results with DNA extracted from whole blood. Only if the amount of starting material is ≤ 0.5µg DNA we observe a slight decrease in the assay performance. In conclusion, we show that high quality methylome-wide investigations using MBD-seq can be conducted in DNA extracted from archived dry blood spots without sacrificing quality and without bias in enrichment profile as long as the amount of starting material is sufficient. In general, the amount of DNA extracted from a single blood spot is sufficient for methylome-wide investigations with the MBD-seq approach.

Biofunctional polymer nanoparticles for intra-articular targeting and retention in cartilage

NASA Astrophysics Data System (ADS)

Rothenfluh, Dominique A.; Bermudez, Harry; O'Neil, Conlin P.; Hubbell, Jeffrey A.

2008-03-01

The extracellular matrix of dense, avascular tissues presents a barrier to entry for polymer-based therapeutics, such as drugs encapsulated within polymeric particles. Here, we present an approach by which polymer nanoparticles, sufficiently small to enter the matrix of the targeted tissue, here articular cartilage, are further modified with a biomolecular ligand for matrix binding. This combination of ultrasmall size and biomolecular binding converts the matrix from a barrier into a reservoir, resisting rapid release of the nanoparticles and clearance from the tissue site. Phage display of a peptide library was used to discover appropriate targeting ligands by biopanning on denuded cartilage. The ligand WYRGRL was selected in 94 of 96 clones sequenced after five rounds of biopanning and was demonstrated to bind to collagen II α1. Peptide-functionalized nanoparticles targeted articular cartilage up to 72-fold more than nanoparticles displaying a scrambled peptide sequence following intra-articular injection in the mouse.

The rise and fall of a human recombination hot spot.

PubMed

Jeffreys, Alec J; Neumann, Rita

2009-05-01

Human meiotic crossovers mainly cluster into narrow hot spots that profoundly influence patterns of haplotype diversity and that may also affect genome instability and sequence evolution. Hot spots also seem to be ephemeral, but processes of hot-spot activation and their subsequent evolutionary dynamics remain unknown. We now analyze the life cycle of a recombination hot spot. Sperm typing revealed a polymorphic hot spot that was activated in cis by a single base change, providing evidence for a primary sequence determinant necessary, though not sufficient, to activate recombination. This activating mutation occurred roughly 70,000 y ago and has persisted to the present, most likely fortuitously through genetic drift despite its systematic elimination by biased gene conversion. Nonetheless, this self-destructive conversion will eventually lead to hot-spot extinction. These findings define a subclass of highly transient hot spots and highlight the importance of understanding hot-spot turnover and how it influences haplotype diversity.

The stability of AID and its function in class-switching are critically sensitive to the identity of its nuclear-export sequence

PubMed Central

Geisberger, Roland; Rada, Cristina; Neuberger, Michael S.

2009-01-01

The carboxyterminal region of activation-induced deaminase (AID) is required for its function in Ig class switch recombination (CSR) and also contains a nuclear-export sequence (NES). Here, based on an extensive fine-structure mutation analysis of the AID NES, as well as from AID chimeras bearing heterologous NESs, we show that while a functional NES is indeed essential for CSR, it is not sufficient. The precise nature of the NES is critical both for AID stabilization and CSR function: minor changes in the NES can perturb stabilization and CSR without jeopardizing nuclear export. The results indicate that the AID NES fulfills a function beyond simply providing a signal for nuclear export and suggest the possibility that the quality of exportin-binding may be critical to the stabilization of AID and its activity in CSR. PMID:19351893

DNA barcoding as a tool for coral reef conservation

NASA Astrophysics Data System (ADS)

Neigel, J.; Domingo, A.; Stake, J.

2007-09-01

DNA Barcoding (DBC) is a method for taxonomic identification of animals that is based entirely on the 5' portion of the mitochondrial gene, cytochrome oxidase subunit I ( COI-5). It can be especially useful for identification of larval forms or incomplete specimens lacking diagnostic morphological characters. DBC can also facilitate the discovery of species and in defining “molecular taxonomic units” in problematic groups. However, DBC is not a panacea for coral reef taxonomy. In two of the most ecologically important groups on coral reefs, the Anthozoa and Porifera, COI-5 sequences have diverged too little to be diagnostic for all species. Other problems for DBC include paraphyly in mitochondrial gene trees and lack of differentiation between hybrids and their maternal ancestors. DBC also depends on the availability of databases of COI-5 sequences, which are still in early stages of development. A global effort to barcode all fish species has demonstrated the importance of large-scale coordination and is yielding promising results. Whether or not COI-5 by itself is sufficient for species assignments has become a contentious question; it is generally advantageous to use sequences from multiple loci.

Targeted gene insertion for molecular medicine.

PubMed

Voigt, Katrin; Izsvák, Zsuzsanna; Ivics, Zoltán

2008-11-01

Genomic insertion of a functional gene together with suitable transcriptional regulatory elements is often required for long-term therapeutical benefit in gene therapy for several genetic diseases. A variety of integrating vectors for gene delivery exist. Some of them exhibit random genomic integration, whereas others have integration preferences based on attributes of the targeted site, such as primary DNA sequence and physical structure of the DNA, or through tethering to certain DNA sequences by host-encoded cellular factors. Uncontrolled genomic insertion bears the risk of the transgene being silenced due to chromosomal position effects, and can lead to genotoxic effects due to mutagenesis of cellular genes. None of the vector systems currently used in either preclinical experiments or clinical trials displays sufficient preferences for target DNA sequences that would ensure appropriate and reliable expression of the transgene and simultaneously prevent hazardous side effects. We review in this paper the advantages and disadvantages of both viral and non-viral gene delivery technologies, discuss mechanisms of target site selection of integrating genetic elements (viruses and transposons), and suggest distinct molecular strategies for targeted gene delivery.

Sequence-engineered mRNA Without Chemical Nucleoside Modifications Enables an Effective Protein Therapy in Large Animals

PubMed Central

Thess, Andreas; Grund, Stefanie; Mui, Barbara L; Hope, Michael J; Baumhof, Patrick; Fotin-Mleczek, Mariola; Schlake, Thomas

2015-01-01

Being a transient carrier of genetic information, mRNA could be a versatile, flexible, and safe means for protein therapies. While recent findings highlight the enormous therapeutic potential of mRNA, evidence that mRNA-based protein therapies are feasible beyond small animals such as mice is still lacking. Previous studies imply that mRNA therapeutics require chemical nucleoside modifications to obtain sufficient protein expression and avoid activation of the innate immune system. Here we show that chemically unmodified mRNA can achieve those goals as well by applying sequence-engineered molecules. Using erythropoietin (EPO) driven production of red blood cells as the biological model, engineered Epo mRNA elicited meaningful physiological responses from mice to nonhuman primates. Even in pigs of about 20 kg in weight, a single adequate dose of engineered mRNA encapsulated in lipid nanoparticles (LNPs) induced high systemic Epo levels and strong physiological effects. Our results demonstrate that sequence-engineered mRNA has the potential to revolutionize human protein therapies. PMID:26050989

Identification by 16S rRNA Gene Sequencing of Lactobacillus salivarius Bacteremic Cholecystitis

PubMed Central

Woo, Patrick C. Y.; Fung, Ami M. Y.; Lau, Susanna K. P.; Yuen, Kwok-Yung

2002-01-01

An anaerobic, nonsporulating, gram-positive bacterium was isolated from blood and bile pus cultures of a 70-year-old man with bacteremic acute cholecystitis. The API 20A system showed that it was 70% Actinomyces naeslundii and 30% Bifidobacterium species, whereas the Vitek ANI system and the ATB ID32A Expression system showed that it was “unidentified.” The 16S rRNA gene of the strain was amplified and sequenced. There were 3 base differences between the nucleotide sequence of the isolate and that of Lactobacillus salivarius subsp. salivarius or L. salivarius subsp. salicinius, indicating that the isolate was a strain of L. salivarius. The patient responded to cholecystectomy and a 2-week course of antibiotic treatment. Identification of the organism in the present study was important because the duration of antibiotic therapy would have been entirely different depending on the organism. If the bacterium had been identified as Actinomyces, penicillin for 6 months would have been the regimen of choice. However, it was Lactobacillus, and a 2-week course of antibiotic was sufficient. PMID:11773128

Rapid Multistep Synthesis of 1,2,4-Oxadiazoles in a Single Continuous Microreactor Sequence

PubMed Central

Grant, Daniel; Dahl, Russell; Cosford, Nicholas D. P.

2009-01-01

A general method for the synthesis of bis-substituted 1,2,4-oxadiazoles from readily available arylnitriles and activated carbonyls in a single continuous microreactor sequence is described. The synthesis incorporates three sequential microreactors to produce 1,2,4-oxadiazoles in ~30 min in quantities (40–80 mg) sufficient for full characterization and rapid library supply. PMID:18687005

Draft genome sequence of Thauera sp. strain SWB20, isolated from a Singapore wastewater treatment facility using gel microdroplets

DOE PAGES

Dichosa, Armand E. K.; Davenport, Karen W.; Li, Po-E; ...

2015-03-19

In this study, we report here the genome sequence of Thauera sp. strain SWB20, isolated from a Singaporean wastewater treatment facility using gel microdroplets (GMDs) and single-cell genomics (SCG). This approach provided a single clonal microcolony that was sufficient to obtain a 4.9-Mbp genome assembly of an ecologically relevant Thauera species.

Microfluidics-Based Lab-on-Chip Systems in DNA-Based Biosensing: An Overview

PubMed Central

Dutse, Sabo Wada; Yusof, Nor Azah

2011-01-01

Microfluidics-based lab-on-chip (LOC) systems are an active research area that is revolutionising high-throughput sequencing for the fast, sensitive and accurate detection of a variety of pathogens. LOCs also serve as portable diagnostic tools. The devices provide optimum control of nanolitre volumes of fluids and integrate various bioassay operations that allow the devices to rapidly sense pathogenic threat agents for environmental monitoring. LOC systems, such as microfluidic biochips, offer advantages compared to conventional identification procedures that are tedious, expensive and time consuming. This paper aims to provide a broad overview of the need for devices that are easy to operate, sensitive, fast, portable and sufficiently reliable to be used as complementary tools for the control of pathogenic agents that damage the environment. PMID:22163925

Predicting discovery rates of genomic features.

PubMed

Gravel, Simon

2014-06-01

Successful sequencing experiments require judicious sample selection. However, this selection must often be performed on the basis of limited preliminary data. Predicting the statistical properties of the final sample based on preliminary data can be challenging, because numerous uncertain model assumptions may be involved. Here, we ask whether we can predict "omics" variation across many samples by sequencing only a fraction of them. In the infinite-genome limit, we find that a pilot study sequencing 5% of a population is sufficient to predict the number of genetic variants in the entire population within 6% of the correct value, using an estimator agnostic to demography, selection, or population structure. To reach similar accuracy in a finite genome with millions of polymorphisms, the pilot study would require ∼15% of the population. We present computationally efficient jackknife and linear programming methods that exhibit substantially less bias than the state of the art when applied to simulated data and subsampled 1000 Genomes Project data. Extrapolating based on the National Heart, Lung, and Blood Institute Exome Sequencing Project data, we predict that 7.2% of sites in the capture region would be variable in a sample of 50,000 African Americans and 8.8% in a European sample of equal size. Finally, we show how the linear programming method can also predict discovery rates of various genomic features, such as the number of transcription factor binding sites across different cell types. Copyright © 2014 by the Genetics Society of America.

Protein 3D Structure Computed from Evolutionary Sequence Variation

PubMed Central

Sheridan, Robert; Hopf, Thomas A.; Pagnani, Andrea; Zecchina, Riccardo; Sander, Chris

2011-01-01

The evolutionary trajectory of a protein through sequence space is constrained by its function. Collections of sequence homologs record the outcomes of millions of evolutionary experiments in which the protein evolves according to these constraints. Deciphering the evolutionary record held in these sequences and exploiting it for predictive and engineering purposes presents a formidable challenge. The potential benefit of solving this challenge is amplified by the advent of inexpensive high-throughput genomic sequencing. In this paper we ask whether we can infer evolutionary constraints from a set of sequence homologs of a protein. The challenge is to distinguish true co-evolution couplings from the noisy set of observed correlations. We address this challenge using a maximum entropy model of the protein sequence, constrained by the statistics of the multiple sequence alignment, to infer residue pair couplings. Surprisingly, we find that the strength of these inferred couplings is an excellent predictor of residue-residue proximity in folded structures. Indeed, the top-scoring residue couplings are sufficiently accurate and well-distributed to define the 3D protein fold with remarkable accuracy. We quantify this observation by computing, from sequence alone, all-atom 3D structures of fifteen test proteins from different fold classes, ranging in size from 50 to 260 residues., including a G-protein coupled receptor. These blinded inferences are de novo, i.e., they do not use homology modeling or sequence-similar fragments from known structures. The co-evolution signals provide sufficient information to determine accurate 3D protein structure to 2.7–4.8 Å Cα-RMSD error relative to the observed structure, over at least two-thirds of the protein (method called EVfold, details at http://EVfold.org). This discovery provides insight into essential interactions constraining protein evolution and will facilitate a comprehensive survey of the universe of protein structures, new strategies in protein and drug design, and the identification of functional genetic variants in normal and disease genomes. PMID:22163331

Mosaic organization of DNA nucleotides

NASA Technical Reports Server (NTRS)

Peng, C. K.; Buldyrev, S. V.; Havlin, S.; Simons, M.; Stanley, H. E.; Goldberger, A. L.

1994-01-01

Long-range power-law correlations have been reported recently for DNA sequences containing noncoding regions. We address the question of whether such correlations may be a trivial consequence of the known mosaic structure ("patchiness") of DNA. We analyze two classes of controls consisting of patchy nucleotide sequences generated by different algorithms--one without and one with long-range power-law correlations. Although both types of sequences are highly heterogenous, they are quantitatively distinguishable by an alternative fluctuation analysis method that differentiates local patchiness from long-range correlations. Application of this analysis to selected DNA sequences demonstrates that patchiness is not sufficient to account for long-range correlation properties.

Time-resolved fluorescence imaging of slab gels for lifetime base-calling in DNA sequencing applications.

PubMed

Lassiter, S J; Stryjewski, W; Legendre, B L; Erdmann, R; Wahl, M; Wurm, J; Peterson, R; Middendorf, L; Soper, S A

2000-11-01

A compact time-resolved near-IR fluorescence imager was constructed to obtain lifetime and intensity images of DNA sequencing slab gels. The scanner consisted of a microscope body with f/1.2 relay optics onto which was mounted a pulsed diode laser (repetition rate 80 MHz, lasing wavelength 680 nm, average power 5 mW), filtering optics, and a large photoactive area (diameter 500 microns) single-photon avalanche diode that was actively quenched to provide a large dynamic operating range. The time-resolved data were processed using electronics configured in a conventional time-correlated single-photon-counting format with all of the counting hardware situated on a PC card resident on the computer bus. The microscope head produced a timing response of 450 ps (fwhm) in a scanning mode, allowing the measurement of subnano-second lifetimes. The time-resolved microscope head was placed in an automated DNA sequencer and translated across a 21-cm-wide gel plate in approximately 6 s (scan rate 3.5 cm/s) with an accumulation time per pixel of 10 ms. The sampling frequency was 0.17 Hz (duty cycle 0.0017), sufficient to prevent signal aliasing during the electrophoresis separation. Software (written in Visual Basic) allowed acquisition of both the intensity image and lifetime analysis of DNA bands migrating through the gel in real time. Using a dual-labeling (IRD700 and Cy5.5 labeling dyes)/two-lane sequencing strategy, we successfully read 670 bases of a control M13mp18 ssDNA template using lifetime identification. Comparison of the reconstructed sequence with the known sequence of the phage indicated the number of miscalls was only 2, producing an error rate of approximately 0.3% (identification accuracy 99.7%). The lifetimes were calculated using maximum likelihood estimators and allowed on-line determinations with high precision, even when short integration times were used to construct the decay profiles. Comparison of the lifetime base calling to a single-dye/four-lane sequencing strategy indicated similar results in terms of miscalls, but reduced insertion and deletion errors using lifetime identification methods, improving the overall read accuracy.

Effects of different preservation methods on inter simple sequence repeat (ISSR) and random amplified polymorphic DNA (RAPD) molecular markers in botanic samples.

PubMed

Wang, Xiaolong; Li, Lin; Zhao, Jiaxin; Li, Fangliang; Guo, Wei; Chen, Xia

2017-04-01

To evaluate the effects of different preservation methods (stored in a -20°C ice chest, preserved in liquid nitrogen and dried in silica gel) on inter simple sequence repeat (ISSR) or random amplified polymorphic DNA (RAPD) analyses in various botanical specimens (including broad-leaved plants, needle-leaved plants and succulent plants) for different times (three weeks and three years), we used a statistical analysis based on the number of bands, genetic index and cluster analysis. The results demonstrate that methods used to preserve samples can provide sufficient amounts of genomic DNA for ISSR and RAPD analyses; however, the effect of different preservation methods on these analyses vary significantly, and the preservation time has little effect on these analyses. Our results provide a reference for researchers to select the most suitable preservation method depending on their study subject for the analysis of molecular markers based on genomic DNA. Copyright © 2017 Académie des sciences. Published by Elsevier Masson SAS. All rights reserved.

Use of novel species-specific PCR primers targeted to DNA gyrase subunit B (gyrB) gene for species identification of the Cronobacter sakazakii and Cronobacter dublinensis.

PubMed

Huang, Chien-Hsun; Chang, Mu-Tzu; Huang, Lina

2013-02-01

Cronobacter sakazakii and its phylogenetically closest species are considered to be an opportunistic pathogens associated with food-borne disease in neonates and infants. Neither phenotypic nor genotypic (16S ribosomal DNA sequence analysis) techniques can provide sufficient resolutions for accurately and rapidly identification of these species. The objective of this study was to develop species-specific PCR based on the gyrB gene sequence for direct species identification of the C. sakazakii and Cronobacter dublinensis within the C. sakazakii group. Two pair of species-specific primers were designed and used to specifically identify C. sakazakii and C. dublinensis, but none of the other C. sakazakii group strains. Our data indicate that the novel species-specific primers could be used to rapidly and accurately identify the species of C. sakazakii and C. dublinensis from C. sakazakii group by the PCR based assays. Copyright © 2012 Elsevier Ltd. All rights reserved.

Development and use of EST-SSR markers for assessing genetic diversity in the brown planthopper (Nilaparvata lugens Stål).

PubMed

Jing, S; Liu, B; Peng, L; Peng, X; Zhu, L; Fu, Q; He, G

2012-02-01

To assess genetic diversity in populations of the brown planthopper (Nilaparvata lugens Stål) (Homoptera: Delphacidae), we have developed and applied microsatellite, or simple sequence repeat (SSR), markers from expressed sequence tags (ESTs). We found that the brown planthopper clusters of ESTs were rich in SSRs with unique frequencies and distributions of SSR motifs. Three hundred and fifty-one EST-SSR markers were developed and yielded clear bands from samples of four brown planthopper populations. High cross-species transferability of these markers was detected in the closely related planthopper N. muiri. The newly developed EST-SSR markers provided sufficient resolution to distinguish within and among biotypes. Analyses based on SSR data revealed host resistance-based genetic differentiation among different brown planthopper populations; the genetic diversity of populations feeding on susceptible rice varieties was lower than that of populations feeding on resistant rice varieties. This is the first large-scale development of brown planthopper SSR markers, which will be useful for future molecular genetics and genomics studies of this serious agricultural pest.

A novel multi-locus sequence typing (MLST) protocol for Leuconostoc lactis isolates from traditional dairy products in China and Mongolia

PubMed Central

2014-01-01

Background Economically, Leuconostoc lactis is one of the most important species in the genus Leuconostoc. It plays an important role in the food industry including the production of dextrans and bacteriocins. Currently, traditional molecular typing approaches for characterisation of this species at the isolate level are either unavailable or are not sufficiently reliable for practical use. Multilocus sequence typing (MLST) is a robust and reliable method for characterising bacterial and fungal species at the molecular level. In this study, a novel MLST protocol was developed for 50 L. lactis isolates from Mongolia and China. Results Sequences from eight targeted genes (groEL, carB, recA, pheS, murC, pyrG, rpoB and uvrC) were obtained. Sequence analysis indicated 20 different sequence types (STs), with 13 of them being represented by a single isolate. Phylogenetic analysis based on the sequences of eight MLST loci indicated that the isolates belonged to two major groups, A (34 isolates) and B (16 isolates). Linkage disequilibrium analyses indicated that recombination occurred at a low frequency in L. lactis, indicating a clonal population structure. Split-decomposition analysis indicated that intraspecies recombination played a role in generating genotypic diversity amongst isolates. Conclusions Our results indicated that MLST is a valuable tool for typing L. lactis isolates that can be used for further monitoring of evolutionary changes and population genetics. PMID:24912963

Cryptic Hepatitis B and E in Patients With Acute Hepatitis of Unknown Etiology.

PubMed

Ganova-Raeva, Lilia; Punkova, Lili; Campo, David S; Dimitrova, Zoya; Skums, Pavel; Vu, Nga H; Dat, Do T; Dalton, Harry R; Khudyakov, Yury

2015-12-15

Up to 30% of acute viral hepatitis has no known etiology. To determine the disease etiology in patients with acute hepatitis of unknown etiology (HUE), serum specimens were obtained from 38 patients residing in the United Kingdom and Vietnam and from 26 healthy US blood donors. All specimens tested negative for known viral infections causing hepatitis, using commercially available serological and nucleic acid assays. Specimens were processed by sequence-independent complementary DNA amplification and next-generation sequencing (NGS). Sufficient material for individual NGS libraries was obtained from 12 HUE cases and 26 blood donors; the remaining HUE cases were sequenced as a pool. Read mapping was done by targeted and de novo assembly. Sequences from hepatitis B virus (HBV) were detected in 7 individuals with HUE (58.3%) and the pooled library, and hepatitis E virus (HEV) was detected in 2 individuals with HUE (16.7%) and the pooled library. Both HEV-positive cases were coinfected with HBV. HBV sequences belonged to genotypes A, D, or G, and HEV sequences belonged to genotype 3. No known hepatotropic viruses were detected in the tested normal human sera. NGS-based detection of HBV and HEV infections is more sensitive than using commercially available assays. HBV and HEV may be cryptically associated with HUE. Published by Oxford University Press on behalf of the Infectious Diseases Society of America 2015. This work is written by (a) US Government employee(s) and is in the public domain in the US.

Genome-wide profiling of DNA-binding proteins using barcode-based multiplex Solexa sequencing.

PubMed

Raghav, Sunil Kumar; Deplancke, Bart

2012-01-01

Chromatin immunoprecipitation (ChIP) is a commonly used technique to detect the in vivo binding of proteins to DNA. ChIP is now routinely paired to microarray analysis (ChIP-chip) or next-generation sequencing (ChIP-Seq) to profile the DNA occupancy of proteins of interest on a genome-wide level. Because ChIP-chip introduces several biases, most notably due to the use of a fixed number of probes, ChIP-Seq has quickly become the method of choice as, depending on the sequencing depth, it is more sensitive, quantitative, and provides a greater binding site location resolution. With the ever increasing number of reads that can be generated per sequencing run, it has now become possible to analyze several samples simultaneously while maintaining sufficient sequence coverage, thus significantly reducing the cost per ChIP-Seq experiment. In this chapter, we provide a step-by-step guide on how to perform multiplexed ChIP-Seq analyses. As a proof-of-concept, we focus on the genome-wide profiling of RNA Polymerase II as measuring its DNA occupancy at different stages of any biological process can provide insights into the gene regulatory mechanisms involved. However, the protocol can also be used to perform multiplexed ChIP-Seq analyses of other DNA-binding proteins such as chromatin modifiers and transcription factors.

Molecular variation and distribution of Anopheles fluviatilis (Diptera: Culicidae) complex in Iran.

PubMed

Naddaf, Saied Reza; Razavi, Mohammad Reza; Bahramali, Golnaz

2010-09-01

Anopheles fluviatilis James (Diptera: Culicidae) is one of the known malaria vectors in south and southeastern Iran. Earlier ITS2 sequences analysis of specimens from Iran demonstrated only a single genotype that was identical to species Y in India, which is also the same as species T. We identified 2 haplotypes in the An. fluviatilis populations of Iran based on differences in nucleotide sequences of D3 domain of the 28S locus of ribosomal DNA (rDNA). Comparison of sequence data from 44 Iranian specimens with those publicly available in the Genbank database showed that all of the 28S-D3 sequences from Kazeroun and Khesht regions in Fars Province were identical to the database entry representing species U in India. In other regions, all the individuals showed heterozygosity at the single nucleotide position, which identifies species U and T. It is argued that the 2 species may co-occur in some regions and hybridize; however, the heterozygosity in the 28S-D3 locus was not reflected in ITS2 sequences and this locus for all individuals was identical to species T. This study shows that in a newly diverged species, like members of An. fluviatilis complex, a single molecular marker may not be sufficiently discriminatory to identify all the taxa over a vast geographical area. In addition, other molecular markers may provide more reliable information for species discrimination.

Isolation and bacterial expression of a sesquiterpene synthase CDNA clone from peppermint(mentha .chi. piperita, L.) that produces the aphid alarm pheromone (E)-.beta.-farnesene

DOEpatents

Croteau, Rodney Bruce; Wildung, Mark Raymond; Crock, John E.

1999-01-01

A cDNA encoding (E)-.beta.-farnesene synthase from peppermint (Mentha piperita) has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Accordingly, an isolated DNA sequence (SEQ ID NO:1) is provided which codes for the expression of (E)-.beta.-farnesene synthase (SEQ ID NO:2), from peppermint (Mentha piperita). In other aspects, replicable recombinant cloning vehicles are provided which code for (E)-.beta.-farnesene synthase, or for a base sequence sufficiently complementary to at least a portion of (E)-.beta.-farnesene synthase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding (E)-.beta.-farnesene synthase. Thus, systems and methods are provided for the recombinant expression of the aforementioned recombinant (E)-.beta.-farnesene synthase that may be used to facilitate its production, isolation and purification in significant amounts. Recombinant (E)-.beta.-farnesene synthase may be used to obtain expression or enhanced expression of (E)-.beta.-farnesene synthase in plants in order to enhance the production of (E)-.beta.-farnesene, or may be otherwise employed for the regulation or expression of (E)-.beta.-farnesene synthase, or the production of its product.

Isolation and bacterial expression of a sesquiterpene synthase cDNA clone from peppermint (Mentha x piperita, L.) that produces the aphid alarm pheromone (E)-.beta.-farnesene

DOEpatents

Croteau, Rodney Bruce; Crock, John E.

2005-01-25

A cDNA encoding (E)-.beta.-farnesene synthase from peppermint (Mentha piperita) has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Accordingly, an isolated DNA sequence (SEQ ID NO:1) is provided which codes for the expression of (E)-.beta.-farnesene synthase (SEQ ID NO:2), from peppermint (Mentha piperita). In other aspects, replicable recombinant cloning vehicles are provided which code for (E)-.beta.-farnesene synthase, or for a base sequence sufficiently complementary to at least a portion of (E)-.beta.-farnesene synthase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding (E)-.beta.-farnesene synthase. Thus, systems and methods are provided for the recombinant expression of the aforementioned recombinant (E)-.beta.-famesene synthase that may be used to facilitate its production, isolation and purification in significant amounts. Recombinant (E)-.beta.-farnesene synthase may be used to obtain expression or enhanced expression of (E)-.beta.-famesene synthase in plants in order to enhance the production of (E)-.beta.-farnesene, or may be otherwise employed for the regulation or expression of (E)-.beta.-farnesene synthase, or the production of its product.

Association analysis using next-generation sequence data from publicly available control groups: the robust variance score statistic

PubMed Central

Derkach, Andriy; Chiang, Theodore; Gong, Jiafen; Addis, Laura; Dobbins, Sara; Tomlinson, Ian; Houlston, Richard; Pal, Deb K.; Strug, Lisa J.

2014-01-01

Motivation: Sufficiently powered case–control studies with next-generation sequence (NGS) data remain prohibitively expensive for many investigators. If feasible, a more efficient strategy would be to include publicly available sequenced controls. However, these studies can be confounded by differences in sequencing platform; alignment, single nucleotide polymorphism and variant calling algorithms; read depth; and selection thresholds. Assuming one can match cases and controls on the basis of ethnicity and other potential confounding factors, and one has access to the aligned reads in both groups, we investigate the effect of systematic differences in read depth and selection threshold when comparing allele frequencies between cases and controls. We propose a novel likelihood-based method, the robust variance score (RVS), that substitutes genotype calls by their expected values given observed sequence data. Results: We show theoretically that the RVS eliminates read depth bias in the estimation of minor allele frequency. We also demonstrate that, using simulated and real NGS data, the RVS method controls Type I error and has comparable power to the ‘gold standard’ analysis with the true underlying genotypes for both common and rare variants. Availability and implementation: An RVS R script and instructions can be found at strug.research.sickkids.ca, and at https://github.com/strug-lab/RVS. Contact: lisa.strug@utoronto.ca Supplementary information: Supplementary data are available at Bioinformatics online. PMID:24733292

Reproducibility and Consistency of In Vitro Nucleosome Reconstitutions Demonstrated by Invitrosome Isolation and Sequencing

PubMed Central

Kempton, Colton E.; Heninger, Justin R.; Johnson, Steven M.

2014-01-01

Nucleosomes and their positions in the eukaryotic genome play an important role in regulating gene expression by influencing accessibility to DNA. Many factors influence a nucleosome's final position in the chromatin landscape including the underlying genomic sequence. One of the primary reasons for performing in vitro nucleosome reconstitution experiments is to identify how the underlying DNA sequence will influence a nucleosome's position in the absence of other compounding cellular factors. However, concerns have been raised about the reproducibility of data generated from these kinds of experiments. Here we present data for in vitro nucleosome reconstitution experiments performed on linear plasmid DNA that demonstrate that, when coverage is deep enough, these reconstitution experiments are exquisitely reproducible and highly consistent. Our data also suggests that a coverage depth of 35X be maintained for maximal confidence when assaying nucleosome positions, but lower coverage levels may be generally sufficient. These coverage depth recommendations are sufficient in the experimental system and conditions used in this study, but may vary depending on the exact parameters used in other systems. PMID:25093869

INTERDISCIPLINARY PHYSICS AND RELATED AREAS OF SCIENCE AND TECHNOLOGY: Relaxation Property and Stability Analysis of the Quasispecies Models

NASA Astrophysics Data System (ADS)

Feng, Xiao-Li; Li, Yu-Xiao; Gu, Jian-Zhong; Zhuo, Yi-Zhong

2009-10-01

The relaxation property of both Eigen model and Crow-Kimura model with a single peak fitness landscape is studied from phase transition point of view. We first analyze the eigenvalue spectra of the replication mutation matrices. For sufficiently long sequences, the almost crossing point between the largest and second-largest eigenvalues locates the error threshold at which critical slowing down behavior appears. We calculate the critical exponent in the limit of infinite sequence lengths and compare it with the result from numerical curve fittings at sufficiently long sequences. We find that for both models the relaxation time diverges with exponent 1 at the error (mutation) threshold point. Results obtained from both methods agree quite well. From the unlimited correlation length feature, the first order phase transition is further confirmed. Finally with linear stability theory, we show that the two model systems are stable for all ranges of mutation rate. The Eigen model is asymptotically stable in terms of mutant classes, and the Crow-Kimura model is completely stable.

A compact, in vivo screen of all 6-mers reveals drivers of tissue-specific expression and guides synthetic regulatory element design.

PubMed

Smith, Robin P; Riesenfeld, Samantha J; Holloway, Alisha K; Li, Qiang; Murphy, Karl K; Feliciano, Natalie M; Orecchia, Lorenzo; Oksenberg, Nir; Pollard, Katherine S; Ahituv, Nadav

2013-07-18

Large-scale annotation efforts have improved our ability to coarsely predict regulatory elements throughout vertebrate genomes. However, it is unclear how complex spatiotemporal patterns of gene expression driven by these elements emerge from the activity of short, transcription factor binding sequences. We describe a comprehensive promoter extension assay in which the regulatory potential of all 6 base-pair (bp) sequences was tested in the context of a minimal promoter. To enable this large-scale screen, we developed algorithms that use a reverse-complement aware decomposition of the de Bruijn graph to design a library of DNA oligomers incorporating every 6-bp sequence exactly once. Our library multiplexes all 4,096 unique 6-mers into 184 double-stranded 15-bp oligomers, which is sufficiently compact for in vivo testing. We injected each multiplexed construct into zebrafish embryos and scored GFP expression in 15 tissues at two developmental time points. Twenty-seven constructs produced consistent expression patterns, with the majority doing so in only one tissue. Functional sequences are enriched near biologically relevant genes, match motifs for developmental transcription factors, and are required for enhancer activity. By concatenating tissue-specific functional sequences, we generated completely synthetic enhancers for the notochord, epidermis, spinal cord, forebrain and otic lateral line, and show that short regulatory sequences do not always function modularly. This work introduces a unique in vivo catalog of short, functional regulatory sequences and demonstrates several important principles of regulatory element organization. Furthermore, we provide resources for designing compact, reverse-complement aware k-mer libraries.

Repetitive DNA in the pea (Pisum sativum L.) genome: comprehensive characterization using 454 sequencing and comparison to soybean and Medicago truncatula

PubMed Central

Macas, Jiří; Neumann, Pavel; Navrátilová, Alice

2007-01-01

Background Extraordinary size variation of higher plant nuclear genomes is in large part caused by differences in accumulation of repetitive DNA. This makes repetitive DNA of great interest for studying the molecular mechanisms shaping architecture and function of complex plant genomes. However, due to methodological constraints of conventional cloning and sequencing, a global description of repeat composition is available for only a very limited number of higher plants. In order to provide further data required for investigating evolutionary patterns of repeated DNA within and between species, we used a novel approach based on massive parallel sequencing which allowed a comprehensive repeat characterization in our model species, garden pea (Pisum sativum). Results Analysis of 33.3 Mb sequence data resulted in quantification and partial sequence reconstruction of major repeat families occurring in the pea genome with at least thousands of copies. Our results showed that the pea genome is dominated by LTR-retrotransposons, estimated at 140,000 copies/1C. Ty3/gypsy elements are less diverse and accumulated to higher copy numbers than Ty1/copia. This is in part due to a large population of Ogre-like retrotransposons which alone make up over 20% of the genome. In addition to numerous types of mobile elements, we have discovered a set of novel satellite repeats and two additional variants of telomeric sequences. Comparative genome analysis revealed that there are only a few repeat sequences conserved between pea and soybean genomes. On the other hand, all major families of pea mobile elements are well represented in M. truncatula. Conclusion We have demonstrated that even in a species with a relatively large genome like pea, where a single 454-sequencing run provided only 0.77% coverage, the generated sequences were sufficient to reconstruct and analyze major repeat families corresponding to a total of 35–48% of the genome. These data provide a starting point for further investigations of legume plant genomes based on their global comparative analysis and for the development of more sophisticated approaches for data mining. PMID:18031571

[Multiplexing mapping of human cDNAs]. Final report, September 1, 1991--February 28, 1994

DOE Office of Scientific and Technical Information (OSTI.GOV)

Not Available

Using PCR with automated product analysis, 329 human brain cDNA sequences have been assigned to individual human chromosomes. Primers were designed from single-pass cDNA sequences expressed sequence tags (ESTs). Primers were used in PCR reactions with DNA from somatic cell hybrid mapping panels as templates, often with multiplexing. Many ESTs mapped match sequence database records. To evaluate of these matches, the position of the primers relative to the matching region (In), the BLAST scores and the Poisson probability values of the EST/sequence record match were determined. In cases where the gene product was stringently identified by the sequence match hadmore » already been mapped, the gene locus determined by EST was consistent with the previous position which strongly supports the validity of assigning unknown genes to human chromosomes based on the EST sequence matches. In the present cases mapping the ESTs to a chromosome can also be considered to have mapped the known gene product: rolipram-sensitive cAMP phosphodiesterase, chromosome 1; protein phosphatase 2A{beta}, chromosome 4; alpha-catenin, chromosome 5; the ELE1 oncogene, chromosome 10q11.2 or q2.1-q23; MXII protein, chromosome l0q24-qter; ribosomal protein L18a homologue, chromosome 14; ribosomal protein L3, chromosome 17; and moesin, Xp11-cen. There were also ESTs mapped that were closely related to non-human sequence records. These matches therefore can be considered to identify human counterparts of known gene products, or members of known gene families. Examples of these include membrane proteins, translation-associated proteins, structural proteins, and enzymes. These data then demonstrate that single pass sequence information is sufficient to design PCR primers useful for assigning cDNA sequences to human chromosomes. When the EST sequence matches previous sequence database records, the chromosome assignments of the EST can be used to make preliminary assignments of the human gene to a chromosome.« less

A phylogenomic approach to bacterial subspecies classification: proof of concept in Mycobacterium abscessus.

PubMed

Tan, Joon Liang; Khang, Tsung Fei; Ngeow, Yun Fong; Choo, Siew Woh

2013-12-13

Mycobacterium abscessus is a rapidly growing mycobacterium that is often associated with human infections. The taxonomy of this species has undergone several revisions and is still being debated. In this study, we sequenced the genomes of 12 M. abscessus strains and used phylogenomic analysis to perform subspecies classification. A data mining approach was used to rank and select informative genes based on the relative entropy metric for the construction of a phylogenetic tree. The resulting tree topology was similar to that generated using the concatenation of five classical housekeeping genes: rpoB, hsp65, secA, recA and sodA. Additional support for the reliability of the subspecies classification came from the analysis of erm41 and ITS gene sequences, single nucleotide polymorphisms (SNPs)-based classification and strain clustering demonstrated by a variable number tandem repeat (VNTR) assay and a multilocus sequence analysis (MLSA). We subsequently found that the concatenation of a minimal set of three median-ranked genes: DNA polymerase III subunit alpha (polC), 4-hydroxy-2-ketovalerate aldolase (Hoa) and cell division protein FtsZ (ftsZ), is sufficient to recover the same tree topology. PCR assays designed specifically for these genes showed that all three genes could be amplified in the reference strain of M. abscessus ATCC 19977T. This study provides proof of concept that whole-genome sequence-based data mining approach can provide confirmatory evidence of the phylogenetic informativeness of existing markers, as well as lead to the discovery of a more economical and informative set of markers that produces similar subspecies classification in M. abscessus. The systematic procedure used in this study to choose the informative minimal set of gene markers can potentially be applied to species or subspecies classification of other bacteria.

Optimisation of DNA extraction from the crustacean Daphnia

PubMed Central

Athanasio, Camila Gonçalves; Chipman, James K.; Viant, Mark R.

2016-01-01

Daphnia are key model organisms for mechanistic studies of phenotypic plasticity, adaptation and microevolution, which have led to an increasing demand for genomics resources. A key step in any genomics analysis, such as high-throughput sequencing, is the availability of sufficient and high quality DNA. Although commercial kits exist to extract genomic DNA from several species, preparation of high quality DNA from Daphnia spp. and other chitinous species can be challenging. Here, we optimise methods for tissue homogenisation, DNA extraction and quantification customised for different downstream analyses (e.g., LC-MS/MS, Hiseq, mate pair sequencing or Nanopore). We demonstrate that if Daphnia magna are homogenised as whole animals (including the carapace), absorbance-based DNA quantification methods significantly over-estimate the amount of DNA, resulting in using insufficient starting material for experiments, such as preparation of sequencing libraries. This is attributed to the high refractive index of chitin in Daphnia’s carapace at 260 nm. Therefore, unless the carapace is removed by overnight proteinase digestion, the extracted DNA should be quantified with fluorescence-based methods. However, overnight proteinase digestion will result in partial fragmentation of DNA therefore the prepared DNA is not suitable for downstream methods that require high molecular weight DNA, such as PacBio, mate pair sequencing and Nanopore. In conclusion, we found that the MasterPure DNA purification kit, coupled with grinding of frozen tissue, is the best method for extraction of high molecular weight DNA as long as the extracted DNA is quantified with fluorescence-based methods. This method generated high yield and high molecular weight DNA (3.10 ± 0.63 ng/µg dry mass, fragments >60 kb), free of organic contaminants (phenol, chloroform) and is suitable for large number of downstream analyses. PMID:27190714

MS/MS-Assisted Design of Sequence-Controlled Synthetic Polymers for Improved Reading of Encoded Information

NASA Astrophysics Data System (ADS)

Charles, Laurence; Cavallo, Gianni; Monnier, Valérie; Oswald, Laurence; Szweda, Roza; Lutz, Jean-François

2017-06-01

In order to improve their MS/MS sequencing, structure of sequence-controlled synthetic polymers can be optimized based on considerations regarding their fragmentation behavior in collision-induced dissociation conditions, as demonstrated here for two digitally encoded polymer families. In poly(triazole amide)s, the main dissociation route proceeded via cleavage of the amide bond in each monomer, hence allowing the chains to be safely sequenced. However, a competitive cleavage of an ether bond in a tri(ethylene glycol) spacer placed between each coding moiety complicated MS/MS spectra while not bringing new structural information. Changing the tri(ethylene glycol) spacer to an alkyl group of the same size allowed this unwanted fragmentation pathway to be avoided, hence greatly simplifying the MS/MS reading step for such undecyl-based poly(triazole amide)s. In poly(alkoxyamine phosphodiester)s, a single dissociation pathway was achieved with repeating units containing an alkoxyamine linkage, which, by very low dissociation energy, made any other chemical bonds MS/MS-silent. Structure of these polymers was further tailored to enhance the stability of those precursor ions with a negatively charged phosphate group per monomer in order to improve their MS/MS readability. Increasing the size of both the alkyl coding moiety and the nitroxide spacer allowed sufficient distance between phosphate groups for all of them to be deprotonated simultaneously. Because the charge state of product ions increased with their polymerization degree, MS/MS spectra typically exhibited groups of fragments at one or the other side of the precursor ion depending on the original α or ω end-group they contain, allowing sequence reconstruction in a straightforward manner. [Figure not available: see fulltext.

A two-locus global DNA barcode for land plants: the coding rbcL gene complements the non-coding trnH-psbA spacer region.

PubMed

Kress, W John; Erickson, David L

2007-06-06

A useful DNA barcode requires sufficient sequence variation to distinguish between species and ease of application across a broad range of taxa. Discovery of a DNA barcode for land plants has been limited by intrinsically lower rates of sequence evolution in plant genomes than that observed in animals. This low rate has complicated the trade-off in finding a locus that is universal and readily sequenced and has sufficiently high sequence divergence at the species-level. Here, a global plant DNA barcode system is evaluated by comparing universal application and degree of sequence divergence for nine putative barcode loci, including coding and non-coding regions, singly and in pairs across a phylogenetically diverse set of 48 genera (two species per genus). No single locus could discriminate among species in a pair in more than 79% of genera, whereas discrimination increased to nearly 88% when the non-coding trnH-psbA spacer was paired with one of three coding loci, including rbcL. In silico trials were conducted in which DNA sequences from GenBank were used to further evaluate the discriminatory power of a subset of these loci. These trials supported the earlier observation that trnH-psbA coupled with rbcL can correctly identify and discriminate among related species. A combination of the non-coding trnH-psbA spacer region and a portion of the coding rbcL gene is recommended as a two-locus global land plant barcode that provides the necessary universality and species discrimination.

DNA Barcoding Green Microalgae Isolated from Neotropical Inland Waters

PubMed Central

Hadi, Sámed I. I. A.; Santana, Hugo; Brunale, Patrícia P. M.; Gomes, Taísa G.; Oliveira, Márcia D.; Matthiensen, Alexandre; Oliveira, Marcos E. C.; Silva, Flávia C. P.; Brasil, Bruno S. A. F.

2016-01-01

This study evaluated the feasibility of using the Ribulose Bisphosphate Carboxylase Large subunit gene (rbcL) and the Internal Transcribed Spacers 1 and 2 of the nuclear rDNA (nuITS1 and nuITS2) markers for identifying a very diverse, albeit poorly known group, of green microalgae from neotropical inland waters. Fifty-one freshwater green microalgae strains isolated from Brazil, the largest biodiversity reservoir in the neotropics, were submitted to DNA barcoding. Currently available universal primers for ITS1-5.8S-ITS2 region amplification were sufficient to successfully amplify and sequence 47 (92%) of the samples. On the other hand, new sets of primers had to be designed for rbcL, which allowed 96% of the samples to be sequenced. Thirty-five percent of the strains could be unambiguously identified to the species level based either on nuITS1 or nuITS2 sequences’ using barcode gap calculations. nuITS2 Compensatory Base Change (CBC) and ITS1-5.8S-ITS2 region phylogenetic analysis, together with morphological inspection, confirmed the identification accuracy. In contrast, only 6% of the strains could be assigned to the correct species based solely on rbcL sequences. In conclusion, the data presented here indicates that either nuITS1 or nuITS2 are useful markers for DNA barcoding of freshwater green microalgae, with advantage for nuITS2 due to the larger availability of analytical tools and reference barcodes deposited at databases for this marker. PMID:26900844

Similarity-based gene detection: using COGs to find evolutionarily-conserved ORFs.

PubMed

Powell, Bradford C; Hutchison, Clyde A

2006-01-19

Experimental verification of gene products has not kept pace with the rapid growth of microbial sequence information. However, existing annotations of gene locations contain sufficient information to screen for probable errors. Furthermore, comparisons among genomes become more informative as more genomes are examined. We studied all open reading frames (ORFs) of at least 30 codons from the genomes of 27 sequenced bacterial strains. We grouped the potential peptide sequences encoded from the ORFs by forming Clusters of Orthologous Groups (COGs). We used this grouping in order to find homologous relationships that would not be distinguishable from noise when using simple BLAST searches. Although COG analysis was initially developed to group annotated genes, we applied it to the task of grouping anonymous DNA sequences that may encode proteins. "Mixed COGs" of ORFs (clusters in which some sequences correspond to annotated genes and some do not) are attractive targets when seeking errors of gene prediction. Examination of mixed COGs reveals some situations in which genes appear to have been missed in current annotations and a smaller number of regions that appear to have been annotated as gene loci erroneously. This technique can also be used to detect potential pseudogenes or sequencing errors. Our method uses an adjustable parameter for degree of conservation among the studied genomes (stringency). We detail results for one level of stringency at which we found 83 potential genes which had not previously been identified, 60 potential pseudogenes, and 7 sequences with existing gene annotations that are probably incorrect. Systematic study of sequence conservation offers a way to improve existing annotations by identifying potentially homologous regions where the annotation of the presence or absence of a gene is inconsistent among genomes.

Similarity-based gene detection: using COGs to find evolutionarily-conserved ORFs

PubMed Central

Powell, Bradford C; Hutchison, Clyde A

2006-01-01

Background Experimental verification of gene products has not kept pace with the rapid growth of microbial sequence information. However, existing annotations of gene locations contain sufficient information to screen for probable errors. Furthermore, comparisons among genomes become more informative as more genomes are examined. We studied all open reading frames (ORFs) of at least 30 codons from the genomes of 27 sequenced bacterial strains. We grouped the potential peptide sequences encoded from the ORFs by forming Clusters of Orthologous Groups (COGs). We used this grouping in order to find homologous relationships that would not be distinguishable from noise when using simple BLAST searches. Although COG analysis was initially developed to group annotated genes, we applied it to the task of grouping anonymous DNA sequences that may encode proteins. Results "Mixed COGs" of ORFs (clusters in which some sequences correspond to annotated genes and some do not) are attractive targets when seeking errors of gene predicion. Examination of mixed COGs reveals some situations in which genes appear to have been missed in current annotations and a smaller number of regions that appear to have been annotated as gene loci erroneously. This technique can also be used to detect potential pseudogenes or sequencing errors. Our method uses an adjustable parameter for degree of conservation among the studied genomes (stringency). We detail results for one level of stringency at which we found 83 potential genes which had not previously been identified, 60 potential pseudogenes, and 7 sequences with existing gene annotations that are probably incorrect. Conclusion Systematic study of sequence conservation offers a way to improve existing annotations by identifying potentially homologous regions where the annotation of the presence or absence of a gene is inconsistent among genomes. PMID:16423288

Numeric promoter description - A comparative view on concepts and general application.

PubMed

Beier, Rico; Labudde, Dirk

2016-01-01

Nucleic acid molecules play a key role in a variety of biological processes. Starting from storage and transfer tasks, this also comprises the triggering of biological processes, regulatory effects and the active influence gained by target binding. Based on the experimental output (in this case promoter sequences), further in silico analyses aid in gaining new insights into these processes and interactions. The numerical description of nucleic acids thereby constitutes a bridge between the concrete biological issues and the analytical methods. Hence, this study compares 26 descriptor sets obtained by applying well-known numerical description concepts to an established dataset of 38 DNA promoter sequences. The suitability of the description sets was evaluated by computing partial least squares regression models and assessing the model accuracy. We conclude that the major importance regarding the descriptive power is attached to positional information rather than to explicitly incorporated physico-chemical information, since a sufficient amount of implicit physico-chemical information is already encoded in the nucleobase classification. The regression models especially benefited from employing the information that is encoded in the sequential and structural neighborhood of the nucleobases. Thus, the analyses of n-grams (short fragments of length n) suggested that they are valuable descriptors for DNA target interactions. A mixed n-gram descriptor set thereby yielded the best description of the promoter sequences. The corresponding regression model was checked and found to be plausible as it was able to reproduce the characteristic binding motifs of promoter sequences in a reasonable degree. As most functional nucleic acids are based on the principle of molecular recognition, the findings are not restricted to promoter sequences, but can rather be transferred to other kinds of functional nucleic acids. Thus, the concepts presented in this study could provide advantages for future nucleic acid-based technologies, like biosensoring, therapeutics and molecular imaging. Copyright © 2015 Elsevier Inc. All rights reserved.

The interplay of descriptor-based computational analysis with pharmacophore modeling builds the basis for a novel classification scheme for feruloyl esterases.

PubMed

Udatha, D B R K Gupta; Kouskoumvekaki, Irene; Olsson, Lisbeth; Panagiotou, Gianni

2011-01-01

One of the most intriguing groups of enzymes, the feruloyl esterases (FAEs), is ubiquitous in both simple and complex organisms. FAEs have gained importance in biofuel, medicine and food industries due to their capability of acting on a large range of substrates for cleaving ester bonds and synthesizing high-added value molecules through esterification and transesterification reactions. During the past two decades extensive studies have been carried out on the production and partial characterization of FAEs from fungi, while much less is known about FAEs of bacterial or plant origin. Initial classification studies on FAEs were restricted on sequence similarity and substrate specificity on just four model substrates and considered only a handful of FAEs belonging to the fungal kingdom. This study centers on the descriptor-based classification and structural analysis of experimentally verified and putative FAEs; nevertheless, the framework presented here is applicable to every poorly characterized enzyme family. 365 FAE-related sequences of fungal, bacterial and plantae origin were collected and they were clustered using Self Organizing Maps followed by k-means clustering into distinct groups based on amino acid composition and physico-chemical composition descriptors derived from the respective amino acid sequence. A Support Vector Machine model was subsequently constructed for the classification of new FAEs into the pre-assigned clusters. The model successfully recognized 98.2% of the training sequences and all the sequences of the blind test. The underlying functionality of the 12 proposed FAE families was validated against a combination of prediction tools and published experimental data. Another important aspect of the present work involves the development of pharmacophore models for the new FAE families, for which sufficient information on known substrates existed. Knowing the pharmacophoric features of a small molecule that are essential for binding to the members of a certain family opens a window of opportunities for tailored applications of FAEs. Copyright © 2010 Elsevier Inc. All rights reserved.

Rational Protein Engineering Guided by Deep Mutational Scanning

PubMed Central

Shin, HyeonSeok; Cho, Byung-Kwan

2015-01-01

Sequence–function relationship in a protein is commonly determined by the three-dimensional protein structure followed by various biochemical experiments. However, with the explosive increase in the number of genome sequences, facilitated by recent advances in sequencing technology, the gap between protein sequences available and three-dimensional structures is rapidly widening. A recently developed method termed deep mutational scanning explores the functional phenotype of thousands of mutants via massive sequencing. Coupled with a highly efficient screening system, this approach assesses the phenotypic changes made by the substitution of each amino acid sequence that constitutes a protein. Such an informational resource provides the functional role of each amino acid sequence, thereby providing sufficient rationale for selecting target residues for protein engineering. Here, we discuss the current applications of deep mutational scanning and consider experimental design. PMID:26404267

Transforming clinical microbiology with bacterial genome sequencing.

PubMed

Didelot, Xavier; Bowden, Rory; Wilson, Daniel J; Peto, Tim E A; Crook, Derrick W

2012-09-01

Whole-genome sequencing of bacteria has recently emerged as a cost-effective and convenient approach for addressing many microbiological questions. Here, we review the current status of clinical microbiology and how it has already begun to be transformed by using next-generation sequencing. We focus on three essential tasks: identifying the species of an isolate, testing its properties, such as resistance to antibiotics and virulence, and monitoring the emergence and spread of bacterial pathogens. We predict that the application of next-generation sequencing will soon be sufficiently fast, accurate and cheap to be used in routine clinical microbiology practice, where it could replace many complex current techniques with a single, more efficient workflow.

Transforming clinical microbiology with bacterial genome sequencing

PubMed Central

2016-01-01

Whole genome sequencing of bacteria has recently emerged as a cost-effective and convenient approach for addressing many microbiological questions. Here we review the current status of clinical microbiology and how it has already begun to be transformed by the use of next-generation sequencing. We focus on three essential tasks: identifying the species of an isolate, testing its properties such as resistance to antibiotics and virulence, and monitoring the emergence and spread of bacterial pathogens. The application of next-generation sequencing will soon be sufficiently fast, accurate and cheap to be used in routine clinical microbiology practice, where it could replace many complex current techniques with a single, more efficient workflow. PMID:22868263

Combinatorial Pooling Enables Selective Sequencing of the Barley Gene Space

PubMed Central

Lonardi, Stefano; Duma, Denisa; Alpert, Matthew; Cordero, Francesca; Beccuti, Marco; Bhat, Prasanna R.; Wu, Yonghui; Ciardo, Gianfranco; Alsaihati, Burair; Ma, Yaqin; Wanamaker, Steve; Resnik, Josh; Bozdag, Serdar; Luo, Ming-Cheng; Close, Timothy J.

2013-01-01

For the vast majority of species – including many economically or ecologically important organisms, progress in biological research is hampered due to the lack of a reference genome sequence. Despite recent advances in sequencing technologies, several factors still limit the availability of such a critical resource. At the same time, many research groups and international consortia have already produced BAC libraries and physical maps and now are in a position to proceed with the development of whole-genome sequences organized around a physical map anchored to a genetic map. We propose a BAC-by-BAC sequencing protocol that combines combinatorial pooling design and second-generation sequencing technology to efficiently approach denovo selective genome sequencing. We show that combinatorial pooling is a cost-effective and practical alternative to exhaustive DNA barcoding when preparing sequencing libraries for hundreds or thousands of DNA samples, such as in this case gene-bearing minimum-tiling-path BAC clones. The novelty of the protocol hinges on the computational ability to efficiently compare hundred millions of short reads and assign them to the correct BAC clones (deconvolution) so that the assembly can be carried out clone-by-clone. Experimental results on simulated data for the rice genome show that the deconvolution is very accurate, and the resulting BAC assemblies have high quality. Results on real data for a gene-rich subset of the barley genome confirm that the deconvolution is accurate and the BAC assemblies have good quality. While our method cannot provide the level of completeness that one would achieve with a comprehensive whole-genome sequencing project, we show that it is quite successful in reconstructing the gene sequences within BACs. In the case of plants such as barley, this level of sequence knowledge is sufficient to support critical end-point objectives such as map-based cloning and marker-assisted breeding. PMID:23592960

Combinatorial pooling enables selective sequencing of the barley gene space.

PubMed

Lonardi, Stefano; Duma, Denisa; Alpert, Matthew; Cordero, Francesca; Beccuti, Marco; Bhat, Prasanna R; Wu, Yonghui; Ciardo, Gianfranco; Alsaihati, Burair; Ma, Yaqin; Wanamaker, Steve; Resnik, Josh; Bozdag, Serdar; Luo, Ming-Cheng; Close, Timothy J

2013-04-01

For the vast majority of species - including many economically or ecologically important organisms, progress in biological research is hampered due to the lack of a reference genome sequence. Despite recent advances in sequencing technologies, several factors still limit the availability of such a critical resource. At the same time, many research groups and international consortia have already produced BAC libraries and physical maps and now are in a position to proceed with the development of whole-genome sequences organized around a physical map anchored to a genetic map. We propose a BAC-by-BAC sequencing protocol that combines combinatorial pooling design and second-generation sequencing technology to efficiently approach denovo selective genome sequencing. We show that combinatorial pooling is a cost-effective and practical alternative to exhaustive DNA barcoding when preparing sequencing libraries for hundreds or thousands of DNA samples, such as in this case gene-bearing minimum-tiling-path BAC clones. The novelty of the protocol hinges on the computational ability to efficiently compare hundred millions of short reads and assign them to the correct BAC clones (deconvolution) so that the assembly can be carried out clone-by-clone. Experimental results on simulated data for the rice genome show that the deconvolution is very accurate, and the resulting BAC assemblies have high quality. Results on real data for a gene-rich subset of the barley genome confirm that the deconvolution is accurate and the BAC assemblies have good quality. While our method cannot provide the level of completeness that one would achieve with a comprehensive whole-genome sequencing project, we show that it is quite successful in reconstructing the gene sequences within BACs. In the case of plants such as barley, this level of sequence knowledge is sufficient to support critical end-point objectives such as map-based cloning and marker-assisted breeding.

Production Scheduling of Sequenced Tapes for Printed Circuit Pack Assembly.

DTIC Science & Technology

1987-07-09

detail. L j 6 The subject matter of this thesis is inspired directly from their technical report. The goals of this research are twofold: 1) Test their...The subject matter of the following chapters describes a heuristic approach to another variation of the sequenced tape production scheduling problem...assignment problem, comprise the subject matter of Chapter 5. It is sufficient to note that the three definitions of the term common correspond to the

Conditions for the invertibility of the nonlinear difference operator (\\mathscr Rx)(n)=H(x(n),x(n+1)), n\\in\\mathbb Z, in the space of bounded number sequences

NASA Astrophysics Data System (ADS)

Slyusarchuk, Vasilii E.

2009-02-01

Necessary and sufficient conditions are found for the invertibility of the nonlinear difference operator \\displaystyle (\\mathscr Rx)(n)=H(x(n),x(n+1)),\\qquad n\\in\\mathbb Z, in the space of bounded two-sided number sequences. Here H\\colon \\mathbb R^2\\to \\mathbb R is a continuous function. Bibliography: 29 titles.

Augmentation of machine structure to improve its diagnosability

NASA Technical Reports Server (NTRS)

Hsieh, L.

1973-01-01

Two methods of augmenting the structure of a sequential machine so that it is diagnosable are presented. The checkable (checking sequences) and repeated symbol distinguishing sequences (RDS) are discussed. It was found that as few as twice the number of outputs of the given machine is sufficient for constructing a state-output augmentation with RDS. Techniques for minimizing the number of states in resolving convergences and in resolving equivalent and nonreduced cycles are developed.

Spatially Resolved Spectroscopy of Narrow-line Seyfert 1 Host Galaxies

NASA Astrophysics Data System (ADS)

Scharwächter, J.; Husemann, B.; Busch, G.; Komossa, S.; Dopita, M. A.

2017-10-01

We present optical integral field spectroscopy for five z< 0.062 narrow-line Seyfert 1 (NLS1) galaxies, probing their host galaxies at ≳ 2{--}3 {kpc} scales. Emission lines from the active galactic nucleus (AGN) and the large-scale host galaxy are analyzed separately, based on an AGN-host decomposition technique. The host galaxy gas kinematics indicates large-scale gas rotation in all five sources. At the probed scales of ≳ 2{--}3 {kpc}, the host galaxy gas is found to be predominantly ionized by star formation without any evidence of a strong AGN contribution. None of the five objects shows specific star formation rates (SFRs) exceeding the main sequence of low-redshift star-forming galaxies. The specific SFRs for MCG-05-01-013 and WPVS 007 are roughly consistent with the main sequence, while ESO 399-IG20, MS 22549-3712, and TON S180 show lower specific SFRs, intermediate to the main sequence and the red quiescent galaxies. The host galaxy metallicities, derived for the two sources with sufficient data quality (ESO 399-IG20 and MCG-05-01-013), indicate central oxygen abundances just below the low-redshift mass-metallicity relation. Based on this initial case study, we outline a comparison of AGN and host galaxy parameters as a starting point for future extended NLS1 studies with similar methods.

Photometric binary stars in Praesepe and the search for globular cluster binaries

NASA Technical Reports Server (NTRS)

Bolte, Michael

1991-01-01

A radial velocity study of the stars which are located on a second sequence above the single-star zero-age main sequence at a given color in the color-magnitude diagram of the open cluster Praesepe, (NGC 2632) shows that 10, and possibly 11, of 17 are binary systems. Of the binary systems, five have full amplitudes for their velocity variations that are greater than 50 km/s. To the extent that they can be applied to globular clusters, these results suggests that (1) observations of 'second-sequence' stars in globular clusters would be an efficient way of finding main-sequence binary systems in globulars, and (2) current instrumentation on large telescopes is sufficient for establishing unambiguously the existence of main-sequence binary systems in nearby globular clusters.

OrthoSelect: a protocol for selecting orthologous groups in phylogenomics.

PubMed

Schreiber, Fabian; Pick, Kerstin; Erpenbeck, Dirk; Wörheide, Gert; Morgenstern, Burkhard

2009-07-16

Phylogenetic studies using expressed sequence tags (EST) are becoming a standard approach to answer evolutionary questions. Such studies are usually based on large sets of newly generated, unannotated, and error-prone EST sequences from different species. A first crucial step in EST-based phylogeny reconstruction is to identify groups of orthologous sequences. From these data sets, appropriate target genes are selected, and redundant sequences are eliminated to obtain suitable sequence sets as input data for tree-reconstruction software. Generating such data sets manually can be very time consuming. Thus, software tools are needed that carry out these steps automatically. We developed a flexible and user-friendly software pipeline, running on desktop machines or computer clusters, that constructs data sets for phylogenomic analyses. It automatically searches assembled EST sequences against databases of orthologous groups (OG), assigns ESTs to these predefined OGs, translates the sequences into proteins, eliminates redundant sequences assigned to the same OG, creates multiple sequence alignments of identified orthologous sequences and offers the possibility to further process this alignment in a last step by excluding potentially homoplastic sites and selecting sufficiently conserved parts. Our software pipeline can be used as it is, but it can also be adapted by integrating additional external programs. This makes the pipeline useful for non-bioinformaticians as well as to bioinformatic experts. The software pipeline is especially designed for ESTs, but it can also handle protein sequences. OrthoSelect is a tool that produces orthologous gene alignments from assembled ESTs. Our tests show that OrthoSelect detects orthologs in EST libraries with high accuracy. In the absence of a gold standard for orthology prediction, we compared predictions by OrthoSelect to a manually created and published phylogenomic data set. Our tool was not only able to rebuild the data set with a specificity of 98%, but it detected four percent more orthologous sequences. Furthermore, the results OrthoSelect produces are in absolut agreement with the results of other programs, but our tool offers a significant speedup and additional functionality, e.g. handling of ESTs, computing sequence alignments, and refining them. To our knowledge, there is currently no fully automated and freely available tool for this purpose. Thus, OrthoSelect is a valuable tool for researchers in the field of phylogenomics who deal with large quantities of EST sequences. OrthoSelect is written in Perl and runs on Linux/Mac OS X. The tool can be downloaded at (http://gobics.de/fabian/orthoselect.php).

Event-triggered consensus tracking of multi-agent systems with Lur'e nonlinear dynamics

NASA Astrophysics Data System (ADS)

Huang, Na; Duan, Zhisheng; Wen, Guanghui; Zhao, Yu

2016-05-01

In this paper, distributed consensus tracking problem for networked Lur'e systems is investigated based on event-triggered information interactions. An event-triggered control algorithm is designed with the advantages of reducing controller update frequency and sensor energy consumption. By using tools of ?-procedure and Lyapunov functional method, some sufficient conditions are derived to guarantee that consensus tracking is achieved under a directed communication topology. Meanwhile, it is shown that Zeno behaviour of triggering time sequences is excluded for the proposed event-triggered rule. Finally, some numerical simulations on coupled Chua's circuits are performed to illustrate the effectiveness of the theoretical algorithms.

Substrate-Driven Mapping of the Degradome by Comparison of Sequence Logos

PubMed Central

Fuchs, Julian E.; von Grafenstein, Susanne; Huber, Roland G.; Kramer, Christian; Liedl, Klaus R.

2013-01-01

Sequence logos are frequently used to illustrate substrate preferences and specificity of proteases. Here, we employed the compiled substrates of the MEROPS database to introduce a novel metric for comparison of protease substrate preferences. The constructed similarity matrix of 62 proteases can be used to intuitively visualize similarities in protease substrate readout via principal component analysis and construction of protease specificity trees. Since our new metric is solely based on substrate data, we can engraft the protease tree including proteolytic enzymes of different evolutionary origin. Thereby, our analyses confirm pronounced overlaps in substrate recognition not only between proteases closely related on sequence basis but also between proteolytic enzymes of different evolutionary origin and catalytic type. To illustrate the applicability of our approach we analyze the distribution of targets of small molecules from the ChEMBL database in our substrate-based protease specificity trees. We observe a striking clustering of annotated targets in tree branches even though these grouped targets do not necessarily share similarity on protein sequence level. This highlights the value and applicability of knowledge acquired from peptide substrates in drug design of small molecules, e.g., for the prediction of off-target effects or drug repurposing. Consequently, our similarity metric allows to map the degradome and its associated drug target network via comparison of known substrate peptides. The substrate-driven view of protein-protein interfaces is not limited to the field of proteases but can be applied to any target class where a sufficient amount of known substrate data is available. PMID:24244149

Development of a massively parallel sequencing assay for investigating sequence polymorphisms of 15 short tandem repeats in a Chinese Northern Han population.

PubMed

Zhang, Qing-Xia; Yang, Meng; Pan, Ya-Jiao; Zhao, Jing; Qu, Bao-Wang; Cheng, Feng; Yang, Ya-Ran; Jiao, Zhang-Ping; Liu, Li; Yan, Jiang-Wei

2018-05-17

Massively parallel sequencing (MPS) has been used in forensic genetics in recent years owing to several advantages, e.g. MPS can provide precise descriptions of the repeat allele structure and variation in the repeat-flanking regions, increasing the discriminating power among loci and individuals. However, it cannot be fully utilized unless sufficient population data are available for all loci. Thus, there is a pressing need to perform population studies providing a basis for the introduction of MPS into forensic practice. Here, we constructed a multiplex PCR system with fusion primers for one-directional PCR for MPS of 15 commonly used forensic autosomal STRs and amelogenin. Samples from 554 unrelated Chinese Northern Han individuals were typed using this MPS assay. In total, 313 alleles obtained by MPS for all 15 STRs were observed, and the corresponding allele frequencies ranged between 0.0009 and 0.5162. Of all 15 loci, the number of alleles identified for 12 loci increased compared to capillary electrophoresis approaches, and for the following six loci more than double the number of alleles was found: D2S1338, D5S818, D21S11, D13S317, vWA, and D3S1358. Forensic parameters were calculated based on length and sequence-based alleles. D21S11 showed the highest heterozygosity (0.8791), discrimination power (0.9865), and paternity exclusion probability in trios (0.7529). The cumulative match probability for MPS was approximately 2.3157 × 10 -20 . © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A conserved intronic U1 snRNP-binding sequence promotes trans-splicing in Drosophila

PubMed Central

Gao, Jun-Li; Fan, Yu-Jie; Wang, Xiu-Ye; Zhang, Yu; Pu, Jia; Li, Liang; Shao, Wei; Zhan, Shuai; Hao, Jianjiang

2015-01-01

Unlike typical cis-splicing, trans-splicing joins exons from two separate transcripts to produce chimeric mRNA and has been detected in most eukaryotes. Trans-splicing in trypanosomes and nematodes has been characterized as a spliced leader RNA-facilitated reaction; in contrast, its mechanism in higher eukaryotes remains unclear. Here we investigate mod(mdg4), a classic trans-spliced gene in Drosophila, and report that two critical RNA sequences in the middle of the last 5′ intron, TSA and TSB, promote trans-splicing of mod(mdg4). In TSA, a 13-nucleotide (nt) core motif is conserved across Drosophila species and is essential and sufficient for trans-splicing, which binds U1 small nuclear RNP (snRNP) through strong base-pairing with U1 snRNA. In TSB, a conserved secondary structure acts as an enhancer. Deletions of TSA and TSB using the CRISPR/Cas9 system result in developmental defects in flies. Although it is not clear how the 5′ intron finds the 3′ introns, compensatory changes in U1 snRNA rescue trans-splicing of TSA mutants, demonstrating that U1 recruitment is critical to promote trans-splicing in vivo. Furthermore, TSA core-like motifs are found in many other trans-spliced Drosophila genes, including lola. These findings represent a novel mechanism of trans-splicing, in which RNA motifs in the 5′ intron are sufficient to bring separate transcripts into close proximity to promote trans-splicing. PMID:25838544

A Proposal for a Genome Similarity-Based Taxonomy for Plant-Pathogenic Bacteria that Is Sufficiently Precise to Reflect Phylogeny, Host Range, and Outbreak Affiliation Applied to Pseudomonas syringae sensu lato as a Proof of Concept.

PubMed

Vinatzer, Boris A; Weisberg, Alexandra J; Monteil, Caroline L; Elmarakeby, Haitham A; Sheppard, Samuel K; Heath, Lenwood S

2017-01-01

Taxonomy of plant pathogenic bacteria is challenging because pathogens of different crops often belong to the same named species but current taxonomy does not provide names for bacteria below the subspecies level. The introduction of the host range-based pathovar system in the 1980s provided a temporary solution to this problem but has many limitations. The affordability of genome sequencing now provides the opportunity for developing a new genome-based taxonomic framework. We already proposed to name individual bacterial isolates based on pairwise genome similarity. Here, we expand on this idea and propose to use genome similarity-based codes, which we now call life identification numbers (LINs), to describe and name bacterial taxa. Using 93 genomes of Pseudomonas syringae sensu lato, LINs were compared with a P. syringae genome tree whereby the assigned LINs were found to be informative of a majority of phylogenetic relationships. LINs also reflected host range and outbreak association for strains of P. syringae pathovar actinidiae, a pathovar for which many genome sequences are available. We conclude that LINs could provide the basis for a new taxonomic framework to address the shortcomings of the current pathovar system and to complement the current taxonomic system of bacteria in general.

HMMerThread: detecting remote, functional conserved domains in entire genomes by combining relaxed sequence-database searches with fold recognition.

PubMed

Bradshaw, Charles Richard; Surendranath, Vineeth; Henschel, Robert; Mueller, Matthias Stefan; Habermann, Bianca Hermine

2011-03-10

Conserved domains in proteins are one of the major sources of functional information for experimental design and genome-level annotation. Though search tools for conserved domain databases such as Hidden Markov Models (HMMs) are sensitive in detecting conserved domains in proteins when they share sufficient sequence similarity, they tend to miss more divergent family members, as they lack a reliable statistical framework for the detection of low sequence similarity. We have developed a greatly improved HMMerThread algorithm that can detect remotely conserved domains in highly divergent sequences. HMMerThread combines relaxed conserved domain searches with fold recognition to eliminate false positive, sequence-based identifications. With an accuracy of 90%, our software is able to automatically predict highly divergent members of conserved domain families with an associated 3-dimensional structure. We give additional confidence to our predictions by validation across species. We have run HMMerThread searches on eight proteomes including human and present a rich resource of remotely conserved domains, which adds significantly to the functional annotation of entire proteomes. We find ∼4500 cross-species validated, remotely conserved domain predictions in the human proteome alone. As an example, we find a DNA-binding domain in the C-terminal part of the A-kinase anchor protein 10 (AKAP10), a PKA adaptor that has been implicated in cardiac arrhythmias and premature cardiac death, which upon stress likely translocates from mitochondria to the nucleus/nucleolus. Based on our prediction, we propose that with this HLH-domain, AKAP10 is involved in the transcriptional control of stress response. Further remotely conserved domains we discuss are examples from areas such as sporulation, chromosome segregation and signalling during immune response. The HMMerThread algorithm is able to automatically detect the presence of remotely conserved domains in proteins based on weak sequence similarity. Our predictions open up new avenues for biological and medical studies. Genome-wide HMMerThread domains are available at http://vm1-hmmerthread.age.mpg.de.

Physical Maps for Genome Analysis of Serotype A and D Strains of the Fungal Pathogen Cryptococcus neoformans

PubMed Central

Schein, Jacqueline E.; Tangen, Kristin L.; Chiu, Readman; Shin, Heesun; Lengeler, Klaus B.; MacDonald, William Kim; Bosdet, Ian; Heitman, Joseph; Jones, Steven J.M.; Marra, Marco A.; Kronstad, James W.

2002-01-01

The basidiomycete fungus Cryptococcus neoformans is an important opportunistic pathogen of humans that poses a significant threat to immunocompromised individuals. Isolates of C. neoformans are classified into serotypes (A, B, C, D, and AD) based on antigenic differences in the polysaccharide capsule that surrounds the fungal cells. Genomic and EST sequencing projects are underway for the serotype D strain JEC21 and the serotype A strain H99. As part of a genomics program for C. neoformans, we have constructed fingerprinted bacterial artificial chromosome (BAC) clone physical maps for strains H99 and JEC21 to support the genomic sequencing efforts and to provide an initial comparison of the two genomes. The BAC clones represented an estimated 10-fold redundant coverage of the genomes of each serotype and allowed the assembly of 20 contigs each for H99 and JEC21. We found that the genomes of the two strains are sufficiently distinct to prevent coassembly of the two maps when combined fingerprint data are used to construct contigs. Hybridization experiments placed 82 markers on the JEC21 map and 102 markers on the H99 map, enabling contigs to be linked with specific chromosomes identified by electrophoretic karyotyping. These markers revealed both extensive similarity in gene order (conservation of synteny) between JEC21 and H99 as well as examples of chromosomal rearrangements including inversions and translocations. Sequencing reads were generated from the ends of the BAC clones to allow correlation of genomic shotgun sequence data with physical map contigs. The BAC maps therefore represent a valuable resource for the generation, assembly, and finishing of the genomic sequence of both JEC21 and H99. The physical maps also serve as a link between map-based and sequence-based data, providing a powerful resource for continued genomic studies. [This paper is dedicated to the memory of Michael Smith, Founding Director of the Biotechnology Laboratory and the BC Cancer Agency Genome Sciences Centre. Supplemental material is available online at http://www.genome.org.] PMID:12213782

HMMerThread: Detecting Remote, Functional Conserved Domains in Entire Genomes by Combining Relaxed Sequence-Database Searches with Fold Recognition

PubMed Central

Bradshaw, Charles Richard; Surendranath, Vineeth; Henschel, Robert; Mueller, Matthias Stefan; Habermann, Bianca Hermine

2011-01-01

Conserved domains in proteins are one of the major sources of functional information for experimental design and genome-level annotation. Though search tools for conserved domain databases such as Hidden Markov Models (HMMs) are sensitive in detecting conserved domains in proteins when they share sufficient sequence similarity, they tend to miss more divergent family members, as they lack a reliable statistical framework for the detection of low sequence similarity. We have developed a greatly improved HMMerThread algorithm that can detect remotely conserved domains in highly divergent sequences. HMMerThread combines relaxed conserved domain searches with fold recognition to eliminate false positive, sequence-based identifications. With an accuracy of 90%, our software is able to automatically predict highly divergent members of conserved domain families with an associated 3-dimensional structure. We give additional confidence to our predictions by validation across species. We have run HMMerThread searches on eight proteomes including human and present a rich resource of remotely conserved domains, which adds significantly to the functional annotation of entire proteomes. We find ∼4500 cross-species validated, remotely conserved domain predictions in the human proteome alone. As an example, we find a DNA-binding domain in the C-terminal part of the A-kinase anchor protein 10 (AKAP10), a PKA adaptor that has been implicated in cardiac arrhythmias and premature cardiac death, which upon stress likely translocates from mitochondria to the nucleus/nucleolus. Based on our prediction, we propose that with this HLH-domain, AKAP10 is involved in the transcriptional control of stress response. Further remotely conserved domains we discuss are examples from areas such as sporulation, chromosome segregation and signalling during immune response. The HMMerThread algorithm is able to automatically detect the presence of remotely conserved domains in proteins based on weak sequence similarity. Our predictions open up new avenues for biological and medical studies. Genome-wide HMMerThread domains are available at http://vm1-hmmerthread.age.mpg.de. PMID:21423752

Sequence-based genotyping clarifies conflicting historical morphometric and biological data for 5 Eimeria species infecting turkeys.

PubMed

El-Sherry, S; Ogedengbe, M E; Hafeez, M A; Sayf-Al-Din, M; Gad, N; Barta, J R

2015-02-01

Unlike with Eimeria species infecting chickens, specific identification and nomenclature of Eimeria species infecting turkeys is complicated, and in the absence of molecular data, imprecise. In an attempt to reconcile contradictory data reported on oocyst morphometrics and biological descriptions of various Eimeria species infecting turkey, we established single oocyst derived lines of 5 important Eimeria species infecting turkeys, Eimeria meleagrimitis (USMN08-01 strain), Eimeria adenoeides (Guelph strain), Eimeria gallopavonis (Weybridge strain), Eimeria meleagridis (USAR97-01 strain), and Eimeria dispersa (Briston strain). Short portions (514 bp) of mitochondrial cytochrome c oxidase subunit I gene (mt COI) from each were amplified and sequenced. Comparison of these sequences showed sufficient species-specific sequence variation to recommend these short mt COI sequences as species-specific markers. Uniformity of oocyst features (dimensions and oocyst structure) of each pure line was observed. Additional morphological features of the oocysts of these species are described as useful for the microscopic differentiation of these Eimeria species. Combined molecular and morphometric data on these single species lines compared with the original species descriptions and more recent data have helped to clarify some confusing, and sometimes conflicting, features associated with these Eimeria spp. For example, these new data suggest that the KCH and KR strains of E. adenoeides reported previously represent 2 distinct species, E. adenoeides and E. meleagridis, respectively. Likewise, analysis of the Weybridge strain of E. adenoeides, which has long been used as a reference strain in various studies conducted on the pathogenicity of E. adenoeides, indicates that this coccidium is actually a strain of E. gallopavonis. We highly recommend mt COI sequence-based genotyping be incorporated into all studies using Eimeria spp. of turkeys to confirm species identifications and so that any resulting data can be associated correctly with a single named Eimeria species. © 2015 Poultry Science Association Inc.

Nullomers and High Order Nullomers in Genomic Sequences

PubMed Central

Vergni, Davide; Santoni, Daniele

2016-01-01

A nullomer is an oligomer that does not occur as a subsequence in a given DNA sequence, i.e. it is an absent word of that sequence. The importance of nullomers in several applications, from drug discovery to forensic practice, is now debated in the literature. Here, we investigated the nature of nullomers, whether their absence in genomes has just a statistical explanation or it is a peculiar feature of genomic sequences. We introduced an extension of the notion of nullomer, namely high order nullomers, which are nullomers whose mutated sequences are still nullomers. We studied different aspects of them: comparison with nullomers of random sequences, CpG distribution and mean helical rise. In agreement with previous results we found that the number of nullomers in the human genome is much larger than expected by chance. Nevertheless antithetical results were found when considering a random DNA sequence preserving dinucleotide frequencies. The analysis of CpG frequencies in nullomers and high order nullomers revealed, as expected, a high CpG content but it also highlighted a strong dependence of CpG frequencies on the dinucleotide position, suggesting that nullomers have their own peculiar structure and are not simply sequences whose CpG frequency is biased. Furthermore, phylogenetic trees were built on eleven species based on both the similarities between the dinucleotide frequencies and the number of nullomers two species share, showing that nullomers are fairly conserved among close species. Finally the study of mean helical rise of nullomers sequences revealed significantly high mean rise values, reinforcing the hypothesis that those sequences have some peculiar structural features. The obtained results show that nullomers are the consequence of the peculiar structure of DNA (also including biased CpG frequency and CpGs islands), so that the hypermutability model, also taking into account CpG islands, seems to be not sufficient to explain nullomer phenomenon. Finally, high order nullomers could emphasize those features that already make simple nullomers useful in several applications. PMID:27906971

Genetic diversity of Burkholderia (Proteobacteria) species from the Caatinga and Atlantic rainforest biomes in Bahia, Brazil.

PubMed

Santini, A C; Santos, H R M; Gross, E; Corrêa, R X

2013-03-11

The genus Burkholderia (β-Proteobacteria) currently comprises more than 60 species, including parasites, symbionts and free-living organisms. Several new species of Burkholderia have recently been described showing a great diversity of phenotypes. We examined the diversity of Burkholderia spp in environmental samples collected from Caatinga and Atlantic rainforest biomes of Bahia, Brazil. Legume nodules were collected from five locations, and 16S rDNA and recA genes of the isolated microorganisms were analyzed. Thirty-three contigs of 16S rRNA genes and four contigs of the recA gene related to the genus Burkholderia were obtained. The genetic dissimilarity of the strains ranged from 0 to 2.5% based on 16S rDNA analysis, indicating two main branches: one distinct branch of the dendrogram for the B. cepacia complex and another branch that rendered three major groups, partially reflecting host plants and locations. A dendrogram designed with sequences of this research and those designed with sequences of Burkholderia-type strains and the first hit BLAST had similar topologies. A dendrogram similar to that constructed by analysis of 16S rDNA was obtained using sequences of the fragment of the recA gene. The 16S rDNA sequences enabled sufficient identification of relevant similarities and groupings amongst isolates and the sequences that we obtained. Only 6 of the 33 isolates analyzed via 16S rDNA sequencing showed high similarity with the B. cepacia complex. Thus, over 3/4 of the isolates have potential for biotechnological applications.

DNA sequence requirements for the accurate transcription of a protein-coding plastid gene in a plastid in vitro system from mustard (Sinapis alba L.)

PubMed Central

Link, Gerhard

1984-01-01

A nuclease-treated plastid extract from mustard (Sinapis alba L.) allows efficient transcription of cloned plastid DNA templates. In this in vitro system, the major runoff transcript of the truncated gene for the 32 000 mol. wt. photosystem II protein was accurately initiated from a site close to or identical with the in vivo start site. By using plasmids with deletions in the 5'-flanking region of this gene as templates, a DNA region required for efficient and selective initiation was detected ˜28-35 nucleotides upstream of the transcription start site. This region contains the sequence element TTGACA, which matches the consensus sequence for prokaryotic `−35' promoter elements. In the absence of this region, a region ˜13-27 nucleotides upstream of the start site still enables a basic level of specific transcription. This second region contains the sequence element TATATAA, which matches the consensus sequence for the `TATA' box of genes transcribed by RNA polymerase II (or B). The region between the `TATA'-like element and the transcription start site is not sufficient but may be required for specific transcription of the plastid gene. This latter region contains the sequence element TATACT, which resembles the prokaryotic `−10' (Pribnow) box. Based on the structural and transcriptional features of the 5' upstream region, a `promoter switch' mechanism is proposed, which may account for the developmentally regulated expression of this plastid gene. ImagesFig. 1.Fig. 2.Fig. 3.Fig. 4.Figure 5. PMID:16453540

The Gap Procedure: for the identification of phylogenetic clusters in HIV-1 sequence data.

PubMed

Vrbik, Irene; Stephens, David A; Roger, Michel; Brenner, Bluma G

2015-11-04

In the context of infectious disease, sequence clustering can be used to provide important insights into the dynamics of transmission. Cluster analysis is usually performed using a phylogenetic approach whereby clusters are assigned on the basis of sufficiently small genetic distances and high bootstrap support (or posterior probabilities). The computational burden involved in this phylogenetic threshold approach is a major drawback, especially when a large number of sequences are being considered. In addition, this method requires a skilled user to specify the appropriate threshold values which may vary widely depending on the application. This paper presents the Gap Procedure, a distance-based clustering algorithm for the classification of DNA sequences sampled from individuals infected with the human immunodeficiency virus type 1 (HIV-1). Our heuristic algorithm bypasses the need for phylogenetic reconstruction, thereby supporting the quick analysis of large genetic data sets. Moreover, this fully automated procedure relies on data-driven gaps in sorted pairwise distances to infer clusters, thus no user-specified threshold values are required. The clustering results obtained by the Gap Procedure on both real and simulated data, closely agree with those found using the threshold approach, while only requiring a fraction of the time to complete the analysis. Apart from the dramatic gains in computational time, the Gap Procedure is highly effective in finding distinct groups of genetically similar sequences and obviates the need for subjective user-specified values. The clusters of genetically similar sequences returned by this procedure can be used to detect patterns in HIV-1 transmission and thereby aid in the prevention, treatment and containment of the disease.

GyrB sequence analysis and MALDI-TOF MS as identification tools for plant pathogenic Clavibacter.

PubMed

Zaluga, Joanna; Heylen, Kim; Van Hoorde, Koenraad; Hoste, Bart; Van Vaerenbergh, Johan; Maes, Martine; De Vos, Paul

2011-09-01

The bacterial genus Clavibacter has only one species, Clavibacter michiganensis, containing five subspecies. All five are plant pathogens, among which three are recognized as quarantine pests (mentioned on the EPPO A2 list). Prevention of their introduction and epidemic outbreaks requires a reliable and accurate identification. Currently, identification of these bacteria is time consuming and often problematic, mainly because of cross-reactions with other plant-associated bacteria in immunological tests and false-negative results in PCR detection methods. Furthermore, distinguishing closely related subspecies is not straightforward. This study aimed at evaluating the use of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and a fragment of the gyrB sequence for the reliable and fast identification of the Clavibacter subspecies. Amplification and sequencing of gyrB using a single primer set had sufficient resolution and specificity to identify each subspecies based on both sequence similarities in cluster analyses and specific signatures within the sequences. All five subspecies also generated distinct and reproducible MALDI-TOF MS profiles, with unique and specific ion peaks for each subspecies, which could be used as biomarkers for identification. Results from both methods were in agreement and were able to distinguish the five Clavibacter subspecies from each other and from representatives of closely related Rathayibacter, Leifsonia or Curtobacterium species. Our study suggests that proteomic analysis using MALDI-TOF MS and gyrB sequence are powerful diagnostic tools for the accurate identification of Clavibacter plant pathogens. Copyright © 2011 Elsevier GmbH. All rights reserved.

Alignment-free sequence comparison (II): theoretical power of comparison statistics.

PubMed

Wan, Lin; Reinert, Gesine; Sun, Fengzhu; Waterman, Michael S

2010-11-01

Rapid methods for alignment-free sequence comparison make large-scale comparisons between sequences increasingly feasible. Here we study the power of the statistic D2, which counts the number of matching k-tuples between two sequences, as well as D2*, which uses centralized counts, and D2S, which is a self-standardized version, both from a theoretical viewpoint and numerically, providing an easy to use program. The power is assessed under two alternative hidden Markov models; the first one assumes that the two sequences share a common motif, whereas the second model is a pattern transfer model; the null model is that the two sequences are composed of independent and identically distributed letters and they are independent. Under the first alternative model, the means of the tuple counts in the individual sequences change, whereas under the second alternative model, the marginal means are the same as under the null model. Using the limit distributions of the count statistics under the null and the alternative models, we find that generally, asymptotically D2S has the largest power, followed by D2*, whereas the power of D2 can even be zero in some cases. In contrast, even for sequences of length 140,000 bp, in simulations D2* generally has the largest power. Under the first alternative model of a shared motif, the power of D2*approaches 100% when sufficiently many motifs are shared, and we recommend the use of D2* for such practical applications. Under the second alternative model of pattern transfer,the power for all three count statistics does not increase with sequence length when the sequence is sufficiently long, and hence none of the three statistics under consideration canbe recommended in such a situation. We illustrate the approach on 323 transcription factor binding motifs with length at most 10 from JASPAR CORE (October 12, 2009 version),verifying that D2* is generally more powerful than D2. The program to calculate the power of D2, D2* and D2S can be downloaded from http://meta.cmb.usc.edu/d2. Supplementary Material is available at www.liebertonline.com/cmb.

Short intronic repeat sequences facilitate circular RNA production

PubMed Central

Liang, Dongming

2014-01-01

Recent deep sequencing studies have revealed thousands of circular noncoding RNAs generated from protein-coding genes. These RNAs are produced when the precursor messenger RNA (pre-mRNA) splicing machinery “backsplices” and covalently joins, for example, the two ends of a single exon. However, the mechanism by which the spliceosome selects only certain exons to circularize is largely unknown. Using extensive mutagenesis of expression plasmids, we show that miniature introns containing the splice sites along with short (∼30- to 40-nucleotide) inverted repeats, such as Alu elements, are sufficient to allow the intervening exons to circularize in cells. The intronic repeats must base-pair to one another, thereby bringing the splice sites into close proximity to each other. More than simple thermodynamics is clearly at play, however, as not all repeats support circularization, and increasing the stability of the hairpin between the repeats can sometimes inhibit circular RNA biogenesis. The intronic repeats and exonic sequences must collaborate with one another, and a functional 3′ end processing signal is required, suggesting that circularization may occur post-transcriptionally. These results suggest detailed and generalizable models that explain how the splicing machinery determines whether to produce a circular noncoding RNA or a linear mRNA. PMID:25281217

Culturable bacteria present in the fluid of the hooded-pitcher plant Sarracenia minor based on 16S rDNA gene sequence data.

PubMed

Siragusa, Alex J; Swenson, Janice E; Casamatta, Dale A

2007-08-01

The culturable microbial community within the pitcher fluid of 93 Sarracenia minor carnivorous plants was examined over a 2-year study. Many aspects of the plant/bacterial/insect interaction within the pitcher fluid are minimally understood because the bacterial taxa present in these pitchers have not been identified. Thirteen isolates were characterized by 16S rDNA sequencing and subsequent phylogenetic analysis. The Proteobacteria were the most abundant taxa and included representatives from Serratia, Achromobacter, and Pantoea. The Actinobacteria Micrococcus was also abundant while Bacillus, Lactococcus, Chryseobacterium, and Rhodococcus were infrequently encountered. Several isolates conformed to species identifiers (>98% rDNA gene sequence similarity) including Serratia marcescens (isolates found in 27.5% of pitchers), Achromobacter xylosoxidans (37.6%), Micrococcus luteus (40.9%), Bacillus cereus (isolates found in 10.2%), Bacillus thuringiensis (5.4%), Lactococcus lactis (17.2%), and Rhodococcus equi (2.2%). Species-area curves suggest that sampling efforts were sufficient to recover a representative culturable bacterial community. The bacteria present represent a diverse community probably as a result of introduction by insect vectors, but the ecological significance remains under explored.

A 20 bp cis-acting element is both necessary and sufficient to mediate elicitor response of a maize PRms gene.

PubMed

Raventós, D; Jensen, A B; Rask, M B; Casacuberta, J M; Mundy, J; San Segundo, B

1995-01-01

Transient gene expression assays in barley aleurone protoplasts were used to identify a cis-regulatory element involved in the elicitor-responsive expression of the maize PRms gene. Analysis of transcriptional fusions between PRms 5' upstream sequences and a chloramphenicol acetyltransferase reporter gene, as well as chimeric promoters containing PRms promoter fragments or repeated oligonucleotides fused to a minimal promoter, delineated a 20 bp sequence which functioned as an elicitor-response element (ERE). This sequence contains a motif (-246 AATTGACC) similar to sequences found in promoters of other pathogen-responsive genes. The analysis also indicated that an enhancing sequence(s) between -397 and -296 is required for full PRms activation by elicitors. The protein kinase inhibitor staurosporine was found to completely block the transcriptional activation induced by elicitors. These data indicate that protein phosphorylation is involved in the signal transduction pathway leading to PRms expression.

Defining epitope coverage requirements for T cell-based HIV vaccines: Theoretical considerations and practical applications

PubMed Central

2011-01-01

Background HIV vaccine development must address the genetic diversity and plasticity of the virus that permits the presentation of diverse genetic forms to the immune system and subsequent escape from immune pressure. Assessment of potential HIV strain coverage by candidate T cell-based vaccines (whether natural sequence or computationally optimized products) is now a critical component in interpreting candidate vaccine suitability. Methods We have utilized an N-mer identity algorithm to represent T cell epitopes and explore potential coverage of the global HIV pandemic using natural sequences derived from candidate HIV vaccines. Breadth (the number of T cell epitopes generated) and depth (the variant coverage within a T cell epitope) analyses have been incorporated into the model to explore vaccine coverage requirements in terms of the number of discrete T cell epitopes generated. Results We show that when multiple epitope generation by a vaccine product is considered a far more nuanced appraisal of the potential HIV strain coverage of the vaccine product emerges. By considering epitope breadth and depth several important observations were made: (1) epitope breadth requirements to reach particular levels of vaccine coverage, even for natural sequence-based vaccine products is not necessarily an intractable problem for the immune system; (2) increasing the valency (number of T cell epitope variants present) of vaccine products dramatically decreases the epitope requirements to reach particular coverage levels for any epidemic; (3) considering multiple-hit models (more than one exact epitope match with an incoming HIV strain) places a significantly higher requirement upon epitope breadth in order to reach a given level of coverage, to the point where low valency natural sequence based products would not practically be able to generate sufficient epitopes. Conclusions When HIV vaccine sequences are compared against datasets of potential incoming viruses important metrics such as the minimum epitope count required to reach a desired level of coverage can be easily calculated. We propose that such analyses can be applied early in the planning stages and during the execution phase of a vaccine trial to explore theoretical and empirical suitability of a vaccine product to a particular epidemic setting. PMID:22152192

A Two-Locus Global DNA Barcode for Land Plants: The Coding rbcL Gene Complements the Non-Coding trnH-psbA Spacer Region

PubMed Central

Kress, W. John; Erickson, David L.

2007-01-01

Background A useful DNA barcode requires sufficient sequence variation to distinguish between species and ease of application across a broad range of taxa. Discovery of a DNA barcode for land plants has been limited by intrinsically lower rates of sequence evolution in plant genomes than that observed in animals. This low rate has complicated the trade-off in finding a locus that is universal and readily sequenced and has sufficiently high sequence divergence at the species-level. Methodology/Principal Findings Here, a global plant DNA barcode system is evaluated by comparing universal application and degree of sequence divergence for nine putative barcode loci, including coding and non-coding regions, singly and in pairs across a phylogenetically diverse set of 48 genera (two species per genus). No single locus could discriminate among species in a pair in more than 79% of genera, whereas discrimination increased to nearly 88% when the non-coding trnH-psbA spacer was paired with one of three coding loci, including rbcL. In silico trials were conducted in which DNA sequences from GenBank were used to further evaluate the discriminatory power of a subset of these loci. These trials supported the earlier observation that trnH-psbA coupled with rbcL can correctly identify and discriminate among related species. Conclusions/Significance A combination of the non-coding trnH-psbA spacer region and a portion of the coding rbcL gene is recommended as a two-locus global land plant barcode that provides the necessary universality and species discrimination. PMID:17551588

Nucleic and amino acid sequences relating to a novel transketolase, and methods for the expression thereof

DOEpatents

Croteau, Rodney Bruce; Wildung, Mark Raymond; Lange, Bernd Markus; McCaskill, David G.

2001-01-01

cDNAs encoding 1-deoxyxylulose-5-phosphate synthase from peppermint (Mentha piperita) have been isolated and sequenced, and the corresponding amino acid sequences have been determined. Accordingly, isolated DNA sequences (SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7) are provided which code for the expression of 1-deoxyxylulose-5-phosphate synthase from plants. In another aspect the present invention provides for isolated, recombinant DXPS proteins, such as the proteins having the sequences set forth in SEQ ID NO:4, SEQ ID NO:6 and SEQ ID NO:8. In other aspects, replicable recombinant cloning vehicles are provided which code for plant 1-deoxyxylulose-5-phosphate synthases, or for a base sequence sufficiently complementary to at least a portion of 1-deoxyxylulose-5-phosphate synthase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding a plant 1-deoxyxylulose-5-phosphate synthase. Thus, systems and methods are provided for the recombinant expression of the aforementioned recombinant 1-deoxyxylulose-5-phosphate synthase that may be used to facilitate its production, isolation and purification in significant amounts. Recombinant 1-deoxyxylulose-5-phosphate synthase may be used to obtain expression or enhanced expression of 1-deoxyxylulose-5-phosphate synthase in plants in order to enhance the production of 1-deoxyxylulose-5-phosphate, or its derivatives such as isopentenyl diphosphate (BP), or may be otherwise employed for the regulation or expression of 1-deoxyxylulose-5-phosphate synthase, or the production of its products.

What caused the outbreak of ESBL-producing Klebsiella pneumoniae in a neonatal intensive care unit, Germany 2009 to 2012? Reconstructing transmission with epidemiological analysis and whole-genome sequencing

PubMed Central

Haller, Sebastian; Eller, Christoph; Hermes, Julia; Kaase, Martin; Steglich, Matthias; Radonić, Aleksandar; Dabrowski, Piotr Wojtek; Nitsche, Andreas; Pfeifer, Yvonne; Werner, Guido; Wunderle, Werner; Velasco, Edward; Abu Sin, Muna; Eckmanns, Tim; Nübel, Ulrich

2015-01-01

Objective We aimed to retrospectively reconstruct the timing of transmission events and pathways in order to understand why extensive preventive measures and investigations were not sufficient to prevent new cases. Methods We extracted available information from patient charts to describe cases and to compare them to the normal population of the ward. We conducted a cohort study to identify risk factors for pathogen acquisition. We sequenced the available isolates to determine the phylogenetic relatedness of Klebsiella pneumoniae isolates on the basis of their genome sequences. Results The investigation comprises 37 cases and the 10 cases with ESBL (extended-spectrum beta-lactamase)-producing K. pneumoniae bloodstream infection. Descriptive epidemiology indicated that a continuous transmission from person to person was most likely. Results from the cohort study showed that ‘frequent manipulation’ (a proxy for increased exposure to medical procedures) was significantly associated with being a case (RR 1.44, 95% CI 1.02 to 2.19). Genome sequences revealed that all 48 bacterial isolates available for sequencing from 31 cases were closely related (maximum genetic distance, 12 single nucleotide polymorphisms). Based on our calculation of evolutionary rate and sequence diversity, we estimate that the outbreak strain was endemic since 2008. Conclusions Epidemiological and phylogenetic analyses consistently indicated that there were additional, undiscovered cases prior to the onset of microbiological screening and that the spread of the pathogen remained undetected over several years, driven predominantly by person-to-person transmission. Whole-genome sequencing provided valuable information on the onset, course and size of the outbreak, and on possible ways of transmission. PMID:25967999

Geranyl diphosphate synthase from mint

DOEpatents

Croteau, Rodney Bruce; Wildung, Mark Raymond; Burke, Charles Cullen; Gershenzon, Jonathan

1999-01-01

A cDNA encoding geranyl diphosphate synthase from peppermint has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Accordingly, an isolated DNA sequence (SEQ ID No:1) is provided which codes for the expression of geranyl diphosphate synthase (SEQ ID No:2) from peppermint (Mentha piperita). In other aspects, replicable recombinant cloning vehicles are provided which code for geranyl diphosphate synthase or for a base sequence sufficiently complementary to at least a portion of the geranyl diphosphate synthase DNA or RNA to enable hybridization therewith (e.g., antisense geranyl diphosphate synthase RNA or fragments of complementary geranyl diphosphate synthase DNA which are useful as polymerase chain reaction primers or as probes for geranyl diphosphate synthase or related genes). In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding geranyl diphosphate synthase. Thus, systems and methods are provided for the recombinant expression of geranyl diphosphate synthase that may be used to facilitate the production, isolation and purification of significant quantities of recombinant geranyl diphosphate synthase for subsequent use, to obtain expression or enhanced expression of geranyl diphosphate synthase in plants in order to enhance the production of monoterpenoids, to produce geranyl diphosphate in cancerous cells as a precursor to monoterpenoids having anti-cancer properties or may be otherwise employed for the regulation or expression of geranyl diphosphate synthase or the production of geranyl diphosphate.

Geranyl diphosphate synthase from mint

DOEpatents

Croteau, R.B.; Wildung, M.R.; Burke, C.C.; Gershenzon, J.

1999-03-02

A cDNA encoding geranyl diphosphate synthase from peppermint has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Accordingly, an isolated DNA sequence (SEQ ID No:1) is provided which codes for the expression of geranyl diphosphate synthase (SEQ ID No:2) from peppermint (Mentha piperita). In other aspects, replicable recombinant cloning vehicles are provided which code for geranyl diphosphate synthase or for a base sequence sufficiently complementary to at least a portion of the geranyl diphosphate synthase DNA or RNA to enable hybridization therewith (e.g., antisense geranyl diphosphate synthase RNA or fragments of complementary geranyl diphosphate synthase DNA which are useful as polymerase chain reaction primers or as probes for geranyl diphosphate synthase or related genes). In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding geranyl diphosphate synthase. Thus, systems and methods are provided for the recombinant expression of geranyl diphosphate synthase that may be used to facilitate the production, isolation and purification of significant quantities of recombinant geranyl diphosphate synthase for subsequent use, to obtain expression or enhanced expression of geranyl diphosphate synthase in plants in order to enhance the production of monoterpenoids, to produce geranyl diphosphate in cancerous cells as a precursor to monoterpenoids having anti-cancer properties or may be otherwise employed for the regulation or expression of geranyl diphosphate synthase or the production of geranyl diphosphate. 5 figs.

Association analysis using next-generation sequence data from publicly available control groups: the robust variance score statistic.

PubMed

Derkach, Andriy; Chiang, Theodore; Gong, Jiafen; Addis, Laura; Dobbins, Sara; Tomlinson, Ian; Houlston, Richard; Pal, Deb K; Strug, Lisa J

2014-08-01

Sufficiently powered case-control studies with next-generation sequence (NGS) data remain prohibitively expensive for many investigators. If feasible, a more efficient strategy would be to include publicly available sequenced controls. However, these studies can be confounded by differences in sequencing platform; alignment, single nucleotide polymorphism and variant calling algorithms; read depth; and selection thresholds. Assuming one can match cases and controls on the basis of ethnicity and other potential confounding factors, and one has access to the aligned reads in both groups, we investigate the effect of systematic differences in read depth and selection threshold when comparing allele frequencies between cases and controls. We propose a novel likelihood-based method, the robust variance score (RVS), that substitutes genotype calls by their expected values given observed sequence data. We show theoretically that the RVS eliminates read depth bias in the estimation of minor allele frequency. We also demonstrate that, using simulated and real NGS data, the RVS method controls Type I error and has comparable power to the 'gold standard' analysis with the true underlying genotypes for both common and rare variants. An RVS R script and instructions can be found at strug.research.sickkids.ca, and at https://github.com/strug-lab/RVS. lisa.strug@utoronto.ca Supplementary data are available at Bioinformatics online. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

No need to replace an "anomalous" primate (Primates) with an "anomalous" bear (Carnivora, Ursidae).

PubMed

Gutiérrez, Eliécer E; Pine, Ronald H

2015-01-01

By means of mitochondrial 12S rRNA sequencing of putative "yeti", "bigfoot", and other "anomalous primate" hair samples, a recent study concluded that two samples, presented as from the Himalayas, do not belong to an "anomalous primate", but to an unknown, anomalous type of ursid. That is, that they match 12S rRNA sequences of a fossil Polar Bear (Ursusmaritimus), but neither of modern Polar Bears, nor of Brown Bears (Ursusarctos), the closest relative of Polar Bears, and one that occurs today in the Himalayas. We have undertaken direct comparison of sequences; replication of the original comparative study; inference of phylogenetic relationships of the two samples with respect to those from all extant species of Ursidae (except for the Giant Panda, Ailuropodamelanoleuca) and two extinct Pleistocene species; and application of a non-tree-based population aggregation approach for species diagnosis and identification. Our results demonstrate that the very short fragment of the 12S rRNA gene sequenced by Sykes et al. is not sufficiently informative to support the hypotheses provided by these authors with respect to the taxonomic identity of the individuals from which these sequences were obtained. We have concluded that there is no reason to believe that the two samples came from anything other than Brown Bears. These analyses afforded an opportunity to test the monophyly of morphologically defined species and to comment on both their phylogenetic relationships and future efforts necessary to advance our understanding of ursid systematics.

Whole genome sequence of an unusual Borrelia burgdorferi sensu lato isolate

DOE Office of Scientific and Technical Information (OSTI.GOV)

Casjens, S.R.; Dunn, J.; Fraser-Liggett, C. M.

2011-03-01

Human Lyme disease is caused by a number of related Borrelia burgdorferi sensu lato species. We report here the complete genome sequence of Borrelia sp. isolate SV1 from Finland. This isolate is to date the closest known relative of B. burgdorferi sensu stricto, but it is sufficiently genetically distinct from that species that it and its close relatives warrant its candidacy for new-species status. We suggest that this isolate should be named 'Borrelia finlandensis.'

A putative N-terminal nuclear export sequence is sufficient for Mps1 nuclear exclusion during interphase.

PubMed

Jia, Haiwei; Zhang, Xiaojuan; Wang, Wenjun; Bai, Yuanyuan; Ling, Youguo; Cao, Cheng; Ma, Runlin Z; Zhong, Hui; Wang, Xue; Xu, Quanbin

2015-02-27

Mps1, an essential component of the mitotic checkpoint, is also an important interphase regulator and has roles in DNA damage response, cytokinesis and centrosome duplication. Mps1 predominantly resides in the cytoplasm and relocates into the nucleus at the late G2 phase. So far, the mechanism underlying the Mps1 translocation between the cytoplasm and nucleus has been unclear. In this work, a dynamic export process of Mps1 from the nucleus to cytoplasm in interphase was revealed- a process blocked by the Crm1 inhibitor, Leptomycin B, suggesting that export of Mps1 is Crm1 dependent. Consistent with this speculation, a direct association between Mps1 and Crm1 was found. Furthermore, a putative nuclear export sequence (pNES) motif at the N-terminal of Mps1 was identified by analyzing the motif of Mps1. This motif shows a high sequence similarity to the classic NES, a fusion of this motif with EGFP results in dramatic exclusion of the fusion protein from the nucleus. Additionally, Mps1 mutant loss of pNES integrity was shown by replacing leucine with alanine which produced a diffused subcellular distribution, compared to the wild type protein which resides predominantly in cytoplasm. Taken these findings together, it was concluded that the pNES sequence is sufficient for the Mps1 export from nucleus during interphase.

Single reaction, real time RT-PCR detection of all known avian and human metapneumoviruses.

PubMed

Lemaitre, E; Allée, C; Vabret, A; Eterradossi, N; Brown, P A

2018-01-01

Current molecular methods for the detection of avian and human metapneumovirus (AMPV, HMPV) are specifically targeted towards each virus species or individual subgroups of these. Here a broad range SYBR Green I real time RT-PCR was developed which amplified a highly conserved fragment of sequence in the N open reading frame. This method was sufficiently efficient and specific in detecting all MPVs. Its validation according to the NF U47-600 norm for the four AMPV subgroups estimated low limits of detection between 1000 and 10copies/μL, similar with detection levels described previously for real time RT-PCRs targeting specific subgroups. RNA viruses present a challenge for the design of durable molecular diagnostic test due to the rate of change in their genome sequences which can vary substantially in different areas and over time. The fact that the regions of sequence for primer hybridization in the described method have remained sufficiently conserved since the AMPV and HMPV diverged, should give the best chance of continued detection of current subgroups and of potential unknown or future emerging MPV strains. Copyright © 2017 Elsevier B.V. All rights reserved.

Development of a novel biosensing system based on the structural change of a polymerized guanine-quadruplex DNA nanostructure.

PubMed

Morita, Yo; Yoshida, Wataru; Savory, Nasa; Han, Sung Woong; Tera, Masayuki; Nagasawa, Kazuo; Nakamura, Chikashi; Sode, Koji; Ikebukuro, Kazunori

2011-08-15

By inserting an adenosine aptamer into an aptamer that forms a G-quadruplex, we developed an adaptor molecule, named the Gq-switch, which links an electrode with flavin adenine dinucleotide-dependent glucose dehydrogenase (FADGDH) that is capable of transferring electron to a electrode directly. First, we selected an FADGDH-binding aptamer and identified that its sequence is composed of two blocks of consecutive six guanine bases and it forms a polymerized G-quadruplex structure. Then, we inserted a sequence of an adenosine aptamer between the two blocks of consecutive guanine bases, and we found it also bound to adenosine. Then we named it as Gq-switch. In the absence of adenosine, the Gq-switch-FADGDH complex forms a 30-nm high bulb-shaped structure that changes in the presence of adenosine to give an 8-nm high wire-shaped structure. This structural change brings the FADGDH sufficiently close to the electrode for electron transfer to occur, and the adenosine can be detected from the current produced by the FADGDH. Adenosine was successfully detected with a concentration dependency using the Gq-switch-FADGDH complex immobilized Au electrode by measuring response current to the addition of glucose. Copyright © 2011 Elsevier B.V. All rights reserved.

Microbial detection with low molecular weight RNA.

PubMed

Kourentzi, K D; Fox, G E; Willson, R C

2001-12-01

The need to monitor microorganisms in the environment has increased interest in assays based on hybridization probes that target nucleic acids (e.g., rRNA). We report the development of liquid-phase assays for specific bacterial 5S rRNA sequences or similarly sized artificial RNAs (aRNAs) using molecular beacon technology. These beacons fluoresce only in the presence of specific target sequences, rendering as much as a 27-fold fluorescence enhancement. The assays can be used with both crude cell lysates and purified total RNA preparations. Minimal sample preparation (e.g., heating to promote leakage from cells) is sufficient to detect many Gram-negative bacteria. Using this approach it was possible to detect an aRNA-labeled Escherichia coli strain in the presence of a large background of an otherwise identical E. coli strain. Finally, by using a longer wavelength carboxytetramethylrhodamine beacon it was possible to reduce the fraction of the signal due to cellular autofluorescence to below 0.5%.

Microbial detection with low molecular weight RNA

NASA Technical Reports Server (NTRS)

Kourentzi, K. D.; Fox, G. E.; Willson, R. C.

2001-01-01

The need to monitor microorganisms in the environment has increased interest in assays based on hybridization probes that target nucleic acids (e.g., rRNA). We report the development of liquid-phase assays for specific bacterial 5S rRNA sequences or similarly sized artificial RNAs (aRNAs) using molecular beacon technology. These beacons fluoresce only in the presence of specific target sequences, rendering as much as a 27-fold fluorescence enhancement. The assays can be used with both crude cell lysates and purified total RNA preparations. Minimal sample preparation (e.g., heating to promote leakage from cells) is sufficient to detect many Gram-negative bacteria. Using this approach it was possible to detect an aRNA-labeled Escherichia coli strain in the presence of a large background of an otherwise identical E. coli strain. Finally, by using a longer wavelength carboxytetramethylrhodamine beacon it was possible to reduce the fraction of the signal due to cellular autofluorescence to below 0.5%.

Motif finding in DNA sequences based on skipping nonconserved positions in background Markov chains.

PubMed

Zhao, Xiaoyan; Sze, Sing-Hoi

2011-05-01

One strategy to identify transcription factor binding sites is through motif finding in upstream DNA sequences of potentially co-regulated genes. Despite extensive efforts, none of the existing algorithms perform very well. We consider a string representation that allows arbitrary ignored positions within the nonconserved portion of single motifs, and use O(2(l)) Markov chains to model the background distributions of motifs of length l while skipping these positions within each Markov chain. By focusing initially on positions that have fixed nucleotides to define core occurrences, we develop an algorithm to identify motifs of moderate lengths. We compare the performance of our algorithm to other motif finding algorithms on a few benchmark data sets, and show that significant improvement in accuracy can be obtained when the sites are sufficiently conserved within a given sample, while comparable performance is obtained when the site conservation rate is low. A software program (PosMotif ) and detailed results are available online at http://faculty.cse.tamu.edu/shsze/posmotif.

All-atom 3D structure prediction of transmembrane β-barrel proteins from sequences.

PubMed

Hayat, Sikander; Sander, Chris; Marks, Debora S; Elofsson, Arne

2015-04-28

Transmembrane β-barrels (TMBs) carry out major functions in substrate transport and protein biogenesis but experimental determination of their 3D structure is challenging. Encouraged by successful de novo 3D structure prediction of globular and α-helical membrane proteins from sequence alignments alone, we developed an approach to predict the 3D structure of TMBs. The approach combines the maximum-entropy evolutionary coupling method for predicting residue contacts (EVfold) with a machine-learning approach (boctopus2) for predicting β-strands in the barrel. In a blinded test for 19 TMB proteins of known structure that have a sufficient number of diverse homologous sequences available, this combined method (EVfold_bb) predicts hydrogen-bonded residue pairs between adjacent β-strands at an accuracy of ∼70%. This accuracy is sufficient for the generation of all-atom 3D models. In the transmembrane barrel region, the average 3D structure accuracy [template-modeling (TM) score] of top-ranked models is 0.54 (ranging from 0.36 to 0.85), with a higher (44%) number of residue pairs in correct strand-strand registration than in earlier methods (18%). Although the nonbarrel regions are predicted less accurately overall, the evolutionary couplings identify some highly constrained loop residues and, for FecA protein, the barrel including the structure of a plug domain can be accurately modeled (TM score = 0.68). Lower prediction accuracy tends to be associated with insufficient sequence information and we therefore expect increasing numbers of β-barrel families to become accessible to accurate 3D structure prediction as the number of available sequences increases.

An ultra-low cost NMR device with arbitrary pulse programming

NASA Astrophysics Data System (ADS)

Chen, Hsueh-Ying; Kim, Yaewon; Nath, Pulak; Hilty, Christian

2015-06-01

Ultra-low cost, general purpose electronics boards featuring microprocessors or field programmable gate arrays (FPGA) are reaching capabilities sufficient for direct implementation of NMR spectrometers. We demonstrate a spectrometer based on such a board, implemented with a minimal need for the addition of custom electronics and external components. This feature allows such a spectrometer to be readily implemented using typical knowledge present in an NMR laboratory. With FPGA technology, digital tasks are performed with precise timing, without the limitation of predetermined hardware function. In this case, the FPGA is used for programming of arbitrarily timed pulse sequence events, and to digitally generate required frequencies. Data acquired from a 0.53 T permanent magnet serves as a demonstration of the flexibility of pulse programming for diverse experiments. Pulse sequences applied include a spin-lattice relaxation measurement using a pulse train with small-flip angle pulses, and a Carr-Purcell-Meiboom-Gill experiment with phase cycle. Mixing of NMR signals with a digitally generated, 4-step phase-cycled reference frequency is further implemented to achieve sequential quadrature detection. The flexibility in hardware implementation permits tailoring this type of spectrometer for applications such as relaxometry, polarimetry, diffusometry or NMR based magnetometry.

Elastin as a self-organizing biomaterial: use of recombinantly expressed human elastin polypeptides as a model for investigations of structure and self-assembly of elastin.

PubMed

Keeley, Fred W; Bellingham, Catherine M; Woodhouse, Kimberley A

2002-02-28

Elastin is the major extracellular matrix protein of large arteries such as the aorta, imparting characteristics of extensibility and elastic recoil. Once laid down in tissues, polymeric elastin is not subject to turnover, but is able to sustain its mechanical resilience through thousands of millions of cycles of extension and recoil. Elastin consists of ca. 36 domains with alternating hydrophobic and cross-linking characteristics. It has been suggested that these hydrophobic domains, predominantly containing glycine, proline, leucine and valine, often occurring in tandemly repeated sequences, are responsible for the ability of elastin to align monomeric chains for covalent cross-linking. We have shown that small, recombinantly expressed polypeptides based on sequences of human elastin contain sufficient information to self-organize into fibrillar structures and promote the formation of lysine-derived cross-links. These cross-linked polypeptides can also be fabricated into membrane structures that have solubility and mechanical properties reminiscent of native insoluble elastin. Understanding the basis of the self-organizational ability of elastin-based polypeptides may provide important clues for the general design of self-assembling biomaterials.

The chemical structure of DNA sequence signals for RNA transcription

NASA Technical Reports Server (NTRS)

George, D. G.; Dayhoff, M. O.

1982-01-01

The proposed recognition sites for RNA transcription for E. coli NRA polymerase, bacteriophage T7 RNA polymerase, and eukaryotic RNA polymerase Pol II are evaluated in the light of the requirements for efficient recognition. It is shown that although there is good experimental evidence that specific nucleic acid sequence patterns are involved in transcriptional regulation in bacteria and bacterial viruses, among the sequences now available, only in the case of the promoters recognized by bacteriophage T7 polymerase does it seem likely that the pattern is sufficient. It is concluded that the eukaryotic pattern that is investigated is not restrictive enough to serve as a recognition site.

A Systems Approach towards an Intelligent and Self-Controlling Platform for Integrated Continuous Reaction Sequences**

PubMed Central

Ingham, Richard J; Battilocchio, Claudio; Fitzpatrick, Daniel E; Sliwinski, Eric; Hawkins, Joel M; Ley, Steven V

2015-01-01

Performing reactions in flow can offer major advantages over batch methods. However, laboratory flow chemistry processes are currently often limited to single steps or short sequences due to the complexity involved with operating a multi-step process. Using new modular components for downstream processing, coupled with control technologies, more advanced multi-step flow sequences can be realized. These tools are applied to the synthesis of 2-aminoadamantane-2-carboxylic acid. A system comprising three chemistry steps and three workup steps was developed, having sufficient autonomy and self-regulation to be managed by a single operator. PMID:25377747

Enhanced sequencing coverage with digital droplet multiple displacement amplification

PubMed Central

Sidore, Angus M.; Lan, Freeman; Lim, Shaun W.; Abate, Adam R.

2016-01-01

Sequencing small quantities of DNA is important for applications ranging from the assembly of uncultivable microbial genomes to the identification of cancer-associated mutations. To obtain sufficient quantities of DNA for sequencing, the small amount of starting material must be amplified significantly. However, existing methods often yield errors or non-uniform coverage, reducing sequencing data quality. Here, we describe digital droplet multiple displacement amplification, a method that enables massive amplification of low-input material while maintaining sequence accuracy and uniformity. The low-input material is compartmentalized as single molecules in millions of picoliter droplets. Because the molecules are isolated in compartments, they amplify to saturation without competing for resources; this yields uniform representation of all sequences in the final product and, in turn, enhances the quality of the sequence data. We demonstrate the ability to uniformly amplify the genomes of single Escherichia coli cells, comprising just 4.7 fg of starting DNA, and obtain sequencing coverage distributions that rival that of unamplified material. Digital droplet multiple displacement amplification provides a simple and effective method for amplifying minute amounts of DNA for accurate and uniform sequencing. PMID:26704978

Low-coverage single-cell mRNA sequencing reveals cellular heterogeneity and activated signaling pathways in developing cerebral cortex.

PubMed

Pollen, Alex A; Nowakowski, Tomasz J; Shuga, Joe; Wang, Xiaohui; Leyrat, Anne A; Lui, Jan H; Li, Nianzhen; Szpankowski, Lukasz; Fowler, Brian; Chen, Peilin; Ramalingam, Naveen; Sun, Gang; Thu, Myo; Norris, Michael; Lebofsky, Ronald; Toppani, Dominique; Kemp, Darnell W; Wong, Michael; Clerkson, Barry; Jones, Brittnee N; Wu, Shiquan; Knutsson, Lawrence; Alvarado, Beatriz; Wang, Jing; Weaver, Lesley S; May, Andrew P; Jones, Robert C; Unger, Marc A; Kriegstein, Arnold R; West, Jay A A

2014-10-01

Large-scale surveys of single-cell gene expression have the potential to reveal rare cell populations and lineage relationships but require efficient methods for cell capture and mRNA sequencing. Although cellular barcoding strategies allow parallel sequencing of single cells at ultra-low depths, the limitations of shallow sequencing have not been investigated directly. By capturing 301 single cells from 11 populations using microfluidics and analyzing single-cell transcriptomes across downsampled sequencing depths, we demonstrate that shallow single-cell mRNA sequencing (~50,000 reads per cell) is sufficient for unbiased cell-type classification and biomarker identification. In the developing cortex, we identify diverse cell types, including multiple progenitor and neuronal subtypes, and we identify EGR1 and FOS as previously unreported candidate targets of Notch signaling in human but not mouse radial glia. Our strategy establishes an efficient method for unbiased analysis and comparison of cell populations from heterogeneous tissue by microfluidic single-cell capture and low-coverage sequencing of many cells.

Discriminating between stabilizing and destabilizing protein design mutations via recombination and simulation.

PubMed

Johnson, Lucas B; Gintner, Lucas P; Park, Sehoo; Snow, Christopher D

2015-08-01

Accuracy of current computational protein design (CPD) methods is limited by inherent approximations in energy potentials and sampling. These limitations are often used to qualitatively explain design failures; however, relatively few studies provide specific examples or quantitative details that can be used to improve future CPD methods. Expanding the design method to include a library of sequences provides data that is well suited for discriminating between stabilizing and destabilizing design elements. Using thermophilic endoglucanase E1 from Acidothermus cellulolyticus as a model enzyme, we computationally designed a sequence with 60 mutations. The design sequence was rationally divided into structural blocks and recombined with the wild-type sequence. Resulting chimeras were assessed for activity and thermostability. Surprisingly, unlike previous chimera libraries, regression analysis based on one- and two-body effects was not sufficient for predicting chimera stability. Analysis of molecular dynamics simulations proved helpful in distinguishing stabilizing and destabilizing mutations. Reverting to the wild-type amino acid at destabilized sites partially regained design stability, and introducing predicted stabilizing mutations in wild-type E1 significantly enhanced thermostability. The ability to isolate stabilizing and destabilizing elements in computational design offers an opportunity to interpret previous design failures and improve future CPD methods. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Systematic discovery of novel eukaryotic transcriptional regulators using sequence homology independent prediction.

PubMed

Bossi, Flavia; Fan, Jue; Xiao, Jun; Chandra, Lilyana; Shen, Max; Dorone, Yanniv; Wagner, Doris; Rhee, Seung Y

2017-06-26

The molecular function of a gene is most commonly inferred by sequence similarity. Therefore, genes that lack sufficient sequence similarity to characterized genes (such as certain classes of transcriptional regulators) are difficult to classify using most function prediction algorithms and have remained uncharacterized. To identify novel transcriptional regulators systematically, we used a feature-based pipeline to screen protein families of unknown function. This method predicted 43 transcriptional regulator families in Arabidopsis thaliana, 7 families in Drosophila melanogaster, and 9 families in Homo sapiens. Literature curation validated 12 of the predicted families to be involved in transcriptional regulation. We tested 33 out of the 195 Arabidopsis putative transcriptional regulators for their ability to activate transcription of a reporter gene in planta and found twelve coactivators, five of which had no prior literature support. To investigate mechanisms of action in which the predicted regulators might work, we looked for interactors of an Arabidopsis candidate that did not show transactivation activity in planta and found that it might work with other members of its own family and a subunit of the Polycomb Repressive Complex 2 to regulate transcription. Our results demonstrate the feasibility of assigning molecular function to proteins of unknown function without depending on sequence similarity. In particular, we identified novel transcriptional regulators using biological features enriched in transcription factors. The predictions reported here should accelerate the characterization of novel regulators.

Strain/species identification in metagenomes using genome-specific markers

PubMed Central

Tu, Qichao; He, Zhili; Zhou, Jizhong

2014-01-01

Shotgun metagenome sequencing has become a fast, cheap and high-throughput technology for characterizing microbial communities in complex environments and human body sites. However, accurate identification of microorganisms at the strain/species level remains extremely challenging. We present a novel k-mer-based approach, termed GSMer, that identifies genome-specific markers (GSMs) from currently sequenced microbial genomes, which were then used for strain/species-level identification in metagenomes. Using 5390 sequenced microbial genomes, 8 770 321 50-mer strain-specific and 11 736 360 species-specific GSMs were identified for 4088 strains and 2005 species (4933 strains), respectively. The GSMs were first evaluated against mock community metagenomes, recently sequenced genomes and real metagenomes from different body sites, suggesting that the identified GSMs were specific to their targeting genomes. Sensitivity evaluation against synthetic metagenomes with different coverage suggested that 50 GSMs per strain were sufficient to identify most microbial strains with ≥0.25× coverage, and 10% of selected GSMs in a database should be detected for confident positive callings. Application of GSMs identified 45 and 74 microbial strains/species significantly associated with type 2 diabetes patients and obese/lean individuals from corresponding gastrointestinal tract metagenomes, respectively. Our result agreed with previous studies but provided strain-level information. The approach can be directly applied to identify microbial strains/species from raw metagenomes, without the effort of complex data pre-processing. PMID:24523352

Strain rates, stress markers and earthquake clustering (Invited)

NASA Astrophysics Data System (ADS)

Fry, B.; Gerstenberger, M.; Abercrombie, R. E.; Reyners, M.; Eberhart-Phillips, D. M.

2013-12-01

The 2010-present Canterbury earthquakes comprise a well-recorded sequence in a relatively low strain-rate shallow crustal region. We present new scientific results to test the hypothesis that: Earthquake sequences in low-strain rate areas experience high stress drop events, low-post seismic relaxation, and accentuated seismic clustering. This hypothesis is based on a physical description of the aftershock process in which the spatial distribution of stress accumulation and stress transfer are controlled by fault strength and orientation. Following large crustal earthquakes, time dependent forecasts are often developed by fitting parameters defined by Omori's aftershock decay law. In high-strain rate areas, simple forecast models utilizing a single p-value fit observed aftershock sequences well. In low-strain rate areas such as Canterbury, assumptions of simple Omori decay may not be sufficient to capture the clustering (sub-sequence) nature exhibited by the punctuated rise in activity following significant child events. In Canterbury, the moment release is more clustered than in more typical Omori sequences. The individual earthquakes in these clusters also exhibit somewhat higher stress drops than in the average crustal sequence in high-strain rate regions, suggesting the earthquakes occur on strong Andersonian-oriented faults, possibly juvenile or well-healed . We use the spectral ratio procedure outlined in (Viegas et al., 2010) to determine corner frequencies and Madariaga stress-drop values for over 800 events in the sequence. Furthermore, we will discuss the relevance of tomographic results of Reyners and Eberhart-Phillips (2013) documenting post-seismic stress-driven fluid processes following the three largest events in the sequence as well as anisotropic patterns in surface wave tomography (Fry et al., 2013). These tomographic studies are both compatible with the hypothesis, providing strong evidence for the presence of widespread and hydrated regional upper crustal cracking parallel to sub-parallel to the dominant transverse failure plane in the sequence. Joint interpretation of the three separate datasets provide a positive first attempt at testing our fundamental hypothesis.

Clinical next-generation sequencing in patients with non-small cell lung cancer.

PubMed

Hagemann, Ian S; Devarakonda, Siddhartha; Lockwood, Christina M; Spencer, David H; Guebert, Kalin; Bredemeyer, Andrew J; Al-Kateb, Hussam; Nguyen, TuDung T; Duncavage, Eric J; Cottrell, Catherine E; Kulkarni, Shashikant; Nagarajan, Rakesh; Seibert, Karen; Baggstrom, Maria; Waqar, Saiama N; Pfeifer, John D; Morgensztern, Daniel; Govindan, Ramaswamy

2015-02-15

A clinical assay was implemented to perform next-generation sequencing (NGS) of genes commonly mutated in multiple cancer types. This report describes the feasibility and diagnostic yield of this assay in 381 consecutive patients with non-small cell lung cancer (NSCLC). Clinical targeted sequencing of 23 genes was performed with DNA from formalin-fixed, paraffin-embedded (FFPE) tumor tissue. The assay used Agilent SureSelect hybrid capture followed by Illumina HiSeq 2000, MiSeq, or HiSeq 2500 sequencing in a College of American Pathologists-accredited, Clinical Laboratory Improvement Amendments-certified laboratory. Single-nucleotide variants and insertion/deletion events were reported. This assay was performed before methods were developed to detect rearrangements by NGS. Two hundred nine of all requisitioned samples (55%) were successfully sequenced. The most common reason for not performing the sequencing was an insufficient quantity of tissue available in the blocks (29%). Excisional, endoscopic, and core biopsy specimens were sufficient for testing in 95%, 66%, and 40% of the cases, respectively. The median turnaround time (TAT) in the pathology laboratory was 21 days, and there was a trend of an improved TAT with more rapid sequencing platforms. Sequencing yielded a mean coverage of 1318×. Potentially actionable mutations (ie, predictive or prognostic) were identified in 46% of 209 samples and were most commonly found in KRAS (28%), epidermal growth factor receptor (14%), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (4%), phosphatase and tensin homolog (1%), and BRAF (1%). Five percent of the samples had multiple actionable mutations. A targeted therapy was instituted on the basis of NGS in 11% of the sequenced patients or in 6% of all patients. NGS-based diagnostics are feasible in NSCLC and provide clinically relevant information from readily available FFPE tissue. The sample type is associated with the probability of successful testing. © 2014 American Cancer Society.

A greedy, graph-based algorithm for the alignment of multiple homologous gene lists.

PubMed

Fostier, Jan; Proost, Sebastian; Dhoedt, Bart; Saeys, Yvan; Demeester, Piet; Van de Peer, Yves; Vandepoele, Klaas

2011-03-15

Many comparative genomics studies rely on the correct identification of homologous genomic regions using accurate alignment tools. In such case, the alphabet of the input sequences consists of complete genes, rather than nucleotides or amino acids. As optimal multiple sequence alignment is computationally impractical, a progressive alignment strategy is often employed. However, such an approach is susceptible to the propagation of alignment errors in early pairwise alignment steps, especially when dealing with strongly diverged genomic regions. In this article, we present a novel accurate and efficient greedy, graph-based algorithm for the alignment of multiple homologous genomic segments, represented as ordered gene lists. Based on provable properties of the graph structure, several heuristics are developed to resolve local alignment conflicts that occur due to gene duplication and/or rearrangement events on the different genomic segments. The performance of the algorithm is assessed by comparing the alignment results of homologous genomic segments in Arabidopsis thaliana to those obtained by using both a progressive alignment method and an earlier graph-based implementation. Especially for datasets that contain strongly diverged segments, the proposed method achieves a substantially higher alignment accuracy, and proves to be sufficiently fast for large datasets including a few dozens of eukaryotic genomes. http://bioinformatics.psb.ugent.be/software. The algorithm is implemented as a part of the i-ADHoRe 3.0 package.

An EOG-Based Human-Machine Interface for Wheelchair Control.

PubMed

Huang, Qiyun; He, Shenghong; Wang, Qihong; Gu, Zhenghui; Peng, Nengneng; Li, Kai; Zhang, Yuandong; Shao, Ming; Li, Yuanqing

2017-07-27

Non-manual human-machine interfaces (HMIs) have been studied for wheelchair control with the aim of helping severely paralyzed individuals regain some mobility. The challenge is to rapidly, accurately and sufficiently produce control commands, such as left and right turns, forward and backward motions, acceleration, deceleration, and stopping. In this paper, a novel electrooculogram (EOG)-based HMI is proposed for wheelchair control. Thirteen flashing buttons are presented in the graphical user interface (GUI), and each of the buttons corresponds to a command. These buttons flash on a one-by-one manner in a pre-defined sequence. The user can select a button by blinking in sync with its flashes. The algorithm detects the eye blinks from a channel of vertical EOG data and determines the user's target button based on the synchronization between the detected blinks and the button's flashes. For healthy subjects/patients with spinal cord injuries (SCIs), the proposed HMI achieved an average accuracy of 96.7%/91.7% and a response time of 3.53 s/3.67 s with 0 false positive rates (FPRs). Using only one channel of vertical EOG signals associated with eye blinks, the proposed HMI can accurately provide sufficient commands with a satisfactory response time. The proposed HMI provides a novel non-manual approach for severely paralyzed individuals to control a wheelchair. Compared with a newly established EOG-based HMI, the proposed HMI can generate more commands with higher accuracy, lower FPR and fewer electrodes.

Trellises and Trellis-Based Decoding Algorithms for Linear Block Codes. Part 3; An Iterative Decoding Algorithm for Linear Block Codes Based on a Low-Weight Trellis Search

NASA Technical Reports Server (NTRS)

Lin, Shu; Fossorier, Marc

1998-01-01

For long linear block codes, maximum likelihood decoding based on full code trellises would be very hard to implement if not impossible. In this case, we may wish to trade error performance for the reduction in decoding complexity. Sub-optimum soft-decision decoding of a linear block code based on a low-weight sub-trellis can be devised to provide an effective trade-off between error performance and decoding complexity. This chapter presents such a suboptimal decoding algorithm for linear block codes. This decoding algorithm is iterative in nature and based on an optimality test. It has the following important features: (1) a simple method to generate a sequence of candidate code-words, one at a time, for test; (2) a sufficient condition for testing a candidate code-word for optimality; and (3) a low-weight sub-trellis search for finding the most likely (ML) code-word.

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

PubMed

Vinner, Lasse; Mourier, Tobias; Friis-Nielsen, Jens; Gniadecki, Robert; Dybkaer, Karen; Rosenberg, Jacob; Langhoff, Jill Levin; Cruz, David Flores Santa; Fonager, Jannik; Izarzugaza, Jose M G; Gupta, Ramneek; Sicheritz-Ponten, Thomas; Brunak, Søren; Willerslev, Eske; Nielsen, Lars Peter; Hansen, Anders Johannes

2015-08-19

Although nearly one fifth of all human cancers have an infectious aetiology, the causes for the majority of cancers remain unexplained. Despite the enormous data output from high-throughput shotgun sequencing, viral DNA in a clinical sample typically constitutes a proportion of host DNA that is too small to be detected. Sequence variation among virus genomes complicates application of sequence-specific, and highly sensitive, PCR methods. Therefore, we aimed to develop and characterize a method that permits sensitive detection of sequences despite considerable variation. We demonstrate that our low-stringency in-solution hybridization method enables detection of <100 viral copies. Furthermore, distantly related proviral sequences may be enriched by orders of magnitude, enabling discovery of hitherto unknown viral sequences by high-throughput sequencing. The sensitivity was sufficient to detect retroviral sequences in clinical samples. We used this method to conduct an investigation for novel retrovirus in samples from three cancer types. In accordance with recent studies our investigation revealed no retroviral infections in human B-cell lymphoma cells, cutaneous T-cell lymphoma or colorectal cancer biopsies. Nonetheless, our generally applicable method makes sensitive detection possible and permits sequencing of distantly related sequences from complex material.

Cloning, expression and characterisation of P450-Hal1 (CYP116B62) from Halomonas sp. NCIMB 172: A self-sufficient P450 with high expression and diverse substrate scope.

PubMed

Porter, Joanne L; Sabatini, Selina; Manning, Jack; Tavanti, Michele; Galman, James L; Turner, Nicholas J; Flitsch, Sabine L

2018-06-01

Cytochrome P450 monooxygenases are able to catalyse a range of synthetically challenging reactions ranging from hydroxylation and demethylation to sulfoxidation and epoxidation. As such they have great potential for biocatalytic applications but are underutilised due to often-poor expression, stability and solubility in recombinant bacterial hosts. The use of self-sufficient P450 s with fused haem and reductase domains has already contributed heavily to improving catalytic efficiency and simplifying an otherwise more complex multi-component system of P450 and redox partners. Herein, we present a new addition to the class VII family with the cloning, sequencing and characterisation of the self-sufficient CYP116B62 Hal1 from Halomonas sp. NCIMB 172, the genome of which has not yet been sequenced. Hal1 exhibits high levels of expression in a recombinant E. coli host and can be utilised from cell lysate or used in purified form. Hal1 favours NADPH as electron donor and displays a diverse range of activities including hydroxylation, demethylation and sulfoxidation. These properties make Hal1 suitable for future biocatalytic applications or as a template for optimisation through engineering. Copyright © 2018 Elsevier Inc. All rights reserved.

NMR-based automated protein structure determination.

PubMed

Würz, Julia M; Kazemi, Sina; Schmidt, Elena; Bagaria, Anurag; Güntert, Peter

2017-08-15

NMR spectra analysis for protein structure determination can now in many cases be performed by automated computational methods. This overview of the computational methods for NMR protein structure analysis presents recent automated methods for signal identification in multidimensional NMR spectra, sequence-specific resonance assignment, collection of conformational restraints, and structure calculation, as implemented in the CYANA software package. These algorithms are sufficiently reliable and integrated into one software package to enable the fully automated structure determination of proteins starting from NMR spectra without manual interventions or corrections at intermediate steps, with an accuracy of 1-2 Å backbone RMSD in comparison with manually solved reference structures. Copyright © 2017 Elsevier Inc. All rights reserved.

Protein abundances can distinguish between naturally-occurring and laboratory strains of Yersinia pestis, the causative agent of plague

DOE PAGES

Merkley, Eric D.; Sego, Landon H.; Lin, Andy; ...

2017-08-30

Adaptive processes in bacterial species can occur rapidly in laboratory culture, leading to genetic divergence between naturally occurring and laboratory-adapted strains. Differentiating wild and closely-related laboratory strains is clearly important for biodefense and bioforensics; however, DNA sequence data alone has thus far not provided a clear signature, perhaps due to lack of understanding of how diverse genome changes lead to adapted phenotypes. Protein abundance profiles from mass spectrometry-based proteomics analyses are a molecular measure of phenotype. Proteomics data contains sufficient information that powerful statistical methods can uncover signatures that distinguish wild strains of Yersinia pestis from laboratory-adapted strains.

Identification of somatic mutations in cancer through Bayesian-based analysis of sequenced genome pairs

PubMed Central

2013-01-01

Background The field of cancer genomics has rapidly adopted next-generation sequencing (NGS) in order to study and characterize malignant tumors with unprecedented resolution. In particular for cancer, one is often trying to identify somatic mutations – changes specific to a tumor and not within an individual’s germline. However, false positive and false negative detections often result from lack of sufficient variant evidence, contamination of the biopsy by stromal tissue, sequencing errors, and the erroneous classification of germline variation as tumor-specific. Results We have developed a generalized Bayesian analysis framework for matched tumor/normal samples with the purpose of identifying tumor-specific alterations such as single nucleotide mutations, small insertions/deletions, and structural variation. We describe our methodology, and discuss its application to other types of paired-tissue analysis such as the detection of loss of heterozygosity as well as allelic imbalance. We also demonstrate the high level of sensitivity and specificity in discovering simulated somatic mutations, for various combinations of a) genomic coverage and b) emulated heterogeneity. Conclusion We present a Java-based implementation of our methods named Seurat, which is made available for free academic use. We have demonstrated and reported on the discovery of different types of somatic change by applying Seurat to an experimentally-derived cancer dataset using our methods; and have discussed considerations and practices regarding the accurate detection of somatic events in cancer genomes. Seurat is available at https://sites.google.com/site/seuratsomatic. PMID:23642077

Identification of somatic mutations in cancer through Bayesian-based analysis of sequenced genome pairs.

PubMed

Christoforides, Alexis; Carpten, John D; Weiss, Glen J; Demeure, Michael J; Von Hoff, Daniel D; Craig, David W

2013-05-04

The field of cancer genomics has rapidly adopted next-generation sequencing (NGS) in order to study and characterize malignant tumors with unprecedented resolution. In particular for cancer, one is often trying to identify somatic mutations--changes specific to a tumor and not within an individual's germline. However, false positive and false negative detections often result from lack of sufficient variant evidence, contamination of the biopsy by stromal tissue, sequencing errors, and the erroneous classification of germline variation as tumor-specific. We have developed a generalized Bayesian analysis framework for matched tumor/normal samples with the purpose of identifying tumor-specific alterations such as single nucleotide mutations, small insertions/deletions, and structural variation. We describe our methodology, and discuss its application to other types of paired-tissue analysis such as the detection of loss of heterozygosity as well as allelic imbalance. We also demonstrate the high level of sensitivity and specificity in discovering simulated somatic mutations, for various combinations of a) genomic coverage and b) emulated heterogeneity. We present a Java-based implementation of our methods named Seurat, which is made available for free academic use. We have demonstrated and reported on the discovery of different types of somatic change by applying Seurat to an experimentally-derived cancer dataset using our methods; and have discussed considerations and practices regarding the accurate detection of somatic events in cancer genomes. Seurat is available at https://sites.google.com/site/seuratsomatic.

Adaptive low-rank subspace learning with online optimization for robust visual tracking.

PubMed

Liu, Risheng; Wang, Di; Han, Yuzhuo; Fan, Xin; Luo, Zhongxuan

2017-04-01

In recent years, sparse and low-rank models have been widely used to formulate appearance subspace for visual tracking. However, most existing methods only consider the sparsity or low-rankness of the coefficients, which is not sufficient enough for appearance subspace learning on complex video sequences. Moreover, as both the low-rank and the column sparse measures are tightly related to all the samples in the sequences, it is challenging to incrementally solve optimization problems with both nuclear norm and column sparse norm on sequentially obtained video data. To address above limitations, this paper develops a novel low-rank subspace learning with adaptive penalization (LSAP) framework for subspace based robust visual tracking. Different from previous work, which often simply decomposes observations as low-rank features and sparse errors, LSAP simultaneously learns the subspace basis, low-rank coefficients and column sparse errors to formulate appearance subspace. Within LSAP framework, we introduce a Hadamard production based regularization to incorporate rich generative/discriminative structure constraints to adaptively penalize the coefficients for subspace learning. It is shown that such adaptive penalization can significantly improve the robustness of LSAP on severely corrupted dataset. To utilize LSAP for online visual tracking, we also develop an efficient incremental optimization scheme for nuclear norm and column sparse norm minimizations. Experiments on 50 challenging video sequences demonstrate that our tracker outperforms other state-of-the-art methods. Copyright © 2017 Elsevier Ltd. All rights reserved.

Comprehensive viral enrichment enables sensitive respiratory virus genomic identification and analysis by next generation sequencing.

PubMed

O'Flaherty, Brigid M; Li, Yan; Tao, Ying; Paden, Clinton R; Queen, Krista; Zhang, Jing; Dinwiddie, Darrell L; Gross, Stephen M; Schroth, Gary P; Tong, Suxiang

2018-06-01

Next generation sequencing (NGS) technologies have revolutionized the genomics field and are becoming more commonplace for identification of human infectious diseases. However, due to the low abundance of viral nucleic acids (NAs) in relation to host, viral identification using direct NGS technologies often lacks sufficient sensitivity. Here, we describe an approach based on two complementary enrichment strategies that significantly improves the sensitivity of NGS-based virus identification. To start, we developed two sets of DNA probes to enrich virus NAs associated with respiratory diseases. The first set of probes spans the genomes, allowing for identification of known viruses and full genome sequencing, while the second set targets regions conserved among viral families or genera, providing the ability to detect both known and potentially novel members of those virus groups. Efficiency of enrichment was assessed by NGS testing reference virus and clinical samples with known infection. We show significant improvement in viral identification using enriched NGS compared to unenriched NGS. Without enrichment, we observed an average of 0.3% targeted viral reads per sample. However, after enrichment, 50%-99% of the reads per sample were the targeted viral reads for both the reference isolates and clinical specimens using both probe sets. Importantly, dramatic improvements on genome coverage were also observed following virus-specific probe enrichment. The methods described here provide improved sensitivity for virus identification by NGS, allowing for a more comprehensive analysis of disease etiology. © 2018 O'Flaherty et al.; Published by Cold Spring Harbor Laboratory Press.

Rapid DNA extraction protocol for detection of alpha-1 antitrypsin deficiency from dried blood spots by real-time PCR.

PubMed

Struniawski, R; Szpechcinski, A; Poplawska, B; Skronski, M; Chorostowska-Wynimko, J

2013-01-01

The dried blood spot (DBS) specimens have been successfully employed for the large-scale diagnostics of α1-antitrypsin (AAT) deficiency as an easy to collect and transport alternative to plasma/serum. In the present study we propose a fast, efficient, and cost effective protocol of DNA extraction from dried blood spot (DBS) samples that provides sufficient quantity and quality of DNA and effectively eliminates any natural PCR inhibitors, allowing for successful AAT genotyping by real-time PCR and direct sequencing. DNA extracted from 84 DBS samples from chronic obstructive pulmonary disease patients was genotyped for AAT deficiency variants by real-time PCR. The results of DBS AAT genotyping were validated by serum IEF phenotyping and AAT concentration measurement. The proposed protocol allowed successful DNA extraction from all analyzed DBS samples. Both quantity and quality of DNA were sufficient for further real-time PCR and, if necessary, for genetic sequence analysis. A 100% concordance between AAT DBS genotypes and serum phenotypes in positive detection of two major deficiency S- and Z- alleles was achieved. Both assays, DBS AAT genotyping by real-time PCR and serum AAT phenotyping by IEF, positively identified PI*S and PI*Z allele in 8 out of the 84 (9.5%) and 16 out of 84 (19.0%) patients, respectively. In conclusion, the proposed protocol noticeably reduces the costs and the hand-on-time of DBS samples preparation providing genomic DNA of sufficient quantity and quality for further real-time PCR or genetic sequence analysis. Consequently, it is ideally suited for large-scale AAT deficiency screening programs and should be method of choice.

Learning multiple variable-speed sequences in striatum via cortical tutoring.

PubMed

Murray, James M; Escola, G Sean

2017-05-08

Sparse, sequential patterns of neural activity have been observed in numerous brain areas during timekeeping and motor sequence tasks. Inspired by such observations, we construct a model of the striatum, an all-inhibitory circuit where sequential activity patterns are prominent, addressing the following key challenges: (i) obtaining control over temporal rescaling of the sequence speed, with the ability to generalize to new speeds; (ii) facilitating flexible expression of distinct sequences via selective activation, concatenation, and recycling of specific subsequences; and (iii) enabling the biologically plausible learning of sequences, consistent with the decoupling of learning and execution suggested by lesion studies showing that cortical circuits are necessary for learning, but that subcortical circuits are sufficient to drive learned behaviors. The same mechanisms that we describe can also be applied to circuits with both excitatory and inhibitory populations, and hence may underlie general features of sequential neural activity pattern generation in the brain.

Molecular detection of bovine Noroviruses in Argentinean dairy calves: Circulation of a tentative new genotype.

PubMed

Ferragut, Fátima; Vega, Celina G; Mauroy, Axel; Conceição-Neto, Nádia; Zeller, Mark; Heylen, Elisabeth; Uriarte, Enrique Louge; Bilbao, Gladys; Bok, Marina; Matthijnssens, Jelle; Thiry, Etienne; Badaracco, Alejandra; Parreño, Viviana

2016-06-01

Bovine noroviruses are enteric pathogens detected in fecal samples of both diarrheic and non-diarrheic calves from several countries worldwide. However, epidemiological information regarding bovine noroviruses is still lacking for many important cattle producing countries from South America. In this study, three bovine norovirus genogroup III sequences were determined by conventional RT-PCR and Sanger sequencing in feces from diarrheic dairy calves from Argentina (B4836, B4848, and B4881, all collected in 2012). Phylogenetic studies based on a partial coding region for the RNA-dependent RNA polymerase (RdRp, 503 nucleotides) of these three samples suggested that two of them (B4836 and B4881) belong to genotype 2 (GIII.2) while the third one (B4848) was more closely related to genotype 1 (GIII.1) strains. By deep sequencing, the capsid region from two of these strains could be determined. This confirmed the circulation of genotype 1 (B4848) together with the presence of another sequence (B4881) sharing its highest genetic relatedness with genotype 1, but sufficiently distant to constitute a new genotype. This latter strain was shown in silico to be a recombinant: phylogenetic divergence was detected between its RNA-dependent RNA polymerase coding sequence (genotype GIII.2) and its capsid protein coding sequence (genotype GIII.1 or a potential norovirus genotype). According to this data, this strain could be the second genotype GIII.2_GIII.1 bovine norovirus recombinant described in literature worldwide. Further analysis suggested that this strain could even be a potential norovirus GIII genotype, tentatively named GIII.4. The data provides important epidemiological and evolutionary information on bovine noroviruses circulating in South America. Copyright © 2016. Published by Elsevier B.V.

Genomic patterns of introgression in rainbow and westslope cutthroat trout illuminated by overlapping paired-end RAD sequencing

USGS Publications Warehouse

Hohenlohe, Paul A.; Day, Mitch D.; Amish, Stephen J.; Miller, Michael R.; Kamps-Hughes, Nick; Boyer, Matthew C.; Muhlfeld, Clint C.; Allendorf, Fred W.; Johnson, Eric A.; Luikart, Gordon

2013-01-01

Rapid and inexpensive methods for genomewide single nucleotide polymorphism (SNP) discovery and genotyping are urgently needed for population management and conservation. In hybridized populations, genomic techniques that can identify and genotype thousands of species-diagnostic markers would allow precise estimates of population- and individual-level admixture as well as identification of 'super invasive' alleles, which show elevated rates of introgression above the genomewide background (likely due to natural selection). Techniques like restriction-site-associated DNA (RAD) sequencing can discover and genotype large numbers of SNPs, but they have been limited by the length of continuous sequence data they produce with Illumina short-read sequencing. We present a novel approach, overlapping paired-end RAD sequencing, to generate RAD contigs of >300–400 bp. These contigs provide sufficient flanking sequence for design of high-throughput SNP genotyping arrays and strict filtering to identify duplicate paralogous loci. We applied this approach in five populations of native westslope cutthroat trout that previously showed varying (low) levels of admixture from introduced rainbow trout (RBT). We produced 77 141 RAD contigs and used these data to filter and genotype 3180 previously identified species-diagnostic SNP loci. Our population-level and individual-level estimates of admixture were generally consistent with previous microsatellite-based estimates from the same individuals. However, we observed slightly lower admixture estimates from genomewide markers, which might result from natural selection against certain genome regions, different genomic locations for microsatellites vs. RAD-derived SNPs and/or sampling error from the small number of microsatellite loci (n = 7). We also identified candidate adaptive super invasive alleles from RBT that had excessively high admixture proportions in hybridized cutthroat trout populations.

Targeted next generation sequencing of the entire vitamin D receptor gene reveals polymorphisms correlated with vitamin D deficiency among older Filipino women with and without fragility fracture.

PubMed

Zumaraga, Mark Pretzel; Medina, Paul Julius; Recto, Juan Miguel; Abrahan, Lauro; Azurin, Edelyn; Tanchoco, Celeste C; Jimeno, Cecilia A; Palmes-Saloma, Cynthia

2017-03-01

This study aimed to discover genetic variants in the entire 101 kB vitamin D receptor (VDR) gene for vitamin D deficiency in a group of postmenopausal Filipino women using targeted next generation sequencing (TNGS) approach in a case-control study design. A total of 50 women with and without osteoporotic fracture seen at the Philippine Orthopedic Center were included. Blood samples were collected for determination of serum vitamin D, calcium, phosphorus, glucose, blood urea nitrogen, creatinine, aspartate aminotransferase, alanine aminotransferase and as primary source for targeted VDR gene sequencing using the Ion Torrent Personal Genome Machine. The variant calling was based on the GATK best practice workflow and annotated using Annovar tool. A total of 1496 unique variants in the whole 101-kb VDR gene were identified. Novel sequence variations not registered in the dbSNP database were found among cases and controls at a rate of 23.1% and 16.6% of total discovered variants, respectively. One disease-associated enhancer showed statistically significant association to low serum 25-hydroxy vitamin D levels (Pearson chi-square P-value=0.009). The transcription factor binding site prediction program PROMO predicted the disruption of three transcription factor binding sites in this enhancer region. These findings show the power of TNGS in identifying sequence variations in a very large gene and the surprising results obtained in this study greatly expand the catalog of known VDR sequence variants that may represent an important clue in the emergence of vitamin D deficiency. Such information will also provide the additional guidance necessary toward a personalized nutritional advice to reach sufficient vitamin D status. Copyright © 2016 Elsevier Inc. All rights reserved.

Comparison of Spectrophotometry, Chromate Inhibition, and Cytofluorometry Versus Gene Sequencing for Detection of Heterozygously Glucose-6-Phosphate Dehydrogenase-Deficient Females.

PubMed

Peters, Anna L; Veldthuis, Martijn; van Leeuwen, Karin; Bossuyt, Patrick M M; Vlaar, Alexander P J; van Bruggen, Robin; de Korte, Dirk; Van Noorden, Cornelis J F; van Zwieten, Rob

2017-11-01

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzyme deficiency worldwide. Detection of heterozygously deficient females can be difficult as residual activity in G6PD-sufficient red blood cells (RBCs) can mask deficiency. In this study, we compared accuracy of 4 methods for detection of G6PD deficiency in females. Blood samples from females more than 3 months of age were used for spectrophotometric measurement of G6PD activity and for determination of the percentage G6PD-negative RBCs by cytofluorometry. An additional sample from females suspected to have G6PD deficiency based on the spectrophotometric G6PD activity was used for measuring chromate inhibition and sequencing of the G6PD gene. Of 165 included females, 114 were suspected to have heterozygous deficiency. From 75 females, an extra sample was obtained. In this group, mutation analysis detected 27 heterozygously deficient females. The sensitivity of spectrophotometry, cytofluorometry, and chromate inhibition was calculated to be 0.52 (confidence interval [CI]: 0.32-0.71), 0.85 (CI: 0.66-0.96), and 0.96 (CI: 0.71-1.00, respectively, and the specificity was 1.00 (CI: 0.93-1.00), 0.88 (CI: 0.75-0.95), and 0.98 (CI: 0.89-1.00), respectively. Heterozygously G6PD-deficient females with a larger percentage of G6PD-sufficient RBCs are missed by routine methods measuring total G6PD activity. However, the majority of these females can be detected with both chromate inhibition and cytofluorometry.

Sequenza: allele-specific copy number and mutation profiles from tumor sequencing data.

PubMed

Favero, F; Joshi, T; Marquard, A M; Birkbak, N J; Krzystanek, M; Li, Q; Szallasi, Z; Eklund, A C

2015-01-01

Exome or whole-genome deep sequencing of tumor DNA along with paired normal DNA can potentially provide a detailed picture of the somatic mutations that characterize the tumor. However, analysis of such sequence data can be complicated by the presence of normal cells in the tumor specimen, by intratumor heterogeneity, and by the sheer size of the raw data. In particular, determination of copy number variations from exome sequencing data alone has proven difficult; thus, single nucleotide polymorphism (SNP) arrays have often been used for this task. Recently, algorithms to estimate absolute, but not allele-specific, copy number profiles from tumor sequencing data have been described. We developed Sequenza, a software package that uses paired tumor-normal DNA sequencing data to estimate tumor cellularity and ploidy, and to calculate allele-specific copy number profiles and mutation profiles. We applied Sequenza, as well as two previously published algorithms, to exome sequence data from 30 tumors from The Cancer Genome Atlas. We assessed the performance of these algorithms by comparing their results with those generated using matched SNP arrays and processed by the allele-specific copy number analysis of tumors (ASCAT) algorithm. Comparison between Sequenza/exome and SNP/ASCAT revealed strong correlation in cellularity (Pearson's r = 0.90) and ploidy estimates (r = 0.42, or r = 0.94 after manual inspecting alternative solutions). This performance was noticeably superior to previously published algorithms. In addition, in artificial data simulating normal-tumor admixtures, Sequenza detected the correct ploidy in samples with tumor content as low as 30%. The agreement between Sequenza and SNP array-based copy number profiles suggests that exome sequencing alone is sufficient not only for identifying small scale mutations but also for estimating cellularity and inferring DNA copy number aberrations. © The Author 2014. Published by Oxford University Press on behalf of the European Society for Medical Oncology.

Strategic approaches to unraveling genetic causes of cardiovascular diseases

USDA-ARS?s Scientific Manuscript database

DNA sequence variants are major components of the "causal field" for virtually all medical phenotypes, whether single gene familial disorders or complex traits without a clear familial aggregation. The causal variants in single gene disorders are necessary and sufficient to impart large effects. In ...

A general method for the purification of restriction enzymes.

PubMed Central

Greene, P J; Heyneker, H L; Bolivar, F; Rodriguez, R L; Betlach, M C; Covarrubias, A A; Backman, K; Russel, D J; Tait, R; Boyer, H W

1978-01-01

An abbreviated procedure has been developed for the purification of restriction endonucleases. This procedure uses chromatography on phosphocellulose and hydroxylapatite and results in enzymes of sufficient purity to permit their use in the sequencing, molecular cloning, and physical mapping of DNA. PMID:673857

Formation of high-field magnetic white dwarfs from common envelopes

PubMed Central

Nordhaus, Jason; Wellons, Sarah; Spiegel, David S.; Metzger, Brian D.; Blackman, Eric G.

2011-01-01

The origin of highly magnetized white dwarfs has remained a mystery since their initial discovery. Recent observations indicate that the formation of high-field magnetic white dwarfs is intimately related to strong binary interactions during post-main-sequence phases of stellar evolution. If a low-mass companion, such as a planet, brown dwarf, or low-mass star, is engulfed by a post-main-sequence giant, gravitational torques in the envelope of the giant lead to a reduction of the companion’s orbit. Sufficiently low-mass companions in-spiral until they are shredded by the strong gravitational tides near the white dwarf core. Subsequent formation of a super-Eddington accretion disk from the disrupted companion inside a common envelope can dramatically amplify magnetic fields via a dynamo. Here, we show that these disk-generated fields are sufficiently strong to explain the observed range of magnetic field strengths for isolated, high-field magnetic white dwarfs. A higher-mass binary analogue may also contribute to the origin of magnetar fields. PMID:21300910

MetaGenSense: A web-application for analysis and exploration of high throughput sequencing metagenomic data

PubMed Central

Denis, Jean-Baptiste; Vandenbogaert, Mathias; Caro, Valérie

2016-01-01

The detection and characterization of emerging infectious agents has been a continuing public health concern. High Throughput Sequencing (HTS) or Next-Generation Sequencing (NGS) technologies have proven to be promising approaches for efficient and unbiased detection of pathogens in complex biological samples, providing access to comprehensive analyses. As NGS approaches typically yield millions of putatively representative reads per sample, efficient data management and visualization resources have become mandatory. Most usually, those resources are implemented through a dedicated Laboratory Information Management System (LIMS), solely to provide perspective regarding the available information. We developed an easily deployable web-interface, facilitating management and bioinformatics analysis of metagenomics data-samples. It was engineered to run associated and dedicated Galaxy workflows for the detection and eventually classification of pathogens. The web application allows easy interaction with existing Galaxy metagenomic workflows, facilitates the organization, exploration and aggregation of the most relevant sample-specific sequences among millions of genomic sequences, allowing them to determine their relative abundance, and associate them to the most closely related organism or pathogen. The user-friendly Django-Based interface, associates the users’ input data and its metadata through a bio-IT provided set of resources (a Galaxy instance, and both sufficient storage and grid computing power). Galaxy is used to handle and analyze the user’s input data from loading, indexing, mapping, assembly and DB-searches. Interaction between our application and Galaxy is ensured by the BioBlend library, which gives API-based access to Galaxy’s main features. Metadata about samples, runs, as well as the workflow results are stored in the LIMS. For metagenomic classification and exploration purposes, we show, as a proof of concept, that integration of intuitive exploratory tools, like Krona for representation of taxonomic classification, can be achieved very easily. In the trend of Galaxy, the interface enables the sharing of scientific results to fellow team members. PMID:28451381

MetaGenSense: A web-application for analysis and exploration of high throughput sequencing metagenomic data.

PubMed

Correia, Damien; Doppelt-Azeroual, Olivia; Denis, Jean-Baptiste; Vandenbogaert, Mathias; Caro, Valérie

2015-01-01

The detection and characterization of emerging infectious agents has been a continuing public health concern. High Throughput Sequencing (HTS) or Next-Generation Sequencing (NGS) technologies have proven to be promising approaches for efficient and unbiased detection of pathogens in complex biological samples, providing access to comprehensive analyses. As NGS approaches typically yield millions of putatively representative reads per sample, efficient data management and visualization resources have become mandatory. Most usually, those resources are implemented through a dedicated Laboratory Information Management System (LIMS), solely to provide perspective regarding the available information. We developed an easily deployable web-interface, facilitating management and bioinformatics analysis of metagenomics data-samples. It was engineered to run associated and dedicated Galaxy workflows for the detection and eventually classification of pathogens. The web application allows easy interaction with existing Galaxy metagenomic workflows, facilitates the organization, exploration and aggregation of the most relevant sample-specific sequences among millions of genomic sequences, allowing them to determine their relative abundance, and associate them to the most closely related organism or pathogen. The user-friendly Django-Based interface, associates the users' input data and its metadata through a bio-IT provided set of resources (a Galaxy instance, and both sufficient storage and grid computing power). Galaxy is used to handle and analyze the user's input data from loading, indexing, mapping, assembly and DB-searches. Interaction between our application and Galaxy is ensured by the BioBlend library, which gives API-based access to Galaxy's main features. Metadata about samples, runs, as well as the workflow results are stored in the LIMS. For metagenomic classification and exploration purposes, we show, as a proof of concept, that integration of intuitive exploratory tools, like Krona for representation of taxonomic classification, can be achieved very easily. In the trend of Galaxy, the interface enables the sharing of scientific results to fellow team members.

Monoterpene synthases from common sage (Salvia officinalis)

DOEpatents

Croteau, Rodney Bruce; Wise, Mitchell Lynn; Katahira, Eva Joy; Savage, Thomas Jonathan

1999-01-01

cDNAs encoding (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase from common sage (Salvia officinalis) have been isolated and sequenced, and the corresponding amino acid sequences has been determined. Accordingly, isolated DNA sequences (SEQ ID No:1; SEQ ID No:3 and SEQ ID No:5) are provided which code for the expression of (+)-bornyl diphosphate synthase (SEQ ID No:2), 1,8-cineole synthase (SEQ ID No:4) and (+)-sabinene synthase SEQ ID No:6), respectively, from sage (Salvia officinalis). In other aspects, replicable recombinant cloning vehicles are provided which code for (+)-bornyl diphosphate synthase, 1,8-cineole synthase or (+)-sabinene synthase, or for a base sequence sufficiently complementary to at least a portion of (+)-bornyl diphosphate synthase, 1,8-cineole synthase or (+)-sabinene synthase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding (+)-bornyl diphosphate synthase, 1,8-cineole synthase or (+)-sabinene synthase. Thus, systems and methods are provided for the recombinant expression of the aforementioned recombinant monoterpene synthases that may be used to facilitate their production, isolation and purification in significant amounts. Recombinant (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase may be used to obtain expression or enhanced expression of (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase in plants in order to enhance the production of monoterpenoids, or may be otherwise employed for the regulation or expression of (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase, or the production of their products.

Sequence alignment visualization in HTML5 without Java.

PubMed

Gille, Christoph; Birgit, Weyand; Gille, Andreas

2014-01-01

Java has been extensively used for the visualization of biological data in the web. However, the Java runtime environment is an additional layer of software with an own set of technical problems and security risks. HTML in its new version 5 provides features that for some tasks may render Java unnecessary. Alignment-To-HTML is the first HTML-based interactive visualization for annotated multiple sequence alignments. The server side script interpreter can perform all tasks like (i) sequence retrieval, (ii) alignment computation, (iii) rendering, (iv) identification of a homologous structural models and (v) communication with BioDAS-servers. The rendered alignment can be included in web pages and is displayed in all browsers on all platforms including touch screen tablets. The functionality of the user interface is similar to legacy Java applets and includes color schemes, highlighting of conserved and variable alignment positions, row reordering by drag and drop, interlinked 3D visualization and sequence groups. Novel features are (i) support for multiple overlapping residue annotations, such as chemical modifications, single nucleotide polymorphisms and mutations, (ii) mechanisms to quickly hide residue annotations, (iii) export to MS-Word and (iv) sequence icons. Alignment-To-HTML, the first interactive alignment visualization that runs in web browsers without additional software, confirms that to some extend HTML5 is already sufficient to display complex biological data. The low speed at which programs are executed in browsers is still the main obstacle. Nevertheless, we envision an increased use of HTML and JavaScript for interactive biological software. Under GPL at: http://www.bioinformatics.org/strap/toHTML/.

A Sequence-Independent Strategy for Detection and Cloning of Circular DNA Virus Genomes by Using Multiply Primed Rolling-Circle Amplification

PubMed Central

Rector, Annabel; Tachezy, Ruth; Van Ranst, Marc

2004-01-01

The discovery of novel viruses has often been accomplished by using hybridization-based methods that necessitate the availability of a previously characterized virus genome probe or knowledge of the viral nucleotide sequence to construct consensus or degenerate PCR primers. In their natural replication cycle, certain viruses employ a rolling-circle mechanism to propagate their circular genomes, and multiply primed rolling-circle amplification (RCA) with φ29 DNA polymerase has recently been applied in the amplification of circular plasmid vectors used in cloning. We employed an isothermal RCA protocol that uses random hexamer primers to amplify the complete genomes of papillomaviruses without the need for prior knowledge of their DNA sequences. We optimized this RCA technique with extracted human papillomavirus type 16 (HPV-16) DNA from W12 cells, using a real-time quantitative PCR assay to determine amplification efficiency, and obtained a 2.4 × 104-fold increase in HPV-16 DNA concentration. We were able to clone the complete HPV-16 genome from this multiply primed RCA product. The optimized protocol was subsequently applied to a bovine fibropapillomatous wart tissue sample. Whereas no papillomavirus DNA could be detected by restriction enzyme digestion of the original sample, multiply primed RCA enabled us to obtain a sufficient amount of papillomavirus DNA for restriction enzyme analysis, cloning, and subsequent sequencing of a novel variant of bovine papillomavirus type 1. The multiply primed RCA method allows the discovery of previously unknown papillomaviruses, and possibly also other circular DNA viruses, without a priori sequence information. PMID:15113879

Molecular characterization and phylogenetic analysis of a yak (Bos grunniens) κ-casein cDNA from lactating mammary gland.

PubMed

Bai, W L; Yin, R H; Dou, Q L; Jiang, W Q; Zhao, S J; Ma, Z J; Luo, G B; Zhao, Z H

2011-04-01

κ-Casein is one of the major proteins in the milk of mammals. It plays an important role in determining the size and specific function of milk micelles. We have previously identified and characterized a genetic variant of yak κ-casein by evaluating genomic DNA. Here, we isolate and characterize a yak κ-casein cDNA harboring the full-length open reading frame (ORF) from lactating mammary gland. Total RNA was extracted from mammary tissue of lactating female yak, and the κ-casein cDNA were synthesized by RT-PCR technique, then cloned and sequenced. The obtained cDNA of 660-bp contained an ORF sufficient to encode the entire amino acid sequence of κ-casein precursor protein consisting of 190 amino acids with a signal peptide of 21 amino acids. Yak κ-casein has a predicted molecular mass of 19,006.588 Da with a calculated isoelectric point of 7.245. Compared with the corresponding sequences in GenBank of cattle, buffalo, sheep, goat, Arabian camel, horse, and rabbit, yak κ-casein sequence had identity of 64.76-98.78% in cDNA, and identity of 44.79-98.42% and similarity of 53.65-98.42% in deduced amino acids, revealing a high homology with the other livestock species. Based on κ-casein cDNA sequences, the phylogenetic analysis indicated that yak κ-casein had a close relationship with that of cattle. This work might be useful in the genetic engineering researches for yak κ-casein.

The 193-base pair Gsg2 (haspin) promoter region regulates germ cell-specific expression bidirectionally and synchronously.

PubMed

Tokuhiro, Keizo; Miyagawa, Yasushi; Yamada, Shuichi; Hirose, Mika; Ohta, Hiroshi; Nishimune, Yoshitake; Tanaka, Hiromitsu

2007-03-01

Haspin is a unique protein kinase expressed predominantly in haploid male germ cells. The genomic structure of haspin (Gsg2) has revealed it to be intronless, and the entire transcription unit is in an intron of the integrin alphaE (Itgae) gene. Transcription occurs from a bidirectional promoter that also generates an alternatively spliced integrin alphaE-derived mRNA (Aed). In mice, the testis-specific alternative splicing of Aed is expressed bidirectionally downstream from the Gsg2 transcription initiation site, and a segment consisting of 26 bp transcribes both genomic DNA strands between Gsg2 and the Aed transcription initiation sites. To investigate the mechanisms for this unique gene regulation, we cloned and characterized the Gsg2 promoter region. The 193-bp genomic fragment from the 5' end of the Gsg2 and Aed genes, fused with EGFP and DsRed genes, drove the expression of both proteins in haploid germ cells of transgenic mice. This promoter element contained only a GC-rich sequence, and not the previously reported DNA sequences known to bind various transcription factors--with the exception of E2F1, TCFAP2A1 (AP2), and SP1. Here, we show that the 193-bp DNA sequence is sufficient for the specific, bidirectional, and synchronous expression in germ cells in the testis. We also demonstrate the existence of germ cell nuclear factors specifically bound to the promoter sequence. This activity may be regulated by binding to the promoter sequence with germ cell-specific nuclear complex(es) without regulation via DNA methylation.

No need to replace an “anomalous” primate (Primates) with an “anomalous” bear (Carnivora, Ursidae)

PubMed Central

Gutiérrez, Eliécer E.; Pine, Ronald H.

2015-01-01

Abstract By means of mitochondrial 12S rRNA sequencing of putative “yeti”, “bigfoot”, and other “anomalous primate” hair samples, a recent study concluded that two samples, presented as from the Himalayas, do not belong to an “anomalous primate”, but to an unknown, anomalous type of ursid. That is, that they match 12S rRNA sequences of a fossil Polar Bear (Ursus maritimus), but neither of modern Polar Bears, nor of Brown Bears (Ursus arctos), the closest relative of Polar Bears, and one that occurs today in the Himalayas. We have undertaken direct comparison of sequences; replication of the original comparative study; inference of phylogenetic relationships of the two samples with respect to those from all extant species of Ursidae (except for the Giant Panda, Ailuropoda melanoleuca) and two extinct Pleistocene species; and application of a non-tree-based population aggregation approach for species diagnosis and identification. Our results demonstrate that the very short fragment of the 12S rRNA gene sequenced by Sykes et al. is not sufficiently informative to support the hypotheses provided by these authors with respect to the taxonomic identity of the individuals from which these sequences were obtained. We have concluded that there is no reason to believe that the two samples came from anything other than Brown Bears. These analyses afforded an opportunity to test the monophyly of morphologically defined species and to comment on both their phylogenetic relationships and future efforts necessary to advance our understanding of ursid systematics. PMID:25829853

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

PubMed

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

2017-09-04

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

Microbial community analysis of the hypersaline water of the Dead Sea using high-throughput amplicon sequencing.

PubMed

Jacob, Jacob H; Hussein, Emad I; Shakhatreh, Muhamad Ali K; Cornelison, Christopher T

2017-10-01

Amplicon sequencing using next-generation technology (bTEFAP ® ) has been utilized in describing the diversity of Dead Sea microbiota. The investigated area is a well-known salt lake in the western part of Jordan found in the lowest geographical location in the world (more than 420 m below sea level) and characterized by extreme salinity (approximately, 34%) in addition to other extreme conditions (low pH, unique ionic composition different from sea water). DNA was extracted from Dead Sea water. A total of 314,310 small subunit RNA (SSU rRNA) sequences were parsed, and 288,452 sequences were then clustered. For alpha diversity analysis, sample was rarefied to 3,000 sequences. The Shannon-Wiener index curve plot reached a plateau at approximately 3,000 sequences indicating that sequencing depth was sufficient to capture the full scope of microbial diversity. Archaea was found to be dominating the sequences (52%), whereas Bacteria constitute 45% of the sequences. Altogether, prokaryotic sequences (which constitute 97% of all sequences) were found to predominate. The findings expand on previous studies by using high-throughput amplicon sequencing to describe the microbial community in an environment which in recent years has been shown to hide some interesting diversity. © 2017 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

Transposon facilitated DNA sequencing

DOE Office of Scientific and Technical Information (OSTI.GOV)

Berg, D.E.; Berg, C.M.; Huang, H.V.

1990-01-01

The purpose of this research is to investigate and develop methods that exploit the power of bacterial transposable elements for large scale DNA sequencing: Our premise is that the use of transposons to put primer binding sites randomly in target DNAs should provide access to all portions of large DNA fragments, without the inefficiencies of methods involving random subcloning and attendant repetitive sequencing, or of sequential synthesis of many oligonucleotide primers that are used to match systematically along a DNA molecule. Two unrelated bacterial transposons, Tn5 and {gamma}{delta}, are being used because they have both proven useful for molecular analyses,more » and because they differ sufficiently in mechanism and specificity of transposition to merit parallel development.« less

A cis-regulatory module activating transcription in the suspensor contains five cis-regulatory elements

DOE PAGES

Henry, Kelli F.; Kawashima, Tomokazu; Goldberg, Robert B.

2015-03-22

Little is known about the molecular mechanisms by which the embryo proper and suspensor of plant embryos activate specific gene sets shortly after fertilization. We analyzed the upstream region of the Scarlet Runner Bean ( Phaseolus coccineus) G564 gene in order to understand how genes are activated specifically in the suspensor during early embryo development. Previously, we showed that a 54-bp fragment of the G564 upstream region is sufficient for suspensor transcription and contains at least three required cis-regulatory sequences, including the 10-bp motif (5'-GAAAAGCGAA-3'), the 10 bp-like motif (5'-GAAAAACGAA-3'), and Region 2 motif (partial sequence 5'-TTGGT-3'). Here, we usemore » site-directed mutagenesis experiments in transgenic tobacco globularstage embryos to identify two additional cis-regulatory elements within the 54-bp cis-regulatory module that are required for G564 suspensor transcription: the Fifth motif (5'-GAGTTA-3') and a third 10-bp-related sequence (5'-GAAAACCACA-3'). Further deletion of the 54-bp fragment revealed that a 47-bp fragment containing the five motifs (the 10-bp, 10-bp-like, 10-bp-related, Region 2 and Fifth motifs) is sufficient for suspensor transcription, and represents a cis-regulatory module. A consensus sequence for each type of motif was determined by comparing motif sequences shown to activate suspensor transcription. Phylogenetic analyses suggest that the regulation of G564 is evolutionarily conserved. Lastly, a homologous cis-regulatory module was found upstream of the G564 ortholog in the Common Bean (Phaseolus vulgaris), indicating that the regulation of G564 is evolutionarily conserved in closely related bean species.« less

A cis-regulatory module activating transcription in the suspensor contains five cis-regulatory elements

DOE Office of Scientific and Technical Information (OSTI.GOV)

Henry, Kelli F.; Kawashima, Tomokazu; Goldberg, Robert B.

Little is known about the molecular mechanisms by which the embryo proper and suspensor of plant embryos activate specific gene sets shortly after fertilization. We analyzed the upstream region of the Scarlet Runner Bean ( Phaseolus coccineus) G564 gene in order to understand how genes are activated specifically in the suspensor during early embryo development. Previously, we showed that a 54-bp fragment of the G564 upstream region is sufficient for suspensor transcription and contains at least three required cis-regulatory sequences, including the 10-bp motif (5'-GAAAAGCGAA-3'), the 10 bp-like motif (5'-GAAAAACGAA-3'), and Region 2 motif (partial sequence 5'-TTGGT-3'). Here, we usemore » site-directed mutagenesis experiments in transgenic tobacco globularstage embryos to identify two additional cis-regulatory elements within the 54-bp cis-regulatory module that are required for G564 suspensor transcription: the Fifth motif (5'-GAGTTA-3') and a third 10-bp-related sequence (5'-GAAAACCACA-3'). Further deletion of the 54-bp fragment revealed that a 47-bp fragment containing the five motifs (the 10-bp, 10-bp-like, 10-bp-related, Region 2 and Fifth motifs) is sufficient for suspensor transcription, and represents a cis-regulatory module. A consensus sequence for each type of motif was determined by comparing motif sequences shown to activate suspensor transcription. Phylogenetic analyses suggest that the regulation of G564 is evolutionarily conserved. Lastly, a homologous cis-regulatory module was found upstream of the G564 ortholog in the Common Bean (Phaseolus vulgaris), indicating that the regulation of G564 is evolutionarily conserved in closely related bean species.« less

A cis-regulatory module activating transcription in the suspensor contains five cis-regulatory elements.

PubMed

Henry, Kelli F; Kawashima, Tomokazu; Goldberg, Robert B

2015-06-01

Little is known about the molecular mechanisms by which the embryo proper and suspensor of plant embryos activate specific gene sets shortly after fertilization. We analyzed the upstream region of the Scarlet Runner Bean (Phaseolus coccineus) G564 gene in order to understand how genes are activated specifically in the suspensor during early embryo development. Previously, we showed that a 54-bp fragment of the G564 upstream region is sufficient for suspensor transcription and contains at least three required cis-regulatory sequences, including the 10-bp motif (5'-GAAAAGCGAA-3'), the 10 bp-like motif (5'-GAAAAACGAA-3'), and Region 2 motif (partial sequence 5'-TTGGT-3'). Here, we use site-directed mutagenesis experiments in transgenic tobacco globular-stage embryos to identify two additional cis-regulatory elements within the 54-bp cis-regulatory module that are required for G564 suspensor transcription: the Fifth motif (5'-GAGTTA-3') and a third 10-bp-related sequence (5'-GAAAACCACA-3'). Further deletion of the 54-bp fragment revealed that a 47-bp fragment containing the five motifs (the 10-bp, 10-bp-like, 10-bp-related, Region 2 and Fifth motifs) is sufficient for suspensor transcription, and represents a cis-regulatory module. A consensus sequence for each type of motif was determined by comparing motif sequences shown to activate suspensor transcription. Phylogenetic analyses suggest that the regulation of G564 is evolutionarily conserved. A homologous cis-regulatory module was found upstream of the G564 ortholog in the Common Bean (Phaseolus vulgaris), indicating that the regulation of G564 is evolutionarily conserved in closely related bean species.

Method and apparatus for biological sequence comparison

DOEpatents

Marr, T.G.; Chang, W.I.

1997-12-23

A method and apparatus are disclosed for comparing biological sequences from a known source of sequences, with a subject (query) sequence. The apparatus takes as input a set of target similarity levels (such as evolutionary distances in units of PAM), and finds all fragments of known sequences that are similar to the subject sequence at each target similarity level, and are long enough to be statistically significant. The invention device filters out fragments from the known sequences that are too short, or have a lower average similarity to the subject sequence than is required by each target similarity level. The subject sequence is then compared only to the remaining known sequences to find the best matches. The filtering member divides the subject sequence into overlapping blocks, each block being sufficiently large to contain a minimum-length alignment from a known sequence. For each block, the filter member compares the block with every possible short fragment in the known sequences and determines a best match for each comparison. The determined set of short fragment best matches for the block provide an upper threshold on alignment values. Regions of a certain length from the known sequences that have a mean alignment value upper threshold greater than a target unit score are concatenated to form a union. The current block is compared to the union and provides an indication of best local alignment with the subject sequence. 5 figs.

Method and apparatus for biological sequence comparison

DOEpatents

Marr, Thomas G.; Chang, William I-Wei

1997-01-01

A method and apparatus for comparing biological sequences from a known source of sequences, with a subject (query) sequence. The apparatus takes as input a set of target similarity levels (such as evolutionary distances in units of PAM), and finds all fragments of known sequences that are similar to the subject sequence at each target similarity level, and are long enough to be statistically significant. The invention device filters out fragments from the known sequences that are too short, or have a lower average similarity to the subject sequence than is required by each target similarity level. The subject sequence is then compared only to the remaining known sequences to find the best matches. The filtering member divides the subject sequence into overlapping blocks, each block being sufficiently large to contain a minimum-length alignment from a known sequence. For each block, the filter member compares the block with every possible short fragment in the known sequences and determines a best match for each comparison. The determined set of short fragment best matches for the block provide an upper threshold on alignment values. Regions of a certain length from the known sequences that have a mean alignment value upper threshold greater than a target unit score are concatenated to form a union. The current block is compared to the union and provides an indication of best local alignment with the subject sequence.

Molecular Divergence and Species Delimitation of the Cultivated Oyster Mushrooms: Integration of IGS1 and ITS

PubMed Central

Bhassu, Subha; Tan, Yee Shin; Vikineswary, Sabaratnam

2014-01-01

Identification of edible mushrooms particularly Pleurotus genus has been restricted due to various obstacles. The present study attempted to use the combination of two variable regions of IGS1 and ITS for classifying the economically cultivated Pleurotus species. Integration of the two regions proved a high ability that not only could clearly distinguish the species but also served sufficient intraspecies variation. Phylogenetic tree (IGS1 + ITS) showed seven distinct clades, each clade belonging to a separate species group. Moreover, the species differentiation was tested by AMOVA and the results were reconfirmed by presenting appropriate amounts of divergence (91.82% among and 8.18% within the species). In spite of achieving a proper classification of species by combination of IGS1 and ITS sequences, the phylogenetic tree showed the misclassification of the species of P. nebrodensis and P. eryngii var. ferulae with other strains of P. eryngii. However, the constructed median joining (MJ) network could not only differentiate between these species but also offer a profound perception of the species' evolutionary process. Eventually, due to the sufficient variation among and within species, distinct sequences, simple amplification, and location between ideal conserved ribosomal genes, the integration of IGS1 and ITS sequences is recommended as a desirable DNA barcode. PMID:24587752

The least channel capacity for chaos synchronization.

PubMed

Wang, Mogei; Wang, Xingyuan; Liu, Zhenzhen; Zhang, Huaguang

2011-03-01

Recently researchers have found that a channel with capacity exceeding the Kolmogorov-Sinai entropy of the drive system (h(KS)) is theoretically necessary and sufficient to sustain the unidirectional synchronization to arbitrarily high precision. In this study, we use symbolic dynamics and the automaton reset sequence to distinguish the information that is required in identifying the current drive word and obtaining the synchronization. Then, we show that the least channel capacity that is sufficient to transmit the distinguished information and attain the synchronization of arbitrarily high precision is h(KS). Numerical simulations provide support for our conclusions.

PulseNet International: Vision for the implementation of whole genome sequencing (WGS) for global food-borne disease surveillance

PubMed Central

Nadon, Celine; Van Walle, Ivo; Gerner-Smidt, Peter; Campos, Josefina; Chinen, Isabel; Concepcion-Acevedo, Jeniffer; Gilpin, Brent; Smith, Anthony M.; Kam, Kai Man; Perez, Enrique; Trees, Eija; Kubota, Kristy; Takkinen, Johanna; Nielsen, Eva Møller; Carleton, Heather

2017-01-01

PulseNet International is a global network dedicated to laboratory-based surveillance for food-borne diseases. The network comprises the national and regional laboratory networks of Africa, Asia Pacific, Canada, Europe, Latin America and the Caribbean, the Middle East, and the United States. The PulseNet International vision is the standardised use of whole genome sequencing (WGS) to identify and subtype food-borne bacterial pathogens worldwide, replacing traditional methods to strengthen preparedness and response, reduce global social and economic disease burden, and save lives. To meet the needs of real-time surveillance, the PulseNet International network will standardise subtyping via WGS using whole genome multilocus sequence typing (wgMLST), which delivers sufficiently high resolution and epidemiological concordance, plus unambiguous nomenclature for the purposes of surveillance. Standardised protocols, validation studies, quality control programmes, database and nomenclature development, and training should support the implementation and decentralisation of WGS. Ideally, WGS data collected for surveillance purposes should be publicly available, in real time where possible, respecting data protection policies. WGS data are suitable for surveillance and outbreak purposes and for answering scientific questions pertaining to source attribution, antimicrobial resistance, transmission patterns, and virulence, which will further enable the protection and improvement of public health with respect to food-borne disease. PMID:28662764

Short intronic repeat sequences facilitate circular RNA production.

PubMed

Liang, Dongming; Wilusz, Jeremy E

2014-10-15

Recent deep sequencing studies have revealed thousands of circular noncoding RNAs generated from protein-coding genes. These RNAs are produced when the precursor messenger RNA (pre-mRNA) splicing machinery "backsplices" and covalently joins, for example, the two ends of a single exon. However, the mechanism by which the spliceosome selects only certain exons to circularize is largely unknown. Using extensive mutagenesis of expression plasmids, we show that miniature introns containing the splice sites along with short (∼ 30- to 40-nucleotide) inverted repeats, such as Alu elements, are sufficient to allow the intervening exons to circularize in cells. The intronic repeats must base-pair to one another, thereby bringing the splice sites into close proximity to each other. More than simple thermodynamics is clearly at play, however, as not all repeats support circularization, and increasing the stability of the hairpin between the repeats can sometimes inhibit circular RNA biogenesis. The intronic repeats and exonic sequences must collaborate with one another, and a functional 3' end processing signal is required, suggesting that circularization may occur post-transcriptionally. These results suggest detailed and generalizable models that explain how the splicing machinery determines whether to produce a circular noncoding RNA or a linear mRNA. © 2014 Liang and Wilusz; Published by Cold Spring Harbor Laboratory Press.

Human Fip1 is a subunit of CPSF that binds to U-rich RNA elements and stimulates poly(A) polymerase.

PubMed

Kaufmann, Isabelle; Martin, Georges; Friedlein, Arno; Langen, Hanno; Keller, Walter

2004-02-11

In mammals, polyadenylation of mRNA precursors (pre-mRNAs) by poly(A) polymerase (PAP) depends on cleavage and polyadenylation specificity factor (CPSF). CPSF is a multisubunit complex that binds to the canonical AAUAAA hexamer and to U-rich upstream sequence elements on the pre-mRNA, thereby stimulating the otherwise weakly active and nonspecific polymerase to elongate efficiently RNAs containing a poly(A) signal. Based on sequence similarity to the Saccharomyces cerevisiae polyadenylation factor Fip1p, we have identified human Fip1 (hFip1) and found that the protein is an integral subunit of CPSF. hFip1 interacts with PAP and has an arginine-rich RNA-binding motif that preferentially binds to U-rich sequence elements on the pre-mRNA. Recombinant hFip1 is sufficient to stimulate the in vitro polyadenylation activity of PAP in a U-rich element-dependent manner. hFip1, CPSF160 and PAP form a ternary complex in vitro, suggesting that hFip1 and CPSF160 act together in poly(A) site recognition and in cooperative recruitment of PAP to the RNA. These results show that hFip1 significantly contributes to CPSF-mediated stimulation of PAP activity.

Human Fip1 is a subunit of CPSF that binds to U-rich RNA elements and stimulates poly(A) polymerase

PubMed Central

Kaufmann, Isabelle; Martin, Georges; Friedlein, Arno; Langen, Hanno; Keller, Walter

2004-01-01

In mammals, polyadenylation of mRNA precursors (pre-mRNAs) by poly(A) polymerase (PAP) depends on cleavage and polyadenylation specificity factor (CPSF). CPSF is a multisubunit complex that binds to the canonical AAUAAA hexamer and to U-rich upstream sequence elements on the pre-mRNA, thereby stimulating the otherwise weakly active and nonspecific polymerase to elongate efficiently RNAs containing a poly(A) signal. Based on sequence similarity to the Saccharomyces cerevisiae polyadenylation factor Fip1p, we have identified human Fip1 (hFip1) and found that the protein is an integral subunit of CPSF. hFip1 interacts with PAP and has an arginine-rich RNA-binding motif that preferentially binds to U-rich sequence elements on the pre-mRNA. Recombinant hFip1 is sufficient to stimulate the in vitro polyadenylation activity of PAP in a U-rich element-dependent manner. hFip1, CPSF160 and PAP form a ternary complex in vitro, suggesting that hFip1 and CPSF160 act together in poly(A) site recognition and in cooperative recruitment of PAP to the RNA. These results show that hFip1 significantly contributes to CPSF-mediated stimulation of PAP activity. PMID:14749727

Measuring Working Memory With Digit Span and the Letter-Number Sequencing Subtests From the WAIS-IV: Too Low Manipulation Load and Risk for Underestimating Modality Effects.

PubMed

Egeland, Jens

2015-01-01

The Wechsler Adult Intelligence Scale (WAIS) is one of the most frequently used tests among psychologists. In the fourth edition of the test (WAIS-IV), the subtests Digit Span and Letter-Number Sequencing are expanded for better measurement of working memory (WM). However, it is not clear whether the new extended tasks contribute sufficient complexity to be sensitive measures of manipulation WM, nor do we know to what degree WM capacity differs between the visual and the auditory modality because the WAIS-IV only tests the auditory modality. Performance by a mixed sample of 226 patients referred for neuropsychological examination on the Digit Span and Letter-Number Sequencing subtests from the WAIS-IV and on Spatial Span from the Wechsler Memory Scale-Third Edition was analyzed in two confirmatory factor analyses to investigate whether a unitary WM model or divisions based on modality or level/complexity best fit the data. The modality model showed the best fit when analyzing summed scores for each task as well as scores for the longest span. The clinician is advised to apply tests with higher manipulation load and to consider testing visual span as well before drawing conclusions about impaired WM from the WAIS-IV.

Preparation by alkaline treatment and detailed characterisation of empty hepatitis B virus core particles for vaccine and gene therapy applications.

PubMed

Strods, Arnis; Ose, Velta; Bogans, Janis; Cielens, Indulis; Kalnins, Gints; Radovica, Ilze; Kazaks, Andris; Pumpens, Paul; Renhofa, Regina

2015-06-26

Hepatitis B virus (HBV) core (HBc) virus-like particles (VLPs) are one of the most powerful protein engineering tools utilised to expose immunological epitopes and/or cell-targeting signals and for the packaging of genetic material and immune stimulatory sequences. Although HBc VLPs and their numerous derivatives are produced in highly efficient bacterial and yeast expression systems, the existing purification and packaging protocols are not sufficiently optimised and standardised. Here, a simple alkaline treatment method was employed for the complete removal of internal RNA from bacteria- and yeast-produced HBc VLPs and for the conversion of these VLPs into empty particles, without any damage to the VLP structure. The empty HBc VLPs were able to effectively package the added DNA and RNA sequences. Furthermore, the alkaline hydrolysis technology appeared efficient for the purification and packaging of four different HBc variants carrying lysine residues on the HBc VLP spikes. Utilising the introduced lysine residues and the intrinsic aspartic and glutamic acid residues exposed on the tips of the HBc spikes for chemical coupling of the chosen peptide and/or nucleic acid sequences ensured a standard and easy protocol for the further development of versatile HBc VLP-based vaccine and gene therapy applications.

Interactions between the promoter and first intron are involved in transcriptional control of alpha 1(I) collagen gene expression.

PubMed Central

Bornstein, P; McKay, J; Liska, D J; Apone, S; Devarayalu, S

1988-01-01

The first intron of the human collagen alpha 1(I) gene contains several positively and negatively acting elements. We have studied the transcription of collagen-human growth hormone fusion genes, containing deletions and rearrangements of collagen intronic sequences, by transient transfection of chick tendon fibroblasts and NIH 3T3 cells. In chick tendon fibroblasts, but not in 3T3 cells, inversion of intronic sequences containing a previously studied 274-base-pair segment, A274, resulted in markedly reduced human growth hormone mRNA levels as determined by an RNase protection assay. This inhibitory effect was largely alleviated when deletions were introduced in the collagen promoter of plasmids containing negatively oriented intronic sequences. Evidence for interaction of the promoter with the intronic segment, A274, was obtained by gel mobility shift assays. We suggest that promoter-intron interactions, mediated by DNA-binding proteins, regulate collagen gene transcription. Inversion of intronic segments containing critical interactive elements might then lead to an altered geometry and reduced activity of a transcriptional complex in those cells with sufficiently high levels of appropriate transcription factors. We further suggest that the deleted promoter segment plays a key role in directing DNA interactions involved in transcriptional control. Images PMID:3211130

Phylogenetic Relationships of Yessotoxin-Producing Dinoflagellates, Based on the Large Subunit and Internal Transcribed Spacer Ribosomal DNA Domains▿

PubMed Central

Howard, Meredith D. A.; Smith, G. Jason; Kudela, Raphael M.

2009-01-01

Yessotoxin (YTX) is a globally distributed marine toxin produced by some isolates of the dinoflagellate species Protoceratium reticulatum, Lingulodinium polyedrum, and Gonyaulax spinifera within the order Gonyaulacales. The process of isolating cells and testing each isolate individually for YTX production during toxic blooms are labor intensive, and this impedes our ability to respond quickly to toxic blooms. In this study, we used molecular sequences from the large subunit and internal transcribed spacer genomic regions in the ribosomal operon of known YTX-producing dinoflagellates to determine if genetic differences exist among geographically distinct populations or between toxic and nontoxic isolates within species. In all analyses, all three YTX-producing species fell within the Gonyaulacales order in agreement with morphological taxonomy. Phylogenetic analyses of available rRNA gene sequences indicate that the capacity for YTX production appears to be confined to the order Gonyaulacales. These findings indicate that Gonyaulacoloid dinoflagellate species are the most likely to produce YTX and thus should be prioritized for YTX screening during events. Dinoflagellate species that fall outside of the Gonyaulacales order are unlikely to produce YTX. Although the rRNA operon offers multiple sequence domains to resolve species level diversification within this dinoflagellate order, these domains are not sufficiently variable to provide robust markers for YTX toxicity. PMID:19011074

Genome-wide association links candidate genes to resistance to Plum Pox Virus in apricot (Prunus armeniaca).

PubMed

Mariette, Stéphanie; Wong Jun Tai, Fabienne; Roch, Guillaume; Barre, Aurélien; Chague, Aurélie; Decroocq, Stéphane; Groppi, Alexis; Laizet, Yec'han; Lambert, Patrick; Tricon, David; Nikolski, Macha; Audergon, Jean-Marc; Abbott, Albert G; Decroocq, Véronique

2016-01-01

In fruit tree species, many important traits have been characterized genetically by using single-family descent mapping in progenies segregating for the traits. However, most mapped loci have not been sufficiently resolved to the individual genes due to insufficient progeny sizes for high resolution mapping and the previous lack of whole-genome sequence resources of the study species. To address this problem for Plum Pox Virus (PPV) candidate resistance gene identification in Prunus species, we implemented a genome-wide association (GWA) approach in apricot. This study exploited the broad genetic diversity of the apricot (Prunus armeniaca) germplasm containing resistance to PPV, next-generation sequence-based genotyping, and the high-quality peach (Prunus persica) genome reference sequence for single nucleotide polymorphism (SNP) identification. The results of this GWA study validated previously reported PPV resistance quantitative trait loci (QTL) intervals, highlighted other potential resistance loci, and resolved each to a limited set of candidate genes for further study. This work substantiates the association genetics approach for resolution of QTL to candidate genes in apricot and suggests that this approach could simplify identification of other candidate genes for other marked trait intervals in this germplasm. © 2015 INRA, UMR 1332 BFP New Phytologist © 2015 New Phytologist Trust.

Preparation by alkaline treatment and detailed characterisation of empty hepatitis B virus core particles for vaccine and gene therapy applications

PubMed Central

Strods, Arnis; Ose, Velta; Bogans, Janis; Cielens, Indulis; Kalnins, Gints; Radovica, Ilze; Kazaks, Andris; Pumpens, Paul; Renhofa, Regina

2015-01-01

Hepatitis B virus (HBV) core (HBc) virus-like particles (VLPs) are one of the most powerful protein engineering tools utilised to expose immunological epitopes and/or cell-targeting signals and for the packaging of genetic material and immune stimulatory sequences. Although HBc VLPs and their numerous derivatives are produced in highly efficient bacterial and yeast expression systems, the existing purification and packaging protocols are not sufficiently optimised and standardised. Here, a simple alkaline treatment method was employed for the complete removal of internal RNA from bacteria- and yeast-produced HBc VLPs and for the conversion of these VLPs into empty particles, without any damage to the VLP structure. The empty HBc VLPs were able to effectively package the added DNA and RNA sequences. Furthermore, the alkaline hydrolysis technology appeared efficient for the purification and packaging of four different HBc variants carrying lysine residues on the HBc VLP spikes. Utilising the introduced lysine residues and the intrinsic aspartic and glutamic acid residues exposed on the tips of the HBc spikes for chemical coupling of the chosen peptide and/or nucleic acid sequences ensured a standard and easy protocol for the further development of versatile HBc VLP-based vaccine and gene therapy applications. PMID:26113394

Preparation by alkaline treatment and detailed characterisation of empty hepatitis B virus core particles for vaccine and gene therapy applications

NASA Astrophysics Data System (ADS)

Strods, Arnis; Ose, Velta; Bogans, Janis; Cielens, Indulis; Kalnins, Gints; Radovica, Ilze; Kazaks, Andris; Pumpens, Paul; Renhofa, Regina

2015-06-01

Hepatitis B virus (HBV) core (HBc) virus-like particles (VLPs) are one of the most powerful protein engineering tools utilised to expose immunological epitopes and/or cell-targeting signals and for the packaging of genetic material and immune stimulatory sequences. Although HBc VLPs and their numerous derivatives are produced in highly efficient bacterial and yeast expression systems, the existing purification and packaging protocols are not sufficiently optimised and standardised. Here, a simple alkaline treatment method was employed for the complete removal of internal RNA from bacteria- and yeast-produced HBc VLPs and for the conversion of these VLPs into empty particles, without any damage to the VLP structure. The empty HBc VLPs were able to effectively package the added DNA and RNA sequences. Furthermore, the alkaline hydrolysis technology appeared efficient for the purification and packaging of four different HBc variants carrying lysine residues on the HBc VLP spikes. Utilising the introduced lysine residues and the intrinsic aspartic and glutamic acid residues exposed on the tips of the HBc spikes for chemical coupling of the chosen peptide and/or nucleic acid sequences ensured a standard and easy protocol for the further development of versatile HBc VLP-based vaccine and gene therapy applications.

A Novel Paramyxovirus?

PubMed Central

García-Sastre, Adolfo; Palese, Peter

2005-01-01

In public databases, we identified sequences reported as human genes expressed in kidney mesangial cells. The similarity of these genes to paramyxovirus matrix, fusion, and phosphoprotein genes suggests that they are derived from a novel paramyxovirus. These genes are sufficiently unique to suggest the existence of a novel paramyxovirus genus. PMID:15705331

Variant Profiling of Candidate Genes in Pancreatic Ductal Adenocarcinoma.

PubMed

Huang, Jiaqi; Löhr, Johannes-Matthias; Nilsson, Magnus; Segersvärd, Ralf; Matsson, Hans; Verbeke, Caroline; Heuchel, Rainer; Kere, Juha; Iafrate, A John; Zheng, Zongli; Ye, Weimin

2015-11-01

Pancreatic ductal adenocarcinoma (PDAC) has a poor prognosis. Variant profiling is crucial for developing personalized treatment and elucidating the etiology of this disease. Patients with PDAC undergoing surgery from 2007 to 2012 (n = 73) were followed from diagnosis until death or the end of the study. We applied an anchored multiplex PCR (AMP)-based next-generation sequencing (NGS) method to a panel of 65 selected genes and assessed analytical performance by sequencing a quantitative multiplex DNA reference standard. In clinical PDAC samples, detection of low-level KRAS (Kirsten rat sarcoma viral oncogene homolog) mutations was validated by allele-specific PCR and digital PCR. We compared overall survival of patients according to KRAS mutation status by log-rank test and applied logistic regression to evaluate the association between smoking and tumor variant types. The AMP-based NGS method could detect variants with allele frequencies as low as 1% given sufficient sequencing depth (>1500×). Low-frequency KRAS G12 mutations (allele frequency 1%-5%) were all confirmed by allele-specific PCR and digital PCR. The most prevalent genetic alterations were in KRAS (78% of patients), TP53 (tumor protein p53) (25%), and SMAD4 (SMAD family member 4) (8%). Overall survival in T3-stage PDAC patients differed among KRAS mutation subtypes (P = 0.019). Transversion variants were more common in ever-smokers than in never-smokers (odds ratio 5.7; 95% CI 1.2-27.8). The AMP-based NGS method is applicable for profiling tumor variants. Using this approach, we demonstrated that in PDAC patients, KRAS mutant subtype G12V is associated with poorer survival, and that transversion variants are more common among smokers. © 2015 American Association for Clinical Chemistry.

Molecular characterization of human T-cell lymphotropic virus type 1 full and partial genomes by Illumina massively parallel sequencing technology.

PubMed

Pessôa, Rodrigo; Watanabe, Jaqueline Tomoko; Nukui, Youko; Pereira, Juliana; Casseb, Jorge; Kasseb, Jorge; de Oliveira, Augusto César Penalva; Segurado, Aluisio Cotrim; Sanabani, Sabri Saeed

2014-01-01

Here, we report on the partial and full-length genomic (FLG) variability of HTLV-1 sequences from 90 well-characterized subjects, including 48 HTLV-1 asymptomatic carriers (ACs), 35 HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and 7 adult T-cell leukemia/lymphoma (ATLL) patients, using an Illumina paired-end protocol. Blood samples were collected from 90 individuals, and DNA was extracted from the PBMCs to measure the proviral load and to amplify the HTLV-1 FLG from two overlapping fragments. The amplified PCR products were subjected to deep sequencing. The sequencing data were assembled, aligned, and mapped against the HTLV-1 genome with sufficient genetic resemblance and utilized for further phylogenetic analysis. A high-throughput sequencing-by-synthesis instrument was used to obtain an average of 3210- and 5200-fold coverage of the partial (n = 14) and FLG (n = 76) data from the HTLV-1 strains, respectively. The results based on the phylogenetic trees of consensus sequences from partial and FLGs revealed that 86 (95.5%) individuals were infected with the transcontinental sub-subtypes of the cosmopolitan subtype (aA) and that 4 individuals (4.5%) were infected with the Japanese sub-subtypes (aB). A comparison of the nucleotide and amino acids of the FLG between the three clinical settings yielded no correlation between the sequenced genotype and clinical outcomes. The evolutionary relationships among the HTLV sequences were inferred from nucleotide sequence, and the results are consistent with the hypothesis that there were multiple introductions of the transcontinental subtype in Brazil. This study has increased the number of subtype aA full-length genomes from 8 to 81 and HTLV-1 aB from 2 to 5 sequences. The overall data confirmed that the cosmopolitan transcontinental sub-subtypes were the most prevalent in the Brazilian population. It is hoped that this valuable genomic data will add to our current understanding of the evolutionary history of this medically important virus.

The current status and portability of our sequence handling software.

PubMed Central

Staden, R

1986-01-01

I describe the current status of our sequence analysis software. The package contains a comprehensive suite of programs for managing large shotgun sequencing projects, a program containing 61 functions for analysing single sequences and a program for comparing pairs of sequences for similarity. The programs that have been described before have been improved by the addition of new functions and by being made very much easier to use. The major interactive programs have 125 pages of online help available from within them. Several new programs are described including screen editing of aligned gel readings for shotgun sequencing projects; a method to highlight errors in aligned gel readings, new methods for searching for putative signals in sequences. We use the programs on a VAX computer but the whole package has been rewritten to make it easy to transport it to other machines. I believe the programs will now run on any machine with a FORTRAN77 compiler and sufficient memory. We are currently putting the programs onto an IBM PC XT/AT and another micro running under UNIX. PMID:3511446

On some new normed sequence spaces

NASA Astrophysics Data System (ADS)

Pranajaya, G.; Herawati, E.

2018-01-01

The sequence spaces (c 0)Λ, c Λ, and (ℓ ∞)Λ was introduced and studied by Mursaleen and Noman [11]. In the present paper, for M is a generalization of Orlicz function, we extend the spaces Mursaleen and Noman’s to [c 0(M)]Λ, [c(M)]Λ, and [ℓ ∞(M)]Λ, respectively, and investigate some topological properties of these spaces. Finally, we determine the necessary and sufficient conditions of an infinite matrix A belonging to classes (c 0(M), c 0(M)), (c(M), c(M)), and (ℓ ∞(M), ℓ ∞(M)).

Introduction of optical tweezers in advanced physics laboratory

NASA Astrophysics Data System (ADS)

Wang, Gang

2017-08-01

Laboratories are an essential part of undergraduate optoelectronics and photonics education. Of particular interest are the sequence of laboratories which offer students meaningful research experience within a reasonable time-frame limited by regular laboratory hours. We will present our introduction of optical tweezers into the upper-level physics laboratory. We developed the sequence of experiments in the Advanced Lab to offer students sufficient freedom to explore, rather than simply setting up a demonstration following certain recipes. We will also present its impact on our current curriculum of optoelectronics concentration within the physics program.

SU-D-18C-01: A Novel 4D-MRI Technology Based On K-Space Retrospective Sorting

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Y; Yin, F; Cai, J

2014-06-01

Purpose: Current 4D-MRI techniques lack sufficient temporal/spatial resolution and consistent tumor contrast. To overcome these limitations, this study presents the development and initial evaluation of an entirely new framework of 4D-MRI based on k-space retrospective sorting. Methods: An important challenge of the proposed technique is to determine the number of repeated scans(NR) required to obtain sufficient k-space data for 4D-MRI. To do that, simulations using 29 cancer patients' respiratory profiles were performed to derive the relationship between data acquisition completeness(Cp) and NR, also relationship between NR(Cp=95%) and the following factors: total slice(NS), respiratory phase bin length(Lb), frame rate(fr), resolution(R) andmore » image acquisition starting-phase(P0). To evaluate our technique, a computer simulation study on a 4D digital human phantom (XCAT) were conducted with regular breathing (fr=0.5Hz; R=256×256). A 2D echo planer imaging(EPI) MRI sequence were assumed to acquire raw k-space data, with respiratory signal and acquisition time for each k-space data line recorded simultaneously. K-space data was re-sorted based on respiratory phases. To evaluate 4D-MRI image quality, tumor trajectories were measured and compared with the input signal. Mean relative amplitude difference(D) and cross-correlation coefficient(CC) are calculated. Finally, phase-sharing sliding window technique was applied to investigate the feasibility of generating ultra-fast 4D-MRI. Result: Cp increased with NR(Cp=100*[1-exp(-0.19*NR)], when NS=30, Lb=100%/6). NR(Cp=95%) was inversely-proportional to Lb (r=0.97), but independent of other factors. 4D-MRI on XCAT demonstrated highly accurate motion information (D=0.67%, CC=0.996) with much less artifacts than those on image-based sorting 4D-MRI. Ultra-fast 4D-MRI with an apparent temporal resolution of 10 frames/second was reconstructed using the phase-sharing sliding window technique. Conclusions: A novel 4D-MRI technology based on k-space sorting has been successfully developed and evaluated on the digital phantom. Framework established can be applied to a variety of MR sequences, showing great promises to develop the optimal 4D-MRI technique for many radiation therapy applications. NIH (1R21CA165384-01A1)« less

Analysis of Rare, Exonic Variation amongst Subjects with Autism Spectrum Disorders and Population Controls

PubMed Central

Liu, Li; Sabo, Aniko; Neale, Benjamin M.; Nagaswamy, Uma; Stevens, Christine; Lim, Elaine; Bodea, Corneliu A.; Muzny, Donna; Reid, Jeffrey G.; Banks, Eric; Coon, Hillary; DePristo, Mark; Dinh, Huyen; Fennel, Tim; Flannick, Jason; Gabriel, Stacey; Garimella, Kiran; Gross, Shannon; Hawes, Alicia; Lewis, Lora; Makarov, Vladimir; Maguire, Jared; Newsham, Irene; Poplin, Ryan; Ripke, Stephan; Shakir, Khalid; Samocha, Kaitlin E.; Wu, Yuanqing; Boerwinkle, Eric; Buxbaum, Joseph D.; Cook, Edwin H.; Devlin, Bernie; Schellenberg, Gerard D.; Sutcliffe, James S.; Daly, Mark J.; Gibbs, Richard A.; Roeder, Kathryn

2013-01-01

We report on results from whole-exome sequencing (WES) of 1,039 subjects diagnosed with autism spectrum disorders (ASD) and 870 controls selected from the NIMH repository to be of similar ancestry to cases. The WES data came from two centers using different methods to produce sequence and to call variants from it. Therefore, an initial goal was to ensure the distribution of rare variation was similar for data from different centers. This proved straightforward by filtering called variants by fraction of missing data, read depth, and balance of alternative to reference reads. Results were evaluated using seven samples sequenced at both centers and by results from the association study. Next we addressed how the data and/or results from the centers should be combined. Gene-based analyses of association was an obvious choice, but should statistics for association be combined across centers (meta-analysis) or should data be combined and then analyzed (mega-analysis)? Because of the nature of many gene-based tests, we showed by theory and simulations that mega-analysis has better power than meta-analysis. Finally, before analyzing the data for association, we explored the impact of population structure on rare variant analysis in these data. Like other recent studies, we found evidence that population structure can confound case-control studies by the clustering of rare variants in ancestry space; yet, unlike some recent studies, for these data we found that principal component-based analyses were sufficient to control for ancestry and produce test statistics with appropriate distributions. After using a variety of gene-based tests and both meta- and mega-analysis, we found no new risk genes for ASD in this sample. Our results suggest that standard gene-based tests will require much larger samples of cases and controls before being effective for gene discovery, even for a disorder like ASD. PMID:23593035

Systematic discovery of novel eukaryotic transcriptional regulators using sequence homology independent prediction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bossi, Flavia; Fan, Jue; Xiao, Jun

Here, the molecular function of a gene is most commonly inferred by sequence similarity. Therefore, genes that lack sufficient sequence similarity to characterized genes (such as certain classes of transcriptional regulators) are difficult to classify using most function prediction algorithms and have remained uncharacterized. As a result, to identify novel transcriptional regulators systematically, we used a feature-based pipeline to screen protein families of unknown function. This method predicted 43 transcriptional regulator families in Arabidopsis thaliana, 7 families in Drosophila melanogaster, and 9 families in Homo sapiens. Literature curation validated 12 of the predicted families to be involved in transcriptional regulation.more » We tested 33 out of the 195 Arabidopsis putative transcriptional regulators for their ability to activate transcription of a reporter gene in planta and found twelve coactivators, five of which had no prior literature support. To investigate mechanisms of action in which the predicted regulators might work, we looked for interactors of an Arabidopsis candidate that did not show transactivation activity in planta and found that it might work with other members of its own family and a subunit of the Polycomb Repressive Complex 2 to regulate transcription. Our results demonstrate the feasibility of assigning molecular function to proteins of unknown function without depending on sequence similarity. In particular, we identified novel transcriptional regulators using biological features enriched in transcription factors. The predictions reported here should accelerate the characterization of novel regulators.« less

Systematic discovery of novel eukaryotic transcriptional regulators using sequence homology independent prediction

DOE PAGES

Bossi, Flavia; Fan, Jue; Xiao, Jun; ...

2017-06-26

Here, the molecular function of a gene is most commonly inferred by sequence similarity. Therefore, genes that lack sufficient sequence similarity to characterized genes (such as certain classes of transcriptional regulators) are difficult to classify using most function prediction algorithms and have remained uncharacterized. As a result, to identify novel transcriptional regulators systematically, we used a feature-based pipeline to screen protein families of unknown function. This method predicted 43 transcriptional regulator families in Arabidopsis thaliana, 7 families in Drosophila melanogaster, and 9 families in Homo sapiens. Literature curation validated 12 of the predicted families to be involved in transcriptional regulation.more » We tested 33 out of the 195 Arabidopsis putative transcriptional regulators for their ability to activate transcription of a reporter gene in planta and found twelve coactivators, five of which had no prior literature support. To investigate mechanisms of action in which the predicted regulators might work, we looked for interactors of an Arabidopsis candidate that did not show transactivation activity in planta and found that it might work with other members of its own family and a subunit of the Polycomb Repressive Complex 2 to regulate transcription. Our results demonstrate the feasibility of assigning molecular function to proteins of unknown function without depending on sequence similarity. In particular, we identified novel transcriptional regulators using biological features enriched in transcription factors. The predictions reported here should accelerate the characterization of novel regulators.« less

ACGT-containing abscisic acid response element (ABRE) and coupling element 3 (CE3) are functionally equivalent.

PubMed

Hobo, T; Asada, M; Kowyama, Y; Hattori, T

1999-09-01

ACGT-containing ABA response elements (ABREs) have been functionally identified in the promoters of various genes. In addition, single copies of ABRE have been found to require a cis-acting, coupling element to achieve ABA induction. A coupling element 3 (CE3) sequence, originally identified as such in the barley HVA1 promoter, is found approximately 30 bp downstream of motif A (ACGT-containing ABRE) in the promoter of the Osem gene. The relationship between these two elements was further defined by linker-scan analyses of a 55 bp fragment of the Osem promoter, which is sufficient for ABA-responsiveness and VP1 activation. The analyses revealed that both motif A and CE3 sequence were required not only for ABA-responsiveness but also for VP1 activation. Since the sequences of motif A and CE3 were found to be similar, motif-exchange experiments were carried out. The experiments demonstrated that motif A and CE3 were interchangeable by each other with respect to both ABA and VP1 regulation. In addition, both sequences were shown to be recognized by a VP1-interacting, ABA-responsive bZIP factor TRAB1. These results indicate that ACGT-containing ABREs and CE3 are functionally equivalent cis-acting elements. Furthermore, TRAB1 was shown to bind two other non-ACGT ABREs. Based on these results, all these ABREs including CE3 are proposed to be categorized into a single class of cis-acting elements.

Personalized genomic analyses for cancer mutation discovery and interpretation

PubMed Central

Jones, Siân; Anagnostou, Valsamo; Lytle, Karli; Parpart-Li, Sonya; Nesselbush, Monica; Riley, David R.; Shukla, Manish; Chesnick, Bryan; Kadan, Maura; Papp, Eniko; Galens, Kevin G.; Murphy, Derek; Zhang, Theresa; Kann, Lisa; Sausen, Mark; Angiuoli, Samuel V.; Diaz, Luis A.; Velculescu, Victor E.

2015-01-01

Massively parallel sequencing approaches are beginning to be used clinically to characterize individual patient tumors and to select therapies based on the identified mutations. A major question in these analyses is the extent to which these methods identify clinically actionable alterations and whether the examination of the tumor tissue alone is sufficient or whether matched normal DNA should also be analyzed to accurately identify tumor-specific (somatic) alterations. To address these issues, we comprehensively evaluated 815 tumor-normal paired samples from patients of 15 tumor types. We identified genomic alterations using next-generation sequencing of whole exomes or 111 targeted genes that were validated with sensitivities >95% and >99%, respectively, and specificities >99.99%. These analyses revealed an average of 140 and 4.3 somatic mutations per exome and targeted analysis, respectively. More than 75% of cases had somatic alterations in genes associated with known therapies or current clinical trials. Analyses of matched normal DNA identified germline alterations in cancer-predisposing genes in 3% of patients with apparently sporadic cancers. In contrast, a tumor-only sequencing approach could not definitively identify germline changes in cancer-predisposing genes and led to additional false-positive findings comprising 31% and 65% of alterations identified in targeted and exome analyses, respectively, including in potentially actionable genes. These data suggest that matched tumor-normal sequencing analyses are essential for precise identification and interpretation of somatic and germline alterations and have important implications for the diagnostic and therapeutic management of cancer patients. PMID:25877891

Mass spectrometry-based identification of allergens from Curvularia pallescens, a prevalent aerospore in India.

PubMed

Dey, Debarati; Saha, Bodhisattwa; Sircar, Gaurab; Ghosal, Kavita; Bhattacharya, Swati Gupta

2016-07-01

The worldwide prevalence of fungal allergy in recent years has augmented mining allergens from yet unexplored ones. Curvularia pallescens (CP) being a dominant aerospore in India and a major sensitiser on a wide range of allergic population, pose a serious threat to human health. Therefore, we aimed to identify novel allergens from CP in our present study. A cohort of 22 CP-sensitised patients was selected by positive Skin prick grade. Individual sera exhibited elevated specific IgE level and significant histamine release on a challenge with antigenic extract of CP. First gel-based profiling of CP proteome was done by 1- and 2-dimensional gel. Parallel 1- and 2-dimensional immunoblot were performed applying individual as well as pooled patient sera. Identification of the sero-reactive spots from the 2-dimensional gel was found to be challenging as CP was not previously sequenced. Hence, mass spectrometry-based proteomic workflow consisting of conventional database search was not alone sufficient. Therefore, de novo sequencing preceded homology search was implemented for further identification. Altogether 11 allergenic proteins including Brn-1, vacuolar protease, and fructose-bis-phosphate aldolase were identified with high statistical confidence (p<0.05). This is the first study to report on any allergens from CP. This kind of proteome-based analysis provided a catalogue of CP allergens that would lead an improved way of diagnosis and therapy of CP-related allergy. Copyright © 2016 Elsevier B.V. All rights reserved.

34 CFR 675.19 - Fiscal procedures and records.

Code of Federal Regulations, 2011 CFR

2011-07-01

... EDUCATION, DEPARTMENT OF EDUCATION FEDERAL WORK-STUDY PROGRAMS Federal Work-Study Program § 675.19 Fiscal... paid on an hourly basis, a time record showing the hours each student worked in clock time sequence, or the total hours worked per day; (ii) Include a payroll voucher containing sufficient information to...

34 CFR 675.19 - Fiscal procedures and records.

Code of Federal Regulations, 2010 CFR

2010-07-01

... EDUCATION, DEPARTMENT OF EDUCATION FEDERAL WORK-STUDY PROGRAMS Federal Work-Study Program § 675.19 Fiscal... paid on an hourly basis, a time record showing the hours each student worked in clock time sequence, or the total hours worked per day; (ii) Include a payroll voucher containing sufficient information to...

Comparative Analysis of Four Buckwheat Species Based on Morphology and Complete Chloroplast Genome Sequences.

PubMed

Wang, Cheng-Long; Ding, Meng-Qi; Zou, Chen-Yan; Zhu, Xue-Mei; Tang, Yu; Zhou, Mei-Liang; Shao, Ji-Rong

2017-07-26

Buckwheat is a nutritional and economically crop belonging to Polygonaceae, Fagopyrum. To better understand the mutation patterns and evolution trend in the chloroplast (cp) genome of buckwheat, and found sufficient number of variable regions to explore the phylogenetic relationships of this genus, two complete cp genomes of buckwheat including Fagopyrum dibotrys (F. dibotrys) and Fagopyrum luojishanense (F. luojishanense) were sequenced, and other two Fagopyrum cp genomes were used for comparative analysis. After morphological analysis, the main difference among these buckwheat were height, leaf shape, seeds and flower type. F. luojishanense was distinguishable from the cultivated species easily. Although the F. dibotrys and two cultivated species has some similarity, they different in habit and component contents. The cp genome of F. dibotrys was 159,320 bp while the F. luojishanense was 159,265 bp. 48 and 61 SSRs were found in F. dibotrys and F. luojishanense respectively. Meanwhile, 10 highly variable regions among these buckwheat species were located precisely. The phylogenetic relationships among four Fagopyrum species based on complete cp genomes was showed. The results suggested that F. dibotrys is more closely related to Fagopyrum tataricum. These data provided valuable genetic information for Fagopyrum species identification, taxonomy, phylogenetic study and molecular breeding.

The Linear Interaction Energy Method for the Prediction of Protein Stability Changes Upon Mutation

PubMed Central

Wickstrom, Lauren; Gallicchio, Emilio; Levy, Ronald M.

2011-01-01

The coupling of protein energetics and sequence changes is a critical aspect of computational protein design, as well as for the understanding of protein evolution, human disease, and drug resistance. In order to study the molecular basis for this coupling, computational tools must be sufficiently accurate and computationally inexpensive enough to handle large amounts of sequence data. We have developed a computational approach based on the linear interaction energy (LIE) approximation to predict the changes in the free energy of the native state induced by a single mutation. This approach was applied to a set of 822 mutations in 10 proteins which resulted in an average unsigned error of 0.82 kcal/mol and a correlation coefficient of 0.72 between the calculated and experimental ΔΔG values. The method is able to accurately identify destabilizing hot spot mutations however it has difficulty in distinguishing between stabilizing and destabilizing mutations due to the distribution of stability changes for the set of mutations used to parameterize the model. In addition, the model also performs quite well in initial tests on a small set of double mutations. Based on these promising results, we can begin to examine the relationship between protein stability and fitness, correlated mutations, and drug resistance. PMID:22038697

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

PubMed

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

2009-02-15

Protein function prediction is a central problem in bioinformatics, increasing in importance recently due to the rapid accumulation of biological data awaiting interpretation. Sequence data represents the bulk of this new stock and is the obvious target for consideration as input, as newly sequenced organisms often lack any other type of biological characterization. We have previously introduced PFP (Protein Function Prediction) as our sequence-based predictor of Gene Ontology (GO) functional terms. PFP interprets the results of a PSI-BLAST search by extracting and scoring individual functional attributes, searching a wide range of E-value sequence matches, and utilizing conventional data mining techniques to fill in missing information. We have shown it to be effective in predicting both specific and low-resolution functional attributes when sufficient data is unavailable. Here we describe (1) significant improvements to the PFP infrastructure, including the addition of prediction significance and confidence scores, (2) a thorough benchmark of performance and comparisons to other related prediction methods, and (3) applications of PFP predictions to genome-scale data. We applied PFP predictions to uncharacterized protein sequences from 15 organisms. Among these sequences, 60-90% could be annotated with a GO molecular function term at high confidence (>or=80%). We also applied our predictions to the protein-protein interaction network of the Malaria plasmodium (Plasmodium falciparum). High confidence GO biological process predictions (>or=90%) from PFP increased the number of fully enriched interactions in this dataset from 23% of interactions to 94%. Our benchmark comparison shows significant performance improvement of PFP relative to GOtcha, InterProScan, and PSI-BLAST predictions. This is consistent with the performance of PFP as the overall best predictor in both the AFP-SIG '05 and CASP7 function (FN) assessments. PFP is available as a web service at http://dragon.bio.purdue.edu/pfp/. (c) 2008 Wiley-Liss, Inc.

DEApp: an interactive web interface for differential expression analysis of next generation sequence data.

PubMed

Li, Yan; Andrade, Jorge

2017-01-01

A growing trend in the biomedical community is the use of Next Generation Sequencing (NGS) technologies in genomics research. The complexity of downstream differential expression (DE) analysis is however still challenging, as it requires sufficient computer programing and command-line knowledge. Furthermore, researchers often need to evaluate and visualize interactively the effect of using differential statistical and error models, assess the impact of selecting different parameters and cutoffs, and finally explore the overlapping consensus of cross-validated results obtained with different methods. This represents a bottleneck that slows down or impedes the adoption of NGS technologies in many labs. We developed DEApp, an interactive and dynamic web application for differential expression analysis of count based NGS data. This application enables models selection, parameter tuning, cross validation and visualization of results in a user-friendly interface. DEApp enables labs with no access to full time bioinformaticians to exploit the advantages of NGS applications in biomedical research. This application is freely available at https://yanli.shinyapps.io/DEAppand https://gallery.shinyapps.io/DEApp.

The heptanucleotide motif GAGACGC is a key component of a cis-acting promoter element that is critical for SnSAG1 expression in Sarcocystis neurona.

PubMed

Gaji, Rajshekhar Y; Howe, Daniel K

2009-07-01

The apicomplexan parasite Sarcocystis neurona undergoes a complex process of intracellular development, during which many genes are temporally regulated. The described study was undertaken to begin identifying the basic promoter elements that control gene expression in S. neurona. Sequence analysis of the 5'-flanking region of five S. neurona genes revealed a conserved heptanucleotide motif GAGACGC that is similar to the WGAGACG motif described upstream of multiple genes in Toxoplasma gondii. The promoter region for the major surface antigen gene SnSAG1, which contains three heptanucleotide motifs within 135 bases of the transcription start site, was dissected by functional analysis using a dual luciferase reporter assay. These analyses revealed that a minimal promoter fragment containing all three motifs was sufficient to drive reporter molecule expression, with the presence and orientation of the 5'-most heptanucleotide motif being absolutely critical for promoter function. Further studies should help to identify additional sequence elements important for promoter function and for controlling gene expression during intracellular development by this apicomplexan pathogen.

Expansion of inverted repeat does not decrease substitution rates in Pelargonium plastid genomes.

PubMed

Weng, Mao-Lun; Ruhlman, Tracey A; Jansen, Robert K

2017-04-01

For species with minor inverted repeat (IR) boundary changes in the plastid genome (plastome), nucleotide substitution rates were previously shown to be lower in the IR than the single copy regions (SC). However, the impact of large-scale IR expansion/contraction on plastid nucleotide substitution rates among closely related species remains unclear. We included plastomes from 22 Pelargonium species, including eight newly sequenced genomes, and used both pairwise and model-based comparisons to investigate the impact of the IR on sequence evolution in plastids. Ten types of plastome organization with different inversions or IR boundary changes were identified in Pelargonium. Inclusion in the IR was not sufficient to explain the variation of nucleotide substitution rates. Instead, the rate heterogeneity in Pelargonium plastomes was a mixture of locus-specific, lineage-specific and IR-dependent effects. Our study of Pelargonium plastomes that vary in IR length and gene content demonstrates that the evolutionary consequences of retaining these repeats are more complicated than previously suggested. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

View-invariant gait recognition method by three-dimensional convolutional neural network

NASA Astrophysics Data System (ADS)

Xing, Weiwei; Li, Ying; Zhang, Shunli

2018-01-01

Gait as an important biometric feature can identify a human at a long distance. View change is one of the most challenging factors for gait recognition. To address the cross view issues in gait recognition, we propose a view-invariant gait recognition method by three-dimensional (3-D) convolutional neural network. First, 3-D convolutional neural network (3DCNN) is introduced to learn view-invariant feature, which can capture the spatial information and temporal information simultaneously on normalized silhouette sequences. Second, a network training method based on cross-domain transfer learning is proposed to solve the problem of the limited gait training samples. We choose the C3D as the basic model, which is pretrained on the Sports-1M and then fine-tune C3D model to adapt gait recognition. In the recognition stage, we use the fine-tuned model to extract gait features and use Euclidean distance to measure the similarity of gait sequences. Sufficient experiments are carried out on the CASIA-B dataset and the experimental results demonstrate that our method outperforms many other methods.

A draft annotation and overview of the human genome

PubMed Central

Wright, Fred A; Lemon, William J; Zhao, Wei D; Sears, Russell; Zhuo, Degen; Wang, Jian-Ping; Yang, Hee-Yung; Baer, Troy; Stredney, Don; Spitzner, Joe; Stutz, Al; Krahe, Ralf; Yuan, Bo

2001-01-01

Background The recent draft assembly of the human genome provides a unified basis for describing genomic structure and function. The draft is sufficiently accurate to provide useful annotation, enabling direct observations of previously inferred biological phenomena. Results We report here a functionally annotated human gene index placed directly on the genome. The index is based on the integration of public transcript, protein, and mapping information, supplemented with computational prediction. We describe numerous global features of the genome and examine the relationship of various genetic maps with the assembly. In addition, initial sequence analysis reveals highly ordered chromosomal landscapes associated with paralogous gene clusters and distinct functional compartments. Finally, these annotation data were synthesized to produce observations of gene density and number that accord well with historical estimates. Such a global approach had previously been described only for chromosomes 21 and 22, which together account for 2.2% of the genome. Conclusions We estimate that the genome contains 65,000-75,000 transcriptional units, with exon sequences comprising 4%. The creation of a comprehensive gene index requires the synthesis of all available computational and experimental evidence. PMID:11516338

Limitations of the Mycobacterium tuberculosis reference genome H37Rv in the detection of virulence-related loci.

PubMed

O'Toole, Ronan F; Gautam, Sanjay S

2017-10-01

The genome sequence of Mycobacterium tuberculosis strain H37Rv is an important and valuable reference point in the study of M. tuberculosis phylogeny, molecular epidemiology, and drug-resistance mutations. However, it is becoming apparent that use of H37Rv as a sole reference genome in analysing clinical isolates presents some limitations to fully investigating M. tuberculosis virulence. Here, we examine the presence of single locus variants and the absence of entire genes in H37Rv with respect to strains that are responsible for cases and outbreaks of tuberculosis. We discuss how these polymorphisms may affect phenotypic properties of H37Rv including pathogenicity. Based on our observations and those of other researchers, we propose that use of a single reference genome, H37Rv, is not sufficient for the detection and characterisation of M. tuberculosis virulence-related loci. We recommend incorporation of genome sequences of other reference strains, in particular, direct clinical isolates, in such analyses in addition to H37Rv. Copyright © 2017 Elsevier Inc. All rights reserved.

Revisiting sample size: are big trials the answer?

PubMed

Lurati Buse, Giovanna A L; Botto, Fernando; Devereaux, P J

2012-07-18

The superiority of the evidence generated in randomized controlled trials over observational data is not only conditional to randomization. Randomized controlled trials require proper design and implementation to provide a reliable effect estimate. Adequate random sequence generation, allocation implementation, analyses based on the intention-to-treat principle, and sufficient power are crucial to the quality of a randomized controlled trial. Power, or the probability of the trial to detect a difference when a real difference between treatments exists, strongly depends on sample size. The quality of orthopaedic randomized controlled trials is frequently threatened by a limited sample size. This paper reviews basic concepts and pitfalls in sample-size estimation and focuses on the importance of large trials in the generation of valid evidence.

eSensor®: A Microarray Technology Based on Electrochemical Detection of Nucleic Acids and Its Application to Cystic Fibrosis Carrier Screening

NASA Astrophysics Data System (ADS)

Reed, Michael R.; Coty, William A.

We have developed a test for identification of carriers for cystic fibrosis using the eSensor® DNA detection technology. Oligonucleotide probes are deposited within self-assembled monolayers on gold electrodes arrayed upon printed circuit boards. These probes allow sequence-specific capture of amplicons containing a panel of mutation sites associated with cystic fibrosis. DNA targets are detected and mutations genotyped using a “sandwich” assay methodology employing electrochemical detection of ferrocene-labeled oligonucleotides for discrimination of carrier and non-carrier alleles. Performance of the cystic fibrosis application demonstrates sufficient accuracy and reliability for clinical diagnostic use, and the procedure can be performed by trained medical technologists available in the hospital laboratory.

Learning Extended Finite State Machines

NASA Technical Reports Server (NTRS)

Cassel, Sofia; Howar, Falk; Jonsson, Bengt; Steffen, Bernhard

2014-01-01

We present an active learning algorithm for inferring extended finite state machines (EFSM)s, combining data flow and control behavior. Key to our learning technique is a novel learning model based on so-called tree queries. The learning algorithm uses the tree queries to infer symbolic data constraints on parameters, e.g., sequence numbers, time stamps, identifiers, or even simple arithmetic. We describe sufficient conditions for the properties that the symbolic constraints provided by a tree query in general must have to be usable in our learning model. We have evaluated our algorithm in a black-box scenario, where tree queries are realized through (black-box) testing. Our case studies include connection establishment in TCP and a priority queue from the Java Class Library.

Atrx promotes heterochromatin formation at retrotransposons

PubMed Central

Sadic, Dennis; Schmidt, Katharina; Groh, Sophia; Kondofersky, Ivan; Ellwart, Joachim; Fuchs, Christiane; Theis, Fabian J; Schotta, Gunnar

2015-01-01

More than 50% of mammalian genomes consist of retrotransposon sequences. Silencing of retrotransposons by heterochromatin is essential to ensure genomic stability and transcriptional integrity. Here, we identified a short sequence element in intracisternal A particle (IAP) retrotransposons that is sufficient to trigger heterochromatin formation. We used this sequence in a genome-wide shRNA screen and identified the chromatin remodeler Atrx as a novel regulator of IAP silencing. Atrx binds to IAP elements and is necessary for efficient heterochromatin formation. In addition, Atrx facilitates a robust and largely inaccessible heterochromatin structure as Atrx knockout cells display increased chromatin accessibility at retrotransposons and non-repetitive heterochromatic loci. In summary, we demonstrate a direct role of Atrx in the establishment and robust maintenance of heterochromatin. PMID:26012739

A novel, privacy-preserving cryptographic approach for sharing sequencing data

PubMed Central

Cassa, Christopher A; Miller, Rachel A; Mandl, Kenneth D

2013-01-01

Objective DNA samples are often processed and sequenced in facilities external to the point of collection. These samples are routinely labeled with patient identifiers or pseudonyms, allowing for potential linkage to identity and private clinical information if intercepted during transmission. We present a cryptographic scheme to securely transmit externally generated sequence data which does not require any patient identifiers, public key infrastructure, or the transmission of passwords. Materials and methods This novel encryption scheme cryptographically protects participant sequence data using a shared secret key that is derived from a unique subset of an individual’s genetic sequence. This scheme requires access to a subset of an individual’s genetic sequence to acquire full access to the transmitted sequence data, which helps to prevent sample mismatch. Results We validate that the proposed encryption scheme is robust to sequencing errors, population uniqueness, and sibling disambiguation, and provides sufficient cryptographic key space. Discussion Access to a set of an individual’s genotypes and a mutually agreed cryptographic seed is needed to unlock the full sequence, which provides additional sample authentication and authorization security. We present modest fixed and marginal costs to implement this transmission architecture. Conclusions It is possible for genomics researchers who sequence participant samples externally to protect the transmission of sequence data using unique features of an individual’s genetic sequence. PMID:23125421

Handling the data management needs of high-throughput sequencing data: SpeedGene, a compression algorithm for the efficient storage of genetic data

PubMed Central

2012-01-01

Background As Next-Generation Sequencing data becomes available, existing hardware environments do not provide sufficient storage space and computational power to store and process the data due to their enormous size. This is and will be a frequent problem that is encountered everyday by researchers who are working on genetic data. There are some options available for compressing and storing such data, such as general-purpose compression software, PBAT/PLINK binary format, etc. However, these currently available methods either do not offer sufficient compression rates, or require a great amount of CPU time for decompression and loading every time the data is accessed. Results Here, we propose a novel and simple algorithm for storing such sequencing data. We show that, the compression factor of the algorithm ranges from 16 to several hundreds, which potentially allows SNP data of hundreds of Gigabytes to be stored in hundreds of Megabytes. We provide a C++ implementation of the algorithm, which supports direct loading and parallel loading of the compressed format without requiring extra time for decompression. By applying the algorithm to simulated and real datasets, we show that the algorithm gives greater compression rate than the commonly used compression methods, and the data-loading process takes less time. Also, The C++ library provides direct-data-retrieving functions, which allows the compressed information to be easily accessed by other C++ programs. Conclusions The SpeedGene algorithm enables the storage and the analysis of next generation sequencing data in current hardware environment, making system upgrades unnecessary. PMID:22591016

Efficient distinction of invasive aquatic plant species from non-invasive related species using DNA barcoding.

PubMed

Ghahramanzadeh, R; Esselink, G; Kodde, L P; Duistermaat, H; van Valkenburg, J L C H; Marashi, S H; Smulders, M J M; van de Wiel, C C M

2013-01-01

Biological invasions are regarded as threats to global biodiversity. Among invasive aliens, a number of plant species belonging to the genera Myriophyllum, Ludwigia and Cabomba, and to the Hydrocharitaceae family pose a particular ecological threat to water bodies. Therefore, one would try to prevent them from entering a country. However, many related species are commercially traded, and distinguishing invasive from non-invasive species based on morphology alone is often difficult for plants in a vegetative stage. In this regard, DNA barcoding could become a good alternative. In this study, 242 samples belonging to 26 species from 10 genera of aquatic plants were assessed using the chloroplast loci trnH-psbA, matK and rbcL. Despite testing a large number of primer sets and several PCR protocols, the matK locus could not be amplified or sequenced reliably and therefore was left out of the analysis. Using the other two loci, eight invasive species could be distinguished from their respective related species, a ninth one failed to produce sequences of sufficient quality. Based on the criteria of universal application, high sequence divergence and level of species discrimination, the trnH-psbA noncoding spacer was the best performing barcode in the aquatic plant species studied. Thus, DNA barcoding may be helpful with enforcing a ban on trade of such invasive species, such as is already in place in the Netherlands. This will become even more so once DNA barcoding would be turned into machinery routinely operable by a nonspecialist in botany and molecular genetics. © 2012 Blackwell Publishing Ltd.

Packaging of Mason-Pfizer monkey virus (MPMV) genomic RNA depends upon conserved long-range interactions (LRIs) between U5 and gag sequences

PubMed Central

Kalloush, Rawan M.; Vivet-Boudou, Valérie; Ali, Lizna M.; Mustafa, Farah; Marquet, Roland; Rizvi, Tahir A.

2016-01-01

MPMV has great potential for development as a vector for gene therapy. In this respect, precisely defining the sequences and structural motifs that are important for dimerization and packaging of its genomic RNA (gRNA) are of utmost importance. A distinguishing feature of the MPMV gRNA packaging signal is two phylogenetically conserved long-range interactions (LRIs) between U5 and gag complementary sequences, LRI-I and LRI-II. To test their biological significance in the MPMV life cycle, we introduced mutations into these structural motifs and tested their effects on MPMV gRNA packaging and propagation. Furthermore, we probed the structure of key mutants using SHAPE (selective 2′hydroxyl acylation analyzed by primer extension). Disrupting base-pairing of the LRIs affected gRNA packaging and propagation, demonstrating their significance to the MPMV life cycle. A double mutant restoring a heterologous LRI-I was fully functional, whereas a similar LRI-II mutant failed to restore gRNA packaging and propagation. These results demonstrate that while LRI-I acts at the structural level, maintaining base-pairing is not sufficient for LRI-II function. In addition, in vitro RNA dimerization assays indicated that the loss of RNA packaging in LRI mutants could not be attributed to the defects in dimerization. Our findings suggest that U5-gag LRIs play an important architectural role in maintaining the structure of the 5′ region of the MPMV gRNA, expanding the crucial role of LRIs to the nonlentiviral group of retroviruses. PMID:27095024

Cataloging the 1811-1812 New Madrid, central U.S., earthquake sequence

USGS Publications Warehouse

Hough, S.E.

2009-01-01

The three principal New Madrid, central U.S., mainshocks of 1811-1812 were followed by extensive aftershock sequences that included numerous felt events. Although no instrumental data are available for the sequence, historical accounts provide information that can be used to estimate magnitudes and locations for the large aftershocks as well as the mainshocks. Several detailed eyewitness accounts of the sequence provide sufficient information to identify times and rough magnitude estimates for a number of aftershocks that have not been analyzed previously. I also use three extended compilations of felt events to explore the overall sequence productivity. Although one generally cannot estimate magnitudes or locations for individual events, the intensity distributions of recent, instrumentally recorded earthquakes in the region provide a basis for estimation of the magnitude distribution of 1811-1812 aftershocks. The distribution is consistent with a b-value distribution. I estimate Mw 6-6.3 for the three largest identifiable aftershocks, apart from the so-called dawn aftershock on 16 December 1811.

Nanoliter reactors improve multiple displacement amplification of genomes from single cells.

PubMed

Marcy, Yann; Ishoey, Thomas; Lasken, Roger S; Stockwell, Timothy B; Walenz, Brian P; Halpern, Aaron L; Beeson, Karen Y; Goldberg, Susanne M D; Quake, Stephen R

2007-09-01

Since only a small fraction of environmental bacteria are amenable to laboratory culture, there is great interest in genomic sequencing directly from single cells. Sufficient DNA for sequencing can be obtained from one cell by the Multiple Displacement Amplification (MDA) method, thereby eliminating the need to develop culture methods. Here we used a microfluidic device to isolate individual Escherichia coli and amplify genomic DNA by MDA in 60-nl reactions. Our results confirm a report that reduced MDA reaction volume lowers nonspecific synthesis that can result from contaminant DNA templates and unfavourable interaction between primers. The quality of the genome amplification was assessed by qPCR and compared favourably to single-cell amplifications performed in standard 50-microl volumes. Amplification bias was greatly reduced in nanoliter volumes, thereby providing a more even representation of all sequences. Single-cell amplicons from both microliter and nanoliter volumes provided high-quality sequence data by high-throughput pyrosequencing, thereby demonstrating a straightforward route to sequencing genomes from single cells.

Development and evaluation of clicker methodology for introductory physics courses

NASA Astrophysics Data System (ADS)

Lee, Albert H.

Many educators understand that lectures are cost effective but not learning efficient, so continue to search for ways to increase active student participation in this traditionally passive learning environment. In-class polling systems, or "clickers", are inexpensive and reliable tools allowing students to actively participate in lectures by answering multiple-choice questions. Students assess their learning in real time by observing instant polling summaries displayed in front of them. This in turn motivates additional discussions which increase the opportunity for active learning. We wanted to develop a comprehensive clicker methodology that creates an active lecture environment for a broad spectrum of students taking introductory physics courses. We wanted our methodology to incorporate many findings of contemporary learning science. It is recognized that learning requires active construction; students need to be actively involved in their own learning process. Learning also depends on preexisting knowledge; students construct new knowledge and understandings based on what they already know and believe. Learning is context dependent; students who have learned to apply a concept in one context may not be able to recognize and apply the same concept in a different context, even when both contexts are considered to be isomorphic by experts. On this basis, we developed question sequences, each involving the same concept but having different contexts. Answer choices are designed to address students preexisting knowledge. These sequences are used with the clickers to promote active discussions and multiple assessments. We have created, validated, and evaluated sequences sufficient in number to populate all of introductory physics courses. Our research has found that using clickers with our question sequences significantly improved student conceptual understanding. Our research has also found how to best measure student conceptual gain using research-based instruments. Finally, we discovered that students need to have full access to the question sequences after lectures to reap the maximum benefit. Chapter 1 provides an introduction to our research. Chapter 2 provides a literature review relevant for our research. Chapter 3 discusses the creation of the clicker question sequences. Chapter 4 provides a picture of the validation process involving both physics experts and the introductory physics students. Chapter 5 describes how the sequences have been used with clickers in lectures. Chapter 6 provides the evaluation of the effectiveness of the clicker methodology. Chapter 7 contains a brief summary of research results and conclusions.

Ground-based detectability of terrestrial and Jovian extrasolar planets: observations of CM Draconis at Lick Observatory.

PubMed

Doyle, L R; Dunham, E T; Deeg, H J; Blue, J E; Jenkins, J M

1996-06-25

The detection of terrestrial-sized extrasolar planets from the ground has been thought to be virtually impossible due to atmospheric scintillation limits. However, we show that this is not the case especially selected (but nevertheless main sequence) stars, namely small eclipsing binaries. For the smallest of these systems, CM Draconis, several months to a few years of photometric observations with 1-m-class telescopes will be sufficient to detect the transits of any short-period planets of sizes > or = 1.5 Earth radii (RE), using cross-correlation analysis with moderately good photometry. Somewhat larger telescopes will be needed to extend this detectability to terrestrial planets in larger eclipsing binary systems. (We arbitrarily define "terrestrial planets" herein as those whose disc areas are closer to that of Earth's than Neptune's i.e., less than about 2.78 RE.) As a "spin-off" of such observations, we will also be able to detect the presence of Jovian-mass planets without transits using the timing of the eclipse minima. Eclipse minima will drift in time as the binary system is offset by a sufficiently massive planet (i.e., one Jupiter mass) about the binary/giant-planet barycenter, causing a periodic variation in the light travel time to the observer. We present here an outline of present observations taking place at the University of California Lick Observatory using the Crossley 0.9-m telescope in collaboration with other observatories (in South Korea, Crete, France, Canary Islands, and New York) to detect or constrain the existence of terrestrial planets around main sequence eclipsing binary star systems, starting with CM Draconis. We demonstrate the applicability of photometric data to the general detection of gas giant planets via eclipse minima timings in many other small-mass eclipsing binary systems as well.

Ground-based detectability of terrestrial and Jovian extrasolar planets: observations of CM Draconis at Lick Observatory

NASA Technical Reports Server (NTRS)

Doyle, L. R.; Dunham, E. T.; Deeg, H. J.; Blue, J. E.; Jenkins, J. M.

1996-01-01

The detection of terrestrial-sized extrasolar planets from the ground has been thought to be virtually impossible due to atmospheric scintillation limits. However, we show that this is not the case especially selected (but nevertheless main sequence) stars, namely small eclipsing binaries. For the smallest of these systems, CM Draconis, several months to a few years of photometric observations with 1-m-class telescopes will be sufficient to detect the transits of any short-period planets of sizes > or = 1.5 Earth radii (RE), using cross-correlation analysis with moderately good photometry. Somewhat larger telescopes will be needed to extend this detectability to terrestrial planets in larger eclipsing binary systems. (We arbitrarily define "terrestrial planets" herein as those whose disc areas are closer to that of Earth's than Neptune's i.e., less than about 2.78 RE.) As a "spin-off" of such observations, we will also be able to detect the presence of Jovian-mass planets without transits using the timing of the eclipse minima. Eclipse minima will drift in time as the binary system is offset by a sufficiently massive planet (i.e., one Jupiter mass) about the binary/giant-planet barycenter, causing a periodic variation in the light travel time to the observer. We present here an outline of present observations taking place at the University of California Lick Observatory using the Crossley 0.9-m telescope in collaboration with other observatories (in South Korea, Crete, France, Canary Islands, and New York) to detect or constrain the existence of terrestrial planets around main sequence eclipsing binary star systems, starting with CM Draconis. We demonstrate the applicability of photometric data to the general detection of gas giant planets via eclipse minima timings in many other small-mass eclipsing binary systems as well.

Species detection and identification in sexual organisms using population genetic theory and DNA sequences.

PubMed

Birky, C William

2013-01-01

Phylogenetic trees of DNA sequences of a group of specimens may include clades of two kinds: those produced by stochastic processes (random genetic drift) within a species, and clades that represent different species. The ratio of the mean pairwise sequence difference between a pair of clades (K) to the mean pairwise sequence difference within a clade (θ) can be used to determine whether the clades are samples from different species (K/θ ≥ 4) or the same species (K/θ<4) with probability ≥ 0.95. Previously I applied this criterion to delimit species of asexual organisms. Here I use data from the literature to show how it can also be applied to delimit sexual species using four groups of sexual organisms as examples: ravens, spotted leopards, sea butterflies, and liverworts. Mitochondrial or chloroplast genes are used because these segregate earlier during speciation than most nuclear genes and hence detect earlier stages of speciation. In several cases the K/θ ratio was greater than 4, confirming the original authors' intuition that the clades were sufficiently different to be assigned to different species. But the K/θ ratio split each of two liverwort species into two evolutionary species, and showed that support for the distinction between the common and Chihuahuan raven species is weak. I also discuss some possible sources of error in using the K/θ ratio; the most significant one would be cases where males migrate between different populations but females do not, making the use of maternally inherited organelle genes problematic. The K/θ ratio must be used with some caution, like all other methods for species delimitation. Nevertheless, it is a simple theory-based quantitative method for using DNA sequences to make rigorous decisions about species delimitation in sexual as well as asexual eukaryotes.

A Poisson hierarchical modelling approach to detecting copy number variation in sequence coverage data.

PubMed

Sepúlveda, Nuno; Campino, Susana G; Assefa, Samuel A; Sutherland, Colin J; Pain, Arnab; Clark, Taane G

2013-02-26

The advent of next generation sequencing technology has accelerated efforts to map and catalogue copy number variation (CNV) in genomes of important micro-organisms for public health. A typical analysis of the sequence data involves mapping reads onto a reference genome, calculating the respective coverage, and detecting regions with too-low or too-high coverage (deletions and amplifications, respectively). Current CNV detection methods rely on statistical assumptions (e.g., a Poisson model) that may not hold in general, or require fine-tuning the underlying algorithms to detect known hits. We propose a new CNV detection methodology based on two Poisson hierarchical models, the Poisson-Gamma and Poisson-Lognormal, with the advantage of being sufficiently flexible to describe different data patterns, whilst robust against deviations from the often assumed Poisson model. Using sequence coverage data of 7 Plasmodium falciparum malaria genomes (3D7 reference strain, HB3, DD2, 7G8, GB4, OX005, and OX006), we showed that empirical coverage distributions are intrinsically asymmetric and overdispersed in relation to the Poisson model. We also demonstrated a low baseline false positive rate for the proposed methodology using 3D7 resequencing data and simulation. When applied to the non-reference isolate data, our approach detected known CNV hits, including an amplification of the PfMDR1 locus in DD2 and a large deletion in the CLAG3.2 gene in GB4, and putative novel CNV regions. When compared to the recently available FREEC and cn.MOPS approaches, our findings were more concordant with putative hits from the highest quality array data for the 7G8 and GB4 isolates. In summary, the proposed methodology brings an increase in flexibility, robustness, accuracy and statistical rigour to CNV detection using sequence coverage data.

Phase 2 of the array automated assembly task for the low cost solar array project

NASA Technical Reports Server (NTRS)

Campbell, R. B.; Davis, J. R.; Ostroski, J. W.; Rai-Choudhury, P.; Rohatgi, A.; Seman, E. J.; Stapleton, R. E.

1979-01-01

The process sequence for the fabrication of dendritic web silicon into solar panels was modified to include aluminum back surface field formation. Plasma etching was found to be a feasible technique for pre-diffusion cleaning of the web. Several contacting systems were studied. The total plated Pd-Ni system was not compatible with the process sequence; however, the evaporated TiPd-electroplated Cu system was shown stable under life testing. Ultrasonic bonding parameters were determined for various interconnect and contact metals but the yield of the process was not sufficiently high to use for module fabrication at this time. Over 400 solar cells were fabricated according to the modified sequence. No sub-process incompatibility was seen. These cells were used to fabricate four demonstration modules. A cost analysis of the modified process sequence resulted in a selling price of $0.75/peak watt.

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

NASA Astrophysics Data System (ADS)

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

2018-07-01

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

Design of nucleic acid strands with long low-barrier folding pathways.

PubMed

Condon, Anne; Kirkpatrick, Bonnie; Maňuch, Ján

2017-01-01

A major goal of natural computing is to design biomolecules, such as nucleic acid sequences, that can be used to perform computations. We design sequences of nucleic acids that are "guaranteed" to have long folding pathways relative to their length. This particular sequences with high probability follow low-barrier folding pathways that visit a large number of distinct structures. Long folding pathways are interesting, because they demonstrate that natural computing can potentially support long and complex computations. Formally, we provide the first scalable designs of molecules whose low-barrier folding pathways, with respect to a simple, stacked pair energy model, grow superlinearly with the molecule length, but for which all significantly shorter alternative folding pathways have an energy barrier that is [Formula: see text] times that of the low-barrier pathway for any [Formula: see text] and a sufficiently long sequence.

Top-down analysis of protein samples by de novo sequencing techniques

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vyatkina, Kira; Wu, Si; Dekker, Lennard J. M.

MOTIVATION: Recent technological advances have made high-resolution mass spectrometers affordable to many laboratories, thus boosting rapid development of top-down mass spectrometry, and implying a need in efficient methods for analyzing this kind of data. RESULTS: We describe a method for analysis of protein samples from top-down tandem mass spectrometry data, which capitalizes on de novo sequencing of fragments of the proteins present in the sample. Our algorithm takes as input a set of de novo amino acid strings derived from the given mass spectra using the recently proposed Twister approach, and combines them into aggregated strings endowed with offsets. Themore » former typically constitute accurate sequence fragments of sufficiently well-represented proteins from the sample being analyzed, while the latter indicate their location in the protein sequence, and also bear information on post-translational modifications and fragmentation patterns.« less

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

NASA Astrophysics Data System (ADS)

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

2017-12-01

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

Non-rigid Motion Correction in 3D Using Autofocusing with Localized Linear Translations

PubMed Central

Cheng, Joseph Y.; Alley, Marcus T.; Cunningham, Charles H.; Vasanawala, Shreyas S.; Pauly, John M.; Lustig, Michael

2012-01-01

MR scans are sensitive to motion effects due to the scan duration. To properly suppress artifacts from non-rigid body motion, complex models with elements such as translation, rotation, shear, and scaling have been incorporated into the reconstruction pipeline. However, these techniques are computationally intensive and difficult to implement for online reconstruction. On a sufficiently small spatial scale, the different types of motion can be well-approximated as simple linear translations. This formulation allows for a practical autofocusing algorithm that locally minimizes a given motion metric – more specifically, the proposed localized gradient-entropy metric. To reduce the vast search space for an optimal solution, possible motion paths are limited to the motion measured from multi-channel navigator data. The novel navigation strategy is based on the so-called “Butterfly” navigators which are modifications to the spin-warp sequence that provide intrinsic translational motion information with negligible overhead. With a 32-channel abdominal coil, sufficient number of motion measurements were found to approximate possible linear motion paths for every image voxel. The correction scheme was applied to free-breathing abdominal patient studies. In these scans, a reduction in artifacts from complex, non-rigid motion was observed. PMID:22307933

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

PubMed

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

2017-11-06

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

Introducing a model of pairing based on base pair specific interactions between identical DNA sequences

NASA Astrophysics Data System (ADS)

(O' Lee, Dominic J.

2018-02-01

At present, there have been suggested two types of physical mechanism that may facilitate preferential pairing between DNA molecules, with identical or similar base pair texts, without separation of base pairs. One mechanism solely relies on base pair specific patterns of helix distortion being the same on the two molecules, discussed extensively in the past. The other mechanism proposes that there are preferential interactions between base pairs of the same composition. We introduce a model, built on this second mechanism, where both thermal stretching and twisting fluctuations are included, as well as the base pair specific helix distortions. Firstly, we consider an approximation for weak pairing interactions, or short molecules. This yields a dependence of the energy on the square root of the molecular length, which could explain recent experimental data. However, analysis suggests that this approximation is no longer valid at large DNA lengths. In a second approximation, for long molecules, we define two adaptation lengths for twisting and stretching, over which the pairing interaction can limit the accumulation of helix disorder. When the pairing interaction is sufficiently strong, both adaptation lengths are finite; however, as we reduce pairing strength, the stretching adaptation length remains finite but the torsional one becomes infinite. This second state persists to arbitrarily weak values of the pairing strength; suggesting that, if the molecules are long enough, the pairing energy scales as length. To probe differences between the two pairing mechanisms, we also construct a model of similar form. However, now, pairing between identical sequences solely relies on the intrinsic helix distortion patterns. Between the two models, we see interesting qualitative differences. We discuss our findings, and suggest new work to distinguish between the two mechanisms.

29 CFR 1926.752 - Site layout, site-specific erection plan and construction sequence.

Code of Federal Regulations, 2011 CFR

2011-07-01

... standard test method of field-cured samples, either 75 percent of the intended minimum compressive design... the basis of an appropriate ASTM standard test method of field-cured samples, either 75 percent of the intended minimum compressive design strength or sufficient strength to support the loads imposed during...

29 CFR 1926.752 - Site layout, site-specific erection plan and construction sequence.

Code of Federal Regulations, 2013 CFR

2013-07-01

... standard test method of field-cured samples, either 75 percent of the intended minimum compressive design... the basis of an appropriate ASTM standard test method of field-cured samples, either 75 percent of the intended minimum compressive design strength or sufficient strength to support the loads imposed during...

29 CFR 1926.752 - Site layout, site-specific erection plan and construction sequence.

Code of Federal Regulations, 2012 CFR

2012-07-01

... standard test method of field-cured samples, either 75 percent of the intended minimum compressive design... the basis of an appropriate ASTM standard test method of field-cured samples, either 75 percent of the intended minimum compressive design strength or sufficient strength to support the loads imposed during...

29 CFR 1926.752 - Site layout, site-specific erection plan and construction sequence.

Code of Federal Regulations, 2010 CFR

2010-07-01

... standard test method of field-cured samples, either 75 percent of the intended minimum compressive design... the basis of an appropriate ASTM standard test method of field-cured samples, either 75 percent of the intended minimum compressive design strength or sufficient strength to support the loads imposed during...

29 CFR 1926.752 - Site layout, site-specific erection plan and construction sequence.

Code of Federal Regulations, 2014 CFR

2014-07-01

... standard test method of field-cured samples, either 75 percent of the intended minimum compressive design... the basis of an appropriate ASTM standard test method of field-cured samples, either 75 percent of the intended minimum compressive design strength or sufficient strength to support the loads imposed during...

Comprehensive Genome-wide Screen for Genes with Cis-acting Regulatory Elements That Respond to Marek's Disease Virus Infection

USDA-ARS?s Scientific Manuscript database

The comprehensive identification of genes underlying phenotypic variation of complex traits such as disease resistance remains one of the greatest challenges in biology despite having genome sequences and more powerful tools. Most genome-wide screens lack sufficient resolving power as they typically...

Pride, Pity, Anger, Guilt: Thought-Affect Sequences in the Classroom.

ERIC Educational Resources Information Center

Weiner, Bernard

A set of prevalent emotions, including pity, anger, guilt, pride (self-esteem), gratitude, and resignation, shares a common characteristic, i.e., causal attributions appear to be sufficient antecedents for their elicitation. Research in the field of emotions has shown that the underlying properties or dimensions of attributions are the significant…

Two proximal activating protein-1-binding sites are sufficient to stimulate transcription of the ovine follicle-stimulating hormone-beta gene

EPA Science Inventory

FSH is an important regulator of mammalian gametogenesis and the female reproductive cycle. Although little is known about the transcriptional regulation of the beta-subunit (the rate-limiting subunit of FSH synthesis), sequence analysis of the ovine FSHbeta promoter has revealed...

Inflammation Thread Runs across Medical Laboratory Specialities.

PubMed

Nydegger, Urs; Lung, Thomas; Risch, Lorenz; Risch, Martin; Medina Escobar, Pedro; Bodmer, Thomas

2016-01-01

We work on the assumption that four major specialities or sectors of medical laboratory assays, comprising clinical chemistry, haematology, immunology, and microbiology, embraced by genome sequencing techniques, are routinely in use. Medical laboratory markers for inflammation serve as model: they are allotted to most fields of medical lab assays including genomics. Incessant coding of assays aligns each of them in the long lists of big data. As exemplified with the complement gene family, containing C2, C3, C8A, C8B, CFH, CFI, and ITGB2, heritability patterns/risk factors associated with diseases with genetic glitch of complement components are unfolding. The C4 component serum levels depend on sufficient vitamin D whilst low vitamin D is inversely related to IgG1, IgA, and C3 linking vitamin sufficiency to innate immunity. Whole genome sequencing of microbial organisms may distinguish virulent from nonvirulent and antibiotic resistant from nonresistant varieties of the same species and thus can be listed in personal big data banks including microbiological pathology; the big data warehouse continues to grow.

Inflammation Thread Runs across Medical Laboratory Specialities

PubMed Central

Lung, Thomas; Risch, Lorenz; Risch, Martin; Medina Escobar, Pedro; Bodmer, Thomas

2016-01-01

We work on the assumption that four major specialities or sectors of medical laboratory assays, comprising clinical chemistry, haematology, immunology, and microbiology, embraced by genome sequencing techniques, are routinely in use. Medical laboratory markers for inflammation serve as model: they are allotted to most fields of medical lab assays including genomics. Incessant coding of assays aligns each of them in the long lists of big data. As exemplified with the complement gene family, containing C2, C3, C8A, C8B, CFH, CFI, and ITGB2, heritability patterns/risk factors associated with diseases with genetic glitch of complement components are unfolding. The C4 component serum levels depend on sufficient vitamin D whilst low vitamin D is inversely related to IgG1, IgA, and C3 linking vitamin sufficiency to innate immunity. Whole genome sequencing of microbial organisms may distinguish virulent from nonvirulent and antibiotic resistant from nonresistant varieties of the same species and thus can be listed in personal big data banks including microbiological pathology; the big data warehouse continues to grow. PMID:27493451

Phylogeny of Lagos bat virus: challenges for lyssavirus taxonomy.

PubMed

Markotter, W; Kuzmin, I; Rupprecht, C E; Nel, L H

2008-07-01

Lagos bat virus (LBV) belongs to genotype 2 of the Lyssavirus genus. The complete nucleoprotein (N), phosphoprotein (P), matrixprotein (M) and glycoprotein (G) genes of 13 LBV isolates were sequenced and phylogenetically compared with other lyssavirus representatives. The results identified three different lineages of LBV. One of these lineages demonstrated sufficient sequence diversity to be considered a new lyssavirus genotype (Dakar bat lyssavirus). The suggested quantitative separation of lyssavirus genotypes using the N, P, M and G genes was also investigated using P-distances matrixes. Results indicated that the current criteria should be revised since overlaps between intergenotypic and intragenotypic variation occur.

Origins of the protein synthesis cycle

NASA Technical Reports Server (NTRS)

Fox, S. W.

1981-01-01

Largely derived from experiments in molecular evolution, a theory of protein synthesis cycles has been constructed. The sequence begins with ordered thermal proteins resulting from the self-sequencing of mixed amino acids. Ordered thermal proteins then aggregate to cell-like structures. When they contained proteinoids sufficiently rich in lysine, the structures were able to synthesize offspring peptides. Since lysine-rich proteinoid (LRP) also catalyzes the polymerization of nucleoside triphosphate to polynucleotides, the same microspheres containing LRP could have synthesized both original cellular proteins and cellular nucleic acids. The LRP within protocells would have provided proximity advantageous for the origin and evolution of the genetic code.

Full genome virus detection in fecal samples using sensitive nucleic acid preparation, deep sequencing, and a novel iterative sequence classification algorithm.

PubMed

Cotten, Matthew; Oude Munnink, Bas; Canuti, Marta; Deijs, Martin; Watson, Simon J; Kellam, Paul; van der Hoek, Lia

2014-01-01

We have developed a full genome virus detection process that combines sensitive nucleic acid preparation optimised for virus identification in fecal material with Illumina MiSeq sequencing and a novel post-sequencing virus identification algorithm. Enriched viral nucleic acid was converted to double-stranded DNA and subjected to Illumina MiSeq sequencing. The resulting short reads were processed with a novel iterative Python algorithm SLIM for the identification of sequences with homology to known viruses. De novo assembly was then used to generate full viral genomes. The sensitivity of this process was demonstrated with a set of fecal samples from HIV-1 infected patients. A quantitative assessment of the mammalian, plant, and bacterial virus content of this compartment was generated and the deep sequencing data were sufficient to assembly 12 complete viral genomes from 6 virus families. The method detected high levels of enteropathic viruses that are normally controlled in healthy adults, but may be involved in the pathogenesis of HIV-1 infection and will provide a powerful tool for virus detection and for analyzing changes in the fecal virome associated with HIV-1 progression and pathogenesis.

Full Genome Virus Detection in Fecal Samples Using Sensitive Nucleic Acid Preparation, Deep Sequencing, and a Novel Iterative Sequence Classification Algorithm

PubMed Central

Cotten, Matthew; Oude Munnink, Bas; Canuti, Marta; Deijs, Martin; Watson, Simon J.; Kellam, Paul; van der Hoek, Lia

2014-01-01

We have developed a full genome virus detection process that combines sensitive nucleic acid preparation optimised for virus identification in fecal material with Illumina MiSeq sequencing and a novel post-sequencing virus identification algorithm. Enriched viral nucleic acid was converted to double-stranded DNA and subjected to Illumina MiSeq sequencing. The resulting short reads were processed with a novel iterative Python algorithm SLIM for the identification of sequences with homology to known viruses. De novo assembly was then used to generate full viral genomes. The sensitivity of this process was demonstrated with a set of fecal samples from HIV-1 infected patients. A quantitative assessment of the mammalian, plant, and bacterial virus content of this compartment was generated and the deep sequencing data were sufficient to assembly 12 complete viral genomes from 6 virus families. The method detected high levels of enteropathic viruses that are normally controlled in healthy adults, but may be involved in the pathogenesis of HIV-1 infection and will provide a powerful tool for virus detection and for analyzing changes in the fecal virome associated with HIV-1 progression and pathogenesis. PMID:24695106

Terminal sequence importance of de novo proteins from binary-patterned library: stable artificial proteins with 11- or 12-amino acid alphabet.

PubMed

Okura, Hiromichi; Takahashi, Tsuyoshi; Mihara, Hisakazu

2012-06-01

Successful approaches of de novo protein design suggest a great potential to create novel structural folds and to understand natural rules of protein folding. For these purposes, smaller and simpler de novo proteins have been developed. Here, we constructed smaller proteins by removing the terminal sequences from stable de novo vTAJ proteins and compared stabilities between mutant and original proteins. vTAJ proteins were screened from an α3β3 binary-patterned library which was designed with polar/ nonpolar periodicities of α-helix and β-sheet. vTAJ proteins have the additional terminal sequences due to the method of constructing the genetically repeated library sequences. By removing the parts of the sequences, we successfully obtained the stable smaller de novo protein mutants with fewer amino acid alphabets than the originals. However, these mutants showed the differences on ANS binding properties and stabilities against denaturant and pH change. The terminal sequences, which were designed just as flexible linkers not as secondary structure units, sufficiently affected these physicochemical details. This study showed implications for adjusting protein stabilities by designing N- and C-terminal sequences.

Polyomavirus BK non-coding control region rearrangements in health and disease.

PubMed

Sharma, Preety M; Gupta, Gaurav; Vats, Abhay; Shapiro, Ron; Randhawa, Parmjeet S

2007-08-01

BK virus is an increasingly recognized pathogen in transplanted patients. DNA sequencing of this virus shows considerable genomic variability. To understand the clinical significance of rearrangements in the non-coding control region (NCCR) of BK virus (BKV), we report a meta-analysis of 507 sequences, including 40 sequences generated in our own laboratory, for associations between rearrangements and disease, tissue tropism, geographic origin, and viral genotype. NCCR rearrangements were less frequent in (a) asymptomatic BKV viruria compared to patients viral nephropathy (1.7% vs. 22.5%), and (b) viral genotype 1 compared to other genotypes (2.4% vs. 11.2%). Rearrangements were commoner in malignancy (78.6%), and Norwegians (45.7%), and less common in East Indians (0%), and Japanese (4.3%). A surprising number of rearranged sequences were reported from mononuclear cells of healthy subjects, whereas most plasma sequences were archetypal. This difference could not be related to potential recombinase activity in lymphocytes, as consensus recombination signal sequences could not be found in the NCCR region. NCCR rearrangements are neither required nor a sufficient condition to produce clinical disease. BKV nephropathy and hemorrhagic cystitis are not associated with any unique NCCR configuration or nucleotide sequence.

A single session of prefrontal cortex transcranial direct current stimulation does not modulate implicit task sequence learning and consolidation.

PubMed

Savic, Branislav; Müri, René; Meier, Beat

Transcranial direct current stimulation (tDCS) is assumed to affect cortical excitability and dependent on the specific stimulation conditions either to increase or decrease learning. The purpose of this study was to modulate implicit task sequence learning with tDCS. As cortico-striatal loops are critically involved in implicit task sequence learning, tDCS was applied above the dorsolateral prefrontal cortex (DLPFC). In Experiment 1, anodal, cathodal, or sham tDCS was applied before the start of the sequence learning task. In Experiment 2, stimulation was applied during the sequence learning task. Consolidation of learning was assessed after 24 h. The results of both experiments showed that implicit task sequence learning occurred consistently but it was not modulated by different tDCS conditions. Similarly, consolidation measured after a 24 h-interval including sleep was also not affected by stimulation. These results indicate that a single session of DLPFC tDCS is not sufficient to modulate implicit task sequence learning. This study adds to the accumulating evidence that tDCS may not be as effective as originally thought. Copyright © 2017 Elsevier Inc. All rights reserved.

The illusion of specific capture: surface and solution studies of suboptimal oligonucleotide hybridization

PubMed Central

2013-01-01

Background Hybridization based assays and capture systems depend on the specificity of hybridization between a probe and its intended target. A common guideline in the construction of DNA microarrays, for instance, is that avoiding complementary stretches of more than 15 nucleic acids in a 50 or 60-mer probe will eliminate sequence specific cross-hybridization reactions. Here we present a study of the behavior of partially matched oligonucleotide pairs with complementary stretches starting well below this threshold complementarity length – in silico, in solution, and at the microarray surface. The modeled behavior of pairs of oligonucleotide probes and their targets suggests that even a complementary stretch of sequence 12 nt in length would give rise to specific cross-hybridization. We designed a set of binding partners to a 50-mer oligonucleotide containing complementary stretches from 6 nt to 21 nt in length. Results Solution melting experiments demonstrate that stable partial duplexes can form when only 12 bp of complementary sequence are present; surface hybridization experiments confirm that a signal close in magnitude to full-strength signal can be obtained from hybridization of a 12 bp duplex within a 50mer oligonucleotide. Conclusions Microarray and other molecular capture strategies that rely on a 15 nt lower complementarity bound for eliminating specific cross-hybridization may not be sufficiently conservative. PMID:23445545

Peptide vaccine against canine parvovirus: identification of two neutralization subsites in the N terminus of VP2 and optimization of the amino acid sequence.

PubMed Central

Casal, J I; Langeveld, J P; Cortés, E; Schaaper, W W; van Dijk, E; Vela, C; Kamstrup, S; Meloen, R H

1995-01-01

The N-terminal domain of the major capsid protein VP2 of canine parvovirus was shown to be an excellent target for development of a synthetic peptide vaccine, but detailed information about number of epitopes, optimal length, sequence choice, and site of coupling to the carrier protein was lacking. Therefore, several overlapping peptides based on this N terminus were synthesized to establish conditions for optimal and reproducible induction of neutralizing antibodies in rabbits. The specificity and neutralizing ability of the antibody response for these peptides were determined. Within the N-terminal 23 residues of VP2, two subsites able to induce neutralizing antibodies and which overlapped by only two glycine residues at positions 10 and 11 could be discriminated. The shortest sequence sufficient for neutralization induction was nine residues. Peptides longer than 13 residues consistently induced neutralization, provided that their N termini were located between positions 1 and 11 of VP2. The orientation of the peptides at the carrier protein was also of importance, being more effective when coupled through the N terminus than through the C terminus to keyhole limpet hemocyanin. The results suggest that the presence of amino acid residues 2 to 21 (and probably 3 to 17) of VP2 in a single peptide is preferable for a synthetic peptide vaccine. PMID:7474152

Peptide vaccine against canine parvovirus: identification of two neutralization subsites in the N terminus of VP2 and optimization of the amino acid sequence.

PubMed

Casal, J I; Langeveld, J P; Cortés, E; Schaaper, W W; van Dijk, E; Vela, C; Kamstrup, S; Meloen, R H

1995-11-01

The N-terminal domain of the major capsid protein VP2 of canine parvovirus was shown to be an excellent target for development of a synthetic peptide vaccine, but detailed information about number of epitopes, optimal length, sequence choice, and site of coupling to the carrier protein was lacking. Therefore, several overlapping peptides based on this N terminus were synthesized to establish conditions for optimal and reproducible induction of neutralizing antibodies in rabbits. The specificity and neutralizing ability of the antibody response for these peptides were determined. Within the N-terminal 23 residues of VP2, two subsites able to induce neutralizing antibodies and which overlapped by only two glycine residues at positions 10 and 11 could be discriminated. The shortest sequence sufficient for neutralization induction was nine residues. Peptides longer than 13 residues consistently induced neutralization, provided that their N termini were located between positions 1 and 11 of VP2. The orientation of the peptides at the carrier protein was also of importance, being more effective when coupled through the N terminus than through the C terminus to keyhole limpet hemocyanin. The results suggest that the presence of amino acid residues 2 to 21 (and probably 3 to 17) of VP2 in a single peptide is preferable for a synthetic peptide vaccine.

PCR Assays for Identification of Coccidioides posadasii Based on the Nucleotide Sequence of the Antigen 2/Proline-Rich Antigen

PubMed Central

Bialek, Ralf; Kern, Jan; Herrmann, Tanja; Tijerina, Rolando; Ceceñas, Luis; Reischl, Udo; González, Gloria M.

2004-01-01

A conventional nested PCR and a real-time LightCycler PCR assay for detection of Coccidioides posadasii DNA were designed and tested in 120 clinical strains. These had been isolated from 114 patients within 10 years in Monterrey, Nuevo Leon, Mexico, known to be endemic for coccidioidomycosis. The gene encoding the specific antigen 2/proline-rich antigen (Ag2/PRA) was used as a target. All strains were correctly identified, whereas DNA from related members of the family Onygenaceae remained negative. Melting curve analysis by LightCycler and sequencing of the 526-bp product of the first PCR demonstrated either 100% identity to the GenBank sequence of the Silveira strain, now known to be C. posadasii (accession number AF013256), or a single silent mutation at position 1228. Length determination of two microsatellite-containing loci (GAC and 621) identified all 120 isolates as C. posadasii. Specific DNA was amplified by conventional nested PCR from three microscopically spherule-positive paraffin-embedded tissue samples, whereas 20 human tissue samples positive for other dimorphic fungi remained negative. Additionally, the safety of each step of a modified commercially available DNA extraction procedure was evaluated by using 10 strains. At least three steps of the protocol were demonstrated to sufficiently kill arthroconidia. This safe procedure is applicable to cultures and to clinical specimens. PMID:14766853

HPMCD: the database of human microbial communities from metagenomic datasets and microbial reference genomes.

PubMed

Forster, Samuel C; Browne, Hilary P; Kumar, Nitin; Hunt, Martin; Denise, Hubert; Mitchell, Alex; Finn, Robert D; Lawley, Trevor D

2016-01-04

The Human Pan-Microbe Communities (HPMC) database (http://www.hpmcd.org/) provides a manually curated, searchable, metagenomic resource to facilitate investigation of human gastrointestinal microbiota. Over the past decade, the application of metagenome sequencing to elucidate the microbial composition and functional capacity present in the human microbiome has revolutionized many concepts in our basic biology. When sufficient high quality reference genomes are available, whole genome metagenomic sequencing can provide direct biological insights and high-resolution classification. The HPMC database provides species level, standardized phylogenetic classification of over 1800 human gastrointestinal metagenomic samples. This is achieved by combining a manually curated list of bacterial genomes from human faecal samples with over 21000 additional reference genomes representing bacteria, viruses, archaea and fungi with manually curated species classification and enhanced sample metadata annotation. A user-friendly, web-based interface provides the ability to search for (i) microbial groups associated with health or disease state, (ii) health or disease states and community structure associated with a microbial group, (iii) the enrichment of a microbial gene or sequence and (iv) enrichment of a functional annotation. The HPMC database enables detailed analysis of human microbial communities and supports research from basic microbiology and immunology to therapeutic development in human health and disease. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

A high-resolution whole genome radiation hybrid map of human chromosome 17q22-q25.3 across the genes for GH and TK

DOE Office of Scientific and Technical Information (OSTI.GOV)

Foster, J.W.; Schafer, A.J.; Critcher, R.

1996-04-15

We have constructed a whole genome radiation hybrid (WG-RH) map across a region of human chromosome 17q, from growth hormone (GH) to thymidine kinase (TK). A panel of 128 WG-RH hybrid cell lines generated by X-irradiation and fusion has been tested for the retention of 39 sequence-tagged site (STS) markers by the polymerase chain reaction. This genome mapping technique has allowed the integration of existing VNTR and microsatellite markers with additional new markers and existing STS markers previously mapped to this region by other means. The WG-RH map includes eight expressed sequence tag (EST) and three anonymous markers developed formore » this study, together with 23 anonymous microsatellites and five existing ESTs. Analysis of these data resulted in a high-density comprehensive map across this region of the genome. A subset of these markers has been used to produce a framework map consisting of 20 loci ordered with odds greater than 1000:1. The markers are of sufficient density to build a YAC contig across this region based on marker content. We have developed sequence tags for both ends of a 2.1-Mb YAC and mapped these using the WG-RH panel, allowing a direct comparison of cRay{sub 6000} to physical distance. 31 refs., 3 figs., 2 tabs.« less

Functional characterization of recombinant bromelain of Ananas comosus expressed in a prokaryotic system.

PubMed

George, Susan; Bhasker, Salini; Madhav, Harish; Nair, Archana; Chinnamma, Mohankumar

2014-02-01

Bromelain (BRM) is a defense protein present in the fruit and stem of pineapple (Ananas comosus) and it is grouped as a cysteine protease enzyme with diversified medicinal uses. Based on its therapeutic applications, bromelain has got sufficient attention in pharmaceutical industries. In the present study, the full coding gene of bromelain in pineapple stem (1,093 bp) was amplified by RT-PCR. The PCR product was cloned, sequenced, and characterized. The sequence analysis of the gene revealed the single nucleotide polymorphism and its phylogenetic relatedness. The peptide sequence deduced from the gene showed the amino acid variations, physicochemical properties and secondary and tertiary structural features of the protein. The full BRM gene was transformed to prokaryotic vector pET32b and expressed in Escherichia coli BL21 DE3pLysS host cells successfully. The identity of the recombinant bromelain (rBRM) protein was confirmed by Western blot analysis using anti-BRM-rabbit IgG antibody. The activity of recombinant bromelain compared with purified native bromelain was determined by protease assay. The inhibitory effect of rBRM compared with native BRM in the growth of Gram-positive and Gram-negative strains of Streptococcus agalactiae and Escherichia coli O111 was evident from the antibacterial sensitivity test. To the best of our knowledge, this is the first report showing the bactericidal property of rBRM expressed in a prokaryotic system.

Methods for determining the genetic affinity of microorganisms and viruses

NASA Technical Reports Server (NTRS)

Fox, George E. (Inventor); Willson, III, Richard C. (Inventor); Zhang, Zhengdong (Inventor)

2012-01-01

Selecting which sub-sequences in a database of nucleic acid such as 16S rRNA are highly characteristic of particular groupings of bacteria, microorganisms, fungi, etc. on a substantially phylogenetic tree. Also applicable to viruses comprising viral genomic RNA or DNA. A catalogue of highly characteristic sequences identified by this method is assembled to establish the genetic identity of an unknown organism. The characteristic sequences are used to design nucleic acid hybridization probes that include the characteristic sequence or its complement, or are derived from one or more characteristic sequences. A plurality of these characteristic sequences is used in hybridization to determine the phylogenetic tree position of the organism(s) in a sample. Those target organisms represented in the original sequence database and sufficient characteristic sequences can identify to the species or subspecies level. Oligonucleotide arrays of many probes are especially preferred. A hybridization signal can comprise fluorescence, chemiluminescence, or isotopic labeling, etc.; or sequences in a sample can be detected by direct means, e.g. mass spectrometry. The method's characteristic sequences can also be used to design specific PCR primers. The method uniquely identifies the phylogenetic affinity of an unknown organism without requiring prior knowledge of what is present in the sample. Even if the organism has not been previously encountered, the method still provides useful information about which phylogenetic tree bifurcation nodes encompass the organism.

Genome sequencing identifies Listeria fleischmannii subsp. coloradonensis subsp. nov., isolated from a ranch.

PubMed

den Bakker, Henk C; Manuel, Clyde S; Fortes, Esther D; Wiedmann, Martin; Nightingale, Kendra K

2013-09-01

Twenty Listeria-like isolates were obtained from environmental samples collected on a cattle ranch in northern Colorado; all of these isolates were found to share an identical partial sigB sequence, suggesting close relatedness. The isolates were similar to members of the genus Listeria in that they were Gram-stain-positive, short rods, oxidase-negative and catalase-positive; the isolates were similar to Listeria fleischmannii because they were non-motile at 25 °C. 16S rRNA gene sequencing for representative isolates and whole genome sequencing for one isolate was performed. The genome of the type strain of Listeria fleischmannii (strain LU2006-1(T)) was also sequenced. The draft genomes were very similar in size and the average MUMmer nucleotide identity across 91% of the genomes was 95.16%. Genome sequence data were used to design primers for a six-gene multi-locus sequence analysis (MLSA) scheme. Phylogenies based on (i) the near-complete 16S rRNA gene, (ii) 31 core genes and (iii) six housekeeping genes illustrated the close relationship of these Listeria-like isolates to Listeria fleischmannii LU2006-1(T). Sufficient genetic divergence of the Listeria-like isolates from the type strain of Listeria fleischmannii and differing phenotypic characteristics warrant these isolates to be classified as members of a distinct infraspecific taxon, for which the name Listeria fleischmannii subsp. coloradonensis subsp. nov. is proposed. The type strain is TTU M1-001(T) ( =BAA-2414(T) =DSM 25391(T)). The isolates of Listeria fleischmannii subsp. coloradonensis subsp. nov. differ from the nominate subspecies by the inability to utilize melezitose, turanose and sucrose, and the ability to utilize inositol. The results also demonstrate the utility of whole genome sequencing to facilitate identification of novel taxa within a well-described genus. The genomes of both subspecies of Listeria fleischmannii contained putative enhancin genes; the Listeria fleischmannii subsp. coloradonensis subsp. nov. genome also encoded a putative mosquitocidal toxin. The presence of these genes suggests possible adaptation to an insect host, and further studies are needed to probe niche adaptation of Listeria fleischmannii.

Archaebacterial phylogeny: perspectives on the urkingdoms

NASA Technical Reports Server (NTRS)

Woese, C. R.; Olsen, G. J.

1986-01-01

Comparisons of complete 16S ribosomal RNA sequences have been used to confirm, refine and extend earlier concepts of archaebacterial phylogeny. The archaebacteria fall naturally into two major branches or divisions, I--the sulfur-dependent thermophilic archaebacteria, and II--the methanogenic archaebacteria and their relatives. Division I comprises a relatively closely related and phenotypically homogeneous collection of thermophilic sulfur-dependent species--encompassing the genera Sulfolobus, Thermoproteus, Pyrodictium and Desulfurococcus. The organisms of Division II, however, form a less compact grouping phylogenetically, and are also more diverse in phenotype. All three of the (major) methanogen groups are found in Division II, as are the extreme halophiles and two types of thermoacidophiles, Thermoplasma acidophilum and Thermococcus celer. This last species branches sufficiently deeply in the Division II line that it might be considered to represent a separate, third Division. However, both the extreme halophiles and Tp. acidophilum branch within the cluster of methanogens. The extreme halophiles are specifically related to the Methanomicrobiales, to the exclusion of both the Methanococcales and the Methanobacteriales. Tp. acidophilum is peripherally related to the halophile-Methanomicrobiales group. By 16S rRNA sequence measure the archaebacteria constitute a phylogenetically coherent grouping (clade), which excludes both the eubacteria and the eukaryotes--a conclusion that is supported by other sequence evidence as well. Alternative proposals for archaebacterial phylogeny, not based upon sequence evidence, are discussed and evaluated. In particular, proposals to rename (reclassify) various subgroups of the archaebacteria as new kingdoms are found wanting, for both their lack of proper experimental support and the taxonomic confusion they introduce.

Informative priors based on transcription factor structural class improve de novo motif discovery.

PubMed

Narlikar, Leelavati; Gordân, Raluca; Ohler, Uwe; Hartemink, Alexander J

2006-07-15

An important problem in molecular biology is to identify the locations at which a transcription factor (TF) binds to DNA, given a set of DNA sequences believed to be bound by that TF. In previous work, we showed that information in the DNA sequence of a binding site is sufficient to predict the structural class of the TF that binds it. In particular, this suggests that we can predict which locations in any DNA sequence are more likely to be bound by certain classes of TFs than others. Here, we argue that traditional methods for de novo motif finding can be significantly improved by adopting an informative prior probability that a TF binding site occurs at each sequence location. To demonstrate the utility of such an approach, we present priority, a powerful new de novo motif finding algorithm. Using data from TRANSFAC, we train three classifiers to recognize binding sites of basic leucine zipper, forkhead, and basic helix loop helix TFs. These classifiers are used to equip priority with three class-specific priors, in addition to a default prior to handle TFs of other classes. We apply priority and a number of popular motif finding programs to sets of yeast intergenic regions that are reported by ChIP-chip to be bound by particular TFs. priority identifies motifs the other methods fail to identify, and correctly predicts the structural class of the TF recognizing the identified binding sites. Supplementary material and code can be found at http://www.cs.duke.edu/~amink/.

De novo characterization of Larimichthys crocea transcriptome for growth-/immune-related gene identification and massive microsatellite (SSR) marker development

NASA Astrophysics Data System (ADS)

Han, Zhaofang; Xiao, Shijun; Liu, Xiande; Liu, Yang; Li, Jiakai; Xie, Yangjie; Wang, Zhiyong

2017-03-01

The large yellow croaker, Larimichthys crocea is an important marine fish in China with a high economic value. In the last decade, the stock conservation and aquaculture industry of this species have been facing severe challenges because of wild population collapse and degeneration of important economic traits. However, genes contributing to growth and immunity in L. crocea have not been thoroughly analyzed, and available molecular markers are still not sufficient for genetic resource management and molecular selection. In this work, we sequenced the transcriptome in L. crocea liver tissue with a Roche 454 sequencing platform and assembled the transcriptome into 93 801 transcripts. Of them, 38 856 transcripts were successfully annotated in nt, nr, Swiss-Prot, InterPro, COG, GO and KEGG databases. Based on the annotation information, 3 165 unigenes related to growth and immunity were identified. Additionally, a total of 6 391 simple sequence repeats (SSRs) were identified from the transcriptome, among which 4 498 SSRs had enough flanking regions to design primers for polymerase chain reactions (PCR). To access the polymorphism of these markers, 30 primer pairs were randomly selected for PCR amplification and validation in 30 individuals, and 12 primer pairs (40.0%) exhibited obvious length polymorphisms. This work applied RNA-Seq to assemble and analyze a live transcriptome in L. crocea. With gene annotation and sequence information, genes related to growth and immunity were identified and massive SSR markers were developed, providing valuable genetic resources for future gene functional analysis and selective breeding of L. crocea.

Phylogenetic relationships between some members of the genera Neisseria, Acinetobacter, Moraxella, and Kingella based on partial 16S ribosomal DNA sequence analysis.

PubMed

Enright, M C; Carter, P E; MacLean, I A; McKenzie, H

1994-07-01

We obtained 16S ribosomal DNA (rDNA) sequence data for strains belonging to 11 species of Proteobacteria, including the type strains of Kingella kingae, Neisseria lactamica, Neisseria meningitidis, Moraxella lacunata subsp. lacunata, [Neisseria] ovis, Moraxella catarrhalis, Moraxella osloensis, [Moraxella] phenylpyruvica, and Acinetobacter lwoffii, as well as strains of Neisseria subflava and Acinetobacter calcoaceticus. The data in a distance matrix constructed by comparing the sequences supported the proposal that the genera Acinetobacter and Moraxella and [N.] ovis should be excluded from the family Neisseriaceae. Our results are consistent with hybridization data which suggest that these excluded taxa should be part of a new family, the Moraxellaceae. The strains that we studied can be divided into the following five groups: (i) M. lacunata subsp. lacunata, [N.] ovis, and M. catarrhalis; (ii) M. osloensis; (iii) [M.] phenylpyruvica; (iv) A. calcoaceticus and A. lwoffii; and (v) N. meningitidis, N. subflava, N. lactamica, and K. kingae. We agree with the previous proposal that [N.] ovis should be renamed Moraxella ovis, as this organism is closely related to Moraxella species and not to Neisseria species. The generically misnamed taxon [M.] phenylpyruvica belongs to the proposed family Moraxellaceae, but it is sufficiently different to warrant exclusion from the genus Moraxella. Further work needs to be done to investigate genetically similar species, such as Psychrobacter immobilis, before the true generic position of this organism can be determined. Automated 16S rDNA sequencing with the PCR allows workers to accurately determine phylogenetic relationships between groups of organisms.(ABSTRACT TRUNCATED AT 250 WORDS)

Shifts in phylogenetic diversity of archaeal communities in mangrove sediments at different sites and depths in southeastern Brazil.

PubMed

Mendes, Lucas William; Taketani, Rodrigo Gouvêa; Navarrete, Acácio Aparecido; Tsai, Siu Mui

2012-06-01

This study focused on the structure and composition of archaeal communities in sediments of tropical mangroves in order to obtain sufficient insight into two Brazilian sites from different locations (one pristine and another located in an urban area) and at different depth levels from the surface. Terminal restriction fragment length polymorphism (T-RFLP) of PCR-amplified 16S rRNA gene fragments was used to scan the archaeal community structure, and 16S rRNA gene clone libraries were used to determine the community composition. Redundancy analysis of T-RFLP patterns revealed differences in archaeal community structure according to location, depth and soil attributes. Parameters such as pH, organic matter, potassium and magnesium presented significant correlation with general community structure. Furthermore, phylogenetic analysis revealed a community composition distributed differently according to depth where, in shallow samples, 74.3% of sequences were affiliated with Euryarchaeota and 25.7% were shared between Crenarchaeota and Thaumarchaeota, while for the deeper samples, 24.3% of the sequences were affiliated with Euryarchaeota and 75.7% with Crenarchaeota and Thaumarchaeota. Archaeal diversity measurements based on 16S rRNA gene clone libraries decreased with increasing depth and there was a greater difference between depths (<18% of sequences shared) than sites (>25% of sequences shared). Taken together, our findings indicate that mangrove ecosystems support a diverse archaeal community; it might possibly be involved in nutrient cycles and are affected by sediment properties, depth and distinct locations. Copyright © 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

A novel human-based receptor antagonist of sustained action reveals body weight control by endogenous GLP-1.

PubMed

Patterson, James T; Ottaway, Nickki; Gelfanov, Vasily M; Smiley, David L; Perez-Tilve, Diego; Pfluger, Paul T; Tschöp, Matthias H; Dimarchi, Richard D

2011-02-18

Ex-4 (9-39)a is a well characterized GLP-1 receptor antagonist that suffers from two notable limitations, its nonhuman amino acid sequence and its relatively short in vivo duration of action. Comparable N-terminal shortening of human GLP-1 lessens agonism but does not provide a high potency antagonist. Through a series of GLP-1/Ex-4 hybrid peptides, the minimal structural changes required to generate a pure GLP-1-based antagonist were identified as Glu16, Val19, and Arg20, yielding an antagonist of approximately 3-fold greater in vitro potency compared with Ex-4 (9-39)a. The structural basis of antagonism appears to result from stabilization of the α helix combined with enhanced electrostatic and hydrophobic interactions with the extracellular domain of the receptor. Site-specific acylation of the human-based antagonist yielded a peptide of increased potency as a GLP-1 receptor antagonist and 10-fold greater selectivity relative to the GIP receptor. The acylated antagonist demonstrated sufficient duration of action to maintain inhibitory activity when administered as a daily subcutaneous injection. The sustained pharmacokinetics and enhanced human sequence combine to form an antagonist optimized for clinical study. Daily administration of this antagonist by subcutaneous injection to diet-induced obese mice for 1 week caused a significant increase in food intake, body weight, and glucose intolerance, demonstrating endogenous GLP-1 as a relevant hormone in mammalian energy balance in the obese state.

Machine learning for classifying tuberculosis drug-resistance from DNA sequencing data.

PubMed

Yang, Yang; Niehaus, Katherine E; Walker, Timothy M; Iqbal, Zamin; Walker, A Sarah; Wilson, Daniel J; Peto, Tim E A; Crook, Derrick W; Smith, E Grace; Zhu, Tingting; Clifton, David A

2018-05-15

Correct and rapid determination of Mycobacterium tuberculosis (MTB) resistance against available tuberculosis (TB) drugs is essential for the control and management of TB. Conventional molecular diagnostic test assumes that the presence of any well-studied single nucleotide polymorphisms is sufficient to cause resistance, which yields low sensitivity for resistance classification. Given the availability of DNA sequencing data from MTB, we developed machine learning models for a cohort of 1839 UK bacterial isolates to classify MTB resistance against eight anti-TB drugs (isoniazid, rifampicin, ethambutol, pyrazinamide, ciprofloxacin, moxifloxacin, ofloxacin, streptomycin) and to classify multi-drug resistance. Compared to previous rules-based approach, the sensitivities from the best-performing models increased by 2-4% for isoniazid, rifampicin and ethambutol to 97% (P < 0.01), respectively; for ciprofloxacin and multi-drug resistant TB, they increased to 96%. For moxifloxacin and ofloxacin, sensitivities increased by 12 and 15% from 83 and 81% based on existing known resistance alleles to 95% and 96% (P < 0.01), respectively. Particularly, our models improved sensitivities compared to the previous rules-based approach by 15 and 24% to 84 and 87% for pyrazinamide and streptomycin (P < 0.01), respectively. The best-performing models increase the area-under-the-ROC curve by 10% for pyrazinamide and streptomycin (P < 0.01), and 4-8% for other drugs (P < 0.01). The details of source code are provided at http://www.robots.ox.ac.uk/~davidc/code.php. david.clifton@eng.ox.ac.uk. Supplementary data are available at Bioinformatics online.

Profiling of potential driver mutations in sarcomas by targeted next generation sequencing.

PubMed

Andersson, Carola; Fagman, Henrik; Hansson, Magnus; Enlund, Fredrik

2016-04-01

Comprehensive genetic profiling by massively parallel sequencing, commonly known as next generation sequencing (NGS), is becoming the foundation of personalized oncology. For sarcomas very few targeted treatments are currently in routine use. In clinical practice the preoperative diagnostic workup of soft tissue tumours largely relies on core needle biopsies. Although mostly sufficient for histopathological diagnosis, only very limited amounts of formalin fixated paraffin embedded tissue are often available for predictive mutation analysis. Targeted NGS may thus open up new possibilities for comprehensive characterization of scarce biopsies. We therefore set out to search for driver mutations by NGS in a cohort of 55 clinically and morphologically well characterized sarcomas using low input of DNA from formalin fixated paraffin embedded tissues. The aim was to investigate if there are any recurrent or targetable aberrations in cancer driver genes in addition to known chromosome translocations in different types of sarcomas. We employed a panel covering 207 mutation hotspots in 50 cancer-associated genes to analyse DNA from nine gastrointestinal stromal tumours, 14 synovial sarcomas, seven myxoid liposarcomas, 22 Ewing sarcomas and three Ewing-like small round cell tumours at a large sequencing depth to detect also mutations that are subclonal or occur at low allele frequencies. We found nine mutations in eight different potential driver genes, some of which are potentially actionable by currently existing targeted therapies. Even though no recurrent mutations in driver genes were found in the different sarcoma groups, we show that targeted NGS-based sequencing is clearly feasible in a diagnostic setting with very limited amounts of paraffin embedded tissue and may provide novel insights into mesenchymal cell signalling and potentially druggable targets. Interestingly, we also identify five non-synonymous sequence variants in 4 established cancer driver genes in DNA from normal tissue from sarcoma patients that may possibly predispose or contribute to neoplastic development. Copyright © 2016 Elsevier Inc. All rights reserved.

Multilocus phylogeny and phylogenomics of Eriochrysis P. Beauv. (Poaceae-Andropogoneae): Taxonomic implications and evidence of interspecific hybridization.

PubMed

Welker, Cassiano A D; Souza-Chies, Tatiana T; Longhi-Wagner, Hilda M; Peichoto, Myriam Carolina; McKain, Michael R; Kellogg, Elizabeth A

2016-06-01

Species delimitation is a vital issue concerning evolutionary biology and conservation of biodiversity. However, it is a challenging task for several reasons, including the low interspecies variability of markers currently used in phylogenetic reconstructions and the occurrence of reticulate evolution and polyploidy in many lineages of flowering plants. The first phylogeny of the grass genus Eriochrysis is presented here, focusing on the New World species, in order to examine its relationships to other genera of the subtribe Saccharinae/tribe Andropogoneae and to define the circumscriptions of its taxonomically complicated species. Molecular cloning and sequencing of five regions of four low-copy nuclear genes (apo1, d8, ep2-ex7 and ep2-ex8, kn1) were performed, as well as complete plastome sequencing. Trees were reconstructed using maximum parsimony, maximum likelihood, and Bayesian inference analyses. The present phylogenetic analyses indicate that Eriochrysis is monophyletic and the Old World E. pallida is sister to the New World species. Subtribe Saccharinae is polyphyletic, as is the genus Eulalia. Based on nuclear and plastome sequences plus morphology, we define the circumscriptions of the New World species of Eriochrysis: E. laxa is distinct from E. warmingiana, and E. villosa is distinct from E. cayennensis. Natural hybrids occur between E. laxa and E. villosa. The hybrids are probably tetraploids, based on the number of paralogues in the nuclear gene trees. This is the first record of a polyploid taxon in the genus Eriochrysis. Some incongruities between nuclear genes and plastome analyses were detected and are potentially caused by incomplete lineage sorting and/or ancient hybridization. The set of low-copy nuclear genes used in this study seems to be sufficient to resolve phylogenetic relationships and define the circumscriptions of other species complexes in the grass family and relatives, even in the presence of polyploidy and reticulate evolution. Complete plastome sequencing is also a promising tool for phylogenetic inference. Copyright © 2016 Elsevier Inc. All rights reserved.

Permeation of internal and external monovalent cations through the catfish cone photoreceptor cGMP-gated channel

PubMed Central

1995-01-01

The permeation of monovalent cations through the cGMP-gated channel of catfish cone outer segments was examined by measuring permeability and conductance ratios under biionic conditions. For monovalent cations presented on the cytoplasmic side of the channel, the permeability ratios with respect to extracellular Na followed the sequence NH4 > K > Li > Rb = Na > Cs while the conductance ratios at +50 mV followed the sequence Na approximately NH4 > K > Rb > Li = Cs. These patterns are broadly similar to the amphibian rod channel. The symmetry of the channel was tested by presenting the test ion on the extracellular side and using Na as the common reference ion on the cytoplasmic side. Under these biionic conditions, the permeability ratios with respect to Na at the intracellular side followed the sequence NH4 > Li > K > Na > Rb > Cs while the conductance ratios at +50 mV followed the sequence NH4 > K approximately Na > Rb > Li > Cs. Thus, the channel is asymmetric with respect to external and internal cations. Under symmetrical 120 mM ionic conditions, the single-channel conductance at +50 mV ranged from 58 pS in NH4 to 15 pS for Cs and was in the order NH4 > Na > K > Rb > Cs. Unexpectedly, the single-channel current-voltage relation showed sufficient outward rectification to account for the rectification observed in multichannel patches without invoking voltage dependence in gating. The concentration dependence of the reversal potential for K showed that chloride was impermeant. Anomalous mole fraction behavior was not observed, nor, over a limited concentration range, were multiple dissociation constants. An Eyring rate theory model with a single binding site was sufficient to explain these observations. PMID:8786344

Next generation sequencing techniques in liquid biopsy: focus on non-small cell lung cancer patients.

PubMed

Malapelle, Umberto; Pisapia, Pasquale; Rocco, Danilo; Smeraglio, Riccardo; di Spirito, Maria; Bellevicine, Claudio; Troncone, Giancarlo

2016-10-01

The advent of genomic based personalized medicine has led to multiple advances in the molecular characterization of many tumor types, such as non-small cell lung cancer (NSCLC). NSCLC is diagnosed in most cases on small tissue samples that may be not always sufficient for EGFR mutational assessment to select patients for first and second generations' tyrosine kinase inhibitors (TKIs) therapy. In patients without tissue availability at presentation, the analysis of cell free DNA (cfDNA) derived from liquid biopsy samples, in particular from plasma, represent an established alternative to provide EGFR mutational testing for treatment decision making. In addition, a new paradigm for TKIs resistance management was recently approved by Food and Drug Administration, supporting the liquid biopsy based genotyping prior to tissue based genotyping for the detection of T790M mutation to select patients for third generation TKIs. In these settings, real time PCR (RT-PCR) and digital PCR 'targeted' methods, which detect known mutations by specific probes, have extensively been adopted. Taking into account the restricted reference range and the limited multiplexing power of these targeted methods, the performance of liquid biopsy analyses may be further improved by next generation sequencing (NGS). While most tissue based NGS genotyping is well established, liquid biopsy NGS application is challenging, requiring a careful validation of the whole process, from blood collection to variant calling. Here we review this evolving field, highlighting those methodological points that are crucial to accurately select NSCLC patients for TKIs treatment administration by NGS on cfDNA.

Reclassification of Bacillus marismortui as Salibacillus marismortui comb. nov.

PubMed

Arahal, D R; Márquez, M C; Volcani, B E; Schleifer, K H; Ventosa, A

2000-07-01

Recently, the features of a group of strains isolated from Dead Sea enrichments obtained in 1936 by one of us (B. E. Volcani) were described. They were gram-positive, moderately halophilic, spore-forming rods, and were placed in a new species, Bacillus marismortui. At the same time, the new genus Salibacillus was proposed for the halophilic species Bacillus salexigens. B. marismortui and Salibacillus salexigens have similar phenotypic characteristics and the same peptidoglycan type. Phylogenetic analysis based on 16S rRNA sequence comparisons showed that they are sufficiently closely related (96.6% similarity) as to warrant placement in the same genus. However, DNA-DNA hybridization experiments showed that they constitute two separate species (41% DNA similarity). Therefore the reclassification of Bacillus marismortui as Salibacillus marismortui comb. nov. is proposed.

An object tracking method based on guided filter for night fusion image

NASA Astrophysics Data System (ADS)

Qian, Xiaoyan; Wang, Yuedong; Han, Lei

2016-01-01

Online object tracking is a challenging problem as it entails learning an effective model to account for appearance change caused by intrinsic and extrinsic factors. In this paper, we propose a novel online object tracking with guided image filter for accurate and robust night fusion image tracking. Firstly, frame difference is applied to produce the coarse target, which helps to generate observation models. Under the restriction of these models and local source image, guided filter generates sufficient and accurate foreground target. Then accurate boundaries of the target can be extracted from detection results. Finally timely updating for observation models help to avoid tracking shift. Both qualitative and quantitative evaluations on challenging image sequences demonstrate that the proposed tracking algorithm performs favorably against several state-of-art methods.

A “Rosetta Stone” for metazoan zooplankton: DNA barcode analysis of species diversity of the Sargasso Sea (Northwest Atlantic Ocean)

NASA Astrophysics Data System (ADS)

Bucklin, Ann; Ortman, Brian D.; Jennings, Robert M.; Nigro, Lisa M.; Sweetman, Christopher J.; Copley, Nancy J.; Sutton, Tracey; Wiebe, Peter H.

2010-12-01

Species diversity of the metazoan holozooplankton assemblage of the Sargasso Sea, Northwest Atlantic Ocean, was examined through coordinated morphological taxonomic identification of species and DNA sequencing of a ˜650 base-pair region of mitochondrial cytochrome oxidase I (mtCOI) as a DNA barcode (i.e., short sequence for species recognition and discrimination). Zooplankton collections were made from the surface to 5,000 meters during April, 2006 on the R/V R.H. Brown. Samples were examined by a ship-board team of morphological taxonomists; DNA barcoding was carried out in both ship-board and land-based DNA sequencing laboratories. DNA barcodes were determined for a total of 297 individuals of 175 holozooplankton species in four phyla, including: Cnidaria (Hydromedusae, 4 species; Siphonophora, 47); Arthropoda (Amphipoda, 10; Copepoda, 34; Decapoda, 9; Euphausiacea, 10; Mysidacea, 1; Ostracoda, 27); and Mollusca (Cephalopoda, 8; Heteropoda, 6; Pteropoda, 15); and Chaetognatha (4). Thirty species of fish (Teleostei) were also barcoded. For all seven zooplankton groups for which sufficient data were available, Kimura-2-Parameter genetic distances were significantly lower between individuals of the same species (mean=0.0114; S.D. 0.0117) than between individuals of different species within the same group (mean=0.3166; S.D. 0.0378). This difference, known as the barcode gap, ensures that mtCOI sequences are reliable characters for species identification for the oceanic holozooplankton assemblage. In addition, DNA barcodes allow recognition of new or undescribed species, reveal cryptic species within known taxa, and inform phylogeographic and population genetic studies of geographic variation. The growing database of "gold standard" DNA barcodes serves as a Rosetta Stone for marine zooplankton, providing the key for decoding species diversity by linking species names, morphology, and DNA sequence variation. In light of the pivotal position of zooplankton in ocean food webs, their usefulness as rapid responders to environmental change, and the increasing scarcity of taxonomists, the use of DNA barcodes is an important and useful approach for rapid analysis of species diversity and distribution in the pelagic community.

Role for cis-acting RNA sequences in the temperature-dependent expression of the multiadhesive lig proteins in Leptospira interrogans.

PubMed

Matsunaga, James; Schlax, Paula J; Haake, David A

2013-11-01

The spirochete Leptospira interrogans causes a systemic infection that provokes a febrile illness. The putative lipoproteins LigA and LigB promote adhesion of Leptospira to host proteins, interfere with coagulation, and capture complement regulators. In this study, we demonstrate that the expression level of the LigA and LigB proteins was substantially higher when L. interrogans proliferated at 37°C instead of the standard culture temperature of 30°C. The RNA comprising the 175-nucleotide 5' untranslated region (UTR) and first six lig codons, whose sequence is identical in ligA and ligB, is predicted to fold into two distinct stem-loop structures separated by a single-stranded region. The ribosome-binding site is partially sequestered in double-stranded RNA within the second structure. Toeprint analysis revealed that in vitro formation of a 30S-tRNA(fMet)-mRNA ternary complex was inhibited unless a 5' deletion mutation disrupted the second stem-loop structure. To determine whether the lig sequence could mediate temperature-regulated gene expression in vivo, the 5' UTR and the first six codons were inserted between the Escherichia coli l-arabinose promoter and bgaB (β-galactosidase from Bacillus stearothermophilus) to create a translational fusion. The lig fragment successfully conferred thermoregulation upon the β-galactosidase reporter in E. coli. The second stem-loop structure was sufficient to confer thermoregulation on the reporter, while sequences further upstream in the 5' UTR slightly diminished expression at each temperature tested. Finally, the expression level of β-galactosidase was significantly higher when point mutations predicted to disrupt base pairs in the second structure were introduced into the stem. Compensatory mutations that maintained base pairing of the stem without restoring the wild-type sequence reinstated the inhibitory effect of the 5' UTR on expression. These results indicate that ligA and ligB expression is limited by double-stranded RNA that occludes the ribosome-binding site. At elevated temperatures, the ribosome-binding site is exposed to promote translation initiation.

SEQUENCING of TSUNAMI WAVES: Why the first wave is not always the largest?

NASA Astrophysics Data System (ADS)

Synolakis, C.; Okal, E.

2016-12-01

We discuss what contributes to the `sequencing' of tsunami waves in the far field, that is, to the distribution of the maximum sea surface amplitude inside the dominant wave packet constituting the primary arrival at a distant harbour. Based on simple models of sources for which analytical solutions are available, we show that, as range is increased, the wave pattern evolves from a regime of maximum amplitude in the first oscillation to one of delayed maximum, where the largest amplitude takes place during a subsequent oscillation. In the case of the simple, instantaneous uplift of a circular disk at the surface of an ocean of constant depth, the critical distance for transition between those patterns scales as r 30 /h2 where r0 is the radius of the disk and h the depth of the ocean. This behaviour is explained from simple arguments based on a model where sequencing results from frequency dispersion in the primary wave packet, as the width of its spectrum around its dominant period T0 becomes dispersed in time in an amount comparable to T0 , the latter being controlled by a combination of source size and ocean depth. The general concepts in this model are confirmed in the case of more realistic sources for tsunami excitation by a finite-time deformation of the ocean floor, as well as in real-life simulations of tsunamis excited by large subduction events, for which we find that the influence of fault width on the distribution of sequencing is more important than that of fault length. Finally, simulation of the major events of Chile (2010) and Japan (2011) at large arrays of virtual gauges in the Pacific Basin correctly predicts the majority of the sequencing patterns observed on DART buoys during these events. By providing insight into the evolution with time of wave amplitudes inside primary wave packets for far field tsunamis generated by large earthquakes, our results stress the importance, for civil defense authorities, of issuing warning and evacuation orders of sufficient duration to avoid the hazard

Sequencing of tsunami waves: why the first wave is not always the largest

NASA Astrophysics Data System (ADS)

Okal, Emile A.; Synolakis, Costas E.

2016-02-01

This paper examines the factors contributing to the `sequencing' of tsunami waves in the far field, that is, to the distribution of the maximum sea surface amplitude inside the dominant wave packet constituting the primary arrival at a distant harbour. Based on simple models of sources for which analytical solutions are available, we show that, as range is increased, the wave pattern evolves from a regime of maximum amplitude in the first oscillation to one of delayed maximum, where the largest amplitude takes place during a subsequent oscillation. In the case of the simple, instantaneous uplift of a circular disk at the surface of an ocean of constant depth, the critical distance for transition between those patterns scales as r_0^3 / h^2 where r0 is the radius of the disk and h the depth of the ocean. This behaviour is explained from simple arguments based on a model where sequencing results from frequency dispersion in the primary wave packet, as the width of its spectrum around its dominant period T0 becomes dispersed in time in an amount comparable to T0, the latter being controlled by a combination of source size and ocean depth. The general concepts in this model are confirmed in the case of more realistic sources for tsunami excitation by a finite-time deformation of the ocean floor, as well as in real-life simulations of tsunamis excited by large subduction events, for which we find that the influence of fault width on the distribution of sequencing is more important than that of fault length. Finally, simulation of the major events of Chile (2010) and Japan (2011) at large arrays of virtual gauges in the Pacific Basin correctly predicts the majority of the sequencing patterns observed on DART buoys during these events. By providing insight into the evolution with time of wave amplitudes inside primary wave packets for far field tsunamis generated by large earthquakes, our results stress the importance, for civil defense authorities, of issuing warning and evacuation orders of sufficient duration to avoid the hazard inherent in premature calls for all-clear.

Design of a tobacco exon array with application to investigate the differential cadmium accumulation property in two tobacco varieties

PubMed Central

2012-01-01

Background For decades the tobacco plant has served as a model organism in plant biology to answer fundamental biological questions in the areas of plant development, physiology, and genetics. Due to the lack of sufficient coverage of genomic sequences, however, none of the expressed sequence tag (EST)-based chips developed to date cover gene expression from the whole genome. The availability of Tobacco Genome Initiative (TGI) sequences provides a useful resource to build a whole genome exon array, even if the assembled sequences are highly fragmented. Here, the design of a Tobacco Exon Array is reported and an application to improve the understanding of genes regulated by cadmium (Cd) in tobacco is described. Results From the analysis and annotation of the 1,271,256 Nicotiana tabacum fasta and quality files from methyl filtered genomic survey sequences (GSS) obtained from the TGI and ~56,000 ESTs available in public databases, an exon array with 272,342 probesets was designed (four probes per exon) and tested on two selected tobacco varieties. Two tobacco varieties out of 45 accumulating low and high cadmium in leaf were identified based on the GGE biplot analysis, which is analysis of the genotype main effect (G) plus analysis of the genotype by environment interaction (GE) of eight field trials (four fields over two years) showing reproducibility across the trials. The selected varieties were grown under greenhouse conditions in two different soils and subjected to exon array analyses using root and leaf tissues to understand the genetic make-up of the Cd accumulation. Conclusions An Affymetrix Exon Array was developed to cover a large (~90%) proportion of the tobacco gene space. The Tobacco Exon Array will be available for research use through Affymetrix array catalogue. As a proof of the exon array usability, we have demonstrated that the Tobacco Exon Array is a valuable tool for studying Cd accumulation in tobacco leaves. Data from field and greenhouse experiments supported by gene expression studies strongly suggested that the difference in leaf Cd accumulation between the two specific tobacco cultivars is dependent solely on genetic factors and genetic variability rather than on the environment. PMID:23190529

The Clinical and Cost Effectiveness of Apremilast for Treating Active Psoriatic Arthritis: A Critique of the Evidence.

PubMed

Sideris, Eleftherios; Corbett, Mark; Palmer, Stephen; Woolacott, Nerys; Bojke, Laura

2016-11-01

As part of the National Institute for Health and Clinical Excellence (NICE) single technology appraisal (STA) process, the manufacturer of apremilast was invited to submit evidence for its clinical and cost effectiveness for the treatment of active psoriatic arthritis (PsA) for whom disease-modifying anti-rheumatic drugs (DMARDs) have been inadequately effective, not tolerated or contraindicated. The Centre for Reviews and Dissemination and Centre for Health Economics at the University of York were commissioned to act as the independent Evidence Review Group (ERG). This paper provides a description of the ERG review of the company's submission, the ERG report and submission and summarises the NICE Appraisal Committee's subsequent guidance (December 2015). In the company's initial submission, the base-case analysis resulted in an incremental cost-effectiveness ratio (ICER) of £14,683 per quality-adjusted life-year (QALY) gained for the sequence including apremilast (positioned before tumour necrosis factor [TNF]-α inhibitors) versus a comparator sequence without apremilast. However, the ERG considered that the base-case sequence proposed by the company represented a limited set of potentially relevant treatment sequences and positions for apremilast. The company's base-case results were therefore not a sufficient basis to inform the most efficient use and position of apremilast. The exploratory ERG analyses indicated that apremilast is more effective (i.e. produces higher health gains) when positioned after TNF-α inhibitor therapies. Furthermore, assumptions made regarding a potential beneficial effect of apremilast on long-term Health Assessment Questionnaire (HAQ) progression, which cannot be substantiated, have a very significant impact on results. The NICE Appraisal Committee (AC), when taking into account their preferred assumptions for HAQ progression for patients on treatment with apremilast, placebo response and monitoring costs for apremilast, concluded that the addition of apremilast resulted in cost savings but also a QALY loss. These cost savings were not high enough to compensate for the clinical effectiveness that would be lost. The AC thus decided that apremilast alone or in combination with DMARD therapy is not recommended for treating adults with active PsA that has not responded to prior DMARD therapy, or where such therapy is not tolerated.

SU-E-J-232: Feasibility of MRI-Based Preplan On Low Dose Rate Prostate Brachytherapy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huang, Y; Tward, J; Rassiah-Szegedi, P

Purpose: To investigate the feasibility of using MRI-based preplan for low dose rate prostate brachytherapy. Methods: 12 patients who received transrectal ultrasound (TRUS) guided prostate brachytherapy with Pd-103 were retrospectively studied. Our care-standard of the TRUS-based preplan served as the control. One or more prostate T2-weighted wide and/or narrow-field of view MRIs obtained within the 3 months prior to the implant were imported into the MIM Symphony software v6.3 (MIM Software Inc., Cleveland, OH) for each patient. In total, 37 MRI preplans (10 different image sequences with average thickness of 4.8mm) were generated. The contoured prostate volume and the seedmore » counts required to achieve adequate dosimetric coverage from TRUS and MRI preplans were compared for each patient. The effects of different MRI sequences and image thicknesses were also investigated statistically using Student’s t-test. Lastly, the nomogram from the MRI preplan and TRUS preplan from our historical treatment data were compared. Results: The average prostate volume contoured on the TRUS and MRI were 26.6cc (range: 12.6∼41.3cc), and 27.4 cc (range: 14.3∼50.0cc), respectively. Axial MRI thicknesses (range: 3.5∼8.1mm) did not significantly affect the contoured volume or the number of seeds required on the preplan (R2 = 0.0002 and 0.0012, respectively). Four of the MRI sequences (AX-T2, AX-T2-Whole-Pelvis, AX-T2-FSE, and AXIALT2- Hi-Res) showed statistically significant better prostate volume agreement with TRUS than the other seven sequences (P <0.01). Nomogram overlay between the MRI and TRUS preplans showed good agreement; indicating volumes contoured on MRI preplan scan reliably predict how many seeds are needed for implant. Conclusion: Although MRI does not allow for determination of the actual implant geometry, it can give reliable volumes for seed ordering purposes. Our future work will investigate if MRI is sufficient to reliably replace TRUS preplanning in patients where preplan TRUS may be technically challenging.« less

Discovery of the porcine NGN3 gene and testing its endocrine function in the pig

USDA-ARS?s Scientific Manuscript database

Neurogenin 3 (NGN3) is a member of the basic helix-loop-helix transcription factor family. NGN3 is both necessary and sufficient to drive endocrine differentiation in the developing pancreas in mouse and humans. Until now, the sequence for NGN3 eluded discovery despite completion of the pig genome a...

Identifying Specific Genes Controlling Complex Traits Through A Genome-Wide Screen For cis-Acting Regulatory Elements - An Example Using Marek's Disease

USDA-ARS?s Scientific Manuscript database

The identification of specific genes underlying phenotypic variation of complex traits remains one of the greatest challenges in biology despite having genome sequences and more powerful tools. Most genome-wide screens lack sufficient resolving power as they typically depend on linkage. One altern...

Pre-processing SAR image stream to facilitate compression for transport on bandwidth-limited-link

DOEpatents

Rush, Bobby G.; Riley, Robert

2015-09-29

Pre-processing is applied to a raw VideoSAR (or similar near-video rate) product to transform the image frame sequence into a product that resembles more closely the type of product for which conventional video codecs are designed, while sufficiently maintaining utility and visual quality of the product delivered by the codec.

Learning viewpoint invariant perceptual representations from cluttered images.

PubMed

Spratling, Michael W

2005-05-01

In order to perform object recognition, it is necessary to form perceptual representations that are sufficiently specific to distinguish between objects, but that are also sufficiently flexible to generalize across changes in location, rotation, and scale. A standard method for learning perceptual representations that are invariant to viewpoint is to form temporal associations across image sequences showing object transformations. However, this method requires that individual stimuli be presented in isolation and is therefore unlikely to succeed in real-world applications where multiple objects can co-occur in the visual input. This paper proposes a simple modification to the learning method that can overcome this limitation and results in more robust learning of invariant representations.

A generalization of Fatou's lemma for extended real-valued functions on σ-finite measure spaces: with an application to infinite-horizon optimization in discrete time.

PubMed

Kamihigashi, Takashi

2017-01-01

Given a sequence [Formula: see text] of measurable functions on a σ -finite measure space such that the integral of each [Formula: see text] as well as that of [Formula: see text] exists in [Formula: see text], we provide a sufficient condition for the following inequality to hold: [Formula: see text] Our condition is considerably weaker than sufficient conditions known in the literature such as uniform integrability (in the case of a finite measure) and equi-integrability. As an application, we obtain a new result on the existence of an optimal path for deterministic infinite-horizon optimization problems in discrete time.

Sma3s: a three-step modular annotator for large sequence datasets.

PubMed

Muñoz-Mérida, Antonio; Viguera, Enrique; Claros, M Gonzalo; Trelles, Oswaldo; Pérez-Pulido, Antonio J

2014-08-01

Automatic sequence annotation is an essential component of modern 'omics' studies, which aim to extract information from large collections of sequence data. Most existing tools use sequence homology to establish evolutionary relationships and assign putative functions to sequences. However, it can be difficult to define a similarity threshold that achieves sufficient coverage without sacrificing annotation quality. Defining the correct configuration is critical and can be challenging for non-specialist users. Thus, the development of robust automatic annotation techniques that generate high-quality annotations without needing expert knowledge would be very valuable for the research community. We present Sma3s, a tool for automatically annotating very large collections of biological sequences from any kind of gene library or genome. Sma3s is composed of three modules that progressively annotate query sequences using either: (i) very similar homologues, (ii) orthologous sequences or (iii) terms enriched in groups of homologous sequences. We trained the system using several random sets of known sequences, demonstrating average sensitivity and specificity values of ~85%. In conclusion, Sma3s is a versatile tool for high-throughput annotation of a wide variety of sequence datasets that outperforms the accuracy of other well-established annotation algorithms, and it can enrich existing database annotations and uncover previously hidden features. Importantly, Sma3s has already been used in the functional annotation of two published transcriptomes. © The Author 2014. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

Haemagglutinin and neuraminidase sequencing delineate nosocomial influenza outbreaks with accuracy equivalent to whole genome sequencing.

PubMed

Houghton, Rebecca; Ellis, Joanna; Galiano, Monica; Clark, Tristan W; Wyllie, Sarah

2017-04-01

We describe haemagglutinin (HA) and neuraminidase (NA) sequencing in an apparent cross-site influenza A(H1N1) outbreak in renal transplant and haemodialysis patients, confirmed with whole genome sequencing (WGS). Isolates were sequenced from influenza positive individuals. Phylogenetic trees were constructed using HA and NA sequencing and subsequently WGS. Sequence data was analysed to determine genetic relatedness of viruses obtained from inpatient and outpatient cohorts and compared with epidemiological outbreak information. There were 6 patient cases of influenza in the inpatient renal ward cohort (associated with 3 deaths) and 9 patient cases in the outpatient haemodialysis unit cohort (no deaths). WGS confirmed clustered transmission of two genetically different influenza A(H1N1)pdm09 strains initially identified by analysis of HA and NA genes. WGS took longer, and in this case was not required to determine whether or not the two seemingly linked outbreaks were related. Rapid sequencing of HA and NA genes may be sufficient to aid early influenza outbreak investigation making it appealing for future outbreak investigation. However, as next generation sequencing becomes cheaper and more widely available and bioinformatics software is now freely accessible next generation whole genome analysis may increasingly become a valuable tool for real-time Influenza outbreak investigation. Crown Copyright © 2017. Published by Elsevier Ltd. All rights reserved.

Packaging of Mason-Pfizer monkey virus (MPMV) genomic RNA depends upon conserved long-range interactions (LRIs) between U5 and gag sequences.

PubMed

Kalloush, Rawan M; Vivet-Boudou, Valérie; Ali, Lizna M; Mustafa, Farah; Marquet, Roland; Rizvi, Tahir A

2016-06-01

MPMV has great potential for development as a vector for gene therapy. In this respect, precisely defining the sequences and structural motifs that are important for dimerization and packaging of its genomic RNA (gRNA) are of utmost importance. A distinguishing feature of the MPMV gRNA packaging signal is two phylogenetically conserved long-range interactions (LRIs) between U5 and gag complementary sequences, LRI-I and LRI-II. To test their biological significance in the MPMV life cycle, we introduced mutations into these structural motifs and tested their effects on MPMV gRNA packaging and propagation. Furthermore, we probed the structure of key mutants using SHAPE (selective 2'hydroxyl acylation analyzed by primer extension). Disrupting base-pairing of the LRIs affected gRNA packaging and propagation, demonstrating their significance to the MPMV life cycle. A double mutant restoring a heterologous LRI-I was fully functional, whereas a similar LRI-II mutant failed to restore gRNA packaging and propagation. These results demonstrate that while LRI-I acts at the structural level, maintaining base-pairing is not sufficient for LRI-II function. In addition, in vitro RNA dimerization assays indicated that the loss of RNA packaging in LRI mutants could not be attributed to the defects in dimerization. Our findings suggest that U5-gag LRIs play an important architectural role in maintaining the structure of the 5' region of the MPMV gRNA, expanding the crucial role of LRIs to the nonlentiviral group of retroviruses. © 2016 Kalloush et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Assessing genetically modified crops to minimize the risk of increased food allergy: a review.

PubMed

Goodman, Richard E; Hefle, Susan L; Taylor, Steven L; van Ree, Ronald

2005-06-01

The first genetically modified (GM) crops approved for food use (tomato and soybean) were evaluated for safety by the United States Food and Drug Administration prior to commercial production. Among other factors, those products and all additional GM crops that have been grown commercially have been evaluated for potential increases in allergenic properties using methods that are consistent with the current understanding of food allergens and knowledge regarding the prediction of allergenic activity. Although there have been refinements, the key aspects of the evaluation have not changed. The allergenic properties of the gene donor and the host (recipient) organisms are considered in determining the appropriate testing strategy. The amino acid sequence of the encoded protein is compared to all known allergens to determine whether the protein is a known allergen or is sufficiently similar to any known allergen to indicate an increased probability of allergic cross-reactivity. Stability of the protein in the presence of acid with the stomach protease pepsin is tested as a risk factor for food allergenicity. In vitro or in vivo human IgE binding are tested when appropriate, if the gene donor is an allergen or the sequence of the protein is similar to an allergen. Serum donors and skin test subjects are selected based on their proven allergic responses to the gene donor or to material containing the allergen that was matched in sequence. While some scientists and regulators have suggested using animal models, performing broadly targeted serum IgE testing or extensive pre- or post-market clinical tests, current evidence does not support these tests as being predictive or practical. Based on the evidence to date, the current assessment process has worked well to prevent the unintended introduction of allergens in commercial GM crops.

Measuring the Electronic Properties of DNA-Specific Schottky Diodes Towards Detecting and Identifying Basidiomycetes DNA

PubMed Central

Periasamy, Vengadesh; Rizan, Nastaran; Al-Ta’ii, Hassan Maktuff Jaber; Tan, Yee Shin; Tajuddin, Hairul Annuar; Iwamoto, Mitsumasa

2016-01-01

The discovery of semiconducting behavior of deoxyribonucleic acid (DNA) has resulted in a large number of literatures in the study of DNA electronics. Sequence-specific electronic response provides a platform towards understanding charge transfer mechanism and therefore the electronic properties of DNA. It is possible to utilize these characteristic properties to identify/detect DNA. In this current work, we demonstrate a novel method of DNA-based identification of basidiomycetes using current-voltage (I-V) profiles obtained from DNA-specific Schottky barrier diodes. Electronic properties such as ideality factor, barrier height, shunt resistance, series resistance, turn-on voltage, knee-voltage, breakdown voltage and breakdown current were calculated and used to quantify the identification process as compared to morphological and molecular characterization techniques. The use of these techniques is necessary in order to study biodiversity, but sometimes it can be misleading and unreliable and is not sufficiently useful for the identification of fungi genera. Many of these methods have failed when it comes to identification of closely related species of certain genus like Pleurotus. Our electronics profiles, both in the negative and positive bias regions were however found to be highly characteristic according to the base-pair sequences. We believe that this simple, low-cost and practical method could be useful towards identifying and detecting DNA in biotechnology and pathology. PMID:27435636

Measuring the Electronic Properties of DNA-Specific Schottky Diodes Towards Detecting and Identifying Basidiomycetes DNA

NASA Astrophysics Data System (ADS)

Periasamy, Vengadesh; Rizan, Nastaran; Al-Ta'Ii, Hassan Maktuff Jaber; Tan, Yee Shin; Tajuddin, Hairul Annuar; Iwamoto, Mitsumasa

2016-07-01

The discovery of semiconducting behavior of deoxyribonucleic acid (DNA) has resulted in a large number of literatures in the study of DNA electronics. Sequence-specific electronic response provides a platform towards understanding charge transfer mechanism and therefore the electronic properties of DNA. It is possible to utilize these characteristic properties to identify/detect DNA. In this current work, we demonstrate a novel method of DNA-based identification of basidiomycetes using current-voltage (I-V) profiles obtained from DNA-specific Schottky barrier diodes. Electronic properties such as ideality factor, barrier height, shunt resistance, series resistance, turn-on voltage, knee-voltage, breakdown voltage and breakdown current were calculated and used to quantify the identification process as compared to morphological and molecular characterization techniques. The use of these techniques is necessary in order to study biodiversity, but sometimes it can be misleading and unreliable and is not sufficiently useful for the identification of fungi genera. Many of these methods have failed when it comes to identification of closely related species of certain genus like Pleurotus. Our electronics profiles, both in the negative and positive bias regions were however found to be highly characteristic according to the base-pair sequences. We believe that this simple, low-cost and practical method could be useful towards identifying and detecting DNA in biotechnology and pathology.

A novel EML4-ALK variant: exon 6 of EML4 fused to exon 19 of ALK.

PubMed

Penzel, Roland; Schirmacher, Peter; Warth, Arne

2012-07-01

Cytotoxic chemotherapy remains the mainstay of treatment for most patients with advanced disease. Recently, anaplastic lymphoma kinase (ALK) expression as a major target for successful treatment with ALK inhibitors was detected in a subset of non-small-cell lung carcinomas, usually as a result of echinoderm microtubule-associated protein-like 4 (EML4)-ALK rearrangements. Although the chromosomal breakpoint within the EML4 gene varied, the breakpoint within ALK was most frequently reported within intron 19 or rarely in exon 20. Therefore, the different EML4-ALK variants so far contain the same 3' portion of ALK starting with exon 20. Here, we report a novel EML4-ALK variant detected by reverse transcription polymerase chain reaction analysis. Subsequent sequencing revealed an EML4-ALK fusion variant in which exon 6 of EML4 was fused to exon 19 of ALK. It occurred in a predominant solid pulmonary adenocarcinoma of a 65-year-old woman with a clear split signal of ALK in fluorescence in situ hybridization analysis and a weakly homogeneous ALK expression in immunohistochemical staining. Because of the growing number of fusion variants a primary reverse transcription polymerase chain reaction-based screening for ALK-positive non-small-cell lung carcinoma patients may not be sufficient for predictive diagnostics but transcript-based approaches and sequencing of ALK fusion variants might finally contribute to an optimized selection of patients.

Experimental Strategies for Functional Annotation and Metabolism Discovery: Targeted Screening of Solute Binding Proteins and Unbiased Panning of Metabolomes

DOE PAGES

Vetting, Matthew W.; Al-Obaidi, Nawar; Zhao, Suwen; ...

2014-12-25

The rate at which genome sequencing data is accruing demands enhanced methods for functional annotation and metabolism discovery. Solute binding proteins (SBPs) facilitate the transport of the first reactant in a metabolic pathway, thereby constraining the regions of chemical space and the chemistries that must be considered for pathway reconstruction. Here in this paper, we describe high-throughput protein production and differential scanning fluorimetry platforms, which enabled the screening of 158 SBPs against a 189 component library specifically tailored for this class of proteins. Like all screening efforts, this approach is limited by the practical constraints imposed by construction of themore » library, i.e., we can study only those metabolites that are known to exist and which can be made in sufficient quantities for experimentation. To move beyond these inherent limitations, we illustrate the promise of crystallographic- and mass spectrometric-based approaches for the unbiased use of entire metabolomes as screening libraries. Together, our approaches identified 40 new SBP ligands, generated experiment-based annotations for 2084 SBPs in 71 isofunctional clusters, and defined numerous metabolic pathways, including novel catabolic pathways for the utilization of ethanolamine as sole nitrogen source and the use of D-Ala-D-Ala as sole carbon source. These efforts begin to define an integrated strategy for realizing the full value of amassing genome sequence data.« less

Holding a manual response sequence in memory can disrupt vocal responses that share semantic features with the manual response.

PubMed

Fournier, Lisa Renee; Wiediger, Matthew D; McMeans, Ryan; Mattson, Paul S; Kirkwood, Joy; Herzog, Theibot

2010-07-01

Holding an action plan in memory for later execution can delay execution of another action if the actions share a similar (compatible) feature. This compatibility interference (CI) occurs for actions that share the same response modality (e.g., manual response). We investigated whether CI can generalize to actions that utilize different response modalities (manual and vocal). In three experiments, participants planned and withheld a sequence of key-presses with the left- or right-hand based on the visual identity of the first stimulus, and then immediately executed a speeded, vocal response ('left' or 'right') to a second visual stimulus. The vocal response was based on discriminating stimulus color (Experiment 1), reading a written word (Experiment 2), or reporting the antonym of a written word (Experiment 3). Results showed that CI occurred when the manual response hand (e.g., left) was compatible with the identity of the vocal response (e.g., 'left') in Experiment 1 and 3, but not in Experiment 2. This suggests that partial overlap of semantic codes is sufficient to obtain CI unless the intervening action can be accessed automatically (Experiment 2). These findings are consistent with the code occupation hypothesis and the general framework of the theory of event coding (Behav Brain Sci 24:849-878, 2001a; Behav Brain Sci 24:910-937, 2001b).

Comparing the normalization methods for the differential analysis of Illumina high-throughput RNA-Seq data.

PubMed

Li, Peipei; Piao, Yongjun; Shon, Ho Sun; Ryu, Keun Ho

2015-10-28

Recently, rapid improvements in technology and decrease in sequencing costs have made RNA-Seq a widely used technique to quantify gene expression levels. Various normalization approaches have been proposed, owing to the importance of normalization in the analysis of RNA-Seq data. A comparison of recently proposed normalization methods is required to generate suitable guidelines for the selection of the most appropriate approach for future experiments. In this paper, we compared eight non-abundance (RC, UQ, Med, TMM, DESeq, Q, RPKM, and ERPKM) and two abundance estimation normalization methods (RSEM and Sailfish). The experiments were based on real Illumina high-throughput RNA-Seq of 35- and 76-nucleotide sequences produced in the MAQC project and simulation reads. Reads were mapped with human genome obtained from UCSC Genome Browser Database. For precise evaluation, we investigated Spearman correlation between the normalization results from RNA-Seq and MAQC qRT-PCR values for 996 genes. Based on this work, we showed that out of the eight non-abundance estimation normalization methods, RC, UQ, Med, TMM, DESeq, and Q gave similar normalization results for all data sets. For RNA-Seq of a 35-nucleotide sequence, RPKM showed the highest correlation results, but for RNA-Seq of a 76-nucleotide sequence, least correlation was observed than the other methods. ERPKM did not improve results than RPKM. Between two abundance estimation normalization methods, for RNA-Seq of a 35-nucleotide sequence, higher correlation was obtained with Sailfish than that with RSEM, which was better than without using abundance estimation methods. However, for RNA-Seq of a 76-nucleotide sequence, the results achieved by RSEM were similar to without applying abundance estimation methods, and were much better than with Sailfish. Furthermore, we found that adding a poly-A tail increased alignment numbers, but did not improve normalization results. Spearman correlation analysis revealed that RC, UQ, Med, TMM, DESeq, and Q did not noticeably improve gene expression normalization, regardless of read length. Other normalization methods were more efficient when alignment accuracy was low; Sailfish with RPKM gave the best normalization results. When alignment accuracy was high, RC was sufficient for gene expression calculation. And we suggest ignoring poly-A tail during differential gene expression analysis.

Randomizer for High Data Rates

NASA Technical Reports Server (NTRS)

Garon, Howard; Sank, Victor J.

2018-01-01

NASA as well as a number of other space agencies now recognize that the current recommended CCSDS randomizer used for telemetry (TM) is too short. When multiple applications of the PN8 Maximal Length Sequence (MLS) are required in order to fully cover a channel access data unit (CADU), spectral problems in the form of elevated spurious discretes (spurs) appear. Originally the randomizer was called a bit transition generator (BTG) precisely because it was thought that its primary value was to insure sufficient bit transitions to allow the bit/symbol synchronizer to lock and remain locked. We, NASA, have shown that the old BTG concept is a limited view of the real value of the randomizer sequence and that the randomizer also aids in signal acquisition as well as minimizing the potential for false decoder lock. Under the guidelines we considered here there are multiple maximal length sequences under GF(2) which appear attractive in this application. Although there may be mitigating reasons why another MLS sequence could be selected, one sequence in particular possesses a combination of desired properties which offsets it from the others.

Facile Recovery of Individual High-Molecular-Weight, Low-Copy-Number Natural Plasmids for Genomic Sequencing

DOE Office of Scientific and Technical Information (OSTI.GOV)

Williams, L.E.; Detter, C,; Barrie, K.

2006-06-01

Sequencing of the large (>50 kb), low-copy-number (<5 per cell) plasmids that mediate horizontal gene transfer has been hindered by the difficulty and expense of isolating DNA from individual plasmids of this class. We report here that a kit method previously devised for purification of bacterial artificial chromosomes (BACs) can be adapted for effective preparation of individual plasmids up to 220 kb from wild gram-negative and gram-positive bacteria. Individual plasmid DNA recovered from less than 10 ml of Escherichia coli, Staphylococcus, and Corynebacterium cultures was of sufficient quantity and quality for construction of highcoverage libraries, as shown by sequencing fivemore » native plasmids ranging in size from 30 kb to 94 kb. We also report recommendations for vector screening to optimize plasmid sequence assembly, preliminary annotation of novel plasmid genomes, and insights on mobile genetic element biology derived from these sequences. Adaptation of this BAC method for large plasmid isolation removes one major technical hurdle to expanding our knowledge of the natural plasmid gene pool.« less

Resolving the Complexity of Human Skin Metagenomes Using Single-Molecule Sequencing

PubMed Central

Tsai, Yu-Chih; Deming, Clayton; Segre, Julia A.; Kong, Heidi H.; Korlach, Jonas

2016-01-01

ABSTRACT Deep metagenomic shotgun sequencing has emerged as a powerful tool to interrogate composition and function of complex microbial communities. Computational approaches to assemble genome fragments have been demonstrated to be an effective tool for de novo reconstruction of genomes from these communities. However, the resultant “genomes” are typically fragmented and incomplete due to the limited ability of short-read sequence data to assemble complex or low-coverage regions. Here, we use single-molecule, real-time (SMRT) sequencing to reconstruct a high-quality, closed genome of a previously uncharacterized Corynebacterium simulans and its companion bacteriophage from a skin metagenomic sample. Considerable improvement in assembly quality occurs in hybrid approaches incorporating short-read data, with even relatively small amounts of long-read data being sufficient to improve metagenome reconstruction. Using short-read data to evaluate strain variation of this C. simulans in its skin community at single-nucleotide resolution, we observed a dominant C. simulans strain with moderate allelic heterozygosity throughout the population. We demonstrate the utility of SMRT sequencing and hybrid approaches in metagenome quantitation, reconstruction, and annotation. PMID:26861018

Investigating Correlation between Protein Sequence Similarity and Semantic Similarity Using Gene Ontology Annotations.

PubMed

Ikram, Najmul; Qadir, Muhammad Abdul; Afzal, Muhammad Tanvir

2018-01-01

Sequence similarity is a commonly used measure to compare proteins. With the increasing use of ontologies, semantic (function) similarity is getting importance. The correlation between these measures has been applied in the evaluation of new semantic similarity methods, and in protein function prediction. In this research, we investigate the relationship between the two similarity methods. The results suggest absence of a strong correlation between sequence and semantic similarities. There is a large number of proteins with low sequence similarity and high semantic similarity. We observe that Pearson's correlation coefficient is not sufficient to explain the nature of this relationship. Interestingly, the term semantic similarity values above 0 and below 1 do not seem to play a role in improving the correlation. That is, the correlation coefficient depends only on the number of common GO terms in proteins under comparison, and the semantic similarity measurement method does not influence it. Semantic similarity and sequence similarity have a distinct behavior. These findings are of significant effect for future works on protein comparison, and will help understand the semantic similarity between proteins in a better way.

Discovery sequence and the nature of low permeability gas accumulations

USGS Publications Warehouse

Attanasi, E.D.

2005-01-01

There is an ongoing discussion regarding the geologic nature of accumulations that host gas in low-permeability sandstone environments. This note examines the discovery sequence of the accumulations in low permeability sandstone plays that were classified as continuous-type by the U.S. Geological Survey for the 1995 National Oil and Gas Assessment. It compares the statistical character of historical discovery sequences of accumulations associated with continuous-type sandstone gas plays to those of conventional plays. The seven sandstone plays with sufficient data exhibit declining size with sequence order, on average, and in three of the seven the trend is statistically significant. Simulation experiments show that both a skewed endowment size distribution and a discovery process that mimics sampling proportional to size are necessary to generate a discovery sequence that consistently produces a statistically significant negative size order relationship. The empirical findings suggest that discovery sequence could be used to constrain assessed gas in untested areas. The plays examined represent 134 of the 265 trillion cubic feet of recoverable gas assessed in undeveloped areas of continuous-type gas plays in low permeability sandstone environments reported in the 1995 National Assessment. ?? 2005 International Association for Mathematical Geology.

SSPACE-LongRead: scaffolding bacterial draft genomes using long read sequence information

PubMed Central

2014-01-01

Background The recent introduction of the Pacific Biosciences RS single molecule sequencing technology has opened new doors to scaffolding genome assemblies in a cost-effective manner. The long read sequence information is promised to enhance the quality of incomplete and inaccurate draft assemblies constructed from Next Generation Sequencing (NGS) data. Results Here we propose a novel hybrid assembly methodology that aims to scaffold pre-assembled contigs in an iterative manner using PacBio RS long read information as a backbone. On a test set comprising six bacterial draft genomes, assembled using either a single Illumina MiSeq or Roche 454 library, we show that even a 50× coverage of uncorrected PacBio RS long reads is sufficient to drastically reduce the number of contigs. Comparisons to the AHA scaffolder indicate our strategy is better capable of producing (nearly) complete bacterial genomes. Conclusions The current work describes our SSPACE-LongRead software which is designed to upgrade incomplete draft genomes using single molecule sequences. We conclude that the recent advances of the PacBio sequencing technology and chemistry, in combination with the limited computational resources required to run our program, allow to scaffold genomes in a fast and reliable manner. PMID:24950923

Effects of temperature and mass conservation on the typical chemical sequences of hydrogen oxidation

NASA Astrophysics Data System (ADS)

Nicholson, Schuyler B.; Alaghemandi, Mohammad; Green, Jason R.

2018-01-01

Macroscopic properties of reacting mixtures are necessary to design synthetic strategies, determine yield, and improve the energy and atom efficiency of many chemical processes. The set of time-ordered sequences of chemical species are one representation of the evolution from reactants to products. However, only a fraction of the possible sequences is typical, having the majority of the joint probability and characterizing the succession of chemical nonequilibrium states. Here, we extend a variational measure of typicality and apply it to atomistic simulations of a model for hydrogen oxidation over a range of temperatures. We demonstrate an information-theoretic methodology to identify typical sequences under the constraints of mass conservation. Including these constraints leads to an improved ability to learn the chemical sequence mechanism from experimentally accessible data. From these typical sequences, we show that two quantities defining the variational typical set of sequences—the joint entropy rate and the topological entropy rate—increase linearly with temperature. These results suggest that, away from explosion limits, data over a narrow range of thermodynamic parameters could be sufficient to extrapolate these typical features of combustion chemistry to other conditions.

On a new class of completely integrable nonlinear wave equations. II. Multi-Hamiltonian structure

NASA Astrophysics Data System (ADS)

Nutku, Y.

1987-11-01

The multi-Hamiltonian structure of a class of nonlinear wave equations governing the propagation of finite amplitude waves is discussed. Infinitely many conservation laws had earlier been obtained for these equations. Starting from a (primary) Hamiltonian formulation of these equations the necessary and sufficient conditions for the existence of bi-Hamiltonian structure are obtained and it is shown that the second Hamiltonian operator can be constructed solely through a knowledge of the first Hamiltonian function. The recursion operator which first appears at the level of bi-Hamiltonian structure gives rise to an infinite sequence of conserved Hamiltonians. It is found that in general there exist two different infinite sequences of conserved quantities for these equations. The recursion relation defining higher Hamiltonian structures enables one to obtain the necessary and sufficient conditions for the existence of the (k+1)st Hamiltonian operator which depends on the kth Hamiltonian function. The infinite sequence of conserved Hamiltonians are common to all the higher Hamiltonian structures. The equations of gas dynamics are discussed as an illustration of this formalism and it is shown that in general they admit tri-Hamiltonian structure with two distinct infinite sets of conserved quantities. The isothermal case of γ=1 is an exceptional one that requires separate treatment. This corresponds to a specialization of the equations governing the expansion of plasma into vacuum which will be shown to be equivalent to Poisson's equation in nonlinear acoustics.

PET Imaging Stability Measurements During Simultaneous Pulsing of Aggressive MR Sequences on the SIGNA PET/MR System.

PubMed

Deller, Timothy W; Khalighi, Mohammad Mehdi; Jansen, Floris P; Glover, Gary H

2018-01-01

The recent introduction of simultaneous whole-body PET/MR scanners has enabled new research taking advantage of the complementary information obtainable with PET and MRI. One such application is kinetic modeling, which requires high levels of PET quantitative stability. To accomplish the required PET stability levels, the PET subsystem must be sufficiently isolated from the effects of MR activity. Performance measurements have previously been published, demonstrating sufficient PET stability in the presence of MR pulsing for typical clinical use; however, PET stability during radiofrequency (RF)-intensive and gradient-intensive sequences has not previously been evaluated for a clinical whole-body scanner. In this work, PET stability of the GE SIGNA PET/MR was examined during simultaneous scanning of aggressive MR pulse sequences. Methods: PET performance tests were acquired with MR idle and during simultaneous MR pulsing. Recent system improvements mitigating RF interference and gain variation were used. A fast recovery fast spin echo MR sequence was selected for high RF power, and an echo planar imaging sequence was selected for its high heat-inducing gradients. Measurements were performed to determine PET stability under varying MR conditions using the following metrics: sensitivity, scatter fraction, contrast recovery, uniformity, count rate performance, and image quantitation. A final PET quantitative stability assessment for simultaneous PET scanning during functional MRI studies was performed with a spiral in-and-out gradient echo sequence. Results: Quantitation stability of a 68 Ge flood phantom was demonstrated within 0.34%. Normalized sensitivity was stable during simultaneous scanning within 0.3%. Scatter fraction measured with a 68 Ge line source in the scatter phantom was stable within the range of 40.4%-40.6%. Contrast recovery and uniformity were comparable for PET images acquired simultaneously with multiple MR conditions. Peak noise equivalent count rate was 224 kcps at an effective activity concentration of 18.6 kBq/mL, and the count rate curves and scatter fraction curve were consistent for the alternating MR pulsing states. A final test demonstrated quantitative stability during a spiral functional MRI sequence. Conclusion: PET stability metrics demonstrated that PET quantitation was not affected during simultaneous aggressive MRI. This stability enables demanding applications such as kinetic modeling. © 2018 by the Society of Nuclear Medicine and Molecular Imaging.

Glyphosate has limited short-term effects on commensal bacterial community composition in the gut environment due to sufficient aromatic amino acid levels.

PubMed

Nielsen, Lene Nørby; Roager, Henrik M; Casas, Mònica Escolà; Frandsen, Henrik L; Gosewinkel, Ulrich; Bester, Kai; Licht, Tine Rask; Hendriksen, Niels Bohse; Bahl, Martin Iain

2018-02-01

Recently, concerns have been raised that residues of glyphosate-based herbicides may interfere with the homeostasis of the intestinal bacterial community and thereby affect the health of humans or animals. The biochemical pathway for aromatic amino acid synthesis (Shikimate pathway), which is specifically inhibited by glyphosate, is shared by plants and numerous bacterial species. Several in vitro studies have shown that various groups of intestinal bacteria may be differently affected by glyphosate. Here, we present results from an animal exposure trial combining deep 16S rRNA gene sequencing of the bacterial community with liquid chromatography mass spectrometry (LC-MS) based metabolic profiling of aromatic amino acids and their downstream metabolites. We found that glyphosate as well as the commercial formulation Glyfonova ® 450 PLUS administered at up to fifty times the established European Acceptable Daily Intake (ADI = 0.5 mg/kg body weight) had very limited effects on bacterial community composition in Sprague Dawley rats during a two-week exposure trial. The effect of glyphosate on prototrophic bacterial growth was highly dependent on the availability of aromatic amino acids, suggesting that the observed limited effect on bacterial composition was due to the presence of sufficient amounts of aromatic amino acids in the intestinal environment. A strong correlation was observed between intestinal concentrations of glyphosate and intestinal pH, which may partly be explained by an observed reduction in acetic acid produced by the gut bacteria. We conclude that sufficient intestinal levels of aromatic amino acids provided by the diet alleviates the need for bacterial synthesis of aromatic amino acids and thus prevents an antimicrobial effect of glyphosate in vivo. It is however possible that the situation is different in cases of human malnutrition or in production animals. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Fe65-PTB2 Dimerization Mimics Fe65-APP Interaction.

PubMed

Feilen, Lukas P; Haubrich, Kevin; Strecker, Paul; Probst, Sabine; Eggert, Simone; Stier, Gunter; Sinning, Irmgard; Konietzko, Uwe; Kins, Stefan; Simon, Bernd; Wild, Klemens

2017-01-01

Physiological function and pathology of the Alzheimer's disease causing amyloid precursor protein (APP) are correlated with its cytosolic adaptor Fe65 encompassing a WW and two phosphotyrosine-binding domains (PTBs). The C-terminal Fe65-PTB2 binds a large portion of the APP intracellular domain (AICD) including the GYENPTY internalization sequence fingerprint. AICD binding to Fe65-PTB2 opens an intra-molecular interaction causing a structural change and altering Fe65 activity. Here we show that in the absence of the AICD, Fe65-PTB2 forms a homodimer in solution and determine its crystal structure at 2.6 Å resolution. Dimerization involves the unwinding of a C-terminal α-helix that mimics binding of the AICD internalization sequence, thus shielding the hydrophobic binding pocket. Specific dimer formation is validated by nuclear magnetic resonance (NMR) techniques and cell-based analyses reveal that Fe65-PTB2 together with the WW domain are necessary and sufficient for dimerization. Together, our data demonstrate that Fe65 dimerizes via its APP interaction site, suggesting that besides intra- also intermolecular interactions between Fe65 molecules contribute to homeostatic regulation of APP mediated signaling.

The mandibular symphysis as a starting point for the occlusal-level reconstruction of panfacial fractures with bicondylar fractures and interruption of the maxillary and mandibular arches: report of two cases.

PubMed

Pau, Mauro; Reinbacher, Knut Ernst; Feichtinger, Matthias; Navysany, Kawe; Kärcher, Hans

2014-06-01

Panfacial fractures represent a challenge, even for experienced maxillofacial surgeons, because all references for reconstructing the facial skeleton are missing. Logical reconstructive sequencing based on a clear understanding of the correlation between projection and the widths and lengths of facial subunits should enable the surgeon to achieve correct realignment of the bony framework of the face and to prevent late deformity and functional impairment. Reconstruction is particularly challenging in patients presenting with concomitant fractures at the Le Fort I level and affecting the palate, condyles, and mandibular symphysis. In cases without bony loss and sufficient dentition, we believe that accurate fixation of the mandibular symphysis can represent the starting point of a reconstructive sequence that allows successful reconstruction at the Le Fort I level. Two patients were treated in our department by reconstruction starting in the occlusal area through repair of the mandibular symphysis. Both patients considered the postoperative facial shape and profile to be satisfactory and comparable to the pre-injury situation. Copyright © 2013 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.

In Vivo Visualization of Alzheimer’s Amyloid Plaques by MRI in Transgenic Mice Without a Contrast Agent

PubMed Central

Jack, Clifford R.; Garwood, Michael; Wengenack, Thomas M.; Borowski, Bret; Curran, Geoffrey L.; Lin, Joseph; Adriany, Gregor; Grohn, Olli H.J.; Grimm, Roger; Poduslo, Joseph F.

2009-01-01

One of the cardinal pathologic features of Alzheimer’s disease (AD) is formation of senile, or amyloid, plaques. Transgenic mice have been developed that express one or more of the genes responsible for familial AD in humans. Doubly transgenic mice develop “human-like” plaques, providing a mechanism to study amyloid plaque biology in a controlled manner. Imaging of labeled plaques has been accomplished with other modalities, but only MRI has sufficient spatial and contrast resolution to visualize individual plaques non-invasively. Methods to optimize visualization of plaques in vivo in transgenic mice at 9.4 T using a spin echo sequence based on adiabatic pulses are described. Preliminary results indicate that a spin echo acquisition more accurately reflects plaque size, while a T2* weighted gradient echo sequence reflects plaque iron content not plaque size. In vivo MRI – ex vivo MRI – in vitro histological correlations are provided. Histologically verified plaques as small as 50 μm in diameter were visualized in the living animal. To our knowledge this work represents the first demonstration of non-invasive in vivo visualization of individual AD plaques without the use of a contrast agent. PMID:15562496

A Driving Bioinformatics Approach to Explore Co-regulation of AOX Gene Family Members During Growth and Development.

PubMed

Costa, José Hélio; Arnholdt-Schmitt, Birgit

2017-01-01

The alternative oxidase (AOX) gene family is a hot candidate for functional marker development that could help plant breeding on yield stability through more robust plants based on multi-stress tolerance. However, there is missing knowledge on the interplay between gene family members that might interfere with the efficiency of marker development. It is common view that AOX1 and AOX2 have different physiological roles. Nevertheless, both family member groups act in terms of molecular-biochemical function as "typical" alternative oxidases and co-regulation of AOX1 and AOX2 had been reported. Although conserved sequence differences had been identified, the basis for differential effects on physiology regulation is not sufficiently explored.This protocol gives instructions for a bioinformatics approach that supports discovering potential interaction of AOX family members in regulating growth and development. It further provides a strategy to elucidate the relevance of gene sequence diversity and copy number variation for final functionality in target tissues and finally the whole plant. Thus, overall this protocol provides the means for efficiently identifying plant AOX variants as functional marker candidates related to growth and development.

A Japanese Encephalitis Virus Peptide Present on Johnson Grass Mosaic Virus-Like Particles Induces Virus-Neutralizing Antibodies and Protects Mice against Lethal Challenge

PubMed Central

Saini, Manisha; Vrati, Sudhanshu

2003-01-01

Protection against Japanese encephalitis virus (JEV) is antibody dependent, and neutralizing antibodies alone are sufficient to impart protection. Thus, we are aiming to develop a peptide-based vaccine against JEV by identifying JEV peptide sequences that could induce virus-neutralizing antibodies. Previously, we have synthesized large amounts of Johnson grass mosaic virus (JGMV) coat protein (CP) in Escherichia coli and have shown that it autoassembled to form virus-like particles (VLPs). The envelope (E) protein of JEV contains the virus-neutralization epitopes. Four peptides from different locations within JEV E protein were chosen, and these were fused to JGMV CP by recombinant DNA methods. The fusion protein autoassembled to form VLPs that could be purified by sucrose gradient centrifugation. Immunization of mice with the recombinant VLPs containing JEV peptide sequences induced anti-peptide and anti-JEV antibodies. A 27-amino-acid peptide containing amino acids 373 to 399 from JEV E protein, present on JGMV VLPs, induced virus-neutralizing antibodies. Importantly, these antibodies were obtained without the use of an adjuvant. The immunized mice showed significant protection against a lethal JEV challenge. PMID:12610124

Fe65-PTB2 Dimerization Mimics Fe65-APP Interaction

PubMed Central

Feilen, Lukas P.; Haubrich, Kevin; Strecker, Paul; Probst, Sabine; Eggert, Simone; Stier, Gunter; Sinning, Irmgard; Konietzko, Uwe; Kins, Stefan; Simon, Bernd; Wild, Klemens

2017-01-01

Physiological function and pathology of the Alzheimer’s disease causing amyloid precursor protein (APP) are correlated with its cytosolic adaptor Fe65 encompassing a WW and two phosphotyrosine-binding domains (PTBs). The C-terminal Fe65-PTB2 binds a large portion of the APP intracellular domain (AICD) including the GYENPTY internalization sequence fingerprint. AICD binding to Fe65-PTB2 opens an intra-molecular interaction causing a structural change and altering Fe65 activity. Here we show that in the absence of the AICD, Fe65-PTB2 forms a homodimer in solution and determine its crystal structure at 2.6 Å resolution. Dimerization involves the unwinding of a C-terminal α-helix that mimics binding of the AICD internalization sequence, thus shielding the hydrophobic binding pocket. Specific dimer formation is validated by nuclear magnetic resonance (NMR) techniques and cell-based analyses reveal that Fe65-PTB2 together with the WW domain are necessary and sufficient for dimerization. Together, our data demonstrate that Fe65 dimerizes via its APP interaction site, suggesting that besides intra- also intermolecular interactions between Fe65 molecules contribute to homeostatic regulation of APP mediated signaling. PMID:28553201

Selection of the initial design for the two-stage continual reassessment method.

PubMed

Jia, Xiaoyu; Ivanova, Anastasia; Lee, Shing M

2017-01-01

In the two-stage continual reassessment method (CRM), model-based dose escalation is preceded by a pre-specified escalating sequence starting from the lowest dose level. This is appealing to clinicians because it allows a sufficient number of patients to be assigned to each of the lower dose levels before escalating to higher dose levels. While a theoretical framework to build the two-stage CRM has been proposed, the selection of the initial dose-escalating sequence, generally referred to as the initial design, remains arbitrary, either by specifying cohorts of three patients or by trial and error through extensive simulations. Motivated by a currently ongoing oncology dose-finding study for which clinicians explicitly stated their desire to assign at least one patient to each of the lower dose levels, we proposed a systematic approach for selecting the initial design for the two-stage CRM. The initial design obtained using the proposed algorithm yields better operating characteristics compared to using a cohort of three initial design with a calibrated CRM. The proposed algorithm simplifies the selection of initial design for the two-stage CRM. Moreover, initial designs to be used as reference for planning a two-stage CRM are provided.

A Comparison of RNA-Seq Results from Paired Formalin-Fixed Paraffin-Embedded and Fresh-Frozen Glioblastoma Tissue Samples

PubMed Central

Esteve-Codina, Anna; Arpi, Oriol; Martinez-García, Maria; Pineda, Estela; Mallo, Mar; Gut, Marta; Carrato, Cristina; Rovira, Anna; Lopez, Raquel; Tortosa, Avelina; Dabad, Marc; Del Barco, Sonia; Heath, Simon; Bagué, Silvia; Ribalta, Teresa; Alameda, Francesc; de la Iglesia, Nuria

2017-01-01

The molecular classification of glioblastoma (GBM) based on gene expression might better explain outcome and response to treatment than clinical factors. Whole transcriptome sequencing using next-generation sequencing platforms is rapidly becoming accepted as a tool for measuring gene expression for both research and clinical use. Fresh frozen (FF) tissue specimens of GBM are difficult to obtain since tumor tissue obtained at surgery is often scarce and necrotic and diagnosis is prioritized over freezing. After diagnosis, leftover tissue is usually stored as formalin-fixed paraffin-embedded (FFPE) tissue. However, RNA from FFPE tissues is usually degraded, which could hamper gene expression analysis. We compared RNA-Seq data obtained from matched pairs of FF and FFPE GBM specimens. Only three FFPE out of eleven FFPE-FF matched samples yielded informative results. Several quality-control measurements showed that RNA from FFPE samples was highly degraded but maintained transcriptomic similarities to RNA from FF samples. Certain issues regarding mutation analysis and subtype prediction were detected. Nevertheless, our results suggest that RNA-Seq of FFPE GBM specimens provides reliable gene expression data that can be used in molecular studies of GBM if the RNA is sufficiently preserved. PMID:28122052

Cell type discovery using single-cell transcriptomics: implications for ontological representation.

PubMed

Aevermann, Brian D; Novotny, Mark; Bakken, Trygve; Miller, Jeremy A; Diehl, Alexander D; Osumi-Sutherland, David; Lasken, Roger S; Lein, Ed S; Scheuermann, Richard H

2018-05-01

Cells are fundamental function units of multicellular organisms, with different cell types playing distinct physiological roles in the body. The recent advent of single-cell transcriptional profiling using RNA sequencing is producing 'big data', enabling the identification of novel human cell types at an unprecedented rate. In this review, we summarize recent work characterizing cell types in the human central nervous and immune systems using single-cell and single-nuclei RNA sequencing, and discuss the implications that these discoveries are having on the representation of cell types in the reference Cell Ontology (CL). We propose a method, based on random forest machine learning, for identifying sets of necessary and sufficient marker genes, which can be used to assemble consistent and reproducible cell type definitions for incorporation into the CL. The representation of defined cell type classes and their relationships in the CL using this strategy will make the cell type classes being identified by high-throughput/high-content technologies findable, accessible, interoperable and reusable (FAIR), allowing the CL to serve as a reference knowledgebase of information about the role that distinct cellular phenotypes play in human health and disease.

Main sequence models for massive zero-metal stars

NASA Technical Reports Server (NTRS)

Cary, N.

1974-01-01

Zero-age main-sequence models for stars of 20, 10, 5, and 2 solar masses with no heavy elements are constructed for three different possible primordial helium abundances: Y=0.00, Y=0.23, and Y=0.30. The latter two values of Y bracket the range of primordial helium abundances cited by Wagoner. With the exceptions of the two 20 solar mass models that contain helium, these models are found to be self-consistent in the sense that the formation of carbon through the triple-alpha process during premain sequence contraction is not sufficient to bring the CN cycle into competition with the proton-proton chain on the ZAMS. The zero-metal models of the present study have higher surface and central temperatures, higher central densities, smaller radii, and smaller convective cores than do the population I models with the same masses.

Space power system scheduling using an expert system

NASA Technical Reports Server (NTRS)

Bahrami, K. A.; Biefeld, E.; Costello, L.; Klein, J. W.

1986-01-01

A most pressing problem in space exploration is timely spacecraft power system sequence generation, which requires the scheduling of a set of loads given a set of resource constraints. This is particularly important after an anomaly or failure. This paper discusses the power scheduling problem and how the software program, Plan-It, can be used as a consultant for scheduling power system activities. Modeling of power activities, human interface, and two of the many strategies used by Plan-It are discussed. Preliminary results showing the development of a conflict-free sequence from an initial sequence with conflicts is presented. It shows that a 4-day schedule can be generated in a matter of a few minutes, which provides sufficient time in many cases to aid the crew in the replanning of loads and generation use following a failure or anomaly.

The low information content of Neurospora splicing signals: implications for RNA splicing and intron origin.

PubMed

Collins, Richard A; Stajich, Jason E; Field, Deborah J; Olive, Joan E; DeAbreu, Diane M

2015-05-01

When we expressed a small (0.9 kb) nonprotein-coding transcript derived from the mitochondrial VS plasmid in the nucleus of Neurospora we found that it was efficiently spliced at one or more of eight 5' splice sites and ten 3' splice sites, which are present apparently by chance in the sequence. Further experimental and bioinformatic analyses of other mitochondrial plasmids, random sequences, and natural nuclear genes in Neurospora and other fungi indicate that fungal spliceosomes recognize a wide range of 5' splice site and branchpoint sequences and predict introns to be present at high frequency in random sequence. In contrast, analysis of intronless fungal nuclear genes indicates that branchpoint, 5' splice site and 3' splice site consensus sequences are underrepresented compared with random sequences. This underrepresentation of splicing signals is sufficient to deplete the nuclear genome of splice sites at locations that do not comprise biologically relevant introns. Thus, the splicing machinery can recognize a wide range of splicing signal sequences, but splicing still occurs with great accuracy, not because the splicing machinery distinguishes correct from incorrect introns, but because incorrect introns are substantially depleted from the genome. © 2015 Collins et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Rescaled earthquake recurrence time statistics: application to microrepeaters

NASA Astrophysics Data System (ADS)

Goltz, Christian; Turcotte, Donald L.; Abaimov, Sergey G.; Nadeau, Robert M.; Uchida, Naoki; Matsuzawa, Toru

2009-01-01

Slip on major faults primarily occurs during `characteristic' earthquakes. The recurrence statistics of characteristic earthquakes play an important role in seismic hazard assessment. A major problem in determining applicable statistics is the short sequences of characteristic earthquakes that are available worldwide. In this paper, we introduce a rescaling technique in which sequences can be superimposed to establish larger numbers of data points. We consider the Weibull and log-normal distributions, in both cases we rescale the data using means and standard deviations. We test our approach utilizing sequences of microrepeaters, micro-earthquakes which recur in the same location on a fault. It seems plausible to regard these earthquakes as a miniature version of the classic characteristic earthquakes. Microrepeaters are much more frequent than major earthquakes, leading to longer sequences for analysis. In this paper, we present results for the analysis of recurrence times for several microrepeater sequences from Parkfield, CA as well as NE Japan. We find that, once the respective sequence can be considered to be of sufficient stationarity, the statistics can be well fitted by either a Weibull or a log-normal distribution. We clearly demonstrate this fact by our technique of rescaled combination. We conclude that the recurrence statistics of the microrepeater sequences we consider are similar to the recurrence statistics of characteristic earthquakes on major faults.

Calculating the quality of public high-throughput sequencing data to obtain a suitable subset for reanalysis from the Sequence Read Archive

PubMed Central

Nakazato, Takeru; Bono, Hidemasa

2017-01-01

Abstract It is important for public data repositories to promote the reuse of archived data. In the growing field of omics science, however, the increasing number of submissions of high-throughput sequencing (HTSeq) data to public repositories prevents users from choosing a suitable data set from among the large number of search results. Repository users need to be able to set a threshold to reduce the number of results to obtain a suitable subset of high-quality data for reanalysis. We calculated the quality of sequencing data archived in a public data repository, the Sequence Read Archive (SRA), by using the quality control software FastQC. We obtained quality values for 1 171 313 experiments, which can be used to evaluate the suitability of data for reuse. We also visualized the data distribution in SRA by integrating the quality information and metadata of experiments and samples. We provide quality information of all of the archived sequencing data, which enable users to obtain sufficient quality sequencing data for reanalyses. The calculated quality data are available to the public in various formats. Our data also provide an example of enhancing the reuse of public data by adding metadata to published research data by a third party. PMID:28449062

Exploring the temporal structure of heterochronous sequences using TempEst (formerly Path-O-Gen).

PubMed

Rambaut, Andrew; Lam, Tommy T; Max Carvalho, Luiz; Pybus, Oliver G

2016-01-01

Gene sequences sampled at different points in time can be used to infer molecular phylogenies on a natural timescale of months or years, provided that the sequences in question undergo measurable amounts of evolutionary change between sampling times. Data sets with this property are termed heterochronous and have become increasingly common in several fields of biology, most notably the molecular epidemiology of rapidly evolving viruses. Here we introduce the cross-platform software tool, TempEst (formerly known as Path-O-Gen), for the visualization and analysis of temporally sampled sequence data. Given a molecular phylogeny and the dates of sampling for each sequence, TempEst uses an interactive regression approach to explore the association between genetic divergence through time and sampling dates. TempEst can be used to (1) assess whether there is sufficient temporal signal in the data to proceed with phylogenetic molecular clock analysis, and (2) identify sequences whose genetic divergence and sampling date are incongruent. Examination of the latter can help identify data quality problems, including errors in data annotation, sample contamination, sequence recombination, or alignment error. We recommend that all users of the molecular clock models implemented in BEAST first check their data using TempEst prior to analysis.

Full genome sequence analysis of a novel adenovirus of rhesus macaque origin indicates a new simian adenovirus type and species.

PubMed

Malouli, Daniel; Howell, Grant L; Legasse, Alfred W; Kahl, Christoph; Axthelm, Michael K; Hansen, Scott G; Früh, Klaus

2014-09-01

Multiple novel simian adenoviruses have been isolated over the past years and their potential to cross the species barrier and infect the human population is an ever present threat. Here we describe the isolation and full genome sequencing of a novel simian adenovirus (SAdV) isolated from the urine of two independent, never co-housed, late stage simian immunodeficiency virus (SIV)-infected rhesus macaques. The viral genome sequences revealed a novel type with a unique genome length, GC content, E3 region and DNA polymerase amino acid sequence that is sufficiently distinct from all currently known human- or simian adenovirus species to warrant classifying these isolates as a novel species of simian adenovirus. This new species, termed Simian mastadenovirus D (SAdV-D), displays the standard genome organization for the genus Mastadenovirus containing only one copy of the fiber gene which sets it apart from the old world monkey adenovirus species HAdV-G, SAdV-B and SAdV-C.

Sequence co-evolution gives 3D contacts and structures of protein complexes

PubMed Central

Hopf, Thomas A; Schärfe, Charlotta P I; Rodrigues, João P G L M; Green, Anna G; Kohlbacher, Oliver; Sander, Chris; Bonvin, Alexandre M J J; Marks, Debora S

2014-01-01

Protein–protein interactions are fundamental to many biological processes. Experimental screens have identified tens of thousands of interactions, and structural biology has provided detailed functional insight for select 3D protein complexes. An alternative rich source of information about protein interactions is the evolutionary sequence record. Building on earlier work, we show that analysis of correlated evolutionary sequence changes across proteins identifies residues that are close in space with sufficient accuracy to determine the three-dimensional structure of the protein complexes. We evaluate prediction performance in blinded tests on 76 complexes of known 3D structure, predict protein–protein contacts in 32 complexes of unknown structure, and demonstrate how evolutionary couplings can be used to distinguish between interacting and non-interacting protein pairs in a large complex. With the current growth of sequences, we expect that the method can be generalized to genome-wide elucidation of protein–protein interaction networks and used for interaction predictions at residue resolution. DOI: http://dx.doi.org/10.7554/eLife.03430.001 PMID:25255213

Mitogenic effect contributes to increased virulence of Streptococcus suis sequence type 7 to cause streptococcal toxic shock-like syndrome.

PubMed

Zheng, H; Ye, C; Segura, M; Gottschalk, M; Xu, J

2008-09-01

Streptococcus suis serotype 2 sequence type 7 strains emerged in 1996 and caused a streptococcal toxic shock-like syndrome in 1998 and 2005 in China. Evidence indicated that the virulence of S. suis sequence type 7 had increased, but the mechanism was unknown. The sequence type 7 strain SC84, isolated from a patient with streptococcal toxic shock-like syndrome during the Sichuan outbreak, and the sequence type 1 strain 31533, a typical highly pathogenic strain isolated from a diseased pig, were used in comparative studies. In this study we show the mechanisms underlying cytokine production differed between the two types of strains. The S. suis sequence type 7 strain SC84 possesses a stronger capacity to stimulate T cells, naive T cells and peripheral blood mononuclear cell proliferation than does S. suis sequence type 1 strain 31533. The T cell response to both strains was dependent upon the presence of antigen-presenting cells. Histo-incompatible antigen-presenting cells were sufficient to provide the accessory signals to naive T cell stimulated by the two strains, indicating that both sequence type 7 and 1 strains possess mitogens; however, the mitogenic effect was different. Therefore, we propose that the difference in the mitogenic effect of sequence type 7 strain SC84 compared with the sequence type 1 strain 31533 of S. suis may be associated with the clinical, epidemiological and microbiological difference, where the ST 7 strains have a larger mitogenic effect.

Characterization and assessment of an avian repetitive DNA sequence as an icterid phylogenetic marker.

PubMed

Quinn, J S; Guglich, E; Seutin, G; Lau, R; Marsolais, J; Parna, L; Boag, P T; White, B N

1992-02-01

The first tandemly repeated sequence examined in a passerine bird, a 431-bp PstI fragment named pMAT1, has been cloned from the genome of the brown-headed cowbird (Molothrus ater). The sequence represents about 5-10% of the genome (about 4 x 10(5) copies) and yields prominent ethidium bromide stained bands when genomic DNA cut with a variety of restriction enzymes is electrophoresed in agarose gels. A particularly striking ladder of fragments is apparent when the DNA is cut with HinfI, indicative of a tandem arrangement of the monomer. The cloned PstI monomer has been sequenced, revealing no internal repeated structure. There are sequences that hybridize with pMAT1 found in related nine-primaried oscines but not in more distantly related oscines, suboscines, or nonpasserine species. Little sequence similarity to tandemly repeated PstI cut sequences from the merlin (Falco columbarius), saurus crane (Grus antigone), or Puerto Rican parrot (Amazona vittata) or to HinfI digested sequence from the Toulouse goose (Anser anser) was detected. The isolated sequence was used as a probe to examine DNA samples of eight members of the tribe Icterini. This examination revealed phylogenetically informative characters. The repeat contains cutting sites from a number of restriction enzymes, which, if sufficiently polymorphic, would provide new phylogenetic characters. Sequences like these, conserved within a species, but variable between closely related species, may be very useful for phylogenetic studies of closely related taxa.

Mitogenic effect contributes to increased virulence of Streptococcus suis sequence type 7 to cause streptococcal toxic shock-like syndrome

PubMed Central

Zheng, H; Ye, C; Segura, M; Gottschalk, M; Xu, J

2008-01-01

Streptococcus suis serotype 2 sequence type 7 strains emerged in 1996 and caused a streptococcal toxic shock-like syndrome in 1998 and 2005 in China. Evidence indicated that the virulence of S. suis sequence type 7 had increased, but the mechanism was unknown. The sequence type 7 strain SC84, isolated from a patient with streptococcal toxic shock-like syndrome during the Sichuan outbreak, and the sequence type 1 strain 31533, a typical highly pathogenic strain isolated from a diseased pig, were used in comparative studies. In this study we show the mechanisms underlying cytokine production differed between the two types of strains. The S. suis sequence type 7 strain SC84 possesses a stronger capacity to stimulate T cells, naive T cells and peripheral blood mononuclear cell proliferation than does S. suis sequence type 1 strain 31533. The T cell response to both strains was dependent upon the presence of antigen-presenting cells. Histo-incompatible antigen-presenting cells were sufficient to provide the accessory signals to naive T cell stimulated by the two strains, indicating that both sequence type 7 and 1 strains possess mitogens; however, the mitogenic effect was different. Therefore, we propose that the difference in the mitogenic effect of sequence type 7 strain SC84 compared with the sequence type 1 strain 31533 of S. suis may be associated with the clinical, epidemiological and microbiological difference, where the ST 7 strains have a larger mitogenic effect. PMID:18803762

Molecular evolution of ependymin and the phylogenetic resolution of early divergences among euteleost fishes.

PubMed

Ortí, G; Meyer, A

1996-04-01

The rate and pattern of DNA evolution of ependymin, a single-copy gene coding for a highly expressed glycoprotein in the brain matrix of teleost fishes, is characterized and its phylogenetic utility for fish systematics is assessed. DNA sequences were determined from catfish, electric fish, and characiforms and compared with published ependymin sequences from cyprinids, salmon, pike, and herring. Among these groups, ependymin amino acid sequences were highly divergent (up to 60% sequence difference), but had surprisingly similar hydropathy profiles and invariant glycosylation sites, suggesting that functional properties of the proteins are conserved. Comparison of base composition at third codon positions and introns revealed AT-rich introns and GC-rich third codon positions, suggesting that the biased codon usage observed might not be due to mutational bias. Phylogenetic information content of third codon positions was surprisingly high and sufficient to recover the most basal nodes of the tree, in spite of the observation that pairwise distances (at third codon positions) were well above the presumed saturation level. This finding can be explained by the high proportion of phylogenetically informative nonsynonymous changes at third codon positions among these highly divergent proteins. Ependymin DNA sequences have established the first molecular evidence for the monophyly of a group containing salmonids and esociforms. In addition, ependymin suggests a sister group relationship of electric fish (Gymnotiformes) and Characiformes, constituting a significant departure from currently accepted classifications. However, relationships among characiform lineages were not completely resolved by ependymin sequences in spite of seemingly appropriate levels of variation among taxa and considerably low levels of homoplasy in the data (consistency index = 0.7). If the diversification of Characiformes took place in an "explosive" manner, over a relatively short period of time this pattern should also be observed using other phylogenetic markers. Poor conservation of ependymin's primary structure hinders the design of efficient primers for PCR that could be used in wide-ranging fish systematic studies. However, alternative methods like PCR amplification from cDNA used here should provide promising comparative sequence data for the resolution of phylogenetic relationships among other basal lineages of teleost fishes.

HCV Genotyping from NGS Short Reads and Its Application in Genotype Detection from HCV Mixed Infected Plasma

PubMed Central

Qiu, Ping; Stevens, Richard; Wei, Bo; Lahser, Fred; Howe, Anita Y. M.; Klappenbach, Joel A.; Marton, Matthew J.

2015-01-01

Genotyping of hepatitis C virus (HCV) plays an important role in the treatment of HCV. As new genotype-specific treatment options become available, it has become increasingly important to have accurate HCV genotype and subtype information to ensure that the most appropriate treatment regimen is selected. Most current genotyping methods are unable to detect mixed genotypes from two or more HCV infections. Next generation sequencing (NGS) allows for rapid and low cost mass sequencing of viral genomes and provides an opportunity to probe the viral population from a single host. In this paper, the possibility of using short NGS reads for direct HCV genotyping without genome assembly was evaluated. We surveyed the publicly-available genetic content of three HCV drug target regions (NS3, NS5A, NS5B) in terms of whether these genes contained genotype-specific regions that could predict genotype. Six genotypes and 38 subtypes were included in this study. An automated phylogenetic analysis based HCV genotyping method was implemented and used to assess different HCV target gene regions. Candidate regions of 250-bp each were found for all three genes that have enough genetic information to predict HCV genotypes/subtypes. Validation using public datasets shows 100% genotyping accuracy. To test whether these 250-bp regions were sufficient to identify mixed genotypes, we developed a random primer-based method to sequence HCV plasma samples containing mixtures of two HCV genotypes in different ratios. We were able to determine the genotypes without ambiguity and to quantify the ratio of the abundances of the mixed genotypes in the samples. These data provide a proof-of-concept that this random primed, NGS-based short-read genotyping approach does not need prior information about the viral population and is capable of detecting mixed viral infection. PMID:25830316

Technical Evaluation: Identification of Pathogenic Mutations in PKD1 and PKD2 in Patients with Autosomal Dominant Polycystic Kidney Disease by Next-Generation Sequencing and Use of a Comprehensive New Classification System.

PubMed

Kinoshita, Moritoshi; Higashihara, Eiji; Kawano, Haruna; Higashiyama, Ryo; Koga, Daisuke; Fukui, Takafumi; Gondo, Nobuhisa; Oka, Takehiko; Kawahara, Kozo; Rigo, Krisztina; Hague, Tim; Katsuragi, Kiyonori; Sudo, Kimiyoshi; Takeshi, Masahiko; Horie, Shigeo; Nutahara, Kikuo

2016-01-01

Genetic testing of PKD1 and PKD2 is expected to play an increasingly important role in determining allelic influences in autosomal dominant polycystic kidney disease (ADPKD) in the near future. However, to date, genetic testing is not commonly employed because it is expensive, complicated because of genetic heterogeneity, and does not easily identify pathogenic variants. In this study, we developed a genetic testing system based on next-generation sequencing (NGS), long-range polymerase chain reaction, and a new software package. The new software package integrated seven databases and provided access to five cloud-based computing systems. The database integrated 241 polymorphic nonpathogenic variants detected in 140 healthy Japanese volunteers aged >35 years, who were confirmed by ultrasonography as having no cysts in either kidney. Using this system, we identified 60 novel and 30 known pathogenic mutations in 101 Japanese patients with ADPKD, with an overall detection rate of 89.1% (90/101) [95% confidence interval (CI), 83.0%-95.2%]. The sensitivity of the system increased to 93.1% (94/101) (95% CI, 88.1%-98.0%) when combined with multiplex ligation-dependent probe amplification analysis, making it sufficient for use in a clinical setting. In 82 (87.2%) of the patients, pathogenic mutations were detected in PKD1 (95% CI, 79.0%-92.5%), whereas in 12 (12.8%) patients pathogenic mutations were detected in PKD2 (95% CI, 7.5%-21.0%); this is consistent with previously reported findings. In addition, we were able to reconfirm our pathogenic mutation identification results using Sanger sequencing. In conclusion, we developed a high-sensitivity NGS-based system and successfully employed it to identify pathogenic mutations in PKD1 and PKD2 in Japanese patients with ADPKD.

High-density genetic map construction and QTLs identification for plant height in white jute (Corchorus capsularis L.) using specific locus amplified fragment (SLAF) sequencing.

PubMed

Tao, Aifen; Huang, Long; Wu, Guifen; Afshar, Reza Keshavarz; Qi, Jianmin; Xu, Jiantang; Fang, Pingping; Lin, Lihui; Zhang, Liwu; Lin, Peiqing

2017-05-08

Genetic mapping and quantitative trait locus (QTL) detection are powerful methodologies in plant improvement and breeding. White jute (Corchorus capsularis L.) is an important industrial raw material fiber crop because of its elite characteristics. However, construction of a high-density genetic map and identification of QTLs has been limited in white jute due to a lack of sufficient molecular markers. The specific locus amplified fragment sequencing (SLAF-seq) strategy combines locus-specific amplification and high-throughput sequencing to carry out de novo single nuclear polymorphism (SNP) discovery and large-scale genotyping. In this study, SLAF-seq was employed to obtain sufficient markers to construct a high-density genetic map for white jute. Moreover, with the development of abundant markers, genetic dissection of fiber yield traits such as plant height was also possible. Here, we present QTLs associated with plant height that were identified using our newly constructed genetic linkage groups. An F 8 population consisting of 100 lines was developed. In total, 69,446 high-quality SLAFs were detected of which 5,074 SLAFs were polymorphic; 913 polymorphic markers were used for the construction of a genetic map. The average coverage for each SLAF marker was 43-fold in the parents, and 9.8-fold in each F 8 individual. A linkage map was constructed that contained 913 SLAFs on 11 linkage groups (LGs) covering 1621.4 cM with an average density of 1.61 cM per locus. Among the 11 LGs, LG1 was the largest with 210 markers, a length of 406.34 cM, and an average distance of 1.93 cM between adjacent markers. LG11 was the smallest with only 25 markers, a length of 29.66 cM, and an average distance of 1.19 cM between adjacent markers. 'SNP_only' markers accounted for 85.54% and were the predominant markers on the map. QTL mapping based on the F 8 phenotypes detected 11 plant height QTLs including one major effect QTL across two cultivation locations, with each QTL accounting for 4.14-15.63% of the phenotypic variance. To our knowledge, the linkage map constructed here is the densest one available to date for white jute. This analysis also identified the first QTL in white jute. The results will provide an important platform for gene/QTL mapping, sequence assembly, genome comparisons, and marker-assisted selection breeding for white jute.

THORIUM DISPERSION IN BISMUTH

DOEpatents

Bryner, J.S.

1961-07-01

The growth of thorium bismutaide particles, which are formed when thorium is suspended in liquid bismuth, is inhibited when the liquid metal suspension is being flowed through a reactor and through a heat exchanger in sequence. It involves the addition of as little as 1 part by weight of tellurium to 100 parts of thorium. This addition is sufficient to inhibit particle growth and agglomeration.

Read clouds uncover variation in complex regions of the human genome

PubMed Central

Bishara, Alex; Liu, Yuling; Weng, Ziming; Kashef-Haghighi, Dorna; Newburger, Daniel E.; West, Robert; Sidow, Arend; Batzoglou, Serafim

2015-01-01

Although an increasing amount of human genetic variation is being identified and recorded, determining variants within repeated sequences of the human genome remains a challenge. Most population and genome-wide association studies have therefore been unable to consider variation in these regions. Core to the problem is the lack of a sequencing technology that produces reads with sufficient length and accuracy to enable unique mapping. Here, we present a novel methodology of using read clouds, obtained by accurate short-read sequencing of DNA derived from long fragment libraries, to confidently align short reads within repeat regions and enable accurate variant discovery. Our novel algorithm, Random Field Aligner (RFA), captures the relationships among the short reads governed by the long read process via a Markov Random Field. We utilized a modified version of the Illumina TruSeq synthetic long-read protocol, which yielded shallow-sequenced read clouds. We test RFA through extensive simulations and apply it to discover variants on the NA12878 human sample, for which shallow TruSeq read cloud sequencing data are available, and on an invasive breast carcinoma genome that we sequenced using the same method. We demonstrate that RFA facilitates accurate recovery of variation in 155 Mb of the human genome, including 94% of 67 Mb of segmental duplication sequence and 96% of 11 Mb of transcribed sequence, that are currently hidden from short-read technologies. PMID:26286554

Definition of Cis-Acting Elements Regulating Expression of the Drosophila Melanogaster Ninae Opsin Gene by Oligonucleotide-Directed Mutagenesis

PubMed Central

Mismer, D.; Rubin, G. M.

1989-01-01

We have analyzed the cis-acting regulatory sequences of the Rh1 (ninaE) gene in Drosophila melanogaster by P-element-mediated germline transformation of indicator genes transcribed from mutant ninaE promoter sequences. We have previously shown that a 200-bp region extending from -120 to +67 relative to the transcription start site is sufficient to obtain eye-specific expression from the ninaE promoter. In the present study, 22 different 4-13-bp sequences in the -120/+67 promoter region were altered by oligonucleotide-directed mutagenesis. Several of these sequences were found to be required for proper promoter function; two of these are conserved in the promoter of the homologous gene isolated from the related species Drosophila virilis. Alteration of a conserved 9-bp sequence results in aberrant, low level expression in the body. Alteration of a separate 11-bp sequence, found in the promoter regions of several photoreceptor-specific genes of Drosophila, results in an approximately 15-fold reduction in promoter efficiency but without apparent alteration of tissue-specificity. A protein factor capable of interacting with this 11-bp sequence has been detected by DNaseI footprinting in embryonic nuclear extracts. Finally, we have further characterized two separable enhancer sequences previously shown to be required for normal levels of expression from this promoter. PMID:2521839

Distribution of genotype network sizes in sequence-to-structure genotype-phenotype maps.

PubMed

Manrubia, Susanna; Cuesta, José A

2017-04-01

An essential quantity to ensure evolvability of populations is the navigability of the genotype space. Navigability, understood as the ease with which alternative phenotypes are reached, relies on the existence of sufficiently large and mutually attainable genotype networks. The size of genotype networks (e.g. the number of RNA sequences folding into a particular secondary structure or the number of DNA sequences coding for the same protein structure) is astronomically large in all functional molecules investigated: an exhaustive experimental or computational study of all RNA folds or all protein structures becomes impossible even for moderately long sequences. Here, we analytically derive the distribution of genotype network sizes for a hierarchy of models which successively incorporate features of increasingly realistic sequence-to-structure genotype-phenotype maps. The main feature of these models relies on the characterization of each phenotype through a prototypical sequence whose sites admit a variable fraction of letters of the alphabet. Our models interpolate between two limit distributions: a power-law distribution, when the ordering of sites in the prototypical sequence is strongly constrained, and a lognormal distribution, as suggested for RNA, when different orderings of the same set of sites yield different phenotypes. Our main result is the qualitative and quantitative identification of those features of sequence-to-structure maps that lead to different distributions of genotype network sizes. © 2017 The Author(s).

Exact Fan-Beam Reconstruction With Arbitrary Object Translations and Truncated Projections

NASA Astrophysics Data System (ADS)

Hoskovec, Jan; Clackdoyle, Rolf; Desbat, Laurent; Rit, Simon

2016-06-01

This article proposes a new method for reconstructing two-dimensional (2D) computed tomography (CT) images from truncated and motion contaminated sinograms. The type of motion considered here is a sequence of rigid translations which are assumed to be known. The algorithm first identifies the sufficiency of angular coverage in each 2D point of the CT image to calculate the Hilbert transform from the local “virtual” trajectory which accounts for the motion and the truncation. By taking advantage of data redundancy in the full circular scan, our method expands the reconstructible region beyond the one obtained with chord-based methods. The proposed direct reconstruction algorithm is based on the Differentiated Back-Projection with Hilbert filtering (DBP-H). The motion is taken into account during backprojection which is the first step of our direct reconstruction, before taking the derivatives and inverting the finite Hilbert transform. The algorithm has been tested in a proof-of-concept study on Shepp-Logan phantom simulations with several motion cases and detector sizes.

Bispecific Antibody Affords Complete Post-Exposure Protection of Mice from Both Ebola (Zaire) and Sudan Viruses.

PubMed

Frei, Julia C; Nyakatura, Elisabeth K; Zak, Samantha E; Bakken, Russell R; Chandran, Kartik; Dye, John M; Lai, Jonathan R

2016-01-13

Filoviruses (Ebola and Marburg) cause severe hemorrhagic fever. There are five species of ebolavirus; among these, the Ebola (Zaire) and Sudan viruses (EBOV and SUDV, respectively) are highly pathogenic and have both caused recurring, large outbreaks. However, the EBOV and SUDV glycoprotein (GP) sequences are 45% divergent and thus antigenically distinct. Few antibodies with cross-neutralizing properties have been described to date. We used antibody engineering to develop novel bispecific antibodies (Bis-mAbs) that are cross-reactive toward base epitopes on GP from EBOV and SUDV. These Bis-mAbs exhibit potent neutralization against EBOV and SUDV GP pseudotyped viruses as well as authentic pathogens, and confer a high degree (in one case 100%) post-exposure protection of mice from both viruses. Our studies show that a single agent that targets the GP base epitopes is sufficient for protection in mice; such agents could be included in panfilovirus therapeutic antibody cocktails.

Autonomous proximity operations using machine vision for trajectory control and pose estimation

NASA Technical Reports Server (NTRS)

Cleghorn, Timothy F.; Sternberg, Stanley R.

1991-01-01

A machine vision algorithm was developed which permits guidance control to be maintained during autonomous proximity operations. At present this algorithm exists as a simulation, running upon an 80386 based personal computer, using a ModelMATE CAD package to render the target vehicle. However, the algorithm is sufficiently simple, so that following off-line training on a known target vehicle, it should run in real time with existing vision hardware. The basis of the algorithm is a sequence of single camera images of the target vehicle, upon which radial transforms were performed. Selected points of the resulting radial signatures are fed through a decision tree, to determine whether the signature matches that of the known reference signatures for a particular view of the target. Based upon recognized scenes, the position of the maneuvering vehicle with respect to the target vehicles can be calculated, and adjustments made in the former's trajectory. In addition, the pose and spin rates of the target satellite can be estimated using this method.

Image Geometric Corrections for a New EMCCD-based Dual Modular X-ray Imager

PubMed Central

Qu, Bin; Huang, Ying; Wang, Weiyuan; Cartwright, Alexander N.; Titus, Albert H.; Bednarek, Daniel R.; Rudin, Stephen

2012-01-01

An EMCCD-based dual modular x-ray imager was recently designed and developed from the component level, providing a high dynamic range of 53 dB and an effective pixel size of 26 μm for angiography and fluoroscopy. The unique 2×1 array design efficiently increased the clinical field of view, and also can be readily expanded to an M×N array implementation. Due to the alignment mismatches between the EMCCD sensors and the fiber optic tapers in each module, the output images or video sequences result in a misaligned 2048×1024 digital display if uncorrected. In this paper, we present a method for correcting display registration using a custom-designed two layer printed circuit board. This board was designed with grid lines to serve as the calibration pattern, and provides an accurate reference and sufficient contrast to enable proper display registration. Results show an accurate and fine stitching of the two outputs from the two modules. PMID:22254882

Structural basis for specific recognition of multiple mRNA targets by a PUF regulatory protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Yeming; Opperman, Laura; Wickens, Marvin

2011-11-02

Caenorhabditis elegans fem-3 binding factor (FBF) is a founding member of the PUMILIO/FBF (PUF) family of mRNA regulatory proteins. It regulates multiple mRNAs critical for stem cell maintenance and germline development. Here, we report crystal structures of FBF in complex with 6 different 9-nt RNA sequences, including elements from 4 natural mRNAs. These structures reveal that FBF binds to conserved bases at positions 1-3 and 7-8. The key specificity determinant of FBF vs. other PUF proteins lies in positions 4-6. In FBF/RNA complexes, these bases stack directly with one another and turn away from the RNA-binding surface. A short regionmore » of FBF is sufficient to impart its unique specificity and lies directly opposite the flipped bases. We suggest that this region imposes a flattened curvature on the protein; hence, the requirement for the additional nucleotide. The principles of FBF/RNA recognition suggest a general mechanism by which PUF proteins recognize distinct families of RNAs yet exploit very nearly identical atomic contacts in doing so.« less

Structural basis for specific recognition of multiple mRNA targets by a PUF regulatory protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Yeming; Opperman, Laura; Wickens, Marvin

2010-08-19

Caenorhabditis elegans fem-3 binding factor (FBF) is a founding member of the PUMILIO/FBF (PUF) family of mRNA regulatory proteins. It regulates multiple mRNAs critical for stem cell maintenance and germline development. Here, we report crystal structures of FBF in complex with 6 different 9-nt RNA sequences, including elements from 4 natural mRNAs. These structures reveal that FBF binds to conserved bases at positions 1-3 and 7-8. The key specificity determinant of FBF vs. other PUF proteins lies in positions 4-6. In FBF/RNA complexes, these bases stack directly with one another and turn away from the RNA-binding surface. A short regionmore » of FBF is sufficient to impart its unique specificity and lies directly opposite the flipped bases. We suggest that this region imposes a flattened curvature on the protein; hence, the requirement for the additional nucleotide. The principles of FBF/RNA recognition suggest a general mechanism by which PUF proteins recognize distinct families of RNAs yet exploit very nearly identical atomic contacts in doing so.« less

Cytokinin signalling inhibitory fields provide robustness to phyllotaxis

NASA Astrophysics Data System (ADS)

Besnard, Fabrice; Refahi, Yassin; Morin, Valérie; Marteaux, Benjamin; Brunoud, Géraldine; Chambrier, Pierre; Rozier, Frédérique; Mirabet, Vincent; Legrand, Jonathan; Lainé, Stéphanie; Thévenon, Emmanuel; Farcot, Etienne; Cellier, Coralie; Das, Pradeep; Bishopp, Anthony; Dumas, Renaud; Parcy, François; Helariutta, Ykä; Boudaoud, Arezki; Godin, Christophe; Traas, Jan; Guédon, Yann; Vernoux, Teva

2014-01-01

How biological systems generate reproducible patterns with high precision is a central question in science. The shoot apical meristem (SAM), a specialized tissue producing plant aerial organs, is a developmental system of choice to address this question. Organs are periodically initiated at the SAM at specific spatial positions and this spatiotemporal pattern defines phyllotaxis. Accumulation of the plant hormone auxin triggers organ initiation, whereas auxin depletion around organs generates inhibitory fields that are thought to be sufficient to maintain these patterns and their dynamics. Here we show that another type of hormone-based inhibitory fields, generated directly downstream of auxin by intercellular movement of the cytokinin signalling inhibitor ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN 6 (AHP6), is involved in regulating phyllotactic patterns. We demonstrate that AHP6-based fields establish patterns of cytokinin signalling in the meristem that contribute to the robustness of phyllotaxis by imposing a temporal sequence on organ initiation. Our findings indicate that not one but two distinct hormone-based fields may be required for achieving temporal precision during formation of reiterative structures at the SAM, thus indicating an original mechanism for providing robustness to a dynamic developmental system.

An Outbreak of Streptococcus pyogenes in a Mental Health Facility: Advantage of Well-Timed Whole-Genome Sequencing Over emm Typing.

PubMed

Bergin, Sarah M; Periaswamy, Balamurugan; Barkham, Timothy; Chua, Hong Choon; Mok, Yee Ming; Fung, Daniel Shuen Sheng; Su, Alex Hsin Chuan; Lee, Yen Ling; Chua, Ming Lai Ivan; Ng, Poh Yong; Soon, Wei Jia Wendy; Chu, Collins Wenhan; Tan, Siyun Lucinda; Meehan, Mary; Ang, Brenda Sze Peng; Leo, Yee Sin; Holden, Matthew T G; De, Partha; Hsu, Li Yang; Chen, Swaine L; de Sessions, Paola Florez; Marimuthu, Kalisvar

2018-05-09

OBJECTIVEWe report the utility of whole-genome sequencing (WGS) conducted in a clinically relevant time frame (ie, sufficient for guiding management decision), in managing a Streptococcus pyogenes outbreak, and present a comparison of its performance with emm typing.SETTINGA 2,000-bed tertiary-care psychiatric hospital.METHODSActive surveillance was conducted to identify new cases of S. pyogenes. WGS guided targeted epidemiological investigations, and infection control measures were implemented. Single-nucleotide polymorphism (SNP)-based genome phylogeny, emm typing, and multilocus sequence typing (MLST) were performed. We compared the ability of WGS and emm typing to correctly identify person-to-person transmission and to guide the management of the outbreak.RESULTSThe study included 204 patients and 152 staff. We identified 35 patients and 2 staff members with S. pyogenes. WGS revealed polyclonal S. pyogenes infections with 3 genetically distinct phylogenetic clusters (C1-C3). Cluster C1 isolates were all emm type 4, sequence type 915 and had pairwise SNP differences of 0-5, which suggested recent person-to-person transmissions. Epidemiological investigation revealed that cluster C1 was mediated by dermal colonization and transmission of S. pyogenes in a male residential ward. Clusters C2 and C3 were genomically diverse, with pairwise SNP differences of 21-45 and 26-58, and emm 11 and mostly emm120, respectively. Clusters C2 and C3, which may have been considered person-to-person transmissions by emm typing, were shown by WGS to be unlikely by integrating pairwise SNP differences with epidemiology.CONCLUSIONSWGS had higher resolution than emm typing in identifying clusters with recent and ongoing person-to-person transmissions, which allowed implementation of targeted intervention to control the outbreak.Infect Control Hosp Epidemiol 2018;1-9.

Effect of laying sequence on egg mercury in captive zebra finches: an interpretation considering individual variation.

PubMed

Ou, Langbo; Varian-Ramos, Claire W; Cristol, Daniel A

2015-08-01

Bird eggs are used widely as noninvasive bioindicators for environmental mercury availability. Previous studies, however, have found varying relationships between laying sequence and egg mercury concentrations. Some studies have reported that the mercury concentration was higher in first-laid eggs or declined across the laying sequence, whereas in other studies mercury concentration was not related to egg order. Approximately 300 eggs (61 clutches) were collected from captive zebra finches dosed throughout their reproductive lives with methylmercury (0.3 μg/g, 0.6 μg/g, 1.2 μg/g, or 2.4 μg/g wet wt in diet); the total mercury concentration (mean ± standard deviation [SD] dry wt basis) of their eggs was 7.03 ± 1.38 μg/g, 14.15 ± 2.52 μg/g, 26.85 ± 5.85 μg/g, and 49.76 ± 10.37 μg/g, respectively (equivalent to fresh wt egg mercury concentrations of 1.24 μg/g, 2.50 μg/g, 4.74 μg/g, and 8.79 μg/g). The authors observed a significant decrease in the mercury concentration of successive eggs when compared with the first egg and notable variation between clutches within treatments. The mercury level of individual females within and among treatments did not alter this relationship. Based on the results, sampling of a single egg in each clutch from any position in the laying sequence is sufficient for purposes of population risk assessment, but it is not recommended as a proxy for individual female exposure or as an estimate of average mercury level within the clutch. © 2015 SETAC.

Ranking viruses: measures of positional importance within networks define core viruses for rational polyvalent vaccine development.

PubMed

Anderson, Tavis K; Laegreid, William W; Cerutti, Francesco; Osorio, Fernando A; Nelson, Eric A; Christopher-Hennings, Jane; Goldberg, Tony L

2012-06-15

The extraordinary genetic and antigenic variability of RNA viruses is arguably the greatest challenge to the development of broadly effective vaccines. No single viral variant can induce sufficiently broad immunity, and incorporating all known naturally circulating variants into one multivalent vaccine is not feasible. Furthermore, no objective strategies currently exist to select actual viral variants that should be included or excluded in polyvalent vaccines. To address this problem, we demonstrate a method based on graph theory that quantifies the relative importance of viral variants. We demonstrate our method through application to the envelope glycoprotein gene of a particularly diverse RNA virus of pigs: porcine reproductive and respiratory syndrome virus (PRRSV). Using distance matrices derived from sequence nucleotide difference, amino acid difference and evolutionary distance, we constructed viral networks and used common network statistics to assign each sequence an objective ranking of relative 'importance'. To validate our approach, we use an independent published algorithm to score our top-ranked wild-type variants for coverage of putative T-cell epitopes across the 9383 sequences in our dataset. Top-ranked viruses achieve significantly higher coverage than low-ranked viruses, and top-ranked viruses achieve nearly equal coverage as a synthetic mosaic protein constructed in silico from the same set of 9383 sequences. Our approach relies on the network structure of PRRSV but applies to any diverse RNA virus because it identifies subsets of viral variants that are most important to overall viral diversity. We suggest that this method, through the objective quantification of variant importance, provides criteria for choosing viral variants for further characterization, diagnostics, surveillance and ultimately polyvalent vaccine development.

Contribution of Peptide Backbone to Anti-Citrullinated Peptide Antibody Reactivity

PubMed Central

Trier, Nicole Hartwig; Dam, Catharina Essendrup; Olsen, Dorthe Tange; Hansen, Paul Robert; Houen, Gunnar

2015-01-01

Rheumatoid arthritis (RA) is one of the most common autoimmune diseases, affecting approximately 1–2% of the world population. One of the characteristic features of RA is the presence of autoantibodies. Especially the highly specific anti-citrullinated peptide antibodies (ACPAs), which have been found in up to 70% of RA patients’ sera, have received much attention. Several citrullinated proteins are associated with RA, suggesting that ACPAs may react with different sequence patterns, separating them from traditional antibodies, whose reactivity usually is specific towards a single target. As ACPAs have been suggested to be involved in the development of RA, knowledge about these antibodies may be crucial. In this study, we examined the influence of peptide backbone for ACPA reactivity in immunoassays. The antibodies were found to be reactive with a central Cit-Gly motif being essential for ACPA reactivity and to be cross-reactive between the selected citrullinated peptides. The remaining amino acids within the citrullinated peptides were found to be of less importance for antibody reactivity. Moreover, these findings indicated that the Cit-Gly motif in combination with peptide backbone is essential for antibody reactivity. Based on these findings it was speculated that any amino acid sequence, which brings the peptide into a properly folded structure for antibody recognition is sufficient for antibody reactivity. These findings are in accordance with the current hypothesis that structural homology rather than sequence homology are favored between citrullinated epitopes. These findings are important in relation to clarifying the etiology of RA and to determine the nature of ACPAs, e.g. why some Cit-Gly-containing sequences are not targeted by ACPAs. PMID:26657009

Exploiting EST databases for the development and characterisation of 3425 gene-tagged CISP markers in biofuel crop sugarcane and their transferability in cereals and orphan tropical grasses.

PubMed

Chandra, Amaresh; Jain, Radha; Solomon, Sushil; Shrivastava, Shiksha; Roy, Ajoy K

2013-02-04

Sugarcane is an important cash crop, providing 70% of the global raw sugar as well as raw material for biofuel production. Genetic analysis is hindered in sugarcane because of its large and complex polyploid genome and lack of sufficiently informative gene-tagged markers. Modern genomics has produced large amount of ESTs, which can be exploited to develop molecular markers based on comparative analysis with EST datasets of related crops and whole rice genome sequence, and accentuate their cross-technical functionality in orphan crops like tropical grasses. Utilising 246,180 Saccharum officinarum EST sequences vis-à-vis its comparative analysis with ESTs of sorghum and barley and the whole rice genome sequence, we have developed 3425 novel gene-tagged markers - namely, conserved-intron scanning primers (CISP) - using the web program GeMprospector. Rice orthologue annotation results indicated homology of 1096 sequences with expressed proteins, 491 with hypothetical proteins. The remaining 1838 were miscellaneous in nature. A total of 367 primer-pairs were tested in diverse panel of samples. The data indicate amplification of 41% polymorphic bands leading to 0.52 PIC and 3.50 MI with a set of sugarcane varieties and Saccharum species. In addition, a moderate technical functionality of a set of such markers with orphan tropical grasses (22%) and fodder cum cereal oat (33%) is observed. Developed gene-tagged CISP markers exhibited considerable technical functionality with varieties of sugarcane and unexplored species of tropical grasses. These markers would thus be particularly useful in identifying the economical traits in sugarcane and developing conservation strategies for orphan tropical grasses.

Ontology and diversity of transcript-associated microsatellites mined from a globe artichoke EST database

PubMed Central

Scaglione, Davide; Acquadro, Alberto; Portis, Ezio; Taylor, Christopher A; Lanteri, Sergio; Knapp, Steven J

2009-01-01

Background The globe artichoke (Cynara cardunculus var. scolymus L.) is a significant crop in the Mediterranean basin. Despite its commercial importance and its both dietary and pharmaceutical value, knowledge of its genetics and genomics remains scant. Microsatellite markers have become a key tool in genetic and genomic analysis, and we have exploited recently acquired EST (expressed sequence tag) sequence data (Composite Genome Project - CGP) to develop an extensive set of microsatellite markers. Results A unigene assembly was created from over 36,000 globe artichoke EST sequences, containing 6,621 contigs and 12,434 singletons. Over 12,000 of these unigenes were functionally assigned on the basis of homology with Arabidopsis thaliana reference proteins. A total of 4,219 perfect repeats, located within 3,308 unigenes was identified and the gene ontology (GO) analysis highlighted some GO term's enrichments among different classes of microsatellites with respect to their position. Sufficient flanking sequence was available to enable the design of primers to amplify 2,311 of these microsatellites, and a set of 300 was tested against a DNA panel derived from 28 C. cardunculus genotypes. Consistent amplification and polymorphism was obtained from 236 of these assays. Their polymorphic information content (PIC) ranged from 0.04 to 0.90 (mean 0.66). Between 176 and 198 of the assays were informative in at least one of the three available mapping populations. Conclusion EST-based microsatellites have provided a large set of de novo genetic markers, which show significant amounts of polymorphism both between and within the three taxa of C. cardunculus. They are thus well suited as assays for phylogenetic analysis, the construction of genetic maps, marker-assisted breeding, transcript mapping and other genomic applications in the species. PMID:19785740

Clinical Application of Picodroplet Digital PCR Technology for Rapid Detection of EGFR T790M in Next-Generation Sequencing Libraries and DNA from Limited Tumor Samples.

PubMed

Borsu, Laetitia; Intrieri, Julie; Thampi, Linta; Yu, Helena; Riely, Gregory; Nafa, Khedoudja; Chandramohan, Raghu; Ladanyi, Marc; Arcila, Maria E

2016-11-01

Although next-generation sequencing (NGS) is a robust technology for comprehensive assessment of EGFR-mutant lung adenocarcinomas with acquired resistance to tyrosine kinase inhibitors, it may not provide sufficiently rapid and sensitive detection of the EGFR T790M mutation, the most clinically relevant resistance biomarker. Here, we describe a digital PCR (dPCR) assay for rapid T790M detection on aliquots of NGS libraries prepared for comprehensive profiling, fully maximizing broad genomic analysis on limited samples. Tumor DNAs from patients with EGFR-mutant lung adenocarcinomas and acquired resistance to epidermal growth factor receptor inhibitors were prepared for Memorial Sloan-Kettering-Integrated Mutation Profiling of Actionable Cancer Targets sequencing, a hybrid capture-based assay interrogating 410 cancer-related genes. Precapture library aliquots were used for rapid EGFR T790M testing by dPCR, and results were compared with NGS and locked nucleic acid-PCR Sanger sequencing (reference high sensitivity method). Seventy resistance samples showed 99% concordance with the reference high sensitivity method in accuracy studies. Input as low as 2.5 ng provided a sensitivity of 1% and improved further with increasing DNA input. dPCR on libraries required less DNA and showed better performance than direct genomic DNA. dPCR on NGS libraries is a robust and rapid approach to EGFR T790M testing, allowing most economical utilization of limited material for comprehensive assessment. The same assay can also be performed directly on any limited DNA source and cell-free DNA. Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

In vitro studies of Rickettsia-host cell interactions: Confocal laser scanning microscopy of Rickettsia helvetica-infected eukaryotic cell lines.

PubMed

Speck, Stephanie; Kern, Tanja; Aistleitner, Karin; Dilcher, Meik; Dobler, Gerhard; Essbauer, Sandra

2018-02-01

Rickettsia (R.) helvetica is the most prevalent rickettsia found in Ixodes ricinus ticks in Germany. Several studies reported antibodies against R. helvetica up to 12.5% in humans investigated, however, fulminant clinical cases are rare indicating a rather low pathogenicity compared to other rickettsiae. We investigated growth characteristics of R. helvetica isolate AS819 in two different eukaryotic cell lines with focus on ultra-structural changes of host cells during infection determined by confocal laser scanning microscopy. Further investigations included partially sequencing of rickA, sca4 and sca2 genes, which have been reported to encode proteins involved in cell-to-cell spread and virulence in some rickettsiae. R. helvetica grew constantly but slowly in both cell lines used. Confocal laser scanning microscopy revealed that the dissemination of R. helvetica AS819 in both cell lines was rather mediated by cell break-down and bacterial release than cell-to-cell spread. The cytoskeleton of both investigated eukaryotic cell lines was not altered. R. helvetica possesses rickA, but its expression is not sufficient to promote actin-based motility as demonstrated by confocal laser scanning microscopy. Hypothetical Sca2 and Sca4 proteins were deduced from nucleotide gene sequences but the predicted amino acid sequences were disrupted or truncated compared to other rickettsiae most likely resulting in non-functional proteins. Taken together, these results might give a first hint to the underlying causes of the reduced virulence and pathogenicity of R. helvetica.

Harnessing dark fermentative hydrogen from pretreated mixture of food waste and sewage sludge under sequencing batch mode.

PubMed

Nam, Joo-Youn; Kim, Dong-Hoon; Kim, Sang-Hyoun; Lee, Wontae; Shin, Hang-Sik; Kim, Hyun-Woo

2016-04-01

Food waste and sewage sludge are the most abundant and problematic organic wastes in any society. Mixture of these two wastes may provide appropriate substrate condition for dark fermentative biohydrogen production based on synergistic mutual benefits. This work evaluates continuous hydrogen production from the cosubstrate of food waste and sewage sludge to verify mechanisms of performance improvement in anaerobic sequencing batch reactors. Volatile solid concentration and mixing ratio of food waste and sludge were adjusted to 5 % and 80:20, respectively. Five different hydraulic retention times (HRT) of 36, 42, 48, 72, and 108 h were tested using anaerobic sequencing batch reactors to find out optimal operating condition. Results show that the best performance was achieved at HRT 72 h, where the hydrogen yield, the hydrogen production rate, and hydrogen content were 62.0 mL H2/g VS, 1.0 L H2/L/day, and ~50 %, respectively. Sufficient solid retention time (143 h) and proper loading rate (8.2 g COD/L/day as carbohydrate) at HRT 72h led to the enhanced performance with better hydrogen production showing appropriate n-butyrate/acetate (B/A) ratio of 2.6. Analytical result of terminal-restriction fragment length polymorphism revealed that specific peaks associated with Clostridium sp. and Bacillus sp. were strongly related to enhanced hydrogen production from the cosubstrate of food waste and sewage sludge.

Assessment of an automated capillary system for Plasmodium vivax microsatellite genotyping.

PubMed

Manrique, Paulo; Hoshi, Mari; Fasabi, Manuel; Nolasco, Oscar; Yori, Pablo; Calderón, Martiza; Gilman, Robert H; Kosek, Margaret N; Vinetz, Joseph M; Gamboa, Dionicia

2015-08-21

Several platforms have been used to generate the primary data for microsatellite analysis of malaria parasite genotypes. Each has relative advantages but share a limitation of being time- and cost-intensive. A commercially available automated capillary gel cartridge system was assessed in the microsatellite analysis of Plasmodium vivax diversity in the Peruvian Amazon. The reproducibility and accuracy of a commercially-available automated capillary system, QIAxcel, was assessed using a sequenced PCR product of 227 base pairs. This product was measured 42 times, then 27 P. vivax samples from Peruvian Amazon subjects were analyzed with this instrument using five informative microsatellites. Results from the QIAxcel system were compared with a Sanger-type sequencing machine, the ABI PRISM(®) 3100 Genetic Analyzer. Significant differences were seen between the sequenced amplicons and the results from the QIAxcel instrument. Different runs, plates and cartridges yielded significantly different results. Additionally, allele size decreased with each run by 0.045, or 1 bp, every three plates. QIAxcel and ABI PRISM systems differed in giving different values than those obtained by ABI PRISM, and too many (i.e. inaccurate) alleles per locus were also seen with the automated instrument. While P. vivax diversity could generally be estimated using an automated capillary gel cartridge system, the data demonstrate that this system is not sufficiently precise for reliably identifying parasite strains via microsatellite analysis. This conclusion reached after systematic analysis was due both to inadequate precision and poor reproducibility in measuring PCR product size.

Volume calculation of CT lung lesions based on Halton low-discrepancy sequences

NASA Astrophysics Data System (ADS)

Li, Shusheng; Wang, Liansheng; Li, Shuo

2017-03-01

Volume calculation from the Computed Tomography (CT) lung lesions data is a significant parameter for clinical diagnosis. The volume is widely used to assess the severity of the lung nodules and track its progression, however, the accuracy and efficiency of previous studies are not well achieved for clinical uses. It remains to be a challenging task due to its tight attachment to the lung wall, inhomogeneous background noises and large variations in sizes and shape. In this paper, we employ Halton low-discrepancy sequences to calculate the volume of the lung lesions. The proposed method directly compute the volume without the procedure of three-dimension (3D) model reconstruction and surface triangulation, which significantly improves the efficiency and reduces the complexity. The main steps of the proposed method are: (1) generate a certain number of random points in each slice using Halton low-discrepancy sequences and calculate the lesion area of each slice through the proportion; (2) obtain the volume by integrating the areas in the sagittal direction. In order to evaluate our proposed method, the experiments were conducted on the sufficient data sets with different size of lung lesions. With the uniform distribution of random points, our proposed method achieves more accurate results compared with other methods, which demonstrates the robustness and accuracy for the volume calculation of CT lung lesions. In addition, our proposed method is easy to follow and can be extensively applied to other applications, e.g., volume calculation of liver tumor, atrial wall aneurysm, etc.

Tuning Selectivity of Fluorescent Carbon Nanotube-Based Neurotransmitter Sensors.

PubMed

Mann, Florian A; Herrmann, Niklas; Meyer, Daniel; Kruss, Sebastian

2017-06-28

Detection of neurotransmitters is an analytical challenge and essential to understand neuronal networks in the brain and associated diseases. However, most methods do not provide sufficient spatial, temporal, or chemical resolution. Near-infrared (NIR) fluorescent single-walled carbon nanotubes (SWCNTs) have been used as building blocks for sensors/probes that detect catecholamine neurotransmitters, including dopamine. This approach provides a high spatial and temporal resolution, but it is not understood if these sensors are able to distinguish dopamine from similar catecholamine neurotransmitters, such as epinephrine or norepinephrine. In this work, the organic phase (DNA sequence) around SWCNTs was varied to create sensors with different selectivity and sensitivity for catecholamine neurotransmitters. Most DNA-functionalized SWCNTs responded to catecholamine neurotransmitters, but both dissociation constants ( K d ) and limits of detection were highly dependent on functionalization (sequence). K d values span a range of 2.3 nM (SWCNT-(GC) 15 + norepinephrine) to 9.4 μM (SWCNT-(AT) 15 + dopamine) and limits of detection are mostly in the single-digit nM regime. Additionally, sensors of different SWCNT chirality show different fluorescence increases. Moreover, certain sensors (e.g., SWCNT-(GT) 10 ) distinguish between different catecholamines, such as dopamine and norepinephrine at low concentrations (50 nM). These results show that SWCNTs functionalized with certain DNA sequences are able to discriminate between catecholamine neurotransmitters or to detect them in the presence of interfering substances of similar structure. Such sensors will be useful to measure and study neurotransmitter signaling in complex biological settings.

DNA-based techniques for authentication of processed food and food supplements.

PubMed

Lo, Yat-Tung; Shaw, Pang-Chui

2018-02-01

Authentication of food or food supplements with medicinal values is important to avoid adverse toxic effects, provide consumer rights, as well as for certification purpose. Compared to morphological and spectrometric techniques, molecular authentication is found to be accurate, sensitive and reliable. However, DNA degradation and inclusion of inhibitors may lead to failure in PCR amplification. This paper reviews on the existing DNA extraction and PCR protocols, and the use of small size DNA markers with sufficient discriminative power for molecular authentication. Various emerging new molecular techniques such as isothermal amplification for on-site diagnosis, next-generation sequencing for high-throughput species identification, high resolution melting analysis for quick species differentiation, DNA array techniques for rapid detection and quantitative determination in food products are also discussed. Copyright © 2017 Elsevier Ltd. All rights reserved.

Novel narrow-host-range vectors for direct cloning of foreign DNA in Pseudomonas.

PubMed

Boivin, R; Bellemare, G; Dion, P

1994-01-01

Narrow-host-range vectors, based on an indigenous replicon and containing a multiple cloning site, have been constructed in a Pseudomonas host capable of growth on unusual substrates. The new cloning vectors yield sufficient amounts of DNA for preparative purposes and belong to an incompatibility group different from that of the incP and incQ broad-host-range vectors. One of these vectors, named pDB47F, was used to clone, directly in Pseudomonas, DNA fragments from Agrobacterium, Pseudomonas, and Rhizobium. A clone containing Agrobacterium and KmR gene sequences was transformed with a higher efficiency than an RSF1010-derived vector (by as much as 1250-fold) in four out of five Pseudomonas strains tested. The considerable efficiency obtained with this system makes possible the direct cloning and phenotypic selection of foreign DNA in Pseudomonas.

Orbital Motion of Young Binaries in Ophiuchus and Upper Centaurus–Lupus

NASA Astrophysics Data System (ADS)

Schaefer, G. H.; Prato, L.; Simon, M.

2018-03-01

We present measurements of the orbital positions and flux ratios of 17 binary and triple systems in the Ophiuchus star-forming region and the Upper Centaurus–Lupus cluster based on adaptive optics imaging at the Keck Observatory. We report the detection of visual companions in MML 50 and MML 53 for the first time, as well as the possible detection of a third component in WSB 21. For six systems in our sample, our measurements provide a second orbital position following their initial discoveries over a decade ago. For eight systems with sufficient orbital coverage, we analyze the range of orbital solutions that fit the data. Ultimately, these observations will help provide the groundwork toward measuring precise masses for these pre-main-sequence stars and understanding the distribution of orbital parameters in young multiple systems.

Fusion of Building Information and Range Imaging for Autonomous Location Estimation in Indoor Environments

PubMed Central

Kohoutek, Tobias K.; Mautz, Rainer; Wegner, Jan D.

2013-01-01

We present a novel approach for autonomous location estimation and navigation in indoor environments using range images and prior scene knowledge from a GIS database (CityGML). What makes this task challenging is the arbitrary relative spatial relation between GIS and Time-of-Flight (ToF) range camera further complicated by a markerless configuration. We propose to estimate the camera's pose solely based on matching of GIS objects and their detected location in image sequences. We develop a coarse-to-fine matching strategy that is able to match point clouds without any initial parameters. Experiments with a state-of-the-art ToF point cloud show that our proposed method delivers an absolute camera position with decimeter accuracy, which is sufficient for many real-world applications (e.g., collision avoidance). PMID:23435055

The yeast p53 functional assay: a new tool for molecular epidemiology. Hopes and facts.

PubMed

Fronza, G; Inga, A; Monti, P; Scott, G; Campomenosi, P; Menichini, P; Ottaggio, L; Viaggi, S; Burns, P A; Gold, B; Abbondandolo, A

2000-04-01

The assumption of molecular epidemiology that carcinogens leave fingerprints has suggested that analysis of the frequency, type, and site of mutations in genes frequently altered in carcinogenesis may provide clues to the identification of the factors contributing to carcinogenesis. In this mini-review, we revise the development, and validation of the yeast-based p53 functional assay as a new tool for molecular epidemiology. We show that this assay has some very interesting virtues but also has some drawbacks. The yeast functional assay can be used to determine highly specific mutation fingerprints in the human p53 cDNA sequence. Discrimination is possible when comparing mutation spectra induced by sufficiently different mutagens. However, we also reported that the same carcinogen may induce distinguishable mutation spectra due to known influencing factors.

Complementarity to an miRNA seed region is sufficient to induce moderate repression of a target transcript in the unicellular green alga Chlamydomonas reinhardtii.

PubMed

Yamasaki, Tomohito; Voshall, Adam; Kim, Eun-Jeong; Moriyama, Etsuko; Cerutti, Heriberto; Ohama, Takeshi

2013-12-01

MicroRNAs (miRNAs) are 20-24 nt non-coding RNAs that play important regulatory roles in a broad range of eukaryotes by pairing with mRNAs to direct post-transcriptional repression. The mechanistic details of miRNA-mediated post-transcriptional regulation have been well documented in multicellular model organisms. However, this process remains poorly studied in algae such as Chlamydomonas reinhardtii, and specific features of miRNA biogenesis, target mRNA recognition and subsequent silencing are not well understood. In this study, we report on the characterization of a Chlamydomonas miRNA, cre-miR1174.2, which is processed from a near-perfect hairpin RNA. Using Gaussia luciferase (gluc) reporter genes, we have demonstrated that cre-miR1174.2 is functional in Chlamydomonas and capable of triggering site-specific cleavage at the center of a perfectly complementary target sequence. A mismatch tolerance test assay, based on pools of transgenic strains, revealed that target hybridization to nucleotides of the seed region, at the 5' end of an miRNA, was sufficient to induce moderate repression of expression. In contrast, pairing to the 3' region of the miRNA was not critical for silencing. Our results suggest that the base-pairing requirements for small RNA-mediated repression in C. reinhardtii are more similar to those of metazoans compared with the extensive complementarity that is typical of land plants. Individual Chlamydomonas miRNAs may potentially modulate the expression of numerous endogenous targets as a result of these relaxed base-pairing requirements. © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

Machine learning for classifying tuberculosis drug-resistance from DNA sequencing data

PubMed Central

Yang, Yang; Niehaus, Katherine E; Walker, Timothy M; Iqbal, Zamin; Walker, A Sarah; Wilson, Daniel J; Peto, Tim E A; Crook, Derrick W; Smith, E Grace; Zhu, Tingting; Clifton, David A

2018-01-01

Abstract Motivation Correct and rapid determination of Mycobacterium tuberculosis (MTB) resistance against available tuberculosis (TB) drugs is essential for the control and management of TB. Conventional molecular diagnostic test assumes that the presence of any well-studied single nucleotide polymorphisms is sufficient to cause resistance, which yields low sensitivity for resistance classification. Summary Given the availability of DNA sequencing data from MTB, we developed machine learning models for a cohort of 1839 UK bacterial isolates to classify MTB resistance against eight anti-TB drugs (isoniazid, rifampicin, ethambutol, pyrazinamide, ciprofloxacin, moxifloxacin, ofloxacin, streptomycin) and to classify multi-drug resistance. Results Compared to previous rules-based approach, the sensitivities from the best-performing models increased by 2-4% for isoniazid, rifampicin and ethambutol to 97% (P < 0.01), respectively; for ciprofloxacin and multi-drug resistant TB, they increased to 96%. For moxifloxacin and ofloxacin, sensitivities increased by 12 and 15% from 83 and 81% based on existing known resistance alleles to 95% and 96% (P < 0.01), respectively. Particularly, our models improved sensitivities compared to the previous rules-based approach by 15 and 24% to 84 and 87% for pyrazinamide and streptomycin (P < 0.01), respectively. The best-performing models increase the area-under-the-ROC curve by 10% for pyrazinamide and streptomycin (P < 0.01), and 4–8% for other drugs (P < 0.01). Availability and implementation The details of source code are provided at http://www.robots.ox.ac.uk/~davidc/code.php. Contact david.clifton@eng.ox.ac.uk Supplementary information Supplementary data are available at Bioinformatics online. PMID:29240876

Map-invariant spectral analysis for the identification of DNA periodicities

PubMed Central

2012-01-01

Many signal processing based methods for finding hidden periodicities in DNA sequences have primarily focused on assigning numerical values to the symbolic DNA sequence and then applying spectral analysis tools such as the short-time discrete Fourier transform (ST-DFT) to locate these repeats. The key results pertaining to this approach are however obtained using a very specific symbolic to numerical map, namely the so-called Voss representation. An important research problem is to therefore quantify the sensitivity of these results to the choice of the symbolic to numerical map. In this article, a novel algebraic approach to the periodicity detection problem is presented and provides a natural framework for studying the role of the symbolic to numerical map in finding these repeats. More specifically, we derive a new matrix-based expression of the DNA spectrum that comprises most of the widely used mappings in the literature as special cases, shows that the DNA spectrum is in fact invariable under all these mappings, and generates a necessary and sufficient condition for the invariance of the DNA spectrum to the symbolic to numerical map. Furthermore, the new algebraic framework decomposes the periodicity detection problem into several fundamental building blocks that are totally independent of each other. Sophisticated digital filters and/or alternate fast data transforms such as the discrete cosine and sine transforms can therefore be always incorporated in the periodicity detection scheme regardless of the choice of the symbolic to numerical map. Although the newly proposed framework is matrix based, identification of these periodicities can be achieved at a low computational cost. PMID:23067324

High density FTA plates serve as efficient long-term sample storage for HLA genotyping.

PubMed

Lange, V; Arndt, K; Schwarzelt, C; Boehme, I; Giani, A S; Schmidt, A H; Ehninger, G; Wassmuth, R

2014-02-01

Storage of dried blood spots (DBS) on high-density FTA(®) plates could constitute an appealing alternative to frozen storage. However, it remains controversial whether DBS are suitable for high-resolution sequencing of human leukocyte antigen (HLA) alleles. Therefore, we extracted DNA from DBS that had been stored for up to 4 years, using six different methods. We identified those extraction methods that recovered sufficient high-quality DNA for reliable high-resolution HLA sequencing. Further, we confirmed that frozen whole blood samples that had been stored for several years can be transferred to filter paper without compromising HLA genotyping upon extraction. Concluding, DNA derived from high-density FTA(®) plates is suitable for high-resolution HLA sequencing, provided that appropriate extraction protocols are employed. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Dynamic epigenetic states of maize centromeres

PubMed Central

Liu, Yalin; Su, Handong; Zhang, Jing; Liu, Yang; Han, Fangpu; Birchler, James A.

2015-01-01

The centromere is a specialized chromosomal region identified as the major constriction, upon which the kinetochore complex is formed, ensuring accurate chromosome orientation and segregation during cell division. The rapid evolution of centromere DNA sequence and the conserved centromere function are two contradictory aspects of centromere biology. Indeed, the sole presence of genetic sequence is not sufficient for centromere formation. Various dicentric chromosomes with one inactive centromere have been recognized. It has also been found that de novo centromere formation is common on fragments in which centromeric DNA sequences are lost. Epigenetic factors play important roles in centromeric chromatin assembly and maintenance. Non-disjunction of the supernumerary B chromosome centromere is independent of centromere function, but centromere pairing during early prophase of meiosis I requires an active centromere. This review discusses recent studies in maize about genetic and epigenetic elements regulating formation and maintenance of centromere chromatin, as well as centromere behavior in meiosis. PMID:26579154

A Gibbs sampler for motif detection in phylogenetically close sequences

NASA Astrophysics Data System (ADS)

Siddharthan, Rahul; van Nimwegen, Erik; Siggia, Eric

2004-03-01

Genes are regulated by transcription factors that bind to DNA upstream of genes and recognize short conserved ``motifs'' in a random intergenic ``background''. Motif-finders such as the Gibbs sampler compare the probability of these short sequences being represented by ``weight matrices'' to the probability of their arising from the background ``null model'', and explore this space (analogous to a free-energy landscape). But closely related species may show conservation not because of functional sites but simply because they have not had sufficient time to diverge, so conventional methods will fail. We introduce a new Gibbs sampler algorithm that accounts for common ancestry when searching for motifs, while requiring minimal ``prior'' assumptions on the number and types of motifs, assessing the significance of detected motifs by ``tracking'' clusters that stay together. We apply this scheme to motif detection in sporulation-cycle genes in the yeast S. cerevisiae, using recent sequences of other closely-related Saccharomyces species.

Earthquake stress via event ratio levels: Application to the 2011 and 2016 Oklahoma seismic sequences

DOE Office of Scientific and Technical Information (OSTI.GOV)

Walter, William R.; Yoo, Seung -Hoon; Mayeda, Kevin

Here, we develop a new methodology for determining earthquake stress drop and apparent stress values via spectral ratio asymptotic levels. With sufficient bandwidth, the stress ratio for a pair of events can be directly related to these low- and high-frequency levels. This avoids the need to assume a particular spectral model and derive stress drop from cubed corner frequency measures. The method can be applied to spectral ratios for any pair of closely related earthquakes and is particularly well suited for coda envelope methods that provide good azimuthally averaged, point-source measures. We apply the new method to the 2011 Praguemore » and 2016 Pawnee earthquake sequences in Oklahoma. The sequences show stress scaling with size and depth, with the largest events having apparent stress levels near 1 MPa and smaller and/or shallower events having systematically lower stress values.« less

Hyperpolarized 13C pyruvate mouse brain metabolism with absorptive-mode EPSI at 1 T

NASA Astrophysics Data System (ADS)

Miloushev, Vesselin Z.; Di Gialleonardo, Valentina; Salamanca-Cardona, Lucia; Correa, Fabian; Granlund, Kristin L.; Keshari, Kayvan R.

2017-02-01

The expected signal in echo-planar spectroscopic imaging experiments was explicitly modeled jointly in spatial and spectral dimensions. Using this as a basis, absorptive-mode type detection can be achieved by appropriate choice of spectral delays and post-processing techniques. We discuss the effects of gradient imperfections and demonstrate the implementation of this sequence at low field (1.05 T), with application to hyperpolarized [1-13C] pyruvate imaging of the mouse brain. The sequence achieves sufficient signal-to-noise to monitor the conversion of hyperpolarized [1-13C] pyruvate to lactate in the mouse brain. Hyperpolarized pyruvate imaging of mouse brain metabolism using an absorptive-mode EPSI sequence can be applied to more sophisticated murine disease and treatment models. The simple modifications presented in this work, which permit absorptive-mode detection, are directly translatable to human clinical imaging and generate improved absorptive-mode spectra without the need for refocusing pulses.

Earthquake stress via event ratio levels: Application to the 2011 and 2016 Oklahoma seismic sequences

DOE PAGES

Walter, William R.; Yoo, Seung -Hoon; Mayeda, Kevin; ...

2017-04-03

Here, we develop a new methodology for determining earthquake stress drop and apparent stress values via spectral ratio asymptotic levels. With sufficient bandwidth, the stress ratio for a pair of events can be directly related to these low- and high-frequency levels. This avoids the need to assume a particular spectral model and derive stress drop from cubed corner frequency measures. The method can be applied to spectral ratios for any pair of closely related earthquakes and is particularly well suited for coda envelope methods that provide good azimuthally averaged, point-source measures. We apply the new method to the 2011 Praguemore » and 2016 Pawnee earthquake sequences in Oklahoma. The sequences show stress scaling with size and depth, with the largest events having apparent stress levels near 1 MPa and smaller and/or shallower events having systematically lower stress values.« less

Dynamic epigenetic states of maize centromeres.

PubMed

Liu, Yalin; Su, Handong; Zhang, Jing; Liu, Yang; Han, Fangpu; Birchler, James A

2015-01-01

The centromere is a specialized chromosomal region identified as the major constriction, upon which the kinetochore complex is formed, ensuring accurate chromosome orientation and segregation during cell division. The rapid evolution of centromere DNA sequence and the conserved centromere function are two contradictory aspects of centromere biology. Indeed, the sole presence of genetic sequence is not sufficient for centromere formation. Various dicentric chromosomes with one inactive centromere have been recognized. It has also been found that de novo centromere formation is common on fragments in which centromeric DNA sequences are lost. Epigenetic factors play important roles in centromeric chromatin assembly and maintenance. Non-disjunction of the supernumerary B chromosome centromere is independent of centromere function, but centromere pairing during early prophase of meiosis I requires an active centromere. This review discusses recent studies in maize about genetic and epigenetic elements regulating formation and maintenance of centromere chromatin, as well as centromere behavior in meiosis.

Identification of a Novel Recycling Sequence in the C-tail of FPR2/ALX Receptor

PubMed Central

Thompson, Dawn; McArthur, Simon; Hislop, James N.; Flower, Roderick J.; Perretti, Mauro

2014-01-01

Formyl-peptide receptor type 2 (FPR2; also called ALX because it is the receptor for lipoxin A4) sustains a variety of biological responses relevant to the development and control of inflammation, yet the cellular regulation of this G-protein-coupled receptor remains unexplored. Here we report that, in response to peptide agonist activation, FPR2/ALX undergoes β-arrestin-mediated endocytosis followed by rapid recycling to the plasma membrane. We identify a transplantable recycling sequence that is both necessary and sufficient for efficient receptor recycling. Furthermore, removal of this C-terminal recycling sequence alters the endocytic fate of FPR2/ALX and evokes pro-apoptotic effects in response to agonist activation. This study demonstrates the importance of endocytic recycling in the anti-apoptotic properties of FPR2/ALX and identifies the molecular determinant required for modulation of this process fundamental for the control of inflammation. PMID:25326384

Karyotype Analysis of Four Vicia Species using In Situ Hybridization with Repetitive Sequences

PubMed Central

NAVRÁTILOVÁ, ALICE; NEUMANN, PAVEL; MACAS, JIŘÍ

2003-01-01

Mitotic chromosomes of four Vicia species (V. sativa, V. grandiflora, V. pannonica and V. narbonensis) were subjected to in situ hybridization with probes derived from conserved plant repetitive DNA sequences (18S–25S and 5S rDNA, telomeres) and genus‐specific satellite repeats (VicTR‐A and VicTR‐B). Numbers and positions of hybridization signals provided cytogenetic landmarks suitable for unambiguous identification of all chromosomes, and establishment of the karyotypes. The VicTR‐A and ‐B sequences, in particular, produced highly informative banding patterns that alone were sufficient for discrimination of all chromosomes. However, these patterns were not conserved among species and thus could not be employed for identification of homologous chromosomes. This fact, together with observed variations in positions and numbers of rDNA loci, suggests considerable divergence between karyotypes of the species studied. PMID:12770847

A Poisson hierarchical modelling approach to detecting copy number variation in sequence coverage data

PubMed Central

2013-01-01

Background The advent of next generation sequencing technology has accelerated efforts to map and catalogue copy number variation (CNV) in genomes of important micro-organisms for public health. A typical analysis of the sequence data involves mapping reads onto a reference genome, calculating the respective coverage, and detecting regions with too-low or too-high coverage (deletions and amplifications, respectively). Current CNV detection methods rely on statistical assumptions (e.g., a Poisson model) that may not hold in general, or require fine-tuning the underlying algorithms to detect known hits. We propose a new CNV detection methodology based on two Poisson hierarchical models, the Poisson-Gamma and Poisson-Lognormal, with the advantage of being sufficiently flexible to describe different data patterns, whilst robust against deviations from the often assumed Poisson model. Results Using sequence coverage data of 7 Plasmodium falciparum malaria genomes (3D7 reference strain, HB3, DD2, 7G8, GB4, OX005, and OX006), we showed that empirical coverage distributions are intrinsically asymmetric and overdispersed in relation to the Poisson model. We also demonstrated a low baseline false positive rate for the proposed methodology using 3D7 resequencing data and simulation. When applied to the non-reference isolate data, our approach detected known CNV hits, including an amplification of the PfMDR1 locus in DD2 and a large deletion in the CLAG3.2 gene in GB4, and putative novel CNV regions. When compared to the recently available FREEC and cn.MOPS approaches, our findings were more concordant with putative hits from the highest quality array data for the 7G8 and GB4 isolates. Conclusions In summary, the proposed methodology brings an increase in flexibility, robustness, accuracy and statistical rigour to CNV detection using sequence coverage data. PMID:23442253

Molecular Characterization of Indolent Prostate Cancer

DTIC Science & Technology

2016-12-01

prostate - specific antigen (PSA) test, and treated aggressively following diagnosis, leading to the contemporary problem of prostate cancer over-diagnosis... specific purpose of comparing low- risk and high-risk prostate cancer. Figure 3 shows the mapping rates for exon, intron, and inter-genic sequences. The...FFPE specimens, for the specific comparison of low-risk and high-risk prostate cancer. 5. Identified sufficient number of biopsy cases and sections

Serendipitous discovery of Wolbachia genomes in multiple Drosophila species.

PubMed

Salzberg, Steven L; Dunning Hotopp, Julie C; Delcher, Arthur L; Pop, Mihai; Smith, Douglas R; Eisen, Michael B; Nelson, William C

2005-01-01

The Trace Archive is a repository for the raw, unanalyzed data generated by large-scale genome sequencing projects. The existence of this data offers scientists the possibility of discovering additional genomic sequences beyond those originally sequenced. In particular, if the source DNA for a sequencing project came from a species that was colonized by another organism, then the project may yield substantial amounts of genomic DNA, including near-complete genomes, from the symbiotic or parasitic organism. By searching the publicly available repository of DNA sequencing trace data, we discovered three new species of the bacterial endosymbiont Wolbachia pipientis in three different species of fruit fly: Drosophila ananassae, D. simulans, and D. mojavensis. We extracted all sequences with partial matches to a previously sequenced Wolbachia strain and assembled those sequences using customized software. For one of the three new species, the data recovered were sufficient to produce an assembly that covers more than 95% of the genome; for a second species the data produce the equivalent of a 'light shotgun' sampling of the genome, covering an estimated 75-80% of the genome; and for the third species the data cover approximately 6-7% of the genome. The results of this study reveal an unexpected benefit of depositing raw data in a central genome sequence repository: new species can be discovered within this data. The differences between these three new Wolbachia genomes and the previously sequenced strain revealed numerous rearrangements and insertions within each lineage and hundreds of novel genes. The three new genomes, with annotation, have been deposited in GenBank.

Statistical tests to compare motif count exceptionalities

PubMed Central

Robin, Stéphane; Schbath, Sophie; Vandewalle, Vincent

2007-01-01

Background Finding over- or under-represented motifs in biological sequences is now a common task in genomics. Thanks to p-value calculation for motif counts, exceptional motifs are identified and represent candidate functional motifs. The present work addresses the related question of comparing the exceptionality of one motif in two different sequences. Just comparing the motif count p-values in each sequence is indeed not sufficient to decide if this motif is significantly more exceptional in one sequence compared to the other one. A statistical test is required. Results We develop and analyze two statistical tests, an exact binomial one and an asymptotic likelihood ratio test, to decide whether the exceptionality of a given motif is equivalent or significantly different in two sequences of interest. For that purpose, motif occurrences are modeled by Poisson processes, with a special care for overlapping motifs. Both tests can take the sequence compositions into account. As an illustration, we compare the octamer exceptionalities in the Escherichia coli K-12 backbone versus variable strain-specific loops. Conclusion The exact binomial test is particularly adapted for small counts. For large counts, we advise to use the likelihood ratio test which is asymptotic but strongly correlated with the exact binomial test and very simple to use. PMID:17346349

Endogenous Hot Spots of De Novo Telomere Addition in the Yeast Genome Contain Proximal Enhancers That Bind Cdc13

PubMed Central

Obodo, Udochukwu C.; Epum, Esther A.; Platts, Margaret H.; Seloff, Jacob; Dahlson, Nicole A.; Velkovsky, Stoycho M.; Paul, Shira R.

2016-01-01

DNA double-strand breaks (DSBs) pose a threat to genome stability and are repaired through multiple mechanisms. Rarely, telomerase, the enzyme that maintains telomeres, acts upon a DSB in a mutagenic process termed telomere healing. The probability of telomere addition is increased at specific genomic sequences termed sites of repair-associated telomere addition (SiRTAs). By monitoring repair of an induced DSB, we show that SiRTAs on chromosomes V and IX share a bipartite structure in which a core sequence (Core) is directly targeted by telomerase, while a proximal sequence (Stim) enhances the probability of de novo telomere formation. The Stim and Core sequences are sufficient to confer a high frequency of telomere addition to an ectopic site. Cdc13, a single-stranded DNA binding protein that recruits telomerase to endogenous telomeres, is known to stimulate de novo telomere addition when artificially recruited to an induced DSB. Here we show that the ability of the Stim sequence to enhance de novo telomere addition correlates with its ability to bind Cdc13, indicating that natural sites at which telomere addition occurs at high frequency require binding by Cdc13 to a sequence 20 to 100 bp internal from the site at which telomerase acts to initiate de novo telomere addition. PMID:27044869

Recognition of the Xenopus ribosomal core promoter by the transcription factor xUBF involves multiple HMG box domains and leads to an xUBF interdomain interaction.

PubMed

Leblanc, B; Read, C; Moss, T

1993-02-01

The interaction of the ribosomal transcription factor xUBF with the RNA polymerase I core promoter of Xenopus laevis has been studied both at the DNA and protein levels. It is shown that a single xUBF-DNA complex forms over the 40S initiation site (+1) and involves at least the DNA sequences between -20 and +60 bp. DNA sequences upstream of +10 and downstream of +18 are each sufficient to direct complex formation independently. HMG box 1 of xUBF independently recognizes the sequences -20 to -1 and +1 to +22 and the addition of the N-terminal dimerization domain to HMG box 1 stabilizes its interaction with these sequences approximately 10-fold. HMG boxes 2/3 interact with the DNA downstream of +22 and can independently position xUBF across the initiation site. The C-terminal segment of xUBF, HMG boxes 4, 5 or the acidic domain, directly or indirectly interact with HMG box 1, making the core promoter sequences between -11 and -15 hypersensitive to DNase. This interaction also requires the DNA sequences between +17 and +32, i.e. the HMG box 2/3 binding site. The data suggest extensive folding of the core promoter within the xUBF complex.

Whole Genome Sequencing versus Traditional Genotyping for Investigation of a Mycobacterium tuberculosis Outbreak: A Longitudinal Molecular Epidemiological Study

PubMed Central

Rückert, Christian; Nübel, Ulrich; Blom, Jochen; Wirth, Thierry; Jaenicke, Sebastian; Schuback, Sieglinde; Rüsch-Gerdes, Sabine; Supply, Philip; Kalinowski, Jörn; Niemann, Stefan

2013-01-01

Background Understanding Mycobacterium tuberculosis (Mtb) transmission is essential to guide efficient tuberculosis control strategies. Traditional strain typing lacks sufficient discriminatory power to resolve large outbreaks. Here, we tested the potential of using next generation genome sequencing for identification of outbreak-related transmission chains. Methods and Findings During long-term (1997 to 2010) prospective population-based molecular epidemiological surveillance comprising a total of 2,301 patients, we identified a large outbreak caused by an Mtb strain of the Haarlem lineage. The main performance outcome measure of whole genome sequencing (WGS) analyses was the degree of correlation of the WGS analyses with contact tracing data and the spatio-temporal distribution of the outbreak cases. WGS analyses of the 86 isolates revealed 85 single nucleotide polymorphisms (SNPs), subdividing the outbreak into seven genome clusters (two to 24 isolates each), plus 36 unique SNP profiles. WGS results showed that the first outbreak isolates detected in 1997 were falsely clustered by classical genotyping. In 1998, one clone (termed “Hamburg clone”) started expanding, apparently independently from differences in the social environment of early cases. Genome-based clustering patterns were in better accordance with contact tracing data and the geographical distribution of the cases than clustering patterns based on classical genotyping. A maximum of three SNPs were identified in eight confirmed human-to-human transmission chains, involving 31 patients. We estimated the Mtb genome evolutionary rate at 0.4 mutations per genome per year. This rate suggests that Mtb grows in its natural host with a doubling time of approximately 22 h (400 generations per year). Based on the genome variation discovered, emergence of the Hamburg clone was dated back to a period between 1993 and 1997, hence shortly before the discovery of the outbreak through epidemiological surveillance. Conclusions Our findings suggest that WGS is superior to conventional genotyping for Mtb pathogen tracing and investigating micro-epidemics. WGS provides a measure of Mtb genome evolution over time in its natural host context. Please see later in the article for the Editors' Summary PMID:23424287

Translation of vph mRNA in Streptomyces lividans and Escherichia coli after removal of the 5' untranslated leader.

PubMed

Wu, C J; Janssen, G R

1996-10-01

The Streptomyces vinaceus viomycin phosphotransferase (vph) mRNA contains an untranslated leader with a conventional Shine-Dalgarno homology. The vph leader was removed by ligation of the vph coding sequence to the transcriptional start site of a Streptomyces or an Escherichia coli promoter, such that transcription would initiate at the first position of the vph start codon. Analysis of mRNA demonstrated that transcription initiated primarily at the A of the vph AUG translational start codon in both Streptomyces lividans and E. coli; cells expressing the unleadered vph mRNA were resistant to viomycin indicating that the Shine-Dalgarno sequence, or other features contained within the leader, was not necessary for vph translation. Addition of four nucleotides (5'-AUGC-3') onto the 5' end of the unleadered vph mRNA resulted in translation initiation from the vph start codon and the AUG triplet contained within the added sequence. Translational fusions of vph sequence to a Tn5 neo reporter gene indicated that the first 16 codons of vph coding sequence were sufficient to specify the translational start site and reading frame for expression of neomycin resistance in both E. coli and S. lividans.

Structure and biochemical functions of four simian virus 40 truncated large-T antigens.

PubMed Central

Chaudry, F; Harvey, R; Smith, A E

1982-01-01

The structure of four abnormal T antigens which are present in different simian virus 40 (SV40)-transformed mouse cell lines was studied by tryptic peptide mapping, partial proteolysis fingerprinting, immunoprecipitation with monoclonal antibodies, and in vitro translation. The results obtained allowed us to deduce that these proteins, which have apparent molecular weights of 15,000, 22,000, 33,000 and 45,000, are truncated forms of large-T antigen extending to different amounts into the amino acid sequences unique to large-T. The proteins are all phosphorylated, probably at a site between amino acids 106 and 123. The mRNAs coding for the proteins probably contain the normal large-T splice but are shorter than the normal transcripts of the SV40 early region. The truncated large-Ts were tested for the ability to bind to double-stranded DNA-cellulose. This showed that the 33,000- and 45,000-molecular-weight polypeptides contained sequences sufficient for binding under the conditions used, whereas the 15,000- and 22,000-molecular-weight forms did not. Together with published data, this allows the tentative mapping of a region of SV40 large-T between amino acids 109 and 272 that is necessary and may be sufficient for the binding to double-stranded DNA-cellulose in vitro. None of the truncated large-T species formed a stable complex with the host cell protein referred to as nonviral T-antigen or p53, suggesting that the carboxy-terminal sequences of large-T are necessary for complex formation. Images PMID:6292504

Tracing the neural basis of auditory entrainment.

PubMed

Lehmann, Alexandre; Arias, Diana Jimena; Schönwiesner, Marc

2016-11-19

Neurons in the auditory cortex synchronize their responses to temporal regularities in sound input. This coupling or "entrainment" is thought to facilitate beat extraction and rhythm perception in temporally structured sounds, such as music. As a consequence of such entrainment, the auditory cortex responds to an omitted (silent) sound in a regular sequence. Although previous studies suggest that the auditory brainstem frequency-following response (FFR) exhibits some of the beat-related effects found in the cortex, it is unknown whether omissions of sounds evoke a brainstem response. We simultaneously recorded cortical and brainstem responses to isochronous and irregular sequences of consonant-vowel syllable /da/ that contained sporadic omissions. The auditory cortex responded strongly to omissions, but we found no evidence of evoked responses to omitted stimuli from the auditory brainstem. However, auditory brainstem responses in the isochronous sound sequence were more consistent across trials than in the irregular sequence. These results indicate that the auditory brainstem faithfully encodes short-term acoustic properties of a stimulus and is sensitive to sequence regularity, but does not entrain to isochronous sequences sufficiently to generate overt omission responses, even for sequences that evoke such responses in the cortex. These findings add to our understanding of the processing of sound regularities, which is an important aspect of human cognitive abilities like rhythm, music and speech perception. Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.