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Sample records for amplicon va derived

  1. Constitutive and Inducible Innate Responses in Cells Infected by HSV-1-Derived Amplicon Vectors

    PubMed Central

    Tsitoura, Eliza; Epstein, Alberto L

    2010-01-01

    Amplicons are helper-dependent herpes simplex virus type 1 (HSV-1)-based vectors that can deliver very large foreign DNA sequences and, as such, are good candidates both for gene delivery and vaccine development. However, many studies have shown that innate constitutive or induced cellular responses, elicited or activated by the entry of HSV-1 particles, can play a significant role in the control of transgenic expression and in the induction of inflammatory responses. Moreover, transgene expression from helper-free amplicon stocks is often weak and transient, depending on the particular type of infected cells, suggesting that cellular responses could be also responsible for the silencing of amplicon-mediated transgene expression. This review summarizes the current experimental evidence underlying these latter concepts, focusing on the impact on transgene expression of very-early interactions between amplicon particles and the infected cells, and speculates on possible ways to counteract the cellular protective mechanisms, thus allowing stable transgene expression without enhancement of vector toxicity. PMID:20811588

  2. Use of Amplicon-6 Vectors Derived from Human Herpesvirus 6 for Efficient Expression of Membrane-Associated and -Secreted Proteins in T Cells

    PubMed Central

    Borenstein, Ronen; Singer, Oded; Moseri, Adi; Frenkel, Niza

    2004-01-01

    The composite amplicon-6 vectors, which are derived from human herpesvirus 6 (HHV-6), can target hematopoietic cells. In the presence of the respective helper viruses, the amplicons are replicated by the rolling circle mechanism, yielding defective genomes of overall size 135 to 150 kb, composed of multiple repeats of units, containing the viral DNA replication origin, packaging signals, and the selected transgene(s). We report the use of amplicon-6 vectors designed for transgene expression in T cells. The selected transgenes included the green fluorescent protein marker, the herpes simplex virus type 1 glycoprotein D (gD), and the gD gene deleted in the transmembrane region (gDsec). The vectors were tested after electroporation and passage in T cells with or without helper HHV-6A superinfections. The results were as follows. (i)The vectors could be passaged both as cell-associated and as cell-free secreted virions infectious to new cells. (ii)The intact gD accumulated at the cell surface, whereas the gDsec was dispersed at internal locations of the cells or was secreted into the medium. (iii)Analyses of amplicon-6-gD expression by flow cytometry have shown significant expression in cultures with reiterated amplicons and helper viruses. The vector has spread to >60% of the cells, and the efficiency of expression per cell increased 15-fold, most likely due to the presence of concatemeric amplicon repeats. Current studies are designed to test whether amplicon-6 vectors can be used for gene therapy in lymphocytes and whether amplicon-6 vectors expressed in T cells and dendritic cells can induce strong cellular and humoral immune responses. PMID:15078955

  3. Functional Overexpression of Vomeronasal Receptors Using a Herpes Simplex Virus Type 1 (HSV-1)-Derived Amplicon.

    PubMed

    Stein, Benjamin; Alonso, María Teresa; Zufall, Frank; Leinders-Zufall, Trese; Chamero, Pablo

    2016-01-01

    In mice, social behaviors such as mating and aggression are mediated by pheromones and related chemosignals. The vomeronasal organ (VNO) detects olfactory information from other individuals by sensory neurons tuned to respond to specific chemical cues. Receptors expressed by vomeronasal neurons are implicated in selective detection of these cues. Nearly 400 receptor genes have been identified in the mouse VNO, but the tuning properties of individual receptors remain poorly understood, in part due to the lack of a robust heterologous expression system. Here we develop a herpes virus-based amplicon delivery system to overexpress three types of vomeronasal receptor genes and to characterize cell responses to their proposed ligands. Through Ca2+ imaging in native VNO cells we show that virus-induced overexpression of V1rj2, V2r1b or Fpr3 caused a pronounced increase of responsivity to sulfated steroids, MHC-binding peptide or the synthetic hexapeptide W-peptide, respectively. Other related ligands were not recognized by infected individual neurons, indicating a high degree of selectivity by the overexpressed receptor. Removal of G-protein signaling eliminates Ca2+ responses, indicating that the endogenous second messenger system is essential for observing receptor activation. Our results provide a novel expression system for vomeronasal receptors that should be useful for understanding the molecular logic of VNO ligand detection. Functional expression of vomeronasal receptors and their deorphanization provides an essential requirement for deciphering the neural mechanisms controlling behavior. PMID:27195771

  4. Functional Overexpression of Vomeronasal Receptors Using a Herpes Simplex Virus Type 1 (HSV-1)-Derived Amplicon

    PubMed Central

    Stein, Benjamin; Alonso, María Teresa; Zufall, Frank; Leinders-Zufall, Trese; Chamero, Pablo

    2016-01-01

    In mice, social behaviors such as mating and aggression are mediated by pheromones and related chemosignals. The vomeronasal organ (VNO) detects olfactory information from other individuals by sensory neurons tuned to respond to specific chemical cues. Receptors expressed by vomeronasal neurons are implicated in selective detection of these cues. Nearly 400 receptor genes have been identified in the mouse VNO, but the tuning properties of individual receptors remain poorly understood, in part due to the lack of a robust heterologous expression system. Here we develop a herpes virus-based amplicon delivery system to overexpress three types of vomeronasal receptor genes and to characterize cell responses to their proposed ligands. Through Ca2+ imaging in native VNO cells we show that virus-induced overexpression of V1rj2, V2r1b or Fpr3 caused a pronounced increase of responsivity to sulfated steroids, MHC-binding peptide or the synthetic hexapeptide W-peptide, respectively. Other related ligands were not recognized by infected individual neurons, indicating a high degree of selectivity by the overexpressed receptor. Removal of G-protein signaling eliminates Ca2+ responses, indicating that the endogenous second messenger system is essential for observing receptor activation. Our results provide a novel expression system for vomeronasal receptors that should be useful for understanding the molecular logic of VNO ligand detection. Functional expression of vomeronasal receptors and their deorphanization provides an essential requirement for deciphering the neural mechanisms controlling behavior. PMID:27195771

  5. Murine Bone Marrow-Derived Dendritic Cells Transduced by Light-Helper-Dependent Herpes Simplex Virus-1 Amplicon Vector Acquire a Mature Dendritic Cell Phenotype.

    PubMed

    Oz-Arslan, Devrim; Tsitoura, Eliza; Kazazi, Dorothea; Kouvatsis, Vlasis; Epstein, Alberto L; Mavromara, Penelope

    2015-06-01

    Dendritic cells (DCs) turn into the most potent antigen-presenting cells following a complex transforming process, which leads to their maturation. Herpes simplex virus-1 (HSV-1) amplicon vectors represent highly versatile viral vector platforms with the ability to transduce immature DCs at exceedingly high efficiencies, while the efficiency of infection of mature DCs is significantly low. However, the bacterial artificial chromosome (BAC)-dependent (BD) amplicon vectors tested so far do not result in the maturation of mouse bone marrow-derived DCs (BMDCs) in vitro. In this study we investigated the effects of light-helper-dependent (LHD) amplicon vectors produced with the replication-defective HSV-1 LaLΔJ helper virus system. First, we observed that transgene expression in BMDC cultures was equally potent between the LHD and the BD amplicon vectors. We determined that the percentage of transduced cells and the duration of transgene expression were negatively influenced by the presence of increasing levels of helper virus. Second, infection by the LHD amplicon vector as well as the helper HSV-1 LaLΔJ virus alone resulted in the phenotypic maturation of BMDCs and the expression of both interferon-stimulated genes and proinflammatory cytokines. Further comparisons of the gene expression of infected DCs showed that while interferon-stimulated genes such as Ifit1, Ifit3, Mx2, Isg15, and Cxcl10 were induced by both BD and LHD amplicon vectors, early proinflammatory cytokine gene expression (Tnfa, Il1a, Il1b, Il6, Il10, Il12b, Cxcl1, and Cxcl16) and DC maturation were mediated only by the LHD amplicons. PMID:26046494

  6. Mechanisms Regulating Acquisition of Platelet-Derived Factor V/Va by Megakaryocytes.

    PubMed

    Gertz, Jacqueline M; Bouchard, Beth A

    2015-10-01

    Factor Va serves as the nonenzymatic protein cofactor for the prothrombinase complex, which converts prothrombin to thrombin in the events leading to formation of a hemostatic plug. Several observations support the concept that platelet-derived factor V/Va is physically and functionally distinct and plays a more important role in thrombin generation at sites of vascular injury as compared to its plasma counterpart. Platelet-derived factor V/Va is generated following endocytosis of the plasma-derived molecule by the platelet precursor cells, megakaryocytes, via a two receptor system consisting of low density lipoprotein (LDL) receptor-related protein-1 (LRP-1) and an unidentified specific "binding site". More recently, it was suggested that a cell surface-expressed β-galactoside binding protein, galectin-8, was involved in factor V endocytosis. Endocytosed factor V is trafficked through the cell and retailored prior to its storage in α-granules. Given the essential role of platelet-derived factor Va in clot formation, understanding the cellular and molecular mechanisms that regulate how platelets acquire this molecule will be important for the treatment of excessive bleeding or clotting. PMID:25800007

  7. Identification of QTL conditioning partial resistance to white mold in kidney bean line VA19 derived from an interspecific population

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Scarlet-runner bean (Phaseolus coccineus L.), a representative species of the secondary gene pool of common bean, is a potential source of white mold resistance for improving dry bean and snap bean. VA19 is a light-red kidney bean line that possesses resistance to white mold putatively derived from...

  8. Effects of an apovincaminic acid derivative VA-045 on neuronal activity in rat brain stem reticular formation.

    PubMed

    Okuyama, S; Kawashima, N; Araki, H; Otomo, S; Shima, K

    1994-01-01

    Extracellular single unit and spontaneous cortical electroencephalographic (ECoG) recordings were made in the brain stem reticular formation (RF) and the frontal cortex, respectively, of urethane-anesthetized rats. VA-045, an apovincaminic acid derivative had no significant effects on the spontaneous firing rate of the RF neurons and ECoG. Closed head injury (CHI) was induced by dropping a 400 g weight through a tube from 70 cm on a steel helmet placed on the vertex. CHI led to a reduction in the firing rate of RF neurons and ECoG synchronization. VA-045 dose-dependently reversed the CHI-induced decrease in the firing rate of RF and led to ECoG desyncronization. Clonidine, an alpha 2 adrenoceptor agonist, and scopolamine, a muscarinic receptor antagonist, also reduced the firing rate of RF neurons and led to ECoG synchronization. VA-045 dose-dependently antagonized the effects of clonidine, but not the effects of scopolamine on RF neuronal activity and ECoG. Thus, VA-045 has an ameliorating effect on the CHI-induced depression of neuronal activity in the RF. A central alpha 2 adrenoceptor antagonistic action may be involved. PMID:7968229

  9. A Novel Ligustrazine Derivative T-VA Prevents Neurotoxicity in Differentiated PC12 Cells and Protects the Brain against Ischemia Injury in MCAO Rats

    PubMed Central

    Li, Guoling; Tian, Yufei; Zhang, Yuzhong; Hong, Ying; Hao, Yingzhi; Chen, Chunxiao; Wang, Penglong; Lei, Haimin

    2015-01-01

    Broad-spectrum drugs appear to be more promising for the treatment of acute ischemic stroke. In our previous work, a new ligustrazine derivative (3,5,6-trimethylpyrazin-2-yl) methyl 3-methoxy-4-[(3,5,6-trimethylpyrazin-2-yl)methoxy]benzoate (T-VA) showed neuroprotective effect on injured PC12 cells (EC50 = 4.249 µM). In the current study, we show that this beneficial effect was due to the modulation of nuclear transcription factor-κB/p65 (NF-κB/p65) and cyclooxygenase-2 (COX-2) expressions. We also show that T-VA exhibited neuroprotective effect in a rat model of ischemic stroke with concomitant improvement of motor functions. We propose that the protective effect observed in vivo is owing to increased vascular endothelial growth factor (VEGF) expression, decreased oxidative stress, and up-regulation of Ca2+–Mg2+ ATP enzyme activity. Altogether, our results warrant further studies on the utility of T-VA for the potential treatment of ischemic brain injuries, such as stroke. PMID:26370988

  10. The herpes simplex virus amplicon: analyses of cis-acting replication functions.

    PubMed Central

    Spaete, R R; Frenkel, N

    1985-01-01

    Previous studies have shown that defective virus vectors (amplicons) derived from herpes simplex viruses could be efficiently propagated in virus stocks in the presence of trans-acting helper virus functions. The present study established that two separate cis-acting functions--a DNA replication origin and a cleavage/packaging signal--are required for amplicon propagation. Using deleted derivatives of cloned amplicons, we mapped one of the viral DNA replication origins (ori-2 or oriL) at coordinate 0.422 of the standard HSV-1 genome and at an equivalent position within the HSV-2 genome. Images PMID:2983310

  11. Silencing Status Epilepticus-Induced BDNF Expression with Herpes Simplex Virus Type-1 Based Amplicon Vectors

    PubMed Central

    Falcicchia, Chiara; Trempat, Pascal; Binaschi, Anna; Perrier-Biollay, Coline; Roncon, Paolo; Soukupova, Marie; Berthommé, Hervé; Simonato, Michele

    2016-01-01

    Brain-derived neurotrophic factor (BDNF) has been found to produce pro- but also anti-epileptic effects. Thus, its validity as a therapeutic target must be verified using advanced tools designed to block or to enhance its signal. The aim of this study was to develop tools to silence the BDNF signal. We generated Herpes simplex virus type 1 (HSV-1) derived amplicon vectors, i.e. viral particles containing a genome of 152 kb constituted of concatameric repetitions of an expression cassette, enabling the expression of the gene of interest in multiple copies. HSV-1 based amplicon vectors are non-pathogenic and have been successfully employed in the past for gene delivery into the brain of living animals. Therefore, amplicon vectors should represent a logical choice for expressing a silencing cassette, which, in multiple copies, is expected to lead to an efficient knock-down of the target gene expression. Here, we employed two amplicon-based BDNF silencing strategies. The first, antisense, has been chosen to target and degrade the cytoplasmic mRNA pool of BDNF, whereas the second, based on the convergent transcription technology, has been chosen to repress transcription at the BDNF gene. Both these amplicon vectors proved to be effective in down-regulating BDNF expression in vitro, in BDNF-expressing mesoangioblast cells. However, only the antisense strategy was effective in vivo, after inoculation in the hippocampus in a model of status epilepticus in which BDNF mRNA levels are strongly increased. Interestingly, the knocking down of BDNF levels induced with BDNF-antisense was sufficient to produce significant behavioral effects, in spite of the fact that it was produced only in a part of a single hippocampus. In conclusion, this study demonstrates a reliable effect of amplicon vectors in knocking down gene expression in vitro and in vivo. Therefore, this approach may find broad applications in neurobiological studies. PMID:26954758

  12. Co-existence of circular and multiple linear amplicons in methotrexate-resistant Leishmania.

    PubMed Central

    Olmo, A; Arrebola, R; Bernier, V; González-Pacanowska, D; Ruiz-Pérez, L M

    1995-01-01

    Circular and linear amplicons were analyzed in detail in Leishmania tropica cells resistant to methotrexate (MTX). Both types of elements presented sequences related to the H locus and coexisted in resistant cells. The linear amplicons appeared first during the selection process (at 10 microM MTX) and varied with regard to size and structure in cells exposed to increasing concentrations of drug. The circular element was evident at higher concentrations (50 microMs) but was the major amplified DNA in cells resistant to 1000 microM MTX while the level of amplification of the linear elements remained low. The extrachromosomal DNAs were unstable in the absence of drug and their disappearance coincided with an increase in sensitivity to MTX. Mapping of the minichromosomes and the circular element showed that they were all constituted by inverted duplications. The circular amplicon contained an inverted repeat derived from the H locus that encompassed the pteridine reductase gene (PTR1) responsible for MTX resistance. The amplified segment in the linear amplicons was longer and included the pgpB and pgpC genes that encode P-glycoproteins of unknown function previously characterized in different Leishmania species. Images PMID:7659507

  13. PCR Amplicon Prediction from Multiplex Degenerate Primer and Probe Sets

    SciTech Connect

    Gardner, S. N.

    2013-08-08

    Assessing primer specificity and predicting both desired and off-target amplification products is an essential step for robust PCR assay design. Code is described to predict potential polymerase chain reaction (PCR) amplicons in a large sequence database such as NCBI nt from either singleplex or a large multiplexed set of primers, allowing degenerate primer and probe bases, with target mismatch annotates amplicons with gene information automatically downloaded from NCBI, and optionally it can predict whether there are also TaqMan/Luminex probe matches within predicted amplicons.

  14. PCR Amplicon Prediction from Multiplex Degenerate Primer and Probe Sets

    Energy Science and Technology Software Center (ESTSC)

    2013-08-08

    Assessing primer specificity and predicting both desired and off-target amplification products is an essential step for robust PCR assay design. Code is described to predict potential polymerase chain reaction (PCR) amplicons in a large sequence database such as NCBI nt from either singleplex or a large multiplexed set of primers, allowing degenerate primer and probe bases, with target mismatch annotates amplicons with gene information automatically downloaded from NCBI, and optionally it can predict whether theremore » are also TaqMan/Luminex probe matches within predicted amplicons.« less

  15. Virion-associated cofactor high-mobility group DNA-binding protein-1 facilitates transposition from the herpes simplex virus/Sleeping Beauty amplicon vector platform.

    PubMed

    de Silva, Suresh; Lotta, Louis T; Burris, Clark A; Bowers, William J

    2010-11-01

    The development of the integration-competent, herpes simplex virus/Sleeping Beauty (HSV/SB) amplicon vector platform has created a means to efficiently and stably deliver therapeutic transcription units (termed "transgenons") to neurons within the mammalian brain. Furthermore, an investigation into the transposition capacity of the HSV/SB vector system revealed that the amplicon genome provides an optimal substrate for the transposition of transgenons at least 12 kb in length [de Silva, S., Mastrangelo, M.A., Lotta, L.T., Jr., Burris, C.A., Federoff, H.J., and Bowers, W.J. ( 2010 ). Gene Ther. 17, 424-431]. These results prompted an investigation into the factors that may contribute toward efficient transposition from the HSV/SB amplicon. One of the cellular cofactors known to play a key role during SB-mediated transposition is the high-mobility group DNA-binding protein-1 (HMGB1). Our present investigation into the role of HMGB1 during amplicon-based transposition revealed that transposition is not strictly dependent on the presence of cellular HMGB1, contrary to what had been previously demonstrated with plasmid-based SB transposition. We have shown for the first time that during amplicon preparation, biologically active HMGB1 derived from the packaging cell line is copackaged into amplicon vector particles. As a result, HSV/SB amplicon virions arrive prearmed with HMGB1 protein at levels sufficient for facilitating SB-mediated transposition in the transduced mammalian cell. PMID:20568967

  16. Herpes simplex virus amplicon: effect of size on replication of constructed defective genomes containing eucaryotic DNA sequences.

    PubMed Central

    Kwong, A D; Frenkel, N

    1984-01-01

    Previous studies (R. R. Spaete and N. Frenkel, Cell 30:295-304, 1982) have documented the potential use of defective virus vectors (amplicons) derived from herpes simplex virus for the efficient introduction of foreign DNA sequences into eucaryotic cells. Specifically, cotransfection of cells with helper virus DNA and cloned amplicons (8 to 10 kilobases [kb]) containing bacterial plasmid DNA sequences linked to a set of herpes simplex virus cis-acting propagation signals (a replication origin and a cleavage-packaging signal) resulted in the generation of virus stocks containing packaged defective genomes that consisted of uniform head-to-tail reiterations of the chimeric seed amplicon sequences. The chimeric defective genomes could be stably propagated in virus stocks and could thus be used to efficiently infect cells. We now report on additional studies designed to propagate relatively large sets of eucaryotic DNA sequences within chimeric packaged defective genomes. These studies have utilized a 12-kb chicken DNA sequence encoding the chicken ovalbumin gene and cloned by Lai et al. (Proc. Natl. Acad. Sci. U.S.A. 77:244-248, 1980) in the plasmid pOV12. Virus stocks derived from cells cotransfected with helper virus DNA and chimeric amplicons (overall size of 19.8 kb, of which 12 kb corresponded to the chicken DNA) contained defective genomes composed of reiterations of the 19.8-kb seed amplicon sequences. However, in addition to the authentically sized repeat units, defective genomes in the derivative virus stocks contained smaller repeat units representing deleted versions of the seed 19.8-kb amplicons. The recombinational events leading to the formation of deleted repeats did not appear to occur at unique sites, as shown by comparative analyses of multiple, independently generated virus series propagated from separate transfections. In contast, seed amplicons ranging in size from 11 to 15 kb and containing subsets of the 12-kb chicken DNA sequences replicated

  17. Induction of humoral responses to BHV-1 glycoprotein D expressed by HSV-1 amplicon vectors

    PubMed Central

    Blanc, Andrea Maria; Berois, Mabel Beatriz; Tomé, Lorena Magalí; Epstein, Alberto L.

    2012-01-01

    Herpes simplex virus type-1 (HSV-1) amplicon vectors are versatile and useful tools for transferring genes into cells that are capable of stimulating a specific immune response to their expressed antigens. In this work, two HSV-1-derived amplicon vectors were generated. One of these expressed the full-length glycoprotein D (gD) of bovine herpesvirus 1 while the second expressed the truncated form of gD (gDtr) which lacked the trans-membrane region. After evaluating gD expression in the infected cells, the ability of both vectors to induce a specific gD immune response was tested in BALB/c mice that were intramuscularly immunized. Specific serum antibody responses were detected in mice inoculated with both vectors, and the response against truncated gD was higher than the response against full-length gD. These results reinforce previous findings that HSV-1 amplicon vectors can potentially deliver antigens to animals and highlight the prospective use of these vectors for treating infectious bovine rhinotracheitis disease. PMID:22437537

  18. Fast, accurate and easy-to-pipeline methods for amplicon sequence processing

    NASA Astrophysics Data System (ADS)

    Antonielli, Livio; Sessitsch, Angela

    2016-04-01

    Next generation sequencing (NGS) technologies established since years as an essential resource in microbiology. While on the one hand metagenomic studies can benefit from the continuously increasing throughput of the Illumina (Solexa) technology, on the other hand the spreading of third generation sequencing technologies (PacBio, Oxford Nanopore) are getting whole genome sequencing beyond the assembly of fragmented draft genomes, making it now possible to finish bacterial genomes even without short read correction. Besides (meta)genomic analysis next-gen amplicon sequencing is still fundamental for microbial studies. Amplicon sequencing of the 16S rRNA gene and ITS (Internal Transcribed Spacer) remains a well-established widespread method for a multitude of different purposes concerning the identification and comparison of archaeal/bacterial (16S rRNA gene) and fungal (ITS) communities occurring in diverse environments. Numerous different pipelines have been developed in order to process NGS-derived amplicon sequences, among which Mothur, QIIME and USEARCH are the most well-known and cited ones. The entire process from initial raw sequence data through read error correction, paired-end read assembly, primer stripping, quality filtering, clustering, OTU taxonomic classification and BIOM table rarefaction as well as alternative "normalization" methods will be addressed. An effective and accurate strategy will be presented using the state-of-the-art bioinformatic tools and the example of a straightforward one-script pipeline for 16S rRNA gene or ITS MiSeq amplicon sequencing will be provided. Finally, instructions on how to automatically retrieve nucleotide sequences from NCBI and therefore apply the pipeline to targets other than 16S rRNA gene (Greengenes, SILVA) and ITS (UNITE) will be discussed.

  19. Systems consequences of amplicon formation in human breast cancer

    PubMed Central

    Inaki, Koichiro; Menghi, Francesca; Woo, Xing Yi; Wagner, Joel P.; Jacques, Pierre-Étienne; Lee, Yi Fang; Shreckengast, Phung Trang; Soon, Wendy WeiJia; Malhotra, Ankit; Teo, Audrey S.M.; Hillmer, Axel M.; Khng, Alexis Jiaying; Ruan, Xiaoan; Ong, Swee Hoe; Bertrand, Denis; Nagarajan, Niranjan; Karuturi, R. Krishna Murthy; Hidalgo Miranda, Alfredo

    2014-01-01

    Chromosomal structural variations play an important role in determining the transcriptional landscape of human breast cancers. To assess the nature of these structural variations, we analyzed eight breast tumor samples with a focus on regions of gene amplification using mate-pair sequencing of long-insert genomic DNA with matched transcriptome profiling. We found that tandem duplications appear to be early events in tumor evolution, especially in the genesis of amplicons. In a detailed reconstruction of events on chromosome 17, we found large unpaired inversions and deletions connect a tandemly duplicated ERBB2 with neighboring 17q21.3 amplicons while simultaneously deleting the intervening BRCA1 tumor suppressor locus. This series of events appeared to be unusually common when examined in larger genomic data sets of breast cancers albeit using approaches with lesser resolution. Using siRNAs in breast cancer cell lines, we showed that the 17q21.3 amplicon harbored a significant number of weak oncogenes that appeared consistently coamplified in primary tumors. Down-regulation of BRCA1 expression augmented the cell proliferation in ERBB2-transfected human normal mammary epithelial cells. Coamplification of other functionally tested oncogenic elements in other breast tumors examined, such as RIPK2 and MYC on chromosome 8, also parallel these findings. Our analyses suggest that structural variations efficiently orchestrate the gain and loss of cancer gene cassettes that engage many oncogenic pathways simultaneously and that such oncogenic cassettes are favored during the evolution of a cancer. PMID:25186909

  20. Systems consequences of amplicon formation in human breast cancer.

    PubMed

    Inaki, Koichiro; Menghi, Francesca; Woo, Xing Yi; Wagner, Joel P; Jacques, Pierre-Étienne; Lee, Yi Fang; Shreckengast, Phung Trang; Soon, Wendy WeiJia; Malhotra, Ankit; Teo, Audrey S M; Hillmer, Axel M; Khng, Alexis Jiaying; Ruan, Xiaoan; Ong, Swee Hoe; Bertrand, Denis; Nagarajan, Niranjan; Karuturi, R Krishna Murthy; Miranda, Alfredo Hidalgo; Liu, Edison T

    2014-10-01

    Chromosomal structural variations play an important role in determining the transcriptional landscape of human breast cancers. To assess the nature of these structural variations, we analyzed eight breast tumor samples with a focus on regions of gene amplification using mate-pair sequencing of long-insert genomic DNA with matched transcriptome profiling. We found that tandem duplications appear to be early events in tumor evolution, especially in the genesis of amplicons. In a detailed reconstruction of events on chromosome 17, we found large unpaired inversions and deletions connect a tandemly duplicated ERBB2 with neighboring 17q21.3 amplicons while simultaneously deleting the intervening BRCA1 tumor suppressor locus. This series of events appeared to be unusually common when examined in larger genomic data sets of breast cancers albeit using approaches with lesser resolution. Using siRNAs in breast cancer cell lines, we showed that the 17q21.3 amplicon harbored a significant number of weak oncogenes that appeared consistently coamplified in primary tumors. Down-regulation of BRCA1 expression augmented the cell proliferation in ERBB2-transfected human normal mammary epithelial cells. Coamplification of other functionally tested oncogenic elements in other breast tumors examined, such as RIPK2 and MYC on chromosome 8, also parallel these findings. Our analyses suggest that structural variations efficiently orchestrate the gain and loss of cancer gene cassettes that engage many oncogenic pathways simultaneously and that such oncogenic cassettes are favored during the evolution of a cancer. PMID:25186909

  1. AmpliconDuo: A Split-Sample Filtering Protocol for High-Throughput Amplicon Sequencing of Microbial Communities

    PubMed Central

    Lange, Anja; Jost, Steffen; Heider, Dominik; Bock, Christina; Budeus, Bettina; Schilling, Elmar; Strittmatter, Axel; Boenigk, Jens; Hoffmann, Daniel

    2015-01-01

    High throughput sequencing (HTSeq) of small ribosomal subunit amplicons has the potential for a comprehensive characterization of microbial community compositions, down to rare species. However, the error-prone nature of the multi-step experimental process requires that the resulting raw sequences are subjected to quality control procedures. These procedures often involve an abundance cutoff for rare sequences or clustering of sequences, both of which limit genetic resolution. Here we propose a simple experimental protocol that retains the high genetic resolution granted by HTSeq methods while effectively removing many low abundance sequences that are likely due to PCR and sequencing errors. According to this protocol, we split samples and submit both halves to independent PCR and sequencing runs. The resulting sequence data is graphically and quantitatively characterized by the discordance between the two experimental branches, allowing for a quick identification of problematic samples. Further, we discard sequences that are not found in both branches (“AmpliconDuo filter”). We show that the majority of sequences removed in this way, mostly low abundance but also some higher abundance sequences, show features expected from random modifications of true sequences as introduced by PCR and sequencing errors. On the other hand, the filter retains many low abundance sequences observed in both branches and thus provides a more reliable census of the rare biosphere. We find that the AmpliconDuo filter increases biological resolution as it increases apparent community similarity between biologically similar communities, while it does not affect apparent community similarities between biologically dissimilar communities. The filter does not distort overall apparent community compositions. Finally, we quantitatively explain the effect of the AmpliconDuo filter by a simple mathematical model. PMID:26523925

  2. DNA-based methods to prepare helper virus-free herpes amplicon vectors and versatile design of amplicon vector plasmids.

    PubMed

    Kasai, Kazue; Saeki, Yoshinaga

    2006-06-01

    The herpes simplex virus (HSV) amplicon vector is a versatile plasmid-based gene delivery vehicle with a large transgene capacity (up to 150 kb) and the ability to infect a broad range of cell types. The vector system was originally developed by Frenkel and her colleagues in 1980. Ever since, a great deal of effort by various investigators has been directed at minimizing the toxicity associated with the inevitable contamination by helper virus. In 1996, Fraefel and his colleagues successfully devised a cosmid-based packaging system that was free of contamination by helper virus (so-called helper virus-free packaging), which utilized as helper a set of 5 overlapping cosmid clones that covered the entire HSV genome, which lacked the DNA packaging/cleavage signals. With the helper virus-free system, broader applications of the vector became possible. Cloning of the entire HSV genome in bacteria artificial chromosome (BAC) plasmids enabled stable maintenance and propagation of the helper HSV genome in bacteria. It also allowed for the development of BAC-based helper virus-free packaging systems. In this article, we review various versions of DNA-based methods to prepare HSV amplicon vectors free of helper virus contamination. We also examine recent advances in vector design, including methods of vector construction, hybrid amplicon vectors, and the infectious BAC system. Future directions in improving packaging systems and vector designs are discussed. PMID:16787182

  3. Multiplex amplicon sequencing for microbe identification in community-based culture collections.

    PubMed

    Armanhi, Jaderson Silveira Leite; de Souza, Rafael Soares Correa; de Araújo, Laura Migliorini; Okura, Vagner Katsumi; Mieczkowski, Piotr; Imperial, Juan; Arruda, Paulo

    2016-01-01

    Microbiome analysis using metagenomic sequencing has revealed a vast microbial diversity associated with plants. Identifying the molecular functions associated with microbiome-plant interaction is a significant challenge concerning the development of microbiome-derived technologies applied to agriculture. An alternative to accelerate the discovery of the microbiome benefits to plants is to construct microbial culture collections concomitant with accessing microbial community structure and abundance. However, traditional methods of isolation, cultivation, and identification of microbes are time-consuming and expensive. Here we describe a method for identification of microbes in culture collections constructed by picking colonies from primary platings that may contain single or multiple microorganisms, which we named community-based culture collections (CBC). A multiplexing 16S rRNA gene amplicon sequencing based on two-step PCR amplifications with tagged primers for plates, rows, and columns allowed the identification of the microbial composition regardless if the well contains single or multiple microorganisms. The multiplexing system enables pooling amplicons into a single tube. The sequencing performed on the PacBio platform led to recovery near-full-length 16S rRNA gene sequences allowing accurate identification of microorganism composition in each plate well. Cross-referencing with plant microbiome structure and abundance allowed the estimation of diversity and abundance representation of microorganism in the CBC. PMID:27404280

  4. Multiplex amplicon sequencing for microbe identification in community-based culture collections

    PubMed Central

    Armanhi, Jaderson Silveira Leite; de Souza, Rafael Soares Correa; de Araújo, Laura Migliorini; Okura, Vagner Katsumi; Mieczkowski, Piotr; Imperial, Juan; Arruda, Paulo

    2016-01-01

    Microbiome analysis using metagenomic sequencing has revealed a vast microbial diversity associated with plants. Identifying the molecular functions associated with microbiome-plant interaction is a significant challenge concerning the development of microbiome-derived technologies applied to agriculture. An alternative to accelerate the discovery of the microbiome benefits to plants is to construct microbial culture collections concomitant with accessing microbial community structure and abundance. However, traditional methods of isolation, cultivation, and identification of microbes are time-consuming and expensive. Here we describe a method for identification of microbes in culture collections constructed by picking colonies from primary platings that may contain single or multiple microorganisms, which we named community-based culture collections (CBC). A multiplexing 16S rRNA gene amplicon sequencing based on two-step PCR amplifications with tagged primers for plates, rows, and columns allowed the identification of the microbial composition regardless if the well contains single or multiple microorganisms. The multiplexing system enables pooling amplicons into a single tube. The sequencing performed on the PacBio platform led to recovery near-full-length 16S rRNA gene sequences allowing accurate identification of microorganism composition in each plate well. Cross-referencing with plant microbiome structure and abundance allowed the estimation of diversity and abundance representation of microorganism in the CBC. PMID:27404280

  5. Mutascope: sensitive detection of somatic mutations from deep amplicon sequencing

    PubMed Central

    Yost, Shawn E.; Alakus, Hakan; Matsui, Hiroko; Schwab, Richard B.; Jepsen, Kristen; Frazer, Kelly A.; Harismendy, Olivier

    2013-01-01

    Summary: We present Mutascope, a sequencing analysis pipeline specifically developed for the identification of somatic variants present at low-allelic fraction from high-throughput sequencing of amplicons from matched tumor-normal specimen. Using datasets reproducing tumor genetic heterogeneity, we demonstrate that Mutascope has a higher sensitivity and generates fewer false-positive calls than tools designed for shotgun sequencing or diploid genomes. Availability: Freely available on the web at http://sourceforge.net/projects/mutascope/. Contact: oharismendy@ucsd.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:23712659

  6. INTERACTIONS BETWEEN FLUORESCENT PSEUDOMONADS AND VA MYCORRHIZAL FUNGI

    EPA Science Inventory

    Cucumber seeds were treated with rifampin-resistant derivatives of Pseudomonas puntida (A12, N1R or R-20) or P. fluorescens (2-79 or 3871) and planted in soils with and without added inoculum of the VA mycorrhizal fungi Glomus intraradices Schenck & Smith or G. etunicatum Becker ...

  7. KaVA ESTEMA project

    NASA Astrophysics Data System (ADS)

    Oyadomari, Miyako; Imai, Hiroshi; Cho, Se-Hyung; Asaki, Yoshiharu; Choi, Yoon-Kyong; Kim, Jaeheon; Yun, Youngjoo; Matsumoto, Naoko; Min, Cheul-Hong; Oyama, Tomoaki; Yoon, Sung-Chul; Yoon, Dong-Hwan; Kim, Dong-Jin; Dodson, Richard; Rioja, Maria; Burns, Ross; Orosz, Gabor; Nakagawa, Akiharu; Chibueze O, James; Nakashima, Jun-ichi; Sobolev, Andrey

    2016-07-01

    The ESTEMA (Expanded Study on Stellar Masers) project is one of three Large Programs of the KaVA (the combined array of the Korean VLBI Network and Japanese VLBI Exploration of Radio Astrometry), and conducted in 2015-2016. It aims to publish a database of the largest sample of VLBI images of circumstellar water (H2O) and silicon-monoxide (SiO) maser sources towards circumstellar envelopes (CSEs) of 80 evolved stars in late AGB to early post-AGB phase. Here we present the specifications of the ESTEMA observations and the planned scientific goals in order to share the basic information of the ESTEMA with astronomical community and encourage future collaborations with the ESTEMA and future follow-up observations for the targeted stars.

  8. The VA-Medical School Partnership: The Medical School Perspective.

    ERIC Educational Resources Information Center

    Petersdorf, Robert G.

    1987-01-01

    Issues in the relationship between the Veterans' Administration (VA) and medical schools are discussed, including VA faculty recruitment and retention, ambulatory care in VA teaching hospitals, governance and growth of research within VA medical centers, and effects of cost containment and competition on teaching and training in VA hospitals. (MSE)

  9. 76 FR 75509 - Autopsies at VA Expense

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-12-02

    ... that amendment, VA promulgated 38 CFR 17.38, on October 6, 1999, 64 FR 54212. Section 17.38, inter alia... procedure; Alcohol abuse; Alcoholism; Claims; Day care; Dental health; Drug abuse; Government contracts...-reference to VA regulations that authorize certain outpatient and ambulatory care. The proposed rule...

  10. 78 FR 32126 - VA Dental Insurance Program

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-05-29

    ... in the Federal Register (77 FR 12517) a proposed rule to amend VA regulations to establish VADIP, a... coverage capabilities as determined during the Federal contracting process. See 77 FR 12518. Although VA... 510(b). See 77 FR 12520. We will conduct the Federal contracting process anticipating this...

  11. Integrated Microfluidic Nucleic Acid Isolation, Isothermal Amplification, and Amplicon Quantification

    PubMed Central

    Mauk, Michael G.; Liu, Changchun; Song, Jinzhao; Bau, Haim H.

    2015-01-01

    Microfluidic components and systems for rapid (<60 min), low-cost, convenient, field-deployable sequence-specific nucleic acid-based amplification tests (NAATs) are described. A microfluidic point-of-care (POC) diagnostics test to quantify HIV viral load from blood samples serves as a representative and instructive example to discuss the technical issues and capabilities of “lab on a chip” NAAT devices. A portable, miniaturized POC NAAT with performance comparable to conventional PCR (polymerase-chain reaction)-based tests in clinical laboratories can be realized with a disposable, palm-sized, plastic microfluidic chip in which: (1) nucleic acids (NAs) are extracted from relatively large (~mL) volume sample lysates using an embedded porous silica glass fiber or cellulose binding phase (“membrane”) to capture sample NAs in a flow-through, filtration mode; (2) NAs captured on the membrane are isothermally (~65 °C) amplified; (3) amplicon production is monitored by real-time fluorescence detection, such as with a smartphone CCD camera serving as a low-cost detector; and (4) paraffin-encapsulated, lyophilized reagents for temperature-activated release are pre-stored in the chip. Limits of Detection (LOD) better than 103 virons/sample can be achieved. A modified chip with conduits hosting a diffusion-mode amplification process provides a simple visual indicator to readily quantify sample NA template. In addition, a companion microfluidic device for extracting plasma from whole blood without a centrifuge, generating cell-free plasma for chip-based molecular diagnostics, is described. Extensions to a myriad of related applications including, for example, food testing, cancer screening, and insect genotyping are briefly surveyed.

  12. Taxonomic Assessment of Rumen Microbiota Using Total RNA and Targeted Amplicon Sequencing Approaches.

    PubMed

    Li, Fuyong; Henderson, Gemma; Sun, Xu; Cox, Faith; Janssen, Peter H; Guan, Le Luo

    2016-01-01

    Taxonomic characterization of active gastrointestinal microbiota is essential to detect shifts in microbial communities and functions under various conditions. This study aimed to identify and quantify potentially active rumen microbiota using total RNA sequencing and to compare the outcomes of this approach with the widely used targeted RNA/DNA amplicon sequencing technique. Total RNA isolated from rumen digesta samples from five beef steers was subjected to Illumina paired-end sequencing (RNA-seq), and bacterial and archaeal amplicons of partial 16S rRNA/rDNA were subjected to 454 pyrosequencing (RNA/DNA Amplicon-seq). Taxonomic assessments of the RNA-seq, RNA Amplicon-seq, and DNA Amplicon-seq datasets were performed using a pipeline developed in house. The detected major microbial phylotypes were common among the three datasets, with seven bacterial phyla, fifteen bacterial families, and five archaeal taxa commonly identified across all datasets. There were also unique microbial taxa detected in each dataset. Elusimicrobia and Verrucomicrobia phyla; Desulfovibrionaceae, Elusimicrobiaceae, and Sphaerochaetaceae families; and Methanobrevibacter woesei were only detected in the RNA-Seq and RNA Amplicon-seq datasets, whereas Streptococcaceae was only detected in the DNA Amplicon-seq dataset. In addition, the relative abundances of four bacterial phyla, eight bacterial families and one archaeal taxon were different among the three datasets. This is the first study to compare the outcomes of rumen microbiota profiling between RNA-seq and RNA/DNA Amplicon-seq datasets. Our results illustrate the differences between these methods in characterizing microbiota both qualitatively and quantitatively for the same sample, and so caution must be exercised when comparing data. PMID:27446027

  13. Taxonomic Assessment of Rumen Microbiota Using Total RNA and Targeted Amplicon Sequencing Approaches

    PubMed Central

    Li, Fuyong; Henderson, Gemma; Sun, Xu; Cox, Faith; Janssen, Peter H.; Guan, Le Luo

    2016-01-01

    Taxonomic characterization of active gastrointestinal microbiota is essential to detect shifts in microbial communities and functions under various conditions. This study aimed to identify and quantify potentially active rumen microbiota using total RNA sequencing and to compare the outcomes of this approach with the widely used targeted RNA/DNA amplicon sequencing technique. Total RNA isolated from rumen digesta samples from five beef steers was subjected to Illumina paired-end sequencing (RNA-seq), and bacterial and archaeal amplicons of partial 16S rRNA/rDNA were subjected to 454 pyrosequencing (RNA/DNA Amplicon-seq). Taxonomic assessments of the RNA-seq, RNA Amplicon-seq, and DNA Amplicon-seq datasets were performed using a pipeline developed in house. The detected major microbial phylotypes were common among the three datasets, with seven bacterial phyla, fifteen bacterial families, and five archaeal taxa commonly identified across all datasets. There were also unique microbial taxa detected in each dataset. Elusimicrobia and Verrucomicrobia phyla; Desulfovibrionaceae, Elusimicrobiaceae, and Sphaerochaetaceae families; and Methanobrevibacter woesei were only detected in the RNA-Seq and RNA Amplicon-seq datasets, whereas Streptococcaceae was only detected in the DNA Amplicon-seq dataset. In addition, the relative abundances of four bacterial phyla, eight bacterial families and one archaeal taxon were different among the three datasets. This is the first study to compare the outcomes of rumen microbiota profiling between RNA-seq and RNA/DNA Amplicon-seq datasets. Our results illustrate the differences between these methods in characterizing microbiota both qualitatively and quantitatively for the same sample, and so caution must be exercised when comparing data. PMID:27446027

  14. A chromosomal region 7p11.2 transcript map: its development and application to the study of EGFR amplicons in glioblastoma.

    PubMed Central

    Eley, Greg D.; Reiter, Jill L.; Pandita, Ajay; Park, Soyeon; Jenkins, Robert B.; Maihle, Nita J.; James, C. David

    2002-01-01

    Cumulative information available about the organization of amplified chromosomal regions in human tumors suggests that the amplification repeat units, or amplicons, can be of a simple or complex nature. For the former, amplified regions generally retain their native chromosomal configuration and involve a single amplification target sequence. For complex amplicons, amplified DNAs usually undergo substantial reorganization relative to the normal chromosomal regions from which they evolve, and the regions subject to amplification may contain multiple target sequences. Previous efforts to characterize the 7p11.2 epidermal growth factor receptor ) amplicon in glioblastoma have relied primarily on the use of markers positioned by linkage analysis and/or radiation hybrid mapping, both of which are known to have the potential for being inaccurate when attempting to order loci over relatively short (<1 Mb) chromosomal regions. Due to the limited resolution of genetic maps that have been established through the use of these approaches, we have constructed a 2-Mb bacterial and P1-derived artificial chromosome (BAC-PAC) contig for the EGFR region and have applied markers positioned on its associated physical map to the analysis of 7p11.2 amplifications in a series of glioblastomas. Our data indicate that EGFR is the sole amplification target within the mapped region, although there are several additional 7p11.2 genes that can be coamplified and overexpressed with EGFR. Furthermore, these results are consistent with EGFR amplicons retaining the same organization as the native chromosome 7p11.2 region from which they are derived. PMID:11916499

  15. Agreement Between HEDIS Performance Assessments in the VA and Medicare Advantage: Is Quality in the Eye of the Beholder?

    PubMed

    Trivedi, Amal N; Wilson, Ira B; Charlton, Mary E; Kizer, Kenneth W

    2016-01-01

    Medicare Advantage (MA) plans and the Veterans Affairs (VA) health care system assess quality of care using standardized Healthcare Effectiveness Data and Information Set (HEDIS) performance measures. Little is known, however, about the relative accuracy of quality indicators for persons receiving care in more than one health care system. Among Veterans dually enrolled in an MA plan, we examined the agreement between MA and VA HEDIS assessments. Our study tested the hypothesis that private health plans underreport quality of care relative to a fully integrated delivery system utilizing a comprehensive electronic health record. Despite assessing the same individuals using identical measure specifications, reported VA performance was significantly better than reported MA performance for all 12 HEDIS measures. The VA's performance advantage ranged from 9.8% (glycosylated hemoglobin [HbA1c] < 7.0% in diabetes) to 54.7% (blood pressure < 140/90 mm Hg in diabetes). The overall agreement between VA and MA HEDIS assessments ranged from 38.5% to 62.6%. Performance rates derived from VA and MA aggregate data were 1.6% to 14.3% higher than those reported by VA alone. This analysis suggests that neither MA plans nor the VA fully capture quality of care information for dually enrolled persons. However, the VA's system-wide electronic health record may allow for more complete capture of quality information across multiple providers and settings. PMID:27033565

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

    PubMed

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

    2015-01-01

    The DNA target sequence is the key element in designing detection methods for genetically modified organisms (GMOs). Unfortunately this information is frequently lacking, especially for unauthorized GMOs. In addition, patent sequences are generally poorly annotated, buried in complex and extensive documentation and hard to link to the corresponding GM event. Here, we present the JRC GMO-Amplicons, a database of amplicons collected by screening public nucleotide sequence databanks by in silico determination of PCR amplification with reference methods for GMO analysis. The European Union Reference Laboratory for Genetically Modified Food and Feed (EU-RL GMFF) provides these methods in the GMOMETHODS database to support enforcement of EU legislation and GM food/feed control. The JRC GMO-Amplicons database is composed of more than 240 000 amplicons, which can be easily accessed and screened through a web interface. To our knowledge, this is the first attempt at pooling and collecting publicly available sequences related to GMOs in food and feed. The JRC GMO-Amplicons supports control laboratories in the design and assessment of GMO methods, providing inter-alia in silico prediction of primers specificity and GM targets coverage. The new tool can assist the laboratories in the analysis of complex issues, such as the detection and identification of unauthorized GMOs. Notably, the JRC GMO-Amplicons database allows the retrieval and characterization of GMO-related sequences included in patents documentation. Finally, it can help annotating poorly described GM sequences and identifying new relevant GMO-related sequences in public databases. The JRC GMO-Amplicons is freely accessible through a web-based portal that is hosted on the EU-RL GMFF website. Database URL: http://gmo-crl.jrc.ec.europa.eu/jrcgmoamplicons/. PMID:26424080

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

    PubMed Central

    2012-01-01

    Background Next-generation sequencing allows the analysis of an unprecedented number of viral sequence variants from infected patients, presenting a novel opportunity for understanding virus evolution, drug resistance and immune escape. However, sequencing in bulk is error prone. Thus, the generated data require error identification and correction. Most error-correction methods to date are not optimized for amplicon analysis and assume that the error rate is randomly distributed. Recent quality assessment of amplicon sequences obtained using 454-sequencing showed that the error rate is strongly linked to the presence and size of homopolymers, position in the sequence and length of the amplicon. All these parameters are strongly sequence specific and should be incorporated into the calibration of error-correction algorithms designed for amplicon sequencing. Results In this paper, we present two new efficient error correction algorithms optimized for viral amplicons: (i) k-mer-based error correction (KEC) and (ii) empirical frequency threshold (ET). Both were compared to a previously published clustering algorithm (SHORAH), in order to evaluate their relative performance on 24 experimental datasets obtained by 454-sequencing of amplicons with known sequences. All three algorithms show similar accuracy in finding true haplotypes. However, KEC and ET were significantly more efficient than SHORAH in removing false haplotypes and estimating the frequency of true ones. Conclusions Both algorithms, KEC and ET, are highly suitable for rapid recovery of error-free haplotypes obtained by 454-sequencing of amplicons from heterogeneous viruses. The implementations of the algorithms and data sets used for their testing are available at: http://alan.cs.gsu.edu/NGS/?q=content/pyrosequencing-error-correction-algorithm PMID:22759430

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

    PubMed Central

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

    2015-01-01

    The DNA target sequence is the key element in designing detection methods for genetically modified organisms (GMOs). Unfortunately this information is frequently lacking, especially for unauthorized GMOs. In addition, patent sequences are generally poorly annotated, buried in complex and extensive documentation and hard to link to the corresponding GM event. Here, we present the JRC GMO-Amplicons, a database of amplicons collected by screening public nucleotide sequence databanks by in silico determination of PCR amplification with reference methods for GMO analysis. The European Union Reference Laboratory for Genetically Modified Food and Feed (EU-RL GMFF) provides these methods in the GMOMETHODS database to support enforcement of EU legislation and GM food/feed control. The JRC GMO-Amplicons database is composed of more than 240 000 amplicons, which can be easily accessed and screened through a web interface. To our knowledge, this is the first attempt at pooling and collecting publicly available sequences related to GMOs in food and feed. The JRC GMO-Amplicons supports control laboratories in the design and assessment of GMO methods, providing inter-alia in silico prediction of primers specificity and GM targets coverage. The new tool can assist the laboratories in the analysis of complex issues, such as the detection and identification of unauthorized GMOs. Notably, the JRC GMO-Amplicons database allows the retrieval and characterization of GMO-related sequences included in patents documentation. Finally, it can help annotating poorly described GM sequences and identifying new relevant GMO-related sequences in public databases. The JRC GMO-Amplicons is freely accessible through a web-based portal that is hosted on the EU-RL GMFF website. Database URL: http://gmo-crl.jrc.ec.europa.eu/jrcgmoamplicons/ PMID:26424080

  19. Women's veteran identity and utilization of VA health services.

    PubMed

    Di Leone, Brooke A L; Wang, Joyce M; Kressin, Nancy; Vogt, Dawne

    2016-02-01

    Women have participated in the United States military since its founding. However, until the mid-20th century, there had been limited recognition of women as official members of the military, and women remain a statistical minority within military and veteran populations. It is therefore important to better understand women's veteran identity (which we define here as one's self-concept as derived from their veteran status) and associated implications for service use and experiences in the Department of Veterans Affairs (VA) health care setting. The present research examined the centrality of, and positive regard for, women's veteran identity among 407 female veterans deployed in support of the recent wars in Iraq and Afghanistan. Data were collected via a mailed national survey. Positive regard for veteran identity, but not veteran identity centrality,was positively associated with participants' age and length of time spent in the military. Results also showed that the centrality of women's veteran identity was positively related to their choice to use VA for health care and their feelings of belonging within VA, and that veteran identity centrality and positive regard for veteran identity are differentially associated with participants' military experiences (e.g., combat exposure, deployment sexual harassment) and mental health symptomatology (e.g., depression). PMID:25729892

  20. Integration of complete chloroplast genome sequences with small amplicon datasets improves phylogenetic resolution in Acacia.

    PubMed

    Williams, Anna V; Miller, Joseph T; Small, Ian; Nevill, Paul G; Boykin, Laura M

    2016-03-01

    Combining whole genome data with previously obtained amplicon sequences has the potential to increase the resolution of phylogenetic analyses, particularly at low taxonomic levels or where recent divergence, rapid speciation or slow genome evolution has resulted in limited sequence variation. However, the integration of these types of data for large scale phylogenetic studies has rarely been investigated. Here we conduct a phylogenetic analysis of the whole chloroplast genome and two nuclear ribosomal loci for 65 Acacia species from across the most recent Acacia phylogeny. We then combine this data with previously generated amplicon sequences (four chloroplast loci and two nuclear ribosomal loci) for 508 Acacia species. We use several phylogenetic methods, including maximum likelihood bootstrapping (with and without constraint) and ExaBayes, in order to determine the success of combining a dataset of 4000bp with one of 189,000bp. The results of our study indicate that the inclusion of whole genome data gave a far better resolved and well supported representation of the phylogenetic relationships within Acacia than using only amplicon sequences, with the greatest support observed when using a whole genome phylogeny as a constraint on the amplicon sequences. Our study therefore provides methods for optimal integration of genomic and amplicon sequences. PMID:26702955

  1. Assessing copy number alterations in targeted, amplicon-based next-generation sequencing data.

    PubMed

    Grasso, Catherine; Butler, Timothy; Rhodes, Katherine; Quist, Michael; Neff, Tanaya L; Moore, Stephen; Tomlins, Scott A; Reinig, Erica; Beadling, Carol; Andersen, Mark; Corless, Christopher L

    2015-01-01

    Changes in gene copy number are important in the setting of precision medicine. Recent studies have established that copy number alterations (CNAs) can be detected in sequencing libraries prepared by hybridization-capture, but there has been comparatively little attention given to CNA assessment in amplicon-based libraries prepared by PCR. In this study, we developed an algorithm for detecting CNAs in amplicon-based sequencing data. CNAs determined from the algorithm mirrored those from a hybridization-capture library. In addition, analysis of 14 pairs of matched normal and breast carcinoma tissues revealed that sequence data pooled from normal samples could be substituted for a matched normal tissue without affecting the detection of clinically relevant CNAs (>|2| copies). Comparison of CNAs identified by array comparative genomic hybridization and amplicon-based libraries across 10 breast carcinoma samples showed an excellent correlation. The CNA algorithm also compared favorably with fluorescence in situ hybridization, with agreement in 33 of 38 assessments across four different genes. Factors that influenced the detection of CNAs included the number of amplicons per gene, the average read depth, and, most important, the proportion of tumor within the sample. Our results show that CNAs can be identified in amplicon-based targeted sequencing data, and that their detection can be optimized by ensuring adequate tumor content and read coverage. PMID:25468433

  2. Do Older Rural and Urban Veterans Experience Different Rates of Unplanned Readmission to VA and Non-VA Hospitals?

    ERIC Educational Resources Information Center

    Weeks, William B.; Lee, Richard E.; Wallace, Amy E.; West, Alan N.; Bagian, James P.

    2009-01-01

    Context: Unplanned readmission within 30 days of discharge is an indicator of hospital quality. Purpose: We wanted to determine whether older rural veterans who were enrolled in the VA had different rates of unplanned readmission to VA or non-VA hospitals than their urban counterparts. Methods: We used the combined VA/Medicare dataset to examine…

  3. 75 FR 61859 - Proposed Information Collection (Create Payment Request for the VA Funding Fee Payment System (VA...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-10-06

    ....kessinger@va.gov . Please refer to ``OMB Control No. 2900-0474'' in any correspondence. During the comment... AFFAIRS Proposed Information Collection (Create Payment Request for the VA Funding Fee Payment System (VA... (VBA), Department of Veterans Affairs (VA), is announcing an opportunity for public comment on...

  4. 75 FR 61252 - Proposed Information Collection (Create Payment Request for the VA Funding Fee Payment System (VA...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-10-04

    ....kessinger@va.gov . Please refer to ``OMB Control No. 2900-0474'' in any correspondence. During the comment... AFFAIRS Proposed Information Collection (Create Payment Request for the VA Funding Fee Payment System (VA... (VBA), Department of Veterans Affairs (VA), is announcing an opportunity for public comment on...

  5. 78 FR 59771 - Proposed Information Collection (Create Payment Request for the VA Funding Fee Payment System (VA...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-09-27

    ...), Department of Veterans Affairs, 810 Vermont Avenue NW., Washington, DC 20420 or email nancy.kessinger@va.gov... AFFAIRS Proposed Information Collection (Create Payment Request for the VA Funding Fee Payment System (VA... (VBA), Department of Veterans Affairs (VA), is announcing an opportunity for public comment on...

  6. KENO V.a certification package

    SciTech Connect

    Niemer, K.A.

    1994-04-01

    KENO V.a has been certified. KENO V.a is a multigroup Monte Carlo criticality program used to calculate the k-effective of a 3-D system. It is part of the SCALE modular code system for performing Standardized Computer Analyses for Licensing Evaluation. SCALE was developed for the Nuclear Regulatory Commission to satisfy a need for a standardized method of analysis for the evaluation of nuclear fuel facility and package designs. In its present form, the system has the capability to perform criticality, shielding, and heat transfer analyses using well established functional modules tailored to the SCALE system. KENO V.a will be used at SRS to perform critical calculations related to nuclear criticality safety.

  7. Characterizing partial AZFc deletions of the Y chromosome with amplicon-specific sequence markers

    PubMed Central

    Navarro-Costa, Paulo; Pereira, Luísa; Alves, Cíntia; Gusmão, Leonor; Proença, Carmen; Marques-Vidal, Pedro; Rocha, Tiago; Correia, Sónia C; Jorge, Sónia; Neves, António; Soares, Ana P; Nunes, Joaquim; Calhaz-Jorge, Carlos; Amorim, António; Plancha, Carlos E; Gonçalves, João

    2007-01-01

    Background The AZFc region of the human Y chromosome is a highly recombinogenic locus containing multi-copy male fertility genes located in repeated DNA blocks (amplicons). These AZFc gene families exhibit slight sequence variations between copies which are considered to have functional relevance. Yet, partial AZFc deletions yield phenotypes ranging from normospermia to azoospermia, thwarting definite conclusions on their real impact on fertility. Results The amplicon content of partial AZFc deletion products was characterized with novel amplicon-specific sequence markers. Data indicate that partial AZFc deletions are a male infertility risk [odds ratio: 5.6 (95% CI: 1.6–30.1)] and although high diversity of partial deletion products and sequence conversion profiles were recorded, the AZFc marker profiles detected in fertile men were also observed in infertile men. Additionally, the assessment of rearrangement recurrence by Y-lineage analysis indicated that while partial AZFc deletions occurred in highly diverse samples, haplotype diversity was minimal in fertile men sharing identical marker profiles. Conclusion Although partial AZFc deletion products are highly heterogeneous in terms of amplicon content, this plasticity is not sufficient to account for the observed phenotypical variance. The lack of causative association between the deletion of specific gene copies and infertility suggests that AZFc gene content might be part of a multifactorial network, with Y-lineage evolution emerging as a possible phenotype modulator. PMID:17903263

  8. Bacterial metabarcoding by 16S rRNA gene ion torrent amplicon sequencing.

    PubMed

    Fantini, Elio; Gianese, Giulio; Giuliano, Giovanni; Fiore, Alessia

    2015-01-01

    Ion Torrent is a next generation sequencing technology based on the detection of hydrogen ions produced during DNA chain elongation; this technology allows analyzing and characterizing genomes, genes, and species. Here, we describe an Ion Torrent procedure applied to the metagenomic analysis of 16S rRNA gene amplicons to study the bacterial diversity in food and environmental samples. PMID:25343859

  9. Evaluation of Hybridization Capture Versus Amplicon-Based Methods for Whole-Exome Sequencing.

    PubMed

    Samorodnitsky, Eric; Jewell, Benjamin M; Hagopian, Raffi; Miya, Jharna; Wing, Michele R; Lyon, Ezra; Damodaran, Senthilkumar; Bhatt, Darshna; Reeser, Julie W; Datta, Jharna; Roychowdhury, Sameek

    2015-09-01

    Next-generation sequencing has aided characterization of genomic variation. While whole-genome sequencing may capture all possible mutations, whole-exome sequencing remains cost-effective and captures most phenotype-altering mutations. Initial strategies for exome enrichment utilized a hybridization-based capture approach. Recently, amplicon-based methods were designed to simplify preparation and utilize smaller DNA inputs. We evaluated two hybridization capture-based and two amplicon-based whole-exome sequencing approaches, utilizing both Illumina and Ion Torrent sequencers, comparing on-target alignment, uniformity, and variant calling. While the amplicon methods had higher on-target rates, the hybridization capture-based approaches demonstrated better uniformity. All methods identified many of the same single-nucleotide variants, but each amplicon-based method missed variants detected by the other three methods and reported additional variants discordant with all three other technologies. Many of these potential false positives or negatives appear to result from limited coverage, low variant frequency, vicinity to read starts/ends, or the need for platform-specific variant calling algorithms. All methods demonstrated effective copy-number variant calling when evaluated against a single-nucleotide polymorphism array. This study illustrates some differences between whole-exome sequencing approaches, highlights the need for selecting appropriate variant calling based on capture method, and will aid laboratories in selecting their preferred approach. PMID:26110913

  10. De novo-generated small palindromes are characteristic of amplicon boundary junction of double minutes.

    PubMed

    Zhu, Jing; Yu, Yang; Meng, Xiangning; Fan, Yihui; Zhang, Yu; Zhou, Chunshui; Yue, Zhichao; Jin, Yan; Zhang, Chunyu; Yu, Lisa; Ji, Wei; Jia, Xueyuan; Guan, Rongwei; Wu, Jie; Yu, Jingcui; Bai, Jing; Guan, Xin-Yuan; Wang, Mingrong; Lee, Ki-Young; Sun, Wenjing; Fu, Songbin

    2013-08-15

    Double minutes (DMs) are hallmarks of gene amplification. However, their molecular structure and the mechanisms of formation are largely unknown. To elucidate the structure and underlying molecular mechanism of DMs, we obtained and cloned DMs using microdissection; and degenerated oligonucleotide primed polymerase chain reaction (DOP-PCR) from the ovarian cancer cell line UACC-1598. Two large amplicons, the 284 kb AmpMYCN, originating from locus 2p24.3 and the 391 kb AmpEIF5A2, from locus 3q26.2, were found co-amplified on the same DMs. The two amplicons are joined through a complex 7 kb junction DNA sequence. Analysis of the junction has revealed three de novo created small palindromes surrounding the six breakpoints. Consistent with these observations, we further found that 70% of the 57 reported DM junction sequences have de novo creation of small palindromic sequences surrounding the breakpoints. Together, our findings indicate that de novo-generated small palindromic sequences are characteristic of amplicon boundary junctions on DMs. It is possible that the de novo-generated small palindromic sequences, which may be generated through non-homologous end joining in concert with a novel DNA repair machinery, play a common role in amplicon rejoining and gene amplification. PMID:23382041

  11. De novo origin of VCY2 from autosome to Y-transposed amplicon.

    PubMed

    Cao, Peng-Rong; Wang, Lei; Jiang, Yu-Chao; Yi, Yin-Sha; Qu, Fang; Liu, Tao-Cheng; Lv, Yuan

    2015-01-01

    The formation of new genes is a primary driving force of evolution in all organisms. The de novo evolution of new genes from non-protein-coding genomic regions is emerging as an important additional mechanism for novel gene creation. Y chromosomes underlie sex determination in mammals and contain genes that are required for male-specific functions. In this study, a search was undertaken for Y chromosome de novo genes derived from non-protein-coding sequences. The Y chromosome orphan gene variable charge, Y-linked (VCY)2, is an autosome-derived gene that has sequence similarity to large autosomal fragments but lacks an autosomal protein-coding homolog. VCY2 locates in the amplicon containing long DNA fragments that were transposed from autosomes to the Y chromosome before the ape-monkey split. We confirmed that VCY2 cannot be encoded by autosomes due to the presence of multiple disablers that disrupt the open reading frame, such as the absence of start or stop codons and the presence of premature stop codons. Similar observations have been made for homologs in the autosomes of the chimpanzee, gorilla, rhesus macaque, baboon and out-group marmoset, which suggests that there was a non-protein-coding ancestral VCY2 that was common to apes and monkeys that predated the transposition event. Furthermore, while protein-coding orthologs are absent, a putative non-protein-coding VCY2 with conserved disablers was identified in the rhesus macaque Y chromosome male-specific region. This finding implies that VCY2 might have not acquired its protein-coding ability before the ape-monkey split. VCY2 encodes a testis-specific expressed protein and is involved in the pathologic process of male infertility, and the acquisition of this gene might improve male fertility. This is the first evidence that de novo genes can be generated from transposed autosomal non-protein-coding segments, and this evidence provides novel insights into the evolutionary history of the Y chromosome. PMID

  12. Estimation of copy number using SYBR Green: confounding by AT-rich DNA and by variation in amplicon length.

    PubMed

    Colborn, James M; Byrd, Brian D; Koita, Ousmane A; Krogstad, Donald J

    2008-12-01

    Although SYBR Green is used to estimate copy number, its fluorescence varies with amplicon length and adenine/thymine (AT) content. As a result, threshold cycle (Ct) values obtained using real-time polymerase chain reaction (PCR) are lower for longer amplicons (P<0.001) and amplicons with greater AT content (P<0.001). In contrast, neither amplicon length nor AT content affects the Ct with TaqMan probes or LUX-labeled primers. Because SYBR Green yields lower Cts with AT-rich templates and longer templates, it overestimates copy number for those templates. Therefore, sequence-specific methods such as TaqMan probes or LUX-labeled primers should be considered when using real-time PCR to estimate copy number if the amplicons generated are AT-rich or vary in length. PMID:19052298

  13. 76 FR 56861 - Virginia Disaster #VA-00038

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-09-14

    ... ADMINISTRATION Virginia Disaster VA-00038 AGENCY: U.S. Small Business Administration. ACTION: Notice. SUMMARY... Commonwealth of Virginia (FEMA-4024-DR), dated 09/03/2011. Incident: Hurricane Irene. Incident Period: 08/26..., Southampton, Suffolk City, Sussex, Virginia Beach City, Westmoreland, Williamsburg City, York. The...

  14. 77 FR 73510 - Virginia Disaster #VA-00052

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-12-10

    ... From the Federal Register Online via the Government Publishing Office SMALL BUSINESS ADMINISTRATION Virginia Disaster VA-00052 AGENCY: U.S. Small Business Administration. ACTION: Notice. SUMMARY... State of Virginia (FEMA- 4092-DR), dated 11/26/2012. Incident: Hurricane Sandy Incident Period:...

  15. 76 FR 70804 - Virginia Disaster #VA-00037

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-11-15

    ... ADMINISTRATION Virginia Disaster VA-00037 AGENCY: U.S. Small Business Administration. ACTION: Notice. SUMMARY: This is a Notice of the Presidential declaration of a major disaster for the Commonwealth of Virginia... Economic Injury Loans): Louisa. Contiguous Counties (Economic Injury Loans Only): Virginia:...

  16. 75 FR 24757 - Virginia Disaster #VA-00029

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-05-05

    ... From the Federal Register Online via the Government Publishing Office SMALL BUSINESS ADMINISTRATION Virginia Disaster VA-00029 AGENCY: U.S. Small Business Administration. ACTION: Notice. SUMMARY... Commonwealth of Virginia (FEMA-1905-DR), dated 04/27/2010. Incident: Severe Winter Storms and...

  17. 77 FR 47489 - Virginia Disaster #VA-00048

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-08-08

    ... From the Federal Register Online via the Government Publishing Office SMALL BUSINESS ADMINISTRATION Virginia Disaster VA-00048 AGENCY: Small Business Administration. ACTION: Notice. SUMMARY: This is... State of Virginia (FEMA- 4072-DR), dated 07/27/2012. Incident: Severe Storms and Straight-line...

  18. 75 FR 9006 - Virginia Disaster #VA-00028

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-02-26

    ... From the Federal Register Online via the Government Publishing Office SMALL BUSINESS ADMINISTRATION Virginia Disaster VA-00028 AGENCY: U.S. Small Business Administration. ACTION: Notice. SUMMARY... Commonwealth of Virginia (FEMA-1874-DR), dated 02/16/2010. Incident: Severe Winter Storm and...

  19. 76 FR 40766 - Virginia Disaster #VA-00032

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-07-11

    ... ADMINISTRATION Virginia Disaster VA-00032 AGENCY: U.S. Small Business Administration. ACTION: Notice. SUMMARY: This is a notice of an Administrative declaration of a disaster for the Commonwealth of Virginia dated... determined to be adversely affected by the disaster: Primary Counties: Pulaski. Contiguous Counties:...

  20. 76 FR 72020 - Virginia Disaster #VA-00039

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-11-21

    ... ADMINISTRATION Virginia Disaster VA-00039 AGENCY: U.S. Small Business Administration. ACTION: Notice. SUMMARY: This is a notice of an Administrative declaration of a disaster for the Commonwealth of Virginia dated... adversely affected by the disaster: Primary Counties: Fairfax, Prince William. Contiguous Counties:...

  1. 76 FR 59765 - Virginia Disaster # VA-00036

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-09-27

    ... ADMINISTRATION Virginia Disaster VA-00036 AGENCY: U.S. Small Business Administration. ACTION: Notice SUMMARY: This is a notice of an Administrative declaration of a disaster for the Commonwealth of Virginia dated...: Virginia: Charles City, Chesterfield, Colonial Heights City, Dinwiddie, Hanover, Henrico, James City,...

  2. 76 FR 72022 - Virginia Disaster #VA-00040

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-11-21

    ... From the Federal Register Online via the Government Publishing Office SMALL BUSINESS ADMINISTRATION Virginia Disaster VA-00040 AGENCY: U.S. Small Business Administration. ACTION: Notice. SUMMARY... State of Virginia (FEMA- 4042-DR), dated 11/10/2011. Incident: Earthquake. Incident Period:...

  3. 76 FR 40765 - Virginia Disaster #VA-00034

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-07-11

    ... ADMINISTRATION Virginia Disaster VA-00034 AGENCY: U.S. Small Business Administration. ACTION: Notice. SUMMARY: This is a notice of an Administrative declaration of a disaster for the Commonwealth of Virginia dated...: Virginia: Bristol City, Grayson, Russell, Scott, Smyth. Tennessee: Johnson, Sullivan. The Interest...

  4. 77 FR 74908 - Virginia Disaster #VA-00051

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-12-18

    ... ADMINISTRATION Virginia Disaster VA-00051 AGENCY: U.S. Small Business Administration. ACTION: Notice. SUMMARY: This is a notice of an Administrative declaration of a disaster for the Commonwealth of Virginia dated... adversely affected by the disaster: Primary Counties: Accomack. Contiguous Counties: Virginia:...

  5. 76 FR 72994 - Virginia Disaster #VA-00041

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-11-28

    ... From the Federal Register Online via the Government Publishing Office SMALL BUSINESS ADMINISTRATION Virginia Disaster VA-00041 AGENCY: U.S. Small Business Administration. ACTION: Notice. SUMMARY... State of Virginia (FEMA- 4045-DR), dated 11/17/2011. Incident: Remnants of Tropical Storm Lee....

  6. 77 FR 38179 - Autopsies at VA Expense

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-06-27

    ... document published in the Federal Register on December 2, 2011 (76 FR 75509), VA proposed the above... abuse; Alcoholism; Claims; Day care; Dental health; Drug abuse; Government contracts; Grant programs... statute that previously authorized certain outpatient and ambulatory care, which included...

  7. Telephone Enrollment in the VA Healthcare System. Interim final rule.

    PubMed

    2016-03-16

    This rulemaking amends VA's medical regulations to allow veterans to complete applications for health care enrollment by telephone by providing application information to a VA employee, agreeing to VA's provisions regarding copayment liability and assignment of third-party insurance benefits, and attesting to the accuracy and authenticity of the information provided over the phone. This action will make it easier for veterans to apply to enroll and will speed VA processing of applications. PMID:26987128

  8. Assessing a new approach to verbal autopsy interpretation in a rural Ethiopian community: the InterVA model.

    PubMed Central

    Fantahun, Mesganaw; Fottrell, Edward; Berhane, Yemane; Wall, Stig; Högberg, Ulf; Byass, Peter

    2006-01-01

    OBJECTIVE: Verbal autopsy (VA) -- the interviewing of family members or caregivers about the circumstances of a death after the event -- is an established tool in areas where routine death registration is non-existent or inadequate. We assessed the performance of a probabilistic model (InterVA) for interpreting community-based VA interviews, in order to investigate patterns of cause-specific mortality in a rural Ethiopian community. We compared results with those obtained after review of the VA by local physicians, with a view to validating the model as a community-based tool. METHODS: Two-hundred and eighty-nine VA interviews were successfully completed; these included most deaths occurring in a defined community over a 1-year period. The VA interviews were interpreted by physicians and by the model, and cause-specific mortality fractions were derived for the whole community and for particular age groups using both approaches. FINDINGS: The results of the two approaches to interpretation correlated well in this example from Ethiopia. Four major cause groups accounted for over 60% of all mortality, and patterns within specific age groups were consistent with expectations for an underdeveloped high-mortality community in sub-Saharan Africa. CONCLUSION: Compared with interpretation by physicians, the InterVA model is much less labour intensive and offers 100% consistency. It is a valuable new tool for characterizing patterns of cause-specific mortality in communities without death registration and for comparing patterns of mortality in different populations. PMID:16583079

  9. 48 CFR 819.7109 - VA review of application.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... SOCIOECONOMIC PROGRAMS SMALL BUSINESS PROGRAMS VA Mentor-Protégé Program 819.7109 VA review of application. (a) VA OSDBU will review the information to establish the mentor and protégé eligibility and to ensure... charge to apply for the Mentor-Protégé Program. (b) After OSDBU completes its review and provides...

  10. 48 CFR 819.7109 - VA review of application.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... SOCIOECONOMIC PROGRAMS SMALL BUSINESS PROGRAMS VA Mentor-Protégé Program 819.7109 VA review of application. (a) VA OSDBU will review the information to establish the mentor and protégé eligibility and to ensure... charge to apply for the Mentor-Protégé Program. (b) After OSDBU completes its review and provides...

  11. 48 CFR 819.7109 - VA review of application.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... SOCIOECONOMIC PROGRAMS SMALL BUSINESS PROGRAMS VA Mentor-Protégé Program 819.7109 VA review of application. (a) VA OSDBU will review the information to establish the mentor and protégé eligibility and to ensure... charge to apply for the Mentor-Protégé Program. (b) After OSDBU completes its review and provides...

  12. 48 CFR 819.7109 - VA review of application.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... SOCIOECONOMIC PROGRAMS SMALL BUSINESS PROGRAMS VA Mentor-Protégé Program 819.7109 VA review of application. (a) VA OSDBU will review the information to establish the mentor and protégé eligibility and to ensure... charge to apply for the Mentor-Protégé Program. (b) After OSDBU completes its review and provides...

  13. 48 CFR 819.7109 - VA review of application.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... SOCIOECONOMIC PROGRAMS SMALL BUSINESS PROGRAMS VA Mentor-Protégé Program 819.7109 VA review of application. (a) VA OSDBU will review the information to establish the mentor and protégé eligibility and to ensure... charge to apply for the Mentor-Protégé Program. (b) After OSDBU completes its review and provides...

  14. Simultaneous Amplicon Sequencing to Explore Co-Occurrence Patterns of Bacterial, Archaeal and Eukaryotic Microorganisms in Rumen Microbial Communities

    PubMed Central

    Kittelmann, Sandra; Seedorf, Henning; Walters, William A.; Clemente, Jose C.; Knight, Rob; Gordon, Jeffrey I.; Janssen, Peter H.

    2013-01-01

    Ruminants rely on a complex rumen microbial community to convert dietary plant material to energy-yielding products. Here we developed a method to simultaneously analyze the community's bacterial and archaeal 16S rRNA genes, ciliate 18S rRNA genes and anaerobic fungal internal transcribed spacer 1 genes using 12 DNA samples derived from 11 different rumen samples from three host species (Ovis aries, Bos taurus, Cervus elephas) and multiplex 454 Titanium pyrosequencing. We show that the mixing ratio of the group-specific DNA templates before emulsion PCR is crucial to compensate for differences in amplicon length. This method, in contrast to using a non-specific universal primer pair, avoids sequencing non-targeted DNA, such as plant- or endophyte-derived rRNA genes, and allows increased or decreased levels of community structure resolution for each microbial group as needed. Communities analyzed with different primers always grouped by sample origin rather than by the primers used. However, primer choice had a greater impact on apparent archaeal community structure than on bacterial community structure, and biases for certain methanogen groups were detected. Co-occurrence analysis of microbial taxa from all three domains of life suggested strong within- and between-domain correlations between different groups of microorganisms within the rumen. The approach used to simultaneously characterize bacterial, archaeal and eukaryotic components of a microbiota should be applicable to other communities occupying diverse habitats. PMID:23408926

  15. Characterization of FGFR1 Locus in sqNSCLC Reveals a Broad and Heterogeneous Amplicon

    PubMed Central

    Rooney, Claire; Geh, Catherine; Williams, Victoria; Heuckmann, Johannes M.; Menon, Roopika; Schneider, Petra; Al-Kadhimi, Katherine; Dymond, Michael; Smith, Neil R.; Baker, Dawn; French, Tim; Smith, Paul D.; Harrington, Elizabeth A.; Barrett, J. Carl; Kilgour, Elaine

    2016-01-01

    FGFR1 amplification occurs in ~20% of sqNSCLC and trials with FGFR inhibitors have selected FGFR1 amplified patients by FISH. Lung cancer cell lines were profiled for sensitivity to AZD4547, a potent, selective inhibitor of FGFRs 1–3. Sensitivity to FGFR inhibition was associated with but not wholly predicted by increased FGFR1 gene copy number. Additional biomarker assays evaluating expression of FGFRs and correlation between amplification and expression in clinical tissues are therefore warranted. We validated nanoString for mRNA expression analysis of 194 genes, including FGFRs, from clinical tumour tissue. In a panel of sqNSCLC tumours 14.4% (13/90) were FGFR1 amplified by FISH. Although mean FGFR1 expression was significantly higher in amplified samples, there was significant overlap in the range of expression levels between the amplified and non-amplified cohorts with several non-amplified samples expressing FGFR1 to levels equivalent to amplified samples. Statistical analysis revealed increased expression of FGFR1 neighboring genes on the 8p12 amplicon (BAG4, LSM1 and WHSC1L1) in FGFR1 amplified tumours, suggesting a broad rather than focal amplicon and raises the potential for codependencies. High resolution aCGH analysis of pre-clinical and clinical samples supported the presence of a broad and heterogeneous amplicon around the FGFR1 locus. In conclusion, the range of FGFR1 expression levels in both FGFR1 amplified and non-amplified NSCLC tissues, together with the breadth and intra-patient heterogeneity of the 8p amplicon highlights the need for gene expression analysis of clinical samples to inform the understanding of determinants of response to FGFR inhibitors. In this respect the nanoString platform provides an attractive option for RNA analysis of FFPE clinical samples. PMID:26905262

  16. DADA2: High-resolution sample inference from Illumina amplicon data.

    PubMed

    Callahan, Benjamin J; McMurdie, Paul J; Rosen, Michael J; Han, Andrew W; Johnson, Amy Jo A; Holmes, Susan P

    2016-07-01

    We present the open-source software package DADA2 for modeling and correcting Illumina-sequenced amplicon errors (https://github.com/benjjneb/dada2). DADA2 infers sample sequences exactly and resolves differences of as little as 1 nucleotide. In several mock communities, DADA2 identified more real variants and output fewer spurious sequences than other methods. We applied DADA2 to vaginal samples from a cohort of pregnant women, revealing a diversity of previously undetected Lactobacillus crispatus variants. PMID:27214047

  17. Molecular cloning and characterization of a 20q13.2 amplicon in breast carcinoma

    SciTech Connect

    Collins, C.; Froula, J.; Kowbel, D.

    1994-09-01

    Comparative genomic hybridization (CGH) has identified an amplification event involving chromosome band 20q13.2 in 15-20% of primary breast carcinomas. The application of FISH to the study of tumor interphase nuclei using 33 locus specific cosmid and P1 probes revealed amplification of band 20q13.2 in 35% of breast cancer cell lines and 8% of primary tumors. Moreover, this study localized the amplification event to the 1.5Mb interval defined by (Flpter 0.80-0.84.) and excluded all known genes in the region as candidates for the putative oncogene(s). To both identify the putative oncogene(s) and characterize the amplicon, a 12 member 4Mb YAC contig has been assembled by STS mapping that spans the core of the amplicon. The YAC contig is now being converted to a P1 contig to facilitate sequencing, exon trapping and direct selection of cDNAs. This is being accomplished by performing interAlu PCR reactions on individual YACs and sequencing the reaction products to create 5-10 new STSs per megaYAC. The DuPont P1 library is then screened for these STSs by the PCR. To date 21 P1 clones, forming 6 contigs, have been isolated by screening the DuPont P1 library for existing and/or newly created STSs. The ends of the 21 P1 clones are being sequenced to facilitate contig alignment and to enable chromosome walking. In collaboration with the Human Genome Center at the Lawrence Berkeley Laboratory we have initiated the directed sequencing of two P1 contigs, localized within the amplicon core, and ultimately will sequence the entire 1-2Mb amplicon.

  18. Massively parallel sequencing of 17 commonly used forensic autosomal STRs and amelogenin with small amplicons.

    PubMed

    Kim, Eun Hye; Lee, Hwan Young; Yang, In Seok; Jung, Sang-Eun; Yang, Woo Ick; Shin, Kyoung-Jin

    2016-05-01

    The next-generation sequencing (NGS) method has been utilized to analyze short tandem repeat (STR) markers, which are routinely used for human identification purposes in the forensic field. Some researchers have demonstrated the successful application of the NGS system to STR typing, suggesting that NGS technology may be an alternative or additional method to overcome limitations of capillary electrophoresis (CE)-based STR profiling. However, there has been no available multiplex PCR system that is optimized for NGS analysis of forensic STR markers. Thus, we constructed a multiplex PCR system for the NGS analysis of 18 markers (13CODIS STRs, D2S1338, D19S433, Penta D, Penta E and amelogenin) by designing amplicons in the size range of 77-210 base pairs. Then, PCR products were generated from two single-sources, mixed samples and artificially degraded DNA samples using a multiplex PCR system, and were prepared for sequencing on the MiSeq system through construction of a subsequent barcoded library. By performing NGS and analyzing the data, we confirmed that the resultant STR genotypes were consistent with those of CE-based typing. Moreover, sequence variations were detected in targeted STR regions. Through the use of small-sized amplicons, the developed multiplex PCR system enables researchers to obtain successful STR profiles even from artificially degraded DNA as well as STR loci which are analyzed with large-sized amplicons in the CE-based commercial kits. In addition, successful profiles can be obtained from mixtures up to a 1:19 ratio. Consequently, the developed multiplex PCR system, which produces small size amplicons, can be successfully applied to STR NGS analysis of forensic casework samples such as mixtures and degraded DNA samples. PMID:26799314

  19. Ioncopy: a novel method for calling copy number alterations in amplicon sequencing data including significance assessment

    PubMed Central

    Budczies, Jan; Pfarr, Nicole; Stenzinger, Albrecht; Treue, Denise; Endris, Volker; Ismaeel, Fakher; Bangemann, Nikola; Blohmer, Jens-Uwe; Dietel, Manfred; Loibl, Sibylle; Klauschen, Frederick; Weichert, Wilko; Denkert, Carsten

    2016-01-01

    Recently, it has been demonstrated that calling of copy number alterations (CNAs) from amplicon sequencing (AS) data is feasible. Most approaches, however, require non-tumor (germline) DNA for data normalization. Here, we present the method Ioncopy for CNA detection which requires no normal controls and includes a significance assessment for each detected alteration. Ioncopy was evaluated in a cohort of 184 clinically annotated breast carcinomas. A total number of 252 amplifications were detected, of which 183 (72.6%) could be validated by a call of an additional amplicon interrogating the same gene. Moreover, a total number of 33 deletions were found, whereof 27 (81.8%) could be validated. Analyzing the 16 most frequently amplified genes, validation rates of over 89% could be achieved for 11 of these genes. 11 of the top 16 genes showed significant overexpression in the amplified tumors. 89.5% of the HER2-amplified tumors were GRB7 and STARD3 co-amplified, whereas 68.4% of the HER2-amplified tumors had additional MED1 amplifications. Correlations between CNAs measured by amplicons in HER2 exons 19, 20 and 21 were strong (all R > 0.93). AS based detection of HER2 amplifications had a sensitivity of 90.0% and a specificity of 98.8% compared to the gold standard of HER2 immunohistochemistry combined with in situ hybridization. In summary, we developed and validated a novel method for detection and significance assessment of CNAs in amplicon sequencing data. Using Ioncopy, AS offers a straightforward and efficient approach to simultaneously analyze gene amplifications and gene deletions together with simple somatic mutations in a single assay. PMID:26910888

  20. Electrochemical DNA sensor for anthrax toxin activator gene atxA-detection of PCR amplicons.

    PubMed

    Das, Ritu; Goel, Ajay K; Sharma, Mukesh K; Upadhyay, Sanjay

    2015-12-15

    We report the DNA probe functionalized electrochemical genosensor for the detection of Bacillus anthracis, specific towards the regulatory gene atxA. The DNA sensor is fabricated on electrochemically deposited gold nanoparticle on self assembled layer of (3-Mercaptopropyl) trimethoxysilane (MPTS) on GC electrode. DNA hybridization is monitored by differential pulse voltammogram (DPV). The modified GC electrode is characterized by atomic force microscopy (AFM), cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS) method. We also quantified the DNA probe density on electrode surface by the chronocoulometric method. The detection is specific and selective for atxA gene by DNA probe on the electrode surface. No report is available for the detection of B. anthracis by using atxA an anthrax toxin activator gene. In the light of real and complex sample, we have studied the PCR amplicons of 303, 361 and 568 base pairs by using symmetric and asymmetric PCR approaches. The DNA probe of atxA gene efficiently hybridizes with different base pairs of PCR amplicons. The detection limit is found to be 1.0 pM (S/N ratio=3). The results indicate that the DNA sensor is able to detect synthetic target as well as PCR amplicons of different base pairs. PMID:26257186

  1. From Benchtop to Desktop: Important Considerations when Designing Amplicon Sequencing Workflows

    PubMed Central

    Murray, Dáithí C.; Coghlan, Megan L.; Bunce, Michael

    2015-01-01

    Amplicon sequencing has been the method of choice in many high-throughput DNA sequencing (HTS) applications. To date there has been a heavy focus on the means by which to analyse the burgeoning amount of data afforded by HTS. In contrast, there has been a distinct lack of attention paid to considerations surrounding the importance of sample preparation and the fidelity of library generation. No amount of high-end bioinformatics can compensate for poorly prepared samples and it is therefore imperative that careful attention is given to sample preparation and library generation within workflows, especially those involving multiple PCR steps. This paper redresses this imbalance by focusing on aspects pertaining to the benchtop within typical amplicon workflows: sample screening, the target region, and library generation. Empirical data is provided to illustrate the scope of the problem. Lastly, the impact of various data analysis parameters is also investigated in the context of how the data was initially generated. It is hoped this paper may serve to highlight the importance of pre-analysis workflows in achieving meaningful, future-proof data that can be analysed appropriately. As amplicon sequencing gains traction in a variety of diagnostic applications from forensics to environmental DNA (eDNA) it is paramount workflows and analytics are both fit for purpose. PMID:25902146

  2. Analysing 454 amplicon resequencing experiments using the modular and database oriented Variant Identification Pipeline

    PubMed Central

    2010-01-01

    Background Next-generation amplicon sequencing enables high-throughput genetic diagnostics, sequencing multiple genes in several patients together in one sequencing run. Currently, no open-source out-of-the-box software solution exists that reliably reports detected genetic variations and that can be used to improve future sequencing effectiveness by analyzing the PCR reactions. Results We developed an integrated database oriented software pipeline for analysis of 454/Roche GS-FLX amplicon resequencing experiments using Perl and a relational database. The pipeline enables variation detection, variation detection validation, and advanced data analysis, which provides information that can be used to optimize PCR efficiency using traditional means. The modular approach enables customization of the pipeline where needed and allows researchers to adopt their analysis pipeline to their experiments. Clear documentation and training data is available to test and validate the pipeline prior to using it on real sequencing data. Conclusions We designed an open-source database oriented pipeline that enables advanced analysis of 454/Roche GS-FLX amplicon resequencing experiments using SQL-statements. This modular database approach allows easy coupling with other pipeline modules such as variant interpretation or a LIMS system. There is also a set of standard reporting scripts available. PMID:20487544

  3. Sample Preparation for Fungal Community Analysis by High-Throughput Sequencing of Barcode Amplicons.

    PubMed

    Clemmensen, Karina Engelbrecht; Ihrmark, Katarina; Durling, Mikael Brandström; Lindahl, Björn D

    2016-01-01

    Fungal species participate in vast numbers of processes in the landscape around us. However, their often cryptic growth, inside various substrates and in highly diverse species assemblages, has been a major obstacle to thorough analysis of fungal communities, hampering exhaustive description of the fungal kingdom. Recent technological developments allowing rapid, high-throughput sequencing of mixed communities from many samples at once are currently having a tremendous impact in fungal community ecology. Universal DNA extraction followed by amplification and sequencing of fungal species-level barcodes such as the nuclear internal transcribed spacer (ITS) region now enable identification and relative quantification of fungal community members across well-replicated experimental settings. Here, we present the sample preparation procedure presently used in our laboratory for fungal community analysis by high-throughput sequencing of amplified ITS2 markers. We focus on the procedure optimized for studies of total fungal communities in humus-rich soils, wood, and litter. However, this procedure can be applied to other sample types and markers. We focus on the laboratory-based part of sample preparation, that is, the procedure from the point where samples enter the laboratory until amplicons are submitted for sequencing. Our procedure comprises four main parts: (1) universal DNA extraction, (2) optimization of PCR conditions, (3) production of tagged ITS amplicons, and (4) preparation of the multiplexed amplicon mix to be sequenced. The presented procedure is independent of the specific high-throughput sequencing technology used, which makes it highly versatile. PMID:26791497

  4. How Involved are Non-VA Chaplains in Supporting Veterans?

    PubMed

    Kopacz, Marek S; Feldstein, Bruce D; Asekoff, Cecille Allman; Kaprow, Maurice S; Smith-Coggins, Rebecca; Rasmussen, Kathy A

    2016-08-01

    In terms of supporting veteran populations, little is known of the experiences of chaplains professionally active outside of Department of Veterans Affairs (VA) healthcare settings. The present study looks to examine how involved non-VA chaplains are in supporting veterans as well as their familiarity with the VA. An online survey was distributed in a convenience sample of chaplains, of which n = 39 met the inclusion criterion for this study (i.e., no past or present VA affiliation). The results find that most of the non-VA chaplains encounter veteran service users either on a weekly or monthly basis. Though familiar with VA services, non-VA chaplains were not sure of their veteran service users' VA enrollment status nor did they feel able to adequately advise their veteran service users on VA enrollment. The results suggest that non-VA chaplains actively support veteran populations. Opportunities for enhancing chaplaincy services and VA outreach programs are discussed. PMID:27023459

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

    PubMed Central

    2012-01-01

    Background High-resolution HLA genotyping is a critical diagnostic and research assay. Current methods rarely achieve unambiguous high-resolution typing without making population-specific frequency inferences due to a lack of locus coverage and difficulty in exon-phase matching. Achieving high-resolution typing is also becoming more challenging with traditional methods as the database of known HLA alleles increases. Results We designed a cDNA amplicon-based pyrosequencing method to capture 94% of the HLA class I open-reading-frame with only two amplicons per sample, and an analogous method for class II HLA genes, with a primary focus on sequencing the DRB loci. We present a novel Galaxy server-based analysis workflow for determining genotype. During assay validation, we performed two GS Junior sequencing runs to determine the accuracy of the HLA class I amplicons and DRB amplicon at different levels of multiplexing. When 116 amplicons were multiplexed, we unambiguously resolved 99%of class I alleles to four- or six-digit resolution, as well as 100% unambiguous DRB calls. The second experiment, with 271 multiplexed amplicons, missed some alleles, but generated high-resolution, concordant typing for 93% of class I alleles, and 96% for DRB1 alleles. In a third, preliminary experiment we attempted to sequence novel amplicons for other class II loci with mixed success. Conclusions The presented assay is higher-throughput and higher-resolution than existing HLA genotyping methods, and suitable for allele discovery or large cohort sampling. The validated class I and DRB primers successfully generated unambiguously high-resolution genotypes, while further work is needed to validate additional class II genotyping amplicons. PMID:22866951

  6. 48 CFR 853.215-70 - VA Form 10-1170, Application for Furnishing Nursing Home Care to Beneficiaries of VA.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ..., Application for Furnishing Nursing Home Care to Beneficiaries of VA. 853.215-70 Section 853.215-70 Federal... 853.215-70 VA Form 10-1170, Application for Furnishing Nursing Home Care to Beneficiaries of VA. VA Form 10-1170, Application for Furnishing Nursing Home Care to Beneficiaries of VA, will be used...

  7. 48 CFR 853.215-70 - VA Form 10-1170, Application for Furnishing Nursing Home Care to Beneficiaries of VA.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ..., Application for Furnishing Nursing Home Care to Beneficiaries of VA. 853.215-70 Section 853.215-70 Federal... 853.215-70 VA Form 10-1170, Application for Furnishing Nursing Home Care to Beneficiaries of VA. VA Form 10-1170, Application for Furnishing Nursing Home Care to Beneficiaries of VA, will be used...

  8. 48 CFR 853.215-70 - VA Form 10-1170, Application for Furnishing Nursing Home Care to Beneficiaries of VA.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ..., Application for Furnishing Nursing Home Care to Beneficiaries of VA. 853.215-70 Section 853.215-70 Federal... 853.215-70 VA Form 10-1170, Application for Furnishing Nursing Home Care to Beneficiaries of VA. VA Form 10-1170, Application for Furnishing Nursing Home Care to Beneficiaries of VA, will be used...

  9. 48 CFR 853.215-70 - VA Form 10-1170, Application for Furnishing Nursing Home Care to Beneficiaries of VA.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ..., Application for Furnishing Nursing Home Care to Beneficiaries of VA. 853.215-70 Section 853.215-70 Federal... 853.215-70 VA Form 10-1170, Application for Furnishing Nursing Home Care to Beneficiaries of VA. VA Form 10-1170, Application for Furnishing Nursing Home Care to Beneficiaries of VA, will be used...

  10. 48 CFR 853.215-70 - VA Form 10-1170, Application for Furnishing Nursing Home Care to Beneficiaries of VA.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ..., Application for Furnishing Nursing Home Care to Beneficiaries of VA. 853.215-70 Section 853.215-70 Federal... 853.215-70 VA Form 10-1170, Application for Furnishing Nursing Home Care to Beneficiaries of VA. VA Form 10-1170, Application for Furnishing Nursing Home Care to Beneficiaries of VA, will be used...

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

    PubMed Central

    2013-01-01

    Background Wheat gluten is important for the industrial quality of bread wheat (Triticum aestivum L.) and durum wheat (T. turgidum L.). Gluten proteins are also the source of immunogenic peptides that can trigger a T cell reaction in celiac disease (CD) patients, leading to inflammatory responses in the small intestine. Various peptides with three major T cell epitopes involved in CD are derived from alpha-gliadin fraction of gluten. Alpha-gliadins are encoded by a large multigene family and amino acid variation in the CD epitopes is known to influence the immunogenicity of individual gene family members. Current commercial methods of gluten detection are unable to distinguish between immunogenic and non-immunogenic CD epitope variants and thus to accurately quantify the overall CD epitope load of a given wheat variety. Such quantification is indispensable for correct selection of wheat varieties with low potential to cause CD. Results A 454 RNA-amplicon sequencing method was developed for alpha-gliadin transcripts encompassing the three major CD epitopes and their variants. The method was used to screen developing grains on plants of 61 different durum wheat cultivars and accessions. A dedicated sequence analysis pipeline returned a total of 304 unique alpha-gliadin transcripts, corresponding to a total of 171 ‘unique deduced protein fragments’ of alpha-gliadins. The numbers of these fragments obtained in each plant were used to calculate quantitative and quantitative differences between the CD epitopes expressed in the endosperm of these wheat plants. A few plants showed a lower fraction of CD epitope-encoding alpha-gliadin transcripts, but none were free of CD epitopes. Conclusions The dedicated 454 RNA-amplicon sequencing method enables 1) the grouping of wheat plants according to the genetic variation in alpha-gliadin transcripts, and 2) the screening for plants which are potentially less CD-immunogenic. The resulting alpha-gliadin sequence database will

  12. Application of PCR amplicon sequencing using a single primer pair in PCR amplification to assess variations in Helicobacter pylori CagA EPIYA tyrosine phosphorylation motifs

    PubMed Central

    2010-01-01

    Background The presence of various EPIYA tyrosine phosphorylation motifs in the CagA protein of Helicobacter pylori has been suggested to contribute to pathogenesis in adults. In this study, a unique PCR assay and sequencing strategy was developed to establish the number and variation of cagA EPIYA motifs. Findings MDA-DNA derived from gastric biopsy specimens from eleven subjects with gastritis was used with M13- and T7-sequence-tagged primers for amplification of the cagA EPIYA motif region. Automated capillary electrophoresis using a high resolution kit and amplicon sequencing confirmed variations in the cagA EPIYA motif region. In nine cases, sequencing revealed the presence of AB, ABC, or ABCC (Western type) cagA EPIYA motif, respectively. In two cases, double cagA EPIYA motifs were detected (ABC/ABCC or ABC/AB), indicating the presence of two H. pylori strains in the same biopsy. Conclusion Automated capillary electrophoresis and Amplicon sequencing using a single, M13- and T7-sequence-tagged primer pair in PCR amplification enabled a rapid molecular typing of cagA EPIYA motifs. Moreover, the techniques described allowed for a rapid detection of mixed H. pylori strains present in the same biopsy specimen. PMID:20181142

  13. Impact of Mutation Type and Amplicon Characteristics on Genetic Diversity Measures Generated Using a High-Resolution Melting Diversity Assay

    PubMed Central

    Cousins, Matthew M.; Donnell, Deborah; Eshleman, Susan H.

    2013-01-01

    We adapted high-resolution melting (HRM) technology to measure genetic diversity without sequencing. Diversity is measured as a single numeric HRM score. Herein, we determined the impact of mutation types and amplicon characteristics on HRM diversity scores. Plasmids were generated with single-base changes, insertions, and deletions. Different primer sets were used to vary the position of mutations within amplicons. Plasmids and plasmid mixtures were analyzed to determine the impact of mutation type, position, and concentration on HRM scores. The impact of amplicon length and G/C content on HRM scores was also evaluated. Different mutation types affected HRM scores to varying degrees (1-bp deletion < 1-bp change < 3-bp insertion < 9-bp insertion). The impact of mutations on HRM scores was influenced by amplicon length and the position of the mutation within the amplicon. Mutations were detected at concentrations of 5% to 95%, with the greatest impact at 50%. The G/C content altered melting temperature values of amplicons but had no impact on HRM scores. These data are relevant to the design of assays that measure genetic diversity using HRM technology. PMID:23178437

  14. Medical Student Psychiatry Examination Performance at VA and Non-VA Clerkship Sites

    ERIC Educational Resources Information Center

    Tucker, Phebe; von Schlageter, Margo Shultes; Park, EunMi; Rosenberg, Emily; Benjamin, Ashley B.; Nawar, Ola

    2009-01-01

    Objective: The authors examined the effects of medical student assignment to U.S. Department of Veterans Affairs (VA) Medical Center inpatient and outpatient psychiatry clerkship sites versus other university and community sites on the performance outcome measure of National Board of Medical Examiners (NBME) subject examination scores. Methods:…

  15. Using Amplicon Sequencing To Characterize and Monitor Bacterial Diversity in Drinking Water Distribution Systems

    PubMed Central

    Shaw, Jennifer L. A.; Weyrich, Laura S.; Sawade, Emma; Drikas, Mary; Cooper, Alan J.

    2015-01-01

    Drinking water assessments use a variety of microbial, physical, and chemical indicators to evaluate water treatment efficiency and product water quality. However, these indicators do not allow the complex biological communities, which can adversely impact the performance of drinking water distribution systems (DWDSs), to be characterized. Entire bacterial communities can be studied quickly and inexpensively using targeted metagenomic amplicon sequencing. Here, amplicon sequencing of the 16S rRNA gene region was performed alongside traditional water quality measures to assess the health, quality, and efficiency of two distinct, full-scale DWDSs: (i) a linear DWDS supplied with unfiltered water subjected to basic disinfection before distribution and (ii) a complex, branching DWDS treated by a four-stage water treatment plant (WTP) prior to disinfection and distribution. In both DWDSs bacterial communities differed significantly after disinfection, demonstrating the effectiveness of both treatment regimes. However, bacterial repopulation occurred further along in the DWDSs, and some end-user samples were more similar to the source water than to the postdisinfection water. Three sample locations appeared to be nitrified, displaying elevated nitrate levels and decreased ammonia levels, and nitrifying bacterial species, such as Nitrospira, were detected. Burkholderiales were abundant in samples containing large amounts of monochloramine, indicating resistance to disinfection. Genera known to contain pathogenic and fecal-associated species were also identified in several locations. From this study, we conclude that metagenomic amplicon sequencing is an informative method to support current compliance-based methods and can be used to reveal bacterial community interactions with the chemical and physical properties of DWDSs. PMID:26162884

  16. Clinical impact of targeted amplicon sequencing for meningioma as a practical clinical-sequencing system.

    PubMed

    Yuzawa, Sayaka; Nishihara, Hiroshi; Yamaguchi, Shigeru; Mohri, Hiromi; Wang, Lei; Kimura, Taichi; Tsuda, Masumi; Tanino, Mishie; Kobayashi, Hiroyuki; Terasaka, Shunsuke; Houkin, Kiyohiro; Sato, Norihiro; Tanaka, Shinya

    2016-07-01

    Recent genetic analyses using next-generation sequencers have revealed numerous genetic alterations in various tumors including meningioma, which is the most common primary brain tumor. However, their use as routine laboratory examinations in clinical applications for tumor genotyping is not cost effective. To establish a clinical sequencing system for meningioma and investigate the clinical significance of genotype, we retrospectively performed targeted amplicon sequencing on 103 meningiomas and evaluated the association with clinicopathological features. We designed amplicon-sequencing panels targeting eight genes including NF2 (neurofibromin 2), TRAF7, KLF4, AKT1, and SMO. Libraries prepared with genomic DNA extracted from PAXgene-fixed paraffin-embedded tissues of 103 meningioma specimens were sequenced using the Illumina MiSeq. NF2 loss in some cases was also confirmed by interphase-fluorescent in situ hybridization. We identified NF2 loss and/or at least one mutation in NF2, TRAF7, KLF4, AKT1, and SMO in 81 out of 103 cases (79%) by targeted amplicon sequencing. On the basis of genetic status, we categorized meningiomas into three genotype groups: NF2 type, TRAKLS type harboring mutation in TRAF7, AKT1, KLF4, and/or SMO, and 'not otherwise classified' type. Genotype significantly correlated with tumor volume, tumor location, and magnetic resonance imaging findings such as adjacent bone change and heterogeneous gadolinium enhancement, as well as histopathological subtypes. In addition, multivariate analysis revealed that genotype was independently associated with risk of recurrence. In conclusion, we established a rapid clinical sequencing system that enables final confirmation of meningioma genotype within 7 days turnaround time. Our method will bring multiple benefits to neuropathologists and neurosurgeons for accurate diagnosis and appropriate postoperative management. PMID:27102344

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

    PubMed

    Ibarbalz, Federico M; Pérez, María Victoria; Figuerola, Eva L M; Erijman, Leonardo

    2014-01-01

    The performance of two sets of primers targeting variable regions of the 16S rRNA gene V1-V3 and V4 was compared in their ability to describe changes of bacterial diversity and temporal turnover in full-scale activated sludge. Duplicate sets of high-throughput amplicon sequencing data of the two 16S rRNA regions shared a collection of core taxa that were observed across a series of twelve monthly samples, although the relative abundance of each taxon was substantially different between regions. A case in point was the changes in the relative abundance of filamentous bacteria Thiothrix, which caused a large effect on diversity indices, but only in the V1-V3 data set. Yet the relative abundance of Thiothrix in the amplicon sequencing data from both regions correlated with the estimation of its abundance determined using fluorescence in situ hybridization. In nonmetric multidimensional analysis samples were distributed along the first ordination axis according to the sequenced region rather than according to sample identities. The dynamics of microbial communities indicated that V1-V3 and the V4 regions of the 16S rRNA gene yielded comparable patterns of: 1) the changes occurring within the communities along fixed time intervals, 2) the slow turnover of activated sludge communities and 3) the rate of species replacement calculated from the taxa-time relationships. The temperature was the only operational variable that showed significant correlation with the composition of bacterial communities over time for the sets of data obtained with both pairs of primers. In conclusion, we show that despite the bias introduced by amplicon sequencing, the variable regions V1-V3 and V4 can be confidently used for the quantitative assessment of bacterial community dynamics, and provide a proper qualitative account of general taxa in the community, especially when the data are obtained over a convenient time window rather than at a single time point. PMID:24923665

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

    PubMed

    Pabinger, Stephan; Ernst, Karina; Pulverer, Walter; Kallmeyer, Rainer; Valdes, Ana M; Metrustry, Sarah; Katic, Denis; Nuzzo, Angelo; Kriegner, Albert; Vierlinger, Klemens; Weinhaeusel, Andreas

    2016-01-01

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

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

    PubMed Central

    Pabinger, Stephan; Ernst, Karina; Pulverer, Walter; Kallmeyer, Rainer; Valdes, Ana M.; Metrustry, Sarah; Katic, Denis; Nuzzo, Angelo; Kriegner, Albert; Vierlinger, Klemens; Weinhaeusel, Andreas

    2016-01-01

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

  20. Quick diagnosis of human brain meningitis using omp85 gene amplicon as a genetic marker.

    PubMed

    Dash, Sandip K; Sharma, Minakshi; Khare, Shashi; Kumar, Ashok

    2013-06-01

    The usual diagnosis of life-threatening human brain bacterial meningitis are expensive, time consuming or non-confirmatory. A quick PCR based diagnosis of meningitis in cerebrospinal fluids (CSF) using specific primers of virulent Omp85 gene of Neisseria meningitidis can detect as low as 1.0 ng of genomic DNA (G-DNA) in 80 min for confirmation of bacterial meningitis caused by N. meningitidis infection. The 257 bp amplicon of Omp85 gene does not show homology with other suspected pathogens in CSF and can be used as a specific genetic marker for diagnosis of the disease. PMID:24426115

  1. Parallel tagged amplicon sequencing of relatively long PCR products using the Illumina HiSeq platform and transcriptome assembly.

    PubMed

    Feng, Yan-Jie; Liu, Qing-Feng; Chen, Meng-Yun; Liang, Dan; Zhang, Peng

    2016-01-01

    In phylogenetics and population genetics, a large number of loci are often needed to accurately resolve species relationships. Normally, loci are enriched by PCR and sequenced by Sanger sequencing, which is expensive when the number of amplicons is large. Next-generation sequencing (NGS) techniques are increasingly used for parallel amplicon sequencing, which reduces sequencing costs tremendously, but has not reduced preparation costs very much. Moreover, for most current NGS methods, amplicons need to be purified and quantified before sequencing and their lengths are also restricted (normally <700 bp). Here, we describe an approach to sequence pooled amplicons of any length using the Illumina platform. Using this method, amplicons are pooled at equal volume rather than at equal concentration, thus eliminating the laborious purification and quantification steps. We then shear the pooled amplicons, repair the ends, add sample identifying linkers and pool multiple samples prior to Illumina library preparation. Data are then assembled using the transcriptome assembly program trinity, which is optimized to deal with templates of highly varying quantities. We demonstrated the utility of our approach by recovering 93.5% of the target amplicons (size up to 1650 bp) in full length for a 16 taxa × 101 loci project, using ~2.0 GB of Illumina HiSeq paired-end 90-bp data. Overall, we validate a rapid, cost-effective and scalable approach to sequence a large number of targeted loci from a large number of samples that is particularly suitable for both phylogenetics and population genetics studies that require a modest scale of data. PMID:25959587

  2. Herpes simplex virus amplicon: cleavage of concatemeric DNA is linked to packaging and involves amplification of the terminally reiterated a sequence.

    PubMed Central

    Deiss, L P; Frenkel, N

    1986-01-01

    Herpes simplex virus-infected cells contain large concatemeric DNA molecules arising from replication of the viral genome. The large concatemers are cleaved to generate unit-length molecules terminating at both ends with the a sequence. We have used constructed defective virus vectors (amplicons) derived from herpes simplex virus to study the mechanism of cleavage of viral DNA concatemers and the packaging of viral DNA into nucleocapsids. These studies revealed that (i) a 248-base-pair a sequence contained the signal(s) required for cleavage-packaging, (ii) the cleavage of viral DNA concatemers was coupled to packaging, (iii) the a sequence contained the information required for its own amplification, and (iv) cleavage-packaging occurred by a novel process involving the amplification of the a sequence. Images PMID:3005637

  3. Bioinformatic Amplicon Read Processing Strategies Strongly Affect Eukaryotic Diversity and the Taxonomic Composition of Communities

    PubMed Central

    Majaneva, Markus; Hyytiäinen, Kirsi; Varvio, Sirkka Liisa; Nagai, Satoshi; Blomster, Jaanika

    2015-01-01

    Amplicon read sequencing has revolutionized the field of microbial diversity studies. The technique has been developed for bacterial assemblages and has undergone rigorous testing with mock communities. However, due to the great complexity of eukaryotes and the numbers of different rDNA copies, analyzing eukaryotic diversity is more demanding than analyzing bacterial or mock communities, so studies are needed that test the methods of analyses on taxonomically diverse natural communities. In this study, we used 20 samples collected from the Baltic Sea ice, slush and under-ice water to investigate three program packages (UPARSE, mothur and QIIME) and 18 different bioinformatic strategies implemented in them. Our aim was to assess the impact of the initial steps of bioinformatic strategies on the results when analyzing natural eukaryotic communities. We found significant differences among the strategies in resulting read length, number of OTUs and estimates of diversity as well as clear differences in the taxonomic composition of communities. The differences arose mainly because of the variable number of chimeric reads that passed the pre-processing steps. Singleton removal and denoising substantially lowered the number of errors. Our study showed that the initial steps of the bioinformatic amplicon read processing strategies require careful consideration before applying them to eukaryotic communities. PMID:26047335

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

    PubMed

    Koch, T; Jacobsen, N; Fensholdt, J; Boas, U; Fenger, M; Jakobsen, M H

    2000-01-01

    Ligand immobilization on solid surfaces is an essential step in fields such as diagnostics, bio sensor manufacturing, and new material sciences in general. In this paper a photochemical approach based on anthraquinone as the chromophore is presented. Photochemical procedures offer special advantages as they are able to generate highly reactive species in an orientation specific manner. As presented here, anthraquinone (AQ) mediated covalent DNA immobilization appears to be superior to currently known procedures. A synthetic procedure providing AQ-phosphoramidites is presented. These reagents facilitate AQ conjugation during routine DNA synthesis, thus enabling the AQ-oligonucleotides to be immobilized in a very convenient and efficient manner. AQ-conjugated PCR primers can be used directly in PCR. When the PCR is performed in solution, the amplicons can be immobilized after the PCR. Moreover, when the primers are immobilized prior to the PCR, a solid-phase PCR can be performed and the amplicons are thus produced directly on the solid support. PMID:10898568

  5. AMPLISAS: a web server for multilocus genotyping using next-generation amplicon sequencing data.

    PubMed

    Sebastian, Alvaro; Herdegen, Magdalena; Migalska, Magdalena; Radwan, Jacek

    2016-03-01

    Next-generation sequencing (NGS) technologies are revolutionizing the fields of biology and medicine as powerful tools for amplicon sequencing (AS). Using combinations of primers and barcodes, it is possible to sequence targeted genomic regions with deep coverage for hundreds, even thousands, of individuals in a single experiment. This is extremely valuable for the genotyping of gene families in which locus-specific primers are often difficult to design, such as the major histocompatibility complex (MHC). The utility of AS is, however, limited by the high intrinsic sequencing error rates of NGS technologies and other sources of error such as polymerase amplification or chimera formation. Correcting these errors requires extensive bioinformatic post-processing of NGS data. Amplicon Sequence Assignment (AMPLISAS) is a tool that performs analysis of AS results in a simple and efficient way, while offering customization options for advanced users. AMPLISAS is designed as a three-step pipeline consisting of (i) read demultiplexing, (ii) unique sequence clustering and (iii) erroneous sequence filtering. Allele sequences and frequencies are retrieved in excel spreadsheet format, making them easy to interpret. AMPLISAS performance has been successfully benchmarked against previously published genotyped MHC data sets obtained with various NGS technologies. PMID:26257385

  6. Amplicon-based metagenomics identified candidate organisms in soils that caused yield decline in strawberry.

    PubMed

    Xu, Xiangming; Passey, Thomas; Wei, Feng; Saville, Robert; Harrison, Richard J

    2015-01-01

    A phenomenon of yield decline due to weak plant growth in strawberry was recently observed in non-chemo-fumigated soils, which was not associated with the soil fungal pathogen Verticillium dahliae, the main target of fumigation. Amplicon-based metagenomics was used to profile soil microbiota in order to identify microbial organisms that may have caused the yield decline. A total of 36 soil samples were obtained in 2013 and 2014 from four sites for metagenomic studies; two of the four sites had a yield-decline problem, the other two did not. More than 2000 fungal or bacterial operational taxonomy units (OTUs) were found in these samples. Relative abundance of individual OTUs was statistically compared for differences between samples from sites with or without yield decline. A total of 721 individual comparisons were statistically significant - involving 366 unique bacterial and 44 unique fungal OTUs. Based on further selection criteria, we focused on 34 bacterial and 17 fungal OTUs and found that yield decline resulted probably from one or more of the following four factors: (1) low abundance of Bacillus and Pseudomonas populations, which are well known for their ability of supressing pathogen development and/or promoting plant growth; (2) lack of the nematophagous fungus (Paecilomyces species); (3) a high level of two non-specific fungal root rot pathogens; and (4) wet soil conditions. This study demonstrated the usefulness of an amplicon-based metagenomics approach to profile soil microbiota and to detect differential abundance in microbes. PMID:26504572

  7. Analysis of the microbiome: Advantages of whole genome shotgun versus 16S amplicon sequencing.

    PubMed

    Ranjan, Ravi; Rani, Asha; Metwally, Ahmed; McGee, Halvor S; Perkins, David L

    2016-01-22

    The human microbiome has emerged as a major player in regulating human health and disease. Translational studies of the microbiome have the potential to indicate clinical applications such as fecal transplants and probiotics. However, one major issue is accurate identification of microbes constituting the microbiota. Studies of the microbiome have frequently utilized sequencing of the conserved 16S ribosomal RNA (rRNA) gene. We present a comparative study of an alternative approach using whole genome shotgun sequencing (WGS). In the present study, we analyzed the human fecal microbiome compiling a total of 194.1 × 10(6) reads from a single sample using multiple sequencing methods and platforms. Specifically, after establishing the reproducibility of our methods with extensive multiplexing, we compared: 1) The 16S rRNA amplicon versus the WGS method, 2) the Illumina HiSeq versus MiSeq platforms, 3) the analysis of reads versus de novo assembled contigs, and 4) the effect of shorter versus longer reads. Our study demonstrates that whole genome shotgun sequencing has multiple advantages compared with the 16S amplicon method including enhanced detection of bacterial species, increased detection of diversity and increased prediction of genes. In addition, increased length, either due to longer reads or the assembly of contigs, improved the accuracy of species detection. PMID:26718401

  8. Bioinformatic Amplicon Read Processing Strategies Strongly Affect Eukaryotic Diversity and the Taxonomic Composition of Communities.

    PubMed

    Majaneva, Markus; Hyytiäinen, Kirsi; Varvio, Sirkka Liisa; Nagai, Satoshi; Blomster, Jaanika

    2015-01-01

    Amplicon read sequencing has revolutionized the field of microbial diversity studies. The technique has been developed for bacterial assemblages and has undergone rigorous testing with mock communities. However, due to the great complexity of eukaryotes and the numbers of different rDNA copies, analyzing eukaryotic diversity is more demanding than analyzing bacterial or mock communities, so studies are needed that test the methods of analyses on taxonomically diverse natural communities. In this study, we used 20 samples collected from the Baltic Sea ice, slush and under-ice water to investigate three program packages (UPARSE, mothur and QIIME) and 18 different bioinformatic strategies implemented in them. Our aim was to assess the impact of the initial steps of bioinformatic strategies on the results when analyzing natural eukaryotic communities. We found significant differences among the strategies in resulting read length, number of OTUs and estimates of diversity as well as clear differences in the taxonomic composition of communities. The differences arose mainly because of the variable number of chimeric reads that passed the pre-processing steps. Singleton removal and denoising substantially lowered the number of errors. Our study showed that the initial steps of the bioinformatic amplicon read processing strategies require careful consideration before applying them to eukaryotic communities. PMID:26047335

  9. Application of novel "mini-amplicon" STR multiplexes to high volume casework on degraded skeletal remains.

    PubMed

    Parsons, Thomas J; Huel, Rene; Davoren, Jon; Katzmarzyk, Cheryl; Milos, Ana; Selmanović, Arijana; Smajlović, Lejla; Coble, Michael D; Rizvić, Adnan

    2007-06-01

    The International Commission on Missing Persons (ICMP) conducts high throughput STR profiling on degraded skeletal remains, primarily recovered from mass graves relating to conflicts from 1992 to 1999 in the former Yugoslavia. To date, over 11,000 individuals have been identified through comparison of bone profiles to a large database of profiles from family members of the missing. To increase success rates in STR recovery, three short amplicon STR multiplexes (a 7-plex, a 6-plex, and a 5-plex) have been devised and implemented. These target loci from large commercial multiplexes, with an average decrease in amplicon size of 144 bp. The ICMP "miniplexes" have proven to provide substantially greater recovery of DNA data from a certain subset of difficult samples. However, the circumstances under which miniplexes provide additional data are restricted, and their advantages do not outweigh those of large commercial multiplexes for a majority of cases. The miniplexes, however, also have a very powerful use in DNA testing to support large scale reassociation of commingled, partial skeletons recovered from secondary mass graves. PMID:19083751

  10. Amplicon-based metagenomics identified candidate organisms in soils that caused yield decline in strawberry

    PubMed Central

    Xu, Xiangming; Passey, Thomas; Wei, Feng; Saville, Robert; Harrison, Richard J.

    2015-01-01

    A phenomenon of yield decline due to weak plant growth in strawberry was recently observed in non-chemo-fumigated soils, which was not associated with the soil fungal pathogen Verticillium dahliae, the main target of fumigation. Amplicon-based metagenomics was used to profile soil microbiota in order to identify microbial organisms that may have caused the yield decline. A total of 36 soil samples were obtained in 2013 and 2014 from four sites for metagenomic studies; two of the four sites had a yield-decline problem, the other two did not. More than 2000 fungal or bacterial operational taxonomy units (OTUs) were found in these samples. Relative abundance of individual OTUs was statistically compared for differences between samples from sites with or without yield decline. A total of 721 individual comparisons were statistically significant – involving 366 unique bacterial and 44 unique fungal OTUs. Based on further selection criteria, we focused on 34 bacterial and 17 fungal OTUs and found that yield decline resulted probably from one or more of the following four factors: (1) low abundance of Bacillus and Pseudomonas populations, which are well known for their ability of supressing pathogen development and/or promoting plant growth; (2) lack of the nematophagous fungus (Paecilomyces species); (3) a high level of two non-specific fungal root rot pathogens; and (4) wet soil conditions. This study demonstrated the usefulness of an amplicon-based metagenomics approach to profile soil microbiota and to detect differential abundance in microbes. PMID:26504572

  11. ICO amplicon NGS data analysis: a Web tool for variant detection in common high-risk hereditary cancer genes analyzed by amplicon GS Junior next-generation sequencing.

    PubMed

    Lopez-Doriga, Adriana; Feliubadaló, Lídia; Menéndez, Mireia; Lopez-Doriga, Sergio; Morón-Duran, Francisco D; del Valle, Jesús; Tornero, Eva; Montes, Eva; Cuesta, Raquel; Campos, Olga; Gómez, Carolina; Pineda, Marta; González, Sara; Moreno, Victor; Capellá, Gabriel; Lázaro, Conxi

    2014-03-01

    Next-generation sequencing (NGS) has revolutionized genomic research and is set to have a major impact on genetic diagnostics thanks to the advent of benchtop sequencers and flexible kits for targeted libraries. Among the main hurdles in NGS are the difficulty of performing bioinformatic analysis of the huge volume of data generated and the high number of false positive calls that could be obtained, depending on the NGS technology and the analysis pipeline. Here, we present the development of a free and user-friendly Web data analysis tool that detects and filters sequence variants, provides coverage information, and allows the user to customize some basic parameters. The tool has been developed to provide accurate genetic analysis of targeted sequencing of common high-risk hereditary cancer genes using amplicon libraries run in a GS Junior System. The Web resource is linked to our own mutation database, to assist in the clinical classification of identified variants. We believe that this tool will greatly facilitate the use of the NGS approach in routine laboratories. PMID:24227591

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

    PubMed

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

    2015-01-01

    The cultivated peanut (Arachis hypogaea L.) is an allotetraploid (AABB) species derived from the A-genome (Arachis duranensis) and B-genome (Arachis ipaensis) progenitors. Presence of two versions of a DNA sequence based on the two progenitor genomes poses a serious technical and analytical problem during single nucleotide polymorphism (SNP) marker identification and analysis. In this context, we have analyzed 200 amplicons derived from expressed sequence tags (ESTs) and genome survey sequences (GSS) to identify SNPs in a panel of genotypes consisting of 12 cultivated peanut varieties and two diploid progenitors representing the ancestral genomes. A total of 18 EST-SNPs and 44 genomic-SNPs were identified in 12 peanut varieties by aligning the sequence of A. hypogaea with diploid progenitors. The average frequency of sequence polymorphism was higher for genomic-SNPs than the EST-SNPs with one genomic-SNP every 1011 bp as compared to one EST-SNP every 2557 bp. In order to estimate the potential and further applicability of these identified SNPs, 96 peanut varieties were genotyped using high resolution melting (HRM) method. Polymorphism information content (PIC) values for EST-SNPs ranged between 0.021 and 0.413 with a mean of 0.172 in the set of peanut varieties, while genomic-SNPs ranged between 0.080 and 0.478 with a mean of 0.249. Total 33 SNPs were used for polymorphism detection among the parents and 10 selected lines from mapping population Y13Zh (Zhenzhuhei × Yueyou13). Of the total 33 SNPs, nine SNPs showed polymorphism in the mapping population Y13Zh, and seven SNPs were successfully mapped into five linkage groups. Our results showed that SNPs can be identified in allotetraploid peanut with high accuracy through amplicon sequencing and HRM assay. The identified SNPs were very informative and can be used for different genetic and breeding applications in peanut. PMID:26697032

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

    PubMed Central

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

    2015-01-01

    The cultivated peanut (Arachis hypogaea L.) is an allotetraploid (AABB) species derived from the A-genome (Arachis duranensis) and B-genome (Arachis ipaensis) progenitors. Presence of two versions of a DNA sequence based on the two progenitor genomes poses a serious technical and analytical problem during single nucleotide polymorphism (SNP) marker identification and analysis. In this context, we have analyzed 200 amplicons derived from expressed sequence tags (ESTs) and genome survey sequences (GSS) to identify SNPs in a panel of genotypes consisting of 12 cultivated peanut varieties and two diploid progenitors representing the ancestral genomes. A total of 18 EST-SNPs and 44 genomic-SNPs were identified in 12 peanut varieties by aligning the sequence of A. hypogaea with diploid progenitors. The average frequency of sequence polymorphism was higher for genomic-SNPs than the EST-SNPs with one genomic-SNP every 1011 bp as compared to one EST-SNP every 2557 bp. In order to estimate the potential and further applicability of these identified SNPs, 96 peanut varieties were genotyped using high resolution melting (HRM) method. Polymorphism information content (PIC) values for EST-SNPs ranged between 0.021 and 0.413 with a mean of 0.172 in the set of peanut varieties, while genomic-SNPs ranged between 0.080 and 0.478 with a mean of 0.249. Total 33 SNPs were used for polymorphism detection among the parents and 10 selected lines from mapping population Y13Zh (Zhenzhuhei × Yueyou13). Of the total 33 SNPs, nine SNPs showed polymorphism in the mapping population Y13Zh, and seven SNPs were successfully mapped into five linkage groups. Our results showed that SNPs can be identified in allotetraploid peanut with high accuracy through amplicon sequencing and HRM assay. The identified SNPs were very informative and can be used for different genetic and breeding applications in peanut. PMID:26697032

  14. Differences in the methylation patterns of the VaSTS1 and VaSTS10 genes of Vitis amurensis Rupr.

    PubMed

    Tyunin, A P; Kiselev, K V; Karetin, Y A

    2013-09-01

    Resveratrol is a plant-derived phenol but the mechanism that regulates its biosynthesis remains unidentified. Stilbene synthase (STS) catalyzes resveratrol formation in vivo and we have proposed that inducers of resveratrol production affect STS expression through an unidentified epigenetic mechanism. To investigate the role of DNA methylation in resveratrol biosynthesis, we treated both rolB transgenic and empty vector control Vitis amurensis cell cultures with the DNA demethylation agent, 5-azacytidine. Treated cells had increased resveratrol production through activation of VaSTS10 expression. The lowest levels of cytosine methylation were at the 5'- and 3'-ends of the VaSTS1 protein-coding sequence. Cytosine methylation decreased mostly at the 5'- and 3'-ends of VaSTS10 after azaC treatment with an intriguing regularity in the number of cytosine nucleotides within the 5'- and 3'- ends of the protein-coding sequences. Thus, cytosine methylation is crucial for the regulation of the resveratrol biosynthetic pathway. PMID:23690043

  15. Home Health Care and Patterns of Subsequent VA and Medicare Health Care Utilization for Veterans

    ERIC Educational Resources Information Center

    Van Houtven, Courtney Harold; Jeffreys, Amy S.; Coffman, Cynthia J.

    2008-01-01

    Purpose: The Veterans Affairs or VA health care system is in the process of significantly expanding home health care (HOC) nationwide. We describe VA HHC use in 2003 for all VA HHC users from 2002; we examine whether VA utilization across a broad spectrum of services differed for a sample of VA HHC users and their propensity-score-matched…

  16. 38 CFR 1.203 - Information to be reported to VA Police.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... reported to VA Police. 1.203 Section 1.203 Pensions, Bonuses, and Veterans' Relief DEPARTMENT OF VETERANS... be reported to VA Police. Information about actual or possible violations of criminal laws related to... occurs on VA premises, will be reported by VA management officials to the VA police component...

  17. 38 CFR 1.203 - Information to be reported to VA Police.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... reported to VA Police. 1.203 Section 1.203 Pensions, Bonuses, and Veterans' Relief DEPARTMENT OF VETERANS... be reported to VA Police. Information about actual or possible violations of criminal laws related to... occurs on VA premises, will be reported by VA management officials to the VA police component...

  18. 38 CFR 1.203 - Information to be reported to VA Police.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... reported to VA Police. 1.203 Section 1.203 Pensions, Bonuses, and Veterans' Relief DEPARTMENT OF VETERANS... be reported to VA Police. Information about actual or possible violations of criminal laws related to... occurs on VA premises, will be reported by VA management officials to the VA police component...

  19. 38 CFR 1.203 - Information to be reported to VA Police.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... reported to VA Police. 1.203 Section 1.203 Pensions, Bonuses, and Veterans' Relief DEPARTMENT OF VETERANS... be reported to VA Police. Information about actual or possible violations of criminal laws related to... occurs on VA premises, will be reported by VA management officials to the VA police component...

  20. 38 CFR 1.203 - Information to be reported to VA Police.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... reported to VA Police. 1.203 Section 1.203 Pensions, Bonuses, and Veterans' Relief DEPARTMENT OF VETERANS... be reported to VA Police. Information about actual or possible violations of criminal laws related to... occurs on VA premises, will be reported by VA management officials to the VA police component...

  1. DOE Spent Nuclear Fuel Information In Support of TSPA-VA

    SciTech Connect

    A. Brewer; D. Cresap; D. Fillmore; H. Loo; M. Ebner; R. McCormack

    1998-09-01

    RW has started the viability assessment (VA) effort to determine the feasibility of Yucca Mountain as the first geologic repository for spent nuclear fuel (SNF) and high-level waste. One component of the viability assessment will be a total system performance assessment (TSPA), based on the design concept and the scientific data and analysis available, describing the repository's probable behavior relative to the overall system performance standards. Thus, all the data collected from the Exploratory Studies Facility to-date have been incorporated into the latest TSPA model. In addition, the Repository Integration Program, an integrated probabilistic simulator, used in the TSPA has also been updated by Golder Associates Incorporated at December 1997. To ensure that the Department of Energy-owned (DOE-owned) SNF continues to be acceptable for disposal in the repository, it will be included in the TSPA-VA evaluation. A number of parameters are needed in the TSPA-VA models to predict the performance of the DOE-owned SNF materials placed into the potential repository. This report documents all of the basis and/or derivation for each of these parameters. A number of properties were not readily available at the time the TSPA-VA data was requested. Thus, expert judgement and opinion was utilized to determine a best property value. The performance of the DOE-owned SNF will be published as part of the TSPA-VA report. Each DOE site will be collecting better data as the DOE SNF program moves closer to repository license application. As required by the RW-0333P, the National Spent Nuclear Fuel Program will be assisting each site in qualifying the information used to support the performance assessment evaluations.

  2. Membrane-dependent Interaction of Factor Xa and Prothrombin with Factor Va in the Prothrombinase Complex

    PubMed Central

    Qureshi, Shabir H.; Yang, Likui; Manithody, Chandrashekhara; Rezaie, Alireza R.

    2009-01-01

    Because all three protein components of prothrombinase, factors (f) Xa and Va and prothrombin, bind to negatively charged membrane phospholipids, the exact role of the membrane in the prothrombinase reaction has not been fully understood. In this study, we prepared deletion derivatives of fXa and prothrombin in which both the Gla and first EGF-like domains of the protease (E2-fXa) as well as the Gla and both kringle domains of the substrate (prethrombin-2) were deleted. The fVa-mediated catalytic activity of E2-fXa toward prethrombin-2 was analyzed in both the absence and presence of phospholipids composed of 80% phosphatidylcholine (PC) and 20% phosphatidylserine (PS). PCPS markedly accelerated the initial rate of prethrombin-2 activation by E2-fXa, with the cofactor exhibiting saturation only in the presence of phospholipids (apparent Kd ∼60 nM). Competitive kinetic studies in the presence of the two exosite-1-specific ligands Tyr63-sulfated hirudin54-65 and TM456 suggested that while both peptides are highly effective inhibitors of the fVa-mediated activation of prethrombin-2 by E2-fXa in the absence of PCPS, they are ineffective competitors in the presence of phospholipids. Since neither E2-fXa nor prethrombin-2 can interact with membranes, these results suggest that fVa interaction with PCPS improves the affinity of the activation complex for the proexosite-1 of the substrate. Direct binding studies employing OG488-EGR-labeled fXa and E2-fXa revealed that the interaction of the Gla-domain of fXa with PCPS also induces conformational changes in the protease to facilitate its high-affinity interaction with fVa. PMID:19378973

  3. 76 FR 52230 - Establishment of Class E Airspace; Forest, VA

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-08-22

    ... Airspace at Forest, VA, to accommodate the new Area Navigation (RNAV) Global Positioning System (GPS..., VA (76 FR 34196) Docket No. FAA-2011-0378. Interested parties were invited to participate in this... Procedures (44 FR 11034; February 26, 1979); and (3) does not warrant preparation of a Regulatory...

  4. FACILITIES FOR EDUCATION IN VA HOSPITALS. FINAL REPORT.

    ERIC Educational Resources Information Center

    GREEN, ALAN C.; AND OTHERS

    THIS STUDY WAS AUTHORIZED BY THE VA DEPARTMENT OF MEDICINE AND SURGERY FOR THE PURPOSE OF IDENTIFYING AND DETERMINING THE FACILITIES NEEDED TO PROPERLY HOUSE AND SUPPORT EDUCATION ACTIVITIES IN EXISTING AND FUTURE VA HOSPITALS AND TO PRODUCE ARCHITECTURAL GUIDANCE IN THE DESIGN OF THE FACILITIES. CURRENT PRACTICES AND SIGNIFICANT TRENDS IN MEDICAL…

  5. 12 CFR 3.205 - VaR-based measure.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... portfolio of correlation trading positions that is modeled under § 3.209. A national bank or Federal savings... proxies are used. (b) Quantitative requirements for VaR-based measure. (1) The VaR-based measure must be... association's trading portfolio over a full business cycle. A national bank or Federal savings...

  6. 12 CFR 324.205 - VaR-based measure.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... of correlation trading positions that is modeled under § 324.209. An FDIC-supervised institution may.... (b) Quantitative requirements for VaR-based measure. (1) The VaR-based measure must be calculated on... of at least six months representing the volatility of the FDIC-supervised institution's...

  7. 38 CFR 17.71 - Revocation of VA approval.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... revocation of VA approval, VA health care personnel shall: (1) Cease referring veterans to the community residential care facility; and, (2) Notify any veteran residing in the community residential care facility of... health care personnel shall notify that person or entity of the community residential care...

  8. 38 CFR 17.71 - Revocation of VA approval.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... revocation of VA approval, VA health care personnel shall: (1) Cease referring veterans to the community residential care facility; and, (2) Notify any veteran residing in the community residential care facility of... health care personnel shall notify that person or entity of the community residential care...

  9. 38 CFR 17.71 - Revocation of VA approval.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... revocation of VA approval, VA health care personnel shall: (1) Cease referring veterans to the community residential care facility; and, (2) Notify any veteran residing in the community residential care facility of... health care personnel shall notify that person or entity of the community residential care...

  10. 38 CFR 17.71 - Revocation of VA approval.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... revocation of VA approval, VA health care personnel shall: (1) Cease referring veterans to the community residential care facility; and, (2) Notify any veteran residing in the community residential care facility of... health care personnel shall notify that person or entity of the community residential care...

  11. 76 FR 60713 - Establishment of Class E Airspace; Bumpass, VA

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-09-30

    ... Airspace at Bumpass, VA, to accommodate the new Standard Instrument Approach Procedures serving Lake Anna... establish Class E airspace at Bumpass, VA (76 FR 45479) Docket No. FAA-2011-0377. Interested parties were... for Lake Anna Airport. This action is necessary for the safety and management of IFR operations at...

  12. 78 FR 76061 - Authorization for Non-VA Medical Services

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-12-16

    ..., Health professions, Health records, Homeless, Mental health programs, Nursing homes, Reporting and.... SUPPLEMENTARY INFORMATION: On November 28, 2012, VA proposed a rule in the Federal Register, at 77 FR 70967, to... for that disability. On the same date, VA published a companion direct final rule at 77 FR 70893...

  13. 77 FR 25592 - Drawbridge Operation Regulations; James River, Hopewell, VA

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-05-01

    ... SECURITY Coast Guard 33 CFR Part 117 Drawbridge Operation Regulations; James River, Hopewell, VA AGENCY... Memorial Bridge, at mile 65.0, across the James River, at Hopewell, VA. This deviation is necessary to... schedule, the SR 156/Benjamin Harrison Memorial Bridge, at mile 65.0, across the James River, at...

  14. 33 CFR 80.510 - Chesapeake Bay Entrance, VA.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 33 Navigation and Navigable Waters 1 2010-07-01 2010-07-01 false Chesapeake Bay Entrance, VA. 80.510 Section 80.510 Navigation and Navigable Waters COAST GUARD, DEPARTMENT OF HOMELAND SECURITY INTERNATIONAL NAVIGATION RULES COLREGS DEMARCATION LINES Fifth District § 80.510 Chesapeake Bay Entrance, VA....

  15. 76 FR 34576 - Amendment of Class E Airspace; Waynesboro, VA

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-06-14

    ...) to amend Class E airspace at Eagle's Nest Airport, Waynesboro, VA (75 FR 14820) Docket No. FAA-2010..., 40113, 40120; E.O. 10854, 24 FR 9565, 3 CFR, 1959-1963 Comp., p. 389. Sec. 71.1 0 2. The incorporation... Federal Aviation Administration 14 CFR Part 71 Amendment of Class E Airspace; Waynesboro, VA...

  16. 78 FR 63143 - VA Dental Insurance Program-Federalism

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-10-23

    ... AFFAIRS 38 CFR Part 17 RIN 2900-AO86 VA Dental Insurance Program--Federalism AGENCY: Department of... its regulations related to the VA Dental Insurance Program (VADIP), a pilot program to offer premium-based dental insurance to enrolled veterans and certain survivors and dependents of...

  17. Single-step conversion of cells to retrovirus vector producers with herpes simplex virus-Epstein-Barr virus hybrid amplicons.

    PubMed

    Sena-Esteves, M; Saeki, Y; Camp, S M; Chiocca, E A; Breakefield, X O

    1999-12-01

    We report here on the development and characterization of a novel herpes simplex virus type 1 (HSV-1) amplicon-based vector system which takes advantage of the host range and retention properties of HSV-Epstein-Barr virus (EBV) hybrid amplicons to efficiently convert cells to retrovirus vector producer cells after single-step transduction. The retrovirus genes gag-pol and env (GPE) and retroviral vector sequences were modified to minimize sequence overlap and cloned into an HSV-EBV hybrid amplicon. Retrovirus expression cassettes were used to generate the HSV-EBV-retrovirus hybrid vectors, HERE and HERA, which code for the ecotropic and the amphotropic envelopes, respectively. Retrovirus vector sequences encoding lacZ were cloned downstream from the GPE expression unit. Transfection of 293T/17 cells with amplicon plasmids yielded retrovirus titers between 10(6) and 10(7) transducing units/ml, while infection of the same cells with amplicon vectors generated maximum titers 1 order of magnitude lower. Retrovirus titers were dependent on the extent of transduction by amplicon vectors for the same cell line, but different cell lines displayed varying capacities to produce retrovirus vectors even at the same transduction efficiencies. Infection of human and dog primary gliomas with this system resulted in the production of retrovirus vectors for more than 1 week and the long-term retention and increase in transgene activity over time in these cell populations. Although the efficiency of this system still has to be determined in vivo, many applications are foreseeable for this approach to gene delivery. PMID:10559361

  18. The Bias Associated with Amplicon Sequencing Does Not Affect the Quantitative Assessment of Bacterial Community Dynamics

    PubMed Central

    Figuerola, Eva L. M.; Erijman, Leonardo

    2014-01-01

    The performance of two sets of primers targeting variable regions of the 16S rRNA gene V1–V3 and V4 was compared in their ability to describe changes of bacterial diversity and temporal turnover in full-scale activated sludge. Duplicate sets of high-throughput amplicon sequencing data of the two 16S rRNA regions shared a collection of core taxa that were observed across a series of twelve monthly samples, although the relative abundance of each taxon was substantially different between regions. A case in point was the changes in the relative abundance of filamentous bacteria Thiothrix, which caused a large effect on diversity indices, but only in the V1–V3 data set. Yet the relative abundance of Thiothrix in the amplicon sequencing data from both regions correlated with the estimation of its abundance determined using fluorescence in situ hybridization. In nonmetric multidimensional analysis samples were distributed along the first ordination axis according to the sequenced region rather than according to sample identities. The dynamics of microbial communities indicated that V1–V3 and the V4 regions of the 16S rRNA gene yielded comparable patterns of: 1) the changes occurring within the communities along fixed time intervals, 2) the slow turnover of activated sludge communities and 3) the rate of species replacement calculated from the taxa–time relationships. The temperature was the only operational variable that showed significant correlation with the composition of bacterial communities over time for the sets of data obtained with both pairs of primers. In conclusion, we show that despite the bias introduced by amplicon sequencing, the variable regions V1–V3 and V4 can be confidently used for the quantitative assessment of bacterial community dynamics, and provide a proper qualitative account of general taxa in the community, especially when the data are obtained over a convenient time window rather than at a single time point. PMID:24923665

  19. 76 FR 79067 - Payment or Reimbursement for Emergency Treatment Furnished by Non-VA Providers in Non-VA...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-12-21

    ...- connected conditions. As explained in a notice of proposed rulemaking published on June 11, 2010 (75 FR... with Non- VA Outpatient Care''. 75 FR 7218 (Feb. 18, 2010). Second, section 402(b)(3) made the... proposed rule published on June 11, 2010 (75 FR 33216), we proposed to amend the following VA...

  20. The depiction of Alboran Sea Gyre during Donde Va? using remote sensing and conventional data

    NASA Technical Reports Server (NTRS)

    Laviolette, P. E.

    1984-01-01

    Experienced oceanographic investigators have come to realize that remote sensing techniques are most successful when applied as part of programs of integrated measurements aimed at solving specific oceanographic problems. A good example of such integration occurred during the multi-platform international experiment, Donde Va? in the Alboran Sea during the period June through October, 1982. The objective of Donde Va? was to derive the interrelationship of the Atlantic waters entering the Mediterranean Sea and the Alboran Sea Gyre. The experimental plan conceived solely with this objective in mind consisted of a variety of remote sensing and conventional platforms: three ships, three aircraft, five current moorings, two satellites and a specialized beach radar (CODAR). Integrated analyses of these multiple-data sets are still being conducted. However, the initial results show detailed structure of the incoming Atlantic jet and Alboran Sea Gyre that would not have been possible by conventional means.

  1. Pitfalls of haplotype phasing from amplicon-based long-read sequencing

    PubMed Central

    Laver, Thomas W.; Caswell, Richard C.; Moore, Karen A.; Poschmann, Jeremie; Johnson, Matthew B.; Owens, Martina M.; Ellard, Sian; Paszkiewicz, Konrad H.; Weedon, Michael N.

    2016-01-01

    The long-read sequencers from Pacific Bioscience (PacBio) and Oxford Nanopore Technologies (ONT) offer the opportunity to phase mutations multiple kilobases apart directly from sequencing reads. In this study, we used long-range PCR with ONT and PacBio sequencing to phase two variants 9 kb apart in the RET gene. We also re-analysed data from a recent paper which had apparently successfully used ONT to phase clinically important haplotypes at the CYP2D6 and HLA loci. From these analyses, we demonstrate PCR-chimera formation during PCR amplification and reference alignment bias are pitfalls that need to be considered when attempting to phase variants using amplicon-based long-read sequencing technologies. These methodological pitfalls need to be avoided if the opportunities provided by long-read sequencers are to be fully exploited. PMID:26883533

  2. Amplicon restriction patterns associated with nitrogenase activity of root nodules for selection of superior Myrica seedlings.

    PubMed

    Yanthan, Mhathung; Misra, Arvind K

    2013-11-01

    Trees of Myrica sp. grow abundantly in the forests of Meghalaya, India. These trees are actinorhizal and harbour nitrogen-fixing Frankia in their root nodules and contribute positively towards the enhancement of nitrogen status of forest areas. They can be used in rejuvenation of mine spoils and nitrogen-depleted fallow lands generated due to slash and burn agriculture practiced in the area. We have studied the association of amplicon restriction patterns (ARPs) of Myrica ribosomal RNA gene and internal transcribed spacer (ITS) region and nitrogenase activity of its root nodules. We found that ARPs thus obtained could be used as markers for early screening of seedlings that could support strains of Frankia that fix atmospheric nitrogen more efficiently. PMID:24287658

  3. 16S rRNA amplicon sequencing dataset for conventionalized and conventionally raised zebrafish larvae.

    PubMed

    Davis, Daniel J; Bryda, Elizabeth C; Gillespie, Catherine H; Ericsson, Aaron C

    2016-09-01

    Data presented here contains metagenomic analysis regarding the sequential conventionalization of germ-free zebrafish embryos. Zebrafish embryos that underwent a germ-free sterilization process immediately after fertilization were promptly exposed to and raised to larval stage in conventional fish water. At 6 days postfertilization (dpf), these "conventionalized" larvae were compared to zebrafish larvae that were raised in conventional fish water never undergoing the initial sterilization process. Bacterial 16S rRNA amplicon sequencing was performed on DNA isolated from homogenates of the larvae revealing distinct microbiota variations between the two groups. The dataset described here is also related to the research article entitled "Microbial modulation of behavior and stress responses in zebrafish larvae" (Davis et al., 2016) [1]. PMID:27508247

  4. Integrating metagenomic and amplicon databases to resolve the phylogenetic and ecological diversity of the Chlamydiae.

    PubMed

    Lagkouvardos, Ilias; Weinmaier, Thomas; Lauro, Federico M; Cavicchioli, Ricardo; Rattei, Thomas; Horn, Matthias

    2014-01-01

    In the era of metagenomics and amplicon sequencing, comprehensive analyses of available sequence data remain a challenge. Here we describe an approach exploiting metagenomic and amplicon data sets from public databases to elucidate phylogenetic diversity of defined microbial taxa. We investigated the phylum Chlamydiae whose known members are obligate intracellular bacteria that represent important pathogens of humans and animals, as well as symbionts of protists. Despite their medical relevance, our knowledge about chlamydial diversity is still scarce. Most of the nine known families are represented by only a few isolates, while previous clone library-based surveys suggested the existence of yet uncharacterized members of this phylum. Here we identified more than 22,000 high quality, non-redundant chlamydial 16S rRNA gene sequences in diverse databases, as well as 1900 putative chlamydial protein-encoding genes. Even when applying the most conservative approach, clustering of chlamydial 16S rRNA gene sequences into operational taxonomic units revealed an unexpectedly high species, genus and family-level diversity within the Chlamydiae, including 181 putative families. These in silico findings were verified experimentally in one Antarctic sample, which contained a high diversity of novel Chlamydiae. In our analysis, the Rhabdochlamydiaceae, whose known members infect arthropods, represents the most diverse and species-rich chlamydial family, followed by the protist-associated Parachlamydiaceae, and a putative new family (PCF8) with unknown host specificity. Available information on the origin of metagenomic samples indicated that marine environments contain the majority of the newly discovered chlamydial lineages, highlighting this environment as an important chlamydial reservoir. PMID:23949660

  5. Long amplicon (LA)-qPCR for the discrimination of infectious and noninfectious phix174 bacteriophages after UV inactivation.

    PubMed

    Ho, Johannes; Seidel, Michael; Niessner, Reinhard; Eggers, Jutta; Tiehm, Andreas

    2016-10-15

    Waterborne viruses are increasingly being considered in risk assessment schemes. In general, virus detection by culture methods is time consuming. In contrast, detection by quantitative polymerase chain reaction (qPCR) is more rapid and therefore, more suitable for monitoring. At present, qPCR lacks the essential ability for discriminating between infectious and non-infectious viruses, thus limiting its applicability for monitoring disinfection processes. In this study, a method was developed to quantify UV inactivation by long amplicon (LA)-qPCR. Bacteriophage phiX174 was used as a surrogate for human pathogenic viruses. A qPCR protocol was developed with new sets of primers, resulting in amplicon lengths of 108, 250, 456, 568, 955, 1063, 1544, and 1764 nucleotides. The log reduction of gene copies increased with increasing amplicon length. Additional treatment with the intercalating dye, PMA, had no effect, indicating that the bacteriophage capsids were not damaged by low pressure UV irradiation. A qPCR of nearly the complete genome (approx. 5000 nucleotides) showed similar results to the plaque assay. The log reduction in qPCR correlates with [specific amplicon length x UV dose]. The normalized DNA effect constant can be applied to calculate phiX174 inactivation based on qPCR detection. PMID:27450352

  6. 76 FR 72838 - Amendment of Class E Airspace; Luray, VA

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-11-28

    ... Luray, VA, to accommodate the new Area Navigation (RNAV) Global Positioning System (GPS) Standard... accommodate the new Area Navigation (RNAV) Global Positioning System (GPS) Standard Instrument Approach... FR 52292). Interested parties were invited to participate in this rulemaking effort by...

  7. Policy Recommendations to VA Leave NAS at Odds with Congress

    ERIC Educational Resources Information Center

    Walsh, John

    1978-01-01

    Describes adverse congressional reaction to a recent National Academy of Sciences (NAS) recommendation that the Veteran's Administration's (VA) health care system be phased into a general health care system. (SL)

  8. Exploration Day at Busch Gardens, Williamsburg, Va. - Aug. 5, 2011

    NASA Video Gallery

    Friday, August 8, was NASA Days at Busch Gardens Williamsburg, Va. NASA exhibits and educational specialists worked to inspire young and old, and NASA astronaut Susan Kilrain -- a veteran of two Sp...

  9. 78 FR 42455 - Medications Prescribed by Non-VA Providers

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-07-16

    ... Administrative practice and procedure, Alcohol abuse, Alcoholism, Claims, Day care, Dental health, Drug abuse... section to read as follows: Sec. 17.96 Medication prescribed by non-VA physicians. * * * * * (a) * * *...

  10. 76 FR 48204 - Fund Availability Under VA's Homeless Providers Grant and Per Diem Program

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-08-08

    ... availability of funds for currently operational fiscal year (FY) 2009 VA Grant and Per Diem Special Need Grant Recipients in conjunction with their collaborative VA Special Need partners and currently operational VA... under VA's Homeless Providers Grant and Per Diem Program for FY 2009 operational Grant and Per...

  11. 78 FR 38452 - Agency Information Collection (VA Police Officer Pre-Employment Screening Checklist) Activities...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-06-26

    ... AFFAIRS Agency Information Collection (VA Police Officer Pre-Employment Screening Checklist) Activities... ``OMB Control No. 2900-0524.'' SUPPLEMENTARY INFORMATION: Title: VA Police Officer Pre-Employment... checks on applicants seeking employment as VA police officers. VA will use the data collected...

  12. 78 FR 18425 - Proposed Information Collection VA Police Officer Pre-Employment Screening Checklist); Comment...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-03-26

    ... AFFAIRS Proposed Information Collection VA Police Officer Pre-Employment Screening Checklist); Comment... applicant's qualification and suitability as a VA police officer. DATES: Written comments and... information technology. Title: VA Police Officer Pre-Employment Screening Checklist, VA Form 0120. OMB...

  13. Polymicrobial Nature of Chronic Diabetic Foot Ulcer Biofilm Infections Determined Using Bacterial Tag Encoded FLX Amplicon Pyrosequencing (bTEFAP)

    PubMed Central

    Dowd, Scot E.; Wolcott, Randall D.; Sun, Yan; McKeehan, Trevor; Smith, Ethan; Rhoads, Daniel

    2008-01-01

    Background Diabetic extremity ulcers are associated with chronic infections. Such ulcer infections are too often followed by amputation because there is little or no understanding of the ecology of such infections or how to control or eliminate this type of chronic infection. A primary impediment to the healing of chronic wounds is biofilm phenotype infections. Diabetic foot ulcers are the most common, disabling, and costly complications of diabetes. Here we seek to derive a better understanding of the polymicrobial nature of chronic diabetic extremity ulcer infections. Methods and Findings Using a new bacterial tag encoded FLX amplicon pyrosequencing (bTEFAP) approach we have evaluated the bacterial diversity of 40 chronic diabetic foot ulcers from different patients. The most prevalent bacterial genus associated with diabetic chronic wounds was Corynebacterium spp. Findings also show that obligate anaerobes including Bacteroides, Peptoniphilus, Fingoldia, Anaerococcus, and Peptostreptococcus spp. are ubiquitous in diabetic ulcers, comprising a significant portion of the wound biofilm communities. Other major components of the bacterial communities included commonly cultured genera such as Streptococcus, Serratia, Staphylococcus and Enterococcus spp. Conclusions In this article, we highlight the patterns of population diversity observed in the samples and introduce preliminary evidence to support the concept of functional equivalent pathogroups (FEP). Here we introduce FEP as consortia of genotypically distinct bacteria that symbiotically produce a pathogenic community. According to this hypothesis, individual members of these communities when they occur alone may not cause disease but when they coaggregate or consort together into a FEP the synergistic effect provides the functional equivalence of well-known pathogens, such as Staphylococcus aureus, giving the biofilm community the factors necessary to maintain chronic biofilm infections. Further work is

  14. Shedding Light on the Microbial Community of the Macropod Foregut Using 454-Amplicon Pyrosequencing

    PubMed Central

    Gulino, Lisa-Maree; Ouwerkerk, Diane; Kang, Alicia Y. H.; Maguire, Anita J.; Kienzle, Marco; Klieve, Athol V.

    2013-01-01

    Twenty macropods from five locations in Queensland, Australia, grazing on a variety of native pastures were surveyed and the bacterial community of the foregut was examined using 454-amplicon pyrosequencing. Specifically, the V3/V4 region of 16S rRNA gene was examined. A total of 5040 OTUs were identified in the data set (post filtering). Thirty-two OTUs were identified as ‘shared’ OTUS (i.e. present in all samples) belonging to either Firmicutes or Bacteroidetes (Clostridiales/Bacteroidales). These phyla predominated the general microbial community in all macropods. Genera represented within the shared OTUs included: unclassified Ruminococcaceae, unclassified Lachnospiraceae, unclassified Clostridiales, Peptococcus sp. Coprococcus spp., Streptococcus spp., Blautia sp., Ruminoccocus sp., Eubacterium sp., Dorea sp., Oscillospira sp. and Butyrivibrio sp. The composition of the bacterial community of the foregut samples of each the host species (Macropus rufus, Macropus giganteus and Macropus robustus) was significantly different allowing differentiation between the host species based on alpha and beta diversity measures. Specifically, eleven dominant OTUs that separated the three host species were identified and classified as: unclassified Ruminococcaceae, unclassified Bacteroidales, Prevotella spp. and a Syntrophococcus sucromutans. Putative reductive acetogens and fibrolytic bacteria were also identified in samples. Future work will investigate the presence and role of fibrolytics and acetogens in these ecosystems. Ideally, the isolation and characterization of these organisms will be used for enhanced feed efficiency in cattle, methane mitigation and potentially for other industries such as the biofuel industry. PMID:23626688

  15. Rare amplicons implicate frequent deregulation of cell fate specification pathways in oral squamous cell carcinoma.

    PubMed

    Snijders, Antoine M; Schmidt, Brian L; Fridlyand, Jane; Dekker, Nusi; Pinkel, Daniel; Jordan, Richard C K; Albertson, Donna G

    2005-06-16

    Genomes of solid tumors are characterized by gains and losses of regions, which may contribute to tumorigenesis by altering gene expression. Often the aberrations are extensive, encompassing whole chromosome arms, which makes identification of candidate genes in these regions difficult. Here, we focused on narrow regions of gene amplification to facilitate identification of genetic pathways important in oral squamous cell carcinoma (SCC) development. We used array comparative genomic hybridization (array CGH) to define minimum common amplified regions and then used expression analysis to identify candidate driver genes in amplicons that spanned <3 Mb. We found genes involved in integrin signaling (TLN1), survival (YAP1, BIRC2), and adhesion and migration (TLN1, LAMA3, MMP7), as well as members of the hedgehog (GLI2) and notch (JAG1, RBPSUH, FJX1) pathways to be amplified and overexpressed. Deregulation of these and other members of the hedgehog and notch pathways (HHIP, SMO, DLL1, NOTCH4) implicates deregulation of developmental and differentiation pathways, cell fate misspecification, in oral SCC development. PMID:15824737

  16. Evaluation of human gene variant detection in amplicon pools by the GS-FLX parallel Pyrosequencer

    PubMed Central

    Bordoni, Roberta; Bonnal, Raoul; Rizzi, Ermanno; Carrera, Paola; Benedetti, Sara; Cremonesi, Laura; Stenirri, Stefania; Colombo, Alessio; Montrasio, Cristina; Bonalumi, Sara; Albertini, Alberto; Bernardi, Luigi Rossi; Ferrari, Maurizio; De Bellis, Gianluca

    2008-01-01

    Background A new priority in genome research is large-scale resequencing of genes to understand the molecular basis of hereditary disease and cancer. We assessed the ability of massively parallel pyrosequencing to identify sequence variants in pools. From a large collection of human PCR samples we selected 343 PCR products belonging to 16 disease genes and including a large spectrum of sequence variations previously identified by Sanger sequencing. The sequence variants included SNPs and small deletions and insertions (up to 44 bp), in homozygous or heterozygous state. Results The DNA was combined in 4 pools containing from 27 to 164 amplicons and from 8,9 to 50,8 Kb to sequence for a total of 110 Kb. Pyrosequencing generated over 80 million base pairs of data. Blind searching for sequence variations with a specifically designed bioinformatics procedure identified 465 putative sequence variants, including 412 true variants, 53 false positives (in or adjacent to homopolymeric tracts), no false negatives. All known variants in positions covered with at least 30× depth were correctly recognized. Conclusion Massively parallel pyrosequencing may be used to simplify and speed the search for DNA variations in PCR products. Our results encourage further studies to evaluate molecular diagnostics applications. PMID:18842124

  17. Flow cytometry community fingerprinting and amplicon sequencing for the assessment of landfill leachate cellulolytic bioaugmentation.

    PubMed

    Kinet, R; Dzaomuho, P; Baert, J; Taminiau, B; Daube, G; Nezer, C; Brostaux, Y; Nguyen, F; Dumont, G; Thonart, P; Delvigne, F

    2016-08-01

    Flow cytometry (FCM) is a high throughput single cell technology that is actually becoming widely used for studying phenotypic and genotypic diversity among microbial communities. This technology is considered in this work for the assessment of a bioaugmentation treatment in order to enhance cellulolytic potential of landfill leachate. The experimental results reveal the relevant increase of leachate cellulolytic potential due to bioaugmentation. Cytometric monitoring of microbial dynamics along these assays is then realized. The flow FP package is used to establish microbial samples fingerprint from initial 2D cytometry histograms. This procedure allows highlighting microbial communities' variation along the assays. Cytometric and 16S rRNA gene sequencing fingerprinting methods are then compared. The two approaches give same evidence about microbial dynamics throughout digestion assay. There are however a lack of significant correlation between cytometric and amplicon sequencing fingerprint at genus or species level. Same phenotypical profiles of microbiota during assays matched to several 16S rRNA gene sequencing ones. Flow cytometry fingerprinting can thus be considered as a promising routine on-site method suitable for the detection of stability/variation/disturbance of complex microbial communities involved in bioprocesses. PMID:27160955

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

    NASA Astrophysics Data System (ADS)

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

    2010-03-01

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

  19. A Multiplexed Amplicon Approach for Detecting Gene Fusions by Next-Generation Sequencing.

    PubMed

    Beadling, Carol; Wald, Abigail I; Warrick, Andrea; Neff, Tanaya L; Zhong, Shan; Nikiforov, Yuri E; Corless, Christopher L; Nikiforova, Marina N

    2016-03-01

    Chromosomal rearrangements that result in oncogenic gene fusions are clinically important drivers of many cancer types. Rapid and sensitive methods are therefore needed to detect a broad range of gene fusions in clinical specimens that are often of limited quantity and quality. We describe a next-generation sequencing approach that uses a multiplex PCR-based amplicon panel to interrogate fusion transcripts that involve 19 driver genes and 94 partners implicated in solid tumors. The panel also includes control assays that evaluate the 3'/5' expression ratios of 12 oncogenic kinases, which might be used to infer gene fusion events when the partner is unknown or not included on the panel. There was good concordance between the solid tumor fusion gene panel and other methods, including fluorescence in situ hybridization, real-time PCR, Sanger sequencing, and other next-generation sequencing panels, because 40 specimens known to harbor gene fusions were correctly identified. No specific fusion reads were observed in 59 fusion-negative specimens. The 3'/5' expression ratio was informative for fusions that involved ALK, RET, and NTRK1 but not for BRAF or ROS1 fusions. However, among 37 ALK or RET fusion-negative specimens, four exhibited elevated 3'/5' expression ratios, indicating that fusions predicted solely by 3'/5' read ratios require confirmatory testing. PMID:26747586

  20. Extracellular DNA amplicon sequencing reveals high levels of benthic eukaryotic diversity in the central Red Sea.

    PubMed

    Pearman, John K; Irigoien, Xabier; Carvalho, Susana

    2016-04-01

    The present study aims to characterize the benthic eukaryotic biodiversity patterns at a coarse taxonomic level in three areas of the central Red Sea (a lagoon, an offshore area in Thuwal and a shallow coastal area near Jeddah) based on extracellular DNA. High-throughput amplicon sequencing targeting the V9 region of the 18S rRNA gene was undertaken for 32 sediment samples. High levels of alpha-diversity were detected with 16,089 operational taxonomic units (OTUs) being identified. The majority of the OTUs were assigned to Metazoa (29.2%), Alveolata (22.4%) and Stramenopiles (17.8%). Stramenopiles (Diatomea) and Alveolata (Ciliophora) were frequent in a lagoon and in shallower coastal stations, whereas metazoans (Arthropoda: Maxillopoda) were dominant in deeper offshore stations. Only 24.6% of total OTUs were shared among all areas. Beta-diversity was generally lower between the lagoon and Jeddah (nearshore) than between either of those and the offshore area, suggesting a nearshore-offshore biodiversity gradient. The current approach allowed for a broad-range of benthic eukaryotic biodiversity to be analysed with significantly less labour than would be required by other traditional taxonomic approaches. Our findings suggest that next generation sequencing techniques have the potential to provide a fast and standardised screening of benthic biodiversity at large spatial and temporal scales. PMID:26525270

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

    PubMed Central

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

    2015-01-01

    Integrity of the mRNA in clinical samples has major impact on the quality of measured expression levels. This is independent of the measurement technique being next generation sequencing (NGS), Quantitative real-time PCR (qPCR) or microarray profiling. If mRNA is highly degraded or damaged, measured data will be very unreliable and the whole study is likely a waste of time and money. It is therefore common strategy to test the quality of RNA in samples before conducting large and costly studies. Most methods today to assess the quality of RNA are ignorant to the nature of the RNA and, therefore, reflect the integrity of ribosomal RNA, which is the dominant species, rather than of mRNAs, microRNAs and long non-coding RNAs, which usually are the species of interest. Here, we present a novel molecular approach to assess the quality of the targeted RNA species by measuring the differential amplification (ΔAmp) of an Endogenous RNase Resistant (ERR) marker relative to a reference gene, optionally combined with the measurement of two amplicons of different lengths. The combination reveals any mRNA degradation caused by ribonucleases as well as physical, chemical or UV damage. ΔAmp has superior sensitivity to common microfluidic electrophoretic methods, senses the integrity of the actual targeted RNA species, and allows for a smoother and more cost efficient workflow. PMID:27077042

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

    PubMed

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

    2011-01-01

    Next-gen sequencing technologies have revolutionized data collection in genetic studies and advanced genome biology to novel frontiers. However, to date, next-gen technologies have been used principally for whole genome sequencing and transcriptome sequencing. Yet many questions in population genetics and systematics rely on sequencing specific genes of known function or diversity levels. Here, we describe a targeted amplicon sequencing (TAS) approach capitalizing on next-gen capacity to sequence large numbers of targeted gene regions from a large number of samples. Our TAS approach is easily scalable, simple in execution, neither time-nor labor-intensive, relatively inexpensive, and can be applied to a broad diversity of organisms and/or genes. Our TAS approach includes a bioinformatic application, BarcodeCrucher, to take raw next-gen sequence reads and perform quality control checks and convert the data into FASTA format organized by gene and sample, ready for phylogenetic analyses. We demonstrate our approach by sequencing targeted genes of known phylogenetic utility to estimate a phylogeny for the Pancrustacea. We generated data from 44 taxa using 68 different 10-bp multiplexing identifiers. The overall quality of data produced was robust and was informative for phylogeny estimation. The potential for this method to produce copious amounts of data from a single 454 plate (e.g., 325 taxa for 24 loci) significantly reduces sequencing expenses incurred from traditional Sanger sequencing. We further discuss the advantages and disadvantages of this method, while offering suggestions to enhance the approach. PMID:22002916

  3. Amplicon pyrosequencing reveals the soil microbial diversity associated with invasive Japanese barberry (Berberis thunbergii DC.).

    PubMed

    Coats, V C; Pelletreau, K N; Rumpho, M E

    2014-03-01

    The soil microbial community acts as a reservoir of microbes that directly influences the structure and composition of the aboveground plant community, promotes plant growth, increases stress tolerance and mediates local patterns of nutrient cycling. Direct interactions between plants and rhizosphere-dwelling microorganisms occur at, or near, the surface of the root. Upon introduction and establishment, invasive plants modify the soil microbial communities and soil biochemistry affecting bioremediation efforts and future plant communities. Here, we used tag-encoded FLX amplicon 454 pyrosequencing (TEFAP) to characterize the bacterial and fungal community diversity in the rhizosphere of Berberis thunbergii DC. (Japanese barberry) from invasive stands in coastal Maine to investigate effects of soil type, soil chemistry and surrounding plant cover on the soil microbial community structure. Acidobacteria, Actinobacteria, Proteobacteria and Verrucomicrobia were the dominant bacterial phyla, whereas fungal communities were comprised mostly of Ascomycota and Basidiomycota phyla members, including Agaricomycetes and Sordariomycetes. Bulk soil chemistry had more effect on the bacterial community structure than the fungal community. An effect of geographic location was apparent in the rhizosphere microbial communities, yet it was less significant than the effect of surrounding plant cover. These data demonstrate a high degree of spatial variation in the rhizosphere microbial communities of Japanese barberry with apparent effects of soil chemistry, location and canopy cover on the microbial community structure. PMID:24118303

  4. Digital fragment analysis of short tandem repeats by high-throughput amplicon sequencing.

    PubMed

    Darby, Brian J; Erickson, Shay F; Hervey, Samuel D; Ellis-Felege, Susan N

    2016-07-01

    High-throughput sequencing has been proposed as a method to genotype microsatellites and overcome the four main technical drawbacks of capillary electrophoresis: amplification artifacts, imprecise sizing, length homoplasy, and limited multiplex capability. The objective of this project was to test a high-throughput amplicon sequencing approach to fragment analysis of short tandem repeats and characterize its advantages and disadvantages against traditional capillary electrophoresis. We amplified and sequenced 12 muskrat microsatellite loci from 180 muskrat specimens and analyzed the sequencing data for precision of allele calling, propensity for amplification or sequencing artifacts, and for evidence of length homoplasy. Of the 294 total alleles, we detected by sequencing, only 164 alleles would have been detected by capillary electrophoresis as the remaining 130 alleles (44%) would have been hidden by length homoplasy. The ability to detect a greater number of unique alleles resulted in the ability to resolve greater population genetic structure. The primary advantages of fragment analysis by sequencing are the ability to precisely size fragments, resolve length homoplasy, multiplex many individuals and many loci into a single high-throughput run, and compare data across projects and across laboratories (present and future) with minimal technical calibration. A significant disadvantage of fragment analysis by sequencing is that the method is only practical and cost-effective when performed on batches of several hundred samples with multiple loci. Future work is needed to optimize throughput while minimizing costs and to update existing microsatellite allele calling and analysis programs to accommodate sequence-aware microsatellite data. PMID:27386092

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

    PubMed Central

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

    2015-01-01

    Previously we presented Swarm v1, a novel and open source amplicon clustering program that produced fine-scale molecular operational taxonomic units (OTUs), free of arbitrary global clustering thresholds and input-order dependency. Swarm v1 worked with an initial phase that used iterative single-linkage with a local clustering threshold (d), followed by a phase that used the internal abundance structures of clusters to break chained OTUs. Here we present Swarm v2, which has two important novel features: (1) a new algorithm for d = 1 that allows the computation time of the program to scale linearly with increasing amounts of data; and (2) the new fastidious option that reduces under-grouping by grafting low abundant OTUs (e.g., singletons and doubletons) onto larger ones. Swarm v2 also directly integrates the clustering and breaking phases, dereplicates sequencing reads with d = 0, outputs OTU representatives in fasta format, and plots individual OTUs as two-dimensional networks. PMID:26713226

  6. Drug and drug-related supply promotion by pharmaceutical company representatives at VA facilities. Final rule.

    PubMed

    2012-03-01

    This final rule amends the Department of Veterans Affairs (VA) regulations regarding access to VA facilities by pharmaceutical company representatives. The purposes of the rule are to reduce or eliminate any potential for disruption in the patient care environment, manage activities and promotions at VA facilities, and provide pharmaceutical company representatives with a consistent standard of permissible business practice at VA facilities. The amendments will facilitate mutually beneficial relationships between VA and pharmaceutical company representatives. PMID:22420057

  7. Groundtruthing Next-Gen Sequencing for Microbial Ecology–Biases and Errors in Community Structure Estimates from PCR Amplicon Pyrosequencing

    PubMed Central

    Polson, Shawn W.; Wommack, K. Eric; Williamson, Shannon J.; McDonald, Ian R.; Cary, S. Craig

    2012-01-01

    Analysis of microbial communities by high-throughput pyrosequencing of SSU rRNA gene PCR amplicons has transformed microbial ecology research and led to the observation that many communities contain a diverse assortment of rare taxa–a phenomenon termed the Rare Biosphere. Multiple studies have investigated the effect of pyrosequencing read quality on operational taxonomic unit (OTU) richness for contrived communities, yet there is limited information on the fidelity of community structure estimates obtained through this approach. Given that PCR biases are widely recognized, and further unknown biases may arise from the sequencing process itself, a priori assumptions about the neutrality of the data generation process are at best unvalidated. Furthermore, post-sequencing quality control algorithms have not been explicitly evaluated for the accuracy of recovered representative sequences and its impact on downstream analyses, reducing useful discussion on pyrosequencing reads to their diversity and abundances. Here we report on community structures and sequences recovered for in vitro-simulated communities consisting of twenty 16S rRNA gene clones tiered at known proportions. PCR amplicon libraries of the V3–V4 and V6 hypervariable regions from the in vitro-simulated communities were sequenced using the Roche 454 GS FLX Titanium platform. Commonly used quality control protocols resulted in the formation of OTUs with >1% abundance composed entirely of erroneous sequences, while over-aggressive clustering approaches obfuscated real, expected OTUs. The pyrosequencing process itself did not appear to impose significant biases on overall community structure estimates, although the detection limit for rare taxa may be affected by PCR amplicon size and quality control approach employed. Meanwhile, PCR biases associated with the initial amplicon generation may impose greater distortions in the observed community structure. PMID:22970184

  8. Ambient temperature detection of PCR amplicons with a novel sequence-specific nucleic acid lateral flow biosensor.

    PubMed

    Ang, Geik Yong; Yu, Choo Yee; Yean, Chan Yean

    2012-01-01

    In the field of diagnostics, molecular amplification targeting unique genetic signature sequences has been widely used for rapid identification of infectious agents, which significantly aids physicians in determining the choice of treatment as well as providing important epidemiological data for surveillance and disease control assessment. We report the development of a rapid nucleic acid lateral flow biosensor (NALFB) in a dry-reagent strip format for the sequence-specific detection of single-stranded polymerase chain reaction (PCR) amplicons at ambient temperature (22-25°C). The NALFB was developed in combination with a linear-after-the-exponential PCR assay and the applicability of this biosensor was demonstrated through detection of the cholera toxin gene from diarrheal-causing toxigenic Vibrio cholerae. Amplification using the advanced asymmetric PCR boosts the production of fluorescein-labeled single-stranded amplicons, allowing capture probes immobilized on the NALFB to hybridize specifically with complementary targets in situ on the strip. Subsequent visual formation of red lines is achieved through the binding of conjugated gold nanoparticles to the fluorescein label of the captured amplicons. The visual detection limit observed with synthetic target DNA was 0.3 ng and 1 pg with pure genomic DNA. Evaluation of the NALFB with 164 strains of V. cholerae and non-V. cholerae bacteria recorded 100% for both sensitivity and specificity. The whole procedure of the low-cost NALFB, which is performed at ambient temperature, eliminates the need for preheated buffers or additional equipment, greatly simplifying the protocol for sequence-specific PCR amplicon analysis. PMID:22705404

  9. VA opsin-based photoreceptors in the hypothalamus of birds.

    PubMed

    Halford, Stephanie; Pires, Susana S; Turton, Michael; Zheng, Lei; González-Menéndez, Irene; Davies, Wayne L; Peirson, Stuart N; García-Fernández, José M; Hankins, Mark W; Foster, Russell G

    2009-08-25

    Studies in the 1930s demonstrated that birds possess photoreceptors that are located within the hypothalamus and regulate photoperiodic responses to day length. Most recently, photoperiod has been shown to alter the activity of the pars tuberalis to release thyrotrophin, which ultimately drives a reproductive response. Despite these significant findings, the cellular and molecular identity of the hypothalamic photoreceptors has remained a mystery. Action spectra implicated an opsin-based photopigment system, but further identification based on rod- or cone-opsin probes failed, suggesting the utilization of a novel opsin. The vertebrate ancient (VA) opsin photopigments were isolated in 1997 but were thought to have a restricted taxonomic distribution, confined to the agnatha and teleost fish. Here, we report the isolation of VA opsin from chicken and show that the two isoforms spliced from this gene (cVAL and cVA) are capable of forming functional photopigments. Further, we show that VA opsin is expressed within a population of hypothalamic neurons with extensive projections to the median eminence. These results provide the most complete cellular and molecular description of a deep brain photoreceptor in any vertebrate and strongly implicate VA opsin in mediating the avian photoperiodic response. PMID:19664923

  10. Analysis of the mouse gut microbiome using full-length 16S rRNA amplicon sequencing

    PubMed Central

    Shin, Jongoh; Lee, Sooin; Go, Min-Jeong; Lee, Sang Yup; Kim, Sun Chang; Lee, Chul-Ho; Cho, Byung-Kwan

    2016-01-01

    Demands for faster and more accurate methods to analyze microbial communities from natural and clinical samples have been increasing in the medical and healthcare industry. Recent advances in next-generation sequencing technologies have facilitated the elucidation of the microbial community composition with higher accuracy and greater throughput than was previously achievable; however, the short sequencing reads often limit the microbial composition analysis at the species level due to the high similarity of 16S rRNA amplicon sequences. To overcome this limitation, we used the nanopore sequencing platform to sequence full-length 16S rRNA amplicon libraries prepared from the mouse gut microbiota. A comparison of the nanopore and short-read sequencing data showed that there were no significant differences in major taxonomic units (89%) except one phylotype and three taxonomic units. Moreover, both sequencing data were highly similar at all taxonomic resolutions except the species level. At the species level, nanopore sequencing allowed identification of more species than short-read sequencing, facilitating the accurate classification of the bacterial community composition. Therefore, this method of full-length 16S rRNA amplicon sequencing will be useful for rapid, accurate and efficient detection of microbial diversity in various biological and clinical samples. PMID:27411898

  11. Analysis of the mouse gut microbiome using full-length 16S rRNA amplicon sequencing.

    PubMed

    Shin, Jongoh; Lee, Sooin; Go, Min-Jeong; Lee, Sang Yup; Kim, Sun Chang; Lee, Chul-Ho; Cho, Byung-Kwan

    2016-01-01

    Demands for faster and more accurate methods to analyze microbial communities from natural and clinical samples have been increasing in the medical and healthcare industry. Recent advances in next-generation sequencing technologies have facilitated the elucidation of the microbial community composition with higher accuracy and greater throughput than was previously achievable; however, the short sequencing reads often limit the microbial composition analysis at the species level due to the high similarity of 16S rRNA amplicon sequences. To overcome this limitation, we used the nanopore sequencing platform to sequence full-length 16S rRNA amplicon libraries prepared from the mouse gut microbiota. A comparison of the nanopore and short-read sequencing data showed that there were no significant differences in major taxonomic units (89%) except one phylotype and three taxonomic units. Moreover, both sequencing data were highly similar at all taxonomic resolutions except the species level. At the species level, nanopore sequencing allowed identification of more species than short-read sequencing, facilitating the accurate classification of the bacterial community composition. Therefore, this method of full-length 16S rRNA amplicon sequencing will be useful for rapid, accurate and efficient detection of microbial diversity in various biological and clinical samples. PMID:27411898

  12. Strengths and Limitations of 16S rRNA Gene Amplicon Sequencing in Revealing Temporal Microbial Community Dynamics

    PubMed Central

    Poretsky, Rachel; Rodriguez-R, Luis M.; Luo, Chengwei; Tsementzi, Despina; Konstantinidis, Konstantinos T.

    2014-01-01

    This study explored the short-term planktonic microbial community structure and resilience in Lake Lanier (GA, USA) while simultaneously evaluating the technical aspects of identifying taxa via 16S rRNA gene amplicon and metagenomic sequence data. 16S rRNA gene amplicons generated from four temporally discrete samples were sequenced with 454 GS-FLX-Ti yielding ∼40,000 rRNA gene sequences from each sample and representing ∼300 observed OTUs. Replicates obtained from the same biological sample clustered together but several biases were observed, linked to either the PCR or sequencing-preparation steps. In comparisons with companion whole-community shotgun metagenome datasets, the estimated number of OTUs at each timepoint was concordant, but 1.5 times and ∼10 times as many phyla and genera, respectively, were identified in the metagenomes. Our analyses showed that the 16S rRNA gene captures broad shifts in community diversity over time, but with limited resolution and lower sensitivity compared to metagenomic data. We also identified OTUs that showed marked shifts in abundance over four close timepoints separated by perturbations and tracked these taxa in the metagenome vs. 16S rRNA amplicon data. A strong summer storm had less of an effect on community composition than did seasonal mixing, which revealed a distinct succession of organisms. This study provides insights into freshwater microbial communities and advances the approaches for assessing community diversity and dynamics in situ. PMID:24714158

  13. The Impact of Private Insurance Coverage on Veterans' Use of VA Care: Insurance and Selection Effects

    PubMed Central

    Shen, Yujing; Hendricks, Ann; Wang, Fenghua; Gardner, John; Kazis, Lewis E

    2008-01-01

    Objective To examine private insurance coverage and its impact on use of Veterans Health Administration (VA) care among VA enrollees without Medicare coverage. Data Sources The 1999 National Health Survey of Veteran Enrollees merged with VA administrative data, with other information drawn from American Hospital Association data and the Area Resource File. Study Design We modeled VA enrollees' decision of having private insurance coverage and its impact on use of VA care controlling for sociodemographic information, patients' health status, VA priority status and access to VA and non-VA alternatives. We estimated the true impact of insurance on the use of VA care by teasing out potential selection bias. Bias came from two sources: a security selection effect (sicker enrollees purchase private insurance for extra security and use more VA and non-VA care) and a preference selection effect (VA enrollees who prefer non-VA care may purchase private insurance and use less VA care). Principal Findings VA enrollees with private insurance coverage were less likely to use VA care. Security selection dominated preference selection and naïve models that did not control for selection effects consistently underestimated the insurance effect. Conclusions Our results indicate that prior research, which has not controlled for insurance selection effects, may have underestimated the potential impact of any private insurance policy change, which may in turn affect VA enrollees' private insurance coverage and consequently their use of VA care. From the decline in private insurance coverage from 1999 to 2002, we projected an increase of 29,400 patients and 158 million dollars for VA health care services. PMID:18211529

  14. The cost of hospital readmissions: evidence from the VA.

    PubMed

    Carey, Kathleen; Stefos, Theodore

    2016-09-01

    This paper is an examination of hospital 30-day readmission costs using data from 119 acute care hospitals operated by the U.S. Veterans Administration (VA) in fiscal year 2011. We applied a two-part model that linked readmission probability to readmission cost to obtain patient level estimates of expected readmission cost for VA patients overall, and for patients discharged for three prevalent conditions with relatively high readmission rates. Our focus was on the variable component of direct patient cost. Overall, managers could expect to save $2140 for the average 30-day readmission avoided. For heart attack, heart failure, and pneumonia patients, expected readmission cost estimates were $3432, $2488 and $2278. Patient risk of illness was the dominant driver of readmission cost in all cases. The VA experience has implications for private sector hospitals that treat a high proportion of chronically ill and/or low income patients, or that are contemplating adopting bundled payment mechanisms. PMID:25576391

  15. Analysis of a Drosophila amplicon in follicle cells highlights the diversity of metazoan replication origins.

    PubMed

    Kim, Jane C; Orr-Weaver, Terry L

    2011-10-01

    To investigate the properties of metazoan replication origins, recent studies in cell culture have adopted the strategy of identifying origins using genome-wide approaches and assessing correlations with such features as transcription and histone modifications. Drosophila amplicon in follicle cells (DAFCs), genomic regions that undergo repeated rounds of DNA replication to increase DNA copy number, serve as powerful in vivo model replicons. Because there are six DAFCs, compared with thousands of origins activated in the typical S phase, close molecular characterization of all DAFCs is possible. To determine the extent to which the six DAFCs are different or similar, we investigated the developmental and replication properties of the newly identified DAFC-34B. DAFC-34B contains two genes expressed in follicle cells, although the timing and spatial patterns of expression suggest that amplification is not a strategy to promote high expression at this locus. Like the previously characterized DAFC-62D, DAFC-34B displays origin activation at two separate stages of development. However, unlike DAFC-62D, amplification at the later stage is not transcription-dependent. We mapped the DAFC-34B amplification origin to 1 kb by nascent strand analysis and delineated cis requirements for origin activation, finding that a 6-kb region, but not the 1-kb origin alone, is sufficient for amplification. We analyzed the developmental localization of the origin recognition complex (ORC) and the minichromosome maintenance (MCM)2-7 complex, the replicative helicase. Intriguingly, the final round of origin activation at DAFC-34B occurs in the absence of detectable ORC, although MCMs are present, suggesting a new amplification initiation mechanism. PMID:21933960

  16. Amplicon-based semiconductor sequencing of human exomes: performance evaluation and optimization strategies.

    PubMed

    Damiati, E; Borsani, G; Giacopuzzi, Edoardo

    2016-05-01

    The Ion Proton platform allows to perform whole exome sequencing (WES) at low cost, providing rapid turnaround time and great flexibility. Products for WES on Ion Proton system include the AmpliSeq Exome kit and the recently introduced HiQ sequencing chemistry. Here, we used gold standard variants from GIAB consortium to assess the performances in variants identification, characterize the erroneous calls and develop a filtering strategy to reduce false positives. The AmpliSeq Exome kit captures a large fraction of bases (>94 %) in human CDS, ClinVar genes and ACMG genes, but with 2,041 (7 %), 449 (13 %) and 11 (19 %) genes not fully represented, respectively. Overall, 515 protein coding genes contain hard-to-sequence regions, including 90 genes from ClinVar. Performance in variants detection was maximum at mean coverage >120×, while at 90× and 70× we measured a loss of variants of 3.2 and 4.5 %, respectively. WES using HiQ chemistry showed ~71/97.5 % sensitivity, ~37/2 % FDR and ~0.66/0.98 F1 score for indels and SNPs, respectively. The proposed low, medium or high-stringency filters reduced the amount of false positives by 10.2, 21.2 and 40.4 % for indels and 21.2, 41.9 and 68.2 % for SNP, respectively. Amplicon-based WES on Ion Proton platform using HiQ chemistry emerged as a competitive approach, with improved accuracy in variants identification. False-positive variants remain an issue for the Ion Torrent technology, but our filtering strategy can be applied to reduce erroneous variants. PMID:27003585

  17. Prerequisites for amplicon pyrosequencing of microbial methanol utilizers in the environment

    PubMed Central

    Kolb, Steffen; Stacheter, Astrid

    2013-01-01

    The commercial availability of next generation sequencing (NGS) technologies facilitated the assessment of functional groups of microorganisms in the environment with high coverage, resolution, and reproducibility. Soil methylotrophs were among the first microorganisms in the environment that were assessed with molecular tools, and nowadays, as well with NGS technologies. Studies in the past years re-attracted notice to the pivotal role of methylotrophs in global conversions of methanol, which mainly originates from plants, and is involved in oxidative reactions and ozone formation in the atmosphere. Aerobic methanol utilizers belong to Bacteria, yeasts, Ascomycota, and molds. Numerous bacterial methylotrophs are facultatively aerobic, and also contribute to anaerobic methanol oxidation in the environment, whereas strict anaerobic methanol utilizers belong to methanogens and acetogens. The diversity of enzymes catalyzing the initial oxidation of methanol is considerable, and comprises at least five different enzyme types in aerobes, and one in strict anaerobes. Only the gene of the large subunit of pyrroloquinoline quinone (PQQ)-dependent methanol dehydrogenase (MDH; mxaF) has been analyzed by environmental pyrosequencing. To enable a comprehensive assessment of methanol utilizers in the environment, new primers targeting genes of the PQQ MDH in Methylibium (mdh2), of the nicotinamide adenine dinucleotide-dependent MDH (mdh), of the methanol oxidoreductase of Actinobacteria (mdo), of the fungal flavin adenine nucleotide-dependent alcohol oxidase (mod1, mod2, and homologs), and of the gene of the large subunit of the methanol:corrinoid methyltransferases (mtaC) in methanogens and acetogens need to be developed. Combined stable isotope probing of nucleic acids or proteins with amplicon-based NGS are straightforward approaches to reveal insights into functions of certain methylotrophic taxa in the global methanol cycle. PMID:24046766

  18. Voltammetric detection of sequence-selective DNA hybridization related to Toxoplasma gondii in PCR amplicons.

    PubMed

    Gokce, Gultekin; Erdem, Arzum; Ceylan, Cagdas; Akgöz, Muslum

    2016-03-01

    This work describes the single-use electrochemical DNA biosensor technology developed for voltammetric detection of sequence selective DNA hybridization related to important human and veterinary pathogen; Toxoplasma gondii. In the principle of electrochemical label-free detection assay, the duplex of DNA hybrid formation was detected by measuring guanine oxidation signal occured in the presence of DNA hybridization. The biosensor design consisted of the immobilization of an inosine-modified (guanine-free) probe onto the surface of pencil graphite electrode (PGE), and the detection of the duplex formation in connection with the differential pulse voltammetry(DPV) by measuring the guanine signal. Toxoplasma gondii capture probe was firstly immobilized onto the surface of the activated PGE by wet adsorption. The extent of hybridization at PGE surface between the probe and the target was then determined by measuring the guanine signal observed at +1.0V. The electrochemical monitoring of optimum DNA hybridization has been performed in the target concentration of 40µg/mL in 50min of hybridization time. The specificity of the electrochemical biosensor was then tested using non-complementary, or mismatch short DNA sequences. Under the optimum conditions, the guanine oxidation signal indicating full hybridization was measured in various target concentration from 0.5 to 25µg/mL and a detection limit was found to be 1.78µg/mL. This single-use biosensor platform was successfully applied for the voltammetric detection of DNA hybridization related to Toxoplasma gondii in PCR amplicons. PMID:26717837

  19. Herpes Simplex Virus Type 1/Adeno-Associated Virus rep+ Hybrid Amplicon Vector Improves the Stability of Transgene Expression in Human Cells by Site-Specific Integration

    PubMed Central

    Wang, Y.; Camp, S. M.; Niwano, M.; Shen, X.; Bakowska, J. C.; Breakefield, X. O.; Allen, P. D.

    2002-01-01

    Herpes simplex virus type 1 (HSV-1) amplicon vectors are promising gene delivery tools, but their utility in gene therapy has been impeded to some extent by their inability to achieve stable transgene expression. In this study, we examined the possibility of improving transduction stability in cultured human cells via site-specific genomic integration mediated by adeno-associated virus (AAV) Rep and inverted terminal repeats (ITRs). A rep− HSV/AAV hybrid amplicon vector was made by inserting a transgene cassette flanked with AAV ITRs into an HSV-1 amplicon backbone, and a rep+ HSV/AAV hybrid amplicon was made by inserting rep68/78 outside the rep− vector 3′ AAV ITR sequence. Both vectors also had a pair of loxP sites flanking the ITRs. The resulting hybrid amplicon vectors were successfully packaged and compared to a standard amplicon vector for stable transduction frequency (STF) in human 293 and Gli36 cell lines and primary myoblasts. The rep+, but not the rep−, hybrid vector improved STF in all three types of cells; 84% of Gli36 and 40% of 293 stable clones transduced by the rep+ hybrid vector integrated the transgene into the AAVS1 site. Due to the difficulty in expanding primary myoblasts, we did not assess site-specific integration in these cells. A strategy to attempt further improvement of STF by “deconcatenating” the hybrid amplicon DNA via Cre-loxP recombination was tested, but it did not increase STF. These data demonstrate that introducing the integrating elements of AAV into HSV-1 amplicon vectors can significantly improve their ability to achieve stable gene transduction by conferring the AAV-like capability of site-specific genomic integration in dividing cells. PMID:12072515

  20. Gene-specific amplicons from metagenomes as an alternative to directed evolution for enzyme screening: a case study using phenylacetaldehyde reductases.

    PubMed

    Itoh, Nobuya; Kazama, Miki; Takeuchi, Nami; Isotani, Kentaro; Kurokawa, Junji

    2016-06-01

    Screening gene-specific amplicons from metagenomes (S-GAM) is a highly promising technique for the isolation of genes encoding enzymes for biochemical and industrial applications. From metagenomes, we isolated phenylacetaldehyde reductase (par) genes, which code for an enzyme that catalyzes the production of various Prelog's chiral alcohols. Nearly full-length par genes were amplified by PCR from metagenomic DNA, the products of which were fused with engineered par sequences at both terminal regions of the expression vector to ensure proper expression and then used to construct Escherichia coli plasmid libraries. Sequence- and activity-based screening of these libraries identified different homologous par genes, Hpar-001 to -036, which shared more than 97% amino acid sequence identity with PAR. Comparative characterization of these active homologs revealed a wide variety of enzymatic properties including activity, substrate specificity, and thermal stability. Moreover, amino acid substitutions in these genes coincided with those of Sar268 and Har1 genes, which were independently engineered by error-prone PCR to exhibit increased activity in the presence of concentrated 2-propanol. The comparative data from both approaches suggest that sequence information from homologs isolated from metagenomes is quite useful for enzyme engineering. Furthermore, by examining the GAM-based sequence dataset derived from soil metagenomes, we easily found amino acid substitutions that increase the thermal stability of PAR/PAR homologs. Thus, GAM-based approaches can provide not only useful homologous enzymes but also an alternative to directed evolution methodologies. PMID:27419059

  1. Analysis of β-Subunit-dependent GABAA Receptor Modulation and Behavioral Effects of Valerenic Acid Derivatives.

    PubMed

    Khom, S; Hintersteiner, J; Luger, D; Haider, M; Pototschnig, G; Mihovilovic, M D; Schwarzer, C; Hering, S

    2016-06-01

    Valerenic acid (VA)-a β2/3-selective GABA type A (GABAA) receptor modulator-displays anxiolytic and anticonvulsive effects in mice devoid of sedation, making VA an interesting drug candidate. Here we analyzed β-subunit-dependent enhancement of GABA-induced chloride currents (IGABA) by a library of VA derivatives and studied their effects on pentylenetetrazole (PTZ)-induced seizure threshold and locomotion. Compound-induced IGABA enhancement was determined in oocytes expressing α1β1γ2S, α1β2γ2S, or α1β3γ2S receptors. Effects on seizure threshold and locomotion were studied using C57BL/6N mice and compared with saline-treated controls. β2/3-selective VA derivatives such as VA-amide (VA-A) modulating α1β3γ2S (VA-A: Emax = 972 ± 69%, n = 6, P < 0.05) and α1β2γ2S receptors (Emax = 1119 ± 72%, n = 6, P < 0.05) more efficaciously than VA (α1β3γ2S: VA: Emax = 632 ± 88%, n = 9 versus α1β2γ2S: VA: Emax = 721 ± 68%, n = 6) displayed significantly more pronounced seizure threshold elevation than VA (saline control: 40.4 ± 1.4 mg/kg PTZ versus VA 10 mg/kg: 49.0 ± 1.8 mg/kg PTZ versus VA-A 3 mg/kg: 57.9 ± 1.9 mg/kg PTZ, P < 0.05). Similarly, VA's methylamide (VA-MA) enhancing IGABA through β3-containing receptors more efficaciously than VA (Emax = 1043 ± 57%, P < 0.01, n = 6) displayed stronger anticonvulsive effects. Increased potency of IGABA enhancement and anticonvulsive effects at lower doses compared with VA were observed for VA-tetrazole (α1β3γ2S: VA-TET: EC50 = 6.0 ± 1.0 μM, P < 0.05; VA-TET: 0.3 mg/kg: 47.3 ± 0.5 mg/kg PTZ versus VA: 10 mg/kg: 49.0 ± 1.8 mg/kg PTZ, P < 0.05). At higher doses (≥10 mg/kg), VA-A, VA-MA, and VA-TET reduced locomotion. In contrast, unselective VA derivatives induced anticonvulsive effects only at high doses (30 mg/kg) or did not display any behavioral effects. Our data indicate that the β2/3-selective compounds VA-A, VA-MA, and VA-TET induce anticonvulsive effects at low doses (≤10 mg/kg), whereas

  2. 76 FR 43575 - Amendment of Class E Airspace; Staunton, VA

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-07-21

    ... Valley Regional Airport, Staunton, VA (76 FR 14822) Docket No. FAA- 2010-1285. Interested parties were... Executive Order 12866; (2) is not a ``significant rule'' under DOT Regulatory Policies and Procedures (44 FR... FR 9565, 3 CFR, 1959-1963 Comp., p. 389. Sec. 71.1 0 2. The incorporation by reference in 14 CFR...

  3. 77 FR 70967 - Authorization for Non-VA Medical Services

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-11-28

    ... regulatory action under Executive Order 12866. Unfunded Mandates Reform Act The Unfunded Mandates Reform Act... specify veterans' eligibility for non-VA care. Administrative Procedure Act Concurrent with this proposed... documents will speed notice and comment rulemaking under section 553 of the Administrative Procedure...

  4. 75 FR 41247 - Virginia Disaster Number VA-00028

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-07-15

    ... ADMINISTRATION Virginia Disaster Number VA-00028 AGENCY: U.S. Small Business Administration. ACTION: Amendment 4... Only for the Commonwealth of Virginia (FEMA-1874-DR), dated 02/16/2010. Incident: Severe Winter Storm... disaster declaration for Private Non-Profit organizations in the Commonwealth of Virginia, dated...

  5. 76 FR 58557 - Virginia Disaster Number VA-00038

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-09-21

    ... ADMINISTRATION Virginia Disaster Number VA-00038 AGENCY: U.S. Small Business Administration. ACTION: Amendment 1... Only for the Commonwealth of Virginia (FEMA-4024-DR), dated 09/03/2011. Incident: Hurricane Irene... Private Non-Profit organizations in the Commonwealth of Virginia, dated 09/03/2011, is hereby amended...

  6. 77 FR 530 - Virginia Disaster Number VA-00037

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-01-05

    ... ADMINISTRATION Virginia Disaster Number VA-00037 AGENCY: U.S. Small Business Administration. ACTION: Amendment 1... Virginia (FEMA-4042-DR), dated 11/04/2011. Incident: Earthquake. Incident Period: 08/23/2011 through 10/25... major disaster declaration for the Commonwealth of Virginia, dated 11/04/2011 is hereby amended...

  7. 77 FR 7229 - Virginia Disaster Number VA-00037

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-02-10

    ... ADMINISTRATION Virginia Disaster Number VA-00037 AGENCY: U.S. Small Business Administration. ACTION: Amendment 3... Virginia (FEMA-4042-DR), dated 11/04/2011. Incident: Earthquake. Incident Period: 08/23/2011 through 10/25... disaster declaration for the Commonwealth of Virginia, dated 11/04/2011 is hereby amended to include...

  8. 76 FR 62132 - Virginia Disaster Number VA-00038

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-10-06

    ... ADMINISTRATION Virginia Disaster Number VA-00038 AGENCY: U.S. Small Business Administration. ACTION: Amendment 2... only for the State of Virginia (FEMA-4024-DR), dated 09/03/2011. Incident: Hurricane Irene. Incident... Non-Profit organizations in the State of Virginia, dated 09/03/2011, is hereby amended to include...

  9. 75 FR 41559 - Virginia Disaster Number VA-00029

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-07-16

    ... ADMINISTRATION Virginia Disaster Number VA-00029 AGENCY: U.S. Small Business Administration. ACTION: Amendment 1... Only for the Commonwealth of Virginia (FEMA-1905-DR), dated 04/27/2010. Incident: Severe winter storms... disaster declaration for Private Non-Profit organizations in the Commonwealth of Virginia, dated...

  10. 75 FR 26813 - VIRGINIA Disaster Number VA-00028

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-05-12

    ... ADMINISTRATION VIRGINIA Disaster Number VA-00028 AGENCY: U.S. Small Business Administration. ACTION: Amendment 2... Only for the Commonwealth of VIRGINIA (FEMA-1874-DR), dated 02/16/2010. Incident: Severe Winter Storm... disaster declaration for Private Non-Profit organizations in the Commonwealth of VIRGINIA, dated...

  11. 77 FR 51100 - Virginia Disaster No. VA-00048

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-08-23

    ... ADMINISTRATION Virginia Disaster No. VA-00048 AGENCY: U.S. Small Business Administration. ACTION: Amendment 1... Only for the State of VIRGINIA (FEMA-4072-DR), dated 07/27/2012. Incident: Severe Storms and Straight... Private Non-Profit organizations in the State of VIRGINIA, dated 07/27/2012, is hereby amended to...

  12. 76 FR 74837 - Virginia Disaster Number VA-00040

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-12-01

    ... ADMINISTRATION Virginia Disaster Number VA-00040 AGENCY: U.S. Small Business Administration. ACTION: Amendment 1... Only for the State of Virginia (FEMA-4042-DR), dated 11/10/2011. Incident: Earthquake. Incident Period... non-profit organizations in the State of Virginia, dated 11/10/2011, is hereby amended to include...

  13. 75 FR 2895 - Virginia Disaster Number VA-00027

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-01-19

    ... ADMINISTRATION Virginia Disaster Number VA-00027 AGENCY: U.S. Small Business Administration. ACTION: Amendment 1... Only for the Commonwealth of Virginia (FEMA-1862-DR), dated 12/09/2009. Incident: Severe Storms and... the Commonwealth of ] Virginia, dated 12/09/2009, is hereby amended to establish the incident...

  14. 77 FR 1547 - Virginia Disaster Number VA-00040

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-01-10

    ... ADMINISTRATION Virginia Disaster Number VA-00040 AGENCY: U.S. Small Business Administration. ACTION: Amendment 3... Only for the Commonwealth of Virginia (FEMA--4042-DR), dated 11/10/2011. Incident: Earthquake. Incident... Non-Profit organizations in the Commonwealth of Virginia, dated 11/10/2011, is hereby amended...

  15. 78 FR 2708 - Virginia Disaster Number VA-00052

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-01-14

    ... ADMINISTRATION Virginia Disaster Number VA-00052 AGENCY: U.S. Small Business Administration. ACTION: Amendment 1... Only for the State of Virginia (FEMA--4092--DR), dated 11/26/2012 . Incident: Hurricane Sandy Incident... Non-Profit organizations in the State of Virginia, dated 11/26/2012, is hereby amended to include...

  16. 75 FR 35511 - Virginia Disaster Number VA-00028

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-06-22

    ... ADMINISTRATION Virginia Disaster Number VA-00028 AGENCY: Small Business Administration. ACTION: Amendment 3... Only for the Commonwealth of Virginia (FEMA-1874-DR), dated 02/16/2010. Incident: Severe Winter Storm... disaster declaration for Private Non-Profit organizations in the Commonwealth of Virginia, dated...

  17. 77 FR 1547 - Virginia Disaster Number VA-00037

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-01-10

    ... ADMINISTRATION Virginia Disaster Number VA-00037 AGENCY: U.S. Small Business Administration. ACTION: Amendment 2... Virginia (FEMA-4042-DR), dated 11/04/ 2011. Incident: Earthquake. Incident Period: 08/23/2011 Through 10/25... disaster declaration for the State of Virginia, dated 11/04/2011 is hereby amended to include the...

  18. 75 FR 21370 - Virginia Disaster Number VA-00028

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-04-23

    ... ADMINISTRATION Virginia Disaster Number VA-00028 AGENCY: U.S. Small Business Administration. ACTION: Amendment 1... Only for the Commonwealth of Virginia (FEMA-1874-DR), dated 02/16/2010. Incident: Severe Winter Storm... disaster declaration for Private Non-Profit organizations in the Commonwealth of Virginia, dated...

  19. 76 FR 78957 - Virginia Disaster Number VA-00040

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-12-20

    ... ADMINISTRATION Virginia Disaster Number VA-00040 AGENCY: U.S. Small Business Administration. ACTION: Amendment 2... Only for the Commonwealth of Virginia (FEMA--4042--DR), dated 11/10/2011. Incident: Earthquake... Private Non-Profit organizations in the Commonwealth of Virginia, dated 11/10/2011, is hereby amended...

  20. Geropsychology Training in a VA Nursing Home Setting

    ERIC Educational Resources Information Center

    Karel, Michele J.; Moye, Jennifer

    2005-01-01

    There is a growing need for professional psychology training in nursing home settings, and nursing homes provide a rich environment for teaching geropsychology competencies. We describe the nursing home training component of our Department of Veterans Affairs (VA) Predoctoral Internship and Geropsychology Postdoctoral Fellowship programs. Our…

  1. Military and VA General Dentistry Training: A National Resource.

    ERIC Educational Resources Information Center

    Atchison, Kathryn A.; Bachand, William; Buchanan, C. Richard; Lefever, Karen H.; Lin, Sylvia; Engelhardt, Rita

    2002-01-01

    Compared the program characteristics of the postgraduate general dentistry (PGD) training programs sponsored by the military and the Veterans Health Administration (VA). Gathered information on program infrastructure and emphasis, resident preparation prior to entering the program, and patients served and types of services provided. Programs…

  2. VA Library Service--Today's look at Tomorrow's Library.

    ERIC Educational Resources Information Center

    Veterans Administration, Washington, DC.

    The Conference Poceedings are divided into three broad topics: systems planning, audiovisuals in biomedical communication, and automation and networking. Speakers from within the Veterans Administration (VA), from the National Medical Audiovisual Center, and the Lister Hill National Center for Biomedical Communications, National Library of…

  3. 77 FR 67063 - VA Directive 0005 on Scientific Integrity

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-11-08

    ... April 9, 2012 (77 FR 21158). FOR FURTHER INFORMATION CONTACT: Billy E. Jones, M.D., Senior Advisor to... was announced in the Federal Register on April 9, 2012 (77 FR 21158). All of the public comments have... AFFAIRS VA Directive 0005 on Scientific Integrity AGENCY: Office of Policy and Planning, Department...

  4. 76 FR 77270 - Board Meeting; January 9, 2012, Arlington, VA

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-12-12

    ... REVIEW BOARD Board Meeting; January 9, 2012, Arlington, VA The U.S. Nuclear Waste Technical Review Board... Waste Technical Review Board will hold a public meeting in Arlington, Virginia, on Monday, January 9... on technical issues related to nuclear waste management and to review the technical validity of...

  5. 78 FR 62441 - VA Dental Insurance Program-Federalism

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-10-22

    ... of veterans. Specifically, this rule will add language to clarify the limited preemptive effect of... CFR 17.169 to add language to clarify the limited preemptive effect of certain criteria in the VA... business of insurance. Although section 510 does not include express preemption language, Congress...

  6. 76 FR 30534 - Establishment of Class E Airspace; Kenbridge, VA

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-05-26

    ... proposed rulemaking to amend Class E airspace 700 feet above the surface, at Kenbridge, VA (75 FR 14823...: Authority: 49 U.S.C. 106(g); 40103, 40113, 40120; E.O. 10854, 24 FR 9565, 3 CFR, 1959-1963 Comp., p. 389... Policies and Procedures (44 FR 11034; February 26, 1979); and (3) does not warrant preparation of...

  7. 75 FR 79295 - Establishment of Class E Airspace; Crewe, VA

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-12-20

    ... Federal Register a notice of proposed rulemaking to establish Class E airspace at Crewe, VA (75 FR 57215...: Authority: 49 U.S.C. 106(g); 40103, 40113, 40120; E.O. 10854, 24 FR 9565, 3 CFR, 1959-1963 Comp., p. 389... ``significant rule'' under DOT Regulatory Policies and Procedures (44 FR 11034; February 26, 1979); and (3)...

  8. 32 CFR 105.10 - SARC and SAPR VA procedures.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... directly to the installation commander in accordance with 32 CFR part 103, to include providing regular... CIVILIAN SEXUAL ASSAULT PREVENTION AND RESPONSE PROGRAM PROCEDURES § 105.10 SARC and SAPR VA procedures. (a) SARC procedures. The SARC shall: (1) Serve as the single point of contact to coordinate sexual...

  9. Genotyping-in-Thousands by sequencing (GT-seq): A cost effective SNP genotyping method based on custom amplicon sequencing.

    PubMed

    Campbell, Nathan R; Harmon, Stephanie A; Narum, Shawn R

    2015-07-01

    Genotyping-in-Thousands by sequencing (GT-seq) is a method that uses next-generation sequencing of multiplexed PCR products to generate genotypes from relatively small panels (50-500) of targeted single-nucleotide polymorphisms (SNPs) for thousands of individuals in a single Illumina HiSeq lane. This method uses only unlabelled oligos and PCR master mix in two thermal cycling steps for amplification of targeted SNP loci. During this process, sequencing adapters and dual barcode sequence tags are incorporated into the amplicons enabling thousands of individuals to be pooled into a single sequencing library. Post sequencing, reads from individual samples are split into individual files using their unique combination of barcode sequences. Genotyping is performed with a simple perl script which counts amplicon-specific sequences for each allele, and allele ratios are used to determine the genotypes. We demonstrate this technique by genotyping 2068 individual steelhead trout (Oncorhynchus mykiss) samples with a set of 192 SNP markers in a single library sequenced in a single Illumina HiSeq lane. Genotype data were 99.9% concordant to previously collected TaqMan(™) genotypes at the same 192 loci, but call rates were slightly lower with GT-seq (96.4%) relative to Taqman (99.0%). Of the 192 SNPs, 187 were genotyped in ≥90% of the individual samples and only 3 SNPs were genotyped in <70% of samples. This study demonstrates amplicon sequencing with GT-seq greatly reduces the cost of genotyping hundreds of targeted SNPs relative to existing methods by utilizing a simple library preparation method and massive efficiency of scale. PMID:25476721

  10. 77 FR 2348 - Agency Information Collection (VA Enrollment Certification): Activity Under OMB Review

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-01-17

    ... or email denise.mclamb@va.gov . Please refer to ``OMB Control No. 2900-0073.'' SUPPLEMENTARY... AFFAIRS Agency Information Collection (VA Enrollment Certification): Activity Under OMB Review AGENCY..., 2012. ADDRESSES: Submit written comments on the collection of information through...

  11. 31. FELGATES CREEK BRIDGE (HAER No. VA48I), VIEW FROM WEST ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    31. FELGATES CREEK BRIDGE (HAER No. VA-48-I), VIEW FROM WEST END. NOTE ALUMINUM RAILING ON EAST SIDE FOR CANTILEVERED BIKE PATH. - Colonial Parkway, Yorktown to Jamestown Island, Yorktown, York County, VA

  12. The cytological manifestation of gene amplification in multidrug-resistant mouse leukemia P388 sublines is correlated with amplicon content

    SciTech Connect

    Il`inskaya, G.V.; Kopnin, B.P.; Demidova, N.S.

    1995-10-01

    Previously, we showed that development of multidrug resistance (MDR) in mouse P388 leukemia cells is often associated with the appearance of newly-formed chromosomelike structures that contain amplified copies of the mdr1 gene. In the present study, we compared amplicon content in P388 sublines showing different types of these structures. A strong correlation between the formation of specific acentric markers consisting of two identical arms and the absence of the sorcin gene coamplification was found. In all the sublines containing other types of chromosomelike structures, the sorcin gene is coamplified. 9 refs., 2 figs., 1 tab.

  13. Analysis of β-Subunit-dependent GABAA Receptor Modulation and Behavioral Effects of Valerenic Acid Derivatives

    PubMed Central

    Hintersteiner, J.; Luger, D.; Haider, M.; Pototschnig, G.; Mihovilovic, M. D.; Schwarzer, C.; Hering, S.

    2016-01-01

    Valerenic acid (VA)—a β2/3-selective GABA type A (GABAA) receptor modulator—displays anxiolytic and anticonvulsive effects in mice devoid of sedation, making VA an interesting drug candidate. Here we analyzed β-subunit-dependent enhancement of GABA-induced chloride currents (IGABA) by a library of VA derivatives and studied their effects on pentylenetetrazole (PTZ)-induced seizure threshold and locomotion. Compound-induced IGABA enhancement was determined in oocytes expressing α1β1γ2S, α1β2γ2S, or α1β3γ2S receptors. Effects on seizure threshold and locomotion were studied using C57BL/6N mice and compared with saline-treated controls. β2/3-selective VA derivatives such as VA-amide (VA-A) modulating α1β3γ2S (VA-A: Emax = 972 ± 69%, n = 6, P < 0.05) and α1β2γ2S receptors (Emax = 1119 ± 72%, n = 6, P < 0.05) more efficaciously than VA (α1β3γ2S: VA: Emax = 632 ± 88%, n = 9 versus α1β2γ2S: VA: Emax = 721 ± 68%, n = 6) displayed significantly more pronounced seizure threshold elevation than VA (saline control: 40.4 ± 1.4 mg/kg PTZ versus VA 10 mg/kg: 49.0 ± 1.8 mg/kg PTZ versus VA-A 3 mg/kg: 57.9 ± 1.9 mg/kg PTZ, P < 0.05). Similarly, VA’s methylamide (VA-MA) enhancing IGABA through β3-containing receptors more efficaciously than VA (Emax = 1043 ± 57%, P < 0.01, n = 6) displayed stronger anticonvulsive effects. Increased potency of IGABA enhancement and anticonvulsive effects at lower doses compared with VA were observed for VA-tetrazole (α1β3γ2S: VA-TET: EC50 = 6.0 ± 1.0 μM, P < 0.05; VA-TET: 0.3 mg/kg: 47.3 ± 0.5 mg/kg PTZ versus VA: 10 mg/kg: 49.0 ± 1.8 mg/kg PTZ, P < 0.05). At higher doses (≥10 mg/kg), VA-A, VA-MA, and VA-TET reduced locomotion. In contrast, unselective VA derivatives induced anticonvulsive effects only at high doses (30 mg/kg) or did not display any behavioral effects. Our data indicate that the β2/3-selective compounds VA-A, VA-MA, and VA-TET induce anticonvulsive effects at low doses (≤10 mg

  14. 48 CFR 852.219-71 - VA mentor-protégé program.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 48 Federal Acquisition Regulations System 5 2011-10-01 2011-10-01 false VA mentor-protégÃ....219-71 VA mentor-protégé program. As prescribed in 819.7115(a), insert the following clause: VA Mentor-Protégé Program (DEC 2009) (a) Large businesses are encouraged to participate in the VA...

  15. 48 CFR 852.219-71 - VA mentor-protégé program.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 48 Federal Acquisition Regulations System 5 2012-10-01 2012-10-01 false VA mentor-protégÃ....219-71 VA mentor-protégé program. As prescribed in 819.7115(a), insert the following clause: VA Mentor-Protégé Program (DEC 2009) (a) Large businesses are encouraged to participate in the VA...

  16. 48 CFR 852.219-71 - VA mentor-protégé program.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 48 Federal Acquisition Regulations System 5 2013-10-01 2013-10-01 false VA mentor-protégÃ....219-71 VA mentor-protégé program. As prescribed in 819.7115(a), insert the following clause: VA Mentor-Protégé Program (DEC 2009) (a) Large businesses are encouraged to participate in the VA...

  17. 48 CFR 852.219-71 - VA mentor-protégé program.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 5 2010-10-01 2010-10-01 false VA mentor-protégÃ....219-71 VA mentor-protégé program. As prescribed in 819.7115(a), insert the following clause: VA Mentor-Protégé Program (DEC 2009) (a) Large businesses are encouraged to participate in the VA...

  18. 48 CFR 852.219-71 - VA mentor-protégé program.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 48 Federal Acquisition Regulations System 5 2014-10-01 2014-10-01 false VA mentor-protégÃ....219-71 VA mentor-protégé program. As prescribed in 819.7115(a), insert the following clause: VA Mentor-Protégé Program (DEC 2009) (a) Large businesses are encouraged to participate in the VA...

  19. 12 CFR 3.206 - Stressed VaR-based measure.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 12 Banks and Banking 1 2014-01-01 2014-01-01 false Stressed VaR-based measure. 3.206 Section 3.206... Risk-Weighted Assets-Market Risk § 3.206 Stressed VaR-based measure. (a) General requirement. At least... calculate its VaR-based measure to calculate a stressed VaR-based measure. (b) Quantitative requirements...

  20. 38 CFR 21.1032 - VA has a duty to assist claimants in obtaining evidence.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 38 Pensions, Bonuses, and Veterans' Relief 2 2010-07-01 2010-07-01 false VA has a duty to assist claimants in obtaining evidence. 21.1032 Section 21.1032 Pensions, Bonuses, and Veterans' Relief DEPARTMENT... Educational Assistance Claims § 21.1032 VA has a duty to assist claimants in obtaining evidence. (a) VA's...

  1. 38 CFR 21.33 - VA has a duty to assist claimants in obtaining evidence.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 38 Pensions, Bonuses, and Veterans' Relief 2 2010-07-01 2010-07-01 false VA has a duty to assist... Employment Under 38 U.S.C. Chapter 31 Claims § 21.33 VA has a duty to assist claimants in obtaining evidence... claimant either orally or in writing when VA: (i) Has made reasonable efforts to obtain relevant...

  2. 75 FR 78808 - Agency Information Collection (VA Request for Determination of Reasonable Value) Activity Under...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-12-16

    ... AFFAIRS Agency Information Collection (VA Request for Determination of Reasonable Value) Activity Under.... SUMMARY: In compliance with the Paperwork Reduction Act (PRA) of 1995 (44 U.S.C. 3501-3521), this notice... INFORMATION: Title: VA Request for Determination of Reasonable Value VA Form 26- 1805 and 26-1805-1....

  3. 38 CFR 26.7 - VA environmental decision making and documents.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 38 Pensions, Bonuses, and Veterans' Relief 2 2014-07-01 2014-07-01 false VA environmental decision making and documents. 26.7 Section 26.7 Pensions, Bonuses, and Veterans' Relief DEPARTMENT OF VETERANS AFFAIRS (CONTINUED) ENVIRONMENTAL EFFECTS OF THE DEPARTMENT OF VETERANS AFFAIRS (VA) ACTIONS § 26.7 VA environmental decision making and...

  4. 48 CFR 853.236-70 - VA Form 10-6298, Architect-Engineer Fee Proposal.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 48 Federal Acquisition Regulations System 5 2011-10-01 2011-10-01 false VA Form 10-6298, Architect... VETERANS AFFAIRS CLAUSES AND FORMS FORMS Prescription of Forms 853.236-70 VA Form 10-6298, Architect-Engineer Fee Proposal. VA Form 10-6298, Architect-Engineer Fee Proposal, shall be used as prescribed in...

  5. 48 CFR 853.236-70 - VA Form 10-6298, Architect-Engineer Fee Proposal.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 5 2010-10-01 2010-10-01 false VA Form 10-6298, Architect... VETERANS AFFAIRS CLAUSES AND FORMS FORMS Prescription of Forms 853.236-70 VA Form 10-6298, Architect-Engineer Fee Proposal. VA Form 10-6298, Architect-Engineer Fee Proposal, shall be used as prescribed in...

  6. The Impact of VA's Geriatric Research, Education and Clinical Centers on Academic Affiliates

    ERIC Educational Resources Information Center

    Bragg, Elizabeth J.; Meganathan, Karthikeyan; Shay, Kenneth; Gilman, Stuart C.; Zeiss, Robert A.; Hettler, Debbie L.

    2011-01-01

    The education mission of the Department of Veterans Affairs (VA) is to train health professionals to benefit VA and the United States. One approach for achieving that mission, along with VA's research and clinical missions, was the establishment of Geriatric Research, Education and Clinical Centers (GRECCs) in 1975. These were developed at VA…

  7. 78 FR 48543 - Veterans Health Administration Fund Availability Under the VA's Homeless Providers Grant and Per...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-08-08

    ... Veterans Affairs (VA) is announcing the availability of 1-year renewal funding for currently operational... Special Need partners and currently operational VA GPD Special Need Grant Recipients which do not involve... under VA's Homeless Providers GPD Program for FY 2011 operational GPD Special Need grant recipients...

  8. 12 CFR 324.206 - Stressed VaR-based measure.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 12 Banks and Banking 5 2014-01-01 2014-01-01 false Stressed VaR-based measure. 324.206 Section 324... Stressed VaR-based measure. (a) General requirement. At least weekly, an FDIC-supervised institution must use the same internal model(s) used to calculate its VaR-based measure to calculate a stressed...

  9. 78 FR 6849 - Agency Information Collection (Verification of VA Benefits) Activity Under OMB Review

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-01-31

    ... AFFAIRS Agency Information Collection (Verification of VA Benefits) Activity Under OMB Review AGENCY... abstracted below to the Office of Management and Budget (OMB) for review and comment. The PRA submission... VA Benefits, VA Form 26-8937. OMB Control Number: 2900-0406. ] Type of Review: Extension of...

  10. 75 FR 17832 - Proposed Information Collection (VA Loan Electronic Reporting Interface (VALERI) System) Activity...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-04-07

    ... nancy.kessinger@va.gov . Please refer to ``OMB Control No. 2900-0021'' in any correspondence. During the... AFFAIRS Proposed Information Collection (VA Loan Electronic Reporting Interface (VALERI) System) Activity.... SUMMARY: The Veterans Benefits Administration (VBA), Department of Veterans Affairs (VA), is announcing...

  11. 77 FR 60746 - Proposed Information Collection (VA/DOD Joint Disability Evaluation Board Claim) Activity...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-10-04

    ... 20420 or email to ] nancy.kessinger@va.gov . Please refer to ``OMB Control No. 2900-0704'' in any... AFFAIRS Proposed Information Collection (VA/DOD Joint Disability Evaluation Board Claim) Activity: Comment...: The Veterans Benefits Administration (VBA), Department of Veterans Affairs (VA), is announcing...

  12. 77 FR 64383 - Proposed Information Collection (Verification of VA Benefits) Activity: Comment Request

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-10-19

    ... AFFAIRS Proposed Information Collection (Verification of VA Benefits) Activity: Comment Request AGENCY... Benefits Administration (VBA), Department of Veterans Affairs (VA), is announcing an opportunity for public... name prior to the closing of any VA-guaranteed loans on an automatic basis. DATES: Written comments...

  13. 75 FR 60171 - Proposed Information Collection (Credit Underwriting Standards and Procedures for Processing VA...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-09-29

    ...., Washington, DC 20420 or e-mail nancy.kessinger@va.gov . Please refer to ``OMB Control No. 2900-0521'' in any... AFFAIRS Proposed Information Collection (Credit Underwriting Standards and Procedures for Processing VA... Affairs (VA), is announcing an opportunity for public comment on the proposed collection of...

  14. 78 FR 79563 - Proposed Information Collection (VA MATIC Authorization); Comment Request

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-12-30

    ...), Department of Veterans Affairs, 810 Vermont Avenue NW., Washington, DC 20420 or email nancy.kessinger@va.gov... AFFAIRS Proposed Information Collection (VA MATIC Authorization); Comment Request AGENCY: Veterans... Administration (VBA), Department of Veterans Affairs (VA), is announcing an opportunity for public comment on...

  15. 78 FR 53506 - Agency Information Collection (HUD/VA Addendum to Uniform Residential Loan Application) Activity...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-08-29

    ....rennie@va.gov . Please refer to ``OMB Control No. 2900-0144.'' SUPPLEMENTARY INFORMATION: Title: HUD/VA... AFFAIRS Agency Information Collection (HUD/VA Addendum to Uniform Residential Loan Application) Activity... information through www.Regulations.gov , or to Office of Information and Regulatory Affairs, Office...

  16. 76 FR 24570 - Proposed Information Collection (Application for VA Education Benefits) Activity; Comment Request

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-05-02

    ...., Washington, DC 20420 or email to nancy.kessinger@va.gov . Please refer to ``OMB Control No. 2900-0154'' in... AFFAIRS Proposed Information Collection (Application for VA Education Benefits) Activity; Comment Request... Veterans Benefits Administration (VBA), Department of Veterans Affairs (VA), is announcing an...

  17. 78 FR 60379 - Proposed Information Collection (Credit Underwriting Standards and Procedures for Processing VA...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-10-01

    ...., Washington, DC 20420 or email nancy.kessinger@va.gov . Please refer to ``OMB Control No. 2900-0521'' in any... AFFAIRS Proposed Information Collection (Credit Underwriting Standards and Procedures for Processing VA... Affairs (VA), is announcing an opportunity for public comment on the proposed collection of...

  18. 76 FR 67561 - Proposed Information Collection (VA Enrollment Certification) Activity: Comment Request

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-11-01

    ..., 810 Vermont Avenue NW., Washington, DC 20420 or email to nancy.kessinger@va.gov . Please refer to... AFFAIRS Proposed Information Collection (VA Enrollment Certification) Activity: Comment Request AGENCY... Benefits Administration (VBA), Department of Veterans Affairs (VA), is announcing an opportunity for...

  19. 75 FR 54965 - Proposed Information Collection (Statement of Purchaser or Owner Assuming Seller's Loans, VA Form...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-09-09

    ...., Washington, DC 20420 or e-mail nancy.kessinger@va.gov . Please refer to ``OMB Control No. 2900-0111'' in any... AFFAIRS Proposed Information Collection (Statement of Purchaser or Owner Assuming Seller's Loans, VA Form... Affairs (VA), is announcing an opportunity for public comment on the proposed collection of...

  20. 76 FR 27381 - Proposed Information Collection (Notice of Waiver of VA Compensation or Pension To Receive...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-05-11

    ...., Washington, DC 20420 or e-mail to nancy.kessinger@va.gov . Please refer to ``OMB Control No. 2900-0463'' in... AFFAIRS Proposed Information Collection (Notice of Waiver of VA Compensation or Pension To Receive... of Veterans Affairs (VA), is announcing an opportunity for public comment on the proposed...

  1. 78 FR 29436 - Proposed Information Collection (HUD/VA Addendum to Uniform Residential Loan Application...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-05-20

    ...), Department of Veterans Affairs, 810 Vermont Avenue NW., Washington, DC 20420 or email nancy.kessinger@va.gov... AFFAIRS Proposed Information Collection (HUD/VA Addendum to Uniform Residential Loan Application) Activity.... SUMMARY: The Veterans Benefits Administration (VBA), Department of Veterans Affairs (VA), is announcing...

  2. 77 FR 56711 - Proposed Information Collection (VA MATIC Enrollment/Change); Comment Request

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-09-13

    ... Affairs, 810 Vermont Avenue NW., Washington, DC 20420 or email nancy.kessinger@va.gov . Please refer to... AFFAIRS Proposed Information Collection (VA MATIC Enrollment/Change); Comment Request AGENCY: Veterans... Administration (VBA), Department of Veterans Affairs (VA), is announcing an opportunity for public comment on...

  3. 76 FR 67557 - Proposed Information Collection (Survey of Veteran Enrollees' Health and Reliance Upon VA...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-11-01

    ..., 810 Vermont Avenue NW., Washington, DC 20420; or email: cynthia.harvey-pryor@va.gov . Please refer to... AFFAIRS Proposed Information Collection (Survey of Veteran Enrollees' Health and Reliance Upon VA...: Notice. SUMMARY: The Veterans Health Administration (VHA), Department of Veterans Affairs (VA),...

  4. 75 FR 33898 - Agency Information Collection (VA Loan Electronic Reporting Interface (VALERI) System) Activity...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-06-15

    ... through http://www.Regulations.gov ; or to VA's OMB Desk Officer, OMB Human Resources and Housing Branch..., (202) 461-7485, FAX (202) 273-0443 or e-mail denise.mclamb@va.gov . Please refer to ``OMB Control No... AFFAIRS Agency Information Collection (VA Loan Electronic Reporting Interface (VALERI) System)...

  5. 78 FR 36642 - Proposed Information Collection (VA Loan Electronic Reporting Interface (VALERI) System) Activity...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-06-18

    ... nancy.kessinger@va.gov . Please refer to ``OMB Control No. 2900-0021'' in any correspondence. During the... AFFAIRS Proposed Information Collection (VA Loan Electronic Reporting Interface (VALERI) System) Activity.... SUMMARY: The Veterans Benefits Administration (VBA), Department of Veterans Affairs (VA), is announcing...

  6. 75 FR 48413 - Agency Information Collection (HUD/VA Addendum to Uniform Residential Loan Application) Activity...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-08-10

    ... information through www.Regulations.gov ; or to VA's OMB Desk Officer, OMB Human Resources and Housing Branch..., (202) 461-7485, FAX (202) 273-0443 or e-mail denise.mclamb@va.gov . Please refer to ``OMB Control No... AFFAIRS Agency Information Collection (HUD/VA Addendum to Uniform Residential Loan Application)...

  7. 78 FR 21817 - Amendment of Restricted Area R-6601; Fort A.P. Hill, VA

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-04-12

    ... limits and increase the time of designation of restricted area R-6601, Fort A.P. Hill, VA, (77 FR 35308... Administration 14 CFR Part 73 RIN 2120-AA66 Amendment of Restricted Area R-6601; Fort A.P. Hill, VA AGENCY... limits and time of designation of restricted area R-6601, Fort A.P. Hill, VA. The U.S. Army...

  8. 77 FR 23204 - VA Acquisition Regulation: Electronic Submission of Payment Requests

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-04-18

    ... using under VA's current interim electronic invoicing clause. See 74 FR 32223. VA's Electronic Invoice... INFORMATION: On July 7, 2009, VA published a notice, in the Federal Register at 74 FR 32223, of a class... interchange (EDI) formats; or (3) another electronic form as prescribed by the contract administration...

  9. 48 CFR 852.219-9 - VA Small business subcontracting plan minimum requirements.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 48 Federal Acquisition Regulations System 5 2011-10-01 2011-10-01 false VA Small business... Provisions and Clauses 852.219-9 VA Small business subcontracting plan minimum requirements. As prescribed in subpart 819.709, insert the following clause: VA Small Business Subcontracting Plan Minimum...

  10. 38 CFR 17.106 - VA collection rules; third-party payers.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 38 Pensions, Bonuses, and Veterans' Relief 1 2013-07-01 2013-07-01 false VA collection rules; third-party payers. 17.106 Section 17.106 Pensions, Bonuses, and Veterans' Relief DEPARTMENT OF VETERANS AFFAIRS MEDICAL Charges, Waivers, and Collections § 17.106 VA collection rules; third-party payers. (a)(1) General rule. VA has the right to...