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Sample records for rapid genomic characterization

  1. Rapid and efficient genome-wide characterization of Xanthomonas TAL effector genes

    PubMed Central

    Yu, Yan-Hua; Lu, Ye; He, Yong-Qiang; Huang, Sheng; Tang, Ji-Liang

    2015-01-01

    Xanthomonas TALE transcriptional activators act as virulence or avirulence factors by activating host disease susceptibility or resistance genes. Their specificity is determined by a tandem repeat domain. Some Xanthomonas pathogens contain 10–30 TALEs per strain. Although TALEs play critical roles in pathogenesis, their studies have so far been limited to a few examples, due to their highly repetitive gene structure and extreme similarity among different members, which constrict sequencing and assembling. To facilitate TALE studies, we developed an efficient and rapid pipeline for genome-wide cloning of tal genes as many as possible from a strain. Here, we report the pipeline and its use to identify all 18 tal genes from a newly isolated strain of the rice pathogen Xathomonas oryzae. Target prediction revealed a number of potential rice targets including several notable genes such as genes encoding SWEET, WRKY, Hen1, and BAK1 proteins, which provide candidates for further experimental functional analysis of the TALEs. PMID:26271455

  2. Rapid and efficient genome-wide characterization of Xanthomonas TAL effector genes.

    PubMed

    Yu, Yan-Hua; Lu, Ye; He, Yong-Qiang; Huang, Sheng; Tang, Ji-Liang

    2015-01-01

    Xanthomonas TALE transcriptional activators act as virulence or avirulence factors by activating host disease susceptibility or resistance genes. Their specificity is determined by a tandem repeat domain. Some Xanthomonas pathogens contain 10-30 TALEs per strain. Although TALEs play critical roles in pathogenesis, their studies have so far been limited to a few examples, due to their highly repetitive gene structure and extreme similarity among different members, which constrict sequencing and assembling. To facilitate TALE studies, we developed an efficient and rapid pipeline for genome-wide cloning of tal genes as many as possible from a strain. Here, we report the pipeline and its use to identify all 18 tal genes from a newly isolated strain of the rice pathogen Xathomonas oryzae. Target prediction revealed a number of potential rice targets including several notable genes such as genes encoding SWEET, WRKY, Hen1, and BAK1 proteins, which provide candidates for further experimental functional analysis of the TALEs. PMID:26271455

  3. Prospective Genomic Characterization of the German Enterohemorrhagic Escherichia coli O104:H4 Outbreak by Rapid Next Generation Sequencing Technology

    PubMed Central

    Zentz, Emily B.; Leopold, Shana R.; Rico, Alain; Prior, Karola; Szczepanowski, Rafael; Ji, Yongmei; Zhang, Wenlan; McLaughlin, Stephen F.; Henkhaus, John K.; Leopold, Benjamin; Bielaszewska, Martina; Prager, Rita; Brzoska, Pius M.; Moore, Richard L.; Guenther, Simone; Rothberg, Jonathan M.; Karch, Helge

    2011-01-01

    An ongoing outbreak of exceptionally virulent Shiga toxin (Stx)-producing Escherichia coli O104:H4 centered in Germany, has caused over 830 cases of hemolytic uremic syndrome (HUS) and 46 deaths since May 2011. Serotype O104:H4, which has not been detected in animals, has rarely been associated with HUS in the past. To prospectively elucidate the unique characteristics of this strain in the early stages of this outbreak, we applied whole genome sequencing on the Life Technologies Ion Torrent PGM™ sequencer and Optical Mapping to characterize one outbreak isolate (LB226692) and a historic O104:H4 HUS isolate from 2001 (01-09591). Reference guided draft assemblies of both strains were completed with the newly introduced PGM™ within 62 hours. The HUS-associated strains both carried genes typically found in two types of pathogenic E. coli, enteroaggregative E. coli (EAEC) and enterohemorrhagic E. coli (EHEC). Phylogenetic analyses of 1,144 core E. coli genes indicate that the HUS-causing O104:H4 strains and the previously published sequence of the EAEC strain 55989 show a close relationship but are only distantly related to common EHEC serotypes. Though closely related, the outbreak strain differs from the 2001 strain in plasmid content and fimbrial genes. We propose a model in which EAEC 55989 and EHEC O104:H4 strains evolved from a common EHEC O104:H4 progenitor, and suggest that by stepwise gain and loss of chromosomal and plasmid-encoded virulence factors, a highly pathogenic hybrid of EAEC and EHEC emerged as the current outbreak clone. In conclusion, rapid next-generation technologies facilitated prospective whole genome characterization in the early stages of an outbreak. PMID:21799941

  4. GeneSippr: A Rapid Whole-Genome Approach for the Identification and Characterization of Foodborne Pathogens such as Priority Shiga Toxigenic Escherichia coli

    PubMed Central

    Koziol, Adam G.; Manninger, Paul; Blais, Burton W.

    2015-01-01

    The timely identification and characterization of foodborne bacteria for risk assessment purposes is a key operation in outbreak investigations. Current methods require several days and/or provide low-resolution characterization. Here we describe a whole-genome-sequencing (WGS) approach (GeneSippr) enabling same-day identification of colony isolates recovered from investigative food samples. The identification of colonies of priority Shiga-toxigenic Escherichia coli (STEC) (i.e., serogroups O26, O45, O103, O111, O121, O145 and O157) served as a proof of concept. Genomic DNA was isolated from single colonies and sequencing was conducted on the Illumina MiSeq instrument with raw data sampling from the instrument following 4.5 hrs of sequencing. Modeling experiments indicated that datasets comprised of 21-nt reads representing approximately 4-fold coverage of the genome were sufficient to avoid significant gaps in sequence data. A novel bioinformatic pipeline was used to identify the presence of specific marker genes based on mapping of the short reads to reference sequence libraries, along with the detection of dispersed conserved genomic markers as a quality control metric to assure the validity of the analysis. STEC virulence markers were correctly identified in all isolates tested, and single colonies were identified within 9 hrs. This method has the potential to produce high-resolution characterization of STEC isolates, and whole-genome sequence data generated following the GeneSippr analysis could be used for isolate identification in place of lengthy biochemical characterization and typing methodologies. Significant advantages of this procedure include ease of adaptation to the detection of any gene marker of interest, as well as to the identification of other foodborne pathogens for which genomic markers have been defined. PMID:25860693

  5. Genomic changes under rapid evolution: selection for parasitoid resistance.

    PubMed

    Jalvingh, Kirsten M; Chang, Peter L; Nuzhdin, Sergey V; Wertheim, Bregje

    2014-03-22

    In this study, we characterize changes in the genome during a swift evolutionary adaptation, by combining experimental selection with high-throughput sequencing. We imposed strong experimental selection on an ecologically relevant trait, parasitoid resistance in Drosophila melanogaster against Asobara tabida. Replicated selection lines rapidly evolved towards enhanced immunity. Larval survival after parasitization increased twofold after just five generations of selection. Whole-genome sequencing revealed that the fast and strong selection response in innate immunity produced multiple, highly localized genomic changes. We identified narrow genomic regions carrying a significant signature of selection, which were present across all chromosomes and covered in total less than 5% of the whole D. melanogaster genome. We identified segregating sites with highly significant changes in frequency between control and selection lines that fell within these narrow 'selected regions'. These segregating sites were associated with 42 genes that constitute possible targets of selection. A region on chromosome 2R was highly enriched in significant segregating sites and may be of major effect on parasitoid defence. The high genetic variability and small linkage blocks in our base population are likely responsible for allowing this complex trait to evolve without causing widespread erosive effects in the genome, even under such a fast and strong selective regime.

  6. Rapid whole genome sequencing and precision neonatology.

    PubMed

    Petrikin, Joshua E; Willig, Laurel K; Smith, Laurie D; Kingsmore, Stephen F

    2015-12-01

    Traditionally, genetic testing has been too slow or perceived to be impractical to initial management of the critically ill neonate. Technological advances have led to the ability to sequence and interpret the entire genome of a neonate in as little as 26 h. As the cost and speed of testing decreases, the utility of whole genome sequencing (WGS) of neonates for acute and latent genetic illness increases. Analyzing the entire genome allows for concomitant evaluation of the currently identified 5588 single gene diseases. When applied to a select population of ill infants in a level IV neonatal intensive care unit, WGS yielded a diagnosis of a causative genetic disease in 57% of patients. These diagnoses may lead to clinical management changes ranging from transition to palliative care for uniformly lethal conditions for alteration or initiation of medical or surgical therapy to improve outcomes in others. Thus, institution of 2-day WGS at time of acute presentation opens the possibility of early implementation of precision medicine. This implementation may create opportunities for early interventional, frequently novel or off-label therapies that may alter disease trajectory in infants with what would otherwise be fatal disease. Widespread deployment of rapid WGS and precision medicine will raise ethical issues pertaining to interpretation of variants of unknown significance, discovery of incidental findings related to adult onset conditions and carrier status, and implementation of medical therapies for which little is known in terms of risks and benefits. Despite these challenges, precision neonatology has significant potential both to decrease infant mortality related to genetic diseases with onset in newborns and to facilitate parental decision making regarding transition to palliative care.

  7. Rapid whole genome sequencing and precision neonatology.

    PubMed

    Petrikin, Joshua E; Willig, Laurel K; Smith, Laurie D; Kingsmore, Stephen F

    2015-12-01

    Traditionally, genetic testing has been too slow or perceived to be impractical to initial management of the critically ill neonate. Technological advances have led to the ability to sequence and interpret the entire genome of a neonate in as little as 26 h. As the cost and speed of testing decreases, the utility of whole genome sequencing (WGS) of neonates for acute and latent genetic illness increases. Analyzing the entire genome allows for concomitant evaluation of the currently identified 5588 single gene diseases. When applied to a select population of ill infants in a level IV neonatal intensive care unit, WGS yielded a diagnosis of a causative genetic disease in 57% of patients. These diagnoses may lead to clinical management changes ranging from transition to palliative care for uniformly lethal conditions for alteration or initiation of medical or surgical therapy to improve outcomes in others. Thus, institution of 2-day WGS at time of acute presentation opens the possibility of early implementation of precision medicine. This implementation may create opportunities for early interventional, frequently novel or off-label therapies that may alter disease trajectory in infants with what would otherwise be fatal disease. Widespread deployment of rapid WGS and precision medicine will raise ethical issues pertaining to interpretation of variants of unknown significance, discovery of incidental findings related to adult onset conditions and carrier status, and implementation of medical therapies for which little is known in terms of risks and benefits. Despite these challenges, precision neonatology has significant potential both to decrease infant mortality related to genetic diseases with onset in newborns and to facilitate parental decision making regarding transition to palliative care. PMID:26521050

  8. Characterization of the Genomic Xist Locus in Rodents Reveals Conservation of Overall Gene Structure and Tandem Repeats but Rapid Evolution of Unique Sequence

    PubMed Central

    Nesterova, Tatyana B.; Slobodyanyuk, Sergey Ya.; Elisaphenko, Eugene A.; Shevchenko, Alexander I.; Johnston, Colette; Pavlova, Marina E.; Rogozin, Igor B.; Kolesnikov, Nikolay N.; Brockdorff, Neil; Zakian, Suren M.

    2001-01-01

    The Xist locus plays a central role in the regulation of X chromosome inactivation in mammals, although its exact mode of action remains to be elucidated. Evolutionary studies are important in identifying conserved genomic regions and defining their possible function. Here we report cloning, sequence analysis, and detailed characterization of the Xist gene from four closely related species of common vole (field mouse), Microtus arvalis. Our analysis reveals that there is overall conservation of Xist gene structure both between different vole species and relative to mouse and human Xist/XIST. Within transcribed sequence, there is significant conservation over five short regions of unique sequence and also over Xist-specific tandem repeats. The majority of unique sequences, however, are evolving at an unexpectedly high rate. This is also evident from analysis of flanking sequences, which reveals a very high rate of rearrangement and invasion of dispersed repeats. We discuss these results in the context of Xist gene function and evolution. [The sequence data described in this paper have been submitted to the GenBank data library under accession nos. AJ310127–AJ310130 and AJ311670.] PMID:11337478

  9. Rapid extraction and preservation of genomic DNA from human samples.

    PubMed

    Kalyanasundaram, D; Kim, J-H; Yeo, W-H; Oh, K; Lee, K-H; Kim, M-H; Ryew, S-M; Ahn, S-G; Gao, D; Cangelosi, G A; Chung, J-H

    2013-02-01

    Simple and rapid extraction of human genomic DNA remains a bottleneck for genome analysis and disease diagnosis. Current methods using microfilters require cumbersome, multiple handling steps in part because salt conditions must be controlled for attraction and elution of DNA in porous silica. We report a novel extraction method of human genomic DNA from buccal swab and saliva samples. DNA is attracted onto a gold-coated microchip by an electric field and capillary action while the captured DNA is eluted by thermal heating at 70 °C. A prototype device was designed to handle four microchips, and a compatible protocol was developed. The extracted DNA using microchips was characterized by qPCR for different sample volumes, using different lengths of PCR amplicon, and nuclear and mitochondrial genes. In comparison with a commercial kit, an equivalent yield of DNA extraction was achieved with fewer steps. Room-temperature preservation for 1 month was demonstrated for captured DNA, facilitating straightforward collection, delivery, and handling of genomic DNA in an environment-friendly protocol.

  10. Complete Genome Sequences of 17 Rapidly Growing Nontuberculous Mycobacterial Strains.

    PubMed

    Caverly, Lindsay J; Spilker, Theodore; LiPuma, John J

    2016-01-01

    We report the complete genome sequences of 17 rapidly growing nontuberculous mycobacterial (NTM) strains, including 16 Mycobacterium abscessus complex strains and one M. immunogenum strain. These sequences add value to studies of the genetic diversity of rapidly growing NTM strains recovered from human specimens. PMID:27660787

  11. Complete Genome Sequences of 17 Rapidly Growing Nontuberculous Mycobacterial Strains

    PubMed Central

    Spilker, Theodore; LiPuma, John J.

    2016-01-01

    We report the complete genome sequences of 17 rapidly growing nontuberculous mycobacterial (NTM) strains, including 16 Mycobacterium abscessus complex strains and one M. immunogenum strain. These sequences add value to studies of the genetic diversity of rapidly growing NTM strains recovered from human specimens. PMID:27660787

  12. In Transition: Primate Genomics at a Time of Rapid Change

    PubMed Central

    Rogers, Jeffrey

    2013-01-01

    The field of nonhuman primate genomics is undergoing rapid change and making impressive progress. Exploiting new technologies for DNA sequencing, researchers have generated new whole-genome sequence assemblies for multiple primate species over the past 6 years. In addition, investigations of within-species genetic variation, gene expression and RNA sequences, conservation of non-protein-coding regions of the genome, and other aspects of comparative genomics are moving at an accelerating speed. This progress is opening a wide array of new research opportunities in the analysis of comparative primate genome content and evolution. It also creates new possibilities for the use of nonhuman primates as model organisms in biomedical research. This transition, based on both new technology and the new information being generated in regard to human genetics, provides an important justification for reevaluating the research goals, strategies, and study designs used in primate genetics and genomics. PMID:24174444

  13. Rapid genome reshaping by multiple-gene loss after whole-genome duplication in teleost fish suggested by mathematical modeling

    PubMed Central

    Sato, Yukuto; Tsukamoto, Katsumi; Nishida, Mutsumi

    2015-01-01

    Whole-genome duplication (WGD) is believed to be a significant source of major evolutionary innovation. Redundant genes resulting from WGD are thought to be lost or acquire new functions. However, the rates of gene loss and thus temporal process of genome reshaping after WGD remain unclear. The WGD shared by all teleost fish, one-half of all jawed vertebrates, was more recent than the two ancient WGDs that occurred before the origin of jawed vertebrates, and thus lends itself to analysis of gene loss and genome reshaping. Using a newly developed orthology identification pipeline, we inferred the post–teleost-specific WGD evolutionary histories of 6,892 protein-coding genes from nine phylogenetically representative teleost genomes on a time-calibrated tree. We found that rapid gene loss did occur in the first 60 My, with a loss of more than 70–80% of duplicated genes, and produced similar genomic gene arrangements within teleosts in that relatively short time. Mathematical modeling suggests that rapid gene loss occurred mainly by events involving simultaneous loss of multiple genes. We found that the subsequent 250 My were characterized by slow and steady loss of individual genes. Our pipeline also identified about 1,100 shared single-copy genes that are inferred to have become singletons before the divergence of clupeocephalan teleosts. Therefore, our comparative genome analysis suggests that rapid gene loss just after the WGD reshaped teleost genomes before the major divergence, and provides a useful set of marker genes for future phylogenetic analysis. PMID:26578810

  14. Rapid genome reshaping by multiple-gene loss after whole-genome duplication in teleost fish suggested by mathematical modeling.

    PubMed

    Inoue, Jun; Sato, Yukuto; Sinclair, Robert; Tsukamoto, Katsumi; Nishida, Mutsumi

    2015-12-01

    Whole-genome duplication (WGD) is believed to be a significant source of major evolutionary innovation. Redundant genes resulting from WGD are thought to be lost or acquire new functions. However, the rates of gene loss and thus temporal process of genome reshaping after WGD remain unclear. The WGD shared by all teleost fish, one-half of all jawed vertebrates, was more recent than the two ancient WGDs that occurred before the origin of jawed vertebrates, and thus lends itself to analysis of gene loss and genome reshaping. Using a newly developed orthology identification pipeline, we inferred the post-teleost-specific WGD evolutionary histories of 6,892 protein-coding genes from nine phylogenetically representative teleost genomes on a time-calibrated tree. We found that rapid gene loss did occur in the first 60 My, with a loss of more than 70-80% of duplicated genes, and produced similar genomic gene arrangements within teleosts in that relatively short time. Mathematical modeling suggests that rapid gene loss occurred mainly by events involving simultaneous loss of multiple genes. We found that the subsequent 250 My were characterized by slow and steady loss of individual genes. Our pipeline also identified about 1,100 shared single-copy genes that are inferred to have become singletons before the divergence of clupeocephalan teleosts. Therefore, our comparative genome analysis suggests that rapid gene loss just after the WGD reshaped teleost genomes before the major divergence, and provides a useful set of marker genes for future phylogenetic analysis.

  15. Neutral and adaptive genomic signatures of rapid poleward range expansion.

    PubMed

    Swaegers, J; Mergeay, J; Van Geystelen, A; Therry, L; Larmuseau, M H D; Stoks, R

    2015-12-01

    Many species are expanding their range polewards, and this has been associated with rapid phenotypic change. Yet, it is unclear to what extent this reflects rapid genetic adaptation or neutral processes associated with range expansion, or selection linked to the new thermal conditions encountered. To disentangle these alternatives, we studied the genomic signature of range expansion in the damselfly Coenagrion scitulum using 4950 newly developed genomic SNPs and linked this to the rapidly evolved phenotypic differences between core and (newly established) edge populations. Most edge populations were genetically clearly differentiated from the core populations and all were differentiated from each other indicating independent range expansion events. In addition, evidence for genetic drift in the edge populations, and strong evidence for adaptive genetic variation in association with the range expansion was detected. We identified one SNP under consistent selection in four of the five edge populations and showed that the allele increasing in frequency is associated with increased flight performance. This indicates collateral, non-neutral evolutionary changes in independent edge populations driven by the range expansion process. We also detected a genomic signature of adaptation to the newly encountered thermal regimes, reflecting a pattern of countergradient variation. The latter signature was identified at a single SNP as well as in a set of covarying SNPs using a polygenic multilocus approach to detect selection. Overall, this study highlights how a strategic geographic sampling design and the integration of genomic, phenotypic and environmental data can identify and disentangle the neutral and adaptive processes that are simultaneously operating during range expansions. PMID:26561985

  16. Genomic characterization of acute leukemias.

    PubMed

    Chiaretti, Sabina; Gianfelici, Valentina; Ceglie, Giulia; Foà, Robin

    2014-01-01

    Over the past two decades, hematologic malignancies have been extensively evaluated due to the introduction of powerful technologies, such as conventional karyotyping, FISH analysis, gene and microRNA expression profiling, array comparative genomic hybridization and SNP arrays, and next-generation sequencing (including whole-exome sequencing and RNA-seq). These analyses have allowed for the refinement of the mechanisms underlying the leukemic transformation in several oncohematologic disorders and, more importantly, they have permitted the definition of novel prognostic algorithms aimed at stratifying patients at the onset of disease and, consequently, treating them in the most appropriate manner. Furthermore, the identification of specific molecular markers is opening the door to targeted and personalized medicine. The most important findings on novel acquisitions in the context of acute lymphoblastic leukemia of both B and T lineage and de novo acute myeloid leukemia are described in this review.

  17. Rapid Characterization of Large Earthquakes in Chile

    NASA Astrophysics Data System (ADS)

    Barrientos, S. E.; Team, C.

    2015-12-01

    Chile, along 3000 km of it 4200 km long coast, is regularly affected by very large earthquakes (up to magnitude 9.5) resulting from the convergence and subduction of the Nazca plate beneath the South American plate. These megathrust earthquakes exhibit long rupture regions reaching several hundreds of km with fault displacements of several tens of meters. Minimum delay characterization of these giant events to establish their rupture extent and slip distribution is of the utmost importance for rapid estimations of the shaking area and their corresponding tsunami-genic potential evaluation, particularly when there are only few minutes to warn the coastal population for immediate actions. The task of a rapid evaluation of large earthquakes is accomplished in Chile through a network of sensors being implemented by the National Seismological Center of the University of Chile. The network is mainly composed approximately by one hundred broad-band and strong motion instruments and 130 GNSS devices; all will be connected in real time. Forty units present an optional RTX capability, where satellite orbits and clock corrections are sent to the field device producing a 1-Hz stream at 4-cm level. Tests are being conducted to stream the real-time raw data to be later processed at the central facility. Hypocentral locations and magnitudes are estimated after few minutes by automatic processing software based on wave arrival; for magnitudes less than 7.0 the rapid estimation works within acceptable bounds. For larger events, we are currently developing automatic detectors and amplitude estimators of displacement coming out from the real time GNSS streams. This software has been tested for several cases showing that, for plate interface events, the minimum magnitude threshold detectability reaches values within 6.2 and 6.5 (1-2 cm coastal displacement), providing an excellent tool for earthquake early characterization from a tsunamigenic perspective.

  18. Rapid evolutionary turnover underlies conserved lncRNA-genome interactions.

    PubMed

    Quinn, Jeffrey J; Zhang, Qiangfeng C; Georgiev, Plamen; Ilik, Ibrahim A; Akhtar, Asifa; Chang, Howard Y

    2016-01-15

    Many long noncoding RNAs (lncRNAs) can regulate chromatin states, but the evolutionary origin and dynamics driving lncRNA-genome interactions are unclear. We adapted an integrative strategy that identifies lncRNA orthologs in different species despite limited sequence similarity, which is applicable to mammalian and insect lncRNAs. Analysis of the roX lncRNAs, which are essential for dosage compensation of the single X chromosome in Drosophila males, revealed 47 new roX orthologs in diverse Drosophilid species across ∼40 million years of evolution. Genetic rescue by roX orthologs and engineered synthetic lncRNAs showed that altering the number of focal, repetitive RNA structures determines roX ortholog function. Genomic occupancy maps of roX RNAs in four species revealed conserved targeting of X chromosome neighborhoods but rapid turnover of individual binding sites. Many new roX-binding sites evolved from DNA encoding a pre-existing RNA splicing signal, effectively linking dosage compensation to transcribed genes. Thus, dynamic change in lncRNAs and their genomic targets underlies conserved and essential lncRNA-genome interactions. PMID:26773003

  19. A rapid whole genome sequencing and analysis system supporting genomic epidemiology (7th Annual SFAF Meeting, 2012)

    SciTech Connect

    FitzGerald, Michael

    2012-06-01

    Michael FitzGerald on "A rapid whole genome sequencing and analysis system supporting genomic epidemiology" at the 2012 Sequencing, Finishing, Analysis in the Future Meeting held June 5-7, 2012 in Santa Fe, New Mexico.

  20. A rapid whole genome sequencing and analysis system supporting genomic epidemiology (7th Annual SFAF Meeting, 2012)

    ScienceCinema

    FitzGerald, Michael [Broad Institute

    2016-07-12

    Michael FitzGerald on "A rapid whole genome sequencing and analysis system supporting genomic epidemiology" at the 2012 Sequencing, Finishing, Analysis in the Future Meeting held June 5-7, 2012 in Santa Fe, New Mexico.

  1. Genomic characterization of the Yersinia genus

    PubMed Central

    2010-01-01

    Background New DNA sequencing technologies have enabled detailed comparative genomic analyses of entire genera of bacterial pathogens. Prior to this study, three species of the enterobacterial genus Yersinia that cause invasive human diseases (Yersinia pestis, Yersinia pseudotuberculosis, and Yersinia enterocolitica) had been sequenced. However, there were no genomic data on the Yersinia species with more limited virulence potential, frequently found in soil and water environments. Results We used high-throughput sequencing-by-synthesis instruments to obtain 25- to 42-fold average redundancy, whole-genome shotgun data from the type strains of eight species: Y. aldovae, Y. bercovieri, Y. frederiksenii, Y. kristensenii, Y. intermedia, Y. mollaretii, Y. rohdei, and Y. ruckeri. The deepest branching species in the genus, Y. ruckeri, causative agent of red mouth disease in fish, has the smallest genome (3.7 Mb), although it shares the same core set of approximately 2,500 genes as the other members of the species, whose genomes range in size from 4.3 to 4.8 Mb. Yersinia genomes had a similar global partition of protein functions, as measured by the distribution of Cluster of Orthologous Groups families. Genome to genome variation in islands with genes encoding functions such as ureases, hydrogeneases and B-12 cofactor metabolite reactions may reflect adaptations to colonizing specific host habitats. Conclusions Rapid high-quality draft sequencing was used successfully to compare pathogenic and non-pathogenic members of the Yersinia genus. This work underscores the importance of the acquisition of horizontally transferred genes in the evolution of Y. pestis and points to virulence determinants that have been gained and lost on multiple occasions in the history of the genus. PMID:20047673

  2. The Capsella rubella genome and the genomic consequences of rapid mating system evolution.

    PubMed

    Slotte, Tanja; Hazzouri, Khaled M; Ågren, J Arvid; Koenig, Daniel; Maumus, Florian; Guo, Ya-Long; Steige, Kim; Platts, Adrian E; Escobar, Juan S; Newman, L Killian; Wang, Wei; Mandáková, Terezie; Vello, Emilio; Smith, Lisa M; Henz, Stefan R; Steffen, Joshua; Takuno, Shohei; Brandvain, Yaniv; Coop, Graham; Andolfatto, Peter; Hu, Tina T; Blanchette, Mathieu; Clark, Richard M; Quesneville, Hadi; Nordborg, Magnus; Gaut, Brandon S; Lysak, Martin A; Jenkins, Jerry; Grimwood, Jane; Chapman, Jarrod; Prochnik, Simon; Shu, Shengqiang; Rokhsar, Daniel; Schmutz, Jeremy; Weigel, Detlef; Wright, Stephen I

    2013-07-01

    The shift from outcrossing to selfing is common in flowering plants, but the genomic consequences and the speed at which they emerge remain poorly understood. An excellent model for understanding the evolution of self fertilization is provided by Capsella rubella, which became self compatible <200,000 years ago. We report a C. rubella reference genome sequence and compare RNA expression and polymorphism patterns between C. rubella and its outcrossing progenitor Capsella grandiflora. We found a clear shift in the expression of genes associated with flowering phenotypes, similar to that seen in Arabidopsis, in which self fertilization evolved about 1 million years ago. Comparisons of the two Capsella species showed evidence of rapid genome-wide relaxation of purifying selection in C. rubella without a concomitant change in transposable element abundance. Overall we document that the transition to selfing may be typified by parallel shifts in gene expression, along with a measurable reduction of purifying selection. PMID:23749190

  3. A Rapid Turnaround Cryogenic Detector Characterization System

    NASA Technical Reports Server (NTRS)

    Benford, Dominic j.; Dipirro, Michael J.; Forgione, Joshua B.; Jackson, Clifton E.; Jackson, Michael L.; Kogut, Al; Moseley, S. Harvey; Shirron, Peter J.

    2004-01-01

    Upcoming major NASA missions such as the Einstein Inflation Probe and the Single Aperture Far-Infrared Observatory require arrays of detectors with thousands of elements, operating at temperatures near l00 mK and sensitive to wavelengths from approx. 100 microns to approx. 3 mm. Such detectors represent a substantial enabling technology for these missions, and must be demonstrated soon in order for them to proceed. In order to make rapid progress on detector development, the cryogenic testing cycle must be made convenient and quick. We have developed a cryogenic detector characterization system capable of testing superconducting detector arrays in formats up to 8 x 32, read out by SQUID multiplexers. The system relies on the cooling of a two-stage adiabatic demagnetization refrigerator immersed in a liquid helium bath. This approach permits a detector to be cooled from 300K to 50 mK in about 4 hours, so that a test cycle begun in the morning will be over by the end of the day. Tine system is modular, with two identical immersible units, so that while one unit is cooling, the second can be reconfigured for the next battery of tests. We describe the design, construction, and performance of this cryogenic detector testing facility.

  4. Mass spectrometry for rapid characterization of microorganisms.

    PubMed

    Demirev, Plamen A; Fenselau, Catherine

    2008-01-01

    Advances in instrumentation, proteomics, and bioinformatics have contributed to the successful applications of mass spectrometry (MS) for detection, identification, and classification of microorganisms. These MS applications are based on the detection of organism-specific biomarker molecules, which allow differentiation between organisms to be made. Intact proteins, their proteolytic peptides, and nonribosomal peptides have been successfully utilized as biomarkers. Sequence-specific fragments for biomarkers are generated by tandem MS of intact proteins or proteolytic peptides, obtained after, for instance, microwave-assisted acid hydrolysis. In combination with proteome database searching, individual biomarker proteins are unambiguously identified from their tandem mass spectra, and from there the source microorganism is also identified. Such top-down or bottom-up proteomics approaches permit rapid, sensitive, and confident characterization of individual microorganisms in mixtures and are reviewed here. Examples of MS-based functional assays for detection of targeted microorganisms, e.g., Bacillus anthracis, in environmental or clinically relevant backgrounds are also reviewed. PMID:20636075

  5. Integrated genomic characterization of endometrial carcinoma.

    PubMed

    Kandoth, Cyriac; Schultz, Nikolaus; Cherniack, Andrew D; Akbani, Rehan; Liu, Yuexin; Shen, Hui; Robertson, A Gordon; Pashtan, Itai; Shen, Ronglai; Benz, Christopher C; Yau, Christina; Laird, Peter W; Ding, Li; Zhang, Wei; Mills, Gordon B; Kucherlapati, Raju; Mardis, Elaine R; Levine, Douglas A

    2013-05-01

    We performed an integrated genomic, transcriptomic and proteomic characterization of 373 endometrial carcinomas using array- and sequencing-based technologies. Uterine serous tumours and ∼25% of high-grade endometrioid tumours had extensive copy number alterations, few DNA methylation changes, low oestrogen receptor/progesterone receptor levels, and frequent TP53 mutations. Most endometrioid tumours had few copy number alterations or TP53 mutations, but frequent mutations in PTEN, CTNNB1, PIK3CA, ARID1A and KRAS and novel mutations in the SWI/SNF chromatin remodelling complex gene ARID5B. A subset of endometrioid tumours that we identified had a markedly increased transversion mutation frequency and newly identified hotspot mutations in POLE. Our results classified endometrial cancers into four categories: POLE ultramutated, microsatellite instability hypermutated, copy-number low, and copy-number high. Uterine serous carcinomas share genomic features with ovarian serous and basal-like breast carcinomas. We demonstrated that the genomic features of endometrial carcinomas permit a reclassification that may affect post-surgical adjuvant treatment for women with aggressive tumours.

  6. Finding and Characterizing Repeats in Plant Genomes.

    PubMed

    Nicolas, Jacques; Peterlongo, Pierre; Tempel, Sébastien

    2016-01-01

    Plant genomes contain a particularly high proportion of repeated structures of various types. This chapter proposes a guided tour of available software that can help biologists to look for these repeats and check some hypothetical models intended to characterize their structures. Since transposable elements are a major source of repeats in plants, many methods have been used or developed for this large class of sequences. They are representative of the range of tools available for other classes of repeats and we have provided a whole section on this topic as well as a selection of the main existing software. In order to better understand how they work and how repeats may be efficiently found in genomes, it is necessary to look at the technical issues involved in the large-scale search of these structures. Indeed, it may be hard to keep up with the profusion of proposals in this dynamic field and the rest of the chapter is devoted to the foundations of the search for repeats and more complex patterns. The second section introduces the key concepts that are useful for understanding the current state of the art in playing with words, applied to genomic sequences. This can be seen as the first stage of a very general approach called linguistic analysis that is interested in the analysis of natural or artificial texts. Words, the lexical level, correspond to simple repeated entities in texts or strings. In fact, biologists need to represent more complex entities where a repeat family is built on more abstract structures, including direct or inverted small repeats, motifs, composition constraints as well as ordering and distance constraints between these elementary blocks. In terms of linguistics, this corresponds to the syntactic level of a language. The last section introduces concepts and practical tools that can be used to reach this syntactic level in biological sequence analysis. PMID:26519414

  7. Finding and Characterizing Repeats in Plant Genomes.

    PubMed

    Nicolas, Jacques; Peterlongo, Pierre; Tempel, Sébastien

    2016-01-01

    Plant genomes contain a particularly high proportion of repeated structures of various types. This chapter proposes a guided tour of available software that can help biologists to look for these repeats and check some hypothetical models intended to characterize their structures. Since transposable elements are a major source of repeats in plants, many methods have been used or developed for this large class of sequences. They are representative of the range of tools available for other classes of repeats and we have provided a whole section on this topic as well as a selection of the main existing software. In order to better understand how they work and how repeats may be efficiently found in genomes, it is necessary to look at the technical issues involved in the large-scale search of these structures. Indeed, it may be hard to keep up with the profusion of proposals in this dynamic field and the rest of the chapter is devoted to the foundations of the search for repeats and more complex patterns. The second section introduces the key concepts that are useful for understanding the current state of the art in playing with words, applied to genomic sequences. This can be seen as the first stage of a very general approach called linguistic analysis that is interested in the analysis of natural or artificial texts. Words, the lexical level, correspond to simple repeated entities in texts or strings. In fact, biologists need to represent more complex entities where a repeat family is built on more abstract structures, including direct or inverted small repeats, motifs, composition constraints as well as ordering and distance constraints between these elementary blocks. In terms of linguistics, this corresponds to the syntactic level of a language. The last section introduces concepts and practical tools that can be used to reach this syntactic level in biological sequence analysis.

  8. A method for rapid characterization of diffusion.

    PubMed

    Song, Y-Q; Hürlimann, M D; Flaum, C

    2003-04-01

    This paper describes a method to determine molecular displacements as a function of time in just two scans: one reference scan using the Carr-Purcell-Meiboom-Gill (CPMG) sequence, a second scan using a modified CPMG sequence (KCPMG). Measurements on free diffusion in bulk fluids, and on restricted diffusion in porous rock samples are reported. This technique can also be used for rapid measurement of flow and chemical exchange. PMID:12713974

  9. Genomic Characterization of Neoparamoeba pemaquidensis (Amoebozoa) and Its Kinetoplastid Endosymbiont▿

    PubMed Central

    Tanifuji, Goro; Kim, Eunsoo; Onodera, Naoko T.; Gibeault, Rebecca; Dlutek, Marlena; Cawthorn, Richard J.; Fiala, Ivan; Lukeš, Julius; Greenwood, Spencer J.; Archibald, John M.

    2011-01-01

    We have performed a genomic characterization of a kinetoplastid protist living within the amoebozoan Neoparamoeba pemaquidensis. The genome of this “Ichthyobodo-related organism” was found to be unexpectedly large, with at least 11 chromosomes between 1.0 and 3.5 Mbp and a total genome size of at least 25 Mbp. PMID:21666073

  10. Rapid lithography: Photopolymerization characterizations and initiation kinetics

    NASA Astrophysics Data System (ADS)

    Stocker, Michael Paul

    In order to improve upon the resolution of photolithography, a technique that is used to produce features for today's micro and nanodevices, techniques must move beyond e-beam and deep-UV sources. Multiphoton absorption polymerization (MAP) uses near-infrared light for the creation of complex, three-dimensional features on the sub-100 nm scale. The resolution of MAP can be enhanced further using a two-beam technique called resolution augmentation through photo-induced deactivation (RAPID) to the reach feature sizes as small as 40 nm. The mechanism and kinetics of photo-induced deactivation are not well understood. To better understand these processes, studies of different photoinitiators have been performed. We find that some photoinitiators are so efficient at deactivation that they are capable of undergoing self-deactivation by addition of another photon from the excitation source. This phenomenon is manifested in a polymerization trend in which feature size has a proportional velocity (PROVE) dependence, the opposite of the conventional velocity dependence. We also demonstrate that the velocity dependence can also be tuned between PROVE and conventional dependences. Kinetic models have been formulated to account for the observed deactivation. By reconciling experimental data for some sample photoinitiators with the kinetic model through the use of simulations, kinetic rate constants are determined. The self-deactivation efficiency of each photoinitiator was determined. The lifetimes of intermediates in the radical photopolymerization process were also determined. The kinetic rate constants associated with photoinitiators should allow for the customization of photoinitiators for specific applications and make RAPID a more efficient process capable of reaching resolution on the level of 30 nm and below.

  11. Genomic diversity, population structure, and migration following rapid range expansion in the Balsam poplar, Populus balsamifera.

    PubMed

    Keller, Stephen R; Olson, Matthew S; Silim, Salim; Schroeder, William; Tiffin, Peter

    2010-03-01

    Rapid range expansions can cause pervasive changes in the genetic diversity and structure of populations. The postglacial history of the Balsam Poplar, Populus balsamifera, involved the colonization of most of northern North America, an area largely covered by continental ice sheets during the last glacial maximum. To characterize how this expansion shaped genomic diversity within and among populations, we developed 412 SNP markers that we assayed for a range-wide sample of 474 individuals sampled from 34 populations. We complemented the SNP data set with DNA sequence data from 11 nuclear loci from 94 individuals, and used coalescent analyses to estimate historical population size, demographic growth, and patterns of migration. Bayesian clustering identified three geographically separated demes found in the Northern, Central, and Eastern portions of the species' range. These demes varied significantly in nucleotide diversity, the abundance of private polymorphisms, and population substructure. Most measures supported the Central deme as descended from the primary refuge of diversity. Both SNPs and sequence data suggested recent population growth, and coalescent analyses of historical migration suggested a massive expansion from the Centre to the North and East. Collectively, these data demonstrate the strong influence that range expansions exert on genomic diversity, both within local populations and across the range. Our results suggest that an in-depth knowledge of nucleotide diversity following expansion requires sampling within multiple populations, and highlight the utility of combining insights from different data types in population genomic studies.

  12. Genomics of Rapid Incipient Speciation in Sympatric Threespine Stickleback

    PubMed Central

    Marques, David A.; Lucek, Kay; Meier, Joana I.; Mwaiko, Salome; Wagner, Catherine E.; Excoffier, Laurent; Seehausen, Ole

    2016-01-01

    Ecological speciation is the process by which reproductively isolated populations emerge as a consequence of divergent natural or ecologically-mediated sexual selection. Most genomic studies of ecological speciation have investigated allopatric populations, making it difficult to infer reproductive isolation. The few studies on sympatric ecotypes have focused on advanced stages of the speciation process after thousands of generations of divergence. As a consequence, we still do not know what genomic signatures of the early onset of ecological speciation look like. Here, we examined genomic differentiation among migratory lake and resident stream ecotypes of threespine stickleback reproducing in sympatry in one stream, and in parapatry in another stream. Importantly, these ecotypes started diverging less than 150 years ago. We obtained 34,756 SNPs with restriction-site associated DNA sequencing and identified genomic islands of differentiation using a Hidden Markov Model approach. Consistent with incipient ecological speciation, we found significant genomic differentiation between ecotypes both in sympatry and parapatry. Of 19 islands of differentiation resisting gene flow in sympatry, all were also differentiated in parapatry and were thus likely driven by divergent selection among habitats. These islands clustered in quantitative trait loci controlling divergent traits among the ecotypes, many of them concentrated in one region with low to intermediate recombination. Our findings suggest that adaptive genomic differentiation at many genetic loci can arise and persist in sympatry at the very early stage of ecotype divergence, and that the genomic architecture of adaptation may facilitate this. PMID:26925837

  13. Rapid Intraoperative Molecular Characterization of Glioma

    PubMed Central

    Shankar, Ganesh M.; Francis, Joshua M.; Rinne, Mikael L.; Ramkissoon, Shakti H.; Huang, Franklin W.; Venteicher, Andrew S.; Akama-Garren, Elliot H.; Kang, Yun Jee; Lelic, Nina; Kim, James C.; Brown, Loreal E.; Charbonneau, Sarah K.; Golby, Alexandra J.; Pedamallu, Chandra Sekhar; Hoang, Mai P.; Sullivan, Ryan J.; Cherniack, Andrew D.; Garraway, Levi A.; Stemmer-Rachamimov, Anat; Reardon, David A.; Wen, Patrick Y.; Brastianos, Priscilla K.; Curry, William T.; Barker, Fred G.; Hahn, William C.; Nahed, Brian V.; Ligon, Keith L.; Louis, David N.; Cahill, Daniel P.; Meyerson, Matthew

    2016-01-01

    IMPORTANCE Conclusive intraoperative pathologic confirmation of diffuse infiltrative glioma guides the decision to pursue definitive neurosurgical resection. Establishing the intraoperative diagnosis by histologic analysis can be difficult in low-cellularity infiltrative gliomas. Therefore, we developed a rapid and sensitive genotyping assay to detect somatic single-nucleotide variants in the telomerase reverse transcriptase (TERT) promoter and isocitrate dehydrogenase 1 (IDH1). OBSERVATIONS This assay was applied to tissue samples from 190 patients with diffuse gliomas, including archived fixed and frozen specimens and tissue obtained intraoperatively. Results demonstrated 96% sensitivity (95% CI, 90%–99%) and 100% specificity (95% CI, 95%–100%) for World Health Organization grades II and III gliomas. In a series of live cases, glioma-defining mutations could be identified within 60 minutes, which could facilitate the diagnosis in an intraoperative timeframe. CONCLUSIONS AND RELEVANCE The genotyping method described herein can establish the diagnosis of low-cellularity tumors like glioma and could be adapted to the point-of-care diagnosis of other lesions that are similarly defined by highly recurrent somatic mutations. PMID:26181761

  14. Technical note: Rapid calculation of genomic evaluations for new animals.

    PubMed

    Wiggans, G R; VanRaden, P M; Cooper, T A

    2015-03-01

    A method was developed to calculate preliminary genomic evaluations daily or weekly before the release of official monthly evaluations by processing only newly genotyped animals using estimates of single nucleotide polymorphism effects from the previous official evaluation. To minimize computing time, reliabilities and genomic inbreeding are not calculated, and fixed weights are used to combine genomic and traditional information. Correlations of preliminary and September official monthly evaluations for animals with genotypes that became usable after the extraction of genotypes for August 2014 evaluations were >0.99 for most Holstein traits. Correlations were lower for breeds with smaller population size. Earlier access to genomic evaluations benefits producers by enabling earlier culling decisions and genotyping laboratories by making workloads more uniform across the month.

  15. Genome Sequence of Rapid Beer-Spoiling Isolate Lactobacillus brevis BSO 464

    PubMed Central

    Bergsveinson, Jordyn; Pittet, Vanessa; Ewen, Emily; Baecker, Nina

    2015-01-01

    The genome of brewery-isolate Lactobacillus brevis BSO 464 was sequenced and assembly produced a chromosome and eight plasmids. This bacterium tolerates dissolved CO2/pressure and can rapidly spoil packaged beer. This genome is useful for analyzing the genetics associated with beer spoilage by lactic acid bacteria. PMID:26634759

  16. Genome Sequence of Rapid Beer-Spoiling Isolate Lactobacillus brevis BSO 464.

    PubMed

    Bergsveinson, Jordyn; Pittet, Vanessa; Ewen, Emily; Baecker, Nina; Ziola, Barry

    2015-01-01

    The genome of brewery-isolate Lactobacillus brevis BSO 464 was sequenced and assembly produced a chromosome and eight plasmids. This bacterium tolerates dissolved CO2/pressure and can rapidly spoil packaged beer. This genome is useful for analyzing the genetics associated with beer spoilage by lactic acid bacteria. PMID:26634759

  17. The population genomics of rapid adaptation: disentangling signatures of selection and demography in white sands lizards.

    PubMed

    Laurent, Stefan; Pfeifer, Susanne P; Settles, Matthew L; Hunter, Samuel S; Hardwick, Kayla M; Ormond, Louise; Sousa, Vitor C; Jensen, Jeffrey D; Rosenblum, Erica Bree

    2016-01-01

    Understanding the process of adaptation during rapid environmental change remains one of the central focal points of evolutionary biology. The recently formed White Sands system of southern New Mexico offers an outstanding example of rapid adaptation, with a variety of species having rapidly evolved blanched forms on the dunes that contrast with their close relatives in the surrounding dark soil habitat. In this study, we focus on two of the White Sands lizard species, Sceloporus cowlesi and Aspidoscelis inornata, for which previous research has linked mutations in the melanocortin-1 receptor gene (Mc1r) to blanched coloration. We sampled populations both on and off the dunes and used a custom sequence capture assay based on probed fosmid libraries to obtain >50 kb of sequence around Mc1r and hundreds of other random genomic locations. We then used model-based statistical inference methods to describe the demographic and adaptive history characterizing the colonization of White Sands. We identified a number of similarities between the two focal species, including strong evidence of selection in the blanched populations in the Mc1r region. We also found important differences between the species, suggesting different colonization times, different genetic architecture underlying the blanched phenotype and different ages of the beneficial alleles. Finally, the beneficial allele is dominant in S. cowlesi and recessive in A. inornata, allowing for a rare empirical test of theoretically expected patterns of selective sweeps under these differing models.

  18. A primer on rapid prototyping of genomic databases in Prolog

    SciTech Connect

    Yoshida, Kaoru; Smith, C.L. ); Overbeek, R. . Mathematics and Computer Science Div.)

    1992-01-01

    This report presents a tutorial on how one might create an integrated database of genomic information. We outline the required steps for implementation, give a brief introduction to Prolog, and discuss the query facility supported by our system. Our goal is to enable researchers to being constructing their own biological information system.

  19. Rapid genome resequencing of an atoxigenic strain of Aspergillus carbonarius

    DOE PAGES

    Cabañes, F. Javier; Sanseverino, Walter; Castellá, Gemma; Bragulat, M. Rosa; Cigliano, Riccardo Aiese; Sánchez, Armand

    2015-03-13

    In microorganisms, Ion Torrent sequencing technology has been proved to be useful in whole-genome sequencing of bacterial genomes (5 Mbp). In our study, for the first time we used this technology to perform a resequencing approach in a whole fungal genome (36 Mbp), a non-ochratoxin A producing strain of Aspergillus carbonarius. Ochratoxin A (OTA) is a potent nephrotoxin which is found mainly in cereals and their products, but it also occurs in a variety of common foods and beverages. Due to the fact that this strain does not produce OTA, we focused some of the bioinformatics analyses in genes involvedmore » in OTA biosynthesis, using a reference genome of an OTA producing strain of the same species. This study revealed that in the atoxigenic strain there is a high accumulation of nonsense and missense mutations in several genes. Importantly, a two fold increase in gene mutation ratio was observed in PKS and NRPS encoding genes which are suggested to be involved in OTA biosynthesis.« less

  20. Rapid genome resequencing of an atoxigenic strain of Aspergillus carbonarius

    PubMed Central

    Cabañes, F. Javier; Sanseverino, Walter; Castellá, Gemma; Bragulat, M. Rosa; Cigliano, Riccardo Aiese; Sánchez, Armand

    2015-01-01

    In microorganisms, Ion Torrent sequencing technology has been proved to be useful in whole-genome sequencing of bacterial genomes (5 Mbp). In our study, for the first time we used this technology to perform a resequencing approach in a whole fungal genome (36 Mbp), a non-ochratoxin A producing strain of Aspergillus carbonarius. Ochratoxin A (OTA) is a potent nephrotoxin which is found mainly in cereals and their products, but it also occurs in a variety of common foods and beverages. Due to the fact that this strain does not produce OTA, we focused some of the bioinformatics analyses in genes involved in OTA biosynthesis, using a reference genome of an OTA producing strain of the same species. This study revealed that in the atoxigenic strain there is a high accumulation of nonsense and missense mutations in several genes. Importantly, a two fold increase in gene mutation ratio was observed in PKS and NRPS encoding genes which are suggested to be involved in OTA biosynthesis. PMID:25765923

  1. Rapid calculation of genomic evaluations for new animals

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A method was developed to calculate preliminary genomic evaluations daily or weekly before the release of official monthly evaluations by processing only newly genotyped animals using estimates of SNP effects from the previous official evaluation. To minimize computing time, reliabilities and genomi...

  2. CONTRAILS: A tool for rapid identification of transgene integration sites in complex, repetitive genomes using low-coverage paired-end sequencing

    PubMed Central

    Lambirth, Kevin C.; Whaley, Adam M.; Schlueter, Jessica A.; Bost, Kenneth L.; Piller, Kenneth J.

    2015-01-01

    Transgenic crops have become a staple in modern agriculture, and are typically characterized using a variety of molecular techniques involving proteomics and metabolomics. Characterization of the transgene insertion site is of great interest, as disruptions, deletions, and genomic location can affect product selection and fitness, and identification of these regions and their integrity is required for regulatory agencies. Here, we present CONTRAILS (Characterization of Transgene Insertion Locations with Sequencing), a straightforward, rapid and reproducible method for the identification of transgene insertion sites in highly complex and repetitive genomes using low coverage paired-end Illumina sequencing and traditional PCR. This pipeline requires little to no troubleshooting and is not restricted to any genome type, allowing use for many molecular applications. Using whole genome sequencing of in-house transgenic Glycine max, a legume with a highly repetitive and complex genome, we used CONTRAILS to successfully identify the location of a single T-DNA insertion to single base resolution. PMID:26697366

  3. CONTRAILS: A tool for rapid identification of transgene integration sites in complex, repetitive genomes using low-coverage paired-end sequencing.

    PubMed

    Lambirth, Kevin C; Whaley, Adam M; Schlueter, Jessica A; Bost, Kenneth L; Piller, Kenneth J

    2015-12-01

    Transgenic crops have become a staple in modern agriculture, and are typically characterized using a variety of molecular techniques involving proteomics and metabolomics. Characterization of the transgene insertion site is of great interest, as disruptions, deletions, and genomic location can affect product selection and fitness, and identification of these regions and their integrity is required for regulatory agencies. Here, we present CONTRAILS (Characterization of Transgene Insertion Locations with Sequencing), a straightforward, rapid and reproducible method for the identification of transgene insertion sites in highly complex and repetitive genomes using low coverage paired-end Illumina sequencing and traditional PCR. This pipeline requires little to no troubleshooting and is not restricted to any genome type, allowing use for many molecular applications. Using whole genome sequencing of in-house transgenic Glycine max, a legume with a highly repetitive and complex genome, we used CONTRAILS to successfully identify the location of a single T-DNA insertion to single base resolution. PMID:26697366

  4. Genomic characterization of recurrent high-grade astroblastoma.

    PubMed

    Bale, Tejus A; Abedalthagafi, Malak; Bi, Wenya Linda; Kang, Yun Jee; Merrill, Parker; Dunn, Ian F; Dubuc, Adrian; Charbonneau, Sarah K; Brown, Loreal; Ligon, Azra H; Ramkissoon, Shakti H; Ligon, Keith L

    2016-01-01

    Astroblastomas are rare primary brain tumors, diagnosed based on histologic features. Not currently assigned a WHO grade, they typically display indolent behavior, with occasional variants taking a more aggressive course. We characterized the immunohistochemical characteristics, copy number (high-resolution array comparative genomic hybridization, OncoCopy) and mutational profile (targeted next-generation exome sequencing, OncoPanel) of a cohort of seven biopsies from four patients to identify recurrent genomic events that may help distinguish astroblastomas from other more common high-grade gliomas. We found that tumor histology was variable across patients and between primary and recurrent tumor samples. No common molecular features were identified among the four tumors. Mutations commonly observed in astrocytic tumors (IDH1/2, TP53, ATRX, and PTEN) or ependymoma were not identified. However one case with rapid clinical progression displayed mutations more commonly associated with GBM (NF1(N1054H/K63)*, PIK3CA(R38H) and ERG(A403T)). Conversely, another case, originally classified as glioblastoma with nine-year survival before recurrence, lacked a GBM mutational profile. Other mutations frequently seen in lower grade gliomas (BCOR, BCORL1, ERBB3, MYB, ATM) were also present in several tumors. Copy number changes were variable across tumors. Our findings indicate that astroblastomas have variable growth patterns and morphologic features, posing significant challenges to accurate classification in the absence of diagnostically specific copy number alterations and molecular features. Their histopathologic overlap with glioblastoma will likely confound the observation of long-term GBM "survivors". Further genomic profiling is needed to determine whether these tumors represent a distinct entity and to guide management strategies. PMID:27425854

  5. The Arabidopsis lyrata genome sequence and the basis of rapid genome size change

    SciTech Connect

    Hu, Tina T.; Pattyn, Pedro; Bakker, Erica G.; Cao, Jun; Cheng, Jan-Fang; Clark, Richard M.; Fahlgren, Noah; Fawcett, Jeffrey A.; Grimwood, Jane; Gundlach, Heidrun; Haberer, Georg; Hollister, Jesse D.; Ossowski, Stephan; Ottilar, Robert P.; Salamov, Asaf A.; Schneeberger, Korbinian; Spannagl, Manuel; Wang, Xi; Yang, Liang; Nasrallah, Mikhail E.; Bergelson, Joy; Carrington, James C.; Gaut, Brandon S.; Schmutz, Jeremy; Mayer, Klaus F. X.; Van de Peer, Yves; Grigoriev, Igor V.; Nordborg, Magnus; Weigel, Detlef; Guo, Ya-Long

    2011-04-29

    In our manuscript, we present a high-quality genome sequence of the Arabidopsis thaliana relative, Arabidopsis lyrata, produced by dideoxy sequencing. We have performed the usual types of genome analysis (gene annotation, dN/dS studies etc. etc.), but this is relegated to the Supporting Information. Instead, we focus on what was a major motivation for sequencing this genome, namely to understand how A. thaliana lost half its genome in a few million years and lived to tell the tale. The rather surprising conclusion is that there is not a single genomic feature that accounts for the reduced genome, but that every aspect centromeres, intergenic regions, transposable elements, gene family number is affected through hundreds of thousands of cuts. This strongly suggests that overall genome size in itself is what has been under selection, a suggestion that is strongly supported by our demonstration (using population genetics data from A. thaliana) that new deletions seem to be driven to fixation.

  6. Eight New Genomes and Synthetic Controls Increase the Accessibility of Rapid Melt-MAMA SNP Typing of Coxiella burnetii

    PubMed Central

    Byström, Mona; Forsman, Mats; Frangoulidis, Dimitrios; Janse, Ingmar; Larsson, Pär; Lindgren, Petter; Öhrman, Caroline; van Rotterdam, Bart; Sjödin, Andreas; Myrtennäs, Kerstin

    2014-01-01

    The case rate of Q fever in Europe has increased dramatically in recent years, mainly because of an epidemic in the Netherlands in 2009. Consequently, there is a need for more extensive genetic characterization of the disease agent Coxiella burnetii in order to better understand the epidemiology and spread of this disease. Genome reference data are essential for this purpose, but only thirteen genome sequences are currently available. Current methods for typing C. burnetii are criticized for having problems in comparing results across laboratories, require the use of genomic control DNA, and/or rely on markers in highly variable regions. We developed in this work a method for single nucleotide polymorphism (SNP) typing of C. burnetii isolates and tissue samples based on new assays targeting ten phylogenetically stable synonymous canonical SNPs (canSNPs). These canSNPs represent previously known phylogenetic branches and were here identified from sequence comparisons of twenty-one C. burnetii genomes, eight of which were sequenced in this work. Importantly, synthetic control templates were developed, to make the method useful to laboratories lacking genomic control DNA. An analysis of twenty-one C. burnetii genomes confirmed that the species exhibits high sequence identity. Most of its SNPs (7,493/7,559 shared by >1 genome) follow a clonal inheritance pattern and are therefore stable phylogenetic typing markers. The assays were validated using twenty-six genetically diverse C. burnetii isolates and three tissue samples from small ruminants infected during the epidemic in the Netherlands. Each sample was assigned to a clade. Synthetic controls (vector and PCR amplified) gave identical results compared to the corresponding genomic controls and are viable alternatives to genomic DNA. The results from the described method indicate that it could be useful for cheap and rapid disease source tracking at non-specialized laboratories, which requires accurate genotyping

  7. Eight new genomes and synthetic controls increase the accessibility of rapid melt-MAMA SNP typing of Coxiella burnetii.

    PubMed

    Karlsson, Edvin; Macellaro, Anna; Byström, Mona; Forsman, Mats; Frangoulidis, Dimitrios; Janse, Ingmar; Larsson, Pär; Lindgren, Petter; Ohrman, Caroline; van Rotterdam, Bart; Sjödin, Andreas; Myrtennäs, Kerstin

    2014-01-01

    The case rate of Q fever in Europe has increased dramatically in recent years, mainly because of an epidemic in the Netherlands in 2009. Consequently, there is a need for more extensive genetic characterization of the disease agent Coxiella burnetii in order to better understand the epidemiology and spread of this disease. Genome reference data are essential for this purpose, but only thirteen genome sequences are currently available. Current methods for typing C. burnetii are criticized for having problems in comparing results across laboratories, require the use of genomic control DNA, and/or rely on markers in highly variable regions. We developed in this work a method for single nucleotide polymorphism (SNP) typing of C. burnetii isolates and tissue samples based on new assays targeting ten phylogenetically stable synonymous canonical SNPs (canSNPs). These canSNPs represent previously known phylogenetic branches and were here identified from sequence comparisons of twenty-one C. burnetii genomes, eight of which were sequenced in this work. Importantly, synthetic control templates were developed, to make the method useful to laboratories lacking genomic control DNA. An analysis of twenty-one C. burnetii genomes confirmed that the species exhibits high sequence identity. Most of its SNPs (7,493/7,559 shared by >1 genome) follow a clonal inheritance pattern and are therefore stable phylogenetic typing markers. The assays were validated using twenty-six genetically diverse C. burnetii isolates and three tissue samples from small ruminants infected during the epidemic in the Netherlands. Each sample was assigned to a clade. Synthetic controls (vector and PCR amplified) gave identical results compared to the corresponding genomic controls and are viable alternatives to genomic DNA. The results from the described method indicate that it could be useful for cheap and rapid disease source tracking at non-specialized laboratories, which requires accurate genotyping

  8. Use of Unamplified RNA/cDNA–Hybrid Nanopore Sequencing for Rapid Detection and Characterization of RNA Viruses

    PubMed Central

    Kilianski, Andy; Roth, Pierce A.; Liem, Alvin T.; Hill, Jessica M.; Willis, Kristen L.; Rossmaier, Rebecca D.; Marinich, Andrew V.; Maughan, Michele N.; Karavis, Mark A.; Kuhn, Jens H.; Honko, Anna N.

    2016-01-01

    Nanopore sequencing, a novel genomics technology, has potential applications for routine biosurveillance, clinical diagnosis, and outbreak investigation of virus infections. Using rapid sequencing of unamplified RNA/cDNA hybrids, we identified Venezuelan equine encephalitis virus and Ebola virus in 3 hours from sample receipt to data acquisition, demonstrating a fieldable technique for RNA virus characterization. PMID:27191483

  9. Use of Unamplified RNA/cDNA-Hybrid Nanopore Sequencing for Rapid Detection and Characterization of RNA Viruses.

    PubMed

    Kilianski, Andy; Roth, Pierce A; Liem, Alvin T; Hill, Jessica M; Willis, Kristen L; Rossmaier, Rebecca D; Marinich, Andrew V; Maughan, Michele N; Karavis, Mark A; Kuhn, Jens H; Honko, Anna N; Rosenzweig, C Nicole

    2016-08-01

    Nanopore sequencing, a novel genomics technology, has potential applications for routine biosurveillance, clinical diagnosis, and outbreak investigation of virus infections. Using rapid sequencing of unamplified RNA/cDNA hybrids, we identified Venezuelan equine encephalitis virus and Ebola virus in 3 hours from sample receipt to data acquisition, demonstrating a fieldable technique for RNA virus characterization. PMID:27191483

  10. Rapid evolution of cheating mitochondrial genomes in small yeast populations.

    PubMed

    Jasmin, Jean-Nicolas; Zeyl, Clifford

    2014-01-01

    Outcrossed sex exposes genes to competition with their homologues, allowing alleles that transmit more often than their competitors to spread despite organismal fitness costs. Mitochondrial populations in species with biparental inheritance are thought to be especially susceptible to such cheaters because they lack strict transmission rules like meiosis or maternal inheritance. Yet the interaction between mutation and natural selection in the evolution of cheating mitochondrial genomes has not been tested experimentally. Using yeast experimental populations, we show that although cheaters were rare in a large sample of spontaneous respiratory-deficient mitochondrial mutations (petites), cheaters evolve under experimentally enforced outcrossing even when mutation supply and selection are restricted by repeatedly bottlenecking populations.

  11. A BIOINFORMATIC STRATEGY TO RAPIDLY CHARACTERIZE CDNA LIBRARIES

    EPA Science Inventory

    A Bioinformatic Strategy to Rapidly Characterize cDNA Libraries

    G. Charles Ostermeier1, David J. Dix2 and Stephen A. Krawetz1.
    1Departments of Obstetrics and Gynecology, Center for Molecular Medicine and Genetics, & Institute for Scientific Computing, Wayne State Univer...

  12. Rapid Whole-Genome Sequencing for Genetic Disease Diagnosis in Neonatal Intensive Care Units

    PubMed Central

    Saunders, Carol Jean; Miller, Neil Andrew; Soden, Sarah Elizabeth; Dinwiddie, Darrell Lee; Noll, Aaron; Alnadi, Noor Abu; Andraws, Nevene; Patterson, Melanie LeAnn; Krivohlavek, Lisa Ann; Fellis, Joel; Humphray, Sean; Saffrey, Peter; Kingsbury, Zoya; Weir, Jacqueline Claire; Betley, Jason; Grocock, Russell James; Margulies, Elliott Harrison; Farrow, Emily Gwendolyn; Artman, Michael; Safina, Nicole Pauline; Petrikin, Joshua Erin; Hall, Kevin Peter; Kingsmore, Stephen Francis

    2014-01-01

    Monogenic diseases are frequent causes of neonatal morbidity and mortality, and disease presentations are often undifferentiated at birth. More than 3500 monogenic diseases have been characterized, but clinical testing is available for only some of them and many feature clinical and genetic heterogeneity. Hence, an immense unmet need exists for improved molecular diagnosis in infants. Because disease progression is extremely rapid, albeit heterogeneous, in newborns, molecular diagnoses must occur quickly to be relevant for clinical decision-making. We describe 50-hour differential diagnosis of genetic disorders by whole-genome sequencing (WGS) that features automated bioinformatic analysis and is intended to be a prototype for use in neonatal intensive care units. Retrospective 50-hour WGS identified known molecular diagnoses in two children. Prospective WGS disclosed potential molecular diagnosis of a severe GJB2-related skin disease in one neonate; BRAT1-related lethal neonatal rigidity and multifocal seizure syndrome in another infant; identified BCL9L as a novel, recessive visceral heterotaxy gene (HTX6) in a pedigree; and ruled out known candidate genes in one infant. Sequencing of parents or affected siblings expedited the identification of disease genes in prospective cases. Thus, rapid WGS can potentially broaden and foreshorten differential diagnosis, resulting in fewer empirical treatments and faster progression to genetic and prognostic counseling. PMID:23035047

  13. Rapid characterization of fuel atomizers using an optical patternator

    SciTech Connect

    Sankar, S.V.; Maher, K.E.; Robart, D.M.; Bachalo, W.D.

    1999-07-01

    Planar laser scattering (PLS) and planar laser-induced fluorescence (PLIF) techniques are currently being used for rapid characterization of fuel sprays associated with gas turbine atomizers, diesel injectors, and automotive fuel injectors. These techniques can be used for qualitative, quantitative, and rapid measurement of fuel mass, spray geometry, and Sauter mean diameters in various sprays. The spatial distribution of the fuel mass can be inferred directly from the PLIF image, and the Sauter mean diameter can be measured by simultaneously recording the PLIF and PLS images and then rationing the two. A spray characterization system incorporating the PLS and/or PLIF techniques has been loosely termed an optical patternator, and in this study, it has been used to characterize both steady and pulsed sprays. The results obtained with the optical patternator have been directly validated using a phase Doppler particle analyzer (PDPA).

  14. Characterizing allelic association in the genome era

    PubMed Central

    WEIR, B. S.; LAURIE, C. C.

    2015-01-01

    Summary Whole genome data are allowing the estimation of population genetic parameters with an accuracy not imagined 50 years ago. Variation in these parameters along the genome is being found empirically where once only approximate theoretical values were available. Along with increased information, however, has come the issue of multiple testing and the realization that high values of the coefficients of variation of quantities such as relatedness measures may make it difficult to draw inferences. This review concentrates on measures of allelic association within and between individuals and within and between populations. PMID:21429275

  15. Polyploid formation in cotton is not accompanied by rapid genomic changes.

    PubMed

    Liu, B; Brubaker, C L; Mergeai, G; Cronn, R C; Wendel, J F

    2001-06-01

    Recent work has demonstrated that allopolyploid speciation in plants may be associated with non-Mendelian genomic changes in the early generations following polyploid synthesis. To address the question of whether rapid genomic changes also occur in allopolyploid cotton (Gossypium) species, amplified fragment length polymorphism (AFLP) analysis was performed to evaluate nine sets of newly synthesized allotetraploid and allohexaploid plants, their parents, and the selfed progeny from colchicine-doubled synthetics. Using both methylation-sensitive and methylation-insensitive enzymes, the extent of fragment additivity in newly combined genomes was ascertained for a total of approximately 22,000 genomic loci. Fragment additivity was observed in nearly all cases, with the few exceptions most likely reflecting parental heterozygosity or experimental error. In addition, genomic Southern analysis on six sets of synthetic allopolyploids probed with five retrotransposons also revealed complete additivity. Because no alterations were observed using methylation-sensitive isoschizomers, epigenetic changes following polyploid synthesis were also minimal. These indications of genomic additivity and epigenetic stasis during allopolyploid formation provide a contrast to recent evidence from several model plant allopolyploids, most notably wheat and Brassica, where rapid and unexplained genomic changes have been reported. In addition, the data contrast with evidence from repetitive DNAs in Gossypium, some of which are subject to non-Mendelian molecular evolutionary phenomena in extant polyploids. These contrasts indicate polyploid speciation in plants is accompanied by a diverse array of molecular evolutionary phenomena, which will vary among both genomic constituents and taxa. PMID:11444689

  16. Rapid detection by multiplex PCR of Genomic Islands, prophages and Integrative Conjugative Elements in V. cholerae 7th pandemic variants.

    PubMed

    Spagnoletti, Matteo; Ceccarelli, Daniela; Colombo, Mauro M

    2012-01-01

    Vibrio cholerae poses a threat to human health, and new epidemic variants have been reported so far. Seventh pandemic V. cholerae strains are characterized by highly related genomic sequences but can be discriminated by a large set of Genomic Islands, phages and Integrative Conjugative Elements. Classical serotyping and biotyping methods do not easily discriminate among new variants arising worldwide, therefore the establishment of new methods for their identification is required. We developed a multiplex PCR assay for the rapid detection of the major 7th pandemic variants of V. cholerae O1 and O139. Three specific genomic islands (GI-12, GI-14 and GI-15), two phages (Kappa and TLC), Vibrio Seventh Pandemic Island 2 (VSP-II), and the ICEs of the SXT/R391 family were selected as targets of our multiplex PCR based on a comparative genomic approach. The optimization and specificity of the multiplex PCR was assessed on 5 V. cholerae 7th pandemic reference strains, and other 34 V. cholerae strains from various epidemic events were analyzed to validate the reliability of our method. This assay had sufficient specificity to identify twelve different V. cholerae genetic profiles, and therefore has the potential to be used as a rapid screening method.

  17. Nuclear DNA content in Sinningia (Gesneriaceae); intraspecific genome size variation and genome characterization in S. speciosa.

    PubMed

    Zaitlin, David; Pierce, Andrew J

    2010-12-01

    The Gesneriaceae (Lamiales) is a family of flowering plants comprising >3000 species of mainly tropical origin, the most familiar of which is the cultivated African violet (Saintpaulia spp.). Species of Gesneriaceae are poorly represented in the lists of taxa sampled for genome size estimation; measurements are available for three species of Ramonda and one each of Haberlea, Saintpaulia, and Streptocarpus, all species of Old World origin. We report here nuclear genome size estimates for 10 species of Sinningia, a neotropical genus largely restricted to Brazil. Flow cytometry of leaf cell nuclei showed that holoploid genome size in Sinningia is very small (approximately two times the size of the Arabidopsis genome), and is small compared to the other six species of Gesneriaceae with genome size estimates. We also documented intraspecific genome size variation of 21%-26% within a group of wild Sinningia speciosa (Lodd.) Hiern collections. In addition, we analyzed 1210 genome survey sequences from S. speciosa to characterize basic features of the nuclear genome such as guanine-cytosine content, types of repetitive elements, numbers of protein-coding sequences, and sequences unique to S. speciosa. We included several other angiosperm species as genome size standards, one of which was the snapdragon (Antirrhinum majus L.; Veronicaceae, Lamiales). Multiple measurements on three accessions indicated that the genome size of A. majus is ~633 × 10⁶ base pairs, which is approximately 40% of the previously published estimate. PMID:21164539

  18. Nuclear DNA content in Sinningia (Gesneriaceae); intraspecific genome size variation and genome characterization in S. speciosa.

    PubMed

    Zaitlin, David; Pierce, Andrew J

    2010-12-01

    The Gesneriaceae (Lamiales) is a family of flowering plants comprising >3000 species of mainly tropical origin, the most familiar of which is the cultivated African violet (Saintpaulia spp.). Species of Gesneriaceae are poorly represented in the lists of taxa sampled for genome size estimation; measurements are available for three species of Ramonda and one each of Haberlea, Saintpaulia, and Streptocarpus, all species of Old World origin. We report here nuclear genome size estimates for 10 species of Sinningia, a neotropical genus largely restricted to Brazil. Flow cytometry of leaf cell nuclei showed that holoploid genome size in Sinningia is very small (approximately two times the size of the Arabidopsis genome), and is small compared to the other six species of Gesneriaceae with genome size estimates. We also documented intraspecific genome size variation of 21%-26% within a group of wild Sinningia speciosa (Lodd.) Hiern collections. In addition, we analyzed 1210 genome survey sequences from S. speciosa to characterize basic features of the nuclear genome such as guanine-cytosine content, types of repetitive elements, numbers of protein-coding sequences, and sequences unique to S. speciosa. We included several other angiosperm species as genome size standards, one of which was the snapdragon (Antirrhinum majus L.; Veronicaceae, Lamiales). Multiple measurements on three accessions indicated that the genome size of A. majus is ~633 × 10⁶ base pairs, which is approximately 40% of the previously published estimate.

  19. Pioglitazone rapidly reduces neuropathic pain through astrocyte and non-genomic PPARγ mechanisms

    PubMed Central

    Griggs, Ryan B.; Donahue, Renee R.; Morgenweck, Jenny; Grace, Peter M.; Sutton, Amanda; Watkins, Linda R.; Taylor, Bradley K.

    2014-01-01

    Repeated administration of peroxisome proliferator-activated receptor gamma (PPARγ) agonists reduces neuropathic pain-like behavior and associated changes in glial activation in the spinal cord dorsal horn. As PPARγ is a nuclear receptor, sustained changes in gene expression are widely believed to be the mechanism of pain reduction. However, we recently reported that a single intrathecal injection of pioglitazone, a PPARγ agonist, reduced hyperalgesia within 30 minutes, a time frame that is typically less than that required for genomic mechanisms. To determine the very rapid anti-hyperalgesic actions of PPARγ activation we administered pioglitazone to rats with spared nerve injury (SNI) and evaluated hyperalgesia. Pioglitazone inhibited hyperalgesia within 5 min of injection, consistent with a non-genomic mechanism. Systemic or intrathecal administration of GW9662, a PPARγ antagonist, inhibited the anti-hyperalgesic actions of intraperitoneal or intrathecal pioglitazone, suggesting a spinal PPARγ-dependent mechanism. To further address the contribution of non-genomic mechanisms, we blocked new protein synthesis in the spinal cord with anisomycin. When co-administered intrathecally, anisomycin did not change pioglitazone anti-hyperalgesia at an early 7.5 min timepoint, further supporting a rapid non-genomic mechanism. At later timepoints anisomycin reduced pioglitazone anti-hyperalgesia, suggesting a delayed recruitment of genomic mechanisms. Pioglitazone reduction of SNI-induced increases in GFAP expression occurred more rapidly than expected, within 60 min. We are the first to show that activation of spinal PPARγ rapidly reduces neuropathic pain independent from canonical genomic activity. We conclude that acute pioglitazone inhibits neuropathic pain in part by reducing astrocyte activation, and via both genomic and non-genomic PPARγ mechanisms. PMID:25599238

  20. Characterizing genomic alterations in cancer by complementary functional associations

    PubMed Central

    Kim, J. W.; Botvinnik, O. B.; Abudayyeh, O.; Birger, C.; Rosenbluh, J.; Shrestha, Y.; Abazeed, M. E.; Hammerman, P. S.; DiCara, D.; Konieczkowski, D. J.; Johannessen, C. M.; Liberzon, A.; Alizad-Rahvar, A. R.; Alexe, G.; Aguirre, A.; Ghandi, M.; Greulich, H.; Vazquez, F.; Weir, B. A.; Van Allen, E. M.; Tsherniak, A.; Shao, D. D.; Zack, T. I.; Noble, M.; Getz, G.; Beroukhim, R.; Garraway, L. A.; Ardakani, M.; Romualdi, C.; Sales, G.; Barbie, D. A.; Boehm, J. S.; Hahn, W. C.; Mesirov, J. P.; Tamayo, P.

    2016-01-01

    Systematic efforts to sequence the cancer genome have identified large numbers of relevant mutations and copy number alterations in human cancers; however, elucidating their functional consequences, and their interactions to drive or maintain oncogenic states, is still a significant challenge. Here we introduce REVEALER, a computational method that identifies combinations of mutually exclusive genomic alterations correlated with functional phenotypes, such as the activation or gene-dependency of oncogenic pathways or the sensitivity to a drug treatment. We use REVEALER to uncover complementary genomic alterations associated with the transcriptional activation of β-catenin and NRF2, MEK-inhibitor sensitivity, and KRAS dependency. REVEALER successfully identified both known and new associations demonstrating the power of combining functional profiles with extensive characterization of genomic alterations in cancer genomes. PMID:27088724

  1. Rapid genome-scale mapping of chromatin accessibility in tissue

    PubMed Central

    2012-01-01

    Background The challenge in extracting genome-wide chromatin features from limiting clinical samples poses a significant hurdle in identification of regulatory marks that impact the physiological or pathological state. Current methods that identify nuclease accessible chromatin are reliant on large amounts of purified nuclei as starting material. This complicates analysis of trace clinical tissue samples that are often stored frozen. We have developed an alternative nuclease based procedure to bypass nuclear preparation to interrogate nuclease accessible regions in frozen tissue samples. Results Here we introduce a novel technique that specifically identifies Tissue Accessible Chromatin (TACh). The TACh method uses pulverized frozen tissue as starting material and employs one of the two robust endonucleases, Benzonase or Cyansase, which are fully active under a range of stringent conditions such as high levels of detergent and DTT. As a proof of principle we applied TACh to frozen mouse liver tissue. Combined with massive parallel sequencing TACh identifies accessible regions that are associated with euchromatic features and accessibility at transcriptional start sites correlates positively with levels of gene transcription. Accessible chromatin identified by TACh overlaps to a large extend with accessible chromatin identified by DNase I using nuclei purified from freshly isolated liver tissue as starting material. The similarities are most pronounced at highly accessible regions, whereas identification of less accessible regions tends to be more divergence between nucleases. Interestingly, we show that some of the differences between DNase I and Benzonase relate to their intrinsic sequence biases and accordingly accessibility of CpG islands is probed more efficiently using TACh. Conclusion The TACh methodology identifies accessible chromatin derived from frozen tissue samples. We propose that this simple, robust approach can be applied across a broad range of

  2. Selection on rapidly evolving proteins in the Arabidopsis genome.

    PubMed Central

    Barrier, Marianne; Bustamante, Carlos D; Yu, Jiaye; Purugganan, Michael D

    2003-01-01

    Genes that have undergone positive or diversifying selection are likely to be associated with adaptive divergence between species. One indicator of adaptive selection at the molecular level is an excess of amino acid replacement fixed differences per replacement site relative to the number of synonymous fixed differences per synonymous site (omega = K(a)/K(s)). We used an evolutionary expressed sequence tag (EST) approach to estimate the distribution of omega among 304 orthologous loci between Arabidopsis thaliana and A. lyrata to identify genes potentially involved in the adaptive divergence between these two Brassicaceae species. We find that 14 of 304 genes (approximately 5%) have an estimated omega > 1 and are candidates for genes with increased selection intensities. Molecular population genetic analyses of 6 of these rapidly evolving protein loci indicate that, despite their high levels of between-species nonsynonymous divergence, these genes do not have elevated levels of intraspecific replacement polymorphisms compared to previously studied genes. A hierarchical Bayesian analysis of protein-coding region evolution within and between species also indicates that the selection intensities of these genes are elevated compared to previously studied A. thaliana nuclear loci. PMID:12618409

  3. Genomic characterization of Italian Clostridium botulinum group I strains.

    PubMed

    Giordani, Francesco; Fillo, Silvia; Anselmo, Anna; Palozzi, Anna Maria; Fortunato, Antonella; Gentile, Bernardina; Azarnia Tehran, Domenico; Ciammaruconi, Andrea; Spagnolo, Ferdinando; Pittiglio, Valentina; Anniballi, Fabrizio; Auricchio, Bruna; De Medici, Dario; Lista, Florigio

    2015-12-01

    Clostridium botulinum is a gram-positive bacterium capable of producing the botulinum neurotoxin, a powerful poison that causes botulism, a severe neuroparalytic disease. Its genome has been sequenced entirely and its gene content has been analyzed. To date, 19 full genomes and 64 draft genomes are available. The geographical origin of these genomes is predominantly from the US. In the present study, 10 Italian genomes of C. botulinum group I were analyzed and compared with previously sequenced group I genomes, in order to genetically characterize the Italian population of C. botulinum group I and to investigate the phylogenetic relationships among different lineages. Using the suites of software ClonalFrame and ClonalOrigin to perform genomic analysis, we demonstrated that Italian C. botulinum group I population is phylogenetically heterogeneous encompassing different and distant lineages including overseas strains, too. Moreover, a high recombination rate was demonstrated in the evolution of C. botulinum group I species. Finally, genome sequencing of the strain 357 led us to identify a novel botulinum neurotoxin subtype, F8. PMID:26341861

  4. Integrated Genomic Characterization of Papillary Thyroid Carcinoma

    PubMed Central

    Agrawal, Nishant; Akbani, Rehan; Aksoy, B. Arman; Ally, Adrian; Arachchi, Harindra; Asa, Sylvia L.; Auman, J. Todd; Balasundaram, Miruna; Balu, Saianand; Baylin, Stephen B.; Behera, Madhusmita; Bernard, Brady; Beroukhim, Rameen; Bishop, Justin A.; Black, Aaron D.; Bodenheimer, Tom; Boice, Lori; Bootwalla, Moiz S.; Bowen, Jay; Bowlby, Reanne; Bristow, Christopher A.; Brookens, Robin; Brooks, Denise; Bryant, Robert; Buda, Elizabeth; Butterfield, Yaron S.N.; Carling, Tobias; Carlsen, Rebecca; Carter, Scott L.; Carty, Sally E.; Chan, Timothy A.; Chen, Amy Y.; Cherniack, Andrew D.; Cheung, Dorothy; Chin, Lynda; Cho, Juok; Chu, Andy; Chuah, Eric; Cibulskis, Kristian; Ciriello, Giovanni; Clarke, Amanda; Clayman, Gary L.; Cope, Leslie; Copland, John; Covington, Kyle; Danilova, Ludmila; Davidsen, Tanja; Demchok, John A.; DiCara, Daniel; Dhalla, Noreen; Dhir, Rajiv; Dookran, Sheliann S.; Dresdner, Gideon; Eldridge, Jonathan; Eley, Greg; El-Naggar, Adel K.; Eng, Stephanie; Fagin, James A.; Fennell, Timothy; Ferris, Robert L.; Fisher, Sheila; Frazer, Scott; Frick, Jessica; Gabriel, Stacey B.; Ganly, Ian; Gao, Jianjiong; Garraway, Levi A.; Gastier-Foster, Julie M.; Getz, Gad; Gehlenborg, Nils; Ghossein, Ronald; Gibbs, Richard A.; Giordano, Thomas J.; Gomez-Hernandez, Karen; Grimsby, Jonna; Gross, Benjamin; Guin, Ranabir; Hadjipanayis, Angela; Harper, Hollie A.; Hayes, D. Neil; Heiman, David I.; Herman, James G.; Hoadley, Katherine A.; Hofree, Matan; Holt, Robert A.; Hoyle, Alan P.; Huang, Franklin W.; Huang, Mei; Hutter, Carolyn M.; Ideker, Trey; Iype, Lisa; Jacobsen, Anders; Jefferys, Stuart R.; Jones, Corbin D.; Jones, Steven J.M.; Kasaian, Katayoon; Kebebew, Electron; Khuri, Fadlo R.; Kim, Jaegil; Kramer, Roger; Kreisberg, Richard; Kucherlapati, Raju; Kwiatkowski, David J.; Ladanyi, Marc; Lai, Phillip H.; Laird, Peter W.; Lander, Eric; Lawrence, Michael S.; Lee, Darlene; Lee, Eunjung; Lee, Semin; Lee, William; Leraas, Kristen M.; Lichtenberg, Tara M.; Lichtenstein, Lee; Lin, Pei; Ling, Shiyun; Liu, Jinze; Liu, Wenbin; Liu, Yingchun; LiVolsi, Virginia A.; Lu, Yiling; Ma, Yussanne; Mahadeshwar, Harshad S.; Marra, Marco A.; Mayo, Michael; McFadden, David G.; Meng, Shaowu; Meyerson, Matthew; Mieczkowski, Piotr A.; Miller, Michael; Mills, Gordon; Moore, Richard A.; Mose, Lisle E.; Mungall, Andrew J.; Murray, Bradley A.; Nikiforov, Yuri E.; Noble, Michael S.; Ojesina, Akinyemi I.; Owonikoko, Taofeek K.; Ozenberger, Bradley A.; Pantazi, Angeliki; Parfenov, Michael; Park, Peter J.; Parker, Joel S.; Paull, Evan O.; Pedamallu, Chandra Sekhar; Perou, Charles M.; Prins, Jan F.; Protopopov, Alexei; Ramalingam, Suresh S.; Ramirez, Nilsa C.; Ramirez, Ricardo; Raphael, Benjamin J.; Rathmell, W. Kimryn; Ren, Xiaojia; Reynolds, Sheila M.; Rheinbay, Esther; Ringel, Matthew D.; Rivera, Michael; Roach, Jeffrey; Robertson, A. Gordon; Rosenberg, Mara W.; Rosenthall, Matthew; Sadeghi, Sara; Saksena, Gordon; Sander, Chris; Santoso, Netty; Schein, Jacqueline E.; Schultz, Nikolaus; Schumacher, Steven E.; Seethala, Raja R.; Seidman, Jonathan; Senbabaoglu, Yasin; Seth, Sahil; Sharpe, Samantha; Mills Shaw, Kenna R.; Shen, John P.; Shen, Ronglai; Sherman, Steven; Sheth, Margi; Shi, Yan; Shmulevich, Ilya; Sica, Gabriel L.; Simons, Janae V.; Sipahimalani, Payal; Smallridge, Robert C.; Sofia, Heidi J.; Soloway, Matthew G.; Song, Xingzhi; Sougnez, Carrie; Stewart, Chip; Stojanov, Petar; Stuart, Joshua M.; Tabak, Barbara; Tam, Angela; Tan, Donghui; Tang, Jiabin; Tarnuzzer, Roy; Taylor, Barry S.; Thiessen, Nina; Thorne, Leigh; Thorsson, Vésteinn; Tuttle, R. Michael; Umbricht, Christopher B.; Van Den Berg, David J.; Vandin, Fabio; Veluvolu, Umadevi; Verhaak, Roel G.W.; Vinco, Michelle; Voet, Doug; Walter, Vonn; Wang, Zhining; Waring, Scot; Weinberger, Paul M.; Weinstein, John N.; Weisenberger, Daniel J.; Wheeler, David; Wilkerson, Matthew D.; Wilson, Jocelyn; Williams, Michelle; Winer, Daniel A.; Wise, Lisa; Wu, Junyuan; Xi, Liu; Xu, Andrew W.; Yang, Liming; Yang, Lixing; Zack, Travis I.; Zeiger, Martha A.; Zeng, Dong; Zenklusen, Jean Claude; Zhao, Ni; Zhang, Hailei; Zhang, Jianhua; Zhang, Jiashan (Julia); Zhang, Wei; Zmuda, Erik; Zou., Lihua

    2014-01-01

    Summary Papillary thyroid carcinoma (PTC) is the most common type of thyroid cancer. Here, we describe the genomic landscape of 496 PTCs. We observed a low frequency of somatic alterations (relative to other carcinomas) and extended the set of known PTC driver alterations to include EIF1AX, PPM1D and CHEK2 and diverse gene fusions. These discoveries reduced the fraction of PTC cases with unknown oncogenic driver from 25% to 3.5%. Combined analyses of genomic variants, gene expression, and methylation demonstrated that different driver groups lead to different pathologies with distinct signaling and differentiation characteristics. Similarly, we identified distinct molecular subgroups of BRAF-mutant tumors and multidimensional analyses highlighted a potential involvement of oncomiRs in less-differentiated subgroups. Our results propose a reclassification of thyroid cancers into molecular subtypes that better reflect their underlying signaling and differentiation properties, which has the potential to improve their pathological classification and better inform the management of the disease. PMID:25417114

  5. Rapid honey characterization and botanical classification by an electronic tongue.

    PubMed

    Major, Nikola; Marković, Ksenija; Krpan, Marina; Sarić, Goran; Hruškar, Mirjana; Vahčić, Nada

    2011-07-15

    In this paper a commercial electronic tongue (αAstree, Alpha M.O.S.) was applied for botanical classification and physicochemical characterization of honey samples. The electronic tongue was comprised of seven potentiometric sensors coupled with an Ag/AgCl reference electrode. Botanical classification was performed by PCA, CCA and ANN modeling on 12 samples of acacia, chestnut and honeydew honey. The physicochemical characterization of honey was obtained by ANN modeling and the parameters included were electrical conductivity, acidity, water content, invert sugar and total sugar. The initial reference values for the physicochemical parameters observed were determined by traditional methods. Botanical classification of honey samples obtained by ANN was 100% accurate while the highest correlation between observed and predicted values was obtained for electrical conductivity (0.999), followed by acidity (0.997), water content (0.994), invert sugar content (0.988) and total sugar content (0.979). All developed ANN models for rapid honey characterization and botanical classification performed excellently showing the potential of the electronic tongue as a tool in rapid honey analysis and characterization. The advantage of using such a technique is a simple sample preparation procedure, there are no chemicals involved and there are no additional costs except the initial measurements required for ANN model development.

  6. Dexamethasone rapidly inhibits glucose uptake via non-genomic mechanisms in contracting myotubes.

    PubMed

    Gong, Hong; Liu, Lei; Ni, Chen-Xu; Zhang, Yi; Su, Wen-Jun; Lian, Yong-Jie; Peng, Wei; Zhang, Jun-Ping; Jiang, Chun-Lei

    2016-08-01

    Glucocorticoids (GCs) are a class of steroid hormones that regulate multiple aspects of glucose homeostasis. In skeletal muscle, it is well established that prolonged GC excess inhibits glucose uptake and utilization through glucocorticoid receptor (GR)-mediated transcriptional changes. However, it remains obscure that whether the rapid non-genomic effects of GC on glucose uptake are involved in acute exercise stress. Therefore, we used electric pulse stimulation (EPS)-evoked contracting myotubes to determine whether the non-genomic actions of GC were involved and its underlying mechanism(s). Pretreatment with dexamethasone (Dex, 10 μM) significantly prevented contraction-stimulated glucose uptake and glucose transporter 4 (Glut4) translocation within 20 min in C2C12 myotubes. Neither GC nuclear receptor antagonist (RU486) nor protein synthesis inhibitor (cycloheximide, Chx) affected the rapid inhibition effects of Dex. AMPK and CaMKII-dependent signaling pathways were associated with the non-genomic effects of Dex. These results provide evidence that GC rapidly suppresses glucose uptake in contracting myotubes via GR-independent non-genomic mechanisms. AMPK and CaMKII-mediated Glut4 translocation may play a critical role in GC-induced rapid inhibition of glucose uptake. PMID:27246478

  7. Draft Genome Sequence of Mycobacterium wolinskyi, a Rapid-Growing Species of Nontuberculous Mycobacteria.

    PubMed

    de Man, Tom J B; Perry, K Allison; Lawsin, Adrian; Coulliette, Angela D; Jensen, Bette; Toney, Nadege C; Limbago, Brandi M; Noble-Wang, Judith

    2016-01-01

    Mycobacterium wolinskyi is a nonpigmented, rapidly growing nontuberculous mycobacterium species that is associated with bacteremia, peritonitis, infections associated with implants/prostheses, and skin and soft tissue infections often following surgical procedures in humans. Here, we report the first functionally annotated draft genome sequence of M. wolinskyi CDC_01. PMID:26988052

  8. Rapid Characterization of Shorelines using a Georeferenced Video Mapping System

    SciTech Connect

    Anderson, Michael G.; Judd, Chaeli; Marcoe, K.

    2012-09-01

    Increased understanding of shoreline conditions is needed, yet current approaches are limited in ability to characterize remote areas or document features at a finer resolution. Documentation using video mapping may provide a rapid and repeatable method for assessing the current state of the environment and determining changes to the shoreline over time. In this study, we compare two studies using boat-based, georeferenced video mapping in coastal Washington and the Columbia River Estuary to map and characterize coastal stressors and functional data. In both areas, mapping multiple features along the shoreline required approximation of the coastline. However, characterization of vertically oriented features such as shoreline armoring and small features such as pilings and large woody debris was possible. In addition, end users noted that geovideo provides a permanent record to allow a user to examine recorded video anywhere along a transect or at discrete points.

  9. Characterizing the citrus cultivar Carrizo genome through 454 shotgun sequencing.

    PubMed

    Belknap, William R; Wang, Yi; Huo, Naxin; Wu, Jiajie; Rockhold, David R; Gu, Yong Q; Stover, Ed

    2011-12-01

    The citrus cultivar Carrizo is the single most important rootstock to the US citrus industry and has resistance or tolerance to a number of major citrus diseases, including citrus tristeza virus, foot rot, and Huanglongbing (HLB, citrus greening). A Carrizo genomic sequence database providing approximately 3.5×genome coverage (haploid genome size approximately 367 Mb) was populated through 454 GS FLX shotgun sequencing. Analysis of the repetitive DNA fraction indicated a total interspersed repeat fraction of 36.5%. Assembly and characterization of abundant citrus Ty3/gypsy elements revealed a novel type of element containing open reading frames encoding a viral RNA-silencing suppressor protein (RNA binding protein, rbp) and a plant cytokinin riboside 5′-monophosphate phosphoribohydrolase-related protein (LONELY GUY, log). Similar gypsy elements were identified in the Populus trichocarpa genome. Gene-coding region analysis indicated that 24.4% of the nonrepetitive reads contained genic regions. The depth of genome coverage was sufficient to allow accurate assembly of constituent genes, including a putative phloem-expressed gene. The development of the Carrizo database (http://citrus.pw.usda.gov/) will contribute to characterization of agronomically significant loci and provide a publicly available genomic resource to the citrus research community.

  10. Characterizing the citrus cultivar Carrizo genome through 454 shotgun sequencing.

    PubMed

    Belknap, William R; Wang, Yi; Huo, Naxin; Wu, Jiajie; Rockhold, David R; Gu, Yong Q; Stover, Ed

    2011-12-01

    The citrus cultivar Carrizo is the single most important rootstock to the US citrus industry and has resistance or tolerance to a number of major citrus diseases, including citrus tristeza virus, foot rot, and Huanglongbing (HLB, citrus greening). A Carrizo genomic sequence database providing approximately 3.5×genome coverage (haploid genome size approximately 367 Mb) was populated through 454 GS FLX shotgun sequencing. Analysis of the repetitive DNA fraction indicated a total interspersed repeat fraction of 36.5%. Assembly and characterization of abundant citrus Ty3/gypsy elements revealed a novel type of element containing open reading frames encoding a viral RNA-silencing suppressor protein (RNA binding protein, rbp) and a plant cytokinin riboside 5′-monophosphate phosphoribohydrolase-related protein (LONELY GUY, log). Similar gypsy elements were identified in the Populus trichocarpa genome. Gene-coding region analysis indicated that 24.4% of the nonrepetitive reads contained genic regions. The depth of genome coverage was sufficient to allow accurate assembly of constituent genes, including a putative phloem-expressed gene. The development of the Carrizo database (http://citrus.pw.usda.gov/) will contribute to characterization of agronomically significant loci and provide a publicly available genomic resource to the citrus research community. PMID:22133378

  11. Evidence from phylogenetic and genome fingerprinting analyses suggests rapidly changing variation in Halorubrum and Haloarcula populations

    PubMed Central

    Ram Mohan, Nikhil; Fullmer, Matthew S.; Makkay, Andrea M.; Wheeler, Ryan; Ventosa, Antonio; Naor, Adit; Gogarten, J. Peter; Papke, R. Thane

    2014-01-01

    Halobacteria require high NaCl concentrations for growth and are the dominant inhabitants of hypersaline environments above 15% NaCl. They are well-documented to be highly recombinogenic, both in frequency and in the range of exchange partners. In this study, we examine the genetic and genomic variation of cultured, naturally co-occurring environmental populations of Halobacteria. Sequence data from multiple loci (~2500 bp) identified many closely and more distantly related strains belonging to the genera Halorubrum and Haloarcula. Genome fingerprinting using a random priming PCR amplification method to analyze these isolates revealed diverse banding patterns across each of the genera and surprisingly even for isolates that are identical at the nucleotide level for five protein coding sequenced loci. This variance in genome structure even between identical multilocus sequence analysis (MLSA) haplotypes indicates that accumulation of genomic variation is rapid: faster than the rate of third codon substitutions. PMID:24782838

  12. Rapid storage and retrieval of genomic intervals from a relational database system using nested containment lists.

    PubMed

    Wiley, Laura K; Sivley, R Michael; Bush, William S

    2013-01-01

    Efficient storage and retrieval of genomic annotations based on range intervals is necessary, given the amount of data produced by next-generation sequencing studies. The indexing strategies of relational database systems (such as MySQL) greatly inhibit their use in genomic annotation tasks. This has led to the development of stand-alone applications that are dependent on flat-file libraries. In this work, we introduce MyNCList, an implementation of the NCList data structure within a MySQL database. MyNCList enables the storage, update and rapid retrieval of genomic annotations from the convenience of a relational database system. Range-based annotations of 1 million variants are retrieved in under a minute, making this approach feasible for whole-genome annotation tasks. Database URL: https://github.com/bushlab/mynclist. PMID:23894185

  13. Rapid storage and retrieval of genomic intervals from a relational database system using nested containment lists.

    PubMed

    Wiley, Laura K; Sivley, R Michael; Bush, William S

    2013-01-01

    Efficient storage and retrieval of genomic annotations based on range intervals is necessary, given the amount of data produced by next-generation sequencing studies. The indexing strategies of relational database systems (such as MySQL) greatly inhibit their use in genomic annotation tasks. This has led to the development of stand-alone applications that are dependent on flat-file libraries. In this work, we introduce MyNCList, an implementation of the NCList data structure within a MySQL database. MyNCList enables the storage, update and rapid retrieval of genomic annotations from the convenience of a relational database system. Range-based annotations of 1 million variants are retrieved in under a minute, making this approach feasible for whole-genome annotation tasks. Database URL: https://github.com/bushlab/mynclist.

  14. Genome Sequencing and Mapping Reveal Loss of Heterozygosity as a Mechanism for Rapid Adaptation in the Vegetable Pathogen Phytophthora capsici

    SciTech Connect

    Lamour, Kurt H.; Mudge, Joann; Gobena, Daniel; Hurtado-Gonzales, Oscar P.; Schmutz, Jeremy; Kuo, Alan; Miller, Neil A.; Rice, Brandon J.; Raffaele, Sylvain; Cano, Liliana M.; Bharti, Arvind K.; Donahoo, Ryan S.; Finely, Sabra; Huitema, Edgar; Hulvey, Jon; Platt, Darren; Salamov, Asaf; Savidor, Alon; Sharma, Rahul; Stam, Remco; Sotrey, Dylan; Thines, Marco; Win, Joe; Haas, Brian J.; Dinwiddie, Darrell L.; Jenkins, Jerry; Knight, James R.; Affourtit, Jason P.; Han, Cliff S.; Chertkov, Olga; Lindquist, Erika A.; Detter, Chris; Grigoriev, Igor V.; Kamoun, Sophien; Kingsmore, Stephen F.

    2012-02-07

    The oomycete vegetable pathogen Phytophthora capsici has shown remarkable adaptation to fungicides and new hosts. Like other members of this destructive genus, P. capsici has an explosive epidemiology, rapidly producing massive numbers of asexual spores on infected hosts. In addition, P. capsici can remain dormant for years as sexually recombined oospores, making it difficult to produce crops at infested sites, and allowing outcrossing populations to maintain significant genetic variation. Genome sequencing, development of a high-density genetic map, and integrative genomic or genetic characterization of P. capsici field isolates and intercross progeny revealed significant mitotic loss of heterozygosity (LOH) in diverse isolates. LOH was detected in clonally propagated field isolates and sexual progeny, cumulatively affecting >30percent of the genome. LOH altered genotypes for more than 11,000 single-nucleotide variant sites and showed a strong association with changes in mating type and pathogenicity. Overall, it appears that LOH may provide a rapid mechanism for fixing alleles and may be an important component of adaptability for P. capsici.

  15. Genome sequencing and mapping reveal loss of heterozygosity as a mechanism for rapid adaptation in the vegetable pathogen Phytophthora capsici

    PubMed Central

    Lamour, Kurt H.; Mudge, Joann; Gobena, Daniel; Hurtado-Gonzales, Oscar P.; Schmutz, Jeremy; Kuo, Alan; Miller, Neil A.; Rice, Brandon J.; Raffaele, Sylvain; Cano, Liliana M.; Bharti, Arvind K.; Donahoo, Ryan S.; Finley, Sabra; Huitema, Edgar; Hulvey, Jon; Platt, Darren; Salamov, Asaf; Savidor, Alon; Sharma, Rahul; Stam, Remco; Storey, Dylan; Thines, Marco; Win, Joe; Haas, Brian J.; Dinwiddie, Darrell L.; Jenkins, Jerry; Knight, James R.; Affourtit, Jason P.; Han, Cliff S.; Chertkov, Olga; Lindquist, Erika A.; Detter, Chris; Grigoriev, Igor V.; Kamoun, Sophien; Kingsmore, Stephen F.

    2013-01-01

    The oomycete vegetable pathogen Phytophthora capsici has shown remarkable adaptation to fungicides and new hosts. Like other members of this destructive genus, P. capsici has an explosive epidemiology, rapidly producing massive numbers of asexual spores on infected hosts. In addition, P. capsici can remain dormant for years as sexually-recombined oospores, making it difficult to produce crops at infested sites, and allowing outcrossing populations to maintain significant genetic variation. Genome sequencing, development of a high-density genetic map, and integrative genomic/genetic characterization of P. capsici field isolates and intercross progeny revealed significant mitotic loss of heterozygosity (LOH) and higher levels of SNVs than those reported for humans, plants, and P. infestans. LOH was detected in clonally propagated field isolates and sexual progeny, cumulatively affecting >30% of the genome. LOH altered genotypes for more than 11,000 single nucleotide variant (SNV) sites and showed a strong association with changes in mating type and pathogenicity. Overall, it appears that LOH may provide a rapid mechanism for fixing alleles and may be an important component of adaptability for P. capsici. PMID:22712506

  16. Genome scan for cognitive trait loci of dyslexia: Rapid naming and rapid switching of letters, numbers, and colors.

    PubMed

    Rubenstein, Kevin B; Raskind, Wendy H; Berninger, Virginia W; Matsushita, Mark M; Wijsman, Ellen M

    2014-06-01

    Dyslexia, or specific reading disability, is a common developmental disorder that affects 5-12% of school-aged children. Dyslexia and its component phenotypes, assessed categorically or quantitatively, have complex genetic bases. The ability to rapidly name letters, numbers, and colors from rows presented visually correlates strongly with reading in multiple languages and is a valid predictor of reading and spelling impairment. Performance on measures of rapid naming and switching, RAN and RAS, is stable throughout elementary school years, with slowed performance persisting in adults who still manifest dyslexia. Targeted analyses of dyslexia candidate regions have included RAN measures, but only one other genome-wide linkage study has been reported. As part of a broad effort to identify genetic contributors to dyslexia, we performed combined oligogenic segregation and linkage analyses of measures of RAN and RAS in a family-based cohort ascertained through probands with dyslexia. We obtained strong evidence for linkage of RAN letters to the DYX3 locus on chromosome 2p and RAN colors to chromosome 10q, but were unable to confirm the chromosome 6p21 linkage detected for a composite measure of RAN colors and objects in the previous genome-wide study. PMID:24807833

  17. Genome Wide Characterization of Simple Sequence Repeats in Cucumber

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The whole genome sequence of the cucumber cultivar Gy14 was recently sequenced at 15× coverage with the Roche 454 Titanium technology. The microsatellite DNA sequences (simple sequence repeats, SSRs) in the assembled scaffolds were computationally explored and characterized. A total of 112,073 SSRs ...

  18. TALEN or Cas9 - rapid, efficient and specific choices for genome modifications.

    PubMed

    Wei, Chuanxian; Liu, Jiyong; Yu, Zhongsheng; Zhang, Bo; Gao, Guanjun; Jiao, Renjie

    2013-06-20

    Precise modifications of complex genomes at the single nucleotide level have been one of the big goals for scientists working in basic and applied genetics, including biotechnology, drug development, gene therapy and synthetic biology. However, the relevant techniques for making these manipulations in model organisms and human cells have been lagging behind the rapid high throughput studies in the post-genomic era with a bottleneck of low efficiency, time consuming and laborious manipulation, and off-targeting problems. Recent discoveries of TALEs (transcription activator-like effectors) coding system and CRISPR (clusters of regularly interspaced short palindromic repeats) immune system in bacteria have enabled the development of customized TALENs (transcription activator-like effector nucleases) and CRISPR/Cas9 to rapidly edit genomic DNA in a variety of cell types, including human cells, and different model organisms at a very high efficiency and specificity. In this review, we first briefly summarize the development and applications of TALENs and CRISPR/Cas9-mediated genome editing technologies; compare the advantages and constraints of each method; particularly, discuss the expected applications of both techniques in the field of site-specific genome modification and stem cell based gene therapy; finally, propose the future directions and perspectives for readers to make the choices.

  19. Rapid enrichment of leucocytes and genomic DNA from blood based on bifunctional core shell magnetic nanoparticles

    NASA Astrophysics Data System (ADS)

    Xie, Xin; Nie, Xiaorong; Yu, Bingbin; Zhang, Xu

    2007-04-01

    A series of protocols are proposed to extract genomic DNA from whole blood at different scales using carboxyl-functionalized magnetic nanoparticles as solid-phase absorbents. The enrichment of leucocytes and the adsorption of genomic DNA can be achieved with the same carboxyl-functionalized magnetic nanoparticles. The DNA bound to the bead surfaces can be used directly as PCR templates. By coupling cell separation and DNA purification, the whole operation can be accomplished in a few minutes. Our simplified protocols proved to be rapid, low cost, and biologically and chemically non-hazardous, and are therefore promising for microfabrication of a DNA-preparation chip and routine laboratory use.

  20. Comprehensive genomic characterization of squamous cell lung cancers.

    PubMed

    2012-09-27

    Lung squamous cell carcinoma is a common type of lung cancer, causing approximately 400,000 deaths per year worldwide. Genomic alterations in squamous cell lung cancers have not been comprehensively characterized, and no molecularly targeted agents have been specifically developed for its treatment. As part of The Cancer Genome Atlas, here we profile 178 lung squamous cell carcinomas to provide a comprehensive landscape of genomic and epigenomic alterations. We show that the tumour type is characterized by complex genomic alterations, with a mean of 360 exonic mutations, 165 genomic rearrangements, and 323 segments of copy number alteration per tumour. We find statistically recurrent mutations in 11 genes, including mutation of TP53 in nearly all specimens. Previously unreported loss-of-function mutations are seen in the HLA-A class I major histocompatibility gene. Significantly altered pathways included NFE2L2 and KEAP1 in 34%, squamous differentiation genes in 44%, phosphatidylinositol-3-OH kinase pathway genes in 47%, and CDKN2A and RB1 in 72% of tumours. We identified a potential therapeutic target in most tumours, offering new avenues of investigation for the treatment of squamous cell lung cancers. PMID:22960745

  1. Comprehensive genomic characterization of squamous cell lung cancers

    PubMed Central

    2012-01-01

    Summary Lung squamous cell carcinoma (lung SqCC) is a common type of lung cancer, causing approximately 400,000 deaths per year worldwide. Genomic alterations in lung SqCC have not been comprehensively characterized and no molecularly targeted agents have been developed specifically for its treatment. As part of The Cancer Genome Atlas (TCGA), we profiled 178 lung SqCCs to provide a comprehensive landscape of genomic and epigenomic alterations. Lung SqCC is characterized by complex genomic alterations, with a mean of 360 exonic mutations, 165 genomic rearrangements, and 323 segments of copy number alteration per tumor. We found statistically recurrent mutations in 18 genes in including mutation of TP53 in nearly all specimens. Previously unreported loss-of-function mutations were seen in the HLA-A class I major histocompatibility gene. Significantly altered pathways included NFE2L2/KEAP1 in 34%, squamous differentiation genes in 44%, PI3K/AKT in 47%, and CDKN2A/RB1 in 72% of tumors. We identified a potential therapeutic target in the majority of tumors, offering new avenues of investigation for lung SqCC treatment. PMID:22960745

  2. Genomic Plasticity in Ralstonia eutropha and Ralstonia pickettii: Evidence for Rapid Genomic Change and Adaptation

    SciTech Connect

    Terence L. MArsh

    2007-06-27

    The proposed foci of our investigations were on Ralstonia eutropha and Rasltonia pickettii. We have 18 derived lineages of the former as well as their progenitor and eleven isolates of the latter. Our goal was to measure the level of plasticity in these strains and attempt to derive a mechanistic understanding of how genomic plasticity formed. Extensive attempts to reproducibly induce conformational changes in the genome of R. eutropha were unsuccessful. We thought that we had a reasonable lead on this inasmuch as we had shown that the ancestral strain along with many of the derivative lineages exhibited “temperature induced mutation and mortality akin to R. metallodurans. However we were unable to get subtractive hybridization working to the degree that it revealed differences between the lineages. During this time the R. pickettii analysis was proving quite fruitful and so we concentrated our efforts on our analyses of R. pickettii. These strains were isolated from a copper-contaminated lake sediment and were resistant to copper at 800 µg/ml (CuSO4). Our results in the investigation of R. pickettii permitted a view into the adaptation of a beta-proteobacteria to an extreme environment. Our worked revealed that within the same ecosystem two genomovars with structurally different genomes and genome sizes were present and apparently filling similar if not identical niches. The genomovars were detected with REP & BOX-PCR, pulse field gel electrophoresis, and DNA:DNA hybridizations. Moreover there were different metal resistance patterns associated with the different genomovars, one showing resistance to Zn and Cd while the other had resistance to Ni. Five of the isolates had a high-copy number extrachromosomal element that was identified as the replicative form of a filamentous phage. Mature virions were isolated from culture broth using PEG precipitation and CsCl density centrifugation. The DNA associated with the filamentous particles was single stranded and had

  3. Rapid diversification of five Oryza AA genomes associated with rice adaptation.

    PubMed

    Zhang, Qun-Jie; Zhu, Ting; Xia, En-Hua; Shi, Chao; Liu, Yun-Long; Zhang, Yun; Liu, Yuan; Jiang, Wen-Kai; Zhao, You-Jie; Mao, Shu-Yan; Zhang, Li-Ping; Huang, Hui; Jiao, Jun-Ying; Xu, Ping-Zhen; Yao, Qiu-Yang; Zeng, Fan-Chun; Yang, Li-Li; Gao, Ju; Tao, Da-Yun; Wang, Yue-Ju; Bennetzen, Jeffrey L; Gao, Li-Zhi

    2014-11-18

    Comparative genomic analyses among closely related species can greatly enhance our understanding of plant gene and genome evolution. We report de novo-assembled AA-genome sequences for Oryza nivara, Oryza glaberrima, Oryza barthii, Oryza glumaepatula, and Oryza meridionalis. Our analyses reveal massive levels of genomic structural variation, including segmental duplication and rapid gene family turnover, with particularly high instability in defense-related genes. We show, on a genomic scale, how lineage-specific expansion or contraction of gene families has led to their morphological and reproductive diversification, thus enlightening the evolutionary process of speciation and adaptation. Despite strong purifying selective pressures on most Oryza genes, we documented a large number of positively selected genes, especially those genes involved in flower development, reproduction, and resistance-related processes. These diversifying genes are expected to have played key roles in adaptations to their ecological niches in Asia, South America, Africa and Australia. Extensive variation in noncoding RNA gene numbers, function enrichment, and rates of sequence divergence might also help account for the different genetic adaptations of these rice species. Collectively, these resources provide new opportunities for evolutionary genomics, numerous insights into recent speciation, a valuable database of functional variation for crop improvement, and tools for efficient conservation of wild rice germplasm.

  4. Characterization of mango (Mangifera indica L.) transcriptome and chloroplast genome.

    PubMed

    Azim, M Kamran; Khan, Ishtaiq A; Zhang, Yong

    2014-05-01

    We characterized mango leaf transcriptome and chloroplast genome using next generation DNA sequencing. The RNA-seq output of mango transcriptome generated >12 million reads (total nucleotides sequenced >1 Gb). De novo transcriptome assembly generated 30,509 unigenes with lengths in the range of 300 to ≥3,000 nt and 67× depth of coverage. Blast searching against nonredundant nucleotide databases and several Viridiplantae genomic datasets annotated 24,593 mango unigenes (80% of total) and identified Citrus sinensis as closest neighbor of mango with 9,141 (37%) matched sequences. The annotation with gene ontology and Clusters of Orthologous Group terms categorized unigene sequences into 57 and 25 classes, respectively. More than 13,500 unigenes were assigned to 293 KEGG pathways. Besides major plant biology related pathways, KEGG based gene annotation pointed out active presence of an array of biochemical pathways involved in (a) biosynthesis of bioactive flavonoids, flavones and flavonols, (b) biosynthesis of terpenoids and lignins and (c) plant hormone signal transduction. The mango transcriptome sequences revealed 235 proteases belonging to five catalytic classes of proteolytic enzymes. The draft genome of mango chloroplast (cp) was obtained by a combination of Sanger and next generation sequencing. The draft mango cp genome size is 151,173 bp with a pair of inverted repeats of 27,093 bp separated by small and large single copy regions, respectively. Out of 139 genes in mango cp genome, 91 found to be protein coding. Sequence analysis revealed cp genome of C. sinensis as closest neighbor of mango. We found 51 short repeats in mango cp genome supposed to be associated with extensive rearrangements. This is the first report of transcriptome and chloroplast genome analysis of any Anacardiaceae family member.

  5. Comprehensive Pan-Genomic Characterization of Adrenocortical Carcinoma.

    PubMed

    Zheng, Siyuan; Cherniack, Andrew D; Dewal, Ninad; Moffitt, Richard A; Danilova, Ludmila; Murray, Bradley A; Lerario, Antonio M; Else, Tobias; Knijnenburg, Theo A; Ciriello, Giovanni; Kim, Seungchan; Assie, Guillaume; Morozova, Olena; Akbani, Rehan; Shih, Juliann; Hoadley, Katherine A; Choueiri, Toni K; Waldmann, Jens; Mete, Ozgur; Robertson, A Gordon; Wu, Hsin-Ta; Raphael, Benjamin J; Shao, Lina; Meyerson, Matthew; Demeure, Michael J; Beuschlein, Felix; Gill, Anthony J; Sidhu, Stan B; Almeida, Madson Q; Fragoso, Maria C B V; Cope, Leslie M; Kebebew, Electron; Habra, Mouhammed A; Whitsett, Timothy G; Bussey, Kimberly J; Rainey, William E; Asa, Sylvia L; Bertherat, Jérôme; Fassnacht, Martin; Wheeler, David A; Hammer, Gary D; Giordano, Thomas J; Verhaak, Roel G W

    2016-05-01

    We describe a comprehensive genomic characterization of adrenocortical carcinoma (ACC). Using this dataset, we expand the catalogue of known ACC driver genes to include PRKAR1A, RPL22, TERF2, CCNE1, and NF1. Genome wide DNA copy-number analysis revealed frequent occurrence of massive DNA loss followed by whole-genome doubling (WGD), which was associated with aggressive clinical course, suggesting WGD is a hallmark of disease progression. Corroborating this hypothesis were increased TERT expression, decreased telomere length, and activation of cell-cycle programs. Integrated subtype analysis identified three ACC subtypes with distinct clinical outcome and molecular alterations which could be captured by a 68-CpG probe DNA-methylation signature, proposing a strategy for clinical stratification of patients based on molecular markers. PMID:27165744

  6. Characterization of the Poplar Pan-Genome by Genome-Wide Identification of Structural Variation.

    PubMed

    Pinosio, Sara; Giacomello, Stefania; Faivre-Rampant, Patricia; Taylor, Gail; Jorge, Veronique; Le Paslier, Marie Christine; Zaina, Giusi; Bastien, Catherine; Cattonaro, Federica; Marroni, Fabio; Morgante, Michele

    2016-10-01

    Many recent studies have emphasized the important role of structural variation (SV) in determining human genetic and phenotypic variation. In plants, studies aimed at elucidating the extent of SV are still in their infancy. Evidence has indicated a high presence and an active role of SV in driving plant genome evolution in different plant species.With the aim of characterizing the size and the composition of the poplar pan-genome, we performed a genome-wide analysis of structural variation in three intercrossable poplar species: Populus nigra, Populus deltoides, and Populus trichocarpa We detected a total of 7,889 deletions and 10,586 insertions relative to the P. trichocarpa reference genome, covering respectively 33.2 Mb and 62.9 Mb of genomic sequence, and 3,230 genes affected by copy number variation (CNV). The majority of the detected variants are inter-specific in agreement with a recent origin following separation of species.Insertions and deletions (INDELs) were preferentially located in low-gene density regions of the poplar genome and were, for the majority, associated with the activity of transposable elements. Genes affected by SV showed lower-than-average expression levels and higher levels of dN/dS, suggesting that they are subject to relaxed selective pressure or correspond to pseudogenes.Functional annotation of genes affected by INDELs showed over-representation of categories associated with transposable elements activity, while genes affected by genic CNVs showed enrichment in categories related to resistance to stress and pathogens. This study provides a genome-wide catalogue of SV and the first insight on functional and structural properties of the poplar pan-genome. PMID:27499133

  7. Characterization of the Poplar Pan-Genome by Genome-Wide Identification of Structural Variation

    PubMed Central

    Pinosio, Sara; Giacomello, Stefania; Faivre-Rampant, Patricia; Taylor, Gail; Jorge, Veronique; Le Paslier, Marie Christine; Zaina, Giusi; Bastien, Catherine; Cattonaro, Federica; Marroni, Fabio; Morgante, Michele

    2016-01-01

    Many recent studies have emphasized the important role of structural variation (SV) in determining human genetic and phenotypic variation. In plants, studies aimed at elucidating the extent of SV are still in their infancy. Evidence has indicated a high presence and an active role of SV in driving plant genome evolution in different plant species. With the aim of characterizing the size and the composition of the poplar pan-genome, we performed a genome-wide analysis of structural variation in three intercrossable poplar species: Populus nigra, Populus deltoides, and Populus trichocarpa. We detected a total of 7,889 deletions and 10,586 insertions relative to the P. trichocarpa reference genome, covering respectively 33.2 Mb and 62.9 Mb of genomic sequence, and 3,230 genes affected by copy number variation (CNV). The majority of the detected variants are inter-specific in agreement with a recent origin following separation of species. Insertions and deletions (INDELs) were preferentially located in low-gene density regions of the poplar genome and were, for the majority, associated with the activity of transposable elements. Genes affected by SV showed lower-than-average expression levels and higher levels of dN/dS, suggesting that they are subject to relaxed selective pressure or correspond to pseudogenes. Functional annotation of genes affected by INDELs showed over-representation of categories associated with transposable elements activity, while genes affected by genic CNVs showed enrichment in categories related to resistance to stress and pathogens. This study provides a genome-wide catalogue of SV and the first insight on functional and structural properties of the poplar pan-genome. PMID:27499133

  8. Genomic characterization provides new insight into Salmonella phage diversity

    PubMed Central

    2013-01-01

    Background Salmonella is a widely distributed foodborne pathogen that causes tens of millions of salmonellosis cases globally every year. While the genomic diversity of Salmonella is increasingly well studied, our knowledge of Salmonella phage genomic diversity is still rather limited, despite the contributions of both lysogenic and lytic phages to Salmonella virulence, diversity and ecology (e.g., through horizontal gene transfer and Salmonella lysis). To gain a better understanding of phage diversity in a specific ecological niche, we sequenced 22 Salmonella phages isolated from a number of dairy farms from New York State (United States) and analyzed them using a comparative genomics approach. Results Classification of the 22 phages according to the presence/absence of orthologous genes allowed for classification into 8 well supported clusters. In addition to two phage clusters that represent novel virulent Salmonella phages, we also identified four phage clusters that each contained previously characterized phages from multiple continents. Our analyses also identified two clusters of phages that carry putative virulence (e.g., adhesins) and antimicrobial resistance (tellurite and bicyclomycin) genes as well as virulent and temperate transducing phages. Insights into phage evolution from our analyses include (i) identification of DNA metabolism genes that may facilitate nucleotide synthesis in phages with a G+C % distinct from Salmonella, and (ii) evidence of Salmonella phage tailspike and fiber diversity due to both single nucleotide polymorphisms and major re-arrangements, which may affect the host specificity of Salmonella phages. Conclusions Genomics-based characterization of 22 Salmonella phages isolated from dairy farms allowed for identification of a number of novel Salmonella phages. While the comparative genomics analyses of these phages provide a number of new insights in the evolution and diversity of Salmonella phages, they only represent a first

  9. Forward Genetics by Genome Sequencing Reveals That Rapid Cyanide Release Deters Insect Herbivory of Sorghum bicolor

    PubMed Central

    Krothapalli, Kartikeya; Buescher, Elizabeth M.; Li, Xu; Brown, Elliot; Chapple, Clint; Dilkes, Brian P.; Tuinstra, Mitchell R.

    2013-01-01

    Whole genome sequencing has allowed rapid progress in the application of forward genetics in model species. In this study, we demonstrated an application of next-generation sequencing for forward genetics in a complex crop genome. We sequenced an ethyl methanesulfonate-induced mutant of Sorghum bicolor defective in hydrogen cyanide release and identified the causal mutation. A workflow identified the causal polymorphism relative to the reference BTx623 genome by integrating data from single nucleotide polymorphism identification, prior information about candidate gene(s) implicated in cyanogenesis, mutation spectra, and polymorphisms likely to affect phenotypic changes. A point mutation resulting in a premature stop codon in the coding sequence of dhurrinase2, which encodes a protein involved in the dhurrin catabolic pathway, was responsible for the acyanogenic phenotype. Cyanogenic glucosides are not cyanogenic compounds but their cyanohydrins derivatives do release cyanide. The mutant accumulated the glucoside, dhurrin, but failed to efficiently release cyanide upon tissue disruption. Thus, we tested the effects of cyanide release on insect herbivory in a genetic background in which accumulation of cyanogenic glucoside is unchanged. Insect preference choice experiments and herbivory measurements demonstrate a deterrent effect of cyanide release capacity, even in the presence of wild-type levels of cyanogenic glucoside accumulation. Our gene cloning method substantiates the value of (1) a sequenced genome, (2) a strongly penetrant and easily measurable phenotype, and (3) a workflow to pinpoint a causal mutation in crop genomes and accelerate in the discovery of gene function in the postgenomic era. PMID:23893483

  10. Rapid Characterization of Vegetation Structure with a Microsoft Kinect Sensor

    PubMed Central

    Azzari, George; Goulden, Michael L.; Rusu, Radu B.

    2013-01-01

    The importance of vegetation structure and biomass in controlling land-atmosphere exchange is widely recognized, but measurements of canopy structure are challenging, time consuming, and often rely on destructive methods. The Microsoft Kinect is an infrared sensor designed for video gaming that outputs synchronized color and depth images and that has the potential to allow rapid characterization of vegetation structure. We compared depth images from a Kinect sensor with manual measurements of plant structure and size for two species growing in a California grassland. The depth images agreed well with the horizontal and vertical measurements of plant size made manually. Similarly, the plant volumes calculated with a three-dimensional convex hulls approach was well related to plant biomass. The Kinect showed some limitations for ecological observation associated with a short measurement range and daytime light contamination. Nonetheless, the Kinect's light weight, fast acquisition time, low power requirement, and cost make it a promising tool for rapid field surveys of canopy structure, especially in small-statured vegetation. PMID:23435053

  11. A genomic selection component analysis characterizes migration-selection balance.

    PubMed

    Monnahan, Patrick J; Colicchio, Jack; Kelly, John K

    2015-07-01

    The genetic differentiation of populations in response to local selection pressures has long been studied by evolutionary biologists, but key details about the process remain obscure. How rapidly can local adaptation evolve, how extensive is the process across the genome, and how strong are the opposing forces of natural selection and gene flow? Here, we combine direct measurement of survival and reproduction with whole-genome genotyping of a plant species (Mimulus guttatus) that has recently invaded a novel habitat (the Quarry population). We renovate the classic selection component method to accommodate genomic data and observe selection at SNPs throughout the genome. SNPs showing viability selection in Quarry exhibit elevated divergence from neighboring populations relative to neutral SNPs. We also find that nonsignificant SNPs exhibit a subtle, but still significant, change in allele frequency toward neighboring populations, a predicted effect of gene flow. Given that the Quarry population is most probably only 30-40 generations old, the alleles conferring local advantage are almost certainly older than the population itself. Thus, local adaptation owes to the recruitment of standing genetic variation.

  12. Genomic characterization and phylogenetic analysis of Zika virus circulating in the Americas.

    PubMed

    Ye, Qing; Liu, Zhong-Yu; Han, Jian-Feng; Jiang, Tao; Li, Xiao-Feng; Qin, Cheng-Feng

    2016-09-01

    The rapid spread and potential link with birth defects have made Zika virus (ZIKV) a global public health problem. The virus was discovered 70years ago, yet the knowledge about its genomic structure and the genetic variations associated with current ZIKV explosive epidemics remains not fully understood. In this review, the genome organization, especially conserved terminal structures of ZIKV genome were characterized and compared with other mosquito-borne flaviviruses. It is suggested that major viral proteins of ZIKV share high structural and functional similarity with other known flaviviruses as shown by sequence comparison and prediction of functional motifs in viral proteins. Phylogenetic analysis demonstrated that all ZIKV strains circulating in the America form a unique clade within the Asian lineage. Furthermore, we identified a series of conserved amino acid residues that differentiate the Asian strains including the current circulating American strains from the ancient African strains. Overall, our findings provide an overview of ZIKV genome characterization and evolutionary dynamics in the Americas and point out critical clues for future virological and epidemiological studies. PMID:27156653

  13. Genomic characterization and phylogenetic analysis of Zika virus circulating in the Americas.

    PubMed

    Ye, Qing; Liu, Zhong-Yu; Han, Jian-Feng; Jiang, Tao; Li, Xiao-Feng; Qin, Cheng-Feng

    2016-09-01

    The rapid spread and potential link with birth defects have made Zika virus (ZIKV) a global public health problem. The virus was discovered 70years ago, yet the knowledge about its genomic structure and the genetic variations associated with current ZIKV explosive epidemics remains not fully understood. In this review, the genome organization, especially conserved terminal structures of ZIKV genome were characterized and compared with other mosquito-borne flaviviruses. It is suggested that major viral proteins of ZIKV share high structural and functional similarity with other known flaviviruses as shown by sequence comparison and prediction of functional motifs in viral proteins. Phylogenetic analysis demonstrated that all ZIKV strains circulating in the America form a unique clade within the Asian lineage. Furthermore, we identified a series of conserved amino acid residues that differentiate the Asian strains including the current circulating American strains from the ancient African strains. Overall, our findings provide an overview of ZIKV genome characterization and evolutionary dynamics in the Americas and point out critical clues for future virological and epidemiological studies.

  14. Rapid geo-acoustic characterization from a seismic survey

    NASA Astrophysics Data System (ADS)

    Heaney, Kevin D.; Sternlicht, Daniel; Teranishi, Arthur; Castille, Brett; Hamilton, Michael

    2002-05-01

    A recent transmission loss experiment was conducted in Long Beach Harbor for the THUMS Long Beach Company. The objective of the experiment was to measure the range at which the received level was 160 dB for compliance with Marine Mammal regulations. This short experiment provided the opportunity to test the rapid geo-acoustic characterization (RGC) algorithm and perform real-time geo-acoustic inversions from a seismic source. The airgun source transmitted pulses every 20 s corresponding to every 45 m. The water depth was 10-15 m and the water was assumed to be iso-velocity. The data quality was excellent, providing clear striation patterns in the broadband frequency display. The RGC algorithm matches the observed time-spread, striation slope, and TL slope to precomputed values using a normal mode algorithm and parametric geo-acoustic profiles based on Hamilton and Bachman's model. Precomputation of the acoustic observables, combined with real-time signal processing permits real time geo-acoustic characterization.

  15. The Salmonella In Silico Typing Resource (SISTR): An Open Web-Accessible Tool for Rapidly Typing and Subtyping Draft Salmonella Genome Assemblies.

    PubMed

    Yoshida, Catherine E; Kruczkiewicz, Peter; Laing, Chad R; Lingohr, Erika J; Gannon, Victor P J; Nash, John H E; Taboada, Eduardo N

    2016-01-01

    For nearly 100 years serotyping has been the gold standard for the identification of Salmonella serovars. Despite the increasing adoption of DNA-based subtyping approaches, serotype information remains a cornerstone in food safety and public health activities aimed at reducing the burden of salmonellosis. At the same time, recent advances in whole-genome sequencing (WGS) promise to revolutionize our ability to perform advanced pathogen characterization in support of improved source attribution and outbreak analysis. We present the Salmonella In Silico Typing Resource (SISTR), a bioinformatics platform for rapidly performing simultaneous in silico analyses for several leading subtyping methods on draft Salmonella genome assemblies. In addition to performing serovar prediction by genoserotyping, this resource integrates sequence-based typing analyses for: Multi-Locus Sequence Typing (MLST), ribosomal MLST (rMLST), and core genome MLST (cgMLST). We show how phylogenetic context from cgMLST analysis can supplement the genoserotyping analysis and increase the accuracy of in silico serovar prediction to over 94.6% on a dataset comprised of 4,188 finished genomes and WGS draft assemblies. In addition to allowing analysis of user-uploaded whole-genome assemblies, the SISTR platform incorporates a database comprising over 4,000 publicly available genomes, allowing users to place their isolates in a broader phylogenetic and epidemiological context. The resource incorporates several metadata driven visualizations to examine the phylogenetic, geospatial and temporal distribution of genome-sequenced isolates. As sequencing of Salmonella isolates at public health laboratories around the world becomes increasingly common, rapid in silico analysis of minimally processed draft genome assemblies provides a powerful approach for molecular epidemiology in support of public health investigations. Moreover, this type of integrated analysis using multiple sequence-based methods of sub

  16. Genomic signatures of rapid adaptive evolution in the bluespotted cornetfish, a Mediterranean Lessepsian invader.

    PubMed

    Bernardi, Giacomo; Azzurro, Ernesto; Golani, Daniel; Miller, Michael Ryan

    2016-07-01

    Biological invasions are increasingly creating ecological and economical problems both on land and in aquatic environments. For over a century, the Mediterranean Sea has steadily been invaded by Indian Ocean/Red Sea species (called Lessepsian invaders) via the Suez Canal, with a current estimate of ~450 species. The bluespotted cornetfish, Fistularia commersonii, considered a 'Lessepsian sprinter', entered the Mediterranean in 2000 and by 2007 had spread through the entire basin from Israel to Spain. The situation is unique and interesting both because of its unprecedented rapidity and by the fact that it took this species c. 130 years to immigrate into the Mediterranean. Using genome scans, with restriction site-associated DNA (RAD) sequencing, we evaluated neutral and selected genomic regions for Mediterranean vs. Red Sea cornetfish individuals. We found that few fixed neutral changes were detectable among populations. However, almost half of the genes associated with the 47 outlier loci (potentially under selection) were related to disease resistance and osmoregulation. Due to the short time elapsed from the beginning of the invasion to our sampling, we interpret these changes as signatures of rapid adaptation that may be explained by several mechanisms including preadaptation and strong local selection. Such genomic regions are therefore good candidates to further study their role in invasion success. PMID:27162055

  17. Genomic characterization of the Atlantic cod sex-locus.

    PubMed

    Star, Bastiaan; Tørresen, Ole K; Nederbragt, Alexander J; Jakobsen, Kjetill S; Pampoulie, Christophe; Jentoft, Sissel

    2016-01-01

    A variety of sex determination mechanisms can be observed in evolutionary divergent teleosts. Sex determination is genetic in Atlantic cod (Gadus morhua), however the genomic location or size of its sex-locus is unknown. Here, we characterize the sex-locus of Atlantic cod using whole genome sequence (WGS) data of 227 wild-caught specimens. Analyzing more than 55 million polymorphic loci, we identify 166 loci that are associated with sex. These loci are located in six distinct regions on five different linkage groups (LG) in the genome. The largest of these regions, an approximately 55 Kb region on LG11, contains the majority of genotypes that segregate closely according to a XX-XY system. Genotypes in this region can be used genetically determine sex, whereas those in the other regions are inconsistently sex-linked. The identified region on LG11 and its surrounding genes have no clear sequence homology with genes or regulatory elements associated with sex-determination or differentiation in other species. The functionality of this sex-locus therefore remains unknown. The WGS strategy used here proved adequate for detecting the small regions associated with sex in this species. Our results highlight the evolutionary flexibility in genomic architecture underlying teleost sex-determination and allow practical applications to genetically sex Atlantic cod. PMID:27499266

  18. Genomic characterization of the Atlantic cod sex-locus

    PubMed Central

    Star, Bastiaan; Tørresen, Ole K.; Nederbragt, Alexander J.; Jakobsen, Kjetill S.; Pampoulie, Christophe; Jentoft, Sissel

    2016-01-01

    A variety of sex determination mechanisms can be observed in evolutionary divergent teleosts. Sex determination is genetic in Atlantic cod (Gadus morhua), however the genomic location or size of its sex-locus is unknown. Here, we characterize the sex-locus of Atlantic cod using whole genome sequence (WGS) data of 227 wild-caught specimens. Analyzing more than 55 million polymorphic loci, we identify 166 loci that are associated with sex. These loci are located in six distinct regions on five different linkage groups (LG) in the genome. The largest of these regions, an approximately 55 Kb region on LG11, contains the majority of genotypes that segregate closely according to a XX-XY system. Genotypes in this region can be used genetically determine sex, whereas those in the other regions are inconsistently sex-linked. The identified region on LG11 and its surrounding genes have no clear sequence homology with genes or regulatory elements associated with sex-determination or differentiation in other species. The functionality of this sex-locus therefore remains unknown. The WGS strategy used here proved adequate for detecting the small regions associated with sex in this species. Our results highlight the evolutionary flexibility in genomic architecture underlying teleost sex-determination and allow practical applications to genetically sex Atlantic cod. PMID:27499266

  19. Characterization of the flamenco region of the Drosophila melanogaster genome.

    PubMed

    Robert, V; Prud'homme, N; Kim, A; Bucheton, A; Pélisson, A

    2001-06-01

    The flamenco gene, located at 20A1-3 in the beta-heterochromatin of the Drosophila X chromosome, is a major regulator of the gypsy/mdg4 endogenous retrovirus. As a first step to characterize this gene, approximately 100 kb of genomic DNA flanking a P-element-induced mutation of flamenco was isolated. This DNA is located in a sequencing gap of the Celera Genomics project, i.e., one of those parts of the genome in which the "shotgun" sequence could not be assembled, probably because it contains long stretches of repetitive DNA, especially on the proximal side of the P insertion point. Deficiency mapping indicated that sequences required for the normal flamenco function are located >130 kb proximal to the insertion site. The distal part of the cloned DNA does, nevertheless, contain several unique sequences, including at least four different transcription units. Dip1, the closest one to the P-element insertion point, might be a good candidate for a gypsy regulator, since it putatively encodes a nuclear protein containing two double-stranded RNA-binding domains. However, transgenes containing dip1 genomic DNA were not able to rescue flamenco mutant flies. The possible nature of the missing flamenco sequences is discussed. PMID:11404334

  20. Genomic allergen rapid detection in-house validation--a proof of concept.

    PubMed

    Johansson, Henrik; Rydnert, Frida; Kühnl, Jochen; Schepky, Andreas; Borrebaeck, Carl; Lindstedt, Malin

    2014-06-01

    Chemical sensitization is an adverse immunologic response to chemical substances, inducing hypersensitivity in exposed individuals. Identifying chemical sensitizers is of great importance for chemical, pharmaceutical, and cosmetic industries, in order to prevent the use of sensitizers in consumer products. Historically, chemical sensitizers have been assessed mainly by in vivo methods, however, recently enforced European legislations urge and promote the development of animal-free test methods able to predict chemical sensitizers. Recently, we presented a predictive biomarker signature in the myeloid cell line MUTZ-3, for assessment of skin sensitizers. The identified genomic biomarkers were found to be involved in immunologically relevant pathways, induced by recognition of foreign substances and regulating dendritic cell maturation and cytoprotective mechanisms. We have developed the usage of this biomarker signature into a novel in vitro assay for assessment of chemical sensitizers, called Genomic Allergen Rapid Detection (GARD). The assay is based on chemical stimulation of MUTZ-3 cultures, using the compounds to be assayed as stimulatory agents. The readout of the assay is a transcriptional quantification of the genomic predictors, collectively termed the GARD Prediction Signature (GPS), using a complete genome expression array. Compounds are predicted as either sensitizers or nonsensitizers by a Support Vector Machine model. In this report, we provide a proof of concept for the functionality of the GARD assay by describing the classification of 26 blinded and 11 nonblinded chemicals as sensitizers or nonsensitizers. Based on these classifications, the accuracy, sensitivity, and specificity of the assay were estimated to 89, 89, and 88%, respectively. PMID:24675087

  1. Sequenced genomes and rapidly emerging technologies pave the way for conifer evolutionary developmental biology.

    PubMed

    Uddenberg, Daniel; Akhter, Shirin; Ramachandran, Prashanth; Sundström, Jens F; Carlsbecker, Annelie

    2015-01-01

    Conifers, Ginkgo, cycads and gnetophytes comprise the four groups of extant gymnosperms holding a unique position of sharing common ancestry with the angiosperms. Comparative studies of gymnosperms and angiosperms are the key to a better understanding of ancient seed plant morphologies, how they have shifted over evolution to shape modern day species, and how the genes governing these morphologies have evolved. However, conifers and other gymnosperms have been notoriously difficult to study due to their long generation times, inaccessibility to genetic experimentation and unavailable genome sequences. Now, with three draft genomes from spruces and pines, rapid advances in next generation sequencing methods for genome wide expression analyses, and enhanced methods for genetic transformation, we are much better equipped to address a number of key evolutionary questions relating to seed plant evolution. In this mini-review we highlight recent progress in conifer developmental biology relevant to evo-devo questions. We discuss how genome sequence data and novel techniques might allow us to explore genetic variation and naturally occurring conifer mutants, approaches to reduce long generation times to allow for genetic studies in conifers, and other potential upcoming research avenues utilizing current and emergent techniques. Results from developmental studies of conifers and other gymnosperms in comparison to those in angiosperms will provide information to trace core molecular developmental control tool kits of ancestral seed plants, but foremost they will greatly improve our understanding of the biology of conifers and other gymnosperms in their own right. PMID:26579190

  2. Rapid and Pervasive Changes in Genome-Wide Enhancer Usage During Mammalian Development

    PubMed Central

    Nord, Alex S.; Blow, Matthew J.; Attanasio, Catia; Akiyama, Jennifer A.; Holt, Amy; Hosseini, Roya; Phouanenavong, Sengthavy; Plajzer-Frick, Ingrid; Shoukry, Malak; Afzal, Veena; Rubenstein, John L. R.; Rubin, Edward M.; Pennacchio, Len A.; Visel, Axel

    2014-01-01

    Summary Enhancers are distal regulatory elements that can activate tissue-specific gene expression and are abundant throughout mammalian genomes. While substantial progress has been made towards genome-wide annotation of mammalian enhancers, their temporal activity patterns and global contributions in the context of developmental in vivo processes remain poorly explored. Here we used epigenomic profiling for H3K27ac, a mark of active enhancers, coupled to transgenic mouse assays to examine the genome-wide utilization of enhancers in three different mouse tissues across seven developmental stages. The majority of the ~90,000 enhancers identified exhibited tightly temporally restricted predicted activity windows and were associated with stage-specific biological functions and regulatory pathways in individual tissues. Comparative genomic analysis revealed that evolutionary conservation of enhancers decreases following mid-gestation across all tissues examined. The dynamic enhancer activities uncovered in this study illuminate rapid and pervasive temporal in vivo changes in enhancer usage underlying processes central to development and disease. PMID:24360275

  3. Sequenced genomes and rapidly emerging technologies pave the way for conifer evolutionary developmental biology

    PubMed Central

    Uddenberg, Daniel; Akhter, Shirin; Ramachandran, Prashanth; Sundström, Jens F.; Carlsbecker, Annelie

    2015-01-01

    Conifers, Ginkgo, cycads and gnetophytes comprise the four groups of extant gymnosperms holding a unique position of sharing common ancestry with the angiosperms. Comparative studies of gymnosperms and angiosperms are the key to a better understanding of ancient seed plant morphologies, how they have shifted over evolution to shape modern day species, and how the genes governing these morphologies have evolved. However, conifers and other gymnosperms have been notoriously difficult to study due to their long generation times, inaccessibility to genetic experimentation and unavailable genome sequences. Now, with three draft genomes from spruces and pines, rapid advances in next generation sequencing methods for genome wide expression analyses, and enhanced methods for genetic transformation, we are much better equipped to address a number of key evolutionary questions relating to seed plant evolution. In this mini-review we highlight recent progress in conifer developmental biology relevant to evo-devo questions. We discuss how genome sequence data and novel techniques might allow us to explore genetic variation and naturally occurring conifer mutants, approaches to reduce long generation times to allow for genetic studies in conifers, and other potential upcoming research avenues utilizing current and emergent techniques. Results from developmental studies of conifers and other gymnosperms in comparison to those in angiosperms will provide information to trace core molecular developmental control tool kits of ancestral seed plants, but foremost they will greatly improve our understanding of the biology of conifers and other gymnosperms in their own right. PMID:26579190

  4. Sequenced genomes and rapidly emerging technologies pave the way for conifer evolutionary developmental biology.

    PubMed

    Uddenberg, Daniel; Akhter, Shirin; Ramachandran, Prashanth; Sundström, Jens F; Carlsbecker, Annelie

    2015-01-01

    Conifers, Ginkgo, cycads and gnetophytes comprise the four groups of extant gymnosperms holding a unique position of sharing common ancestry with the angiosperms. Comparative studies of gymnosperms and angiosperms are the key to a better understanding of ancient seed plant morphologies, how they have shifted over evolution to shape modern day species, and how the genes governing these morphologies have evolved. However, conifers and other gymnosperms have been notoriously difficult to study due to their long generation times, inaccessibility to genetic experimentation and unavailable genome sequences. Now, with three draft genomes from spruces and pines, rapid advances in next generation sequencing methods for genome wide expression analyses, and enhanced methods for genetic transformation, we are much better equipped to address a number of key evolutionary questions relating to seed plant evolution. In this mini-review we highlight recent progress in conifer developmental biology relevant to evo-devo questions. We discuss how genome sequence data and novel techniques might allow us to explore genetic variation and naturally occurring conifer mutants, approaches to reduce long generation times to allow for genetic studies in conifers, and other potential upcoming research avenues utilizing current and emergent techniques. Results from developmental studies of conifers and other gymnosperms in comparison to those in angiosperms will provide information to trace core molecular developmental control tool kits of ancestral seed plants, but foremost they will greatly improve our understanding of the biology of conifers and other gymnosperms in their own right.

  5. Next Generation Sequencing to Characterize Mitochondrial Genomic DNA Heteroplasmy

    PubMed Central

    Huang, Taosheng

    2015-01-01

    This protocol is to describe the methodology to characterize mitochondria DNA (mtDNA) heteroplasmy with parallel sequencing. Mitochondria play a very important role in important cellular functions. Each eukaryotic cell contains hundreds of mitochondria with hundreds of mitochondria genomes. The mutant mtDNA and the wild type may co-exist as heteroplasmy, and cause human disease. The purpose of this methodology is to simultaneously determine mtDNA sequence and to quantify the heteroplasmy level. The protocol includes two-fragment mitochondria genome DNA PCR amplification. The PCR product is then mixed at an equimolar ratio. The samples will be barcoded and sequenced with high-throughput next-generation sequencing technology. We found that this technology is highly sensitive, specific, and accurate in determining mtDNA mutations and the degree of heteroplasmic level. PMID:21975941

  6. Characterization of a Rapidly Solidified Iron-Based Superalloy

    NASA Astrophysics Data System (ADS)

    Smugeresky, J. E.

    1982-09-01

    Rapidly-solidified powders of an iron-based superalloy were characterized before and after consolidation by hot isostatic pressing. Powders made by inert gas atomization were compared to powders made by centrifugal atomization. Although many of the powder characteristics were similar, the microstructures were not. The inert gas atomized powder structure is cellular while the centrifugally atomized powder structure is dendritic. In general the finer powder particles have the finer micro-structure with the effect more noticeable in centrifugally atomized powders. After consolidation, the differences in microstructure are more dependent on the consolidation temperature and post-consolidation heat treatment than in the powder type or size. Higher consolidation temperatures and/or post-consolidation heat treatment will result in transformation of the as-solidified microstructures. The transformed microstructure and the mechanical properties can in some cases be related to the as-solidified structure. Heat treatment is needed to obtain mechanical properties equivalent to those of ingot metallurgy processed material.

  7. Rapid Whole-Genome Sequencing of Mycobacterium tuberculosis Isolates Directly from Clinical Samples

    PubMed Central

    Brown, Amanda C.; Einer-Jensen, Katja; Holdstock, Jolyon; Houniet, Darren T.; Chan, Jacqueline Z. M.; Depledge, Daniel P.; Nikolayevskyy, Vladyslav; Broda, Agnieszka; Stone, Madeline J.; Christiansen, Mette T.; Williams, Rachel; McAndrew, Michael B.; Tutill, Helena; Brown, Julianne; Melzer, Mark; Rosmarin, Caryn; McHugh, Timothy D.; Shorten, Robert J.; Drobniewski, Francis; Speight, Graham; Breuer, Judith

    2015-01-01

    The rapid identification of antimicrobial resistance is essential for effective treatment of highly resistant Mycobacterium tuberculosis. Whole-genome sequencing provides comprehensive data on resistance mutations and strain typing for monitoring transmission, but unlike for conventional molecular tests, this has previously been achievable only from cultures of M. tuberculosis. Here we describe a method utilizing biotinylated RNA baits designed specifically for M. tuberculosis DNA to capture full M. tuberculosis genomes directly from infected sputum samples, allowing whole-genome sequencing without the requirement of culture. This was carried out on 24 smear-positive sputum samples, collected from the United Kingdom and Lithuania where a matched culture sample was available, and 2 samples that had failed to grow in culture. M. tuberculosis sequencing data were obtained directly from all 24 smear-positive culture-positive sputa, of which 20 were of high quality (>20× depth and >90% of the genome covered). Results were compared with those of conventional molecular and culture-based methods, and high levels of concordance between phenotypical resistance and predicted resistance based on genotype were observed. High-quality sequence data were obtained from one smear-positive culture-negative case. This study demonstrated for the first time the successful and accurate sequencing of M. tuberculosis genomes directly from uncultured sputa. Identification of known resistance mutations within a week of sample receipt offers the prospect for personalized rather than empirical treatment of drug-resistant tuberculosis, including the use of antimicrobial-sparing regimens, leading to improved outcomes. PMID:25972414

  8. Evaluating and Characterizing Ancient Whole-Genome Duplications in Plants with Gene Count Data

    PubMed Central

    Tiley, George P.; Ané, Cécile; Burleigh, J. Gordon

    2016-01-01

    Whole-genome duplications (WGDs) have helped shape the genomes of land plants, and recent evidence suggests that the genomes of all angiosperms have experienced at least two ancient WGDs. In plants, WGDs often are followed by rapid fractionation, in which many homeologous gene copies are lost. Thus, it can be extremely difficult to identify, let alone characterize, ancient WGDs. In this study, we use a new maximum likelihood estimator to test for evidence of ancient WGDs in land plants and estimate the fraction of new genes copies that are retained following a WGD using gene count data, the number of gene copies in gene families. We identified evidence of many putative ancient WGDs in land plants and found that the genome fractionation rates vary tremendously among ancient WGDs. Analyses of WGDs within Brassicales also indicate that background gene duplication and loss rates vary across land plants, and different gene families have different probabilities of being retained following a WGD. Although our analyses are largely robust to errors in duplication and loss rates and the choice of priors, simulations indicate that this method can have trouble detecting multiple WGDs that occur on the same branch, especially when the gene retention rates for ancient WGDs are very low. They also suggest that we should carefully evaluate evidence for some ancient plant WGD hypotheses. PMID:26988251

  9. Characterization of copy number variation in genomic regions containing STR loci using array comparative genomic hybridization.

    PubMed

    Repnikova, Elena A; Rosenfeld, Jill A; Bailes, Andrea; Weber, Cecilia; Erdman, Linda; McKinney, Aimee; Ramsey, Sarah; Hashimoto, Sayaka; Lamb Thrush, Devon; Astbury, Caroline; Reshmi, Shalini C; Shaffer, Lisa G; Gastier-Foster, Julie M; Pyatt, Robert E

    2013-09-01

    Short tandem repeat (STR) loci are commonly used in forensic casework, familial analysis for human identification, and for monitoring hematopoietic cell engraftment after bone marrow transplant. Unexpected genetic variation leading to sequence and length differences in STR loci can complicate STR typing, and presents challenges in casework interpretation. Copy number variation (CNV) is a relatively recently identified form of genetic variation consisting of genomic regions present at variable copy numbers within an individual compared to a reference genome. Large scale population studies have demonstrated that likely all individuals carry multiple regions with CNV of 1kb in size or greater in their genome. To date, no study correlating genomic regions containing STR loci with CNV has been conducted. In this study, we analyzed results from 32,850 samples sent for clinical array comparative genomic hybridization (CGH) analysis for the presence of CNV at regions containing the 13 CODIS (Combined DNA Index System) STR, and the Amelogenin X (AMELX) and Amelogenin Y (AMELY) loci. Thirty-two individuals with CNV involving STR loci on chromosomes 2, 4, 7, 11, 12, 13, 16, and 21, and twelve with CNV involving the AMELX/AMELY loci were identified. These results were correlated with data from publicly available databases housing information on CNV identified in normal populations and additional clinical cases. These collective results demonstrate the presence of CNV in regions containing 9 of the 13 CODIS STR and AMELX/Y loci. Further characterization of STR profiles within regions of CNV, additional cataloging of these variants in multiple populations, and contributing such examples to the public domain will provide valuable information for reliable use of these loci.

  10. Genomic Characterization of Large Heterochromatic Gaps in the Human Genome Assembly

    PubMed Central

    Altemose, Nicolas; Miga, Karen H.; Maggioni, Mauro; Willard, Huntington F.

    2014-01-01

    The largest gaps in the human genome assembly correspond to multi-megabase heterochromatic regions composed primarily of two related families of tandem repeats, Human Satellites 2 and 3 (HSat2,3). The abundance of repetitive DNA in these regions challenges standard mapping and assembly algorithms, and as a result, the sequence composition and potential biological functions of these regions remain largely unexplored. Furthermore, existing genomic tools designed to predict consensus-based descriptions of repeat families cannot be readily applied to complex satellite repeats such as HSat2,3, which lack a consistent repeat unit reference sequence. Here we present an alignment-free method to characterize complex satellites using whole-genome shotgun read datasets. Utilizing this approach, we classify HSat2,3 sequences into fourteen subfamilies and predict their chromosomal distributions, resulting in a comprehensive satellite reference database to further enable genomic studies of heterochromatic regions. We also identify 1.3 Mb of non-repetitive sequence interspersed with HSat2,3 across 17 unmapped assembly scaffolds, including eight annotated gene predictions. Finally, we apply our satellite reference database to high-throughput sequence data from 396 males to estimate array size variation of the predominant HSat3 array on the Y chromosome, confirming that satellite array sizes can vary between individuals over an order of magnitude (7 to 98 Mb) and further demonstrating that array sizes are distributed differently within distinct Y haplogroups. In summary, we present a novel framework for generating initial reference databases for unassembled genomic regions enriched with complex satellite DNA, and we further demonstrate the utility of these reference databases for studying patterns of sequence variation within human populations. PMID:24831296

  11. The evolutionary analysis of "orphans" from the Drosophila genome identifies rapidly diverging and incorrectly annotated genes.

    PubMed

    Schmid, K J; Aquadro, C F

    2001-10-01

    In genome projects of eukaryotic model organisms, a large number of novel genes of unknown function and evolutionary history ("orphans") are being identified. Since many orphans have no known homologs in distant species, it is unclear whether they are restricted to certain taxa or evolve rapidly, either because of a lack of constraints or positive Darwinian selection. Here we use three criteria for the selection of putatively rapidly evolving genes from a single sequence of Drosophila melanogaster. Thirteen candidate genes were chosen from the Adh region on the second chromosome and 1 from the tip of the X chromosome. We succeeded in obtaining sequence from 6 of these in the closely related species D. simulans and D. yakuba. Only 1 of the 6 genes showed a large number of amino acid replacements and in-frame insertions/deletions. A population survey of this gene suggests that its rapid evolution is due to the fixation of many neutral or nearly neutral mutations. Two other genes showed "normal" levels of divergence between species. Four genes had insertions/deletions that destroy the putative reading frame within exons, suggesting that these exons have been incorrectly annotated. The evolutionary analysis of orphan genes in closely related species is useful for the identification of both rapidly evolving and incorrectly annotated genes.

  12. DNA immunoprecipitation semiconductor sequencing (DIP-SC-seq) as a rapid method to generate genome wide epigenetic signatures.

    PubMed

    Thomson, John P; Fawkes, Angie; Ottaviano, Raffaele; Hunter, Jennifer M; Shukla, Ruchi; Mjoseng, Heidi K; Clark, Richard; Coutts, Audrey; Murphy, Lee; Meehan, Richard R

    2015-05-14

    Modification of DNA resulting in 5-methylcytosine (5 mC) or 5-hydroxymethylcytosine (5hmC) has been shown to influence the local chromatin environment and affect transcription. Although recent advances in next generation sequencing technology allow researchers to map epigenetic modifications across the genome, such experiments are often time-consuming and cost prohibitive. Here we present a rapid and cost effective method of generating genome wide DNA modification maps utilising commercially available semiconductor based technology (DNA immunoprecipitation semiconductor sequencing; "DIP-SC-seq") on the Ion Proton sequencer. Focussing on the 5hmC mark we demonstrate, by directly comparing with alternative sequencing strategies, that this platform can successfully generate genome wide 5hmC patterns from as little as 500 ng of genomic DNA in less than 4 days. Such a method can therefore facilitate the rapid generation of multiple genome wide epigenetic datasets.

  13. A rapid noninvasive characterization of CT x-ray sources

    SciTech Connect

    Randazzo, Matt; Tambasco, Mauro

    2015-07-15

    Purpose: The aim of this study is to generate spatially varying half value layers (HVLs) that can be used to construct virtual equivalent source models of computed tomography (CT) x-ray sources for use in Monte Carlo CT dose computations. Methods: To measure the spatially varying HVLs, the authors combined a cylindrical HVL measurement technique with the characterization of bowtie filter relative attenuation (COBRA) geometry. An apparatus given the name “HVL Jig” was fabricated to accurately position a real-time dosimeter off-isocenter while surrounded by concentric cylindrical aluminum filters (CAFs). In this geometry, each projection of the rotating x-ray tube is filtered by an identical amount of high-purity (type 1100 H-14) aluminum while the stationary radiation dose probe records an air kerma rate versus time waveform. The CAFs were progressively nested to acquire exposure data at increasing filtrations to calculate the HVL. Using this dose waveform and known setup geometry, each timestamp was related to its corresponding fan angle. Data were acquired using axial CT protocols (i.e., rotating tube and stationary patient table) at energies of 80, 100, and 120 kVp on a single CT scanner. These measurements were validated against the more laborious conventional step-and-shoot approach (stationary x-ray tube). Results: At each energy, HVL data points from the COBRA-cylinder technique were fit to a trendline and compared with the conventional approach. The average relative difference in HVL between the two techniques was 1.3%. There was a systematic overestimation in HVL due to scatter contamination. Conclusions: The described method is a novel, rapid, accurate, and noninvasive approach that allows one to acquire the spatially varying fluence and HVL data using a single experimental setup in a minimum of three scans. These measurements can be used to characterize the CT beam in terms of the angle-dependent fluence and energy spectra along the bowtie filter

  14. Genomic Analysis of the Emergence and Rapid Global Dissemination of the Clonal Group 258 Klebsiella pneumoniae Pandemic.

    PubMed

    Bowers, Jolene R; Kitchel, Brandon; Driebe, Elizabeth M; MacCannell, Duncan R; Roe, Chandler; Lemmer, Darrin; de Man, Tom; Rasheed, J Kamile; Engelthaler, David M; Keim, Paul; Limbago, Brandi M

    2015-01-01

    Multidrug-resistant Klebsiella pneumoniae producing the KPC carbapenemase have rapidly spread throughout the world, causing severe healthcare-associated infections with limited antimicrobial treatment options. Dissemination of KPC-producing K. pneumoniae is largely attributed to expansion of a single dominant strain, ST258. In this study, we explore phylogenetic relationships and evolution within ST258 and its clonal group, CG258, using whole genome sequence analysis of 167 isolates from 20 countries collected over 17 years. Our results show a common ST258 ancestor emerged from its diverse parental clonal group around 1995 and likely acquired blaKPC prior to dissemination. Over the past two decades, ST258 has remained highly clonal despite diversity in accessory elements and divergence in the capsule polysaccharide synthesis locus. Apart from the large recombination event that gave rise to ST258, few mutations set it apart from its clonal group. However, one mutation occurs in a global transcription regulator. Characterization of outer membrane protein sequences revealed a profile in ST258 that includes a truncated OmpK35 and modified OmpK37. Our work illuminates potential genomic contributors to the pathogenic success of ST258, helps us better understand the global dissemination of this strain, and identifies genetic markers unique to ST258.

  15. Genomic Analysis of the Emergence and Rapid Global Dissemination of the Clonal Group 258 Klebsiella pneumoniae Pandemic

    PubMed Central

    Driebe, Elizabeth M.; MacCannell, Duncan R.; Roe, Chandler; Lemmer, Darrin; de Man, Tom; Rasheed, J. Kamile; Engelthaler, David M.; Keim, Paul; Limbago, Brandi M.

    2015-01-01

    Multidrug-resistant Klebsiella pneumoniae producing the KPC carbapenemase have rapidly spread throughout the world, causing severe healthcare-associated infections with limited antimicrobial treatment options. Dissemination of KPC-producing K. pneumoniae is largely attributed to expansion of a single dominant strain, ST258. In this study, we explore phylogenetic relationships and evolution within ST258 and its clonal group, CG258, using whole genome sequence analysis of 167 isolates from 20 countries collected over 17 years. Our results show a common ST258 ancestor emerged from its diverse parental clonal group around 1995 and likely acquired blaKPC prior to dissemination. Over the past two decades, ST258 has remained highly clonal despite diversity in accessory elements and divergence in the capsule polysaccharide synthesis locus. Apart from the large recombination event that gave rise to ST258, few mutations set it apart from its clonal group. However, one mutation occurs in a global transcription regulator. Characterization of outer membrane protein sequences revealed a profile in ST258 that includes a truncated OmpK35 and modified OmpK37. Our work illuminates potential genomic contributors to the pathogenic success of ST258, helps us better understand the global dissemination of this strain, and identifies genetic markers unique to ST258. PMID:26196384

  16. Rapid Identification of Potential Drugs for Diabetic Nephropathy Using Whole-Genome Expression Profiles of Glomeruli

    PubMed Central

    Shi, Jingsong; Jiang, Song; Qiu, Dandan; Le, Weibo; Wang, Xiao; Lu, Yinhui; Liu, Zhihong

    2016-01-01

    Objective. To investigate potential drugs for diabetic nephropathy (DN) using whole-genome expression profiles and the Connectivity Map (CMAP). Methodology. Eighteen Chinese Han DN patients and six normal controls were included in this study. Whole-genome expression profiles of microdissected glomeruli were measured using the Affymetrix human U133 plus 2.0 chip. Differentially expressed genes (DEGs) between late stage and early stage DN samples and the CMAP database were used to identify potential drugs for DN using bioinformatics methods. Results. (1) A total of 1065 DEGs (FDR < 0.05 and fold change > 1.5) were found in late stage DN patients compared with early stage DN patients. (2) Piperlongumine, 15d-PGJ2 (15-delta prostaglandin J2), vorinostat, and trichostatin A were predicted to be the most promising potential drugs for DN, acting as NF-κB inhibitors, histone deacetylase inhibitors (HDACIs), PI3K pathway inhibitors, or PPARγ agonists, respectively. Conclusion. Using whole-genome expression profiles and the CMAP database, we rapidly predicted potential DN drugs, and therapeutic potential was confirmed by previously published studies. Animal experiments and clinical trials are needed to confirm both the safety and efficacy of these drugs in the treatment of DN. PMID:27069916

  17. Rapid and simple methodology for isolation of high quality genomic DNA from coniferous tissues (Taxus baccata).

    PubMed

    Barzegari, Abolfazl; Vahed, Sepideh Zununi; Atashpaz, Sina; Khani, Sajjad; Omidi, Yadollah

    2010-02-01

    Various investigations have been so far performed for extraction of genomic DNA from plant tissues, in which the extracted intact DNA can be exploited for a diverse range of biological studies. Extraction of high quality DNA from leathery plant tissues (e.g., coniferous organs) appears to be a critical stage. Moreover, for some species such as Taxus trees, bioprocess engineering and biosynthesis of secondary metabolites (e.g., paclitaxel) is a crucial step due to the restrictions associated with extinction of these species. However, extraction of intact genomic DNA from these plants still demands a rapid, easy and efficient protocol. To pursue such aim, in the current work, we report on the development of a simple and highly efficient method for the extraction of DNA from Taxus baccata. Based upon our protocol, interfering phenolic compounds were removed from extraction using polyvinylpyrrolidone and RNA contamination was resolved using LiCl. By employing this method, high quality genomic DNA was successfully extracted from leaves of T. baccata. The quality of extracted DNA was validated by various techniques such as RAPD marker, restriction digestions and pre-AFLP. Upon our findings, we propose this simple method to be considered for extraction of DNA from leathery plant tissues.

  18. Isothermal rolling circle amplification of virus genomes for rapid antigen detection and typing.

    PubMed

    Brasino, Michael D; Cha, Jennifer N

    2015-08-01

    In this work, isothermal rolling circle amplification (RCA) of the multi-kilobase genome of engineered filamentous bacteriophage is used to report the presence and identification of specific protein analytes in solution. First, bacteriophages were chosen as sensing platforms because peptides or antibodies that bind medically relevant targets can be isolated through phage display or expressed as fusions to their p3 and p8 coat proteins. Second, the circular, single-stranded genome contained within the phage serves as a natural large DNA template for a RCA reaction to rapidly generate exponential amounts of double stranded DNA in a single isothermal step that can be easily detected using low-cost fluorescent nucleic acid stains. Amplifying the entire phage genome also provides high detection sensitivities. Furthermore, since the sequence of the viral DNA can be easily modified with multiple restriction enzyme sites, a simple DNA digest can be applied to detect and identify multiple antigens simultaneously. The methods developed here will lead to protein sensors that are highly scalable to produce, can be run without complex biological equipment and do not require the use of multiple antibodies or high-cost fluorescent DNA probes or nucleotides.

  19. Rapid Evolution of Genomic Imprinting in Two Species of the Brassicaceae.

    PubMed

    Hatorangan, Marcelinus R; Laenen, Benjamin; Steige, Kim A; Slotte, Tanja; Köhler, Claudia

    2016-08-01

    Genomic imprinting is an epigenetic phenomenon occurring in mammals and flowering plants that causes genes to adopt a parent-of-origin-specific mode of expression. While the imprinting status of genes is well conserved in mammals, clear estimates for the degree of conservation were lacking in plants. We therefore analyzed the genome-wide imprinting status of Capsella rubella, which shared a common recent ancestor with Arabidopsis thaliana ∼10 to 14 million years ago. However, only ∼14% of maternally expressed genes (MEGs) and ∼29% of paternally expressed genes (PEGs) in C. rubella were commonly imprinted in both species, revealing that genomic imprinting is a rapidly evolving phenomenon in plants. Nevertheless, conserved PEGs exhibited signs of selection, suggesting that a subset of imprinted genes play an important functional role and are therefore maintained in plants. Like in Arabidopsis, PEGs in C. rubella are frequently associated with the presence of transposable elements that preferentially belong to helitron and MuDR families. Our data further reveal that MEGs and PEGs differ in their targeting by 24-nucleotide small RNAs and asymmetric DNA methylation, suggesting different mechanisms establishing DNA methylation at MEGs and PEGs. PMID:27465027

  20. Microstructural characterization of rapidly solidified Al-Li-Co powders

    NASA Astrophysics Data System (ADS)

    Samuel, Fawzy H.

    1986-01-01

    A study of the combined effect of alloying elements and melt superheat has been carried out on the as-solidified structure of rapidly solidified Al-Li-Co powders. Three alloys, viz., Al-3 pct Li, Al-3 pct Li-0.4 pct Co, and Al-3 pct Li-0.8 pct Co were chosen, and the liquid melt in each alloy atomized from the temperatures 1173 and 1073 K, using the centrifugal atomization technique. The microstructural characterization was done using light, scanning, and transmission electron microscopy. Four types of microstructures, viz., dendritic, cellular, equiaxed-type, and featureless structures, were observed by light microscopy. The cooling rate, as determined from the same, lay in the range 104 to 106 Ks-1, but was seen to go beyond 107 Ks-1 when estimated from TEM micrographs. On the micro-level, the Al-Li powders were found to exhibit dendritic structures with differing morphologies, whereas low-angle cell walls with perturbed interfaces were the main structural features observed in the Al-Li-Co alloys. Increasing both cobalt content and powder particle diameter favored transition from dendritic into cellular structure. The featureless zone was comprised mainly of elongated columnar grains (0.2 μm width and 1.5 μm length). A mechanism describing the cellular structure formation has been proposed. Aging of the melt-quenched powders at 473 K for times up to 100 hours results in the dissolution of the cellular structure. A mechanism for the same has been postulated. The difference in the superheats chosen in the present work is found not sufficient to cause drastic microstructural changes.

  1. Genomic Sequencing and Characterization of Cynomolgus Macaque Cytomegalovirus▿

    PubMed Central

    Marsh, Angie K.; Willer, David O.; Ambagala, Aruna P. N.; Dzamba, Misko; Chan, Jacqueline K.; Pilon, Richard; Fournier, Jocelyn; Sandstrom, Paul; Brudno, Michael; MacDonald, Kelly S.

    2011-01-01

    Cytomegalovirus (CMV) infection is the most common opportunistic infection in immunosuppressed individuals, such as transplant recipients or people living with HIV/AIDS, and congenital CMV is the leading viral cause of developmental disabilities in infants. Due to the highly species-specific nature of CMV, animal models that closely recapitulate human CMV (HCMV) are of growing importance for vaccine development. Here we present the genomic sequence of a novel nonhuman primate CMV from cynomolgus macaques (Macaca fascicularis; CyCMV). CyCMV (Ottawa strain) was isolated from the urine of a healthy, captive-bred, 4-year-old cynomolgus macaque of Philippine origin, and the viral genome was sequenced using next-generation Illumina sequencing to an average of 516-fold coverage. The CyCMV genome is 218,041 bp in length, with 49.5% G+C content and 84% protein-coding density. We have identified 262 putative open reading frames (ORFs) with an average coding length of 789 bp. The genomic organization of CyCMV is largely colinear with that of rhesus macaque CMV (RhCMV). Of the 262 CyCMV ORFs, 137 are homologous to HCMV genes, 243 are homologous to RhCMV 68.1, and 200 are homologous to RhCMV 180.92. CyCMV encodes four ORFs that are not present in RhCMV strain 68.1 or 180.92 but have homologies with HCMV (UL30, UL74A, UL126, and UL146). Similar to HCMV, CyCMV does not produce the RhCMV-specific viral homologue of cyclooxygenase-2. This newly characterized CMV may provide a novel model in which to study CMV biology and HCMV vaccine development. PMID:21994460

  2. Genomic and phenotypic characterization of the species Acinetobacter venetianus

    PubMed Central

    Fondi, Marco; Maida, Isabel; Perrin, Elena; Orlandini, Valerio; La Torre, Laura; Bosi, Emanuele; Negroni, Andrea; Zanaroli, Giulio; Fava, Fabio; Decorosi, Francesca; Giovannetti, Luciana; Viti, Carlo; Vaneechoutte, Mario; Dijkshoorn, Lenie; Fani, Renato

    2016-01-01

    Crude oil is a complex mixture of hydrocarbons and other organic compounds that can produce serious environmental problems and whose removal is highly demanding in terms of human and technological resources. The potential use of microbes as bioremediation agents is one of the most promising fields in this area. Members of the species Acinetobacter venetianus have been previously characterized for their capability to degrade n-alkanes and thus may represent interesting model systems to implement this process. Although a preliminary experimental characterization of the overall hydrocarbon degradation capability has been performed for five of them, to date, the genetic/genomic features underlying such molecular processes have not been identified. Here we have integrated genomic and phenotypic information for six A. venetianus strains, i.e. VE-C3, RAG-1T, LUH 13518, LUH 7437, LUH 5627 and LUH 8758. Besides providing a thorough description of the A. venetianus species, these data were exploited to infer the genetic features (presence/absence patterns of genes) and the short-term evolutionary events possibly responsible for the variability in n-alkane degradation efficiency of these strains, including the mechanisms of interaction with the fuel droplet and the subsequent catabolism of this pollutant. PMID:26902269

  3. Whole-Genome Sequencing of Native Sheep Provides Insights into Rapid Adaptations to Extreme Environments.

    PubMed

    Yang, Ji; Li, Wen-Rong; Lv, Feng-Hua; He, San-Gang; Tian, Shi-Lin; Peng, Wei-Feng; Sun, Ya-Wei; Zhao, Yong-Xin; Tu, Xiao-Long; Zhang, Min; Xie, Xing-Long; Wang, Yu-Tao; Li, Jin-Quan; Liu, Yong-Gang; Shen, Zhi-Qiang; Wang, Feng; Liu, Guang-Jian; Lu, Hong-Feng; Kantanen, Juha; Han, Jian-Lin; Li, Meng-Hua; Liu, Ming-Jun

    2016-10-01

    Global climate change has a significant effect on extreme environments and a profound influence on species survival. However, little is known of the genome-wide pattern of livestock adaptations to extreme environments over a short time frame following domestication. Sheep (Ovis aries) have become well adapted to a diverse range of agroecological zones, including certain extreme environments (e.g., plateaus and deserts), during their post-domestication (approximately 8-9 kya) migration and differentiation. Here, we generated whole-genome sequences from 77 native sheep, with an average effective sequencing depth of ∼5× for 75 samples and ∼42× for 2 samples. Comparative genomic analyses among sheep in contrasting environments, that is, plateau (>4,000 m above sea level) versus lowland (<100 m), high-altitude region (>1500 m) versus low-altitude region (<1300 m), desert (<10 mm average annual precipitation) versus highly humid region (>600 mm), and arid zone (<400 mm) versus humid zone (>400 mm), detected a novel set of candidate genes as well as pathways and GO categories that are putatively associated with hypoxia responses at high altitudes and water reabsorption in arid environments. In addition, candidate genes and GO terms functionally related to energy metabolism and body size variations were identified. This study offers novel insights into rapid genomic adaptations to extreme environments in sheep and other animals, and provides a valuable resource for future research on livestock breeding in response to climate change. PMID:27401233

  4. Whole-Genome Sequencing of Native Sheep Provides Insights into Rapid Adaptations to Extreme Environments

    PubMed Central

    Yang, Ji; Li, Wen-Rong; Lv, Feng-Hua; He, San-Gang; Tian, Shi-Lin; Peng, Wei-Feng; Sun, Ya-Wei; Zhao, Yong-Xin; Tu, Xiao-Long; Zhang, Min; Xie, Xing-Long; Wang, Yu-Tao; Li, Jin-Quan; Liu, Yong-Gang; Shen, Zhi-Qiang; Wang, Feng; Liu, Guang-Jian; Lu, Hong-Feng; Kantanen, Juha; Han, Jian-Lin; Li, Meng-Hua; Liu, Ming-Jun

    2016-01-01

    Global climate change has a significant effect on extreme environments and a profound influence on species survival. However, little is known of the genome-wide pattern of livestock adaptations to extreme environments over a short time frame following domestication. Sheep (Ovis aries) have become well adapted to a diverse range of agroecological zones, including certain extreme environments (e.g., plateaus and deserts), during their post-domestication (approximately 8–9 kya) migration and differentiation. Here, we generated whole-genome sequences from 77 native sheep, with an average effective sequencing depth of ∼5× for 75 samples and ∼42× for 2 samples. Comparative genomic analyses among sheep in contrasting environments, that is, plateau (>4,000 m above sea level) versus lowland (<100 m), high-altitude region (>1500 m) versus low-altitude region (<1300 m), desert (<10 mm average annual precipitation) versus highly humid region (>600 mm), and arid zone (<400 mm) versus humid zone (>400 mm), detected a novel set of candidate genes as well as pathways and GO categories that are putatively associated with hypoxia responses at high altitudes and water reabsorption in arid environments. In addition, candidate genes and GO terms functionally related to energy metabolism and body size variations were identified. This study offers novel insights into rapid genomic adaptations to extreme environments in sheep and other animals, and provides a valuable resource for future research on livestock breeding in response to climate change. PMID:27401233

  5. Rapid and highly efficient construction of TALE-based transcriptional regulators and nucleases for genome modification.

    PubMed

    Li, Lixin; Piatek, Marek J; Atef, Ahmed; Piatek, Agnieszka; Wibowo, Anjar; Fang, Xiaoyun; Sabir, J S M; Zhu, Jian-Kang; Mahfouz, Magdy M

    2012-03-01

    Transcription activator-like effectors (TALEs) can be used as DNA-targeting modules by engineering their repeat domains to dictate user-selected sequence specificity. TALEs have been shown to function as site-specific transcriptional activators in a variety of cell types and organisms. TALE nucleases (TALENs), generated by fusing the FokI cleavage domain to TALE, have been used to create genomic double-strand breaks. The identity of the TALE repeat variable di-residues, their number, and their order dictate the DNA sequence specificity. Because TALE repeats are nearly identical, their assembly by cloning or even by synthesis is challenging and time consuming. Here, we report the development and use of a rapid and straightforward approach for the construction of designer TALE (dTALE) activators and nucleases with user-selected DNA target specificity. Using our plasmid set of 100 repeat modules, researchers can assemble repeat domains for any 14-nucleotide target sequence in one sequential restriction-ligation cloning step and in only 24 h. We generated several custom dTALEs and dTALENs with new target sequence specificities and validated their function by transient expression in tobacco leaves and in vitro DNA cleavage assays, respectively. Moreover, we developed a web tool, called idTALE, to facilitate the design of dTALENs and the identification of their genomic targets and potential off-targets in the genomes of several model species. Our dTALE repeat assembly approach along with the web tool idTALE will expedite genome-engineering applications in a variety of cell types and organisms including plants.

  6. Comparative Genomic Analysis of Rapid Evolution of an Extreme-Drug-Resistant Acinetobacter baumannii Clone

    PubMed Central

    Tan, Sean Yang-Yi; Chua, Song Lin; Liu, Yang; Høiby, Niels; Andersen, Leif Percival; Givskov, Michael; Song, Zhijun; Yang, Liang

    2013-01-01

    The emergence of extreme-drug-resistant (EDR) bacterial strains in hospital and nonhospital clinical settings is a big and growing public health threat. Understanding the antibiotic resistance mechanisms at the genomic levels can facilitate the development of next-generation agents. Here, comparative genomics has been employed to analyze the rapid evolution of an EDR Acinetobacter baumannii clone from the intensive care unit (ICU) of Rigshospitalet at Copenhagen. Two resistant A. baumannii strains, 48055 and 53264, were sequentially isolated from two individuals who had been admitted to ICU within a 1-month interval. Multilocus sequence typing indicates that these two isolates belonged to ST208. The A. baumannii 53264 strain gained colistin resistance compared with the 48055 strain and became an EDR strain. Genome sequencing indicates that A. baumannii 53264 and 48055 have almost identical genomes—61 single-nucleotide polymorphisms (SNPs) were found between them. The A. baumannii 53264 strain was assembled into 130 contigs, with a total length of 3,976,592 bp with 38.93% GC content. The A. baumannii 48055 strain was assembled into 135 contigs, with a total length of 4,049,562 bp with 39.00% GC content. Genome comparisons showed that this A. baumannii clone is classified as an International clone II strain and has 94% synteny with the A. baumannii ACICU strain. The ResFinder server identified a total of 14 antibiotic resistance genes in the A. baumannii clone. Proteomic analyses revealed that a putative porin protein was down-regulated when A. baumannii 53264 was exposed to antimicrobials, which may reduce the entry of antibiotics into the bacterial cell. PMID:23538992

  7. Whole-Genome Sequencing of Native Sheep Provides Insights into Rapid Adaptations to Extreme Environments.

    PubMed

    Yang, Ji; Li, Wen-Rong; Lv, Feng-Hua; He, San-Gang; Tian, Shi-Lin; Peng, Wei-Feng; Sun, Ya-Wei; Zhao, Yong-Xin; Tu, Xiao-Long; Zhang, Min; Xie, Xing-Long; Wang, Yu-Tao; Li, Jin-Quan; Liu, Yong-Gang; Shen, Zhi-Qiang; Wang, Feng; Liu, Guang-Jian; Lu, Hong-Feng; Kantanen, Juha; Han, Jian-Lin; Li, Meng-Hua; Liu, Ming-Jun

    2016-10-01

    Global climate change has a significant effect on extreme environments and a profound influence on species survival. However, little is known of the genome-wide pattern of livestock adaptations to extreme environments over a short time frame following domestication. Sheep (Ovis aries) have become well adapted to a diverse range of agroecological zones, including certain extreme environments (e.g., plateaus and deserts), during their post-domestication (approximately 8-9 kya) migration and differentiation. Here, we generated whole-genome sequences from 77 native sheep, with an average effective sequencing depth of ∼5× for 75 samples and ∼42× for 2 samples. Comparative genomic analyses among sheep in contrasting environments, that is, plateau (>4,000 m above sea level) versus lowland (<100 m), high-altitude region (>1500 m) versus low-altitude region (<1300 m), desert (<10 mm average annual precipitation) versus highly humid region (>600 mm), and arid zone (<400 mm) versus humid zone (>400 mm), detected a novel set of candidate genes as well as pathways and GO categories that are putatively associated with hypoxia responses at high altitudes and water reabsorption in arid environments. In addition, candidate genes and GO terms functionally related to energy metabolism and body size variations were identified. This study offers novel insights into rapid genomic adaptations to extreme environments in sheep and other animals, and provides a valuable resource for future research on livestock breeding in response to climate change.

  8. Rapid detection, characterization, and enrumeration of food-borne pathogens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In recent years, there has been much research activity on the development of methodologies that are rapid, accurate, and ultrasensitive for detecting pathogenic microorganisms in food. Rapid methods include immunological systems such as the lateral flow assays and enzyme-linked immunosorbent assays...

  9. Centromere and telomere sequence alterations reflect the rapid genome evolution within the carnivorous plant genus Genlisea.

    PubMed

    Tran, Trung D; Cao, Hieu X; Jovtchev, Gabriele; Neumann, Pavel; Novák, Petr; Fojtová, Miloslava; Vu, Giang T H; Macas, Jiří; Fajkus, Jiří; Schubert, Ingo; Fuchs, Joerg

    2015-12-01

    Linear chromosomes of eukaryotic organisms invariably possess centromeres and telomeres to ensure proper chromosome segregation during nuclear divisions and to protect the chromosome ends from deterioration and fusion, respectively. While centromeric sequences may differ between species, with arrays of tandemly repeated sequences and retrotransposons being the most abundant sequence types in plant centromeres, telomeric sequences are usually highly conserved among plants and other organisms. The genome size of the carnivorous genus Genlisea (Lentibulariaceae) is highly variable. Here we study evolutionary sequence plasticity of these chromosomal domains at an intrageneric level. We show that Genlisea nigrocaulis (1C = 86 Mbp; 2n = 40) and G. hispidula (1C = 1550 Mbp; 2n = 40) differ as to their DNA composition at centromeres and telomeres. G. nigrocaulis and its close relative G. pygmaea revealed mainly 161 bp tandem repeats, while G. hispidula and its close relative G. subglabra displayed a combination of four retroelements at centromeric positions. G. nigrocaulis and G. pygmaea chromosome ends are characterized by the Arabidopsis-type telomeric repeats (TTTAGGG); G. hispidula and G. subglabra instead revealed two intermingled sequence variants (TTCAGG and TTTCAGG). These differences in centromeric and, surprisingly, also in telomeric DNA sequences, uncovered between groups with on average a > 9-fold genome size difference, emphasize the fast genome evolution within this genus. Such intrageneric evolutionary alteration of telomeric repeats with cytosine in the guanine-rich strand, not yet known for plants, might impact the epigenetic telomere chromatin modification.

  10. Centromere and telomere sequence alterations reflect the rapid genome evolution within the carnivorous plant genus Genlisea.

    PubMed

    Tran, Trung D; Cao, Hieu X; Jovtchev, Gabriele; Neumann, Pavel; Novák, Petr; Fojtová, Miloslava; Vu, Giang T H; Macas, Jiří; Fajkus, Jiří; Schubert, Ingo; Fuchs, Joerg

    2015-12-01

    Linear chromosomes of eukaryotic organisms invariably possess centromeres and telomeres to ensure proper chromosome segregation during nuclear divisions and to protect the chromosome ends from deterioration and fusion, respectively. While centromeric sequences may differ between species, with arrays of tandemly repeated sequences and retrotransposons being the most abundant sequence types in plant centromeres, telomeric sequences are usually highly conserved among plants and other organisms. The genome size of the carnivorous genus Genlisea (Lentibulariaceae) is highly variable. Here we study evolutionary sequence plasticity of these chromosomal domains at an intrageneric level. We show that Genlisea nigrocaulis (1C = 86 Mbp; 2n = 40) and G. hispidula (1C = 1550 Mbp; 2n = 40) differ as to their DNA composition at centromeres and telomeres. G. nigrocaulis and its close relative G. pygmaea revealed mainly 161 bp tandem repeats, while G. hispidula and its close relative G. subglabra displayed a combination of four retroelements at centromeric positions. G. nigrocaulis and G. pygmaea chromosome ends are characterized by the Arabidopsis-type telomeric repeats (TTTAGGG); G. hispidula and G. subglabra instead revealed two intermingled sequence variants (TTCAGG and TTTCAGG). These differences in centromeric and, surprisingly, also in telomeric DNA sequences, uncovered between groups with on average a > 9-fold genome size difference, emphasize the fast genome evolution within this genus. Such intrageneric evolutionary alteration of telomeric repeats with cytosine in the guanine-rich strand, not yet known for plants, might impact the epigenetic telomere chromatin modification. PMID:26485466

  11. Rapid microsatellite identification from Illumina paired-end genomic sequencing in two birds and a snake

    USGS Publications Warehouse

    Castoe, Todd A.; Poole, Alexander W.; de Koning, A. P. Jason; Jones, Kenneth L.; Tomback, Diana F.; Oyler-McCance, Sara J.; Fike, Jennifer A.; Lance, Stacey L.; Streicher, Jeffrey W.; Smith, Eric N.; Pollock, David D.

    2012-01-01

    Identification of microsatellites, or simple sequence repeats (SSRs), can be a time-consuming and costly investment requiring enrichment, cloning, and sequencing of candidate loci. Recently, however, high throughput sequencing (with or without prior enrichment for specific SSR loci) has been utilized to identify SSR loci. The direct "Seq-to-SSR" approach has an advantage over enrichment-based strategies in that it does not require a priori selection of particular motifs, or prior knowledge of genomic SSR content. It has been more expensive per SSR locus recovered, however, particularly for genomes with few SSR loci, such as bird genomes. The longer but relatively more expensive 454 reads have been preferred over less expensive Illumina reads. Here, we use Illumina paired-end sequence data to identify potentially amplifiable SSR loci (PALs) from a snake (the Burmese python, Python molurus bivittatus), and directly compare these results to those from 454 data. We also compare the python results to results from Illumina sequencing of two bird genomes (Gunnison Sage-grouse, Centrocercus minimus, and Clark's Nutcracker, Nucifraga columbiana), which have considerably fewer SSRs than the python. We show that direct Illumina Seq-to-SSR can identify and characterize thousands of potentially amplifiable SSR loci for as little as $10 per sample – a fraction of the cost of 454 sequencing. Given that Illumina Seq-to-SSR is effective, inexpensive, and reliable even for species such as birds that have few SSR loci, it seems that there are now few situations for which prior hybridization is justifiable.

  12. Rapid microsatellite identification from illumina paired-end genomic sequencing in two birds and a snake

    USGS Publications Warehouse

    Castoe, T.A.; Poole, A.W.; de Koning, A. P. J.; Jones, K.L.; Tomback, D.F.; Oyler-McCance, S.J.; Fike, J.A.; Lance, S.L.; Streicher, J.W.; Smith, E.N.; Pollock, D.D.

    2012-01-01

    Identification of microsatellites, or simple sequence repeats (SSRs), can be a time-consuming and costly investment requiring enrichment, cloning, and sequencing of candidate loci. Recently, however, high throughput sequencing (with or without prior enrichment for specific SSR loci) has been utilized to identify SSR loci. The direct "Seq-to-SSR" approach has an advantage over enrichment-based strategies in that it does not require a priori selection of particular motifs, or prior knowledge of genomic SSR content. It has been more expensive per SSR locus recovered, however, particularly for genomes with few SSR loci, such as bird genomes. The longer but relatively more expensive 454 reads have been preferred over less expensive Illumina reads. Here, we use Illumina paired-end sequence data to identify potentially amplifiable SSR loci (PALs) from a snake (the Burmese python, Python molurus bivittatus), and directly compare these results to those from 454 data. We also compare the python results to results from Illumina sequencing of two bird genomes (Gunnison Sage-grouse, Centrocercus minimus, and Clark's Nutcracker, Nucifraga columbiana), which have considerably fewer SSRs than the python. We show that direct Illumina Seq-to-SSR can identify and characterize thousands of potentially amplifiable SSR loci for as little as $10 per sample - a fraction of the cost of 454 sequencing. Given that Illumina Seq-to-SSR is effective, inexpensive, and reliable even for species such as birds that have few SSR loci, it seems that there are now few situations for which prior hybridization is justifiable. ?? 2012 Castoe et al.

  13. Genomic Characterization of the Genus Nairovirus (Family Bunyaviridae)

    PubMed Central

    Kuhn, Jens H.; Wiley, Michael R.; Rodriguez, Sergio E.; Bào, Yīmíng; Prieto, Karla; Travassos da Rosa, Amelia P. A.; Guzman, Hilda; Savji, Nazir; Ladner, Jason T.; Tesh, Robert B.; Wada, Jiro; Jahrling, Peter B.; Bente, Dennis A.; Palacios, Gustavo

    2016-01-01

    Nairovirus, one of five bunyaviral genera, includes seven species. Genomic sequence information is limited for members of the Dera Ghazi Khan, Hughes, Qalyub, Sakhalin, and Thiafora nairovirus species. We used next-generation sequencing and historical virus-culture samples to determine 14 complete and nine coding-complete nairoviral genome sequences to further characterize these species. Previously unsequenced viruses include Abu Mina, Clo Mor, Great Saltee, Hughes, Raza, Sakhalin, Soldado, and Tillamook viruses. In addition, we present genomic sequence information on additional isolates of previously sequenced Avalon, Dugbe, Sapphire II, and Zirqa viruses. Finally, we identify Tunis virus, previously thought to be a phlebovirus, as an isolate of Abu Hammad virus. Phylogenetic analyses indicate the need for reassignment of Sapphire II virus to Dera Ghazi Khan nairovirus and reassignment of Hazara, Tofla, and Nairobi sheep disease viruses to novel species. We also propose new species for the Kasokero group (Kasokero, Leopards Hill, Yogue viruses), the Ketarah group (Gossas, Issyk-kul, Keterah/soft tick viruses) and the Burana group (Wēnzhōu tick virus, Huángpí tick virus 1, Tǎchéng tick virus 1). Our analyses emphasize the sister relationship of nairoviruses and arenaviruses, and indicate that several nairo-like viruses (Shāyáng spider virus 1, Xīnzhōu spider virus, Sānxiá water strider virus 1, South Bay virus, Wǔhàn millipede virus 2) require establishment of novel genera in a larger nairovirus-arenavirus supergroup. PMID:27294949

  14. A Comprehensive Characterization of Mitochondrial Genome in Papillary Thyroid Cancer

    PubMed Central

    Su, Xingyun; Wang, Weibin; Ruan, Guodong; Liang, Min; Zheng, Jing; Chen, Ye; Wu, Huiling; Fahey, Thomas J.; Guan, Minxin; Teng, Lisong

    2016-01-01

    Nuclear genetic alterations have been widely investigated in papillary thyroid cancer (PTC), however, the characteristics of the mitochondrial genome remain uncertain. We sequenced the entire mitochondrial genome of 66 PTCs, 16 normal thyroid tissues and 376 blood samples of healthy individuals. There were 2508 variations (543 sites) detected in PTCs, among which 33 variations were novel. Nearly half of the PTCs (31/66) had heteroplasmic variations. Among the 31 PTCs, 28 specimens harbored a total of 52 somatic mutations distributed in 44 sites. Thirty-three variations including seven nonsense, 11 frameshift and 15 non-synonymous variations selected by bioinformatic software were regarded as pathogenic. These 33 pathogenic mutations were associated with older age (p = 0.0176) and advanced tumor stage (p = 0.0218). In addition, they tended to be novel (p = 0.0003), heteroplasmic (p = 0.0343) and somatic (p = 0.0018). The mtDNA copy number increased in more than two-third (46/66) of PTCs, and the average content in tumors was nearly four times higher than that in adjacent normal tissues (p < 0.0001). Three sub-haplogroups of N (A4, B4a and B4g) and eight single-nucleotide polymorphisms (mtSNPs) (A16164G, C16266T, G5460A, T6680C, G9123A, A14587G, T16362C, and G709A) were associated with the occurrence of PTC. Here we report a comprehensive characterization of the mitochondrial genome and demonstrate its significance in pathogenesis and progression of PTC. This can help to clarify the molecular mechanisms underlying PTC and offer potential biomarkers or therapeutic targets for future clinical practice. PMID:27735863

  15. Genomic Characterization of the Genus Nairovirus (Family Bunyaviridae).

    PubMed

    Kuhn, Jens H; Wiley, Michael R; Rodriguez, Sergio E; Bào, Yīmíng; Prieto, Karla; Travassos da Rosa, Amelia P A; Guzman, Hilda; Savji, Nazir; Ladner, Jason T; Tesh, Robert B; Wada, Jiro; Jahrling, Peter B; Bente, Dennis A; Palacios, Gustavo

    2016-01-01

    Nairovirus, one of five bunyaviral genera, includes seven species. Genomic sequence information is limited for members of the Dera Ghazi Khan, Hughes, Qalyub, Sakhalin, and Thiafora nairovirus species. We used next-generation sequencing and historical virus-culture samples to determine 14 complete and nine coding-complete nairoviral genome sequences to further characterize these species. Previously unsequenced viruses include Abu Mina, Clo Mor, Great Saltee, Hughes, Raza, Sakhalin, Soldado, and Tillamook viruses. In addition, we present genomic sequence information on additional isolates of previously sequenced Avalon, Dugbe, Sapphire II, and Zirqa viruses. Finally, we identify Tunis virus, previously thought to be a phlebovirus, as an isolate of Abu Hammad virus. Phylogenetic analyses indicate the need for reassignment of Sapphire II virus to Dera Ghazi Khan nairovirus and reassignment of Hazara, Tofla, and Nairobi sheep disease viruses to novel species. We also propose new species for the Kasokero group (Kasokero, Leopards Hill, Yogue viruses), the Ketarah group (Gossas, Issyk-kul, Keterah/soft tick viruses) and the Burana group (Wēnzhōu tick virus, Huángpí tick virus 1, Tǎchéng tick virus 1). Our analyses emphasize the sister relationship of nairoviruses and arenaviruses, and indicate that several nairo-like viruses (Shāyáng spider virus 1, Xīnzhōu spider virus, Sānxiá water strider virus 1, South Bay virus, Wǔhàn millipede virus 2) require establishment of novel genera in a larger nairovirus-arenavirus supergroup. PMID:27294949

  16. Genome-Wide Tuning of Protein Expression Levels to Rapidly Engineer Microbial Traits.

    PubMed

    Freed, Emily F; Winkler, James D; Weiss, Sophie J; Garst, Andrew D; Mutalik, Vivek K; Arkin, Adam P; Knight, Rob; Gill, Ryan T

    2015-11-20

    The reliable engineering of biological systems requires quantitative mapping of predictable and context-independent expression over a broad range of protein expression levels. However, current techniques for modifying expression levels are cumbersome and are not amenable to high-throughput approaches. Here we present major improvements to current techniques through the design and construction of E. coli genome-wide libraries using synthetic DNA cassettes that can tune expression over a ∼10(4) range. The cassettes also contain molecular barcodes that are optimized for next-generation sequencing, enabling rapid and quantitative tracking of alleles that have the highest fitness advantage. We show these libraries can be used to determine which genes and expression levels confer greater fitness to E. coli under different growth conditions. PMID:26478262

  17. Rapid dissection of a complex phenotype through genomic-scale mapping of fitness altering genes.

    PubMed

    Warnecke, T E; Lynch, M D; Karimpour-Fard, A; Lipscomb, M L; Handke, P; Mills, T; Ramey, C J; Hoang, T; Gill, R T

    2010-05-01

    The understanding and engineering of complex phenotypes is a critical issue in biotechnology. Conventional approaches for engineering such phenotypes are often resource intensive, marginally effective, and unable to generate the level of biological understanding desired. Here, we report a new approach for rapidly dissecting a complex phenotype that is based upon the combination of genome-scale growth phenotype data, precisely targeted growth selections, and informatic strategies for abstracting and summarizing data onto coherent biological processes. We measured at high resolution (125 NT) and for the entire genome the effect of increased gene copy number on overall biological fitness corresponding to the expression of a complex phenotype (tolerance to 3-hydroxypropionic acid (3-HP) in Escherichia coli). Genetic level fitness data were then mapped according to various definitions of gene-gene interaction in order to generate network-level fitness data. When metabolic pathways were used to define interactions, we observed that genes within the chorismate and threonine super-pathways were disproportionately enriched throughout selections for 3-HP tolerance. Biochemical and genetic studies demonstrated that alleviation of inhibition of either of these super-pathways was sufficient to mitigate 3-HP toxicity. These data enabled the design of combinatorial modifications that almost completely offset 3-HP toxicity in minimal medium resulting in a 20 g/L and 25-fold increase in tolerance and specific growth, respectively.

  18. Optical Whole-Genome Restriction Mapping as a Tool for Rapidly Distinguishing and Identifying Bacterial Contaminants in Clinical Samples

    PubMed Central

    Baldwin, James C.

    2015-01-01

    Introduction Optical restriction genome mapping is a technology in which a genome is linearized on a surface and digested with specific restriction enzymes, giving an arrangement of the genome with gaps whose order and size are unique for a given organism. Current applications of this technology include assisting with the correct scaffolding and ordering of genomes in conjunction with whole-genome sequencing, observation of genetic drift and evolution using comparative genomics and epidemiological monitoring of the spread of infections. Here, we investigated the suitability of genome mapping for use in clinical labs as a potential diagnostic tool. Materials and Methods Using whole genome mapping, we investigated the basic performance of the technology for identifying two bacteria of interest for food-safety (Lactobacilli spp. and Enterohemorrhagic Escherichia coli). We further evaluated the performance for identifying multiple organisms from both simple and complex mixtures. Results We were able to successfully generate optical restriction maps of four Lactobacillus species as well as a strain of Enterohemorrhagic Escherichia coli from within a mixed solution, each distinguished using a common compatible restriction enzyme. Finally, we demonstrated that optical restriction maps were successfully obtained and the correct organism identified within a clinical matrix. Conclusion With additional development, whole genome mapping may be a useful clinical tool for rapid invitro diagnostics. PMID:26435946

  19. Rapid pair-wise synteny analysis of large bacterial genomes using web-based GeneOrder4.0

    PubMed Central

    2010-01-01

    Background The growing whole genome sequence databases necessitate the development of user-friendly software tools to mine these data. Web-based tools are particularly useful to wet-bench biologists as they enable platform-independent analysis of sequence data, without having to perform complex programming tasks and software compiling. Findings GeneOrder4.0 is a web-based "on-the-fly" synteny and gene order analysis tool for comparative bacterial genomics (ca. 8 Mb). It enables the visualization of synteny by plotting protein similarity scores between two genomes and it also provides visual annotation of "hypothetical" proteins from older archived genomes based on more recent annotations. Conclusions The web-based software tool GeneOrder4.0 is a user-friendly application that has been updated to allow the rapid analysis of synteny and gene order in large bacterial genomes. It is developed with the wet-bench researcher in mind. PMID:20178631

  20. Design of Genomic Signatures of Pathogen Identification & Characterization

    SciTech Connect

    Slezak, T; Gardner, S; Allen, J; Vitalis, E; Jaing, C

    2010-02-09

    This chapter will address some of the many issues associated with the identification of signatures based on genomic DNA/RNA, which can be used to identify and characterize pathogens for biodefense and microbial forensic goals. For the purposes of this chapter, we define a signature as one or more strings of contiguous genomic DNA or RNA bases that are sufficient to identify a pathogenic target of interest at the desired resolution and which could be instantiated with particular detection chemistry on a particular platform. The target may be a whole organism, an individual functional mechanism (e.g., a toxin gene), or simply a nucleic acid indicative of the organism. The desired resolution will vary with each program's goals but could easily range from family to genus to species to strain to isolate. The resolution may not be taxonomically based but rather pan-mechanistic in nature: detecting virulence or antibiotic-resistance genes shared by multiple microbes. Entire industries exist around different detection chemistries and instrument platforms for identification of pathogens, and we will only briefly mention a few of the techniques that we have used at Lawrence Livermore National Laboratory (LLNL) to support our biosecurity-related work since 2000. Most nucleic acid based detection chemistries involve the ability to isolate and amplify the signature target region(s), combined with a technique to detect the amplification. Genomic signature based identification techniques have the advantage of being precise, highly sensitive and relatively fast in comparison to biochemical typing methods and protein signatures. Classical biochemical typing methods were developed long before knowledge of DNA and resulted in dozens of tests (Gram's stain, differential growth characteristics media, etc.) that could be used to roughly characterize the major known pathogens (of course some are uncultivable). These tests could take many days to complete and precise resolution of species

  1. TARGeT: a web-based pipeline for retrieving and characterizing gene and transposable element families from genomic sequences.

    PubMed

    Han, Yujun; Burnette, James M; Wessler, Susan R

    2009-06-01

    Gene families compose a large proportion of eukaryotic genomes. The rapidly expanding genomic sequence database provides a good opportunity to study gene family evolution and function. However, most gene family identification programs are restricted to searching protein databases where data are often lagging behind the genomic sequence data. Here, we report a user-friendly web-based pipeline, named TARGeT (Tree Analysis of Related Genes and Transposons), which uses either a DNA or amino acid 'seed' query to: (i) automatically identify and retrieve gene family homologs from a genomic database, (ii) characterize gene structure and (iii) perform phylogenetic analysis. Due to its high speed, TARGeT is also able to characterize very large gene families, including transposable elements (TEs). We evaluated TARGeT using well-annotated datasets, including the ascorbate peroxidase gene family of rice, maize and sorghum and several TE families in rice. In all cases, TARGeT rapidly recapitulated the known homologs and predicted new ones. We also demonstrated that TARGeT outperforms similar pipelines and has functionality that is not offered elsewhere.

  2. Genomic Characterization of Phenylalanine Ammonia Lyase Gene in Buckwheat.

    PubMed

    Thiyagarajan, Karthikeyan; Vitali, Fabio; Tolaini, Valentina; Galeffi, Patrizia; Cantale, Cristina; Vikram, Prashant; Singh, Sukhwinder; De Rossi, Patrizia; Nobili, Chiara; Procacci, Silvia; Del Fiore, Antonella; Antonini, Alessandro; Presenti, Ombretta; Brunori, Andrea

    2016-01-01

    Phenylalanine Ammonia Lyase (PAL) gene which plays a key role in bio-synthesis of medicinally important compounds, Rutin/quercetin was sequence characterized for its efficient genomics application. These compounds possessing anti-diabetic and anti-cancer properties and are predominantly produced by Fagopyrum spp. In the present study, PAL gene was sequenced from three Fagopyrum spp. (F. tataricum, F. esculentum and F. dibotrys) and showed the presence of three SNPs and four insertion/deletions at intra and inter specific level. Among them, the potential SNP (position 949th bp G>C) with Parsimony Informative Site was selected and successfully utilised to individuate the zygosity/allelic variation of 16 F. tataricum varieties. Insertion mutations were identified in coding region, which resulted the change of a stretch of 39 amino acids on the putative protein. Our Study revealed that autogamous species (F. tataricum) has lower frequency of observed SNPs as compared to allogamous species (F. dibotrys and F. esculentum). The identified SNPs in F. tataricum didn't result to amino acid change, while in other two species it caused both conservative and non-conservative variations. Consistent pattern of SNPs across the species revealed their phylogenetic importance. We found two groups of F. tataricum and one of them was closely related with F. dibotrys. Sequence characterization information of PAL gene reported in present investigation can be utilized in genetic improvement of buckwheat in reference to its medicinal value. PMID:26990297

  3. Genomic Characterization of Phenylalanine Ammonia Lyase Gene in Buckwheat

    PubMed Central

    Thiyagarajan, Karthikeyan; Vitali, Fabio; Tolaini, Valentina; Galeffi, Patrizia; Cantale, Cristina; Vikram, Prashant; Singh, Sukhwinder; De Rossi, Patrizia; Nobili, Chiara; Procacci, Silvia; Del Fiore, Antonella; Antonini, Alessandro; Presenti, Ombretta; Brunori, Andrea

    2016-01-01

    Phenylalanine Ammonia Lyase (PAL) gene which plays a key role in bio-synthesis of medicinally important compounds, Rutin/quercetin was sequence characterized for its efficient genomics application. These compounds possessing anti-diabetic and anti-cancer properties and are predominantly produced by Fagopyrum spp. In the present study, PAL gene was sequenced from three Fagopyrum spp. (F. tataricum, F. esculentum and F. dibotrys) and showed the presence of three SNPs and four insertion/deletions at intra and inter specific level. Among them, the potential SNP (position 949th bp G>C) with Parsimony Informative Site was selected and successfully utilised to individuate the zygosity/allelic variation of 16 F. tataricum varieties. Insertion mutations were identified in coding region, which resulted the change of a stretch of 39 amino acids on the putative protein. Our Study revealed that autogamous species (F. tataricum) has lower frequency of observed SNPs as compared to allogamous species (F. dibotrys and F. esculentum). The identified SNPs in F. tataricum didn’t result to amino acid change, while in other two species it caused both conservative and non-conservative variations. Consistent pattern of SNPs across the species revealed their phylogenetic importance. We found two groups of F. tataricum and one of them was closely related with F. dibotrys. Sequence characterization information of PAL gene reported in present investigation can be utilized in genetic improvement of buckwheat in reference to its medicinal value. PMID:26990297

  4. Genomic Characterization of Phenylalanine Ammonia Lyase Gene in Buckwheat.

    PubMed

    Thiyagarajan, Karthikeyan; Vitali, Fabio; Tolaini, Valentina; Galeffi, Patrizia; Cantale, Cristina; Vikram, Prashant; Singh, Sukhwinder; De Rossi, Patrizia; Nobili, Chiara; Procacci, Silvia; Del Fiore, Antonella; Antonini, Alessandro; Presenti, Ombretta; Brunori, Andrea

    2016-01-01

    Phenylalanine Ammonia Lyase (PAL) gene which plays a key role in bio-synthesis of medicinally important compounds, Rutin/quercetin was sequence characterized for its efficient genomics application. These compounds possessing anti-diabetic and anti-cancer properties and are predominantly produced by Fagopyrum spp. In the present study, PAL gene was sequenced from three Fagopyrum spp. (F. tataricum, F. esculentum and F. dibotrys) and showed the presence of three SNPs and four insertion/deletions at intra and inter specific level. Among them, the potential SNP (position 949th bp G>C) with Parsimony Informative Site was selected and successfully utilised to individuate the zygosity/allelic variation of 16 F. tataricum varieties. Insertion mutations were identified in coding region, which resulted the change of a stretch of 39 amino acids on the putative protein. Our Study revealed that autogamous species (F. tataricum) has lower frequency of observed SNPs as compared to allogamous species (F. dibotrys and F. esculentum). The identified SNPs in F. tataricum didn't result to amino acid change, while in other two species it caused both conservative and non-conservative variations. Consistent pattern of SNPs across the species revealed their phylogenetic importance. We found two groups of F. tataricum and one of them was closely related with F. dibotrys. Sequence characterization information of PAL gene reported in present investigation can be utilized in genetic improvement of buckwheat in reference to its medicinal value.

  5. Rapid detection, characterization, and enumeration of foodborne pathogens.

    PubMed

    Hoorfar, J

    2011-11-01

    As food safety management further develops, microbiological testing will continue to play an important role in assessing whether Food Safety Objectives are achieved. However, traditional microbiological culture-based methods are limited, particularly in their ability to provide timely data. The present review discusses the reasons for the increasing interest in rapid methods, current developments in the field, the research needs, and the future trends. The advent of biotechnology has introduced new technologies that led to the emergence of rapid diagnostic methods and altered food testing practices. Rapid methods are comprised of many different detection technologies, including specialized enzyme substrates, antibodies and DNA, ranging from simple differential plating media to the use of sophisticated instruments. The use of non-invasive sampling techniques for live animals especially came into focus with the 1990s outbreak of bovine spongiform encephalopathy that was linked to the human outbreak of Creutzfeldt Jakob's Disease. Serology is still an important tool in preventing foodborne pathogens to enter the human food supply through meat and milk from animals. One of the primary uses of rapid methods is for fast screening of large number of samples, where most of them are expected to be test-negative, leading to faster product release for sale. This has been the main strength of rapid methods such as real-time Polymerase Chain Reaction (PCR). Enrichment PCR, where a primary culture broth is tested in PCR, is the most common approach in rapid testing. Recent reports show that it is possible both to enrich a sample and enumerate by pathogen-specific real-time PCR, if the enrichment time is short. This can be especially useful in situations where food producers ask for the level of pathogen in a contaminated product. Another key issue is automation, where the key drivers are miniaturization and multiple testing, which mean that not only one instrument is flexible

  6. The Salmonella In Silico Typing Resource (SISTR): An Open Web-Accessible Tool for Rapidly Typing and Subtyping Draft Salmonella Genome Assemblies

    PubMed Central

    Laing, Chad R.; Lingohr, Erika J.; Gannon, Victor P. J.; Nash, John H. E.; Taboada, Eduardo N.

    2016-01-01

    For nearly 100 years serotyping has been the gold standard for the identification of Salmonella serovars. Despite the increasing adoption of DNA-based subtyping approaches, serotype information remains a cornerstone in food safety and public health activities aimed at reducing the burden of salmonellosis. At the same time, recent advances in whole-genome sequencing (WGS) promise to revolutionize our ability to perform advanced pathogen characterization in support of improved source attribution and outbreak analysis. We present the Salmonella In Silico Typing Resource (SISTR), a bioinformatics platform for rapidly performing simultaneous in silico analyses for several leading subtyping methods on draft Salmonella genome assemblies. In addition to performing serovar prediction by genoserotyping, this resource integrates sequence-based typing analyses for: Multi-Locus Sequence Typing (MLST), ribosomal MLST (rMLST), and core genome MLST (cgMLST). We show how phylogenetic context from cgMLST analysis can supplement the genoserotyping analysis and increase the accuracy of in silico serovar prediction to over 94.6% on a dataset comprised of 4,188 finished genomes and WGS draft assemblies. In addition to allowing analysis of user-uploaded whole-genome assemblies, the SISTR platform incorporates a database comprising over 4,000 publicly available genomes, allowing users to place their isolates in a broader phylogenetic and epidemiological context. The resource incorporates several metadata driven visualizations to examine the phylogenetic, geospatial and temporal distribution of genome-sequenced isolates. As sequencing of Salmonella isolates at public health laboratories around the world becomes increasingly common, rapid in silico analysis of minimally processed draft genome assemblies provides a powerful approach for molecular epidemiology in support of public health investigations. Moreover, this type of integrated analysis using multiple sequence-based methods of sub

  7. The Salmonella In Silico Typing Resource (SISTR): An Open Web-Accessible Tool for Rapidly Typing and Subtyping Draft Salmonella Genome Assemblies.

    PubMed

    Yoshida, Catherine E; Kruczkiewicz, Peter; Laing, Chad R; Lingohr, Erika J; Gannon, Victor P J; Nash, John H E; Taboada, Eduardo N

    2016-01-01

    For nearly 100 years serotyping has been the gold standard for the identification of Salmonella serovars. Despite the increasing adoption of DNA-based subtyping approaches, serotype information remains a cornerstone in food safety and public health activities aimed at reducing the burden of salmonellosis. At the same time, recent advances in whole-genome sequencing (WGS) promise to revolutionize our ability to perform advanced pathogen characterization in support of improved source attribution and outbreak analysis. We present the Salmonella In Silico Typing Resource (SISTR), a bioinformatics platform for rapidly performing simultaneous in silico analyses for several leading subtyping methods on draft Salmonella genome assemblies. In addition to performing serovar prediction by genoserotyping, this resource integrates sequence-based typing analyses for: Multi-Locus Sequence Typing (MLST), ribosomal MLST (rMLST), and core genome MLST (cgMLST). We show how phylogenetic context from cgMLST analysis can supplement the genoserotyping analysis and increase the accuracy of in silico serovar prediction to over 94.6% on a dataset comprised of 4,188 finished genomes and WGS draft assemblies. In addition to allowing analysis of user-uploaded whole-genome assemblies, the SISTR platform incorporates a database comprising over 4,000 publicly available genomes, allowing users to place their isolates in a broader phylogenetic and epidemiological context. The resource incorporates several metadata driven visualizations to examine the phylogenetic, geospatial and temporal distribution of genome-sequenced isolates. As sequencing of Salmonella isolates at public health laboratories around the world becomes increasingly common, rapid in silico analysis of minimally processed draft genome assemblies provides a powerful approach for molecular epidemiology in support of public health investigations. Moreover, this type of integrated analysis using multiple sequence-based methods of sub

  8. Endometrial and acute myeloid leukemia cancer genomes characterized

    Cancer.gov

    Two studies from The Cancer Genome Atlas (TCGA) program reveal details about the genomic landscapes of acute myeloid leukemia (AML) and endometrial cancer. Both provide new insights into the molecular underpinnings of these cancers with the potential to i

  9. Genome-wide mining, characterization, and development of microsatellite markers in Marsupenaeus japonicus by genome survey sequencing

    NASA Astrophysics Data System (ADS)

    Lu, Xia; Luan, Sheng; Kong, Jie; Hu, Longyang; Mao, Yong; Zhong, Shengping

    2015-12-01

    The kuruma prawn, Marsupenaeus japonicus, is one of the most cultivated and consumed species of shrimp. However, very few molecular genetic/genomic resources are publically available for it. Thus, the characterization and distribution of simple sequence repeats (SSRs) remains ambiguous and the use of SSR markers in genomic studies and marker-assisted selection is limited. The goal of this study is to characterize and develop genome-wide SSR markers in M. japonicus by genome survey sequencing for application in comparative genomics and breeding. A total of 326 945 perfect SSRs were identifi ed, among which dinucleotide repeats were the most frequent class (44.08%), followed by mononucleotides (29.67%), trinucleotides (18.96%), tetranucleotides (5.66%), hexanucleotides (1.07%), and pentanucleotides (0.56%). In total, 151 541 SSR loci primers were successfully designed. A subset of 30 SSR primer pairs were synthesized and tested in 42 individuals from a wild population, of which 27 loci (90.0%) were successfully amplifi ed with specifi c products and 24 (80.0%) were polymorphic. For the amplifi ed polymorphic loci, the alleles ranged from 5 to 17 (with an average of 9.63), and the average PIC value was 0.796. A total of 58 256 SSR-containing sequences had signifi cant Gene Ontology annotation; these are good functional molecular marker candidates for association studies and comparative genomic analysis. The newly identifi ed SSRs signifi cantly contribute to the M. japonicus genomic resources and will facilitate a number of genetic and genomic studies, including high density linkage mapping, genome-wide association analysis, marker-aided selection, comparative genomics analysis, population genetics, and evolution.

  10. Rapid molecular strategy for orbivirus detection and characterization.

    PubMed

    Palacios, Gustavo; Cowled, Chris; Bussetti, Ana V; Savji, Nazir; Weir, Richard; Wick, Ivan; Travassos da Rosa, Amelia; Calisher, Charles H; Tesh, Robert B; Boyle, David; Lipkin, W Ian

    2011-06-01

    Orbiviruses infect a wide range of hosts, including humans. The ability to detect them has been hampered by their diversity. Here we present a simple consensus reverse transcription (RT)-PCR method targeting the polymerase gene for orbivirus recognition and characterization. Phylogenetic assignment is achieved by automated Web-based sequence analysis of amplification products.

  11. Rapid molecular strategy for filovirus detection and characterization.

    PubMed

    Zhai, Junhui; Palacios, Gustavo; Towner, Jonathan S; Jabado, Omar; Kapoor, Vishal; Venter, Marietjie; Grolla, Allen; Briese, Thomas; Paweska, Janusz; Swanepoel, Robert; Feldmann, Heinz; Nichol, Stuart T; Lipkin, W Ian

    2007-01-01

    Filoviruses have the capacity to cause lethal outbreaks of hemorrhagic fever in primates. Here we present a simple consensus reverse transcription-PCR method for filovirus recognition and characterization and demonstrate its utility with all known filovirus strains. Phylogenetic assignment is achieved by automated web-based sequence analysis of amplification products.

  12. Complete Genome Sequence of Mycobacterium chelonae Type Strain CCUG 47445, a Rapidly Growing Species of Nontuberculous Mycobacteria

    PubMed Central

    Jaén-Luchoro, Daniel; Salvà-Serra, Francisco; Aliaga-Lozano, Francisco; Seguí, Carolina; Busquets, Antonio; Ramírez, Antonio; Ruíz, Mikel; Gomila, Margarita; Lalucat, Jorge

    2016-01-01

    Mycobacterium chelonae strains are ubiquitous rapidly growing mycobacteria associated with skin and soft tissue infections, cellulitis, abscesses, osteomyelitis, catheter infections, disseminated diseases, and postsurgical infections after implants with prostheses, transplants, and even hemodialysis procedures. Here, we report the complete genome sequence of M. chelonae type strain CCUG 47445. PMID:27284158

  13. The Genomics, Epigenomics, and Transcriptomics of HPV-Associated Oropharyngeal Cancer--Understanding the Basis of a Rapidly Evolving Disease.

    PubMed

    Lechner, M; Fenton, T R

    2016-01-01

    Human papillomavirus (HPV) has been shown to represent a major independent risk factor for head and neck squamous cell cancer, in particular for oropharyngeal carcinoma. This type of cancer is rapidly evolving in the Western world, with rising trends particularly in the young, and represents a distinct epidemiological, clinical, and molecular entity. It is the aim of this review to give a detailed description of genomic, epigenomic, transcriptomic, and posttranscriptional changes that underlie the phenotype of this deadly disease. The review will also link these changes and examine what is known about the interactions between the host genome and viral genome, and investigate changes specific for the viral genome. These data are then integrated into an updated model of HPV-induced head and neck carcinogenesis.

  14. Rice hoja blanca virus genome characterization and expression in vitro.

    PubMed

    Ramirez, B C; Macaya, G; Calvert, L A; Haenni, A L

    1992-06-01

    No information exists on the organization and mechanisms of expression of the genome of rice hoja blanca virus (RHBV), a member of the tenuivirus group, but here we describe the first steps in its characterization. RHBV contains four ssRNA and three dsRNA species, the sizes of which were estimated by native and denaturing gel electrophoresis. Hybridization analyses using 32P-labelled riboprobes of viral and viral complementary polarities showed that unequal amounts of the two polarities of at least the smallest RNA are present in the virion, and indicated that the dsRNA species contain the same information as the ssRNA species of corresponding size. Total RHBV RNA directs the synthesis of two major proteins of 23K and 21K in vitro. RNA3 directs the synthesis of a 23K protein designated NS3, and RNA4 of a 21K protein designated NS4. The NS4 protein corresponds to the non-structural protein that accumulates in RHBV-infected rice tissue. The nuclecocapsid protein is not translated from either total RHBV RNA or any individual RHBV RNA in vitro. PMID:1607863

  15. Mitochondrial Genomes Suggest Rapid Evolution of Dwarf California Channel Islands Foxes (Urocyon littoralis)

    PubMed Central

    Hofman, Courtney A.; Rick, Torben C.; Hawkins, Melissa T. R.; Funk, W. Chris; Ralls, Katherine; Boser, Christina L.; Collins, Paul W.; Coonan, Tim; King, Julie L.; Morrison, Scott A.; Newsome, Seth D.; Sillett, T. Scott; Fleischer, Robert C.; Maldonado, Jesus E.

    2015-01-01

    Island endemics are typically differentiated from their mainland progenitors in behavior, morphology, and genetics, often resulting from long-term evolutionary change. To examine mechanisms for the origins of island endemism, we present a phylogeographic analysis of whole mitochondrial genomes from the endangered island fox (Urocyon littoralis), endemic to California’s Channel Islands, and mainland gray foxes (U. cinereoargenteus). Previous genetic studies suggested that foxes first appeared on the islands >16,000 years ago, before human arrival (~13,000 cal BP), while archaeological and paleontological data supported a colonization >7000 cal BP. Our results are consistent with initial fox colonization of the northern islands probably by rafting or human introduction ~9200–7100 years ago, followed quickly by human translocation of foxes from the northern to southern Channel Islands. Mitogenomes indicate that island foxes are monophyletic and most closely related to gray foxes from northern California that likely experienced a Holocene climate-induced range shift. Our data document rapid morphological evolution of island foxes (in ~2000 years or less). Despite evidence for bottlenecks, island foxes have generated and maintained multiple mitochondrial haplotypes. This study highlights the intertwined evolutionary history of island foxes and humans, and illustrates a new approach for investigating the evolutionary histories of other island endemics. PMID:25714775

  16. Mitochondrial genomes suggest rapid evolution of dwarf California Channel Islands foxes (Urocyon littoralis).

    PubMed

    Hofman, Courtney A; Rick, Torben C; Hawkins, Melissa T R; Funk, W Chris; Ralls, Katherine; Boser, Christina L; Collins, Paul W; Coonan, Tim; King, Julie L; Morrison, Scott A; Newsome, Seth D; Sillett, T Scott; Fleischer, Robert C; Maldonado, Jesus E

    2015-01-01

    Island endemics are typically differentiated from their mainland progenitors in behavior, morphology, and genetics, often resulting from long-term evolutionary change. To examine mechanisms for the origins of island endemism, we present a phylogeographic analysis of whole mitochondrial genomes from the endangered island fox (Urocyon littoralis), endemic to California's Channel Islands, and mainland gray foxes (U. cinereoargenteus). Previous genetic studies suggested that foxes first appeared on the islands >16,000 years ago, before human arrival (~13,000 cal BP), while archaeological and paleontological data supported a colonization >7000 cal BP. Our results are consistent with initial fox colonization of the northern islands probably by rafting or human introduction ~9200-7100 years ago, followed quickly by human translocation of foxes from the northern to southern Channel Islands. Mitogenomes indicate that island foxes are monophyletic and most closely related to gray foxes from northern California that likely experienced a Holocene climate-induced range shift. Our data document rapid morphological evolution of island foxes (in ~2000 years or less). Despite evidence for bottlenecks, island foxes have generated and maintained multiple mitochondrial haplotypes. This study highlights the intertwined evolutionary history of island foxes and humans, and illustrates a new approach for investigating the evolutionary histories of other island endemics.

  17. Genomic Evidence of Rapid and Stable Adaptive Oscillations over Seasonal Time Scales in Drosophila

    PubMed Central

    Bergland, Alan O.; Behrman, Emily L.; O'Brien, Katherine R.; Schmidt, Paul S.; Petrov, Dmitri A.

    2014-01-01

    In many species, genomic data have revealed pervasive adaptive evolution indicated by the fixation of beneficial alleles. However, when selection pressures are highly variable along a species' range or through time adaptive alleles may persist at intermediate frequencies for long periods. So called “balanced polymorphisms” have long been understood to be an important component of standing genetic variation, yet direct evidence of the strength of balancing selection and the stability and prevalence of balanced polymorphisms has remained elusive. We hypothesized that environmental fluctuations among seasons in a North American orchard would impose temporally variable selection on Drosophila melanogaster that would drive repeatable adaptive oscillations at balanced polymorphisms. We identified hundreds of polymorphisms whose frequency oscillates among seasons and argue that these loci are subject to strong, temporally variable selection. We show that these polymorphisms respond to acute and persistent changes in climate and are associated in predictable ways with seasonally variable phenotypes. In addition, our results suggest that adaptively oscillating polymorphisms are likely millions of years old, with some possibly predating the divergence between D. melanogaster and D. simulans. Taken together, our results are consistent with a model of balancing selection wherein rapid temporal fluctuations in climate over generational time promotes adaptive genetic diversity at loci underlying polygenic variation in fitness related phenotypes. PMID:25375361

  18. Genomic evidence of rapid and stable adaptive oscillations over seasonal time scales in Drosophila.

    PubMed

    Bergland, Alan O; Behrman, Emily L; O'Brien, Katherine R; Schmidt, Paul S; Petrov, Dmitri A

    2014-11-01

    In many species, genomic data have revealed pervasive adaptive evolution indicated by the fixation of beneficial alleles. However, when selection pressures are highly variable along a species' range or through time adaptive alleles may persist at intermediate frequencies for long periods. So called "balanced polymorphisms" have long been understood to be an important component of standing genetic variation, yet direct evidence of the strength of balancing selection and the stability and prevalence of balanced polymorphisms has remained elusive. We hypothesized that environmental fluctuations among seasons in a North American orchard would impose temporally variable selection on Drosophila melanogaster that would drive repeatable adaptive oscillations at balanced polymorphisms. We identified hundreds of polymorphisms whose frequency oscillates among seasons and argue that these loci are subject to strong, temporally variable selection. We show that these polymorphisms respond to acute and persistent changes in climate and are associated in predictable ways with seasonally variable phenotypes. In addition, our results suggest that adaptively oscillating polymorphisms are likely millions of years old, with some possibly predating the divergence between D. melanogaster and D. simulans. Taken together, our results are consistent with a model of balancing selection wherein rapid temporal fluctuations in climate over generational time promotes adaptive genetic diversity at loci underlying polygenic variation in fitness related phenotypes.

  19. Construction and characterization of a valve for rapid gas injection

    NASA Astrophysics Data System (ADS)

    Uedo, M.; Rossi, J. O.; Aso, Y.; Mangueira, L. S.; Pereira, C. A.

    1989-01-01

    An electromagnetic valve for fast gas injection was built and characterized. This type of gas injection valve is routinely applied to various plasma experiments: in magnetic confinement devices as TOKAMAK, FRP, and Compact Toroids as well as intense ion beam and neutral particle generators. The valve is capable of injecting gas pulses with up to 80 m Torr peak pressure, rising time less than 400 microsec and duration time of 40 ms, in the present experimental set-up. It is easy to build and its components can be totally acquired in the country.

  20. Genome skimming: A rapid approach to gaining diverse biological insights into multicellular pathogens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    New genome sequence information can now be generated very quickly and cheaply for virtually any organism. The dive into genomics is increasingly tempting to scientists studying plant pathogens and other eukaryotic species without reference genomes. The ease of data collection, however, is tempered ...

  1. Comprehensive characterization of human genome variation by high coverage whole-genome sequencing of forty four Caucasians.

    PubMed

    Shen, Hui; Li, Jian; Zhang, Jigang; Xu, Chao; Jiang, Yan; Wu, Zikai; Zhao, Fuping; Liao, Li; Chen, Jun; Lin, Yong; Tian, Qing; Papasian, Christopher J; Deng, Hong-Wen

    2013-01-01

    Whole genome sequencing studies are essential to obtain a comprehensive understanding of the vast pattern of human genomic variations. Here we report the results of a high-coverage whole genome sequencing study for 44 unrelated healthy Caucasian adults, each sequenced to over 50-fold coverage (averaging 65.8×). We identified approximately 11 million single nucleotide polymorphisms (SNPs), 2.8 million short insertions and deletions, and over 500,000 block substitutions. We showed that, although previous studies, including the 1000 Genomes Project Phase 1 study, have catalogued the vast majority of common SNPs, many of the low-frequency and rare variants remain undiscovered. For instance, approximately 1.4 million SNPs and 1.3 million short indels that we found were novel to both the dbSNP and the 1000 Genomes Project Phase 1 data sets, and the majority of which (∼96%) have a minor allele frequency less than 5%. On average, each individual genome carried ∼3.3 million SNPs and ∼492,000 indels/block substitutions, including approximately 179 variants that were predicted to cause loss of function of the gene products. Moreover, each individual genome carried an average of 44 such loss-of-function variants in a homozygous state, which would completely "knock out" the corresponding genes. Across all the 44 genomes, a total of 182 genes were "knocked-out" in at least one individual genome, among which 46 genes were "knocked out" in over 30% of our samples, suggesting that a number of genes are commonly "knocked-out" in general populations. Gene ontology analysis suggested that these commonly "knocked-out" genes are enriched in biological process related to antigen processing and immune response. Our results contribute towards a comprehensive characterization of human genomic variation, especially for less-common and rare variants, and provide an invaluable resource for future genetic studies of human variation and diseases.

  2. Characterization of rapidly solidified powder of high-speed steel

    NASA Astrophysics Data System (ADS)

    Miglierini, Marcel; Lančok, Adriana; Kusý, Martin

    2009-04-01

    Rapidly solidified particles of high-speed steel were classified into several granulometric fractions ranging from less than 25 μm up to more than 160 μm in diameter and studied by transmission and conversion electron Mössbauer spectrometry. The former was applied at 300, 77, and 5 K. Presence of magnetic and a non-magnetic crystallographic phase was observed. They were identified by X-ray diffraction as ferrite (bcc-Fe) and austenite (fcc-Fe), respectively. In addition, M4C3 and M2C carbides were found. The magnetic phase diminishes in the bulk of the particles bigger than 63 μm (transmission Mössbauer spectroscopy) and/or 80 μm (X-ray diffraction). Its contribution is higher at the surface of the particles (conversion electron Mössbauer spectrometry). The origin of the non-magnetic phase is not changed even at 5 K. Reasonable agreement is achieved between Mössbauer and X-ray diffraction data as far as the fraction of Fe-containing phases is concerned.

  3. [Isolation and characterization of the centromeric BAC clones from different genomes in genus Oryza].

    PubMed

    Yi, Chuan-Deng; Gong, Zhi-Yun; Liang, Guo-Hua; Wang, Fei-Hua; Tang, Shu-Zhu; Gu, Ming-Hong

    2007-07-01

    Centromeres play an important role in ensuring the correct segregation and transmission of chromosome during mitosis and meiosis in eukaryotes. In this research, we constructed five BAC libraries for diploid wild rice with different genomes. Together with the technique of colony blot hybridization and fluorescence in situ hybridization (FISH), centromere-related BAC clones were screened and characterized from different genomes. Meanwhile, co-hybridization was detected between these clones and the five genomes. The results from this study demonstrated that: (1) there were centromere-specific satellite repeat in Oryza officinalis (CC genome) and O. brachyantha (FF genome), respectively, and centromere-specific CRR-related sequence was found in O. brachyantha; (2) homology sequences of CentO and CRR of O. sativa (AA genome) were detected on all centromeres of O. glaberrima (AA genome), O. punctata (BB genome) and O. australiensis (EE genome); And (3) the two somatic chromosomes of O. officinalis comprised of homology sequences of CentO satellites as revealed FISH analysis probed with RCS2. Homology sequences of CRR of O. sativa were also detected on all centromeres of O. officinalis. The results provided a foundation toward cloning the centromeric sequences from different genomes of genus Oryza, studying centromere organization and evolution of different genome, analyzing the relationship between centromeric structure and function among different genome.

  4. Genome-Based Selection and Characterization of Fusarium circinatum-Specific Sequences.

    PubMed

    Maphosa, Mkhululi N; Steenkamp, Emma T; Wingfield, Brenda D

    2016-03-01

    Fusarium circinatum is an important pathogen of pine trees and its management in the commercial forestry environment relies largely on early detection, particularly in seedling nurseries. The fact that the entire genome of this pathogen is available opens new avenues for the development of diagnostic tools for this fungus. In this study we identified open reading frames (ORFs) unique to F. circinatum and determined that they were specific to the pathogen. The ORF identification process involved bioinformatics-based screening of all the putative F. circinatum ORFs against public databases. This was followed by functional characterization of ORFs found to be unique to F. circinatum. We used PCR- and hybridization-based approaches to confirm the presence of selected unique genes in different strains of F. circinatum and their absence from other Fusarium species for which genome sequence data are not yet available. These included species that are closely related to F. circinatum as well as those that are commonly encountered in the forestry environment. Thirty-six ORFs were identified as potentially unique to F. circinatum. Nineteen of these encode proteins with known domains while the other 17 encode proteins of unknown function. The results of our PCR analyses and hybridization assays showed that three of the selected genes were present in all of the strains of F. circinatum tested and absent from the other Fusarium species screened. These data thus indicate that the selected genes are common and unique to F. circinatum. These genes thus could be good candidates for use in rapid, in-the-field diagnostic assays specific to F. circinatum. Our study further demonstrates how genome sequence information can be mined for the identification of new diagnostic markers for the detection of plant pathogens. PMID:26888868

  5. Field methods for rapidly characterizing paint waste during bridge rehabilitation.

    PubMed

    Shu, Zhan; Axe, Lisa; Jahan, Kauser; Ramanujachary, Kandalam V

    2015-09-01

    For Department of Transportation (DOT) agencies, bridge rehabilitation involving paint removal results in waste that is often managed as hazardous. Hence, an approach that provides field characterization of the waste classification would be beneficial. In this study, an analysis of variables critical to the leaching process was conducted to develop a predictive tool for waste classification. This approach first involved identifying mechanistic processes that control leaching. Because steel grit is used to remove paint, elevated iron concentrations remain in the paint waste. As such, iron oxide coatings provide an important surface for metal adsorption. The diffuse layer model was invoked (logKMe=4.65 for Pb and logKMe=2.11 for Cr), where 90% of the data were captured within the 95% confidence level. Based on an understanding of mechanistic processes along with principal component analysis (PCA) of data obtained from field-portable X-ray fluorescence (FP-XRF), statistically-based models for leaching from paint waste were developed. Modeling resulted in 96% of the data falling within the 95% confidence level for Pb (R(2) 0.6-0.9, p ⩽ 0.04), Ba (R(2) 0.5-0.7, p ⩽ 0.1), and Zn (R(2) 0.6-0.7, p ⩽ 0.08). However, the regression model obtained for Cr leaching was not significant (R(2) 0.3-0.5, p ⩽ 0.75). The results of this work may assist DOT agencies with applying a predictive tool in the field that addresses the mobility of trace metals as well as disposal and management of paint waste during bridge rehabilitation.

  6. Reverse transcription genome exponential amplification reaction assay for rapid and universal detection of human rhinoviruses.

    PubMed

    Guan, Li; Zhao, Lin-Qing; Zhou, Hang-Yu; Nie, Kai; Li, Xin-Na; Zhang, Dan; Song, Juan; Qian, Yuan; Ma, Xue-Jun

    2016-07-01

    Human rhinoviruses (HRVs) have long been recognized as the cause of more than one-half of acute viral upper respiratory illnesses, and they are associated with more-serious diseases in children, such as asthma, acute otitis media and pneumonia. A rapid and universal test for of HRV infection is in high demand. In this study, a reverse transcription genome exponential amplification reaction (RT-GEAR) assay targeting the HRV 5' untranslated region (UTR) was developed for pan-HRV detection. The reaction was performed in a single tube in one step at 65 °C for 60 min using a real-time fluorometer (Genie(®)II; Optigene). The RT-GEAR assay showed no cross-reactivity with common human enteroviruses, including HEV71, CVA16, CVA6, CVA10, CVA24, CVB5, Echo30, and PV1-3 or with other common respiratory viruses including FluA H3, FluB, PIV1-4, ADV3, RSVA, RSVB and HMPV. With in vitro-transcribed RNA containing the amplified regions of HRV-A60, HRV-B06 and HRV-C07 as templates, the sensitivity of the RT-GEAR assay was 5, 50 and 5 copies/reaction, respectively. Experiments to evaluate the clinical performance of the RT-GEAR assay were also carried out with a panel of 143 previously verified samples, and the results were compared with those obtained using a published semi-nested PCR assay followed by sequencing. The tested panel comprised 91 HRV-negative samples and 52 HRV-positive samples (18 HRV-A-positive samples, 3 HRV-B-positive samples and 31 HRV-C-positive samples). The sensitivity and specificity of the pan-HRVs RT-GEAR assay was 98.08 % and 100 %, respectively. The kappa correlation between the two methods was 0.985. The RT-GEAR assay based on a portable Genie(®)II fluorometer is a sensitive, specific and rapid assay for the universal detection of HRV infection. PMID:27132014

  7. Reverse transcription genome exponential amplification reaction assay for rapid and universal detection of human rhinoviruses.

    PubMed

    Guan, Li; Zhao, Lin-Qing; Zhou, Hang-Yu; Nie, Kai; Li, Xin-Na; Zhang, Dan; Song, Juan; Qian, Yuan; Ma, Xue-Jun

    2016-07-01

    Human rhinoviruses (HRVs) have long been recognized as the cause of more than one-half of acute viral upper respiratory illnesses, and they are associated with more-serious diseases in children, such as asthma, acute otitis media and pneumonia. A rapid and universal test for of HRV infection is in high demand. In this study, a reverse transcription genome exponential amplification reaction (RT-GEAR) assay targeting the HRV 5' untranslated region (UTR) was developed for pan-HRV detection. The reaction was performed in a single tube in one step at 65 °C for 60 min using a real-time fluorometer (Genie(®)II; Optigene). The RT-GEAR assay showed no cross-reactivity with common human enteroviruses, including HEV71, CVA16, CVA6, CVA10, CVA24, CVB5, Echo30, and PV1-3 or with other common respiratory viruses including FluA H3, FluB, PIV1-4, ADV3, RSVA, RSVB and HMPV. With in vitro-transcribed RNA containing the amplified regions of HRV-A60, HRV-B06 and HRV-C07 as templates, the sensitivity of the RT-GEAR assay was 5, 50 and 5 copies/reaction, respectively. Experiments to evaluate the clinical performance of the RT-GEAR assay were also carried out with a panel of 143 previously verified samples, and the results were compared with those obtained using a published semi-nested PCR assay followed by sequencing. The tested panel comprised 91 HRV-negative samples and 52 HRV-positive samples (18 HRV-A-positive samples, 3 HRV-B-positive samples and 31 HRV-C-positive samples). The sensitivity and specificity of the pan-HRVs RT-GEAR assay was 98.08 % and 100 %, respectively. The kappa correlation between the two methods was 0.985. The RT-GEAR assay based on a portable Genie(®)II fluorometer is a sensitive, specific and rapid assay for the universal detection of HRV infection.

  8. Rapid coastal spread of First Americans: Novel insights from South America's Southern Cone mitochondrial genomes

    PubMed Central

    Bodner, Martin; Perego, Ugo A.; Huber, Gabriela; Fendt, Liane; Röck, Alexander W.; Zimmermann, Bettina; Olivieri, Anna; Gómez-Carballa, Alberto; Lancioni, Hovirag; Angerhofer, Norman; Bobillo, Maria Cecilia; Corach, Daniel; Woodward, Scott R.; Salas, Antonio; Achilli, Alessandro; Torroni, Antonio; Bandelt, Hans-Jürgen; Parson, Walther

    2012-01-01

    It is now widely agreed that the Native American founders originated from a Beringian source population ∼15–18 thousand years ago (kya) and rapidly populated all of the New World, probably mainly following the Pacific coastal route. However, details about the migration into the Americas and the routes pursued on the continent still remain unresolved, despite numerous genetic, archaeological, and linguistic investigations. To examine the pioneering peopling phase of the South American continent, we screened literature and mtDNA databases and identified two novel mitochondrial DNA (mtDNA) clades, here named D1g and D1j, within the pan-American haplogroup D1. They both show overall rare occurrences but local high frequencies, and are essentially restricted to populations from the Southern Cone of South America (Chile and Argentina). We selected and completely sequenced 43 D1g and D1j mtDNA genomes applying highest quality standards. Molecular and phylogeographic analyses revealed extensive variation within each of the two clades and possibly distinct dispersal patterns. Their age estimates agree with the dating of the earliest archaeological sites in South America and indicate that the Paleo-Indian spread along the entire longitude of the American double continent might have taken even <2000 yr. This study confirms that major sampling and sequencing efforts are mandatory for uncovering all of the most basal variation in the Native American mtDNA haplogroups and for clarification of Paleo-Indian migrations, by targeting, if possible, both the general mixed population of national states and autochthonous Native American groups, especially in South America. PMID:22333566

  9. Rapid coastal spread of First Americans: novel insights from South America's Southern Cone mitochondrial genomes.

    PubMed

    Bodner, Martin; Perego, Ugo A; Huber, Gabriela; Fendt, Liane; Röck, Alexander W; Zimmermann, Bettina; Olivieri, Anna; Gómez-Carballa, Alberto; Lancioni, Hovirag; Angerhofer, Norman; Bobillo, Maria Cecilia; Corach, Daniel; Woodward, Scott R; Salas, Antonio; Achilli, Alessandro; Torroni, Antonio; Bandelt, Hans-Jürgen; Parson, Walther

    2012-05-01

    It is now widely agreed that the Native American founders originated from a Beringian source population ~15-18 thousand years ago (kya) and rapidly populated all of the New World, probably mainly following the Pacific coastal route. However, details about the migration into the Americas and the routes pursued on the continent still remain unresolved, despite numerous genetic, archaeological, and linguistic investigations. To examine the pioneering peopling phase of the South American continent, we screened literature and mtDNA databases and identified two novel mitochondrial DNA (mtDNA) clades, here named D1g and D1j, within the pan-American haplogroup D1. They both show overall rare occurrences but local high frequencies, and are essentially restricted to populations from the Southern Cone of South America (Chile and Argentina). We selected and completely sequenced 43 D1g and D1j mtDNA genomes applying highest quality standards. Molecular and phylogeographic analyses revealed extensive variation within each of the two clades and possibly distinct dispersal patterns. Their age estimates agree with the dating of the earliest archaeological sites in South America and indicate that the Paleo-Indian spread along the entire longitude of the American double continent might have taken even <2000 yr. This study confirms that major sampling and sequencing efforts are mandatory for uncovering all of the most basal variation in the Native American mtDNA haplogroups and for clarification of Paleo-Indian migrations, by targeting, if possible, both the general mixed population of national states and autochthonous Native American groups, especially in South America. PMID:22333566

  10. Next-generation sequencing strategies for characterizing the turkey genome.

    PubMed

    Dalloul, Rami A; Zimin, Aleksey V; Settlage, Robert E; Kim, Sungwon; Reed, Kent M

    2014-02-01

    The turkey genome sequencing project was initiated in 2008 and has relied primarily on next-generation sequencing (NGS) technologies. Our first efforts used a synergistic combination of 2 NGS platforms (Roche/454 and Illumina GAII), detailed bacterial artificial chromosome (BAC) maps, and unique assembly tools to sequence and assemble the genome of the domesticated turkey, Meleagris gallopavo. Since the first release in 2010, efforts to improve the genome assembly, gene annotation, and genomic analyses continue. The initial assembly build (2.01) represented about 89% of the genome sequence with 17X coverage depth (931 Mb). Sequence contigs were assigned to 30 of the 40 chromosomes with approximately 10% of the assembled sequence corresponding to unassigned chromosomes (ChrUn). The sequence has been refined through both genome-wide and area-focused sequencing, including shotgun and paired-end sequencing, and targeted sequencing of chromosomal regions with low or incomplete coverage. These additional efforts have improved the sequence assembly resulting in 2 subsequent genome builds of higher genome coverage (25X/Build3.0 and 30X/Build4.0) with a current sequence totaling 1,010 Mb. Further, BAC with end sequences assigned to the Z/W and MG18 (MHC) chromosomes, ChrUn, or not placed in the previous build were isolated, deeply sequenced (Hi-Seq), and incorporated into the latest build (5.0). To aid in the annotation and to generate a gene expression atlas of major tissues, a comprehensive set of RNA samples was collected at various developmental stages of female and male turkeys. Transcriptome sequencing data (using Illumina Hi-Seq) will provide information to enhance the final assembly and ultimately improve sequence annotation. The most current sequence covers more than 95% of the turkey genome and should yield a much improved gene level of annotation, making it a valuable resource for studying genetic variations underlying economically important traits in poultry.

  11. PNA-based microbial pathogen identification and resistance marker detection: an accurate, isothermal rapid assay based on genome-specific features

    PubMed Central

    Smolina, Irina; Miller, Nancy S.; Frank-Kamenetskii, Maxim

    2010-01-01

    With the rapidly growing availability of the entire genome sequences of microbial pathogens, there is unmet need for increasingly sensitive systems to monitor the gene-specific markers for diagnosis of bacteremia that enables an earlier detection of causative agent and determination of drug resistance. To address these challenges, a novel FISH-type genomic sequence-based molecular technique is proposed that can identify bacteria and simultaneously detect antibiotic resistance markers for rapid and accurate testing of pathogens. The approach is based on a synergistic combination of advanced Peptide Nucleic Acid (PNA)-based technology and signal-enhancing Rolling Circle Amplification (RCA) reaction to achieve a highly specific and sensitive assay. A specific PNA-DNA construct serves as an exceedingly selective and very effective biomarker, while RCA enhances detection sensitivity and provide with a highly multiplexed assay system. Distinct-color fluorescent decorator probes are used to identify about 20-nucleotide-long signature sequences in bacterial genomic DNA and/or key genetic markers of drug resistance in order to identify and characterize various pathogens. The technique's potential and its utility for clinical diagnostics are illustrated by identification of S. aureus with simultaneous discrimination of methicillin-sensitive (MSSA) versus methicillin-resistant (MRSA) strains. Overall these promising results hint to the adoption of PNA-based rapid sensitive detection for diagnosis of other clinically relevant organisms. Thereby, new assay enables significantly earlier administration of appropriate antimicrobial therapy and may, thus have a positive impact on the outcome of the patient. PMID:20953307

  12. Preliminary Genomic Characterization of Ten Hardwood Tree Species from Multiplexed Low Coverage Whole Genome Sequencing.

    PubMed

    Staton, Margaret; Best, Teodora; Khodwekar, Sudhir; Owusu, Sandra; Xu, Tao; Xu, Yi; Jennings, Tara; Cronn, Richard; Arumuganathan, A Kathiravetpilla; Coggeshall, Mark; Gailing, Oliver; Liang, Haiying; Romero-Severson, Jeanne; Schlarbaum, Scott; Carlson, John E

    2015-01-01

    Forest health issues are on the rise in the United States, resulting from introduction of alien pests and diseases, coupled with abiotic stresses related to climate change. Increasingly, forest scientists are finding genetic/genomic resources valuable in addressing forest health issues. For a set of ten ecologically and economically important native hardwood tree species representing a broad phylogenetic spectrum, we used low coverage whole genome sequencing from multiplex Illumina paired ends to economically profile their genomic content. For six species, the genome content was further analyzed by flow cytometry in order to determine the nuclear genome size. Sequencing yielded a depth of 0.8X to 7.5X, from which in silico analysis yielded preliminary estimates of gene and repetitive sequence content in the genome for each species. Thousands of genomic SSRs were identified, with a clear predisposition toward dinucleotide repeats and AT-rich repeat motifs. Flanking primers were designed for SSR loci for all ten species, ranging from 891 loci in sugar maple to 18,167 in redbay. In summary, we have demonstrated that useful preliminary genome information including repeat content, gene content and useful SSR markers can be obtained at low cost and time input from a single lane of Illumina multiplex sequence.

  13. Preliminary Genomic Characterization of Ten Hardwood Tree Species from Multiplexed Low Coverage Whole Genome Sequencing

    PubMed Central

    Staton, Margaret; Best, Teodora; Khodwekar, Sudhir; Owusu, Sandra; Xu, Tao; Xu, Yi; Jennings, Tara; Cronn, Richard; Arumuganathan, A. Kathiravetpilla; Coggeshall, Mark; Gailing, Oliver; Liang, Haiying; Romero-Severson, Jeanne; Schlarbaum, Scott; Carlson, John E.

    2015-01-01

    Forest health issues are on the rise in the United States, resulting from introduction of alien pests and diseases, coupled with abiotic stresses related to climate change. Increasingly, forest scientists are finding genetic/genomic resources valuable in addressing forest health issues. For a set of ten ecologically and economically important native hardwood tree species representing a broad phylogenetic spectrum, we used low coverage whole genome sequencing from multiplex Illumina paired ends to economically profile their genomic content. For six species, the genome content was further analyzed by flow cytometry in order to determine the nuclear genome size. Sequencing yielded a depth of 0.8X to 7.5X, from which in silico analysis yielded preliminary estimates of gene and repetitive sequence content in the genome for each species. Thousands of genomic SSRs were identified, with a clear predisposition toward dinucleotide repeats and AT-rich repeat motifs. Flanking primers were designed for SSR loci for all ten species, ranging from 891 loci in sugar maple to 18,167 in redbay. In summary, we have demonstrated that useful preliminary genome information including repeat content, gene content and useful SSR markers can be obtained at low cost and time input from a single lane of Illumina multiplex sequence. PMID:26698853

  14. Preliminary Genomic Characterization of Ten Hardwood Tree Species from Multiplexed Low Coverage Whole Genome Sequencing.

    PubMed

    Staton, Margaret; Best, Teodora; Khodwekar, Sudhir; Owusu, Sandra; Xu, Tao; Xu, Yi; Jennings, Tara; Cronn, Richard; Arumuganathan, A Kathiravetpilla; Coggeshall, Mark; Gailing, Oliver; Liang, Haiying; Romero-Severson, Jeanne; Schlarbaum, Scott; Carlson, John E

    2015-01-01

    Forest health issues are on the rise in the United States, resulting from introduction of alien pests and diseases, coupled with abiotic stresses related to climate change. Increasingly, forest scientists are finding genetic/genomic resources valuable in addressing forest health issues. For a set of ten ecologically and economically important native hardwood tree species representing a broad phylogenetic spectrum, we used low coverage whole genome sequencing from multiplex Illumina paired ends to economically profile their genomic content. For six species, the genome content was further analyzed by flow cytometry in order to determine the nuclear genome size. Sequencing yielded a depth of 0.8X to 7.5X, from which in silico analysis yielded preliminary estimates of gene and repetitive sequence content in the genome for each species. Thousands of genomic SSRs were identified, with a clear predisposition toward dinucleotide repeats and AT-rich repeat motifs. Flanking primers were designed for SSR loci for all ten species, ranging from 891 loci in sugar maple to 18,167 in redbay. In summary, we have demonstrated that useful preliminary genome information including repeat content, gene content and useful SSR markers can be obtained at low cost and time input from a single lane of Illumina multiplex sequence. PMID:26698853

  15. Non-genomic rapid inhibition of Na+/H+-exchange 1 and apoptotic immunosuppression in human T cells by glucocorticoids.

    PubMed

    Chang, Ching-Pang; Wang, Shyi-Wu; Huang, Zih-Ling; Wang, Olivia Ya-Hsuan; Huang, Michael I-Ta; Lu, Li-Ming; Tarng, Der-Cherng; Chien, Chau-Heng; Chien, Eileen Jea

    2010-06-01

    Glucocorticoids (GCs) have been employed as immunosuppressive agents for many years. However, it is still unclear how GCs instantly uncouple T cells from acute stressful inflammatory. In terms of time scale, the genomic activity of the classic GC receptor cannot fulfill this role under crisis; but a rapid non-genomic response can. In a previous study, intracellular acidification was found to be due to a rapid non-genomic inhibition of Na(+)/H(+)-exchange 1 (NHE1) and this event led to the immunosuppression of T cell proliferation by progesterone. The aim of this study was to examine whether there is a rapid acidification response caused by an inhibition of NHE1 activity and to explore the differential non-genomic effect on immunosuppression of hydrocortisone and dexamethasone. The IC(50) values for NHE1-dependent pH(i) recovery by hydrocortisone and dexamethasone are 250 and 1 nM, respectively. Co-stimulation of GCs with phytohemagglutinin (PHA) is able to inhibit PHA-induced IL-2 secretion, IL-4 secretion, and T-cell proliferation. Furthermore, apoptosis in PHA-activated T cells is not induced by hydrocortisone but by dexamethasone. The mechanism of immunosuppression on proliferation by dexamethasone was found to be different of hydrocortisone and seems to involve cytotoxicity against T cells. Moreover, apoptosis induced by dexamethasone and impermeable dexamethasone-bovine serum albumin suggests that the apoptotic immunosuppression occurs through both the plasma membrane and cytoplasmic sites. The rapid inhibitory responses triggered by GCs would seem to release T cells instantly when an acute stress-related response is needed. Nonetheless, the apoptotic immunosuppression by dexamethasone is attributable to its severe cytotoxicity.

  16. Rapid and Inexpensive Whole-Genome Genotyping-by-Sequencing for Crossover Localization and Fine-Scale Genetic Mapping

    PubMed Central

    Rowan, Beth A.; Patel, Vipul; Weigel, Detlef; Schneeberger, Korbinian

    2015-01-01

    The reshuffling of existing genetic variation during meiosis is important both during evolution and in breeding. The reassortment of genetic variants relies on the formation of crossovers (COs) between homologous chromosomes. The pattern of genome-wide CO distributions can be rapidly and precisely established by the short-read sequencing of individuals from F2 populations, which in turn are useful for quantitative trait locus (QTL) mapping. Although sequencing costs have decreased precipitously in recent years, the costs of library preparation for hundreds of individuals have remained high. To enable rapid and inexpensive CO detection and QTL mapping using low-coverage whole-genome sequencing of large mapping populations, we have developed a new method for library preparation along with Trained Individual GenomE Reconstruction, a probabilistic method for genotype and CO predictions for recombinant individuals. In an example case with hundreds of F2 individuals from two Arabidopsis thaliana accessions, we resolved most CO breakpoints to within 2 kb and reduced a major flowering time QTL to a 9-kb interval. In addition, an extended region of unusually low recombination revealed a 1.8-Mb inversion polymorphism on the long arm of chromosome 4. We observed no significant differences in the frequency and distribution of COs between F2 individuals with and without a functional copy of the DNA helicase gene RECQ4A. In summary, we present a new, cost-efficient method for large-scale, high-precision genotyping-by-sequencing. PMID:25585881

  17. Rapid and inexpensive whole-genome genotyping-by-sequencing for crossover localization and fine-scale genetic mapping.

    PubMed

    Rowan, Beth A; Patel, Vipul; Weigel, Detlef; Schneeberger, Korbinian

    2015-03-01

    The reshuffling of existing genetic variation during meiosis is important both during evolution and in breeding. The reassortment of genetic variants relies on the formation of crossovers (COs) between homologous chromosomes. The pattern of genome-wide CO distributions can be rapidly and precisely established by the short-read sequencing of individuals from F2 populations, which in turn are useful for quantitative trait locus (QTL) mapping. Although sequencing costs have decreased precipitously in recent years, the costs of library preparation for hundreds of individuals have remained high. To enable rapid and inexpensive CO detection and QTL mapping using low-coverage whole-genome sequencing of large mapping populations, we have developed a new method for library preparation along with Trained Individual GenomE Reconstruction, a probabilistic method for genotype and CO predictions for recombinant individuals. In an example case with hundreds of F2 individuals from two Arabidopsis thaliana accessions, we resolved most CO breakpoints to within 2 kb and reduced a major flowering time QTL to a 9-kb interval. In addition, an extended region of unusually low recombination revealed a 1.8-Mb inversion polymorphism on the long arm of chromosome 4. We observed no significant differences in the frequency and distribution of COs between F2 individuals with and without a functional copy of the DNA helicase gene RECQ4A. In summary, we present a new, cost-efficient method for large-scale, high-precision genotyping-by-sequencing.

  18. Rapid identification of thousands of copperhead snake (Agkistrodon contortrix) microsatellite loci from modest amounts of 454 shotgun genome sequence.

    PubMed

    Castoe, Todd A; Poole, Alexander W; Gu, Wanjun; Jason de Koning, A P; Daza, Juan M; Smith, Eric N; Pollock, David D

    2010-03-01

    Optimal integration of next-generation sequencing into mainstream research requires re-evaluation of how problems can be reasonably overcome and what questions can be asked. One potential application is the rapid acquisition of genomic information to identify microsatellite loci for evolutionary, population genetic and chromosome linkage mapping research on non-model and not previously sequenced organisms. Here, we report on results using high-throughput sequencing to obtain a large number of microsatellite loci from the venomous snake Agkistrodon contortrix, the copperhead. We used the 454 Genome Sequencer FLX next-generation sequencing platform to sample randomly ∼27 Mbp (128 773 reads) of the copperhead genome, thus sampling about 2% of the genome of this species. We identified microsatellite loci in 11.3% of all reads obtained, with 14 612 microsatellite loci identified in total, 4564 of which had flanking sequences suitable for polymerase chain reaction primer design. The random sequencing-based approach to identify microsatellites was rapid, cost-effective and identified thousands of useful microsatellite loci in a previously unstudied species. PMID:21565030

  19. Identification and characterization of a de novo partial trisomy 10p by comparative genomic hybridization (CGH).

    PubMed

    Benzacken, B; Lapierre, J M; Siffroi, J P; Chalvon, A; Tachdjian, G

    1998-10-01

    We report the characterization of a de novo unbalanced chromosome rearrangement by comparative genomic hybridization (CGH) in a 15-day-old child with hypotonia and dysmorphia. We describe the combined use of CGH and fluorescence in situ hybridization (FISH) to identify the origin of the additional chromosomal material on the short arm of chromosome 6. Investigation with FISH revealed that the excess material was not derived from chromosome 6. Identification of unknown unbalanced aberrations that could not be identified by traditional cytogenetics procedures is possible by CGH analysis. Visual analysis of digital images from CGH-metaphase spreads revealed a predominantly green signal on the telomeric region of chromosome 10p. After quantitative digital ratio imaging of 10 CGH-metaphase spreads, a region of gain was found in the chromosome band 10p14-pter. The CGH finding was confirmed by FISH analysis, using a whole chromosome 10 paint probe. These results show the usefulness of CGH for a rapid characterization of de novo unbalanced translocation, unidentifiable by karyotype alone.

  20. Rapid genome-wide evolution in Brassica rapa populations following drought revealed by sequencing of ancestral and descendant gene pools.

    PubMed

    Franks, Steven J; Kane, Nolan C; O'Hara, Niamh B; Tittes, Silas; Rest, Joshua S

    2016-08-01

    There is increasing evidence that evolution can occur rapidly in response to selection. Recent advances in sequencing suggest the possibility of documenting genetic changes as they occur in populations, thus uncovering the genetic basis of evolution, particularly if samples are available from both before and after selection. Here, we had a unique opportunity to directly assess genetic changes in natural populations following an evolutionary response to a fluctuation in climate. We analysed genome-wide differences between ancestors and descendants of natural populations of Brassica rapa plants from two locations that rapidly evolved changes in multiple phenotypic traits, including flowering time, following a multiyear late-season drought in California. These ancestor-descendant comparisons revealed evolutionary shifts in allele frequencies in many genes. Some genes showing evolutionary shifts have functions related to drought stress and flowering time, consistent with an adaptive response to selection. Loci differentiated between ancestors and descendants (FST outliers) were generally different from those showing signatures of selection based on site frequency spectrum analysis (Tajima's D), indicating that the loci that evolved in response to the recent drought and those under historical selection were generally distinct. Very few genes showed similar evolutionary responses between two geographically distinct populations, suggesting independent genetic trajectories of evolution yielding parallel phenotypic changes. The results show that selection can result in rapid genome-wide evolutionary shifts in allele frequencies in natural populations, and highlight the usefulness of combining resurrection experiments in natural populations with genomics for studying the genetic basis of adaptive evolution. PMID:27072809

  1. Rapid genome-wide evolution in Brassica rapa populations following drought revealed by sequencing of ancestral and descendant gene pools.

    PubMed

    Franks, Steven J; Kane, Nolan C; O'Hara, Niamh B; Tittes, Silas; Rest, Joshua S

    2016-08-01

    There is increasing evidence that evolution can occur rapidly in response to selection. Recent advances in sequencing suggest the possibility of documenting genetic changes as they occur in populations, thus uncovering the genetic basis of evolution, particularly if samples are available from both before and after selection. Here, we had a unique opportunity to directly assess genetic changes in natural populations following an evolutionary response to a fluctuation in climate. We analysed genome-wide differences between ancestors and descendants of natural populations of Brassica rapa plants from two locations that rapidly evolved changes in multiple phenotypic traits, including flowering time, following a multiyear late-season drought in California. These ancestor-descendant comparisons revealed evolutionary shifts in allele frequencies in many genes. Some genes showing evolutionary shifts have functions related to drought stress and flowering time, consistent with an adaptive response to selection. Loci differentiated between ancestors and descendants (FST outliers) were generally different from those showing signatures of selection based on site frequency spectrum analysis (Tajima's D), indicating that the loci that evolved in response to the recent drought and those under historical selection were generally distinct. Very few genes showed similar evolutionary responses between two geographically distinct populations, suggesting independent genetic trajectories of evolution yielding parallel phenotypic changes. The results show that selection can result in rapid genome-wide evolutionary shifts in allele frequencies in natural populations, and highlight the usefulness of combining resurrection experiments in natural populations with genomics for studying the genetic basis of adaptive evolution.

  2. BAC-pool 454-sequencing: A rapid and efficient approach to sequence complex tetraploid cotton genomes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    New and emerging next generation sequencing technologies have been promising in reducing sequencing costs, but not significantly for complex polyploid plant genomes such as cotton. Large and highly repetitive genome of G. hirsutum (~2.5GB) is less amenable and cost-intensive with traditional BAC-by...

  3. The SEED and the Rapid Annotation of microbial genomes using Subsystems Technology (RAST)

    PubMed Central

    Overbeek, Ross; Olson, Robert; Pusch, Gordon D.; Olsen, Gary J.; Davis, James J.; Disz, Terry; Edwards, Robert A.; Gerdes, Svetlana; Parrello, Bruce; Shukla, Maulik; Vonstein, Veronika; Wattam, Alice R.; Xia, Fangfang; Stevens, Rick

    2014-01-01

    In 2004, the SEED (http://pubseed.theseed.org/) was created to provide consistent and accurate genome annotations across thousands of genomes and as a platform for discovering and developing de novo annotations. The SEED is a constantly updated integration of genomic data with a genome database, web front end, API and server scripts. It is used by many scientists for predicting gene functions and discovering new pathways. In addition to being a powerful database for bioinformatics research, the SEED also houses subsystems (collections of functionally related protein families) and their derived FIGfams (protein families), which represent the core of the RAST annotation engine (http://rast.nmpdr.org/). When a new genome is submitted to RAST, genes are called and their annotations are made by comparison to the FIGfam collection. If the genome is made public, it is then housed within the SEED and its proteins populate the FIGfam collection. This annotation cycle has proven to be a robust and scalable solution to the problem of annotating the exponentially increasing number of genomes. To date, >12 000 users worldwide have annotated >60 000 distinct genomes using RAST. Here we describe the interconnectedness of the SEED database and RAST, the RAST annotation pipeline and updates to both resources. PMID:24293654

  4. Rapid development of PCR-based genome-specific repetitive DNA junction markers in wheat

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In hexaploid wheat (Triticum aestivum L.) (AABBDD, C=17,000Mb), repeat DNA accounts for ~ 90% of the genome of which transposable elements (TEs) constitute 60-80 %. Despite the dynamic evolution of TEs, our previous study indicated that the majority of TEs between the homologous wheat genomes are co...

  5. Eco-genomic analysis of the poleward range expansion of the wasp spider Argiope bruennichi shows rapid adaptation and genomic admixture.

    PubMed

    Krehenwinkel, Henrik; Rödder, Dennis; Tautz, Diethard

    2015-12-01

    Poleward range expansions are commonly attributed to global change, but could alternatively be driven by rapid evolutionary adaptation. A well-documented example of a range expansion during the past decades is provided by the European wasp spider Argiope bruennichi. Using ecological niche modeling, thermal tolerance experiments and a genome-wide analysis of gene expression divergence, we show that invasive populations have adapted to novel climatic conditions in the course of their expansion. Their climatic niche shift is mirrored in an increased cold tolerance and a population-specific and functionally differentiated gene expression response. We generated an Argiope reference genome sequence and used population genome resequencing to assess genomic changes associated with the new climatic adaptations. We find clear genetic differentiation and a significant admixture with alleles from East Asian populations in the invasive Northern European populations. Population genetic modeling suggests that at least some of these introgressing alleles have contributed to the new adaptations during the expansion. Our results thus confirm the notion that range expansions are not a simple consequence of climate change, but are accompanied by fast genetic changes and adaptations that may be fuelled through admixture between long separated lineages. PMID:26183328

  6. Analyses of Charophyte Chloroplast Genomes Help Characterize the Ancestral Chloroplast Genome of Land Plants

    PubMed Central

    Civáň, Peter; Foster, Peter G.; Embley, Martin T.; Séneca, Ana; Cox, Cymon J.

    2014-01-01

    Despite the significance of the relationships between embryophytes and their charophyte algal ancestors in deciphering the origin and evolutionary success of land plants, few chloroplast genomes of the charophyte algae have been reconstructed to date. Here, we present new data for three chloroplast genomes of the freshwater charophytes Klebsormidium flaccidum (Klebsormidiophyceae), Mesotaenium endlicherianum (Zygnematophyceae), and Roya anglica (Zygnematophyceae). The chloroplast genome of Klebsormidium has a quadripartite organization with exceptionally large inverted repeat (IR) regions and, uniquely among streptophytes, has lost the rrn5 and rrn4.5 genes from the ribosomal RNA (rRNA) gene cluster operon. The chloroplast genome of Roya differs from other zygnematophycean chloroplasts, including the newly sequenced Mesotaenium, by having a quadripartite structure that is typical of other streptophytes. On the basis of the improbability of the novel gain of IR regions, we infer that the quadripartite structure has likely been lost independently in at least three zygnematophycean lineages, although the absence of the usual rRNA operonic synteny in the IR regions of Roya may indicate their de novo origin. Significantly, all zygnematophycean chloroplast genomes have undergone substantial genomic rearrangement, which may be the result of ancient retroelement activity evidenced by the presence of integrase-like and reverse transcriptase-like elements in the Roya chloroplast genome. Our results corroborate the close phylogenetic relationship between Zygnematophyceae and land plants and identify 89 protein-coding genes and 22 introns present in the chloroplast genome at the time of the evolutionary transition of plants to land, all of which can be found in the chloroplast genomes of extant charophytes. PMID:24682153

  7. Analyses of charophyte chloroplast genomes help characterize the ancestral chloroplast genome of land plants.

    PubMed

    Civaň, Peter; Foster, Peter G; Embley, Martin T; Séneca, Ana; Cox, Cymon J

    2014-04-01

    Despite the significance of the relationships between embryophytes and their charophyte algal ancestors in deciphering the origin and evolutionary success of land plants, few chloroplast genomes of the charophyte algae have been reconstructed to date. Here, we present new data for three chloroplast genomes of the freshwater charophytes Klebsormidium flaccidum (Klebsormidiophyceae), Mesotaenium endlicherianum (Zygnematophyceae), and Roya anglica (Zygnematophyceae). The chloroplast genome of Klebsormidium has a quadripartite organization with exceptionally large inverted repeat (IR) regions and, uniquely among streptophytes, has lost the rrn5 and rrn4.5 genes from the ribosomal RNA (rRNA) gene cluster operon. The chloroplast genome of Roya differs from other zygnematophycean chloroplasts, including the newly sequenced Mesotaenium, by having a quadripartite structure that is typical of other streptophytes. On the basis of the improbability of the novel gain of IR regions, we infer that the quadripartite structure has likely been lost independently in at least three zygnematophycean lineages, although the absence of the usual rRNA operonic synteny in the IR regions of Roya may indicate their de novo origin. Significantly, all zygnematophycean chloroplast genomes have undergone substantial genomic rearrangement, which may be the result of ancient retroelement activity evidenced by the presence of integrase-like and reverse transcriptase-like elements in the Roya chloroplast genome. Our results corroborate the close phylogenetic relationship between Zygnematophyceae and land plants and identify 89 protein-coding genes and 22 introns present in the chloroplast genome at the time of the evolutionary transition of plants to land, all of which can be found in the chloroplast genomes of extant charophytes. PMID:24682153

  8. Analyses of charophyte chloroplast genomes help characterize the ancestral chloroplast genome of land plants.

    PubMed

    Civaň, Peter; Foster, Peter G; Embley, Martin T; Séneca, Ana; Cox, Cymon J

    2014-04-01

    Despite the significance of the relationships between embryophytes and their charophyte algal ancestors in deciphering the origin and evolutionary success of land plants, few chloroplast genomes of the charophyte algae have been reconstructed to date. Here, we present new data for three chloroplast genomes of the freshwater charophytes Klebsormidium flaccidum (Klebsormidiophyceae), Mesotaenium endlicherianum (Zygnematophyceae), and Roya anglica (Zygnematophyceae). The chloroplast genome of Klebsormidium has a quadripartite organization with exceptionally large inverted repeat (IR) regions and, uniquely among streptophytes, has lost the rrn5 and rrn4.5 genes from the ribosomal RNA (rRNA) gene cluster operon. The chloroplast genome of Roya differs from other zygnematophycean chloroplasts, including the newly sequenced Mesotaenium, by having a quadripartite structure that is typical of other streptophytes. On the basis of the improbability of the novel gain of IR regions, we infer that the quadripartite structure has likely been lost independently in at least three zygnematophycean lineages, although the absence of the usual rRNA operonic synteny in the IR regions of Roya may indicate their de novo origin. Significantly, all zygnematophycean chloroplast genomes have undergone substantial genomic rearrangement, which may be the result of ancient retroelement activity evidenced by the presence of integrase-like and reverse transcriptase-like elements in the Roya chloroplast genome. Our results corroborate the close phylogenetic relationship between Zygnematophyceae and land plants and identify 89 protein-coding genes and 22 introns present in the chloroplast genome at the time of the evolutionary transition of plants to land, all of which can be found in the chloroplast genomes of extant charophytes.

  9. Genomics-enabled sensor platform for rapid detection of viruses related to disease outbreak.

    SciTech Connect

    Brozik, Susan M; Manginell, Ronald P; Moorman, Matthew W; Xiao, Xiaoyin; Edwards, Thayne L.; Anderson, John Moses; Pfeifer, Kent Bryant; Branch, Darren W.; Wheeler, David Roger; Polsky, Ronen; Lopez, DeAnna M.; Ebel, Gregory D.; Prasad, Abhishek N.; Brozik, James A.; Rudolph, Angela R.; Wong, Lillian P.

    2013-09-01

    Bioweapons and emerging infectious diseases pose growing threats to our national security. Both natural disease outbreak and outbreaks due to a bioterrorist attack are a challenge to detect, taking days after the outbreak to identify since most outbreaks are only recognized through reportable diseases by health departments and reports of unusual diseases by clinicians. In recent decades, arthropod-borne viruses (arboviruses) have emerged as some of the most significant threats to human health. They emerge, often unexpectedly, from cryptic transmission foci causing localized outbreaks that can rapidly spread to multiple continents due to increased human travel and trade. Currently, diagnosis of acute infections requires amplification of viral nucleic acids, which can be costly, highly specific, technically challenging and time consuming. No diagnostic devices suitable for use at the bedside or in an outbreak setting currently exist. The original goals of this project were to 1) develop two highly sensitive and specific diagnostic assays for detecting RNA from a wide range of arboviruses; one based on an electrochemical approach and the other a fluorescent based assay and 2) develop prototype microfluidic diagnostic platforms for preclinical and field testing that utilize the assays developed in goal 1. We generated and characterized suitable primers for West Nile Virus RNA detection. Both optical and electrochemical transduction technologies were developed for DNA-RNA hybridization detection and were implemented in microfluidic diagnostic sensing platforms that were developed in this project.

  10. Rapid evolution of the compact and unusual mitochondrial genome in the ctenophore, Pleurobrachia bachei.

    PubMed

    Kohn, Andrea B; Citarella, Mathew R; Kocot, Kevin M; Bobkova, Yelena V; Halanych, Kenneth M; Moroz, Leonid L

    2012-04-01

    Ctenophores are one of the most basally branching lineages of metazoans with the largest mitochondrial organelles in the animal kingdom. We sequenced the mitochondrial (mtDNA) genome from the Pacific cidipid ctenophore, Pleurobrachia bachei. The circular mitochondrial genome is 11,016 nts, with only 12 genes, and one of the smallest metazoan mtDNA genomes recorded. The protein coding genes are intronless cox1-3, cob, nad1, 3, 4, 4L and 5. The nad2 and 6 genes are represented as short fragments whereas the atp6 gene was found in the nuclear genome. Only the large ribosomal RNA subunit and two tRNAs were present with possibly the small subunit unidentifiable due to extensive fragmentation. The observed unique features of this mitochondrial genome suggest that nuclear and mitochondrial genomes have evolved at very different rates. This reduced mtDNA genome sharply contrasts with the very large sizes of mtDNA found in other basal metazoans including Porifera (sponges), and Placozoa (Trichoplax). PMID:22201557

  11. Characterization of evolutionary rates and constraints in three mammalian genomes

    SciTech Connect

    Cooper, Gregory M.; Brudno, Michael; Stone, Eric A.; Dubchak, Inna; Batzoglou, Serafim; Sidow, Arend

    2004-02-15

    We present an analysis of rates and patterns of microevolutionary phenomena that have shaped the human, mouse, and rat genomes since their last common ancestor. We find evidence for a shift in the mutational spectrum between the mouse and rat lineages, with the net effect being a relative increase in GC content in the rat genome. Our estimate for the neutral point substitution rate separating the two rodents is 0.196 substitutions per site, and 0.65 substitutions per site for the tree relating all three mammals. Small insertions and deletions of 1-10 bp in length (''microindels'') occur at approximately 5 percent of the point substitution rate. Inferred regional correlations in evolutionary rates between lineages and between types of sites support the idea that rates of evolution are influenced by local genomic or cell biological context. No substantial correlations between rates of point substitutions and rates of microindels are found, however, implying that the influences that affect these processes are distinct. Finally, we have identified those regions in the human genome that are evolving slowly, which are likely to include functional elements important to human biology. At least 5 percent of the human genome is under substantial constraint, most of which is noncoding.

  12. Comprehensive characterization of complex structural variations in cancer by directly comparing genome sequence reads.

    PubMed

    Moncunill, Valentí; Gonzalez, Santi; Beà, Sílvia; Andrieux, Lise O; Salaverria, Itziar; Royo, Cristina; Martinez, Laura; Puiggròs, Montserrat; Segura-Wang, Maia; Stütz, Adrian M; Navarro, Alba; Royo, Romina; Gelpí, Josep L; Gut, Ivo G; López-Otín, Carlos; Orozco, Modesto; Korbel, Jan O; Campo, Elias; Puente, Xose S; Torrents, David

    2014-11-01

    The development of high-throughput sequencing technologies has advanced our understanding of cancer. However, characterizing somatic structural variants in tumor genomes is still challenging because current strategies depend on the initial alignment of reads to a reference genome. Here, we describe SMUFIN (somatic mutation finder), a single program that directly compares sequence reads from normal and tumor genomes to accurately identify and characterize a range of somatic sequence variation, from single-nucleotide variants (SNV) to large structural variants at base pair resolution. Performance tests on modeled tumor genomes showed average sensitivity of 92% and 74% for SNVs and structural variants, with specificities of 95% and 91%, respectively. Analyses of aggressive forms of solid and hematological tumors revealed that SMUFIN identifies breakpoints associated with chromothripsis and chromoplexy with high specificity. SMUFIN provides an integrated solution for the accurate, fast and comprehensive characterization of somatic sequence variation in cancer. PMID:25344728

  13. Characterizing genomic alterations in cancer by complementary functional associations | Office of Cancer Genomics

    Cancer.gov

    Systematic efforts to sequence the cancer genome have identified large numbers of mutations and copy number alterations in human cancers. However, elucidating the functional consequences of these variants, and their interactions to drive or maintain oncogenic states, remains a challenge in cancer research. We developed REVEALER, a computational method that identifies combinations of mutually exclusive genomic alterations correlated with functional phenotypes, such as the activation or gene dependency of oncogenic pathways or sensitivity to a drug treatment.

  14. Genomic Characterization of Burkholderia pseudomallei Isolates Selected for Medical Countermeasures Testing: Comparative Genomics Associated with Differential Virulence

    PubMed Central

    Sahl, Jason W.; Allender, Christopher J.; Colman, Rebecca E.; Califf, Katy J.; Schupp, James M.; Currie, Bart J.; Van Zandt, Kristopher E.; Gelhaus, H. Carl; Keim, Paul; Tuanyok, Apichai

    2015-01-01

    Burkholderia pseudomallei is the causative agent of melioidosis and a potential bioterrorism agent. In the development of medical countermeasures against B. pseudomallei infection, the US Food and Drug Administration (FDA) animal Rule recommends using well-characterized strains in animal challenge studies. In this study, whole genome sequence data were generated for 6 B. pseudomallei isolates previously identified as candidates for animal challenge studies; an additional 5 isolates were sequenced that were associated with human inhalational melioidosis. A core genome single nucleotide polymorphism (SNP) phylogeny inferred from a concatenated SNP alignment from the 11 isolates sequenced in this study and a diverse global collection of isolates demonstrated the diversity of the proposed Animal Rule isolates. To understand the genomic composition of each isolate, a large-scale blast score ratio (LS-BSR) analysis was performed on the entire pan-genome; this demonstrated the variable composition of genes across the panel and also helped to identify genes unique to individual isolates. In addition, a set of ~550 genes associated with pathogenesis in B. pseudomallei were screened against the 11 sequenced genomes with LS-BSR. Differential gene distribution for 54 virulence-associated genes was observed between genomes and three of these genes were correlated with differential virulence observed in animal challenge studies using BALB/c mice. Differentially conserved genes and SNPs associated with disease severity were identified and could be the basis for future studies investigating the pathogenesis of B. pseudomallei. Overall, the genetic characterization of the 11 proposed Animal Rule isolates provides context for future studies involving B. pseudomallei pathogenesis, differential virulence, and efficacy to therapeutics. PMID:25803742

  15. Genomic characterization of Burkholderia pseudomallei isolates selected for medical countermeasures testing: comparative genomics associated with differential virulence.

    PubMed

    Sahl, Jason W; Allender, Christopher J; Colman, Rebecca E; Califf, Katy J; Schupp, James M; Currie, Bart J; Van Zandt, Kristopher E; Gelhaus, H Carl; Keim, Paul; Tuanyok, Apichai

    2015-01-01

    Burkholderia pseudomallei is the causative agent of melioidosis and a potential bioterrorism agent. In the development of medical countermeasures against B. pseudomallei infection, the US Food and Drug Administration (FDA) animal Rule recommends using well-characterized strains in animal challenge studies. In this study, whole genome sequence data were generated for 6 B. pseudomallei isolates previously identified as candidates for animal challenge studies; an additional 5 isolates were sequenced that were associated with human inhalational melioidosis. A core genome single nucleotide polymorphism (SNP) phylogeny inferred from a concatenated SNP alignment from the 11 isolates sequenced in this study and a diverse global collection of isolates demonstrated the diversity of the proposed Animal Rule isolates. To understand the genomic composition of each isolate, a large-scale blast score ratio (LS-BSR) analysis was performed on the entire pan-genome; this demonstrated the variable composition of genes across the panel and also helped to identify genes unique to individual isolates. In addition, a set of ~550 genes associated with pathogenesis in B. pseudomallei were screened against the 11 sequenced genomes with LS-BSR. Differential gene distribution for 54 virulence-associated genes was observed between genomes and three of these genes were correlated with differential virulence observed in animal challenge studies using BALB/c mice. Differentially conserved genes and SNPs associated with disease severity were identified and could be the basis for future studies investigating the pathogenesis of B. pseudomallei. Overall, the genetic characterization of the 11 proposed Animal Rule isolates provides context for future studies involving B. pseudomallei pathogenesis, differential virulence, and efficacy to therapeutics.

  16. Whole-Genome Comparison Reveals Novel Genetic Elements That Characterize the Genome of Industrial Strains of Saccharomyces cerevisiae

    PubMed Central

    Borneman, Anthony R.; Desany, Brian A.; Riches, David; Affourtit, Jason P.; Forgan, Angus H.; Pretorius, Isak S.; Egholm, Michael; Chambers, Paul J.

    2011-01-01

    Human intervention has subjected the yeast Saccharomyces cerevisiae to multiple rounds of independent domestication and thousands of generations of artificial selection. As a result, this species comprises a genetically diverse collection of natural isolates as well as domesticated strains that are used in specific industrial applications. However the scope of genetic diversity that was captured during the domesticated evolution of the industrial representatives of this important organism remains to be determined. To begin to address this, we have produced whole-genome assemblies of six commercial strains of S. cerevisiae (four wine and two brewing strains). These represent the first genome assemblies produced from S. cerevisiae strains in their industrially-used forms and the first high-quality assemblies for S. cerevisiae strains used in brewing. By comparing these sequences to six existing high-coverage S. cerevisiae genome assemblies, clear signatures were found that defined each industrial class of yeast. This genetic variation was comprised of both single nucleotide polymorphisms and large-scale insertions and deletions, with the latter often being associated with ORF heterogeneity between strains. This included the discovery of more than twenty probable genes that had not been identified previously in the S. cerevisiae genome. Comparison of this large number of S. cerevisiae strains also enabled the characterization of a cluster of five ORFs that have integrated into the genomes of the wine and bioethanol strains on multiple occasions and at diverse genomic locations via what appears to involve the resolution of a circular DNA intermediate. This work suggests that, despite the scrutiny that has been directed at the yeast genome, there remains a significant reservoir of ORFs and novel modes of genetic transmission that may have significant phenotypic impact in this important model and industrial species. PMID:21304888

  17. Genome-culture coevolution promotes rapid divergence of killer whale ecotypes.

    PubMed

    Foote, Andrew D; Vijay, Nagarjun; Ávila-Arcos, María C; Baird, Robin W; Durban, John W; Fumagalli, Matteo; Gibbs, Richard A; Hanson, M Bradley; Korneliussen, Thorfinn S; Martin, Michael D; Robertson, Kelly M; Sousa, Vitor C; Vieira, Filipe G; Vinař, Tomáš; Wade, Paul; Worley, Kim C; Excoffier, Laurent; Morin, Phillip A; Gilbert, M Thomas P; Wolf, Jochen B W

    2016-01-01

    Analysing population genomic data from killer whale ecotypes, which we estimate have globally radiated within less than 250,000 years, we show that genetic structuring including the segregation of potentially functional alleles is associated with socially inherited ecological niche. Reconstruction of ancestral demographic history revealed bottlenecks during founder events, likely promoting ecological divergence and genetic drift resulting in a wide range of genome-wide differentiation between pairs of allopatric and sympatric ecotypes. Functional enrichment analyses provided evidence for regional genomic divergence associated with habitat, dietary preferences and post-zygotic reproductive isolation. Our findings are consistent with expansion of small founder groups into novel niches by an initial plastic behavioural response, perpetuated by social learning imposing an altered natural selection regime. The study constitutes an important step towards an understanding of the complex interaction between demographic history, culture, ecological adaptation and evolution at the genomic level. PMID:27243207

  18. Genome-culture coevolution promotes rapid divergence of killer whale ecotypes.

    PubMed

    Foote, Andrew D; Vijay, Nagarjun; Ávila-Arcos, María C; Baird, Robin W; Durban, John W; Fumagalli, Matteo; Gibbs, Richard A; Hanson, M Bradley; Korneliussen, Thorfinn S; Martin, Michael D; Robertson, Kelly M; Sousa, Vitor C; Vieira, Filipe G; Vinař, Tomáš; Wade, Paul; Worley, Kim C; Excoffier, Laurent; Morin, Phillip A; Gilbert, M Thomas P; Wolf, Jochen B W

    2016-05-31

    Analysing population genomic data from killer whale ecotypes, which we estimate have globally radiated within less than 250,000 years, we show that genetic structuring including the segregation of potentially functional alleles is associated with socially inherited ecological niche. Reconstruction of ancestral demographic history revealed bottlenecks during founder events, likely promoting ecological divergence and genetic drift resulting in a wide range of genome-wide differentiation between pairs of allopatric and sympatric ecotypes. Functional enrichment analyses provided evidence for regional genomic divergence associated with habitat, dietary preferences and post-zygotic reproductive isolation. Our findings are consistent with expansion of small founder groups into novel niches by an initial plastic behavioural response, perpetuated by social learning imposing an altered natural selection regime. The study constitutes an important step towards an understanding of the complex interaction between demographic history, culture, ecological adaptation and evolution at the genomic level.

  19. Genome-culture coevolution promotes rapid divergence of killer whale ecotypes

    PubMed Central

    Foote, Andrew D.; Vijay, Nagarjun; Ávila-Arcos, María C.; Baird, Robin W.; Durban, John W.; Fumagalli, Matteo; Gibbs, Richard A.; Hanson, M. Bradley; Korneliussen, Thorfinn S.; Martin, Michael D.; Robertson, Kelly M.; Sousa, Vitor C.; Vieira, Filipe G.; Vinař, Tomáš; Wade, Paul; Worley, Kim C.; Excoffier, Laurent; Morin, Phillip A.; Gilbert, M. Thomas P.; Wolf, Jochen B.W.

    2016-01-01

    Analysing population genomic data from killer whale ecotypes, which we estimate have globally radiated within less than 250,000 years, we show that genetic structuring including the segregation of potentially functional alleles is associated with socially inherited ecological niche. Reconstruction of ancestral demographic history revealed bottlenecks during founder events, likely promoting ecological divergence and genetic drift resulting in a wide range of genome-wide differentiation between pairs of allopatric and sympatric ecotypes. Functional enrichment analyses provided evidence for regional genomic divergence associated with habitat, dietary preferences and post-zygotic reproductive isolation. Our findings are consistent with expansion of small founder groups into novel niches by an initial plastic behavioural response, perpetuated by social learning imposing an altered natural selection regime. The study constitutes an important step towards an understanding of the complex interaction between demographic history, culture, ecological adaptation and evolution at the genomic level. PMID:27243207

  20. Development and characterization of genomic and expressed SSRs in citrus by genome-wide analysis.

    PubMed

    Liu, Sheng-Rui; Li, Wen-Yang; Long, Dang; Hu, Chun-Gen; Zhang, Jin-Zhi

    2013-01-01

    Microsatellites or simple sequence repeats (SSRs) are one of the most popular sources of genetic markers and play a significant role in plant genetics and breeding. In this study, we identified citrus SSRs in the genome of Clementine mandarin and analyzed their frequency and distribution in different genomic regions. A total of 80,708 SSRs were detected in the genome with an overall density of 268 SSRs/Mb. While di-nucleotide repeats were the most frequent microsatellites in genomic DNA sequence, tetra-nucleotides, which had more repeat units than any other SSR types, had the highest cumulative sequence length. We identified 6,834 transcripts as containing 8,989 SSRs in 33,929 Clementine mandarin transcripts, among which, tri-nucleotide motifs (36.0%) were the most common, followed by di-nucleotide (26.9%) and hexa-nucleotide motifs (15.1%). The motif AG (16.7%) was most abundant among these SSRs, while motifs AAG (6.6%), AAT (5.0%), and TAG (2.2%) were most common among tri-nucleotides. Functional categorization of transcripts containing SSRs revealed that 5,879 (86.0%) of such transcripts had homology with known proteins, GO and KEGG annotation revealed that transcripts containing SSRs were those implicated in diverse biological processes in plants, including binding, development, transcription, and protein degradation. When 27 genomic and 78 randomly selected SSRs were tested on Clementine mandarin, 95 SSRs revealed polymorphism. These 95 SSRs were further deployed on 18 genotypes of the three generas of Rutaceae for the genetic diversity assessment, genomic SSRs generally show low transferability in comparison to SSRs developed from expressed sequences. These transcript-markers identified in our study may provide a valuable genetic and genomic tool for further genetic research and varietal development in citrus, such as diversity study, QTL mapping, molecular breeding, comparative mapping and other genetic analyses.

  1. Characterization of reniform nematode genome through shotgun sequencing

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The reniform nematode (RN), a major agricultural pest particularly on cotton in the United States(U.S.), is among the major plant parasitic nematodes for which limited genomic information exists. In this study, over 380 Mb of sequence data were generated from four pooled adult female RN and assembl...

  2. Characterizing the walnut genome through analyses of BAC end sequences

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Persian walnut (Juglans regia L.) is an economically important tree for its nut crop and timber. To gain insight into the structure and evolution of the walnut genome, we constructed two bacterial artificial chromosome (BAC) libraries, containing a total of 129,024 clones, from in vitro-grown shoots...

  3. FusX: A Rapid One-Step Transcription Activator-Like Effector Assembly System for Genome Science.

    PubMed

    Ma, Alvin C; McNulty, Melissa S; Poshusta, Tanya L; Campbell, Jarryd M; Martínez-Gálvez, Gabriel; Argue, David P; Lee, Han B; Urban, Mark D; Bullard, Cassandra E; Blackburn, Patrick R; Man, Toni K; Clark, Karl J; Ekker, Stephen C

    2016-06-01

    Transcription activator-like effectors (TALEs) are extremely effective, single-molecule DNA-targeting molecular cursors used for locus-specific genome science applications, including high-precision molecular medicine and other genome engineering applications. TALEs are used in genome engineering for locus-specific DNA editing and imaging, as artificial transcriptional activators and repressors, and for targeted epigenetic modification. TALEs as nucleases (TALENs) are effective editing tools and offer high binding specificity and fewer sequence constraints toward the targeted genome than other custom nuclease systems. One bottleneck of broader TALE use is reagent accessibility. For example, one commonly deployed method uses a multitube, 5-day assembly protocol. Here we describe FusX, a streamlined Golden Gate TALE assembly system that (1) is backward compatible with popular TALE backbones, (2) is functionalized as a single-tube 3-day TALE assembly process, (3) requires only commonly used basic molecular biology reagents, and (4) is cost-effective. More than 100 TALEN pairs have been successfully assembled using FusX, and 27 pairs were quantitatively tested in zebrafish, with each showing high somatic and germline activity. Furthermore, this assembly system is flexible and is compatible with standard molecular biology laboratory tools, but can be scaled with automated laboratory support. To demonstrate, we use a highly accessible and commercially available liquid-handling robot to rapidly and accurately assemble TALEs using the FusX TALE toolkit. Together, the FusX system accelerates TALE-based genomic science applications from basic science screening work for functional genomics testing and molecular medicine applications. PMID:26854857

  4. FusX: A Rapid One-Step Transcription Activator-Like Effector Assembly System for Genome Science

    PubMed Central

    Ma, Alvin C.; McNulty, Melissa S.; Poshusta, Tanya L.; Campbell, Jarryd M.; Martínez-Gálvez, Gabriel; Argue, David P.; Lee, Han B.; Urban, Mark D.; Bullard, Cassandra E.; Blackburn, Patrick R.; Man, Toni K.; Clark, Karl J.; Ekker, Stephen C.

    2016-01-01

    Transcription activator-like effectors (TALEs) are extremely effective, single-molecule DNA-targeting molecular cursors used for locus-specific genome science applications, including high-precision molecular medicine and other genome engineering applications. TALEs are used in genome engineering for locus-specific DNA editing and imaging, as artificial transcriptional activators and repressors, and for targeted epigenetic modification. TALEs as nucleases (TALENs) are effective editing tools and offer high binding specificity and fewer sequence constraints toward the targeted genome than other custom nuclease systems. One bottleneck of broader TALE use is reagent accessibility. For example, one commonly deployed method uses a multitube, 5-day assembly protocol. Here we describe FusX, a streamlined Golden Gate TALE assembly system that (1) is backward compatible with popular TALE backbones, (2) is functionalized as a single-tube 3-day TALE assembly process, (3) requires only commonly used basic molecular biology reagents, and (4) is cost-effective. More than 100 TALEN pairs have been successfully assembled using FusX, and 27 pairs were quantitatively tested in zebrafish, with each showing high somatic and germline activity. Furthermore, this assembly system is flexible and is compatible with standard molecular biology laboratory tools, but can be scaled with automated laboratory support. To demonstrate, we use a highly accessible and commercially available liquid-handling robot to rapidly and accurately assemble TALEs using the FusX TALE toolkit. Together, the FusX system accelerates TALE-based genomic science applications from basic science screening work for functional genomics testing and molecular medicine applications. PMID:26854857

  5. Genome-Wide Analysis in Three Fusarium Pathogens Identifies Rapidly Evolving Chromosomes and Genes Associated with Pathogenicity.

    PubMed

    Sperschneider, Jana; Gardiner, Donald M; Thatcher, Louise F; Lyons, Rebecca; Singh, Karam B; Manners, John M; Taylor, Jennifer M

    2015-05-19

    Pathogens and hosts are in an ongoing arms race and genes involved in host-pathogen interactions are likely to undergo diversifying selection. Fusarium plant pathogens have evolved diverse infection strategies, but how they interact with their hosts in the biotrophic infection stage remains puzzling. To address this, we analyzed the genomes of three Fusarium plant pathogens for genes that are under diversifying selection. We found a two-speed genome structure both on the chromosome and gene group level. Diversifying selection acts strongly on the dispensable chromosomes in Fusarium oxysporum f. sp. lycopersici and on distinct core chromosome regions in Fusarium graminearum, all of which have associations with virulence. Members of two gene groups evolve rapidly, namely those that encode proteins with an N-terminal [SG]-P-C-[KR]-P sequence motif and proteins that are conserved predominantly in pathogens. Specifically, 29 F. graminearum genes are rapidly evolving, in planta induced and encode secreted proteins, strongly pointing toward effector function. In summary, diversifying selection in Fusarium is strongly reflected as genomic footprints and can be used to predict a small gene set likely to be involved in host-pathogen interactions for experimental verification.

  6. Genome-Wide Analysis in Three Fusarium Pathogens Identifies Rapidly Evolving Chromosomes and Genes Associated with Pathogenicity

    PubMed Central

    Sperschneider, Jana; Gardiner, Donald M.; Thatcher, Louise F.; Lyons, Rebecca; Singh, Karam B.; Manners, John M.; Taylor, Jennifer M.

    2015-01-01

    Pathogens and hosts are in an ongoing arms race and genes involved in host–pathogen interactions are likely to undergo diversifying selection. Fusarium plant pathogens have evolved diverse infection strategies, but how they interact with their hosts in the biotrophic infection stage remains puzzling. To address this, we analyzed the genomes of three Fusarium plant pathogens for genes that are under diversifying selection. We found a two-speed genome structure both on the chromosome and gene group level. Diversifying selection acts strongly on the dispensable chromosomes in Fusarium oxysporum f. sp. lycopersici and on distinct core chromosome regions in Fusarium graminearum, all of which have associations with virulence. Members of two gene groups evolve rapidly, namely those that encode proteins with an N-terminal [SG]-P-C-[KR]-P sequence motif and proteins that are conserved predominantly in pathogens. Specifically, 29 F. graminearum genes are rapidly evolving, in planta induced and encode secreted proteins, strongly pointing toward effector function. In summary, diversifying selection in Fusarium is strongly reflected as genomic footprints and can be used to predict a small gene set likely to be involved in host–pathogen interactions for experimental verification. PMID:25994930

  7. Collision-induced fragmentation accurate mass spectrometric analysis methods to rapidly characterize plant extracts

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The rapid advances in analytical chromatography equipment have made the reliable and reproducible measurement of a wide range of plant chemical components possible. Full chemical characterization of a given plant material is possible with the new mass spectrometers currently available. However, th...

  8. Collision-induced fragmentation accurate mass spectrometric analysis methods to rapidly characterize phytochemicals in plant extracts

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The rapid advances in analytical chromatography equipment have made the reliable and reproducible measurement of a wide range of plant chemical components possible. Full chemical characterization of a given plant material is possible with the new mass spectrometers currently available. New methods a...

  9. Collision-induced fragmentation accurate mass spectrometric analysis methods to rapidly characterize plant extracts

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The rapid advances in analytical chromatography equipment have made the reliable and reproducible measurement of a wide range of plant chemical components possible. Full chemical characterization of a given plant material is possible with the new mass spectrometers currently available. For phytochem...

  10. Rapid, Enhanced IV Characterization of Multi-Junction PV Devices under One Sun at NREL

    SciTech Connect

    Moriarty, Tom; France, Ryan; Steiner, Myles

    2015-06-14

    Multi-junction technology is rapidly advancing, which puts increasing demands on IV characterization resources. We report on a tool and procedure for fast turn-around of IV data under the reference conditions, but also under controlled variations from the reference conditions. This enhanced data set can improve further iterations of device optimization.

  11. Rapid, Enhanced IV Characterization of Multi-Junction PV Devices under One Sun at NREL: Preprint

    SciTech Connect

    Moriarty, Tom; France, Ryan; Steiner, Myles

    2015-09-15

    Multi-junction technology is rapidly advancing, which puts increasing demands on IV characterization resources. We report on a tool and procedure for fast turn-around of IV data under the reference conditions, but also under controlled variations from the reference conditions. This enhanced data set can improve further iterations of device optimization.

  12. Three-Enzyme Cascade Bioreactor for Rapid Digestion of Genomic DNA into Single Nucleosides.

    PubMed

    Yin, Junfa; Xu, Tian; Zhang, Ning; Wang, Hailin

    2016-08-01

    Structure-based DNA modification analysis provides accurate and important information on genomic DNA changes from epigenetic modifications to various DNA lesions. However, genomic DNA strands are often required to be efficiently digested into single nucleosides. It is an arduous task because of the involvement of multiple enzymes with different catalytic acitivities. Here we constructed a three-enzyme cascade capillary monolithic bioreactor that consists of immobilized deoxyribonuclease I (DNase I), snake venom phosphodiesterase (SVP), and alkaline phosphatase (ALPase). By the use of this cascade capillary bioreactor, genomic DNA can be efficiently digested into single nucleosides with an increasing rate of ∼20 folds. The improvement is mainly attributed to dramatically increase enzymatic capacity and activity. With a designed macro-porous structure, genomic DNA of 5-30 Kb (∼1.6-10 million Daltons) can be directly passed through the bioreactor simply by hand pushing or a low-pressure microinjection pump. By coupling with liquid chromatography-tandem mass spectrometry (LC-MS/MS), we further developed a sensitive assay for detection of an oxidative stress biomarker 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in DNA. The proposed three-enzyme cascade bioreactor is also potentially applicable for fast identification and quantitative detection of other lesions and modifications in genomic DNA. PMID:27416319

  13. Identification, characterization and comparative genomics of chimpanzee endogenous retroviruses

    PubMed Central

    Polavarapu, Nalini; Bowen, Nathan J; McDonald, John F

    2006-01-01

    Background Retrotransposons, the most abundant and widespread class of eukaryotic transposable elements, are believed to play a significant role in mutation and disease and to have contributed significantly to the evolution of genome structure and function. The recent sequencing of the chimpanzee genome is providing an unprecedented opportunity to study the functional significance of these elements in two closely related primate species and to better evaluate their role in primate evolution. Results We report here that the chimpanzee genome contains at least 42 separate families of endogenous retroviruses, nine of which were not previously identified. All but two (CERV 1/PTERV1 and CERV 2) of the 42 families of chimpanzee endogenous retroviruses were found to have orthologs in humans. Molecular analysis (PCR and Southern hybridization) of CERV 2 elements demonstrates that this family is present in chimpanzee, bonobo, gorilla and old-world monkeys but absent in human, orangutan and new-world monkeys. A survey of endogenous retroviral positional variation between chimpanzees and humans determined that approximately 7% of all chimpanzee-human INDEL variation is associated with endogenous retroviral sequences. Conclusion Nine families of chimpanzee endogenous retroviruses have been transpositionally active since chimpanzees and humans diverged from a common ancestor. Seven of these transpositionally active families have orthologs in humans, one of which has also been transpositionally active in humans since the human-chimpanzee divergence about six million years ago. Comparative analyses of orthologous regions of the human and chimpanzee genomes have revealed that a significant portion of INDEL variation between chimpanzees and humans is attributable to endogenous retroviruses and may be of evolutionary significance. PMID:16805923

  14. Final progress report, Construction of a genome-wide highly characterized clone resource for genome sequencing

    SciTech Connect

    Nierman, William C.

    2000-02-14

    At TIGR, the human Bacterial Artificial Chromosome (BAC) end sequencing and trimming were with an overall sequencing success rate of 65%. CalTech human BAC libraries A, B, C and D as well as Roswell Park Cancer Institute's library RPCI-11 were used. To date, we have generated >300,000 end sequences from >186,000 human BAC clones with an average read length {approx}460 bp for a total of 141 Mb covering {approx}4.7% of the genome. Over sixty percent of the clones have BAC end sequences (BESs) from both ends representing over five-fold coverage of the genome by the paired-end clones. The average phred Q20 length is {approx}400 bp. This high accuracy makes our BESs match the human finished sequences with an average identity of 99% and a match length of 450 bp, and a frequency of one match per 12.8 kb contig sequence. Our sample tracking has ensured a clone tracking accuracy of >90%, which gives researchers a high confidence in (1) retrieving the right clone from the BA C libraries based on the sequence matches; and (2) building a minimum tiling path of sequence-ready clones across the genome and genome assembly scaffolds.

  15. Genomics-assisted characterization of a breeding collection of Apios americana, an edible tuberous legume

    PubMed Central

    Belamkar, Vikas; Farmer, Andrew D.; Weeks, Nathan T.; Kalberer, Scott R.; Blackmon, William J.; Cannon, Steven B.

    2016-01-01

    For species with potential as new crops, rapid improvement may be facilitated by new genomic methods. Apios (Apios americana Medik.), once a staple food source of Native American Indians, produces protein-rich tubers, tolerates a wide range of soils, and symbiotically fixes nitrogen. We report the first high-quality de novo transcriptome assembly, an expression atlas, and a set of 58,154 SNP and 39,609 gene expression markers (GEMs) for characterization of a breeding collection. Both SNPs and GEMs identify six genotypic clusters in the collection. Transcripts mapped to the Phaseolus vulgaris genome–another phaseoloid legume with the same chromosome number–provide provisional genetic locations for 46,852 SNPs. Linkage disequilibrium decays within 10 kb (based on the provisional genetic locations), consistent with outcrossing reproduction. SNPs and GEMs identify more than 21 marker-trait associations for at least 11 traits. This study demonstrates a holistic approach for mining plant collections to accelerate crop improvement. PMID:27721469

  16. Contributing to Tumor Molecular Characterization Projects with a Global Impact | Office of Cancer Genomics

    Cancer.gov

    My name is Nicholas Griner and I am the Scientific Program Manager for the Cancer Genome Characterization Initiative (CGCI) in the Office of Cancer Genomics (OCG). Until recently, I spent most of my scientific career working in a cancer research laboratory. In my postdoctoral training, my research focused on identifying novel pathways that contribute to both prostate and breast cancers and studying proteins within these pathways that may be targeted with cancer drugs.

  17. Construction of the BAC Library of Small Abalone (Haliotis diversicolor) for Gene Screening and Genome Characterization.

    PubMed

    Jiang, Likun; You, Weiwei; Zhang, Xiaojun; Xu, Jian; Jiang, Yanliang; Wang, Kai; Zhao, Zixia; Chen, Baohua; Zhao, Yunfeng; Mahboob, Shahid; Al-Ghanim, Khalid A; Ke, Caihuan; Xu, Peng

    2016-02-01

    The small abalone (Haliotis diversicolor) is one of the most important aquaculture species in East Asia. To facilitate gene cloning and characterization, genome analysis, and genetic breeding of it, we constructed a large-insert bacterial artificial chromosome (BAC) library, which is an important genetic tool for advanced genetics and genomics research. The small abalone BAC library includes 92,610 clones with an average insert size of 120 Kb, equivalent to approximately 7.6× of the small abalone genome. We set up three-dimensional pools and super pools of 18,432 BAC clones for target gene screening using PCR method. To assess the approach, we screened 12 target genes in these 18,432 BAC clones and identified 16 positive BAC clones. Eight positive BAC clones were then sequenced and assembled with the next generation sequencing platform. The assembled contigs representing these 8 BAC clones spanned 928 Kb of the small abalone genome, providing the first batch of genome sequences for genome evaluation and characterization. The average GC content of small abalone genome was estimated as 40.33%. A total of 21 protein-coding genes, including 7 target genes, were annotated into the 8 BACs, which proved the feasibility of PCR screening approach with three-dimensional pools in small abalone BAC library. One hundred fifty microsatellite loci were also identified from the sequences for marker development in the future. The BAC library and clone pools provided valuable resources and tools for genetic breeding and conservation of H. diversicolor.

  18. Characterization of Genomic Alterations in Radiation-Associated Breast Cancer among Childhood Cancer Survivors, Using Comparative Genomic Hybridization (CGH) Arrays

    PubMed Central

    Yang, Xiaohong R.; Killian, J. Keith; Hammond, Sue; Burke, Laura S.; Bennett, Hunter; Wang, Yonghong; Davis, Sean R.; Strong, Louise C.; Neglia, Joseph; Stovall, Marilyn; Weathers, Rita E.; Robison, Leslie L.; Bhatia, Smita; Mabuchi, Kiyohiko; Inskip, Peter D.; Meltzer, Paul

    2015-01-01

    Ionizing radiation is an established risk factor for breast cancer. Epidemiologic studies of radiation-exposed cohorts have been primarily descriptive; molecular events responsible for the development of radiation-associated breast cancer have not been elucidated. In this study, we used array comparative genomic hybridization (array-CGH) to characterize genome-wide copy number changes in breast tumors collected in the Childhood Cancer Survivor Study (CCSS). Array-CGH data were obtained from 32 cases who developed a second primary breast cancer following chest irradiation at early ages for the treatment of their first cancers, mostly Hodgkin lymphoma. The majority of these cases developed breast cancer before age 45 (91%, n = 29), had invasive ductal tumors (81%, n = 26), estrogen receptor (ER)-positive staining (68%, n = 19 out of 28), and high proliferation as indicated by high Ki-67 staining (77%, n = 17 out of 22). Genomic regions with low-copy number gains and losses and high-level amplifications were similar to what has been reported in sporadic breast tumors, however, the frequency of amplifications of the 17q12 region containing human epidermal growth factor receptor 2 (HER2) was much higher among CCSS cases (38%, n = 12). Our findings suggest that second primary breast cancers in CCSS were enriched for an “amplifier” genomic subgroup with highly proliferative breast tumors. Future investigation in a larger irradiated cohort will be needed to confirm our findings. PMID:25764003

  19. Characterization of genomic alterations in radiation-associated breast cancer among childhood cancer survivors, using comparative genomic hybridization (CGH) arrays.

    PubMed

    Yang, Xiaohong R; Killian, J Keith; Hammond, Sue; Burke, Laura S; Bennett, Hunter; Wang, Yonghong; Davis, Sean R; Strong, Louise C; Neglia, Joseph; Stovall, Marilyn; Weathers, Rita E; Robison, Leslie L; Bhatia, Smita; Mabuchi, Kiyohiko; Inskip, Peter D; Meltzer, Paul

    2015-01-01

    Ionizing radiation is an established risk factor for breast cancer. Epidemiologic studies of radiation-exposed cohorts have been primarily descriptive; molecular events responsible for the development of radiation-associated breast cancer have not been elucidated. In this study, we used array comparative genomic hybridization (array-CGH) to characterize genome-wide copy number changes in breast tumors collected in the Childhood Cancer Survivor Study (CCSS). Array-CGH data were obtained from 32 cases who developed a second primary breast cancer following chest irradiation at early ages for the treatment of their first cancers, mostly Hodgkin lymphoma. The majority of these cases developed breast cancer before age 45 (91%, n = 29), had invasive ductal tumors (81%, n = 26), estrogen receptor (ER)-positive staining (68%, n = 19 out of 28), and high proliferation as indicated by high Ki-67 staining (77%, n = 17 out of 22). Genomic regions with low-copy number gains and losses and high-level amplifications were similar to what has been reported in sporadic breast tumors, however, the frequency of amplifications of the 17q12 region containing human epidermal growth factor receptor 2 (HER2) was much higher among CCSS cases (38%, n = 12). Our findings suggest that second primary breast cancers in CCSS were enriched for an "amplifier" genomic subgroup with highly proliferative breast tumors. Future investigation in a larger irradiated cohort will be needed to confirm our findings. PMID:25764003

  20. A novel high-throughput multi-parameter flow cytometry based method for monitoring and rapid characterization of microbiome dynamics in anaerobic systems.

    PubMed

    Dhoble, Abhishek S; Bekal, Sadia; Dolatowski, William; Yanz, Connor; Lambert, Kris N; Bhalerao, Kaustubh D

    2016-11-01

    A novel multidimensional flow cytometry based method has been demonstrated to monitor and rapidly characterize the dynamics of the complex anaerobic microbiome associated with perturbations in external environmental factors. While community fingerprinting provides an estimate of the meta genomic structure, flow cytometry provides a fingerprint of the community morphology including its autofluorescence spectrum in a high-throughput manner. Using anaerobic microbial consortia perturbed with the controlled addition of various carbon sources, it is possible to quantitatively discriminate between divergent microbiome analogous to community fingerprinting techniques using automated ribosomal intergenic spacer analysis (ARISA). The utility of flow cytometry based method has also been demonstrated in a fully functional industry scale anaerobic digester to distinguish between microbiome composition caused by varying hydraulic retention time (HRT). This approach exploits the rich multidimensional information from flow cytometry for rapid characterization of the dynamics of microbial communities. PMID:27614579

  1. Rapid and efficient clathrin-mediated endocytosis revealed in genome-edited mammalian cells.

    PubMed

    Doyon, Jeffrey B; Zeitler, Bryan; Cheng, Jackie; Cheng, Aaron T; Cherone, Jennifer M; Santiago, Yolanda; Lee, Andrew H; Vo, Thuy D; Doyon, Yannick; Miller, Jeffrey C; Paschon, David E; Zhang, Lei; Rebar, Edward J; Gregory, Philip D; Urnov, Fyodor D; Drubin, David G

    2011-03-01

    Clathrin-mediated endocytosis (CME) is the best-studied pathway by which cells selectively internalize molecules from the plasma membrane and surrounding environment. Previous live-cell imaging studies using ectopically overexpressed fluorescent fusions of endocytic proteins indicated that mammalian CME is a highly dynamic but inefficient and heterogeneous process. In contrast, studies of endocytosis in budding yeast using fluorescent protein fusions expressed at physiological levels from native genomic loci have revealed a process that is very regular and efficient. To analyse endocytic dynamics in mammalian cells in which endogenous protein stoichiometry is preserved, we targeted zinc finger nucleases (ZFNs) to the clathrin light chain A and dynamin-2 genomic loci and generated cell lines expressing fluorescent protein fusions from each locus. The genome-edited cells exhibited enhanced endocytic function, dynamics and efficiency when compared with previously studied cells, indicating that CME is highly sensitive to the levels of its protein components. Our study establishes that ZFN-mediated genome editing is a robust tool for expressing protein fusions at endogenous levels to faithfully report subcellular localization and dynamics.

  2. Rapid genome resequencing of an atoxigenic strain of Aspergillus carbonarius

    SciTech Connect

    Cabañes, F. Javier; Sanseverino, Walter; Castellá, Gemma; Bragulat, M. Rosa; Cigliano, Riccardo Aiese; Sánchez, Armand

    2015-03-13

    In microorganisms, Ion Torrent sequencing technology has been proved to be useful in whole-genome sequencing of bacterial genomes (5 Mbp). In our study, for the first time we used this technology to perform a resequencing approach in a whole fungal genome (36 Mbp), a non-ochratoxin A producing strain of Aspergillus carbonarius. Ochratoxin A (OTA) is a potent nephrotoxin which is found mainly in cereals and their products, but it also occurs in a variety of common foods and beverages. Due to the fact that this strain does not produce OTA, we focused some of the bioinformatics analyses in genes involved in OTA biosynthesis, using a reference genome of an OTA producing strain of the same species. This study revealed that in the atoxigenic strain there is a high accumulation of nonsense and missense mutations in several genes. Importantly, a two fold increase in gene mutation ratio was observed in PKS and NRPS encoding genes which are suggested to be involved in OTA biosynthesis.

  3. Rapid and Easy In Silico Serotyping of Escherichia coli Isolates by Use of Whole-Genome Sequencing Data

    PubMed Central

    Joensen, Katrine G.; Tetzschner, Anna M. M.; Iguchi, Atsushi; Aarestrup, Frank M.

    2015-01-01

    Accurate and rapid typing of pathogens is essential for effective surveillance and outbreak detection. Conventional serotyping of Escherichia coli is a delicate, laborious, time-consuming, and expensive procedure. With whole-genome sequencing (WGS) becoming cheaper, it has vast potential in routine typing and surveillance. The aim of this study was to establish a valid and publicly available tool for WGS-based in silico serotyping of E. coli applicable for routine typing and surveillance. A FASTA database of specific O-antigen processing system genes for O typing and flagellin genes for H typing was created as a component of the publicly available Web tools hosted by the Center for Genomic Epidemiology (CGE) (www.genomicepidemiology.org). All E. coli isolates available with WGS data and conventional serotype information were subjected to WGS-based serotyping employing this specific SerotypeFinder CGE tool. SerotypeFinder was evaluated on 682 E. coli genomes, 108 of which were sequenced for this study, where both the whole genome and the serotype were available. In total, 601 and 509 isolates were included for O and H typing, respectively. The O-antigen genes wzx, wzy, wzm, and wzt and the flagellin genes fliC, flkA, fllA, flmA, and flnA were detected in 569 and 508 genome sequences, respectively. SerotypeFinder for WGS-based O and H typing predicted 560 of 569 O types and 504 of 508 H types, consistent with conventional serotyping. In combination with other available WGS typing tools, E. coli serotyping can be performed solely from WGS data, providing faster and cheaper typing than current routine procedures and making WGS typing a superior alternative to conventional typing strategies. PMID:25972421

  4. Fast-scan Cyclic Voltammetry for the Characterization of Rapid Adenosine Release

    PubMed Central

    Nguyen, Michael D.; Venton, B. Jill

    2014-01-01

    Adenosine is a signaling molecule and downstream product of ATP that acts as a neuromodulator. Adenosine regulates physiological processes, such as neurotransmission and blood flow, on a time scale of minutes to hours. Recent developments in electrochemical techniques, including fast-scan cyclic voltammetry (FSCV), have allowed direct detection of adenosine with sub-second temporal resolution. FSCV studies have revealed a novel mode of rapid signaling that lasts only a few seconds. This rapid release of adenosine can be evoked by electrical or mechanical stimulations or it can be observed spontaneously without stimulation. Adenosine signaling on this time scale is activity dependent; however, the mode of release is not fully understood. Rapid adenosine release modulates oxygen levels and evoked dopamine release, indicating that adenosine may have a rapid modulatory role. In this review, we outline how FSCV can be used to detect adenosine release, compare FSCV with other techniques used to measure adenosine, and present an overview of adenosine signaling that has been characterized using FSCV. These studies point to a rapid mode of adenosine modulation, whose mechanism and function will continue to be characterized in the future. PMID:26900429

  5. Genome Characterization of the Oleaginous Fungus Mortierella alpina

    PubMed Central

    Feng, Yun; Ren, Yan; Gu, Zhennan; Chen, Haiqin; Wang, Hongchao; Thomas, Michael J.; Zhang, Baixi; Berquin, Isabelle M.; Li, Yang; Wu, Jiansheng; Zhang, Huanxin; Song, Yuanda; Liu, Xiang; Norris, James S.; Wang, Suriguga; Du, Peng; Shen, Junguo; Wang, Na; Yang, Yanlin; Wang, Wei; Feng, Lu; Ratledge, Colin; Zhang, Hao; Chen, Yong Q.

    2011-01-01

    Mortierella alpina is an oleaginous fungus which can produce lipids accounting for up to 50% of its dry weight in the form of triacylglycerols. It is used commercially for the production of arachidonic acid. Using a combination of high throughput sequencing and lipid profiling, we have assembled the M. alpina genome, mapped its lipogenesis pathway and determined its major lipid species. The 38.38 Mb M. alpina genome shows a high degree of gene duplications. Approximately 50% of its 12,796 gene models, and 60% of genes in the predicted lipogenesis pathway, belong to multigene families. Notably, M. alpina has 18 lipase genes, of which 11 contain the class 2 lipase domain and may share a similar function. M. alpina's fatty acid synthase is a single polypeptide containing all of the catalytic domains required for fatty acid synthesis from acetyl-CoA and malonyl-CoA, whereas in many fungi this enzyme is comprised of two polypeptides. Major lipids were profiled to confirm the products predicted in the lipogenesis pathway. M. alpina produces a complex mixture of glycerolipids, glycerophospholipids and sphingolipids. In contrast, only two major sterol lipids, desmosterol and 24(28)-methylene-cholesterol, were detected. Phylogenetic analysis based on genes involved in lipid metabolism suggests that oleaginous fungi may have acquired their lipogenic capacity during evolution after the divergence of Ascomycota, Basidiomycota, Chytridiomycota and Mucoromycota. Our study provides the first draft genome and comprehensive lipid profile for M. alpina, and lays the foundation for possible genetic engineering of M. alpina to produce higher levels and diverse contents of dietary lipids. PMID:22174787

  6. Complete genome assemblies and methylome characterization in infectious diseases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Understanding the genetic basis of infectious diseases is a critical component to effective treatments. Because of the rapid evolution of bacterial strains and frequent horizontal transfer of DNA between them, resequencing of new isolates against known reference strains often provides an incomplete ...

  7. Mitochondrial genome of the Torpedo scad Megalaspis cordyla (Perciformes: Carangidae): genome characterization and phylogenetic consideration.

    PubMed

    Li, Min; Li, Yufang; Chen, Zuozhi

    2016-05-01

    This study presented the complete mitochondrial genome of the Torpedo scad Megalaspis cordyla, the only member of its genus, as well as its phylogenetic position in Carangidae. The genome is 16,566 bp containing the usual 2 rRNA genes, 13 protein-coding genes, 22 tRNA genes, and 1 control region. Gene organization is similar to that observed in most other vertebrates. Gene overlapping and separating were also observed in M. cordyla mitogenome. The overall base compositions of mitogenome was 28.83% A, 25.81% T, 15.93% G, and 29.43% C. Phylogenetic analyses using the concatenated sequence of the protein-coding genes of the reported Carangidae mitogenome showed similar results in the neighbour-joining and Bayesian inference trees. Three clades were formed as Subfamilies Caranginae, Seriolinae and Trachinotinae in Carangidae. M. cordyla was most closely related to the species in genus Caranx. PMID:25319290

  8. Complete mitochondrial genome of Anodontostoma chacunda (Clupeiformes: Clupeidae): genome characterization and phylogenetic consideration.

    PubMed

    Li, Min; Zou, Keshu

    2013-10-01

    The complete mitochondrial genome sequence of Anodontostoma chacunda was determined in this paper. The genome is 16,772 bp in length and contains 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and 2 noncoding regions. With the exception of ND6 and eight tRNA genes, all other genes are encoded on the heavy strand. Gene organization is similar to that observed in most other vertebrates. Overall base compositions of mitogenome are 27.4% of A, 28.7% of C, 25.1% of T, and 18.8% of G. Phylogenetic analyses using the concatenated nucleotide sequence of 12 protein-coding genes on the heavy strand reveal that genus Anodontostoma is the sister group to the assemblage (Nematalosa, (Clupanodon, Konosirus)) within subfamily Dorosomatinae.

  9. Mitochondrial genome of the Torpedo scad Megalaspis cordyla (Perciformes: Carangidae): genome characterization and phylogenetic consideration.

    PubMed

    Li, Min; Li, Yufang; Chen, Zuozhi

    2016-05-01

    This study presented the complete mitochondrial genome of the Torpedo scad Megalaspis cordyla, the only member of its genus, as well as its phylogenetic position in Carangidae. The genome is 16,566 bp containing the usual 2 rRNA genes, 13 protein-coding genes, 22 tRNA genes, and 1 control region. Gene organization is similar to that observed in most other vertebrates. Gene overlapping and separating were also observed in M. cordyla mitogenome. The overall base compositions of mitogenome was 28.83% A, 25.81% T, 15.93% G, and 29.43% C. Phylogenetic analyses using the concatenated sequence of the protein-coding genes of the reported Carangidae mitogenome showed similar results in the neighbour-joining and Bayesian inference trees. Three clades were formed as Subfamilies Caranginae, Seriolinae and Trachinotinae in Carangidae. M. cordyla was most closely related to the species in genus Caranx.

  10. Development of a set of public SSR markers derived from genomic sequence of a rapid cycling Brassica oleracea L. genotype.

    PubMed

    Iniguez-Luy, Federico L; Voort, Amy V; Osborn, Thomas C

    2008-10-01

    The traditional development of simple sequence repeat (SSR) or microsatellite markers by probe hybridization can be time-consuming and requires the use of specialized laboratory equipment. In this study, probe hybridization was circumvented by using sequence information on 3,500 genomic clones mainly from Brassica oleracea to identify di, tri, tetra and penta-nucleotide repeats. A total of 587 primer pairs flanking SSR were developed using this approach. From these, 420 SSR markers amplified DNA in two parental lines of B. rapa (26% were polymorphic) and 523 in two parental lines of B. oleracea (32% were polymorphic). A diverse array of motif types was identified, characterized and compared with traditional SSR detection methods. The most abundant motifs found were di- (38%) and trinucleotides (33%) followed by penta- (16%) and tetranucleotide (13%) motifs. The type of motif class, motif length and repeat were not indicative of polymorphisms. The frequency of B. oleracea SSRs in genomic shotgun sequence was estimated to be 1 every 4 Kb. In general, the average motif length and repeat numbers were shorter than those obtained previously by probe hybridization, and they contained a more balanced representation of SSR motif types in the genome by identifying those that do not hybridize well to DNA probes. Brassica genomic DNA sequence information is a promising resource for developing a large number of SSR molecular markers in Brassica species.

  11. Characterization of Three Mycobacterium spp. with Potential Use in Bioremediation by Genome Sequencing and Comparative Genomics

    PubMed Central

    Das, Sarbashis; Pettersson, B.M. Fredrik; Behra, Phani Rama Krishna; Ramesh, Malavika; Dasgupta, Santanu; Bhattacharya, Alok; Kirsebom, Leif A.

    2015-01-01

    We provide the genome sequences of the type strains of the polychlorophenol-degrading Mycobacterium chlorophenolicum (DSM43826), the degrader of chlorinated aliphatics Mycobacterium chubuense (DSM44219) and Mycobacterium obuense (DSM44075) that has been tested for use in cancer immunotherapy. The genome sizes of M. chlorophenolicum, M. chubuense, and M. obuense are 6.93, 5.95, and 5.58 Mb with GC-contents of 68.4%, 69.2%, and 67.9%, respectively. Comparative genomic analysis revealed that 3,254 genes are common and we predicted approximately 250 genes acquired through horizontal gene transfer from different sources including proteobacteria. The data also showed that the biodegrading Mycobacterium spp. NBB4, also referred to as M. chubuense NBB4, is distantly related to the M. chubuense type strain and should be considered as a separate species, we suggest it to be named Mycobacterium ethylenense NBB4. Among different categories we identified genes with potential roles in: biodegradation of aromatic compounds and copper homeostasis. These are the first nonpathogenic Mycobacterium spp. found harboring genes involved in copper homeostasis. These findings would therefore provide insight into the role of this group of Mycobacterium spp. in bioremediation as well as the evolution of copper homeostasis within the Mycobacterium genus. PMID:26079817

  12. Characterization of Three Mycobacterium spp. with Potential Use in Bioremediation by Genome Sequencing and Comparative Genomics.

    PubMed

    Das, Sarbashis; Pettersson, B M Fredrik; Behra, Phani Rama Krishna; Ramesh, Malavika; Dasgupta, Santanu; Bhattacharya, Alok; Kirsebom, Leif A

    2015-07-01

    We provide the genome sequences of the type strains of the polychlorophenol-degrading Mycobacterium chlorophenolicum (DSM43826), the degrader of chlorinated aliphatics Mycobacterium chubuense (DSM44219) and Mycobacterium obuense (DSM44075) that has been tested for use in cancer immunotherapy. The genome sizes of M. chlorophenolicum, M. chubuense, and M. obuense are 6.93, 5.95, and 5.58 Mb with GC-contents of 68.4%, 69.2%, and 67.9%, respectively. Comparative genomic analysis revealed that 3,254 genes are common and we predicted approximately 250 genes acquired through horizontal gene transfer from different sources including proteobacteria. The data also showed that the biodegrading Mycobacterium spp. NBB4, also referred to as M. chubuense NBB4, is distantly related to the M. chubuense type strain and should be considered as a separate species, we suggest it to be named Mycobacterium ethylenense NBB4. Among different categories we identified genes with potential roles in: biodegradation of aromatic compounds and copper homeostasis. These are the first nonpathogenic Mycobacterium spp. found harboring genes involved in copper homeostasis. These findings would therefore provide insight into the role of this group of Mycobacterium spp. in bioremediation as well as the evolution of copper homeostasis within the Mycobacterium genus.

  13. Integrative Genomic Characterization and a Genomic Staging System for Gastrointestinal Stromal Tumors

    PubMed Central

    Ylipää, Antti; Hunt, Kelly K.; Yang, Jilong; Lazar, Alexander J. F.; Torres, Keila E.; Lev, Dina Chelouche; Nykter, Matti; Pollock, Raphael E.; Trent, Jonathan; Zhang, Wei

    2010-01-01

    Gastrointestinal stromal tumors (GISTs) were historically grouped with leiomyosarcomas (LMSs) based on their morphological similarities, but recently they have been unequivocally established as a distinct type of sarcoma based on the molecular features and response to imatinib treatment. To gain further insight into the genomic differences between GISTs and LMSs, we mapped gene copy number aberrations (CNAs) in 42 GISTs and 30 LMSs and integrated them with gene expression profiles. Our studies revealed distinct patterns of CNAs between GISTs and LMSs. Losses in chromosomes 1p, 14q, 15q, and 22q were significantly more frequent in GISTs than in LMSs (P < 0.001), whereas losses in chromosomes 10 and 16 as well as gains in 1q, 14q, and 15q (P < 0.001) were more common in LMSs. By integrating CNAs with gene expression data and clinical information, we found several clinically relevant CNAs that were prognostic of survival in patients with GIST. Furthermore, GISTs were categorized into four groups according to an accumulating pattern of genetic alterations. Many key cellular pathways were differently expressed in the four groups and the patients had increasingly worse prognosis as the extent of genomic alterations increased. These findings lead us to propose a new tumor-progression genetic staging system termed Genomic Instability Stage (GIS) to complement the current prognostic predictive system based on tumor size, mitotic index (MI), and KIT mutation. PMID:20818650

  14. Rapid Characterization of Microorganisms by Mass Spectrometry—What Can Be Learned and How?

    NASA Astrophysics Data System (ADS)

    Fenselau, Catherine C.

    2013-08-01

    Strategies for the rapid and reliable analysis of microorganisms have been sought to meet national needs in defense, homeland security, space exploration, food and water safety, and clinical diagnosis. Mass spectrometry has long been a candidate technique because it is extremely rapid and can provide highly specific information. It has excellent sensitivity. Molecular and fragment ion masses provide detailed fingerprints, which can also be interpreted. Mass spectrometry is also a broad band method—everything has a mass—and it is automatable. Mass spectrometry is a physiochemical method that is orthogonal and complementary to biochemical and morphological methods used to characterize microorganisms.

  15. Comprehensive genomic characterization of head and neck squamous cell carcinomas.

    PubMed

    2015-01-29

    The Cancer Genome Atlas profiled 279 head and neck squamous cell carcinomas (HNSCCs) to provide a comprehensive landscape of somatic genomic alterations. Here we show that human-papillomavirus-associated tumours are dominated by helical domain mutations of the oncogene PIK3CA, novel alterations involving loss of TRAF3, and amplification of the cell cycle gene E2F1. Smoking-related HNSCCs demonstrate near universal loss-of-function TP53 mutations and CDKN2A inactivation with frequent copy number alterations including amplification of 3q26/28 and 11q13/22. A subgroup of oral cavity tumours with favourable clinical outcomes displayed infrequent copy number alterations in conjunction with activating mutations of HRAS or PIK3CA, coupled with inactivating mutations of CASP8, NOTCH1 and TP53. Other distinct subgroups contained loss-of-function alterations of the chromatin modifier NSD1, WNT pathway genes AJUBA and FAT1, and activation of oxidative stress factor NFE2L2, mainly in laryngeal tumours. Therapeutic candidate alterations were identified in most HNSCCs. PMID:25631445

  16. Genomic Characterization of Methanomicrobiales Reveals Three Classes of Methanogens

    SciTech Connect

    Anderson, Iain; Ulrich, Luke; Lupa, Boguslaw; Susanti, Dwi; Porat, I.; Hooper, Sean; Lykidis, A; Sieprawska-Lupa, Magdalena; Dharmarajan, Lakshmi; Goltsman, Eugene; Lapidus, Alla L.; Saunders, Elizabeth H; Han, Cliff; Land, Miriam L; Lucas, Susan; Mukhopadhyay, Biswarup; Whitman, William; Woese, Carl; Bristow, James; Kyrpides, Nikos C

    2009-01-01

    Background Methanomicrobiales is the least studied order of methanogens. While these organisms appear to be more closely related to the Methanosarcinales in ribosomal-based phylogenetic analyses, they are metabolically more similar to Class I methanogens. Methodology/Principal Findings In order to improve our understanding of this lineage, we have completely sequenced the genomes of two members of this order, Methanocorpusculum labreanum Z and Methanoculleus marisnigri JR1, and compared them with the genome of a third, Methanospirillum hungatei JF-1. Similar to Class I methanogens, Methanomicrobiales use a partial reductive citric acid cycle for 2-oxoglutarate biosynthesis, and they have the Eha energy-converting hydrogenase. In common with Methanosarcinales, Methanomicrobiales possess the Ech hydrogenase and at least some of them may couple formylmethanofuran formation and heterodisulfide reduction to transmembrane ion gradients. Uniquely, M. labreanum and M. hungatei contain hydrogenases similar to the Pyrococcus furiosus Mbh hydrogenase, and all three Methanomicrobiales have anti-sigma factor and anti-anti-sigma factor regulatory proteins not found in other methanogens. Phylogenetic analysis based on seven core proteins of methanogenesis and cofactor biosynthesis places the Methanomicrobiales equidistant from Class I methanogens and Methanosarcinales. Conclusions/Significance Our results indicate that Methanomicrobiales, rather than being similar to Class I methanogens or Methanomicrobiales, share some features of both and have some unique properties. We find that there are three distinct classes of methanogens: the Class I methanogens, the Methanomicrobiales (Class II), and the Methanosarcinales (Class III).

  17. Comprehensive genomic characterization of head and neck squamous cell carcinomas

    PubMed Central

    2014-01-01

    The Cancer Genome Atlas profiled 279 head and neck squamous cell carcinomas (HNSCCs) to provide a comprehensive landscape of somatic genomic alterations. We find that human papillomavirus-associated (HPV) tumors are dominated by helicase domain mutations of the oncogene PIK3CA, novel alterations involving loss of TRAF3, and amplification of the cell cycle gene E2F1. Smoking-related HNSCCs demonstrate near universal loss of TP53 mutations and CDKN2A with frequent copy number alterations including a novel amplification of 11q22. A subgroup of oral cavity tumors with favorable clinical outcomes displayed infrequent CNAs in conjunction with activating mutations of HRAS or PIK3CA, coupled with inactivating mutations of CASP8, NOTCH1 and wild-type TP53. Other distinct subgroups harbored novel loss of function alterations of the chromatin modifier NSD1, Wnt pathway genes AJUBA and FAT1, and activation of oxidative stress factor NFE2L2, mainly in laryngeal tumors. Therapeutic candidate alterations were identified in the majority of HNSCC's. PMID:25631445

  18. Comprehensive genomic characterization of head and neck squamous cell carcinomas.

    PubMed

    2015-01-29

    The Cancer Genome Atlas profiled 279 head and neck squamous cell carcinomas (HNSCCs) to provide a comprehensive landscape of somatic genomic alterations. Here we show that human-papillomavirus-associated tumours are dominated by helical domain mutations of the oncogene PIK3CA, novel alterations involving loss of TRAF3, and amplification of the cell cycle gene E2F1. Smoking-related HNSCCs demonstrate near universal loss-of-function TP53 mutations and CDKN2A inactivation with frequent copy number alterations including amplification of 3q26/28 and 11q13/22. A subgroup of oral cavity tumours with favourable clinical outcomes displayed infrequent copy number alterations in conjunction with activating mutations of HRAS or PIK3CA, coupled with inactivating mutations of CASP8, NOTCH1 and TP53. Other distinct subgroups contained loss-of-function alterations of the chromatin modifier NSD1, WNT pathway genes AJUBA and FAT1, and activation of oxidative stress factor NFE2L2, mainly in laryngeal tumours. Therapeutic candidate alterations were identified in most HNSCCs.

  19. Genomic Characterization of Methanomicrobiales Reveals Three Classes of Methanogens

    SciTech Connect

    Anderson, Iain; Ulrich, Luke E.; Lupa, Boguslaw; Susanti, Dwi; Porat, Iris; Hooper, Sean D.; Lykidis, Athanasios; Sieprawska-Lupa, Magdalena; Dharmarajan, Lakshmi; Goltsman, Eugene; Lapidus, Alla; Saunders, Elizabeth; Han, Cliff; Land, Miriam; Lucas, Susan; Mukhopadhyay, Biswarup; Whitman, William B.; Woese, Carl; Bristow, James; Kyrpides, Nikos

    2009-05-01

    Methanomicrobiales is the least studied order of methanogens. While these organisms appear to be more closely related to the Methanosarcinales in ribosomal-based phylogenetic analyses, they are metabolically more similar to Class I methanogens. In order to improve our understanding of this lineage, we have completely sequenced the genomes of two members of this order, Methanocorpusculum labreanum Z and Methanoculleus marisnigri JR1, and compared them with the genome of a third, Methanospirillum hungatei JF-1. Similar to Class I methanogens, Methanomicrobiales use a partial reductive citric acid cycle for 2-oxoglutarate biosynthesis, and they have the Eha energy-converting hydrogenase. In common with Methanosarcinales, Methanomicrobiales possess the Ech hydrogenase and at least some of them may couple formylmethanofuran formation and heterodisulfide reduction to transmembrane ion gradients. Uniquely, M. labreanum and M. hungatei contain hydrogenases similar to the Pyrococcus furiosus Mbh hydrogenase, and all three Methanomicrobiales have anti-sigma factor and anti-anti-sigma factor regulatory proteins not found in other methanogens. Phylogenetic analysis based on seven core proteins of methanogenesis and cofactor biosynthesis places the Methanomicrobiales equidistant from Class I methanogens and Methanosarcinales. Our results indicate that Methanomicrobiales, rather than being similar to Class I methanogens or Methanomicrobiales, share some features of both and have some unique properties. We find that there are three distinct classes of methanogens: the Class I methanogens, the Methanomicrobiales (Class II), and the Methanosarcinales (Class III).

  20. Expedited Site Characterization: A rapid, cost-effective process for preremedial site characterization

    SciTech Connect

    Burton, J.C.; Walker, J.L.; Jennings, T.V.; Aggarwal, P.K.; Hastings, B.; Meyer, W.T.; Rose, C.M.; Rosignolo, C.L.

    1993-11-01

    Argonne National Laboratory has developed a unique, cost- and time-effective, technically innovative process for preremedial site characterization, referred to as Expedited Site Characterization (ESC). The cost of the ESC field sampling process ranges from 1/10 to 1/5 of the cost of traditional site characterization. The time required for this ESC field activity is approximately 1/30 of that for current methods. Argonne`s preremedial site investigations based on this approach have been accepted by the appropriate regulatory agencies. The ESC process is flexible and neither site nor contaminant dependent. The process has been successfully tested and applied in site investigations of multiple contaminated landfills in New Mexico (for the US Department of the Interior`s Bureau of Land Management [BLM]) and at former grain storage facilities in Nebraska and Kansas, contaminated with carbon tetrachloride (for the Department of Agriculture`s Commodity Credit Corporation [CCC/USDA]). A working demonstration of this process was sponsored by the US Department of Energy (DOE) Office of Technology Development as a model of the methodology needed to accelerate site characterizations at DOE facilities. This report describes the application of the process in New Mexico, Nebraska and Kansas.

  1. Two Rapidly Growing Mycobacterial Species Isolated from a Brain Abscess: First Whole-Genome Sequences of Mycobacterium immunogenum and Mycobacterium llatzerense

    PubMed Central

    Greninger, Alexander L.; Langelier, Charles; Cunningham, Gail; Keh, Chris; Melgar, Michael; Chiu, Charles Y.

    2015-01-01

    Rapidly growing mycobacteria are rarely found in central nervous system infections. We describe a case of polymicrobial infection in a brain abscess including two rapidly growing Mycobacterium species, M. immunogenum and M. llatzerense. The Mycobacterium isolates were distinguishable by molecular methods, and whole-genome sequencing showed <60% pairwise nucleotide identity. PMID:25926490

  2. Enabling functional genomics with genome engineering.

    PubMed

    Hilton, Isaac B; Gersbach, Charles A

    2015-10-01

    Advances in genome engineering technologies have made the precise control over genome sequence and regulation possible across a variety of disciplines. These tools can expand our understanding of fundamental biological processes and create new opportunities for therapeutic designs. The rapid evolution of these methods has also catalyzed a new era of genomics that includes multiple approaches to functionally characterize and manipulate the regulation of genomic information. Here, we review the recent advances of the most widely adopted genome engineering platforms and their application to functional genomics. This includes engineered zinc finger proteins, TALEs/TALENs, and the CRISPR/Cas9 system as nucleases for genome editing, transcription factors for epigenome editing, and other emerging applications. We also present current and potential future applications of these tools, as well as their current limitations and areas for future advances.

  3. Enabling functional genomics with genome engineering.

    PubMed

    Hilton, Isaac B; Gersbach, Charles A

    2015-10-01

    Advances in genome engineering technologies have made the precise control over genome sequence and regulation possible across a variety of disciplines. These tools can expand our understanding of fundamental biological processes and create new opportunities for therapeutic designs. The rapid evolution of these methods has also catalyzed a new era of genomics that includes multiple approaches to functionally characterize and manipulate the regulation of genomic information. Here, we review the recent advances of the most widely adopted genome engineering platforms and their application to functional genomics. This includes engineered zinc finger proteins, TALEs/TALENs, and the CRISPR/Cas9 system as nucleases for genome editing, transcription factors for epigenome editing, and other emerging applications. We also present current and potential future applications of these tools, as well as their current limitations and areas for future advances. PMID:26430154

  4. Efficient CRISPR/Cas9 plasmids for rapid and versatile genome editing in Drosophila.

    PubMed

    Gokcezade, Joseph; Sienski, Grzegorz; Duchek, Peter

    2014-11-01

    The CRISPR-associated RNA-guided nuclease Cas9 has emerged as a powerful tool for genome engineering in a variety of organisms. To achieve efficient gene targeting rates in Drosophila, current approaches require either injection of in vitro transcribed RNAs or injection into transgenic Cas9-expressing embryos. We report a simple and versatile alternative method for CRISPR-mediated genome editing in Drosophila using bicistronic Cas9/sgRNA expression vectors. Gene targeting with this single-plasmid injection approach is as efficient as in transgenic nanos-Cas9 embryos and allows the isolation of targeted knock-out and knock-in alleles by molecular screening within 2 months. Our strategy is independent of genetic background and does not require prior establishment of transgenic flies. PMID:25236734

  5. Population genomics reveal recent speciation and rapid evolutionary adaptation in polar bears.

    PubMed

    Liu, Shiping; Lorenzen, Eline D; Fumagalli, Matteo; Li, Bo; Harris, Kelley; Xiong, Zijun; Zhou, Long; Korneliussen, Thorfinn Sand; Somel, Mehmet; Babbitt, Courtney; Wray, Greg; Li, Jianwen; He, Weiming; Wang, Zhuo; Fu, Wenjing; Xiang, Xueyan; Morgan, Claire C; Doherty, Aoife; O'Connell, Mary J; McInerney, James O; Born, Erik W; Dalén, Love; Dietz, Rune; Orlando, Ludovic; Sonne, Christian; Zhang, Guojie; Nielsen, Rasmus; Willerslev, Eske; Wang, Jun

    2014-05-01

    Polar bears are uniquely adapted to life in the High Arctic and have undergone drastic physiological changes in response to Arctic climates and a hyper-lipid diet of primarily marine mammal prey. We analyzed 89 complete genomes of polar bear and brown bear using population genomic modeling and show that the species diverged only 479-343 thousand years BP. We find that genes on the polar bear lineage have been under stronger positive selection than in brown bears; nine of the top 16 genes under strong positive selection are associated with cardiomyopathy and vascular disease, implying important reorganization of the cardiovascular system. One of the genes showing the strongest evidence of selection, APOB, encodes the primary lipoprotein component of low-density lipoprotein (LDL); functional mutations in APOB may explain how polar bears are able to cope with life-long elevated LDL levels that are associated with high risk of heart disease in humans. PMID:24813606

  6. POPULATION GENOMICS REVEAL RECENT SPECIATION AND RAPID EVOLUTIONARY ADAPTATION IN POLAR BEARS

    PubMed Central

    Liu, Shiping; Lorenzen, Eline D.; Fumagalli, Matteo; Li, Bo; Harris, Kelley; Xiong, Zijun; Zhou, Long; Korneliussen, Thorfinn Sand; Somel, Mehmet; Babbitt, Courtney; Wray, Greg; Li, Jianwen; He, Weiming; Wang, Zhuo; Fu, Wenjing; Xiang, Xueyan; Morgan, Claire C.; Doherty, Aoife; O’Connell, Mary J.; McInerney, James O.; Born, Erik W.; Dalén, Love; Dietz, Rune; Orlando, Ludovic; Sonne, Christian; Zhang, Guojie; Nielsen, Rasmus; Willerslev, Eske; Wang, Jun

    2014-01-01

    SUMMARY Polar bears are uniquely adapted to life in the High Arctic and have undergone drastic physiological changes in response to Arctic climates and a hyperlipid diet of primarily marine mammal prey. We analyzed 89 complete genomes of polar bear and brown bear using population genomic modeling and show that the species diverged only 479–343 thousand years BP. We find that genes on the polar bear lineage have been under stronger positive selection than in brown bears; nine of the top 16 genes under strong positive selection are associated with cardiomyopathy and vascular disease, implying important reorganization of the cardio-vascular system. One of the genes showing the strongest evidence of selection, APOB, encodes the primary lipoprotein component of low-density lipoprotein (LDL); functional mutations in APOB may explain how polar bears are able to cope with life-long elevated LDL levels that are associated with high risk of heart disease in humans. PMID:24813606

  7. Universal and rapid salt-extraction of high quality genomic DNA for PCR-based techniques.

    PubMed

    Aljanabi, S M; Martinez, I

    1997-11-15

    A very simple, fast, universally applicable and reproducible method to extract high quality megabase genomic DNA from different organisms is described. We applied the same method to extract high quality complex genomic DNA from different tissues (wheat, barley, potato, beans, pear and almond leaves as well as fungi, insects and shrimps' fresh tissue) without any modification. The method does not require expensive and environmentally hazardous reagents and equipment. It can be performed even in low technology laboratories. The amount of tissue required by this method is approximately 50-100 mg. The quantity and the quality of the DNA extracted by this method is high enough to perform hundreds of PCR-based reactions and also to be used in other DNA manipulation techniques such as restriction digestion, Southern blot and cloning.

  8. Population genomics reveal recent speciation and rapid evolutionary adaptation in polar bears.

    PubMed

    Liu, Shiping; Lorenzen, Eline D; Fumagalli, Matteo; Li, Bo; Harris, Kelley; Xiong, Zijun; Zhou, Long; Korneliussen, Thorfinn Sand; Somel, Mehmet; Babbitt, Courtney; Wray, Greg; Li, Jianwen; He, Weiming; Wang, Zhuo; Fu, Wenjing; Xiang, Xueyan; Morgan, Claire C; Doherty, Aoife; O'Connell, Mary J; McInerney, James O; Born, Erik W; Dalén, Love; Dietz, Rune; Orlando, Ludovic; Sonne, Christian; Zhang, Guojie; Nielsen, Rasmus; Willerslev, Eske; Wang, Jun

    2014-05-01

    Polar bears are uniquely adapted to life in the High Arctic and have undergone drastic physiological changes in response to Arctic climates and a hyper-lipid diet of primarily marine mammal prey. We analyzed 89 complete genomes of polar bear and brown bear using population genomic modeling and show that the species diverged only 479-343 thousand years BP. We find that genes on the polar bear lineage have been under stronger positive selection than in brown bears; nine of the top 16 genes under strong positive selection are associated with cardiomyopathy and vascular disease, implying important reorganization of the cardiovascular system. One of the genes showing the strongest evidence of selection, APOB, encodes the primary lipoprotein component of low-density lipoprotein (LDL); functional mutations in APOB may explain how polar bears are able to cope with life-long elevated LDL levels that are associated with high risk of heart disease in humans.

  9. Rapid Genome Assembly and Comparison Decode Intrastrain Variation in Human Alphaherpesviruses

    PubMed Central

    Tafuri, Yolanda R.; Shreve, Jacob T.; Bowen, Christopher D.; Shipley, Mackenzie M.; Enquist, L. W.

    2015-01-01

    ABSTRACT Herpes simplex virus (HSV) is a widespread pathogen that causes epithelial lesions with recurrent disease that manifests over a lifetime. The lifelong aspect of infection results from latent viral infection of neurons, a reservoir from which the virus reactivates periodically. Recent work has demonstrated the breadth of genetic variation in globally distributed HSV strains. However, the amount of variation or capacity for mutation within one strain has not been well studied. Here we developed and applied a streamlined new approach for assembly and comparison of large DNA viral genomes such as HSV-1. This viral genome assembly (VirGA) workflow incorporates a combination of de novo assembly, alignment, and annotation strategies to automate the generation of draft genomes for large viruses. We applied this approach to quantify the amount of variation between clonal derivatives of a common parental virus stock. In addition, we examined the genetic basis for syncytial plaque phenotypes displayed by a subset of these strains. In each of the syncytial strains, we found an identical DNA change, affecting one residue in the gB (UL27) fusion protein. Since these identical mutations could have appeared after extensive in vitro passaging, we applied the VirGA sequencing and comparison approach to two clinical HSV-1 strains isolated from the same patient. One of these strains was syncytial upon first culturing; its sequence revealed the same gB mutation. These data provide insight into the extent and origin of genome-wide intrastrain HSV-1 variation and present useful methods for expansion to in vivo patient infection studies. PMID:25827418

  10. A New Method for Rapid Screening of End-Point PCR Products: Application to Single Genome Amplified HIV and SIV Envelope Amplicons

    PubMed Central

    Houzet, Laurent; Deleage, Claire; Satie, Anne-Pascale; Merlande, Laetitia; Mahe, Dominique; Dejucq-Rainsford, Nathalie

    2015-01-01

    PCR is the most widely applied technique for large scale screening of bacterial clones, mouse genotypes, virus genomes etc. A drawback of large PCR screening is that amplicon analysis is usually performed using gel electrophoresis, a step that is very labor intensive, tedious and chemical waste generating. Single genome amplification (SGA) is used to characterize the diversity and evolutionary dynamics of virus populations within infected hosts. SGA is based on the isolation of single template molecule using limiting dilution followed by nested PCR amplification and requires the analysis of hundreds of reactions per sample, making large scale SGA studies very challenging. Here we present a novel approach entitled Long Amplicon Melt Profiling (LAMP) based on the analysis of the melting profile of the PCR reactions using SYBR Green and/or EvaGreen fluorescent dyes. The LAMP method represents an attractive alternative to gel electrophoresis and enables the quick discrimination of positive reactions. We validate LAMP for SIV and HIV env-SGA, in 96- and 384-well plate formats. Because the melt profiling allows the screening of several thousands of PCR reactions in a cost-effective, rapid and robust way, we believe it will greatly facilitate any large scale PCR screening. PMID:26053379

  11. Molecular Characterization of a Beak and Feather Disease Virus Genome from a Purple Crowned Lorikeet (Glossopsitta porphyrocephala)

    PubMed Central

    Subir, Sarker; Fearnside, Kathleen; Forwood, Jade K.; Ghorashi, Seyed A.; Raidal, Shane R.

    2016-01-01

    The complete genome sequence of beak and feather disease virus (BFDV) from a purple crowned lorikeet (Glossopsitta porphyrocephala) was characterized. The genome consists of 2,010 nucleotides and encodes replicase-associated protein and capsid protein. This is the first evidence of BFDV infectivity and complete genome sequence for this novel host. PMID:27795264

  12. Analysis of complete mitochondrial genome sequences increases phylogenetic resolution of bears (Ursidae), a mammalian family that experienced rapid speciation

    PubMed Central

    Yu, Li; Li, Yi-Wei; Ryder, Oliver A; Zhang, Ya-Ping

    2007-01-01

    Background Despite the small number of ursid species, bear phylogeny has long been a focus of study due to their conservation value, as all bear genera have been classified as endangered at either the species or subspecies level. The Ursidae family represents a typical example of rapid evolutionary radiation. Previous analyses with a single mitochondrial (mt) gene or a small number of mt genes either provide weak support or a large unresolved polytomy for ursids. We revisit the contentious relationships within Ursidae by analyzing complete mt genome sequences and evaluating the performance of both entire mt genomes and constituent mtDNA genes in recovering a phylogeny of extremely recent speciation events. Results This mitochondrial genome-based phylogeny provides strong evidence that the spectacled bear diverged first, while within the genus Ursus, the sloth bear is the sister taxon of all the other five ursines. The latter group is divided into the brown bear/polar bear and the two black bears/sun bear assemblages. These findings resolve the previous conflicts between trees using partial mt genes. The ability of different categories of mt protein coding genes to recover the correct phylogeny is concordant with previous analyses for taxa with deep divergence times. This study provides a robust Ursidae phylogenetic framework for future validation by additional independent evidence, and also has significant implications for assisting in the resolution of other similarly difficult phylogenetic investigations. Conclusion Identification of base composition bias and utilization of the combined data of whole mitochondrial genome sequences has allowed recovery of a strongly supported phylogeny that is upheld when using multiple alternative outgroups for the Ursidae, a mammalian family that underwent a rapid radiation since the mid- to late Pliocene. It remains to be seen if the reliability of mt genome analysis will hold up in studies of other difficult phylogenetic

  13. A Novel Method of Characterizing Genetic Sequences: Genome Space with Biological Distance and Applications

    PubMed Central

    Liang, Qian; He, Rong L.; Yau, Stephen S.-T.

    2011-01-01

    Background Most existing methods for phylogenetic analysis involve developing an evolutionary model and then using some type of computational algorithm to perform multiple sequence alignment. There are two problems with this approach: (1) different evolutionary models can lead to different results, and (2) the computation time required for multiple alignments makes it impossible to analyse the phylogeny of a whole genome. This motivates us to create a new approach to characterize genetic sequences. Methodology To each DNA sequence, we associate a natural vector based on the distributions of nucleotides. This produces a one-to-one correspondence between the DNA sequence and its natural vector. We define the distance between two DNA sequences to be the distance between their associated natural vectors. This creates a genome space with a biological distance which makes global comparison of genomes with same topology possible. We use our proposed method to analyze the genomes of the new influenza A (H1N1) virus, human rhinoviruses (HRV) and mammalian mitochondrial. The result shows that a triple-reassortant swine virus circulating in North America and the Eurasian swine virus belong to the lineage of the influenza A (H1N1) virus. For the HRV and mammalian mitochondrial genomes, the results coincide with biologists' analyses. Conclusions Our approach provides a powerful new tool for analyzing and annotating genomes and their phylogenetic relationships. Whole or partial genomes can be handled more easily and more quickly than using multiple alignment methods. Once a genome space has been constructed, it can be stored in a database. There is no need to reconstruct the genome space for subsequent applications, whereas in multiple alignment methods, realignment is needed to add new sequences. Furthermore, one can make a global comparison of all genomes simultaneously, which no other existing method can achieve. PMID:21399690

  14. Comparative genomics and stx phage characterization of LEE-negative Shiga toxin-producing Escherichia coli.

    PubMed

    Steyert, Susan R; Sahl, Jason W; Fraser, Claire M; Teel, Louise D; Scheutz, Flemming; Rasko, David A

    2012-01-01

    Infection by Escherichia coli and Shigella species are among the leading causes of death due to diarrheal disease in the world. Shiga toxin-producing E. coli (STEC) that do not encode the locus of enterocyte effacement (LEE-negative STEC) often possess Shiga toxin gene variants and have been isolated from humans and a variety of animal sources. In this study, we compare the genomes of nine LEE-negative STEC harboring various stx alleles with four complete reference LEE-positive STEC isolates. Compared to a representative collection of prototype E. coli and Shigella isolates representing each of the pathotypes, the whole genome phylogeny demonstrated that these isolates are diverse. Whole genome comparative analysis of the 13 genomes revealed that in addition to the absence of the LEE pathogenicity island, phage-encoded genes including non-LEE encoded effectors, were absent from all nine LEE-negative STEC genomes. Several plasmid-encoded virulence factors reportedly identified in LEE-negative STEC isolates were identified in only a subset of the nine LEE-negative isolates further confirming the diversity of this group. In combination with whole genome analysis, we characterized the lambdoid phages harboring the various stx alleles and determined their genomic insertion sites. Although the integrase gene sequence corresponded with genomic location, it was not correlated with stx variant, further highlighting the mosaic nature of these phages. The transcription of these phages in different genomic backgrounds was examined. Expression of the Shiga toxin genes, stx(1) and/or stx(2), as well as the Q genes, were examined with quantitative reverse transcriptase polymerase chain reaction assays. A wide range of basal and induced toxin induction was observed. Overall, this is a first significant foray into the genome space of this unexplored group of emerging and divergent pathogens.

  15. Improved Method for Rapid and Efficient Determination of Genome Replication and Protein Expression of Clinical Hepatitis B Virus Isolates▿

    PubMed Central

    Qin, Yanli; Zhang, Jiming; Garcia, Tamako; Ito, Kiyoaki; Gutelius, Danielle; Li, Jisu; Wands, Jack; Tong, Shuping

    2011-01-01

    Different hepatitis B virus (HBV) genotypes and variants are associated with different clinical outcomes and/or response to antiviral therapy, yet the comparison of the in vitro replication capacity of a large number of clinical isolates remains technically challenging and time-consuming. Although the full-length HBV genome can be amplified from high-titer blood samples by PCR using High Fidelityplus DNA polymerase and primers targeting the conserved precore region, the HBV clones thus generated are replication deficient due to the inability to generate the terminally redundant pregenomic RNA essential for genome replication. The transfection experiment is further complicated by PCR errors and the presence of viral quasispecies. A previous study found that the precise removal of non-HBV sequence by SapI digestion led to HBV replication in transfected cells, possibly due to low-level genome circularization by a cellular enzyme. We released HBV genome from the cloning vector using BspQI, an inexpensive isoschizomer of SapI, and increased the efficiency of genome replication by an extra step of in vitro DNA ligation. The uncut plasmid DNA can be used for transfection if the sole purpose is to study envelope protein expression. We found significant PCR errors associated with the High Fidelityplus DNA polymerase, which could be greatly diminished using Phusion DNA polymerase or masked by the use of a clone pool. The reduced PCR error and modified enzymatic steps prior to transfection should facilitate a more widespread functional characterization of clinical HBV isolates, while the clone pool approach is useful for samples with significant sequence heterogeneity. PMID:21289153

  16. Improved method for rapid and efficient determination of genome replication and protein expression of clinical hepatitis B virus isolates.

    PubMed

    Qin, Yanli; Zhang, Jiming; Garcia, Tamako; Ito, Kiyoaki; Gutelius, Danielle; Li, Jisu; Wands, Jack; Tong, Shuping

    2011-04-01

    Different hepatitis B virus (HBV) genotypes and variants are associated with different clinical outcomes and/or response to antiviral therapy, yet the comparison of the in vitro replication capacity of a large number of clinical isolates remains technically challenging and time-consuming. Although the full-length HBV genome can be amplified from high-titer blood samples by PCR using High Fidelity(plus) DNA polymerase and primers targeting the conserved precore region, the HBV clones thus generated are replication deficient due to the inability to generate the terminally redundant pregenomic RNA essential for genome replication. The transfection experiment is further complicated by PCR errors and the presence of viral quasispecies. A previous study found that the precise removal of non-HBV sequence by SapI digestion led to HBV replication in transfected cells, possibly due to low-level genome circularization by a cellular enzyme. We released HBV genome from the cloning vector using BspQI, an inexpensive isoschizomer of SapI, and increased the efficiency of genome replication by an extra step of in vitro DNA ligation. The uncut plasmid DNA can be used for transfection if the sole purpose is to study envelope protein expression. We found significant PCR errors associated with the High Fidelity(plus) DNA polymerase, which could be greatly diminished using Phusion DNA polymerase or masked by the use of a clone pool. The reduced PCR error and modified enzymatic steps prior to transfection should facilitate a more widespread functional characterization of clinical HBV isolates, while the clone pool approach is useful for samples with significant sequence heterogeneity. PMID:21289153

  17. Genome-scale functional characterization of Drosophila developmental enhancers in vivo.

    PubMed

    Kvon, Evgeny Z; Kazmar, Tomas; Stampfel, Gerald; Yáñez-Cuna, J Omar; Pagani, Michaela; Schernhuber, Katharina; Dickson, Barry J; Stark, Alexander

    2014-08-01

    Transcriptional enhancers are crucial regulators of gene expression and animal development and the characterization of their genomic organization, spatiotemporal activities and sequence properties is a key goal in modern biology. Here we characterize the in vivo activity of 7,705 Drosophila melanogaster enhancer candidates covering 13.5% of the non-coding non-repetitive genome throughout embryogenesis. 3,557 (46%) candidates are active, suggesting a high density with 50,000 to 100,000 developmental enhancers genome-wide. The vast majority of enhancers display specific spatial patterns that are highly dynamic during development. Most appear to regulate their neighbouring genes, suggesting that the cis-regulatory genome is organized locally into domains, which are supported by chromosomal domains, insulator binding and genome evolution. However, 12 to 21 per cent of enhancers appear to skip non-expressed neighbours and regulate a more distal gene. Finally, we computationally identify cis-regulatory motifs that are predictive and required for enhancer activity, as we validate experimentally. This work provides global insights into the organization of an animal regulatory genome and the make-up of enhancer sequences and confirms and generalizes principles from previous studies. All enhancer patterns are annotated manually with a controlled vocabulary and all results are available through a web interface (http://enhancers.starklab.org), including the raw images of all microscopy slides for manual inspection at arbitrary zoom levels.

  18. Characterization of the mitochondrial genome of Amolops tuberodepressus (Anura: Ranidae).

    PubMed

    Zhang, Chaohua; Xia, Yun; Zeng, Xiaomao

    2016-07-01

    Amolops tuberodepressus is a vulnerable torrent frog, and only know distributed in the Wuliang Mountain in southwestern China. In the present study, the mitochondrial DNA (mtDNA) sequence of A. tuberodepressus was determined. The genome was 18 348 bp in length, and it contained 37 genes (13 protein-coding genes, two ribosomal RNAs, and 22 transfer RNAs), one partial control region and one light strand replication origin. The gene rearrangement was observed within the WANCY tRNA gene cluster region, which similar to other Amolops species. In this paper, we utilized 13 protein-coding genes of A. tuberodepressus and other 10 closely ranid species to construct the species phylogenetic tree to verify the A. tuberodepressus was accuracy.

  19. Cloning and genomic characterization of Felis domesticus papillomavirus type 1.

    PubMed

    Tachezy, Ruth; Duson, Griet; Rector, Annabel; Jenson, A Bennett; Sundberg, John P; Van Ranst, Marc

    2002-09-30

    A novel papillomavirus was cloned from hyperkeratotic cutaneous lesions of a Persian domestic cat. The Felis domesticus papillomavirus (FdPV-1) genome counts 8300 bp and has a typical genome structure with an early region (E1, E2, E4, E6, E7), a late region (L1, L2), and a noncoding upstream regulatory region (URR or NCR1) between the end of L1 and the beginning of E6. The FdPV-1 also shows an unusual second noncoding region (NCR2) of 1.3 kb, situated between the end of E2 and the beginning of L2. This NCR2 is uniquely related to a similar region in the canine oral papillomavirus (COPV). Phylogenetic analysis places FdPV-1 together with COPV, the cottontail rabbit papillomavirus, human papillomavirus type 1 (HPV-1), and HPV-63 in the group of the benign cutaneous papillomaviruses. The position of FdPV-1 in the phylogenetic tree allows us to hypothesize that already in an early phase of the papillomavirus molecular evolution, a split occurred into viruses with a dual tropism primarily for cutaneous epithelia but also secondarily for mucosal surfaces, and viruses with a specific monotropism for mucosal surfaces. The close relationship between FdPV-1 and COPV, and between their Canidae and Felidae hosts, supports the hypothesis that papillomaviruses have speciated and coevolved together with their hosts throughout vertebrate evolution. A papillomavirus mutation rate of 0.73 to 0.96 x 10(-8) nucleotide substitutions per base per year was calculated. PMID:12359433

  20. NUT Midline Carcinoma: Morphoproteomic Characterization with Genomic and Therapeutic Correlates.

    PubMed

    Sun, Hongxia; McGuire, Mary F; Zhang, Songlin; Brown, Robert E

    2015-01-01

    NUT midline carcinoma is a rare entity arising primarily in the midline of teenagers and young adults. Genomically, it is associated with a translocation involving a nuclear protein in testis (NUT) gene with other genes, most commonly, the BRD4 gene. The resultant is a partial or near total block in differentiation of tumor cells into mature squamous elements. Such tumors are resistant to conventional therapy with a reported mean survival at less than 1 year. In this study, we investigated two cases with genomic confirmation as NUT midline carcinoma by morphoproteomic analysis using immunohistochemical antibodies. Our results showed overexpression, largely in the undifferentiated cells of the tumors of: 1) Stemness marker, SRY (sex determining region Y)-box 2 (Sox2); 2) Constitutive activation of the mTORC2 pathway with expression of total insulin-like growth factor-1 receptor (IGF-1R[Tyr1165/1166]), and nuclear p-mTOR (Ser 2448) and p-Akt (Ser 473); and 3) c-Myc, silent mating type information regulation 2 homolog 1 (Sirt1) and histone methyltransferase enhancer of Zeste, Drosophila, homolog 2 (EZH2) as molecular impediments to differentiation. These data were analyzed through the use of QIAGEN's Ingenuity(®) Pathway Analysis (IPA(®), QIAGEN Redwood City, www.qiagen.com/ingenuity). The results established the interconnection of these pathways and molecules, and identified several pharmacogenomic agents--melatonin, metformin, vorinostat, curcumin, and sulforaphane--that have the potential to remove the block in differentiation and lead to the establishment of a more benign form of NUT midline carcinoma.

  1. Rapid Identification and Characterization of Francisella by Molecular Biology and Other Techniques

    PubMed Central

    Lai, Xin-He; Zhao, Long-Fei; Chen, Xiao-Ming; Ren, Yi

    2016-01-01

    Francisella tularensis is the causative pathogen of tularemia and a Tier 1 bioterror agent on the CDC list. Considering the fact that some subpopulation of the F. tularensis strains is more virulent, more significantly associated with mortality, and therefore poses more threat to humans, rapid identification and characterization of this subpopulation strains is of invaluable importance. This review summarizes the up-to-date developments of assays for mainly detecting and characterizing F. tularensis and a touch of caveats of some of the assays. PMID:27335619

  2. Using genomics to characterize evolutionary potential for conservation of wild populations

    PubMed Central

    Harrisson, Katherine A; Pavlova, Alexandra; Telonis-Scott, Marina; Sunnucks, Paul

    2014-01-01

    Genomics promises exciting advances towards the important conservation goal of maximizing evolutionary potential, notwithstanding associated challenges. Here, we explore some of the complexity of adaptation genetics and discuss the strengths and limitations of genomics as a tool for characterizing evolutionary potential in the context of conservation management. Many traits are polygenic and can be strongly influenced by minor differences in regulatory networks and by epigenetic variation not visible in DNA sequence. Much of this critical complexity is difficult to detect using methods commonly used to identify adaptive variation, and this needs appropriate consideration when planning genomic screens, and when basing management decisions on genomic data. When the genomic basis of adaptation and future threats are well understood, it may be appropriate to focus management on particular adaptive traits. For more typical conservations scenarios, we argue that screening genome-wide variation should be a sensible approach that may provide a generalized measure of evolutionary potential that accounts for the contributions of small-effect loci and cryptic variation and is robust to uncertainty about future change and required adaptive response(s). The best conservation outcomes should be achieved when genomic estimates of evolutionary potential are used within an adaptive management framework. PMID:25553064

  3. Sequence Analysis and Characterization of Active Human Alu Subfamilies Based on the 1000 Genomes Pilot Project.

    PubMed

    Konkel, Miriam K; Walker, Jerilyn A; Hotard, Ashley B; Ranck, Megan C; Fontenot, Catherine C; Storer, Jessica; Stewart, Chip; Marth, Gabor T; Batzer, Mark A

    2015-08-29

    The goal of the 1000 Genomes Consortium is to characterize human genome structural variation (SV), including forms of copy number variations such as deletions, duplications, and insertions. Mobile element insertions, particularly Alu elements, are major contributors to genomic SV among humans. During the pilot phase of the project we experimentally validated 645 (611 intergenic and 34 exon targeted) polymorphic "young" Alu insertion events, absent from the human reference genome. Here, we report high resolution sequencing of 343 (322 unique) recent Alu insertion events, along with their respective target site duplications, precise genomic breakpoint coordinates, subfamily assignment, percent divergence, and estimated A-rich tail lengths. All the sequenced Alu loci were derived from the AluY lineage with no evidence of retrotransposition activity involving older Alu families (e.g., AluJ and AluS). AluYa5 is currently the most active Alu subfamily in the human lineage, followed by AluYb8, and many others including three newly identified subfamilies we have termed AluYb7a3, AluYb8b1, and AluYa4a1. This report provides the structural details of 322 unique Alu variants from individual human genomes collectively adding about 100 kb of genomic variation. Many Alu subfamilies are currently active in human populations, including a surprising level of AluY retrotransposition. Human Alu subfamilies exhibit continuous evolution with potential drivers sprouting new Alu lineages.

  4. Using genomics to characterize evolutionary potential for conservation of wild populations.

    PubMed

    Harrisson, Katherine A; Pavlova, Alexandra; Telonis-Scott, Marina; Sunnucks, Paul

    2014-11-01

    Genomics promises exciting advances towards the important conservation goal of maximizing evolutionary potential, notwithstanding associated challenges. Here, we explore some of the complexity of adaptation genetics and discuss the strengths and limitations of genomics as a tool for characterizing evolutionary potential in the context of conservation management. Many traits are polygenic and can be strongly influenced by minor differences in regulatory networks and by epigenetic variation not visible in DNA sequence. Much of this critical complexity is difficult to detect using methods commonly used to identify adaptive variation, and this needs appropriate consideration when planning genomic screens, and when basing management decisions on genomic data. When the genomic basis of adaptation and future threats are well understood, it may be appropriate to focus management on particular adaptive traits. For more typical conservations scenarios, we argue that screening genome-wide variation should be a sensible approach that may provide a generalized measure of evolutionary potential that accounts for the contributions of small-effect loci and cryptic variation and is robust to uncertainty about future change and required adaptive response(s). The best conservation outcomes should be achieved when genomic estimates of evolutionary potential are used within an adaptive management framework.

  5. Genomics-informed isolation and characterization of a symbiotic Nanoarchaeota system from a terrestrial geothermal environment.

    PubMed

    Wurch, Louie; Giannone, Richard J; Belisle, Bernard S; Swift, Carolyn; Utturkar, Sagar; Hettich, Robert L; Reysenbach, Anna-Louise; Podar, Mircea

    2016-01-01

    Biological features can be inferred, based on genomic data, for many microbial lineages that remain uncultured. However, cultivation is important for characterizing an organism's physiology and testing its genome-encoded potential. Here we use single-cell genomics to infer cultivation conditions for the isolation of an ectosymbiotic Nanoarchaeota ('Nanopusillus acidilobi') and its host (Acidilobus, a crenarchaeote) from a terrestrial geothermal environment. The cells of 'Nanopusillus' are among the smallest known cellular organisms (100-300 nm). They appear to have a complete genetic information processing machinery, but lack almost all primary biosynthetic functions as well as respiration and ATP synthesis. Genomic and proteomic comparison with its distant relative, the marine Nanoarchaeum equitans illustrate an ancient, common evolutionary history of adaptation of the Nanoarchaeota to ectosymbiosis, so far unique among the Archaea. PMID:27378076

  6. Genetic Characterization and Comparative Genome Analysis of Brucella melitensis Isolates from India

    PubMed Central

    Azam, Sarwar; Rao, Sashi Bhushan; Jakka, Padmaja; NarasimhaRao, Veera; Bhargavi, Bindu; Gupta, Vivek Kumar

    2016-01-01

    Brucellosis is the most frequent zoonotic disease worldwide, with over 500,000 new human infections every year. Brucella melitensis, the most virulent species in humans, primarily affects goats and the zoonotic transmission occurs by ingestion of unpasteurized milk products or through direct contact with fetal tissues. Brucellosis is endemic in India but no information is available on population structure and genetic diversity of Brucella spp. in India. We performed multilocus sequence typing of four B. melitensis strains isolated from naturally infected goats from India. For more detailed genetic characterization, we carried out whole genome sequencing and comparative genome analysis of one of the B. melitensis isolates, Bm IND1. Genome analysis identified 141 unique SNPs, 78 VNTRs, 51 Indels, and 2 putative prophage integrations in the Bm IND1 genome. Our data may help to develop improved epidemiological typing tools and efficient preventive strategies to control brucellosis. PMID:27525259

  7. Chromosomal localization and characterization of rDNA loci in the Brassica A and C genomes.

    PubMed

    Snowdon, R J; Köhler, W; Köhler, A

    1997-08-01

    Using fluorescence in situ hybridization, we located ribosomal DNA loci on prometaphase chromosomes of the diploid species Brassica rapa and Brassica oleracea and their amphidiploid Brassica napus. Based on comparisons of chromosome morphology and hybridization patterns, we characterized the individual B. napus rDNA loci according to their presumed origins in the Brassica A and C genomes. As reported in other studies, the sum of rDNA loci observed on B. rapa (AA genome) and B. oleracea (CC genome) chromosomes was one greater than the total number of loci seen in their amphidiploid B. napus (AACC). Evidence is presented that this reduction in B. napus rDNA locus number results from the loss of the smallest A genome rDNA site in the amphidiploid.

  8. Genomics-informed isolation and characterization of a symbiotic Nanoarchaeota system from a terrestrial geothermal environment

    DOE PAGES

    Wurch, Louie; Giannone, Richard J.; Belisle, Bernard S.; Swift, Carolyn; Utturkar, Sagar; Hettich, Robert L.; Reysenbach, Anna-Louise; Podar, Mircea

    2016-07-05

    Biological features can be inferred, based on genomic data, for many microbial lineages that remain uncultured. However, cultivation is important for characterizing an organism’s physiology and testing its genome-encoded potential. Here we use single-cell genomics to infer cultivation conditions for the isolation of an ectosymbiotic Nanoarchaeota (‘Nanopusillus acidilobi’) and its host (Acidilobus, a crenarchaeote) from a terrestrial geothermal environment. The cells of ‘Nanopusillus’ are among the smallest known cellular organisms (100–300 nm). They appear to have a complete genetic information processing machinery, but lack almost all primary biosynthetic functions as well as respiration and ATP synthesis. Lastly, genomic and proteomicmore » comparison with its distant relative, the marine Nanoarchaeum equitans illustrate an ancient, common evolutionary history of adaptation of the Nanoarchaeota to ectosymbiosis, so far unique among the Archaea.« less

  9. Genomics-informed isolation and characterization of a symbiotic Nanoarchaeota system from a terrestrial geothermal environment

    PubMed Central

    Wurch, Louie; Giannone, Richard J.; Belisle, Bernard S.; Swift, Carolyn; Utturkar, Sagar; Hettich, Robert L.; Reysenbach, Anna-Louise; Podar, Mircea

    2016-01-01

    Biological features can be inferred, based on genomic data, for many microbial lineages that remain uncultured. However, cultivation is important for characterizing an organism's physiology and testing its genome-encoded potential. Here we use single-cell genomics to infer cultivation conditions for the isolation of an ectosymbiotic Nanoarchaeota (‘Nanopusillus acidilobi') and its host (Acidilobus, a crenarchaeote) from a terrestrial geothermal environment. The cells of ‘Nanopusillus' are among the smallest known cellular organisms (100–300 nm). They appear to have a complete genetic information processing machinery, but lack almost all primary biosynthetic functions as well as respiration and ATP synthesis. Genomic and proteomic comparison with its distant relative, the marine Nanoarchaeum equitans illustrate an ancient, common evolutionary history of adaptation of the Nanoarchaeota to ectosymbiosis, so far unique among the Archaea. PMID:27378076

  10. Genomics-informed isolation and characterization of a symbiotic Nanoarchaeota system from a terrestrial geothermal environment.

    PubMed

    Wurch, Louie; Giannone, Richard J; Belisle, Bernard S; Swift, Carolyn; Utturkar, Sagar; Hettich, Robert L; Reysenbach, Anna-Louise; Podar, Mircea

    2016-07-05

    Biological features can be inferred, based on genomic data, for many microbial lineages that remain uncultured. However, cultivation is important for characterizing an organism's physiology and testing its genome-encoded potential. Here we use single-cell genomics to infer cultivation conditions for the isolation of an ectosymbiotic Nanoarchaeota ('Nanopusillus acidilobi') and its host (Acidilobus, a crenarchaeote) from a terrestrial geothermal environment. The cells of 'Nanopusillus' are among the smallest known cellular organisms (100-300 nm). They appear to have a complete genetic information processing machinery, but lack almost all primary biosynthetic functions as well as respiration and ATP synthesis. Genomic and proteomic comparison with its distant relative, the marine Nanoarchaeum equitans illustrate an ancient, common evolutionary history of adaptation of the Nanoarchaeota to ectosymbiosis, so far unique among the Archaea.

  11. Genetic Characterization and Comparative Genome Analysis of Brucella melitensis Isolates from India.

    PubMed

    Azam, Sarwar; Rao, Sashi Bhushan; Jakka, Padmaja; NarasimhaRao, Veera; Bhargavi, Bindu; Gupta, Vivek Kumar; Radhakrishnan, Girish

    2016-01-01

    Brucellosis is the most frequent zoonotic disease worldwide, with over 500,000 new human infections every year. Brucella melitensis, the most virulent species in humans, primarily affects goats and the zoonotic transmission occurs by ingestion of unpasteurized milk products or through direct contact with fetal tissues. Brucellosis is endemic in India but no information is available on population structure and genetic diversity of Brucella spp. in India. We performed multilocus sequence typing of four B. melitensis strains isolated from naturally infected goats from India. For more detailed genetic characterization, we carried out whole genome sequencing and comparative genome analysis of one of the B. melitensis isolates, Bm IND1. Genome analysis identified 141 unique SNPs, 78 VNTRs, 51 Indels, and 2 putative prophage integrations in the Bm IND1 genome. Our data may help to develop improved epidemiological typing tools and efficient preventive strategies to control brucellosis. PMID:27525259

  12. Rapid Screening for CRISPR-Directed Editing of the Drosophila Genome Using white Coconversion

    PubMed Central

    Ge, Daniel Tianfang; Tipping, Cindy; Brodsky, Michael H.; Zamore, Phillip D.

    2016-01-01

    Adoption of a streamlined version of the bacterial clustered regular interspersed short palindromic repeat (CRISPR)/Cas9 defense system has accelerated targeted genome engineering. The Streptococcus pyogenes Cas9 protein, directed by a simplified, CRISPR-like single-guide RNA, catalyzes a double-stranded DNA break at a specific genomic site; subsequent repair by end joining can introduce mutagenic insertions or deletions, while repair by homologous recombination using an exogenous DNA template can incorporate new sequences at the target locus. However, the efficiency of Cas9-directed mutagenesis is low in Drosophila melanogaster. Here, we describe a strategy that reduces the time and effort required to identify flies with targeted genomic changes. The strategy uses editing of the white gene, evidenced by altered eye color, to predict successful editing of an unrelated gene-of-interest. The red eyes of wild-type flies are readily distinguished from white-eyed (end-joining-mediated loss of White function) or brown-eyed (recombination-mediated conversion to the whitecoffee allele) mutant flies. When single injected G0 flies produce individual G1 broods, flies carrying edits at a gene-of-interest were readily found in broods in which all G1 offspring carried white mutations. Thus, visual assessment of eye color substitutes for wholesale PCR screening of large numbers of G1 offspring. We find that end-joining-mediated mutations often show signatures of microhomology-mediated repair and that recombination-based mutations frequently involve donor plasmid integration at the target locus. Finally, we show that gap repair induced by two guide RNAs more reliably converts the intervening target sequence, whereas the use of Lig4169 mutants to suppress end joining does not improve recombination efficacy. PMID:27543296

  13. Rapid extraction of genomic DNA from saliva for HLA typing on microarray based on magnetic nanobeads

    NASA Astrophysics Data System (ADS)

    Xie, Xin; Zhang, Xu; Yu, Bingbin; Gao, Huafang; Zhang, Huan; Fei, Weiyang

    2004-09-01

    A series of simplified protocols are developed for extracting genomic DNA from saliva by using the magnetic nanobeads as absorbents. In these protocols, both the enrichment of the target cells and the adsorption of DNA can be achieved simultaneously by our functionally modified magnetic beads in one step, and the DNA-nanobeads complex can be used as PCR templates. HLA typing based on an oligonucleotide array was conducted by hybridization with the PCR products. The result shows that the protocols are robust and sensitive.

  14. Rapid response near-infrared spectrophotometric characterization of Near Earth Objects

    NASA Astrophysics Data System (ADS)

    Mommert, Michael; Trilling, David; Axelrod, Tim; Butler, Nat; Jedicke, Robert; Moskovitz, Nicholas; Pichardo, Barbara; Reyes, Mauricio

    2014-11-01

    Small NEOs are, as a whole, poorly characterized, and we know nothing about the physical properties of the majority of all NEOs. The rate of NEO discoveries is increasing each year, and projects to determine the physical properties of NEOs are lagging behind. NEOs are faint, and generally even fainter by the time that follow-up characterizations can be made days or weeks later. There is a need for a high-throughput, high-efficiency physical characterization strategy in which hundreds of faint NEOs can be characterized each year. Broadband photometry in the near-infrared is sufficiently diagnostic to assign taxonomic types, and hence constrain both the individual and ensemble properties of NEOs. We will present results from our recently initiated program of rapid response near-infrared spectrophotometric characterization of NEOs. We are using UKIRT (on Mauna Kea) and the RATIR instrument on the 1.5m telescope at the San Pedro Martir Observatory (Mexico) to allow us to make observations most nights of the year in robotic/queue mode. This technique is powerful and fast. We have written automated software that allows us to observe NEOs very soon after discovery. Our targets are NEOs that are generally too faint for other characterization techniques. We are on pace to characterize hundreds of NEOs per year.

  15. Rapid and sensitive detection of foodborne pathogenic bacteria (Staphylococcus aureus) using an electrochemical DNA genomic biosensor and its application in fresh beef.

    PubMed

    Abdalhai, Mandour H; Fernandes, António Maximiano; Bashari, Mohand; Ji, Jian; He, Qian; Sun, Xiulan

    2014-12-31

    Rapid early detection of food contamination is the main key in food safety and quality control. Biosensors are emerging as a vibrant area of research, and the use of DNA biosensor recognition detectors is relatively new. In this study a genomic DNA biosensor system with a fixing and capture probe was modified by a sulfhydryl and amino group, respectively, as complementary with target DNA. After immobilization and hybridization, the following sandwich structure fixing DNA-target DNA-capture DNA-PbS NPs was formed to detect pathogenic bacteria (Staphylococuus aureus EF529607.1) by using GCE modified with (multiwalled carbon nanotubes-chitosan-bismuth) to increase the sensitivity of the electrode. The modification procedure was characterized by cyclic voltammetry and electrochemical impedance spectroscopy. The sandwich structure was dissolved in 1 M nitric acid to become accessible to the electrode, and the PbS NPs was measured in solution by differential pulse voltammetry (DPV). The results showed that the detection limit of the DNA sensor was 3.17 × 10(-14) M S. aureus using PbS NPs, whereas the result for beef samples was 1.23 ng/mL. Thus, according to the experimental results presented, the DNA biosensor exhibited high sensitivity and rapid response, and it will be useful for the food matrix.

  16. Genomic characterization of a core set of the USDA-NPGS Ethiopian sorghum germplasm collection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The USDA Agriculture Research Service National Plant Germplasm System (NPGS) preserves the largest sorghum germplasm collection in the world, which includes 7,217 accessions from the center of diversity in Ethiopia. The characterization of this exotic germplasm at a genome-wide scale will improve co...

  17. The evolution of genomic GC content undergoes a rapid reversal within the genus Plasmodium.

    PubMed

    Nikbakht, Hamid; Xia, Xuhua; Hickey, Donal A

    2014-09-01

    The genome of the malarial parasite Plasmodium falciparum is extremely AT rich. This bias toward a low GC content is a characteristic of several, but not all, species within the genus Plasmodium. We compared 4283 orthologous pairs of protein-coding sequences between Plasmodium falciparum and the less AT-biased Plasmodium vivax. Our results indicate that the common ancestor of these two species was also extremely AT rich. This means that, although there was a strong bias toward A+T during the early evolution of the ancestral Plasmodium lineage, there was a subsequent reversal of this trend during the more recent evolution of some species, such as P. vivax. Moreover, we show that not only is the P. vivax genome losing its AT richness, it is actually gaining a very significant degree of GC richness. This example illustrates the potential volatility of nucleotide content during the course of molecular evolution. Such reversible fluxes in nucleotide content within lineages could have important implications for phylogenetic reconstruction based on molecular sequence data.

  18. HYBRIDCHECK: software for the rapid detection, visualization and dating of recombinant regions in genome sequence data.

    PubMed

    Ward, Ben J; van Oosterhout, Cock

    2016-03-01

    HYBRIDCHECK is a software package to visualize the recombination signal in large DNA sequence data set, and it can be used to analyse recombination, genetic introgression, hybridization and horizontal gene transfer. It can scan large (multiple kb) contigs and whole-genome sequences of three or more individuals. HYBRIDCHECK is written in the r software for OS X, Linux and Windows operating systems, and it has a simple graphical user interface. In addition, the r code can be readily incorporated in scripts and analysis pipelines. HYBRIDCHECK implements several ABBA-BABA tests and visualizes the effects of hybridization and the resulting mosaic-like genome structure in high-density graphics. The package also reports the following: (i) the breakpoint positions, (ii) the number of mutations in each introgressed block, (iii) the probability that the identified region is not caused by recombination and (iv) the estimated age of each recombination event. The divergence times between the donor and recombinant sequence are calculated using a JC, K80, F81, HKY or GTR correction, and the dating algorithm is exceedingly fast. By estimating the coalescence time of introgressed blocks, it is possible to distinguish between hybridization and incomplete lineage sorting. HYBRIDCHECK is libré software and it and its manual are free to download from http://ward9250.github.io/HybridCheck/. PMID:26394708

  19. HYBRIDCHECK: software for the rapid detection, visualization and dating of recombinant regions in genome sequence data.

    PubMed

    Ward, Ben J; van Oosterhout, Cock

    2016-03-01

    HYBRIDCHECK is a software package to visualize the recombination signal in large DNA sequence data set, and it can be used to analyse recombination, genetic introgression, hybridization and horizontal gene transfer. It can scan large (multiple kb) contigs and whole-genome sequences of three or more individuals. HYBRIDCHECK is written in the r software for OS X, Linux and Windows operating systems, and it has a simple graphical user interface. In addition, the r code can be readily incorporated in scripts and analysis pipelines. HYBRIDCHECK implements several ABBA-BABA tests and visualizes the effects of hybridization and the resulting mosaic-like genome structure in high-density graphics. The package also reports the following: (i) the breakpoint positions, (ii) the number of mutations in each introgressed block, (iii) the probability that the identified region is not caused by recombination and (iv) the estimated age of each recombination event. The divergence times between the donor and recombinant sequence are calculated using a JC, K80, F81, HKY or GTR correction, and the dating algorithm is exceedingly fast. By estimating the coalescence time of introgressed blocks, it is possible to distinguish between hybridization and incomplete lineage sorting. HYBRIDCHECK is libré software and it and its manual are free to download from http://ward9250.github.io/HybridCheck/.

  20. Identification and characterization of essential genes in the human genome

    PubMed Central

    Wang, Tim; Birsoy, Kıvanç; Hughes, Nicholas W.; Krupczak, Kevin M.; Post, Yorick; Wei, Jenny J.; Lander, Eric S.; Sabatini, David M.

    2015-01-01

    Large-scale genetic analysis of lethal phenotypes has elucidated the molecular underpinnings of many biological processes. Using the bacterial clustered regularly interspaced short palindromic repeats (CRISPR) system, we constructed a genome-wide single-guide RNA (sgRNA) library to screen for genes required for proliferation and survival in a human cancer cell line. Our screen revealed the set of cell-essential genes, which was validated by an orthogonal gene-trap-based screen and comparison with yeast gene knockouts. This set is enriched for genes that encode components of fundamental pathways, are expressed at high levels, and contain few inactivating polymorphisms in the human population. We also uncovered a large group of uncharacterized genes involved in RNA processing, a number of whose products localize to the nucleolus. Lastly, screens in additional cell lines showed a high degree of overlap in gene essentiality, but also revealed differences specific to each cell line and cancer type that reflect the developmental origin, oncogenic drivers, paralogous gene expression pattern, and chromosomal structure of each line. These results demonstrate the power of CRISPR-based screens and suggest a general strategy for identifying liabilities in cancer cells. PMID:26472758

  1. Rapid characterization of titanium microstructural features for specific modelling of mechanical properties

    NASA Astrophysics Data System (ADS)

    Searles, T.; Tiley, J.; Tanner, A.; Williams, R.; Rollins, B.; Lee, E.; Kar, S.; Banerjee, R.; Fraser, H. L.

    2005-01-01

    Mechanical properties of α/β Ti alloys are closely related to their microstructure. The complexity of the microstructural features involved makes it rather difficult to develop models for predicting properties of these alloys. Advances in stereology and microscopy permit rapid characterization of various features in Ti alloys including Widmanstätten α-laths, grain sizes, grain shapes, colony structures and volume fractions of different phases. This research documents the stereology procedures for characterizing microstructural features in Ti alloys, including the use of three-dimensional serial sectioning and reconstruction procedures for developing through material measurements. The resulting data indicate the powerful characterization processes now available, and the ability to rapidly assess microstructural features in Ti alloys. The processes were tested using Ti-62222 by serial sectioning the sample and conducting automated stereology protocols to determine features. In addition, three-dimensional reconstruction was completed on a Ti-6242 sample to evaluate lath interactions within the alloy. Results indicate the tremendous potential for characterizing microstructures using advanced techniques.

  2. A rapid method to characterize seabed habitats and associated macro-organisms

    USGS Publications Warehouse

    Anderson, T.J.; Cochrane, G.R.; Roberts, D.A.; Chezar, H.; Hatcher, G.; ,

    2007-01-01

    This study presents a method for rapidly collecting, processing, and interrogating real-time abiotic and biotic seabed data to determine seabed habitat classifications. This is done from data collected over a large area of an acoustically derived seabed map, along multidirectional transects, using a towed small camera-sled. The seabed, within the newly designated Point Harris Marine Reserve on the northern coast of San Miguel Island, California, was acoustically imaged using sidescan sonar then ground-truthed using a towed small camera-sled. Seabed characterizations were made from video observations, and were logged to a laptop computer (PC) in real time. To ground-truth the acoustic mosaic, and to characterize abiotic and biotic aspects of the seabed, a three-tiered characterization scheme was employed that described the substratum type, physical structure (i.e., bedform or vertical relief), and the occurrence of benthic macrofauna and flora. A crucial advantage of the method described here, is that preliminary seabed characterizations can be interrogated and mapped over the sidescan mosaic and other seabed information within hours of data collection. This ability to rapidly process seabed data is invaluable to scientists and managers, particularly in modifying concurrent or planning subsequent surveys.

  3. The large-scale blast score ratio (LS-BSR) pipeline: a method to rapidly compare genetic content between bacterial genomes.

    PubMed

    Sahl, Jason W; Caporaso, J Gregory; Rasko, David A; Keim, Paul

    2014-01-01

    Background. As whole genome sequence data from bacterial isolates becomes cheaper to generate, computational methods are needed to correlate sequence data with biological observations. Here we present the large-scale BLAST score ratio (LS-BSR) pipeline, which rapidly compares the genetic content of hundreds to thousands of bacterial genomes, and returns a matrix that describes the relatedness of all coding sequences (CDSs) in all genomes surveyed. This matrix can be easily parsed in order to identify genetic relationships between bacterial genomes. Although pipelines have been published that group peptides by sequence similarity, no other software performs the rapid, large-scale, full-genome comparative analyses carried out by LS-BSR. Results. To demonstrate the utility of the method, the LS-BSR pipeline was tested on 96 Escherichia coli and Shigella genomes; the pipeline ran in 163 min using 16 processors, which is a greater than 7-fold speedup compared to using a single processor. The BSR values for each CDS, which indicate a relative level of relatedness, were then mapped to each genome on an independent core genome single nucleotide polymorphism (SNP) based phylogeny. Comparisons were then used to identify clade specific CDS markers and validate the LS-BSR pipeline based on molecular markers that delineate between classical E. coli pathogenic variant (pathovar) designations. Scalability tests demonstrated that the LS-BSR pipeline can process 1,000 E. coli genomes in 27-57 h, depending upon the alignment method, using 16 processors. Conclusions. LS-BSR is an open-source, parallel implementation of the BSR algorithm, enabling rapid comparison of the genetic content of large numbers of genomes. The results of the pipeline can be used to identify specific markers between user-defined phylogenetic groups, and to identify the loss and/or acquisition of genetic information between bacterial isolates. Taxa-specific genetic markers can then be translated into clinical

  4. First results from the rapid-response spectrophotometric characterization of Near-Earth objects using RATIR

    NASA Astrophysics Data System (ADS)

    Navarro-Meza, Samuel; Mommert, Michael; Reyes-Ruiz, Mauricio; Trilling, David E.; Butler, Nathaniel; Pichardo, Barbara; Moskovitz, Nicholas; Jedicke, Robert

    2016-10-01

    We are carrying out a program to obtain rapid-response spectrophotometric characterization of newly discovered Near Earth Objects. Our first results, based on observations made with WFCAM on UKIRT, are presented in Mommert et al. (2016). Here we present a preliminary analysis of the r-i distribution of ~140 small (<500m) NEOs observed with the RATIR instrument on the 1.5-m telescope on San Pedro Martir. The observations are made in queue mode, and the data processing is carried out autonomously. Our goals are to derive coarse taxonomic and therefore compositional classifications for each of these objects, which will allow us to derive composition as a function of NEO size. This work is part of a collaboration in which we will characterize hundreds of NEOs that are generally too faint for other characterization techniques (down to V~21). This work is supported by funding from NASA's Solar System Observations program.

  5. Isolation and characterization of novel microsatellite markers from the sika deer (Cervus nippon) genome.

    PubMed

    Li, Y M; Bai, C Y; Niu, W P; Yu, H; Yang, R J; Yan, S Q; Zhang, J Y; Zhang, M J; Zhao, Z H

    2015-01-01

    Microsatellite markers are widely and evenly distributed, and are highly polymorphic. Rapid and convenient detection through automated analysis means that microsatellite markers are widely used in the construction of plant and animal genetic maps, in quantitative trait loci localization, marker-assisted selection, identification of genetic relationships, and genetic diversity and phylogenetic tree construction. However, few microsatellite markers remain to be isolated. We used streptavidin magnetic beads to affinity-capture and construct a (CA)n microsatellite DNA-enriched library from sika deer. We selected sequences containing more than six repeats to design primers. Clear bands were selected, which were amplified using non-specific primers following PCR amplification to screen polymorphisms in a group of 65 unrelated sika deer. The positive clone rate reached 82.9% by constructing the enriched library, and we then selected positive clones for sequencing. There were 395 sequences with CA repeats, and the CA repeat number was 4-105. We selected sequences containing more than six repeats to design primers, of which 297 pairs were designed. We next selected clear bands and used non-specific primers to amplify following PCR amplification. In total, 245 pairs of primers were screened. We then selected 50 pairs of primers to randomly screen for polymorphisms. We detected 47 polymorphic and 3 monomorphic loci in 65 unrelated sika deer. These newly isolated and characterized microsatellite loci can be used to construct genetic maps and for lineage testing in deer. In addition, they can be used for comparative genomics between Cervidae species. PMID:26436393

  6. Rapid and Inexpensive Screening of Genomic Copy Number Variations Using a Novel Quantitative Fluorescent PCR Method

    PubMed Central

    Han, Joan C.; Elsea, Sarah H.; Pena, Heloísa B.; Pena, Sérgio Danilo Junho

    2013-01-01

    Detection of human microdeletion and microduplication syndromes poses significant burden on public healthcare systems in developing countries. With genome-wide diagnostic assays frequently inaccessible, targeted low-cost PCR-based approaches are preferred. However, their reproducibility depends on equally efficient amplification using a number of target and control primers. To address this, the recently described technique called Microdeletion/Microduplication Quantitative Fluorescent PCR (MQF-PCR) was shown to reliably detect four human syndromes by quantifying DNA amplification in an internally controlled PCR reaction. Here, we confirm its utility in the detection of eight human microdeletion syndromes, including the more common WAGR, Smith-Magenis, and Potocki-Lupski syndromes with 100% sensitivity and 100% specificity. We present selection, design, and performance evaluation of detection primers using variety of approaches. We conclude that MQF-PCR is an easily adaptable method for detection of human pathological chromosomal aberrations. PMID:24288428

  7. Integrated Genomic Characterization Reveals Novel, Therapeutically Relevant Drug Targets in FGFR and EGFR Pathways in Sporadic Intrahepatic Cholangiocarcinoma

    PubMed Central

    Liang, Winnie S.; Fonseca, Rafael; Bryce, Alan H.; McCullough, Ann E.; Barrett, Michael T.; Hunt, Katherine; Patel, Maitray D.; Young, Scott W.; Collins, Joseph M.; Silva, Alvin C.; Condjella, Rachel M.; Block, Matthew; McWilliams, Robert R.; Lazaridis, Konstantinos N.; Klee, Eric W.; Bible, Keith C.; Harris, Pamela; Oliver, Gavin R.; Bhavsar, Jaysheel D.; Nair, Asha A.; Middha, Sumit; Asmann, Yan; Kocher, Jean-Pierre; Schahl, Kimberly; Kipp, Benjamin R.; Barr Fritcher, Emily G.; Baker, Angela; Aldrich, Jessica; Kurdoglu, Ahmet; Izatt, Tyler; Christoforides, Alexis; Cherni, Irene; Nasser, Sara; Reiman, Rebecca; Phillips, Lori; McDonald, Jackie; Adkins, Jonathan; Mastrian, Stephen D.; Placek, Pamela; Watanabe, Aprill T.; LoBello, Janine; Han, Haiyong; Von Hoff, Daniel; Craig, David W.; Stewart, A. Keith; Carpten, John D.

    2014-01-01

    Advanced cholangiocarcinoma continues to harbor a difficult prognosis and therapeutic options have been limited. During the course of a clinical trial of whole genomic sequencing seeking druggable targets, we examined six patients with advanced cholangiocarcinoma. Integrated genome-wide and whole transcriptome sequence analyses were performed on tumors from six patients with advanced, sporadic intrahepatic cholangiocarcinoma (SIC) to identify potential therapeutically actionable events. Among the somatic events captured in our analysis, we uncovered two novel therapeutically relevant genomic contexts that when acted upon, resulted in preliminary evidence of anti-tumor activity. Genome-wide structural analysis of sequence data revealed recurrent translocation events involving the FGFR2 locus in three of six assessed patients. These observations and supporting evidence triggered the use of FGFR inhibitors in these patients. In one example, preliminary anti-tumor activity of pazopanib (in vitro FGFR2 IC50≈350 nM) was noted in a patient with an FGFR2-TACC3 fusion. After progression on pazopanib, the same patient also had stable disease on ponatinib, a pan-FGFR inhibitor (in vitro, FGFR2 IC50≈8 nM). In an independent non-FGFR2 translocation patient, exome and transcriptome analysis revealed an allele specific somatic nonsense mutation (E384X) in ERRFI1, a direct negative regulator of EGFR activation. Rapid and robust disease regression was noted in this ERRFI1 inactivated tumor when treated with erlotinib, an EGFR kinase inhibitor. FGFR2 fusions and ERRFI mutations may represent novel targets in sporadic intrahepatic cholangiocarcinoma and trials should be characterized in larger cohorts of patients with these aberrations. PMID:24550739

  8. Integrated genomic characterization reveals novel, therapeutically relevant drug targets in FGFR and EGFR pathways in sporadic intrahepatic cholangiocarcinoma.

    PubMed

    Borad, Mitesh J; Champion, Mia D; Egan, Jan B; Liang, Winnie S; Fonseca, Rafael; Bryce, Alan H; McCullough, Ann E; Barrett, Michael T; Hunt, Katherine; Patel, Maitray D; Young, Scott W; Collins, Joseph M; Silva, Alvin C; Condjella, Rachel M; Block, Matthew; McWilliams, Robert R; Lazaridis, Konstantinos N; Klee, Eric W; Bible, Keith C; Harris, Pamela; Oliver, Gavin R; Bhavsar, Jaysheel D; Nair, Asha A; Middha, Sumit; Asmann, Yan; Kocher, Jean-Pierre; Schahl, Kimberly; Kipp, Benjamin R; Barr Fritcher, Emily G; Baker, Angela; Aldrich, Jessica; Kurdoglu, Ahmet; Izatt, Tyler; Christoforides, Alexis; Cherni, Irene; Nasser, Sara; Reiman, Rebecca; Phillips, Lori; McDonald, Jackie; Adkins, Jonathan; Mastrian, Stephen D; Placek, Pamela; Watanabe, Aprill T; Lobello, Janine; Han, Haiyong; Von Hoff, Daniel; Craig, David W; Stewart, A Keith; Carpten, John D

    2014-02-01

    Advanced cholangiocarcinoma continues to harbor a difficult prognosis and therapeutic options have been limited. During the course of a clinical trial of whole genomic sequencing seeking druggable targets, we examined six patients with advanced cholangiocarcinoma. Integrated genome-wide and whole transcriptome sequence analyses were performed on tumors from six patients with advanced, sporadic intrahepatic cholangiocarcinoma (SIC) to identify potential therapeutically actionable events. Among the somatic events captured in our analysis, we uncovered two novel therapeutically relevant genomic contexts that when acted upon, resulted in preliminary evidence of anti-tumor activity. Genome-wide structural analysis of sequence data revealed recurrent translocation events involving the FGFR2 locus in three of six assessed patients. These observations and supporting evidence triggered the use of FGFR inhibitors in these patients. In one example, preliminary anti-tumor activity of pazopanib (in vitro FGFR2 IC50≈350 nM) was noted in a patient with an FGFR2-TACC3 fusion. After progression on pazopanib, the same patient also had stable disease on ponatinib, a pan-FGFR inhibitor (in vitro, FGFR2 IC50≈8 nM). In an independent non-FGFR2 translocation patient, exome and transcriptome analysis revealed an allele specific somatic nonsense mutation (E384X) in ERRFI1, a direct negative regulator of EGFR activation. Rapid and robust disease regression was noted in this ERRFI1 inactivated tumor when treated with erlotinib, an EGFR kinase inhibitor. FGFR2 fusions and ERRFI mutations may represent novel targets in sporadic intrahepatic cholangiocarcinoma and trials should be characterized in larger cohorts of patients with these aberrations. PMID:24550739

  9. Genomic identification, rapid evolution, and expression of Argonaute genes in the tilapia, Oreochromis niloticus.

    PubMed

    Tao, Wenjing; Sun, Lina; Chen, Jinlin; Shi, Hongjuan; Wang, Deshou

    2016-09-01

    Argonaute proteins are key components of the small RNA-induced silencing complex and have multiple roles in RNA-directed regulatory pathways. Argonaute genes can be divided into two subfamilies: the Ago (interacting with microRNA/small interfering RNA) and Piwi subfamilies (interacting with piwi-interacting RNAs (piRNAs)). In the present study, genome-wide analyses firstly yielded the identification of different members of Agos and Piwis in the tilapia, coelacanth, spotted gar, and elephant shark. The additional teleost Ago3b was generated following the fish-specific genome duplication event. Selective pressure analysis on Agos and Piwis between cichlids and other teleosts showed an accelerated evolution of Piwil1 in the cichlid lineages, and the positive selected sites were located in the region of PIWI domain, suggesting that these amino acid substitutions are adapt to targeted cleavage of messenger RNA (mRNA) in cichlids. Ago1 and Ago4 were detected at higher levels at 5 days after hatching (dah) in both ovaries and testes compared with other stages, supporting the previously reported requirement of Ago-mediated pathways to clear the maternal mRNAs during the early embryogenesis. The Piwis were abundantly expressed in tilapia testes, indicating their essential roles in male germline, especially in spermatogenesis. Notable expression of Piwis was also detected in skeletal muscle, indicating that piRNA pathway may not only be confined to development and maintenance of the germline but may also play important roles in somatic tissues. The expression of Piwil1 and Piwil2 was examined by quantitative PCR (qPCR) and in situ hybridization (ISH) to validate the spatial and temporal expression profiles. Taken together, these results present a thorough overview of tilapia Argonaute family and provide a new perspective on the evolution and function of this family in teleosts. PMID:27491892

  10. Construction of the BAC Library of Small Abalone (Haliotis diversicolor) for Gene Screening and Genome Characterization.

    PubMed

    Jiang, Likun; You, Weiwei; Zhang, Xiaojun; Xu, Jian; Jiang, Yanliang; Wang, Kai; Zhao, Zixia; Chen, Baohua; Zhao, Yunfeng; Mahboob, Shahid; Al-Ghanim, Khalid A; Ke, Caihuan; Xu, Peng

    2016-02-01

    The small abalone (Haliotis diversicolor) is one of the most important aquaculture species in East Asia. To facilitate gene cloning and characterization, genome analysis, and genetic breeding of it, we constructed a large-insert bacterial artificial chromosome (BAC) library, which is an important genetic tool for advanced genetics and genomics research. The small abalone BAC library includes 92,610 clones with an average insert size of 120 Kb, equivalent to approximately 7.6× of the small abalone genome. We set up three-dimensional pools and super pools of 18,432 BAC clones for target gene screening using PCR method. To assess the approach, we screened 12 target genes in these 18,432 BAC clones and identified 16 positive BAC clones. Eight positive BAC clones were then sequenced and assembled with the next generation sequencing platform. The assembled contigs representing these 8 BAC clones spanned 928 Kb of the small abalone genome, providing the first batch of genome sequences for genome evaluation and characterization. The average GC content of small abalone genome was estimated as 40.33%. A total of 21 protein-coding genes, including 7 target genes, were annotated into the 8 BACs, which proved the feasibility of PCR screening approach with three-dimensional pools in small abalone BAC library. One hundred fifty microsatellite loci were also identified from the sequences for marker development in the future. The BAC library and clone pools provided valuable resources and tools for genetic breeding and conservation of H. diversicolor. PMID:26438131

  11. Genome-wide microsatellite characterization and marker development in the sequenced Brassica crop species.

    PubMed

    Shi, Jiaqin; Huang, Shunmou; Zhan, Jiepeng; Yu, Jingyin; Wang, Xinfa; Hua, Wei; Liu, Shengyi; Liu, Guihua; Wang, Hanzhong

    2014-02-01

    Although much research has been conducted, the pattern of microsatellite distribution has remained ambiguous, and the development/utilization of microsatellite markers has still been limited/inefficient in Brassica, due to the lack of genome sequences. In view of this, we conducted genome-wide microsatellite characterization and marker development in three recently sequenced Brassica crops: Brassica rapa, Brassica oleracea and Brassica napus. The analysed microsatellite characteristics of these Brassica species were highly similar or almost identical, which suggests that the pattern of microsatellite distribution is likely conservative in Brassica. The genomic distribution of microsatellites was highly non-uniform and positively or negatively correlated with genes or transposable elements, respectively. Of the total of 115 869, 185 662 and 356 522 simple sequence repeat (SSR) markers developed with high frequencies (408.2, 343.8 and 356.2 per Mb or one every 2.45, 2.91 and 2.81 kb, respectively), most represented new SSR markers, the majority had determined physical positions, and a large number were genic or putative single-locus SSR markers. We also constructed a comprehensive database for the newly developed SSR markers, which was integrated with public Brassica SSR markers and annotated genome components. The genome-wide SSR markers developed in this study provide a useful tool to extend the annotated genome resources of sequenced Brassica species to genetic study/breeding in different Brassica species.

  12. Genome-Wide Microsatellite Characterization and Marker Development in the Sequenced Brassica Crop Species

    PubMed Central

    Shi, Jiaqin; Huang, Shunmou; Zhan, Jiepeng; Yu, Jingyin; Wang, Xinfa; Hua, Wei; Liu, Shengyi; Liu, Guihua; Wang, Hanzhong

    2014-01-01

    Although much research has been conducted, the pattern of microsatellite distribution has remained ambiguous, and the development/utilization of microsatellite markers has still been limited/inefficient in Brassica, due to the lack of genome sequences. In view of this, we conducted genome-wide microsatellite characterization and marker development in three recently sequenced Brassica crops: Brassica rapa, Brassica oleracea and Brassica napus. The analysed microsatellite characteristics of these Brassica species were highly similar or almost identical, which suggests that the pattern of microsatellite distribution is likely conservative in Brassica. The genomic distribution of microsatellites was highly non-uniform and positively or negatively correlated with genes or transposable elements, respectively. Of the total of 115 869, 185 662 and 356 522 simple sequence repeat (SSR) markers developed with high frequencies (408.2, 343.8 and 356.2 per Mb or one every 2.45, 2.91 and 2.81 kb, respectively), most represented new SSR markers, the majority had determined physical positions, and a large number were genic or putative single-locus SSR markers. We also constructed a comprehensive database for the newly developed SSR markers, which was integrated with public Brassica SSR markers and annotated genome components. The genome-wide SSR markers developed in this study provide a useful tool to extend the annotated genome resources of sequenced Brassica species to genetic study/breeding in different Brassica species. PMID:24130371

  13. Genomic characterization of a temperate phage of the psychrotolerant deep-sea bacterium Aurantimonas sp.

    PubMed

    Yoshida, Mitsuhiro; Yoshida-Takashima, Yukari; Nunoura, Takuro; Takai, Ken

    2015-01-01

    A temperate phage (termed AmM-1) was identified from the psychrotolerant Rhizobiales bacterium Aurantimonas sp. C5-1, which was isolated from bathypelagic water (water depth = 1,500 m) in the northwest Pacific. The AmM-1 genome is 47,800 bp in length and contains 67 coding sequences. Although phage AmM-1 morphologically belongs to the family Myoviridae, its genomic structure, particularly modular genome organization, is similar to that of lambda-type phages of Siphoviridae. Genetic and phylogenetic analyses of the structural core genes also revealed that AmM-1 has a mosaic genomic structure that includes a lambda-like head (Siphoviridae) and P2-like tail (Myoviridae). The sequences of the structural core genes of AmM-1 are distinct from those of previously characterized phage groups but similar to those of recently identified one prophage element and one phage of marine Rhizobiales bacteria: a potential prophage element in the marine psychrotolerant Aureimonas ureilytica DSM 18598 genome and the temperate phage RR-1A infecting Rhizobium radiobacter P007 isolated from deep subseafloor sediment. The mosaic genome structure of AmM-1 suggests the occurrence of genetic exchange between distinct temperate phages in marine Rhizobiales populations.

  14. An integrated genomic approach for rapid delineation of candidate genes regulating agro-morphological traits in chickpea.

    PubMed

    Saxena, Maneesha S; Bajaj, Deepak; Das, Shouvik; Kujur, Alice; Kumar, Vinod; Singh, Mohar; Bansal, Kailash C; Tyagi, Akhilesh K; Parida, Swarup K

    2014-12-01

    The identification and fine mapping of robust quantitative trait loci (QTLs)/genes governing important agro-morphological traits in chickpea still lacks systematic efforts at a genome-wide scale involving wild Cicer accessions. In this context, an 834 simple sequence repeat and single-nucleotide polymorphism marker-based high-density genetic linkage map between cultivated and wild parental accessions (Cicer arietinum desi cv. ICC 4958 and Cicer reticulatum wild cv. ICC 17160) was constructed. This inter-specific genetic map comprising eight linkage groups spanned a map length of 949.4 cM with an average inter-marker distance of 1.14 cM. Eleven novel major genomic regions harbouring 15 robust QTLs (15.6-39.8% R(2) at 4.2-15.7 logarithm of odds) associated with four agro-morphological traits (100-seed weight, pod and branch number/plant and plant hairiness) were identified and mapped on chickpea chromosomes. Most of these QTLs showed positive additive gene effects with effective allelic contribution from ICC 4958, particularly for increasing seed weight (SW) and pod and branch number. One robust SW-influencing major QTL region (qSW4.2) has been narrowed down by combining QTL mapping with high-resolution QTL region-specific association analysis, differential expression profiling and gene haplotype-based association/LD mapping. This enabled to delineate a strong SW-regulating ABI3VP1 transcription factor (TF) gene at trait-specific QTL interval and consequently identified favourable natural allelic variants and superior high seed weight-specific haplotypes in the upstream regulatory region of this gene showing increased transcript expression during seed development. The genes (TFs) harbouring diverse trait-regulating QTLs, once validated and fine-mapped by our developed rapid integrated genomic approach and through gene/QTL map-based cloning, can be utilized as potential candidates for marker-assisted genetic enhancement of chickpea.

  15. Comparative genomics of chemosensory protein genes reveals rapid evolution and positive selection in ant-specific duplicates.

    PubMed

    Kulmuni, J; Wurm, Y; Pamilo, P

    2013-06-01

    Gene duplications can have a major role in adaptation, and gene families underlying chemosensation are particularly interesting due to their essential role in chemical recognition of mates, predators and food resources. Social insects add yet another dimension to the study of chemosensory genomics, as the key components of their social life rely on chemical communication. Still, chemosensory gene families are little studied in social insects. Here we annotated chemosensory protein (CSP) genes from seven ant genomes and studied their evolution. The number of functional CSP genes ranges from 11 to 21 depending on species, and the estimated rates of gene birth and death indicate high turnover of genes. Ant CSP genes include seven conservative orthologous groups present in all the ants, and a group of genes that has expanded independently in different ant lineages. Interestingly, the expanded group of genes has a differing mode of evolution from the orthologous groups. The expanded group shows rapid evolution as indicated by a high dN/dS (nonsynonymous to synonymous changes) ratio, several sites under positive selection and many pseudogenes, whereas the genes in the seven orthologous groups evolve slowly under purifying selection and include only one pseudogene. These results show that adaptive changes have played a role in ant CSP evolution. The expanded group of ant-specific genes is phylogenetically close to a conservative orthologous group CSP7, which includes genes known to be involved in ant nestmate recognition, raising an interesting possibility that the expanded CSPs function in ant chemical communication.

  16. Comparative genomics of chemosensory protein genes reveals rapid evolution and positive selection in ant-specific duplicates.

    PubMed

    Kulmuni, J; Wurm, Y; Pamilo, P

    2013-06-01

    Gene duplications can have a major role in adaptation, and gene families underlying chemosensation are particularly interesting due to their essential role in chemical recognition of mates, predators and food resources. Social insects add yet another dimension to the study of chemosensory genomics, as the key components of their social life rely on chemical communication. Still, chemosensory gene families are little studied in social insects. Here we annotated chemosensory protein (CSP) genes from seven ant genomes and studied their evolution. The number of functional CSP genes ranges from 11 to 21 depending on species, and the estimated rates of gene birth and death indicate high turnover of genes. Ant CSP genes include seven conservative orthologous groups present in all the ants, and a group of genes that has expanded independently in different ant lineages. Interestingly, the expanded group of genes has a differing mode of evolution from the orthologous groups. The expanded group shows rapid evolution as indicated by a high dN/dS (nonsynonymous to synonymous changes) ratio, several sites under positive selection and many pseudogenes, whereas the genes in the seven orthologous groups evolve slowly under purifying selection and include only one pseudogene. These results show that adaptive changes have played a role in ant CSP evolution. The expanded group of ant-specific genes is phylogenetically close to a conservative orthologous group CSP7, which includes genes known to be involved in ant nestmate recognition, raising an interesting possibility that the expanded CSPs function in ant chemical communication. PMID:23403962

  17. Rapid geo-acoustic characterization from a surface ship of opportunity

    NASA Astrophysics Data System (ADS)

    Heaney, Kevin D.

    2002-05-01

    The recent shift from deep water antisubmarine warfare to the littoral has brought about a dramatic change in the requirements of our understanding of the acoustic environment. In particular, shallow water acoustic propagation is dominated by the interaction of sound with the bottom and is therefore very sensitive to the geo-acoustic parameters of the local sea-floor. The rapid geo-acoustic characterization (RGC) algorithm is presented, that determines a geo-acoustic model that best matches the general acoustic propagation parameters. Using data from a passing surface ship, the time spread, TL versus range and striation slope are measured at various frequencies. These are then matched to a simple two-layer geo-acoustic model based on the empirical curves of Hamilton and Bachman. The resulting estimate does reproduce the global features of the acoustic field that are relevant to acoustic prediction. This method is robust, rapid, and produces the relevant acoustic predictions.

  18. PRIMUS: Rapid Reconstruction of Pedigrees from Genome-wide Estimates of Identity by Descent

    PubMed Central

    Staples, Jeffrey; Qiao, Dandi; Cho, Michael H.; Silverman, Edwin K.; Nickerson, Deborah A.; Below, Jennifer E.

    2014-01-01

    Understanding and correctly utilizing relatedness among samples is essential for genetic analysis; however, managing sample records and pedigrees can often be error prone and incomplete. Data sets ascertained by random sampling often harbor cryptic relatedness that can be leveraged in genetic analyses for maximizing power. We have developed a method that uses genome-wide estimates of pairwise identity by descent to identify families and quickly reconstruct and score all possible pedigrees that fit the genetic data by using up to third-degree relatives, and we have included it in the software package PRIMUS (Pedigree Reconstruction and Identification of the Maximally Unrelated Set). Here, we validate its performance on simulated, clinical, and HapMap pedigrees. Among these samples, we demonstrate that PRIMUS can verify reported pedigree structures and identify cryptic relationships. Finally, we show that PRIMUS reconstructed pedigrees, all of which were previously unknown, for 203 families from a cohort collected in Starr County, TX (1,890 samples). PMID:25439724

  19. PRIMUS: rapid reconstruction of pedigrees from genome-wide estimates of identity by descent.

    PubMed

    Staples, Jeffrey; Qiao, Dandi; Cho, Michael H; Silverman, Edwin K; Nickerson, Deborah A; Below, Jennifer E

    2014-11-01

    Understanding and correctly utilizing relatedness among samples is essential for genetic analysis; however, managing sample records and pedigrees can often be error prone and incomplete. Data sets ascertained by random sampling often harbor cryptic relatedness that can be leveraged in genetic analyses for maximizing power. We have developed a method that uses genome-wide estimates of pairwise identity by descent to identify families and quickly reconstruct and score all possible pedigrees that fit the genetic data by using up to third-degree relatives, and we have included it in the software package PRIMUS (Pedigree Reconstruction and Identification of the Maximally Unrelated Set). Here, we validate its performance on simulated, clinical, and HapMap pedigrees. Among these samples, we demonstrate that PRIMUS can verify reported pedigree structures and identify cryptic relationships. Finally, we show that PRIMUS reconstructed pedigrees, all of which were previously unknown, for 203 families from a cohort collected in Starr County, TX (1,890 samples). PMID:25439724

  20. Genome-wide characterization of the routes to pluripotency.

    PubMed

    Hussein, Samer M I; Puri, Mira C; Tonge, Peter D; Benevento, Marco; Corso, Andrew J; Clancy, Jennifer L; Mosbergen, Rowland; Li, Mira; Lee, Dong-Sung; Cloonan, Nicole; Wood, David L A; Munoz, Javier; Middleton, Robert; Korn, Othmar; Patel, Hardip R; White, Carl A; Shin, Jong-Yeon; Gauthier, Maely E; Lê Cao, Kim-Anh; Kim, Jong-Il; Mar, Jessica C; Shakiba, Nika; Ritchie, William; Rasko, John E J; Grimmond, Sean M; Zandstra, Peter W; Wells, Christine A; Preiss, Thomas; Seo, Jeong-Sun; Heck, Albert J R; Rogers, Ian M; Nagy, Andras

    2014-12-11

    Somatic cell reprogramming to a pluripotent state continues to challenge many of our assumptions about cellular specification, and despite major efforts, we lack a complete molecular characterization of the reprograming process. To address this gap in knowledge, we generated extensive transcriptomic, epigenomic and proteomic data sets describing the reprogramming routes leading from mouse embryonic fibroblasts to induced pluripotency. Through integrative analysis, we reveal that cells transition through distinct gene expression and epigenetic signatures and bifurcate towards reprogramming transgene-dependent and -independent stable pluripotent states. Early transcriptional events, driven by high levels of reprogramming transcription factor expression, are associated with widespread loss of histone H3 lysine 27 (H3K27me3) trimethylation, representing a general opening of the chromatin state. Maintenance of high transgene levels leads to re-acquisition of H3K27me3 and a stable pluripotent state that is alternative to the embryonic stem cell (ESC)-like fate. Lowering transgene levels at an intermediate phase, however, guides the process to the acquisition of ESC-like chromatin and DNA methylation signature. Our data provide a comprehensive molecular description of the reprogramming routes and is accessible through the Project Grandiose portal at http://www.stemformatics.org.

  1. Genomic DNA characterization of pork spleen by Raman spectroscopy

    NASA Astrophysics Data System (ADS)

    Guzmán-Embús, D. A.; Orrego Cardozo, M.; Vargas-Hernández, C.

    2013-11-01

    In this paper, the study of Raman signal enhancement due to interaction between ZnO rods and pork spleen DNA is reported. ZnO microstructures were synthesized by the Sol-Gel method and afterward combined with porcine spleen DNA extracted in the previous stages, following standardized cell lysis, deproteinization, and precipitation processes. Raman spectroscopy was used for the characterization of structures of ZnO and ZnO-DNA complex, and the results show the respective bands of ZnO wurtzite hexagonal phase for modes E2 (M), A1(TO), E2(High), E1(LO), and 2LO. Due to the SERS effect in the spectral range from 200 to 1800 cm,-1 Raman bands caused by vibrations of the deoxyribose C-O-C binding were also observed, producing deformation of the ring as shown in the 559 cm-1 peak. The broad band at 782 cm-1, together with the complex vibration of the string 5'-COPO-C3', is over a wide band of thymine (790 cm-1) or cytosine (780 cm-1). A prominent band near 1098 cm-1 assigned to symmetric stretching vibration phosphodioxy group (PO2-) DNA backbone is most favoured in intensity by the addition of ZnO particles originated by the SERS effect. This effect suggests a possible mechanism for enhancing the Raman signal due to the electromagnetic interaction between a DNA molecule and the flat surface of the ZnO rod.

  2. Identification and Characterization of Microsatellite Markers Derived from the Whole Genome Analysis of Taenia solium

    PubMed Central

    Pajuelo, Mónica J.; Eguiluz, María; Dahlstrom, Eric; Requena, David; Guzmán, Frank; Ramirez, Manuel; Sheen, Patricia; Frace, Michael; Sammons, Scott; Cama, Vitaliano; Anzick, Sarah; Bruno, Dan; Mahanty, Siddhartha; Wilkins, Patricia; Nash, Theodore; Gonzalez, Armando; García, Héctor H.; Gilman, Robert H.; Porcella, Steve; Zimic, Mirko

    2015-01-01

    Background Infections with Taenia solium are the most common cause of adult acquired seizures worldwide, and are the leading cause of epilepsy in developing countries. A better understanding of the genetic diversity of T. solium will improve parasite diagnostics and transmission pathways in endemic areas thereby facilitating the design of future control measures and interventions. Microsatellite markers are useful genome features, which enable strain typing and identification in complex pathogen genomes. Here we describe microsatellite identification and characterization in T. solium, providing information that will assist in global efforts to control this important pathogen. Methods For genome sequencing, T. solium cysts and proglottids were collected from Huancayo and Puno in Peru, respectively. Using next generation sequencing (NGS) and de novo assembly, we assembled two draft genomes and one hybrid genome. Microsatellite sequences were identified and 36 of them were selected for further analysis. Twenty T. solium isolates were collected from Tumbes in the northern region, and twenty from Puno in the southern region of Peru. The size-polymorphism of the selected microsatellites was determined with multi-capillary electrophoresis. We analyzed the association between microsatellite polymorphism and the geographic origin of the samples. Results The predicted size of the hybrid (proglottid genome combined with cyst genome) T. solium genome was 111 MB with a GC content of 42.54%. A total of 7,979 contigs (>1,000 nt) were obtained. We identified 9,129 microsatellites in the Puno-proglottid genome and 9,936 in the Huancayo-cyst genome, with 5 or more repeats, ranging from mono- to hexa-nucleotide. Seven microsatellites were polymorphic and 29 were monomorphic within the analyzed isolates. T. solium tapeworms were classified into two genetic groups that correlated with the North/South geographic origin of the parasites. Conclusions/Significance The availability of draft

  3. Rapid mechanisms for generating genome diversity: whole ploidy shifts, aneuploidy, and loss of heterozygosity.

    PubMed

    Bennett, Richard J; Forche, Anja; Berman, Judith

    2014-10-01

    Human fungal pathogens can exist in a variety of ploidy states, including euploid and aneuploid forms. Ploidy change has a major impact on phenotypic properties, including the regulation of interactions with the human host. In addition, the rapid emergence of drug-resistant isolates is often associated with the formation of specific supernumerary chromosomes. Pathogens such as Candida albicans and Cryptococcus neoformans appear particularly well adapted for propagation in multiple ploidy states with novel pathways driving ploidy variation. In both species, heterozygous cells also readily undergo loss of heterozygosity (LOH), leading to additional phenotypic changes such as altered drug resistance. Here, we examine the sexual and parasexual cycles that drive ploidy variation in human fungal pathogens and discuss ploidy and LOH events with respect to their far-reaching roles in fungal adaptation and pathogenesis. PMID:25081629

  4. The Swedish new variant of Chlamydia trachomatis: genome sequence, morphology, cell tropism and phenotypic characterization

    PubMed Central

    Unemo, Magnus; Seth-Smith, Helena M. B.; Cutcliffe, Lesley T.; Skilton, Rachel J.; Barlow, David; Goulding, David; Persson, Kenneth; Harris, Simon R.; Kelly, Anne; Bjartling, Carina; Fredlund, Hans; Olcén, Per; Thomson, Nicholas R.; Clarke, Ian N.

    2010-01-01

    Chlamydia trachomatis is a major cause of bacterial sexually transmitted infections worldwide. In 2006, a new variant of C. trachomatis (nvCT), carrying a 377 bp deletion within the plasmid, was reported in Sweden. This deletion included the targets used by the commercial diagnostic systems from Roche and Abbott. The nvCT is clonal (serovar/genovar E) and it spread rapidly in Sweden, undiagnosed by these systems. The degree of spread may also indicate an increased biological fitness of nvCT. The aims of this study were to describe the genome of nvCT, to compare the nvCT genome to all available C. trachomatis genome sequences and to investigate the biological properties of nvCT. An early nvCT isolate (Sweden2) was analysed by genome sequencing, growth kinetics, microscopy, cell tropism assay and antimicrobial susceptibility testing. It was compared with relevant C. trachomatis isolates, including a similar serovar E C. trachomatis wild-type strain that circulated in Sweden prior to the initially undetected expansion of nvCT. The nvCT genome does not contain any major genetic polymorphisms – the genes for central metabolism, development cycle and virulence are conserved – or phenotypic characteristics that indicate any altered biological fitness. This is supported by the observations that the nvCT and wild-type C. trachomatis infections are very similar in terms of epidemiological distribution, and that differences in clinical signs are only described, in one study, in women. In conclusion, the nvCT does not appear to have any altered biological fitness. Therefore, the rapid transmission of nvCT in Sweden was due to the strong diagnostic selective advantage and its introduction into a high-frequency transmitting population. PMID:20093289

  5. Rapid evolution of a recently retroposed transcription factor YY2 in mammalian genomes

    SciTech Connect

    Luo, C; Lu, X; Stubbs, L; Kim, J

    2005-11-11

    YY2 was originally identified due to its unusual similarity to the evolutionarily well conserved, zinc-finger gene YY1. In this study, we have determined the evolutionary origin and conservation of YY2 using comparative genomic approaches. Our results indicate that YY2 is a retroposed copy of YY1 that has been inserted into another gene locus named Mbtps2 (membrane-bound transcription factor protease site 2). This retroposition is estimated to have occurred after the divergence of placental mammals from other vertebrates based on the detection of YY2 only in the placental mammals. The N-terminal and C-terminal regions of YY2 have evolved under different selection pressures. The N-terminal region has evolved at a very fast pace with very limited functional constraints whereas the DNA-binding, C-terminal region still maintains very similar sequence structure as YY1 and is also well conserved among placental mammals. In situ hybridizations using different adult mouse tissues indicate that mouse YY2 is expressed at relatively low levels in Purkinje and granular cells of cerebellum, and neuronal cells of cerebrum, but at very high levels in testis. The expression levels of YY2 is much lower than YY1, but the overall spatial expression patterns are similar to those of Mbtps2, suggesting a possible shared transcriptional control between YY2 and Mbtps2. Taken together, the formation and evolution of YY2 represent a very unusual case where a transcription factor was first retroposed into another gene locus encoding a protease and survived with different selection schemes and expression patterns.

  6. Rapid, Multiplexed Characterization of Shiga Toxin-Producing Escherichia coli (STEC) Isolates Using Suspension Array Technology

    PubMed Central

    Carter, John M.; Lin, Andrew; Clotilde, Laurie; Lesho, Matthew

    2016-01-01

    Molecular methods have emerged as the most reliable techniques to detect and characterize pathogenic Escherichia coli. These molecular techniques include conventional single analyte and multiplex PCR, PCR followed by microarray detection, pulsed-field gel electrophoresis (PFGE), and whole genome sequencing. The choice of methods used depends upon the specific needs of the particular study. One versatile method involves detecting serogroup-specific markers by hybridization or binding to encoded microbeads in a suspension array. This molecular serotyping method has been developed and adopted for investigating E. coli outbreaks. The major advantages of this technique are the ability to simultaneously serotype E. coli and detect the presence of virulence and pathogenicity markers. Here, we describe the development of a family of multiplex molecular serotyping methods for Shiga toxin-producing E. coli, compare their performance to traditional serotyping methods, and discuss the cost-benefit balance of these methods in the context of various food safety objectives. PMID:27242670

  7. Rapid, Multiplexed Characterization of Shiga Toxin-Producing Escherichia coli (STEC) Isolates Using Suspension Array Technology.

    PubMed

    Carter, John M; Lin, Andrew; Clotilde, Laurie; Lesho, Matthew

    2016-01-01

    Molecular methods have emerged as the most reliable techniques to detect and characterize pathogenic Escherichia coli. These molecular techniques include conventional single analyte and multiplex PCR, PCR followed by microarray detection, pulsed-field gel electrophoresis (PFGE), and whole genome sequencing. The choice of methods used depends upon the specific needs of the particular study. One versatile method involves detecting serogroup-specific markers by hybridization or binding to encoded microbeads in a suspension array. This molecular serotyping method has been developed and adopted for investigating E. coli outbreaks. The major advantages of this technique are the ability to simultaneously serotype E. coli and detect the presence of virulence and pathogenicity markers. Here, we describe the development of a family of multiplex molecular serotyping methods for Shiga toxin-producing E. coli, compare their performance to traditional serotyping methods, and discuss the cost-benefit balance of these methods in the context of various food safety objectives. PMID:27242670

  8. Characterization of the Genome, Proteome, and Structure of Yersiniophage ϕR1-37

    PubMed Central

    Hyytiäinen, Heidi J.; Happonen, Lotta J.; Kiljunen, Saija; Datta, Neeta; Mattinen, Laura; Williamson, Kirsty; Kristo, Paula; Szeliga, Magdalena; Kalin-Mänttäri, Laura; Ahola-Iivarinen, Elina; Kalkkinen, Nisse; Butcher, Sarah J.

    2012-01-01

    The bacteriophage vB_YecM-ϕR1-37 (ϕR1-37) is a lytic yersiniophage that can propagate naturally in different Yersinia species carrying the correct lipopolysaccharide receptor. This large-tailed phage has deoxyuridine (dU) instead of thymidine in its DNA. In this study, we determined the genomic sequence of phage ϕR1-37, mapped parts of the phage transcriptome, characterized the phage particle proteome, and characterized the virion structure by cryo-electron microscopy and image reconstruction. The 262,391-bp genome of ϕR1-37 is one of the largest sequenced phage genomes, and it contains 367 putative open reading frames (ORFs) and 5 tRNA genes. Mass-spectrometric analysis identified 69 phage particle structural proteins with the genes scattered throughout the genome. A total of 269 of the ORFs (73%) lack homologues in sequence databases. Based on terminator and promoter sequences identified from the intergenic regions, the phage genome was predicted to consist of 40 to 60 transcriptional units. Image reconstruction revealed that the ϕR1-37 capsid consists of hexameric capsomers arranged on a T=27 lattice similar to the bacteriophage ϕKZ. The tail of ϕR1-37 has a contractile sheath. We conclude that phage ϕR1-37 is a representative of a novel phage type that carries the dU-containing genome in a ϕKZ-like head. PMID:22973030

  9. Characterization and Calibration of Lightpipe Radiation Thermometers for Use in Rapid Thermal Processing

    NASA Astrophysics Data System (ADS)

    Tsai, B. K.; DeWitt, D. P.

    2003-09-01

    Lightpipe radiation thermometers (LPRTs) are the sensor of choice for temperature measurements in Rapid Thermal Processing (RTP) applications. At the National Institute of Standards and Technology (NIST), we have developed protocols for calibrating and characterizing LPRTs for use in RTP and other applications. In this paper, the LPRTs and the sodium heat pipe blackbody (Na-HPBB) used in the calibration process at NIST will be introduced. The calibration and characterization methods (spatial response, spectral response, temporal response, and optical inspection) of the LPRTs will be described also. Finally, a discussion of the application of LPRTs in an environment outside of the calibration laboratory, along with a list of recommendations for proper use of LPRTs, will be presented.

  10. Rapid, simple and efficient method for detection of viral genomes on raspberries.

    PubMed

    Perrin, A; Loutreul, J; Boudaud, N; Bertrand, I; Gantzer, C

    2015-11-01

    In recent years, foodborne viruses, especially human noroviruses (NoV) and hepatitis A virus (HAV), have been increasingly reported as the causes of foodborne disease outbreaks. Soft red fruits, especially raspberries, have a high incidence among the types of food concerned. Due to low infectious doses and low concentrations of enteric viruses in food samples, it is necessary to have an efficient and rapid detection method to implement prevention measures. A standard method for virus detection and quantification in food, including raspberries (XP CEN ISO/TS 15216-1 and -2, 2013) is currently available. This method proposes a consensus detection approach by RT-real time PCR (RT-qPCR) but also a virus extraction procedure based on the elution-concentration principle. In this study, an alternative method of extraction in which RNAs are directly extracted from food matrices (based on direct RNA extraction) has been optimized. First, each step was improved to make it a highly rapid, specific and simple method. Second, the standard virus concentration method was compared with the optimized direct RNA extraction one. Human enteric viral surrogates, Murine Norovirus (MNV) and F-specific RNA bacteriophage GA, were selected according to their adhesion properties and resistance to pH close to our main targets (NoV and HAV). Raspberries were artificially contaminated using two different techniques (immersion and spotting) in order to define a recovery rate and the amounts of virus recovered. Results showed that the direct RNA extraction method revealed significantly higher viral extraction efficiency (46.2%) than the elution-concentration method (20.3%), with similar proportions of inhibitors for both. In the same way with inoculation by spotting, the best recovery rate of GA phage (39.7% against 0.7%) and MNV (42.8% against 0.5%) was observed by direct RNA extraction. For the lowest concentrations of phage and virus in the immersion bath, only the direct RNA extraction method

  11. Identification and characterization of potential drug targets by subtractive genome analyses of methicillin resistant Staphylococcus aureus.

    PubMed

    Uddin, Reaz; Saeed, Kiran

    2014-02-01

    Methicillin resistant Staphylococcus aureus (MRSA) causes serious infections in humans and becomes resistant to a number of antibiotics. Due to the emergence of antibiotic resistance strains, there is an essential need to develop novel drug targets to address the challenge of multidrug-resistant bacteria. In current study, the idea was to utilize the available genome or proteome in a subtractive genome analyses protocol to identify drug targets within two of the MRSA types, i.e., MRSA ST398 and MRSA 252. Recently, the use of subtractive genomic approaches helped in the identification and characterization of novel drug targets of a number of pathogens. Our protocol involved a similarity search between pathogen and host, essentiality study using the database of essential genes, metabolic functional association study using Kyoto Encyclopedia of Genes and Genomes database (KEGG), cellular membrane localization analysis and Drug Bank database. Functional family characterizations of the identified non homologous hypothetical essential proteins were done by SVMProt server. Druggability potential of each of the identified drug targets was also evaluated by Drug Bank database. Moreover, metabolic pathway analysis of the identified druggable essential proteins with KEGG revealed that the identified proteins are participating in unique and essential metabolic pathways amongst MRSA strains. In short, the complete proteome analyses by the use of advanced computational tools, databases and servers resulted in identification and characterization of few nonhomologous/hypothetical and essential proteins which are not homologous to the host genome. Therefore, these non-homologous essential targets ensure the survival of the pathogen and hence can be targeted for drug discovery.

  12. Genomic characterization of novel marine vesiviruses from Steller sea lions (Eumetopias jubatus) from Alaska.

    PubMed

    McClenahan, Shasta D; Burek, Kathy A; Beckmen, Kimberlee B; Knowles, Nick J; Neill, John D; Romero, Carlos H

    2008-12-01

    Marine vesiviruses were isolated in cell culture from oral and rectal swabs and vesicular fluid from Alaskan Steller sea lions (SSL; Eumetopias jubatus). Further characterization by RT-PCR, complete genomic sequencing, and phylogenetic analyses indicated that these viruses are most closely related to the marine vesiviruses, but are distinct viruses and represent two novel genotypes. The complete genome of these two SSL isolates was sequenced after cloning their viral cDNA. The genomes were found to be 8302 and 8305 nucleotides in length, organized in three open reading frames and contained 5' and 3' untranslated regions (UTR) of 19 and 180 nucleotides, respectively. The complete genomes of both SSL viruses were most closely related to each other and shared 83.0% nucleotide identity. Using the very limited number of complete genomic vesivirus sequences available in the NCBI database, these novel SSL vesiviruses seem most closely related to vesicular exanthema of swine virus-A48 and least related to rabbit vesivirus and walrus calicivirus. Specific antiserum against some evolutionary closer marine vesiviruses did not neutralize these isolates supporting the novel nature of these SSL viruses. PMID:18765261

  13. Characterization and genomic analysis of two Staphylococcus aureus bacteriophages isolated from poultry/livestock farms.

    PubMed

    Yoon, Hyunjin; Yun, Jiae; Lim, Jeong-A; Roh, Eunjung; Jung, Kyu-Seok; Chang, Yoonjee; Ryu, Sangryeol; Heu, Sunggi

    2013-11-01

    Staphylococcus aureus is one of the most important pathogens, causing various diseases in humans and animals. As methicillin-resistant S. aureus (MRSA) has become increasingly prevalent, controlling this pathogen with standard antibiotic treatment has become challenging. Bacteriophages (phages) have attracted interest as alternative antibacterial agents to control MRSA. In this study, we isolated six S. aureus phages from soils of poultry/livestock farms. Based on the results of host range determination with 150 S. aureus strains and restriction enzyme treatment of phage DNA, two phages, designated SP5 and SP6, were selected for further characterization and genome sequencing. Both SP5 and SP6 were classified as members of the family Siphoviridae. The genome of SP5 comprises 43 305 bp and contains 63 ORFs, while the SP6 genome comprises 42 902 bp and contains 61 ORFs. Although they have different host spectra, the phage genomes exhibit high nucleotide similarity to each other. Adsorption assay results suggested that the host range determinants of the two phages are involved in both adsorption and infection. Comparative genomic analyses of the two phages provided evidence that the lysogenic/lytic control module and tail proteins may be important for host specificity. PMID:23973965

  14. An Empirical Strategy for Characterizing Bacterial Proteomes across Species in the Absence of Genomic Sequences

    PubMed Central

    Turse, Joshua E.; Marshall, Matthew J.; Fredrickson, James K.; Lipton, Mary S.; Callister, Stephen J.

    2010-01-01

    Global protein identification through current proteomics methods typically depends on the availability of sequenced genomes. In spite of increasingly high throughput sequencing technologies, this information is not available for every microorganism and rarely available for entire microbial communities. Nevertheless, the protein-level homology that exists between related bacteria makes it possible to extract biological information from the proteome of an organism or microbial community by using the genomic sequences of a near neighbor organism. Here, we demonstrate a trans-organism search strategy for determining the extent to which near-neighbor genome sequences can be applied to identify proteins in unsequenced environmental isolates. In proof of concept testing, we found that within a CLUSTAL W distance of 0.089, near-neighbor genomes successfully identified a high percentage of proteins within an organism. Application of this strategy to characterize environmental bacterial isolates lacking sequenced genomes, but having 16S rDNA sequence similarity to Shewanella resulted in the identification of 300–500 proteins in each strain. The majority of identified pathways mapped to core processes, as well as to processes unique to the Shewanellae, in particular to the presence of c-type cytochromes. Examples of core functional categories include energy metabolism, protein and nucleotide synthesis and cofactor biosynthesis, allowing classification of bacteria by observation of conserved processes. Additionally, within these core functionalities, we observed proteins involved in the alternative lactate utilization pathway, recently described in Shewanella. PMID:21103051

  15. Characterization of Equine Infectious Anemia Virus Integration in the Horse Genome

    PubMed Central

    Liu, Qiang; Wang, Xue-Feng; Ma, Jian; He, Xi-Jun; Wang, Xiao-Jun; Zhou, Jian-Hua

    2015-01-01

    Human immunodeficiency virus (HIV)-1 has a unique integration profile in the human genome relative to murine and avian retroviruses. Equine infectious anemia virus (EIAV) is another well-studied lentivirus that can also be used as a promising retro-transfection vector, but its integration into its native host has not been characterized. In this study, we mapped 477 integration sites of the EIAV strain EIAVFDDV13 in fetal equine dermal (FED) cells during in vitro infection. Published integration sites of EIAV and HIV-1 in the human genome were also analyzed as references. Our results demonstrated that EIAVFDDV13 tended to integrate into genes and AT-rich regions, and it avoided integrating into transcription start sites (TSS), which is consistent with EIAV and HIV-1 integration in the human genome. Notably, the integration of EIAVFDDV13 favored long interspersed elements (LINEs) and DNA transposons in the horse genome, whereas the integration of HIV-1 favored short interspersed elements (SINEs) in the human genome. The chromosomal environment near LINEs or DNA transposons potentially influences viral transcription and may be related to the unique EIAV latency states in equids. The data on EIAV integration in its natural host will facilitate studies on lentiviral infection and lentivirus-based therapeutic vectors. PMID:26102582

  16. Characterization of the chloroplast genome sequence of oil palm (Elaeis guineensis Jacq.).

    PubMed

    Uthaipaisanwong, P; Chanprasert, J; Shearman, J R; Sangsrakru, D; Yoocha, T; Jomchai, N; Jantasuriyarat, C; Tragoonrung, S; Tangphatsornruang, S

    2012-06-01

    Oil palm (Elaeis guineensis Jacq.) is an economically important crop, which is grown for oil production. To better understand the molecular basis of oil palm chloroplasts, we characterized the complete chloroplast (cp) genome sequence obtained from 454 pyrosequencing. The oil palm cp genome is 156,973 bp in length consisting of a large single-copy region of 85,192 bp flanked on each side by inverted repeats of 27,071 bp with a small single-copy region of 17,639 bp joining the repeats. The genome contains 112 unique genes: 79 protein-coding genes, 4 ribosomal RNA genes and 29 tRNA genes. By aligning the cp genome sequence with oil palm cDNA sequences, we observed 18 non-silent and 10 silent RNA editing events among 19 cp protein-coding genes. Creation of an initiation codon by RNA editing in rpl2 has been reported in several monocots and was also found in the oil palm cp genome. Fifty common chloroplast protein-coding genes from 33 plant taxa were used to construct ML and MP phylogenetic trees. Their topologies are similar and strongly support for the position of E. guineensis as the sister of closely related species Phoenix dactylifera in Arecaceae (palm families) of monocot subtrees.

  17. Rapid antibiotic-resistance predictions from genome sequence data for Staphylococcus aureus and Mycobacterium tuberculosis

    PubMed Central

    Bradley, Phelim; Gordon, N. Claire; Walker, Timothy M.; Dunn, Laura; Heys, Simon; Huang, Bill; Earle, Sarah; Pankhurst, Louise J.; Anson, Luke; de Cesare, Mariateresa; Piazza, Paolo; Votintseva, Antonina A.; Golubchik, Tanya; Wilson, Daniel J.; Wyllie, David H.; Diel, Roland; Niemann, Stefan; Feuerriegel, Silke; Kohl, Thomas A.; Ismail, Nazir; Omar, Shaheed V.; Smith, E. Grace; Buck, David; McVean, Gil; Walker, A. Sarah; Peto, Tim E. A.; Crook, Derrick W.; Iqbal, Zamin

    2015-01-01

    The rise of antibiotic-resistant bacteria has led to an urgent need for rapid detection of drug resistance in clinical samples, and improvements in global surveillance. Here we show how de Bruijn graph representation of bacterial diversity can be used to identify species and resistance profiles of clinical isolates. We implement this method for Staphylococcus aureus and Mycobacterium tuberculosis in a software package (‘Mykrobe predictor') that takes raw sequence data as input, and generates a clinician-friendly report within 3 minutes on a laptop. For S. aureus, the error rates of our method are comparable to gold-standard phenotypic methods, with sensitivity/specificity of 99.1%/99.6% across 12 antibiotics (using an independent validation set, n=470). For M. tuberculosis, our method predicts resistance with sensitivity/specificity of 82.6%/98.5% (independent validation set, n=1,609); sensitivity is lower here, probably because of limited understanding of the underlying genetic mechanisms. We give evidence that minor alleles improve detection of extremely drug-resistant strains, and demonstrate feasibility of the use of emerging single-molecule nanopore sequencing techniques for these purposes. PMID:26686880

  18. Rapid antibiotic-resistance predictions from genome sequence data for Staphylococcus aureus and Mycobacterium tuberculosis.

    PubMed

    Bradley, Phelim; Gordon, N Claire; Walker, Timothy M; Dunn, Laura; Heys, Simon; Huang, Bill; Earle, Sarah; Pankhurst, Louise J; Anson, Luke; de Cesare, Mariateresa; Piazza, Paolo; Votintseva, Antonina A; Golubchik, Tanya; Wilson, Daniel J; Wyllie, David H; Diel, Roland; Niemann, Stefan; Feuerriegel, Silke; Kohl, Thomas A; Ismail, Nazir; Omar, Shaheed V; Smith, E Grace; Buck, David; McVean, Gil; Walker, A Sarah; Peto, Tim E A; Crook, Derrick W; Iqbal, Zamin

    2015-01-01

    The rise of antibiotic-resistant bacteria has led to an urgent need for rapid detection of drug resistance in clinical samples, and improvements in global surveillance. Here we show how de Bruijn graph representation of bacterial diversity can be used to identify species and resistance profiles of clinical isolates. We implement this method for Staphylococcus aureus and Mycobacterium tuberculosis in a software package ('Mykrobe predictor') that takes raw sequence data as input, and generates a clinician-friendly report within 3 minutes on a laptop. For S. aureus, the error rates of our method are comparable to gold-standard phenotypic methods, with sensitivity/specificity of 99.1%/99.6% across 12 antibiotics (using an independent validation set, n=470). For M. tuberculosis, our method predicts resistance with sensitivity/specificity of 82.6%/98.5% (independent validation set, n=1,609); sensitivity is lower here, probably because of limited understanding of the underlying genetic mechanisms. We give evidence that minor alleles improve detection of extremely drug-resistant strains, and demonstrate feasibility of the use of emerging single-molecule nanopore sequencing techniques for these purposes. PMID:26686880

  19. Rapid antibiotic-resistance predictions from genome sequence data for Staphylococcus aureus and Mycobacterium tuberculosis.

    PubMed

    Bradley, Phelim; Gordon, N Claire; Walker, Timothy M; Dunn, Laura; Heys, Simon; Huang, Bill; Earle, Sarah; Pankhurst, Louise J; Anson, Luke; de Cesare, Mariateresa; Piazza, Paolo; Votintseva, Antonina A; Golubchik, Tanya; Wilson, Daniel J; Wyllie, David H; Diel, Roland; Niemann, Stefan; Feuerriegel, Silke; Kohl, Thomas A; Ismail, Nazir; Omar, Shaheed V; Smith, E Grace; Buck, David; McVean, Gil; Walker, A Sarah; Peto, Tim E A; Crook, Derrick W; Iqbal, Zamin

    2015-01-01

    The rise of antibiotic-resistant bacteria has led to an urgent need for rapid detection of drug resistance in clinical samples, and improvements in global surveillance. Here we show how de Bruijn graph representation of bacterial diversity can be used to identify species and resistance profiles of clinical isolates. We implement this method for Staphylococcus aureus and Mycobacterium tuberculosis in a software package ('Mykrobe predictor') that takes raw sequence data as input, and generates a clinician-friendly report within 3 minutes on a laptop. For S. aureus, the error rates of our method are comparable to gold-standard phenotypic methods, with sensitivity/specificity of 99.1%/99.6% across 12 antibiotics (using an independent validation set, n=470). For M. tuberculosis, our method predicts resistance with sensitivity/specificity of 82.6%/98.5% (independent validation set, n=1,609); sensitivity is lower here, probably because of limited understanding of the underlying genetic mechanisms. We give evidence that minor alleles improve detection of extremely drug-resistant strains, and demonstrate feasibility of the use of emerging single-molecule nanopore sequencing techniques for these purposes.

  20. RapTOR: Automated sequencing library preparation and suppression for rapid pathogen characterization ( 7th Annual SFAF Meeting, 2012)

    ScienceCinema

    Lane, Todd [SNL

    2016-07-12

    Todd Lane on "RapTOR: Automated sequencing library preparation and suppression for rapid pathogen characterization" at the 2012 Sequencing, Finishing, Analysis in the Future Meeting held June 5-7, 2012 in Santa Fe, New Mexico.

  1. RapTOR: Automated sequencing library preparation and suppression for rapid pathogen characterization ( 7th Annual SFAF Meeting, 2012)

    SciTech Connect

    Lane, Todd

    2012-06-01

    Todd Lane on "RapTOR: Automated sequencing library preparation and suppression for rapid pathogen characterization" at the 2012 Sequencing, Finishing, Analysis in the Future Meeting held June 5-7, 2012 in Santa Fe, New Mexico.

  2. Rapidly-Deposited Polydopamine Coating via High Temperature and Vigorous Stirring: Formation, Characterization and Biofunctional Evaluation

    PubMed Central

    Zhou, Ping; Deng, Yi; Lyu, Beier; Zhang, Ranran; Zhang, Hai; Ma, Hongwei; Lyu, Yalin; Wei, Shicheng

    2014-01-01

    Polydopamine (PDA) coating provides a promising approach for immobilization of biomolecules onto almost all kinds of solid substrates. However, the deposition kinetics of PDA coating as a function of temperature and reaction method is not well elucidated. Since dopamine self-polymerization usually takes a long time, therefore, rapid-formation of PDA film becomes imperative for surface modification of biomaterials and medical devices. In the present study, a practical method for preparation of rapidly-deposited PDA coating was developed using a uniquely designed device, and the kinetics of dopamine self-polymerization was investigated by QCM sensor system. It was found that high temperature and vigorous stirring could dramatically speed up the formation of PDA film on QCM chip surface. Surface characterization, BSA binding study, cell viability assay and antibacterial test demonstrates that the polydopamine coating after polymerization for 30 min by our approach exhibits similar properties to those of 24 h counterpart. The method has a great potential for rapid-deposition of polydopamine films to modify biomaterial surfaces. PMID:25415328

  3. Characterizing rapid-onset vasodilation to single muscle contractions in the human leg.

    PubMed

    Credeur, Daniel P; Holwerda, Seth W; Restaino, Robert M; King, Phillip M; Crutcher, Kiera L; Laughlin, M Harold; Padilla, Jaume; Fadel, Paul J

    2015-02-15

    Rapid-onset vasodilation (ROV) following single muscle contractions has been examined in the forearm of humans, but has not yet been characterized in the leg. Given known vascular differences between the arm and leg, we sought to characterize ROV following single muscle contractions in the leg. Sixteen healthy men performed random ordered single contractions at 5, 10, 20, 40, and 60% of their maximum voluntary contraction (MVC) using isometric knee extension made with the leg above and below heart level, and these were compared with single isometric contractions of the forearm (handgrip). Single thigh cuff compressions (300 mmHg) were utilized to estimate the mechanical contribution to leg ROV. Continuous blood flow was determined by duplex-Doppler ultrasound and blood pressure via finger photoplethysmography (Finometer). Single isometric knee extensor contractions produced intensity-dependent increases in peak leg vascular conductance that were significantly greater than the forearm in both the above- and below-heart level positions (e.g., above heart level: leg 20% MVC, +138 ± 28% vs. arm 20% MVC, +89 ± 17%; P < 0.05). Thigh cuff compressions also produced a significant hyperemic response, but these were brief and smaller in magnitude compared with single isometric contractions in the leg. Collectively, these data demonstrate the presence of a rapid and robust vasodilation to single muscle contractions in the leg that is largely independent of mechanical factors, thus establishing the leg as a viable model to study ROV in humans. PMID:25539935

  4. Characterizing rapid-onset vasodilation to single muscle contractions in the human leg.

    PubMed

    Credeur, Daniel P; Holwerda, Seth W; Restaino, Robert M; King, Phillip M; Crutcher, Kiera L; Laughlin, M Harold; Padilla, Jaume; Fadel, Paul J

    2015-02-15

    Rapid-onset vasodilation (ROV) following single muscle contractions has been examined in the forearm of humans, but has not yet been characterized in the leg. Given known vascular differences between the arm and leg, we sought to characterize ROV following single muscle contractions in the leg. Sixteen healthy men performed random ordered single contractions at 5, 10, 20, 40, and 60% of their maximum voluntary contraction (MVC) using isometric knee extension made with the leg above and below heart level, and these were compared with single isometric contractions of the forearm (handgrip). Single thigh cuff compressions (300 mmHg) were utilized to estimate the mechanical contribution to leg ROV. Continuous blood flow was determined by duplex-Doppler ultrasound and blood pressure via finger photoplethysmography (Finometer). Single isometric knee extensor contractions produced intensity-dependent increases in peak leg vascular conductance that were significantly greater than the forearm in both the above- and below-heart level positions (e.g., above heart level: leg 20% MVC, +138 ± 28% vs. arm 20% MVC, +89 ± 17%; P < 0.05). Thigh cuff compressions also produced a significant hyperemic response, but these were brief and smaller in magnitude compared with single isometric contractions in the leg. Collectively, these data demonstrate the presence of a rapid and robust vasodilation to single muscle contractions in the leg that is largely independent of mechanical factors, thus establishing the leg as a viable model to study ROV in humans.

  5. A genomic selection component analysis characterizes migration-selection balance within a hybrid Mimulus population

    PubMed Central

    Monnahan, Patrick J.; Colicchio, Jack; Kelly, John K.

    2016-01-01

    The genetic differentiation of populations in response to local selection pressures has long been studied by evolutionary biologists, but key details about the process remain obscure. How rapidly can local adaptation evolve, how extensive is the process across the genome, and how strong are the opposing forces of natural selection and gene flow? Here, we combine direct measurement of survival and reproduction with whole-genome genotyping of a plant species (Mimulus guttatus) that has recently invaded a novel habitat (the Quarry population). We renovate the classic selection component method to accommodate genomic data and observe selection at SNPs throughout the genome. SNPs showing viability selection in Quarry exhibit elevated divergence from neighboring populations relative to neutral SNPs. We also find that non-significant SNPs exhibit a subtle, but still significant, change in allele frequency towards neighboring populations, a predicted effect of gene flow. Given that the Quarry population is most probably only 30–40 generations old, the alleles conferring local advantage are almost certainly older than the population itself. Thus, local adaptation owes to the recruitment of standing genetic variation. PMID:26082096

  6. Genome sequence of E. coli O104:H4 leads to rapid development of a targeted antimicrobial agent against this emerging pathogen.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A recent widespread outbreak of Escherichia coli O104:H4 in Germany demonstrates the dynamic nature of emerging and re-emerging food-borne pathogens, particularly STECs and related pathogenic E. coli. Rapid genomic sequencing and public availability of these data from the German outbreak strain allo...

  7. Genome Sequence of E. coli O104:H4 Leads to Rapid Development of a Targeted Antimicrobial Agent against This Emerging Pathogen

    PubMed Central

    Scholl, Dean; Gebhart, Dana; Williams, Steven R.; Bates, Anna; Mandrell, Robert

    2012-01-01

    A recent widespread outbreak of Escherichia coli O104:H4 in Germany demonstrates the dynamic nature of emerging and re-emerging food-borne pathogens, particularly STECs and related pathogenic E. coli. Rapid genome sequencing and public availability of these data from the German outbreak strain allowed us to identify an O-antigen-specific bacteriophage tail spike protein encoded in the genome. We synthesized this gene and fused it to the tail fiber gene of an R-type pyocin, a phage tail-like bacteriocin, and expressed the novel bacteriocin such that the tail fiber fusion was incorporated into the bacteriocin structure. The resulting particles have bactericidal activity specifically against E. coli strains that produce the O104 lipopolysaccharide antigen, including the outbreak strain. This O-antigen tailspike-R-type pyocin strategy provides a platform to respond rapidly to emerging pathogens upon the availability of the pathogen's genome sequence. PMID:22432037

  8. An Integrated Genomic Approach for Rapid Delineation of Candidate Genes Regulating Agro-Morphological Traits in Chickpea

    PubMed Central

    Saxena, Maneesha S.; Bajaj, Deepak; Das, Shouvik; Kujur, Alice; Kumar, Vinod; Singh, Mohar; Bansal, Kailash C.; Tyagi, Akhilesh K.; Parida, Swarup K.

    2014-01-01

    The identification and fine mapping of robust quantitative trait loci (QTLs)/genes governing important agro-morphological traits in chickpea still lacks systematic efforts at a genome-wide scale involving wild Cicer accessions. In this context, an 834 simple sequence repeat and single-nucleotide polymorphism marker-based high-density genetic linkage map between cultivated and wild parental accessions (Cicer arietinum desi cv. ICC 4958 and Cicer reticulatum wild cv. ICC 17160) was constructed. This inter-specific genetic map comprising eight linkage groups spanned a map length of 949.4 cM with an average inter-marker distance of 1.14 cM. Eleven novel major genomic regions harbouring 15 robust QTLs (15.6–39.8% R2 at 4.2–15.7 logarithm of odds) associated with four agro-morphological traits (100-seed weight, pod and branch number/plant and plant hairiness) were identified and mapped on chickpea chromosomes. Most of these QTLs showed positive additive gene effects with effective allelic contribution from ICC 4958, particularly for increasing seed weight (SW) and pod and branch number. One robust SW-influencing major QTL region (qSW4.2) has been narrowed down by combining QTL mapping with high-resolution QTL region-specific association analysis, differential expression profiling and gene haplotype-based association/LD mapping. This enabled to delineate a strong SW-regulating ABI3VP1 transcription factor (TF) gene at trait-specific QTL interval and consequently identified favourable natural allelic variants and superior high seed weight-specific haplotypes in the upstream regulatory region of this gene showing increased transcript expression during seed development. The genes (TFs) harbouring diverse trait-regulating QTLs, once validated and fine-mapped by our developed rapid integrated genomic approach and through gene/QTL map-based cloning, can be utilized as potential candidates for marker-assisted genetic enhancement of chickpea. PMID:25335477

  9. Genetic and Phenotypic Characterization of a Pseudomonas aeruginosa Population with High Frequency of Genomic Islands

    PubMed Central

    Morales-Espinosa, Rosario; Soberón-Chávez, Gloria; Delgado-Sapién, Gabriela; Sandner-Miranda, Luisa; Méndez, José L.; González-Valencia, Gerardo; Cravioto, Alejandro

    2012-01-01

    Various genomic islands, PAPI-1, PAPI-2, PAGI-1, PAGI-2, PAGI-3, and PAGI-4, and the element pKLC102 have been characterized in different P. aeruginosa strains from diverse habitats and geographical locations. Chromosomal DNA macroarray of 100 P. aeruginosa strains isolated from 85 unrelated patients hospitalized in an intensive care unit was created to assess the occurrence of these genomic islands (GEIs). The macroarray was then hybridized with labeled probes derived from each genomic island. In addition, PFGE patterns with SpeI, frequency of virulence genes, and antimicrobial resistance patterns of the strains were studied. Our results showed that almost all P. aeruginosa strains presented up to eight virulence genes. By SpeI macrorestriction fragment analysis we were able to identify 49 restriction patterns; 35 patterns correspond to single strains and the remaining 14 to strains subgroup (a–n). Most of the strains showed variation in number or composition of GEIs and a specific antimicrobial pattern indicating that each strain was an unrelated isolate. In terms of the number of genomic islands per strain, 7 GEIs were found in 34% of the strains, 6 in 18%, 5 in 12%, 4 in 14%, 3 in 10%, 2 in 7%, and 1 in 4%; only one isolate did not present any GEI. The genomic islands PAPI-1 and PAPI-2 and the element pKLC102 were the most frequently detected. The analysis of the location of each GEI in the chromosome of two strains show that the islands PAGI-3, PAPI-1, PAPI-2 and pKLC102 are present in the insertion site previously reported, but that PAGI-2 and PAGI-4 are inserted in another chromosome place in a site not characterized yet. In conclusion our data show that P. aeruginosa strains exhibited an epidemic population structure with horizontal transfer of DNA resulting in a high frequency of GEIs. PMID:22662157

  10. Enhancing genome investigations in the mosquito Culex quinquefasciatus via BAC library construction and characterization

    PubMed Central

    2011-01-01

    Background Culex quinquefasciatus (Say) is a major species in the Culex pipiens complex and an important vector for several human pathogens including West Nile virus and parasitic filarial nematodes causing lymphatic filariasis. It is common throughout tropical and subtropical regions and is among the most geographically widespread mosquito species. Although the complete genome sequence is now available, additional genomic tools are needed to improve the sequence assembly. Findings We constructed a bacterial artificial chromosome (BAC) library using the pIndigoBAC536 vector and HindIII partially digested DNA isolated from Cx. quinquefasciatus pupae, Johannesburg strain (NDJ). Insert size was estimated by NotI digestion and pulsed-field gel electrophoresis of 82 randomly selected clones. To estimate genome coverage, each 384-well plate was pooled for screening with 29 simple sequence repeat (SSR) and five gene markers. The NDJ library consists of 55,296 clones arrayed in 144 384-well microplates. Fragment insert size ranged from 50 to 190 kb in length (mean = 106 kb). Based on a mean insert size of 106 kb and a genome size of 579 Mbp, the BAC library provides ~10.1-fold coverage of the Cx. quinquefasciatus genome. PCR screening of BAC DNA plate pools for SSR loci from the genetic linkage map and for four genes associated with reproductive diapause in Culex pipiens resulted in a mean of 9.0 positive plate pools per locus. Conclusion The NDJ library represents an excellent resource for genome assembly enhancement and characterization in Culex pipiens complex mosquitoes. PMID:21914202

  11. Genome wide characterization of short tandem repeat markers in sweet orange (Citrus sinensis).

    PubMed

    Biswas, Manosh Kumar; Xu, Qiang; Mayer, Christoph; Deng, Xiuxin

    2014-01-01

    Sweet orange (Citrus sinensis) is one of the major cultivated and most-consumed citrus species. With the goal of enhancing the genomic resources in citrus, we surveyed, developed and characterized microsatellite markers in the ≈347 Mb sequence assembly of the sweet orange genome. A total of 50,846 SSRs were identified with a frequency of 146.4 SSRs/Mbp. Dinucleotide repeats are the most frequent repeat class and the highest density of SSRs was found in chromosome 4. SSRs are non-randomly distributed in the genome and most of the SSRs (62.02%) are located in the intergenic regions. We found that AT-rich SSRs are more frequent than GC-rich SSRs. A total number of 21,248 SSR primers were successfully developed, which represents 89 SSR markers per Mb of the genome. A subset of 950 developed SSR primer pairs were synthesized and tested by wet lab experiments on a set of 16 citrus accessions. In total we identified 534 (56.21%) polymorphic SSR markers that will be useful in citrus improvement. The number of amplified alleles ranges from 2 to 12 with an average of 4 alleles per marker and an average PIC value of 0.75. The newly developed sweet orange primer sequences, their in silico PCR products, exact position in the genome assembly and putative function are made publicly available. We present the largest number of SSR markers ever developed for a citrus species. Almost two thirds of the markers are transferable to 16 citrus relatives and may be used for constructing a high density linkage map. In addition, they are valuable for marker-assisted selection studies, population structure analyses and comparative genomic studies of C. sinensis with other citrus related species. Altogether, these markers provide a significant contribution to the citrus research community.

  12. Complete Genome Sequence of Hepatitis B Virus Genotype E: The First Molecular Characterization from an Imported Case in Mexico

    PubMed Central

    Escobar-Escamilla, Noé; Fragoso-Fonseca, David Esaú; Arreguín-Porras, Dulce María; Esteban-Valencia, María del Carmen; Corona-Valdespino, Estela; Falcón-Acosta, Jaime Israel; Vázquez-Campuzano, Roberto; Garcés-Ayala, Fabiola; Ortiz-Alcantara, Joanna María; López-Martinez, Irma

    2016-01-01

    Hepatitis B virus infection is currently a global public health problem. Here, we present the first characterization and complete genome sequence of a strain belonging to genotype E in Mexico, obtained from a foreign carrier with chronic infection. PMID:27034495

  13. Genome differentiation in a species pair of coregonine fishes: an extremely rapid speciation driven by stress-activated retrotransposons mediating extensive ribosomal DNA multiplications

    PubMed Central

    2013-01-01

    Background Sympatric species pairs are particularly common in freshwater fishes associated with postglacial lakes in northern temperate environments. The nature of divergences between co-occurring sympatric species, factors contributing to reproductive isolation and modes of genome evolution is a much debated topic in evolutionary biology addressed by various experimental tools. To the best of our knowledge, nobody approached this field using molecular cytogenetics. We examined chromosomes and genomes of one postglacial species pair, sympatric European winter-spawning Coregonus albula and the local endemic dwarf-sized spring-spawning C. fontanae, both originating in Lake Stechlin. We have employed molecular cytogenetic tools to identify the genomic differences between the two species of the sympatric pair on the sub-chromosomal level of resolution. Results Fluorescence in situ hybridization (FISH) experiments consistently revealed a distinct variation in the copy number of loci of the major ribosomal DNA (the 45S unit) between C. albula and C. fontanae genomes. In C. fontanae, up to 40 chromosomes were identified to bear a part of the major ribosomal DNA, while in C. albula only 8–10 chromosomes possessed these genes. To determine mechanisms how such extensive genome alternation might have arisen, a PCR screening for retrotransposons from genomic DNA of both species was performed. The amplified retrotransposon Rex1 was used as a probe for FISH mapping onto chromosomes of both species. These experiments showed a clear co-localization of the ribosomal DNA and the retrotransposon Rex1 in a pericentromeric region of one or two acrocentric chromosomes in both species. Conclusion We demonstrated genomic consequences of a rapid ecological speciation on the level undetectable by neither sequence nor karyotype analysis. We provide indirect evidence that ribosomal DNA probably utilized the spreading mechanism of retrotransposons subsequently affecting recombination rates

  14. Genome-wide characterization of genetic variation in the unicellular, green alga Chlamydomonas reinhardtii.

    PubMed

    Jang, Hyosik; Ehrenreich, Ian M

    2012-01-01

    Chlamydomonas reinhardtii is a model system for studying cilia, photosynthesis, and other core features of eukaryotes, and is also an emerging source of biofuels. Despite its importance to basic and applied biological research, the level and pattern of genetic variation in this haploid green alga has yet to be characterized on a genome-wide scale. To improve understanding of C. reinhardtii's genetic variability, we generated low coverage whole genome resequencing data for nearly all of the available isolates of this species, which were sampled from a number of sites in North America over the past ∼70 years. Based on the analysis of more than 62,000 single nucleotide polymorphisms, we identified two groups of isolates that represent geographical subpopulations of the species. We also found that measurements of genetic diversity were highly variable throughout the genome, in part due to technical factors. We studied the level and pattern of linkage disequilibrium (LD), and observed one chromosome that exhibits elevated LD. Furthermore, we detected widespread evidence of recombination across the genome, which implies that outcrossing occurs in natural populations of this species. In summary, our study provides multiple insights into the sequence diversity of C. reinhardtii that will be useful to future studies of natural genetic variation in this organism.

  15. Characterizing genomic variation of Arabidopsis thaliana: the roles of geography and climate.

    PubMed

    Lasky, Jesse R; Des Marais, David L; McKay, John K; Richards, James H; Juenger, Thomas E; Keitt, Timothy H

    2012-11-01

    Arabidopsis thaliana inhabits diverse climates and exhibits varied phenology across its range. Although A. thaliana is an extremely well-studied model species, the relationship between geography, growing season climate and its genetic variation is poorly characterized. We used redundancy analysis (RDA) to quantify the association of genomic variation [214 051 single nucleotide polymorphisms (SNPs)] with geography and climate among 1003 accessions collected from 447 locations in Eurasia. We identified climate variables most correlated with genomic variation, which may be important selective gradients related to local adaptation across the species range. Climate variation among sites of origin explained slightly more genomic variation than geographical distance. Large-scale spatial gradients and early spring temperatures explained the most genomic variation, while growing season and summer conditions explained the most after controlling for spatial structure. SNP variation in Scandinavia showed the greatest climate structure among regions, possibly because of relatively consistent phenology and life history of populations in this region. Climate variation explained more variation among nonsynonymous SNPs than expected by chance, suggesting that much of the climatic structure of SNP correlations is due to changes in coding sequence that may underlie local adaptation.

  16. Rapid characterization of GC-FTIR spectra by a PCA-based expert system

    NASA Astrophysics Data System (ADS)

    Hasenoehrl, Erik J.; Perkins, Jonathan H.; Griffiths, Peter R.

    1992-03-01

    A capillary gas chromatographic separation with Fourier transform infrared identification (GC/FT-IR) can yield structural information about volatile and semivolatile components of a complex mixture to a minimum identifiable quantity of about 5 ng for polar compounds using a lightpipe detector. Three procedures for analyzing GC/FT-IR data are currently in common use. In order of increasing selectivity they are Gram-Schmidt (GS) reconstructions, wavelength-selective chromatograms, and spectral library searching. This paper describes an approach by which functional and structural information can be extracted rapidly and on-line in a GC analysis with a specificity that is intermediate between that of selective wavelength chromatograms and library searching. In this approach principal components analysis (PCA) is used to build expert system rules to characterize molecular structures in a similar manner to those which we have recently described.

  17. Rapid Detection and Immune Characterization of Mycobacterium abscessus Infection in Cystic Fibrosis Patients

    PubMed Central

    Mayatepek, Ertan; Mackenzie, Colin R.; Schramm, Dirk; Jacobsen, Marc

    2015-01-01

    Cystic fibrosis patients are highly susceptible to infections with non-tuberculous mycobacteria. Especially Mycobacterium abscessus infections are common but reliable diagnosis is hampered by non-specific clinical symptoms and insensitive mycobacterial culture. In the present study we established novel methods for rapid detection and immune characterization of Mycobacterium abscessus infection in cystic fibrosis patients. We performed Mycobacterium abscessus specific DNA-strip- and quantitative PCR-based analyses of non-cultured sputum samples to detect and characterize Mycobacterium abscessus infections. Concomitantly in vitro T-cell reactivation with purified protein derivatives (PPDs) from different mycobacterial species was used to determine Mycobacterium abscessus specific T-cell cytokine expression of infected cystic fibrosis patients. Four of 35 cystic fibrosis patients (11.4%) were Mycobacterium abscessus culture positive and showed concordant DNA-strip-test results. Quantitative PCR revealed marked differences of mycobacterial burden between cystic fibrosis patients and during disease course. Tandem-repeat analysis classified distinct Mycobacterium abscessus strains of infected cystic fibrosis patients and excluded patient-to-patient transmission. Mycobacterium abscessus specific T-cells were detected in the blood of cystic fibrosis patients with confirmed chronic infection and a subgroup of patients without evidence of Mycobacterium abscessus infection. Comparison of cytokine expression and phenotypic markers revealed increased proportions of CD40L positive T-cells that lack Interleukin-2 expression as a marker for chronic Mycobacterium abscessus infections in cystic fibrosis patients. Direct sputum examination enabled rapid diagnosis and quantification of Mycobacterium abscessus in cystic fibrosis patients. T-cell in vitro reactivation and cytokine expression analyses may contribute to diagnosis of chronic Mycobacterium abscessus infection. PMID:25742660

  18. Rapid detection and immune characterization of Mycobacterium abscessus infection in cystic fibrosis patients.

    PubMed

    Steindor, Mathis; Nkwouano, Vanesa; Mayatepek, Ertan; Mackenzie, Colin R; Schramm, Dirk; Jacobsen, Marc

    2015-01-01

    Cystic fibrosis patients are highly susceptible to infections with non-tuberculous mycobacteria. Especially Mycobacterium abscessus infections are common but reliable diagnosis is hampered by non-specific clinical symptoms and insensitive mycobacterial culture. In the present study we established novel methods for rapid detection and immune characterization of Mycobacterium abscessus infection in cystic fibrosis patients. We performed Mycobacterium abscessus specific DNA-strip- and quantitative PCR-based analyses of non-cultured sputum samples to detect and characterize Mycobacterium abscessus infections. Concomitantly in vitro T-cell reactivation with purified protein derivatives (PPDs) from different mycobacterial species was used to determine Mycobacterium abscessus specific T-cell cytokine expression of infected cystic fibrosis patients. Four of 35 cystic fibrosis patients (11.4%) were Mycobacterium abscessus culture positive and showed concordant DNA-strip-test results. Quantitative PCR revealed marked differences of mycobacterial burden between cystic fibrosis patients and during disease course. Tandem-repeat analysis classified distinct Mycobacterium abscessus strains of infected cystic fibrosis patients and excluded patient-to-patient transmission. Mycobacterium abscessus specific T-cells were detected in the blood of cystic fibrosis patients with confirmed chronic infection and a subgroup of patients without evidence of Mycobacterium abscessus infection. Comparison of cytokine expression and phenotypic markers revealed increased proportions of CD40L positive T-cells that lack Interleukin-2 expression as a marker for chronic Mycobacterium abscessus infections in cystic fibrosis patients. Direct sputum examination enabled rapid diagnosis and quantification of Mycobacterium abscessus in cystic fibrosis patients. T-cell in vitro reactivation and cytokine expression analyses may contribute to diagnosis of chronic Mycobacterium abscessus infection. PMID:25742660

  19. Roughness gradients on zirconia for rapid screening of cell-surface interactions: Fabrication, characterization and application.

    PubMed

    Flamant, Quentin; Stanciuc, Ana-Maria; Pavailler, Hugo; Sprecher, Christoph Martin; Alini, Mauro; Peroglio, Marianna; Anglada, Marc

    2016-10-01

    Roughness is one of the key parameters for successful osseointegration of dental implants. The understanding of how roughness affects cell response is thus crucial to improve implant performance. Surface gradients, which allow rapid and systematic investigations of cell-surface interactions, have the potential to facilitate this task. In this study, a novel method aiming to produce roughness gradients at the surface of zirconia using hydrofluoric acid etching was implemented. The topography was exhaustively characterized at the microscale and nanoscale by white light interferometry and atomic force microscopy, including the analysis of amplitude, spatial, hybrid, functional, and fractal parameters. A rapid screening of the influence of roughness on human mesenchymal stem cell morphology was conducted and potential correlations between roughness parameters and cell morphology were investigated. The roughness gradient induced significant changes in cell area (p < 0.001), aspect ratio (p = 0.01), and solidity (p = 0.026). Nanoroughness parameters were linearly correlated to cell solidity (p < 0.005), while microroughness parameters appeared nonlinearly correlated to cell area, highlighting the importance of multiscale optimization of implant topography to induce the desired cell response. The gradient method proposed here drastically reduces the efforts and resources necessary to study cell-surface interactions and provides results directly transferable to industry. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2502-2514, 2016. PMID:27227541

  20. Solidification characterization of a new rapidly solidified Ni-Cr-Co based superalloy

    SciTech Connect

    Wu, Kai; Liu, Guoquan; Hu, Benfu; Li, Feng; Zhang, Yiwen; Tao, Yu; Liu, Jiantao

    2012-11-15

    The solidification characterization of a new rapidly solidified Ni-Cr-Co based superalloy prepared by plasma rotating electrode process was investigated by means of optical microscope, scanning electron microscope, and transmission electron microscope. The results show that the solidification microstructure changes from dendrites to cellular and microcrystal structures with decreasing powder size. The elements of Co, Cr, W and Ni are enriched in the dendrites, while Mo, Nb and Ti are higher in the interdendritic regions. The relationships between powder size with the average solid-liquid interface moving rate, the average interface temperature gradient and the average cooling rate are established. Microsegregation is increased with larger powder size. The geometric integrity of MC Prime type carbides in the powders changes from regular to diverse with decreasing powder size. The morphology and quantity of carbides depend on the thermal parameters and non-equilibrium solute partition coefficients during rapid solidification. - Highlights: Black-Right-Pointing-Pointer The relations of solidification thermal parameters with powder size are established. Black-Right-Pointing-Pointer The relation of non-equilibrium solute partition with powder size is investigated. Black-Right-Pointing-Pointer The solidification microstructure is related to thermal parameters. Black-Right-Pointing-Pointer The segregation behavior is linked to non-equilibrium partition coefficients. Black-Right-Pointing-Pointer The morphology and quantity of carbides depend on the above combined factors.

  1. A breast cancer cell microarray (CMA) as a rapid method to characterize candidate biomarkers.

    PubMed

    Wu, Xinyan; Zahari, Muhammad S; Renuse, Santosh; Jacob, Harrys K C; Sakamuri, Sruthi; Singal, Mukul; Gabrielson, Edward; Sukumar, Saraswati; Pandey, Akhilesh

    2014-01-01

    Tissue microarrays (TMAs) have become an invaluable tool in cancer research to evaluate expression and subcellular localization of proteins in cells and tissues. As the catalogs of candidate biomarkers and therapeutic targets become more extensive, there is a need to characterize and validate these targets and biomarkers in cell lines as a primary biological system in research laboratories. Thus, cell microarrays (CMAs) are useful as a high-throughput screening tool. Here, we constructed a CMA containing 32 publicly available immortalized breast cell lines with the goal of creating a method to rapidly screen for antigens of interest in breast cancer research in a relatively easy, rapid and cost-effective manner. As proof of concept, we performed immunocytochemical staining of the HER2 receptor, as the status of this protein is relevant to breast cancer and has previously been reported for these cell lines. We observed a complete concordance of our staining with the published status of HER2 in these cell lines. In addition, we examined the expression of CD44, epithelial markers EpCAM and E-cadherin and tyrosine phosphoproteins. The labeling of these proteins correlates with the known biology of the cell lines. Our results demonstrate the utility of our method to screen for potential biomarkers and therapeutic targets in breast cancer and we suggest that CMAs be used as a general approach in breast cancer research.

  2. A breast cancer cell microarray (CMA) as a rapid method to characterize candidate biomarkers

    PubMed Central

    Wu, Xinyan; Zahari, Muhammad S; Renuse, Santosh; Jacob, Harrys KC; Sakamuri, Sruthi; Singal, Mukul; Gabrielson, Edward; Sukumar, Saraswati; Pandey, Akhilesh

    2014-01-01

    Tissue microarrays (TMAs) have become an invaluable tool in cancer research to evaluate expression and subcellular localization of proteins in cells and tissues. As the catalogs of candidate biomarkers and therapeutic targets become more extensive, there is a need to characterize and validate these targets and biomarkers in cell lines as a primary biological system in research laboratories. Thus, cell microarrays (CMAs) are useful as a high-throughput screening tool. Here, we constructed a CMA containing 32 publicly available immortalized breast cell lines with the goal of creating a method to rapidly screen for antigens of interest in breast cancer research in a relatively easy, rapid and cost-effective manner. As proof of concept, we performed immunocytochemical staining of the HER2 receptor, as the status of this protein is relevant to breast cancer and has previously been reported for these cell lines. We observed a complete concordance of our staining with the published status of HER2 in these cell lines. In addition, we examined the expression of CD44, epithelial markers EpCAM and E-cadherin and tyrosine phosphoproteins. The labeling of these proteins correlates with the known biology of the cell lines. Our results demonstrate the utility of our method to screen for potential biomarkers and therapeutic targets in breast cancer and we suggest that CMAs be used as a general approach in breast cancer research. PMID:25535895

  3. Bioinformatic tools for using whole genome sequencing as a rapid high resolution diagnostic typing tool when tracing bioterror organisms in the food and feed chain.

    PubMed

    Segerman, Bo; De Medici, Dario; Ehling Schulz, Monika; Fach, Patrick; Fenicia, Lucia; Fricker, Martina; Wielinga, Peter; Van Rotterdam, Bart; Knutsson, Rickard

    2011-03-01

    The rapid technological development in the field of parallel sequencing offers new opportunities when tracing and tracking microorganisms in the food and feed chain. If a bioterror organism is deliberately spread it is of crucial importance to get as much information as possible regarding the strain as fast as possible to aid the decision process and select suitable controls, tracing and tracking tools. A lot of efforts have been made to sequence multiple strains of potential bioterror organisms so there is a relatively large set of reference genomes available. This study is focused on how to use parallel sequencing for rapid phylogenomic analysis and screen for genetic modifications. A bioinformatic methodology has been developed to rapidly analyze sequence data with minimal post-processing. Instead of assembling the genome, defining genes, defining orthologous relations and calculating distances, the present method can achieve a similar high resolution directly from the raw sequence data. The method defines orthologous sequence reads instead of orthologous genes and the average similarity of the core genome (ASC) is calculated. The sequence reads from the core and from the non-conserved genomic regions can also be separated for further analysis. Finally, the comparison algorithm is used to visualize the phylogenomic diversity of the bacterial bioterror organisms Bacillus anthracis and Clostridium botulinum using heat plot diagrams.

  4. Rapid genome mapping in nano channel array for highly complete and accurate de novo sequence assembly of the complex Aegilops tauschii genome

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Next-generation sequencing (NGS) technologies have enabled high-throughput and low-cost generation of sequence data; however, de novo genome assembly remains a great challenge, particularly for large genomes. NGS short reads are often insufficient to create large contigs that span repeat sequences...

  5. Use of Alignment-Free Phylogenetics for Rapid Genome Sequence-Based Typing of Helicobacter pylori Virulence Markers and Antibiotic Susceptibility

    PubMed Central

    Kusters, Johannes G.

    2015-01-01

    Whole-genome sequencing is becoming a leading technology in the typing and epidemiology of microbial pathogens, but the increase in genomic information necessitates significant investment in bioinformatic resources and expertise, and currently used methodologies struggle with genetically heterogeneous bacteria such as the human gastric pathogen Helicobacter pylori. Here we demonstrate that the alignment-free analysis method feature frequency profiling (FFP) can be used to rapidly construct phylogenetic trees of draft bacterial genome sequences on a standard desktop computer and that coupling with in silico genotyping methods gives useful information for comparative and clinical genomic and molecular epidemiology applications. FFP-based phylogenetic trees of seven gastric Helicobacter species matched those obtained by analysis of 16S rRNA genes and ribosomal proteins, and FFP- and core genome single nucleotide polymorphism-based analysis of 63 H. pylori genomes again showed comparable phylogenetic clustering, consistent with genomotypes assigned by using multilocus sequence typing (MLST). Analysis of 377 H. pylori genomes highlighted the conservation of genomotypes and linkage with phylogeographic characteristics and predicted the presence of an incomplete or nonfunctional cag pathogenicity island in 18/276 genomes. In silico analysis of antibiotic susceptibility markers suggests that most H. pylori hspAmerind and hspEAsia isolates are predicted to carry the T2812C mutation potentially conferring low-level clarithromycin resistance, while levels of metronidazole resistance were similar in all multilocus sequence types. In conclusion, the use of FFP phylogenetic clustering and in silico genotyping allows determination of genome evolution and phylogeographic clustering and can contribute to clinical microbiology by genomotyping for outbreak management and the prediction of pathogenic potential and antibiotic susceptibility. PMID:26135867

  6. Rapid Separation Methods to Characterize Actinides and Metallic Impurities in Plutonium Scrap Materials at SRS

    SciTech Connect

    Maxwell, S.L. III; Jones, V.D.

    1998-07-01

    The Nuclear Materials Stabilization and Storage Division at SRS plans to stabilize selected plutonium scrap residue materials for long term storage by dissolution processing and plans to stabilize other plutonium vault materials via high-temperature furnace processing. To support these nuclear material stabilization activities, the SRS Analytical Laboratories Department (ALD) will provide characterization of materials required prior to the dissolution or the high-firing of these materials. Lab renovations to install new analytical instrumentation are underway to support these activities that include glove boxes with simulated-process dissolution and high- pressure microwave dissolution capability. Inductively-coupled plasma atomic emission spectrometry (ICP-AES), inductively- coupled mass spectrometry (ICP-MS) and thermal-ionization mass spectrometry (TIMS) will be used to measure actinide isotopics and metallic impurities. New high-speed actinide separation methods have been developed that will be applied to isotopic characterization of nuclear materials by TIMS and ICP-MS to eliminate isobaric interferences between Pu-238 /U- 238 and Pu-241/Am-241. TEVA Resin, UTEVA Resin, and TRU Resin columns will be used with vacuum-assisted flow rates to minimize TIMS and ICP-MS sample turnaround times. For metallic impurity analysis, rapid column removal methods using UTEVA Resin, AGMP-1 anion resin and AG MP-50 cation resin have also been developed to remove plutonium and uranium matrix interferences prior to ICP-AES and ICP- MS measurements.

  7. Genome-wide characterization and analysis of F-box protein-encoding genes in the Malus domestica genome.

    PubMed

    Cui, Hao-Ran; Zhang, Zheng-Rong; Lv, Wei; Xu, Jia-Ning; Wang, Xiao-Yun

    2015-08-01

    The F-box protein family is a large family that is characterized by conserved F-box domains of approximately 40-50 amino acids in the N-terminus. F-box proteins participate in diverse cellular processes, such as development of floral organs, signal transduction and response to stress, primarily as a component of the Skp1-cullin-F-box (SCF) complex. In this study, using a global search of the apple genome, 517 F-box protein-encoding genes (F-box genes for short) were identified and further subdivided into 12 groups according to the characterization of known functional domains, which suggests the different potential functions or processes that they were involved in. Among these domains, the galactose oxidase domain was analyzed for the first time in plants, and this domain was present with or without the Kelch domain. The F-box genes were distributed in all 17 apple chromosomes with various densities and tended to form gene clusters. Spatial expression profile analysis revealed that F-box genes have organ-specific expression and are widely expressed in all organs. Proteins that contained the galactose oxidase domain were highly expressed in leaves, flowers and seeds. From a fruit ripening expression profile, 166 F-box genes were identified. The expressions of most of these genes changed little during maturation, but five of them increased significantly. Using qRT-PCR to examine the expression of F-box genes encoding proteins with domains related to stress, the results revealed that F-box proteins were up- or down-regulated, which suggests that F-box genes were involved in abiotic stress. The results of this study helped to elucidate the functions of F-box proteins, especially in Rosaceae plants.

  8. Characterization and comparative genomic analysis of bacteriophages infecting members of the Bacillus cereus group.

    PubMed

    Lee, Ju-Hoon; Shin, Hakdong; Ryu, Sangryeol

    2014-05-01

    The Bacillus cereus group phages infecting B. cereus, B. anthracis, and B. thuringiensis (Bt) have been studied at the molecular level and, recently, at the genomic level to control the pathogens B. cereus and B. anthracis and to prevent phage contamination of the natural insect pesticide Bt. A comparative phylogenetic analysis has revealed three different major phage groups with different morphologies (Myoviridae for group I, Siphoviridae for group II, and Tectiviridae for group III), genome size (group I > group II > group III), and lifestyle (virulent for group I and temperate for group II and III). A subsequent phage genome comparison using a dot plot analysis showed that phages in each group are highly homologous, substantiating the grouping of B. cereus phages. Endolysin is a host lysis protein that contains two conserved domains: a cell-wall-binding domain (CBD) and an enzymatic activity domain (EAD). In B. cereus sensu lato phage group I, four different endolysin groups have been detected, according to combinations of two types of CBD and four types of EAD. Group I phages have two copies of tail lysins and one copy of endolysin, but the functions of the tail lysins are still unknown. In the B. cereus sensu lato phage group II, the B. anthracis phages have been studied and applied for typing and rapid detection of pathogenic host strains. In the B. cereus sensu lato phage group III, the B. thuringiensis phages Bam35 and GIL01 have been studied to understand phage entry and lytic switch regulation mechanisms. In this review, we suggest that further study of the B. cereus group phages would be useful for various phage applications, such as biocontrol, typing, and rapid detection of the pathogens B. cereus and B. anthracis and for the prevention of phage contamination of the natural insect pesticide Bt.

  9. Complete genome and clinicopathological characterization of a virulent Newcastle disease virus isolate from South America.

    PubMed

    Diel, Diego G; Susta, Leonardo; Cardenas Garcia, Stivalis; Killian, Mary L; Brown, Corrie C; Miller, Patti J; Afonso, Claudio L

    2012-02-01

    Newcastle disease (ND) is one of the most important diseases of poultry, negatively affecting poultry production worldwide. The disease is caused by Newcastle disease virus (NDV) or avian paramyxovirus type 1 (APMV-1), a negative-sense single-stranded RNA virus of the genus Avulavirus, family Paramyxoviridae. Although all NDV isolates characterized to date belong to a single serotype of APMV-1, significant genetic diversity has been described between different NDV isolates. Here we present the complete genome sequence and the clinicopathological characterization of a virulent Newcastle disease virus isolate (NDV-Peru/08) obtained from poultry during an outbreak of ND in Peru in 2008. Phylogenetic reconstruction and analysis of the evolutionary distances between NDV-Peru/08 and other isolates representing established NDV genotypes revealed the existence of large genomic and amino differences that clearly distinguish this isolate from viruses of typical NDV genotypes. Although NDV-Peru/08 is a genetically distinct virus, pathogenesis studies conducted with chickens revealed that NDV-Peru/08 infection results in clinical signs characteristic of velogenic viscerotropic NDV strains. Additionally, vaccination studies have shown that an inactivated NDV-LaSota/46 vaccine conferred full protection from NDV-Peru/08-induced clinical disease and mortality. This represents the first complete characterization of a virulent NDV isolate from South America.

  10. Rapid characterization of the biomechanical properties of drug-treated cells in a microfluidic device

    NASA Astrophysics Data System (ADS)

    Zhang, Xiaofei; Chu, Henry K.; Zhang, Yang; Bai, Guohua; Wang, Kaiqun; Tan, Qiulin; Sun, Dong

    2015-10-01

    Cell mechanics is closely related to many cell functions. Recent studies have suggested that the deformability of cells can be an effective biomarker to indicate the onset and progression of diseases. In this paper, a microfluidic chip is designed for rapid characterization of the mechanics of drug-treated cells through stretching with dielectrophoresis (DEP) force. This chip was fabricated using PDMS and micro-electrodes were integrated and patterned on the ITO layer of the chip. Leukemia NB4 cells were considered and the effect of all-trans retinoic acid (ATRA) drug on NB4 cells were examined via the microfluidic chip. To induce a DEP force onto the cell, a relatively weak ac voltage was utilized to immobilize a cell at one side of the electrodes. The applied voltage was then increased to 3.5 V pp and the cell started to be stretched along the applied electric field lines. The elongation of the cell was observed using an optical microscope and the results showed that both types of cells were deformed by the induced DEP force. The strain of the NB4 cell without the drug treatment was recorded to be about 0.08 (time t = 180 s) and the drug-treated NB4 cell was about 0.21 (time t = 180 s), indicating a decrease in the stiffness after drug treatment. The elastic modulus of the cell was also evaluated and the modulus changed from 140 Pa to 41 Pa after drug treatment. This microfluidic chip can provide a simple and rapid platform for measuring the change in the biomechanical properties of cells and can potentially be used as the tool to determine the biomechanical effects of different drug treatments for drug discovery and development applications.

  11. A rapid solvothermal synthesis of cerium oxide hollow spheres and characterization

    SciTech Connect

    Kempaiah Devaraju, Murukanahally; Liu, Xiangwen; Yin, Shu; Sato, Tsugio

    2012-10-15

    An easy and size controlled solvothermal synthesis of CeO{sub 2} hollow spheres is still a challenge in the area of materials synthesis. Here, CeO{sub 2} hollow spheres have been synthesized using PVA500 as a surfactant via solvothermal reaction followed by calcinations. The size of CeO{sub 2} hollow spheres could be controlled from 500 to 150 nm by changing the amounts of Ce(NO{sub 3}){sub 3}{center_dot}6H{sub 2}O and PVA500. The possible growth mechanism of CeO{sub 2} hollow sphere was explained. The CO oxidation catalytic activity of the CeO{sub 2} hollow spheres were superior to that of the commercial CeO{sub 2} powder due to the high specific surface area and small crystallite size. - Graphical abstract: A rapid and easy way to prepare CeO{sub 2} hollow sphere with 150-500 nm in diameter was successfully achieved by solvothermal reaction. The prepared particles showed hollowness due to Ostwald ripening process. An improved catalytic activity was observed and discussed. Highlights: A rapid synthesis of CeO{sub 2} hollow spheres with diameter size from 15 to 500 nm. Black-Right-Pointing-Pointer Cheap surfactant was used to prepare hollow spheres. Black-Right-Pointing-Pointer Effect of temperature and surfactant ratio were investigated. Black-Right-Pointing-Pointer Systematic characterization by XRD, FESEM, TEM, TG, FTIR and UV. Black-Right-Pointing-Pointer CO oxidation analysis results showed better catalytic activity.

  12. Hydrodynamic trapping for rapid assembly and in situ electrical characterization of droplet interface bilayer arrays.

    PubMed

    Nguyen, Mary-Anne; Srijanto, Bernadeta; Collier, C Patrick; Retterer, Scott T; Sarles, Stephen A

    2016-09-21

    The droplet interface bilayer (DIB) is a modular technique for assembling planar lipid membranes between water droplets in oil. The DIB method thus provides a unique capability for developing digital, droplet-based membrane platforms for rapid membrane characterization, drug screening and ion channel recordings. This paper demonstrates a new, low-volume microfluidic system that automates droplet generation, sorting, and sequential trapping in designated locations to enable the rapid assembly of arrays of DIBs. The channel layout of the device is guided by an equivalent circuit model, which predicts that a serial arrangement of hydrodynamic DIB traps enables sequential droplet placement and minimizes the hydrodynamic pressure developed across filled traps to prevent squeeze-through of trapped droplets. Furthermore, the incorporation of thin-film electrodes fabricated via evaporation metal deposition onto the glass substrate beneath the channels allows for the first time in situ, simultaneous electrical interrogation of multiple DIBs within a sealed device. Combining electrical measurements with imaging enables measurements of membrane capacitance and resistance and bilayer area, and our data show that DIBs formed in different trap locations within the device exhibit similar sizes and transport properties. Simultaneous, single channel recordings of ion channel gating in multiple membranes are obtained when alamethicin peptides are incorporated into the captured droplets, qualifying the thin-film electrodes as a means for measuring stimuli-responsive functions of membrane-bound biomolecules. This novel microfluidic-electrophysiology platform provides a reproducible, high throughput method for performing electrical measurements to study transmembrane proteins and biomembranes in low-volume, droplet-based membranes. PMID:27513561

  13. Well-characterized sequence features of eukaryote genomes and implications for ab initio gene prediction.

    PubMed

    Huang, Ying; Chen, Shi-Yi; Deng, Feilong

    2016-01-01

    In silico analysis of DNA sequences is an important area of computational biology in the post-genomic era. Over the past two decades, computational approaches for ab initio prediction of gene structure from genome sequence alone have largely facilitated our understanding on a variety of biological questions. Although the computational prediction of protein-coding genes has already been well-established, we are also facing challenges to robustly find the non-coding RNA genes, such as miRNA and lncRNA. Two main aspects of ab initio gene prediction include the computed values for describing sequence features and used algorithm for training the discriminant function, and by which different combinations are employed into various bioinformatic tools. Herein, we briefly review these well-characterized sequence features in eukaryote genomes and applications to ab initio gene prediction. The main purpose of this article is to provide an overview to beginners who aim to develop the related bioinformatic tools. PMID:27536341

  14. Characterization of apparently balanced chromosomal rearrangements from the developmental genome anatomy project.

    PubMed

    Higgins, Anne W; Alkuraya, Fowzan S; Bosco, Amy F; Brown, Kerry K; Bruns, Gail A P; Donovan, Diana J; Eisenman, Robert; Fan, Yanli; Farra, Chantal G; Ferguson, Heather L; Gusella, James F; Harris, David J; Herrick, Steven R; Kelly, Chantal; Kim, Hyung-Goo; Kishikawa, Shotaro; Korf, Bruce R; Kulkarni, Shashikant; Lally, Eric; Leach, Natalia T; Lemyre, Emma; Lewis, Janine; Ligon, Azra H; Lu, Weining; Maas, Richard L; MacDonald, Marcy E; Moore, Steven D P; Peters, Roxanna E; Quade, Bradley J; Quintero-Rivera, Fabiola; Saadi, Irfan; Shen, Yiping; Shendure, Jay; Williamson, Robin E; Morton, Cynthia C

    2008-03-01

    Apparently balanced chromosomal rearrangements in individuals with major congenital anomalies represent natural experiments of gene disruption and dysregulation. These individuals can be studied to identify novel genes critical in human development and to annotate further the function of known genes. Identification and characterization of these genes is the goal of the Developmental Genome Anatomy Project (DGAP). DGAP is a multidisciplinary effort that leverages the recent advances resulting from the Human Genome Project to increase our understanding of birth defects and the process of human development. Clinically significant phenotypes of individuals enrolled in DGAP are varied and, in most cases, involve multiple organ systems. Study of these individuals' chromosomal rearrangements has resulted in the mapping of 77 breakpoints from 40 chromosomal rearrangements by FISH with BACs and fosmids, array CGH, Southern-blot hybridization, MLPA, RT-PCR, and suppression PCR. Eighteen chromosomal breakpoints have been cloned and sequenced. Unsuspected genomic imbalances and cryptic rearrangements were detected, but less frequently than has been reported previously. Chromosomal rearrangements, both balanced and unbalanced, in individuals with multiple congenital anomalies continue to be a valuable resource for gene discovery and annotation.

  15. Characterization of the complete mitochondrial genome of flower-breeding Drosophila incompta (Diptera, Drosophilidae).

    PubMed

    De Ré, F C; Wallau, G L; Robe, L J; Loreto, E L S

    2014-12-01

    Drosophila incompta belongs to the flavopilosa group of Drosophila, and has a restricted ecology, being adapted to flowers of Cestrum as feeding and oviposition sites. We sequenced, assembled, and characterized the complete mitochondrial genome (mtDNA) of D. incompta. In addition, we performed phylogenomic and polymorphism analyses to assess evolutionary diversification of this species. Our results suggest that this genome is syntenic with the other published mtDNA of Drosophila. This molecule contains 15,641 bp and encompasses two rRNA, 22 tRNA and 13 protein-coding genes. Regarding nucleotide composition, we found a high A-T bias (76.6 %). The recovered phylogenies indicate D. incompta in the virilis-repleta radiation, as sister to the virilis or repleta groups. The most interesting result is the high degree of polymorphism found throughout the D. incompta mitogenome, revealing pronounced intrapopulational variation. Furthermore, intraspecific nucleotide diversity levels varied between different regions of the genome, thus allowing the use of different mitochondrial molecular markers for analysis of population structure of this species.

  16. Genome-wide characterization of the Pectate Lyase-like (PLL) genes in Brassica rapa.

    PubMed

    Jiang, Jingjing; Yao, Lina; Miao, Ying; Cao, Jiashu

    2013-11-01

    Pectate lyases (PL) depolymerize demethylated pectin (pectate, EC 4.2.2.2) by catalyzing the eliminative cleavage of α-1,4-glycosidic linked galacturonan. Pectate Lyase-like (PLL) genes are one of the largest and most complex families in plants. However, studies on the phylogeny, gene structure, and expression of PLL genes are limited. To understand the potential functions of PLL genes in plants, we characterized their intron-exon structure, phylogenetic relationships, and protein structures, and measured their expression patterns in various tissues, specifically the reproductive tissues in Brassica rapa. Sequence alignments revealed two characteristic motifs in PLL genes. The chromosome location analysis indicated that 18 of the 46 PLL genes were located in the least fractionated sub-genome (LF) of B. rapa, while 16 were located in the medium fractionated sub-genome (MF1) and 12 in the more fractionated sub-genome (MF2). Quantitative RT-PCR analysis showed that BrPLL genes were expressed in various tissues, with most of them being expressed in flowers. Detailed qRT-PCR analysis identified 11 pollen specific PLL genes and several other genes with unique spatial expression patterns. In addition, some duplicated genes showed similar expression patterns. The phylogenetic analysis identified three PLL gene subfamilies in plants, among which subfamily II might have evolved from gene neofunctionalization or subfunctionalization. Therefore, this study opens the possibility for exploring the roles of PLL genes during plant development.

  17. Genome-wide identification and characterization of aquaporin gene family in moso bamboo (Phyllostachys edulis).

    PubMed

    Sun, Huayu; Li, Lichao; Lou, Yongfeng; Zhao, Hansheng; Gao, Zhimin

    2016-05-01

    Aquaporins (AQPs) are known to play a major role in maintaining water and hydraulic conductivity balance in the plant system. Numerous studies have showed AQPs execute multi-function throughout plant growth and development, including water transport, nitrogen, carbon, and micronutrient acquisition etc. However, little information on AQPs is known in bamboo. In this study, we present the first genome-wide identification and characterization of AQP genes in moso bamboo (Phyllostachys edulis) using bioinformatics. In total, 26 AQP genes were identified by homologous analysis, which were divided into four groups (PIPs, TIPs, NIPs, and SIPs) based on the phylogenetic analysis. All the genes were located on 26 different scaffolds respectively on basis of the gene mapped to bamboo genome. Evolutionary analysis indicated that Ph. edulis was more close to Oryza sativa than Zea mays in the genetic relationship. Besides, qRT-PCR was used to analyze gene expression profiles, which revealed that AQP genes were expressed constitutively in all the detected tissues, and were all responsive to the environmental cues such as drought, water, and NaCl stresses. This data suggested that AQPs may play fundamental roles in maintaining normal growth and development of bamboo, which would contribute to better understanding for the complex regulation mechanism involved in the fast-growing process of bamboo. Furthermore, the result could provide valuable information for further research on bamboo functional genomics. PMID:26993482

  18. GeneSV - an Approach to Help Characterize Possible Variations in Genomic and Protein Sequences.

    PubMed

    Zemla, Adam; Kostova, Tanya; Gorchakov, Rodion; Volkova, Evgeniya; Beasley, David W C; Cardosa, Jane; Weaver, Scott C; Vasilakis, Nikos; Naraghi-Arani, Pejman

    2014-01-01

    A computational approach for identification and assessment of genomic sequence variability (GeneSV) is described. For a given nucleotide sequence, GeneSV collects information about the permissible nucleotide variability (changes that potentially preserve function) observed in corresponding regions in genomic sequences, and combines it with conservation/variability results from protein sequence and structure-based analyses of evaluated protein coding regions. GeneSV was used to predict effects (functional vs. non-functional) of 37 amino acid substitutions on the NS5 polymerase (RdRp) of dengue virus type 2 (DENV-2), 36 of which are not observed in any publicly available DENV-2 sequence. 32 novel mutants with single amino acid substitutions in the RdRp were generated using a DENV-2 reverse genetics system. In 81% (26 of 32) of predictions tested, GeneSV correctly predicted viability of introduced mutations. In 4 of 5 (80%) mutants with double amino acid substitutions proximal in structure to one another GeneSV was also correct in its predictions. Predictive capabilities of the developed system were illustrated on dengue RNA virus, but described in the manuscript a general approach to characterize real or theoretically possible variations in genomic and protein sequences can be applied to any organism. PMID:24453480

  19. Biological characterization and complete genomic sequence of Apium virus Y infecting celery.

    PubMed

    Xu, Donglin; Liu, Hsing-Yeh; Koike, Steven T; Li, Fan; Li, Ruhui

    2011-01-01

    A celery isolate of Apium virus Y (ApVY-Ce) from diseased plants in a commercial field in California was characterized. The experimental host range of the virus included 13 plant species in the families Apiaceae, Chenopodiaceae and Solanaceae. Almost all infected plant species showed foliar chlorosis and distortion or severe stunting and systemic chlorosis. ApVY-Ce was transmitted to all 10 host species in the Apiaceae by green peach aphids. It reacted with the potyvirus group antibody and Celery mosaic virus (CeMV) antiserum. The complete genomic sequence of ApVY-Ce was determined to be 9917 nucleotides, excluding the 3' poly(A) tail, and it comprises a large open reading frame encoding a polyprotein of 3184 amino acid residues. Its genomic organization is typical of potyviruses, and contains conserved motifs found in the genus Potyvirus. Comparisons with available genomic sequences of other potyviruses indicate that ApVY-Ce shares 26.1-52.9% identities with species of the existing genera and unassigned viruses in the Potyviridae at the polyprotein sequence level. Extensive phylogenetic analysis based on the 3'-partial sequences confirms that ApVY-Ce is most closely related to CeMV and is a distinct species of the genus Potyvirus.

  20. Genomic and proteomic characterization of SE-I, a temperate bacteriophage infecting Erysipelothrix rhusiopathiae.

    PubMed

    Yuan, Wentao; Zhang, Yaning; Wang, Guangcao; Bai, Juan; Wang, Xianwei; Li, Yufeng; Jiang, Ping

    2016-11-01

    A bacteriophage infecting pathogenic Erysipelothrix rhusiopathiae was isolated from a swine farm experiencing an outbreak of acute swine erysipelas; we designated this phage SE-I. SE-I has an icosahedral head, a long tail and a double-stranded DNA genome. The 34,997-bp genome has a GC content of 34 % and contains 43 open reading frames (ORFs) encoding packaging, structural, lysin-holin, and hypothetical proteins. Components of purified SE-I were separated using SDS-PAGE and analyzed using liquid chromatography-mass spectrometry. Nine proteins were identified, encoded by ORF9, ORF15, ORF23, ORF30, ORF31, ORF33, ORF39, ORF40 and ORF 42. A phylogenetic tree constructed based on the sequence of the large terminase subunit revealed that SE-I is closely related to Staphylococcus phages P954 and phi3396. The CHAP-domain-containing protein encoded by ORF25 was expressed in E. coli and which was able to inactivate host bacteria. SE-I was able to infect 7 of 13 E. rhusiopathiae strains, but was unable to infect Salmonella, Streptococcus suis, and Staphylococcus aureus. This is the first report of the isolation, characterization, and genomic and proteomic analysis of a temperate phage infecting E. rhusiopathiae, and it might lead to the development of new anti- E. rhusiopathiae agents. PMID:27541818

  1. Full-length sequencing and genomic characterization of Bagaza, Kedougou, and Zika viruses.

    PubMed

    Kuno, G; Chang, G-J J

    2007-01-01

    Many members of the genus Flavivirus are the agents of important diseases of humans, livestock, and wildlife. Currently, no complete genome sequence is available for the three African viruses, Bagaza, Zika, and Kedougou viruses, each representing a distinct virus subgroup according to the latest virus classification. In this study, we obtained a complete genome sequence of each of those three viruses and characterized the open reading frames (ORFs) with respect to gene sizes, cleavage sites, potential glycosylation sites, distribution of cysteine residues, and unique motifs. The sequences of the three viruses were then scanned across the entire length of the ORF against available sequences of other African flaviviruses and selected reference viruses for genetic relatedness. The data collectively indicated that Kedougou virus was close to dengue viruses but nonetheless distinct, while Bagaza virus shared genetic relatedness with West Nile virus in several genomic regions. In the non-coding regions, it was found that a particular organizational pattern of conserved sequences in the 3' terminal region generally correlated with the current virus grouping.

  2. Characterization and distribution of repetitive elements in association with genes in the human genome.

    PubMed

    Liang, Kai-Chiang; Tseng, Joseph T; Tsai, Shaw-Jenq; Sun, H Sunny

    2015-08-01

    Repetitive elements constitute more than 50% of the human genome. Recent studies implied that the complexity of living organisms is not just a direct outcome of a number of coding sequences; the repetitive elements, which do not encode proteins, may also play a significant role. Though scattered studies showed that repetitive elements in the regulatory regions of a gene control gene expression, no systematic survey has been done to report the characterization and distribution of various types of these repetitive elements in the human genome. Sequences from 5' and 3' untranslated regions and upstream and downstream of a gene were downloaded from the Ensembl database. The repetitive elements in the neighboring of each gene were identified and classified using cross-matching implemented in the RepeatMasker. The annotation and distribution of distinct classes of repetitive elements associated with individual gene were collected to characterize genes in association with different types of repetitive elements using systems biology program. We identified a total of 1,068,400 repetitive elements which belong to 37-class families and 1235 subclasses that are associated with 33,761 genes and 57,365 transcripts. In addition, we found that the tandem repeats preferentially locate proximal to the transcription start site (TSS) of genes and the major function of these genes are involved in developmental processes. On the other hand, interspersed repetitive elements showed a tendency to be accumulated at distal region from the TSS and the function of interspersed repeat-containing genes took part in the catabolic/metabolic processes. Results from the distribution analysis were collected and used to construct a gene-based repetitive element database (GBRED; http://www.binfo.ncku.edu.tw/GBRED/index.html). A user-friendly web interface was designed to provide the information of repetitive elements associated with any particular gene(s). This is the first study focusing on the gene

  3. Genome wide characterization of simple sequence repeats in watermelon genome and their application in comparative mapping and genetic diversity analysis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Simple sequence repeats (SSR) or microsatellite markers are one of the most informative and versatile DNA-based markers. The use of next-generation sequencing technologies allow whole genome sequencing and make it possible to develop large numbers of SSRs through bioinformatic analysis of genome da...

  4. Identification and Characterization of Shared Duplications between Rice and Wheat Provide New Insight into Grass Genome Evolution[W

    PubMed Central

    Salse, Jérôme; Bolot, Stéphanie; Throude, Michaël; Jouffe, Vincent; Piegu, Benoît; Quraishi, Umar Masood; Calcagno, Thomas; Cooke, Richard; Delseny, Michel; Feuillet, Catherine

    2008-01-01

    The grass family comprises the most important cereal crops and is a good system for studying, with comparative genomics, mechanisms of evolution, speciation, and domestication. Here, we identified and characterized the evolution of shared duplications in the rice (Oryza sativa) and wheat (Triticum aestivum) genomes by comparing 42,654 rice gene sequences with 6426 mapped wheat ESTs using improved sequence alignment criteria and statistical analysis. Intraspecific comparisons identified 29 interchromosomal duplications covering 72% of the rice genome and 10 duplication blocks covering 67.5% of the wheat genome. Using the same methodology, we assessed orthologous relationships between the two genomes and detected 13 blocks of colinearity that represent 83.1 and 90.4% of the rice and wheat genomes, respectively. Integration of the intraspecific duplications data with colinearity relationships revealed seven duplicated segments conserved at orthologous positions. A detailed analysis of the length, composition, and divergence time of these duplications and comparisons with sorghum (Sorghum bicolor) and maize (Zea mays) indicated common and lineage-specific patterns of conservation between the different genomes. This allowed us to propose a model in which the grass genomes have evolved from a common ancestor with a basic number of five chromosomes through a series of whole genome and segmental duplications, chromosome fusions, and translocations. PMID:18178768

  5. Rapid parallel evolution of standing variation in a single, complex, genomic region is associated with life history in steelhead/rainbow trout.

    PubMed

    Pearse, Devon E; Miller, Michael R; Abadía-Cardoso, Alicia; Garza, John Carlos

    2014-05-22

    Rapid adaptation to novel environments may drive changes in genomic regions through natural selection. Such changes may be population-specific or, alternatively, may involve parallel evolution of the same genomic region in multiple populations, if that region contains genes or co-adapted gene complexes affecting the selected trait(s). Both quantitative and population genetic approaches have identified associations between specific genomic regions and the anadromous (steelhead) and resident (rainbow trout) life-history strategies of Oncorhynchus mykiss. Here, we use genotype data from 95 single nucleotide polymorphisms and show that the distribution of variation in a large region of one chromosome, Omy5, is strongly associated with life-history differentiation in multiple above-barrier populations of rainbow trout and their anadromous steelhead ancestors. The associated loci are in strong linkage disequilibrium, suggesting the presence of a chromosomal inversion or other rearrangement limiting recombination. These results provide the first evidence of a common genomic basis for life-history variation in O. mykiss in a geographically diverse set of populations and extend our knowledge of the heritable basis of rapid adaptation of complex traits in novel habitats.

  6. Genome-wide characterization of microsatellites and marker development in the carcinogenic liver fluke Clonorchis sinensis.

    PubMed

    Nguyen, Thao T B; Arimatsu, Yuji; Hong, Sung-Jong; Brindley, Paul J; Blair, David; Laha, Thewarach; Sripa, Banchob

    2015-06-01

    Clonorchis sinensis is an important carcinogenic human liver fluke endemic in East and Southeast Asia. There are several conventional molecular markers that have been used for identification and genetic diversity; however, no information about microsatellites of this liver fluke is published so far. We here report microsatellite characterization and marker development for a genetic diversity study in C. sinensis, using a genome-wide bioinformatics approach. Based on our search criteria, a total of 256,990 microsatellites (≥12 base pairs) were identified from a genome database of C. sinensis, with hexanucleotide motif being the most abundant (51%) followed by pentanucleotide (18.3%) and trinucleotide (12.7%). The tetranucleotide, dinucleotide, and mononucleotide motifs accounted for 9.75, 7.63, and 0.14%, respectively. The total length of all microsatellites accounts for 0. 72% of 547 Mb of the whole genome size, and the frequency of microsatellites was found to be one microsatellite in every 2.13 kb of DNA. For the di-, tri-, and tetranucleotide, the repeat numbers redundant are six (28%), four (45%), and three (76%), respectively. The ATC repeat is the most abundant microsatellites followed by AT, AAT, and AC, respectively. Within 40 microsatellite loci developed, 24 microsatellite markers showed potential to differentiate between C. sinensis and Opisthorchis viverrini. Seven out of 24 loci showed to be heterozygous with observed heterozygosity that ranged from 0.467 to 1. Four primer sets could amplify both C. sinensis and O. viverrini DNA with different sizes. This study provides basic information of C. sinensis microsatellites, and the genome-wide markers developed may be a useful tool for the genetic study of C. sinensis. PMID:25782682

  7. Genomic characterization of sex-identification markers in Sebastes carnatus and Sebastes chrysomelas rockfishes.

    PubMed

    Fowler, Benjamin L S; Buonaccorsi, Vincent P

    2016-05-01

    Fish have evolved a variety of sex-determining (SD) systems including male heterogamy (XY), female heterogamy (ZW) and environmental SD. Little is known about SD mechanisms of Sebastes rockfishes, a highly speciose genus of importance to evolutionary and conservation biology. Here, we characterize the sex determination system in the sympatrically distributed sister species Sebastes chrysomelas and Sebastes carnatus. To identify sex-specific genotypic markers, double digest restriction site - associated DNA sequencing (ddRAD-seq) of genomic DNA from 40 sexed individuals of both species was performed. Loci were filtered for presence in all of the individuals of one sex, absence in the other sex and no heterozygosity. Of the 74 965 loci present in all males, 33 male-specific loci met the criteria in at least one species and 17 in both. Conversely, no female-specific loci were detected, together providing evidence of an XY sex determination system in both species. When aligned to a draft reference genome from Sebastes aleutianus, 26 sex-specific loci were interspersed among 1168 loci that were identical between sexes. The nascent Y chromosome averaged 5% divergence from the X chromosome and mapped to reference Sebastes genome scaffolds totalling 6.9Mbp in length. These scaffolds aligned to a single chromosome in three model fish genomes. Read coverage differences were also detected between sex-specific and autosomal loci. A PCR-RFLP assay validated the bioinformatic results and correctly identified sex of five additional individuals of known sex. A sex-determining gene in other teleosts gonadal soma-derived factor (gsdf) was present in the model fish chromosomes that spanned our sex-specific markers. PMID:26923740

  8. Identification and complete genome characterization of a novel picornavirus in turkey (Meleagris gallopavo)

    PubMed Central

    Boros, Ákos; Nemes, Csaba; Pankovics, Péter; Kapusinszky, Beatrix; Delwart, Eric

    2012-01-01

    Members of the family Picornaviridae are important pathogens of humans and animals, although compared with the thousands of known bird species (>10 000), only a few (n = 11) picornaviruses have been identified from avian sources. This study reports the metagenomic detection and complete genome characterization of a novel turkey picornavirus from faecal samples collected from eight turkey farms in Hungary. Using RT-PCR, both healthy (two of three) and affected (seven of eight) commercial turkeys with enteric and/or stunting syndrome were shown to be shedding viruses in seven (88 %) of the eight farms. The viral genome sequence (turkey/M176/2011/HUN; GenBank accession no. JQ691613) shows a high degree of amino acid sequence identity (96 %) to the partial P3 genome region of a picornavirus reported recently in turkey and chickens from the USA and probably belongs to the same species. In the P1 and P2 regions, turkey/M176/2011/HUN is related most closely to, but distinct from, the kobuviruses and turdivirus 1. Complete genome analysis revealed the presence of characteristic picornaviral amino acid motifs, a potential type II-like 5′ UTR internal ribosome entry site (first identified among avian-origin picornaviruses) and a conserved, 48 nt long ‘barbell-like’ structure found at the 3′ UTR of turkey/M176/2011/HUN and members of the picornavirus genera Avihepatovirus and Kobuvirus. The general presence of turkey picornavirus – a novel picornavirus species – in faecal samples from healthy and affected turkeys in Hungary and in the USA suggests the worldwide occurrence and endemic circulation of this virus in turkey farms. Further studies are needed to investigate the aetiological role and pathogenic potential of this picornavirus in food animals. PMID:22875254

  9. Identification and complete genome characterization of a novel picornavirus in turkey (Meleagris gallopavo).

    PubMed

    Boros, Ákos; Nemes, Csaba; Pankovics, Péter; Kapusinszky, Beatrix; Delwart, Eric; Reuter, Gábor

    2012-10-01

    Members of the family Picornaviridae are important pathogens of humans and animals, although compared with the thousands of known bird species (>10 000), only a few (n = 11) picornaviruses have been identified from avian sources. This study reports the metagenomic detection and complete genome characterization of a novel turkey picornavirus from faecal samples collected from eight turkey farms in Hungary. Using RT-PCR, both healthy (two of three) and affected (seven of eight) commercial turkeys with enteric and/or stunting syndrome were shown to be shedding viruses in seven (88 %) of the eight farms. The viral genome sequence (turkey/M176/2011/HUN; GenBank accession no. JQ691613) shows a high degree of amino acid sequence identity (96 %) to the partial P3 genome region of a picornavirus reported recently in turkey and chickens from the USA and probably belongs to the same species. In the P1 and P2 regions, turkey/M176/2011/HUN is related most closely to, but distinct from, the kobuviruses and turdivirus 1. Complete genome analysis revealed the presence of characteristic picornaviral amino acid motifs, a potential type II-like 5' UTR internal ribosome entry site (first identified among avian-origin picornaviruses) and a conserved, 48 nt long 'barbell-like' structure found at the 3' UTR of turkey/M176/2011/HUN and members of the picornavirus genera Avihepatovirus and Kobuvirus. The general presence of turkey picornavirus - a novel picornavirus species - in faecal samples from healthy and affected turkeys in Hungary and in the USA suggests the worldwide occurrence and endemic circulation of this virus in turkey farms. Further studies are needed to investigate the aetiological role and pathogenic potential of this picornavirus in food animals.

  10. Genome-wide characterization of microsatelittes and marker development in the carcinogenic liver fluke Clonorchis sinensis

    PubMed Central

    Nguyen, Thao T.B.; Arimatsu, Yuji; Hong, Sung-Jong; Brindley, Paul J.; Blair, David; Laha, Thewarach; Sripa, Banchob

    2015-01-01

    Clonorchis sinensis is an important carcinogenic human liver fluke endemic in East and Southeast Asia. There are several conventional molecular markers have been used for identification and genetic diversity, however, no information about microsatellites of this liver fluke published so far. We here report microsatellite characterization and marker development for genetic diversity study in C. sinensis using genome-wide bioinformatics approach. Based on our search criteria, a total of 256,990 microsatellites (≥ 12 base pairs) were identified from genome database of C. sinensis with hexa-nucleotide motif being the most abundant (51%) followed by penta-nucleotide (18.3%) and tri-nucleotide (12.7%). The tetra-nucleotide, di-nucleotide and mononucleotide motifs accounted for 9.75 %, 7.63% and 0.14%, respectively. The total length of all microsatellites accounts for 0. 72 % of 547 Mb of the whole genome size and the frequency of microsatellites were found to be one microsatellite in every 2.13 kb of DNA. For the di-, tri, and tetra-nucleotide, the repeat numbers redundant are six (28%), four (45%) and three (76%), respectively. The ATC repeat is the most abundant microsatellites followed by AT, AAT and AC, respectively. Within 40 microsatellite loci developed, 24 microsatellite markers showed potential to differentiate between C. sinensis and O. viverrini. Seven out of 24 loci showed heterozygous with observed heterozygosity ranged from 0.467 to 1. Four-primer sets could amplify both C. sinensis and O. viverrini DNA with different sizes. This study provides basic information of C. sinensis microsatellites and the genome-wide markers developed may be a useful tool for genetic study of C. sinensis. PMID:25782682

  11. Genomic characterization of viral integration sites in HPV-related cancers.

    PubMed

    Bodelon, Clara; Untereiner, Michael E; Machiela, Mitchell J; Vinokurova, Svetlana; Wentzensen, Nicolas

    2016-11-01

    Persistent infection with carcinogenic human papillomaviruses (HPV) causes the majority of anogenital cancers and a subset of head and neck cancers. The HPV genome is frequently found integrated into the host genome of invasive cancers. The mechanisms of how it may promote disease progression are not well understood. Thoroughly characterizing integration events can provide insights into HPV carcinogenesis. Individual studies have reported limited number of integration sites in cell lines and human samples. We performed a systematic review of published integration sites in HPV-related cancers and conducted a pooled analysis to formally test for integration hotspots and genomic features enriched in integration events using data from the Encyclopedia of DNA Elements (ENCODE). Over 1,500 integration sites were reported in the literature, of which 90.8% (N = 1,407) were in human tissues. We found 10 cytobands enriched for integration events, three previously reported ones (3q28, 8q24.21 and 13q22.1) and seven additional ones (2q22.3, 3p14.2, 8q24.22, 14q24.1, 17p11.1, 17q23.1 and 17q23.2). Cervical infections with HPV18 were more likely to have breakpoints in 8q24.21 (p = 7.68 × 10(-4) ) than those with HPV16. Overall, integration sites were more likely to be in gene regions than expected by chance (p = 6.93 × 10(-9) ). They were also significantly closer to CpG regions, fragile sites, transcriptionally active regions and enhancers. Few integration events occurred within 50 Kb of known cervical cancer driver genes. This suggests that HPV integrates in accessible regions of the genome, preferentially genes and enhancers, which may affect the expression of target genes. PMID:27343048

  12. Variability among the Most Rapidly Evolving Plastid Genomic Regions is Lineage-Specific: Implications of Pairwise Genome Comparisons in Pyrus (Rosaceae) and Other Angiosperms for Marker Choice

    PubMed Central

    Ter-Voskanyan, Hasmik; Allgaier, Martin; Borsch, Thomas

    2014-01-01

    Plastid genomes exhibit different levels of variability in their sequences, depending on the respective kinds of genomic regions. Genes are usually more conserved while noncoding introns and spacers evolve at a faster pace. While a set of about thirty maximum variable noncoding genomic regions has been suggested to provide universally promising phylogenetic markers throughout angiosperms, applications often require several regions to be sequenced for many individuals. Our project aims to illuminate evolutionary relationships and species-limits in the genus Pyrus (Rosaceae)—a typical case with very low genetic distances between taxa. In this study, we have sequenced the plastid genome of Pyrus spinosa and aligned it to the already available P. pyrifolia sequence. The overall p-distance of the two Pyrus genomes was 0.00145. The intergenic spacers between ndhC–trnV, trnR–atpA, ndhF–rpl32, psbM–trnD, and trnQ–rps16 were the most variable regions, also comprising the highest total numbers of substitutions, indels and inversions (potentially informative characters). Our comparative analysis of further plastid genome pairs with similar low p-distances from Oenothera (representing another rosid), Olea (asterids) and Cymbidium (monocots) showed in each case a different ranking of genomic regions in terms of variability and potentially informative characters. Only two intergenic spacers (ndhF–rpl32 and trnK–rps16) were consistently found among the 30 top-ranked regions. We have mapped the occurrence of substitutions and microstructural mutations in the four genome pairs. High AT content in specific sequence elements seems to foster frequent mutations. We conclude that the variability among the fastest evolving plastid genomic regions is lineage-specific and thus cannot be precisely predicted across angiosperms. The often lineage-specific occurrence of stem-loop elements in the sequences of introns and spacers also governs lineage-specific mutations

  13. Ultra-rapid preparation of total genomic DNA from isolates of yeast and mould using Whatman FTA filter paper technology - a reusable DNA archiving system.

    PubMed

    Borman, Andrew M; Linton, Christopher J; Miles, Sarah-Jane; Campbell, Colin K; Johnson, Elizabeth M

    2006-08-01

    Conventional methods for purifying PCR-grade fungal genomic DNA typically require cell disruption (either physical or enzymatic) coupled with laborious organic extraction and precipitation stages, or expensive column-based technologies. Here we present an easy and extremely rapid method of preparing yeast and mould genomic DNAs from living cultures using Whatman FTA filter matrix technology. Aqueous suspensions of yeast cells or hyphal fragments and conidia (in the case of moulds) are applied directly (or after freeze-thawing) to dry FTA filters. Inoculated filters are then subjected to brief microwave treatment, to dry the filters and inactivate the organisms. Filter punches are removed, washed rapidly, dried and placed directly into PCR reactions. We show that this procedure inactivated all of the 38 yeast and 75 mould species tested, and generated PCR-grade DNA preparations in around 15 minutes. A total of 218 out of 226 fungal isolates tested liberated amplifiable DNA after application to FTA filters. Detection limits with yeast cultures were approximately 10 colony-forming units per punch. Moreover, we demonstrate that filter punches can be recovered after PCR, washed and used in fresh PCR reactions without detectable cross-contamination. Whatman FTA technology thus represents a cheap, ultra-rapid method of fungal genomic DNA preparation, and also potentially represents a powerful fungal DNA archiving and storage system. PMID:16882605

  14. Genomic characterization, expression analysis, and antimicrobial function of a glyrichin homologue from rock bream, Oplegnathus fasciatus.

    PubMed

    Kasthuri, Saranya Revathy; Wan, Qiang; Umasuthan, Navaneethaiyer; Bathige, S D N K; Lim, Bong-Soo; Jung, Hyung-Bok; Lee, Jehee; Whang, Ilson

    2013-11-01

    Antimicrobial peptides are important innate effector molecules, playing a vital role in antimicrobial immunity in all species. Glyrichin is a transmembrane protein and an antibacterial peptide, exerting its functions against a wide range of pathogenic bacteria. In this study, cDNA and a BAC clone harboring the glyrichin gene were identified from rock bream and characterized. Genomic characterization showed that the OfGlyrichin gene exhibited a 3 exon-2 intron structure. OfGlyrichin is a 79-amino-acid protein with a transmembrane domain at (22)GFMMGFAVGMAAGAMFGTFSCLR(44). Pairwise and multiple sequence alignments showed high identity and conservation with mammalian orthologues. Phylogenetic analysis showed a close relationship with fish species. Higher levels of OfGlyrichin transcripts were detected in the liver from healthy rock bream which were induced by immunogens like lipopolysaccharide, poly I:C, rock bream irido virus, Edwardsiella tarda and Streptococcus iniae. The synthetic peptide (pOf19) showed antibacterial activity against Escherichia coli, E. tarda, and S. iniae. Analysis of the bacterial morphological features after pOf19 peptide treatment showed breakage of the cell membrane, affirming that antibacterial function is accomplished through membrane lysis. The pOf19 peptide also showed antiviral activity against RBIV infection. The high conservation of the genomic structure and protein, together with the antimicrobial roles of OfGlyrichin, provide evidence for the evolutionary existence of this protein playing a vital role in innate immune defense in rock bream.

  15. Phenotypic and genomic characterization of human coxsackievirus A16 strains with distinct virulence in mice.

    PubMed

    Han, Jian-Feng; Yu, Nan; Pan, Yu-Xian; He, Si-Jie; Xu, Li-Juan; Cao, Rui-Yuan; Li, Yue-Xiang; Zhu, Shun-Ya; Zhang, Yu; Qin, E-De; Che, Xiao-Yan; Qin, Cheng-Feng

    2014-01-22

    Human coxsackievirus A16 (CA16) infection results in hand, foot, and mouth disease (HFMD) along with other severe neurological diseases in children and poses an important public health threat in Asian countries. During an HFMD epidemic in 2009 in Guangdong, China, two CA16 strains (GD09/119 and GD09/24) were isolated and characterized. Although both strains were similar in plaque morphology and growth properties in vitro, the two isolates exhibited distinct pathogenicity in neonatal mice upon intraperitoneal or intracranial injection. Complete genome sequences of both CA16 strains were determined, and the possible virulence determinants were analyzed and predicted. Phylogenetic analysis revealed that these CA16 isolates from Guangdong belonged to the B1b genotype and were closely related to other recent CA16 strains isolated in mainland China. Similarity and bootscanning analyses of these CA16 strains detected homologous recombination with the EV71 prototype strain BrCr in the non-structural gene regions and the 3'-untranslated regions. Together, the phenotypic and genomic characterizations of the two clinical CA16 isolates circulating in China were compared in detail, and the potential amino acid residues responsible for CA16 virulence in mice were predicted. These findings will help explain the evolutionary relationship of the CA16 strains circulating in China, warranting future studies investigating enterovirus virulence.

  16. Physiological and genomic characterization of Arcobacter anaerophilus IR-1 reveals new metabolic features in Epsilonproteobacteria

    PubMed Central

    Roalkvam, Irene; Drønen, Karine; Stokke, Runar; Daae, Frida L.; Dahle, Håkon; Steen, Ida H.

    2015-01-01

    In this study we characterized and sequenced the genome of Arcobacter anaerophilus strain IR-1 isolated from enrichment cultures used in nitrate-amended corrosion experiments. A. anaerophilus IR-1 could grow lithoautotrophically on hydrogen and hydrogen sulfide and lithoheterothrophically on thiosulfate and elemental sulfur. In addition, the strain grew organoheterotrophically on yeast extract, peptone, and various organic acids. We show for the first time that Arcobacter could grow on the complex organic substrate tryptone and oxidize acetate with elemental sulfur as electron acceptor. Electron acceptors utilized by most Epsilonproteobacteria, such as oxygen, nitrate, and sulfur, were also used by A. anaerophilus IR-1. Strain IR-1 was also uniquely able to use iron citrate as electron acceptor. Comparative genomics of the Arcobacter strains A. butzleri RM4018, A. nitrofigilis CI and A. anaerophilus IR-1 revealed that the free-living strains had a wider metabolic range and more genes in common compared to the pathogen strain. The presence of genes for NAD+-reducing hydrogenase (hox) and dissimilatory iron reduction (fre) were unique for A. anaerophilus IR-1 among Epsilonproteobacteria. Finally, the new strain had an incomplete denitrification pathway where the end product was nitrite, which is different from other Arcobacter strains where the end product is ammonia. Altogether, our study shows that traditional characterization in combination with a modern genomics approach can expand our knowledge on free-living Arcobacter, and that this complementary approach could also provide invaluable knowledge about the physiology and metabolic pathways in other Epsilonproteobacteria from various environments. PMID:26441916

  17. Genome-Wide Characterization of Pancreatic Adenocarcinoma Patients Using Next Generation Sequencing

    PubMed Central

    Demeure, Michael J.; Weiss, Glen J.; Izatt, Tyler; Sinari, Shripad; Christoforides, Alexis; Aldrich, Jessica; Kurdoglu, Ahmet; Barrett, Michael; Phillips, Lori; Benson, Hollie; Tembe, Waibhav; Braggio, Esteban; Kiefer, Jeffrey A.; Legendre, Christophe; Posner, Richard; Hostetter, Galen H.; Baker, Angela; Egan, Jan B.; Han, Haiyong; Lake, Douglas; Stites, Edward C.; Ramanathan, Ramesh K.; Fonseca, Rafael; Stewart, A. Keith; Von Hoff, Daniel

    2012-01-01

    Pancreatic adenocarcinoma (PAC) is among the most lethal malignancies. While research has implicated multiple genes in disease pathogenesis, identification of therapeutic leads has been difficult and the majority of currently available therapies provide only marginal benefit. To address this issue, our goal was to genomically characterize individual PAC patients to understand the range of aberrations that are occurring in each tumor. Because our understanding of PAC tumorigenesis is limited, evaluation of separate cases may reveal aberrations, that are less common but may provide relevant information on the disease, or that may represent viable therapeutic targets for the patient. We used next generation sequencing to assess global somatic events across 3 PAC patients to characterize each patient and to identify potential targets. This study is the first to report whole genome sequencing (WGS) findings in paired tumor/normal samples collected from 3 separate PAC patients. We generated on average 132 billion mappable bases across all patients using WGS, and identified 142 somatic coding events including point mutations, insertion/deletions, and chromosomal copy number variants. We did not identify any significant somatic translocation events. We also performed RNA sequencing on 2 of these patients' tumors for which tumor RNA was available to evaluate expression changes that may be associated with somatic events, and generated over 100 million mapped reads for each patient. We further performed pathway analysis of all sequencing data to identify processes that may be the most heavily impacted from somatic and expression alterations. As expected, the KRAS signaling pathway was the most heavily impacted pathway (P<0.05), along with tumor-stroma interactions and tumor suppressive pathways. While sequencing of more patients is needed, the high resolution genomic and transcriptomic information we have acquired here provides valuable information on the molecular

  18. Characterization of a genomic divergence island between black-and-yellow and gopher Sebastes rockfishes.

    PubMed

    Buonaccorsi, Vincent P; Narum, Shawn R; Karkoska, Kristine A; Gregory, Steven; Deptola, Travis; Weimer, Alexander B

    2011-06-01

    Islands of high genomic divergence that contain genes of evolutionary significance may form between diverging species. The gopher rockfish, Sebastes carnatus, and black-and-yellow rockfish, S. chrysomelas, are sympatrically distributed temperate marine species inhabiting rocky reefs and kelp forests on the west coast of the United States. Prior studies documented low levels of genetic divergence between the two species, except at a single microsatellite locus that displayed high divergence, Sra.7-2. To better characterize genome wide divergence, we scored 25 additional microsatellite loci. Mean neutral divergence between species (F(ST) = 0.01) changed little from prior estimates. Sra.7-2 continued to be an extreme divergence outlier. Five novel microsatellites within ± 15 kb of Sra.7-2 were characterized. High divergence, consistently low diversity in S. chrysomelas, and linkage disequilibrium were detected at these loci, suggesting the influence of recent selection. However, coalescent modelling of divergence at neutral and Sra.7-2 regions showed that initial divergence at Sra.7-2 was ancient, likely predating divergence at neutral regions. It is therefore unlikely that Sra.7-2 divergence represents solely recent ecological divergence within one species and may represent the action of recurrent selection. Introgressive gene flow (2N(E) m) was much higher (>1) at neutral than Sra.7-2 regions (<1) despite evidence that two S. carnatus individuals have recently mixed ancestry at the Sra.7-2 region. The Sra.7-2 genomic region is likely one of several regions containing genes involved in initiating and maintaining species integrity. Completion of the final stages of speciation appears to be a slow and ongoing process for these species. PMID:21557784

  19. Development and characterization of a rapid polymerizing collagen for soft tissue augmentation

    PubMed Central

    Devore, Dale; Zhu, Jiaxun; Brooks, Robert; McCrate, Rebecca Rone; Grant, David A.

    2015-01-01

    Abstract A liquid collagen has been developed that fibrilizes upon injection. Rapid polymerizing collagen (RPC) is a type I porcine collagen that undergoes fibrillization upon interaction with ionic solutions, such as physiological solutions. The ability to inject liquid collagen would be beneficial for many soft tissue augmentation applications. In this study, RPC was synthesized and characterized as a possible dermal filler. Transmission electron microscopy, ion induced RPC fibrillogenesis tests, collagenase resistance assay, and injection force studies were performed to assess RPC's physicochemical properties. An in vivo study was performed which consisted of a 1‐, 3‐, and 6‐month study where RPC was injected into the ears of miniature swine. The results demonstrated that the liquid RPC requires low injection force (<7 N); fibrillogenesis and banding of collagen occurs when RPC is injected into ionic solutions, and RPC has enhanced resistance to collagenase breakdown. The in vivo study demonstrated long‐term biocompatibility with low irritation scores. In conclusion RPC possesses many of the desirable properties of a soft tissue augmentation material. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 758–767, 2016. PMID:26488368

  20. Rapid Discovery and Functional Characterization of Terpene Synthases from Four Endophytic Xylariaceae

    PubMed Central

    Wu, Weihua; Tran, William; Taatjes, Craig A.; Alonso-Gutierrez, Jorge; Lee, Taek Soon; Gladden, John M.

    2016-01-01

    Endophytic fungi are ubiquitous plant endosymbionts that establish complex and poorly understood relationships with their host organisms. Many endophytic fungi are known to produce a wide spectrum of volatile organic compounds (VOCs) with potential energy applications, which have been described as "mycodiesel". Many of these mycodiesel hydrocarbons are terpenes, a chemically diverse class of compounds produced by many plants, fungi, and bacteria. Due to their high energy densities, terpenes, such as pinene and bisabolene, are actively being investigated as potential "drop-in" biofuels for replacing diesel and aviation fuel. In this study, we rapidly discovered and characterized 26 terpene synthases (TPSs) derived from four endophytic fungi known to produce mycodiesel hydrocarbons. The TPS genes were expressed in an E. coli strain harboring a heterologous mevalonate pathway designed to enhance terpene production, and their product profiles were determined using Solid Phase Micro-Extraction (SPME) and GC-MS. Out of the 26 TPS’s profiled, 12 TPS’s were functional, with the majority of them exhibiting both monoterpene and sesquiterpene synthase activity. PMID:26885833

  1. Rapid discovery and functional characterization of terpene synthases from four endophytic xylariaceae

    DOE PAGES

    Wu, Weihua; Tran, William; Taatjes, Craig A.; Alonso-Gutierrez, Jorge; Lee, Taek Soon; Gladden, John M.; Hamberger, Bjorn

    2016-02-17

    Endophytic fungi are ubiquitous plant endosymbionts that establish complex and poorly understood relationships with their host organisms. Many endophytic fungi are known to produce a wide spectrum of volatile organic compounds (VOCs) with potential energy applications, which have been described as "mycodiesel". Many of these mycodiesel hydrocarbons are terpenes, a chemically diverse class of compounds produced by many plants, fungi, and bacteria. Due to their high energy densities, terpenes, such as pinene and bisabolene, are actively being investigated as potential "drop-in" biofuels for replacing diesel and aviation fuel. In this study, we rapidly discovered and characterized 26 terpene synthases (TPSs)more » derived from four endophytic fungi known to produce mycodiesel hydrocarbons. The TPS genes were expressed in an E. coli strain harboring a heterologous mevalonate pathway designed to enhance terpene production, and their product profiles were determined using Solid Phase Micro-Extraction (SPME) and GC-MS. Lastly, out of the 26 TPS’s profiled, 12 TPS’s were functional, with the majority of them exhibiting both monoterpene and sesquiterpene synthase activity.« less

  2. Preparation and characterization of guluronic acid oligosaccharides degraded by a rapid microwave irradiation method.

    PubMed

    Hu, Ting; Li, Chunxia; Zhao, Xia; Li, Guangsheng; Yu, Guangli; Guan, Huashi

    2013-05-24

    Guluronic acid oligosaccharides (GOS) with degree of polymerization (DP) ranging from 1 to 10 were prepared by a rapid microwave degradation method. Polyguluronic acid, fractionated from alginate hydrolysate, was dissolved in dilute ammonia water at a concentration of 20 mg/mL (pH 5) and then hydrolyzed under microwave irradiation (1600 W) at 130°C for 15 min to produce GOS mixture. The GOS mixture was separated by a Bio-Gel P6 column and ten fractions were obtained. Each GOS fraction was further characterized by electrospray ionization mass spectrometry, (1)H NMR, (13)C NMR, and 2D NMR spectroscopy techniques. The data showed that the GOS fractions were saturated oligoguluronates with general molecular formula C(6n)H(8n+2)O(6n+1) (n=1-10). This microwave degradation method was not only convenient, less time consuming, and environment-friendly, but also produced GOS with high yield (71%) and eliminating a desalting procedure compared to conventional acid hydrolysis method. PMID:23584235

  3. Characterization and identification of clinically relevant microorganisms using rapid evaporative ionization mass spectrometry.

    PubMed

    Strittmatter, Nicole; Rebec, Monica; Jones, Emrys A; Golf, Ottmar; Abdolrasouli, Alireza; Balog, Julia; Behrends, Volker; Veselkov, Kirill A; Takats, Zoltan

    2014-07-01

    Rapid evaporative ionization mass spectrometry (REIMS) was investigated for its suitability as a general identification system for bacteria and fungi. Strains of 28 clinically relevant bacterial species were analyzed in negative ion mode, and corresponding data was subjected to unsupervised and supervised multivariate statistical analyses. The created supervised model yielded correct cross-validation results of 95.9%, 97.8%, and 100% on species, genus, and Gram-stain level, respectively. These results were not affected by the resolution of the mass spectral data. Blind identification tests were performed for strains cultured on different culture media and analyzed using different instrumental platforms which led to 97.8-100% correct identification. Seven different Escherichia coli strains were subjected to different culture conditions and were distinguishable with 88% accuracy. In addition, the technique proved suitable to distinguish five pathogenic Candida species with 98.8% accuracy without any further modification to the experimental workflow. These results prove that REIMS is sufficiently specific to serve as a culture condition-independent tool for the identification and characterization of microorganisms.

  4. Characterization of Rapidly Solidified Al-27 Si Hypereutectic Alloy: Effect of Solidification Condition

    NASA Astrophysics Data System (ADS)

    Cai, Zhiyong; Wang, Richu; Zhang, Chun; Peng, Chaoqun; Xie, Lichuan; Wang, Linqian

    2015-03-01

    Rapidly solidified Al-27wt.%Si hypereutectic alloy was fabricated by gas atomization, and its characterization as a function of particle size was investigated. A relationship between the particle size and solidification condition was established to understand the microstructural characteristics. While the irregular primary Si phase transformed to quasi-spherical shape, and its size decreased gradually with the particle size, the primary Si morphology similar to that in ingot metallurgy sample was found from the deep-etched images. In the fine powder, the eutectic Si phase formed a network structure densely distributed in the matrix, while a tangled dendritic formed at the surface. From the distribution of the Si phase, it is suggested that the microstructure inhomogeneity increased as the particle size decreases. The structural distortion of the Al matrix was observed from x-ray diffraction patterns and differential scanning calorimetry curves. From the calculated results, an undercooling of 33 K (or interface velocity of 8 mm/s) was sufficient to suppress the primary Si to less than 2 μm in the present composition. The microhardness increased significantly while the particle size decreases. The microstructure and properties of the bulk material consolidated by hot pressing of the powders obtained were also conducted.

  5. Rapid characterization of candidate biomarkers for pancreatic cancer using cell microarrays (CMAs).

    PubMed

    Kim, Min-Sik; Kuppireddy, Sarada V; Sakamuri, Sruthi; Singal, Mukul; Getnet, Derese; Harsha, H C; Goel, Renu; Balakrishnan, Lavanya; Jacob, Harrys K C; Kashyap, Manoj K; Tankala, Shantal G; Maitra, Anirban; Iacobuzio-Donahue, Christine A; Jaffee, Elizabeth; Goggins, Michael G; Velculescu, Victor E; Hruban, Ralph H; Pandey, Akhilesh

    2012-11-01

    Tissue microarrays have become a valuable tool for high-throughput analysis using immunohistochemical labeling. However, the large majority of biochemical studies are carried out in cell lines to further characterize candidate biomarkers or therapeutic targets with subsequent studies in animals or using primary tissues. Thus, cell line-based microarrays could be a useful screening tool in some situations. Here, we constructed a cell microarray (CMA) containing a panel of 40 pancreatic cancer cell lines available from American Type Culture Collection in addition to those locally available at Johns Hopkins. As proof of principle, we performed immunocytochemical labeling of an epithelial cell adhesion molecule (Ep-CAM), a molecule generally expressed in the epithelium, on this pancreatic cancer CMA. In addition, selected molecules that have been previously shown to be differentially expressed in pancreatic cancer in the literature were validated. For example, we observed strong labeling of CA19-9 antigen, a prognostic and predictive marker for pancreatic cancer. We also carried out a bioinformatics analysis of a literature curated catalog of pancreatic cancer biomarkers developed previously by our group and identified two candidate biomarkers, HLA class I and transmembrane protease, serine 4 (TMPRSS4), and examined their expression in the cell lines represented on the pancreatic cancer CMAs. Our results demonstrate the utility of CMAs as a useful resource for rapid screening of molecules of interest and suggest that CMAs can become a universal standard platform in cancer research.

  6. Rapid Discovery and Functional Characterization of Terpene Synthases from Four Endophytic Xylariaceae.

    PubMed

    Wu, Weihua; Tran, William; Taatjes, Craig A; Alonso-Gutierrez, Jorge; Lee, Taek Soon; Gladden, John M

    2016-01-01

    Endophytic fungi are ubiquitous plant endosymbionts that establish complex and poorly understood relationships with their host organisms. Many endophytic fungi are known to produce a wide spectrum of volatile organic compounds (VOCs) with potential energy applications, which have been described as "mycodiesel". Many of these mycodiesel hydrocarbons are terpenes, a chemically diverse class of compounds produced by many plants, fungi, and bacteria. Due to their high energy densities, terpenes, such as pinene and bisabolene, are actively being investigated as potential "drop-in" biofuels for replacing diesel and aviation fuel. In this study, we rapidly discovered and characterized 26 terpene synthases (TPSs) derived from four endophytic fungi known to produce mycodiesel hydrocarbons. The TPS genes were expressed in an E. coli strain harboring a heterologous mevalonate pathway designed to enhance terpene production, and their product profiles were determined using Solid Phase Micro-Extraction (SPME) and GC-MS. Out of the 26 TPS's profiled, 12 TPS's were functional, with the majority of them exhibiting both monoterpene and sesquiterpene synthase activity. PMID:26885833

  7. Total Antioxidant Capacity and Characterization of Nitraria tangutorum Fruit Extract by Rapid Bioassay-Directed Fractionation.

    PubMed

    Rana, Jat; Missler, Stephen R; Persons, Kathryn; Han, Johnson; Li, Teric

    2016-09-01

    In recent years, the role of reactive nitrogen and oxygen species (RNOS) in human disease has been the subject of considerable study. This has led to research on the potential benefit of natural products as dietary antioxidants to mitigate oxidative stress caused by increased RNOS associated with tissue damage. Five physiologically relevant reactive species include peroxyl radical, hydroxyl radical, peroxynitrite anion, superoxide radical anion, and singlet oxygen. Excessive amounts of these species can lead to the degradation of important biomolecules in vivo, and dietary antioxidants have been shown to inhibit damage both in vitro and in vivo. In this investigation, we have discovered that an extract of the fruit from Nitraria tangutorum Bobr. (Tangut white thorn) demonstrates significant antioxidant capacity against all five reactive species. Rapid bioassay-directed fractionation was used to identify antioxidant phytochemicals by collecting fractions from HPLC effluent into 96 well microplates and testing for antioxidant activity against the 2,2-diphenyl-1-picrylhydrazyl radical. Two different classes of phytochemicals, anthocyanins and flavonoids, were associated with antioxidant activity. Active components were further characterized by UV-Vis spectroscopy and high-resolution MS.

  8. Implementation of Whole Genome Sequencing (WGS) for Identification and Characterization of Shiga Toxin-Producing Escherichia coli (STEC) in the United States

    PubMed Central

    Lindsey, Rebecca L.; Pouseele, Hannes; Chen, Jessica C.; Strockbine, Nancy A.; Carleton, Heather A.

    2016-01-01

    Shiga toxin-producing Escherichia coli (STEC) is an important foodborne pathogen capable of causing severe disease in humans. Rapid and accurate identification and characterization techniques are essential during outbreak investigations. Current methods for characterization of STEC are expensive and time-consuming. With the advent of rapid and cheap whole genome sequencing (WGS) benchtop sequencers, the potential exists to replace traditional workflows with WGS. The aim of this study was to validate tools to do reference identification and characterization from WGS for STEC in a single workflow within an easy to use commercially available software platform. Publically available serotype, virulence, and antimicrobial resistance databases were downloaded from the Center for Genomic Epidemiology (CGE) (www.genomicepidemiology.org) and integrated into a genotyping plug-in with in silico PCR tools to confirm some of the virulence genes detected from WGS data. Additionally, down sampling experiments on the WGS sequence data were performed to determine a threshold for sequence coverage needed to accurately predict serotype and virulence genes using the established workflow. The serotype database was tested on a total of 228 genomes and correctly predicted from WGS for 96.1% of O serogroups and 96.5% of H serogroups identified by conventional testing techniques. A total of 59 genomes were evaluated to determine the threshold of coverage to detect the different WGS targets, 40 were evaluated for serotype and virulence gene detection and 19 for the stx gene subtypes. For serotype, 95% of the O and 100% of the H serogroups were detected at > 40x and ≥ 30x coverage, respectively. For virulence targets and stx gene subtypes, nearly all genes were detected at > 40x, though some targets were 100% detectable from genomes with coverage ≥20x. The resistance detection tool was 97% concordant with phenotypic testing results. With isolates sequenced to > 40x coverage, the different

  9. Rapid genomic changes in allopolyploids of Carassius auratus red var. (♀) × Megalobrama amblycephala (♂)

    PubMed Central

    Qin, Qinbo; Lai, Zhengfa; Cao, Liu; Xiao, Qiong; Wang, YuDe; Liu, Shaojun

    2016-01-01

    To better understand genomic changes in the early generations after polyploidisation, we examined the chromosomal consequences of genomic merger in allotetraploid hybrids (4 nF1) (AABB, 4n = 148) of Carassius auratus red var. (RCC) (AA, 2n = 100) (♀) × Megalobrama amblycephala (BSB) (BB, 2n = 48) (♂). Complete loss of the paternal 5S rDNA sequence and the expected number of maternal chromosomal loci were found in 4 nF1, suggesting directional genomic changes occurred in the first generations after polyploidisation. Recent studies have reported instability of newly established allotetraploid genomes. To assess this in the newly formed 4 nF1 genome, we performed fluorescence in situ hybridisation on an allotetraploid gynogenetic hybrid (4 nG) (AABB, 4n = 148) and an allopentaploid hybrid (5 nH) (AABBB, 5n = 172) from 4 nF1 (♀) × BSB (♂) with 5S rDNA gene and centromere probes from RCC, the original diploid parent. The expected numbers of maternal chromosomal loci were found in 4 nG, while chromosomal locus deletions and chromosome recombinations were detected in 5 nH. These observations suggest that abnormal meiosis did not lead to obvious genomic changes in the newly established allotetraploid genomes, but hybridisation with the original diploid parent resulted in obvious genomic changes in the newly established allotetraploid genomes, as was found for the maternal genome. PMID:27703178

  10. BlastKOALA and GhostKOALA: KEGG Tools for Functional Characterization of Genome and Metagenome Sequences.

    PubMed

    Kanehisa, Minoru; Sato, Yoko; Morishima, Kanae

    2016-02-22

    BlastKOALA and GhostKOALA are automatic annotation servers for genome and metagenome sequences, which perform KO (KEGG Orthology) assignments to characterize individual gene functions and reconstruct KEGG pathways, BRITE hierarchies and KEGG modules to infer high-level functions of the organism or the ecosystem. Both servers are made freely available at the KEGG Web site (http://www.kegg.jp/blastkoala/). In BlastKOALA, the KO assignment is performed by a modified version of the internally used KOALA algorithm after the BLAST search against a non-redundant dataset of pangenome sequences at the species, genus or family level, which is generated from the KEGG GENES database by retaining the KO content of each taxonomic category. In GhostKOALA, which utilizes more rapid GHOSTX for database search and is suitable for metagenome annotation, the pangenome dataset is supplemented with Cd-hit clusters including those for viral genes. The result files may be downloaded and manipulated for further KEGG Mapper analysis, such as comparative pathway analysis using multiple BlastKOALA results. PMID:26585406

  11. Comparative genomic and phylogenetic approaches to characterize the role of genetic recombination in mycobacterial evolution.

    PubMed

    Smith, Silvia E; Showers-Corneli, Patrice; Dardenne, Caitlin N; Harpending, Henry H; Martin, Darren P; Beiko, Robert G

    2012-01-01

    The genus Mycobacterium encompasses over one hundred named species of environmental and pathogenic organisms, including the causative agents of devastating human diseases such as tuberculosis and leprosy. The success of these human pathogens is due in part to their ability to rapidly adapt to their changing environment and host. Recombination is the fastest way for bacterial genomes to acquire genetic material, but conflicting results about the extent of recombination in the genus Mycobacterium have been reported. We examined a data set comprising 18 distinct strains from 13 named species for evidence of recombination. Genomic regions common to all strains (accounting for 10% to 22% of the full genomes of all examined species) were aligned and concatenated in the chromosomal order of one mycobacterial reference species. The concatenated sequence was screened for evidence of recombination using a variety of statistical methods, with each proposed event evaluated by comparing maximum-likelihood phylogenies of the recombinant section with the non-recombinant portion of the dataset. Incongruent phylogenies were identified by comparing the site-wise log-likelihoods of each tree using multiple tests. We also used a phylogenomic approach to identify genes that may have been acquired through horizontal transfer from non-mycobacterial sources. The most frequent associated lineages (and potential gene transfer partners) in the Mycobacterium lineage-restricted gene trees are other members of suborder Corynebacterinae, but more-distant partners were identified as well. In two examined cases of potentially frequent and habitat-directed transfer (M. abscessus to Segniliparus and M. smegmatis to Streptomyces), observed sequence distances were small and consistent with a hypothesis of transfer, while in a third case (M. vanbaalenii to Streptomyces) distances were larger. The analyses described here indicate that whereas evidence of recombination in core regions within the genus is

  12. Cloning and Characterization of a Human Genomic Sequence that Alleviates Repeat-Induced Gene Silencing

    PubMed Central

    Miura, Osamu; Ohyama, Takashi; Shimizu, Noriaki

    2016-01-01

    Plasmids bearing a mammalian replication initiation region (IR) and a nuclear matrix attachment region (MAR) are spontaneously amplified in transfected mammalian cells, and such amplification generates chromosomal homogeneously staining regions (HSRs) or extrachromosomal double minutes (DMs). This method provides a novel, efficient, and rapid way to establish cells that stably produce high levels of recombinant proteins. However, because IR/MAR plasmids are amplified as repeats, they are frequently targeted by repeat-induced gene silencing (RIGS), which silences a variety of repeated sequences in transgenes and the genome. To address this problem, we developed a novel screening system using the IR/MAR plasmid to isolate human genome sequences that alleviate RIGS. The screen identified a 3,271 bp sequence (B-3-31) that elevated transgene expression without affecting the amplification process. Neither non-B structure (i.e., the inverted repeats or bending) nor known epigenetic modifier elements such as MARs, insulators, UCOEs, or STARs could explain the anti-silencing activity of B-3-31. Instead, the activity was distributed throughout the entire B-3-31 sequence, which was extremely A/T-rich and CpG-poor. Because B-3-31 effectively and reproducibly alleviated RIGS of repeated genes, it could be used to increase recombinant protein production. PMID:27078685

  13. Cloning and Characterization of a Human Genomic Sequence that Alleviates Repeat-Induced Gene Silencing.

    PubMed

    Fukuma, Miki; Ganmyo, Yuto; Miura, Osamu; Ohyama, Takashi; Shimizu, Noriaki

    2016-01-01

    Plasmids bearing a mammalian replication initiation region (IR) and a nuclear matrix attachment region (MAR) are spontaneously amplified in transfected mammalian cells, and such amplification generates chromosomal homogeneously staining regions (HSRs) or extrachromosomal double minutes (DMs). This method provides a novel, efficient, and rapid way to establish cells that stably produce high levels of recombinant proteins. However, because IR/MAR plasmids are amplified as repeats, they are frequently targeted by repeat-induced gene silencing (RIGS), which silences a variety of repeated sequences in transgenes and the genome. To address this problem, we developed a novel screening system using the IR/MAR plasmid to isolate human genome sequences that alleviate RIGS. The screen identified a 3,271 bp sequence (B-3-31) that elevated transgene expression without affecting the amplification process. Neither non-B structure (i.e., the inverted repeats or bending) nor known epigenetic modifier elements such as MARs, insulators, UCOEs, or STARs could explain the anti-silencing activity of B-3-31. Instead, the activity was distributed throughout the entire B-3-31 sequence, which was extremely A/T-rich and CpG-poor. Because B-3-31 effectively and reproducibly alleviated RIGS of repeated genes, it could be used to increase recombinant protein production.

  14. Cloning and Characterization of a Human Genomic Sequence that Alleviates Repeat-Induced Gene Silencing.

    PubMed

    Fukuma, Miki; Ganmyo, Yuto; Miura, Osamu; Ohyama, Takashi; Shimizu, Noriaki

    2016-01-01

    Plasmids bearing a mammalian replication initiation region (IR) and a nuclear matrix attachment region (MAR) are spontaneously amplified in transfected mammalian cells, and such amplification generates chromosomal homogeneously staining regions (HSRs) or extrachromosomal double minutes (DMs). This method provides a novel, efficient, and rapid way to establish cells that stably produce high levels of recombinant proteins. However, because IR/MAR plasmids are amplified as repeats, they are frequently targeted by repeat-induced gene silencing (RIGS), which silences a variety of repeated sequences in transgenes and the genome. To address this problem, we developed a novel screening system using the IR/MAR plasmid to isolate human genome sequences that alleviate RIGS. The screen identified a 3,271 bp sequence (B-3-31) that elevated transgene expression without affecting the amplification process. Neither non-B structure (i.e., the inverted repeats or bending) nor known epigenetic modifier elements such as MARs, insulators, UCOEs, or STARs could explain the anti-silencing activity of B-3-31. Instead, the activity was distributed throughout the entire B-3-31 sequence, which was extremely A/T-rich and CpG-poor. Because B-3-31 effectively and reproducibly alleviated RIGS of repeated genes, it could be used to increase recombinant protein production. PMID:27078685

  15. Unexpected structural complexity of supernumerary marker chromosomes characterized by microarray comparative genomic hybridization

    PubMed Central

    Tsuchiya, Karen D; Opheim, Kent E; Hannibal, Mark C; Hing, Anne V; Glass, Ian A; Raff, Michael L; Norwood, Thomas; Torchia, Beth A

    2008-01-01

    Background Supernumerary marker chromosomes (SMCs) are structurally abnormal extra chromosomes that cannot be unambiguously identified by conventional banding techniques. In the past, SMCs have been characterized using a variety of different molecular cytogenetic techniques. Although these techniques can sometimes identify the chromosome of origin of SMCs, they are cumbersome to perform and are not available in many clinical cytogenetic laboratories. Furthermore, they cannot precisely determine the region or breakpoints of the chromosome(s) involved. In this study, we describe four patients who possess one or more SMCs (a total of eight SMCs in all four patients) that were characterized by microarray comparative genomic hybridization (array CGH). Results In at least one SMC from all four patients, array CGH uncovered unexpected complexity, in the form of complex rearrangements, that could have gone undetected using other molecular cytogenetic techniques. Although array CGH accurately defined the chromosome content of all but two minute SMCs, fluorescence in situ hybridization was necessary to determine the structure of the markers. Conclusion The increasing use of array CGH in clinical cytogenetic laboratories will provide an efficient method for more comprehensive characterization of SMCs. Improved SMC characterization, facilitated by array CGH, will allow for more accurate SMC/phenotype correlation. PMID:18471320

  16. Characterizing neutral genomic diversity and selection signatures in indigenous populations of Moroccan goats (Capra hircus) using WGS data

    PubMed Central

    Benjelloun, Badr; Alberto, Florian J.; Streeter, Ian; Boyer, Frédéric; Coissac, Eric; Stucki, Sylvie; BenBati, Mohammed; Ibnelbachyr, Mustapha; Chentouf, Mouad; Bechchari, Abdelmajid; Leempoel, Kevin; Alberti, Adriana; Engelen, Stefan; Chikhi, Abdelkader; Clarke, Laura; Flicek, Paul; Joost, Stéphane; Taberlet, Pierre; Pompanon, François

    2015-01-01

    Since the time of their domestication, goats (Capra hircus) have evolved in a large variety of locally adapted populations in response to different human and environmental pressures. In the present era, many indigenous populations are threatened with extinction due to their substitution by cosmopolitan breeds, while they might represent highly valuable genomic resources. It is thus crucial to characterize the neutral and adaptive genetic diversity of indigenous populations. A fine characterization of whole genome variation in farm animals is now possible by using new sequencing technologies. We sequenced the complete genome at 12× coverage of 44 goats geographically representative of the three phenotypically distinct indigenous populations in Morocco. The study of mitochondrial genomes showed a high diversity exclusively restricted to the haplogroup A. The 44 nuclear genomes showed a very high diversity (24 million variants) associated with low linkage disequilibrium. The overall genetic diversity was weakly structured according to geography and phenotypes. When looking for signals of positive selection in each population we identified many candidate genes, several of which gave insights into the metabolic pathways or biological processes involved in the adaptation to local conditions (e.g., panting in warm/desert conditions). This study highlights the interest of WGS data to characterize livestock genomic diversity. It illustrates the valuable genetic richness present in indigenous populations that have to be sustainably managed and may represent valuable genetic resources for the long-term preservation of the species. PMID:25904931

  17. Characterizing neutral genomic diversity and selection signatures in indigenous populations of Moroccan goats (Capra hircus) using WGS data.

    PubMed

    Benjelloun, Badr; Alberto, Florian J; Streeter, Ian; Boyer, Frédéric; Coissac, Eric; Stucki, Sylvie; BenBati, Mohammed; Ibnelbachyr, Mustapha; Chentouf, Mouad; Bechchari, Abdelmajid; Leempoel, Kevin; Alberti, Adriana; Engelen, Stefan; Chikhi, Abdelkader; Clarke, Laura; Flicek, Paul; Joost, Stéphane; Taberlet, Pierre; Pompanon, François

    2015-01-01

    Since the time of their domestication, goats (Capra hircus) have evolved in a large variety of locally adapted populations in response to different human and environmental pressures. In the present era, many indigenous populations are threatened with extinction due to their substitution by cosmopolitan breeds, while they might represent highly valuable genomic resources. It is thus crucial to characterize the neutral and adaptive genetic diversity of indigenous populations. A fine characterization of whole genome variation in farm animals is now possible by using new sequencing technologies. We sequenced the complete genome at 12× coverage of 44 goats geographically representative of the three phenotypically distinct indigenous populations in Morocco. The study of mitochondrial genomes showed a high diversity exclusively restricted to the haplogroup A. The 44 nuclear genomes showed a very high diversity (24 million variants) associated with low linkage disequilibrium. The overall genetic diversity was weakly structured according to geography and phenotypes. When looking for signals of positive selection in each population we identified many candidate genes, several of which gave insights into the metabolic pathways or biological processes involved in the adaptation to local conditions (e.g., panting in warm/desert conditions). This study highlights the interest of WGS data to characterize livestock genomic diversity. It illustrates the valuable genetic richness present in indigenous populations that have to be sustainably managed and may represent valuable genetic resources for the long-term preservation of the species.

  18. A genome-wide 3C-method for characterizing the three-dimensional architectures of genomes

    PubMed Central

    Duan, Zhijun; Andronescu, Mirela; Schutz, Kevin; Lee, Choli; Shendure, Jay; Fields, Stanley; Noble, William S.; Blau, C. Anthony

    2012-01-01

    Accumulating evidence demonstrates that the three-dimensional (3D) organization of chromosomes within the eukaryotic nucleus reflects and influences genomic activities, including transcription, DNA replication, recombination and DNA repair. In order to uncover structure-function relationships, it is necessary first to understand the principles underlying the folding and the 3D arrangement of chromosomes. Chromosome conformation capture (3C) provides a powerful tool for detecting interactions within and between chromosomes. A high throughput derivative of 3C, chromosome conformation capture on chip (4C), executes a genome-wide interrogation of interaction partners for a given locus. We recently developed a new method, a derivative of 3C and 4C, which, similar to Hi-C, is capable of comprehensively identifying long-range chromosome interactions throughout a genome in an unbiased fashion. Hence, our method can be applied to decipher the 3D architectures of genomes. Here, we provide a detailed protocol for this method. PMID:22776363

  19. Comparative genomics of the bacterial genus Listeria: Genome evolution is characterized by limited gene acquisition and limited gene loss

    PubMed Central

    2010-01-01

    Background The bacterial genus Listeria contains pathogenic and non-pathogenic species, including the pathogens L. monocytogenes and L. ivanovii, both of which carry homologous virulence gene clusters such as the prfA cluster and clusters of internalin genes. Initial evidence for multiple deletions of the prfA cluster during the evolution of Listeria indicates that this genus provides an interesting model for studying the evolution of virulence and also presents practical challenges with regard to definition of pathogenic strains. Results To better understand genome evolution and evolution of virulence characteristics in Listeria, we used a next generation sequencing approach to generate draft genomes for seven strains representing Listeria species or clades for which genome sequences were not available. Comparative analyses of these draft genomes and six publicly available genomes, which together represent the main Listeria species, showed evidence for (i) a pangenome with 2,032 core and 2,918 accessory genes identified to date, (ii) a critical role of gene loss events in transition of Listeria species from facultative pathogen to saprotroph, even though a consistent pattern of gene loss seemed to be absent, and a number of isolates representing non-pathogenic species still carried some virulence associated genes, and (iii) divergence of modern pathogenic and non-pathogenic Listeria species and strains, most likely circa 47 million years ago, from a pathogenic common ancestor that contained key virulence genes. Conclusions Genome evolution in Listeria involved limited gene loss and acquisition as supported by (i) a relatively high coverage of the predicted pan-genome by the observed pan-genome, (ii) conserved genome size (between 2.8 and 3.2 Mb), and (iii) a highly syntenic genome. Limited gene loss in Listeria did include loss of virulence associated genes, likely associated with multiple transitions to a saprotrophic lifestyle. The genus Listeria thus provides

  20. First genome analysis and molecular characterization of Chickpea chlorotic dwarf virus Egyptian isolate infecting squash.

    PubMed

    Fahmy, Inas Farouk; Taha, Omnia; El-Ashry, Abdel Nasser

    2015-06-01

    This study aims to identifying and characterizing some molecular properties of geminiviruses co-infection in squash field crop cultivated in Egypt. Squash crops observed to be heavily infected with several insect vectors, also severe chlorosis and stunting was observed. Electron microscopic analysis has revealed geminate capsid particles which indicate the infection of Geminiviruses, especially SqLCV which represent an economic problem to squash filed crop in Egypt. We have investigated possible mixed infections with different plant viruses associated with chlorotic stunt diseases and or other genus groups of geminiviruses. The main objective of this study is to investigate the recombination events, possible recombinants and variants among these genera in the same family differing in vector transmission. This is the first report of the molecular characterization, phylogenetic analysis and putative recombination events of the full length genome of the Chickpea Chlorotic Dwarf Mastrevirus in Egypt. And the first report of co-infection with another begomovirus infecting squash plants. A full length clone of both viruses were isolated and characterized at the molecular level. The complete nucleotide sequence of DNA-A was determined (2,572 bp) and submitted to the genbank under accession no. KF692356. The isolate from Egypt has about 97.8 % homology with the Chickpea chlorotic dwarf virus (CpCDV) isolate from Syria DNA-A isolate FR687959, a 83.2 % homology with the Sudan isolate AM933134 and a 82.7 % homology with Pakistan isolate FR687960. To best of our knowledge this is the first report of complete genome of CpCDV that infect squash plants in Egypt and worldwide.

  1. Leveraging Comparative Genomics to Identify and Functionally Characterize Genes Associated with Sperm Phenotypes in Python bivittatus (Burmese Python).

    PubMed

    Irizarry, Kristopher J L; Rutllant, Josep

    2016-01-01

    Comparative genomics approaches provide a means of leveraging functional genomics information from a highly annotated model organism's genome (such as the mouse genome) in order to make physiological inferences about the role of genes and proteins in a less characterized organism's genome (such as the Burmese python). We employed a comparative genomics approach to produce the functional annotation of Python bivittatus genes encoding proteins associated with sperm phenotypes. We identify 129 gene-phenotype relationships in the python which are implicated in 10 specific sperm phenotypes. Results obtained through our systematic analysis identified subsets of python genes exhibiting associations with gene ontology annotation terms. Functional annotation data was represented in a semantic scatter plot. Together, these newly annotated Python bivittatus genome resources provide a high resolution framework from which the biology relating to reptile spermatogenesis, fertility, and reproduction can be further investigated. Applications of our research include (1) production of genetic diagnostics for assessing fertility in domestic and wild reptiles; (2) enhanced assisted reproduction technology for endangered and captive reptiles; and (3) novel molecular targets for biotechnology-based approaches aimed at reducing fertility and reproduction of invasive reptiles. Additional enhancements to reptile genomic resources will further enhance their value. PMID:27200191

  2. Leveraging Comparative Genomics to Identify and Functionally Characterize Genes Associated with Sperm Phenotypes in Python bivittatus (Burmese Python).

    PubMed

    Irizarry, Kristopher J L; Rutllant, Josep

    2016-01-01

    Comparative genomics approaches provide a means of leveraging functional genomics information from a highly annotated model organism's genome (such as the mouse genome) in order to make physiological inferences about the role of genes and proteins in a less characterized organism's genome (such as the Burmese python). We employed a comparative genomics approach to produce the functional annotation of Python bivittatus genes encoding proteins associated with sperm phenotypes. We identify 129 gene-phenotype relationships in the python which are implicated in 10 specific sperm phenotypes. Results obtained through our systematic analysis identified subsets of python genes exhibiting associations with gene ontology annotation terms. Functional annotation data was represented in a semantic scatter plot. Together, these newly annotated Python bivittatus genome resources provide a high resolution framework from which the biology relating to reptile spermatogenesis, fertility, and reproduction can be further investigated. Applications of our research include (1) production of genetic diagnostics for assessing fertility in domestic and wild reptiles; (2) enhanced assisted reproduction technology for endangered and captive reptiles; and (3) novel molecular targets for biotechnology-based approaches aimed at reducing fertility and reproduction of invasive reptiles. Additional enhancements to reptile genomic resources will further enhance their value.

  3. Leveraging Comparative Genomics to Identify and Functionally Characterize Genes Associated with Sperm Phenotypes in Python bivittatus (Burmese Python)

    PubMed Central

    Rutllant, Josep

    2016-01-01

    Comparative genomics approaches provide a means of leveraging functional genomics information from a highly annotated model organism's genome (such as the mouse genome) in order to make physiological inferences about the role of genes and proteins in a less characterized organism's genome (such as the Burmese python). We employed a comparative genomics approach to produce the functional annotation of Python bivittatus genes encoding proteins associated with sperm phenotypes. We identify 129 gene-phenotype relationships in the python which are implicated in 10 specific sperm phenotypes. Results obtained through our systematic analysis identified subsets of python genes exhibiting associations with gene ontology annotation terms. Functional annotation data was represented in a semantic scatter plot. Together, these newly annotated Python bivittatus genome resources provide a high resolution framework from which the biology relating to reptile spermatogenesis, fertility, and reproduction can be further investigated. Applications of our research include (1) production of genetic diagnostics for assessing fertility in domestic and wild reptiles; (2) enhanced assisted reproduction technology for endangered and captive reptiles; and (3) novel molecular targets for biotechnology-based approaches aimed at reducing fertility and reproduction of invasive reptiles. Additional enhancements to reptile genomic resources will further enhance their value. PMID:27200191

  4. Rapid characterization of earthquake disasters using real-time sensors and imagery differencing

    NASA Astrophysics Data System (ADS)

    Hudnut, K. W.

    2011-12-01

    Recent major disasters have resulted from diverse earthquakes and related phenomena, hence a daunting array of observational challenges was encountered in the past few years. Rapid characterization of damage to provide situational awareness, and to inform disaster management decisions, has repeatedly proven important after recent events. The 2004 M9.0 Sumatra-Andaman earthquake caused entire islands to be elevated by more than two meters. As a result, corals and other biota were bleached in the sun, which made the uplift apparent in satellite imagery. In the 2010 M8.8 Chile earthquake, similar uplift occurred, and in both cases imagery taken before and afterwards revealed coherent long-wavelength patterns of coastal uplift and submergence. In the 2011 M9.0 Tohoku, Japan earthquake, widespread coastal subsidence occurred, as documented by data from the dense GPS network operated by GSI. These observed patterns indicate along-strike and down-dip maximum co-seismic slip areas on the plate interface. Along with these coastal vertical deformations, all three of these megathrust events also were accompanied by long-wavelength (hundreds of km's) horizontal deformations, also up to several meters. As the upper plate wedges of these subduction zones displaced elastically and rapidly towards the trenches, tsunamigenic strain was released co-seismically. GPS and differential imagery methods (e.g., InSAR and electro-optical) were also used to map the detailed patterns of these large crustal deformations, allowing estimation of slip on the plate interface by many investigators. The down-dip and along-strike slip variations are of interest because the slip pattern strongly influences tsunamigenesis. From these models of spatial and temporal slip on the plate interface, the process of tsunami and strong ground motion generation may then also be better understood and related to observed patterns of shaking and inundation, and to observed damage. If such modeling could be done even

  5. Temperature control and characterization of silicon-germanium growth by rapid thermal chemical vapor deposition

    NASA Astrophysics Data System (ADS)

    Hwang, Sung-Bo

    Rapid thermal chemical vapor deposition (RTCVD) is an emerging technology to utilize low thermal budgets required to grow silicon-germanium alloys in a coherent way. However, the current state-of-the-art in RTCVD technique lacks some key elements required for acceptance of RTCVD in mainstream IC fabrication. These shortcomings include adequate control of wafer temperature during processing, and sufficient understanding of the growth kinetics. This dissertation describes and discusses the temperature control in RTCVD, the growth, and characterization of silicon-germanium alloys. The RTCVD system provides very reliable temperature-measurements, for a range of 480˜820°C, based on infrared-light (1.3 or 1.55mum) absorption in the silicon wafer during the growth of silicon-germanium alloys. A wafer heat transfer model developed using the view-factor analysis is used to investigate temperature distributions with respect to lamp configurations in RTCVD system. For a precise temperature control, a neural model-based controller in single-input-single-output (SISO) system is proposed, and compared with other controllers. Silicon-germanium alloys, in various semiconductor structures including dots, have been grown by RTCVD where temperature is well-controlled by the model-based controller. The structural and chemical properties of silicon-germanium alloys are characterized by X-ray diffraction, atomic force microscopy (AFM), transmission electron microscopy (TEM), and secondary ion mass spectrometry (SIMS). The different growth characteristics dominated by a silicon-source gas are exploited, and their process models are developed with the experimental data utilizing neural networks employed the Bayesian framework to accurately describe the process behaviors such as growth rate and Ge fraction in alloys with respect to process variables (to capture the process nonlinearity). By controlling growth rate and Ge fraction, a uniform and a grading Ge profile in silicon

  6. Formation and Characterization of Beclomethasone Dipropionate Nanoparticles Using Rapid Expansion of Supercritical Solution

    PubMed Central

    Hosseinpour, Mohsen; Vatanara, Alireza; Zarghami, Reza

    2015-01-01

    Purpose: Particle size of Beclometasone Dipropionate (BDP) was reduced by the rapid expansion of supercritical solution (RESS) process, using CO2 as supercritical solvent. Also, the effect of RESS parameters such as extraction pressure, pre-expansion temperature, and weight fraction of co-solvent on the size and distribution of BDP particles were investigated. Methods: The effects of extraction pressure (200-260 bar), pre-expansion temperature (70-110 °C) and weight fraction of menthol as a co-solvent on mean particle size (MPS) of BDP were investigated by design of experiment (DOE). Particles were characterized using Scanning Electron Microscopy (SEM) and Dynamic Light Scattering (DLS). Results: The average sizes of precipitated BDP were between 64.1 and 294 nm. Analysis of variance (ANOVA) showed that extraction pressure was the most significant parameter and a higher extraction pressure caused production of smaller particles. Also, it was found that higher temperature and weight fraction of co-solvent increased the MPS. The interaction effects of extraction pressure-pre-expansion temperature and pre-expansion temperature-co-solvent ratio were significant through the analysis of variance. It was observed that the MPS of precipitated particles was mostly influenced by pressure. Conclusion: The smallest MPS of BDP obtained from the RESS process was 65 nm that revealed a significant size reduction from its original MPS of 9 μm. Moreover, a slight change was observed for precipitated particles of BDP into spherical form while the original particles were irregular in shape. RESS process showed as a promising method for production of BDP nanoparticles that may results in improvement of drug’s physicochemical properties. PMID:26504756

  7. Applying polarity rapid assessment method and ultrafiltration to characterize NDMA precursors in wastewater effluents.

    PubMed

    Chen, Chao; Leavey, Shannon; Krasner, Stuart W; Mel Suffet, I H

    2014-06-15

    Certain nitrosamines in water are disinfection byproducts that are probable human carcinogens. Nitrosamines have diverse and complex precursors that include effluent organic matter, some anthropogenic chemicals, and natural (likely non-humic) substances. An easy and selective tool was first developed to characterize nitrosamine precursors in treated wastewaters, including different process effluents. This tool takes advantages of the polarity rapid assessment method (PRAM) and ultrafiltration (UF) (molecular weight distribution) to locate the fractions with the strongest contributions to the nitrosamine precursor pool in the effluent organic matter. Strong cation exchange (SCX) and C18 solid-phase extraction cartridges were used for their high selectivity for nitrosamine precursors. The details of PRAM operation, such as cartridge clean-up, capacity, pH influence, and quality control were included in this paper, as well as the main parameters of UF operation. Preliminary testing of the PRAM/UF method with effluents from one wastewater treatment plant gave very informative results. SCX retained 45-90% of the N-nitrosodimethylamine (NDMA) formation potential (FP)-a measure of the precursors-in secondary and tertiary wastewater effluents. These results are consistent with NDMA precursors likely having a positively charged amine group. C18 adsorbed 30-45% of the NDMAFP, which indicates that a substantial portion of these precursors were non-polar. The small molecular weight (MW) (<1 kDa) and large MW (>10 kDa) fractions obtained from UF were the primary contributors to NDMAFP. The combination of PRAM and UF brings important information on the characteristics of nitrosamine precursors in water with easy operation.

  8. Rapid characterization of triterpene saponins from Conyza blinii by liquid chromatography coupled with mass spectrometry.

    PubMed

    Qiao, Xue; Zhang, Xing; Ye, Min; Su, Yan-fang; Dong, Jing; Han, Jian; Yin, Jun; Guo, De-an

    2010-11-30

    Conyza blinii Le'vl is a medicinal herb used for the treatment of inflammation in Chinese folk medicine. Its major bioactive constituents are triterpene saponins, most of which contain 6-8 sugar residues. In this report, electrospray ionization tandem mass spectrometry fragmentation behaviors of bisdesmosidic triterpene saponins (conyzasaponin A, B, and C) were studied in both positive and negative ion modes with an ion-trap mass spectrometer. In full scan mass spectrometry, these saponins gave predominant [M-H](-) and [M+Na](+) ions, which determined the molecular weights. In tandem mass spectrometry (MS(n), n = 2-4), the [M-H](-) and [M+Na](+) ions yielded fragments [Y(0α)-H](-) and [B(α)+Na](+), which were diagnostic for the structures of the triterpene skeleton and sugar chains. The structural elucidation was approved by accurate mass data using IT-TOF-MS. An interpretation guideline based on MS(n) (n = 2-4) diagnostic ions was proposed in order to elucidate the chemical structures of unknown triterpene saponins in C. blinii extract. The saponins in C. blinii were separated by liquid chromatography with a methanol/acetonitrile/water solvent system, and then analyzed by ion-trap and IT-TOF mass spectrometers. Based on the interpretation guideline, a total of 35 triterpenoid saponins were tentatively identified. Among them, 15 saponins had been previously reported, and the other 20 saponins were reported from Conyza species for the first time. This study indicates that LC/MS is a powerful technology for the rapid characterization of complicated saponins in herbal extracts.

  9. Full-genome identification and characterization of NBS-encoding disease resistance genes in wheat.

    PubMed

    Bouktila, Dhia; Khalfallah, Yosra; Habachi-Houimli, Yosra; Mezghani-Khemakhem, Maha; Makni, Mohamed; Makni, Hanem

    2015-02-01

    Host resistance is the most economical, effective and ecologically sustainable method of controlling diseases in crop plants. In bread wheat, despite the high number of resistance loci that have been cataloged to date, only few have been cloned, underlying the need for genomics-guided investigations capable of providing a prompt and acute knowledge on the identity of effective resistance genes that can be used in breeding programs. Proteins with a nucleotide-binding site (NBS) encoded by the major plant disease resistance (R) genes play an important role in the responses of plants to various pathogens. In this study, a comprehensive analysis of NBS-encoding genes within the whole wheat genome was performed, and the genome scale characterization of this gene family was established. From the recently published wheat genome sequence, we used a data mining and automatic prediction pipeline to identify 580 complete ORF candidate NBS-encoding genes and 1,099 partial-ORF ones. Among complete gene models, 464 were longer than 200 aa, among them 436 had less than 70 % of sequence identity to each other. This gene models set was deeply characterized. (1) First, we have analyzed domain architecture and identified, in addition to typical domain combinations, the presence of particular domains like signal peptides, zinc fingers, kinases, heavy-metal-associated and WRKY DNA-binding domains. (2) Functional and expression annotation via homology searches in protein and transcript databases, based on sufficient criteria, enabled identifying similar proteins for 60 % of the studied gene models and expression evidence for 13 % of them. (3) Shared orthologous groups were defined using NBS-domain proteins of rice and Brachypodium distachyon. (4) Finally, alignment of the 436 NBS-containing gene models to the full set of scaffolds from the IWGSC's wheat chromosome survey sequence enabled high-stringence anchoring to chromosome arms. The distribution of the R genes was found balanced

  10. Context based computational analysis and characterization of ARS consensus sequences (ACS) of Saccharomyces cerevisiae genome.

    PubMed

    Singh, Vinod Kumar; Krishnamachari, Annangarachari

    2016-09-01

    Genome-wide experimental studies in Saccharomyces cerevisiae reveal that autonomous replicating sequence (ARS) requires an essential consensus sequence (ACS) for replication activity. Computational studies identified thousands of ACS like patterns in the genome. However, only a few hundreds of these sites act as replicating sites and the rest are considered as dormant or evolving sites. In a bid to understand the sequence makeup of replication sites, a content and context-based analysis was performed on a set of replicating ACS sequences that binds to origin-recognition complex (ORC) denoted as ORC-ACS and non-replicating ACS sequences (nrACS), that are not bound by ORC. In this study, DNA properties such as base composition, correlation, sequence dependent thermodynamic and DNA structural profiles, and their positions have been considered for characterizing ORC-ACS and nrACS. Analysis reveals that ORC-ACS depict marked differences in nucleotide composition and context features in its vicinity compared to nrACS. Interestingly, an A-rich motif was also discovered in ORC-ACS sequences within its nucleosome-free region. Profound changes in the conformational features, such as DNA helical twist, inclination angle and stacking energy between ORC-ACS and nrACS were observed. Distribution of ACS motifs in the non-coding segments points to the locations of ORC-ACS which are found far away from the adjacent gene start position compared to nrACS thereby enabling an accessible environment for ORC-proteins. Our attempt is novel in considering the contextual view of ACS and its flanking region along with nucleosome positioning in the S. cerevisiae genome and may be useful for any computational prediction scheme. PMID:27508123

  11. Genome characterization and population genetic structure of the zoonotic pathogen, Streptococcus canis

    PubMed Central

    2012-01-01

    Background Streptococcus canis is an important opportunistic pathogen of dogs and cats that can also infect a wide range of additional mammals including cows where it can cause mastitis. It is also an emerging human pathogen. Results Here we provide characterization of the first genome sequence for this species, strain FSL S3-227 (milk isolate from a cow with an intra-mammary infection). A diverse array of putative virulence factors was encoded by the S. canis FSL S3-227 genome. Approximately 75% of these gene sequences were homologous to known Streptococcal virulence factors involved in invasion, evasion, and colonization. Present in the genome are multiple potentially mobile genetic elements (MGEs) [plasmid, phage, integrative conjugative element (ICE)] and comparison to other species provided convincing evidence for lateral gene transfer (LGT) between S. canis and two additional bovine mastitis causing pathogens (Streptococcus agalactiae, and Streptococcus dysgalactiae subsp. dysgalactiae), with this transfer possibly contributing to host adaptation. Population structure among isolates obtained from Europe and USA [bovine = 56, canine = 26, and feline = 1] was explored. Ribotyping of all isolates and multi locus sequence typing (MLST) of a subset of the isolates (n = 45) detected significant differentiation between bovine and canine isolates (Fisher exact test: P = 0.0000 [ribotypes], P = 0.0030 [sequence types]), suggesting possible host adaptation of some genotypes. Concurrently, the ancestral clonal complex (54% of isolates) occurred in many tissue types, all hosts, and all geographic locations suggesting the possibility of a wide and diverse niche. Conclusion This study provides evidence highlighting the importance of LGT in the evolution of the bacteria S. canis, specifically, its possible role in host adaptation and acquisition of virulence factors. Furthermore, recent LGT detected between S. canis and human bacteria (Streptococcus

  12. Characterization of Genomic Island 3 and Genetic Variability of Chilean Field Strains of Brucella abortus▿

    PubMed Central

    Céspedes, Sandra; Salgado, Paulina; Valenzuela, Patricio; Vidal, Roberto; Oñate, Angel A.

    2011-01-01

    One of the capabilities developed by bacteria is the ability to gain large fragments of DNA from other bacteria or to lose portions of their own genomes. Among these exchangeable fragments are the genomic islands (GIs). Nine GIs have been identified in Brucella, and genomic island 3 (GI-3) is shared by two pathogenic species, B. melitensis and B. abortus. GI-3 encodes mostly unknown proteins. One of the aims of this study was to perform pulsed-field gel electrophoresis (PFGE) on field isolates of B. abortus from Chile to determine whether these isolates are clonally related. Furthermore, we focused on the characterization of GI-3, studying its organization and the genetic conservation of the GI-3 sequence using techniques such as tiling-path PCR (TP-PCR) and restriction fragment length polymorphism-PCR (RFLP-PCR). Our results, after PFGE was performed on 69 field isolates of B. abortus from Chile, showed that the strains were genetically homogeneous. To increase the power of genetic discrimination among these strains, we used multiple locus variable-number tandem-repeat (VNTR) analysis with 16 loci (MLVA-16). The results obtained by MLVA-16 showed that the strains of B. abortus were genetically heterogeneous and that most of them clustered according to their geographic origin. Of the genetic loci studied, panel 2B was the one describing the highest diversity in the analysis, as well as locus Bruce19 in panel 2A. In relation to the study of GI-3, our experimental analysis by TP-PCR identified and confirmed that GI-3 is present in all wild strains of B. abortus, demonstrating the high stability of gene cluster GI-3 in Chilean field strains. PMID:21543580

  13. Development and characterization of genomic SSR markers in Cynodon transvaalensis Burtt-Davy.

    PubMed

    Tan, Chengcheng; Wu, Yanqi; Taliaferro, Charles M; Bell, Greg E; Martin, Dennis L; Smith, Mike W

    2014-08-01

    Simple sequence repeat (SSR) markers are a major molecular tool for genetic and genomic research that have been extensively developed and used in major crops. However, few are available in African bermudagrass (Cynodon transvaalensis Burtt-Davy), an economically important warm-season turfgrass species. African bermudagrass is mainly used for hybridizations with common bermudagrass [C. dactylon var. dactylon (L.) Pers.] in the development of superior interspecific hybrid turfgrass cultivars. Accordingly, the major objective of this study was to develop and characterize a large set of SSR markers. Genomic DNA of C. transvaalensis '4200TN 24-2' from an Oklahoma State University (OSU) turf nursery was extracted for construction of four SSR genomic libraries enriched with [CA](n), [GA](n), [AAG](n), and [AAT](n) as core repeat motifs. A total of 3,064 clones were sequenced at the OSU core facility. The sequences were categorized into singletons and contiguous sequences to exclude redundancy. From the two sequence categories, 1,795 SSR loci were identified. After excluding duplicate SSRs by comparison with previously developed SSR markers using a nucleotide basic local alignment tool, 1,426 unique primer pairs (PPs) were designed. Out of the 1,426 designed PPs, 981 (68.8 %) amplified alleles of the expected size in the donor DNA. Polymorphisms of the SSR PPs tested in eight C. transvaalensis plants were 93 % polymorphic with 544 markers effective in all genotypes. Inheritance of the SSRs was examined in six F(1) progeny of African parents 'T577' × 'Uganda', indicating 917 markers amplified heritable alleles. The SSR markers developed in the study are the first large set of co-dominant markers in African bermudagrass and should be highly valuable for molecular and traditional breeding research.

  14. Transposable element junctions in marker development and genomic characterization of barley

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Barley is a model plant in genomic studies of Triticeae species. A complete barley genome sequence will facilitate not only barley breeding programs, but also those for related species. However, the large genome size and high repetitive sequence content complicate the barley genome assembly. The ma...

  15. Rapidly expanding genetic diversity and host range of the Circoviridae viral family and other Rep encoding small circular ssDNA genomes

    PubMed Central

    Delwart, Eric; Li, Linlin

    2011-01-01

    The genomes of numerous circoviruses and distantly related circular DNA viruses encoding a rolling circle replication initiator protein (Rep) have been characterized from the tissues of mammals, fish, insects, and plants (geminivirus and nanovirus), human and animal feces, in an algae cell, and in diverse environmental samples. We review the genome organization, phylogenetic relationships and initial prevalence studies of cycloviruses, a proposed new genus in the Circoviridae family. Viral fossil rep sequences were also identified integrated on the chromosomes of mammals, frogs, lancelets, crustaceans, mites, gastropods, roundworms, placozoans, hydrozoans, protozoans, land plants, fungi, algae, and phytoplasma bacterias and their plasmids, reflecting their past host range. An ancient origin for viruses with rep-encoding single stranded small circular genomes, predating the diversification of eukaryotes, is discussed. The cellular hosts and pathogenicity of many recently described rep-containing circular genomes remain to be determined. Future studies of the virome of single cell and multi-cellular eukaryotes are likely to further extend the known diversity and host-range of small rep-containing circular viral genomes. PMID:22155583

  16. Discovery and Genomic Characterization of a Novel Ovine Partetravirus and a New Genotype of Bovine Partetravirus

    PubMed Central

    Tse, Herman; Tsoi, Hoi-Wah; Teng, Jade L. L.; Chen, Xin-Chun; Liu, Haiying; Zhou, Boping; Zheng, Bo-Jian; Woo, Patrick C. Y.; Lau, Susanna K. P.; Yuen, Kwok-Yung

    2011-01-01

    Partetravirus is a recently described group of animal parvoviruses which include the human partetravirus, bovine partetravirus and porcine partetravirus (previously known as human parvovirus 4, bovine hokovirus and porcine hokovirus respectively). In this report, we describe the discovery and genomic characterization of partetraviruses in bovine and ovine samples from China. These partetraviruses were detected by PCR in 1.8% of bovine liver samples, 66.7% of ovine liver samples and 71.4% of ovine spleen samples. One of the bovine partetraviruses detected in the present samples is phylogenetically distinct from previously reported bovine partetraviruses and likely represents a novel genotype. The ovine partetravirus is a novel partetravirus and phylogenetically most related to the bovine partetraviruses. The genome organization is conserved amongst these viruses, including the presence of a putative transmembrane protein encoded by an overlapping reading frame in ORF2. Results from the present study provide further support to the classification of partetraviruses as a separate genus in Parvovirinae. PMID:21980506

  17. Genomic Characterization of Esophageal Squamous Cell Carcinoma Reveals Critical Genes Underlying Tumorigenesis and Poor Prognosis.

    PubMed

    Qin, Hai-De; Liao, Xiao-Yu; Chen, Yuan-Bin; Huang, Shao-Yi; Xue, Wen-Qiong; Li, Fang-Fang; Ge, Xiao-Song; Liu, De-Qing; Cai, Qiuyin; Long, Jirong; Li, Xi-Zhao; Hu, Ye-Zhu; Zhang, Shao-Dan; Zhang, Lan-Jun; Lehrman, Benjamin; Scott, Alan F; Lin, Dongxin; Zeng, Yi-Xin; Shugart, Yin Yao; Jia, Wei-Hua

    2016-04-01

    The genetic mechanisms underlying the poor prognosis of esophageal squamous cell carcinoma (ESCC) are not well understood. Here, we report somatic mutations found in ESCC from sequencing 10 whole-genome and 57 whole-exome matched tumor-normal sample pairs. Among the identified genes, we characterized mutations in VANGL1 and showed that they accelerated cell growth in vitro. We also found that five other genes, including three coding genes (SHANK2, MYBL2, FADD) and two non-coding genes (miR-4707-5p, PCAT1), were involved in somatic copy-number alterations (SCNAs) or structural variants (SVs). A survival analysis based on the expression profiles of 321 individuals with ESCC indicated that these genes were significantly associated with poorer survival. Subsequently, we performed functional studies, which showed that miR-4707-5p and MYBL2 promoted proliferation and metastasis. Together, our results shed light on somatic mutations and genomic events that contribute to ESCC tumorigenesis and prognosis and might suggest therapeutic targets.

  18. Characterization and comparative analysis of a simian foamy virus complete genome isolated from Brazilian capuchin monkeys.

    PubMed

    Troncoso, Lian L; Muniz, Cláudia P; Siqueira, Juliana D; Curty, Gislaine; Schrago, Carlos G; Augusto, Anderson; Fedullo, Luiz; Soares, Marcelo A; Santos, André F

    2015-10-01

    Foamy viruses infect a wide range of placental mammals, including primates. However, despite of great diversity of New World primates, only three strains of neotropical simian foamy viruses (SFV) have been described. Only after 40 years since serological characterization, the complete sequence of an SFVcap strain infecting a family of six capuchin monkeys (Sapajus xanthosternos) was obtained. Co-culture of primate peripheral blood mononuclear cells with Cf2Th canine cells was established and monitored for the appearance of cytopathic effects, PCR amplification of integrated SFV proviral genome and viral reverse transcriptase activity. The novel SFVcap was fully sequenced through a next-generation sequencing protocol. Phylogenetic analysis of the complete genome grouped SFVcap and SFVmar, both infecting primate species of the Cebidae family with a genetic similarity of approximately 85%. Similar ORF sizes were observed among SFV from neotropical primates, and env and pol genes were the most conserved. Neotropical SFV presented the smallest LTRs among exogenous mammalians. The novel SFVcap strain provides a valuable research tool for the FV community.

  19. Characterization, correction and de novo assembly of an Oxford Nanopore genomic dataset from Agrobacterium tumefaciens.

    PubMed

    Deschamps, Stéphane; Mudge, Joann; Cameron, Connor; Ramaraj, Thiruvarangan; Anand, Ajith; Fengler, Kevin; Hayes, Kevin; Llaca, Victor; Jones, Todd J; May, Gregory

    2016-01-01

    The MinION is a portable single-molecule DNA sequencing instrument that was released by Oxford Nanopore Technologies in 2014, producing long sequencing reads by measuring changes in ionic flow when single-stranded DNA molecules translocate through the pores. While MinION long reads have an error rate substantially higher than the ones produced by short-read sequencing technologies, they can generate de novo assemblies of microbial genomes, after an initial correction step that includes alignment of Illumina sequencing data or detection of overlaps between Oxford Nanopore reads to improve accuracy. In this study, MinION reads were generated from the multi-chromosome genome of Agrobacterium tumefaciens strain LBA4404. Errors in the consensus two-directional (sense and antisense) "2D" sequences were first characterized by way of comparison with an internal reference assembly. Both Illumina-based correction and self-correction were performed and the resulting corrected reads assembled into high-quality hybrid and non-hybrid assemblies. Corrected read datasets and assemblies were subsequently compared. The results shown here indicate that both hybrid and non-hybrid methods can be used to assemble Oxford Nanopore reads into informative multi-chromosome assemblies, each with slightly different outcomes in terms of contiguity and accuracy. PMID:27350167

  20. Deep subsurface life from North Pond: Enrichment, isolation, characterization and genomes of heterotrophic bacteria

    DOE PAGES

    Russell, Joseph A.; Leon-Zayas, Rosa; Wrighton, Kelly; Biddle, Jennifer F.

    2016-05-10

    Studies of subsurface microorganisms have yielded few environmentally relevant isolates for laboratory studies. In order to address this lack of cultivated microorganisms, we initiated several enrichments on sediment and underlying basalt samples from North Pond, a sediment basin ringed by basalt outcrops underlying an oligotrophic watercolumn west of the Mid-Atlantic Ridge at 22° N. In contrast to anoxic enrichments, growth was observed in aerobic, heterotrophic enrichments from sediment of IODP Hole U1382B at 4 and 68 m below seafloor (mbsf). These sediment depths, respectively, correspond to the fringes of oxygen penetration from overlying seawater in the top of the sedimentmore » column and upward migration of oxygen from oxic seawater from the basalt aquifer below the sediment. Here we report the enrichment, isolation, initial characterization and genomes of three isolated aerobic heterotrophs from North Pond sediments; an Arthrobacter species from 4 mbsf, and Paracoccus and Pseudomonas species from 68 mbsf. These cultivated bacteria are represented in the amplicon 16S rRNA gene libraries created from whole sediments, albeit at low (up to 2%) relative abundance. We provide genomic evidence from our isolates demonstrating that the Arthrobacter and Pseudomonas isolates have the potential to respire nitrate and oxygen, though dissimilatory nitrate reduction could not be confirmed in laboratory cultures. Furthermore, the cultures from this study represent members of abundant phyla, as determined by amplicon sequencing of environmental DNA extracts, and allow for further studies into geochemical factors impacting life in the deep subsurface.« less

  1. Deep Subsurface Life from North Pond: Enrichment, Isolation, Characterization and Genomes of Heterotrophic Bacteria

    PubMed Central

    Russell, Joseph A.; León-Zayas, Rosa; Wrighton, Kelly; Biddle, Jennifer F.

    2016-01-01

    Studies of subsurface microorganisms have yielded few environmentally relevant isolates for laboratory studies. In order to address this lack of cultivated microorganisms, we initiated several enrichments on sediment and underlying basalt samples from North Pond, a sediment basin ringed by basalt outcrops underlying an oligotrophic water-column west of the Mid-Atlantic Ridge at 22°N. In contrast to anoxic enrichments, growth was observed in aerobic, heterotrophic enrichments from sediment of IODP Hole U1382B at 4 and 68 m below seafloor (mbsf). These sediment depths, respectively, correspond to the fringes of oxygen penetration from overlying seawater in the top of the sediment column and upward migration of oxygen from oxic seawater from the basalt aquifer below the sediment. Here we report the enrichment, isolation, initial characterization and genomes of three isolated aerobic heterotrophs from North Pond sediments; an Arthrobacter species from 4 mbsf, and Paracoccus and Pseudomonas species from 68 mbsf. These cultivated bacteria are represented in the amplicon 16S rRNA gene libraries created from whole sediments, albeit at low (up to 2%) relative abundance. We provide genomic evidence from our isolates demonstrating that the Arthrobacter and Pseudomonas isolates have the potential to respire nitrate and oxygen, though dissimilatory nitrate reduction could not be confirmed in laboratory cultures. The cultures from this study represent members of abundant phyla, as determined by amplicon sequencing of environmental DNA extracts, and allow for further studies into geochemical factors impacting life in the deep subsurface. PMID:27242705

  2. Deep Subsurface Life from North Pond: Enrichment, Isolation, Characterization and Genomes of Heterotrophic Bacteria.

    PubMed

    Russell, Joseph A; León-Zayas, Rosa; Wrighton, Kelly; Biddle, Jennifer F

    2016-01-01

    Studies of subsurface microorganisms have yielded few environmentally relevant isolates for laboratory studies. In order to address this lack of cultivated microorganisms, we initiated several enrichments on sediment and underlying basalt samples from North Pond, a sediment basin ringed by basalt outcrops underlying an oligotrophic water-column west of the Mid-Atlantic Ridge at 22°N. In contrast to anoxic enrichments, growth was observed in aerobic, heterotrophic enrichments from sediment of IODP Hole U1382B at 4 and 68 m below seafloor (mbsf). These sediment depths, respectively, correspond to the fringes of oxygen penetration from overlying seawater in the top of the sediment column and upward migration of oxygen from oxic seawater from the basalt aquifer below the sediment. Here we report the enrichment, isolation, initial characterization and genomes of three isolated aerobic heterotrophs from North Pond sediments; an Arthrobacter species from 4 mbsf, and Paracoccus and Pseudomonas species from 68 mbsf. These cultivated bacteria are represented in the amplicon 16S rRNA gene libraries created from whole sediments, albeit at low (up to 2%) relative abundance. We provide genomic evidence from our isolates demonstrating that the Arthrobacter and Pseudomonas isolates have the potential to respire nitrate and oxygen, though dissimilatory nitrate reduction could not be confirmed in laboratory cultures. The cultures from this study represent members of abundant phyla, as determined by amplicon sequencing of environmental DNA extracts, and allow for further studies into geochemical factors impacting life in the deep subsurface. PMID:27242705

  3. Preliminary characterization of mitochondrial genome of Melipona scutellaris, a Brazilian stingless bee.

    PubMed

    Silverio, Manuella Souza; Rodovalho, Vinícius de Rezende; Bonetti, Ana Maria; de Oliveira, Guilherme Corrêa; Cuadros-Orellana, Sara; Ueira-Vieira, Carlos; Rodrigues dos Santos, Anderson

    2014-01-01

    Bees are manufacturers of relevant economical products and have a pollinator role fundamental to ecosystems. Traditionally, studies focused on the genus Melipona have been mostly based on behavioral, and social organization and ecological aspects. Only recently the evolutionary history of this genus has been assessed using molecular markers, including mitochondrial genes. Even though these studies have shed light on the evolutionary history of the Melipona genus, a more accurate picture may emerge when full nuclear and mitochondrial genomes of Melipona species become available. Here we present the assembly, annotation, and characterization of a draft mitochondrial genome of the Brazilian stingless bee Melipona scutellaris using Melipona bicolor as a reference organism. Using Illumina MiSeq data, we achieved the annotation of all protein coding genes, as well as the genes for the two ribosomal subunits (16S and 12S) and transfer RNA genes as well. Using the COI sequence as a DNA barcode, we found that M. cramptoni is the closest species to M. scutellaris.

  4. Genome-wide identification, characterization, and expression analysis of the MLO gene family in Cucumis sativus.

    PubMed

    Zhou, S J; Jing, Z; Shi, J L

    2013-12-11

    Mildew resistance locus o (MLO) is a plant-specific seven-transmembrane (TM) gene family. Several studies have revealed that certain members of the MLO gene family mediate powdery mildew susceptibility in three plant species, namely, Arabidopsis, barley, and tomato. The sequenced cucumber genome provides an opportunity to conduct a comprehensive overview of the MLO gene family. Fourteen genes (designated CsMLO01 through CsMLO14) have been identified within the Cucumis sativus genome by using an in silico cloning method with the MLO amino acid sequences of Arabidopsis thaliana and rice as probes. Sequence alignment revealed that numerous features of the gene family, such as TMs, a calmodulin-binding domain, peptide domains I and II, and 30 important amino acid residues for MLO function, are well conserved. Phylogenetic analysis of the MLO genes from cucumber and other plant species reveals seven different clades (I through VII). Three of these clades comprised MLO genes from A. thaliana, rice, maize, and cucumber, suggesting that these genes may have evolved after the divergence of monocots and dicots. In silico mapping showed that these CsMLOs were located on chromosomes 1, 2, 3, 4, 5, and 6 without any obvious clustering, except CsMLO01. To our knowledge, this paper is the first comprehensive report on MLO genes in C. sativus. These findings will facilitate the functional characterization of the MLOs related to powdery mildew susceptibility and assist in the development of disease resistance in cucumber.

  5. Deep Subsurface Life from North Pond: Enrichment, Isolation, Characterization and Genomes of Heterotrophic Bacteria.

    PubMed

    Russell, Joseph A; León-Zayas, Rosa; Wrighton, Kelly; Biddle, Jennifer F

    2016-01-01

    Studies of subsurface microorganisms have yielded few environmentally relevant isolates for laboratory studies. In order to address this lack of cultivated microorganisms, we initiated several enrichments on sediment and underlying basalt samples from North Pond, a sediment basin ringed by basalt outcrops underlying an oligotrophic water-column west of the Mid-Atlantic Ridge at 22°N. In contrast to anoxic enrichments, growth was observed in aerobic, heterotrophic enrichments from sediment of IODP Hole U1382B at 4 and 68 m below seafloor (mbsf). These sediment depths, respectively, correspond to the fringes of oxygen penetration from overlying seawater in the top of the sediment column and upward migration of oxygen from oxic seawater from the basalt aquifer below the sediment. Here we report the enrichment, isolation, initial characterization and genomes of three isolated aerobic heterotrophs from North Pond sediments; an Arthrobacter species from 4 mbsf, and Paracoccus and Pseudomonas species from 68 mbsf. These cultivated bacteria are represented in the amplicon 16S rRNA gene libraries created from whole sediments, albeit at low (up to 2%) relative abundance. We provide genomic evidence from our isolates demonstrating that the Arthrobacter and Pseudomonas isolates have the potential to respire nitrate and oxygen, though dissimilatory nitrate reduction could not be confirmed in laboratory cultures. The cultures from this study represent members of abundant phyla, as determined by amplicon sequencing of environmental DNA extracts, and allow for further studies into geochemical factors impacting life in the deep subsurface.

  6. Characterization, correction and de novo assembly of an Oxford Nanopore genomic dataset from Agrobacterium tumefaciens

    PubMed Central

    Deschamps, Stéphane; Mudge, Joann; Cameron, Connor; Ramaraj, Thiruvarangan; Anand, Ajith; Fengler, Kevin; Hayes, Kevin; Llaca, Victor; Jones, Todd J.; May, Gregory

    2016-01-01

    The MinION is a portable single-molecule DNA sequencing instrument that was released by Oxford Nanopore Technologies in 2014, producing long sequencing reads by measuring changes in ionic flow when single-stranded DNA molecules translocate through the pores. While MinION long reads have an error rate substantially higher than the ones produced by short-read sequencing technologies, they can generate de novo assemblies of microbial genomes, after an initial correction step that includes alignment of Illumina sequencing data or detection of overlaps between Oxford Nanopore reads to improve accuracy. In this study, MinION reads were generated from the multi-chromosome genome of Agrobacterium tumefaciens strain LBA4404. Errors in the consensus two-directional (sense and antisense) “2D” sequences were first characterized by way of comparison with an internal reference assembly. Both Illumina-based correction and self-correction were performed and the resulting corrected reads assembled into high-quality hybrid and non-hybrid assemblies. Corrected read datasets and assemblies were subsequently compared. The results shown here indicate that both hybrid and non-hybrid methods can be used to assemble Oxford Nanopore reads into informative multi-chromosome assemblies, each with slightly different outcomes in terms of contiguity and accuracy. PMID:27350167

  7. Preliminary Characterization of Mitochondrial Genome of Melipona scutellaris, a Brazilian Stingless Bee

    PubMed Central

    Silverio, Manuella Souza; Rodovalho, Vinícius de Rezende; Bonetti, Ana Maria; de Oliveira, Guilherme Corrêa; Cuadros-Orellana, Sara; Ueira-Vieira, Carlos; Rodrigues dos Santos, Anderson

    2014-01-01

    Bees are manufacturers of relevant economical products and have a pollinator role fundamental to ecosystems. Traditionally, studies focused on the genus Melipona have been mostly based on behavioral, and social organization and ecological aspects. Only recently the evolutionary history of this genus has been assessed using molecular markers, including mitochondrial genes. Even though these studies have shed light on the evolutionary history of the Melipona genus, a more accurate picture may emerge when full nuclear and mitochondrial genomes of Melipona species become available. Here we present the assembly, annotation, and characterization of a draft mitochondrial genome of the Brazilian stingless bee Melipona scutellaris using Melipona bicolor as a reference organism. Using Illumina MiSeq data, we achieved the annotation of all protein coding genes, as well as the genes for the two ribosomal subunits (16S and 12S) and transfer RNA genes as well. Using the COI sequence as a DNA barcode, we found that M. cramptoni is the closest species to M. scutellaris. PMID:25019088

  8. Characterization of the Genomic Architecture and Mutational Spectrum of a Small Cell Prostate Carcinoma

    PubMed Central

    Scott, Alan F.; Mohr, David W.; Ling, Hua; Scharpf, Robert B.; Zhang, Peng; Liptak, Gregory S.

    2014-01-01

    We present the use of a series of laboratory, analytical and interpretation methods to investigate personalized cancer care for a case of small cell prostate carcinoma (SCPC), a rare and aggressive tumor with poor prognosis, for which the underlying genomic architecture and mutational spectrum has not been well characterized. We performed both SNP genotyping and exome sequencing of a Virchow node metastasis from a patient with SCPC. A variety of methods were used to analyze and interpret the tumor genome for copy number variation, loss of heterozygosity (LOH), somatic mosaicism and mutations in genes from known cancer pathways. The combination of genotyping and exome sequencing approaches provided more information than either technique alone. The results showed widespread evidence of copy number changes involving most chromosomes including the possible loss of both alleles of CDKN1B (p27/Kip1). LOH was observed for the regions encompassing the tumor suppressors TP53, RB1, and CHD1. Predicted damaging somatic mutations were observed in the retained TP53 and RB1 alleles. Mutations in other genes that may be functionally relevant were noted, especially the recently reported high confidence cancer drivers FOXA1 and CCAR1. The disruption of multiple cancer drivers underscores why SCPC may be such a difficult cancer to manage. PMID:24823478

  9. Genomic Characterization of Esophageal Squamous Cell Carcinoma Reveals Critical Genes Underlying Tumorigenesis and Poor Prognosis

    PubMed Central

    Qin, Hai-De; Liao, Xiao-Yu; Chen, Yuan-Bin; Huang, Shao-Yi; Xue, Wen-Qiong; Li, Fang-Fang; Ge, Xiao-Song; Liu, De-Qing; Cai, Qiuyin; Long, Jirong; Li, Xi-Zhao; Hu, Ye-Zhu; Zhang, Shao-Dan; Zhang, Lan-Jun; Lehrman, Benjamin; Scott, Alan F.; Lin, Dongxin; Zeng, Yi-Xin; Shugart, Yin Yao; Jia, Wei-Hua

    2016-01-01

    The genetic mechanisms underlying the poor prognosis of esophageal squamous cell carcinoma (ESCC) are not well understood. Here, we report somatic mutations found in ESCC from sequencing 10 whole-genome and 57 whole-exome matched tumor-normal sample pairs. Among the identified genes, we characterized mutations in VANGL1 and showed that they accelerated cell growth in vitro. We also found that five other genes, including three coding genes (SHANK2, MYBL2, FADD) and two non-coding genes (miR-4707-5p, PCAT1), were involved in somatic copy-number alterations (SCNAs) or structural variants (SVs). A survival analysis based on the expression profiles of 321 individuals with ESCC indicated that these genes were significantly associated with poorer survival. Subsequently, we performed functional studies, which showed that miR-4707-5p and MYBL2 promoted proliferation and metastasis. Together, our results shed light on somatic mutations and genomic events that contribute to ESCC tumorigenesis and prognosis and might suggest therapeutic targets. PMID:27058444

  10. The mouse gene for vascular endothelial growth factor. Genomic structure, definition of the transcriptional unit, and characterization of transcriptional and post-transcriptional regulatory sequences.

    PubMed

    Shima, D T; Kuroki, M; Deutsch, U; Ng, Y S; Adamis, A P; D'Amore, P A

    1996-02-16

    We describe the genomic organization and functional characterization of the mouse gene encoding vascular endothelial growth factor (VEGF), a polypeptide implicated in embryonic vascular development and postnatal angiogenesis. The coding region for mouse VEGF is interrupted by seven introns and encompasses approximately 14 kilobases. Organization of exons suggests that, similar to the human VEGF gene, alternative splicing generates the 120-, 164-, and 188-amino acid isoforms, but does not predict a fourth VEGF isoform corresponding to human VEGF206. Approximately 1. 2 kilobases of 5'-flanking region have been sequenced, and primer extension analysis identified a single major transcription initiation site, notably lacking TATA or CCAT consensus sequences. The 5'-flanking region is sufficient to promote a 7-fold induction of basal transcription. The genomic region encoding the 3'-untranslated region was determined by Northern and nuclease mapping analysis. Investigation of mRNA sequences responsible for the rapid turnover of VEGF mRNA (mRNA half-life, <1 h) (Shima, D. T. , Deutsch, U., and D'Amore, P. A. (1995) FEBS Lett. 370, 203-208) revealed that the 3'-untranslated region was sufficient to trigger the rapid turnover of a normally long-lived reporter mRNA in vitro. These data and reagents will allow the molecular and genetic analysis of mechanisms that control the developmental and pathological expression of VEGF.

  11. Characterization of a Single Genomic Locus Encoding the Clustered Protocadherin Receptor Diversity in Xenopus tropicalis

    PubMed Central

    Etlioglu, Hakki E.; Sun, Wei; Huang, Zengjin; Chen, Wei; Schmucker, Dietmar

    2016-01-01

    Clustered protocadherins (cPcdhs) constitute the largest subgroup of the cadherin superfamily, and in mammals are grouped into clusters of α-, β-, and γ-types. Tens of tandemly arranged paralogous Pcdh genes of the Pcdh clusters generate a substantial diversity of receptor isoforms. cPcdhs are known to have important roles in neuronal development, and genetic alterations of cPcdhs have been found to be associated with several neurological diseases. Here, we present a first characterization of cPcdhs in Xenopus tropicalis. We determined and annotated all cPcdh isoforms, revealing that they are present in a single chromosomal locus. We validated a total of 96 isoforms, which we show are organized in three distinct clusters. The X. tropicalis cPcdh locus is composed of one α- and two distinct γ-Pcdh clusters (pcdh-γ1 and pcdh-γ2). Bioinformatics analyses assisted by genomic BAC clone sequencing showed that the X. tropicalis α- and γ-Pcdhs are conserved at the cluster level, but, unlike mammals, X. tropicalis does not contain a β-Pcdh cluster. In contrast, the number of γ-Pcdh isoforms has expanded, possibly due to lineage-specific gene duplications. Interestingly, the number of X. tropicalis α-Pcdhs is identical between X. tropicalis and mouse. Moreover, we find highly conserved as well as novel promoter elements potentially involved in regulating the cluster-specific expression of cPcdh isoforms. This study provides important information for the understanding of the evolutionary history of cPcdh genes and future mechanistic studies. It provides an annotated X. tropicalis cPcdh genomic map and a first molecular characterization essential for functional and comparative studies. PMID:27261006

  12. Characterization of Five Novel Brevibacillus Bacteriophages and Genomic Comparison of Brevibacillus Phages.

    PubMed

    Berg, Jordan A; Merrill, Bryan D; Crockett, Justin T; Esplin, Kyle P; Evans, Marlee R; Heaton, Karli E; Hilton, Jared A; Hyde, Jonathan R; McBride, Morgan S; Schouten, Jordan T; Simister, Austin R; Thurgood, Trever L; Ward, Andrew T; Breakwell, Donald P; Hope, Sandra; Grose, Julianne H

    2016-01-01

    Brevibacillus laterosporus is a spore-forming bacterium that causes a secondary infection in beehives following European Foulbrood disease. To better understand the contributions of Brevibacillus bacteriophages to the evolution of their hosts, five novel phages (Jenst, Osiris, Powder, SecTim467, and Sundance) were isolated and characterized. When compared with the five Brevibacillus phages currently in NCBI, these phages were assigned to clusters based on whole genome and proteome synteny. Powder and Osiris, both myoviruses, were assigned to the previously described Jimmer-like cluster. SecTim467 and Jenst, both siphoviruses, formed a novel phage cluster. Sundance, a siphovirus, was assigned as a singleton phage along with the previously isolated singleton, Emery. In addition to characterizing the basic relationships between these phages, several genomic features were observed. A motif repeated throughout phages Jenst and SecTim467 was frequently upstream of genes predicted to function in DNA replication, nucleotide metabolism, and transcription, suggesting transcriptional co-regulation. In addition, paralogous gene pairs that encode a putative transcriptional regulator were identified in four Brevibacillus phages. These paralogs likely evolved to bind different DNA sequences due to variation at amino acid residues predicted to bind specific nucleotides. Finally, a putative transposable element was identified in SecTim467 and Sundance that carries genes homologous to those found in Brevibacillus chromosomes. Remnants of this transposable element were also identified in phage Jenst. These discoveries provide a greater understanding of the diversity of phages, their behavior, and their evolutionary relationships to one another and to their host. In addition, they provide a foundation with which further Brevibacillus phages can be compared.

  13. Characterization of Five Novel Brevibacillus Bacteriophages and Genomic Comparison of Brevibacillus Phages

    PubMed Central

    Berg, Jordan A.; Merrill, Bryan D.; Crockett, Justin T.; Esplin, Kyle P.; Evans, Marlee R.; Heaton, Karli E.; Hilton, Jared A.; Hyde, Jonathan R.; McBride, Morgan S.; Schouten, Jordan T.; Simister, Austin R.; Thurgood, Trever L.; Ward, Andrew T.; Breakwell, Donald P.; Hope, Sandra; Grose, Julianne H.

    2016-01-01

    Brevibacillus laterosporus is a spore-forming bacterium that causes a secondary infection in beehives following European Foulbrood disease. To better understand the contributions of Brevibacillus bacteriophages to the evolution of their hosts, five novel phages (Jenst, Osiris, Powder, SecTim467, and Sundance) were isolated and characterized. When compared with the five Brevibacillus phages currently in NCBI, these phages were assigned to clusters based on whole genome and proteome synteny. Powder and Osiris, both myoviruses, were assigned to the previously described Jimmer-like cluster. SecTim467 and Jenst, both siphoviruses, formed a novel phage cluster. Sundance, a siphovirus, was assigned as a singleton phage along with the previously isolated singleton, Emery. In addition to characterizing the basic relationships between these phages, several genomic features were observed. A motif repeated throughout phages Jenst and SecTim467 was frequently upstream of genes predicted to function in DNA replication, nucleotide metabolism, and transcription, suggesting transcriptional co-regulation. In addition, paralogous gene pairs that encode a putative transcriptional regulator were identified in four Brevibacillus phages. These paralogs likely evolved to bind different DNA sequences due to variation at amino acid residues predicted to bind specific nucleotides. Finally, a putative transposable element was identified in SecTim467 and Sundance that carries genes homologous to those found in Brevibacillus chromosomes. Remnants of this transposable element were also identified in phage Jenst. These discoveries provide a greater understanding of the diversity of phages, their behavior, and their evolutionary relationships to one another and to their host. In addition, they provide a foundation with which further Brevibacillus phages can be compared. PMID:27304881

  14. Rapid confirmation of suspected methicillin-resistant Staphylococcus aureus colonies on chromogenic agars by a new commercial PCR assay, the GenomEra MRSA/SA Diagnose.

    PubMed

    Hirvonen, J J; Nevalainen, M; Tissari, P; Salmenlinna, S; Rantakokko-Jalava, K; Kaukoranta, S-S

    2012-08-01

    A new automated closed tube PCR assay, the GenomEra(™) MRSA/SA Diagnose (Abacus Diagnostica Oy, Finland) was evaluated for rapid confirmation of methicillin-resistant Staphylococcus aureus (MRSA) from cultured screening specimens. The ability of the assay to detect genotypically different MRSA strains was studied with a collection of 304 MRSA isolates covering 68 spa types. The specificity was investigated with a collection of 146 non-MRSA staphylococcus isolates. The usefulness of the assay for clinical purposes was assessed by a sequential combination of MRSA screening culture and confirmation of the colonies with the GenomEra MRSA/SA Diagnose assay. A total of 145 suspected MRSA colonies on chromogenic plates were analyzed this way. All MRSA isolates from the culture collection and from the clinical screening specimens were confirmed as MRSA with the GenomEra MRSA/SA Diagnose assay and none of the non-MRSA staphylococci caused false-positive results, which indicates both sensitivity and specificity of 100%. The combination of GenomEra MRSA/SA Diagnose with preceding culture on selective MRSA agar permitted MRSA confirmation within 24 h. This practice offers a reliable and quick detection of MRSA that is also suitable in areas where several strain types cause epidemics.

  15. Rapid and accurate species and genomic species identification and exhaustive population diversity assessment of Agrobacterium spp. using recA-based PCR.

    PubMed

    Shams, M; Vial, L; Chapulliot, D; Nesme, X; Lavire, C

    2013-07-01

    Agrobacteria are common soil bacteria that interact with plants as commensals, plant growth promoting rhizobacteria or alternatively as pathogens. Indigenous agrobacterial populations are composites, generally with several species and/or genomic species and several strains per species. We thus developed a recA-based PCR approach to accurately identify and specifically detect agrobacteria at various taxonomic levels. Specific primers were designed for all species and/or genomic species of Agrobacterium presently known, including 11 genomic species of the Agrobacterium tumefaciens complex (G1-G9, G13 and G14, among which only G2, G4, G8 and G14 still received a Latin epithet: pusense, radiobacter, fabrum and nepotum, respectively), A. larrymoorei, A. rubi, R. skierniewicense, A. sp. 1650, and A. vitis, and for the close relative Allorhizobium undicola. Specific primers were also designed for superior taxa, Agrobacterium spp. and Rhizobiaceace. Primer specificities were assessed with target and non-target pure culture DNAs as well as with DNAs extracted from composite agrobacterial communities. In addition, we showed that the amplicon cloning-sequencing approach used with Agrobacterium-specific or Rhizobiaceae-specific primers is a way to assess the agrobacterial diversity of an indigenous agrobacterial population. Hence, the agrobacterium-specific primers designed in the present study enabled the first accurate and rapid identification of all species and/or genomic species of Agrobacterium, as well as their direct detection in environmental samples.

  16. Characterization of genomic instability in Saccharomyces cerevisiae and engaging teaching strategies described in two curricula

    NASA Astrophysics Data System (ADS)

    Keller, Alexandra P.

    Cancer arises through an accumulation of mutations in the genome. In cancer cells, mutations are frequently caused by DNA rearrangements, which include chromosomal breakages, deletions, insertions, and translocations. Such events contribute to genomic instability, a known hallmark of cancer. To study cycles of chromosomal instability, we are using baker's yeast as a model organism. In yeast, a ChrVII system was previously developed (Admire et al., 2006), in which a disomic yeast strain was used to identify regions of instability on ChrVII. Using this system, a fragile site on the left arm of ChrVII (Admire et al., 2006) was identified and characterized. This study led to insight into mechanisms involved in chromosomal rearrangements and mutations that arise from them as well as to an understanding of mechanisms involved in genomic instability. To further our understanding of genomic instability, I devised a strategy to study instability on a different chromosome (ChrV) (Figure 3), so that we could determine whether lessons learned from the ChrVII system are applicable to other chromosomes, and/or whether other mechanisms of instability could be identified. A suitable strain was generated and analyzed, and our findings suggest that frequencies of instability on the right arm of ChrV are similar to those found in ChrVII. The results from the work in ChrV described in this paper support the idea that the instability found on ChrVII is not an isolated occurrence. My research was supported by an NSF GK-12 grant. The aim of this grant is to improve science education in middle schools, and as part of my participation in this program, I studied and practiced effective science communication methodologies. In attempts to explain my research to middle school students, I collaborated with others to develop methods for explaining genetics and the most important techniques I used in my research. While developing these methods, I learned more about what motivates people to learn

  17. Genome-wide molecular characterization of central nervous system primitive neuroectodermal tumor and pineoblastoma

    PubMed Central

    Miller, Suzanne; Rogers, Hazel A.; Lyon, Paul; Rand, Vikki; Adamowicz-Brice, Martyna; Clifford, Steven C.; Hayden, James T.; Dyer, Sara; Pfister, Stefan; Korshunov, Andrey; Brundler, Marie-Anne; Lowe, James; Coyle, Beth; Grundy, Richard G.

    2011-01-01

    Central nervous system primitive neuroectodermal tumor (CNS PNET) and pineoblastoma are highly malignant embryonal brain tumors with poor prognoses. Current therapies are based on the treatment of pediatric medulloblastoma, even though these tumors are distinct at both the anatomical and molecular level. CNS PNET and pineoblastoma have a worse clinical outcome than medulloblastoma; thus, improved therapies based on an understanding of the underlying biology of CNS PNET and pineoblastoma are needed. To this end, we characterized the genomic alterations of 36 pediatric CNS PNETs and 8 pineoblastomas using Affymetrix single nucleotide polymorphism arrays. Overall, the majority of CNS PNETs contained a greater degree of genomic imbalance than pineoblastomas, with gain of 19p (8 [27.6%] of 29), 2p (7 [24.1%] of 29), and 1q (6 [20.7%] of 29) common events in primary CNS PNETs. Novel gene copy number alterations were identified and corroborated by Genomic Identification of Significant Targets In Cancer (GISTIC) analysis: gain of PCDHGA3, 5q31.3 in 62.1% of primary CNS PNETs and all primary pineoblastomas and FAM129A, 1q25 in 55.2% of primary CNS PNETs and 50% of primary pineoblastomas. Comparison of our GISTIC data with publically available data for medulloblastoma confirmed these CNS PNET–specific copy number alterations. With use of the collection of 5 primary and recurrent CNS PNET pairs, we found that gain of 2p21 was maintained at relapse in 80% of cases. Novel gene copy number losses included OR4C12, 11p11.12 in 48.2% of primary CNS PNETs and 50% of primary pineoblastomas. Loss of CDKN2A/B (9p21.3) was identified in 14% of primary CNS PNETs and was significantly associated with older age among children (P = .05). CADPS, 3p14.2 was lost in 27.6% of primary CNS PNETs and was associated with poor prognosis (P = .043). This genome-wide analysis revealed the marked molecular heterogeneity of CNS PNETs and enabled the identification of novel genes and clinical

  18. Bulk Combinatorial Synthesis and High Throughput Characterization for Rapid Assessment of Magnetic Materials: Application of Laser Engineered Net Shaping (LENS™)

    NASA Astrophysics Data System (ADS)

    Geng, J.; Nlebedim, I. C.; Besser, M. F.; Simsek, E.; Ott, R. T.

    2016-07-01

    A bulk combinatorial approach for synthesizing alloy libraries using laser engineered net shaping (LENS™; i.e., 3D printing) was utilized to rapidly assess material systems for magnetic applications. The LENS™ system feeds powders in different ratios into a melt pool created by a laser to synthesize samples with bulk (millimeters) dimensions. By analyzing these libraries with autosampler differential scanning calorimeter/thermal gravimetric analysis and vibrating sample magnetometry, we are able to rapidly characterize the thermodynamic and magnetic properties of the libraries. The Fe-Co binary alloy was used as a model system and the results were compared with data in the literature.

  19. Rapid Characterization of Near-Surface Seafloor Sediment using a Free Fall Penetrometer

    NASA Astrophysics Data System (ADS)

    Mulukutla, G. K.; Melton, J.

    2010-12-01

    The assessment of the mechanical properties of near-surface sediment is of critical importance to several studies of the seafloor. Key properties such as sediment type, grain size, shear strength or bearing capacity are required for most geophysical and geotechnical studies of the seafloor. Recently developed Free Fall Penetrometers (FFP), instrumented not only with accelerometers but also with pressure sensors and optical backscatter sensors, are deployable from an underway vessel to provide rapid data to characterize the seafloor over a wide areal extent. In existing practice FFP data is used to provide a qualitative description of the seabed sediment using models from quasi-static penetrometer testing or as soft, medium or hard and provide an estimate of penetration resistance. The mechanics of FFP impact and embedment is considerably different from quasi-static penetration of a Cone Penetrometer Testing (CPT) as result this study takes a different approach to formulating a model. In many cases the information on the bottom is not sufficient or the penetration resistance is overestimated due to the dilatory effects observed particularly in sediment with a coarse-grained fraction. In this study a model is described that uses acceleration (i.e the deceleration)-time histories to identify the sediment type and provide an estimate of grain size. Estimate of the sediment shear strength for soft-sediment is provided by the formulation of a strain-rate dependent model that accounts for the varying velocity during embedment. The response of the pressure and optical backscatter sensors to embedment are used to identify the mudline and understand the drainage conditions during the embedment and provide a picture of the bottom conditions. The work describes original analysis and uses data from more than 200 drops of two FFPs deployed in the Bering Sea (conducted using the NOAA ship Fairweather) and Gulf of Maine, along with groundtruthing from a bottom sampler and field

  20. Characterization of genome-wide microsatellites of Saccharina japonica based on a preliminary assembly of Illumina sequencing reads

    NASA Astrophysics Data System (ADS)

    Zhang, Linan; Peng, Jie; Li, Xiaojie; Cui, Cuiju; Sun, Juan; Yang, Guanpin

    2016-06-01

    Microsatellites or simple sequence repeats (SSR) function widely and locate dependently in genome. However, their characteristics are often ignored due to the lack of genomic sequences of most species. Kelp ( Saccharina japonica), a brown macroalga, is extensively cultured in China. In this study, the genome of S. japonica was surveyed using an Illumina sequencing platform, and its microsatellites were characterized. The preliminarily assembled genome was 469.4 Mb in size, with a scaffold N50 of 20529 bp. Among the 128370 identified microsatellites, 90671, 25726 and 11973 were found in intergenic regions, introns and exons, averaging 339.3, 178.8 and 205.4 microsatellites per Mb, respectively. These microsatellites distributed unevenly in S. japonica genome. Mononucleotide motifs were the most abundant in the genome, while trinucleotide ones were the most prevalent in exons. The microsatellite abundance decreased significantly with the increase of motif repeat numbers, and the microsatellites with a small number of repeats accounted for a higher proportion of the exons than those of the intergenic regions and introns. C/G-rich motifs were more common in exons than in intergenic regions and introns. These characteristics of microsatellites in S. japonica genome may associate with their functions, and ultimately their adaptation and evolution. Among the 120140 pairs of designed microsatellite primers, approximately 75% were predicted to be able to amplify S. japonica DNA. These microsatellite markers will be extremely useful for the genetic breeding and population evolution studies of kelp.

  1. GenomEra MRSA/SA, a fully automated homogeneous PCR assay for rapid detection of Staphylococcus aureus and the marker of methicillin resistance in various sample matrixes.

    PubMed

    Hirvonen, Jari J; Kaukoranta, Suvi-Sirkku

    2013-09-01

    The GenomEra MRSA/SA assay (Abacus Diagnostica, Turku, Finland) is the first commercial homogeneous PCR assay using thermally stable, intrinsically fluorescent time-resolved fluorometric (TRF) labels resistant to autofluorescence and other background effects. This fully automated closed tube PCR assay simultaneously detects Staphylococcus aureus specific DNA and the mecA gene within 50 min. It can be used for both screening and confirmation of methicillin-resistant and -sensitive S. aureus (MRSA and MSSA) directly in different specimen types or from preceding cultures. The assay has shown excellent performance in comparisons with other diagnostic methods in all the sample types tested. The GenomEra MRSA/SA assay provides rapid assistance for the detection of MRSA as well as invasive staphylococcal infections and helps the early targeting of antimicrobial therapy to patients with potential MRSA infection.

  2. Rapid Characterization of Molecular Chemistry, Nutrient Make-Up and Microlocation of Internal Seed Tissue

    SciTech Connect

    Yu,P.; Block, H.; Niu, Z.; Doiron, K.

    2007-01-01

    Wheat differs from corn in biodegradation kinetics and fermentation characteristics. Wheat exhibits a relatively high rate (23% h{sup 01}) and extent (78% DM) of biodegradation, which can lead to metabolic problems such as acidosis and bloat in ruminants. The objective of this study was to rapidly characterize the molecular chemistry of the internal structure of wheat (cv. AC Barrie) and reveal both its structural chemical make-up and nutrient component matrix by analyzing the intensity and spatial distribution of molecular functional groups within the intact seed using advanced synchrotron-powered Fourier transform infrared (FTIR) microspectroscopy. The experiment was performed at the U2B station of the National Synchrotron Light Source at Brookhaven National Laboratory, New York, USA. The wheat tissue was imaged systematically from the pericarp, seed coat, aleurone layer and endosperm under the peaks at {approx}1732 (carbonyl C{double_bond}O ester), 1515 (aromatic compound of lignin), 1650 (amide I), 1025 (non-structural CHO), 1550 (amide II), 1246 (cellulosic material), 1160, 1150, 1080, 930, 860 (all CHO), 3350 (OH and NH stretching), 2928 (CH{sub 2} stretching band) and 2885 cm{sup -1} (CH{sub 3} stretching band). Hierarchical cluster analysis and principal component analysis were applied to analyze the molecular FTIR spectra obtained from the different inherent structures within the intact wheat tissues. The results showed that, with synchrotron-powered FTIR microspectroscopy, images of the molecular chemistry of wheat could be generated at an ultra-spatial resolution. The features of aromatic lignin, structural and non-structural carbohydrates, as well as nutrient make-up and interactions in the seeds, could be revealed. Both principal component analysis and hierarchical cluster analysis methods are conclusive in showing that they can discriminate and classify the different inherent structures within the seed tissue. The wheat exhibited distinguishable

  3. Investigation on microstructure characterization and property of rapidly solidified Mg-Zn-Ca-Ce-La alloys

    SciTech Connect

    Zhou Tao; Chen Zhenhua; Yang Mingbo; Hu Jianjun; Xia Hua

    2012-01-15

    Rapidly solidified (RS) Mg-Zn-Ca-Ce-La (wt.%) alloys have been produced via atomizing the alloy melt and subsequent splat-quenching on the water-cooled copper twin-rollers in the form of flakes. Microstructure characterization, phase compositions and thermal stability of the alloys have been systematically investigated. The results showed that with addition of RE (Ce and La) to the Mg-6Zn-5Ca alloy, the stable intermetallic compounds i.e. the Mg{sub x}Zn{sub y}RE{sub z} phase with a few Ca (about 3 at.%), shortened as the T Prime phase, were formed at the expense of the binary Mg-Zn and Ca{sub 2}Mg{sub 6}Zn{sub 3} phases, which was possibly beneficial to the enhanced thermal stability of the alloy. In the Mg-6Zn-5Ca-3Ce-0.5La alloy, the composition of the T Prime phase in the grain interior was different from that at the grain boundaries, in which the segregation of the La elements was found, and the atomic percentage ratio of Zn to Ce in the T Prime phase within the grains was close to 2. Moreover, the stable Mg{sub 2}Ca phases were detected around the T Prime phases at the grain boundaries in the alloy. - Research Highlights: Black-Right-Pointing-Pointer The phase constitution of RS Mg-6Zn-5Ca alloy can be improved by RE additions. Black-Right-Pointing-Pointer In the Mg-Zn-Ca-Ce-La alloys, the Mg{sub x}Zn{sub y}RE{sub z} phase with a few Ca (T Prime phase) is formed. Black-Right-Pointing-Pointer The formation of the T Prime phase leads to the loss of the Mg-Zn and Ca{sub 2}Mg{sub 6}Zn{sub 3} phases. Black-Right-Pointing-Pointer The composition of the T Prime phase differs from the grain interior to the grain boundary.

  4. Characterization of RNA isolated from eighteen different human tissues: results from a rapid human autopsy program.

    PubMed

    Walker, Douglas G; Whetzel, Alexis M; Serrano, Geidy; Sue, Lucia I; Lue, Lih-Fen; Beach, Thomas G

    2016-09-01

    Many factors affect the integrity of messenger RNA from human autopsy tissues including postmortem interval (PMI) between death and tissue preservation and the pre-mortem agonal and disease states. In this communication, we describe RNA isolation and characterization of 389 samples from 18 different tissues from elderly donors who were participants in a rapid whole-body autopsy program located in Sun City, Arizona ( www.brainandbodydonationprogram.org ). Most tissues were collected within a PMI of 2-6 h (median 3.15 h; N = 455), but for this study, tissue from cases with longer PMIs (1.25-29.25 h) were included. RNA quality was assessed by RNA integrity number (RIN) and total yield (ng RNA/mg tissue). RIN correlated with PMI for heart (r = -0.531, p = 0.009) and liver (r = -558, p = 0.0017), while RNA yield correlated with PMI for colon (r = -485, p = 0.016) and skin (r = -0.460, p = 0.031). RNAs with the lowest integrity were from skin and cervix where 22.7 and 31.4 % of samples respectively failed to produce intact RNA; by contrast all samples from esophagus, lymph node, jejunum, lung, stomach, submandibular gland and kidney produced RNA with measurable RINs. Expression levels in heart RNA of 4 common housekeeping normalization genes showed significant correlations of Ct values with RIN, but only one gene, glyceraldehyde-3 phosphate dehydrogenase, showed a correlation of Ct with PMI. There were no correlations between RIN values obtained for liver, adrenal, cervix, esophagus and lymph node and those obtained from corresponding brain samples. We show that high quality RNA can be produced from most human autopsy tissues, though with significant differences between tissues and donors. The RNA stability and yield did not depend solely on PMI; other undetermined factors are involved, but these do not include the age of the donor.

  5. Characterization of genome-wide SNPs for the water flea Daphnia pulicaria generated by genotyping-by-sequencing (GBS)

    PubMed Central

    Muñoz, Joaquín; Chaturvedi, Anurag; De Meester, Luc; Weider, Lawrence J.

    2016-01-01

    The keystone aquatic herbivore Daphnia has been studied for more than 150 years in the context of evolution, ecology and ecotoxicology. Although it is rapidly becoming an emergent model for environmental and population genomics, there have been limited genome-wide level studies in natural populations. We report a unique resource of novel Single Nucleotide Polymorphic (SNP) markers for Daphnia pulicaria using the reduction in genomic complexity with the restriction enzymes approach, genotyping-by-sequencing. Using the genome of D. pulex as a reference, SNPs were scored for 53 clones from five natural populations that varied in lake trophic status. Our analyses resulted in 32,313 highly confident and bi-allelic SNP markers. 1,364 outlier SNPs were mapped on the annotated D. pulex genome, which identified 2,335 genes, including 565 within functional genes. Out of 885 EuKaryotic Orthologous Groups that we found from outlier SNPs, 294 were involved in three metabolic and four regulatory pathways. Bayesian-clustering analyses showed two distinct population clusters representing the possible combined effects of geography and lake trophic status. Our results provide an invaluable tool for future population genomics surveys in Daphnia targeting informative regions related to physiological processes that can be linked to the ecology of this emerging eco-responsive taxon. PMID:27346179

  6. Characterization of genome-wide SNPs for the water flea Daphnia pulicaria generated by genotyping-by-sequencing (GBS).

    PubMed

    Muñoz, Joaquín; Chaturvedi, Anurag; De Meester, Luc; Weider, Lawrence J

    2016-06-27

    The keystone aquatic herbivore Daphnia has been studied for more than 150 years in the context of evolution, ecology and ecotoxicology. Although it is rapidly becoming an emergent model for environmental and population genomics, there have been limited genome-wide level studies in natural populations. We report a unique resource of novel Single Nucleotide Polymorphic (SNP) markers for Daphnia pulicaria using the reduction in genomic complexity with the restriction enzymes approach, genotyping-by-sequencing. Using the genome of D. pulex as a reference, SNPs were scored for 53 clones from five natural populations that varied in lake trophic status. Our analyses resulted in 32,313 highly confident and bi-allelic SNP markers. 1,364 outlier SNPs were mapped on the annotated D. pulex genome, which identified 2,335 genes, including 565 within functional genes. Out of 885 EuKaryotic Orthologous Groups that we found from outlier SNPs, 294 were involved in three metabolic and four regulatory pathways. Bayesian-clustering analyses showed two distinct population clusters representing the possible combined effects of geography and lake trophic status. Our results provide an invaluable tool for future population genomics surveys in Daphnia targeting informative regions related to physiological processes that can be linked to the ecology of this emerging eco-responsive taxon.

  7. Characterization of genome-wide SNPs for the water flea Daphnia pulicaria generated by genotyping-by-sequencing (GBS).

    PubMed

    Muñoz, Joaquín; Chaturvedi, Anurag; De Meester, Luc; Weider, Lawrence J

    2016-01-01

    The keystone aquatic herbivore Daphnia has been studied for more than 150 years in the context of evolution, ecology and ecotoxicology. Although it is rapidly becoming an emergent model for environmental and population genomics, there have been limited genome-wide level studies in natural populations. We report a unique resource of novel Single Nucleotide Polymorphic (SNP) markers for Daphnia pulicaria using the reduction in genomic complexity with the restriction enzymes approach, genotyping-by-sequencing. Using the genome of D. pulex as a reference, SNPs were scored for 53 clones from five natural populations that varied in lake trophic status. Our analyses resulted in 32,313 highly confident and bi-allelic SNP markers. 1,364 outlier SNPs were mapped on the annotated D. pulex genome, which identified 2,335 genes, including 565 within functional genes. Out of 885 EuKaryotic Orthologous Groups that we found from outlier SNPs, 294 were involved in three metabolic and four regulatory pathways. Bayesian-clustering analyses showed two distinct population clusters representing the possible combined effects of geography and lake trophic status. Our results provide an invaluable tool for future population genomics surveys in Daphnia targeting informative regions related to physiological processes that can be linked to the ecology of this emerging eco-responsive taxon. PMID:27346179

  8. Accumulation and rapid decay of non-LTR retrotransposons in the genome of the three-spine stickleback.

    PubMed

    Blass, Eryn; Bell, Michael; Boissinot, Stéphane

    2012-01-01

    The diversity and abundance of non-long terminal repeat (LTR) retrotransposons (nLTR-RT) differ drastically among vertebrate genomes. At one extreme, the genome of placental mammals is littered with hundreds of thousands of copies resulting from the activity of a single clade of nLTR-RT, the L1 clade. In contrast, fish genomes contain a much more diverse repertoire of nLTR-RT, represented by numerous active clades and families. Yet, the number of nLTR-RT copies in teleostean fish is two orders of magnitude smaller than in mammals. The vast majority of insertions appear to be very recent, suggesting that nLTR-RT do not accumulate in fish genomes. This pattern had previously been explained by a high rate of turnover, in which the insertion of new elements is offset by the selective loss of deleterious inserts. The turnover model was proposed because of the similarity between fish and Drosophila genomes with regard to their nLTR-RT profile. However, it is unclear if this model applies to fish. In fact, a previous study performed on the puffer fish suggested that transposable element insertions behave as neutral alleles. Here we examined the dynamics of amplification of nLTR-RT in the three-spine stickleback (Gasterosteus aculeatus). In this species, the vast majority of nLTR-RT insertions are relatively young, as suggested by their low level of divergence. Contrary to expectations, a majority of these insertions are fixed in lake and oceanic populations; thus, nLTR-RT do indeed accumulate in the genome of their fish host. This is not to say that nLTR-RTs are fully neutral, as the lack of fixed long elements in this genome suggests a deleterious effect related to their length. This analysis does not support the turnover model and strongly suggests that a much higher rate of DNA loss in fish than in mammals is responsible for the relatively small number of nLTR-RT copies and for the scarcity of ancient elements in fish genomes. We further demonstrate that nLTR-RT decay

  9. Whole Genome Sequencing and a New Bioinformatics Platform Allow for Rapid Gene Identification in D. melanogaster EMS Screens.

    PubMed

    Gonzalez, Michael A; Van Booven, Derek; Hulme, William; Ulloa, Rick H; Lebrigio, Rafael F Acosta; Osterloh, Jeannette; Logan, Mary; Freeman, Marc; Zuchner, Stephan

    2012-01-01

    Forward genetic screens in Drosophila melanogaster using ethyl methanesulfonate (EMS) mutagenesis are a powerful approach for identifying genes that modulate specific biological processes in an in vivo setting. The mapping of genes that contain randomly-induced point mutations has become more efficient in Drosophila thanks to the maturation and availability of many types of genetic tools. However, classic approaches to gene mapping are relatively slow and ultimately require extensive Sanger sequencing of candidate chromosomal loci. With the advent of new high-throughput sequencing techniques, it is increasingly efficient to directly re-sequence the whole genome of model organisms. This approach, in combination with traditional chromosomal mapping, has the potential to greatly simplify and accelerate mutation identification in mutants generated in EMS screens. Here we show that next-generation sequencing (NGS) is an accurate and efficient tool for high-throughput sequencing and mutation discovery in Drosophila melanogaster. As a test case, mutant strains of Drosophila that exhibited long-term survival of severed peripheral axons were identified in a forward EMS mutagenesis. All mutants were recessive and fell into a single lethal complementation group, which suggested that a single gene was responsible for the protective axon degenerative phenotype. Whole genome sequencing of these genomes identified the underlying gene ect4. To improve the process of genome wide mutation identification, we developed Genomes Management Application (GEM.app, https://genomics.med.miami.edu), a graphical online user interface to a custom query framework. Using a custom GEM.app query, we were able to identify that each mutant carried a unique non-sense mutation in the gene ect4 (dSarm), which was recently shown by Osterloh et al. to be essential for the activation of axonal degeneration. Our results demonstrate the current advantages and limitations of NGS in Drosophila and we introduce

  10. Characterization of full-length HIV-1 CRF17_BF genomes and comparison to the prototype CRF12_BF strains.

    PubMed

    Aulicino, Paula C; Gómez-Carrillo, Manuel; Bello, Gonzalo; Rocco, Carlos; Mangano, Andrea; Carr, Jean; Sen, Luisa; Foley, Brian

    2012-03-01

    The aim of this work is to characterize the full-length intersubtype recombinant structure of the HIV-1 Circulating Recombinant Form CRF17_BF. A single genome of CRF17_BF was originally described in 2001 as being largely similar to CRF12_BF. Since then, more genomes of CRF17_BF have been sequenced but not adequately described in publications. Here we describe CRF17_BF as a genuine CRF, and analyze its recombination pattern based on bootscan analyses, subtype signature patterns, and phylogenetic reconstruction of subtype-delimited segments. We show that CRF17_BF can be distinguished from CRF12_BF in several regions of the genome, including vpu, pol, env and nef. A complete and accurate characterization and description of recombination breakpoints in CRFs is required for a proper surveillance of HIV-1 genotypes, and important for epidemiological purposes. PMID:22266022

  11. Rapid Characterization of Polyalcohols by Silylation and MALDI-TOF Mass Spectrometry

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A method for rapid enumerating hydroxyl groups in analytes is described and applied to common polyalcohols (erythritol, mannitol, and xylitol). Polyalcohols were derivatized with trimethylsilylimidazole (TMSI) either separately or as mixtures, and were analyzed without chromatographic separation or...

  12. Using a Microscale Approach to Rapidly Separate and Characterize Three Photosynthetic Pigment Species from Fern

    ERIC Educational Resources Information Center

    Ayudhya, Theppawut Israsena Na; Posey, Frederick T.; Tyus, Jessica C.; Dingra, Nin N.

    2015-01-01

    A rapid separation of three photosynthetic pigments (chlorophyll "a" and "b" and xanthophyll) from fern ("Polystichum acrostichoides") is described using microscale solvent extraction and traditional thin layer chromatography that minimizes use of harmful chemicals and lengthy procedures. The experiment introduces…

  13. Exploiting genotyping by sequencing to characterize the genomic structure of the American cranberry through high-density linkage mapping

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The application of genotyping by sequencing (GBS) approaches, combined with data imputation methodologies, is narrowing the genetic knowledge gap between major and understudied, minor crops. GBS is an excellent tool to characterize the genomic structure of recently domesticated (~200 years) and unde...

  14. Complete Genome Sequence of an NADC30-Like Porcine Reproductive and Respiratory Syndrome Virus Characterized by Recombination with Other Strains

    PubMed Central

    Li, Yingying; Ji, Guobiao; Wang, Juan; Tan, Feifei; Zhuang, Jinshan

    2016-01-01

    We report here the complete genome sequence of an NADC30-like porcine reproductive and respiratory syndrome virus (PRRSV), HNyc15, which was characterized by recombination with VR-2332 and CH-1a PRRSV strains in open reading frames (ORFs) 2 to 4. PMID:27151798

  15. Rapid characterization of the sucrose esters from oriental tobacco using liquid chromatography/ion trap mass spectrometry.

    PubMed

    Ding, Li; Xie, Fuwei; Zhao, Mingyue; Xie, Jianping; Xu, Guowang

    2006-01-01

    Sucrose esters (SEs) from oriental tobacco are normally characterized by gas chromatography/mass spectrometry (GC/MS) after a long saponification and derivatization procedure. To simplify the process, a rapid method has been developed by using liquid chromatography coupled with electrospray ion trap mass spectrometry (LC/ESI-MSn). Using the characteristic fragmentation behavior of abundant SEs identified by GC/MS after purification by gel permeation chromatography (GPC) from cuticular waxes of green oriental tobacco leaf, two types of SEs from green and cured oriental tobacco were identified by MSn analysis. The first is one of three types reported formerly and has 13 SE homologues. However, the presence of unsaturation in one of the acyl substituents of this first type gave rise to a new series with three homologues. The other was found to be a new type and had three homologues. The proposed method enables the rapid and sensitive characterization of SEs from oriental tobacco.

  16. Genome wide in silico characterization of Dof gene families of pigeonpea (Cajanus cajan (L) Millsp.).

    PubMed

    Malviya, N; Gupta, S; Singh, V K; Yadav, M K; Bisht, N C; Sarangi, B K; Yadav, D

    2015-02-01

    The DNA binding with One Finger (Dof) protein is a plant specific transcription factor involved in the regulation of wide range of processes. The analysis of whole genome sequence of pigeonpea has identified 38 putative Dof genes (CcDof) distributed on 8 chromosomes. A total of 17 out of 38 CcDof genes were found to be intronless. A comprehensive in silico characterization of CcDof gene family including the gene structure, chromosome location, protein motif, phylogeny, gene duplication and functional divergence has been attempted. The phylogenetic analysis resulted in 3 major clusters with closely related members in phylogenetic tree revealed common motif distribution. The in silico cis-regulatory element analysis revealed functional diversity with predominance of light responsive and stress responsive elements indicating the possibility of these CcDof genes to be associated with photoperiodic control and biotic and abiotic stress. The duplication pattern showed that tandem duplication is predominant over segmental duplication events. The comparative phylogenetic analysis of these Dof proteins along with 78 soybean, 36 Arabidopsis and 30 rice Dof proteins revealed 7 major clusters. Several groups of orthologs and paralogs were identified based on phylogenetic tree constructed. Our study provides useful information for functional characterization of CcDof genes. PMID:25344821

  17. Sequencing and characterizing the genome of Estrella lausannensis as an undergraduate project: training students and biological insights.

    PubMed

    Bertelli, Claire; Aeby, Sébastien; Chassot, Bérénice; Clulow, James; Hilfiker, Olivier; Rappo, Samuel; Ritzmann, Sébastien; Schumacher, Paolo; Terrettaz, Céline; Benaglio, Paola; Falquet, Laurent; Farinelli, Laurent; Gharib, Walid H; Goesmann, Alexander; Harshman, Keith; Linke, Burkhard; Miyazaki, Ryo; Rivolta, Carlo; Robinson-Rechavi, Marc; van der Meer, Jan Roelof; Greub, Gilbert

    2015-01-01

    With the widespread availability of high-throughput sequencing technologies, sequencing projects have become pervasive in the molecular life sciences. The huge bulk of data generated daily must be analyzed further by biologists with skills in bioinformatics and by "embedded bioinformaticians," i.e., bioinformaticians integrated in wet lab research groups. Thus, students interested in molecular life sciences must be trained in the main steps of genomics: sequencing, assembly, annotation and analysis. To reach that goal, a practical course has been set up for master students at the University of Lausanne: the "Sequence a genome" class. At the beginning of the academic year, a few bacterial species whose genome is unknown are provided to the students, who sequence and assemble the genome(s) and perform manual annotation. Here, we report the progress of the first class from September 2010 to June 2011 and the results obtained by seven master students who specifically assembled and annotated the genome of Estrella lausannensis, an obligate intracellular bacterium related to Chlamydia. The draft genome of Estrella is composed of 29 scaffolds encompassing 2,819,825 bp that encode for 2233 putative proteins. Estrella also possesses a 9136 bp plasmid that encodes for 14 genes, among which we found an integrase and a toxin/antitoxin module. Like all other members of the Chlamydiales order, Estrella possesses a highly conserved type III secretion system, considered as a key virulence factor. The annotation of the Estrella genome also allowed the characterization of the metabolic abilities of this strictly intracellular bacterium. Altogether, the students provided the scientific community with the Estrella genome sequence and a preliminary understanding of the biology of this recently-discovered bacterial genus, while learning to use cutting-edge technologies for sequencing and to perform bioinformatics analyses.

  18. Identification of Variant-Specific Functions of PIK3CA by Rapid Phenotyping of Rare Mutations | Office of Cancer Genomics

    Cancer.gov

    Large-scale sequencing efforts are uncovering the complexity of cancer genomes, which are composed of causal "driver" mutations that promote tumor progression along with many more pathologically neutral "passenger" events. The majority of mutations, both in known cancer drivers and uncharacterized genes, are generally of low occurrence, highlighting the need to functionally annotate the long tail of infrequent mutations present in heterogeneous cancers.

  19. Genome-wide characterization of genetic diversity and population structure in Secale

    PubMed Central

    2014-01-01

    Background Numerous rye accessions are stored in ex situ genebanks worldwide. Little is known about the extent of genetic diversity contained in any of them and its relation to contemporary varieties, since to date rye genetic diversity studies had a very limited scope, analyzing few loci and/ or few accessions. Development of high throughput genotyping methods for rye opened the possibility for genome wide characterizations of large accessions sets. In this study we used 1054 Diversity Array Technology (DArT) markers with defined chromosomal location to characterize genetic diversity and population structure in a collection of 379 rye accessions including wild species, landraces, cultivated materials, historical and contemporary rye varieties. Results Average genetic similarity (GS) coefficients and average polymorphic information content (PIC) values varied among chromosomes. Comparison of chromosome specific average GS within and between germplasm sub-groups indicated regions of chromosomes 1R and 4R as being targeted by selection in current breeding programs. Bayesian clustering, principal coordinate analysis and Neighbor Joining clustering demonstrated that source and improvement status contributed significantly to the structure observed in the analyzed set of Secale germplasm. We revealed a relatively limited diversity in improved rye accessions, both historical and contemporary, as well as lack of correlation between clustering of improved accessions and geographic origin, suggesting common genetic background of rye accessions from diverse geographic regions and extensive germplasm exchange. Moreover, contemporary varieties were distinct from the remaining accessions. Conclusions Our results point to an influence of reproduction methods on the observed diversity patterns and indicate potential of ex situ collections for broadening the genetic diversity in rye breeding programs. Obtained data show that DArT markers provide a realistic picture of the genetic

  20. Characterization of a defective interfering RNA that contains a mosaic of a plant viral genome

    SciTech Connect

    Morris, T.J.; Jackson, A.O.

    1991-01-01

    Our lab was the first to describe and characterize a defective interfering RNA (DI RNAs or DIs) in association with a small RNA plant virus. The features of the DIs that we discovered in infections of tomato bushy stunt virus were compatible with the properties of DIs identified in many animal virus infections. Animal virologists have generally recognized the importance of studying DIs because they are invaluable tools for identifying cis-acting sequences important in virus multiplication and because they offer the opportunity to elucidate mechanisms involved in viral persistence and disease attenuation. Hence our discovery offered a comparably valuable tool for use in plant virus studies for the first time. Since then, we have also discovered the second example of plant viral DI RNAs associated with turnip crinkle virus (TCV), a virus structurally related to TBSV. We proposed a thorough characterization of this unique class of symptom modulating RNAs with the overall objective of identifying viral RNA nucleotide, sequences involved in such fundamental processes as virus replication and encapsidation as well as the degree of symptom expression resulting from the viral-DI-host interaction. The proposed research focused on the molecular characterization of the DI RNAs and the helper virus. We had demonstrated that the DIs were collinear deletion mutants of the genome of a cherry strain of tomato bushy stunt virus (TBSV). We had also shown that these low molecular weight RNAs interfered with the helper plant virus and modulated disease expression by preventing the development of a lethal necrotic disease in susceptible host plants. We also suggested that by exploring the mechanisms associated with the symptom attenuation effect, we might be able to devise novel strategies useful for engineering viral disease resistance.

  1. Identification and characterization of insect-specific proteins by genome data analysis

    PubMed Central

    Zhang, Guojie; Wang, Hongsheng; Shi, Junjie; Wang, Xiaoling; Zheng, Hongkun; Wong, Gane Ka-Shu; Clark, Terry; Wang, Wen; Wang, Jun; Kang, Le

    2007-01-01

    Background Insects constitute the vast majority of known species with their importance including biodiversity, agricultural, and human health concerns. It is likely that the successful adaptation of the Insecta clade depends on specific components in its proteome that give rise to specialized features. However, proteome determination is an intensive undertaking. Here we present results from a computational method that uses genome analysis to characterize insect and eukaryote proteomes as an approximation complementary to experimental approaches. Results Homologs in common to Drosophila melanogaster, Anopheles gambiae, Bombyx mori, Tribolium castaneum, and Apis mellifera were compared to the complete genomes of three non-insect eukaryotes (opisthokonts) Homo sapiens, Caenorhabditis elegans and Saccharomyces cerevisiae. This operation yielded 154 groups of orthologous proteins in Drosophila to be insect-specific homologs; 466 groups were determined to be common to eukaryotes (represented by three opisthokonts). ESTs from the hemimetabolous insect Locust migratoria were also considered in order to approximate their corresponding genes in the insect-specific homologs. Stress and stimulus response proteins were found to consti