RESUMO
SUMMARY: The JCVI pan-genome pipeline is a collection of programs to run PanOCT and tools that support and extend the capabilities of PanOCT. PanOCT (pan-genome ortholog clustering tool) is a tool for pan-genome analysis of closely related prokaryotic species or strains. The JCVI Pan-Genome Pipeline wrapper invokes command-line utilities that prepare input genomes, invoke third-party tools such as NCBI Blast+, run PanOCT, generate a consensus pan-genome, annotate features of the pan-genome, detect sets of genes of interest such as antimicrobial resistance (AMR) genes and generate figures, tables and html pages to visualize the results. The pipeline can run in a hierarchical mode, lowering the RAM and compute resources used. AVAILABILITY AND IMPLEMENTATION: Source code, demo data, and detailed documentation are freely available at https://github.com/JCVenterInstitute/PanGenomePipeline.
Assuntos
Genoma Bacteriano , Genoma Microbiano , Análise por Conglomerados , Células Procarióticas , SoftwareRESUMO
BACKGROUND: Bacterial pan-genomes, comprised of conserved and variable genes across multiple sequenced bacterial genomes, allow for identification of genomic regions that are phylogenetically discriminating or functionally important. Pan-genomes consist of large amounts of data, which can restrict researchers ability to locate and analyze these regions. Multiple software packages are available to visualize pan-genomes, but currently their ability to address these concerns are limited by using only pre-computed data sets, prioritizing core over variable gene clusters, or by not accounting for pan-chromosome positioning in the viewer. RESULTS: We introduce PanACEA (Pan-genome Atlas with Chromosome Explorer and Analyzer), which utilizes locally-computed interactive web-pages to view ordered pan-genome data. It consists of multi-tiered, hierarchical display pages that extend from pan-chromosomes to both core and variable regions to single genes. Regions and genes are functionally annotated to allow for rapid searching and visual identification of regions of interest with the option that user-supplied genomic phylogenies and metadata can be incorporated. PanACEA's memory and time requirements are within the capacities of standard laptops. The capability of PanACEA as a research tool is demonstrated by highlighting a variable region important in differentiating strains of Enterobacter hormaechei. CONCLUSIONS: PanACEA can rapidly translate the results of pan-chromosome programs into an intuitive and interactive visual representation. It will empower researchers to visually explore and identify regions of the pan-chromosome that are most biologically interesting, and to obtain publication quality images of these regions.
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Cromossomos/genética , Biologia Computacional/métodos , Genômica/métodos , HumanosRESUMO
SUMMARY: LOCUST is a custom sequence locus typer tool for classifying microbial genomes. It provides a fully automated opportunity to customize the classification of genome-wide nucleotide variant data most relevant to biological research. AVAILABILITY AND IMPLEMENTATION: Source code, demo data, and detailed documentation are freely available at http://sourceforge.net/projects/locustyper . CONTACT: lbrinkac@jcvi.org. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
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Bactérias/classificação , Genoma Bacteriano , Tipagem Molecular/métodos , Análise de Sequência de DNA/métodos , Software , Bactérias/genética , Genômica/métodosRESUMO
Pan-genome ortholog clustering tool (PanOCT) is a tool for pan-genomic analysis of closely related prokaryotic species or strains. PanOCT uses conserved gene neighborhood information to separate recently diverged paralogs into orthologous clusters where homology-only clustering methods cannot. The results from PanOCT and three commonly used graph-based ortholog-finding programs were compared using a set of four publicly available strains of the same bacterial species. All four methods agreed on â¼70% of the clusters and â¼86% of the proteins. The clusters that did not agree were inspected for evidence of correctness resulting in 85 high-confidence manually curated clusters that were used to compare all four methods.
Assuntos
Proteínas de Bactérias/genética , Genes Bacterianos , Genoma Bacteriano , Software , Bactérias/classificação , Proteínas de Bactérias/classificação , Análise por Conglomerados , Genômica/métodosRESUMO
The hypothalamus, composed of several nuclei, is essential for maintaining our body's homeostasis. The arcuate nucleus (ARC), located in the mediobasal hypothalamus, contains neuronal populations with eminent roles in energy and glucose homeostasis as well as reproduction. These neuronal populations are of great interest for translational research. To fulfill this promise, we used a robotic cell culture platform to provide a scalable and chemically defined approach for differentiating human pluripotent stem cells (hPSCs) into pro-opiomelanocortin (POMC), somatostatin (SST), tyrosine hydroxylase (TH) and gonadotropin-releasing hormone (GnRH) neuronal subpopulations with an ARC-like signature. This robust approach is reproducible across several distinct hPSC lines and exhibits a stepwise induction of key ventral diencephalon and ARC markers in transcriptomic profiling experiments. This is further corroborated by direct comparison to human fetal hypothalamus, and the enriched expression of genes implicated in obesity and type 2 diabetes (T2D). Genome-wide chromatin accessibility profiling by ATAC-seq identified accessible regulatory regions that can be utilized to predict candidate enhancers related to metabolic disorders and hypothalamic development. In depth molecular, cellular, and functional experiments unveiled the responsiveness of the hPSC-derived hypothalamic neurons to hormonal stimuli, such as insulin, neuropeptides including kisspeptin, and incretin mimetic drugs such as Exendin-4, highlighting their potential utility as physiologically relevant cellular models for disease studies. In addition, differential glucose and insulin treatments uncovered adaptability within the generated ARC neurons in the dynamic regulation of POMC and insulin receptors. In summary, the establishment of this model represents a novel, chemically defined, and scalable platform for manufacturing large numbers of hypothalamic arcuate neurons and serves as a valuable resource for modeling metabolic and reproductive disorders.
RESUMO
Embryoid bodies (EBs) and self-organizing organoids derived from human pluripotent stem cells (hPSCs) recapitulate tissue development in a dish and hold great promise for disease modeling and drug development. However, current protocols are hampered by cellular stress and apoptosis during cell aggregation, resulting in variability and impaired cell differentiation. Here, we demonstrate that EBs and various organoid models (e.g., brain, gut, kidney) can be optimized by using the small molecule cocktail named CEPT (chroman 1, emricasan, polyamines, trans-ISRIB), a polypharmacological approach that ensures cytoprotection and cell survival. Application of CEPT for just 24 h during cell aggregation has long-lasting consequences affecting morphogenesis, gene expression, cellular differentiation, and organoid function. Various qualification methods confirmed that CEPT treatment enhanced experimental reproducibility and consistently improved EB and organoid fitness as compared to the widely used ROCK inhibitor Y-27632. Collectively, we discovered that stress-free cell aggregation and superior cell survival in the presence of CEPT are critical quality control determinants that establish a robust foundation for bioengineering complex tissue and organ models.
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Corpos Embrioides , Células-Tronco Pluripotentes , Humanos , Corpos Embrioides/metabolismo , Reprodutibilidade dos Testes , Organoides , Diferenciação CelularRESUMO
Human gliogenesis remains poorly understood, and derivation of astrocytes from human pluripotent stem cells (hPSCs) is inefficient and cumbersome. Here, we report controlled glial differentiation from hPSCs that bypasses neurogenesis, which otherwise precedes astrogliogenesis during brain development and in vitro differentiation. hPSCs were first differentiated into radial glial cells (RGCs) resembling resident RGCs of the fetal telencephalon, and modulation of specific cell signaling pathways resulted in direct and stepwise induction of key astroglial markers (NFIA, NFIB, SOX9, CD44, S100B, glial fibrillary acidic protein [GFAP]). Transcriptomic and genome-wide epigenetic mapping and single-cell analysis confirmed RGC-to-astrocyte differentiation, obviating neurogenesis and the gliogenic switch. Detailed molecular and cellular characterization experiments uncovered new mechanisms and markers for human RGCs and astrocytes. In summary, establishment of a glia-exclusive neural lineage progression model serves as a unique serum-free platform of manufacturing large numbers of RGCs and astrocytes for neuroscience, disease modeling (e.g., Alexander disease), and regenerative medicine.
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Astrócitos , Células-Tronco Pluripotentes , Humanos , Astrócitos/metabolismo , Células Ependimogliais/metabolismo , Células-Tronco Pluripotentes/metabolismo , Neurogênese , Diferenciação Celular , Proteína Glial Fibrilar Ácida/metabolismoRESUMO
Development of new non-addictive analgesics requires advanced strategies to differentiate human pluripotent stem cells (hPSCs) into relevant cell types. Following principles of developmental biology and translational applicability, here we developed an efficient stepwise differentiation method for peptidergic and non-peptidergic nociceptors. By modulating specific cell signaling pathways, hPSCs were first converted into SOX10+ neural crest, followed by differentiation into sensory neurons. Detailed characterization, including ultrastructural analysis, confirmed that the hPSC-derived nociceptors displayed cellular and molecular features comparable to native dorsal root ganglion (DRG) neurons, and expressed high-threshold primary sensory neuron markers, transcription factors, neuropeptides, and over 150 ion channels and receptors relevant for pain research and axonal growth/regeneration studies (e.g., TRPV1, NAV1.7, NAV1.8, TAC1, CALCA, GAP43, DPYSL2, NMNAT2). Moreover, after confirming robust functional activities and differential response to noxious stimuli and specific drugs, a robotic cell culture system was employed to produce large quantities of human sensory neurons, which can be used to develop nociceptor-selective analgesics.
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Neurônios , Células-Tronco Pluripotentes , Humanos , Neurônios/metabolismo , Nociceptores , Diferenciação Celular , Transdução de Sinais , Gânglios Espinais/metabolismo , Células Receptoras SensoriaisRESUMO
BACKGROUND: Ureaplasma urealyticum (UUR) and Ureaplasma parvum (UPA) are sexually transmitted bacteria among humans implicated in a variety of disease states including but not limited to: nongonococcal urethritis, infertility, adverse pregnancy outcomes, chorioamnionitis, and bronchopulmonary dysplasia in neonates. There are 10 distinct serotypes of UUR and 4 of UPA. Efforts to determine whether difference in pathogenic potential exists at the ureaplasma serovar level have been hampered by limitations of antibody-based typing methods, multiple cross-reactions and poor discriminating capacity in clinical samples containing two or more serovars. RESULTS: We determined the genome sequences of the American Type Culture Collection (ATCC) type strains of all UUR and UPA serovars as well as four clinical isolates of UUR for which we were not able to determine serovar designation. UPA serovars had 0.75-0.78 Mbp genomes and UUR serovars were 0.84-0.95 Mbp. The original classification of ureaplasma isolates into distinct serovars was largely based on differences in the major ureaplasma surface antigen called the multiple banded antigen (MBA) and reactions of human and animal sera to the organisms. Whole genome analysis of the 14 serovars and the 4 clinical isolates showed the mba gene was part of a large superfamily, which is a phase variable gene system, and that some serovars have identical sets of mba genes. Most of the differences among serovars are hypothetical genes, and in general the two species and 14 serovars are extremely similar at the genome level. CONCLUSIONS: Comparative genome analysis suggests UUR is more capable of acquiring genes horizontally, which may contribute to its greater virulence for some conditions. The overwhelming evidence of extensive horizontal gene transfer among these organisms from our previous studies combined with our comparative analysis indicates that ureaplasmas exist as quasi-species rather than as stable serovars in their native environment. Therefore, differential pathogenicity and clinical outcome of a ureaplasmal infection is most likely not on the serovar level, but rather may be due to the presence or absence of potential pathogenicity factors in an individual ureaplasma clinical isolate and/or patient to patient differences in terms of autoimmunity and microbiome.
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DNA Bacteriano/química , DNA Bacteriano/genética , Genoma Bacteriano , Análise de Sequência de DNA , Ureaplasma urealyticum/genética , Ureaplasma/genética , Animais , Evolução Molecular , Transferência Genética Horizontal , Humanos , Dados de Sequência Molecular , Ureaplasma/isolamento & purificação , Ureaplasma urealyticum/isolamento & purificação , Fatores de Virulência/genéticaRESUMO
Pathema (http://pathema.jcvi.org) is one of the eight Bioinformatics Resource Centers (BRCs) funded by the National Institute of Allergy and Infectious Disease (NIAID) designed to serve as a core resource for the bio-defense and infectious disease research community. Pathema strives to support basic research and accelerate scientific progress for understanding, detecting, diagnosing and treating an established set of six target NIAID Category A-C pathogens: Category A priority pathogens; Bacillus anthracis and Clostridium botulinum, and Category B priority pathogens; Burkholderia mallei, Burkholderia pseudomallei, Clostridium perfringens and Entamoeba histolytica. Each target pathogen is represented in one of four distinct clade-specific Pathema web resources and underlying databases developed to target the specific data and analysis needs of each scientific community. All publicly available complete genome projects of phylogenetically related organisms are also represented, providing a comprehensive collection of organisms for comparative analyses. Pathema facilitates the scientific exploration of genomic and related data through its integration with web-based analysis tools, customized to obtain, display, and compute results relevant to ongoing pathogen research. Pathema serves the bio-defense and infectious disease research community by disseminating data resulting from pathogen genome sequencing projects and providing access to the results of inter-genomic comparisons for these organisms.
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Infecções Bacterianas/microbiologia , Doenças Transmissíveis/microbiologia , Biologia Computacional/métodos , Bases de Dados Genéticas , Sequência de Aminoácidos , Animais , Infecções Bacterianas/diagnóstico , Biologia Computacional/tendências , Genoma Bacteriano , Humanos , Armazenamento e Recuperação da Informação/métodos , Internet , Dados de Sequência Molecular , National Institute of Allergy and Infectious Diseases (U.S.) , Homologia de Sequência de Aminoácidos , Software , Estados UnidosRESUMO
MOTIVATION: The growth of sequence data has been accompanied by an increasing need to analyze data on distributed computer clusters. The use of these systems for routine analysis requires scalable and robust software for data management of large datasets. Software is also needed to simplify data management and make large-scale bioinformatics analysis accessible and reproducible to a wide class of target users. RESULTS: We have developed a workflow management system named Ergatis that enables users to build, execute and monitor pipelines for computational analysis of genomics data. Ergatis contains preconfigured components and template pipelines for a number of common bioinformatics tasks such as prokaryotic genome annotation and genome comparisons. Outputs from many of these components can be loaded into a Chado relational database. Ergatis was designed to be accessible to a broad class of users and provides a user friendly, web-based interface. Ergatis supports high-throughput batch processing on distributed compute clusters and has been used for data management in a number of genome annotation and comparative genomics projects. AVAILABILITY: Ergatis is an open-source project and is freely available at http://ergatis.sourceforge.net.
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Biologia Computacional/métodos , Internet , Software , Bases de Dados Genéticas , Bases de Dados de Proteínas , Fluxo de TrabalhoRESUMO
We present the genome sequences of a new clinical isolate of the important human pathogen, Aspergillus fumigatus, A1163, and two closely related but rarely pathogenic species, Neosartorya fischeri NRRL181 and Aspergillus clavatus NRRL1. Comparative genomic analysis of A1163 with the recently sequenced A. fumigatus isolate Af293 has identified core, variable and up to 2% unique genes in each genome. While the core genes are 99.8% identical at the nucleotide level, identity for variable genes can be as low 40%. The most divergent loci appear to contain heterokaryon incompatibility (het) genes associated with fungal programmed cell death such as developmental regulator rosA. Cross-species comparison has revealed that 8.5%, 13.5% and 12.6%, respectively, of A. fumigatus, N. fischeri and A. clavatus genes are species-specific. These genes are significantly smaller in size than core genes, contain fewer exons and exhibit a subtelomeric bias. Most of them cluster together in 13 chromosomal islands, which are enriched for pseudogenes, transposons and other repetitive elements. At least 20% of A. fumigatus-specific genes appear to be functional and involved in carbohydrate and chitin catabolism, transport, detoxification, secondary metabolism and other functions that may facilitate the adaptation to heterogeneous environments such as soil or a mammalian host. Contrary to what was suggested previously, their origin cannot be attributed to horizontal gene transfer (HGT), but instead is likely to involve duplication, diversification and differential gene loss (DDL). The role of duplication in the origin of lineage-specific genes is further underlined by the discovery of genomic islands that seem to function as designated "gene dumps" and, perhaps, simultaneously, as "gene factories".
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Aspergillus fumigatus/genética , Ilhas Genômicas , Alérgenos/genética , Aspergillus/classificação , Aspergillus/genética , Aspergillus/fisiologia , Aspergillus fumigatus/classificação , Aspergillus fumigatus/patogenicidade , Aspergillus fumigatus/fisiologia , Cromossomos Fúngicos/genética , Eurotiales/classificação , Eurotiales/genética , Eurotiales/fisiologia , Evolução Molecular , Proteínas Fúngicas/genética , Proteínas Fúngicas/imunologia , Genoma Fúngico , Humanos , Filogenia , Especificidade da Espécie , Virulência/genéticaRESUMO
BACKGROUND: Burkholderia species exhibit enormous phenotypic diversity, ranging from the nonpathogenic, soil- and water-inhabiting Burkholderia thailandensis to the virulent, host-adapted mammalian pathogen B. mallei. Genomic diversity is evident within Burkholderia species as well. Individual isolates of Burkholderia pseudomallei and B. thailandensis, for example, carry a variety of strain-specific genomic islands (GIs), including putative pathogenicity and metabolic islands, prophage-like islands, and prophages. These GIs may provide some strains with a competitive advantage in the environment and/or in the host relative to other strains. RESULTS: Here we present the results of analysis of 37 prophages, putative prophages, and prophage-like elements from six different Burkholderia species. Five of these were spontaneously induced to form bacteriophage particles from B. pseudomallei and B. thailandensis strains and were isolated and fully sequenced; 24 were computationally predicted in sequenced Burkholderia genomes; and eight are previously characterized prophages or prophage-like elements. The results reveal numerous differences in both genome structure and gene content among elements derived from different species as well as from strains within species, due in part to the incorporation of additional DNA, or 'morons' into the prophage genomes. Implications for pathogenicity are also discussed. Lastly, RNAseq analysis of gene expression showed that many of the genes in varphi1026b that appear to contribute to phage and lysogen fitness were expressed independently of the phage structural and replication genes. CONCLUSIONS: This study provides the first estimate of the relative contribution of prophages to the vast phenotypic diversity found among the Burkholderiae.
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Bacteriófagos/fisiologia , Burkholderia/genética , Burkholderia/virologia , Variação Genética , Prófagos/fisiologia , Bacteriófagos/classificação , Bacteriófagos/genética , Burkholderia/classificação , Genoma Bacteriano , Genoma Viral , Interações Hospedeiro-Patógeno , Dados de Sequência Molecular , Filogenia , Prófagos/classificação , Prófagos/genética , Especificidade da EspécieRESUMO
To address the question of how microbial diversity and function in the oral cavities of children relates to caries diagnosis, we surveyed the supragingival plaque biofilm microbiome in 44 juvenile twin pairs. Using shotgun sequencing, we constructed a genome encyclopedia describing the core supragingival plaque microbiome. Caries phenotypes contained statistically significant enrichments in specific genome abundances and distinct community composition profiles, including strain-level changes. Metabolic pathways that are statistically associated with caries include several sugar-associated phosphotransferase systems, antimicrobial resistance, and metal transport. Numerous closely related previously uncharacterized microbes had substantial variation in central metabolism, including the loss of biosynthetic pathways resulting in auxotrophy, changing the ecological role. We also describe the first complete Gracilibacteria genomes from the human microbiome. Caries is a microbial community metabolic disorder that cannot be described by a single etiology, and our results provide the information needed for next-generation diagnostic tools and therapeutics for caries.IMPORTANCE Oral health has substantial economic importance, with over $100 billion spent on dental care in the United States annually. The microbiome plays a critical role in oral health, yet remains poorly classified. To address the question of how microbial diversity and function in the oral cavities of children relate to caries diagnosis, we surveyed the supragingival plaque biofilm microbiome in 44 juvenile twin pairs. Using shotgun sequencing, we constructed a genome encyclopedia describing the core supragingival plaque microbiome. This unveiled several new previously uncharacterized but ubiquitous microbial lineages in the oral microbiome. Caries is a microbial community metabolic disorder that cannot be described by a single etiology, and our results provide the information needed for next-generation diagnostic tools and therapeutics for caries.
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Bactérias/classificação , Bactérias/genética , Cárie Dentária/microbiologia , Placa Dentária/microbiologia , Microbiota , Austrália , Criança , Pré-Escolar , Humanos , Redes e Vias Metabólicas/genética , Metagenômica , Análise de Sequência de DNARESUMO
DNA microarrays have been used to study the expression of thousands of genes at the same time in a variety of cells and tissues. The methods most commonly used to label probes for microarray studies require a minimum of 20 microg of total RNA or 2 microg of poly(A) RNA. This has made it difficult to study small and rare tissue samples. RNA amplification techniques and improved labeling methods have recently been described. These new procedures and reagents allow the use of less input RNA, but they are relatively time-consuming and expensive. Here we introduce a technique for preparing fluorescent probes that can be used to label as little as 1 microg of total RNA. The method is based on priming cDNA synthesis with random hexamer oligonucleotides, on the 5' ends of which are bases with free amino groups. These amine-modified primers are incorporated into the cDNA along with aminoallyl nucleotides, and fluorescent dyes are then chemically added to the free amines. The method is simple to execute, and amine-reactive dyes are considerably less expensive than dye-labeled bases or dendrimers.
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Primers do DNA/análise , Corantes Fluorescentes/análise , Análise de Sequência com Séries de Oligonucleotídeos/métodos , RNA/isolamento & purificação , Células 3T3 , Animais , Primers do DNA/genética , Análise de Falha de Equipamento , Expressão Gênica , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos/instrumentação , RNA/química , RNA/genética , Reprodutibilidade dos TestesRESUMO
Host-associated microbial communities are influenced by both host genetics and environmental factors. However, factors controlling the human oral microbiome and their impact on disease remain to be investigated. To determine the combined and relative effects of host genotype and environment on oral microbiome composition and caries phenotypes, we profiled the supragingival plaque microbiome of 485 dizygotic and monozygotic twins aged 5-11. Oral microbiome similarity always increased with shared host genotype, regardless of caries state. Additionally, although most of the variation in the oral microbiome was determined by environmental factors, highly heritable oral taxa were identified. The most heritable oral bacteria were not associated with caries state, did not tend to co-occur with other taxa, and decreased in abundance with age and sugar consumption frequency. Thus, while the human oral microbiome composition is influenced by host genetic background, potentially cariogenic taxa are likely not controlled by genetic factors.
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Bactérias/isolamento & purificação , Cárie Dentária/genética , Cárie Dentária/microbiologia , Microbiota , Boca/microbiologia , Fatores Etários , Bactérias/classificação , Bactérias/genética , Criança , Pré-Escolar , Ecossistema , Feminino , Predisposição Genética para Doença , Genótipo , Humanos , Masculino , Filogenia , Gêmeos/genéticaRESUMO
cDNA sequences are important for defining the coding region of genes, and full-length cDNA clones have proven to be useful for investigation of the function of gene products. We produced cDNA libraries containing 3.5-5 x 10(5) primary transformants, starting with 5 mug of total RNA prepared from mouse pituitary, adrenal, thymus, and pineal tissue, using a vector-primed cDNA synthesis method. Of approximately 1000 clones sequenced, approximately 20% contained the full open reading frames (ORFs) of known transcripts, based on the presence of the initiating methionine residue codon. The libraries were complex, with 94, 91, 83 and 55% of the clones from the thymus, adrenal, pineal and pituitary libraries, respectively, represented only once. Twenty-five full-length clones, not yet represented in the Mammalian Gene Collection, were identified. Thus, we have produced useful cDNA libraries for the isolation of full-length cDNA clones that are not yet available in the public domain, and demonstrated the utility of a simple method for making high-quality libraries from small amounts of starting material.
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DNA Complementar/biossíntese , Biblioteca Gênica , Vetores Genéticos , Animais , DNA Complementar/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , RNA/metabolismo , Análise de Sequência de DNARESUMO
RNA amplification methods have been used to facilitate making probes from small tissue samples for microarray studies. Our original amplification technique relied on driving the first reverse transcription with oligo(dT) with a T7 RNA polymerase promoter (T7dT) on the 5' end, and subsequent transcriptions with random 9mers with a T3 RNA polymerase promoter (T3N9). Thus, initially, poly(A)(+) RNA is amplified. This creates a potential problem: amplifications based on oligo(dT) priming could be sensitive to RNA degradation; broken mRNA strands should give rise to shorter cDNAs than those seen when intact templates are used. This would be especially troublesome when targets other than those corresponding to the 3' ends of transcripts are printed on an array. To solve this problem, we elected to prime cDNA synthesis with T3N9 at the beginning of each amplification cycle. Following two rounds of amplification, the resulting probes were comparable to those obtained with our original protocol or the Arcturus RiboAmp kit. We show below that as many as four rounds of amplification can be performed reliably. In addition, as predicted, the method works well with degraded templates.
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Análise de Sequência com Séries de Oligonucleotídeos/métodos , RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Células 3T3 , Animais , Linhagem Celular , DNA Complementar/genética , DNA Complementar/metabolismo , Camundongos , RNA/metabolismo , Sensibilidade e EspecificidadeRESUMO
The purpose of this study was to examine individual differences in eye-movement behavior. Six metrics (Fixation Rate, Duration, and Size; Saccade Amplitude; Micro-Saccade Rate and Amplitude) were used to measure individuals' eye-movement behavior profiles (EmBP). We replicate previous research (Andrews & Coppola, 1999; Castelhano & Henderson, 2008) by finding consistent individual differences in fixation duration and saccade amplitude across tasks, and present new findings of stable idiosyncrasies in measures of fixational eye-movement (Fixation Size, Micro-Saccade Rate and Amplitude). Moreover, we observed consistent inter-metric correlations across tasks (e.g., individuals that exhibited relatively high Fixation Rates also presented relative low Micro-Saccade Rates and relatively high Micro-Saccade Amplitudes). Factor Analysis linked the six EmBP metrics together with a single factor, which we speculate might be related to the operational effectiveness of the attentional system, given that individual factor scores were correlated with scores on a self-report measure of attentional function. Normal subjects with relatively high scores on this attention-deficit measure exhibited relatively frequent fixations of short duration and large spatial extent, and relatively infrequent micro-saccades of large amplitude. This EmBP is similar to a general pattern of eye-movement behavior observed with ADHD individuals - difficulty controlling eye movements, maintaining fixation, and inhibiting intrusive saccades. Results of this study indicate that normal individuals exhibit idiosyncratic EmBPs that are quite stable across tasks and are related to attentional ability.
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Atenção/fisiologia , Fixação Ocular/fisiologia , Movimentos Sacádicos/fisiologia , Adolescente , Adulto , Análise Fatorial , Feminino , Humanos , Masculino , Análise de Regressão , Análise e Desempenho de Tarefas , Adulto JovemRESUMO
In the past few years, the field of metagenomics has been growing at an accelerated pace, particularly in response to advancements in new sequencing technologies. The large volume of sequence data from novel organisms generated by metagenomic projects has triggered the development of specialized databases and tools focused on particular groups of organisms or data types. Here we describe a pipeline for the functional annotation of viral metagenomic sequence data. The Viral MetaGenome Annotation Pipeline (VMGAP) pipeline takes advantage of a number of specialized databases, such as collections of mobile genetic elements and environmental metagenomes to improve the classification and functional prediction of viral gene products. The pipeline assigns a functional term to each predicted protein sequence following a suite of comprehensive analyses whose results are ranked according to a priority rules hierarchy. Additional annotation is provided in the form of enzyme commission (EC) numbers, GO/MeGO terms and Hidden Markov Models together with supporting evidence.