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Systematic mappings of protein interactome networks have provided invaluable functional information for numerous model organisms. Here we develop PCR-mediated Linkage of barcoded Adapters To nucleic acid Elements for sequencing (PLATE-seq) that serves as a general tool to rapidly sequence thousands of DNA elements. We validate its utility by generating the ORFeome for Oryza sativa covering 2,300 genes and constructing a high-quality protein-protein interactome map consisting of 322 interactions between 289 proteins, expanding the known interactions in rice by roughly 50%. Our work paves the way for high-throughput profiling of protein-protein interactions in a wide range of organisms.
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Sistemas de Lectura Abierta/genética , Oryza/genética , Mapeo de Interacción de Proteínas/métodos , Mapas de Interacción de Proteínas/genética , Análisis de Secuencia de ADN/métodos , Biología Computacional/métodos , ADN de Plantas/genética , Bases de Datos Genéticas , Genoma de Planta/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodosRESUMEN
The Indigenous village of Bykovskiy is located 40 km from Tiksi, the administrative center of Bulunskiy District (Ulus), in the northern part of the Republic of Sakha (Yakutiya), Russia. Founded as a Soviet fishing cooperative, it became home to Indigenous Sakha, Evenkis, Evens, as well as to Russian settlers and political prisoners from the Baltic states. Post-Soviet transformations, coupled with escalating environmental change processes, has been altering the local economy and subsistence activities since the 1990s. Although our interlocutors directly observed and experienced such changes, they seemed to ignore the visible problem of severe coastal erosion that was destroying a local cemetery. This article is based on ethnographic fieldwork conducted in the study region in 2019, and combines approaches from the anthropology of climate change with reception and communication studies. It examines "ignorance" as a strategy of adaptation to multiple stressors under historically reproduced colonial structures of governance.
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Cambio Climático , Caza , Países Bálticos , Federación de RusiaRESUMEN
OBJECTIVE: To determine whether a mutation in the fibrillin 2 gene (FBN2) is associated with canine hip dysplasia (CHD) and osteoarthritis in dogs. ANIMALS: 1,551 dogs. Procedures-Hip conformation was measured radiographically. The FBN2 was sequenced from genomic DNA of 21 Labrador Retrievers and 2 Greyhounds, and a haplotype in intron 30 of FBN2 was sequenced in 90 additional Labrador Retrievers and 143 dogs of 6 other breeds. Steady-state values of FBN2 mRNA and control genes were measured in hip joint tissues of fourteen 8-month-old Labrador Retriever-Greyhound crossbreeds. RESULTS: The Labrador Retrievers homozygous for a 10-bp deletion haplotype in intron 30 of FBN2 had significantly worse CHD as measured via higher distraction index and extended-hip joint radiograph score and a lower Norberg angle and dorsolateral subluxation score. Among 143 dogs of 6 other breeds, those homozygous for the same deletion haplotype also had significantly worse radiographic CHD. Among the 14 crossbred dogs, as the dorsolateral subluxation score decreased, the capsular FBN2 mRNA increased significantly. Those dogs with incipient hip joint osteoarthritis had significantly increased capsular FBN2 mRNA, compared with those dogs without osteoarthritis. Dogs homozygous for the FBN2 deletion haplotype had significantly less FBN2 mRNA in their femoral head articular cartilage. CONCLUSIONS AND CLINICAL RELEVANCE: The FBN2 deletion haplotype was associated with CHD. Capsular gene expression of FBN2 was confounded by incipient secondary osteoarthritis in dysplastic hip joints. Genes influencing complex traits in dogs can be identified by genome-wide screening, fine mapping, and candidate gene screening.
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Enfermedades de los Perros/genética , Displasia Pélvica Canina/genética , Proteínas de Microfilamentos/genética , Osteoartritis/veterinaria , Animales , Enfermedades de los Perros/diagnóstico por imagen , Perros/genética , Perros/fisiología , Femenino , Fibrilinas , Predisposición Genética a la Enfermedad , Haplotipos , Displasia Pélvica Canina/diagnóstico por imagen , Masculino , Proteínas de Microfilamentos/fisiología , Mutación , Osteoartritis/diagnóstico por imagen , Osteoartritis/genética , ARN Mensajero/genética , RadiografíaRESUMEN
BACKGROUND: Climate change is a major global challenge, especially for Indigenous communities. It can have extensive impacts on peoples' lives that may occur through the living environment, health and mental well-being, and which are requiring constant adaptation. OBJECTIVES: The overall purpose of this research was to evaluate the impacts of climate change and permafrost thaw on mental wellness in Disko Bay, Greenland. It contained two parts: multidisciplinary fieldwork and a questionnaire survey. The aim of the fieldwork was to learn about life and living conditions and to understand what it is like to live in a community that faces impacts of climate change and permafrost thaw. For the questionnaire the aim was to find out which perceived environmental and adaptation factors relate to very good self-rated well-being, quality of life and satisfaction with life. ANALYSIS: Fieldwork data was analyzed by following a thematic analysis, and questionnaire data statistically by cross-tabulation. First, the associations between perceived environmental and adaptation factors were studied either by the Pearson χ2 test or by Fisher's exact test. Second, binary logistic regression analysis was applied to examine more in depth the associations between perceived environmental/adaptation variables and self-rated very good well-being, satisfaction with life and quality of life. The binary logistic regression analysis was conducted in two phases: as univariate and multivariate analyses. RESULTS: Nature and different activities in nature were found to be important to local people, and results suggest that they increase mental wellness, specifically well-being and satisfaction with life. Challenges associated with permafrost thaw, such as changes in the physical environment, infrastructure and impacts on culture were recognized in everyday life. CONCLUSIONS: The results offer relevant information for further plans and actions in this field of research and at the policy level. Our study shows the importance of multidisciplinary research which includes the voice of local communities.
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To fully understand how bacteria respond to their environment, it is essential to assess genome-wide transcriptional activity. New high-throughput sequencing technologies make it possible to query the transcriptome of an organism in an efficient unbiased manner. We applied a strand-specific method to sequence bacterial transcripts using Illumina's high-throughput sequencing technology. The resulting sequences were used to construct genome-wide transcriptional profiles. Novel bioinformatics analyses were developed and used in combination with proteomics data for the qualitative classification of transcriptional activity in defined regions. As expected, most transcriptional activity was consistent with predictions from the genome annotation. Importantly, we identified and confirmed transcriptional activity in areas of the genome inconsistent with the annotation and in unannotated regions. Further analyses revealed potential RpoN-dependent promoter sequences upstream of several noncoding RNAs (ncRNAs), suggesting a role for these ncRNAs in RpoN-dependent phenotypes. We were also able to validate a number of transcriptional start sites, many of which were consistent with predicted promoter motifs. Overall, our approach provides an efficient way to survey global transcriptional activity in bacteria and enables rapid discovery of specific areas in the genome that merit further investigation.
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Perfilación de la Expresión Génica , Pseudomonas syringae/genética , ARN sin Sentido/genética , ARN no Traducido/genética , Dicroismo Circular , Biología Computacional , Genoma Bacteriano/genética , Modelos Genéticos , Técnicas de Amplificación de Ácido Nucleico , Regiones Promotoras Genéticas/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Espectrometría de Masas en Tándem , Sitio de Iniciación de la Transcripción , Transcripción Genética/genéticaRESUMEN
OBJECTIVE: To identify the quantitative trait loci (QTL) that contribute to hip dysplasia in dogs. ANIMALS: 192 Labrador Retrievers. PROCEDURES: Hip dysplasia was measured by use of the Norberg angle (NA), dorsolateral subluxation (DLS) score, and distraction index (DI). Genome-wide screening was conducted by use of 276 unique microsatellites. Linkage analysis was performed with a variance-based linear model. Logarithm of the odds (LOD) scores were reported when values were > 2.0. RESULTS: Canis familiaris autosomes (CFAs) 01, 02, 10, 20, 22, and 32 harbored significant QTL at LOD scores > 2.0. Among the 6 QTL, the QTL on CFA02 had not been reported to harbor QTL for hip dysplasia. The highest LOD score of 3.32 on CFA20 contributed to the second principal component of the DLS score and NA of the right hip joint. The QTL that was mapped on CFA01 (LOD score of 3.13 at 55 centimorgans) was located on the same chromosome reported to harbor a QTL for hip dysplasia in Portuguese Water Dogs and German Shepherd Dogs. In this study, CFAs 10, 20, 22, and 32 harbored QTL for hip dysplasia that have been identified in a Labrador Retriever-Greyhound pedigree and in German Shepherd Dogs. CONCLUSIONS AND CLINICAL RELEVANCE: Multiple QTL were clearly involved with hip dysplasia. Identification of these QTL will enable fine-resolution mapping and subsequent assessment of candidate genes within the refined intervals to enable researchers to develop genetic screening tests and preventative and novel therapeutic regimens.
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Enfermedades de los Perros/genética , Displasia Pélvica Canina/genética , Sitios de Carácter Cuantitativo , Animales , ADN/genética , ADN/aislamiento & purificación , Perros , Femenino , Genotipo , Articulación de la Cadera/patología , Tamaño de la Camada , Masculino , Repeticiones de Microsatélite/genética , Linaje , Fenotipo , Especificidad de la EspecieRESUMEN
The Soviet Union and its successor states have been avid supporters of a modernisation paradigm aimed at 'overcoming remoteness' and 'bringing civilisation' to the periphery and its 'backward' indigenous people. The Baikal-Amur Mainline (BAM) railroad, built as a much-hyped prestige project of late socialism, is a good example of that. The BAM has affected indigenous communities and reconfigured the geographic and social space of East Siberia. Our case study, an Evenki village located fairly close to the BAM, is (in)famous today for its supposed refusal to get connected via a bridge to the nearby railroad town. Some actors portray this disconnection as a sign of backwardness, while others celebrate it as the main reason for native language retention and cultural preservation. Focusing on discourses linking the notions of remoteness and cultural revitalisation, the article argues for conceptualising the story of the missing bridge not as the result of political resistance but rather as an articulation of indigeneity, which foregrounds cultural rights over more contentious political claims. Thus, the article explores constellations of remoteness and indigeneity, posing the question whether there might be a moral right to remoteness to be claimed by those who view spatial distance as a potential resource.
L'Union soviétique et ses États successeurs ont été de fervents partisans d'un paradigme de modernisation visant à « surmonter l'éloignement ¼ et à « amener la civilisation ¼ à la périphérie et à son peuple indigène « arriéré ¼. Le chemin de fer Magistrale BaïkalAmour (la BAM), construit en tant que projet prestigieux très médiatisé du socialisme tardif, en est un bon exemple. La BAM a impacté les communautés autochtones et reconfiguré l'espace géographique et social de la Sibérie orientale. Notre étude de cas se focalise sur un village d'Evenki situé assez près de la BAM, célèbre aujourd'hui pour son refus supposé de se connecter par un pont à la ville ferroviaire à proximité. Certains acteurs considèrent cette déconnexion comme un signe de retard, tandis que d'autres la célèbrent comme la raison principale de la préservation de la langue maternelle et de la culture. Se focalisant sur des discours reliant les notions d'éloignement/isolement et de revitalisation culturelle, l'article plaide en faveur d'une conceptualisation du récit du pont manquant, non comme le résultat de résistance politique, mais plutôt comme une articulation de l'indigénéité mettant en avant des droits culturels plutôt que des revendications politiques plus controversées. L'article examine ainsi des constellations d'éloignement et d'indigénéité soulevant la question d'un droit moral à l'isolement qui serait revendiqué par ceux qui conçoivent la distance spatiale comme ressource potentielle.
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Over the past few years, technological advances in automated DNA sequencing have had a profound effect on the nature of DNA sequencing laboratories. To characterize the changes occurring within DNA sequencing facilities, the DNA Sequencing Research Group conducted three previous studies, in 1998, 2000, and 2003. A new general survey has been designed and conducted by the DSRG to capture the current status of DNA sequencing facilities in all sectors. Included were questions regarding facility administration, pricing, instrumentation, technology, protocols, and operation. The results of the survey are presented here, accompanied by comparisons to the previous surveys. These comparisons formed a basis for the discussion of trends within the facilities in response to the dynamics of a changing technology.
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Laboratorios/tendencias , Análisis de Secuencia de ADN/tendencias , Encuestas y Cuestionarios , Laboratorios/economía , Análisis de Secuencia de ADN/economía , Análisis de Secuencia de ADN/instrumentación , Recursos HumanosRESUMEN
Public and academic discourses about the Polar regions typically focus on the so-called natural environment. While, these discourses and inquiries continue to be relevant, the current article asks the question how to conceptualize the on-going industrial and infrastructural build-up of the Arctic. Acknowledging that the "built environment" is not an invention of modernity, the article nevertheless focuses on large-scale infrastructural projects of the twentieth century, which marks a watershed of industrial and infrastructural development in the north. Given that the Soviet Union was at the vanguard of these developments, the focus will be on Soviet and Russian large-scale projects. We will be discussing two cases of transportation infrastructure, one of them based on an on-going research project being conducted by the authors along the Baikal-Amur Mainline (BAM) and the other focused on the so-called Northern Sea Route, the marine passage with a long history that has recently been regaining public and academic attention. The concluding section will argue for increased attention to the interactions between humans and the built environment, serving as a kind of programmatic call for more anthropological attention to infrastructure in the Russian north and other polar regions.
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Hip dysplasia (HD), elbow dysplasia (ED), and rupture of the cranial (anterior) cruciate ligament (RCCL) are the most common complex orthopedic traits of dogs and all result in debilitating osteoarthritis. We reanalyzed previously reported data: the Norberg angle (a quantitative measure of HD) in 921 dogs, ED in 113 cases and 633 controls, and RCCL in 271 cases and 399 controls and their genotypes at ~185,000 single nucleotide polymorphisms. A novel fixed and random model with a circulating probability unification (FarmCPU) function, with marker-based principal components and a kinship matrix to correct for population stratification, was used. A Bonferroni correction at p<0.01 resulted in a P< 6.96 ×10-8. Six loci were identified; three for HD and three for RCCL. An associated locus at CFA28:34,369,342 for HD was described previously in the same dogs using a conventional mixed model. No loci were identified for RCCL in the previous report but the two loci for ED in the previous report did not reach genome-wide significance using the FarmCPU model. These results were supported by simulation which demonstrated that the FarmCPU held no power advantage over the linear mixed model for the ED sample but provided additional power for the HD and RCCL samples. Candidate genes for HD and RCCL are discussed. When using FarmCPU software, we recommend a resampling test, that a positive control be used to determine the optimum pseudo quantitative trait nucleotide-based covariate structure of the model, and a negative control be used consisting of permutation testing and the identical resampling test as for the non-permuted phenotypes.
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Lesiones del Ligamento Cruzado Anterior/veterinaria , Miembro Anterior/lesiones , Luxación de la Cadera/veterinaria , Polimorfismo de Nucleótido Simple , Animales , Perros , Estudios de Asociación Genética , Genotipo , Funciones de Verosimilitud , Modelos Genéticos , Sitios de Carácter Cuantitativo , Programas InformáticosRESUMEN
PURPOSE: Identification of causative mutations for retinal blinding disorders is often limited by restricted understanding of gene expression and underlying molecular mechanisms that trigger degenerative processes. This study was conducted to develop a catalog of canine retina-expressed genes that would provide a unique tool to investigate normal and altered function in the adult retina. Because of the conserved syntenies between the dog and human, this approach would identify new potential disease candidate genes for both species. METHODS: A canine normalized retinal cDNA library was produced and analyzed by using a modified PhredPhrap algorithm. Computerized annotation provided gene homology and chromosomal location for individual clones and contigs in a Web-accessible database. RESULTS: From 6316 cDNA clones, 3980 retinal expressed sequence tags (ESTs) were derived. Homology to the canine genome draft sequence was found for more than 99% of all ESTs, but only for 32% when compared with annotated canine cDNAs. Functional analysis suggests an enrichment of this library for genes involved with eye function and development, chaperone, or ribosomal functions when compared with mouse and human National Center for Biotechnology Information (NCBI) RefSeq entries. CONCLUSIONS: A combination of annotation approaches with ongoing mapping and expression studies provide functional data covering at least 27% to 30% of the currently proposed canine catalog of genes expressed in the retina. This is an essential first step toward establishing an integrated network for gene identification and expression patterns suitable for functional genetics, comparative genomics and evolutionary analysis of genes and gene families with respect to the developmental and degenerative processes of the retina.
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ADN Complementario/metabolismo , Perros/genética , Expresión Génica , Biblioteca de Genes , Genómica , Retina/metabolismo , Animales , Bases de Datos Factuales , Etiquetas de Secuencia Expresada , Proteínas del Ojo/genéticaRESUMEN
Marker-assisted selection (MAS) is often employed in crop breeding programs to accelerate and enhance cultivar development, via selection during the juvenile phase and parental selection prior to crossing. Next-generation sequencing and its derivative technologies have been used for genome-wide molecular marker discovery. To bridge the gap between marker development and MAS implementation, this study developed a novel practical strategy with a semi-automated pipeline that incorporates trait-associated single nucleotide polymorphism marker discovery, low-cost genotyping through amplicon sequencing (AmpSeq) and decision making. The results document the development of a MAS package derived from genotyping-by-sequencing using three traits (flower sex, disease resistance and acylated anthocyanins) in grapevine breeding. The vast majority of sequence reads (⩾99%) were from the targeted regions. Across 380 individuals and up to 31 amplicons sequenced in each lane of MiSeq data, most amplicons (83 to 87%) had <10% missing data, and read depth had a median of 220-244×. Several strengths of the AmpSeq platform that make this approach of broad interest in diverse crop species include accuracy, flexibility, speed, high-throughput, low-cost and easily automated analysis.
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BACKGROUND: Microarrays are an important tool with which to examine coordinated gene expression. Soybean (Glycine max) is one of the most economically valuable crop species in the world food supply. In order to accelerate both gene discovery as well as hypothesis-driven research in soybean, global expression resources needed to be developed. The applications of microarray for determining patterns of expression in different tissues or during conditional treatments by dual labeling of the mRNAs are unlimited. In addition, discovery of the molecular basis of traits through examination of naturally occurring variation in hundreds of mutant lines could be enhanced by the construction and use of soybean cDNA microarrays. RESULTS: We report the construction and analysis of a low redundancy 'unigene' set of 27,513 clones that represent a variety of soybean cDNA libraries made from a wide array of source tissue and organ systems, developmental stages, and stress or pathogen-challenged plants. The set was assembled from the 5' sequence data of the cDNA clones using cluster analysis programs. The selected clones were then physically reracked and sequenced at the 3' end. In order to increase gene discovery from immature cotyledon libraries that contain abundant mRNAs representing storage protein gene families, we utilized a high density filter normalization approach to preferentially select more weakly expressed cDNAs. All 27,513 cDNA inserts were amplified by polymerase chain reaction. The amplified products, along with some repetitively spotted control or 'choice' clones, were used to produce three 9,728-element microarrays that have been used to examine tissue specific gene expression and global expression in mutant isolines. CONCLUSIONS: Global expression studies will be greatly aided by the availability of the sequence-validated and low redundancy cDNA sets described in this report. These cDNAs and ESTs represent a wide array of developmental stages and physiological conditions of the soybean plant. We also demonstrate that the quality of the data from the soybean cDNA microarrays is sufficiently reliable to examine isogenic lines that differ with respect to a mutant phenotype and thereby to define a small list of candidate genes potentially encoding or modulated by the mutant phenotype.
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ADN Complementario/genética , Perfilación de la Expresión Génica/métodos , Regulación del Desarrollo de la Expresión Génica/genética , Regulación de la Expresión Génica de las Plantas/genética , Glycine max/genética , Glycine max/fisiología , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Análisis por Conglomerados , Cotiledón/genética , ADN de Plantas/genética , Perfilación de la Expresión Génica/estadística & datos numéricos , Biblioteca de Genes , Mutación/genética , Análisis de Secuencia por Matrices de Oligonucleótidos/estadística & datos numéricos , Especificidad de Órganos/genética , FenotipoRESUMEN
Whole genome sequencing revealed the presence of a genomic anomaly in the region of 4.7 to 4.9 Mb of the Pseudomonas syringae pv. tomato (Pst) DC3000 genome. The average read depth coverage of Pst DC3000 whole genome sequencing results suggested that a 165 kb segment of the chromosome had doubled in copy number. Further analysis confirmed the 165 kb duplication and that the two copies were arranged as a direct tandem repeat. Examination of the corresponding locus in Pst NCPPB1106, the parent strain of Pst DC3000, suggested that the 165 kb duplication most likely formed after the two strains diverged via transposition of an ISPsy5 insertion sequence (IS) followed by unequal crossing over between ISPsy5 elements at each end of the duplicated region. Deletion of one copy of the 165 kb region demonstrated that the duplication facilitated enhanced growth in some culture conditions, but did not affect pathogenic growth in host tomato plants. These types of chromosomal structures are predicted to be unstable and we have observed resolution of the 165 kb duplication to single copy and its subsequent re-duplication. These data demonstrate the role of IS elements in recombination events that facilitate genomic reorganization in P. syringae.
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Genoma Bacteriano/genética , Pseudomonas syringae/citología , Pseudomonas syringae/genética , Alelos , Emparejamiento Base/genética , Secuencia de Bases , Duplicación de Gen/genética , Genes Bacterianos , Sitios Genéticos , Solanum lycopersicum/microbiología , Datos de Secuencia Molecular , Fenotipo , Reacción en Cadena de la Polimerasa , Pseudomonas syringae/crecimiento & desarrollo , Pseudomonas syringae/aislamiento & purificación , Análisis de Secuencia de ADNRESUMEN
High-throughput RNA sequencing (RNA-seq) greatly expands the potential for genomics discoveries, but the wide variety of platforms, protocols and performance capabilitites has created the need for comprehensive reference data. Here we describe the Association of Biomolecular Resource Facilities next-generation sequencing (ABRF-NGS) study on RNA-seq. We carried out replicate experiments across 15 laboratory sites using reference RNA standards to test four protocols (poly-A-selected, ribo-depleted, size-selected and degraded) on five sequencing platforms (Illumina HiSeq, Life Technologies PGM and Proton, Pacific Biosciences RS and Roche 454). The results show high intraplatform (Spearman rank R > 0.86) and inter-platform (R > 0.83) concordance for expression measures across the deep-count platforms, but highly variable efficiency and cost for splice junction and variant detection between all platforms. For intact RNA, gene expression profiles from rRNA-depletion and poly-A enrichment are similar. In addition, rRNA depletion enables effective analysis of degraded RNA samples. This study provides a broad foundation for cross-platform standardization, evaluation and improvement of RNA-seq.
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Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , TranscriptomaRESUMEN
PURPOSE: To identify the causative mutations in two early-onset canine retinal degenerations, crd1 and crd2, segregating in the American Staffordshire terrier and the Pit Bull Terrier breeds, respectively. METHODS: Retinal morphology of crd1- and crd2-affected dogs was evaluated by light microscopy. DNA was extracted from affected and related unaffected controls. Association analysis was undertaken using the Illumina Canine SNP array and PLINK (crd1 study), or the Affymetrix Version 2 Canine array, the "MAGIC" genotype algorithm, and Fisher's Exact test for association (crd2 study). Positional candidate genes were evaluated for each disease. RESULTS: Structural photoreceptor abnormalities were observed in crd1-affected dogs as young as 11-weeks old. Rod and cone inner segment (IS) and outer segments (OS) were abnormal in size, shape, and number. In crd2-affected dogs, rod and cone IS and OS were abnormal as early as 3 weeks of age, progressing with age to severe loss of the OS, and thinning of the outer nuclear layer (ONL) by 12 weeks of age. Genome-wide association study (GWAS) identified association at the telomeric end of CFA3 in crd1-affected dogs and on CFA33 in crd2-affected dogs. Candidate gene evaluation identified a three bases deletion in exon 21 of PDE6B in crd1-affected dogs, and a cytosine insertion in exon 10 of IQCB1 in crd2-affected dogs. CONCLUSIONS: Identification of the mutations responsible for these two early-onset retinal degenerations provides new large animal models for comparative disease studies and evaluation of potential therapeutic approaches for the homologous human diseases.
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Proteínas de Unión a Calmodulina/genética , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/genética , ADN/genética , Predisposición Genética a la Enfermedad , Células Fotorreceptoras de Vertebrados/metabolismo , Degeneración Retiniana/genética , Animales , Proteínas de Unión a Calmodulina/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/metabolismo , Análisis Mutacional de ADN , Modelos Animales de Enfermedad , Perros , Femenino , Estudio de Asociación del Genoma Completo , Genotipo , Masculino , Mutación , Linaje , Células Fotorreceptoras de Vertebrados/patología , Degeneración Retiniana/metabolismo , Degeneración Retiniana/patologíaRESUMEN
BACKGROUND: A-to-I RNA editing is found in all phyla of animals and contributes to transcript diversity that may have profound impacts on behavior and physiology. Many transcripts of genes involved in axonal conductance, synaptic transmission and modulation are the targets of A-to-I RNA editing. There are a number of methods to measure the extent of A-to-I RNA editing, but they are generally costly and time consuming. One way to determine the frequency of A-to-I RNA editing is the peak height ratio method, which compares the size of peaks on electropherograms that represent unedited and edited sites. FINDINGS: Sequencing of 4 editing sites of the Dα6 nicotinic acetylcholine receptor subunit with an antisense primer (which uses T/C peaks to measure unedited and edited sites, respectively) showed very accurate and precise measurements of A-to-I RNA editing. The accuracy and precision were excellent for all editing sites, including those edited with high or low frequencies. The frequency of A-to-I RNA editing was comparable to the editing frequency as measured by clone counting from the same sample. Sequencing these same sites with the sense primer (which uses A/G peaks) yielded inaccurate and imprecise measurements. CONCLUSIONS: We have validated and improved the accuracy and precision of the peak height ratio method to measure the frequency of A-to-I RNA editing, and shown that results are primer specific. Thus, the correct sequencing primer must be utilized for the most dependable data. When compared to other methods used to measure the frequency of A-to-I RNA editing, the major benefits of the peak height ratio are that this method is inexpensive, fast, non-labor intensive and easily adaptable to many laboratory and field settings.
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RNA-Seq has provided valuable insights into global gene expression in a wide variety of organisms. Using a modified RNA-Seq approach and Illumina's high-throughput sequencing technology, we globally identified 5'-ends of transcripts for the plant pathogen Pseudomonas syringae pv. tomato str. DC3000. A substantial fraction of 5'-ends obtained by this method were consistent with results obtained using global RNA-Seq and 5'RACE. As expected, many 5'-ends were positioned a short distance upstream of annotated genes. We also captured 5'-ends within intergenic regions, providing evidence for the expression of un-annotated genes and non-coding RNAs, and detected numerous examples of antisense transcription, suggesting additional levels of complexity in gene regulation in DC3000. Importantly, targeted searches for sequence patterns in the vicinity of 5'-ends revealed over 1200 putative promoters and other regulatory motifs, establishing a broad foundation for future investigations of regulation at the genomic and single gene levels.
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Genoma de Planta , Pseudomonas syringae/genética , Solanum lycopersicum/genética , Transcripción Genética , Secuencia de Bases , Cartilla de ADN , ARN de Planta/genética , Reproducibilidad de los ResultadosRESUMEN
The 2008 ABRF DNA Sequencing Research Group (DSRG) difficult template sequencing study was designed to identify a general set of guidelines that would constitute the best approaches for sequencing difficult templates. This was a continuation of previous DSRG difficult template studies performed in 1996, 1997, and 2003. The distinguishing factors in the present study were the number of DNA templates used, the number of different types of difficult regions tested, and the inclusion of a follow-up phase of the study to identify optimal protocols for each type of difficult template. DNA templates with associated sequencing primers were distributed to participating laboratories and each laboratory returned their sequencing results along with descriptions of the experimental conditions used. The data were analyzed and the best protocols were identified for each difficult template. This information was subsequently distributed to the participating laboratories for a second round of sequencing to evaluate the general applicability of the optimized protocols. The average improvements in sequencing results were 11% overall, with a range of -25% to +43% using the optimized protocols. The full results from this study are presented here and they demonstrate that general experimental protocols and common additives can be used to improve the sequencing success for many difficult templates.