29 resultados para Next generation sequencing


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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

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The desert locust (Schistocerca gregaria) has been used as material for numerous cytogenetic studies. Its genome size is estimated to be 8.55 Gb of DNA comprised in 11 autosomes and the X chromosome. Its X0/XX sex chromosome determinism therefore results in females having 24 chromosomes whereas males have 23. Surprisingly, little is known about the DNA content of this locust's huge chromosomes. Here, we use the Feulgen Image Analysis Densitometry and C-banding techniques to respectively estimate the DNA quantity and heterochromatin content of each chromosome. We also identify three satellite DNAs using both restriction endonucleases and next-generation sequencing. We then use fluorescent in situ hybridization to determine the chromosomal location of these satellite DNAs as well as that of six tandem repeat DNA gene families. The combination of the results obtained in this work allows distinguishing between the different chromosomes not only by size, but also by the kind of repetitive DNAs that they contain. The recent publication of the draft genome of the migratory locust (Locusta migratoria), the largest animal genome hitherto sequenced, invites for sequencing even larger genomes. S. gregaria is a pest that causes high economic losses. It is thus among the primary candidates for genome sequencing. But this species genome is about 50 % larger than that of L. migratoria, and although next-generation sequencing currently allows sequencing large genomes, sequencing it would mean a greater challenge. The chromosome sizes and markers provided here should not only help planning the sequencing project and guide the assembly but would also facilitate assigning assembled linkage groups to actual chromosomes.

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Empirical phylogeographic studies have progressively sampled greater numbers of loci over time, in part motivated by theoretical papers showing that estimates of key demographic parameters improve as the number of loci increases. Recently, next-generation sequencing has been applied to questions about organismal history, with the promise of revolutionizing the field. However, no systematic assessment of how phylogeographic data sets have changed over time with respect to overall size and information content has been performed. Here, we quantify the changing nature of these genetic data sets over the past 20years, focusing on papers published in Molecular Ecology. We found that the number of independent loci, the total number of alleles sampled and the total number of single nucleotide polymorphisms (SNPs) per data set has improved over time, with particularly dramatic increases within the past 5years. Interestingly, uniparentally inherited organellar markers (e.g. animal mitochondrial and plant chloroplast DNA) continue to represent an important component of phylogeographic data. Single-species studies (cf. comparative studies) that focus on vertebrates (particularly fish and to some extent, birds) represent the gold standard of phylogeographic data collection. Based on the current trajectory seen in our survey data, forecast modelling indicates that the median number of SNPs per data set for studies published by the end of the year 2016 may approach similar to 20000. This survey provides baseline information for understanding the evolution of phylogeographic data sets and underscores the fact that development of analytical methods for handling very large genetic data sets will be critical for facilitating growth of the field.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

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There has been a lot of advance in genomics since 1975 when the possibility to determine the nucleotide sequence of a genome was described. In the 90’s the human genome sequencing was started and it was greatly favored by advances in computer technologies. In the last ten years the development of next generation sequencing technologies allowed the sequencing of millions of bases at low cost and in a shorter time compared to the previous technologies. After the conclusion of the human genome project, several initiatives to sequence the genome of domestic animal species were taken, resulting in a large amount of data that is redirecting the goals of genetic studies in domestic animals. The aim of this review was to describe the present situation of the sequencing initiatives on the main domestic animal species of economical interest as well as to list the most important tools available to access the genomic information.

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Although equines have participated in the forming and development of several civilizations around the world since their domestication 6,000 years ago in comparison to other species that have zootechnical interest, few researches have been done related to animal breeding area, especially in Brazil. Some reasons for that are difficulties associated with the species as well as operational aspects. However, developments in genetics in the last decades contributed to a better understanding of the traits related to reproduction, heath, behavior and performance of domestic animals, including equines. Recent technologies as next generation sequencing methods and the high density chips of SNPs for genotyping allowed some advances in the researches already done. These researches used basically the candidate gene strategy, and identified genomic regions related to diseases and syndromes and, more recently, the performance in sport competition and specific abilities. Using these genomic analysis tools, some regions related to race performance have been identified and based on this information; genetic tests to select superior animals for racing performance have started to be available in the market.

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As plataformas de sequenciamento de nova geração são uma alternativa poderosa para estudos de genômica estrutural e funcional. Na genômica de plantas, os trabalhos com as novas plataformas têm sido destinados ao sequenciamento de transcritos, ressequenciamento ou sequenciamento de novo de genomas plastidiais. Neste trabalho, são detalhadas as tecnologias das plataformas mais utilizadas atualmente, bem como é revisada a aplicação dessas tecnologias na genômica estrutural e funcional de plantas.

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Background: The intestinal microbiome (IM) has extensively been studied in the search for a link of bacteria with the cause of Crohn`s disease (CD). The association might result from the action of a specific pathogen and/or an eventual imbalance in bacterial species composition of the gut. The innumerous virulence associated markers and strategies described for adherent and invasive Escherichia coli (AIEC) have made them putative candidate pathogens for CD. IM of CD patients shows dysbiosis, manifested by the proliferation of bacterial groups such as Enterobacteriaceae and reduction of others such as Lactobacillus and Bifidobacterium. The augmented bacterial population comprising of commensal and/or pathogenic organisms super stimulates the immune system, triggering the inflammatory reactions responsible for the clinical manifestations of the disease. Considering the role played by IM in CD and the multiple variables influencing its species composition, resulting in differences among populations, the objective of this study was to determine the bacterial biodiversity in the mucosa associated microbiome of CD patients from a population not previously subject to this analysis, living in the middle west region of Sao Paulo state. Methods: A total of 4 CD patients and 5 controls subjects attending the Botucatu Medical School of the Sao Paulo State University (UNESP) for routine colonoscopy and who signed an informed consent were included in the study. A number of 2 biopsies, one from the ileum and other from any part of the terminal colon, were taken from each subject and immediately frozen at -70[degrees]C until DNA purification. The bacterial biodiversity was assessed by next generation (ion torrent) sequencing of PCR amplicons of the ribosomal DNA 16S V6 region (16S V6 rDNA). The bacterial identification was performed at the genus level, by alignment of the generated DNA sequences with those available at the ribosomal database project (RDP) website. Results: The overall DNA sequence output was based on an average number of 526,427 reads per run, matching 50 bacterial genus 16SrDNA sequences available at the RDB website, and 22 non matching sequences. Over 95% of the sequences corresponded to taxa belonging to the major phyla: Firmicutes, Bacterioidetes, Proteobacteria and Actinobacteria. Irrespective of the intestinal site analyzed, no case-control differences could be observed in the prevalence of Actinobacteria and Firmicutes. The prevalence of Proteobacteria was higher (40%) in the biopsies of control subjects as compared to that of DC patients (16%). For Bacterioidetes, the higher prevalence was observed among DC patients (33% as opposed to 14,5% in controls). The significance for all comparisons considered a p value < 0,05 in a Chi2 test. No mucosal site specific differences could be observed in IM comparisons of CD and control subjects. Conclusions: The rise in the number of Bacterioidetes observed here among CD patients seems to be in agreement with most of studies published thus far. Yet, the reduction in the number of Proteobacteria along with an apparently unaltered population of Actinobacteria and Firmicutes, which include the so called "beneficial" organisms Bifidobacterium and Lactobacillus were rather surprising. These data suggest that the analyses on the role of IM in CD should consider the multiple variables that may influence its species composition.