Sequenciamento e análise do genoma de derxia lacustris HL 12

Orientador : Prof. Dr. Emanuel Maltempi de SouzaCoorientador : Dr. Arnaldo GlogauerDissertação (mestrado) - Universidade Federal do Paraná, Setor de Educação Profissional e Tecnológica, Programa de Pós-Graduação em Bioinformática. Defesa: Curitiba, 29/10/2010Inclui referências : f. 40-43;49-51Resumo: As rizobactérias são bactérias de vida livre que habitam as raízes das plantas, desenvolvem uma relação de simbiose e geram benefícios aos organismos vegetais. A existência desses organismos é de grande importância na agricultura, já que a carência na absorção de nitrogênio impacta diretamente na taxa de crescimento das plantas, diminui a produtividade agrícola e originam problemas de ordem ambiental com a utilização de fertilizantes que são poluentes ao meio. A fixação biológica de nitrogênio é realizada por uma ampla gama de bactérias. Derxia lacustris HL12 é uma bactéria fixadora de nitrogênio, pertencente à classe das Betaproteobacterias, ordem Burkholderiales, família Alcaligenaceae, gênero Derxia. Esse organismo foi isolado de amostras de água provenientes de um lago na região de Taiwan. D. lacustris HL12 é a segunda bactéria inserida no gênero Derxia, de acordo com a comparação entre as sequências do gene do RNA ribossomal 16S desta e de Derxia gummosa DSM 723, que é considerado o organismo de referência. Esse organismo está depositado no NCBI e possui uma montagem com 19 contigs e um contig separado da sequencia do 16S rRNA . D. lacustris HL12 teve seu genoma sequenciado por duas vezes na plataforma Illumina Miseq. O processo de sequenciamento resultou num total de 3.532.510 reads. Seus reads possuem um tamanho médio de 300pb. Os dados brutos foram montados com os seguintes softwares: CLC, Velvet, Celera, Spades, Masurca. A melhor montagem obtida foi a realizada no software Celera, possuindo 129 contigs e percentual de GC igual a 69,20% . O software Matlab foi utilizado em ensaios de ordenação e cálculos do genoma. O software Fgap foi utilizado com os dados provenientes de todas as montagens realizadas para o fechamento dos gaps existentes no genoma. O software Bowtie foi responsável por mapear o genoma com os dados brutos. O software Mummer foi utilizado na construção do dotplot entre D. lacustris HL12 e D. gummosa DSM 723. O software Fastqc foi utilizado para analisar a qualidade dos dados brutos. O genoma de D. lacustris HL12 possui um draft com 28 contigs. Foi localizado um operon ribossomal completo.O genoma passou por uma análise de anotação para identificação de genes em algumas vias metabólicas do organismo. Foram encontrados os genes do cluster Nif, reforçando o indício do organismo fixar nitrogênio com os anotadores Sila e Rast. Palavras-chave: Fixação de nitrogênio, Sequência genômica, Derxia lacustris HL12.Abstract: Rhizobacteria are bacteria which inhabit plant roots developing a symbiosis relationship that benefits those organisms. The existence of this kind of bacteria is of great importance to agriculture, once the need for nitrogen absorption impacts directly on plant growth rate, decreases agricultural productivity and generates environmental problems caused by the use of fertilizers. The biological nitrogen fixation is made by a large number of species. Derxia lacustris HL12 is a nitrogen fixing bacterium belonging to the class Betaproteobacterias, order Burkholderiales, family Alcaligenaceae, genus Derxia. This organism was isolated from fresh water collected from a lake in Taiwan. D. lacustris HL12 is the second bacteria inserted in the genus Derxia, in accordance with the comparison between its gene sequence of RNA ribosomal 16S and that of Derxia gummosa DSM 723, considered the reference organism. This organism is stored at the NCBI and has an assembly with 19 contigs. and one additional contig separated form 16S rRNA's sequence. D. lacustris HL12 had its genome sequenced twice using the Illumina Miseq platform. The sequencing process resulted in a total of 3,532,510 reads. The average size of its reads is of 300 pb. Raw data were assembled using the following softwares: CLC, Velvet, Celera, Spades, Masurca. The best assembly was obtained by using the Celera software and has 129 contigs and a CG percentage of 69.20%. Matlab was used in collation test runs and genome calculations. The software Fgap used the data collected from all the assemblies obtained for closing the existing gaps in the genome. The software Bowtie scanned the genome using raw data. The software Mummer was used in the construction of the dotplot between D. lacustris HL12 and D. gummosa DSM 723. The software Fastqc was employed to analyze the quality of the raw data. D. lacustris HL12's genome has a draft with 28 contigs. A complete ribosomal operon was located. The genome was submitted through an annotation analysis for the identification of the genes in some of the organism's metabolic pathways. Nif cluster genes were found using Sila and Rast annotation tools, which evinces D. lacustris HL12 as a nitrogen fixing bacteria. Key-words: Nitrogen fixation, Geonomic sequencing, Derxia lacustris HL12

RTNduals an R/Bioconductor package for analysis of co-regulation and inference of dual regulons

MOTIVATION: Transcription factors (TFs) are key regulators of gene expression, and can activate or repress multiple target genes, forming regulatory units, or regulons. Understanding downstream effects of these regulators includes evaluating how TFs cooperate or compete within regulatory networks. Here we present RTNduals, an R/Bioconductor package that implements a general method for analyzing pairs of regulons. RESULTS: RTNduals identifies a dual regulon when the number of targets shared between a pair of regulators is statistically significant. The package extends the RTN (Reconstruction of Transcriptional Networks) package, and uses RTN transcriptional networks to identify significant co-regulatory associations between regulons. The Supplementary Information reports two case studies for TFs using the METABRIC and TCGA breast cancer cohorts. AVAILABILITY AND IMPLEMENTATION: RTNduals is written in the R language, and is available from the Bioconductor project at http://bioconductor.org/packages/RTNduals/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online

Novel lncRNAs Co-Expression Networks Identifies LINC00504 with Oncogenic Role in Luminal A Breast Cancer Cells

Long non-coding RNAs (lncRNAs) are functional transcripts with more than 200 nucleotides. These molecules exhibit great regulatory capacity and may act at different levels of gene expression regulation. Despite this regulatory versatility, the biology of these molecules is still poorly understood. Computational approaches are being increasingly used to elucidate biological mechanisms in which these lncRNAs may be involved. Co-expression networks can serve as great allies in elucidating the possible regulatory contexts in which these molecules are involved. Herein, we propose the use of the pipeline deposited in the RTN package to build lncRNAs co-expression networks using TCGA breast cancer (BC) cohort data. Worldwide, BC is the most common cancer in women and has great molecular heterogeneity. We identified an enriched co-expression network for the validation of relevant cell processes in the context of BC, including LINC00504. This lncRNA has increased expression in luminal subtype A samples, and is associated with prognosis in basal-like subtype. Silencing this lncRNA in luminal A cell lines resulted in decreased cell viability and colony formation. These results highlight the relevance of the proposed method for the identification of lncRNAs in specific biological contexts