RNA‐seq: Applications and Best Practices

RNA‐sequencing (RNA‐seq) is the state‐of‐the‐art technique for transcriptome analysis that takes advantage of high‐throughput next‐generation sequencing. Although being a powerful approach, RNA‐seq imposes major challenges throughout its steps with numerous caveats. There are currently many experimental options available, and a complete comprehension of each step is critical to make right decisions and avoid getting into inconclusive results. A complete workflow consists of: (1) experimental design; (2) sample and library preparation; (3) sequencing; and (4) data analysis. RNA‐seq enables a wide range of applications such as the discovery of novel genes, gene/transcript quantification, and differential expression and functional analysis. This chapter will encompass the main aspects from sample preparation to downstream data analysis. It will be discussed how to obtain high‐quality samples, replicates amount, library preparation, sequencing platforms and coverage, focusing on best recommended practices based on specialized literature. Basic techniques and well‐known algorithms are presented and discussed, guiding both beginners and experienced users in the implementation of reliable experiments

Cestode strobilation: prediction of developmental genes and pathways

Background: Cestoda is a class of endoparasitic worms in the flatworm phylum (Platyhelminthes). During the course of their evolution cestodes have evolved some interesting aspects, such as their increased reproductive capacity. In this sense, they have serial repetition of their reproductive organs in the adult stage, which is often associated with external segmentation in a developmental process called strobilation. However, the molecular basis of strobilation is poorly understood. To assess this issue, an evolutionary comparative study among strobilated and non-strobilated flatworm species was conducted to identify genes and proteins related to the strobilation process. Results: We compared the genomic content of 10 parasitic platyhelminth species; five from cestode species, representing strobilated parasitic platyhelminths, and five from trematode species, representing non-strobilated parasitic platyhelminths. This dataset was used to identify 1813 genes with orthologues that are present in all cestode (strobilated) species, but absent from at least one trematode (non-strobilated) species. Development- related genes, along with genes of unknown function (UF), were then selected based on their transcriptional profiles, resulting in a total of 34 genes that were differentially expressed between the larval (pre-strobilation) and adult (strobilated) stages in at least one cestode species. These 34 genes were then assumed to be strobilation related; they included 12 encoding proteins of known function, with 6 related to the Wnt, TGF-β/BMP, or G-protein coupled receptor signaling pathways; and 22 encoding UF proteins. In order to assign function to at least some of the UF genes/proteins, a global gene co-expression analysis was performed for the cestode species Echinococcus multilocularis. This resulted in eight UF genes/proteins being predicted as related to developmental, reproductive, vesicle transport, or signaling processes. Conclusions: Overall, the described in silico data provided evidence of the involvement of 34 genes/proteins and at least 3 developmental pathways in the cestode strobilation process. These results highlight on the molecular mechanisms and evolution of the cestode strobilation process, and point to several interesting proteins as potential developmental markers and/or targets for the development of novel antihelminthic drugs

Secondary metabolite gene clusters in the entomopathogen fungus Metarhizium anisopliae : genome identification and patterns of expression in a cuticle infection model

Background: The described species from the Metarhizium genus are cosmopolitan fungi that infect arthropod hosts. Interestingly, while some species infect a wide range of hosts (host-generalists), other species infect only a few arthropods (host-specialists). This singular evolutionary trait permits unique comparisons to determine how pathogens and virulence determinants emerge. Among the several virulence determinants that have been described, secondary metabolites (SMs) are suggested to play essential roles during fungal infection. Despite progress in the study of pathogen-host relationships, the majority of genes related to SM production in Metarhizium spp. are uncharacterized, and little is known about their genomic organization, expression and regulation. To better understand how infection conditions may affect SM production in Metarhizium anisopliae, we have performed a deep survey and description of SM biosynthetic gene clusters (BGCs) in M. anisopliae, analyzed RNA-seq data from fungi grown on cattle-tick cuticles, evaluated the differential expression of BGCs, and assessed conservation among the Metarhizium genus. Furthermore, our analysis extended to the construction of a phylogeny for the following three BGCs: a tropolone/citrinin-related compound (MaPKS1), a pseurotin-related compound (MaNRPS-PKS2), and a putative helvolic acid (MaTERP1). Results: Among 73 BGCs identified in M. anisopliae, 20 % were up-regulated during initial tick cuticle infection and presumably possess virulence-related roles. These up-regulated BGCs include known clusters, such as destruxin, NG39x and ferricrocin, together with putative helvolic acid and, pseurotin and tropolone/citrinin-related compound clusters as well as uncharacterized clusters. Furthermore, several previously characterized and putative BGCs were silent or down-regulated in initial infection conditions, indicating minor participation over the course of infection. Interestingly, several up-regulated BGCs were not conserved in host-specialist species from the Metarhizium genus, indicating differences in the metabolic strategies employed by generalist and specialist species to overcome and kill their host. These differences in metabolic potential may have been partially shaped by horizontal gene transfer (HGT) events, as our phylogenetic analysis provided evidence that the putative helvolic acid cluster in Metarhizium spp. originated from an HGT event. Conclusions: Several unknown BGCs are described, and aspects of their organization, regulation and origin are discussed, providing further support for the impact of SM on the Metarhizium genus lifestyle and infection process

Genome-wide DNA methylation analysis of Metarhizium anisopliae during tick mimicked infection condition

Background: The Metarhizium genus harbors important entomopathogenic fungi. These species have been widely explored as biological control agents, and strategies to improve the fungal virulence are under investigation. Thus, the interaction between Metarhizium species and susceptible hosts have been explored employing different methods in order to characterize putative virulence determinants. However, the impact of epigenetic modulation on the infection cycle of Metarhizium is still an open topic. Among the different epigenetic modifications, DNA methylation of cytosine bases is an important mechanism to control gene expression in several organisms. To better understand if DNA methylation can govern Metarhizium-host interactions, the genome-wide DNA methylation profile of Metarhizium anisopliae was explored in two conditions: tick mimicked infection and a saprophytic-like control. Results: Using a genome wide DNA methylation profile based on bisulfite sequencing (BS-Seq), approximately 0.60% of the total cytosines were methylated in saprophytic-like condition, which was lower than the DNA methylation level (0.89%) in tick mimicked infection condition. A total of 670 mRNA genes were found to be putatively methylated, with 390 mRNA genes uniquely methylated in the tick mimicked infection condition. GO terms linked to response to stimuli, cell wall morphogenesis, cytoskeleton morphogenesis and secondary metabolism biosynthesis were over-represented in the tick mimicked infection condition, suggesting that energy metabolism is directed towards the regulation of genes associated with infection. However, recognized virulence determinants known to be expressed at distinct infection steps, such as the destruxin backbone gene and the collagen-like protein gene Mcl1, were found methylated, suggesting that a dynamic pattern of methylation could be found during the infectious process. These results were further endorsed employing RT-qPCR from cultures treated or not with the DNA methyltransferase inhibitor 5-Azacytidine. Conclusions: The set of genes here analyzed focused on secondary metabolites associated genes, known to be involved in several processes, including virulence. The BS-Seq pipeline and RT-qPCR analysis employing 5- Azacytidine led to identification of methylated virulence genes in M. anisopliae. The results provided evidences that DNA methylation in M. anisopliae comprises another layer of gene expression regulation, suggesting a main role of DNA methylation regulating putative virulence determinants during M. anisopliae infection cycle

Desenvolvimento das ferramentas SeedServer, para agrupamento de seqüências protéicas homólogas e U-MAGE, para propagação de ontologia funcional

Exportado OPUSMade available in DSpace on 2019-08-14T15:22:13Z (GMT). No. of bitstreams: 1 teserafaelguedes.pdf: 5195773 bytes, checksum: a137ad32a51dab7a00f9f241da67a7b7 (MD5) Previous issue date: 30Com o avanço das tecnologias de seqüenciamento, a organização das bases de dados secundárias é uma contribuição importante, nas quais o conhecimento existente é organizado com base em informações biológicas. Agrupamento de genes homólogos e a atribuição de termos de ontologia funcional envolvendo genes já conhecidos sãoformas de acelerar a análise de dados de novos seqüenciamentos. Neste trabalho relatamos o desenvolvimento do SeedServer, U-MAGE e aplicações. A integração de bancos de dados e programa agrupador de proteínas homólogas capazes de incluir seqüências provenientes de genomas incompletos, conjuntamente com metodologias de validação e comparação de estruturas secundárias resultou no desenvolvimento da ferramenta SeedServer. Grupos de homólogos criados a partir de seqüências de interesse do usuário são gerados com auxílio de uma interface web onde também é possível descarregar as seqüências agrupadas, obter relatórios com taxonomia completa e estimar a origem do gene em questão através da determinação do ancestral comum mais recente. O programa SeedServer, após ser testado e avaliado foi então utilizado em um estudo da heterotrofia de aminoácidos através da formação de grupos de homólogos das enzimas presentes nas vias biossintéticas de aminoácidos essenciais, demonstrando um quadro denominado de Grande Deleção Genômica em diferentes grupos de eucariotos e procariotos. A esse evento pode se suceder a perda da capacidade de assimilação de nitrogênio, componente essencial na formação dos aminoácidos. Estudos filogenéticos mostraram uma maior taxa de mutação dentre as enzimas remanescentes de vias incompletas quando comparadas com outras de vias completas. Adicionalmente, para melhorar a qualidade da anotação funcional de seqüências protéicas, foi criada a ferramenta denominada UMAGE (UniRef50 Matrices for Annotation of Gene Ontology Entries) que utiliza como base de propagação de termos de ontologia funcional, matrizes de recobrimento entre seqüências de mesmo UniRef50. O U-MAGE demonstrou uma melhora qualitativa significativa na anotação de ontologia funcional de diversos organismos. As duas ferramentas SeedServer e U-MAGE contribuem para a aceleração da propagação de informação de proteínas conhecidas, um desafio atual imposto à Bioinformática para fazer frente à intensa produção de novas seqüênciasWith advances in sequencing technologies, an important contribution is the organization of secondary databases, where existing knowledge is organized based on biological information. Grouping of homologous genes and assigning terms of functional ontology involving already known genes are ways to speed the analysis of data from new sequencing. We report the development of SeedServer, U-MAGE and applications. The integration of databases and a program capable of clustering homologous proteins including sequences derived from incomplete genomes, together with validation methods and comparison of secondary structures resulted in the development of SeedServer tool. Groups of homologous sequences chosen from user interest are generated with the aid of a web interface where you can also download the grouped sequences, get taxonomy reports and estimate the origin of the gene in question by determining the lowest common ancestor. The program SeedServer after being tested and evaluated was then used in a study of amino acid heterotrophy by forming groups of homologous enzymes present in the essential amino acids biosynthetic pathways, showing a scenario called the Great Genomic Deletion in different groups of eukaryotes and prokaryotes. Following that event may be the loss of assimilative capacity of nitrogen, an essential component in the formation of amino acids. Phylogenetic studies showed a higher rate of mutation among the enzymes remaining in incomplete pathways when compared with others from complete pathways. Additionally, to improve the quality of functional annotation of protein sequences, we created the tool called U-MAGE (UniRef50 Matrices for Annotation of Gene Ontology Entries) that uses as the basis of propagation of functional ontology terms the coverage between sequences within a UniRef50 organized in matrices. The U-MAGE demonstrated a significant qualitative improvement in functional ontology annotation of various organisms. Both tools SeedServer and U-MAGE contribute to the acceleration of the information spread from known proteins, a challenge to the current Bioinformatics to face the intense production of new sequence

Repetitive Elements in <i>Mycoplasma hyopneumoniae</i> Transcriptional Regulation

<div><p>Transcriptional regulation, a multiple-step process, is still poorly understood in the important pig pathogen <i>Mycoplasma hyopneumoniae</i>. Basic motifs like promoters and terminators have already been described, but no other cis-regulatory elements have been found. DNA repeat sequences have been shown to be an interesting potential source of cis-regulatory elements. In this work, a genome-wide search for tandem and palindromic repetitive elements was performed in the intergenic regions of all coding sequences from <i>M</i>. <i>hyopneumoniae</i> strain 7448. Computational analysis demonstrated the presence of 144 tandem repeats and 1,171 palindromic elements. The DNA repeat sequences were distributed within the 5’ upstream regions of 86% of transcriptional units of <i>M</i>. <i>hyopneumoniae</i> strain 7448. Comparative analysis between distinct repetitive sequences found in related mycoplasma genomes demonstrated different percentages of conservation among pathogenic and nonpathogenic strains. qPCR assays revealed differential expression among genes showing variable numbers of repetitive elements. In addition, repeats found in 206 genes already described to be differentially regulated under different culture conditions of <i>M</i>. <i>hyopneumoniae</i> strain 232 showed almost 80% conservation in relation to <i>M</i>. <i>hyopneumoniae</i> strain 7448 repeats. Altogether, these findings suggest a potential regulatory role of tandem and palindromic DNA repeats in the <i>M</i>. <i>hyopneumoniae</i> transcriptional profile.</p></div

Influence of repeat composition on mycoplasma gene expression.

<p>Influence of repeat composition on mycoplasma gene expression.</p

Pipeline of tandem repeat comparison analysis among mycoplasma genomes.

<p>An example of nonconserved SSR between <i>M</i>. <i>hyopneumoniae</i> strain 7448 (MHP_7448), and <i>M</i>. <i>hyopneumoniae</i> strain J (MHP_J) can be observed above. A (TAT)₉ repeat found in the MHP_7448 gene intergenic region was classified as nonconserved, as a (TAT)₁₁ repeat was localized in the respective orthologous intergenic region in MHP_J. Abbreviations: <i>M</i>. <i>hyopneumoniae</i> strain 7422 (MHP_7422); <i>M</i>. <i>flocculare</i> (MFL). *First CDS of the transcription unit.</p

Pipeline of repeat conservation in putative adhesin-coding genes and differentially expressed genes from strains of <i>M</i>. <i>hyopneumoniae</i>.

<p>(A) Comparison analysis of adhesins, with an example of relative conservation between MHP_7448 genes that have 5 PALG repeats and the respective orthologues in MHP_J that have only 3. The percentage (%) conservation between MHP_7448 and MHP_J repeats was obtained by dividing the number of repeat elements. (B) Repeats present in differentially expressed (DE) genes found in <i>M</i>. <i>hyopneumoniae</i> strain 232 (MHP_232) were compared with those found in MHP_7448 in the same way as in (A). Abbreviations as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168626#pone.0168626.g002" target="_blank">Fig 2</a>.</p

Pipeline of repeat search strategy.

<p>Up to 500 bp of 5’ intergenic regions (IR) of all <i>M</i>. <i>hyopneumoniae</i> strain 7448 (MHP_7448) CDSs were used for <i>in silico</i> prediction of tandem and palindromic DNA repeats.</p