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The global lack of donor shortage poses a major limitation for heart transplantation. New concepts with expanded donor inclusion criteria comprise extended transport distances and prolonged ischemic times with the aim of reaching a larger number of potential donors. Recent developments in cold storage solutions may allow more donor hearts with prolonged ischemic times to be use for transplantation in the future. We present our experience during a long-distance donor heart procurement with the longest reported transport distance and transport time in the current literature. This was made possible through the use of SherpaPak™, an innovative cold storage system which allows for controlled temperatures during transportation.
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MOTIVATION: DNA and RNA modifications can now be identified using nanopore sequencing. However, we currently lack a flexible software to efficiently encode, store, analyze and visualize DNA and RNA modification data. RESULTS: Here, we present ModPhred, a versatile toolkit that facilitates DNA and RNA modification analysis from nanopore sequencing reads in a user-friendly manner. ModPhred integrates probabilistic DNA and RNA modification information within the FASTQ and BAM file formats, can be used to encode multiple types of modifications simultaneously, and its output can be easily coupled to genomic track viewers, facilitating the visualization and analysis of DNA and RNA modification information in individual reads in a simple and computationally efficient manner. AVAILABILITY AND IMPLEMENTATION: ModPhred is available at https://github.com/novoalab/modPhred, is implemented in Python3, and is released under an MIT license. Docker images with all dependencies preinstalled are also provided. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
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Sequenciamento por Nanoporos , Nanoporos , Análise de Sequência de DNA/métodos , Software , DNA , RNA , Sequenciamento de Nucleotídeos em Larga EscalaRESUMO
Inferring homology relationships across genes in different species is a central task in comparative genomics. Therefore, a large number of resources and methods have been developed over the years. Some public databases include phylogenetic trees of homologous gene families which can be used to further differentiate homology relationships into orthology and paralogy. MetaPhOrs is a web server that integrates phylogenetic information from different sources to provide orthology and paralogy relationships based on a common phylogeny-based predictive algorithm and associated with a consistency-based confidence score. Here we describe the latest version of the web server which includes major new implementations and provides orthology and paralogy relationships derived from â¼8.2 million gene family trees-from 13 different source repositories across â¼4000 species with sequenced genomes. MetaPhOrs server is freely available, without registration, at http://orthology.phylomedb.org/.
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Família Multigênica , Filogenia , Software , Homologia de SequênciaRESUMO
Restriction-modification (R-M) systems represent an effective mechanism of defence against invading bacteriophages, and are widely spread among bacteria and archaea. In acquiring a Type II R-M system via horizontal gene transfer, the new hosts become more resistant to phage infection, through the action of a restriction endonuclease (REase), which recognizes and cleaves specific target DNAs. To protect the host cell's DNA, there is also a methyltransferase (MTase), which prevents DNA cleavage by the cognate REase. In some R-M systems, the host also accepts a cis-acting transcription factor (C protein), which regulates the counteracting activities of REase and MTase to avoid host self-restriction. Our study characterized the unexpected phenotype of Escherichia coli cells, which manifested as extensive cell filamentation triggered by acquiring the Csp231I R-M system from Citrobacter sp. Surprisingly, we found that the cell morphology defect was solely dependent on the C regulator. Our transcriptome analysis supported by in vivo and in vitro assays showed that C protein directly silenced the expression of the RacR repressor to affect the Rac prophage-related genes. The rac locus ydaST genes, when derepressed, exerted a toxicity indicated by cell filamentation through an unknown mechanism. These results provide an apparent example of transcription factor cross-talk, which can have significant consequences for the host, and may represent a constraint on lateral gene transfer.
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Bacteriófagos/genética , Enzimas de Restrição-Modificação do DNA/genética , Escherichia coli/genética , Interações Hospedeiro-Patógeno/genética , Sequência de Aminoácidos/genética , Bacteriófagos/patogenicidade , Citrobacter/genética , Enzimas de Restrição do DNA/genética , DNA Bacteriano/química , DNA Bacteriano/genética , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Escherichia coli/virologia , Perfilação da Expressão Gênica/métodos , Regulação Bacteriana da Expressão Gênica , Transferência Genética Horizontal/genética , Fenótipo , Fatores de Transcrição/genética , Proteínas Virais/genéticaRESUMO
RNA molecules play important roles in virtually every cellular process. These functions are often mediated through the adoption of specific structures that enable RNAs to interact with other molecules. Thus, determining the secondary structures of RNAs is central to understanding their function and evolution. In recent years several sequencing-based approaches have been developed that allow probing structural features of thousands of RNA molecules present in a sample. Here, we describe nextPARS, a novel Illumina-based implementation of in vitro parallel probing of RNA structures. Our approach achieves comparable accuracy to previous implementations, while enabling higher throughput and sample multiplexing.
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Sequenciamento de Nucleotídeos em Larga Escala/métodos , Conformação de Ácido Nucleico , RNA Mensageiro/química , Análise de Sequência de RNA/métodos , Biologia ComputacionalRESUMO
Pulsars' signals reaching the atmosphere can be considered being stable under certain assumptions. In such a case the ionosphere remains the main factor distorting signal from the extraterrestrial sources, particularly if we observe them at long radio waves. In this article we present the results of the analysis of relative peak flux changes for two selected pulsars: PSR J0332+5434 (B0329+54) and PSR J1509+5531 (B1508+55), observed with the long radio wave sensor (The PL612 Low Frequency Array (LOFAR) station in Baldy), together with the analysis of Rate of TEC (ROT) parameter changes measured with the Global Navigation Satellite Systems (GNSS) sensor (IGS LAMA station (IGS: International GSSN Service)). The main objective of the work is to find if the rapid plasma density (observed with the Rate of Total Electron Content (TEC)) has a counterpart in the pulsar observation characteristics. This focuses the attention on ionosphere influence during pulsar investigations at low radio frequencies. Additionally, what was the aim of this work, our results give reasons for using pulsar signals from LOFAR together with GNSS data as multi instrumental ionosphere state probes. Our results show a clear anti-correlation between the ROT and the pulsar profile's peak flux trends.
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Achieving high accuracy in orthology inference is essential for many comparative, evolutionary and functional genomic analyses, yet the true evolutionary history of genes is generally unknown and orthologs are used for very different applications across phyla, requiring different precision-recall trade-offs. As a result, it is difficult to assess the performance of orthology inference methods. Here, we present a community effort to establish standards and an automated web-based service to facilitate orthology benchmarking. Using this service, we characterize 15 well-established inference methods and resources on a battery of 20 different benchmarks. Standardized benchmarking provides a way for users to identify the most effective methods for the problem at hand, sets a minimum requirement for new tools and resources, and guides the development of more accurate orthology inference methods.
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Biologia Computacional/normas , Genômica/normas , Filogenia , Proteômica/normas , Archaea/classificação , Archaea/genética , Bactérias/classificação , Bactérias/genética , Biologia Computacional/métodos , Bases de Dados Genéticas , Eucariotos/classificação , Eucariotos/genética , Ontologia Genética , Genômica/métodos , Modelos Genéticos , Proteômica/métodos , Análise de Sequência de Proteína , Homologia de Sequência , Especificidade da EspécieRESUMO
[This corrects the article DOI: 10.1371/journal.pgen.1005626.].
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Many genomes display high levels of heterozygosity (i.e. presence of different alleles at the same loci in homologous chromosomes), being those of hybrid organisms an extreme such case. The assembly of highly heterozygous genomes from short sequencing reads is a challenging task because it is difficult to accurately recover the different haplotypes. When confronted with highly heterozygous genomes, the standard assembly process tends to collapse homozygous regions and reports heterozygous regions in alternative contigs. The boundaries between homozygous and heterozygous regions result in multiple assembly paths that are hard to resolve, which leads to highly fragmented assemblies with a total size larger than expected. This, in turn, causes numerous problems in downstream analyses such as fragmented gene models, wrong gene copy number, or broken synteny. To circumvent these caveats we have developed a pipeline that specifically deals with the assembly of heterozygous genomes by introducing a step to recognise and selectively remove alternative heterozygous contigs. We tested our pipeline on simulated and naturally-occurring heterozygous genomes and compared its accuracy to other existing tools. Our method is freely available at https://github.com/Gabaldonlab/redundans.
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Genoma , Heterozigoto , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Sequência de DNA/métodos , Algoritmos , Genômica/métodos , Homozigoto , Software , Sintenia/genéticaRESUMO
Candida metapsilosis is a rarely-isolated, opportunistic pathogen that belongs to a clade of pathogenic yeasts known as the C. parapsilosis sensu lato species complex. To gain insight into the recent evolution of C. metapsilosis and the genetic basis of its virulence, we sequenced the genome of 11 clinical isolates from various locations, which we compared to each other and to the available genomes of the two remaining members of the complex: C. orthopsilosis and C. parapsilosis. Unexpectedly, we found compelling genomic evidence that C. metapsilosis is a highly heterozygous hybrid species, with all sequenced clinical strains resulting from the same past hybridization event involving two parental lineages that were approximately 4.5% divergent in sequence. This result indicates that the parental species are non-pathogenic, but that hybridization between them formed a new opportunistic pathogen, C. metapsilosis, that has achieved a worldwide distribution. We show that these hybrids are diploid and we identified strains carrying loci for both alternative mating types, which supports mating as the initial mechanism for hybrid formation. We trace the aftermath of this hybridization at the genomic level, and reconstruct the evolutionary relationships among the different strains. Recombination and introgression -resulting in loss of heterozygosis- between the two subgenomes have been rampant, and includes the partial overwriting of the MTLa mating locus in all strains. Collectively, our results shed light on the recent genomic evolution within the C. parapsilosis sensu lato complex, and argue for a re-definition of species within this clade, with at least five distinct homozygous lineages, some of which having the ability to form hybrids.
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Candida/genética , Evolução Molecular , Infecções Oportunistas/genética , Virulência/genética , Candida/patogenicidade , Genoma , Heterozigoto , Humanos , Hibridização Genética , Infecções Oportunistas/microbiologiaRESUMO
Phylogenetic trees representing the evolutionary relationships of homologous genes are the entry point for many evolutionary analyses. For instance, the use of a phylogenetic tree can aid in the inference of orthology and paralogy relationships, and in the detection of relevant evolutionary events such as gene family expansions and contractions, horizontal gene transfer, recombination or incomplete lineage sorting. Similarly, given the plurality of evolutionary histories among genes encoded in a given genome, there is a need for the combined analysis of genome-wide collections of phylogenetic trees (phylomes). Here, we introduce a new release of PhylomeDB (http://phylomedb.org), a public repository of phylomes. Currently, PhylomeDB hosts 120 public phylomes, comprising >1.5 million maximum likelihood trees and multiple sequence alignments. In the current release, phylogenetic trees are annotated with taxonomic, protein-domain arrangement, functional and evolutionary information. PhylomeDB is also a major source for phylogeny-based predictions of orthology and paralogy, covering >10 million proteins across 1059 sequenced species. Here we describe newly implemented PhylomeDB features, and discuss a benchmark of the orthology predictions provided by the database, the impact of proteome updates and the use of the phylome approach in the analysis of newly sequenced genomes and transcriptomes.
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Bases de Dados Genéticas , Genoma , Filogenia , Perfilação da Expressão Gênica , Humanos , Internet , Proteínas/classificação , Proteínas/genética , ProteomaRESUMO
Comparative genomics, metagenomics, and single-cell technologies have shown that populations of microbial species encompass assemblages of closely related strains. This raises the question of whether individual bacterial lineages respond to the presence of their close relatives by modifying their gene expression or, instead, whether assemblages simply act as the arithmetic addition of their individual components. Here, we took advantage of transcriptome sequencing to address this question. For this, we analyzed the transcriptomes of two closely related strains of the extremely halophilic bacterium Salinibacter ruber grown axenically and in coculture. These organisms dominate bacterial assemblages in hypersaline environments worldwide. The strains used here cooccurred in the natural environment and are 100% identical in their 16S rRNA genes, and each strain harbors an accessory genome representing 10% of its complete genome. Overall, transcriptomic patterns from pure cultures were very similar for both strains. Expression was detected along practically the whole genome albeit with some genes at low levels. A subset of genes was very highly expressed in both strains, including genes coding for the light-driven proton pump xanthorhodopsin, genes involved in the stress response, and genes coding for transcriptional regulators. Expression differences between pure cultures affected mainly genes involved in environmental sensing. When the strains were grown in coculture, there was a modest but significant change in their individual transcription patterns compared to those in pure culture. Each strain sensed the presence of the other and responded in a specific manner, which points to fine intraspecific transcriptomic modulation.
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Bacteroidetes/classificação , Bacteroidetes/genética , Sequenciamento de Nucleotídeos em Larga Escala , Interações Microbianas/genética , Transcriptoma/genética , Proteínas de Bactérias/genética , Bacteroidetes/metabolismo , Sequência de Bases , Técnicas de Cocultura , DNA Bacteriano/genética , Genoma Bacteriano/genética , Metagenômica , RNA Ribossômico 16S/genética , Rodopsinas Microbianas/genética , Análise de Sequência de DNA , Transcrição GênicaRESUMO
The pathogenic yeast Candida albicans utilizes hydroxyderivatives of benzene via the catechol and hydroxyhydroquinone branches of the 3-oxoadipate pathway. The genetic basis and evolutionary origin of this catabolic pathway in yeasts are unknown. In this study, we identified C. albicans genes encoding the enzymes involved in the degradation of hydroxybenzenes. We found that the genes coding for core components of the 3-oxoadipate pathway are arranged into two metabolic gene clusters. Our results demonstrate that C. albicans cells cultivated in media containing hydroxybenzene substrates highly induce the transcription of these genes as well as the corresponding enzymatic activities. We also found that C. albicans cells assimilating hydroxybenzenes cope with the oxidative stress by upregulation of cellular antioxidant systems such as alternative oxidase and catalase. Moreover, we investigated the evolution of the enzymes encoded by these clusters and found that most of them share a particularly sparse phylogenetic distribution among Saccharomycotina, which is likely to have been caused by extensive gene loss. We exploited this fact to find co-evolving proteins that are suitable candidates for the missing enzymes of the pathway.
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Adipatos/metabolismo , Candida albicans/genética , Candida albicans/metabolismo , Enzimas/genética , Redes e Vias Metabólicas/genética , Família Multigênica , Fenol/metabolismo , Antioxidantes/metabolismo , Biotransformação , Enzimas/metabolismo , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Ordem dos Genes , Estresse Oxidativo , Filogenia , SinteniaRESUMO
Transfer RNAs (tRNAs) play a central role in protein translation. Studying them has been difficult in part because a simple method to simultaneously quantify their abundance and chemical modifications is lacking. Here we introduce Nano-tRNAseq, a nanopore-based approach to sequence native tRNA populations that provides quantitative estimates of both tRNA abundances and modification dynamics in a single experiment. We show that default nanopore sequencing settings discard the vast majority of tRNA reads, leading to poor sequencing yields and biased representations of tRNA abundances based on their transcript length. Re-processing of raw nanopore current intensity signals leads to a 12-fold increase in the number of recovered tRNA reads and enables recapitulation of accurate tRNA abundances. We then apply Nano-tRNAseq to Saccharomyces cerevisiae tRNA populations, revealing crosstalks and interdependencies between different tRNA modification types within the same molecule and changes in tRNA populations in response to oxidative stress.
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Sequenciamento por Nanoporos , Nanoporos , RNA , RNA de Transferência/química , Análise de Sequência de RNA/métodosRESUMO
Reliable prediction of orthology is central to comparative genomics. Approaches based on phylogenetic analyses closely resemble the original definition of orthology and paralogy and are known to be highly accurate. However, the large computational cost associated to these analyses is a limiting factor that often prevents its use at genomic scales. Recently, several projects have addressed the reconstruction of large collections of high-quality phylogenetic trees from which orthology and paralogy relationships can be inferred. This provides us with the opportunity to infer the evolutionary relationships of genes from multiple, independent, phylogenetic trees. Using such strategy, we combine phylogenetic information derived from different databases, to predict orthology and paralogy relationships for 4.1 million proteins in 829 fully sequenced genomes. We show that the number of independent sources from which a prediction is made, as well as the level of consistency across predictions, can be used as reliable confidence scores. A webserver has been developed to easily access these data (http://orthology.phylomedb.org), which provides users with a global repository of phylogeny-based orthology and paralogy predictions.
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Genômica/métodos , Filogenia , Genoma , Proteínas/classificação , Proteínas/genética , SoftwareRESUMO
The growing availability of complete genomic sequences from diverse species has brought about the need to scale up phylogenomic analyses, including the reconstruction of large collections of phylogenetic trees. Here, we present the third version of PhylomeDB (http://phylomeDB.org), a public database for genome-wide collections of gene phylogenies (phylomes). Currently, PhylomeDB is the largest phylogenetic repository and hosts 17 phylomes, comprising 416,093 trees and 165,840 alignments. It is also a major source for phylogeny-based orthology and paralogy predictions, covering about 5 million proteins in 717 fully-sequenced genomes. For each protein-coding gene in a seed genome, the database provides original and processed alignments, phylogenetic trees derived from various methods and phylogeny-based predictions of orthology and paralogy relationships. The new version of phylomeDB has been extended with novel data access and visualization features, including the possibility of programmatic access. Available seed species include model organisms such as human, yeast, Escherichia coli or Arabidopsis thaliana, but also alternative model species such as the human pathogen Candida albicans, or the pea aphid Acyrtosiphon pisum. Finally, PhylomeDB is currently being used by several genome sequencing projects that couple the genome annotation process with the reconstruction of the corresponding phylome, a strategy that provides relevant evolutionary insights.
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Bases de Dados Genéticas , Genômica , Filogenia , Alinhamento de Sequência , Humanos , Anotação de Sequência MolecularRESUMO
DNA methylation is a common, but not universal, epigenetic modification that plays an important role in multiple cellular processes. While definitely settled for numerous plant, mammalian, and bacterial species, the genome methylation in different fungal species, including widely studied and industrially-relevant yeast species, Yarrowia lipolytica, is still a matter of debate. In this paper, we report a differential DNA methylation level in the genome of Y. lipolytica subjected to sequential subculturing and to heat stress conditions. To this end, we adopted repeated batch bioreactor cultivations of Y. lipolytica subjected to thermal stress in specific time intervals. To analyze the variation in DNA methylation between stressed and control cultures, we (a) quantified the global DNA methylation status using an immuno-assay, and (b) studied DNA methylation patterns through whole-genome sequencing. Primarily, we demonstrated that 5 mC modification can be detected using a commercial immuno-assay, and that the modifications are present in Y. lipolytica's genome at ~0.5% 5 mC frequency. On the other hand, we did not observe any changes in the epigenetic response of Y. lipolytica to heat shock (HS) treatment. Interestingly, we identified a general phenomenon of decreased 5 mC level in Y. lipolytica's genome in the stationary phase of growth, when compared to a late-exponential epigenome. While this study provides an insight into the subculturing stress response and adaptation to the stress at epigenetic level by Y. lipolytica, it also leaves an open question of inability to detect any genomic DNA methylation level (either in CpG context or context-less) through whole-genome sequencing. The results of ONT sequencing, suggesting that 5 mC modification is either rare or non-existent in Y. lipolytica genome, are contradicted with the results of the immunoassay.
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Candida subhashii belongs to the CUG-Ser clade, a group of phylogenetically closely related yeast species that includes some human opportunistic pathogens, such as Candida albicans. Despite being present in the environment, C. subhashii was initially described as the causative agent of a case of peritonitis. Considering the relevance of whole-genome sequencing and analysis for our understanding of genome evolution and pathogenicity, we sequenced, assembled and annotated the genome of C. subhashii type strain. Our results show that C. subhashii presents a highly heterozygous genome and other signatures that point to a hybrid ancestry. The presence of functional pathways for assimilation of hydroxyaromatic compounds goes in line with the affiliation of this yeast with soil microbial communities involved in lignin decomposition. Furthermore, we observed that different clones of this strain may present circular or linear mitochondrial DNA. Re-sequencing and comparison of strains with differential mitochondrial genome topology revealed five candidate genes potentially associated with this conformational change: MSK1, SSZ1, ALG5, MRPL9 and OYE32.
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Candida/genética , Núcleo Celular/genética , Genoma Fúngico , Genoma Mitocondrial , Redes e Vias Metabólicas , Fenóis/metabolismo , Candida/metabolismo , Sequenciamento Completo do GenomaRESUMO
Nanopore RNA sequencing shows promise as a method for discriminating and identifying different RNA modifications in native RNA. Expanding on the ability of nanopore sequencing to detect N6-methyladenosine, we show that other modifications, in particular pseudouridine (Ψ) and 2'-O-methylation (Nm), also result in characteristic base-calling 'error' signatures in the nanopore data. Focusing on Ψ modification sites, we detected known and uncovered previously unreported Ψ sites in mRNAs, non-coding RNAs and rRNAs, including a Pus4-dependent Ψ modification in yeast mitochondrial rRNA. To explore the dynamics of pseudouridylation, we treated yeast cells with oxidative, cold and heat stresses and detected heat-sensitive Ψ-modified sites in small nuclear RNAs, small nucleolar RNAs and mRNAs. Finally, we developed a software, nanoRMS, that estimates per-site modification stoichiometries by identifying single-molecule reads with altered current intensity and trace profiles. This work demonstrates that Nm and Ψ RNA modifications can be detected in cellular RNAs and that their modification stoichiometry can be quantified by nanopore sequencing of native RNA.