Genome Diversity, Recombination, and Virulence across the Major Lineages of Paracoccidioides

We thank Angela Restrepo, Rosana Puccia, Zoilo Pires de Camargo, and Maria Sueli Felipe for kindly providing the isolates for this study. This project has been funded in whole or in part with federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under contract no. HHSN272200900018C. This work was partly supported by Colciencias via the grants “A Gene Atlas for Human Pathogenic Fungi” (122256934875) and “A Comprehensive Genomic and Transcriptomic Analysis of Dimorphic Human Pathogen Fungi and Its Relation with Virulence” (221365842971) and by the Universidad de Antioquia via a “Sostenibilidad 2015/2016” grant. Colciencias National Doctorate Program funding supported J.F.M.; Enlaza Mundos partly supported his fellowship. The Wellcome Trust supported R.A.F.Peer reviewedPublisher PD

The Dynamic Genome and Transcriptome of the Human Fungal Pathogen Blastomyces and Close Relative Emmonsia

Three closely related thermally dimorphic pathogens are causal agents of major fungal diseases affecting humans in the Americas: blastomycosis, histoplasmosis and paracoccidioidomycosis. Here we report the genome sequence and analysis of four strains of the etiological agent of blastomycosis, Blastomyces, and two species of the related genus Emmonsia, typically pathogens of small mammals. Compared to related species, Blastomyces genomes are highly expanded, with long, often sharply demarcated tracts of low GC-content sequence. These GC-poor isochore-like regions are enriched for gypsy elements, are variable in total size between isolates, and are least expanded in the avirulent B. dermatitidis strain ER-3 as compared with the virulent B. gilchristii strain SLH14081. The lack of similar regions in related species suggests these isochore-like regions originated recently in the ancestor of the Blastomyces lineage. While gene content is highly conserved between Blastomyces and related fungi, we identified changes in copy number of genes potentially involved in host interaction, including proteases and characterized antigens. In addition, we studied gene expression changes of B. dermatitidis during the interaction of the infectious yeast form with macrophages and in a mouse model. Both experiments highlight a strong antioxidant defense response in Blastomyces, and upregulation of dioxygenases in vivo suggests that dioxide produced by antioxidants may be further utilized for amino acid metabolism. We identify a number of functional categories upregulated exclusively in vivo, such as secreted proteins, zinc acquisition proteins, and cysteine and tryptophan metabolism, which may include critical virulence factors missed before in in vitro studies. Across the dimorphic fungi, loss of certain zinc acquisition genes and differences in amino acid metabolism suggest unique adaptations of Blastomyces to its host environment. These results reveal the dynamics of genome evolution and of factors contributing to virulence in Blastomyces.Author SummaryDimorphic fungal pathogens including Blastomyces are the cause of major fungal diseases in North and South America. The genus Emmonsia includes species infecting small mammals as well as a newly emerging pathogenic species recently reported in HIV-positive patients in South Africa. Here, we synthesize both genome sequencing of four isolates of Blastomyces and two species of Emmonsia as well as deep sequencing of Blastomyces RNA to draw major new insights into the evolution of this group and the pathogen response to infection. We investigate the trajectory of genome evolution of this group, characterizing the phylogenetic relationships of these species, a remarkable genome expansion that formed large isochore-like regions of low GC content in Blastomyces, and variation of gene content, related to host interaction, among the dimorphic fungal pathogens. Using RNA-Seq, we profile the response of Blastomyces to macrophage and mouse pulmonary infection, identifying key pathways and novel virulence factors. The identification of key fungal genes involved in adaptation to the host suggests targets for further study and therapeutic intervention in Blastomyces and related dimorphic fungal pathogens

Hacia múltiples análisis de motivos SNP de loci asociados con rasgos fenotípicos

Un artículo reciente en el Journal (1) y otros estudios (2) están mejorando en gran medida nuestra visión general de los codeterminantes genéticos candidatos del riesgo cardiovascular o la presión arterial y también están generando curiosidad sobre las fuentes de fuerte observación constante (p. Ej., P <10 15) asociaciones. En 12q24, un polimorfismo de un solo nucleótido (SNP) (rs3184504) (1) en 1 de 6 loci asociado con enfermedad de la arteria coronaria también estuvo fuertemente asociado con la presión arterial (2); un resumen de Internet (3) señaló sus asociaciones con 17 enfermedades o características relacionadas con enfermedades. Tenga en cuenta: Este trabajo fue financiado por la subvención 221356934877 de COLCIENCIAS. Los autores informaron que no tienen relaciones relevantes para revelar el contenido de este documento.A recent paper in the Journal (1) and other studies (2) are greatly improving our overview of candidate genetic codeterminants of cardiovascular risk or blood pressure and are also raising curiosity about sources of consistently strong (e.g., p < 10 15) observed associations. In 12q24, a single nucleotide polymorphism (SNP) (rs3184504) (1) in 1 of 6 loci associated with coronary artery disease was also strongly associated with blood pressure (2); an Internet summary (3) noted its associations with 17 diseases or disease-related characteristics. Please note: This work was funded by COLCIENCIAS grant 221356934877. The authors have reported that they have no relationships relevant to the contents of this paper to disclo

The eukaryotic genome, its reads, and the unfinished assembly

In recent years, readily affordable short read sequences provided by next-generation sequencing (NGS) have become longer and more accurate. This has led to a jump in interest in the utility of NGS-only approaches for exploring eukaryotic genomes. The concept of a static, 'finished' genome assembly, which still appears to be a faraway goal for many eukaryotes, is yielding to new paradigms. We here motivate an object-view concept where the raw reads are the main, fixed object, and assemblies with their annotations take a role of dynamically changing and modifiable views of that object. © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved

The eukaryotic genome, its reads, and the unfinished assembly

In recent years, readily affordable short read sequences provided by next-generation sequencing (NGS) have become longer and more accurate. This has led to a jump in interest in the utility of NGS-only approaches for exploring eukaryotic genomes. The concept of a static, 'finished' genome assembly, which still appears to be a faraway goal for many eukaryotes, is yielding to new paradigms. We here motivate an object-view concept where the raw reads are the main, fixed object, and assemblies with their annotations take a role of dynamically changing and modifiable views of that object. © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved

The complex task of choosing a de novo assembly: Lessons from fungal genomes

Selecting the values of parameters used by de novo genomic assembly programs, or choosing an optimal de novo assembly from several runs obtained with different parameters or programs, are tasks that can require complex decision-making. A key parameter that must be supplied to typical next generation sequencing (NGS) assemblers is the k-mer length, i.e., the word size that determines which de Bruijn graph the program should map out and use. The topic of assembly selection criteria was recently revisited in the Assemblathon 2 study (Bradnam et al., 2013). Although no clear message was delivered with regard to optimal k-mer lengths, it was shown with examples that it is sometimes important to decide if one is most interested in optimizing the sequences of protein-coding genes (the gene space) or in optimizing the whole genome sequence including the intergenic DNA, as what is best for one criterion may not be best for the other. In the present study, our aim was to better understand how the assembly of unicellular fungi (which are typically intermediate in size and complexity between prokaryotes and metazoan eukaryotes) can change as one varies the k-mer values over a wide range. We used two different de novo assembly programs (SOAPdenovo2 and ABySS), and simple assembly metrics that also focused on success in assembling the gene space and repetitive elements. A recent increase in Illumina read length to around 150 bp allowed us to attempt de novo assemblies with a larger range of k-mers, up to 127 bp. We applied these methods to Illumina paired-end sequencing read sets of fungal strains of Paracoccidioides brasiliensis and other species. By visualizing the results in simple plots, we were able to track the effect of changing k-mer size and assembly program, and to demonstrate how such plots can readily reveal discontinuities or other unexpected characteristics that assembly programs can present in practice, especially when they are used in a traditional molecular microbiology laboratory with a 'genomics corner'. Here we propose and apply a component of a first pass validation methodology for benchmarking and understanding fungal genome de novo assembly processes. © 2014 Elsevier Ltd. All rights reserved

The complex task of choosing a de novo assembly: Lessons from fungal genomes

Selecting the values of parameters used by de novo genomic assembly programs, or choosing an optimal de novo assembly from several runs obtained with different parameters or programs, are tasks that can require complex decision-making. A key parameter that must be supplied to typical next generation sequencing (NGS) assemblers is the k-mer length, i.e., the word size that determines which de Bruijn graph the program should map out and use. The topic of assembly selection criteria was recently revisited in the Assemblathon 2 study (Bradnam et al., 2013). Although no clear message was delivered with regard to optimal k-mer lengths, it was shown with examples that it is sometimes important to decide if one is most interested in optimizing the sequences of protein-coding genes (the gene space) or in optimizing the whole genome sequence including the intergenic DNA, as what is best for one criterion may not be best for the other. In the present study, our aim was to better understand how the assembly of unicellular fungi (which are typically intermediate in size and complexity between prokaryotes and metazoan eukaryotes) can change as one varies the k-mer values over a wide range. We used two different de novo assembly programs (SOAPdenovo2 and ABySS), and simple assembly metrics that also focused on success in assembling the gene space and repetitive elements. A recent increase in Illumina read length to around 150 bp allowed us to attempt de novo assemblies with a larger range of k-mers, up to 127 bp. We applied these methods to Illumina paired-end sequencing read sets of fungal strains of Paracoccidioides brasiliensis and other species. By visualizing the results in simple plots, we were able to track the effect of changing k-mer size and assembly program, and to demonstrate how such plots can readily reveal discontinuities or other unexpected characteristics that assembly programs can present in practice, especially when they are used in a traditional molecular microbiology laboratory with a 'genomics corner'. Here we propose and apply a component of a first pass validation methodology for benchmarking and understanding fungal genome de novo assembly processes. © 2014 Elsevier Ltd. All rights reserved

The eukaryotic genome, its reads, and the unfinished assembly

AbstractIn recent years, readily affordable short read sequences provided by next-generation sequencing (NGS) have become longer and more accurate. This has led to a jump in interest in the utility of NGS-only approaches for exploring eukaryotic genomes. The concept of a static, ‘finished’ genome assembly, which still appears to be a faraway goal for many eukaryotes, is yielding to new paradigms. We here motivate an object-view concept where the raw reads are the main, fixed object, and assemblies with their annotations take a role of dynamically changing and modifiable views of that object

Improving the Annotation of the Venom Gland Transcriptome of <i>Pamphobeteus verdolaga</i>, Prospecting Novel Bioactive Peptides

Spider venoms constitute a trove of novel peptides with biotechnological interest. Paucity of next-generation-sequencing (NGS) data generation has led to a description of less than 1% of these peptides. Increasing evidence supports the underestimation of the assembled genes a single transcriptome assembler can predict. Here, the transcriptome of the venom gland of the spider Pamphobeteus verdolaga was re-assembled, using three free access algorithms, Trinity, SOAPdenovo-Trans, and SPAdes, to obtain a more complete annotation. Assembler’s performance was evaluated by contig number, N50, read representation on the assembly, and BUSCO’s terms retrieval against the arthropod dataset. Out of all the assembled sequences with all software, 39.26% were common between the three assemblers, and 27.88% were uniquely assembled by Trinity, while 27.65% were uniquely assembled by SPAdes. The non-redundant merging of all three assemblies’ output permitted the annotation of 9232 sequences, which was 23% more when compared to each software and 28% more when compared to the previous P. verdolaga annotation; moreover, the description of 65 novel theraphotoxins was possible. In the generation of data for non-model organisms, as well as in the search for novel peptides with biotechnological interest, it is highly recommended to employ at least two different transcriptome assemblers