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Histoplasmosis is a systemic fungal disease caused by the pathogen Histoplasma spp. that results in significant morbidity and mortality in persons with HIV/AIDS and can also affect immunocompetent individuals. Although some PCR and antigen-detection assays have been developed, conventional diagnosis has largely relied on culture, which can take weeks. Our aim was to provide a proof of principle for rationally designing and standardizing PCR assays based on Histoplasma-specific genomic sequences. Via automated comparisons of aligned genome contigs/scaffolds and gene (sub)sequences, we identified protein-coding genes that are present in existing sequences of Histoplasma strains but not in other genera. Two of the genes, PPK and CFP4, were used for designing primer sets for conventional and real-time PCR assays. Both resulted in a 100% analytical specificity in vitro and detected 62/62 H. capsulatum isolates using purified DNA. We also obtained positive detections of 2/2 confirmed H. capsulatum clinical FFPE (formalin-fixed paraffin-embedded) samples using both primer sets. Positive control plasmid 10-fold serial dilutions confirmed the analytical sensitivity of the assays. The findings suggest that these novel primer sets should allow for detection sensitivity and reduce false positive results/cross-reactions. New assays for detecting pathogenic fungi, constructed along these lines, could be simple and affordable to implement.

Histoplasmosis is a systemic fungal disease caused by the pathogen Histoplasma spp. that results in significant morbidity and mortality in persons with HIV/AIDS and can also affect immunocompetent individuals. Although some PCR and antigen-detection assays have been developed, conventional diagnosis has largely relied on culture, which can take weeks. Our aim was to provide a proof of principle for rationally designing and standardizing PCR assays based on Histoplasma-specific genomic sequences. Via automated comparisons of aligned genome contigs/scaffolds and gene (sub)sequences, we identified protein-coding genes that are present in existing sequences of Histoplasma strains but not in other genera. Two of the genes, PPK and CFP4, were used for designing primer sets for conventional and real-time PCR assays. Both resulted in a 100% analytical specificity in vitro and detected 62/62 H. capsulatum isolates using purified DNA. We also obtained positive detections of 2/2 confirmed H. capsulatum clinical FFPE (formalin-fixed paraffin-embedded) samples using both primer sets. Positive control plasmid 10-fold serial dilutions confirmed the analytical sensitivity of the assays. The findings suggest that these novel primer sets should allow for detection sensitivity and reduce false positive results/cross-reactions. New assays for detecting pathogenic fungi, constructed along these lines, could be simple and affordable to implement.

Candida auris is an emerging fungal pathogen capable of causing invasive infections in humans. Since its first appearance around 1996, it has been isolated in countries spanning five continents. C. auris is a yeast that has the potential to cause outbreaks in hospitals, can survive in adverse conditions, including dry surfaces and high temperatures, and has been frequently misidentified by traditional methods. Furthermore, strains have been identified that are resistant to two and even all three of the main classes of antifungals currently in use. Several nuclear genome assemblies of C. auris have been published representing different clades and continents, yet until recently, the mitochondrial genomes (mtDNA chromosomes) of this species and the closely related species of C. haemulonii, C. duobushaemulonii, and C. pseudohaemulonii had not been analyzed in depth. We used reads from PacBio and Illumina sequencing to obtain a de novo reference assembly of the mitochondrial genome of the C. auris clade I isolate B8441 from Pakistan. This assembly has a total size of 28.2 kb and contains 13 core protein-coding genes, 25 tRNAs and the 12S and 16S ribosomal subunits. We then performed a comparative analysis by aligning Illumina reads of 129 other isolates from South Asia, Japan, South Africa, and South America with the B8441 reference. The clades of the phylogenetic tree we obtained from the aligned mtDNA sequences were consistent with those derived from the nuclear genome. The mitochondrial genome revealed a generally low genetic variation within clades, although the South Asian clade displayed two sub-branches including strains from both Pakistan and India. In particular, the 86 isolates from Colombia and Venezuela had mtDNA sequences that were all identical at the base level, i.e., a single conserved haplotype or mitochondrial background that exhibited characteristic differences from the Pakistan reference isolate B8441, such as a unique 25-nt insert that may affect function.

Background The band 9p21.3 contains an established genomic risk zone for cardiovascular disease (CVD). Since the initial 2007 Wellcome Trust Case Control Consortium study (WTCCC), the increased CVD risk associated with 9p21.3 has been confirmed by multiple studies in different continents. However, many years later there was still no confirmed report of a corresponding association of 9p21.3 with hypertension, a major CV risk factor, nor with blood pressure (BP). Theory In this contribution, we review the bipartite haplotype structure of the 9p21.3 risk locus: one block is devoid of protein-coding genes but contains the lead CVD risk SNPs, while the other block contains the first exon and regulatory DNA of the gene for the cell cycle inhibitor p15. We consider how findings from molecular biology offer possibilities of an involvement of p15 in hypertension etiology, with expression of the p15 gene modulated by genetic variation from within the 9p21.3 risk locus. Results We present original results from a Colombian study revealing moderate but persistent association signals for BP and hypertension within the classic 9p21.3 CVD risk locus. These SNPs are mostly confined to a ‘hypertension island’ that spans less than 60 ​kb and coincides with the p15 haplotype block. We find confirmation in data originating from much larger, recent European BP studies, albeit with opposite effect directions. Conclusion Although more work will be needed to elucidate possible mechanisms, previous findings and new data prompt reconsidering the question of how variation in 9p21.3 might influence hypertension components of cardiovascular risk., Graphical abstract Schematic depiction of the main observation from association data presented or discussed in this article. Two adjacent haplotype blocks characterize the 9p21.3 cardiovascular risk locus: left, the block or island containing the first part of the p15 gene and its well-studied promoter, characterized by elevated associations (red) with blood pressure (DBP, SBP) and/or hypertension in Colombian and European studies, and right, the block containing lead cardiovascular risk-associated SNPs such as rs1333049 and rs1333050 (blue) but containing no protein-coding genes.Image 1

The mitochondrial genome of the Paracoccidioides brasiliensis reference isolate Pb18 was first sequenced and described by Cardoso et al. (2007), as a circular genome with a size of 71.3 kb and containing 14 protein coding genes, 25 tRNAs, and the large and small subunits of ribosomal RNA. Later in 2011, Desjardins et al. (2011) obtained partial assemblies of mitochondrial genomes of P. lutzii (Pb01), P. americana (Pb03), and P. brasiliensis sensu stricto (Pb18), although with a size of only 43.1 kb for Pb18. Sequencing errors or other limitations resulting from earlier technologies, and the advantages of NGS (short and long reads), prompted us to improve and update the mtDNA sequences and annotations of two Paracoccidioides species. Using Oxford Nanopore and Illumina read sequencing, we generated high-quality complete de novo mitochondrial genome assemblies and annotations for P. brasiliensis (Pb18) and P. americana (Pb03). Both assemblies were characterized by an unusually long spacer or intron region (>50 kb) between exons 2 and 3 of the nad5 gene, which was moderately conserved between Pb03 and Pb18 but not similar to other reported sequences, except for an unassigned contig in the 2011 assembly of Pb03. The reliability of the insert missing from previous mtDNA genome assemblies was confirmed by inspection of the individual Nanopore read sequences containing nad5 coding DNA, and experimentally by PCR for Pb18. We propose that the insert may aid replication initiation and may be excised to produce a smaller structural variant. The updated mtDNA genomes should enable more accurate SNP and other comparative or evolutionary analyses and primer/probe designs. A comparative analysis of the mtDNA from 32 isolates of Paracoccidioides spp., using the SNPs of the aligned mitochondrial genomes, showed groupings within the brasiliensis species complex that were largely consistent with previous findings from only five mitochondrial loci.

Dimorphic fungal pathogens cause a significant human disease burden and unlike most fungal pathogens affect immunocompetent hosts. Most dimorphic fungi are found in the family Ajellomycetaceae, including the genera Histoplasma, Blastomyces, Paracoccidioides, and the recently described Emergomyces. To examine the origin of virulence and host adaptation in these fungal pathogens, we compared the gene content of classic systemic, opportunistic, and non-pathogenic species, including new genomes for Emmonsia species and two closely non-pathogenic species, Helicocarpus griseus and Polytolypa hystricis. We examined differences in gene content between pathogens and environmental fungi, and found that gene families related to plant degradation, synthesis of secondary metabolites, and amino acid and lipid metabolism are retained in H. griseus and P. hystricis. Genes of central importance in dimorphic pathogenic fungi such as heat shock response proteins, morphological switch related proteins are conserved in non-pathogenic species. However, we found that changes in the copy number of proteases, phosphotransferases and transcription factors in systemic dimorphic relative to non-dimorphic species may have aided the evolution of specialized gene regulatory programs to rapidly adapt to higher temperatures and new nutritional environments. Notably, we found that both more basal non-pathogenic species appear homothallic, with both mating type locus idiomorphs fused at a single locus, whereas all related pathogenic species are heterothallic. These differences revealed that independent changes in nutrient acquisition capacity but similar host/substrate shift from plants to animals have occurred in the Onygenaceae and Ajellomycetaceae, and highlighted how the dimorphic pathogens from Ajellomycetaceae have adapted to the host immune response and decreased their capacity for life stages in environmental niches.

The LUFS domain (LUG/LUH, Flo8, single-strand DNA-binding protein [SSBP]) is a well-conserved and apparently ancient region found in diverse proteins and taxa. This domain, which has as its most obvious structural feature a series of three helices, has been identified in transcriptional regulator proteins of animals, plants, and fungi. Recently, in these pages (Wang et al., Protein Sci., 2019, 28:788–793), the first crystal structure of a LUFS domain was reported, for the human SSBP2, a transcriptional repressor. We briefly address how the new insights into LUFS structures might contribute to a better understanding of an important transcriptional activator of yeasts that contains the LUFS domain, Flo8, and consider how a focus on the LUFS domain and its variation could help us to understand etiologies of drug resistance in a recently emerged pathogenic fungus, Candida auris. © 2019 The Protein Society

Graphical abstractMitochondrial genomes can contain repeat landscapes ranging from notable absence of repeats, as in human and fission yeast, to rich and complex repeat systems as in baker's yeast. In this article we characterize exact repetitions of 17-mers in bona fide complete mitochondrial genome sequences of 11 fungi. This strategy allowed us to view and analyze a diversity of repeat landscapes, each with their own repeat 'fauna and flora'. The figure depicts three mitochondrial genomes that contain amply repeated 17-mers (horizontal axis: number of occurrences; vertical axis: number of instances of that number of occurrences); examples of repeats with strong presence in the mitochondrial genomes are represented by sequences in corresponding colors. Display Omitted HighlightsFungal mitochondrial genomes can have complex repeat systems.Repeats in mitochondrial genomes can mislead short-read assembly programs.Repeats can also lead to illegitimate intrachromosomal recombination and large-scale deletions.Where persistent repeats are stably present, their locations can indicate functional or structural importance. The presence of repetitive or non-unique DNA persisting over sizable regions of a eukaryotic genome can hinder the genome's successful de novo assembly from short reads: ambiguities in assigning genome locations to the non-unique subsequences can result in premature termination of contigs and thus overfragmented assemblies. Fungal mitochondrial (mtDNA) genomes are compact (typically less than 100kb), yet often contain short non-unique sequences that can be shown to impede their successful de novo assembly in silico. Such repeats can also confuse processes in the cell in vivo. A well-studied example is ectopic (out-of-register, illegitimate) recombination associated with repeat pairs, which can lead to deletion of functionally important genes that are located between the repeats. Repeats that remain conserved over micro- or macroevolutionary timescales despite such risks may indicate functionally or structurally (e.g., for replication) important regions. This principle could form the basis of a mining strategy for accelerating discovery of function in genome sequences. We present here our screening of a sample of 11 fully sequenced fungal mitochondrial genomes by observing where exact k-mer repeats occurred several times; initial analyses motivated us to focus on 17-mers occurring more than three times. Based on the diverse repeats we observe, we propose that such screening may serve as an efficient expedient for gaining a rapid but representative first insight into the repeat landscapes of sparsely characterized mitochondrial chromosomes. Our matching of the flagged repeats to previously reported regions of interest supports the idea that systems of persisting, non-trivial repeats in genomes can often highlight features meriting further attention.

Sporothrix schenckii is a thermodimorphic fungal pathogen with a high genetic diversity. In this work, we present the assembly and similarity analysis of the whole-genome sequences of two clinical isolates from Colombia of S. schenckii sensu stricto .

Background Genome-wide association studies have so far identified 56 loci associated with risk of coronary artery disease (CAD). Many CAD loci show pleiotropy; that is, they are also associated with other diseases or traits. Objectives This study sought to systematically test if genetic variants identified for non-CAD diseases/traits also associate with CAD and to undertake a comprehensive analysis of the extent of pleiotropy of all CAD loci. Methods In discovery analyses involving 42,335 CAD cases and 78,240 control subjects we tested the association of 29,383 common (minor allele frequency >5%) single nucleotide polymorphisms available on the exome array, which included a substantial proportion of known or suspected single nucleotide polymorphisms associated with common diseases or traits as of 2011. Suggestive association signals were replicated in an additional 30,533 cases and 42,530 control subjects. To evaluate pleiotropy, we tested CAD loci for association with cardiovascular risk factors (lipid traits, blood pressure phenotypes, body mass index, diabetes, and smoking behavior), as well as with other diseases/traits through interrogation of currently available genome-wide association study catalogs. Results We identified 6 new loci associated with CAD at genome-wide significance: on 2q37 (KCNJ13-GIGYF2), 6p21 (C2), 11p15 (MRVI1-CTR9), 12q13 (LRP1), 12q24 (SCARB1), and 16q13 (CETP). Risk allele frequencies ranged from 0.15 to 0.86, and odds ratio per copy of the risk allele ranged from 1.04 to 1.09. Of 62 new and known CAD loci, 24 (38.7%) showed statistical association with a traditional cardiovascular risk factor, with some showing multiple associations, and 29 (47%) showed associations at p < 1 × 10−4 with a range of other diseases/traits. Conclusions We identified 6 loci associated with CAD at genome-wide significance. Several CAD loci show substantial pleiotropy, which may help us understand the mechanisms by which these loci affect CAD risk., Central Illustration

Recent discoveries of novel systemic fungal pathogens with thermally dimorphic yeast-like phases have challenged the current taxonomy of the Ajellomycetaceae, a family currently comprising the genera Blastomyces, Emmonsia, Emmonsiellopsis, Helicocarpus, Histoplasma, Lacazia and Paracoccidioides. Our morphological, phylogenetic and phylogenomic analyses demonstrated species relationships and their specific phenotypes, clarified generic boundaries and provided the first annotated genome assemblies to support the description of two new species. A new genus, Emergomyces, accommodates Emmonsia pasteuriana as type species, and the new species Emergomyces africanus, the aetiological agent of case series of disseminated infections in South Africa. Both species produce small yeast cells that bud at a narrow base at 37°C and lack adiaspores, classically associated with the genus Emmonsia. Another novel dimorphic pathogen, producing broad-based budding cells at 37°C and occurring outside North America, proved to belong to the genus Blastomyces, and is described as Blastomyces percursus. © 2017 Blackwell Verlag GmbH

Histoplasmosis is considered one of the most important endemic and systemic mycoses worldwide. Until now few molecular techniques have been developed for its diagnosis. The aim of this study was to develop and evaluate three real time PCR (qPCR) protocols for different protein-coding genes (100-kDa, H and M antigens) using an animal model. Fresh and formalin-fixed and paraffin-embedded (FFPE) lung tissues from BALB/c mice inoculated i.n. with 2.5x106 Histoplasma capsulatum yeast or PBS were obtained at 1, 2, 3, 4, 8, 12 and 16 weeks post-infection. A collection of DNA from cultures representing different clades of H. capsulatum (30 strains) and other medically relevant pathogens (36 strains of related fungi and Mycobacterium tuberculosis) were used to analyze sensitivity and specificity. Analytical sensitivity and specificity were 100% when DNAs from the different strains were tested. The highest fungal burden occurred at first week post-infection and complete fungal clearance was observed after the third week; similar results were obtained when the presence of H. capsulatum yeast cells was demonstrated in histopathological analysis. In the first week post-infection, all fresh and FFPE lung tissues from H. capsulatum-infected animals were positive for the qPCR protocols tested except for the M antigen protocol, which gave variable results when fresh lung tissue samples were analyzed. In the second week, all qPCR protocols showed variable results for both fresh and FFPE tissues. Samples from the infected mice at the remaining times post-infection and uninfected mice (controls) were negative for all protocols. Good agreement was observed between CFUs, histopathological analysis and qPCR results for the 100-kDa and H antigen protocols. We successfully standardized and validated three qPCR assays for detecting H. capsulatum DNA in fresh and FFPE tissues, and conclude that the 100-kDa and H antigen molecular assays are promising tests for diagnosing this mycosis. © This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

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 Summary Dimorphic 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.

Gene prediction relies on the identification of characteristic features of coding sequences that distinguish them from non-coding DNA. The recent large-scale sequencing of entire genomes from higher eukaryotes, in conjunction with currently used gene prediction algorithms, has provided an abundance of putative genes that can now be analysed for their compositional properties. Strong, systematic differences still exist, in several species, between the compositional properties of sets of ex novo predicted genes and genes that have been experimentally detected and/or verified. This is particularly evident in the estimated gene set (>45,000 genes) of the recently sequenced rice genome, where roughly half the predicted genes are compositionally unusual and have no known orthologues in the dicot Arabidopsis. In a few cases such differences might suggest a bias in experimental gene-finding protocols, but the quasi-random nature of the compositionally aberrant predicted genes is a strong indication that many, if not most, of them are false positives. It therefore appears that some important features of coding regions have not yet been taken into account in existing gene prediction programs. Statistical base compositional properties of curated gene data sets from vertebrates, which we briefly review here, should therefore provide a useful benchmark for fine-tuning probabilistic gene models and model parameters that are currently in use.

Paracoccidiodomycosis (PCM) is a clinically important fungal disease that can acquire serious systemic forms and is caused by the thermodimorphic fungal Paracoccidioides spp. PCM is a tropical disease that is endemic in Latin America, where up to ten million people are infected; 80% of reported cases occur in Brazil, followed by Colombia and Venezuela. To enable genomic studies and to better characterize the pathogenesis of this dimorphic fungus, two reference strains of P. brasiliensis (Pb03, Pb18) and one strain of P. lutzii (Pb01) were sequenced [1]. While the initial draft assemblies were accurate in large scale structure and had high overall base quality, the sequences had frequent small scale defects such as poor quality stretches, unknown bases (N's), and artifactual deletions or nucleotide duplications, all of which caused larger scale errors in predicted gene structures. Since assembly consensus errors can now be addressed using next generation sequencing (NGS) in combination with recent methods allowing systematic assembly improvement, we re-sequenced the three reference strains of Paracoccidioides spp. using Illumina technology. We utilized the high sequencing depth to re-evaluate and improve the original assemblies generated from Sanger sequence reads, and obtained more complete and accurate reference assemblies. The new assemblies led to improved transcript predictions for the vast majority of genes of these reference strains, and often substantially corrected gene structures. These include several genes that are central to virulence or expressed during the pathogenic yeast stage in Paracoccidioides and other fungi, such as HSP90, RYP1-3, BAD1, catalase B, alpha-1,3-glucan synthase and the beta glucan synthase target gene FKS1. The improvement and validation of these reference sequences will now allow more accurate genome-based analyses. To our knowledge, this is one of the first reports of a fully automated and quality-assessed upgrade of a genome assembly and annotation for a non-model fungus., Author Summary The fungal genus Paracoccidioides is the causal agent of paracoccidioidomycosis (PCM), a neglected tropical disease that is endemic in several countries of South America. Paracoccidioides is a pathogenic dimorphic fungus that is capable of converting to a virulent yeast form after inhalation by the host. Therefore the molecular biology of the switch to the yeast phase is of particular interest for understanding the virulence of this and other human pathogenic fungi, and ultimately for reducing the morbidity and mortality caused by such fungal infections. We here present the strategy and methods we used to update and improve accuracy of three reference genome sequences of Paracoccidioides spp. utilizing state-of-the-art Illumina re-sequencing, assembly improvement, re-annotation, and quality assessment. The resulting improved genome resource should be of wide use not solely for advancing research on the genetics and molecular biology of Paracoccidioides and the closely related pathogenic species Histoplasma and Blastomyces, but also for fungal diagnostics based on sequencing or molecular assays, characterizing rapidly changing proteins that may be involved in virulence, SNP-based population analyses and other tasks that require high sequence accuracy. The genome update and underlying strategy and methods also serve as a proof of principle that could encourage similar improvements of other draft genomes.

Graphical abstractThe success of a short-read based genome assembly process in faithfully reproducing the sequences of a real genome, or its genes, can be modulated by some or all of three key parameters: read length r, insert size I, and a bioinformatics parameter, the word length k (k-mer length), which is used in most modern assembly tools based on de Bruijn graphs. The present study focuses on how plots of simple assembly success metrics, and their variation as a function of k, can serve as succinct graphical representations of how the assembly process deals with a given genomic context. Display Omitted HighlightsThree key parameters of a typical short read sequencing and de novo assembly design are the read length, the insert size and a bioinformatic parameter, the k-mer length (k); k is easy to modify, and one can monitor the effects of its variation before choosing a final k-mer length or assembly.The success and the complexity of a de novo genome assembly task depend on one's needs or priorities: gene space, all the unique DNA, or entire genome with all its repeats.When one restricts a fungal de novo assembly task to a mapping of the gene space by reliable sequences, k often plays only a very minor role, but a correct choice of k can be far from trivial if one needs to map the genome's repetitive regions.Plotting metrics that assess presence of DNA and its genes and repeats in a de novo assembly versus k-mer length used can give valuable insight into an assembly process and its variation. 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 150bp allowed us to attempt de novo assemblies with a larger range of k-mers, up to 127bp. 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.

Planning of pipelines for next-generation sequencing (NGS) projects could be facilitated by using simple DNA sequence benchmarks, i.e., standard test sequences that could monitor or help to predict ease or difficulty of (a) short-read sequencing and (b) de novo assembly of the sequenced reads. We propose that familiar, gene-sized sequences, including but not limited to nuclear protein-coding genes, would provide feasible consensus benchmarks allowing simple visualization. We illustrate our proposal for fungi with candidates from ribosomal DNA (rDNA, used in phylogeny and identification/diagnostics), mitochondrial DNA (mtDNA), and combinatorially constructed conceptual (synthetic) DNA sequences. The exploratory analysis of such familiar candidate loci could be a step toward finding, testing and establishing familiar, biologically interpretable consensus benchmark sequences for fungal and other eukaryotic genomes.

In an article published in these pages, Elhaik et al. (Elhaik E, Landan G, Graur D. 2009. Can GC content at third-codon positions be used as a proxy for isochore composition? Mol Biol Evol. 26:1829-1833) asked if GC3, the GC level of the third-codon positions in protein-coding genes, can be used as a 'proxy' to estimate the GC level of the surrounding isochore. We use available data to directly answer this simple question in the affirmative and show how the use of indirect methods can lead to apparently conflicting conclusions. The answer reasserts that in human and other vertebrates, genes have a strong tendency to reside in compositionally corresponding isochores, which has far-reaching implications for genome structure and evolution. © 2010 The Author.

Well before genome sequencing, ultracentrifugation in Cs2SO4 density gradients in the presence of sequence-specific ligands (e.g., Ag+) was shown to lead to a high resolution of mammalian DNAs according to base composition (Corneo et al. 1968). These findings opened a new inroad in the study of the organization of eukaryotic genomes, superseding DNA reassociation kinetics (Britten and Kohne 1968), which was based on the separation of single- and double-stranded DNA on hydroxyapatite (Bernardi 1965). The new density gradient approach showed that, neglecting satellite DNAs, the genomes of warm-blooded vertebrates were characterized by a striking long-range compositional heterogeneity (Filipski et al. 1973; Macaya et al. 1976; Thiery et al. 1976). Indeed, these genomes are mosaics of isochores, long (≫300 kb), compositionally fairly homogeneous regions that belong to a small number of families characterized by different average GC levels (Macaya et al. 1976), and are associated with basic biological properties (for reviews, see Bernardi et al. 1985; Bernardi 1995, 2004). A quarter of a century after the original studies that had defined the approximate sizes and compositions of isochores as well as the compositions and relative amounts of isochore families, it was reported that isochores could not be identified in the draft sequence of the human genome (Lander et al. 2001), starting a debate that is still ongoing. The different computational approaches used to disprove or redefine isochores (Eyre-Walker and Hurst 2001; Haring and Kypr 2001; Lander et al. 2001; Nekrutenko and Li 2001; Cohen et al. 2005) were, however, shown to be inadequate (Bernardi 2001; Clay and Bernardi 2001a, b, 2005; Li 2002; Oliver et al. 2002, 2004; Li et al. 2003; Melodelima et al. 2005), even if some of them led to a partial identification of isochores. This debate prompted us to map the isochores, as originally defined (Macaya et al. 1976), in the finished sequence of the human genome (International Human Genome Sequencing Consortium 2004). Average GC levels were, therefore, assessed over long DNA stretches (>200 kb), while GC variation was estimated by measuring standard deviations of GC over such stretches using a 100-kb moving window. The findings reported here are in agreement with previous results obtained by equilibrium sedimentation and confirm the existence of five isochore families (see below), but they go much farther in that they directly identify and map isochores on chromosomes, thus leading to a resolution of >3000 chromosomal bands. Almost 50 years ago, calf thymus DNA, the standard eukaryotic DNA, was shown to be remarkably more heterogeneous in base composition than bacterial DNAs (Meselson et al. 1957). In fact, the very strong heterogeneity was largely, but not entirely, due to the presence of GC-rich satellites that represent 23% of the bovine genome (see Bernardi 2004). Interestingly, high-resolution ultracentrifugation of bovine DNA was not only able to separate the GC-rich satellites, but also showed a discontinuous compositional heterogeneity of the main band (Filipski et al. 1973), consisting of three families of DNA molecules. This was in contrast with the then predominant view (still defended by some authors) (Galtier et al. 2002) of a continuous heterogeneity of main-band DNA. The families of DNA molecules were then shown to be present in the other mammalian genomes explored and to derive from longer, fairly homogeneous DNA stretches (Macaya et al. 1976; Thiery et al. 1976) that were called isochores (Cuny et al. 1981) for (compositionally) equal landscapes. Later work resolved the first family into two families, L1 and L2, named the second and the third families H1 and H2, respectively, and identified another quantitatively small family, H3 (Zerial et al. 1986). Most mammalian genomes, including the human genome, are made up of five families of isochores—L1, L2, H1, H2, and H3—in order of increasing GC levels, if satellite DNAs (∼2% of the genome) and ribosomal DNAs (∼0.5% of the genome) are neglected. Incidentally, satellite and ribosomal DNAs may also be considered isochores, because of their compositional homogeneity (Bernardi 1995). Isochores were shown to be tightly linked to basic biological properties, such as gene density, replication timing, and recombination (see Bernardi 2004), and have, therefore, been considered “a fundamental level of genome organization” (Eyre-Walker and Hurst 2001). In order to characterize isochores, the present work did not take into account their biological properties (as done by Melodelima et al. 2005) but only relied on two parameters, GC levels and their standard deviations.

The existence of a well conserved linear relationship between GC levels of genes' second and third codon positions (GC2, GC3) prompted us to focus on the landscape, or joint distribution, spanned by these two variables. In human, well curated coding sequences now cover at least 15%–30% of the estimated total gene set. Our analysis of the landscape defined by this gene set revealed not only the well documented linear crest, but also the presence of several peaks and valleys along that crest, a property that was also indicated in two other warm-blooded vertebrates represented by large gene databases, that is, mouse and chicken. GC2 is the sum of eight amino acid frequencies, whereas GC3 is linearly related to the GC level of the chromosomal region containing the gene. The landscapes therefore portray relations between proteins and the DNA environments of the genes that encode them.

This review briefly describes the compositional approach to the animals of vertebrate genomes. This approach involves the study of distributions of, and correlations among, the base compositions (GC levels) of different parts of these genomes, such as exons, introns, third codon positions, flanking of genes, and long genomic sequences or fragments spanning genic and intergenic DNA. Properties of the human genome that were inferred using the compositional approach include its organization into isochores, the presence of much higher gene densities in GC rich than in GC poor regions, and the non-uniform concentration of genes in the chromosomal bands.

Linear correlations exist between the GC levels of third codon positions (GC3) of individual human genes and the GC levels of long genomic sequences and DNA molecules (50-100 kb in size) embedding the genes. These linear relationships allow the positioning of the GC3 histogram of cDNA sequences from the databases relative to the CsCl profile of human DNA. In turn, this allows an estimate of the relative concentrations of genes in genomic regions of different GC content. An estimate obtained by using current sequence data and Gaussian decompositions of the GC3 histogram and of the CsCl profile indicates that the GC-richest (non-ribosomal) component of the human genome is at least 17 times as gene-rich as the GC-poor regions. Moreover, our results suggest that the most recent physical maps of the human genome consisting of overlapping YACs cover less than 50% of the genes.

As the correlations between GC levels in third codon positions (GC3) and intergenic sequence GC levels can be used to assess the distribution of genes in the human genome, they were studied in detail. Previous work from our laboratory has demonstrated the existence of linear correlations between GC levels of exons, introns, third codon positions, 5' flanking regions of genes, and long genomic DNA sequences (> or = 10 kb) or DNA molecules (50-100 kb) in which the genes are embedded. The present study confirms and extends the previous results using a larger set of data. Furthermore, an analysis of 4270 human genomic DNA and cDNA sequences has allowed us to confirm a correlation of GC3 against GC1+2. Recent additions to the sequence database have also allowed separate analyses of the 5' flanking regions of CpG island and non-CpG island genes as well as analyses of 3' flanking regions, which suggest that the GC levels of 3' flanking regions are closer to those of intergenic DNA than are those of other regions of genes.

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.