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Aspergillus fumigatus, an environmental mold, causes life-threatening infections. Studies on the phylogenetic structure of human clinical A. fumigatus isolates are limited. Here, we performed whole genome sequencing of 24 A. fumigatus isolates collected from 18 patients in U.S. healthcare facilities in California. Single-nucleotide polymorphism (SNP) differences between patient isolates ranged from 187 to 70 829 SNPs. For five patients with multiple isolates, we calculated the within-host diversities. Three patients had a within-host diversity that ranged from 4 to 10 SNPs and two patients ranged from 2 to 16 977 SNPs. Findings revealed highly diverse A. fumigatus strains among patients and two patterns of diversity for isolates that come from the same patient, low and extremely high diversity.

Candida auris, an emerging multi-drug resistant organism (MDRO), is an urgent public health threat. We report on a C. auris outbreak investigation at a Virginia ventilator skilled nursing facility (vSNF). During October 2020-June 2021, we identified 28 cases among residents in the ventilator unit. Genomic evidence suggested ≥2 distinct C. auris introductions to the facility. We identified multiple infection and prevention control challenges, highlighting the importance of strengthening MDRO prevention efforts at vSNFs.

Candida auris is an urgent public health threat characterized by high drug-resistant rates and rapid spread in healthcare settings worldwide. As part of the C. auris response, molecular surveillance has helped public health officials track the global spread and investigate local outbreaks. Here, we describe whole-genome sequencing analysis methods used for routine C. auris molecular surveillance in the United States; methods include reference selection, reference preparation, quality assessment and control of sequencing reads, read alignment, and single-nucleotide polymorphism calling and filtration. We also describe the newly developed pipeline MycoSNP, a portable workflow for performing whole-genome sequencing analysis of fungal organisms including C. auris.

Candida auris is a multidrug-resistant pathogen that represents a serious public health threat due to its rapid global emergence, increasing incidence of healthcare-associated outbreaks, and high rates of antifungal resistance. Whole-genome sequencing and genomic surveillance have the potential to bolster C. auris surveillance networks moving forward. Laboratories conducting genomic surveillance need to be able to compare analyses from various national and international surveillance partners to ensure that results are mutually trusted and understood. Therefore, we established an empirical outbreak benchmark dataset consisting of 23 C. auris genomes to help validate comparisons of genomic analyses and facilitate communication among surveillance networks. Our outbreak benchmark dataset represents a polyclonal phylogeny with three subclades. The genomes in this dataset are from well-vetted studies that are supported by multiple lines of evidence, which demonstrate that the whole-genome sequencing data, phylogenetic tree, and epidemiological data are all in agreement. This C. auris benchmark set allows for standardized comparisons of phylogenomic pipelines, ultimately promoting effective C. auris collaborations.

Background: Spiders are among the most venomous animals in nature. Their venom constitutes a source of novel and innovative peptides and proteins with medicinal and biotechnological interest. However, their potential as antimicrobial, anti-cancerous, anti-hypertensive and even in the modulation of nociception is under-studied, mainly because handling the venom is technically challenging and there is paucity of next-generation-sequencing (NGS) data. Due to the increasing evidence of underestimation of the number of genes by the use of a single transcriptome assembler, we re-assembled and optimized the de novo transcriptome of the venom gland of the recently described Colombian spider P. verdolaga, by using three free access algorithms: Trinity, Soapdenovo and SPAdes. All the assemblies were evaluated by statistical parameters (e.g. contigs, GC%, max and min length and N50), by applying BUSCO´s terms retrieval against the arthropod data set to determine the best assembly for each software.Results: Our analyses show that while approximately 54% of all the assembled and structurally annotated sequences could be found in all three algorithms, around 23% of these were unique for Trinity and 21% were unique for SPAdes. The non-redundant merge of all three assemblies’ output permitted the annotation of 8640 sequences; at least 15% more when compared to each software separately, and an increase of 20% when compared to a previous P. verdolaga assembly. Analysis of the annotated genes allowed the identification of unreported lectins, kinins and over 200 peptides and proteins with potential antimicrobial and protease inhibition activities. Furthermore, homology search against the Arachnoserver database and the EROP knowledgebase allowed the identification of 135 novel theraphotoxins of biotechnological interest.Conclusion: Transcriptomic data is of utmost importance for spiders, as it is one of the more feasible and scalable ways to characterize these organisms. However, the use of a single de novo assembler implies an under representation of the expressed sequences, as it has been shown here. In the generation of data for non-model organisms as well as in the search for novel peptides and proteins with biotechnological interest, it is highly recommended that at least two different assemblers are employed.

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 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.

Background Candida auris is a multidrug-resistant yeast associated with hospital outbreaks worldwide. During 2015-2016, multiple outbreaks were reported in Colombia. We aimed to understand the extent of contamination in healthcare settings and to characterize the molecular epidemiology of C. auris in Colombia. Methods We sampled patients, patient contacts, healthcare workers, and the environment in 4 hospitals with recent C. auris outbreaks. Using standardized protocols, people were swabbed at different body sites. Patient and procedure rooms were sectioned into 4 zones and surfaces were swabbed. We performed whole-genome sequencing (WGS) and antifungal susceptibility testing (AFST) on all isolates. Results Seven of the 17 (41%) people swabbed were found to be colonized. Candida auris was isolated from 37 of 322 (11%) environmental samples. These were collected from a variety of items in all 4 zones. WGS and AFST revealed that although isolates were similar throughout the country, isolates from the northern region were genetically distinct and more resistant to amphotericin B (AmB) than the isolates from central Colombia. Four novel nonsynonymous mutations were found to be significantly associated with AmB resistance. Conclusions Our results show that extensive C. auris contamination can occur and highlight the importance of adherence to appropriate infection control practices and disinfection strategies. Observed genetic diversity supports healthcare transmission and a recent expansion of C. auris within Colombia with divergent AmB susceptibility.

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

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.

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 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.