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Amphibian declines are a threat to global biodiversity and part of Earth’s sixth mass extinction. Amphibian extinctions and extirpations are caused by a variety of factors, including the Chytridiomycosis panzootic and its causative agents: the batrachochytridsBatrachochytrium salamandrivorans(Bsal)andBatrachochytrium dendrobatidis(Bd).Until recently, the genomic basis for the batrachochytrids’ evolution and their virulence was largely unknown. To investigate their virulence and evolution, high-quality genome assemblies were needed. Prior to 2022, only a highly fragmented short-read assembly ofBsal’s genome assembly had been generated and made available, largely owing to its repeat-richness. In 2022, a new assembly based on deep nanopore long-read sequencing provided a much-needed improvement in both contiguity and completeness. The new assembly revealed thatBsalhas undergone a repeat-driven genome expansion and has a highly compartmentalized genome architecture. Here, the road map to assembling and polishing a highly repeat-rich chytrid fungus genome using both long and short reads is described, detailing the methodology for each step, and explaining the rationales underpinning those steps. Considerations for tailoring this methodology to other similar genome assemblies are illustrated. Additionally, we describe how a high-quality assembly can be analysed to provide insight into the genomic architecture and the underlying evolutionary processes.&nbsp

Candida glabrata is the second most common etiological cause of worldwide systemic candidiasis in adult patients. Genome analysis of 68 isolates from 8 hospitals across Scotland, together with 83 global isolates, revealed insights into the population genetics and evolution of C. glabrata. Clinical isolates of C. glabrata from across Scotland are highly-genetically diverse, including at least 19 separate sequence types (STs) that have been recovered previously in globally diverse locations, and one newly discovered ST. Several STs had evidence for ancestral recombination, suggesting transmission between distinct geographical regions has coincided with genetic exchange arising in new clades. Three isolates were missing MATα1, potentially representing a second mating type. Signatures of positive selection were identified in every ST including enrichment for Epithelial Adhesins (EPA) thought to facilitate fungal adhesion to human epithelial cells. In patent microevolution was identified from seven sets of recurrent cases of candidiasis, revealing an enrichment for non-synonymous and frameshift indels in cell surface proteins. Microevolution within patients also affected EPA genes, and several genes involved in drug resistance including the ergosterol synthesis gene ERG4 and the echinocandin target FKS1/2, the latter coinciding with a marked drop in fluconazole MIC. In addition to nuclear genome diversity, the C. glabrata mitochondrial genome was particularly diverse, with reduced conserved sequence and conserved protein encoding genes in all non-reference ST15 isolates. Together, this study highlights the genetic diversity within the C. glabrata population that may impact virulence and drug resistance, and two major mechanisms generating this diversity: microevolution and genetic exchange/recombination.

Over the past half century, the chytridiomycosis panzootic has led to the decline of over 500 amphibian species with 90 attributed extinctions. Chytridiomycosis of amphibians is caused by two fungal species Batrachochytrium dendrobatidis (Bd) and Batrachochytrium salamandrivorans (Bsal). The genetic mechanisms underlying host-specificity and pathology in the Batrachochytrium genus remain elusive and their evolution and origins of virulence are largely unknown. Using deep nanopore sequencing, we found that Bsal is extremely repeat-rich with high numbers of long terminal repeats, long interspersed nuclear elements and transposable elements. This repeat-driven genome expansion in Bsal has resulted in a tripling of its length compared with Bd. Key pathogenicity genes including M36 metalloproteases have expanded compared with Bd, and are enriched for flanking transposable elements, suggesting its genome expansion is connected to selective evolutionary processes. Both batrachochytrids have evidence of a two speed genome architecture, including an enrichment of functional categories in compartments of repeat richness or sparsity. Furthermore, among Bd lineages, M36 metalloproteases with signatures of positive selection and, both in Bsal and Bd, genes upregulated during infection in vivo are enriched in repeat-rich and gene-sparse compartment of the genome. This is the first evidence for a two-speed genome in an animal pathogen, shedding new light on the role of repetitive sequences on the evolution of fungal pathogens driving global declines and extinctions of amphibians., {"references":["Scheele, B. C.et al., Science 363, 1459-1463 (2019)","Carey, C.et al., EcoHealth 3, 5-21 (2006)","Stegen, G.et al., Nature 544, 353-356 (2017)","Martel, A.et al., Proc. Natl. Acad. Sci. 110, 15325-15329 (2013)","Farrer, R. A. , Trends Microbiol. 27, 892-893 (2019)","Sabino-Pinto, J. et al. , AMRE 36, 411-416 (2015)","Wang, Y. et al. , New Phytol. 220, 922-935 (2018)","Zhang, S.-J. et al., Genomics Proteomics Bioinformatics 18, 321-332 (2020)","Wos, G. et al., Mob. DNA 12, 7 (2021)","Klein, S. J. & O'Neill, R. J. , Chromosome Res. 26, 5-23 (2018)","Oliver, K. R. & Greene, W. K. , BioEssays 31, 703-714 (2009)"]}

Background Identifying haplotypes is central to sequence analysis in diploid or polyploid genomes. Despite this, there remains a lack of research and tools designed for physical phasing and its downstream analysis. Results HaplotypeTools is a new toolset to phase variant sites using VCF and BAM files and to analyse phased VCFs. Phasing is achieved via the identification of reads overlapping ≥ 2 heterozygous positions and then extended by additional reads, a process that can be parallelized across a computer cluster. HaplotypeTools includes various utility scripts for downstream analysis including crossover detection and phylogenetic placement of haplotypes to other lineages or species. HaplotypeTools was assessed for accuracy against WhatsHap using simulated short and long reads, demonstrating higher accuracy, albeit with reduced haplotype length. HaplotypeTools was also tested on real Illumina data to determine the ancestry of hybrid fungal isolate Batrachochytrium dendrobatidis (Bd) SA-EC3, finding 80% of haplotypes across the genome phylogenetically cluster with parental lineages BdGPL (39%) and BdCAPE (41%), indicating those are the parental lineages. Finally, ~ 99% of phasing was conserved between overlapping phase groups between SA-EC3 and either parental lineage, indicating mitotic gene conversion/parasexuality as the mechanism of recombination for this hybrid isolate. HaplotypeTools is open source and freely available from https://github.com/rhysf/HaplotypeTools under the MIT License. Conclusions HaplotypeTools is a powerful resource for analyzing hybrid or recombinant diploid or polyploid genomes and identifying parental ancestry for sub-genomic regions.

Additional file 2: Fig. S1. Core genome phylogeny for ST239 and ST59. Fig. S2. Regression of root-to-tip distance against sampling time for core genome alignments. Fig. S3. Global core genome phylogenies for ST239 and ST59. Fig. S4. Shared core and accessory genes amongst Chinese ST239 and ST59 isolates. Fig. S5. Conservation of accessory genome homology groups (HGs) across the Chinese ST239 and ST59 isolates. Fig. S6. Presence/absence of mobile genetic elements (MGEs) amongst Chinese ST239 and ST59 isolates. Fig. S7. Presence/absence of antimicrobial resistance genes amongst Chinese ST239 and ST59 isolates. Fig. S8. Presence/absence of virulence genes amongst Chinese ST239 and ST59 isolates. Fig. S9. Number of resistance (AMR) genes carried between ST sub-lineages. Fig. S10. Changing trends of antimicrobial resistance (AMR) mutations/elements within ST239 and ST59 lineages over time. Fig. S11. Number of virulence genes carried between ST sub-lineages and within ST239 and ST59 sub-lineages. Fig. S12. Trends of virulence mutations/elements within Chinese ST239 and ST59 lineages over time. Fig. S13. Core genome phylogeny for ST239 annotated for sasX. Fig. S14. Methicillin resistance acquisition analyses. Fig. S15. Presence absence of virulence genes against cell lysis phenotype. Fig. S16. Colony counts at 0 hours and 96 hours for three ST59 isolates (wild-type and knock-out) compared to two possible ST239 control strains. Table S2. Inferred tMRCA, clock rates and marginal likelihoods inferred by Bayesian dating analyses. Table S3. The distribution of mobile genetic elements (MGEs) in different sub-lineages. Table S4. Bonferroni corrected p-values following fitting of a generalized linear model (GLM) for VFDB virulence gene presence / absence against cell lysis phenotype. Table S5. Relative adaptive difference (S), fitness (F) and fitness cost (C) estimated from the in vitro competition experiment between ST59-WT and ST59 ��chp.

Background Ortholog prediction and synteny visualization across whole genomes are valuable methods for detecting and representing a range of evolutionary processes such as genome expansion, chromosomal rearrangement, and chromosomal translocation. Few standalone methods are currently available to visualize synteny across any number of annotated genomes. Results Here, I present a Synteny Imaging tool (Synima) written in Perl, which uses the graphical features of R. Synima takes orthologues computed from reciprocal best BLAST hits or OrthoMCL, and DAGchainer, and outputs an overview of genome-wide synteny in PDF. Each of these programs are included with the Synima package, and a pipeline for their use. Synima has a range of graphical parameters including size, colours, order, and labels, which are specified in a config file generated by the first run of Synima – and can be subsequently edited. Synima runs quickly on a command line to generate informative and publication quality figures. Synima is open source and freely available from https://github.com/rhysf/Synima under the MIT License. Conclusions Synima should be a valuable tool for visualizing synteny between two or more annotated genome assemblies.

The pathogenic species of Cryptococcus are a major cause of mortality owing to severe infections in immunocompromised as well as immunocompetent individuals. Although antifungal treatment is usually effective, many patients relapse after treatment, and in such cases, comparative analyses of the genomes of incident and relapse isolates may reveal evidence of determinative, microevolutionary changes within the host. Here, we analyzed serial isolates cultured from cerebrospinal fluid specimens of 18 South African patients with recurrent cryptococcal meningitis. The time between collection of the incident isolates and collection of the relapse isolates ranged from 124 days to 290 days, and the analyses revealed that, during this period within the patients, the isolates underwent several genetic and phenotypic changes. Considering the vast genetic diversity of cryptococcal isolates in sub-Saharan Africa, it was not surprising to find that the relapse isolates had acquired different genetic and correlative phenotypic changes. They exhibited various mechanisms for enhancing virulence, such as growth at 39°C, adaptation to stress, and capsule production; a remarkable amplification of ERG11 at the native and unlinked locus may provide stable resistance to fluconazole. Our data provide a deeper understanding of the microevolution of Cryptococcus species under pressure from antifungal chemotherapy and host immune responses. This investigation clearly suggests a promising strategy to identify novel targets for improved diagnosis, therapy, and prognosis. IMPORTANCE Opportunistic infections caused by species of the pathogenic yeast Cryptococcus lead to chronic meningoencephalitis and continue to ravage thousands of patients with HIV/AIDS. Despite receiving antifungal treatment, over 10% of patients develop recurrent disease. In this study, we collected isolates of Cryptococcus from cerebrospinal fluid specimens of 18 patients at the time of their diagnosis and when they relapsed several months later. We then sequenced and compared the genomic DNAs of each pair of initial and relapse isolates. We also tested the isolates for several key properties related to cryptococcal virulence as well as for their susceptibility to the antifungal drug fluconazole. These analyses revealed that the relapsing isolates manifested multiple genetic and chromosomal changes that affected a variety of genes implicated in the pathogenicity of Cryptococcus or resistance to fluconazole. This application of comparative genomics to serial clinical isolates provides a blueprint for identifying the mechanisms whereby pathogenic microbes adapt within patients to prolong disease., National Institute of Allergy and Infectious Diseases (U.S.) (U19 AI110818)

BackgroundCandida auris was first described in 2009, and has since caused nosocomial outbreaks, invasive infections and fungaemia across 11 countries in five continents. An outbreak of C. auris occurred in a specialised cardiothoracic London hospital between April 2015 and November 2016, which to date has been the largest outbreak reported worldwide, involving a total of 72 patients.MethodsTo understand the epidemiology of C. auris infection within this hospital, we sequenced the genomes of outbreak isolates using Oxford Nanopore Technologies and Illumina in order to type antifungal resistance alleles and to explore the outbreak within its local and global context.FindingsPhylogenomic analysis placed the UK outbreak in the India/Pakistan clade, demonstrating an Asian origin. The outbreak showed similar diversity to that of the entire clade and limited local spatiotemporal clustering was observed. One isolate displayed resistance to both echinocandins and 5-flucytosine; the former was associated with a serine to tyrosine amino acid substitution in the gene FKS1, and the latter was associated with a phenylalanine to isoleucine substitution in the gene FUR1. These mutations are novel for this pathogen.InterpretationMultiple differential episodic selection of antifungal resistant genotypes has occurred within a genetically heterogenous population across this outbreak, creating a resilient pathogen and making it difficult to define local-scale patterns of transmission as well as implementing outbreak control measures.FundingAntimicrobial Research Collaborative, Imperial College London