HaplotypeTools: a toolkit for accurately identifying recombination and recombinant genotypes.

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.

Microevolution of Serial Clinical Isolates of Cryptococcus neoformans var. grubii and C. gattii.

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.

Zinc Cluster Transcription Factors Alter Virulence in Candida albicans.

Almost all humans are colonized with Candida albicans However, in immunocompromised individuals, this benign commensal organism becomes a serious, life-threatening pathogen. Here, we describe and analyze the regulatory networks that modulate innate responses in the host niches. We identified Zcf15 and Zcf29, two Zinc Cluster transcription Factors (ZCF) that are required for C. albicans virulence. Previous sequence analysis of clinical C. albicans isolates from immunocompromised patients indicates that both ZCF genes diverged during clonal evolution. Using in vivo animal models, ex vivo cell culture methods, and in vitro sensitivity assays, we demonstrate that knockout mutants of both ZCF15 and ZCF29 are hypersensitive to reactive oxygen species (ROS), suggesting they help neutralize the host-derived ROS produced by phagocytes, as well as establish a sustained infection in vivo Transcriptomic analysis of mutants under resting conditions where cells were not experiencing oxidative stress revealed a large network that control macro and micronutrient homeostasis, which likely contributes to overall pathogen fitness in host niches. Under oxidative stress, both transcription factors regulate a separate set of genes involved in detoxification of ROS and down-regulating ribosome biogenesis. ChIP-seq analysis, which reveals vastly different binding partners for each transcription factor (TF) before and after oxidative stress, further confirms these results. Furthermore, the absence of a dominant binding motif likely facilitates their mobility, and supports the notion that they represent a recent expansion of the ZCF family in the pathogenic Candida species. Our analyses provide a framework for understanding new aspects of the interface between C. albicans and host defense response, and extends our understanding of how complex cell behaviors are linked to the evolution of TFs.