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The protozoan Trypanosoma cruzi almost invariably establishes life-long infections in humans and other mammals, despite the development of potent host immune responses that constrain parasite numbers. The consistent, decades-long persistence of T . cruzi in human hosts arises at least in part from the remarkable level of genetic diversity in multiple families of genes encoding the primary target antigens of anti-parasite immune responses. However, the highly repetitive nature of the genome–largely a result of these same extensive families of genes–have prevented a full understanding of the extent of gene diversity and its maintenance in T . cruzi . In this study, we have combined long-read sequencing and proximity ligation mapping to generate very high-quality assemblies of two T . cruzi strains representing the apparent ancestral lineages of the species. These assemblies reveal not only the full repertoire of the members of large gene families in the two strains, demonstrating extreme diversity within and between isolates, but also provide evidence of the processes that generate and maintain that diversity, including extensive gene amplification, dispersion of copies throughout the genome and diversification via recombination and in situ mutations. Gene amplification events also yield significant copy number variations in a substantial number of genes presumably not required for or involved in immune evasion, thus forming a second level of strain-dependent variation in this species. The extreme genome flexibility evident in T . cruzi also appears to create unique challenges with respect to preserving core genome functions and gene expression that sets this species apart from related kinetoplastids.

Parasitic diseases caused by kinetoplastid parasites are a burden to public health throughout tropical and subtropical regions of the world. TriTrypDB ( https://tritrypdb.org ) is a free online resource for data mining of genomic and functional data from these kinetoplastid parasites and is part of the VEuPathDB Bioinformatics Resource Center ( https://veupathdb.org ). As of release 59, TriTrypDB hosts 83 kinetoplastid genomes, nine of which, including Trypanosoma brucei brucei TREU927, Trypanosoma cruzi CL Brener and Leishmania major Friedlin, undergo manual curation by integrating information from scientific publications, high-throughput assays and user submitted comments. TriTrypDB also integrates transcriptomic, proteomic, epigenomic, population-level and isolate data, functional information from genome-wide RNAi knock-down and fluorescent tagging, and results from automated bioinformatics analysis pipelines. TriTrypDB offers a user-friendly web interface embedded with a genome browser, search strategy system and bioinformatics tools to support custom in silico experiments that leverage integrated data. A Galaxy workspace enables users to analyze their private data (e.g., RNA-sequencing, variant calling, etc.) and explore their results privately in the context of publicly available information in the database. The recent addition of an annotation platform based on Apollo enables users to provide both functional and structural changes that will appear as ‘community annotations’ immediately and, pending curatorial review, will be integrated into the official genome annotation.

Advances in genomics have made whole genome studies increasingly feasible across the life sciences. However, new technologies and algorithmic advances do not guarantee flawless genomic sequences or annotation. Bias, errors, and artifacts can enter at any stage of the process from library preparation to annotation. When planning an experiment that utilizes a genome sequence as the basis for the design, there are a few basic checks that, if performed, may better inform the experimental design and ideally help avoid a failed experiment or inconclusive result.

Toxoplasma gondii is among the most prevalent parasites worldwide, infecting many wild and domestic animals and causing zoonotic infections in humans. T. gondii differs substantially in its broad distribution from closely related parasites that typically have narrow, specialized host ranges. To elucidate the genetic basis for these differences, we compared the genomes of 62 globally distributed T. gondii isolates to several closely related coccidian parasites. Our findings reveal that tandem amplification and diversification of secretory pathogenesis determinants is the primary feature that distinguishes the closely related genomes of these biologically diverse parasites. We further show that the unusual population structure of T. gondii is characterized by clade-specific inheritance of large conserved haploblocks that are significantly enriched in tandemly clustered secretory pathogenesis determinants. The shared inheritance of these conserved haploblocks, which show a different ancestry than the genome as a whole, may thus influence transmission, host range and pathogenicity. Toxoplasma gondii is a parasite that causes zoonotic infections in humans. Here, the authors identify tandem amplification and diversification of secretory pathogenesis determinants in the T. gondii genome and show that clade-specific inheritance of conserved haploblocks enriched for these determinants shapes population structure.

Recent advances in the in vitro cultivation of Cryptosporidium parvum using hollow fiber bioreactor technology (HFB) have permitted continuous growth of parasites that complete all life cycle stages. The method provides access to all stages of the parasite and provides a method for non-animal production of oocysts for use in clinical trials. Here we examined the effect of long-term (>20 months) in vitro culture on virulence-factors, genome conservation, and in vivo pathogenicity of the host by in vitro cultured parasites. We find low-level sequence variation that is consistent with that observed in calf-passaged parasites. Further using a calf model infection, oocysts obtained from the HFB caused diarrhea of the same volume, duration and oocyst shedding intensity as in vivo passaged parasites.

FungiDB (fungidb.org) is a free online resource for data mining and functional genomics analysis for fungal and oomycete species. FungiDB is part of the Eukaryotic Pathogen Genomics Database Resource (EuPathDB, eupathdb.org) platform that integrates genomic, transcriptomic, proteomic, and phenotypic datasets, and other types of data for pathogenic and nonpathogenic, free-living and parasitic organisms. FungiDB is one of the largest EuPathDB databases containing nearly 100 genomes obtained from GenBank, Aspergillus Genome Database (AspGD), The Broad Institute, Joint Genome Institute (JGI), Ensembl, and other sources. FungiDB offers a user-friendly web interface with embedded bioinformatics tools that support custom in silico experiments that leverage FungiDB-integrated data. In addition, a Galaxy-based workspace enables users to generate custom pipelines for large-scale data analysis (e.g., RNA-Seq, variant calling, etc.). This review provides an introduction to the FungiDB resources and focuses on available features, tools, and queries and how they can be used to mine data across a diverse range of integrated FungiDB datasets and records.

Cryptosporidium is a eukaryotic parasite and a leading global cause of waterborne diarrhea, with over 40 recognized species infecting livestock, wildlife, and people. While we have effective tools for detecting Cryptosporidium in clinical and agricultural water samples, there is still a need for a method that can efficiently identify known species as well as infections with multiple Cryptosporidium species, which are increasingly being reported. In this study, we utilized sequencing of a specific region to develop a sensitive and accurate identification workflow for Cryptosporidium species based on high-throughput sequencing. This method can distinguish between all 40 recognized species and accurately detect mixed infections. Our approach provides a sensitive and reliable means to identify Cryptosporidium species in complex clinical and agricultural samples. This has important implications for clinical diagnostics, biosurveillance, and understanding disease transmission, ultimately benefiting clinicians and produce growers.

Cryptosporidium parvum is a significant pathogen causing gastrointestinal infections in humans and animals. It is spread through ingesting contaminated food and water. Despite its global health significance, generating a C. parvum genome sequence has been challenging for many reasons including cloning and challenging subtelomeric regions. A new, gapless, hybrid, telomere-to-telomere genome assembly was created for C. parvum IOWA II, here termed Cp BGF. It reveals 8 chromosomes, a genome size of 9,259,183 bp, and resolves complex subtelomeric regions. To facilitate ease of use and consistency with the literature, the chromosomes have been oriented, and genes in this annotation have been given similar gene IDs as those used in the 2004, C. parvum IOWA II reference genome sequence. The new annotation utilized considerable RNA expression evidence including single-molecule Iso-Seq data; thus, untranslated regions, long noncoding RNAs, and antisense RNAs are annotated. The Cp BGF genome assembly serves as a valuable resource for understanding the biology, pathogenesis, and transmission of C. parvum , and it facilitates the development of diagnostics, drugs, and vaccines against cryptosporidiosis.

Cryptosporidium is a protist parasite that has been identified as the second leading cause of moderate to severe diarrhea in children younger than two and a significant cause of mortality worldwide. Cryptosporidium has a complex, obligate, intracellular but extra cytoplasmic lifecycle in a single host. How genes are regulated in this parasite remains largely unknown. Long non-coding RNAs (lncRNAs) play critical regulatory roles, including gene expression across a broad range of organisms. Cryptosporidium lncRNAs have been reported to enter the host cell nucleus and affect the host response. However, no systematic study of lncRNAs in Cryptosporidium has been conducted to identify additional lncRNAs. In this study, we analyzed a C. parvum in vitro strand-specific RNA-seq developmental time series covering both asexual and sexual stages to identify lncRNAs associated with parasite development. In total, we identified 396 novel lncRNAs, mostly antisense, with 86% being differentially expressed. Surprisingly, nearly 10% of annotated mRNAs have an antisense transcript. lncRNAs occur most often at the 3′ end of their corresponding sense mRNA. Putative lncRNA regulatory regions were identified and many appear to encode bidirectional promoters. A positive correlation between lncRNA and upstream mRNA expression was observed. Evolutionary conservation and expression of lncRNA candidates was observed between C. parvum , C. hominis and C. baileyi . Ten C. parvum protein-encoding genes with antisense transcripts have P. falciparum orthologs that also have antisense transcripts. Three  C. parvum lncRNAs with exceptional properties (e.g., intron splicing) were experimentally validated using RT-PCR and RT-qPCR. This initial characterization of the C. parvum non-coding transcriptome facilitates further investigations into the roles of lncRNAs in parasite development and host-pathogen interactions.

Plasmodium vivax infections remain a major source of malaria-related morbidity and mortality. Early and accurate diagnosis is an integral component of effective malaria control programs. Conventional molecular diagnostic methods provide accurate results but are often resource-intensive, expensive, have a long turnaround time and are beyond the capacity of most malaria-endemic countries. Our laboratory has recently developed a new platform called RealAmp, which combines loop-mediated isothermal amplification (LAMP) with a portable tube scanner real-time isothermal instrument for the rapid detection of malaria parasites. Here we describe new primers for the detection of P. vivax using the RealAmp method. Three pairs of amplification primers required for this method were derived from a conserved DNA sequence unique to the P. vivax genome. The amplification was carried out at 64°C using SYBR Green or SYTO-9 intercalating dyes for 90 minutes with the tube scanner set to collect fluorescence signals at 1-minute intervals. Clinical samples of P. vivax and other human-infecting malaria parasite species were used to determine the sensitivity and specificity of the primers by comparing with an 18S ribosomal RNA-based nested PCR as the gold standard. The new set of primers consistently detected laboratory-maintained isolates of P. vivax from different parts of the world. The primers detected P. vivax in the clinical samples with 94.59% sensitivity (95% CI: 87.48-98.26%) and 100% specificity (95% CI: 90.40-100%) compared to the gold standard nested-PCR method. The new primers also proved to be more sensitive than the published species-specific primers specifically developed for the LAMP method in detecting P. vivax.