Giants' cooperation: a draft genome of the giant ciliate Muniziella cunhai suggests its ecological role in the capybara's digestive metabolism.

Several hundred ciliate species live in animals' guts as a part of their microbiome. Among them, Muniziella cunhai (Trichostomatia, Pycnotrichidae), the largest described ciliate, is found exclusively associated with Hydrochoerus hydrochaeris (capybara), the largest known rodent reaching up to 90 kg. Here, we present the sequence, structural and functional annotation of this giant microeukaryote macronuclear genome and discuss its phylogenetic placement. The 85 Mb genome is highly AT rich (GC content 25.71 %) and encodes a total of 11 397 protein-coding genes, of which 2793 could have their functions predicted with automated functional assignments. Functional annotation showed that M. cunhai can digest recalcitrant structural carbohydrates, non-structural carbohydrates, and microbial cell walls, suggesting a role in diet metabolization and in microbial population control in the capybara's intestine. Moreover, the phylogenetic placement of M. cunhai provides insights on the origins of gigantism in the subclass Trichostomatia.

Expression and biochemical characterization of the putative insulinase enzyme PF11_0189 found in the Plasmodium falciparum genome.

PF11_0189 is a putative insulin degrading enzyme present in Plasmodium falciparum genome. The catalytic domain of PF11_0189 is about 27 kDa. Substrate specificity study shows PF11_0189 acts upon different types of proteins. The substrate specificity is found to be highest when insulin is used as a substrate. Metal dependency study shows highest dependency of PF11_0189 towards zinc metal for its proteolytic activity. Chelation of zinc metal with EDTA shows complete absence of PF11_0189 activity. Peptide inhibitors, P-70 and P-121 from combinatorial peptide library prepared against PF11_0189 show inhibition with an IC50 value of 4.8 µM and 7.5 µM respectively. A proven natural anti-malarial peptide cyclosporin A shows complete inhibition against PF11_0189 with an IC50 value of 0.75 µM suggesting PF11_0189 as a potential target for peptide inhibitors. The study implicates that PF11_0189 is a zinc metalloprotease involved in catalysis of insulin. The study gives a preliminary insight into the mechanism of complications arising from glucose abnormalities during severe malaria.

A Proteogenomic Approach to Unravel New Proteins Encoded in the Leishmania donovani (HU3) Genome.

The high-throughput proteomics data generated by increasingly more sensible mass spectrometers greatly contribute to our better understanding of molecular and cellular mechanisms operating in live beings. Nevertheless, proteomics analyses are based on accurate genomic and protein annotations, and some information may be lost if these resources are incomplete. Here, we show that most proteomics data may be recovered by interconnecting genomics and proteomics approaches (i.e., following a proteogenomic strategy), resulting, in turn, in an improvement of gene/protein models. In this study, we generated proteomics data from Leishmania donovani (HU3 strain) promastigotes that allowed us to detect 1908 proteins in this developmental stage on the basis of the currently annotated proteins available in public databases. However, when the proteomics data were searched against all possible open reading frames existing in the L. donovani genome, twenty new protein-coding genes could be annotated. Additionally, 43 previously annotated proteins were extended at their N-terminal ends to accommodate peptides detected in the proteomics data. Also, different post-translational modifications (phosphorylation, acetylation, methylation, among others) were found to occur in a large number of Leishmania proteins. Finally, a detailed comparative analysis of the L. donovani and Leishmania major experimental proteomes served to illustrate how inaccurate conclusions can be raised if proteomes are compared solely on the basis of the listed proteins identified in each proteome. Finally, we have created data entries (based on freely available repositories) to provide and maintain updated gene/protein models. Raw data are available via ProteomeXchange with the identifier PXD051920.

Chromosome-level genome assembly and annotation of the social amoeba Dictyostelium firmibasis.

Dicytostelium firmibasis is a member of Dictyostelia, a group of social amoebae that upon starvation display aggregative multicellularity where the amoebae transition from uni- to multicellular life. The D. firmibasis genome assembly that is currently available is of limited use due to its low contiguity, large number of undetermined bases, and lack of annotations. Here we used Nanopore long read sequencing, complemented with Illumina sequencing, and developmental transcriptomics as well as small RNA-sequencing, to present a new, fully annotated, chromosome-level D. firmibasis genome assembly. The new assembly contains no undetermined bases, and consists mainly of six large contigs representing the chromosomes, as well as a complete mitochondrial genome. This new genome assembly will be a valuable tool, allowing comprehensive comparison to Dictyostelium discoideum, the dictyostelid genetically tractable model. Further, the new genome will be important for studies of evolutionary processes governing the transition from unicellular to multicellular organisms and will aid in the sequencing and annotation of other dictyostelids genomes, many of which are currently of poor quality.

Telomere-to-telomere Genome Assembly of the Clubroot Pathogen Plasmodiophora Brassicae.

Plasmodiophora brassicae (Woronin, 1877), a biotrophic, obligate parasite, is the causal agent of clubroot disease in brassicas. The clubroot pathogen has been reported in more than 80 countries worldwide, causing economic losses of hundreds of millions every year. Despite its widespread impact, very little is known about the molecular strategies it employs to induce the characteristic clubs in the roots of susceptible hosts during infection, nor about the mechanisms it uses to overcome genetic resistance. Here, we provide the first telomere-to-telomere complete genome of P. brassicae. We generated ∼27 Gb of Illumina, Oxford Nanopore, and PacBio HiFi data from resting spores of strain Pb3A and produced a 25.3 Mb assembly comprising 20 chromosomes, with an N50 of 1.37 Mb. The BUSCO score, the highest reported for any member of the group Rhizaria (Eukaryota: 88.2%), highlights the limitations within the Eukaryota database for members of this lineage. Using available transcriptomic data and protein evidence, we annotated the Pb3A genome, identifying 10,521 protein-coding gene models. This high-quality, complete genome of P. brassicae will serve as a crucial resource for the plant pathology community to advance the much-needed understanding of the evolution of the clubroot pathogen.

Ancient Plasmodium genomes shed light on the history of human malaria.

Malaria-causing protozoa of the genus Plasmodium have exerted one of the strongest selective pressures on the human genome, and resistance alleles provide biomolecular footprints that outline the historical reach of these species1. Nevertheless, debate persists over when and how malaria parasites emerged as human pathogens and spread around the globe1,2. To address these questions, we generated high-coverage ancient mitochondrial and nuclear genome-wide data from P. falciparum, P. vivax and P. malariae from 16 countries spanning around 5,500 years of human history. We identified P. vivax and P. falciparum across geographically disparate regions of Eurasia from as early as the fourth and first millennia BCE, respectively; for P. vivax, this evidence pre-dates textual references by several millennia3. Genomic analysis supports distinct disease histories for P. falciparum and P. vivax in the Americas: similarities between now-eliminated European and peri-contact South American strains indicate that European colonizers were the source of American P. vivax, whereas the trans-Atlantic slave trade probably introduced P. falciparum into the Americas. Our data underscore the role of cross-cultural contacts in the dissemination of malaria, laying the biomolecular foundation for future palaeo-epidemiological research into the impact of Plasmodium parasites on human history. Finally, our unexpected discovery of P. falciparum in the high-altitude Himalayas provides a rare case study in which individual mobility can be inferred from infection status, adding to our knowledge of cross-cultural connectivity in the region nearly three millennia ago.

Comparative genomics of Giardia duodenalis sub-assemblage AI beaver (Be-2) and human (WB-C6) strains show remarkable homozygosity, sequence similarity, and conservation of VSP genes.

Giardia duodenalis, a major cause of waterborne infection, infects a wide range of mammalian hosts and is subdivided into eight genetically well-defined assemblages named A through H. However, fragmented genomes and a lack of comparative analysis within and between the assemblages render unclear the molecular mechanisms controlling host specificity and differential disease outcomes. To address this, we generated a near-complete de novo genome of AI assemblage using the Oxford Nanopore platform by sequencing the Be-2 genome. We generated 148,144 long-reads with quality scores of > 7. The final genome assembly consists of only nine contigs with an N50 of 3,045,186 bp. This assembly agrees closely with the assembly of another strain in the AI assemblage (WB-C6). However, a critical difference is that a region previously placed in the five-prime region of Chr5 belongs to Chr4 of Be-2. We find a high degree of conservation in the ploidy, homozygosity, and the presence of cysteine-rich variant-specific surface proteins (VSPs) within the AI assemblage. Our assembly provides a nearly complete genome of a member of the AI assemblage of G. duodenalis, aiding population genomic studies capable of elucidating Giardia transmission, host range, and pathogenicity.

Genomic and epidemiological evidence for the emergence of a L. infantum/L. donovani hybrid with unusual epidemiology in northern Italy.

Leishmania (L.) infantum is one of the main causative agents of animal and human leishmaniasis across many endemic areas in South America, Europe, North Africa, and Asia. Despite its clinical significance, little is known about the genetic diversity of L. infantum circulating in a given endemic area. Here, we investigate this important open question by applying a comparative genomics approach to seven L. infantum isolates from different hosts and Italian regions, including the northern part of the country (Emilia-Romagna, RER), Sicily, and Sardinia, as an initial attempt to explore the breadth of parasite genetic heterogeneity in Italy. Additionally, microsatellite analysis was carried out to compare the isolates from RER with other 70 L. infantum strains from the same region as well as 65 strains belonging to the L. donovani complex from other countries. We revealed important karyotypic instability and identified strain-specific changes in gene dosage, which affected important virulence factors such as amastins and surface antigen-like proteins. Single nucleotide polymorphism-based clustering analysis of these genomes together with over 80 publicly available L. infantum and L. donovani genomes placed the Italian isolates into three geographically distinct clusters within the Mediterranean basin and uncovered three isolates clustering with putative L. infantum/L. donovani hybrids isolated in Cyprus. As judged by microsatellite profiling, these hybrid isolates are representative of a sub-population of parasites circulating in northern Italy that preferentially infect humans but not dogs. Our results place Italy at the crossroads of L. infantum infection in the Mediterranean and call attention to the public health risk represented by the introduction of non-European Leishmania species.IMPORTANCEThis study closes important knowledge gaps with respect to Leishmania (L.) infantum genetic heterogeneity in a given endemic country, as exemplified here for Italy, and reveals genetic hybridization as a main cause for re-emerging human leishmaniasis in northern Italy. The observed high diversity of Leishmania parasites on the Italian peninsula suggests different geographical origins, with genomic adaptation to various ecologies affecting both pathogenicity and transmission potential. This is documented by the discovery of a putative L. infantum/L. donovani hybrid strain, which has been shown to preferentially infect humans but not dogs. Our results provide important information to health authorities, which need to consider the public health risk represented by the introduction of new Leishmania species into EU countries due to population displacement or travel from countries where exotic/allochthonous parasite species are endemic.

CRISPR-Cas9 high-throughput screening to study drug resistance in Leishmania infantum.

Parasites of the genus Leishmania pose a global health threat with limited treatment options. New drugs are urgently needed, and genomic screens have the potential to accelerate target discovery, mode of action, and resistance mechanisms against these new drugs. We describe here our effort in developing a genome-wide CRISPR-Cas9 screen in Leishmania, an organism lacking a functional nonhomologous end joining system that must rely on microhomology-mediated end joining, single-strand annealing, or homologous recombination for repairing Cas9-induced double-stranded DNA breaks. A new vector for cloning and expressing single guide RNAs (sgRNAs) was designed and proven to be effective in a small pilot project while enriching specific sgRNAs during drug selection. We then developed a whole-genome library of 49,754 sgRNAs, targeting all the genes of Leishmania infantum. This library was transfected in L. infantum expressing Cas9, and these cells were selected for resistance to two antileishmanials, miltefosine and amphotericin B. The sgRNAs the most enriched in the miltefosine screen targeted the miltefosine transporter gene, but sgRNAs targeting genes coding for a RING-variant protein and a transmembrane protein were also enriched. The sgRNAs the most enriched by amphotericin B targeted the sterol 24 C methyltransferase genes and a hypothetical gene. Through gene disruption experiments, we proved that loss of function of these genes was associated with resistance. This study describes the feasibility of carrying out whole-genome CRISPR-Cas9 screens in Leishmania provided that a strong selective pressure is applied. Such a screen can be used for accelerating the development of urgently needed antileishmanial drugs.IMPORTANCELeishmaniasis, a global health threat, lacks adequate treatment options and drug resistance exacerbates the challenge. This study introduces a CRISPR-Cas9 screening approach in Leishmania infantum, unraveling mechanisms of drug resistance at a genome-wide scale. Our screen was applied against two main antileishmanial drugs, and guides were enriched upon drug selection. These guides targeted known and new targets, hence validating the use of this screen against Leishmania. This strategy provides a powerful tool to expedite drug discovery as well as potential therapeutic targets against this neglected tropical disease.

Genomic insights into the cellular specialization of predation in raptorial protists.

BACKGROUND: Predation is a fundamental mechanism for organisms to acquire energy, and various species have evolved diverse tools to enhance their hunting abilities. Among protozoan predators, raptorial Haptorian ciliates are particularly fascinating as they possess offensive extrusomes known as toxicysts, which are rapidly discharged upon prey contact. However, our understanding of the genetic processes and specific toxins involved in toxicyst formation and discharge is still limited. RESULTS: In this study, we investigated the predation strategies and subcellular structures of seven Haptoria ciliate species and obtained their genome sequences using single-cell sequencing technology. Comparative genomic analysis revealed distinct gene duplications related to membrane transport proteins and hydrolytic enzymes in Haptoria, which play a crucial role in the production and discharge of toxicysts. Transcriptomic analysis further confirmed the abundant expression of genes related to membrane transporters and cellular toxins in Haptoria compared to Trichostomatia. Notably, polyketide synthases (PKS) and L-amino acid oxidases (LAAO) were identified as potentially toxin genes that underwent extensive duplication events in Haptoria. CONCLUSIONS: Our results shed light on the evolutionary and genomic adaptations of Haptorian ciliates for their predation strategies in evolution and provide insights into their toxic mechanisms.

Far-East Asian Toxoplasma isolates share ancestry with North and South/Central American recombinant lineages.

Toxoplasma gondii is a global protozoan pathogen. Clonal lineages predominate in Europe, North America, Africa, and China, whereas highly recombinant parasites are endemic in South/Central America. Far East Asian T. gondii isolates are not included in current global population genetic structure analyses at WGS resolution. Here we report a genome-wide population study that compared eight Japanese and two Chinese isolates against representative worldwide T. gondii genomes using POPSICLE, a novel population structure analyzing software. Also included were 7 genomes resurrected from non-viable isolates by target enrichment sequencing. Visualization of the genome structure by POPSICLE shows a mixture of Chinese haplogroup (HG) 13 haploblocks introgressed within the genomes of Japanese HG2 and North American HG12. Furthermore, two ancestral lineages were identified in the Japanese strains; one lineage shares a common ancestor with HG11 found in both Japanese strains and North American HG12. The other ancestral lineage, found in T. gondii isolates from a small island in Japan, is admixed with genetically diversified South/Central American strains. Taken together, this study suggests multiple ancestral links between Far East Asian and American T. gondii strains and provides insight into the transmission history of this cosmopolitan organism.

Integrative analyses on the ciliates Colpoda illuminate the life history evolution of soil microorganisms.

Microorganisms play a central role in sustaining soil ecosystems and agriculture, and these functions are usually associated with their complex life history. Yet, the regulation and evolution of life history have remained enigmatic and poorly understood, especially in protozoa, the third most abundant group of organisms in the soil. Here, we explore the life history of a cosmopolitan species-Colpoda steinii. Our analysis has yielded a high-quality macronuclear genome for C. steinii, with size of 155 Mbp and 37,123 protein-coding genes, as well as mean intron length of ~93 bp, longer than most other studied ciliates. Notably, we identify two possible whole-genome duplication events in C. steinii, which may account for its genome being about twice the size of C. inflata's, another co-existing species. We further resolve the gene expression profiles in diverse life stages of C. steinii, which are also corroborated in C. inflata. During the resting cyst stage, genes associated with cell death and vacuole formation are upregulated, and translation-related genes are downregulated. While the translation-related genes are upregulated during the excystment of resting cysts. Reproductive cysts exhibit a significant reduction in cell adhesion. We also demonstrate that most genes expressed in specific life stages are under strong purifying selection. This study offers a deeper understanding of the life history evolution that underpins the extraordinary success and ecological functions of microorganisms in soil ecosystems.IMPORTANCEColpoda species, as a prominent group among the most widely distributed and abundant soil microorganisms, play a crucial role in sustaining soil ecosystems and promoting plant growth. This investigation reveals their exceptional macronuclear genomic features, including significantly large genome size, long introns, and numerous gene duplications. The gene expression profiles and the specific biological functions associated with the transitions between various life stages are also elucidated. The vast majority of genes linked to life stage transitions are subject to strong purifying selection, as inferred from multiple natural strains newly isolated and deeply sequenced. This substantiates the enduring and conservative nature of Colpoda's life history, which has persisted throughout the extensive evolutionary history of these highly successful protozoa in soil. These findings shed light on the evolutionary dynamics of microbial eukaryotes in the ever-fluctuating soil environments. This integrative research represents a significant advancement in understanding the life histories of these understudied single-celled eukaryotes.

Genome-wide distribution of 5-hydroxymethyluracil and chromatin accessibility in the Breviolum minutum genome.

BACKGROUND: In dinoflagellates, a unique and extremely divergent genomic and nuclear organization has evolved. The highly unusual features of dinoflagellate nuclei and genomes include permanently condensed liquid crystalline chromosomes, primarily packaged by proteins other than histones, genes organized in very long unidirectional gene arrays, a general absence of transcriptional regulation, high abundance of the otherwise very rare DNA modification 5-hydroxymethyluracil (5-hmU), and many others. While most of these fascinating properties are originally identified in the 1970s and 1980s, they have not yet been investigated using modern genomic tools. RESULTS: In this work, we address some of the outstanding questions regarding dinoflagellate genome organization by mapping the genome-wide distribution of 5-hmU (using both immunoprecipitation-based and basepair-resolution chemical mapping approaches) and of chromatin accessibility in the genome of the Symbiodiniaceae dinoflagellate Breviolum minutum. We find that the 5-hmU modification is preferentially enriched over certain classes of repetitive elements, often coincides with the boundaries between gene arrays, and is generally correlated with decreased chromatin accessibility, the latter otherwise being largely uniform along the genome. We discuss the potential roles of 5-hmU in the functional organization of dinoflagellate genomes and its relationship to the transcriptional landscape of gene arrays. CONCLUSIONS: Our results provide the first window into the 5-hmU and chromatin accessibility landscapes in dinoflagellates.

Massive genome reduction predates the divergence of Symbiodiniaceae dinoflagellates.

Dinoflagellates in the family Symbiodiniaceae are taxonomically diverse, predominantly symbiotic lineages that are well-known for their association with corals. The ancestor of these taxa is believed to have been free-living. The establishment of symbiosis (i.e. symbiogenesis) is hypothesized to have occurred multiple times during Symbiodiniaceae evolution, but its impact on genome evolution of these taxa is largely unknown. Among Symbiodiniaceae, the genus Effrenium is a free-living lineage that is phylogenetically positioned between two robustly supported groups of genera within which symbiotic taxa have emerged. The apparent lack of symbiogenesis in Effrenium suggests that the ancestral features of Symbiodiniaceae may have been retained in this lineage. Here, we present de novo assembled genomes (1.2-1.9 Gbp in size) and transcriptome data from three isolates of Effrenium voratum and conduct a comparative analysis that includes 16 Symbiodiniaceae taxa and the other dinoflagellates. Surprisingly, we find that genome reduction, which is often associated with a symbiotic lifestyle, predates the origin of Symbiodiniaceae. The free-living lifestyle distinguishes Effrenium from symbiotic Symbiodiniaceae vis-à-vis their longer introns, more-extensive mRNA editing, fewer (~30%) lineage-specific gene sets, and lower (~10%) level of pseudogenization. These results demonstrate how genome reduction and the adaptation to distinct lifestyles intersect to drive diversification and genome evolution of Symbiodiniaceae.

Whole-genome assembly of a hybrid Trypanosoma cruzi strain assembled with Nanopore sequencing alone.

Trypanosoma cruzi is the causative agent of Chagas disease, which causes 10,000 deaths per year. Despite the high mortality associated with Chagas, relatively few parasite genomes have been assembled to date, with genome assemblies unavailable even for some commonly used laboratory strains. This is at least partially due to T. cruzi's highly complex and highly repetitive genome, which defies investigation using traditional short-read sequencing methods. In this study, we have generated a high-quality whole-genome assembly of the hybrid Tulahuen strain, a commercially available type VI strain, using long-read Nanopore sequencing without short-read scaffolding. The assembled genome contains 25% repeat regions, 17% variable multigene family members, and 27% transposable elements (TEs) and is of comparable quality with T. cruzi genome assemblies that utilized both long- and short-read data. Notably, we find that regions with TEs are significantly enriched for multicopy surface proteins, and that surface proteins are, on average, closer to TEs than to other coding regions. This finding suggests that mobile genetic elements such as transposons may drive recombination within surface protein gene families. This work demonstrates the feasibility of Nanopore sequencing to resolve complex regions of T. cruzi genomes, and with these resolved regions, provides support for a possible mechanism for genomic diversification.

Ancestral aneuploidy and stable chromosomal duplication resulting in differential genome structure and gene expression control in trypanosomatid parasites.

Aneuploidy is widely observed in both unicellular and multicellular eukaryotes, usually associated with adaptation to stress conditions. Chromosomal duplication stability is a tradeoff between the fitness cost of having unbalanced gene copies and the potential fitness gained from increased dosage of specific advantageous genes. Trypanosomatids, a family of protozoans that include species that cause neglected tropical diseases, are a relevant group to study aneuploidies. Their life cycle has several stressors that could select for different patterns of chromosomal duplications and/or losses, and their nearly universal use of polycistronic transcription increases their reliance on gene expansion/contraction, as well as post-transcriptional control as mechanisms for gene expression regulation. By evaluating the data from 866 isolates covering seven trypanosomatid genera, we have revealed that aneuploidy tolerance is an ancestral characteristic of trypanosomatids but has a reduced occurrence in a specific monophyletic clade that has undergone large genomic reorganization and chromosomal fusions. We have also identified an ancient chromosomal duplication that was maintained across these parasite's speciation, named collectively as the trypanosomatid ancestral supernumerary chromosome (TASC). TASC has most genes in the same coding strand, is expressed as a disomic chromosome (even having four copies), and has increased potential for functional variation, but it purges highly deleterious mutations more efficiently than other chromosomes. The evidence of stringent control over gene expression in this chromosome suggests that these parasites have adapted to mitigate the fitness cost associated with this ancient chromosomal duplication.

Uncoupling programmed DNA cleavage and repair scrambles the Paramecium somatic genome.

In the ciliate Paramecium, precise excision of numerous internal eliminated sequences (IESs) from the somatic genome is essential at each sexual cycle. DNA double-strands breaks (DSBs) introduced by the PiggyMac endonuclease are repaired in a highly concerted manner by the non-homologous end joining (NHEJ) pathway, illustrated by complete inhibition of DNA cleavage when Ku70/80 proteins are missing. We show that expression of a DNA-binding-deficient Ku70 mutant (Ku70-6E) permits DNA cleavage but leads to the accumulation of unrepaired DSBs. We uncoupled DNA cleavage and repair by co-expressing wild-type and mutant Ku70. High-throughput sequencing of the developing macronucleus genome in these conditions identifies the presence of extremities healed by de novo telomere addition and numerous translocations between IES-flanking sequences. Coupling the two steps of IES excision ensures that both extremities are held together throughout the process, suggesting that DSB repair proteins are essential for assembly of a synaptic precleavage complex.

Genome assembly of a symbiotic balantidia (Balantidium ctenopharyngodoni) in fish hindgut.

Balantidium ctenopharyngodoni is identified as the sole ciliate species that exclusively resides within the hindgut of grass carp with high prevalence and intensity. In this study, the successful cultivation of B. ctenopharyngodoni enabled us to collect enough cells for genome sequencing. Consequently, we acquired a high-quality genome assembly spanning 68.66 Mb, encompassing a total of 22,334 nanochromosomes. Furthermore, we predicted 29,348 protein-coding genes, and 95.5% of them was supported by the RNA-seq data. The trend of GC content in the subtelomeric regions of single-gene chromosomes was similar to other ciliates containing nanochromosomes. A large number of genes encoding carbohydrate-binding modules with affinities for starch and peptidoglycans was identified. The identification of mitochondrion-related organelles (MROs) within genome indicates its well-suited adaptation to the anaerobic conditions in the hindgut environment. In summary, our results will offer resources for understanding the genetic basis and molecular adaptations of balantidia to hindgut of herbivorous fish.

Protocol for mapping the three-dimensional organization of dinoflagellate genomes.

Dinoflagellate genomes often are very large and difficult to assemble, which has until recently precluded their analysis with modern functional genomic tools. Here, we present a protocol for mapping three-dimensional (3D) genome organization in dinoflagellates and using it for scaffolding their genome assemblies. We describe steps for crosslinking, nuclear lysis, denaturation, restriction digest, ligation, and DNA shearing and purification. We then detail procedures sequencing library generation and computational analysis, including initial Hi-C read mapping and 3D-DNA scaffolding/assembly correction. For complete details on the use and execution of this protocol, please refer to Marinov et al.1.

Comparative genomics analysis reveals sequence characteristics potentially related to host preference in Cryptosporidium xiaoi.

Cryptosporidium spp. are important diarrhea-associated pathogens in humans and livestock. Among the known species, Cryptosporidium xiaoi, which causes cryptosporidiosis in sheep and goats, was previously recognized as a genotype of the bovine-specific Cryptosporidium bovis based on their high sequence identity in the ssrRNA gene. However, the lack of genomic data has limited characterization of the genetic differences between the two closely related species. In this study, we sequenced the genomes of two C. xiaoi isolates and performed comparative genomic analysis to identify the sequence uniqueness of this ovine-adapted species compared with other Cryptosporidium spp. Our results showed that C. xiaoi is genetically related to C. bovis as shown by their 95.8% genomic identity and similar gene content. Consistent with this, both C. xiaoi and C. bovis appear to have fewer genes encoding mitochondrial metabolic enzymes and invasion-related protein families. However, they appear to possess several species-specific genes. Further analysis indicates that the sequence differences between these two Cryptosporidium spp. are mainly in 24 highly polymorphic genes, half of which are located in the subtelomeric regions. Some of these subtelomeric genes encode secretory proteins that have undergone positive selection. In addition, the genomes of two C. xiaoi isolates, identified as subtypes XXIIIf and XXIIIh, share 99.9% nucleotide sequence identity, with six highly divergent genes encoding putative secretory proteins. Therefore, these species-specific genes and sequence polymorphism in subtelomeric genes probably contribute to the different host preference of C. xiaoi and C. bovis.