Reevaluation of the Toxoplasma gondii and Neospora caninum genomes reveals misassembly, karyotype differences, and chromosomal rearrangements.

Neospora caninum primarily infects cattle, causing abortions, with an estimated impact of a billion dollars on the worldwide economy annually. However, the study of its biology has been unheeded by the established paradigm that it is virtually identical to its close relative, the widely studied human pathogen Toxoplasma gondii By revisiting the genome sequence, assembly, and annotation using third-generation sequencing technologies, here we show that the N. caninum genome was originally incorrectly assembled under the presumption of synteny with T. gondii We show that major chromosomal rearrangements have occurred between these species. Importantly, we show that chromosomes originally named Chr VIIb and VIII are indeed fused, reducing the karyotype of both N. caninum and T. gondii to 13 chromosomes. We reannotate the N. caninum genome, revealing more than 500 new genes. We sequence and annotate the nonphotosynthetic plastid and mitochondrial genomes and show that although apicoplast genomes are virtually identical, high levels of gene fragmentation and reshuffling exist between species and strains. Our results correct assembly artifacts that are currently widely distributed in the genome database of N. caninum and T. gondii and, more importantly, highlight the mitochondria as a previously oversighted source of variability and pave the way for a change in the paradigm of synteny, encouraging rethinking the genome as basis of the comparative unique biology of these pathogens.

Comparative genomics of drug-resistant strains of Mycobacterium tuberculosis in Ecuador.

BACKGROUND: Tuberculosis is a serious infectious disease affecting millions of people. In spite of efforts to reduce the disease, increasing antibiotic resistance has contributed to persist in the top 10 causes of death worldwide. In fact, the increased cases of multi (MDR) and extreme drug resistance (XDR) worldwide remains the main challenge for tuberculosis control. Whole genome sequencing is a powerful tool for predicting drug resistance-related variants, studying lineages, tracking transmission, and defining outbreaks. This study presents the identification and characterization of resistant clinical isolates of Mycobacterium tuberculosis including a phylogenetic and molecular resistance profile study by sequencing the complete genome of 24 strains from different provinces of Ecuador. RESULTS: Genomic sequencing was used to identify the variants causing resistance. A total of 15/21 isolates were identified as MDR, 4/21 as pre-XDR and 2/21 as XDR, with three isolates discarded due to low quality; the main sub-lineage was LAM (61.9%) and Haarlem (19%) but clades X, T and S were identified. Of the six pre-XDR and XDR strains, it is noteworthy that five come from females; four come from the LAM sub-lineage and two correspond to the X-class sub-lineage. A core genome of 3,750 genes, distributed in 295 subsystems, was determined. Among these, 64 proteins related to virulence and implicated in the pathogenicity of M. tuberculosis and 66 possible pharmacological targets stand out. Most variants result in nonsynonymous amino acid changes and the most frequent genotypes were identified as conferring resistance to rifampicin, isoniazid, ethambutol, para-aminosalicylic acid and streptomycin. However, an increase in the resistance to fluoroquinolones was detected. CONCLUSION: This work shows for the first time the variability of circulating resistant strains between men and women in Ecuador, highlighting the usefulness of genomic sequencing for the identification of emerging resistance. In this regard, we found an increase in fluoroquinolone resistance. Further sampling effort is needed to determine the total variability and associations with the metadata obtained to generate better health policies.

Population genomics reveals evolution and variation of Saccharomyces cerevisiae in the human and insects gut.

The quest to discover the variety of ecological niches inhabited by Saccharomyces cerevisiae has led to research in areas as diverse as wineries, oak trees and insect guts. The discovery of fungal communities in the human gastrointestinal tract suggested the host's gut as a potential reservoir for yeast adaptation. Here, we report the existence of yeast populations associated with the human gut (HG) that differ from those isolated from other human body sites. Phylogenetic analysis on 12 microsatellite loci and 1715 combined CDSs from whole-genome sequencing revealed three subclusters of HG strains with further evidence of clonal colonization within the host's gut. The presence of such subclusters was supported by other genomic features, such as copy number variation, absence/introgressions of CDSs and relative polymorphism frequency. Functional analysis of CDSs specific of the different subclusters suggested possible alterations in cell wall composition and sporulation features. The phenotypic analysis combined with immunological profiling of these strains further showed that sporulation was related with strain-specific genomic characteristics in the immune recognition pattern. We conclude that both genetic and environmental factors involved in cell wall remodelling and sporulation are the main drivers of adaptation in S. cerevisiae populations in the human gut.

Genomic and Transcriptomic Basis of Hanseniaspora vineae's Impact on Flavor Diversity and Wine Quality.

Hanseniaspora is the main genus of the apiculate yeast group that represents approximately 70% of the grape-associated microflora. Hanseniaspora vineae is emerging as a promising species for quality wine production compared to other non-Saccharomyces species. Wines produced by H. vineae with Saccharomyces cerevisiae consistently exhibit more intense fruity flavors and complexity than wines produced by S. cerevisiae alone. In this work, genome sequencing, assembling, and phylogenetic analysis of two strains of H. vineae showed that it is a member of the Saccharomyces complex and it diverged before the whole-genome duplication (WGD) event from this clade. Specific flavor gene duplications and absences were identified in the H. vineae genome compared to 14 fully sequenced industrial S. cerevisiae genomes. The increased formation of 2-phenylethyl acetate and phenylpropanoids such as 2-phenylethyl and benzyl alcohols might be explained by gene duplications of H. vineae aromatic amino acid aminotransferases (ARO8 and ARO9) and phenylpyruvate decarboxylases (ARO10). Transcriptome and aroma profiles under fermentation conditions confirmed these genes were highly expressed at the beginning of stationary phase coupled to the production of their related compounds. The extremely high level of acetate esters produced by H. vineae compared to that by S. cerevisiae is consistent with the identification of six novel proteins with alcohol acetyltransferase (AATase) domains. The absence of the branched-chain amino acid transaminases (BAT2) and acyl coenzyme A (acyl-CoA)/ethanol O-acyltransferases (EEB1) genes correlates with H. vineae's reduced production of branched-chain higher alcohols, fatty acids, and ethyl esters, respectively. Our study provides sustenance for understanding and potentially utilizing genes that determine fermentation aromas.IMPORTANCE The huge diversity of non-Saccharomyces yeasts in grapes is dominated by the apiculate genus Hanseniaspora Two native strains of Hanseniaspora vineae applied to winemaking because of their high oenological potential in aroma and fermentation performance were selected to obtain high-quality genomes. Here, we present a phylogenetic analysis and the complete transcriptome and aroma metabolome of H. vineae during three fermentation steps. This species produced significantly richer flavor compound diversity than Saccharomyces, including benzenoids, phenylpropanoids, and acetate-derived compounds. The identification of six proteins, different from S. cerevisiae ATF, with diverse acetyltransferase domains in H. vineae offers a relevant source of native genetic variants for this enzymatic activity. The discovery of benzenoid synthesis capacity in H. vineae provides a new eukaryotic model to dilucidate an alternative pathway to that catalyzed by plants' phenylalanine lyases.

Social wasps are a Saccharomyces mating nest.

The reproductive ecology of Saccharomyces cerevisiae is still largely unknown. Recent evidence of interspecific hybridization, high levels of strain heterozygosity, and prion transmission suggest that outbreeding occurs frequently in yeasts. Nevertheless, the place where yeasts mate and recombine in the wild has not been identified. We found that the intestine of social wasps hosts highly outbred S. cerevisiae strains as well as a rare S. cerevisiae×S. paradoxus hybrid. We show that the intestine of Polistes dominula social wasps favors the mating of S. cerevisiae strains among themselves and with S. paradoxus cells by providing a succession of environmental conditions prompting cell sporulation and spores germination. In addition, we prove that heterospecific mating is the only option for European S. paradoxus strains to survive in the gut. Taken together, these findings unveil the best hidden secret of yeast ecology, introducing the insect gut as an environmental alcove in which crosses occur, maintaining and generating the diversity of the ascomycetes.

Isolation and molecular characterization of four novel Neospora caninum strains.

Neospora caninum causes neosporosis, a leading cause of bovine abortion worldwide. Uruguay is a developing economy in South America that produces milk to feed seven times its population annually. Naturally, dairy production is paramount to the country's economy, and bovine reproductive failure impacts it profoundly. Recent studies demonstrated that the vast majority of infectious abortions in dairy cows are caused by N. caninum. To delve into the local situation and contextualize it within the international standing, we set out to characterize the Uruguayan N. caninum strains. For this, we isolated four distinct strains and determined by microsatellite typing that these represent three unique genetic lineages, distinct from those reported previously in the region or elsewhere. An unbiased analysis of the current worldwide genetic diversity of N. caninum strains known, whereby six typing clusters can be resolved, revealed that three of the four Uruguayan strains group closely with regional strains from Argentina and Brazil. The remaining strain groups in an unrelated genetic cluster, suggesting multiple origins of the local strains. Microsatellite typing of N. caninum DNA from fetuses opportunistically collected from local dairy farms correlated more often with one of the isolates. Overall, our results contribute to further understanding of genetic diversity among strains of N. caninum both regionally and worldwide.

Whole genome sequencing of the monomorphic pathogen Mycobacterium bovis reveals local differentiation of cattle clinical isolates.

BACKGROUND: Bovine tuberculosis (bTB) poses serious risks to animal welfare and economy, as well as to public health as a zoonosis. Its etiological agent, Mycobacterium bovis, belongs to the Mycobacterium tuberculosis complex (MTBC), a group of genetically monomorphic organisms featured by a remarkably high overall nucleotide identity (99.9%). Indeed, this characteristic is of major concern for correct typing and determination of strain-specific traits based on sequence diversity. Due to its historical economic dependence on cattle production, Uruguay is deeply affected by the prevailing incidence of Mycobacterium bovis. With the world's highest number of cattle per human, and its intensive cattle production, Uruguay represents a particularly suited setting to evaluate genomic variability among isolates, and the diversity traits associated to this pathogen. RESULTS: We compared 186 genomes from MTBC strains isolated worldwide, and found a highly structured population in M. bovis. The analysis of 23 new M. bovis genomes, belonging to strains isolated in Uruguay evidenced three groups present in the country. Despite presenting an expected highly conserved genomic structure and sequence, these strains segregate into a clustered manner within the worldwide phylogeny. Analysis of the non-pe/ppe differential areas against a reference genome defined four main sources of variability, namely: regions of difference (RD), variable genes, duplications and novel genes. RDs and variant analysis segregated the strains into clusters that are concordant with their spoligotype identities. Due to its high homoplasy rate, spoligotyping failed to reflect the true genomic diversity among worldwide representative strains, however, it remains a good indicator for closely related populations. CONCLUSIONS: This study introduces a comprehensive population structure analysis of worldwide M. bovis isolates. The incorporation and analysis of 23 novel Uruguayan M. bovis genomes, sheds light onto the genomic diversity of this pathogen, evidencing the existence of greater genetic variability among strains than previously contemplated.

Hsp12p and PAU genes are involved in ecological interactions between natural yeast strains.

The coexistence of different yeasts in a single vineyard raises the question on how they communicate and why slow growers are not competed out. Genetically modified laboratory strains of Saccharomyces cerevisiae are extensively used to investigate ecological interactions, but little is known about the genes regulating cooperation and competition in ecologically relevant settings. Here, we present evidences of Hsp12p-dependent altruistic and contact-dependent competitive interactions between two natural yeast isolates. Hsp12p is released during cell death for public benefit by a fast-growing strain that also produces a killer toxin to inhibit growth of a slow grower that can enjoy the benefits of released Hsp12p. We also show that the protein Pau5p is essential in the defense against the killer effect. Our results demonstrate that the combined action of Hsp12p, Pau5p and a killer toxin is sufficient to steer a yeast community.

A computational pipeline to discover highly phylogenetically informative genes in sequenced genomes: application to Saccharomyces cerevisiae natural strains.

The quest for genes representing genetic relationships of strains or individuals within populations and their evolutionary history is acquiring a novel dimension of complexity with the advancement of next-generation sequencing (NGS) technologies. In fact, sequencing an entire genome uncovers genetic variation in coding and non-coding regions and offers the possibility of studying Saccharomyces cerevisiae populations at the strain level. Nevertheless, the disadvantageous cost-benefit ratio (the amount of details disclosed by NGS against the time-expensive and expertise-demanding data assembly process) still precludes the application of these techniques to the routinely assignment of yeast strains, making the selection of the most reliable molecular markers greatly desirable. In this work we propose an original computational approach to discover genes that can be used as a descriptor of the population structure. We found 13 genes whose variability can be used to recapitulate the phylogeny obtained from genome-wide sequences. The same approach that we prove to be successful in yeasts can be generalized to any other population of individuals given the availability of high-quality genomic sequences and of a clear population structure to be targeted.

New insights into phenotype and genotype relationships in Neospora caninum.

The successful isolation of four new Neospora caninum strains from different regions and with different backgrounds (obtained from an abortion storm or congenitally infected and asymptomatic calves) allowed us previously to characterize natural isolates, finding differences in phenotype and microsatellites. Given the variability observed, we wondered in this work whether these differences had consequences in virulence, invasion and vertical transmission using cell cultures and murine neosporosis models. In addition, we performed the genomic analysis and SNP comparative studies of the NcURU isolates. The results obtained in this work allowed us to establish that NcURU isolates are of low virulence and have unique phenotypic characteristics. Likewise, sequencing their genomes has allowed us to delve into the genetic singularities underlying these phenotypes, as well as the common mutated genes. This work opens a new perspective for diagnostic purposes and formulating possible vaccines based on attenuated strains.

Deep serological profiling of the Trypanosoma cruzi TSSA antigen reveals different epitopes and modes of recognition by Chagas disease patients.

BACKGROUND: Trypanosoma cruzi, the agent of Chagas disease, displays a highly structured population, with multiple strains that can be grouped into 6-7 evolutionary lineages showing variable eco-epidemiological traits and likely also distinct disease-associated features. Previous works have shown that antibody responses to 'isoforms' of the polymorphic parasite antigen TSSA enable robust and sensitive identification of the infecting strain with near lineage-level resolution. To optimize the serotyping performance of this molecule, we herein used a combination of immunosignaturing approaches based on peptide microarrays and serum samples from Chagas disease patients to establish a deep linear B-cell epitope profiling of TSSA. METHODS/PRINCIPLE FINDINGS: Our assays revealed variations in the seroprevalence of TSSA isoforms among Chagas disease populations from different settings, hence strongly supporting the differential distribution of parasite lineages in domestic cycles across the Americas. Alanine scanning mutagenesis and the use of peptides of different lengths allowed us to identify key residues involved in antibody pairing and the presence of three discrete B-cell linear epitopes in TSSAII, the isoform with highest seroprevalence in human infections. Comprehensive screening of parasite genomic repositories led to the discovery of 9 novel T. cruzi TSSA variants and one TSSA sequence from the phylogenetically related bat parasite T. cruzi marinkellei. Further residue permutation analyses enabled the identification of diagnostically relevant or non-relevant substitutions among TSSA natural polymorphisms. Interestingly, T. cruzi marinkellei TSSA displayed specific serorecognition by one chronic Chagas disease patient from Colombia, which warrant further investigations on the diagnostic impact of such atypical TSSA. CONCLUSIONS/SIGNIFICANCE: Overall, our findings shed new light into TSSA evolution, epitope landscape and modes of recognition by Chagas disease patients; and have practical implications for the design and/or evaluation of T. cruzi serotyping strategies.

A precision overview of genomic resistance screening in Ecuadorian isolates of Mycobacterium tuberculosis using web-based bioinformatics tools.

INTRODUCTION: Tuberculosis (TB) is among the deadliest diseases worldwide, and its impact is mainly due to the continuous emergence of resistant isolates during treatment due to the laborious process of resistance diagnosis, nonadherence to treatment and circulation of previously resistant isolates of Mycobacterium tuberculosis. In this study, we evaluated the performance and functionalities of web-based tools, including Mykrobe, TB-profiler, PhyResSE, KvarQ, and SAM-TB, for detecting resistance in 88 Ecuadorian isolates of Mycobacterium tuberculosis drug susceptibility tested previously. Statistical analysis was used to determine the correlation between genomic and phenotypic analysis. Our results showed that with the exception of KvarQ, all tools had the highest correlation with the conventional drug susceptibility test (DST) for global resistance detection (98% agreement and 0.941 Cohen's kappa), while SAM-TB, PhyResSE, TB-profiler and Mykrobe had better correlations with DST for first-line drug analysis individually. We also identified that in our study, only 50% of mutations characterized by the web-based tools in the rpoB, katG, embB, pncA, gyrA and rrs regions were canonical and included in the World Health Organization (WHO) catalogue. Our findings suggest that SAM-TB, PhyResSE, TB-profiler and Mykrobe were efficient in determining canonical resistance-related mutations, but more analysis is needed to improve second-line detection. Improving surveillance programs using whole-genome sequencing tools for first-line drugs, MDR-TB and XDR-TB is essential to understand the molecular epidemiology of TB in Ecuador. IMPORTANCE: Tuberculosis, an infectious disease caused by Mycobacterium tuberculosis, most commonly affects the lungs and is often spread through the air when infected people cough, sneeze, or spit. However, despite the existence of effective drug treatment, patient adherence, long duration of treatment, and late diagnosis have reduced the effectiveness of therapy and increased drug resistance. The increase in resistant cases, added to the impact of the COVID-19 pandemic, has highlighted the importance of implementing efficient and timely diagnostic methodologies worldwide. The significance of our research is in evaluating and identifying a more efficient and user-friendly web-based tool to characterize resistance in Mycobacterium tuberculosis by whole-genome sequencing, which will allow more routine application to improve TB strain surveillance programs locally.

The footprint of metabolism in the organization of mammalian genomes.

BACKGROUND: At present five evolutionary hypotheses have been proposed to explain the great variability of the genomic GC content among and within genomes: the mutational bias, the biased gene conversion, the DNA breakpoints distribution, the thermal stability and the metabolic rate. Several studies carried out on bacteria and teleostean fish pointed towards the critical role played by the environment on the metabolic rate in shaping the base composition of genomes. In mammals the debate is still open, and evidences have been produced in favor of each evolutionary hypothesis. Human genes were assigned to three large functional categories (as well as to the corresponding functional classes) according to the KOG database: (i) information storage and processing, (ii) cellular processes and signaling, and (iii) metabolism. The classification was extended to the organisms so far analyzed performing a reciprocal Blastp and selecting the best reciprocal hit. The base composition was calculated for each sequence of the whole CDS dataset. RESULTS: The GC3 level of the above functional categories was increasing from (i) to (iii). This specific compositional pattern was found, as footprint, in all mammalian genomes, but not in frog and lizard ones. Comparative analysis of human versus both frog and lizard functional categories showed that genes involved in the metabolic processes underwent the highest GC3 increment. Analyzing the KOG functional classes of genes, again a well defined intra-genomic pattern was found in all mammals. Not only genes of metabolic pathways, but also genes involved in chromatin structure and dynamics, transcription, signal transduction mechanisms and cytoskeleton, showed an average GC3 level higher than that of the whole genome. In the case of the human genome, the genes of the aforementioned functional categories showed a high probability to be associated with the chromosomal bands. CONCLUSIONS: In the light of different evolutionary hypotheses proposed so far, and contributing with different potential to the genome compositional heterogeneity of mammalian genomes, the one based on the metabolic rate seems to play not a minor role. Keeping in mind similar results reported in bacteria and in teleosts, the specific compositional patterns observed in mammals highlight metabolic rate as unifying factor that fits over a wide range of living organisms.

Peculiar patterns of amino acid substitution and conservation in the fast evolving tunicate Oikopleura dioica.

We analyze the patterns and rates of amino acid evolution in tunicates with special interest on the extremely fast evolving Oikopleura dioica. We show that this species, on average, is twice as fast as the already fast evolving Ciona intestinalis. The acceleration in both species seems to be affected by similar evolutionary forces yet to different extent, since a substantial proportion of the most and less accelerated genes are orthologous between the two species. Among the possible causes that underlie the genome wide acceleration in Oikopleura, relaxation of functional constraints appears to be an important one, since all amino acids exhibit surprisingly homogenous levels of divergence. Such homogeneity, however, is not observed in Ciona. Apart from the genome wide acceleration, detailed analysis of functional groups of genes revealed that genes associated with regulatory functions (transcription regulators, chromatin remodeling proteins and metabolic regulators), have been subjected to an even more extreme process of acceleration, suggesting that adaptive evolution is the most probable cause of their unusual exacerbated rates. Another remarkable observation is that cysteine is among the less conserved amino acids, contrary to what is commonly observed in other species. The possible causes of this particular behavior are discussed.

The Elusive Mitochondrial Genomes of Apicomplexa: Where Are We Now?

Mitochondria are vital organelles of eukaryotic cells, participating in key metabolic pathways such as cellular respiration, thermogenesis, maintenance of cellular redox potential, calcium homeostasis, cell signaling, and cell death. The phylum Apicomplexa is entirely composed of obligate intracellular parasites, causing a plethora of severe diseases in humans, wild and domestic animals. These pathogens include the causative agents of malaria, cryptosporidiosis, neosporosis, East Coast fever and toxoplasmosis, among others. The mitochondria in Apicomplexa has been put forward as a promising source of undiscovered drug targets, and it has been validated as the target of atovaquone, a drug currently used in the clinic to counter malaria. Apicomplexans present a single tubular mitochondria that varies widely both in structure and in genomic content across the phylum. The organelle is characterized by massive gene migrations to the nucleus, sequence rearrangements and drastic functional reductions in some species. Recent third generation sequencing studies have reignited an interest for elucidating the extensive diversity displayed by the mitochondrial genomes of apicomplexans and their intriguing genomic features. The underlying mechanisms of gene transcription and translation are also ill-understood. In this review, we present the state of the art on mitochondrial genome structure, composition and organization in the apicomplexan phylum revisiting topological and biochemical information gathered through classical techniques. We contextualize this in light of the genomic insight gained by second and, more recently, third generation sequencing technologies. We discuss the mitochondrial genomic and mechanistic features found in evolutionarily related alveolates, and discuss the common and distinct origins of the apicomplexan mitochondria peculiarities.

Nanopore Long Read DNA Sequencing of Protozoan Parasites: Hybrid Genome Assembly of Trypanosoma cruzi.

Due to highly repetitive genome sequences, short-read-based Trypanosoma cruzi genomes are extremely fragmented. Contiguous trypanosomatid genomes assemblies have resulted in the advent of third-generation sequencing technologies. Long reads span several to hundreds of kbps allowing to correct assemblies of repeated and low complexity DNA regions. However, these techniques present higher error rates. Hybrid assembly strategies that combine error-prone long reads with much more accurate Illumina short reads represent a very convenient approach for enhancing genome completeness. Here, we describe how to perform a hybrid assembly for genomic analysis of protozoan pathogens using Illumina and Oxford Nanopore sequencing.

Maxicircle architecture and evolutionary insights into Trypanosoma cruzi complex.

We sequenced maxicircles from T. cruzi strains representative of the species evolutionary diversity by using long-read sequencing, which allowed us to uncollapse their repetitive regions, finding that their real lengths range from 35 to 50 kb. T. cruzi maxicircles have a common architecture composed of four regions: coding region (CR), AT-rich region, short (SR) and long repeats (LR). Distribution of genes, both in order and in strand orientation are conserved, being the main differences the presence of deletions affecting genes coding for NADH dehydrogenase subunits, reinforcing biochemical findings that indicate that complex I is not functional in T. cruzi. Moreover, the presence of complete minicircles into maxicircles of some strains lead us to think about the origin of minicircles. Finally, a careful phylogenetic analysis was conducted using coding regions of maxicircles from up to 29 strains, and 1108 single copy nuclear genes from all of the DTUs, clearly establishing that taxonomically T. cruzi is a complex of species composed by group 1 that contains clades A (TcI), B (TcIII) and D (TcIV), and group 2 (1 and 2 do not coincide with groups I and II described decades ago) containing clade C (TcII), being all hybrid strains of the BC type. Three variants of maxicircles exist in T. cruzi: a, b and c, in correspondence with clades A, B, and C from mitochondrial phylogenies. While A and C carry maxicircles a and c respectively, both clades B and D carry b maxicircle variant; hybrid strains also carry the b- variant. We then propose a new nomenclature that is self-descriptive and makes use of both the phylogenetic relationships and the maxicircle variants present in T. cruzi.

How fast is the sessile ciona?

Genomewide analyses of distances between orthologous gene pairs from the ascidian species Ciona intestinalis and Ciona savignyi were compared with those of vertebrates. Combining this data with a detailed and careful use of vertebrate fossil records, we estimated the time of divergence between the two ascidians nearly 180 My. This estimation was obtained after correcting for the different substitution rates found comparing several groups of chordates; indeed we determine here that on average Ciona species evolve 50% faster than vertebrates.

Transcriptome Studies in Trypanosoma cruzi Using RNA-seq.

RNA-seq is a powerful method to study in detail transcriptome changes in defined conditions, providing enormous amount of information on RNA stability and gene regulation. In this chapter, we describe a directional and a nondirectional library preparation protocol for RNA-seq in Trypanosoma cruzi, as well as a pipeline for bioinformatic analysis, which includes read trimming, alignment to a reference genome, and differential expression.

Expanding an expanded genome: long-read sequencing of Trypanosoma cruzi.

Although the genome of Trypanosoma cruzi, the causative agent of Chagas disease, was first made available in 2005, with additional strains reported later, the intrinsic genome complexity of this parasite (the abundance of repetitive sequences and genes organized in tandem) has traditionally hindered high-quality genome assembly and annotation. This also limits diverse types of analyses that require high degrees of precision. Long reads generated by third-generation sequencing technologies are particularly suitable to address the challenges associated with T. cruzi's genome since they permit direct determination of the full sequence of large clusters of repetitive sequences without collapsing them. This, in turn, not only allows accurate estimation of gene copy numbers but also circumvents assembly fragmentation. Here, we present the analysis of the genome sequences of two T. cruzi clones: the hybrid TCC (TcVI) and the non-hybrid Dm28c (TcI), determined by PacBio Single Molecular Real-Time (SMRT) technology. The improved assemblies herein obtained permitted us to accurately estimate gene copy numbers, abundance and distribution of repetitive sequences (including satellites and retroelements). We found that the genome of T. cruzi is composed of a 'core compartment' and a 'disruptive compartment' which exhibit opposite GC content and gene composition. Novel tandem and dispersed repetitive sequences were identified, including some located inside coding sequences. Additionally, homologous chromosomes were separately assembled, allowing us to retrieve haplotypes as separate contigs instead of a unique mosaic sequence. Finally, manual annotation of surface multigene families, mucins and trans-sialidases allows now a better overview of these complex groups of genes.