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.

In this paper, we determine by fluorescent in situ hybridization the variability in the chromosomal location of 45S rDNA clusters in 38 species belonging to 7 genera of the Triatominae subfamily, using a triatomine-specific 18S rDNA probe. Our results show a striking variability at the inter- and intraspecific level, never reported so far in holocentric chromosomes, revealing the extraordinary genomic dynamics that occurred during the evolution in this group of insects. Our results also demonstrate that the chromosomal position of rDNA clusters is an important marker to disclose chromosomal differentiation in species karyotypically homogenous in their chromosome number.

Triatoma infestans is the most important Chagas disease vector in South America. Two main evolutionary lineages, named Andean and non-Andean, have been recognized by geographical distribution, phenetic and genetic characteristics. One of the main differences is the genomic size, varying over 30% in their haploid DNA content. Here we realize a genome wide analysis to compare the repetitive genome fraction (repeatome) between both lineages in order to identify the main repetitive DNA changes occurred during T. infestans differentiation process. RepeatExplorer analysis using Illumina reads showed that both lineages exhibit the same amount of non-repeat sequences, and that satellite DNA is by far the major component of repetitive DNA and the main responsible for the genome size differentiation between both lineages. We characterize 42 satellite DNA families, which are virtually all present in both lineages but with different amount in each lineage. Furthermore, chromosomal location of satellite DNA by fluorescence in situ hybridization showed that genomic variations in T. infestans are mainly due to satellite DNA families located on the heterochromatic regions. The results also show that many satDNA families are located on the euchromatic regions of the chromosomes.

Triatoma bahiensis Sherlock & Serafim, 1967, T. lenti Sherlock & Serafim, 1967, and T. pessoai Sherlock & Serafim, 1967 were described based on material collected in the Brazilian state of Bahia. These species were later included in the T. brasiliensis complex based on their geographic distribution. Triatoma bahiensis and T. pessoai were subsequently synonymized with T. lenti. However, the phylogenetic position of T. lenti within the T. brasiliensis complex has remained doubtful. This study aims to assess the taxonomic status of T. bahiensis and to infer the phylogenetic relationships between T. lenti, T. bahiensis and the other members of the T. brasiliensis species complex. The identities of the species in concern were confirmed by comparisons with high resolution photos of the respective type materials; lectotypes are designated for T. pessoai and T. bahiensis. Morphological, morphometric, molecular, and cytogenetic approaches as well as experimental crosses were used. The low viability of experimental crosses combined with morphological and morphometric data allow the differentiation of T. bahiensis and T. lenti. Pairwise cyt b sequence divergence between T. lenti and T. bahiensis was 2.5%. Cytogenetic and molecular analyses grouped T. lenti and T. bahiensis as members of the T. brasiliensis complex. These results revalidate the specific status of T. bahiensis.

Chagas disease was described by Carlos Chagas, who first identified the parasite Trypanosoma cruzi from a 2-year-old girl called Berenice. Many T. cruzi sequencing projects based on short reads have demonstrated that genome assembly and downstream comparative analyses are extremely challenging in this species, given that half of its genome is composed of repetitive sequences. Here, we report de novo assemblies, annotation, and comparative analyses of the Berenice strain using a combination of Illumina short reads and MinION long reads. Our work demonstrates that Nanopore sequencing improves T. cruzi assembly contiguity and increases the assembly size in ∼16 Mb. Specifically, we found that assembly improvement also refines the completeness of coding regions for both single-copy genes and repetitive transposable elements. Beyond its historical and epidemiological importance, Berenice constitutes a fundamental resource because it now constitutes a high-quality assembly available for TcII (clade C), a prevalent lineage causing human infections in South America. The availability of Berenice genome expands the known genetic diversity of these parasites and reinforces the idea that T. cruzi is intraspecifically divided in three main clades. Finally, this work represents the introduction of Nanopore technology to resolve complex protozoan genomes, supporting its subsequent application for improving trypanosomatid and other highly repetitive genomes.