A divide-and-conquer approach to uncover the genomic structure of the highly virulent RA strain of Trypanosoma cruzi.

Trypanosoma cruzi, the causative agent of Chagas disease, remains a major health and socioeconomic concern in Latin America. Despite its remarkable genetic diversity, high-quality genome assemblies are still limited, and even widely used laboratory strains remain uncharacterized. Here, we present a high-quality genome assembly of the highly virulent RA strain (TcVI), generated using PacBio RSII long-read sequencing. Through the integration of exhaustively curated protein databases and custom-built bioinformatic tools, we improved gene annotation and achieved a comprehensive characterization of the genome. Within the RA genome we identified 17,037 genes conserved across the trypanosomatid clade and 6897 genes and pseudogenes belonging to T. cruzi-specific, rapidly evolving multigene families associated with host adaptation and pathogenicity. Leveraging our recently developed tool for high-throughput GC content profiling, we revealed that the RA genome is organized into 1331 isochore-like regions. This allowed, for the first time, a precise delineation of the so-called "core" and "disruptive" genomic compartments, refining previously proposed models through the identification of their exact genomic coordinates. Regions with GC content < 51%, representing approximately 45% of the genome and enriched in conserved, single-copy genes, were classified as "core". In contrast, GC-rich regions (≥ 51%), enriched in multigene families and transposable elements, were defined as "disruptive". Furthermore, our analysis revealed that the disruptive compartment is not homogeneous: we identified four distinct subtypes within this compartment, each characterized by specific genomic distributions, sequence compositions, and likely distinct evolutionary trajectories. This level of resolution uncovers an additional layer of genome organization complexity previously unrecognized in T. cruzi. The complete and deeply annotated RA strain genome thus provides a valuable resource for the research community and offers new insights into the genome architecture and evolutionary dynamics of this neglected parasite.