Abstract Chagas is an endemic disease in tropical regions of Latin America, caused by the parasite Trypanosoma cruzi . High intraspecies variability and genome complexity have been challenges for the development of genomic variation databases, needed to conduct studies in evolution, population genomics, and identification of genomic elements related to virulence and drug resistance in T. cruzi . Here we present a chromosome-level phased assembly of a T. cruzi strain (Dm25), isolated from a reservoir of the species Didelphis marsupialis located at the Tolima department in Colombia, and belonging to the TcI DTU. We obtained a primary haplotype composed of 32 chromosomes, 30 of them assembled in a single contig, and one complete copy of the maxicircle. While 29 chromosomes show a large collinearity with the assembly of the Brazil A4 strain, three chromosomes with a high density of repeat elements show a large divergence, compared to the Brazil A4 assembly. Considering that the distribution of heterozygous sites suggest that Dm25 is diploid, we assembled a second haplotype for 31 chromosomes, achieving an average of three contigs per chromosome. Nucleotide and protein evolution statistics indicate that T. cruzi Marinkellei separated before the diversification of T. cruzi in the known DTUs. Interchromosomal paralogs of dispersed gene families and histones appeared before but at the same time have a more strict purifying selection, compared to other repeat families. Previously unreported large tandem arrays of protein kinases and histones were identified in this assembly. Over one million variants obtained from Illumina reads aligned to the primary assembly clearly separate the main DTUs. We expect that this new assembly will be a valuable resource for further studies on evolution and functional genomics of Trypanosomatids .

A nearly complete and phased genome assembly of a ColombianTrypanosoma cruziTcI strain and the evolution of gene families