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BACKGROUND Trypanosoma cruzi is an important human pathogen in Latin America with nearly seven million people infected. It has a large degree of genetic diversity, classified into six discrete typing units (DTUs), which probably influences its physiological behavior and clinical manifestations. Several genotyping methods are available, with distinct performance on easiness, cost, resolution and applicability; no method excels in all parameters. OBJECTIVES AND METHODS To devise a molecular method for T. cruzi genotyping, based on polymerase chain reaction (PCR) amplification of a single target with multiple copies in the nuclear genome by large scale sequencing. We have applied this method to 29 T. cruzi isolates, comprising all described DTUs. FINDINGS We were able to classify all samples into sub DTU level with high robustness. Evolutionary relationship between DTUs were ascertained, suggesting that TcIII and TcIV DTUs are non-hybrid, and DTU IV is more similar to the common ancestral. CONCLUSION As the TS-LSS method is based on a single PCR reaction, comprising several copies of the target, it is probably useful for clinical samples, when the amount of DNA is a limiting factor. As large scale sequencing systems become more common, the TS-LSS method can be increasingly applied for T. cruzi genotyping.

Trans‐sialidase (TS) is a membrane glycoprotein belonging to a family of genes from multiple copies involved in the process of cell invasion of the vertebrate host by the Trypanosoma cruzi (T. cruzi). Previous studies have shown that 5′ untranslated region is responsible for controlling gene regulation. In this work, we evaluated segments from TS cDNAs deposited in the Genbank‐dbEST such as the trans‐splicing additional or multiple sites, and their respective signals. We also investigated the compositional variation and the size of the sequences among T. cruzi epimastigotes strains: Dm28c (TcI), Y and CL‐Brener (TcII) and 4167 (Zymodeme 3). To corroborate these theoretical sequences, we acquired new test sequences based on RT‐PCR and qPCR. The fragments obtained from the different T. cruzi strains were cloned, sequenced and compositionally analyzed. CLUSTAL X was used to edit and align both theoretical and obtained sequences. All the TS theoretical sequences were searched against the dbEST database using BLAST program, which resulted in 735 cDNAs sequences. We also estimated the size of the mini‐exon‐containing fragments as 312 base pairs found in Z3, 209 in TcI and 218 in TcII. No mutations in the gene duplication presented in the trans‐splicing site were observed in the different strains. The qPCR analysis detected no differences in TS genes expression, although these genes are involved in host infectivity.