A new Trypanosoma cruzi genotyping method enables high resolution evolutionary analyses

Abstract

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.

Fig 1:. diversity of the trans-sialidase potential targets in CL Brener genome assembly. (A) Fragment size distribution of all trans-sialidase copies identified. (B) Overview of nucleotide alignment of all trans-sialidase targets identified, where each nucleotide has a distinct colour and gaps are represented as gray; the gray box in the bottom represents the CDS; (C) Logoplot of the alignment depicted in B, where the height of each position represents its conservation; the CDS start codon is identified by a rounded box; (D) Overview of the amino acid alignment of all trans-sialidase targets identified with an open reading frame, where each amino acid has a distinct colour and gaps (indels) are represented in gray; (E) Logoplot of the alignment depicted in D, where it is possible to observe that some positions have non-synonymous mutations.

Fig. 2:. phylogenetic tree based on the K20 set. All branches showed 100% bootstrap support, with the exception of the branches depicted in green (from 86.2% to 86.7%).

Fig. 3:. heatmap representation of selected DTU-specific k-mers and their identification in all genome assemblies available at NCBI. Selected k-mers are represented in each row and the genome assemblies are in the columns; the black colour represents absence of that k-mer in the trans-sialidase identified in silico in each genome assembly, and distinct shades of yellow give a estimative of how many copies share that k-mer.