Species Typing of Trypanosomatids Using Amplicon-Based Sequencing

Amplicon-based sequencing has emerged as an exceptionally powerful tool for the identification and characterization of microorganisms, offering enhanced sensitivity and specificity in molecular diagnostics. In this chapter, we detail the amplification and sequencing protocols for two critical genetic markers: the HSP70 gene, essential for identifying Leishmania species, and the 18S ribosomal RNA gene, a well-established marker for detecting Trypanosoma species. We provide a comprehensive overview of the entire workflow, beginning with the optimization of PCR conditions to ensure robust gene amplification. The subsequent sequencing was performed using Oxford Nanopore Technologies, a platform renowned for generating long reads in real time, which facilitates a detailed analysis of genetic material and supports high-resolution taxonomic assignments. Following sequencing, we describe the bioinformatics pipeline used to process and analyze the data. This pipeline incorporates stringent quality control measures and phylogenetic analysis. Together, these steps not only enhance the accuracy of pathogen detection but also allow for an in-depth assessment of genetic diversity among trypanosomatids.