Surveying armadillo and bat trypanosomes by DNA metabarcoding with Oxford Nanopore Technologies sequencing: the importance of fine-tuning parameters to identify mixed infections

Partial 18S rRNA gene sequences for the scientific publication titled "Surveying armadillo and bat trypanosomes by DNA metabarcoding with Oxford Nanopore Technologies sequencing: the importance of fine-tuning parameters to identify mixed infections". Each file represents the results of a step for the creation of a reference database for the identification of Trypanosoma species based on the 18S rRNA region. trypanosoma_18S_db_2097.fas <= all available 18S rRNA sequences associated with the genus Trypanosoma retrieved from the National Center for Biotechnology Information (NCBI) GenBank database in July 2025. trypanosoma_18S_db_336.fas <= The initial FASTA dataset (trypanosoma_18S_db_2097.fas) was processed utilizing the CD-HIT-EST sequence clustering algorithm (Fu et al., 2012). A stringent sequence identity threshold of 99% (-c 0.99) was applied to cluster highly similar sequences and extract a single representative sequence per cluster. This rigorous clustering procedure effectively collapsed the initial dataset, yielding a curated, non-redundant reference database comprising 336 unique 18S rRNA Trypanosoma sequences trypanosoma_18S_db_113.fas <= We manually curated this dataset by interactions of alignments and removing redundant sequences (assumed to be the same species), with the exception of Trypanosoma cruzi were one sequence of each of the nine known main lineages was retained (i.e., DTUI to DTUVI, Tcbat, T. cruzi marinkellei I, and T. cruzi marinkellei II) (Barnabe et al., 2003; Lima et al., 2015). We also trimmed the alignment at both extremes of the sequence to cover a fragment of ca. 560 bp (unaligned) that was amplified by the nested PCR protocol of (Noyes et al. 1999, Noyes et al. 2000). The resulting final reference database consisted of 113 unique sequences. This optimized, dereplicated, and manually curated database served as the foundational reference architecture for all subsequent nanopore read mapping and taxonomic identification procedures. References: Barnabe, C., Brisse, S., Tibayrenc, M., 2003. Phylogenetic diversity of bat trypanosomes of subgenus Schizotrypanum based on multilocus enzyme electrophoresis, random amplified polymorphic DNA, and cytochrome b nucleotide sequence analyses. Infect., Genet. Evol. 2, 201–208. https://doi.org/10.1016/s1567-1348(02)00130-2 Fu, L., Niu, B., Zhu, Z., Wu, S., Li, W., 2012. CD-HIT: accelerated for clustering the next-generation sequencing data. Bioinformatics 28, 3150–3152. https://doi.org/10.1093/bioinformatics/bts565 Lima, L., Espinosa-Álvarez, O., Ortiz, P.A., Trejo-Varón, J.A., Carranza, J.C., Pinto, C.M., Serrano, M.G., Buck, G.A., Camargo, E.P., Teixeira, M.M.G., 2015. Genetic diversity of Trypanosoma cruzi in bats, and multilocus phylogenetic and phylogeographical analyses supporting Tcbat as an independent DTU (discrete typing unit). Acta Trop. 151, 166–177. https://doi.org/10.1016/j.actatropica.2015.07.015 Noyes, H.A., Stevens, J.R., Teixeira, M., Phelan, J., Holz, P., 1999. A nested PCR for the ssrRNA gene detects Trypanosoma binneyi in the platypus and Trypanosoma sp. in wombats and kangaroos in Australia1. Int. J. Parasitol. 29, 331–339. https://doi.org/10.1016/s0020-7519(98)00167-2 Noyes, H.A., Stevens, J.R., Teixeira, M., Phelan, J., Holz, P., 2000. Corrigendum to “A nested PCR for the ssrRNA gene detects Trypanosoma binneyi in the platypus and Trypanosoma sp. in wombats and kangaroos in Australia” [International Journal for Parasitology 29 (2) (1999) 331–339]. Int. J. Parasitol. 30, 228. https://doi.org/10.1016/s0020-7519(00)00026-6