*4.7. Sequence and Phylogenetic Analyses*

Raw sequencing data in both forward and reverse directions were viewed using the Chromas Lite version 2.1 sequence analysis program (https://technelysium.com. au/wp/chromas/ (accessed on 1 February 2021)). The Basic Local Alignment Search Tool (BLAST) (http://blast.ncbi.nlm.nih.gov/Blast.cgi (accessed on 1 February 2021)) was used to compare nucleotide sequences with sequences retrieved from the NCBI GenBank database. Generated DNA consensus sequences were aligned to appropriate reference sequences using the MEGA 6free software [60] for species confirmation and assemblage/sub-assemblage identification.

For the estimation of the phylogenetic relationships among the identified Giardiapositive samples, gdh sequences generated in this study and human- and animal-derived homologue sequences mostly from Brazil retrieved from GenBank were aligned using Clustal X and adjusted manually with GeneDoc [61,62]. Inferences by maximum parsimony (MP) were constructed by PAUP version 4.0b10 using a heuristic search in 1000 replicates, 500 bootstrap replicates, random stepwise addition starting trees (with random addition sequences), and tree bisection and reconnection branch swapping [63]. MrBayes v3.1.2 was used to perform Bayesian analyses with four independent Markov chain runs for 1,000,000 metropolis-coupled MCMC generations, sampling a tree every 100th generation [64]. References [65–109] are cited in the supplementary materials. The first 25% of trees represented burn-in and the remaining trees were used to calculate Bayesian posterior probability. The GTR +I + G substitution model was used. The gdh sequence of G. ardeae was used as the outgroup.

The sequences obtained in this study have been deposited in GenBank under accession numbers MT542718-MT542765 (gdh), MT542766-MT542794 (bg), and MT542795-MT542829 (tpi).
