“…PCR amplifications of all subsamples and negative controls targeting the Cytochrome C oxidase subunit I marker (130 bp) were performed using the primers Minibar-Mod-F and Minibar-Mod-R (Berry et al, 2015), following index combinations as described in Fadrosh et al (2014). PCRs were carried out in 25 μl final volumes with final concentration of 1X Q5 High-Fidelity master mix (New England Biolabs), 2X Q5 enhancer (New England Biolabs), 100 nM of each primer, and 2-3 ng of template DNA (including unique combinations of dualindexing barcodes) (Ribas et al, 2021;Rosa et al, 2024). The PCR amplification protocol was as follows: initial denaturation at 98 C for 30 s; followed by 30 cycles of denaturation at 98 C for 10 s, annealing at 45 C for 15 s, and extension at 72 C for 15 s; and final extension of 2 min at 72 C. Amplicons were visualized on agarose gels and quantified using ImageLab Software v6.0.…”