Evaluation of nanopore-based diatom metabarcoding in tropical freshwater ecosystems

DNA metabarcoding for diatoms has emerged as a promising alternative, offering rapid and comprehensive biodiversity assessments, especially as bioindicators of water quality. However, its performance in tropical Andean river systems remains largely unexplored. This study evaluates the effectiveness of Nanopore-based DNA metabarcoding in characterizing epilithic diatom communities in the upper Guayllabamba river basin (Ecuador). We amplified a long rbcL barcode region (1350–1550 bp) and compared four bioinformatic strategies: three direct-read taxonomic assignment methods (USEARCH Global, Minimap2, and Kraken2) and one OTU-based approach (MetONTIIME), to evaluate differences in species detection, diversity metrics, and ecological assessment outcomes. The results revealed moderate concordance with microscopy-based inventories, which improved when focusing on abundant taxa. Despite some discrepancies in abundance estimates, DNA-based methods successfully captured similar ecological patterns, including trophic status and pollution gradients. Our findings demonstrate the potential of Nanopore-based metabarcoding as a reliable tool for diatom-based biomonitoring in tropical Andean rivers and underscore the advantages of direct-read taxonomic assignment strategies over OTU clustering. They also highlight the need for continued improvement of reference databases to enhance the accuracy and applicability of DNA-based monitoring in underexplored regions.