Assessment of diatom diversity in freshwater ecosystems of Ecuador using Nanopore-Based DNA Metabarcoding
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2023
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Abstract
Our study aimed to compare the effectiveness of microscopic and DNA metabarcoding approaches in assessing the abundance and diversity of epilithic diatoms, while also exploring the suitability of metabarcoding as a reliable biomonitoring tool in Andean freshwater ecosystems. For this, 16 samples from the Guayllabamba River Basin in Ecuador were analyzed. Microscopic analysis involved the taxonomic identification of at least 600 valves under a light microscope. The molecular approach involved amplifying a rbcL region of approximately 1500 bp and conducting high-throughput DNA sequencing using Oxford Nanopore technology.
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We used the primers DPrbcL1 (5´AAGGAGAAATHAATGTCT 3´) and DPrbcL7 (5´AARCAACCTTGTGTAAGTCTC 3´) (Jones et al., 2005) to amplify a region of ~1,400 bp, covering the rbcL gene. pooled PCR products for each sample were sent to Secugen, DNA Sequencing Service (Madrid, Spain) for next-generation sequencing. The sequencing service performed purification and quality analysis of the PCR samples and prepared the library. Briefly, the amplification products were purified through ultrafiltration using NucleoFast 96PCR plates. The purified products were recovered in nuclease-free water and quantified through fluorimetry. DNA ends were repaired using the NEBNext Ultra II End repair enzyme, and the kit labels (EXP-NBD104 and EXP-NBD114) were added to each sample through ligation (SQK-LSK109). The labeled products were sequenced on an Oxford Nanopore MinION R9.4.1 flow cell, obtaining a minimum of 15,000 reads analyzed with a high-accuracy base-calling model. The reads were filtered to select reads between 1350 to 1550 pb.