RT Generic
T1 Assessment of diatom diversity in freshwater ecosystems of Ecuador using Nanopore-Based DNA Metabarcoding
A1 Castillejo Pons, Pablo
A1 Lobo, Eduardo
A1 Heinrich, Carla
A1 Perdiguero Jiménez, Pedro
A1 Frandsen, Paul
A1 Rios-Touma, Blanca
A1 Ballesteros Redondo, María Isabel
AB 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.
YR 2023
FD 2023
LK https://hdl.handle.net/20.500.14352/107425
UL https://hdl.handle.net/20.500.14352/107425
LA eng
NO 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.
DS Docta Complutense
RD 6 abr 2025