16S_nanopore sequencing_metagenome_sediment
Environmental DNA (eDNA) from the sediment samples was obtained using an aliquot from the same day of the sampling, it was conserved at -20C and then was isolated using a kit. Total DNA was extracted following a modified version of the protocol provided by Macherey-NagelTM NucleoSpinTM Soil Kit. DNA integrity was checked using gel electrophoresis, while DNA concentration was measured using Qubit Fluorometer and the Qubit dsDNA BR and HR Assay Kits following their respective protocols. 16S rRNA genes were amplified using the universal bacterial primers 27F 5-AGRGTTYGATYMTGGCT- CAG-3) and 519R (5-G(A/T)ATTACCGCGGC(G/T)GCTG-3). Regions V1 - V9 of 16S rRNA were sequenced using MinION Mk1C (Nanopore Oxford) with the 16S Metabarcoding kit SQK-NBD114.24 with the incorporated primers 27F and 1492R (Rozas at al., 2022) according to the manufacturer.