title: ONT Duplex versus Simplex reads for eDNA Based Prediction of Salmon Aquaculture Benthic Impacts
design: Environmental DANN was extracted from sediment samples using the PowerSoil DNA Kit (Qiagen, Hilden, Germany), following the manufacturers protocol. The concentration and purity of the extracted DNA were assessed with a NanoDrop 2000 spectrophotometer (Peqlab, Erlangen, Germany). Full-length 16S rRNA genes were amplified for Nanopore sequencing using the universal primer pair Bac27f (5-AGAGTTTGATCMTGGCTCAG-3; Frank et al., 2008) and U1492R (5-ACCTTGTTACGRCTT-3; Dawson and Pace, 2002). Amplification was performed with LongAmp Taq polymerase (New England Biolabs, Ipswich, MA, USA) under the following cycling conditions: initial denaturation at 95C for 1 minute; 30 cycles of denaturation at 95C for 20 seconds, annealing at 55C for 30 seconds, and extension at 65C for 2 minutes; and a final extension at 65C for 5 minutes. For each sampling site, three technical PCR replicates were performed on each of the two sediment grab replicates. PCR products were purified with the MinElute PCR Purification Kit (Qiagen) according to the manufacturers instructions. The six PCR replicates per sampling site (2 sediment grabs  3 technical replicates each) were pooled in equimolar amounts prior to library preparation for sequencing. Library preparation for Nanopore sequencing was performed using the Duplex-enabled Native Barcoding Kit 96 V14 (SQK-NBD114.96; Oxford Nanopore Technologies, ONT), adhering strictly to the manufacturers protocol. Sequencing libraries, each containing 15 ng of DNA as quantified by a Quantus Fluorometer (Promega), were loaded onto a MinION sequencing device (MIN-101B, ONT) equipped with an R10.4.1 flow cell (FLO-MIN114, ONT).