Nanopore Amplicon Sequencing of 16S rRNA

We used a protocol based on a recent publication describing monitoring of fresh water for pathogens for this procedure [11 (link)]. Briefly, extracted DNA samples were amplified using the full length of 16s rRNA gene primers with common primer binding sequences 27f and 1492r, attached to unique 24 bp barcodes and nanopore motor protein tether sequence. PCR was performed with 600 nM of each forward and reverse primer, 25 μL of Premix Taq DNA Polymerase (TakaraBio, Shiga, Japan), and a 10 μL DNA template in a 50 μL reaction. The amplification cycles used the following conditions: 94°C for 2 minutes, followed by 35 cycles of 94°C for 30 seconds, 60°C for 30 seconds, and 72°C for 45 seconds with final elongation at 72°C for 5 minutes. The amplicons from the PCR step were purified using NucleoSpin Gel and PCR Clean-up (Macherey Nagel, Duren, Germany) following the manufacturer’s protocol. The barcoded amplicon samples were pooled in equimolar ratios, and library preparation and sequencing were conducted using Ligation Sequencing Kit SQK-LSK-109 (Oxford Nanopore Technologies, Oxford, UK) on the MinION (Oxford Nanopore Technologies, Oxford, UK) sequencing platform following the manufacturer’s instructions.