Nanopore Sequencing of 16S rRNA Gene

The 16S Barcoding Kit (SQK-RAB204; Oxford Nanopore Technologies, Oxford, United Kingdom) [12 (link)] was used for DNA library preparation. PCR amplification was conducted with LongAmp™ Taq 2× Master Mix (New England Biolabs, Ipswich, MA, USA). Amplification was performed under the following conditions: initial denaturation at 95 °C for 1 min, 25 cycles of 95 °C for 20 s, 55 °C for 30 s, and 65 °C for 2 min, followed by a final extension at 65 °C for 5 min. The PCR product of each sample was cleaned up and concentrated with AMPure XP (Beckman Coulter, Indianapolis, IN, USA). A total of 10 μL purified DNA was used for library preparation. MinION Mk1C sequencing was performed by using R9.4.1 flow cells (ONT) [5 (link), 13 (link)].