title: 16S rRNA gene sequencing from forest soil using Oxford Nanopore Technologies protocol 16S Barcoding Kit 1-24 (SQK-16S024)
design: Whole DNA isolated in triplicates was mixed in equal amounts (20 ng)  and then used for microbiota profiling using Oxford Nanopore technology (ONT) (Oxford Nanopore Technologies plc, Oxford, United Kingdom). The library was prepared strictly following the ONT protocol 16S Barcoding Kit 1-24 (SQK-16S024) (16S_9086_v1_revH_14Aug2019) with recommended third-party consumables. Specific primers 27F (5-AGAGTTTGATCMTGGCTCAG-3) and 1492R (5-TACGGYTACCTTGTTACGACTT-3), containing 5 tags that facilitate the ligase-free attachment of Rapid Sequencing Adapters, allow amplification by polymerase chain reaction (PCR) for the entire ~1,500 bp 16S rRNA gene. PCR amplifications were performed in 50 l reactions containing 25 l LongAmp Hot Start 2X Master Mix (NEB, M0533S), 10 ng input DNA (previously adjusted with nuclease-free water to 1 ng/l), 5 l nuclease-free water and 10 l of individual unique 16S barcode. Cycling conditions were 95C for 1 min, 25 cycles of 95C for 20 s, 55C for 30 s, and 65C for 2 min; followed by a final extension at 65C for 5 min. Then, PCR products were purified using Agencourt AMPure XP (Beckman Coulter, Beverly, USA). The concentration of PCR products was quantified using the Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, USA). The resulting libraries were sequenced with the MinION Mk 1B using flowcells FC-106D-R9.4.1 for 72 h (Oxford Nanopore Technologies).