Metagenome of soil: Untreated soil (Control)-T1-R2
The 16S rRNA gene amplicon libraries were prepared using the ONT 16S Barcoding Kit 24 V14 (SQK-16S024) from Oxford Nanopore Technologies, following the manufacturers instructions. Briefly, 1050 ng of purified genomic DNA was used as input for PCR amplification targeting the full-length 16S rRNA gene (V1V9 regions) using primers 27F (AGAGTTTGATCCTGGCTCAG) and 1492R (TACGGYTACCTTGTTACGACTT). The expected amplicon size was approximately 1.5 kb. The PCR amplification was carried out using LongAmp Taq 2X Master Mix (New England Biolabs) in a final volume of 50 L per reaction, under the following thermal cycling conditions: initial denaturation at 95C for 3 min; followed by 35 cycles of 95C for 20 sec, 55C for 30 sec, and 65C for 2 min; and a final extension at 65C for 5 min. The pooled amplicons were purified using AMPure XP beads (Beckman Coulter) at a 1:1 bead-to-sample ratio and quantified using Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific). The final pooled library was prepared by adding the sequencing adapters using the ONT ligation-based chemistry. Sequencing was performed on PromethION 2 Solo platform (Oxford Nanopore Technologies, Oxford, UK) using R10.4.1 flow cells (FLO-PRO114) on MinKNOW (version 24.06.14, ONT).