Non-saline soil microbiota from coastal agricultural land, Bangladesh Site 5
The V1-V9 hypervariable region of the 16S rRNA gene was amplified using the 27F and 1492R primer pair. Polymerase chain reaction (PCR) was performed using Q5 High-Fidelity 2X Master Mix (New England Biolabs, USA) following the manufacturers guidelines. The thermal cycling protocol consisted of an initial denaturation step at 95C for 3 minutes, followed by 30 cycles of denaturation at 95C for 15 seconds, annealing at 60C for 15 seconds, and extension at 72C for 80 seconds, with a final elongation step at 72C for 10 minutes. Post-amplification, the DNA was purified using AMPure XP reagent (Beckman Coulter, Inc., USA) and quantified using the Qubit 4 fluorometer with Qubit 1X dsDNA High Sensitivity (HS) reagent (Thermo Fisher Scientific, USA).For library preparation, 200 ng of purified DNA was processed for end repair and dA-tailing, followed by barcode ligation using the Native Barcoding Kit 24 V14 (SQK-NBD114.24) from Oxford Nanopore. The barcoded libraries were pooled, and sequencing adapters were ligated using the NEBNext Quick Ligation Module (NEB, E6056). The adapter-ligated library were loaded onto the R10.4.1 flow cell (FLO-MIN114; Oxford Nanopore Technologies). Sequencing was conducted on the MinION Mk1C device, with data acquisition performed using MINKNOW software version 24.02.16 (Oxford Nanopore Technologies).