Metagenomics of Environmental sample: Sewage water
Genomic DNA from water sample was subjected to PCR amplification using universal full-length 16S rRNA primers with tailed adaptor sequences. PCR was performed with 2550 ng of DNA using KAPA HiFi HotStart ReadyMix, producing an expected amplicon of ~1.5 kb. Amplified products were visualized on agarose gel, yielded the expected band. Sample was barcoded using the ONT PCR Barcoding Kit (EXP-PBC096) with LongAmp Taq Master Mix. Barcoded amplicons were quantified using Qubit, pooled in equimolar concentrations, and subjected to end-prep, adapter ligation, and AMPure XP bead purification following ONTs LSK114 library preparation workflow. The final library was quantified and loaded onto a PromethION flow cell (FLO-PRO114M). Sequencing was performed on the ONT PromethION 24 platform according to the recommended protocol.