Shotgun metagenomics of activated sludge
Library preparation of the extracted DNA was performed using the Ligation Sequencing Kit V14 (Oxford Nanopore Technologies Ltd). The NEBNext Companion Module for Oxford Nanopore Technologies Ligation Sequencing (New England BioLabs Inc.) and UltraPureTM BSA (50 mg/mL) (Thermo Fisher Scientific) were additionally used for the DNA repair and end-prep and the flow cell priming steps. All steps were performed as instructed by the manufacturer, except the incubations in the Hula mixer were replaced with slow manual inversions (~5 s per inversion). All resuspension steps were performed by flicking the tube. MinION R10.4 version flow cells (Oxford Nanopore) were loaded with the DNA and samples were sequenced in accurate mode (260 bps) for 89-96h.