Ampure xp bead solution

DNA extracts were prepared using Illumina’s 16S Metagenomic Sequencing Library Preparation guidelines. DNA samples were amplified using primers for the V3-V4 16S gene [33 (link)]. All PCR reactions were carried out using a 2720 thermal cycler (Life Technologies) with KAPA HiFi HotStart ReadyMix. The amplicon PCR program was 95 °C for 5 min, followed by 25 cycles of 95 °C for 30 s, 55 °C for 30 s and 72 °C for 30 s followed by a 5 min 72 °C elongation step before ramp down to 4 °C. The Index PCR program was the same as the amplicon step except it was carried out for 8 cycles instead of 25. All PCR products were visualised on agarose gels (1.5%, Sigma-Merck) stained with SYBR safe (Thermofisher, MA, United States). PCR reactions were cleaned using AMPure XP bead solution (Beckman Coulter, CA, USA) according to Illumina’s guidelines. Final DNA products were quantified using the Qubit dsDNA high-sensitivity kit (Thermofisher) and measured on a Qubit 3.0 fluorometer (Thermofisher).

Genomic DNA was subjected to a magnetic bead clean-up before library preparation. Accordingly, DNA was mixed with an equal amount of AMPure XP bead solution (Beckman Coulter) and incubated for 5 min on a rotator mixer at room temperature. Afterward, beads were pelleted on a magnetic rack and washed twice with freshly prepared 70% ethanol, and genomic DNA was eluted with high-quality water by incubation for 10 min at room temperature.
DNA concentrations were determined using the Qubit BR assay kit (Thermo Fisher Scientific) on a Qubit 4 fluorometer (Thermo Fisher Scientific).
Libraries for nanopore sequencing were prepared using the native barcoding kit 24 (Q20+) (catalog no. SQK-NBD112.24; Oxford Nanopore) according to the manufacturer’s instructions.