title: 16S-seq of drinking water
design: Amplicon libraries targeting the 16S rRNA bV18A region were prepared from up to 25 ng of DNA using PCR with Platinum SuperFi DNA Polymerase (Thermo Fisher) and 24-nt barcoded primers ([8F], [1391R]). Each 50-L reaction contained 0.2 mM dNTPs, 0.01 U polymerase, and 500 nM of each primer in SuperFi Buffer. Thermal cycling was 98 C for 3 min; then 25 cycles at 98 C for 30 s, 62 C for 20 s, 72 C for 2 min; and a final elongation at 72 C for 5 min. Amplicons were purified (CleanNGS SPRI beads, 3:5 bead:sample), eluted in 25 L nuclease-free water, and used for library preparation (SQK-LSK114 kit, Oxford Nanopore) with 500 ng total DNA and CleanNGS beads for cleanup. DNA concentration was measured by Qubit dsDNA HS, and library size/purity verified with Tapestation 2200. The final library was loaded onto a MinION R10.4.1 flowcell and sequenced using MinKNOW 23.11.4. Reads were basecalled and demultiplexed with MinKNOW Dorado 7.2.13 (super-accurate basecalling, config r10.4.1_400bps_sup.cfg) using custom barcodes.