16Seq of Shower sample
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.