TWW & DS irrigation
For each sample, multiple hypervariable regions of 16S rRNA genes were amplified with the 16S Ion Metagenomics Kit (Thermo Fisher Scientific) by two separate PCR reactions, amplifying the V2, V4, V8 and V3, V67, V9 hypervariable regions, according to the kit protocol9. Equal volumes of V2, V4, V8 and V3, V67, V9 amplicons were combined. 100 nanograms of pooled amplicons were processed to the amplicon library using the Ion Xpress Plus Fragment Library Kit, and each sample was tagged using the Ion Xpress Barcodes Adapters (Thermo Fisher Scientific), according to the manufacturers protocol. Each sample was adjusted to a 10 picomolar concentration. All three samples were pooled, in equal volumes, and processed with One-Touch 2 and One-Touch ES systems (Thermo Fisher Scientific) according to the manufacturers instructions.