design: After amplification of the V4 16S region, post_amplification purification was performed using Agencourt AMPure XP beads (Beckman Coulter, Cat. No. A63881) to remove free primers and primer dimer species as per manufacturers instructions. Dual indices and Illumina sequencing adaptors were attached using the Nextera XT v2 Index kit (Illumina, Cat. No. 15052163). Lastly, final sequencing libraries were purified using Agencourt AMPure XP beads. The respective 16S V4 libraries were pooled at different yields (150 ng, 10 ng and 2 ng) to account for variability across libraries. Library sizes were determined using the D1000 ScreenTape Assay (Agilent, Cat. No. 5067_5583 and Cat. No. 5067_5582) and the Agilent 4200 TapeStation was used to verify the success of the indexing reaction. Final library concentrations were determined using the Qubit 1x dsDNA High Sensitivity Assay (Thermo Fisher Scientific, Cat. No. Q33231). All libraries were of expected size and were diluted 4 nm in 10 mM Tris (pH 8.5) and pooled for further denaturation and dilution before sequencing. The manufacturers recommendations (Illumina) were used to dilute sequencing libraries and the PhiX control. The respective 16S V4 libraries were pooled, denatured and diluted to 5 pM and 6 pM, and were spiked with a 10% PhiX sequencing control (Illumina, Cat. No. 15017666) at these concentrations. The 16S V4 libraries were sequenced using two MiSeq Reagent v2 Kits (500 cycles) with 2x251 paired_end reads.