Amplicon library preparation and sequencing

Amplicon libraries were generated as described previously^{29 (link),45 (link),46 (link)}. The variable 4 (V4) region of the 16S rRNA gene was amplified in duplicate with the following PCR reaction components: 1× 5Prime Hotstart mastermix, 1× Evagreen, 500 nM forward primer, and 500 nM reverse primer. Amplification was monitored in a CFX96 RT–PCR machine (Bio-Rad) and samples were removed once fluorescence measurements reached ~10,000 RFU (late exponential phase). Cycling conditions were as follows: 94 °C for 3 min, up to 45 cycles of 94 °C for 45 s, 54 °C for 60 s, and 72 °C for 90 s. Duplicate reactions that amplified were pooled together and quantified with Kapa library quantification kit (Kapa Biosystems, KK4824) before equimolar sample mixing. Libraries were concentrated and cleaned using AMPureXP beads (Beckman Coulter). The final library was quantified using a High Sensitivity D1000 Tapestation (Agilent) chip. Sequencing was performed by Fulgent Genetics using the Illumina MiSeq platform and 2 × 300-bp reagent kit for paired-end sequencing.