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DNA concentrations were measured by nanodrop, and 20 ng of DNA was used for 16s library preparation. 16s libraries for Illumina sequencing were prepared using a two-step PCR protocol. The first PCR was done using the pool of 5 primers, to each primer we added a tail (CS1 for the forward primers, CS2 for the reverse primers) that is used in the second PCR reaction.  The PCR program used was as follows: 95 3-minutes (activation of enzyme), 20 cycles of 98 10 seconds; 55 10 seconds, 72 20 seconds. For the second PCR to add the adaptor and index primers, we use the Fluidigm Access Array index primers for Illumina, the PCR program is the same as in the first step, but only 10 cycles are done instead of 20. The primers were mixed together.  After the second PCR, samples are cleaned using Kapa beads (identical to AmpureXP beads) and concentrations were measured by Qubit. The samples were then pooled together in equal amounts, and the pooled sample was run on the Tapestation (Agilent) using a DNA 1000 screentape to determine the size of the pooled libraries. The pooled library was then loaded on the Illumina Miseq and sequenced using a Miseq v2 Kit (500 cycles) to generate 2x250 PE reads. The data were de-multiplexed using Basespace, the Illumina cloud software to generate 2 FASTQ files per sample.