ec1056_id_id
We performed 16S rRNA V4 marker gene sequencing using a previously described dual-barcoding approach (Kozich, 2013), and these are the positive DNA amplification controls (Zymo D6331).  We normalized the reactions using the Just-a-Plate normalization kit (Charm Biotech, JN-120-10), and combined equivalent volumes of 96 samples into single pools. The pools were concentrated using a Zymo gDNA Clean & Concentrator 11-302C kit, fragment size distributions were evaluated on an Agilent FemtoPulse (Agilent Technologies, Santa Clara, CA, USA), and fragments in the 250-450 bp range were selected on a Sage Science Blue Pippin (Sage Science, Beverly, MA, USA). The final molarity of the pool was estimated via qPCR at the BYU DNA sequencing center. Then, we combined the pools and sequenced them on an Illumina MiSeq using 500 cycle v2 chemistry as described previously (77).