title: eti531_title
design: In the laboratory, DNA was extracted from each sample using the Zymo Quick-DNA Fecal/Soil Microbe 96 Kit (D6011) in 50microliters elution buffer: individual flies, first examined under a microscope for morphological confirmation as male D. simulans by examining to determine presence/absence of a distinct genital arch. On each 96-well plate were four controls: a positive extraction control (Zymo D6300), a negative reagent-only control and two empty wells for PCR amplification controls. We performed 16S rRNA V4 marker gene sequencing using a previously described dual-barcoding approach (Kozich, 2013), including a negative control with no template and a positive control (Zymo D6331) for each 96-well plate. Negative controls did not yield visible bands by gel electrophoresis. 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).