Sample C51_AOC_AH V3-V5 region, Ahero, Aedes ochraceus
Adult females that were not visibly bloodengorged were processed for microbial analysis. Prior to genomic DNA extraction, adult female mosquitoes were surface sterilized by rinsing them in 2% bleach solution for 1 min, followed by 70% ethanol for 5 min and then three rinses in sterile phosphate-buffered saline solution each lasting 10 sec. Genomic DNA was extracted from individual mosquitoes using the Qiagen DNeasy Blood and Tissue Kit (Qiagen, GmbH Hilden, Germany) following the manufacturers recommendations and stored at -20oC until further processing. In total, 153 mosquito samples from two mosquito species, Ae. mcintoshi (n = 70) and Ae. ochraceus (n = 83) were processed. All DNA samples were shipped to the W.M Keck Center for Comparative and Functional Genomics at the University of Illinois for sequencing using Illumina MiSeq Bulk V3 platform. The PCR amplification and sequencing procedures targeting the V3-V5 hypervariable region of bacterial 16S rRNA gene were performed. In brief, all DNA samples were measured on a Qubit (Life Technologies) using High Sensitivity DNA kit and diluted to 2 ng/l. PCR master mix was prepared using the Roche High Fidelity Start Kit and 20x Access Array loading reagent and aliquoted into 48 well PCR plates along with 1 l DNA sample and 1 l Fluidigm Illumina linkers (V3-V5-F357: ACACTGACGACATGGTTCTACA and V3-V5-R926:TACGGTAGCAGAGACTTG-GTCT) with unique barcode. In a separate plate, 20x solutions of the following primer pairs: forward 5`-CCTACGGGAGGCAGCAG-3`and reverse 5`-CCGTCAATTCMTTTRAGT-3` were prepared by adding 2 l of forward and reverse primer, 5 l of 20x Access Array Loading Reagent and water to a final volume of 100 l. A 4 l aliquot of sample was loaded in the sample inlets and 4 l of primer loaded in primer inlets of a previously primed Fluidigm 48.48 Access Array IFC, and samples on the array were then amplified on the Fluidigm Biomark HD PCR machine using the following Access Array cycling program without imaging: 50C for 2 min (1 cycle), 70C for 20 min (1 cycle), 95C for 10 min (1 cycle), followed by 10 cycles at 95C for 15 sec, 60C for 30 sec, and 72C for 1 min, 2 cycles at 95C for 15 sec, 80C for 30 sec, 60C for 30 seconds, and 72C for 1 min, 8 cycles at 95C for 15 sec, 60C for 30 sec, and 72 for 1 min, 2 cycles at 95C for 15 sec, 80C for 30 sec, 60C for 30 sec, and 72C for 1 min, 8 cycles at 95C for 15 sec, 60C for 30 sec, and 72C for 1 min, and 5 cycles at  95C for 15 sec, 80C for 30 sec, 60C for 30 sec, and 72C for 1 min. The PCR product was transferred to a new 96 well plate, quantified on a Qubit fluorimeter (Thermo-Fisher) and stored at -20C. All samples were run on a Fragment Analyzer (Advanced Analytics, Ames, IA) and amplicon regions and expected sizes confirmed. Samples were then pooled in equal amounts according to product concentration. The pooled products were size selected on a 2% agarose E-gel (Life Technologies) and extracted from the isolated gel slice with QIAquick gel extraction kit (QIAGEN). Cleaned size selected products were run on an Agilent Bioanalyzer to confirm appropriate profile and determination of average size. The final library pool was spiked with 10% non-indexed PhiX control library (Illumina) and sequenced using Illumina MiSeq V3 Bulk system. The libraries were sequenced from both ends of the molecules to a total read length of 300nt from each end. Cluster density was 964k/mm2 with 85.9% of clusters passing filter.