16S amlicon sequences of Ae. aegypti in different water types and Zika infection states
DNA from mosquitoes was extracted with a Quick-DNA Tissue/Insect Microprep Kit, using the manufacturer instructions, and eluted in 30 L elution buffer. DNA extracted from individual mosquitoes was PCR-amplified in the V4 hypervariable region of the 16S rRNA gene. The presence and size of amplicons were confirmed by gel electrophoresis using a DNA ladder to identify amplicon size. Negative controls, including DNA extraction controls (extraction protocol with sterile PBS) and PCR controls (PCR reaction with molecular-grade H2O) were included in each library preparation. 16S amplicon libraries were prepared by addition of Nextera XT Index Kit v2 Set A adapter sequences which were cleaned using KAPA Pure Beads, quantified by Qubit dsDNA HS Assay, pooled to equimolar concentrations of 5nM per sample, and sequenced at the University of California, Davis, DNA Core Laboratory using Illumina MiSeq PE250.