DNA extraction, amplification, and sequencing
Prior to DNA extraction, we macerated pooled samples using ceramic beads (sandflies), disposable tissue grinding pestles (mosquitos), and manual maceration using the blunt-end of a disposable tipped applicator (carrion flies). We extracted DNA from sandflies and carrion flies with the Qiagen Blood and Tissue Kit (Qiagen, Hilden, Germany) with slight modifications to the manufacturer’s specifications. Briefly, 200 uL of Buffer ATL and 20 uL of Proteinase K were added to the sample in a 1.7 mL Eppendorf tube and the sample incubated for 3-5 hours at 56°C. Post-incubation, samples were vortexed for 10 minutes and then purified through washing. The DNA was eluted in a final volume of 100 uL. Because we wanted to isolate both DNA and RNA from mosquitos for separate studies of viral surveillance, we extracted nucleic acids following a phenol-chloroform protocol modified from Griffiths et al. (2000) and Simister et al. (2011) with final ethanol precipitation and purification to isolate DNA. To purify for DNA, we resuspended the nucleic acid pellet in 50 uL of RNase-free water and incubated half the sample (25 uL) with RNase A in a final concentration of 100 ug/mL for 10 minutes at 37°C. The other half of the sample was purified for RNA and then synthesized for cDNA.
We amplified the extracted DNA from each iDNA sampler type in two separate reactions using a slight modification of the pan-vertebrate primer pair 12SV5F/12SV5R (Riaz et al., 2011), which targets approximately 100 base pairs in the 12S region of the vertebrate mitochondrial genome. We used the reverse primer 12SV5R (TTAGATACCCCACTATGC) as Riaz et al. (2011) and a modified version (we change the thymine to a degenerate base shown underlined) of the forward primer 12SV5F to allow for broader binding of vertebrate targets (Y AGAACAGGCTCCTCTAG). In brief, PCR reactions were carried out in a volume of 20 uL using 10 uL AmpliTaq Gold 360 Master Mix (final concentration of 1x), 5 uL of forward and reverse primers (final concentration of 0.25 uM), 3 uL of water, and 2 uL of DNA template. PCR cycling was as follows: initial denaturing at 95°C for 10 minutes followed by 40 cycles of 95°C for 30 seconds and 58°C for 30 seconds and 72°C for 30 seconds, and a final extension at 72°C for 7 minutes.
PCR amplicons were cleaned using PCRClean DX solid-phase reversible immobilization magnetic beads (Aline Biosciences, Woburn, MA, USA). Each PCR reaction was quantified using Accublue High Sensitivity dsDNA Quantitation kit (Biotium, Fremont, CA, USA) and normalized to 6 ng/uL. Each group of 384 PCR products was then pooled into a single library, for a total of two libraries for carrion flies, four libraries for sandflies, and two libraries for mosquitos. Individual libraries were then tagged with an additional 6 base pair identifying index using the NEBnext Ultra II DNA Library Prep kit (New England Biolabs, Ipswich, MA, USA). Pooled samples were analyzed on a bioanalyzer to confirm fragment size. The libraries were then sequenced using the Illumina HiSeq 3000 (Illumina, San Diego, CA, USA) at the Center for Genome Research and Biocomputing at Oregon State University.