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.