Metagenomic Approach to Characterizing Disease Epidemiology in a Disease-Endemic Environment in Northern Thailand

Takhampunya, Ratree; Korkusol, Achareeya; Pongpichit, Chalermpol; Yodin, Komsan; Rungrojn, Artharee; Chanarat, Nitima; Promsathaporn, Sommai; Monkanna, Taweesak; Thaloengsok, Sasikanya; Tippayachai, Bousaraporn; Kumfao, Naruemon; Richards, Allen L.; Davidson, Silas A.

For DNA from all ectoparasites, a fragment of 16S rDNA (V1-V6) region (1,016 bp) was amplified in triplicate in a first-round PCR using primers; 27F-Y (5 -GAGTTTGATCCTGGCTYAG-3 ), 1061R (5 -CRRCACGAGCTGACGAC-3 ) (Ong et al., 2013) , and 2.5 µM MidBlocker oligonucleotide to inhibit 16S Candidatus Midichloria mitochondrii amplification (Gofton et al., 2015b) . The reaction was performed in a 20-µl volume containing 3 µl of ectoparasite DNA, 400 nM each primer, 200 µM dNTPs, 1.5 mM MgCl 2 , 1× PCR buffer, and 0.4 U of iProof High-Fidelity DNA Polymerase (Bio-Rad). Amplification was performed using a T100 DNA thermal cycler (Bio-Rad) under the following conditions: initial denaturation at 98 • C for 3 min; 40 cycles of 98 • C for 10 s, 60 • C for 20 s, and 72 • C for 30 s; and a final extension at 72 • C for 5 min. The second amplification was performed as described above for human and rodent samples. Negative control PCR reactions were included in every experimental run using Ultrapure DNA/RNA-free distilled water in place of DNA template. PCR reactions were also performed with eluates from mock DNA extractions. Three PCR products from each sample were pooled and cleaned using AMPure magnetic bead-based purification system (Beckman Coulter, United Kingdom) following the manufacturer's instructions. Purified PCR products were eluted and quantified using the Quant-iT PicoGreen dsDNA assay (Invitrogen Life Technologies, MA) according to the manufacturer's protocol. Each purified PCR was normalized and then pooled again with other purified PCR products from other samples by; (i) gender and age group for UFI patients, (ii) season of collection, location (sub-district/district), and rodent genera/species for rodents and rodent chiggers, and (iii) the host type they were collected from, genus/species and stages of ectoparasites for all other ectoparasites (ticks, fleas, and lice) collected from rodents and domesticated mammals (dogs and cows). Additional details on sample pooling for NGS are provided in Supplementary Table S2 .