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.

Real-time PCR and PCR assays were performed on positive NGS pools to confirm the detection of pathogen and the taxonomic species assignment generated by NGS analysis. The detail of assays and target gene(s) for selected pathogens was provided as online Supplementary Data (Supplementary Table S1 ) (Norman et al., 1995; Barbour et al., 1996; Horinouchi et al., 1996; Schwaiger et al., 2001; Scoles et al., 2001; Smythe et al., 2002; Park et al., 2004; Klee et al., 2006; Chmielewski et al., 2009; Colborn et al., 2010; Ganoza et al., 2010; Billeter et al., 2011; Parola et al., 2011; Diaz et al., 2012; Lalzar et al., 2012; Shakya et al., 2013; Gofton et al., 2015a; Pereira et al., 2018) . For all real-time PCR, the reaction consisted of 1X Platinum quantitative PCR SuperMix-UDG (Invitrogen) using standard real-time PCR conditions with primer/probe concentrations and annealing temperatures as indicated in Supplementary Table S1 . For conventional PCR, the assay was carried out in a 50 µl reaction volume containing 0.5 U of iProof High-Fidelity DNA Polymerase, 200 µM dNTPs, MgCl 2 and primer concentration as indicated (Supplementary Table S1 ). The PCR conditions consisted of 98 • C for 3 min, followed by 40 cycles of 98 • C for 10 s, annealing temperature for 30 s, and 72 • C for 45 s.