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.

Samples included in this study were from UFI patients (n = 200), rodents and small mammals (n = 309), rodent-associated ectoparasites (chiggers = 199 pools, ticks = 59 pools, fleas = 23 pools, and lice = 4 pools), and ectoparasites collected from larger animals including dogs, cats, and cattle (ticks = 35 pools, fleas = 88 pools and lice = 12 pools) ( Table 1) . Samples were collected mainly from Bo Kluea and from nearby districts of Nan province (Figure 1) , Thailand. UFI samples were from inpatients and outpatients visiting the hospital with symptoms similar to scrub typhus infection or fever of unknown origin throughout the year 2017. The sampling of rodents and ectoparasites took place twice each year (wet and dry seasons) in 2014 and 2017, and only once in 2018 (dry season). Each sample was amplified in triplicate reactions to minimize PCR bias (Acinas et al., 2005; Sipos et al., 2007; Aird et al., 2011) and PCR products from the three reactions were pooled for each sample and purified before pooling with other samples for library preparation before NGS. All totaled, 929 samples were pooled according to their sample type, area of collection, season of collection, and host species into 183 NGS pools ( Table 2) . After NGS quality control procedures, 13,225,584 16S sequences from the field-collected samples and 38 control samples (6 extraction controls, 25 PCR controls, and 7 index controls) were used for analysis. From the 38 control samples, 153 bacterial genera were detected and then subtracted from the field samples' 16S sequence dataset before conducting downstream analysis. These bacterial genera were considered to be contaminants from molecular reagents, the environment (water), or from cross-contamination during sample processing and between NGS runs (Salter et al., 2014) . The number of pass-filtered raw reads per NGS pool ranged from 24,939 to 627,297, with the highest read (1,625,690) belonging to an ectoparasite pool collected from domesticated mammals (mean ± SD = 294,518 ± 152,314). The number of reads per NGS pool used in OTU assignment ranged from 1,583 to 160,184 (mean ± SD = 110,802 ± 34,095) ( Table 1 and Figure 2A ). The majority of samples with low numbers of reads were from chigger samples. Overall, 7.8% of OTUs (1,032,389 reads) were unclassifiable at the phylum level. Rarefaction curves demonstrated that sequence data for all samples approached completeness as indicated by the curve plateaus ( Figure 2B) . These data suggest that most bacterial profiles of all samples studied were nearly complete.