PCR data and comparative performance of Bacteroidales microbial source tracking genetic markers

We reported modified endpoint PCR results analyzed by universal and human-, swine-, and cattle-specific Bacteroidales gene markers with human sewage and animal fecal samples (i.e., swine, cattle, chicken, goat, sheep, buffalo, and duck) from Tha Chin and Chao Phraya watersheds. Annealing locations of PCR primers were illustrated by maps of 16s rRNA Bacteroidales genes. We also summarized previously published work on the performance of the PCR assays. For further discussion of the data presented here, please refer to Somnark et al., Performance evaluation of Bacteroidales genetic markers for human and animal microbial source tracking in tropical agricultural watersheds, Environ. Pollut. 236 (2018) 100–110.


a b s t r a c t
We reported modified endpoint PCR results analyzed by universal and human-, swine-, and cattle-specific Bacteroidales gene markers with human sewage and animal fecal samples (i.e., swine, cattle, chicken, goat, sheep, buffalo, and duck) from Tha Chin and Chao Phraya watersheds. Annealing locations of PCR primers were illustrated by maps of 16s rRNA Bacteroidales genes. We also summarized previously published work on the performance of the PCR assays. For further discussion of the data presented here, please refer to Somnark  Composite fecal and sewage samples were collected, and DNA extraction was performed Experimental features PCR primers originally designed as endpoint and quantitative PCR were used in the modified endpoint PCR assays.

Data source location
Samples were collected from Tha Chin (Chai Nat, Suphan Buri, Nakhon Pathom, and Samut Sakhon provinces) and Chao Phraya (Phra Nakhon Si Ayutthaya, Pathum Thani, and Bangkok provinces) watersheds, located in the central part of Thailand.

Data accessibility
Data are with this article Value of the data -PCR results of Bacteroidales-modified endpoint PCR markers could be compared with microbial source tracking (MST) studies in other geographic areas for further development of region-specific MST methods. -Bacteroidales PCR primer maps could offer an insight into annealing regions of primers for further design of new primers or evaluating currently available primers with their performance. -A summary of PCR assays that are originally designed and adopted to other regions could serve as a database for comparing the MST method performance in different geographical areas.

Data
We performed endpoint PCR assays modified from published methods originally in PCR and qPCR platforms. PCR results of ten good-performing modified endpoint PCR assays against human sewage and animal fecal samples from Tha Chin and Chao Phraya watersheds are shown (Table 1). There were six modified endpoint PCR assays that demonstrated potentially low sensitivity or specificity during the process of testing against a limited number of samples and therefore were not further tested with total samples (Table 2). We also compiled sensitivity and specificity data of previously published Bacteroidales genetic markers from both studies that originally designed the assays and studies that adopted the designed assays to be used in another geographic location (Table 3). To provide further insight into PCR performance, we mapped PCR primers to 16 s rRNA gene of human-, swine-, and cattle-associated Bacteroidales (Figs. 1 and 2). Amplified PCR products with universal and human-, swine-, and cattle-specific Bacteroidales PCR assays were presented (Fig. 3).

Sample collection and DNA extraction
Raw human sewage and non-human fecal samples were collected from Tha Chin and Chao Phraya watersheds. One composite fecal sample was prepared by mixing fresh feces of at least 20 individuals. Samples were transported on ice to the laboratory. DNA extraction of composite fecal samples and 0.22-µm-pore-size mixed cellulose ester membrane (Merck Millipore, Billerica, MA, USA) after 50-

PCR method modification and performance criteria
PCR primers targeting universal and human-, swine-, and cattle-specific fecal markers were selected from both endpoint and quantitative PCR platforms (Table 4). A 10-μL PCR is composed of 0.5 μL each of 10 μM forward primers and 10 μM reverse primers, 1-μL of DNA template (corresponding to 0.2, 2.0 or 20 ng total DNA), 5 μL of DreamTaq PCR Master Mix (2×; Thermo Fisher Scientific, Waltham, MA, USA), and sterile water. The reaction was processed in a Mastercycler Pro thermocycler (Eppendorf, Hamburg, Germany). PCR cycling conditions were modified as follows: initial denaturation at 95°C for 3 min; 30 cycles of a denaturation step at 95°C for 30 s, an annealing step at varying temperature and time (Table 4), and an elongation step at 72°C for 30 s; and a final extension at 72°C for 10 min. PCR products were visualized with a Gel Doc XR system (BIO-RAD, Hercules, CA, USA). PCRs were run in duplicate. No-template controls and extraction blanks were included for quality control. Performance criteria including sensitivity, specificity, and accuracy were calculated as TP/(TPþ FN), TN/(TN þFP), and (TP þTN)/(TP þFP þTN þFN), respectively, where TP, FN, TN, and FP, are true positive, false negative, true negative, and false positive, respectively.
Limited number of animal samples tested for certain assays due to potentially low sensitivity or specificity. Table 3 Sensitivity and specificity of Bacteroidales markers in studied in which the assays were originally designed and adopted to other geographic regions.    All primers were BLASTed against the NCBI database. The representative sequences from human feces (Accession no. AB242143.1 [24]) and swine feces (AB506329.1 [25]) were selected to align with specific primers. Human-specific, swine-specific and universal Bacteroidales primers are indicated in dotted, dashed and solid arrows, respectively.