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.

The sequence reads generated by the 16S rRNA on MiSeq sequencers were processed on the CLC Genomics workbench v 11.0.1 (Qiagen, Aarhus A/S 1 ). High-throughput sequences were imported into CLC Genomics Workbench according to quality scores of Illumina pipeline 1.8. In order to achieve the highest quality sequences for clustering, paired reads were merged in CLC microbial genomics module v3.0 using default settings (mismatch cost = 1; minimum score = 40; gap cost = 4 and maximum unaligned end mismatch = 5). Primer sequences were trimmed from merged reads using parameters (trim using quality scores = 0.01, trim ambiguous nucleotides = 2, and discard read length shorter than 150 bp). Samples were removed from analysis if the number of reads was less than 100 or less than 25% from the median (the median number of reads across all samples).of minimum read from the median. Chimeric sequences were detected and removed. Filtered sequences were clustered into operational taxonomic units (OTUs) according to a threshold of 97% sequence identity. All such processes were performed using CLC microbial genomics module v3.0. Reference OTU data used in the present study were downloaded from the Greengenes database V13.8 (DeSantis et al., 2006) and SILVA 16S V132 (Quast et al., 2013) . Alpha rarefaction curve plots were generated among samples using CLC Microbial Genomics Module v3.0 with default parameter settings (minimum depth = 1, maximum depth = 100,000 and number of point = 20).