title: Microbial community analysis
design: This study have sequenced different kinds of samples: fecal samples from human, beef cow, milk cow, pig, chicken, duck; rice paddy soil in Vietnam; and freshwater randomly obtained from the Yeongsan River (South Korea) in 2014. Feces and paddy soil (50 mg each samples) were directly processed for DNA extraction using MOBIO Power Fecal DNA isolation kit and MOBIO Power Soil DNA isolation kit (MO BIO Laboratories Inc., CA USA). While, 500 ml of each freshwater samples were filtrated through 0.45μm pore-size membranes (Advantec, Japan) then were sliced with sterile blades prior to the DNA extraction using GeneAll Exgene Soil DNA isolation kit (GeneAll Biotechnology Co. Ltd, Republic of Korea).  PCR was carried out to amplify v4 region of 16S rRNA gene of bacteria and archaea as previously described (Kozich et al., 2013). Two μl of the total DNA from each sample was used as a template, and amplification was done in triplicates using Maxime PCR PreMix Kit (iNtRON Biotechnology Inc., Republic of Korea) with the following conditions: 95oC 2 minutes; 30 cycles: 95oC 20 seconds, 55oC 15 seconds, 72oC 1 minute; 72oC 5 minutes. Obtained PCR products were further gel-purified using AccuPrep Gel Purification kit (Bioneer Inc., Republic of Korea). All obtained DNA was quantified using Qubit (Invitrogen, CA, USA), and equimolar purified amplicons were pooled and stored at -20°C until sequenced. Amplicons were sequenced using Illumina MiSeq platforms according to the manufacturer’s instructions (Illumina Inc., USA).  MiSeq Sequence adapters, unique molecular identifier (MID) tags, pads and linkers were incorporated at the 5’ end of both target-specific primers to allow identification of individual sequences from pooled amplicons (Forward primer: 5’-GTGTGCCAGCMGCCGCGGTAA-3’; Reverse primer: 5’-CCGGACTACHVGGGTWTCTAAT-3’).
attributes.tag: loader
attributes.value: latf-load
attributes.tag: quality
attributes.value: PHRED_33