title: 16s rRNA amplicon sequencing of human gut metagenome: OPG1029-01
design: The 16S rRNA V4-V5 genomic regions from total gut DNA samples were amplified with 515f-y/926r primer pair via PCR (98 C for 3 min, followed by 27 cycles at 98 C for 20 s, 55 C for 30 s, and 72 C for 30 s, and a final extension at 72 C for 2 min). PCR products were purified using AMPure XP Beads (Beckman, A63880) and amplified with Illumina P7 and P5 primer via PCR (98 C for 30 s, followed by 6 cycles at 98 C for 20 s, 60 C for 30 s, and 72 C for 30 s, and a final extension at 72 C for 2 min). The post PCR products were purified again using AMPure XP Beads (Beckman, A63889). Library DNA was mixed with fluorescent dye (Qubit dsDNA HS Reagent) for quantitative quality control (concentration 2 ng / L) using a Qubit 3.0 fluorometer. Library fragment size was detected by 2% agarose gel electrophoresis and the library was qualified with no primer dimer contamination below 100 bp and library main band around 500 bp. The DNA library was pooled and sequenced on Illumina Miseq sequencing platform with PE 332 bp protocol with overlapping reads.