The V4 region of the 16S ribosomal RNA from human stool sample (omnivorous)
The hypervariable region V4 of the 16S rRNA gene was amplified  with fusion primers that produced a barcoded sequencing library. Primer 806R contained the sequence of the P1 adapter at its 5 end, while primer 515F contained the sequence of the A adapter, barcode and linker upstream of the target specific sequence. Each sample was amplified in triplicate. PCR reaction mixture and temperature profile were set as described in Earth Microbiome Project . DNA in pooled triplicate PCR reactions from each sample was measured using the Ion Quantitation Library Kit (Thermo Fisher Scientific, Vilnius, Lithuania). The same amount of DNA from the amplicons was pooled to form the final pool and purified using Agencourt AMPure XP beads (Beckman Coulter, USA), with a bead to DNA ratio of 0.7:1. The concentration of final pooled amplicon library was determined with the Agilent 2100 Bioanalyzer using the High Sensitivity DNA Assay Kit (Agilent Technologies, CA, USA). The template for sequencing  on the Ion GeneStudio S5TM System was prepared using the Ion 520TM & Ion 530TM Kit-OT2. Samples were sequenced on three Ion 530TM chips (Thermo Fisher Scientific, CA, USA).