title: Microbial Ecology Diversity of Natural Habitats
design: Amplicon library preparation of the 16S Bacteria rRNA gene hypervariable v6 region was performed by amplification by using custom fusion primers consisting of the illumina bridge adaptor, 12 different inline barcodes on the forward primer and 8 different indices on the reverse primer, and the 16S specific target sequence; Forward Primers was (967F) and reverse primer was (1064R), Table 2 4.The PCR was carried out in triplicate reactions in a 33 ?L reaction volume of 1.0 U Platinum Taq Hi-Fidelity Polymerase (Life technologies, Carlsbad CA), 1X Hi-Fidelity Buffer, 200 ?M dNTP PurePeak DNA Polymerase mix (Pierce Nucleic acid Technologies, Milwaukee, WI), 1.5 mM MgSO4, 0.2 ?M of each primer and ~10 ng template DNA. Each primer pair had a no template control. Cycling conditions were; an initial 94°C 3 minute denaturation, 30 cycles of; 94°C for 30s, 60°C for 60s and 72°C for 90s followed by a final 10 minute extension at 72°C.The triplicate PCR reactions were pooled before purification on a QIAquick PCR clean-up 96 well plate (Qiagen, Valencia Ca). Purified DNA was quantified by using Picogreen dsDNA reagens (Life Technologies, Carlsbad, CA) and pooled in equimolar amounts. Further amplicons were size selected for products in the range of 260 bp on 1% agarose by using Pippin prep (SageScience Beverly MA). qPCR was used to measure concentrations prior the 150 nucleotide paired-end run sequencing on Illumina NextSeq500 medium output mod. PhiX DNA at 25 % served as the control DNA for the run. Amplicon library preparation of the 16S Bacteria rRNA gene hypervariable v6 region was performed by amplification by using custom fusion primers consisting of the illumina bridge adaptor, 12 different inline barcodes on the forward primer and 8 different indices on the reverse primer, and the 16S specific target sequence; Forward Primers was (967F) and reverse primer was (1064R), Table 2 4.The PCR was carried out in triplicate reactions in a 33 ?L reaction volume of 1.0 U Platinum Taq Hi-Fidelity Polymerase (Life technologies, Carlsbad CA), 1X Hi-Fidelity Buffer, 200 ?M dNTP PurePeak DNA Polymerase mix (Pierce Nucleic acid Technologies, Milwaukee, WI), 1.5 mM MgSO4, 0.2 ?M of each primer and ~10 ng template DNA. Each primer pair had a no template control. Cycling conditions were; an initial 94°C 3 minute denaturation, 30 cycles of; 94°C for 30s, 60°C for 60s and 72°C for 90s followed by a final 10 minute extension at 72°C.The triplicate PCR reactions were pooled before purification on a QIAquick PCR clean-up 96 well plate (Qiagen, Valencia Ca). Purified DNA was quantified by using Picogreen dsDNA reagens (Life Technologies, Carlsbad, CA) and pooled in equimolar amounts. Further amplicons were size selected for products in the range of 260 bp on 1% agarose by using Pippin prep (SageScience Beverly MA). qPCR was used to measure concentrations prior the 150 nucleotide paired-end run sequencing on Illumina NextSeq500 medium output mod. PhiX DNA at 25 % served as the control DNA for the run.