title: Microbial Ecology Diversity of Natural Habitats
design: A Bacterial 16S rDNA v4v6 amplicon library for sequencing was generated by         using fusion primers containing either the A or B 454 Titanium amplicon adaptor from Roche         Diagnostics, (Indianapolis, IN, USA) followed by a multiplex identifier (MID) and ending         with a 16S specific sequence primer. The 16S primers were used in a mixture of one forward         and four reverse primers all in the 5´ to 3´ direction. Forward primer (565F) TGGGCGTAAAG         and reverse primers (1064R) were 1064R-1 CGACAGCCATGCANCACCT, 1064R-2 CGACAACCATGCANCACCT,         1064R-3 CGACGGCCATGCANCACCT and 1064R-4 CGACGACCATGCANCACCT. Conditions for the PCR reaction         was; 1X Platinum HiFi Taq polymerase buffer, 1.6 units Platinum HiFi polymerase, 3.7 mM         MgSO4, 200 lM dNTPs (PurePeak polymerization mix, ThermoFisher), and 400 nM primers. 5-25 ng         of sample DNA was added to a master mix to a final volume of 100 ll and this was divided         into three replicate 33 ll reactions. A no-template negative control for each MID was         included. Cycling conditions included an initial denaturation at 94 °C for 3 minutes; 30         cycles of 94 °C for 30 seconds, 57?60 °C for 45 seconds, and 72 °C for 1 minute; and a final         extension at 72 °C for 2 minutes using a Bio-Rad my-cycler. The quality and concentration of         the amplicon library was evaluated by using the Agilent Tapestation 2000 instrument from         Agilent according to manufacturer's protocol. The reactions were cleaned and products under         300 nucleic acid base pairs were removed using Ampure beads at 0.75X volume (Beckman         Coulter, Brea CA). The final products were re-suspended in 50 ll of 10 mM Tris-EDTA + 0.05%         Tween-20, quantitated using PicoGreen Quant-IT assay (LifeTechnologies), and assayed once         again on the Tapestation 2000 instrument. Amplicons were further titrated in equimolar         concentration before emulsion-PCR based on their double stranded DNA         concentrations.