design: The V4 region of the 16s rRNA gene was amplified from each sample using the Dual indexing sequencing strategy developed by Dr. Patrick D. Schloss (1).  Sequencing was done on the Illumina MiSeq platform, using a MiSeq Reagent Kit V2 500 cycles (Illumina cat# MS-102-2003), according to the manufacturers instructions with modifications found in the Schloss SOP.  Accuprime High Fidelity Taq (Life Technologies cat # 12346094 ) was used instead of Accuprime Pfx supermix.  PCR was performed using the following conditions (Standard or Touch Down) shown below.  If additional template was used the water volume is changed accordingly.  PCR products are visualized using an E-Gel 96 with SYBR Safe DNA Gel Stain, 2% (Life technologies cat# G7208-02).  Libraries are normalized using SequalPrep Normalization Plate Kit (Life technologies cat # A10510-01) following the manufactures protocol for sequential elution.  The concentration of the pooled samples is determined using Kapa Biosystems Library Quantification kit for Illumina platforms (KapaBiosystems KK4824).  The sizes of the amplicons in the library are determined using the Agilent Bioanalyzer High Sensitivity DNA analysis kit (cat# 5067-4626).  The final library consists of equal molar amounts from each of the plates, normalized to the pooled plate at the lowest concentration.  For more information visit: https://github.com/SchlossLab/MiSeq_WetLab_SOP