V4 region of the 16S rRNA gene, technical control: distilled water
The V4 region of the 16s rRNA gene was amplified from each sample. 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. Accuprime High Fidelity Taq (Life Technologies cat # 12346094) was used. PCR was performed using the following conditions: initial denaturation at 95C for 2 min, 30 cycles including 95C for 20 sec, 55C for 15 sec, and 72C for 5 min, and final elongation at 72C for 10 min. PCR products were visualized using an E-Gel 96 with SYBR Safe DNA Gel Stain, 2% (Life technologies cat# G7208-02). Libraries were normalized using SequalPrep Normalization Plate Kit (Life technologies cat # A10510-01) following the manufacturers protocol for sequential elution. The concentration of the pooled samples was determined using Kapa Biosystems Library Quantification kit for Illumina platforms (KapaBiosystems KK4824). The sizes of the amplicons in the library were determined using the Agilent Bioanalyzer High Sensitivity DNA analysis kit (cat# 5067-4626). The final library consisted of equal molar amounts from each of the plates, normalized to the pooled plate at the lowest concentration. Library Preparation for Sequencing and Sequencing Libraries were prepared according to Illuminas protocol for Preparing Libraries for Sequencing on the MiSeq (part# 15039740 Rev. D) for 2nM or 4nM libraries. PhiX and genomes were added in 16S amplicon sequencing to create diversity.