title: 16S rRNA V3-V4 amplicon sequencing of bacteria
design: 16S rRNA V3-V4 amplicon sequencing library preparation and Illumina MiSeq sequencing were conducted at GENEWIZ, Inc. (South Plainfield, NJ, USA). Briefly, microbial genomic DNA for each sample was normalized to 20 ng/l using a Qubit 2.0 Fluorometer (Invitrogen) and was used to generate amplicons using a MetaVx Library Preparation kit (GENEWIZ). The V3-V4 region of the 16S rRNA gene was amplified using the forward and reverse primers (341 Forward: CCTACGGRRBGCASCAGKVRVGAAT, 806 Reverse: GGACTACNVGGGTWTCTAATCC). Second limitedcycle amplification was performed to add multiplexing indices (barcode) and Illumina sequencing adapters. DNA libraries were validated by Agilent 2100 Bioanalyzer (Agilent Technologies) and quantified by Qubit 2.0 Fluorometer (Invitrogen). DNA libraries were multiplexed and loaded on an Illumina MiSeq instrument according to manufacturers instructions (Illumina). Sequencing was performed using the 2x250 paired-end configuration, and image analysis and base calling were conducted by the MiSeq Control Software embedded in the Illumina MiSeq instrument.