CL-16S
Next generation sequencing library preparations and Illumina MiSeq sequencing were conducted at GENEWIZ, Inc. (Beijing, China). DNA samples were quantified using a Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA) and DNA quality was checked on a 0.8% agarose gel.5-50 ng DNA was used to generate amplicons using a MetaVx™ Library Preparation kit (GENEWIZ, Inc., South Plainfield, NJ, USA).A panel of proprietary primers was designed to anneal to the relatively conserved regions bordering V3and V4hypervariable regions. The v3 and v4 regions were amplified using forward primers containing the sequence “CCTACGGRRBGCASCAGKVRVGAAT” and reverse primers containing the sequence “GGACTACNVGGGTWTCTAATCC”. Besides the 16S target-specific sequence, the primers also contain adaptor sequences allowing uniform amplification of the library with high complexity ready for downstream NGS sequencing on IlluminaMiseq.  DNA libraries were validated using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA), and quantified by Qubit and real time PCR (Applied Biosystems, Carlsbad, CA, USA). DNA libraries were multiplexed and loaded on an Illumina MiSeq instrument according to manufacturer’s instructions (Illumina, San Diego, CA, USA).  Sequencing was performed using a 2x300paired-end (PE) configuration; image analysis and base calling were conducted by the MiSeq Control Software (MCS) on the MiSeq instrument.