Qubit and real time pcr

Next-generation sequencing library preparations and Illumina MiSeq sequencing were conducted at GENEWIZ, Inc. DNA samples were precisely quantified using a Qubit 2•0 Fluorometer (Invitrogen) and DNA quality was checked on a 0•8 % agarose gel once again. DNA, 5-50 ng, was used to generate amplicons using a MetaVxTM Library Preparation kit (GENEWIZ, Inc.). A panel of proprietary primers was designed to anneal to the relatively conserved regions bordering V3-V4 hypervariable 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 contained adaptor sequences allowing uniform amplification of the library with high complexity ready for downstream NGS sequencing on Illumina Miseq (31) . DNA libraries were validated using an Agilent 2100 Bioanalyzer (Agilent Technologies) and quantified by Qubit and real-time PCR (Applied Biosystems). DNA libraries were multiplexed and loaded on an Illumina MiSeq instrument, according to the manufacturer's instructions (Illumina). Sequencing was performed using a 2 × 250 or 2 × 300 paired-end configuration; image analysis and base calling were conducted by the MiSeq Control Software on the MiSeq instrument.