Qubit and real time pcr

The library preparations for next-generation sequencing and Illumina MiSeq sequencing were conducted by GENEWIZ, Inc. (South Plainfield, NJ). The DNA samples were quantified using a Qubit 2.0 fluorometer (Invitrogen, Carlsbad, CA) and DNA quality was confirmed by electrophoresis (0.8 % agarose gel). The sequencing library was constructed using a MetaVx™ Library Preparation kit (GENEWIZ, Inc., South Plainfield, NJ). In brief, 5–50 ng of DNA was used for amplicon generation to cover the V3 and V4 hypervariable regions of 16S rDNA. Indexed and universal adapters were added to the ends of the 16S rDNA amplicons by limited-cycle PCR. The DNA libraries were validated with an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA) and quantified using Qubit and real-time PCR (Applied Biosystems, Carlsbad, CA). The DNA libraries were multiplexed and loaded on an Illumina MiSeq following the instructions from the manufacturer (Illumina, San Diego, CA). A 2 × 150 paired-end (PE) configuration was used for sequencing. The image analysis and base calling were processed using MiSeq Control Software. The initial taxonomy was performed on Illumina’s BaseSpace cloud computing platform.

Paired-end index libraries were constructed according to the manufacturer’s instructions (NEBNext® Ultra^™ DNA Library Prep Kit for Illumina®) with minor modifications. Briefly, the LG-PCR products were randomly fragmented into sizes <400 bp by sonication (Diagenode Bioruptor UCD-200). The fragments were treated with End Prep Enzyme Mix. Size selection was then performed for the adaptor-ligated DNA using AxyPrep Mag PCR Clean-up (Axygen), and fragments of ~400 bp (with approximate insert sizes of 250 bp) were recovered. Each sample was amplified by PCR for eight cycles using P5 and P7 primers. The PCR products were cleaned using AxyPrep Mag PCR Clean-up (Axygen), validated with an Agilent 2100 Bioanalyzer (Agilent Technologies), and quantified by Qubit and real-time PCR (Applied Biosystems). Libraries with different indexes were multiplexed and loaded onto an Illumina HiSeq instrument according to the manufacturer’s instructions (Illumina, San Diego, CA, USA). Sequencing was performed using a 2 × 100 paired-end (PE) configuration. Image analysis and base calling were conducted with HiSeq Control Software (HCS) + OLB + GAPipeline-1.6 (Illumina) on the HiSeq instrument. The sequences were processed and analyzed by GENEWIZ using NGSQCToolkit (v2.3).

Next generation sequencing library preparations and Illumina MiSeq sequencing were conducted at GENEWIZ, Inc. (Beijing, China). DNA samples were quantified using a Qubit2.0 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. A panel of proprietary primers was designed to anneal to the relatively conserved regions bordering V3, V4, and V5 hypervariable regions. The V3 and V4 regions were amplified using forward primer in sequence of CCTACGGRRBGCASCAGKVRVGAAT and reverse primer in sequence of GGACTACNVGGGTWTCTAATCC. The V4 and V5 regions were amplified using forward primer in sequence of GTGYCAGCMGCCGCGGTAA and reverse primer in sequence of CTTGTGCGGKCCCCCGYCAATTC. All PCR products were purified with the QIAgen DNA Mini Stool Kit. 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) by GENEWIZ.