11D
DNA extraction was performed using the Eppedorf EpMotion liquid handling system following the Qiagen MagAttract PowerMicrobiome kit (Qiagen, catalog no. 27500-4-EP). Sequencing libraries were prepared by the University of Michigan Host Microbiome Core as described previously [120]. Following extraction, samples were quantify using the Quant-iT PicoGreen dsDNA Assay kit (Thermo Fisher, catalog no. P7589). The V4 region of the 16s rRNA gene was amplified from each sample using a dual indexing sequencing strategy in a 20-l PCR reaction containing 2 l of 10X Accuprime PCR II buffer (Life Technologies, catalog no. 12346-094), 5 l of 4 M universal primers 515-F (5-GTGCCAGCMGCCGCGGTAA-3) and reverse 806-R (5-GGACTACHVGGGTWTCTAAT-3, 0.15 l of Accuprime High-Fidelity Polymerase, and 1 l of DNA template and 11.85 l of sterile PCR-grade water. PCR cycling conditions were as follows: 2 min at 95C, 30 cycles at 95C for 20 s, 55C for 15 s, and 72C for 5 min, with a final extension at 72C for 10 min [121]. PCR products were visualized using an E-Gel 96 with SYBR Safe DNA Gel Stain, 2% (Life technologies, catalog no. G7208-02).  Libraries were normalized using SequalPrep Normalization Plate Kit (Life technologies, catalog no. A10510-01), the concentration of the pooled samples determined using Kapa Biosystems Library Quantification kit for Illumina platforms (KapaBiosystems, catalog no. KK4824) and the sizes of the amplicons were determined using the Agilent Bioanalyzer High Sensitivity DNA analysis kit (Agilent, catalog no. 5067-4626).  The final library consisted of equal molar amounts, normalized to the pooled plate at the lowest concentration and were prepared according to Illuminas protocol for preparing libraries for sequencing on the MiSeq (Illumina, catalog no. 15039740 Rev. D) for 2nM libraries. Sequencing was done on the Illumina MiSeq platform, using a MiSeq Reagent Kit V2 500 cycles (Illumina, catalog no. MS102-2003), according to the manufacturers instructions with minor modifications [121] including the use of Accuprime High Fidelity Taq (Life Technologies, catalog no. 12346094) in a 5.5 pM reaction to which a 15% PhiX spike was added. Sequencing reagents were prepared according to the Schloss SOP. Custom read 1, read 2 and index primers are added to the reagent cartridge and FASTQ files were generated for paired end reads [121].