16s_rRNA_v1v3
DNA from human stool using the PSP Spin Stool DNA Basic Kit (Invitek Diagnostics, Germany, #1038120300) with Stool DNA Stabilizer tubes (Invitek Diagnostics, Germany, #1038111100). The V1V3 hypervariable region of the 16S rRNA gene was amplified using barcoded primers 27F (5-AGAGTTTGATCCTGGCTCAG-3) and 534R (5-ATTACCGCGGCTGCTGG-3). Amplicons were verified by 1% SYBR Safe agarose gel electrophoresis, gel-purified using the NucleoSpin Gel and PCR Clean-Up Kit (Qiagen, Valencia, CA, USA, #740609) and quantified using the Qubit HS DNA quantification kit (Invitrogen, Carlsbad, CA, USA). Equimolar amplicons were pooled, library concentration was determined by qPCR (Library Quant Kit, Cat#KK4824, Roche Diagnostics), and the multiplexed library was sequenced on an Illumina MiSeq using the MiSeq Reagent Kit v3 (600-cycle, Illumina, San Diego, CA, USA).