16S rRNA Gene Sequencing Protocol

For each sample, 16S rRNA DNA libraries were generated following a protocol described previously (47 (link)). Briefly, the V4 region of the 16S rRNA gene was targeted for PCR amplification in triplicate using 5Prime Hot master mix (QuantaBio, USA) with a modified universal bacterial 16S primer pair (515F/806R) (48 (link)). PCR products for each amplicon were pooled, run on a 2% agarose gel for visual confirmation of the libraries, and quantified with a Quant-iT PicoGreen double-stranded DNA (dsDNA) assay kit (Life Technologies, USA). One hundred nanograms of each amplicon was pooled and cleaned using a QIAquick PCR purification kit (Qiagen, USA). The library was then quantified using a Qubit dsDNA BR assay kit (Invitrogen, USA) and mixed with bacteriophage phiX DNA (10%). Paired-end sequencing (250 bp) was performed on a MiSeq platform (Illumina, USA) in iGE3, Institute of Genetics and Genomics in Geneva, CMU, University of Geneva. Sequencing results were obtained and demultiplexed using the standard method supplied by the MiSeq Illumina platform.