## 2 Materials and Methods
### Amplicon-Based Metagenomic Sequencing (Illumina MiSeq) and Metagenomic Data Processing  
The V3 and V4 regions of the bacterial 16S rRNA gene were sequenced by creating two-step and three-step, Nextera barcoded, PCR libraries using the locus specific primer pair 341F (5\({}^{\prime}\)-CCT ACG GGN GGC WGC AG-3\({}^{\prime}\)) and 805R (5\({}^{\prime}\)-GAC TAC HVG GGT ATC TAAC TCC-3\({}^{\prime}\)). The PCR libraries were sequenced on an Illumina MiSeq platform using a v3 600 Cycle kit (Illumina Inc., San Diego, CA, USA) to produce paired-end reads. The reads that passed Illuminas chastity filter were subject to demultiplexing and trimming of any Illumina adaptor residuals by Illuminas own real-time analysis software (MiSeq reporter software v2.6 (Illumina Inc., San Diego, CA, USA); no further refinement or selection). The FastQC software version 0.11.7 (Babraham Institute, Cambridge, UK) was used for determining the quality of the reads [19]. Paired-end reads were discarded if the locus-specific V3-V4 primer could not be trimmed from the sequencing reads with the software cutadapt v1.18 [20]. The sequenced molecule was reformed in silico with the software USEARCH version 11.0.667 by merging the trimmed forward and reverse reads of each paired-end read with a minimal overlap of 15 bp [21]. After a quality filtration step of the merged sequences, whereby maximal one expected erroneous base per merged read was accepted other reads including ambiguous bases or outliers regarding the expected amplicon size distribution were discarded. The surviving reads were denoised using the UNOISE algorithm implemented in USEARCH to form operational taxonomic units (OTUs) with a similarity level of 97% discarding singletons and chimeras in the process [22]. After filtering the resulting OTU abundance table for possible barcode bleed-in contaminations with the UNCROSS algorithm, the UNBIAS algorithm was used for adjusting the abundances for 16S copy numbers [23,24]. OTUs were compared against the reference sequences of the RDP 16S database release 11. The determination of the taxonomies was done with the SINTAX algorithm implemented in USEARCH whereby a minimum confidence threshold of 0.6 was set [25,26]. For visualizing the metagenome krona charts were used [27]. Alpha diversity was calculated by using the richness (observed), Simpson and Shannon indices. The weighted UniFrac distance method based on rarefied OTU abundance counts per sample was used for the calculation of the beta diversity. To expose possible patterns of inter-sample relations, these sample distances were then used in a detrended correspondence analysis (DCA). Further analyses of the tested groups such as the variance (PERMANOVA) and similarities (ANOSIM) were done. Alpha diversity calculations (Simpson and Shannon indices) beta diversity calculations including DCA and rarefaction analysis were performed with the R software packages phyloseq v1.22.3 and vegan v2.5-1. The Simpson index can take values between 0 and 1.0; the closer the value is to 1.0, the less diverse the microbial community is. This index is contrary to the Shannon index, where a high value means a high diversity of the microbial population [28, 29, 30]. The library construction, sequencing and data analysis described in this section were performed by Microsynth AG (Balgach, Switzerland).