*4.4. Bioinformatic Processing of the Sequencing Data*

For bioinformatic processing, the terminal 15 bases of both forward and reverse reads were removed before merging and quality filtering using fastq\_mergepairs and fastq\_filter\_options from Usearch 10 [58]. Subsequently, merged fastq files were demultiplexed and trimmed using Cutadapt [59]. For 16S sequence determination, the Uparse and Sintax algorithms within Usearch using the Silva 16S rRNA database (v123) were applied. All reads were mapped to OTUs, and an OTU table was created using a Qubit device (Thermo Fisher Scientific). The V3+4 region of the 16S rRNA gene was amplified using 10 ng of bacterial template DNA with degenerate region-specific primers (341F: 5 -ACTCCTACGGGAGGCAGCAG-3 ; 806R: 5 -123 GGACTACHVGGGTWTCTAAT-3 ) containing barcodes and Illumina flow cell adaptor sequences in a reaction consisting of 25 (stool) or 35 (tissue) PCR cycles (98 ◦C 15 s, 58 ◦C 20 s, 72 ◦C 40 s) using the NEBNext Ultra II Q5 Master Mix (New England Biolabs, Ipswich, MA, USA). Amplicons were purified with Agencourt AMPure XP Beads (Beckmann Coulter, Brea, CA, USA), normalized and pooled before sequencing on an Illumina MiSeq device using a 600-cycle paired-end kit and the standard Illumina HP10 and HP11 sequencing primers. For bioinformatic processing, the terminal 15 bases of both forward and reverse reads were removed before merging and quality filtering using fastq\_mergepairs and fastq\_filter\_options from Usearch 10 [58]. Subsequently, merged fastq files were demultiplexed and trimmed using Cutadapt [59]. The 16S Uparse and Sintax [60] algorithms were performed within Usearch using the silva 16S rRNA database (v123) [61,62].
