Illumina MiSeq paired end sequencing
For microbiome analysis, skin swabs were taken and analysed using 16S (variable region V1-V3) amplicon based next generation sequencing. Skin swabs for NGS were taken with swabs (Sigma-swab, MWE) and stored in 500 ?L Stool DNA Stabilizer solution (Stratec, Berlin, Germany) at -80 �C. For DNA extraction, the QIAamp UCP Pathogen kit (Qiagen: Hilden, Germany) was used; 650�l of buffer ATL were added to the tube containing the swab and the stabilizing solution and lysis was performed in the presence of 500mg of 100�m diameter zirconia-silica beads (BioSpec: Bartlesville, USA) using Precellys Evolution (Bertin technologies: Montigny-le-Bretonneux, France) for 90 seconds twice with 15 seconds break. The entire amount of lysate was then purified according to the manufacturer�s instructions and the DNA was then eluted twice with 50�l TRIS 10mM EDTA free. Each samples DNA was amplified with the primers 27F-YM (5 -AGAGTTTGATYMTGGCTCAG-3) and 534R (5 -ATTACCGCGGCTGCTGG-3). In a second PCR step, double barcodes were added. The amplicons were purified using AMPure XP beads (Beckman Coulter, Fullerton, Calif) with a bead/DNA ratio of 0.7:1 (vol/vol), according to the manufacturer's instructions. The purified indexed amplicons were quantified using the fluorescent dye-based Qubit� dsDNA HS Assay Kit (Invitrogen) and all samples were pooled equimolarly. Additionally, 12 biological and 4 technical negative controls as well as 2 positive controls treated the same as the samples were added to the library. Subsequently, the pool was purified once more with AMPure XP as described before. Before and after pooling, the High Sensitivity DNA LabChip Kit (Agilent Technologies) was used on the 2100 Bioanalyzer (Agilent Technologies) to analyze the integrity and peak distribution of the library pool. The pool was denatured using NaOH to ensure single stranded DNA. The 16S rRNA sequencing was performed with a final concentration of 8 pM of the library and 20% PhiX control library spike-in (Illumina Inc.) on Illumina MiSeq� platform (Illumina Inc.) using 2x300 bp pair-end reads (MiSeq� Reagent Kit v3 600 cycles; Illumina Inc.). For imaging, data processing and evaluation, the Illumina software MiSeq� Reporter (MSR) v 2.5.1.3 on the MiSeq� system and the Illumina Sequence Analysis Viewer (SAV) v 2.4.5 were used. OTU clustering against the SILVA database was performed using CLC Genomics Workbench 11.0.1 and its microbial genomics module. After quality control (merging and chimera reduction), samples with less than 1000 reads were excluded from analysis (2 samples from HE participant ID 6, week 1 pH 5.5 treated and week 6 pH 8.5 treated body site). The ?-diversity indices richness, evenness, shannon and simpson as well as the ?-diversity and frequency of the top 10 families and species were analyzed based on the RHEA R-scripts published by Lagkouvardos et al 2017.