Gut Microbiome Profiling via Illumina Sequencing

Patients received written instructions for stool collection and immediate transport to the study center or stool samples were directly picked up by the study team. Stool samples were frozen at −80 °C upon arrival in the study center. Total DNA was extracted from frozen stool samples using the MagnaPure LC DNA Isolation Kit III (Bacteria, Fungi) (Roche, Mannheim, Germany) according to the manufacturer’s directive including mechanic and enzymatic lysis [32 (link)]. Hypervariable regions V1–V2 were amplified in a target-specific PCR using the primers 27F and R357 (27F-AGAGTTTGATCCTGGCTCAG; R357-CTGCTGCCTYCCGTA) and sequenced with the Illumina MiSeq technique (Illumina, Eindhoven, The Netherlands) [32 (link)]. Resulting FASTQ files were taken for data analysis. Sequencing was done with the help of the Core Facility for Molecular Biology at the Center for Medical Research in Graz.