Bacterial Profiling of Fecal Samples

DNA was extracted from faecal samples using QIAamp DNA Stool Minikit (Qiagen, Hilden, Germany). The DNA isolation followed the instructions of the manufacturer, but with an extra mechanical lysis step, using 0.1 mm Zirconium/Silica beads (Biospec products, Bartlesville, USA), 2 × 1 min at 6000 rpm with a Precellys evolution (Bertin Instruments, Montigny-le-Bretonneux, France). The isolated DNA was stored at − 20 °C until analysis. 16S rRNA gene amplicons were generated from the V3 and V4 region using the primers (341F 5′-CCTACGGGAGGCAGCAG-3′ and 806R 5′-GGACTACNNGGGTATCTAAT-3′). Sequencing libraries were generated using NEB Next^® Ultra™ DNA Library Prep Kit for Illumina (NEB, USA) and the amplicon library was sequenced on an Illumina HiSeq 2500 platform at Novogene. The generated paired-end reads were merged with FLASH (V1.2.7, http://ccb.jhu.edu/software/FLASH/)⁴¹ and assigned to each sample according to the sample specific barcodes. Quality filtering of sequence data was performed with QIIME (V1.7.0)^{42 (link)} and UCHIME^{43 (link)} was used to detect and remove chimeric sequences^{44 (link)}. The reads were clustered using Uparse software (Uparse v7.0.1001)^{45 (link)} and OTUs (Operational Taxonomic Units) were generated based on 97% sequence homology.