title: 16S rRNA library
design: DNA was amplified using universal bacterial primers targeting the V4 region of the 16S rRNA gene, with both forward and reverse primers being used. Each primer was uniquely barcoded to match the sequences to their respective samples. The PCR mixture was composed of Q5 High-Fidelity 1X Master Mix (New England Biolabs GmbH, Germany) along with 0.5 M of each primer, 2 L of DNA, and sterile water, resulting in a 25 L final volume. Thermal cycling conditions consisted of an initial denaturation step at 94C for 3 minutes, followed by 30 amplification cycles (94C for 45 seconds, 50C for 60 seconds, 72C for 90 seconds), and an extension at 72C for 10 minutes (Primus 25, Peqlab Biotechnologie GmbH, Germany or FlexCycler2, Analytik Jena AG). Negative controls were performed simultaneously using sterile water as a template to monitor and control contamination. Positive controls from a mock community (HM-782D) were included to assess the PCR and sequencing error rates. The PCR products were checked for amplicon presence through agarose gel electrophoresis (2%) and underwent subsequent purification using Agencourt AMPure XP beads (Beckman Coulter, Germany) according to the manufacturer's instructions. The quality and concentration of the products were assessed using the Quant-iT PicoGreen dsDNA Assay Kit (ThermoFisher scientific GmbH, Dreieich, Germany) and the Bioanalyzer (Agilent Technologies Inc., Bblingen, Germany). In the final step, sequencing adapters were ligated to the library, followed by paired-end sequencing on a 250-cycle Illumina MiSeq system.