Total DNA Isolation and 16S rRNA Gene PCR

For total DNA isolation, frozen samples were thawed, treated with DTT (50 µg/ml at 37°C for 20 min) and 250 µl of the sample was used for isolation according to published protocols in a MagnaPure LC device (Roche, Mannheim, Germany) with the MagnaPure LC DNA III Isolation Kit (Bacteria, Fungi) (Roche, Mannheim, Germany) (28 (link)). DNA was eluted in 50-µl elution buffer (Roche, Mannheim, Germany) and stored at -20°C till analysis. For 16S rRNA gene PCR, 2 µl of template DNA were used in a standard PCR setup in 25 µl reactions in triplicates with Roche High Fidelity Polymerase (Mannheim, Germany) and the target-specific primers 515F (GTGYCAGCMGCCGCGGTAA) and 926R (CCGYCAATTYMTTTRAGTTT), 30 amplification cycles, according to published protocols (28 (link)). The indexing of samples, pooling, purification, and quality control were performed as published elsewhere, and the final library was sequenced at 9pM with 20% PhiX on an Illumina MiSeq desktop sequencer (Illumina, Eindhoven, Netherlands) with v3 600 cycles chemistry in 2×300 sequencing mode. FASTQ files were used for data analysis, and raw data were archived in the European Nucleotide Archive (ENA) and can be downloaded with the accession number PRJEB51689 (https://www.ebi.ac.uk/ena/; last access March 2022).