16s sRNA sequencing of control sample
Whole 16S fragment (1500bp) was amplified by conventional PCR using universal primers 27F (5-AGAGTTTGATCCTGGCTCAG-3) and 1541R (5-AAGGAGGTGATCCAGCCGCA-3). Reaction conditions were conducted in a final volume of 25 l Lusing LongAmp Taq 2X Master Mix (New England Biolabs, Massachusetts, USA), primers at 400 nM each, and 3 l LDNA. Run cycles consisted of an initial denaturation step at 94C for 30 secondss followed by 30 cycles of amplification, consisting of denaturation at 94C for 20 secondss, annealing at 56C for 40 secondss, extension at 65C for 75 secondss and a final extension step at 65C for 10 minutes. Amplicons were visualized on a GelRed Nucleic Acid Stained 2% agarose gel, and if visible, they were used for sequencing. Oxford Nanopore ONT sequencing was realised with the MinIon Mk1C device and run on R10.4.1 flow cells. Native barcoding kit (SQK-NBD114.24) allowing the sequencing of the 1500bp 16S fragment in the 24 samples w was used according to the manufacturers protocol. The Minion was running for 48 hours, reads smaller than 200 bp were not taken into accountconsidered.