Metabarcoding of Hungarian sourdough samples

Total DNA from the sourdough samples wasextracted by the Macherey-Nagel (Duren, Germany) Genomic DNA From Food kitfollowing the manufacturers' protocols. 16Smetabarcoding was carried out using the 16S long-read metabarcoding kit(SQK-16S024) of Oxford Nanopore Technologies (Oxford, UK) according to themanufacturer's instructions. In the first step of the library preparation aPCR was performed for the amplification of the 16S rRNA gene target region(~1500 bp) and to add a unique barcode to each sample. DNA concentrations werequantified by Qubit fluorometer (Invitrogen, Waltham, MA). The initial DNAconcentration was 10 ng per sample, the final PCR mix contained 25 ul LongAmpHot Start Taq 2x Master Mix, 10 ul input DNA, 10 ul 16S barcode primers (each)and 5 ul nuclease free water. The reaction was performed using a thermal cycler(Biometra TAdvanced, Analytik Jena, Jena, Germany) with the following PCRconditions: initial denaturation at 95C for 1 min, 25 cycles of 95C for 20 s,55C for 30 s, 65C for 2 min, followed by a final extension step at 65C for 5min. The amplicons were purified with AMPure XP beads (Beckman Coulter, Brea,CA, USA) and eluted in 10 mM Tris-HCl pH 8.0 with 50 mM NaCl. DNA concentrationof the samples was quantified with a Nabi spectrophotometer (MicroDigital,Seongnam-si, South Korea). Approximately 100 fmol of the combined library wasloaded into an ONT SpotON flow cell. Super accurate basecalling andde-barcoding in the Guppy v6.4 software were performed after the sequencing.