title: 16S sequencing of animal feed
design: Total DNA from the feedstuff samples was extracted by E.Z.N.A. Soil DNA Kit and Macherey-Nagel (Dren, Germany) Bioanalysis Genomic DNA From Soil kit following the manufacturers protocols. 16S metabarcoding was carried out using the 16S long-read metabarcoding kit (SQK-16S024) of Oxford Nanopore Technologies (Oxford, UK) according to the manufacturers instructions. In the first step of the library preparation a PCR 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 were quantified by Qubit fluorometer (Invitrogen, Waltham, MA). The initial DNA concentration was 10 ng per sample, the final PCR mix contained 25 l LongAmp Hot Start Taq 2 Master Mix, 10 l input DNA, 10 l 16S barcode primers (each) and 5 l nuclease free water. The reaction was performed using a thermal cycler (Biometra TAdvanced, Analytik Jena, Jena, Germany) with the following PCR conditions: 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 5 min. The amplicons were purified with AMPure XP beads (Beckman Coulter, Brea, CA, USA) and eluted in 10mM Tris-HCl pH 8.0 with 50mM NaCl. DNA concentration of the samples were quantified with a Nabi spectrophotometer (MicroDigital, Seongnam-si, South Korea). Approximately 100 fmol of combined library was loaded into an ONT SpotON flow cell.