PacBio metabarcoding (rRNA ITS) of Alnus glutinosa root samples, Algeria

DNA from Alnus roots was extracted from 0.25 g of roots from each composite sample using the PowerMax Soil DNA Isolation kit (Qiagen. Before extraction, roots were pulverized by bead beating (Retsch MM400), weighted and 520 µl of proteinase K (600 units per ml) was added to the samples. The full ITS region (ITS1-5.8S-ITS2) was amplified via PCR using the primer pair ITS9mun and ITS4ngsuni (Tedersoo & Anslan, 2019). PCR reactions contained the following components: 5 µl of 5x HOT FIREPol Blend Master Mix, 0.5 µl of each 20 mM primer, 1 µl of DNA extract, and 18 µl of ddH2O. The cycling conditions were as follows: initial denaturation at 95 °C for 15 min, followed by 30 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min, with a final extension at 72 °C for 10 min, and a hold at 4 °C. PCR products from duplicate reactions were pooled and visualized on a 1% agarose gel to ensure appropriate amplicon size (600-800 bp). Negative controls were implemented to detect potential contamination. Pooled amplicons were purified with FavorPrepTM kit (Favorgen) and sent to the Norwegian Sequencing Centre for PacBio SMRTbell library preparation and sequencing on a Sequel II instrument, following the manufacturer's protocol.