Amplicon sequencing
PCR amplification was done by kit KAPA HiFi HotStart ReadyMix (Roche, Basel, Switzerland) using an input DNA concentration of 50 ng/µl. We used ITS2 and 16S markers for the identification of fungi and bacteria, respectively. Genetic markers were amplified using labeled pairs of primers ITS3_KYO2 and ITS4_KYO3 (Toju et al., 2012) for ITS2 marker and primer pair 799F (Chelius and Triplett 2001) and 1115R (Redford et a. 2010) for 16S marker V5-V6 region. Each DNA sample was amplified in triplicates in separate 96-microtiter plates, which were subsequently pooled into one sample. Each 96-microtiter plate also included three negative controls (PCR grade water used as a template) and one positive control for fungal/bacterial species (a random DNA sample of one of our pure bacterial/fungal cultures was used as a template). Amplicons were then purified from oligonucleotides by SAP-Exo kit (Jena Bioscience GmbH, Germany). 1 µg of purified amplicon served as a template for library construction using KAPA HyperPlus Kit in combination with KAPA UDI primer mixes (Kapa Biosystems, Massachusetts, USA). Amplicon’s size selection of the final libraries was done by KAPA Pure Beads (Kapa Biosystems) and its effectiveness was checked on 1% agarose gel (SeaKem® LE Agarose, Lonza Group Ltd, Basel Switzerland). The amplicons size for ITS and 16S were around 450 bp and 300 bp, respectively. The quality of the ligated library was quantified using the EliZyme Library Quantification Kit (Elisabeth Pharmacon, Brno, Czechia). Library sequencing was done on the Illumina MiSeq platform (San Diego, California, USA) on a 2x300 bp paired-end reads run performed at CEITEC institute (Brno, Czechia).