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).