16S rRNA gene library preparation and sequencing
ALDRICH
The sequencing library was prepared according to Earth Microbiome Project’s Protocol [The PCR reactions for library preparation were carried out in triplicates as follows: 20 ng of template DNA was mixed with 5PRIME HotMasterMix (Quantabio, USA) consisting of 1U Taq polymerase, 45 nM Cl, 2.5 mM Mg2Then, libraries were pooled in equimolar concentrations, and the amplicon pool was purified according to the protocol using AMPure XP beads (Beckman Coulter, USA). Prior to amplicon sequencing, the amplicon pool was diluted in 10 mM Tris–HCl (pH 8.5) to a final concentration of 4 nM. Following the Illumina recommendations, the amplicon pool was denaturated using an equal amount of 0.2 M NaOH (BioUltra) (Sigma Aldrich Chemie GmbH, Germany) and further diluted to 12 pM in hybridization buffer (HT1 buffer included in the Reagents Kit v3, Illumina, USA). The pool was finally spiked with 5% denaturated PhiX control library (Illumina, USA). The sequencing was performed using the MiSeq sequencing platform with the Reagents Kit v3, 600 cycles (Illumina, USA).