DNA Extraction, Sequencing, and Ribotype Diversity Analysis

Total genomic DNA was extracted in triplicate for the studied microbial mats. The DNAeasy Power Biofilm kit (Qiagen) was used for DNA extraction, using the protocol supplied with the kit. Bacterial 16S rRNA marker gene was amplified from the cellular fractions using the set of primers 8F15B (5′-AGAGTTTGATCCTGG-3′) and 515R14AM (5-TTACCGCGGCTGCT-3′) (Aguirre de Cárcer et al., 2011). The pool of samples with the prepared libraries was sequenced by Illumina MiSeq platform.

Bacterial 16S rRNA gene diversity was assessed with QIIME v2-2019.4 (Bolyen et al., 2019). Briefly, cleaned and trimmed paired reads were filtered and denoised using DADA2 plug-in (Callahan et al., 2016). For chimera identification, 300,000 training sequences were used. Identified amplicon sequence variants (ASVs) were aligned using MAFFT (Katoh et al., 2002) and further processed to construct a phylogeny with fasttree2 (Price et al., 2010). Taxonomy was assigned to ASVs using the q2-feature-classifier (Bokulich et al., 2018) and blasted against the SILVA v132 99% 16S sequence database (Quast et al., 2012). Sequences assigned to chloroplasts were removed from the dataset.

Sequences generated by this study were deposited to GenBank under the BioProject accession number PRJNA795814.