16S rRNA T2-Lake
For the sequencing, the 16S rRNA genes, V3V4 hypervariable regions (amplicons of approximately 459 bp) were sequenced. PCR amplification was carried out using Q5 Hot Start High-Fidelity 2X Master Mix using reaction conditions as recommended by the manufacturer (95 C for 3 minutes, 25 cycles of 95 C for 30 seconds, 55 C for 30 seconds, 72 C for 30 seconds and, ater the last cycle, 72 C for 5 minutes) with region-specific (341F and 785R)57 primers that include the Illumina flowcell adapter sequences. The primer sequence was as fol- lows: forward primer: 5 CGGGNGGCWGCAG 3, reverse primer: 5 GACTACHVGGGTATCTAATCC 3. The Illumina overhang adapter sequences added to the locus-specific-sequences were as follows: forward over- hang: 5 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG (locus-specific sequence), reverse overhang: 5 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG (locus-specific sequence).