16S Illumina amplicon seq of Siofok basin, Lake Balaton planktonic bacterial community
For paired-end 16S rDNA amplicon sequencing, the variable V3 and V4 regions of the 16S rRNA gene were amplified by using forward (5-TCGT CGGCAGCGTCAGATGTG TATAAGAGACAGCCTA CGGGNGGCWGCAG-3) and reverse (5-GTCT CGTGGGCT CGGAGATGTGTATAAGAGAC AGGACTACHVGGGTATCTAATCC-3) (Klindworth et al. 2013). PCR reaction was contained 12.5 ng of DNA, 0.2 M of each Illumina 16S primers and 12.5 l of 2X KAPA HiFi HotStart Ready Mix supplemented with molecular grade water to 25 l final volume. The temperature profile was the following: initial denaturation for 5min at 95 C, 25 cycles of amplification (30 s at 95 C, 30 s at 55 C, 30 s at 72 C) The last step was a final extension for 5 min at 72 C. Amplicons were analysed by agarose gel electrophoresis. Paired-end fragment reads were generated on an Illumina MiSeq sequencer using MiSeq Reagent Kit v3 (600-cycle). Primary data analysis (base-calling) was carried out with Bbcl2fastq^ software (v2.17.1.14, Illumina). Reads were quality and length trimmed in CLC Genomics Workbench Tool 9.5.1 using an error probability of 0.05 (Q13) and a minimum length of 50 nucleotides as threshold.