Bacterial community composition of Pan no. 60 (VBK) soda pan water collected on 15 Jun 2017
Bacterial primers 341F (CCTACGGGNGGCWGCAG) (Herlemann et al., 2011) and 805NR (GACTACHVGGGTATCTAATCC) (Apprill et al., 2015) were used for PCR amplifications. All PCR amplification was performed in duplicates in 20 l of final volume, which contained 4 l of 5xQ5 reaction buffer, 2 l of dNTP (2mM), 0.2 l of Q5 High Fidelity DNA polymerase (2U/l) (New England Biolabs), 0.5 l of each primer (10M), 11.8 l of nuclease free water and 1 l of template DNA. The following thermal cycle conditions were used for 16S rRNA gene amplification: initial denaturation at 98 C for 30 sec and 98 C for 10 sec, followed by 20 cycles (annealing at 48 C for 30 sec, extension at 72 C for 30 sec) and a final elongation step at 72 C for 2 min. Amplicons were pooled and subsequently purified using magnetic beads (Agencourt AMPure XP PCR Purification, 2013).  To prepare libraries for Illumina sequencing, primers were prolonged by illumina handles and index primers. This second PCR reaction contained 4 l of 5xQ5 Reaction buffer, 2 l of dNTP (2mM), 0.2 l of Q5 High Fidelity DNA polymerase, 1 l of each index primer (5 M), 9.8 l of nuclease free water and 1 l of template from the first PCR reaction. The following thermal cycle was used: initial denaturation 98 C for 30 sec and additional 98 C for 10 sec, followed by 15 cycles of denaturation 98 C for 10 sec, annealing 66 C for 30 sec, extension 72 C for 30 sec/kb and the final extension 72C for 2 min. Resulting amplicon libraries were purified again with magnetic beads (Agencourt AMPure) as described previously. Quantification of the libraries were carried out using a PicoGreen assay (Quant-iT PicoGreen dsDNA Assay Kit, Invitrogen). Sequencing  was performed at the Swedish National Genomics Infrastructure (NGI) (Uppsala, Sweden) on Illumina MiSeq platform (Illumina Inc, San Diego, CA, USA) in 2300 bp paired-end format and with v3 chemistry.