16S PacBio sequencing water - waterhole Mongolia GGBW1
Microbiomes from water were obtained using a 0.2 m Sterivex filter unit (Millipore, USA) that was cut into pieces with a sterile scalpel blade. The resulting filter pieces were placed into 1.5 ml microfuge tubes with 5 g of low-binding zirconium glass beads (OPS Diagnostics, NJ, USA). The QIAamp DNA Mini Kit (Qiagen, Germany) was then used to extract DNA with the following modifications: 350 l ATL lysis buffer plus 50 l of proteinase K were added in the initial lysis and vortexed at 3,000 rpm for 5 minutes at 56 C with an Eppendorf MixMate 175 (Eppendorf, Hamburg, Germany). Following centrifugation, the DNA was eluted in 100 l. The DNA from sediment was extracted using the Machery-Nagel NucleoSpin Soil kit. The recommended protocol for the kit was followed using 500 mg of sediment. The DNA concentration was determined using the Agilent Tapestation with Genomic tapes and reagents (Agilent Technologies, USA). Specific primers, originally designed by Wagner et al. 2016 (10), were used to amplify the full 16s rRNA gene (~1,500 bp). Each forward and reverse primer was tagged with a specific 16 bp sequence to identify samples when pooled for PacBio sequencing (Table S1). The PCR reactions were run in triplicate, with a total reaction volume of 20 l per sample. Each reaction contained the following per sample: 0.75 l of each forward primer 5`-AG RGT TYG ATY MTG GCT CAG-3` and reverse primer 5`-RG YTA CCT TGT TAC GAC TT-3`, 0.7 l BSA, 12.5 l of 2X MyFi mix (Bioline, Germany) and 5.3 l of nuclease-free water. The following thermal cycler conditions were used to amplify a ~1,500 bp product: 95C for 3 min, followed by 25 cycles denaturation 95C for 30 s, annealing 57C for 30 s, and extension 72C for 60 s and a final extension at 72C for 3 min. The triplicate PCR products were then pooled. Eight equally pooled samples were purified using 20 l AMPure XP beads (Agencourt Bioscience) according to Illuminas 16S metagenomic sequencing library preparation protocol. The DNA concentration was determined with the Agilent Tapestation using the D5000 tapes and reagents (Agilent Technologies, USA). PCR products from each of the three sample types (sediment, water and swabs) were pooled together, resulting in three pooled samples. The three pools were processed by the Max Delbrck Center, Berlin, for PacBio library construction and sequencing. Pools were individually purified using AMPure XP beads (Beckman Coulter) at a concentration of 0.9X. Libraries were then created for each sample pool using the PacBio (Pacific Biosciences, Menlo Park, CA) 5 kb template preparation protocol and the SMRTbell Template Prep Kit 1.0, following the manufacturers guidelines. The length and concentration of the libraries were then determined with the 2100 Agilent Bioanalyzer using the 1200 DNA chemistry (Agilent Technologies). Sequencing on the PacBio Sequel system was performed using the MagBead Standard protocol, C4 chemistry and P6 polymerase on a single v3 Single-Molecule Real-Time (SMRT) cell with 1  180 min movie for each sample. Each individual pool was run on a SMRT cell.