Methodology:

Melted snow and ice (500-1500 mL) was filtered onto 0.2µm Whatman® Nucleopore using a sterilized filtration unit. DNA was isolated from the filter using MoBio PowerWater® DNA isolation kit and following the instruction manuals.

The highly variable V3/V4 region of the 16S rDNA was amplified with bacterial primers S-D-Bact-0341-b-S-17 forward and S-D-Bact-0785-a-A-21 reverse, with overhang illumina adaptor attached to the primer sequences, creating a single amplicon of ~460 bp (Klindworth et al., 2013). The reaction was carried out in 50 µl volumes containing 0.3 mg/ml Bovine Serum Albumin, 250 uM dNTPs, 0.5 uM of each primer, 0.02 U Phusion High-Fidelity DNA Polymerase (Finnzymes OY, Espoo, Finland) and 5x Phusion HF Buffer containing 1.5mM MgCl2. The following PCR conditions were used: initial denaturation at 95C for 5min, followed by 25 cycles consisting of denaturation (95oC for 40 s), annealing (55oC for 2 min) and extension (72oC for 1 min) and a final extension step at 72oC for 7 min. Samples were sequenced using illumina MiSeq platform at Liverpool Centre for Genomics Research and generated 2 x 300 bp overlapping pair-end reads.

The 16S sequences were further processed using mothur (v. 1.35) pipeline (Schloss et al., 2009). Chimeric sequences were identified and removed using UCHIME (Edgar et al., 2011). Reads were clustered into operational taxonomical units (OTUs), based on at least 97% sequence similarity, and assigned taxonomical identification against SILVA bacterial database (Quast et al., 2013).

Data collection:

Instruments used include:

PCR machine

MiSeq illumina sequencer