IMIS - Marine Research Groups

Samples in all lakes were obtained from surface depths (0.5–1 m), and approximately in the center of the lake. Samples were then filtered with a vacuum system onto 0.2 μm polycarbonate filters (Nucleopore, Whatman) and stored frozen (−20°C) in Allprotect Tissue Reagent (QIAGEN).
Water samples (n=9) were collected during January and February 2008, in lakes Chester Cone, Midge Lake, Escondido, Limnopolar, Turbio, Somero, and Refugio (Beyers Peninsula, Antarctica)
The extracted and amplified DNA pool was subjected to an analysis with a Qubit dsDNA HS, Agilent 4200 TapeStation High Sensitivity DNA and Kapa Illumina Library Quantification qPCR assays.
DNA extraction from each filter was performed with the EZNA Soil DNA isolation kit (Omega Bio-Tek, Inc., Norcross, GA, United States) following the instructions given by the supplier. Sequencing of the region V4 of the 16S rRNA gene was done using the Illumina MiSeq system (2×250 bp) at the genomics facilities of the Research Technology Support Facility of the Michigan State University, United States. For each sample, Illumina compatible, dual indexed amplicon libraries of the 16S-V4 rRNA hypervariable region were created with primers 515f/806r. PCR reactions are composed of 5 μL of 4 μM equimolar primer set, 0.15 μL of AccuPrime Taq DNA High Fidelity Polymerase, 2 μL of 10× AccuPrime PCR Buffer II (Thermo Fisher Scientific, catalog no. 12346094), 11.85 μL of PCR-grade water, and 1 μL of DNA template. The PCR conditions used consisted of 2 min at 95°C, followed by 30 cycles of 95°C for 20 s, 55°C for 15 s, and 72°C for 5 min, followed by 72°C for 10 min.
Completed libraries were batch normalized using Invitrogen SequalPrep DNA Normalization Plates. The DNA was sequenced on an 2x250bp Illumina MiSeq v2 flow cell using a MiSeq v2 500 cycle reagent cartridge.