IMIS - Marine Research Groups

Geographic coverage: pond, stream and wet soil samples from Sweden, Bulgaria, Antarctica, New Zealand, Canada and Svalbard.
Taxonomic coverage: Amplicon sequencing dataset of Bacteria 16S ssu rRNA gene.
Samples were collected in sterile tubes or bags using a sterile spatula or similar equipment. All samples were stored frozen (−20°C) or freeze-dried and stored frozen until further use.
Samples from microbial communities growing on wet soil, in small streams and ponds were collected during several field campaigns (three in each of the Arctic, the Antarctic and non-polar regions) between 2007 and 2014.
Method step description:

1. Microbial DNA was extracted from 0.05 to 0.1 g subsamples using the PowerSoil® DNA Isolation Kit (Qiagen, Germantown, USA) following the manufacturer's recommendations and DNA eluted in sterile DNAse-free water. The DNA quality and quantity was assessed using a NanoDrop (NanoDrop 3300 Fluorospectrometer, ThermoScientific). DNA extracts were stored frozen (−20°C) for no longer than three months before subsequent processing. Samples from Northern Canada were extracted as described in Jungblut et al. (2010), dried and stored frozen.
2. DNA obtained from 90 environmental samples was amplified using primers targeting the V3-V4 region of the 16S rRNA gene (F319 5′-ACTCCTACGGGAGGCAGCAG-3′, R806 5′-GGACTACHVGGGTWTCTAAT-3′)., using a dual multiplexing approach and a “heterogeneity spacer” of 0–3 bp length between the Illumina adapter and the forward/reverse primer sequence to increase read quality. PCR was carried out according to the manual of the Q5 high-fidelity polymerase (New England BioLabs, Ipswich, USA) with a final primer concentration of 0.2 μM and an annealing temperature of 65°C for 15 cycles.
3. The PCR product (1 μl) was used in the second PCR. This PCR was performed using the forward and barcoded reverse primers from the NEXTflex™ 16S V1-V3 Amplicon-Seq Kit (Bioo Scientific, Austin, Texas, USA) at final concentrations of 0.15 μM and an annealing temperature of 65°C for 25 cycles. The remaining PCR conditions were as indicated in manufacturer's instructions for the Q5 high-fidelity polymerase. PCR products were cleaned up with the Agencourt AMPure XP—PCR Purification system (Beckman Coulter, Brea, USA) according to the manufacturer's instructions, and multiplexed at equal concentration. Sequencing was performed using a 300 bp paired-end sequencing protocol on the Illumina MiSeq platform (Illumina, San Diego, USA) at the Genomics Core Facility, European Molecular Biology Laboratory, Heidelberg.