ena-STUDY-CBMSO-18-05-2015-10:40:53:712-14
Spitsbergen Island and Antarctic Peninsula Metagenomic Analysis
Samples were taken from freshwater bodies in the Arctic (Spitsbergen Island, Svalbard archipelago, Norway), and along the Antarctic Peninsula  Ninety liters from each water body were pre-filtered through a 30 ?m nylon mesh. Subsequent filtration by 0.45?m tangential flow filtration (TFF) using a Centramate holder (Pall) separated the free viral community (defined as the <0.45?m fraction) from the cellular (and associated virus) fraction (defined as the fraction between 0.45 and 30 ?m). Viral fractions were subsequently concentrated 100 times by 70-kDa TFF. All stocks were preserved at -20ºC/-80ºC prior to DNA extraction. Cellular fractions community DNA was extracted using PowerSoil DNA isolation kit according to the manufacturer’s instructions. Free viral DNA was obtained from frozen stocks thawed at 4ºC, and passed through a 25% sucrose cushion by centrifugation for 16 h at 60,000 g and 4ºC. The pellets were re-suspended in 10 mM Tris pH 8, 1 mM EDTA, and filtered using a 0.45 ?m syringe filter. Viral concentrates were then treated with DNAse I (500  U.ml-1), Nuclease S7 (420 U.ml-1), RNAse A (100 ?g.ml-1) and RNAse H (2 U per reaction) for 30 min at room temperature to remove free nucleic acids. Nuclease reactions were stopped with 12 mM EDTA/2 mM EGTA, and viral capsids and envelopes were then disrupted with SDS (0.5%) and proteinase K (200 µg.ml-1) treatment. Finally, viral DNA was extracted with phenol-chloroform and ethanol precipitated. Bacterial 16S rRNA marker genes were amplified from the cellular fractions using primers 8F15B and 515R14AM. All PCR reactions were carried out using 1?l of template DNA, 0.5 ?l Phusion High-fidelity polymerase (NEB), 20 nmol dNTPs, 20 pmol of each primer, 1.5?l DMSO, 0.4 mM MgCl2, in a final volume of 50 ?l. Reaction conditions included an initial denaturation step of 30s at 98ºC, followed by 25 cycles of 10s at 98ºC, 30s at 53ºC, 30s at 72ºC, and a final elongation step of 5 min at 72ºC. For the analysis of the microeukaryotic and T4-like  myovirus communities we followed a two-step barcoding strategy that allows a universal set of bar-coded sequencing primers to be appended to an amplified PCR product. In the first step, one of the target-specific primers is modified to include a linker sequence. After amplification, a second primer consisting of the bar code and linker is used to tag the amplicon. The eukaryotic community in the cellular fractions was analyzed using primers targeting the 18S rRNA gene modified from those described by Bates et al. The myovirus communities in both cellular and free viral fractions were assessed using primers modified from those reported by Filée et al. targeting the g23 major capsid protein gene. In this case, proper amplification using the two-step approach for all samples proved difficult. Nevertheless, we were able in most cases to produce the desired products performing an initial amplification using the unmodified primers (45 cycles, melting temperature of 50ºC), followed by agarose gel extraction of DNA bands of appropriate size. The resulting products were re-amplified using the modified primers (10 cycles), at this point linking with the two-steps protocol. Final concentrations of PCR products were measured using a PicoGreen dsDNA Assay Kit (Life Tech.), equal amounts for each sample pooled, agarose gel-extracted using the QIAquick Gel Extraction Kit (QIAGEN), and sequenced using a Roche 454 FLX sequencer with titanium chemistry. Bacterial and Eukaryotic profiles were generated in triplicate (hence three different barcodes/reactions per sample) to mitigate potential reaction-level PCR biases (Bates et al, 2013).
Samples were taken from freshwater bodies in the Arctic (Spitsbergen Island, Svalbard archipelago, Norway), and along the Antarctic Peninsula  Ninety liters from each water body were pre-filtered through a 30 ?m nylon mesh. Subsequent filtration by 0.45?m tangential flow filtration (TFF) using a Centramate holder (Pall) separated the free viral community (defined as the <0.45?m fraction) from the cellular (and associated virus) fraction (defined as the fraction between 0.45 and 30 ?m). Viral fractions were subsequently concentrated 100 times by 70-kDa TFF. All stocks were preserved at -20ºC/-80ºC prior to DNA extraction. Cellular fractions community DNA was extracted using PowerSoil DNA isolation kit according to the manufacturer’s instructions. Free viral DNA was obtained from frozen stocks thawed at 4ºC, and passed through a 25% sucrose cushion by centrifugation for 16 h at 60,000 g and 4ºC. The pellets were re-suspended in 10 mM Tris pH 8, 1 mM EDTA, and filtered using a 0.45 ?m syringe filter. Viral concentrates were then treated with DNAse I (500  U.ml-1), Nuclease S7 (420 U.ml-1), RNAse A (100 ?g.ml-1) and RNAse H (2 U per reaction) for 30 min at room temperature to remove free nucleic acids. Nuclease reactions were stopped with 12 mM EDTA/2 mM EGTA, and viral capsids and envelopes were then disrupted with SDS (0.5%) and proteinase K (200 µg.ml-1) treatment. Finally, viral DNA was extracted with phenol-chloroform and ethanol precipitated. Bacterial 16S rRNA marker genes were amplified from the cellular fractions using primers 8F15B and 515R14AM. All PCR reactions were carried out using 1?l of template DNA, 0.5 ?l Phusion High-fidelity polymerase (NEB), 20 nmol dNTPs, 20 pmol of each primer, 1.5?l DMSO, 0.4 mM MgCl2, in a final volume of 50 ?l. Reaction conditions included an initial denaturation step of 30s at 98ºC, followed by 25 cycles of 10s at 98ºC, 30s at 53ºC, 30s at 72ºC, and a final elongation step of 5 min at 72ºC. For the analysis of the microeukaryotic and T4-like  myovirus communities we followed a two-step barcoding strategy that allows a universal set of bar-coded sequencing primers to be appended to an amplified PCR product. In the first step, one of the target-specific primers is modified to include a linker sequence. After amplification, a second primer consisting of the bar code and linker is used to tag the amplicon. The eukaryotic community in the cellular fractions was analyzed using primers targeting the 18S rRNA gene modified from those described by Bates et al. The myovirus communities in both cellular and free viral fractions were assessed using primers modified from those reported by Filée et al. targeting the g23 major capsid protein gene. In this case, proper amplification using the two-step approach for all samples proved difficult. Nevertheless, we were able in most cases to produce the desired products performing an initial amplification using the unmodified primers (45 cycles, melting temperature of 50ºC), followed by agarose gel extraction of DNA bands of appropriate size. The resulting products were re-amplified using the modified primers (10 cycles), at this point linking with the two-steps protocol. Final concentrations of PCR products were measured using a PicoGreen dsDNA Assay Kit (Life Tech.), equal amounts for each sample pooled, agarose gel-extracted using the QIAquick Gel Extraction Kit (QIAGEN), and sequenced using a Roche 454 FLX sequencer with titanium chemistry. Bacterial and Eukaryotic profiles were generated in triplicate (hence three different barcodes/reactions per sample) to mitigate potential reaction-level PCR biases (Bates et al, 2013).