Samples were obtained from Stations P1 (48.58 N, 125.50 W), P4 (48.65 N, 126.67 W), P6 (48.74 N, 127.67 W) and P8 (48.82 N, 128.66 W) on cruise TN280 between May 17-20 2012.
Water from all samples was obtained from Niskin bottles on the CTD Rosette. Plankton were collected on a 2.0 μm pore-size filter (142 mm diameter polycarbonate) and a 0.2 μm pore-size filter (142 mm diameter Supor) following prefiltration through a 53 μm pore-size filter. 20L of the <0.2 μm filtrate was amended with iron chloride (1g L-1) and incubated for at least 1 hour at room temperature to flocculate viruses (John et al. 2011). Viral flocculates were filtered onto 0.6 μm polycarbonate filters and stored at 4C. The GenBank BioSample accession numbers are SAMN08663113- SAMN08663116.
Viruses were resuspended from filters by incubating them with 0.2M ascorbate-0.25M EDTA-Mg2-0.25M Tris-HCL for at least 24 hours with periodic shaking (John et al. 2011). Viral suspensions were incubated for 2 hours at room temperature with DNase 1 (100 U ml-1, Ambion) to remove contaminating extracellular DNA. Viral DNA was extracted using a MoBio PowerSoil Total RNA isolation kit with the DNA elution column accessory, according to the manufacturers instructions. To convert single stranded DNA (ssDNA) viruses to double stranded DNA (dsDNA), complementary strands were synthesized using a modified random priming-mediated sequence-independent single-primer amplification (RP-SISPA) method designed to generate quantitative whole genome shotgun libraries (Djikeng et al. 2008, Culley et al. 2010). Triplicate 20 μl reactions for each sample and a no template control containing 10 μl template (or water), 0.2mM dNTPs, 0.5 mM DTT, 1 μM FR26RV-N primer (5-GCCGGAGCTCTGCAGATATCNNNNNN -3), 1mM MgCl2, and 1X Klenow buffer were heated to 94 C for 3 minutes and snap cooled on ice. 2.5 U of Klenow Fragment, 3-5 exo- (New England Biolands) was added to each reaction then incubated at 37 C for 60 minutes followed by 75 C for 10 minutes. Reactions were pooled, cleaned with ethanol precipitation, and resuspended in T low-E (10mM Tris-HCl, 0.1 mM EDTA pH 8.0).
To construct Illumina shotgun metagenome libraries, viral DNA was sheared to 800-1000 bp using a Covaris nebulizer and cleaned with AmPure XP beads. Viral metagenomes (viromes) were constructed using the Rubicon ThruPLEX-FD Kit, which uses qPCR to linearly amplify low concentration DNA templates. Libraries were sequenced on an Illumina HiSeq 2500 with 150 paired end reads at the Michigan State University Sequencing Center. FASTQ files of the raw sequence reads are deposited in the Sequence Read Archive under study SRP134205 and the individual file accession numbers SRR6819460-SRR6819463.
Virome reads were trimmed of adapter sequences and low quality regions and filtered to remove low quality reads with the Trimmomatic pipeline (Bolger et al., 2014). Sequences were de novo assembled into contigs by first using diginorm (Brown et al., 2012) followed by the velvet assembler (Zerbino and Birney, 2008) with k-mer length set at 25, 29, 33, 51 for P1, P4, P6, and P8 respectively. Assemblies have been deposited in the Whole Genome Shotgun archive under the accession numbers
GeoMICS_DNAvirome_P1 PYID00000000
GeoMICS_DNAvirome_P4 PYIE00000000
GeoMICS_DNAvirome_P6 PYIF00000000
GeoMICS_DNAvirome_P8 PYIG00000000
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