Dataset: Accession numbers (P. carterae viruses and field samples)
Deployment: Bigelow_Martinez_2015-2016

Laboratory cultured P. carterae CCMP 645 viruses

A 2 L culture of P. carterae CCMP 645 in exponential growth phase, grown in F/2 medium was filtered through a 0.2 um PES membrane under sterile conditions. The filtrate was then initially concentrated down to approximately 25 ml by tangential flow filtration using a Vivaflow 50 cartridge (Sartorius) and further concentrated down to approximately 1 ml using a 30 kDa MWCO AmiconUltra-15 column (Millipore).

DNA from the viral concentrate was extracted using the QiaAMP MinElute Virus spin kit. DNA was tested with universal 16S and 18S primer sets and determined to be free of host, or prokaryotic contamination prior to sequencing.

Two independent sequencing libraries were prepared and sequenced by Illumina MiSeq platforms through Bigelow Laboratory for Ocean Sciences’ Single Cell Genomic Center (150 bp paired-end reads) and through the Sequencing Facility at the University of Wisconsin-Madison (300 bp paired-end reads). In addition, PCR products generated to join contig ends were pooled and sequenced in 3 batches (MiSeq), also generating 300 bp paired ends through the University of Wisconsin-Madison. 

Virus-enriched Bigelow Dock sample metagenome:

Six large, land-based, 2460 dm-3 volume mesocosm tanks were filled on September 24, 2015, from a seawater supply at Bigelow Laboratory’s deep-water dock site at the Damariscotta River Estuary, Maine. The samples were screened through 3 mm mesh, with care taken to ensure that each tank was filled simultaneously and contained the same starting phytoplankton composition. The mesocosms were aimed for testing the effects of climate change by simulating predicted increasing temperature and pCO2 environments in the Gulf of Maine over the next several centuries. However, for our study 1 L samples were collected from each of the mesocosms on Day 0 prior to any further experimental manipulation. All six liters were combined and virus-enriched by filtering through a 0.45 um PES filter to remove most cellular organisms, and concentrated down to 45 ml by tangential flow filtration. Total DNA was extracted from the sample using the MasterPure(TM) Complete DNA and RNA Purification kit (Epicentre) following manufacturer’s recommendations. Total DNA was eluted in 20 ul of nuclease-free water. A DNA library was prepared and sequenced by Illumina MiSeq. 

laboratory experiment