Boothbay Harbor viral-fraction seawater metagenome
A total of 40 L of freshly collected seawater [AW1]was filtered through a 0.2 m mesh size filter, concentrated to 250 ml using a REXEED 30kMWCO tangential flow filter system (Asahi Kasei Medical) (Hill et al. 2005), [AW2]and then stored at 4C overnight. The following morning, this primary concentrate was further concentrated to ~1.5 mL final volume (~30,000x concentration) using Amicon 100 kMWCO ultracentrifugation filters (Millipore Sigma) in a refrigerated centrifuge. The collected material was resuspended off the filter in DNA-free, deionized water and stored at -80 C. The DNA was extracted from a 30 L aliquot of this secondary concentrate using previously described protocols (Thurber et al. 2009). Extracted DNA was subjected to an additional clean and concentration step using a Genomic DNA Clean and Concentrator Kit (Zymo Research), eluting the purified DNA with elution buffer heated to 65C. Libraries for DNA sequencing were created with Nextera XT (Illumina) reagents following the manufacturers instructions, except for purification steps, which were done with column cleanup kits (QIAGEN)[RS1] and library size selection, which was done with BluePippin (Sage Science, Beverly, MA) with a target size of 37043 bp. DNA concentration measurements were performed with Quant-iT dsDNA Assay Kits (Thermo Fisher Scientific) following the manufacturers instructions. Libraries were sequenced with NextSeq 2000 (Illumina) and P1 reagents in 2150 bp mode.
SUB15157420
220909_vDNA