Sampling and Analytical Methodology:
Seawater was collected in 12-L polyvinylchloride bottles affixed to a 24-bottle rosette
sampler, equipped with a Sea-Bird 911+ conductivity, temperature, and pressure profiler. Seawater samples were sequentially filtered using a peristaltic pump onto 25-mm diameter, 3-μm pore size polycarbonate membranes (Millipore IsoporeTM), followed by filtration through 25-mm diameter, 0.2-μm pore size polyethersulfone filters (Pall Supor®). After filtration, filters were placed in sterile 1.5-mL microcentrifuge tubes, immediately flash-frozen in liquid nitrogen, and stored at -80°C until analysis. Back in the shore-based laboratory, DNA from the 0.2-μm filter membranes was extracted and purified using the QIAGEN DNeasy Plant Mini Kit including a bead-beating step (with 0.1- and 0.5-mm beads) and Proteinase K (QIAGEN) for additional cell disruption and lysing. Extracts were eluted in 200 μL of nuclease-free PCR grade water and quantified using the Qubit® fluorometer. Triplicate PCR products were pooled, fragment size was determined using 1.5% agarose gel electrophoresis, and quantity was measured with the Qubit® 2.0 fluorometer with Qubit® dsDNA High Sensitivity Assay kit (Molecular Probes). From each sample, ~50 ng of PCR product was combined and purified using the UltraClean PCR Clean-Up Kit (MoBio).
Data Processing:
Sequences were merged using PEAR (Zhang et al., 2014) and quality-filtered with reads trimmed to 100-150 bp, a maximum expected error of 1%, no ambiguous bases allowed, and an
average Phred quality threshold >34. Reference-based and de novo 70 chimeras were detected using
USEARCH v7.0.1090 (Edgar et al., 2011) with the SILVA 119 database pre-clustered at 97% sequence identity (Quast et al., 2013) and removed. Sequences were deposited into
NCBI’s Sequence Read Archive as BioProject ID PRJNA351881 (accession SRP092782).
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