Dataset: 16S rRNA gene Amplicon Library
Deployment: PICO_1-301

Seawater was collected at ~10:30 AM local time using a 5-L Niskin bottle centered at 1 m or a peristaltic pump with the tubing open at 1 m, and processed immediately. To characterize particle-associated and free-living microbial communities, the large-particle-associated bacterial communities (>63um), small-particle-associated bacterial communities (63 to 5 um), ambiguous bacterial communities (5 to 1-um), and free-living bacterial communities (<1 um) were separated by sequential gravity filtration using a 63-um-pore-size plankton net followed by 5-um-pore-size (Advantec MFS), 1-um-pore-size (GE Water& Process Technologies), and 0.2-um-pore-size (Pall Supor-200) filters. Large particles collected on the 63-um-pore-size plankton net from 10 liters of seawater were resuspended in autoclaved artificial seawater and concentrated on 0.22-um-pore-size filters. Filtered volumes were chosen such that the filtration rate did not become visibly lower during collection, and filters were rinsed with autoclaved artificial seawater to remove additional material smaller than the filter pore size. All filters were stored at -80 deg C until DNA extraction.

DNA extraction and library preparation. We extracted DNA from the size-fractionated samples using a Puregene Yeast/Bacteria kit (Qiagen) according to the manufacturer’s instructions supplemented with three rounds (60 s each) of bead beating. Microbial communities were characterized using a dual index sequencing approach (Kozich et al. 2013) with the following portions of the primers targeting the V3-to-V4 region of the bacterial and archaeal 16S rRNA genes: for 16S F V3, CCTACGGGNGGCWSCAG; and for 16S R V4, GGACTACNVGGGTWTCTAAT (Hugoni et al. 2013). PCR mixtures contained 0.4Uof Q5DNApolymerase (NEB) as well as a final concentration of 200µM deoxynucleoside triphosphates (dNTPs), 2mM MgCl₂, and 0.5 uM primers. PCRs were performed using thermocycling with the following protocol: 98 deg C for 30 s followed by 35 cycles at 98 deg C for 10 s, 55 deg C for 30 s, and 72 deg C for 30 s, with a final extension at 72 deg C for 2 min. Triplicate reaction mixtures per sample were pooled and gel purified.

DNA extraction and sequencing

Microbial biomass was collected by filtering ~1 L of seawater through a 0.22-micron Sterivex filter (Millipore) and filters were stored at -80 deg C until extraction. Nucleic acids were extracted as described previously (Massana et al 1997), with some modifications. In brief, cells were lysed by bead-beating on ice three times for 30 secs in lysis solution (0.75 M sucrose, 40 mM EDTA, 50 mM Tris pH 8.0), followed by consecutive incubations with lysozyme (60 mg/mL; 37 deg C) and SDS (1%; 55 deg C). DNA was purified by phenol-chloroform extraction, RNase treatment, isopropanol precipitation, and PCR inhibitor removal (Zymo). DNA concentration was measured using a NanoDrop ND-1000.

Microbial communities were characterized using a dual index amplicon library approach targeting the 16S rRNA gene V3-V4 region (Kozich et al 2013, Yung et al 2016). PCR reactions contained 20 ng of template gDNA and 0.4 U of Q5 DNA polymerase (NEB) as well as a final concentration of 200 uM dNTPs, 2 mM MgCl2, and 0.5 uM of each primer. PCR reactions were thermocycled using the following protocol: 98 deg C for 30 sec, and 28 cycles at 98 deg C for 10 sec, 55 deg C for 30 sec and 72 deg C for 30 sec, with a final extension at 72 deg C for 2 min. Triplicate reactions per sample were pooled and gel-purified. In total, 151 libraries were paired end (2x 250bp) sequenced on the MiSeq (Illumina) at Duke’s Genome Sequencing and Analysis Core Facility. 

This data set and associated analysis is fully described in Yung et al. (2016) and Ward et al. (2017)