Dataset: V4 SSU rRNA gene primer validation for reef seawater
Deployment: Apprill_2013

Seawater sample collection

Nucleic acids were collected from surface waters along the coastline of the southeastern Red Sea and from coral reefs surrounding the islands and atolls in the Federated States of Micronesia (Supplementary Table 1). Samples from an aquaria-based experiment housed at the Bermuda Institute of Ocean Sciences (BIOS) were also utilized in this study.  Seawater from Ferry Reach, Bermuda (32.37035°N, 64.69545°W) was sampled from a direct inflow line. In addition, this water was also held in separate aerated 30 liter aquaria (similar to de Putron et al. 2011) where it was sampled for nucleic acids (500 ml) and fluorescence in situ hybridization (FISH) analyses (50 ml) over the course of 12 days.

DNA analysis

Seawater collected from the Red Sea (20 liters) and Micronesia (2 liters) was filtered onto 142 mm, 0.22 µm Durapore membrane filters (Millipore, Boston, MA, USA) and 25 mm 0.2 µm polyethersulfone membranes (Supor, Pall, East Hills, NY, USA), respectively, and immediately frozen in liquid nitrogen.  Total genomic DNA was extracted from these samples using previously reported methods (Santoro et al. 2010).  Similarly, microbial biomass originating from the BIOS seawater inflow line and aquaria seawater was concentrated onto 0.2 µm polyethersulfone membranes (Supor) using a 47 mm support filter and a gelman rig under gentle vacuum (~100mm Hg).  Each filter was stored in 1 ml of sterile sucrose lysis buffer (20mM EDTA, 400mM NaCl, 0.75M sucrose, 50mM Tris.HCl) at –80°C. DNA was extracted using the phenol-chloroform method (Giovannoni et al. 1990).  Primers targeting the V4 region of the SSU rRNA gene, 515F (5’-GTGCCAGCMGCCGCGGTAA-3’) and 806R (5’-GGACTACHVGGGTWTCTAAT-3’) (Caporaso et al. 2011), were utilized for PCR amplification using unique barcoded primer combinations for each sample.  In addition, the same DNA samples were amplified separately with the 515F primer and a modified 806RB primer using the identical barcoding approach. This modified reverse primer replaces the ‘H’ degeneracy in the original 806R primer with a ‘N’, and is designed to enhance SAR11 targets (revised primer 806RB, 5’-GGACTACNVGGGTWTCTAAT-3’).  The primers were designed after Kozich and colleagues (2013) and were each equipped with a unique 8-bp barcode, 10-bp pad and 2-bp link that followed the above-mentioned primers (see Supplementary Materials and Methods). Triplicate 25 µl PCR reactions were conducted per sample and each reaction contained 1.25 U of GoTaq Flexi DNA Polymerase (Promega Cooperation, Madison, WI, U.S.A.), 5X Colorless GoTaq Flexi Buffer, 2.5 mM MgCl2, 200 uM dNTP mix (Promega Cooperation, Madison, WI, U.S.A.), 200 nM of each barcoded primer, and 1 - 4 ng of genomic template. The reaction conditions consisted of an initial denaturation step at 95ºC for 2 min, followed by 27-38 cycles of 95ºC for 20 s, 55ºC for 15 s, and 72ºC for 5 min, concluding with an extension step at 72ºC for 10 min. The reactions were carried out on a Bio-Rad thermocycler (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Reaction products (5 µl) were screened on a 1% agarose/TBE gel. The HyperLadder 50bp DNA ladder (generally 5ng µl-1) (Bioline, London, UK) was used to confirm appropriate amplicon size.  The number of PCR cycles varied between samples in order to produce similar, minimal yields, but each sample was subjected to nearly identical PCR cycles with both primer sets. The three replicate reactions were pooled and subsequently purified using the QIAquick Purification Kit (Qiagen, Valencia, CA, USA), and quantified using the Qubit 2.0 Fluorometer with the dsDNA High Sensitivity Assay (Life Technologies, Grand Island, NY USA).  For each primer set, barcoded amplicons were pooled into equimolar ratios. These amplicon pools were then shipped to the University of Illinois W.M. Keck Center for Comparative and Functional Genomics where they were used for construction of two separate libraries which were subsequently sequenced using 2x250bp paired-end MiSeq (Illumina, San Diego, CA, USA), as detailed previously (similar to Kozich et al. 2013). Control samples included sterile water (negative controls) in which PCR did not yield any detectable amplification with either primer set.  A mock community sample (positive control, obtained through BEI Resources, NIAID and NIH as part of the Human Microbiome Project: Genomic DNA from Microbial Mock Community B (even, low concentration), v5.1L, HM-782D) was amplified with the 515F/806RB primers and sequenced to assess amplification and sequencing error rate.

Microbial abundances and FISH

To determine microbial abundances from the BIOS inflow and aquaria samples, the seawater was fixed to 10% formalin and stored at -80°C.  Upon analysis, the samples were thawed and filtered onto 0.2µm filters pre-stained with Irgalan black (0.2g in 2% acetic acid) under gentle vacuum (~100mm Hg) and post-stained with 0, 6-diamidino-2-phenyl dihydrochloride (5µg ml-1, DAPI, SIGMA-Aldrich, St. Louis, MO, USA) (Porter and Feig 1980). Slides were then enumerated using an AX70 epifluorescent microscope (Olympus, Tokyo, Japan) under ultraviolet excitation at 100x magnification as previously described (Parsons et al. 2014).  At least 500 cells per filter (12 fields) were counted.

Fluorescence in situ hybridization (FISH) was used to quantify the abundance of SAR11 in the BIOS aquaria samples using a probe suite (152R-Cy3, 441R-Cy3, 542R-Cy3, 732R-Cy3) and was conducted as previously described (Morris et al. 2002, Parsons et al. 2012). Image analysis coupled with epifluorescence microscopy was used to process FISH slides excited with Cy3 (550nm) and UV wavelengths. The image capturing was performed using a Retiga Exi CCD digital camera with QCapture software version 2.0 (QImaging, Burnaby, BC, Canada) and processed with Image Pro software (version 7.0; Media Cybernetics, Bethesda, MD, USA) as previously described (Parsons et al. 2014).  SAR11 percentages were calculated as SAR11 FISH abundances compared to total cellular abundances.

References:

Caporaso, J. G., C. L. Lauber, W. A. Walters, D. Berg-Lyons, C. A. Lozupone, P. J. Turnbaugh, N. Fierer, and R. Knight. 2011. Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample. Proceedings of the National Academy of Sciences 108:4516-4522.

Giovannoni, S. J., E. F. DeLong, T. M. Schmidt, and N. R. Pace. 1990. Tangential flow filtration and preliminary phylogenetic analysis of marine picoplankton. Applied and Environmental Microbiology 56:2572-2575.

Kozich, J. J., S. L. Westcott, N. T. Baxter, S. Highlander, and P. D. Schloss. 2013. Development of a dual-index sequencing strategy and curation pipeline for analyzing amplicon sequence data on the MiSeq Illumina sequencing platform. Applied and Environmental Microbiology 79:5112-5120.

Morris, R. M., M. S. Rappé, S. A. Connon, K. L. Vergin, W. A. Siebold, C. A. Carlson, and S. J. Giovannoni. 2002. SAR11 clade dominates ocean surface bacterioplankton communities. Nature 420:806-810.

Parsons, R. J., M. Breitbart, M. W. Lomas, and C. A. Carlson. 2012. Ocean time-series reveals recurring seasonal patterns of virioplankton dynamics in the northwestern Sargasso Sea. ISME J 6:273-284.

Parsons, R. J., C. E. Nelson, C. A. Carlson, C. C. Denman, A. J. Andersson, A. L. Kledzik, K. L. Vergin, S. P. McNally, A. H. Treusch, and S. J. Giovannoni. 2014. Marine bacterioplankton community turnover within seasonally hypoxic waters of a subtropical sound: Devil's Hole, Bermuda. Environmental Microbiology:DOI: 10.1111/1462-2920.12445.

Porter, K. G. and Y. S. Feig. 1980. The use of DAPI for identifying and counting aquatic microflora. Limnol. Oceanogr 25:943-948.

Santoro, A. E., K. L. Casciotti, and C. A. Francis. 2010. Activity, abundance and diversity of nitrifying archaea and bacteria in the central California Current. Environmental Microbiology 12:1989-2006.