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doi:10.2204/iodp.proc.327.201.2013

Methods and materials

Sample collection and processing

Samples were cored and collected from Holes U1363B and U1363G as described previously (see the “Methods” chapter [Expedition 327 Scientists, 2011b]); the subset of samples analyzed in this study is summarized in Table T1. Within the sample name, H and X indicate the two different coring methods that were employed: advanced piston corer or extended core barrel, respectively. For processing raw sediment samples, a sterile scoopula was used to transfer up to 20 cm³ of raw sediment into a 50 mL Falcon tube. DNA lysis buffer (20 mM tris-HCl, 2 mM EDTA, 1.2% Triton X-100, and 2% lysozyme (w/v); pH 8) was added to just cover all of the sample material (10–20 mL) and subsequently stored at –80°C until further processing. In order to sample sediment interstitial water, sediment cores were pressed with 11 µm pore-size grade Number 1 Whatman filter paper (Whatman, Maidstone, United Kingdom) in order to collect ~10 mL of fluid that was subsequently filtered through a 0.1 µm pore-size Supor membrane filter (Pall Corporation, Port Washington, NY, USA) by a sterile 20 mL syringe. Sterile forceps were used to transfer the membrane to a microcentrifuge tube, enough DNA lysis buffer was added to cover the membrane (~2 mL), and the sample was stored at –80°C until further processing. The pressed sediment cores, referred hereafter as squeeze cake samples, were preserved without addition of any buffers or reagents, wrapped in foil, and stored at –80°C until further processing.

DNA extraction

All samples were thawed to room temperature and environmental DNA was extracted using the PowerMax Soil DNA isolation kit (MO BIO Laboratories, Carlsbad, CA, USA) following the manufacturer’s protocol, with the following modifications (Mason et al., 2010):

1. After the addition of solution C1, two 5 min incubations at 65°C were performed with intermittent vortexing.

2. Bead beating was performed in 50 mL Falcon tubes using a Vortex Genie 2 benchtop vortexer (Scientific Industries, Bohemia, NY, USA) and a custom-made adapter for 1 h at maximum speed.

3. Nucleic acids were eluted using ultrapure water.

Bead beating for a lesser time was not attempted, and therefore some DNA shearing may have occurred. Up to 10 g of sediment and squeeze cake sample were used for nucleic acid extraction. For the interstitial water sample, the entire filter and associated lysis buffer were added to the bead beating tube and processed identically to other samples. Negative DNA extractions consisting of only kit reagents were processed in parallel to sediment and interstitial water sample extractions.

Nucleic acids were concentrated as outlined in the PowerMax Soil DNA isolation kit manual. Sodium chloride was added to the eluted nucleic acid extracts to a final concentration of 0.2 M and mixed, followed by the addition of 100% ethanol at a ratio of 2:1 and further mixing. The mixture was centrifuged at 2500 × g for 30 min at room temperature. The supernatant was decanted, and the resulting pellet was washed with 70% cold ethanol before evaporation overnight in a sterile laminar flow hood. Following evaporation, nucleic acid pellets were dissolved in ultrapure water and quantified by Picogreen fluorometric assay (Quant-iT dsDNA kit, Invitrogen, Carlsbad, CA, USA).

SSU rRNA gene cloning and sequencing

Small subunit rRNA gene fragments were amplified by polymerase chain reaction (PCR) using universal oligonucleotide primers 519F (5’-CAGCMGCCGCGGTAATWC-3’) and 1406R (5’-ACGGGCGGTGTGTRC-3’) (Lane et al., 1985). Each 20 µL PCR reaction contained 0.5 U of SpeedSTAR HS DNA polymerase (Takara Bio, Inc., Otsu, Shiga, Japan), 1× fast buffer I, 200 µM of each of the four deoxynucleoside triphosphate (dNTPs), 200 nM of both forward and reverse primer, and ~0.5–4 ng of environmental DNA template. PCR cycling conditions consisted of an initial denaturation step at 95°C for 4 min followed by 25 cycles (Samples 1363B8X1_SC, 1363G1H2_SC, 1363G3H1_SC) or 34 cycles (Samples 1363B1H1_SC, 1363B1H1_RS, 1363B7X2_IW, extraction control) of 95°C denaturation for 30 s, 55°C annealing for 1 min, 72°C extension for 2 min, and a final extension step at 72°C for 20 min. Amplification products of the anticipated length were excised from an agarose gel and subsequently purified using the QIAquick gel extraction kit (Qiagen, Valencia, CA, USA). Products were cloned using the TOPO TA cloning kit (Invitrogen) following the manufacturer’s instructions. Clones were sequenced unidirectionally with primer T7 on an ABI 3730XL DNA analyzer (Applied Biosystems, Carlsbad, CA, USA).

Phylogenetic analysis

DNA sequences were trimmed of vector sequence and manually curated using Sequencher version 5.1 (GeneCodes, Ann Arbor, MI, USA). Curated clone sequences were first aligned using the online SINA tool version 1.2.11 (Pruesse et al., 2012) before importing into the ARB software package (Ludwig et al., 2004; Westram et al., 2011) for manual curation of the multiple species alignment and taxonomic classification using version SSURef_111 of the SILVA ARB database (Pruesse et al., 2007; Prüsse et al., 2011). Clone sequences released to GenBank after the release of ARB database SSURef_111 that were highly related to clone sequences obtained in this study were downloaded and included in the relevant phylogenetic analyses. Maximum likelihood phylogenetic analyses were performed by RAxML using the GTR model of nucleotide substitution under the gamma model of rate heterogeneity (Stamatakis, 2006). Bootstrap analyses for phylogenetic trees containing large (>100 sequences; Chloroflexi and MCG) were determined by RAxML using 1000 bootstrap iterations by the CIPRES Science Gateway version 3.3 (Miller et al., 2010). All other bootstrap analyses were determined by RAxML using the rapid bootstrap analysis algorithm (1000 bootstraps) implemented within ARB (Stamatakis et al., 2008). All sequences generated in this study have been deposited in GenBank under accession numbers KC990940–KC991019.

Microbial community analysis

Microbial community analyses were performed using sequences containing >700 unambiguously aligned nucleotides (132 of 140 sequences). The UniFrac significance test (Lozupone et al., 2005) was used to evaluate whether the microbial community structure was different between sets of samples. A maximum likelihood–based phylogenetic tree containing all environmental sequences derived in this study was evaluated using an online implementation of the hypothesis testing approach (Lozupone et al., 2006). Significant differences in community structure within pairs of samples were evaluated using 100 permutations of both the weighted and unweighted implementations of the UniFrac algorithm. Bonferroni-corrected p-values are reported. Microbial community α-diversity estimators, rarefaction curves, and community relatedness were generated or assessed using lane-masked clone sequences grouped into operational taxonomic units (OTUs) defined at 99% and 97% SSU rRNA gene sequence similarity cut-off values using the average neighbor clustering method as implemented by the mothur software package (Schloss et al., 2009). Microbial richness, evenness, and diversity were assessed by the Chao1 richness estimator (S_chao1; Chao, 1984), Simpson evenness index (E_simpson; Simpson, 1949), and the nonparametric Shannon diversity index (_shannon; Shannon, 1948), respectively, as implemented in mothur (Schloss et al., 2009).

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