DNA extraction and purification

DNA was extracted, in duplicate, from 8 to 20 g of “Mobio” IODP sediment samples previously frozen at -80C using a Powersoil total RNA Isolation Kit with the DNA Elution Accessory Kit (MO-BIO Laboratories, Solana Beach, CA, USA) following the manufacturer protocol but without beads. Approximately 2 grams of sediments were utilized for each extraction, and DNA pellets from each depth were pooled together. DNA concentrations were measured using a Qubit 2.0 fluorometer with dsDNA High Sensitivity reagents (Invitrogen, Grand Island, NY, USA). The DNA from the extractions that yielded robust 16S rRNA and metagenomic sequences ranged from 3.75-15 ng of DNA per gram of sediments.

16s rRNA gene PCR and amplicon sequencing

The V4 hypervariable region of the 16S rRNA gene was amplified from extracted DNA with barcoded Illumina-specific adapters (38) with technical replication. DNA template (2 ng) was mixed with 5 uL of 10x Taq Mutant reaction buffer, 0.4 uL of Klentaq LA Taq Polymerase (DNA Polymerase Technology, St. Louis, MO, USA), 2 uL of 10 mM dNTP mix (Sigma Aldrich, St. Louis, MO, USA), 2 uL of reverse and forward V4-specific primers F515 and R806 (39) and the remainder DNA-free water to 50 uL (Ambion, Grand Island, NY, USA). PCR was performed on a Bio-Rad C1000 Touch thermal cycler (Bio-Rad, Hercules, CA, USA) with the following conditions: initial denaturation at 94oC (5 min), followed by 35 cycles of denaturation at 94oC (40 s), primer annealing at 55oC (40 s) and primer extension at 68oC (30 s). The ~400 bp PCR size fragment was confirmed on a 1% agarose gel and replicated samples were pooled. A 50 uL fraction of each pooled sample was purified using a QIAquick PCR Purification Kit (Qiagen, Germantown, MD, USA).  Barcoded amplicons for each sample were pooled in equimolar concentrations and sequenced on an Illumina MiSeq (School of Biology, Georgia Institute of Technology), running MiSeq Control Software v.2.4.0.4, using MiSeq reagent kit v2 (500 cycles) with 5% PhiX genomic library control.