This study included cold seep and hydrothermal vent sediments from along the Pacific coast of North America. Sediments were collected via 30 cm long polycarbonate pushcores from a cold methane seep at Hydrate Ridge (44\u00b034'10. 20\"N, 125\u00b0 8'48. 48\"W) at 777 m water depth with the ROV Ropos (Dive 1458); hydrothermal vents at Guaymas Basin, Gulf of California (27\u00b00'27.84\"N, 111\u00b024'27.84\"W) at 2000 m with the DSV Alvin (Dive 4486); and hydrothermal vents at Middle Valley, Juna de Fuca Ridge (48\u00b027'26.40\"N, 128\u00b042'30.60\"W) at 2413 m with the DSV Alvin (Dive 4625). For this study, a total of three pushcores were collected from Hydrate Ridge at 4\u00b0C, one from Guaymas Basin at 30-35\u00b0C, and one from Middle Valley at 5\u201357\u00b0C (Table 4.1). Total genomic DNA was extracted using a phenol-chloroform protocol modified to prevent nucleic acid loss and eliminate potential inhibitors of downstream PCR, and which has been very successful in studies of low biomass sediments (Adams et al. 2013). PCR amplification was performed with primers designed to be universal to both archaea and bacteria (515F/806R) (Caporaso et al., 2012), containing attached Illumina adaptors and barcodes (Kozich et al., 2013). All DNA extracts were amplified in duplicate with OmniTaq (Taq mutant) polymerase according to the manufacturer\u2019s instructions (DNA Polymerase Technologies, St. Louis, MO, USA), with a final concentration of 0.2 \u03bcM for each primer. For each PCR, 1 \u03bcL template DNA was added to the final reaction mixture for a final volume of 50 \u03bcl. Amplification conditions were as follows: 94\u00b0C for 3 min to denature DNA; 30 cycles at 94\u00b0C for 45 s, 50\u00b0C for 60 s, and 72\u00b0C for 60 s; and a final extension of 10 min at 72 \u00b0C.
Raw sequences were first demultiplexed and quality filtered using the QIIME V. 1.8.0 pipeline (Caporaso et al., 2010a). Sequences of poor quality were filtered based on quality scores (< 25), the presence of homopolymers (> 6 nt), and length (< 250 nt).\u00a0 After quality filtering, the sequencing depth was rarified to the least robust sample (4000 nt) for even sub-sampling and maximum rarefaction depth to avoid biases in all downstream analyses (Lundin et al., 2012).
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BCO-DMO Processing:
\n- modified parameter names (replaced spaces with underscores);
\n- split lat/lon columns into two each; made longitude negative (for West);
\n- removed degrees, minutes, seconds symbols.