For time series collected at Tiny Towers hydrothermal vent with the PPS instrument, hydrothermal fluid samples were collected remotely and filtered through 0.4 micrometer (μm) polycarbonate filters, then preserved in RNALater in the instrument at the seafloor until instrument recovery. Upon recovery, filters were preserved at -80 degrees Celsius (C).

Other samples were collected using the UFO fluid sampler mounted aboard ROV Jason. Hydrothermal fluid samples were pumped through 0.2 μm Sterivex filters or into sterile 10 liter (L) bags mounted to the vehicle. Upon ROV recovery, Sterivex filters were preserved at -80 degrees C. Fluid from bags was filtered through 0.2 μm Sterivex filters, which were later preserved at -80 degrees C, and the filtrate was treated with iron chloride to precipitate viral particles. Precipitated viral particles were filtered through 1.0 μm polycarbonate filters and preserved at 4 degrees C until processing.

DNA was extracted from all filters using the MasterPure Complete DNA and RNA Purification Kit. Sterivex filters were cracked open prior to processing. PPS filters were cut in half prior to processing, and the second half was archived.

For viral metagenomes, viruses were reconstituted through an ascorbic acid buffer and then extracted using the Qiagen UltraSens Virus spin kit.

All samples were sequenced at the Northwest Genomics Center. Library construction was automated in 96-well plate format. At least 25 nanograms (ng) of genomic DNA was subjected to enzymatic fragmentation and a series of library construction steps utilizing the KAPA HyperPlus kit. All library construction steps were automated on the Perkin Elmer Janus platform. Libraries were validated using the Biorad CFX384 Real-Time System and KAPA Library Quantification Kit. Barcoded genome libraries were pooled using liquid handling robotics prior to loading. Massively parallel sequencing-by-synthesis with fluorescently labeled, reversibly terminating nucleotides was carried out on the NovaSeq sequencer.