Dataset: 16S rRNA gene amplicon sequences metadata collected from water samples and biofilms on mineral substrates deployed in the Lau Basin (Tonga) during R/V Thompson cruise TN401 from Mar to Apr 2022

We sequenced the 16S V4 hypervariable region of deep-water samples collected with a Suspended Particulate Rosette Sampler (SuPR) (Mclane Research Laboratories, Inc. Falmouth, MA USA) or Universal Fluid Obtainer (UFO) (National Deep Submergence Facility, Falmouth, MA, USA) using the ROV Jason. At each vent field 15.71–47.51 L of water were pumped through 2–4 0.22 µM Express Plus Membrane filters (MilliporeSigma, MA, USA) both within close proximity to hydrothermal vent animal communities (~10-20 cm above animal assemblages) and away from hydrothermal activity (~10s to 100s of meters distance). A 10 µM custom nylon mesh pre-filter (Sefar Inc., NY, USA) was assembled in front of each sample filter to prevent collection of microbes associated with shed animal cells. Because the SuPR device failed during deployment at Tu’i Malila and during one dive at each ABE and Kilo Moana, 10.5 L deep water samples were additionally taken with the UFO at these localities. To account for cross-contamination using this device, empty filters were deployed but not pumped and used as negative controls. On board ship, filters were preserved in RNALater™ (Thermo Fisher Scientific, Inc., Waltham, MA, USA) and frozen at –80°C. DNA from filter samples was extracted with a phenol:chloroform protocol. The 3 Strain Tagged Genomic DNA Even Mix (ATCC, VA, USA) was added as spike-in control and dilutions of the spike-in without DNA extract were prepared to assess cross-contamination during sample processing in the laboratory. 

For the collection of microbial biofilms on various mineral and other substrates, we used weighted PVC bait cages containing vertically held open-ended 2-mL polypropylene tubes packed with crushed mineral substrates (andesite, basalt, glass beads or Alviniconcha shells) and contained by window screen mesh on either end for microbial colonization. Three of these cages were deployed during the first dive at each vent field and recovered approximately two weeks later using the ROV Jason. On board ship, water and biofilm samples were preserved in RNALater™ (Thermo Fisher Scientific, Inc., Waltham, MA, USA) and frozen at –80°C. DNA from substrate biofilms was extracted with the E.Z.N.A. Soil DNA Kit (Omega Bio-Tek, Norcross, GA, USA), including an additional 45 second bead beating step prior to proteinase K digestion.

Samples were sequenced at the Argonne National Laboratory (Lemont, IL, USA) using amplicon library preparation with the 515F/806R primer pairs (Apprill et al. 2015; Parada et al. 2016). All libraries were 2x150 bp paired-end sequenced on a Illumina NextSeq2000 instrument.