Dataset: Bacterial 16S rRNA barcodes associated with methane seep invertebrates collected off of the Aleutian Islands, Gulf of Alaska on R/V Atlantis cruise AT50-24 in May to June 2024

Sampling

Seep invertebrates were collected from the Sanak seep off of Alaska in June 2024 (53.74865 N/ −162.58970 W; 2,020 m) aboard the R/V Atlantis (expedition AT50-24) with HOV Alvin (operated by the Woods Hole Oceanographic Institution). Specimens for molecular analysis were preserved within 2 h of collection in ~90% ethanol and stored at 4 °C. Total genomic DNA was extracted from whole specimens preserved in ethanol using the Qiagen DNeasy kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions.

Data Collection Methods

Total DNA extracted was quantified via a Qubit 3.0 fluorometer using the dsDNA BR Assay Kit (Thermo Fisher Scientific). The V4-V5 hypervariable region of the 16S rRNA gene was PCR amplified using bacterial primers [515F: GTGYCAGCMGCCGCGGTAA and 806R: GGACTACHVGGGTWTCTAAT] with Illumina adapters on the 5′ end (San Diego, CA). Each PCR product was secondarily barcoded with Illumina NexteraXT index v2 Primers that included unique 8-bp barcodes, with NEB Q5 Hot Start High-Fidelity Mix at an annealing temperature of 66 °C for 11 cycles. Barcoded products were purified using Millipore-Sigma (St. Louis, MO) MultiScreen Plate MSNU03010 with a vacuum manifold and quantified using the QuantIT PicoGreen dsDNA Assay Kit (ThermoFisher Scientific) on a BioRad CFX96 Touch Real-Time PCR Detection System. Barcoded samples were combined in approximately equimolar amounts, purified again with Promega’s Wizard SV Gel and PCR Clean-up System (#A9281), and quantified again using the QuBit system. This sample was submitted to Laragen, Inc. (Culver City, CA) for 2× 250 bp paired-end analysis on the Illumina MiSeq platform with 20% PhiX addition.