Multilayer Sediment Profiling for Microbial Analysis

Freshly recovered sediment cores were divided into three layers (near-surface, middle, bottom) of 6 to 10 cm thickness each (Table 1) for DNA extraction and sequence-based analysis. This approach covered the available thermal range of cored sediment, while providing sufficient material for coordinated experiments with other cruise participants, and accommodating temperature fluctuations due to pulsating hydrothermal flows that impact surficial (5 to 10 cmbsf) sediments [14 (link)]. Total DNA was extracted using the FastDNA^TM Spin Kit for Soil (MP Biomedicals), as recommended for marine sediment [35 (link)], following the manufacturer’s protocol. Bacterial and archaeal SSU rRNA hypervariable region 4–5 (V4-V5) amplicons were generated using the 515F-Y (5′‐GTGYCAGCMGCCGCGGTAA-3’) and 926R (5′‐CCGYCAATTYMTTTRAGTTT-3’) primers and customized thermocycling parameters [36 (link)]. Fungal ITS2 region amplicons were generated using the 5.8S‐Fun (5′‐AACTTTYRRCAAYGGATCWCT‐3′) and ITS4‐Fun (5′‐AGCCTCCGCTTATTGATAT-GCTTAART‐3′) primers and customized thermocycling parameters [37 (link)]. All amplicons were generated and sequenced at Georgia Genomics and Bioinformatics Core, University of Georgia, using Illumina MiSeq PE 300 chemistry.