Microbial Community Profiling of MEDBlack Cruise

For the MEDBlack cruise DNA samples, the hypervariable V4-V5 region of the 16S rRNA gene from Bacteria and Archaea was amplified with high-fidelity Phusion Hot Start II DNA polymerase (Thermo Scientific, Waltham, MA, USA) using universal primers 515F and 928R (34 (link)) and a two-step PCR protocol (35 (link)), as detailed in Supplemental Information 1.2. and Table S1. The correct amplicon size and the absence of non-specific bands were checked by agarose gel electrophoresis. Amplicons were sequenced using a 2 × 250 bp paired-end MiSeq system (Illumina, USA) at the Genotoul platform (Toulouse, France). The raw sequences have been deposited at NCBI GenBank, SRA database, under the BioProject accession number PRJNA895066.
Raw sequences were analyzed on the Galaxy bioinformatics platform through the FROGS pipeline, version 3.2.3, as detailed in the Supplemental Information. Especially, operational taxonomic units (OTUs) were defined by sequence clustering, using the high-resolution SWARM algorithm v3.2.3 (36 (link)). After filtering at 0.005% of abundance, OTUs were taxonomically annotated with the SILVA 16S database (version 138.1).
Bacterial and archaeal abundances were quantified in the MEDBlack cruise DNA samples by quantitative PCR with Takyon No Rox SYBR 2X Master Mix (Eurogentec, Seraing, Belgium). Protocol details are provided in Table S1.