Samples from two different depths (20 and 40 m) were collected on February 15, 2019 from the epipelagic Mediterranean Sea at 20 nautical miles off the coast of Alicante (Spain) (37.35361°N, 0.286194°W) during winter where the water column is mixed. This location has been studied previously by metagenomic approaches (Ghai et al., 2010 (link); Mizuno et al., 2013 (link); Haro-Moreno et al., 2017 (link), 2018 (link), 2019 (link); López-Pérez et al., 2017 (link)). For each depth, 200 L were collected and filtered on board as described in the study of Haro-Moreno et al. (2018) (link). Briefly, seawater samples were sequentially filtered through 20-, 5-, and 0.22-μm pore filter polycarbonate filters (Millipore). Water was directly pumped onto the series of filters to minimize the bottle effect. Filters were immediately frozen on dry ice and stored at −80°C until processing.
DNA extraction was performed from the 0.22-μm filter (free-living bacteria) following the phenol/chloroform extraction. Given the large amount of DNA needed for sequencing, DNA from the two samples (20 and 40 m) was pooled together. Metagenomes were sequenced using Illumina Nextseq (100 bp, paired-end reads) (Macrogen, South Korea) and using PacBio Sequel II (one 8M SMRT Cell Run, 30-h movie) (Genomics Resource Center, University of Maryland, United States).
DNA extraction was performed from the 0.22-μm filter (free-living bacteria) following the phenol/chloroform extraction. Given the large amount of DNA needed for sequencing, DNA from the two samples (20 and 40 m) was pooled together. Metagenomes were sequenced using Illumina Nextseq (100 bp, paired-end reads) (Macrogen, South Korea) and using PacBio Sequel II (one 8M SMRT Cell Run, 30-h movie) (Genomics Resource Center, University of Maryland, United States).