title: NovaSeq of prokaryotes in seawater samples from the benthic boundary layer
design: DNA was extracted from 360 seawater samples with the DNeasy 96 Blood & Tissue Kit (Qiagen) with a modified protocol. For prokaryotes, the V4-V5 regions of the 16S rRNA gene were amplified for a higher resolution (Parada et al., 2016). Amplifications for the library preparation were performed in a two-step polymerase chain reaction (PCR) to first amplify the target region and second, to label the DNA using IDT 10-bp unique dual indexes (Integrated DNA Technologies, Inc., United States of America). The reaction mix for the first PCR consisted of 2.5 L of DNA template for 12.5 L KAPA HiFi HotStart ReadyMix (Roche, Basel, Switzerland), 1 L forward primer 10 M, 1 L reverse primer 10 M and 3 L MiliQ. The program was set to 3 min at 95 C, followed by 35 cycles of 20 sec at 98 C, 30 sec at 50 C, 30 sec at 72 C, followed by 5 min at 72 C. The reaction mix for the second PCR consisted of 10 L KAPA HiFi HotStart ReadyMix (Roche, Basel, Switzerland), 1 L of each tagged primer (forward and reverse) and 5 L of MilliQ for 3 L of cleaned DNA product. The PCR program for the second PCR consisted of the same steps but only repeated for 8 (instead of 35) cycles.  After successful amplification, all PCR products (after the first step PCR and the second step PCR) were cleaned with magnetic NucleoMag NGS-Beads (bead volume at 0.9 times the total volume of the sample, Macherey Nagel, Dren, Germany). DNA fragments were quantified using the Agilent 5300 Fragment analyzer with the DNF-910-33 dsDNA Reagent Kit (35-1500 bp) protocol (Agilent Technologies, Santa Clara, California, United States) to confirm successful labelling. To ensure even representation of all samples during sequencing, one equimolar library per target group was prepared for a NovaSeq 6000 (Illumina, United States of America) sequencing run. The sequencing was performed at BaseClear B.V., Leiden, the Netherlands.