title: NovaSeq of diatoms 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 diatoms, an equimolar mix of three forward primers and two reverse primers was used to maximise the amplification potential of the rcbL gene  (Vasselon et al., 2017).  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 4 L 5x Phire Green PCR buffer by ThermoFisher Scientific (Waltham, MA, USA), 0.8 L BSA 10 mg/mL, 0.4 L dNTPs 2.5 mM, 0.4 L Phire II Hotstart Taq-Polymerase (ThermoFisher Scientific), 1 L forward primer 10 M, 1 L reverse primer 10 M and 9.9 L MiliQ. The program was set to 30 sec at 98 C, followed by 35 cycles of 5 sec at 98 C, 5 sec at 50 C, 15 sec at 72 C, followed by 5 min at 72 C. The reaction mix for the second PCR was the same as for the first, but using 9.4 L of MilliQ and 3 L of cleaned DNA product with IDT10 primer labels. 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.