negative control for sediment and foraminifera samples
DNA was extracted with the NucleoSpin Soil Kit (Macherey-Nagel) following the protocol described in Girard et al. (in press) (see supplementary Table S2 for protocol modifications). To serve as negative controls, for seawater, substrate and foraminifera extractions, the same method was carried out in tubes without any biological samples (empty or with preservation buffer). For foraminifera, the cytochrome c oxidase subunit 1 (COI) from the Leray-region was chosen as a marker following Macher et al. (2021) (Table 1). 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 of the first PCR consisted of 2.5 L of DNA template for 11.7 L Milli-Q water, 2 L PCR buffer CL 10X (Qiagen), 0.4 L MgCl2 25 mM, 0.8 L BSA 10 mg/mL, 0.4 L dNTP 25 mM, 0.2 L Taq-polymerase (Qiagen) 5 U/L, 1 L forward primer 10 M and 1 L reverse primer 10 M for a total volume of 20 L. The program was set to 3 min at 96 C, followed by 35 cycles of 15 s at 96 C, 30 s at 50 C, 40 s at 72 C, followed by 5 min at 72 C. After successful amplification, the PCR products were cleaned with magnetic NucleoMag NGS-Beads (bead volume at 0.9 times the total volume of the sample, Macherey Nagel, Dren, Germany). The reaction mix for the second PCR was the same as the first, using a cleaned DNA product as a template and the 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.