Library preparation
We used a two step amplification protocol, which has been shown to be a robust and cost effective method for DNA metabarcoding (Zizka, Elbrecht, Macher, & Leese, 2019). Each of the 24 DNA extracts (12 from ethanol and 12 from bulk) were PCR amplified in triplicates. The PCR mix was prepared in 25 µl reactions consisting of 12.5 µl 2x KAPA HiFi Hotstart ReadyMix, 0.75 µl of forward and reverse primer (10 µM), 8.5 µl nuclease free water and 2.5 µl of DNA from the bulk samples. For the eDNA samples from the ethanol preservative, the same mix was prepared but with 6 µl nuclease free water and 5 µL of eDNA. PCR cycling conditions were: initial denaturation for 3 min at 95 °C, 35 cycles of denaturation for 30 s at 98 °C, annealing for 30 s at 57 °C and extension for 30 s at 72 °C and a final extension for 1 min at 72°C. The three PCR replicates were pooled and 37.5 µl of the pooled PCR product was purified using 30 µl CleanNGS beads (GC Biotech) according to the manufacturer’s protocol and eluted in 40 µL TE-buffer. We screened the literature to identify primers that have been successfully used in metabarcoding studies for marine species (Table 1). These primer sets were first tested in silico using EcoPCR (Ficetola et al., 2010) and the MIDORI_UNIQUE_COI_MARINE_20180221 reference dataset (Machida, Leray, Ho, & Knowlton, 2017) (maximum errors set at 3, minimum length of the amplicon set at 100, maximum length of the amplicon set at 900). Six primer sets were then tested in the wetlab, and five primer sets yielded a PCR product for our macrobenthos samples (Table 1). Four primer sets (A,B,C and E) amplify the 3’ region and one primer set (D) targets the 5’ region of the Folmer region (Folmer, Black, Hoeh, Lutz, & R, 1994) (Table 1). All 24 samples were amplified with these five primer sets. The purified PCR product was then used as template for the index PCR using the Nextera XT Index kit v2 from Illumina. The reaction mix consisted of 5 µl nuclease free water, 12.5 µl 2X KAPA HiFi HotStart ReadyMix, 2.5 µl of each index primer (10µM) and 2.5 µL purified PCR product. The PCR cycling conditions were: initial denaturation for 3 min at 95°C, 8 cycles of denaturation for 30 s at 95 °C, annealing for 30 s at 55 °C and extension for 30 s at 72 °C and a final extension for 5 min at 72°C. Indexed PCR products were purified with Clean NGS beads. Successful indexing was checked by loading PCR1 and index PCR products on the Qiaxcel (Qiagen). Indexed PCR products were measured twice with the Quantus and equimolarly pooled. The library was sequenced on two Illumina Miseq runs (300bp, PE, sequenced by Admera Health Biopharma Services): primer sets A-D were run together, while primer set E was added to a second run containing the same macrobenthos samples for a study on the variation between biological, DNA and PCR replicates (Van Den Bulcke et al, in preparation). For both runs, 20% PhiX was added.