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.