eDNA metabarcoding
Environmental DNA was extracted from approximate 0.25 g of sediment
using the DNeasy PowerSoil Kit (QIAGEN), with one DNA extraction control
for every 11 sediment samples. For metabarcoding, the V1 and V2 regions
of the 18S rRNA gene were amplified using primers (F04mod:
GCTTGWCTCAAAGATTAAGCC; R22mod: CCTGCTGCCTTCCTTDGA)
(Blaxter et al.,
1998). We used this 18S rRNA marker rather than a COI marker for eDNA
metabarcoding because: (1) this marker has been used successfully to
characterize benthic metazoans
(e.g. Fonseca et
al., 2010; Sinniger et al., 2016); and (2) it amplifies eukaryotes only,
whereas amplicon sequencing targeting eukaryotes using COI markers
typically returns a high percentage of off-target bacterial reads due to
non-specific amplification
(Collins et al.,
2019; Weigand & Macher, 2018). To multiplex, each synthesized primer
starts with two or three Ns, followed by an eight‐base tag and the
gene‐specific primer sequences
(He, Sutherland,
Pawlowski, & Abbott, 2019). PCR protocol and program followed
He et al.
(submitted). Three PCR replicates were used to amplify each eDNA sample
(using the same tagged primers) and replicates were combined after PCR.
PCR products from 21 eDNA samples, one DNA extraction control and one
PCR negative control were combined into a mixture that was then purified
using Agencourt AMPure XP beads (Beckman Coulter) and quantified using
SYBR Green I (Thermo Fisher Scientific) with a Tecan Infinite M200PRO
microplate reader. In total, 12 PCR product mixtures were made. Then, 1
µg of purified PCR mixture was used for library preparation using the
TruSeq DNA PCR-Free Library Prep Kit (Illumina). After library
preparation and quantification, four libraries were pooled for
sequencing using one MiSeq Reagent Kit v3 (600-cycles) (Illumina). In
total, three MiSeq Reagent Kit v3 (600-cycles) were used to sequence the
252 sediment samples.