DNA metabarcode amplification for fish detection
We followed the same procedures
used by Bessey et al. (2021). One-step quantitative polymerase chain
reactions (qPCR) were performed in duplicate for each sample using 2 µL
of extracted DNA and a mitochondrial DNA 16S rDNA universal primer set
targeting fish taxa (16SF/D
5ʹ GACCCTATGGAGCTTTAGAC 3ʹ and 16S2R-degenerate 5ʹ
CGCTGTTATCCCTADRGTAACT 3ʹ; Berry et al. 2017, Deagle et al. 2007), with
the addition of fusion tag primers unique to each sample that included
Illumina P5 and P7 adaptors. A single round of qPCR was performed in a
dedicated PCR laboratory. Quantitative PCR reagents were combined in a
dedicated clean room and included 5 μL AllTaq PCR Buffer (QIAGEN; Venlo,
Netherlands), 0.5 μL AllTaq DNA Polymerase, 0.5 μL dNTPs (10 mM), 1.0 μL
Ultra BSA (500 μg/μL), SYBR Green I (10 units/μL), 0.5 μL forward primer
(20 μM) and 5.0 μL reverse primer (20 μM) , 2 μL of DNA and
Ultrapure™Distilled Water (Life Technologies) made up to 25 μL total
volume. Mastermix was dispensed manually and qPCR was performed on a
CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad, California, USA)
using the following conditions: initial denaturation at 95°C for 5 min,
followed by 40 cycles of 30 s at 95°C, 30 s at the primer annealing
temperature 54°C, and 45 s at 72°C, with a final extension for 10 min at
72°C. All duplicate qPCR products from the same subsample were combined
prior to library pooling. The mean Cq value from qPCR duplicates was
used as an indication of initial DNA copy number. A sequencing library
was made by pooling amplicons into equimolar ratios based on qPCR Ct
values and sequenced on an Illumina Miseq platform (Illumina; San Diego,
USA). The libraries were size selected using a Pippin Prep (Sage
Science, Beverly, USA) and purified using the Qiaquick PCR Purification
Kit (Qiagen; Venlo, Netherlands). The volume of purified library added
to the sequencing run was determined by quantifying the concentration
(Murray et al. 2015) using a Qubit 4 fluorometer (ThermoFisher
Scientific). The library was unidirectionally sequenced using a 300
cycle MiSeq® V2 Reagent Kit and standard flow cell.
PCR plates included blank laboratory extraction controls (extraction
reagents used with no DNA template), PCR negative controls (2 μL of DI
water used rather than DNA template) and positive controls (dhufish;Glaucosoma hebraicum and swordfish; Xiphias gladius ).
Dhufish inhabit the mesocosm, whereas swordfish do not, so the latter
was a more appropriate positive control. No negative control (extraction
or PCR) contained more than 17 reads, with the maximum number of reads
per fish species being four. Therefore, we used a detection rate of
greater than five sequences to classify something as a positive
detection. All positive controls amplified multiple reads identifying
dhufish and swordfish with 100% identity. Swordfish was not detected in
any sample except for our positive PCR control.