Library preparation and high-throughput sequencing
To assess fish diversity in shrimp dDNA, we employed three mitochondrial
markers (Table S2). The first two markers were specifically designed for
fish DNA metabarcoding: the 12S-Teleo (64 bp, Valentini et al. 2016,
hereafter Teleo ) and the 12S-MiFish-U (163-185 bp, Miya et al.
2015, hereafter MiFish ). These markers target distinct short
hypervariable regions of the 12S rRNA gene, and their combined use has
been recommended to enhance fish species detection (Polanco F. et al.,
2021). The third marker, MG2 (Tournayre et al., 2020), targets a
133 bp region of the Cytochrome Oxidase I (COI) gene using highly
degenerate primers. This marker was designed to amplify a wide range of
invertebrates and vertebrates. It allows for the verification of the
consumer species identity and has demonstrated its effectiveness in
assessing Guyanese fish diversity when combined with other primers
(Quéméré et al., 2021).
Metabarcoding libraries were prepared using a two-step PCR strategy with
dual-indexed combinations for tagging, following the protocol outlined
in Galan et al. (2018). For each sample and marker, three independent
amplicon sequencing libraries were built (PCR replicates). Multiple
negative controls for extraction, PCR and indexing controls were
included on each 96-well PCR plate. PCR1 (gene-specific amplifications)
and PCR2 (indexing) were performed using 2X QIAGEN Multiplex Kit Master
Mix (QIAGEN). PCR products were checked by electrophoresis in 1.75%
agarose gel before being pooled (one pool per marker). Once pooled,
non-specific PCR products and primer dimers were removed through size
selection by excision on low-melting agarose gel (1.25%), followed by
purification using the PCR Clean-up Gel Extraction Kit (Macherey-Nagel).
Pool libraries were verified on a fragment analyzer, quantified by qPCR
using the KAPA library quantification kit (Roche), and normalized to 4
nM. Sequencing was performed using a V2 500 cycle-kit reagent cartridge
(Illumina) for 2 × 200 bp paired-end sequencing on an Illumina MiSeq
platform.