Sequencing library preparation
Metabarcoding sequencing libraries were prepared from each pool using a
two-step amplicon protocol (D’Aloia, Bogdanowicz, Harrison, & Buston,
2017) in which an initial 34-cycle PCR targets the gene region of
interest using locus-specific primers with Nextera 5’ tails (5’-TC
GTCGGCAGCGTCAGATGTGTATAAGAGACAG appended to each forward primer and 5’
-GTCTCGTGGGCTC GGAGATGTGTATAAGAGACAG to each reverse primer, full
reaction details in the Supporting information). Equal volumes of the
locus-specific PCR products for each sample were then pooled and a
second 5-cycle PCR further amplified the product and added Nextera-style
sequencing adapters with unique i5 and i7 indexes that allow sequencing
reads to be assigned to samples during analysis (details about reagent
concentrations and PCR conditions in the SI). Rather than using
combinatorial indexing, which can lead to mis-assigned reads caused by
index-swapping (Carøe & Bohmann, 2020; Schnell, Bohmann, & Gilbert,
2015), we used custom-synthesized adapters with unique dual indexes
(Table S1) that can unequivocally identify samples by 8-base indexes on
both ends of the molecule. For the initial screening with the FR and VR
pools, 16 of the markers were amplified in individual PCRs and six of
the markers were duplexed in three reactions – each with two markers.
Duplexed markers amplified fragments from different barcoding genes with
>75 bp between expected amplicon sizes, which allowed for
visualization of two bands, one for each marker, on an agarose gel (see
Supporting Information for details). For each sample, PCR products for
all markers were pooled into a single indexed library. Three
individually indexed PCR replicates were performed for each of the three
replicate FR and VR DNA pools, for nine total replicates per DNA pool
(the experimental design is illustrated in Fig. S1). The experimental
feeds and fishmeal species DNA pools were only analysed with the top
four markers (see results), each amplified in a separate PCR. Three
individually indexed PCR replicates were performed for each of two DNA
extracts from an experimental feed, resulting in six replicates per
experimental feed (see SI, Fig. S1 for schematic). One negative control
(no template) was included for every 14 PCR reactions and extraction
blanks and negative library preparation controls were carried through to
sequencing.
Following the indexing PCRs, all libraries were pooled in equal volume,
cleaned using 1.8x AMPure XP beads (Beckman Coulter), and then eluted
with 50 ul 10 uM Tris-HCl pH 8. A 2% agarose gel was used to confirm
that the indexing PCR was successful based on a size shift after the
addition of indexes and adapters (~130 additional bases)
when compared to pooled non-indexed samples. The libraries were
sequenced using paired-end 150-bp on one lane of a HiSeq X Ten
(Novogene, Inc.) with 15% PhiX to account for moderately low library
complexity (following Aizpurua et al., 2018).