Laboratory protocol
Eleven of the vouchered samples were obtained as DNA extracts from
museums (Table 2). For the remaining samples, DNA was extracted from
muscle tissue preserved in ethanol (vouchered specimens), frozen muscle
tissue (vouchered specimens and market samples), or dried ground tissue
(tissue mixture samples) with single tube column extractions (Omega
BioTek EZNA Tissue kits) following the manufacturer’s instructions.
Extraction blanks were included for each batch of extractions.
DNA extracts were quantified by a Qubit fluorometer (high-sensitivity or
broad-range dsDNA assay depending on concentration range), diluted with
DNAse-free water, and added in equal proportion to the FR and VR DNA
pools. To account for pipetting error, three replicates of the FR and VR
DNA pools were constructed by pooling individual DNA extracts in
triplicate. For the experimental feeds, to disentangle whether biases in
the proportion of sequencing reads attributed to each species were
caused by variation in amplification or DNA extraction efficiency, DNA
extracts from the 30 fishmeal species were combined in two additional
mock DNA pools: one with equal concentration among all taxa (mock equal,
ME) and the other in which DNA extract concentration was proportionate
to the amount of tissue included in the fishmeal (mock variable, MV).
Similar to the previous reference DNA pools, DNA pools for the ME and MV
were prepared in triplicate (Fig. S1).