Experimental tissue mixture samples
Metabarcoding is typically used to detect both rare and abundant
constituents in mixtures, and most application cases (diets, larval
assemblages, net tows, etc.) include species in unequal proportions
along with varying amounts and types of non-target material. To evaluate
detection power in this scenario, we used fishmeal mixed with non-target
tissue composed of different fillers. The purpose of the non-target
tissue (filler) was to test whether metabarcoding markers are negatively
impacted by fillers, either because of a loss of on-target sequencing
reads or because of potential PCR inhibition. Our experimental mixtures
emulated aquaculture feeds; but the experimental feeds have similarities
to other mixed sample types, for example, stomach contents of an
omnivore where fish prey items may be mixed with other (non-fish)
constituents. In our test case, we freeze-dried muscle tissue from 30 of
the unvouchered fish species in the FR pool, coarsely homogenized each
sample in a coffee grinder, and then freezer milled samples flash-frozen
with liquid nitrogen into a powder. Powdered samples were then assigned
to one of six abundance levels and added to make up either 13.33%,
3.65%, 1.91%, 1%, 0.1%, or 0.01% of the total mixture (by weight),
thus spanning four orders of magnitude in relative representation from
abundant-to-rare (Table 3). Each abundance level was represented by five
species. This experimental design allowed us to assess how dominant and
rare taxa added at discrete proportions to a heterogenous mixture relate
to the proportion of sequencing reads attributed to each taxon and to
compare amplification biases across multiple taxa added in the same
amount to the fishmeal.
To test how nontarget material or mixture matrix could affect
metabarcoding efficiency and therefore, species recovery, the
multi-species fishmeal mixtures were combined with two unique filler
types to make seven individual experimental feeds with low (2%), medium
(10%), and high (25%) ratios of fishmeal-to-filler (Table 3). Fillers
for experimental feeds included plant-derived materials – grain and
grass flours – and animal byproducts – bloodmeal and feathermeal – to
emulate mixture constituents used in fish production (i.e., aquaculture
feeds), but are also representative of non-fish diet components.
Fishmeal proportions also mimicked potential levels of fish tissue added
to aquaculture feeds, from low (0%-2%) to high (25%) proportions of
fish in the feed mixture. By multiplying the ratio of fishmeal-to-filler
by the fishmeal tissue in the experimental feed, we could test the
detection threshold for individual taxa in the overall tissue mixture
down to 0.0002% of the experimental feed by mass (i.e., the smallest
tissue input – 0.01% of fishmeal – at 2% fishmeal-to-filler).