What influences prey sequence recovery?
There are many factors that can influence the recovery of prey sequences
and thus the success of the brute force approach. The length of the
target sequence is important, with small amplicons being favored due to
a longer half-life and hence an increased detectability (Kamenova et
al., 2018). Whether the extracts are based upon recently ingested or
heavily digested materials (e.g. feces) will also have an impact on
recovery (Kamenova et al., 2018). For small invertebrates, whole-body
extraction is us usually the only feasible alternative. With whole-body
samples, we find it probable that the consumer DNA has the greatest
impact on prey sequence recovery as it competes for amplification and
detection during PCR and sequencing. The severity of the overabundance
problem is difficult to predict in advance, however, given that
different consumers have variable genome sizes, cell numbers and target
gene copy-numbers. Indeed, the ratio of the prey to consumer sequence
may also vary from one season to the next, between different sexes,
feeding or life stages. The exact ratios acquired here may thus not be
representative for studies of other invertebrates although the study
design is otherwise identical. Nonetheless, we show that the problem of
consumer DNA overabundance may be overcome by sequencing deeper.
With a great overabundance of putative consumer DNA (98%), our results
underline the problem of dietary samples from small animals. In fact,
over 3.8 billion of the sequence reads belonged to the top three
abundant zOTUs, recruiting 1.6, 1.4 and 0.8 billion reads, respectively.
Although we could not resolve the taxonomy of these zOTUs beyond
Maxillopoda, we are confident that these represent the three sampled
copepod species just based on the abundance of their read counts. The
first sequence variant dominated samples of Oithona similis , the
second dominated samples from Microcalanus spp., and the third
dominated samples from Microsetella norvegica . Other abundant
maxillopod zOTUs with similar distribution patterns were also
identified. These may be artifacts of the dominant sequences, real
variants of the gene sequenced, or remnants of copepod prey. As we are
unable to distinguish between these alternatives, we conservatively
chose to discard all Maxillopoda sequence reads from our analyses.
It is however not just consumer DNA that is problematic as contaminants
may further dilute samples, making finding enough prey data more
difficult. In the datasets analyzed in this study, we observed more
reads from the four extraction negatives sequenced for the pilot dataset
(10.8 mill. reads) than in the combined twenty extraction negatives from
the full dataset (9.3 mill. reads). This holds also at the level of real
samples, given that the pilot dataset contains a higher contaminant
percentage (1.6%) than the full dataset (1.1%). If we somehow “worked
cleaner” during preparation of the second sequencing run is not known,
but we hypothesize that increased contamination could explain some of
the observed gap in prey recovery (0.4% in pilot versus 1.2% in full
dataset), since also contaminant sequences may compete for amplification
and sequencing. A lot of non-prey sequences would inevitably lead to a
lower output of prey, and the more contaminated samples are, the greater
the problem. Future dietary studies may arguably increase the yield of
prey sequences by paying attention to lab routines and making them as
clean as possible.
Other possible influences on prey sequence recovery could be technical
– for instance that deeper sequencing leads to a greater number of rare
prey zOTUs surpassing a set cutoff-value (we denoised each sample
individually with a four reads cutoff), or related to the biology of the
consumer. Several Arctic copepod species may for example enter diapause
prior to the polar night. A dormant state ensues, where energy spent on
motility, reproduction and feeding is drastically lowered (Conover,
1988). Because internal wax ester storages are used for energy, feeding
activities are no longer required. Consequently, some copepod species
may be expected to contain little or no prey DNA during certain periods
of the year. The copepods studied here are not expected to enter
diapause, but may to some extent reduce metabolic rates during winter
(Conover & Huntley, 1991).