Discussion
We found that the abundance of eDNA in aquaria was highest just after
fish were added and tended to decrease with time. This is consistent
with a previous study which showed that the highest abundance of fish
eDNA was at the start of experiments, due to the struggles during the
acclimation of introduced fish to the environment (Andersen et al.,
2012; Barnes et al., 2014; Takahara, Minamoto, Yamanaka Doi, and
Kawabata, 2012; Jo, Murakami, Yamamoto, Masuda, and Minamoto, 2019;
Sassoubre, Yamahara, Gardner, Block, and Boehm, 2016). However, we found
that different mock fish communities had different peaks in eDNA
abundance, with MC1 peaking on day 0, and MC2 peaking on day 1. The
reason for this is not known, indicating that further studies of eDNA
and fish behaviour are needed. The abundances of fish species obtained
by HTS were very different from the absolute copy numbers of eDNA
quantified with dPCR and qSeq. In general, PCR efficiency can affect the
proportions of sequences in sequence libraries. PCR efficiency is
affected by primer-target mismatches, composition (e.g., GC content) of
sequences, and the bases adjacent to primers (Ben-Dov et al., 2012;
Ruijter et al., 2009; Salipante et al., 2014; Sipos et al, 2007).
Therefore, these factors may have influenced the proportions of
sequences in our fish sequencing library, resulting in different
proportions compared with in the original eDNA samples.
In this study, the values quantified by dPCR tended to be slightly
higher than those by qSeq. It might be possible that these results are
due to non-specific binding of the probes and subsequent digestion,
resulting in the overestimation of dPCR. Another possible explanation
might be that qSeq-MiFish-U-F did not hybridize to all target eDNA
during single primer extension, causing underestimation. Indeed, the
sequence of the qSeq-MiFish-U-F primer used for SPE in this study has
two mismatches to the sequence of O. latipes . In PCR, this
mismatch resulted in low PCR efficiency; however, the mismatch
disappeared once the primer synthesized template DNA because the
synthesized DNA did not have a mismatch. In this study, we amplified DNA
by a 50-cycle PCR, and thus the effect of the mismatch would not be
significant and even DNA with two mismatch sequences was amplified and
quantified. In contrast, the SPE reaction in the qSeq protocol occurs
only once, and DNA is not amplified in the next PCR step if the first
single-stranded DNA failed to be synthesized. Thus, since SPE would be
more sensitive to mismatches in the template DNA than PCR, increasing
the specificity of the SPE reaction would prevent qSeq from amplifying
and quantifying environmental DNA containing mismatches.
This is the first study to employ the qSeq technique for quantifying
eDNA in fish species. Using an aquarium experiment with five fish
species, we demonstrated that qSeq could quantify eDNA from fish,
verified by dPCR. Quantitative sequencing can simultaneously quantify
multiple species by adding just one step to the standard iTag-sequencing
procedure without the need to establish specific assays for individual
target species like in dPCR or qPCR, which require design of primers and
probes, and condition optimization. Comprehensive quantitative data sets
of eDNA abundance can be obtained by applying qSeq to various
environments and species, providing datasets that will deepen our
knowledge of natural communities and the distribution of species.