5- Conclusion
Here, we provide a thorough examination of the influence of PCR
replication and read sampling depth on two common measures of diversity
in environmental DNA metabarcoding research: α diversity and β
diversity. We find that metabarcoding PCR robustly estimates measures of
diversity that rely on high abundance taxa, and that low numbers of PCR
replicates are sufficient to distinguish sites from each other. However,
we also find that low abundance taxa tend to occur in only one or a few
replicate PCRs, and often require deeper sequencing of PCR amplicon
pools to be counted, in particular when read cutoff thresholds are high.
Importantly, the challenges of counting rare taxa are so great, in
particular in environmentally diverse sites, that even large numbers of
PCR replicates and relatively deep sequencing are insufficient to
guarantee that they will be recorded. Experiments aiming to catalogue
diversity should consider using replicate PCRs, replicate extracts, and
even replicate field sampling. While this problem necessarily limits the
utility of environmental DNA as a mechanisms to catalogue the full
diversity of taxa present at a given site, it also shows the potential
power of eDNA to recover even the rarest of taxa, although the
authenticity of exceptional taxa may need to be explored using a
different enrichment approach, such as hybridization capture or targeted
amplification.