Revisiting the effect of PCR replication and sequencing depth on biodiversity metrics in environmental DNA metabarcoding

Environmental DNA (eDNA) metabarcoding is an increasingly popular tool for measuring and cataloguing biodiversity. Because the environments and substrates in which DNA is preserved differ considerably, eDNA research often requires bespoke approaches to generating eDNA data.Here, we explore how two experimental choices in eDNA study design- the number of PCR replicates and the depth of sequencing of PCR replicates- influence the composition and consistency of taxa recovered from eDNA extracts. We perform 24 PCR replicates from each of six soil samples using two of the most common metabarcodes for Fungi and Viridiplantae (ITS1 and ITS2), and sequence each replicate to an average depth of 84,000 reads. We find that PCR replicates were consistent in composition and relative abundance of taxa assigned at least 3.22% of the reads for Plant ITS2 and 13.58% of Fungal ITS1, and that rare taxa are often unique to one or a few PCR replicates. Stochasticity in detection of rare taxa introduces significant variation in alpha but not beta diversity. Our results suggest that low sequencing depths (1,000 assigned reads) and few PCR replicates may be sufficient for many biological applications, but DNA metabarcoding may never fully recover the true alpha diversity in a DNA extract.