Replication levels, false presences and the estimation of the presence/absence from eDNA metabarcoding data

Abstract

Environmental DNA (eDNA) metabarcoding is increasingly used to study the present and past biodiversity. eDNA analyses often rely on amplification of very small quantities or degraded DNA. To avoid missing detection of taxa that are actually present (false negatives), multiple extractions and amplifications of the same samples are often performed. However, the level of replication needed for reliable estimates of the presence/absence patterns remains an unaddressed topic. Furthermore, degraded DNA and PCR/sequencing errors might produce false positives. We used simulations and empirical data to evaluate the level of replication required for accurate detection of targeted taxa in different contexts and to assess the performance of methods used to reduce the risk of false detections. Furthermore, we evaluated whether statistical approaches developed to estimate occupancy in the presence of observational errors can successfully estimate true prevalence, detection probability and false-positive rates. Replications reduced the rate of false negatives; the optimal level of replication was strongly dependent on the detection probability of taxa. Occupancy models successfully estimated true prevalence, detection probability and false-positive rates, but their performance increased with the number of replicates. At least eight PCR replicates should be performed if detection probability is not high, such as in ancient DNA studies. Multiple DNA extractions from the same sample yielded consistent results; in some cases, collecting multiple samples from the same locality allowed detecting more species. The optimal level of replication for accurate species detection strongly varies among studies and could be explicitly estimated to improve the reliability of results.

Assessing vertebrate biodiversity in a kelp forest ecosystem using environmental DNA

“…2013; Ficetola et al . 2015), this approach may improve low detection rates and the reliability of eDNA metabarcoding studies.…”

“…2014; Ficetola et al . 2015). When eDNA is rare variability across replicates may be expected, but our approach aimed to balance capturing sample heterogeneity with minimizing spurious assignments due to PCR or sequencing errors, primer tag bias (J. L. O'Donnell, R. P. Kelly, N. C. Lowell & J.…”

“…Sample heterogeneity in this study underscores the need for a larger number of replicates (perhaps as much as >8), especially for species with low detection probabilities and field studies lacking ground‐truthing data (Ficetola et al . 2015), but see Lahoz‐Monfort et al . 2015.…”

Effect of marker choice and thermal cycling protocol on zooplankton DNA metabarcoding studies

“…Amplification and sequencing of COI amplicons generated using the low annealing temperature protocol was highly repeatable in spite of using different sequencing chemistries (v2 and v3) and number of sequencing cycles (2 × 250 and 2 × 300), with OTUs representing >0.1% of reads in one replicate almost always detected in the corresponding replicate. Estimating the reproducibility of rare OTUs by sequencing a set of technical replicates in each study could provide a means to establish an abundance threshold for OTU retention, as nonreproducible OTUs are more likely to represent PCR or sequencing artifacts (De Barba et al., 2014; Ficetola et al., 2015). …”

Sedimentary DNA versus morphology in the analysis of diatom-environment relationships

“…Finally, the validated sequence types are taxonomically assigned using ecotag. This algorithm assigns the query sequence to a taxon based on sequence similarity by using a group-specific reference library, which was created using the program ecoPCR, which extracts artificially targeted sequences using an in silico-PCR (Ficetola et al 2015) with the specific rbcL diatom primers (see above) from the EMBL database (release 124, July 2015). Ecotag first searches for the primary and secondary reference(s) that show highest similarity to the query sequence and then assigns it (them) to the most recent common ancestor of the primary and secondary reference sequence(s).…”