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Metabarcoding of environmental DNA constitutes a state-of-the-art tool for environmental studies. One fundamental principle implicit in most metabarcoding studies is that individual sample amplicons can still be identified after being pooled with others—based on their unique combinations of tags—during the so-called demultiplexing step that follows sequencing. Nevertheless, it has been recognized that tags can sometimes be changed (i.e., tag jumping), which ultimately leads to sample crosstalk. Here, using four DNA metabarcoding data sets derived from the analysis of soils and sediments, we show that tag jumping follows very specific and systematic patterns. Specifically, we find a strong correlation between the number of reads in blank samples and their topological position in the tag matrix (described by vertical and horizontal vectors). This observed spatial pattern of artefactual sequences could be explained by polymerase activity, which leads to the exchange of the 3′ tag of single stranded tagged sequences through the formation of heteroduplexes with mixed barcodes. Importantly, tag jumping substantially distorted our data sets—despite our use of methods suggested to minimize this error. We developed a topological model to estimate the noise based on the counts in our blanks, which suggested that 40%–80% of the taxa in our soil and sedimentary samples were likely false positives introduced through tag jumping. We highlight that the amount of false positive detections caused by tag jumping strongly biased our community analyses.

Analysis of environmental DNA detected in lake sediments shows promise to become a great paleoecological technique that can provide detailed information about organism communities living in past environments. However, when interpreting sedimentary environmental DNA records, it is of crucial importance to separate ecosystem responses to large-scale environmental change from “noise” caused by changes in sediment provenance or potential post-depositional DNA mobility. In this study, we show that plant and mammalian communities reconstructed from sediments are strongly affected by sediment provenance, but unaffected by vertical mobility of DNA after sediment deposition. We observe that DNA from aquatic plants was abundant in background sediment, while embedded detrital event layers (sediment deposited under erosion events) primarily contained terrestrial plants; hence, vertical mobility of aquatic plant DNA across sediment layers was negligible within our studied lakes. About 33% of the identified terrestrial plant genera were only found in detrital sediment, suggesting that sediment origin had a strong impact on the reconstructed plant community. Similarly, DNA of some mammalian taxa (Capra hircus, Ursus arctos, Lepus, and Felis) were only or preferentially found in detrital event layers. Temporal changes across the Holocene were the main drivers of change for reconstructed plant communities, but sediment type was the second most important factor of variance. Our results highlight that erosion and sediment provenance need to be considered when reconstructing past mammalian and plant communities using environmental DNA from lake sediments.