Data from: Non-specific amplification compromises environmental DNA metabarcoding with COI

1. Metabarcoding extra-organismal DNA from environmental samples is now a
key technique in aquatic biomonitoring and ecosystem health assessment.
However, choice of genetic marker and primer set is a critical
consideration when designing experiments, especially so when developing
community standards and legislative frameworks. Mitochondrial cytochrome c
oxidase subunit I (COI), the standard DNA barcode marker for animals, with
its extensive reference library, taxonomic discriminatory power, and
predictable sequence variation, is the natural choice for many
metabarcoding applications such as the bulk sequencing of invertebrates.
However, the overall utility of COI for environmental sequencing of
targeted taxonomic groups has yet to be fully scrutinised. 2. Here, by
using a case study of marine and freshwater fishes from the British Isles,
we quantify the in silico performance of twelve mitochondrial primer pairs
from COI, cytochrome b, 12S and 16S, in terms of reference library
coverage, taxonomic discriminatory power, and primer universality. We
subsequently test in vitro three COI primer pairs and one 12S pair for
their specificity, reproducibility, and congruence with independent
datasets derived from traditional survey methods at five estuarine and
coastal sites in the English Channel and North Sea coast. 3. Our results
show that for aqueous extra-organismal DNA at low template concentrations,
both metazoan and fish-targeted COI primers perform poorly in comparison
to 12S, exhibiting low levels of reproducibility due to non-specific
amplification of prokaryotic and non-target eukaryotic DNAs. 4. An ideal
metabarcode would have an extensive reference library for which custom
primer sets can be designed for either broad assessments of biodiversity
or taxon specific surveys, but unfortunately, low primer specificity
hinders the use of COI, while the paucity of reference sequences is
problematic for 12S. The latter, however, can be mitigated by expanding
the concept of DNA barcodes to include whole mitochondrial genomes
generated by genome-skimming existing tissue collections.