Collins, RA, Bakker, J, Wangensteen, OS, Soto, AZ, Corrigan, L, Sims, DW, Genner, MJ and Mariani, S (2019) Non‐specific amplification compromises environmental DNA metabarcoding with COI. Methods in Ecology and Evolution. ISSN 2041-210X

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Abstract

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.

Item Type:	Article
Additional Information:	This is the peer reviewed version of the following article: Collins, RA, Bakker, J, Wangensteen, OS, et al. Non‐specific amplification compromises environmental DNA metabarcoding with COI. Methods Ecol Evol. 2019; 00: 1– 17. https://doi.org/10.1111/2041-210X.13276, which has been published in final form at http://dx.doi.org/10.1111/2041-210x.13276. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions.
Uncontrolled Keywords:	0602 Ecology, 0603 Evolutionary Biology, 0502 Environmental Science and Management
Subjects:	G Geography. Anthropology. Recreation > GE Environmental Sciences Q Science > QH Natural history > QH301 Biology Q Science > QH Natural history > QH426 Genetics
Divisions:	Biological & Environmental Sciences (from Sep 19)
Publisher:	Wiley
Date Deposited:	15 Oct 2019 11:36
Last Modified:	04 Sep 2021 08:39
DOI or ID number:	10.1111/2041-210x.13276
URI:	https://researchonline.ljmu.ac.uk/id/eprint/11575

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