DOI: 10.5061/dryad.b8f6s44
title: Data from: Non-specific amplification compromises environmental DNA metabarcoding with COI

description: 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.
description: COI-SeaDNA-mid fastq
 (R2)Illumina MiSeq reads in
 FASTQ format for the COI-SeaDNA-mid primer set, amplifying environmental
 DNA samples collected at five UK estuary and coastal sites in 2016.
 MD5SUM='f5eeffbc35b0cbde979c3b16f573c87e'.COI-seamid-R2.fastq.gzCOI-SeaDNA-mid fastq (R1)Illumina MiSeq reads in FASTQ format for the COI-SeaDNA-mid primer set, amplifying environmental DNA samples collected at five UK estuary and coastal sites in 2016. MD5SUM='39e8766cf74966a573689c4a3126bd28'.COI-seamid-R1.fastq.gzCOI-Leray-XT fastq (R1)Illumina MiSeq reads in FASTQ format for the COI-Leray-XT primer set, amplifying environmental DNA samples collected at five UK estuary and coastal sites in 2016. MD5SUM='12be4dacc54a38ad87e684d158fc0ffe'.COI-lerayxt-R1.fastq.gzCOI-Leray-XT fastq (R2)Illumina MiSeq reads in FASTQ format for the COI-Leray-XT primer set, amplifying environmental DNA samples collected at five UK estuary and coastal sites in 2016. MD5SUM='39fce75cece80396e835ce91c59a9301'.COI-lerayxt-R2.fastq.gzCOI-SeaDNA-short fastq (R2)Illumina MiSeq reads in FASTQ format for the COI-SeaDNA-short primer set, amplifying environmental DNA samples collected at five UK estuary and coastal sites in 2016. MD5SUM='1256951c64d5912bdf797a76e79c664c'.COI-seashort-R2.fastq.gzCOI-SeaDNA-short fastq (R1)Illumina MiSeq reads in FASTQ format for the COI-SeaDNA-short primer set, amplifying environmental DNA samples collected at five UK estuary and coastal sites in 2016. MD5SUM='01066f1d34d60c5d0f042e6f4ced51bf'.COI-seashort-R1.fastq.gz12S-MiFish-U fastq (R2)Illumina MiSeq reads in FASTQ format for the 12S-MiFish-U primer set, amplifying environmental DNA samples collected at five UK estuary and coastal sites in 2016. MD5SUM='b565669fdd91dad2633444966838cc3f'.12S-mifishu-R2.fastq.gz12S-MiFish-U fastq (R1)Illumina MiSeq reads in FASTQ format for the 12S-MiFish-U primer set, amplifying environmental DNA samples collected at five UK estuary and coastal sites in 2016. MD5SUM='7bcdd03c9853c6af19884cc732dd25ad'.12S-mifishu-R1.fastq.gz