Testing the potential of a ribosomal 16S marker for DNA metabarcoding of insects

Elbrecht, Vasco; Taberlet, Pierre; Déjean, Tony; Valentini, Alice; Usseglio‐Polatera, Philippe; Beisel, Jean‐Nicolas; Coissac, Éric; Boyer, Frédéric; Leese, Florian

doi:10.7717/peerj.1966

Cited by 123 publications

(102 citation statements)

References 35 publications

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“…For DNA metabarcoding, we used two different primer sets targeting two mitochondrial DNA (mtDNA) genes commonly used in arthropod studies: cytochrome c oxidase subunit I (COI) (~211 bp fragment) (Zeale, Butlin, Barker, Lees, & Jones, 2011) and 16S ribosomal RNA (~160 bp fragment) (Elbrecht et al, 2016) (Supporting Information Figure S1). Primers were uniquely tagged.…”

Section: Pcr Amplificationmentioning

confidence: 99%

“…The generic primers used in this study have been designed for metabarcoding of degraded DNA, and tested previously (Elbrecht et al, 2016;Zeale et al, 2011). Given the short amplicon size, they perform comparatively well by resolving most taxa to species level (Tables S2-S3).…”

Section: Choice Of Primers and Database Coverage In Metabarcoding Smentioning

confidence: 99%

“…Hence, several new molecular approaches have recently been suggested for obtaining fast and efficient data on arthropod communities and their interactions through non-invasive genetic techniques. This includes extracting DNA from sources such as bulk samples or insect soups (Arribas, Andújar, Hopkins, Shepherd, & Vogler, 2016;Elbrecht et al, 2016;Hajibabaei, Shokralla, Zhou, Singer, & Baird, 2011;Yu et al, 2012), empty leaf mines (Derocles, Evans, Nichols, Evans, & Lunt, 2015), spider webs (Blake, McKeown, Bushell, & Shaw, 2016;Xu, Yen, Bowman, & Turner, 2015), pitcher plant fluid (Bittleston, Baker, Strominger, Pringle, & Pierce, 2015), environmental samples like soil and water (environmental DNA [eDNA]) (Taberlet, Coissac, Hajibabaei, & Rieseberg, 2012;Thomsen et al, 2012;Thomsen & Willerslev, 2015;Zinger et al, 2018), host plant and predatory diet identification from insect DNA extracts (Jurado-Rivera, Vogler, Reid, Petitpierre, & Gómez-Zurita, 2009;Paula et al, 2016), and predator scat from bats (Bohmann et al, 2011;Vesterinen, Lilley, Laine, & Wahlberg, 2013). Recently, also DNA from pollen attached to insects has been used for retrieving information on plant-pollinator interactions (Bell et al, 2017;Pornon et al, 2016).…”

Section: Introductionmentioning

confidence: 99%

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Environmental DNA metabarcoding of wild flowers reveals diverse communities of terrestrial arthropods

Thomsen

Sigsgaard

2019

Ecology and Evolution

159

207

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Terrestrial arthropods comprise the most species‐rich communities on Earth, and grassland flowers provide resources for hundreds of thousands of arthropod species. Diverse grassland ecosystems worldwide are threatened by various types of environmental change, which has led to decline in arthropod diversity. At the same time, monitoring grassland arthropod diversity is time‐consuming and strictly dependent on declining taxonomic expertise. Environmental DNA (eDNA) metabarcoding of complex samples has demonstrated that information on species compositions can be efficiently and non‐invasively obtained. Here, we test the potential of wild flowers as a novel source of arthropod eDNA. We performed eDNA metabarcoding of flowers from several different plant species using two sets of generic primers, targeting the mitochondrial genes 16S rRNA and COI. Our results show that terrestrial arthropod species leave traces of DNA on the flowers that they interact with. We obtained eDNA from at least 135 arthropod species in 67 families and 14 orders, together representing diverse ecological groups including pollinators, parasitoids, gall inducers, predators, and phytophagous species. Arthropod communities clustered together according to plant species. Our data also indicate that this experiment was not exhaustive, and that an even higher arthropod richness could be obtained using this eDNA approach. Overall, our results demonstrate that it is possible to obtain information on diverse communities of insects and other terrestrial arthropods from eDNA metabarcoding of wild flowers. This novel source of eDNA represents a vast potential for addressing fundamental research questions in ecology, obtaining data on cryptic and unknown species of plant‐associated arthropods, as well as applied research on pest management or conservation of endangered species such as wild pollinators.

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Section: Pcr Amplificationmentioning

confidence: 99%

Section: Choice Of Primers and Database Coverage In Metabarcoding Smentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Environmental DNA metabarcoding of wild flowers reveals diverse communities of terrestrial arthropods

Thomsen

Sigsgaard

2019

Ecology and Evolution

159

207

View full text Add to dashboard Cite

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“…Using multiple primer pairs is expected to reduce amplification biases and increase the opportunities of detecting all targeted taxonomic groups. To fully understand the performance of a multigene metabarcoding approach, mock communities are ideal because the expected number of species is known a priori (Clarke, Soubrier, Weyrich, Weyrich, & Cooper, 2014;Elbrecht et al, 2016;Kermarrec et al, 2013). Nonetheless, there are few studies that have taken this approach (but see Clarke et al, 2014).…”

Section: Introductionmentioning

confidence: 99%

Metabarcoding using multiplexed markers increases species detection in complex zooplankton communities

Zhang

Chain

Abbott

et al. 2018

Evolutionary Applications

140

146

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Metabarcoding combines DNA barcoding with high‐throughput sequencing, often using one genetic marker to understand complex and taxonomically diverse samples. However, species‐level identification depends heavily on the choice of marker and the selected primer pair, often with a trade‐off between successful species amplification and taxonomic resolution. We present a versatile metabarcoding protocol for biomonitoring that involves the use of two barcode markers (COI and 18S) and four primer pairs in a single high‐throughput sequencing run, via sample multiplexing. We validate the protocol using a series of 24 mock zooplanktonic communities incorporating various levels of genetic variation. With the use of a single marker and single primer pair, the highest species recovery was 77%. With all three COI fragments, we detected 62%–83% of species across the mock communities, while the use of the 18S fragment alone resulted in the detection of 73%–75% of species. The species detection level was significantly improved to 89%–93% when both markers were used. Furthermore, multiplexing did not have a negative impact on the proportion of reads assigned to each species and the total number of species detected was similar to when markers were sequenced alone. Overall, our metabarcoding approach utilizing two barcode markers and multiple primer pairs per barcode improved species detection rates over a single marker/primer pair by 14% to 35%, making it an attractive and relatively cost‐effective method for biomonitoring natural zooplankton communities. We strongly recommend combining evolutionary independent markers and, when necessary, multiple primer pairs per marker to increase species detection (i.e., reduce false negatives) in metabarcoding studies.

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“…However, by using barcoding regions with less variability, taxonomic resolution will decrease and therefore it is as mentioned, desirable to attempt to reduce these biases in order to retain the use of COI. Fortunately, a number of recent studies have shown that while COI is variable, with some of the degenerate primers available such as mCOIintF (Leray et al, 2013) and jgHCO2198 (Geller et al, 2013), it is nevertheless possible to get at least for some groups a comparable taxonomic coverage to alternative barcoding regions (Clarke, Beard, Swadling, & Deagle, 2017;Elbrecht et al, 2016). The variation within COI does, however, mean that a large amount of degeneracy is needed.…”

Section: Introductionmentioning

confidence: 99%

A broadly applicable COI primer pair and an efficient single‐tube amplicon library preparation protocol for metabarcoding

Rubbmark

Sint

Horngacher

et al. 2018

Ecology and Evolution

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The nucleotide variation in the cytochrome c oxidase subunit I (COI) gene makes it ideal for assigning sequences to species. However, this variability also makes it difficult to design truly universal primers. Here, we present the forward primer “Sauron‐S878,” specifically designed to facilitate library preparation for metabarcoding. This primer is modified to improve the coverage of terrestrial species compared to the primer mCOIintF, optimized for aquatic systems, which raised the in silico coverage from 74.4% to 98.3% of available NCBI sequences (perfect match in 3′ region, up to three mismatches in remaining primer). When paired with the reverse primer “jgHCO2198” (fragment length ~313 bp), these primers amplified 98.4% of 255 tested DNA extracts from various taxa, which are better than many other common COI barcoding primers. Furthermore, a single‐tube protocol was developed, wherein these primers amplify the target gene, and attach MIDs and Illumina sequencing adapters in one reaction. This eliminates the need for re‐amplification or enzymatic ligation during library preparation while keeping the flexibility to modularly combine primers and MIDs. Using the single‐tube approach, three replicates of three mock samples were sequenced on a MiSeq platform with no adverse effects compared to commercial Nextera indexing kits. From this run, 75% of all included taxa could be recovered, with no considerable bias among taxonomic groups. Despite the fact that 98.4% of the extracts were confirmed to amplify in vitro, this number was lower than expected. A reason for this discrepancy was a clear link between the relative concentration of a specific DNA type in the template and the number of returned reads for this DNA. We would argue that such a bias may be especially problematic in metabarcoding where samples usually contain trace DNA in unknown amounts. However, how this affects the completeness of metabarcoding results has yet been poorly investigated.

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Testing the potential of a ribosomal 16S marker for DNA metabarcoding of insects

Cited by 123 publications

References 35 publications

Environmental DNA metabarcoding of wild flowers reveals diverse communities of terrestrial arthropods

Environmental DNA metabarcoding of wild flowers reveals diverse communities of terrestrial arthropods

Metabarcoding using multiplexed markers increases species detection in complex zooplankton communities

A broadly applicable COI primer pair and an efficient single‐tube amplicon library preparation protocol for metabarcoding

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