Environmental metabarcodes for insects: in silico PCR reveals potential for taxonomic bias

July 18, 2017 | Autor: Laura Weyrich | Categoria: Computational Biology, Biological Sciences, Animals, Insects, Polymerase Chain Reaction
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Molecular Ecology Resources (2014) 14, 1160–1170

doi: 10.1111/1755-0998.12265

Environmental metabarcodes for insects: in silico PCR reveals potential for taxonomic bias LAURENCE J. CLARKE, JULIEN SOUBRIER, LAURA S. WEYRICH and A L A N C O O P E R Australian Centre for Ancient DNA, University of Adelaide, Adelaide, SA 5005, Australia

Abstract Studies of insect assemblages are suited to the simultaneous DNA-based identification of multiple taxa known as metabarcoding. To obtain accurate estimates of diversity, metabarcoding markers ideally possess appropriate taxonomic coverage to avoid PCR-amplification bias, as well as sufficient sequence divergence to resolve species. We used in silico PCR to compare the taxonomic coverage and resolution of newly designed insect metabarcodes (targeting 16S) with that of existing markers [16S and cytochrome oxidase c subunit I (COI)] and then compared their efficiency in vitro. Existing metabarcoding primers amplified in silico 90% coverage. Furthermore, metabarcodes targeting COI appeared to introduce taxonomic PCR-amplification bias, typically amplifying a greater percentage of Lepidoptera and Diptera species, while failing to amplify certain orders in silico. To test whether bias predicted in silico was observed in vitro, we created an artificial DNA blend containing equal amounts of DNA from 14 species, representing 11 insect orders and one arachnid. We PCR-amplified the blend using five primer sets, targeting either COI or 16S, with high-throughput amplicon sequencing yielding more than 6 million reads. In vitro results typically corresponded to in silico PCR predictions, with newly designed 16S primers detecting 11 insect taxa present, thus providing equivalent or better taxonomic coverage than COI metabarcodes. Our results demonstrate that in silico PCR is a useful tool for predicting taxonomic bias in mixed template PCR and that researchers should be wary of potential bias when selecting metabarcoding markers. Keywords: environmental DNA, insect, metabarcoding, PCR bias Received 19 December 2012; revision received 10 April 2014; accepted 11 April 2014

Introduction DNA-based identification of multiple taxa simultaneously from environmental samples (‘metabarcoding’, Epp et al. 2012; Yoccoz et al. 2012) shows the potential to revolutionize biodiversity research. Metabarcoding has provided valuable insights into prokaryote and fungal community ecology (e.g. Qian et al. 2011; Blaalid et al. 2012) and has more recently been applied to plants (e.g. Sønstebø et al. 2010; Hiiesalu et al. 2012; Yoccoz et al. 2012) and animals (e.g. Thomsen et al. 2011). In order to provide taxonomic resolution, metabarcoding markers ideally show little or no variability within species but vary between species, and are flanked by PCR primer-binding sites that are sufficiently conserved to minimize taxonomic bias (Bellemain et al. 2010; Epp et al. 2012). Metabarcoding primers often target short regions Correspondence: Laurence J. Clarke, Fax: +61-8-8313-4364; E-mail: [email protected]

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