Accepted Article
Received Date : 19-Dec-2012 Revised Date : 10-Apr-2014 Accepted Date : 11-Apr-2014 Article type : Resource Article
Environmental metabarcodes for insects: in silico PCR reveals potential for taxonomic bias
Laurence J. Clarke, Julien Soubrier, Laura S. Weyrich and Alan Cooper
Address: Australian Centre for Ancient DNA, University of Adelaide, Adelaide, SA 5005 Australia
Keywords: insect, metabarcoding, PCR bias, environmental DNA.
Corresponding author: Laurence J. Clarke, Australian Centre for Ancient DNA, University of Adelaide, Adelaide, SA 5005 Australia Fax: +61 8 8313 4364 E-mail: [email protected]
Running title: PCR bias in insect metabarcoding primers
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 PCRThis article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/1755-0998.12265 This article is protected by copyright. All rights reserved.
Accepted Article
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 COI) and then compared their efficiency in vitro. Existing metabarcoding primers amplified in silico less than 75% of insect species with complete mitochondrial genomes available, whereas new primers targeting 16S provided greater than 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 different insect orders and one arachnid. We PCR-amplified the blend using five primers 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.
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. Blaalid et al. 2012; Qian et al. 2011), and has more recently been applied to plants (e.g. Hiiesalu et al. 2012; Sønstebø et al. 2010; Yoccoz et al. 2012) and
This article is protected by copyright. All rights reserved.
Accepted Article
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 (
Received Date : 19-Dec-2012 Revised Date : 10-Apr-2014 Accepted Date : 11-Apr-2014 Article type : Resource Article
Environmental metabarcodes for insects: in silico PCR reveals potential for taxonomic bias
Laurence J. Clarke, Julien Soubrier, Laura S. Weyrich and Alan Cooper
Address: Australian Centre for Ancient DNA, University of Adelaide, Adelaide, SA 5005 Australia
Keywords: insect, metabarcoding, PCR bias, environmental DNA.
Corresponding author: Laurence J. Clarke, Australian Centre for Ancient DNA, University of Adelaide, Adelaide, SA 5005 Australia Fax: +61 8 8313 4364 E-mail: [email protected]
Running title: PCR bias in insect metabarcoding primers
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 PCRThis article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/1755-0998.12265 This article is protected by copyright. All rights reserved.
Accepted Article
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 COI) and then compared their efficiency in vitro. Existing metabarcoding primers amplified in silico less than 75% of insect species with complete mitochondrial genomes available, whereas new primers targeting 16S provided greater than 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 different insect orders and one arachnid. We PCR-amplified the blend using five primers 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.
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. Blaalid et al. 2012; Qian et al. 2011), and has more recently been applied to plants (e.g. Hiiesalu et al. 2012; Sønstebø et al. 2010; Yoccoz et al. 2012) and
This article is protected by copyright. All rights reserved.
Accepted Article
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 (
