New mitochondrial primers for metabarcoding of insects, designed and evaluated using in silico methods

Abstract

Insect metabarcoding has been mainly based on PCR amplification of short fragments within the "barcoding region" of the gene cytochrome oxidase I (COI). However, because of the variability of this gene, it has been difficult to design good universal PCR primers. Most primers used today are associated with gaps in the taxonomic coverage or amplification biases that make the results less reliable and impede the detection of species that are present in the sample. We identify new primers for insect metabarcoding using computational approaches (ecoprimers and degeprime) applied to the most comprehensive reference databases of mitochondrial genomes of Hexapoda assembled to date. New primers are evaluated in silico against previously published primers in terms of taxonomic coverage and resolution of the corresponding amplicons. For the latter criterion, we propose a new index, exclusive taxonomic resolution, which is a more biologically meaningful measure than the standard index used today. Our results show that the best markers are found in the ribosomal RNA genes (12S and 16S); they resolve about 90% of the genetically distinct species in the reference database. Some markers in protein-coding genes provide similar performance but only at much higher levels of primer degeneracy. Combining two of the best individual markers improves the effective taxonomic resolution with up to 10%. The resolution is strongly dependent on insect taxon: COI primers detect 40% of Hymenoptera, while 12S primers detect 12% of Collembola. Our results indicate that amplicon-based metabarcoding of insect samples can be improved by choosing other primers than those commonly used today.

Establishing arthropod community composition using metabarcoding: Surprising inconsistencies between soil samples and preservative ethanol and homogenate from Malaise trap catches

“…In this study, we extend a previous in silico study of markers for insect metabarcoding (Marquina et al, 2018) by testing the optimal 16S and COI markers identified there in three different empirical settings. Targeting Malaise trap catches from three different sites at four different time points, we analysed homogenized insects and preservative ethanol from the same samples, and environmental DNA from soil samples taken at the same sites.…”

“…Two mitochondrial markers were selected for PCR amplification based on the findings of Marquina et al (2018). The COI marker was amplified using the primer pair BF2-BR1 ; see also Marquina et al, 2018), resulting in an amplicon of approximately 320 bp.…”

“…In response, some authors have advocated the use of an alternative marker of intermediate variability: more conserved than the high-resolution marker (typically COI), but more variable than the nuclear rRNA genes.The mitochondrial rRNA gene 16S has been offered as such a marker (Clarke et al, 2014;Deagle, Jarman, Coissac, Pompanon, & Taberlet, 2014;Marquina, Andersson, & Ronquist, 2018). To our knowledge, only a few studies have tested the potential of using 16S as a replacement for COI in insects in vitro (Clarke et al, 2014;Elbrecht et al, 2016;Epp et al, 2012), or as a complement (Alberdi, Aizpurua, Gilbert, & Bohmann, 2018), and only one study made use of both markers simultaneously in resolving ecological rather than methodological questions (Kaunisto, Roslin, Sääksjärvi, & Vesterinen, 2017).…”

“…These studies have reported that the performance of 16S is considerably poorer compared to COI (Alberdi et al, 2018;Elbrecht et al, 2016), but this discrepancy may be attributed to the fragment of the 16S gene amplified being very short (160-190 bp approximately), and to the fact that the reference library of the 16S gene is substantially smaller than that of COI (Andújar et al, 2018;Deagle et al, 2014). Using longer fragments of the 16S gene, the taxonomic resolution increases to levels comparable to COI (Clarke et al, 2014;Marquina et al, 2018).…”

Establishing insect community composition using metabarcoding of soil samples, and preservative ethanol and homogenate from Malaise trap catches: surprising inconsistencies between methods

“…An ideal complementary marker should be easily amplified through the different subtaxa of the target group, while providing enough taxonomic resolution to discriminate between related species. The mitochondrial rRNA gene 16S has been offered as such a marker (Clarke et al, 2014;Deagle, Jarman, Coissac, Pompanon, & Taberlet, 2014;Marquina, Andersson, & Ronquist, 2018). To our knowledge, only a few studies have tested the potential of using 16S as a replacement for COI in insects in vitro (Clarke et al, 2014;Elbrecht et al, 2016;Epp et al, 2012), or as a complement (Alberdi, Aizpurua, Gilbert, & Bohmann, 2018), and only one study made use of both markers simultaneously in resolving ecological rather than methodological questions (Kaunisto, Roslin, Sääksjärvi, & Vesterinen, 2017).…”