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

Abstract

1!Insect metabarcoding has been mainly based on PCR amplification of short fragments 2! within the 'barcoding region' of the gene COI. However, because of the variability of 3! this gene, it has been difficult to design good universal PCR primers. Most primers 4! used today are associated with gaps in the taxonomic coverage or amplification biases 5! that make the results less reliable and impede the detection of species that are present 6! in the sample. We identify new primers for insect metabarcoding using computational 7! approaches (ECOPRIMERS and DEGEPRIME) applied to the most comprehensive 8! reference databases of mitochondrial genomes of Hexapoda assembled to date. New 9! primers are evaluated in silico against previously published primers in terms of 10! taxonomic coverage and resolution of the corresponding amplicons. For the latter 11! criterion, we propose a new index, exclusive taxonomic resolution, which is a more 12! biologically meaningful measure than the standard index used today. Our results show 13! that the best markers are found in the ribosomal RNA genes (12S and 16S); they 14! resolve about 90% of the genetically distinct species in the reference database. Some 15! markers in protein-coding genes provide similar performance but only at much higher 16! levels of primer degeneracy. Combining two of the best individual markers improves 17! the effective taxonomic resolution with up to 10%. The resolution is strongly 18! dependent on insect taxon: COI primers detect 40% of Hymenoptera, while 12S 19! primers detect 12% of Collembola. Our results indicate that amplicon-based 20! metabarcoding of insect samples can be improved by choosing other primers than 21! those commonly used today.

Metabarcoding Malaise traps and soil eDNA reveals seasonal and local arthropod diversity shifts

“…Former studies showed that hymenopteran sequences tend to have a lower affinity to universal COI primers compared to other arthropod groups 23 . In this study, only one COI primer pair was used which could have impaired the amplification of problematic arthropod groups such as Hymenoptera but also the Acari and Collembola due to primer bias and the use of target taxon-specific primers could have circumvented this 31 . The European web-spinning larch sawfly, Cephalcia lariciphila was found in soil samples taken at the pure spruce sites in autumn.…”

Metabarcoding and ecological interaction networks for selecting candidate biological control agents

“…As presented in Supplementary Figure S1, we then performed metabarcoding on each arthropod sample, with a two-step DNA amplification and high-throughput sequencing method adapted from the procedure described by Galan et al (2017). We sequenced three short COI fragments, with primer combinations and PCR protocols developed elsewhere (HCO forward: Leray et al 2013, HCO reverse: Folmer et al 1994, LEP F. and R.: Brandon-Mong et al 2015, HEX F. and R.: Marquina et al 2019), to overcome the problem of the lack of primer universality among arthropods. Error-proof indices for individual sample identification were developed with the high-throughput sequencing process described by Martin (2019).…”

Connecting high‐throughput biodiversity inventories: Opportunities for a site‐based genomic framework for global integration and synthesis

“…We view this critical mass, which is complemented by various national‐level efforts, as a solid platform for the development of a terrestrial arthropod module, where passive sampling methods (e.g., pitfall, light and malaise traps, the last of which is already standardized within the BIOSCAN initiative) can be implemented individually, or in combination. Recent efforts toward developing best practices for COI community metabarcoding (e.g., Andújar et al, 2018; Elbrecht et al, 2019; Marquina et al, 2019) can be further developed for harmonized protocols for subsequent sample processing.…”