Evaluating next‐generation sequencing (NGS) methods for routine monitoring of wild bees: Metabarcoding, mitogenomics or NGS barcoding

Abstract

Implementing cost‐effective monitoring programs for wild bees remains challenging due to the high costs of sampling and specimen identification. To reduce costs, next‐generation sequencing (NGS)‐based methods have lately been suggested as alternatives to morphology‐based identifications. To provide a comprehensive presentation of the advantages and weaknesses of different NGS‐based identification methods, we assessed three of the most promising ones, namely metabarcoding, mitogenomics and NGS barcoding. Using a regular monitoring data set (723 specimens identified using morphology), we found that NGS barcoding performed best for both species presence/absence and abundance data, producing only few false positives (3.4%) and no false negatives. In contrast, the proportion of false positives and false negatives was higher using metabarcoding and mitogenomics. Although strong correlations were found between biomass and read numbers, abundance estimates significantly skewed the communities' composition in these two techniques. NGS barcoding recovered the same ecological patterns as morphology. Ecological conclusions based on metabarcoding and mitogenomics were similar to those based on morphology when using presence/absence data, but different when using abundance data. In terms of workload and cost, we show that metabarcoding and NGS barcoding can compete with morphology, but not mitogenomics which was consistently more expensive. Based on these results, we advocate that NGS barcoding is currently the seemliest NGS method for monitoring of wild bees. Furthermore, this method has the advantage of potentially linking DNA sequences with preserved voucher specimens, which enable morphological re‐examination and will thus produce verifiable records which can be fed into faunistic databases.

Ultraconserved yet informative for species delimitation: Ultraconserved elements resolve long‐standing systematic enigma in Central European bees

“…The COI barcode fragment of all specimens was sequenced either by Sanger or NGS‐barcode sequencing using the primer pairs LepF/LepR (Hebert et al., 2004) or mlCOIntF/HCO (Leray et al., 2013) (following protocols in Gueuning et al., 2019). Sequences were assembled and edited in geneious version 11.0.5 and consensus sequences were aligned per species complex using mafft version 7.308 (Katoh & Standley, 2013).…”

Estimation of the relative abundance of species in artificial mixtures of insects using low-coverage shotgun metagenomics

“…Mito-metagenomics has proved to be better than amplicon metabarcoding for quantification purposes (Gómez-Rodríguez et al 2015;Bista et al 2018;Gueuning et al 2019), but estimation of relative abundance amongst species (i.e. in a given sample, species A is more abundant than species B) is not always high (Tang et al 2015;Krehenwinkel et al 2017).…”

“…The quantification power of mito-metagenomics is likely bounded for two reasons. First, when there is no mitochondrion enrichment (as in Zhou et al 2013), only a small proportion of the shotgun reads map into the mitogenome (~0.5 % in insects; Tang et al 2014), so a high sequencing depth is necessary to obtain good quantitative results (Gueuning et al 2019). Second and most important, the number of mitogenomes per nuclear genome (mitochondrial copy number) is variable amongst species and even between tissues.…”

Efficient assessment of nocturnal flying insect communities by combining automatic light traps and DNA metabarcoding

“…The advent of next‐generation DNA sequencing coupled with metabarcoding approaches is revolutionizing the study of diverse insect communities by overcoming taxonomic impediment, allowing the cost‐effective processing of hundreds to thousands of complex mixed community samples at high taxonomic resolution (Barsoum, Bruce, Forster, Ji, & Yu, 2019; Gueuning et al., 2019; Piper et al., 2019; Yu et al., 2012). Typically, metabarcoding studies of communities of insects and other invertebrates involve the collection of field samples using a variety of methods, and then, DNA from multispecies samples is extracted, amplified using PCR and sequenced using a next‐generation sequencing platform (e.g., Braukmann et al., 2019; Marquina, Esparza‐Salas, Roslin, & Ronquist, 2019).…”

“…Applications of this general approach are increasing, particularly in the case of freshwater communities (e.g., Bush et al., 2020; Elbrecht, Vamos, Meissner, Aroviita, & Leese, 2017), where efforts are underway to develop standardized metabarcoding approaches to be used in official monitoring programs such as the European Water Framework Directive (Hering et al., 2018). The use of metabarcoding in studies of terrestrial insects has lagged behind that of freshwater communities, but the technique has already been tested, for instance, in the monitoring of wild bees (Gueuning et al., 2019), invasive species (Piper et al., 2019), and dung insects for ecotoxicological assessments (Blanckenhorn, Rohner, Bernasconi, Haugstetter, & Buser, 2016), among many others. Despite these advances, considerable efforts are still needed to develop, optimize, and standardize efficient methods to collect and process insect samples for DNA metabarcoding studies and monitoring programs, as results are conditional on methodological alternatives adopted, including DNA extraction, primer sets, and bioinformatic pipelines (Brandon‐Mong et al., 2015; Braukmann et al., 2019; Elbrecht et al., 2019).…”