Optimizing insect metabarcoding using replicated mock communities

Abstract

Metabarcoding (high‐throughput sequencing of marker gene amplicons) has emerged as a promising and cost‐effective method for characterizing insect community samples. Yet, the methodology varies greatly among studies and its performance has not been systematically evaluated to date. In particular, it is unclear how accurately metabarcoding can resolve species communities in terms of presence‐absence, abundance and biomass. Here we use mock community experiments and a simple probabilistic model to evaluate the effect of different DNA extraction protocols on metabarcoding performance. Specifically, we ask four questions: (Q1) How consistent are the recovered community profiles across replicate mock communities?; (Q2) How does the choice of lysis buffer affect the recovery of the original community?; (Q3) How are community estimates affected by differing lysis times and homogenization? and (Q4) Is it possible to obtain adequate species abundance estimates through the use of biological spike‐ins? We show that estimates are quite variable across community replicates. In general, a mild lysis protocol is better at reconstructing species lists and approximate counts, while homogenization is better at retrieving biomass composition. Small insects are more likely to be detected in lysates, while some tough species require homogenization to be detected. Results are less consistent across biological replicates for lysates than for homogenates. Some species are associated with strong PCR amplification bias, which complicates the reconstruction of species counts. Yet, with adequate spike‐in data, species abundance can be determined with roughly 40% standard error for homogenates, and with roughly 50% standard error for lysates, under ideal conditions. In the latter case, however, this often requires species‐specific reference data, while spike‐in data generalize better across species for homogenates. We conclude that a non‐destructive, mild lysis approach shows the highest promise for the presence/absence description of the community, while also allowing future morphological or molecular work on the material. However, homogenization protocols perform better for characterizing community composition, in particular in terms of biomass.

Upscaling biodiversity monitoring: Metabarcoding estimates 31,846 insect species from Malaise traps across Germany

“…Firstly, our approach is a compromise between improving feasibility for large-scale monitoring and the chances of detecting all taxa. Additional sample processing, such as complementary mild lysis (Marquina et al 2022, Iwaszkiewicz-Eggebrecht et al 2023), greater replication (Zizka et al 2022b) and using multiple gene markers or primers (Elbrecht et al 2019, Hajibabaei et al 2019), as well as increasing sequencing depth (this study), would undoubtedly recover more species. However, the additional effort required must be weighed against the extra information gained, which may not be substantial (Buchner et al 2021a) Secondly, the incompleteness of regional and interregional reference sequence databases, and the generation of reference sequences of unknown species, remains a persistent challenge (e.g.…”

Effects of livestock on arthropod biodiversity in Iberian Holm oak savannas revealed by metabarcoding

“…For this work, we chose a non-destructive approach for the DNA extraction from the bulk samples, as we were interested in keeping the individual arthropods for further morphological analysis. A series of recent studies have tested the suitability of these non-destructive DNA extraction protocols, finding them as a promising alternative to sample homogenization in terms of biodiversity detection (Batovska et al, 2021;Iwaszkiewicz-Eggebrecht et al, 2023;Marquina et al, 2022;Nielsen et al, 2019). However, there may be limitations relevant for the analysis of our samples: for instance, both Iwaszkiewicz-Eggebrecht et al (2023) and Marquina et al (2022) found that ants (Hymenoptera: Formicidae) were detected in their mock communities in much lower proportion and occasions than other taxa.…”

“…A series of recent studies have tested the suitability of these non-destructive DNA extraction protocols, finding them as a promising alternative to sample homogenization in terms of biodiversity detection (Batovska et al, 2021;Iwaszkiewicz-Eggebrecht et al, 2023;Marquina et al, 2022;Nielsen et al, 2019). However, there may be limitations relevant for the analysis of our samples: for instance, both Iwaszkiewicz-Eggebrecht et al (2023) and Marquina et al (2022) found that ants (Hymenoptera: Formicidae) were detected in their mock communities in much lower proportion and occasions than other taxa. This could have affected our results, as we worked with ground-dwelling arthropod communities, in which ants are prominent members.…”

Three steps towards comparability and standardization among molecular methods for characterizing insect communities

“…Here, all calibration is based on comparison to a set of samples of known contents-and in support of one's findings, one would naturally report on the precision of one's measures. In metabarcoding, such calibration is typically made against a study-specific sample (or 'mock community'; [19][20][21]) of a composition purpose-invented by the laboratory in question. In the end, we face a high probability of comparing apples to oranges.…”