Pooling size sorted Malaise trap fractions to maximize taxon recovery with metabarcoding

Abstract

Background Small and rare specimens can remain undetected when metabarcoding is applied on bulk samples with a high specimen size heterogeneity. This is especially critical for Malaise trap samples, where most of the biodiversity is contributed by small taxa with low biomass. The separation of samples in different size fractions for downstream analysis is one possibility to increase detection of small and rare taxa. However, experiments systematically testing different size sorting approaches and subsequent proportional pooling of fractions are lacking, but would provide important information for the optimization of metabarcoding protocols. We set out to find a size sorting strategy for Malaise trap samples that maximizes taxonomic recovery but remains scalable and time efficient. Methods Three Malaise trap samples were sorted into four size classes using dry sieving. Each fraction was homogenized and lysed. The corresponding lysates were pooled to simulate unsorted samples. Pooling was additionally conducted in equal proportions and in four different proportions enriching the small size fraction of samples. DNA from the individual size classes as well as the pooled fractions was extracted and metabarcoded using the FwhF2 and Fol-degen-rev primer set. Additionally, alternative wet sieving strategies were explored. Results The small size fractions harboured the highest diversity and were best represented when pooling in favour of small specimens. Metabarcoding of unsorted samples decreases taxon recovery compared to size sorted samples. A size separation into only two fractions (below 4 mm and above) can double taxon recovery compared to not size sorting. However, increasing the sequencing depth 3- to 4-fold can also increase taxon recovery to levels comparable with size sorting, but remains biased towards biomass rich taxa in the sample. Conclusion We demonstrate that size fractionation of Malaise trap bulk samples can increase taxon recovery. While results show distinct patterns, the lack of statistical support due to the limited number of samples processed is a limitation. Due to increased speed and lower risk of cross-contamination as well as specimen damage we recommend wet sieving and proportional pooling of the lysates in favour of the small size fraction (80–90% volume). However, for large-scale projects with time constraints, increasing sequencing depth is an alternative solution.

Metabarcoding hyperdiverse kelp holdfast communities on temperate reefs: An experimental approach to inform future studies

“…In our study, the smaller size fraction had greater taxonomic richness than the larger size fraction at the ASV and OTU levels, considering all reads. Similar to previous studies, our results suggest that without size fractioning, it may be difficult to recover the presence of small biomass organisms in taxonomically diverse communities (Elbrecht et al, 2021; Rex & Etter, 2010; Wangensteen, Cebrian, et al, 2018). It is worth noting that each individual holdfast contained a very different assemblage; thus, although we were able to differentiate among subsamples of the large and small fraction confidently, differences in community composition among holdfasts were substantial (Figure 4).…”

“…In our study, the smaller size fraction had greater taxonomic richness than the larger size fraction at the ASV and OTU levels, considering all reads. Similar to previous studies, our results suggest that without size fractioning, it may be difficult to recover the presence of small biomass organisms in taxonomically diverse communities (Elbrecht et al, 2021;Rex & Etter, 2010;Wangensteen, Cebrian, et al, 2018).…”

A practical guide to DNA-based methods for biodiversity assessment

“…Size sorting the organisms prior to extraction helps to fully recover the diversity of a sample, but it also requires a significant time investment as well as multiple DNA extractions per sample, which adds substantially to the cost, so there is a trade-off between taxonomic completeness and processing time/ cost. Different sorting schemes have been proposed, ranging from very strict (separate into multiple size classes, extract DNA from each separately, and then pool the DNA extracts proportionally) to very coarse (remove large individuals, leaving only a leg or other body part in the bulk sample) (Elbrecht et al 2017a(Elbrecht et al , 2021 or even sorting according to taxonomic group (Moriniere et al 2016, Beentjes et al 2019). An alternative to size sorting is to greatly increase sequencing depth during metabarcoding to improve recovery of small organisms.…”

DNA‐based assessment of environmental degradation in an unknown fauna: The freshwater macroinvertebrates of the Indo‐Burmese hotspot

“…All large‐sized (body length > 15 mm) individuals were separated to avoid overrepresentation in mixed community sequencing (Creedy et al, 2019; Elbrecht et al, 2021). For these individuals, a tissue sample (~20 mg) was obtained, and pooled tissues were subsequently dried at room temperature and homogenised using a Tissue Lyser (Qiagen, Hilden, Germany).…”