Abstract
Environmental bulk samples often contain many different taxa that vary several orders of magnitude in biomass. This can be problematic in DNA metabarcoding and metagenomic high‐throughput sequencing approaches, as large specimens contribute disproportionately high amounts of DNA template. Thus, a few specimens of high biomass will dominate the dataset, potentially leading to smaller specimens remaining undetected. Sorting of samples by specimen size (as a proxy for biomass) and balancing the amounts of tissue used per size fraction should improve detection rates, but this approach has not been systematically tested. Here, we explored the effects of size sorting on taxa detection using two freshwater macroinvertebrate bulk samples, collected from a low‐mountain stream in Germany. Specimens were morphologically identified and sorted into three size classes (body size < 2.5 × 5, 5 × 10, and up to 10 × 20 mm). Tissue powder from each size category was extracted individually and pooled based on tissue weight to simulate samples that were not sorted by biomass (“Unsorted”). Additionally, size fractions were pooled so that each specimen contributed approximately equal amounts of biomass (“Sorted”). Mock samples were amplified using four different DNA metabarcoding primer sets targeting the Cytochrome c oxidase I (COI) gene. Sorting taxa by size and pooling them proportionately according to their abundance lead to a more equal amplification of taxa compared to the processing of complete samples without sorting. The sorted samples recovered 30% more taxa than the unsorted samples at the same sequencing depth. Our results imply that sequencing depth can be decreased approximately fivefold when sorting the samples into three size classes and pooling by specimen abundance. Even coarse size sorting can substantially improve taxa detection using DNA metabarcoding. While high‐throughput sequencing will become more accessible and cheaper within the next years, sorting bulk samples by specimen biomass or size is a simple yet efficient method to reduce current sequencing costs.
Highlights
Recent advancements in high-throughput sequencing (HTS) and DNA barcoding have improved our ability to rapidly assess biodiversity
In DNA-based ecosystem assessment, we can distinguish between two approaches: (1) A target gene fragment is amplified and compared to a DNA barcoding database, or (2) the extracted DNA from the bulk sample is shotgun sequenced directly without PCR and can be optionally enriched for target genes
Our results demonstrate that read abundances of the unsorted samples were dominated by few biomass rich taxa that contribute the majority of DNA in the bulk extraction
Summary
Recent advancements in high-throughput sequencing (HTS) and DNA barcoding have improved our ability to rapidly assess biodiversity. While metagenomics might overcome these PCR-based problems, this approach is currently limited because only little reference data is available (e.g., mitochondrial genomes), and a high sequencing depth is required (Crampton-Platt et al, 2016) Both approaches are likely affected by variable cell densities and types, as well as variable mitochondrial genome copy numbers between taxa and specimen life stages (Ballard & Whitlock, 2004; Moraes, 2001), which is potentially affecting taxa detection. While these problems might be solved at least partially by optimized degenerate primers (Elbrecht & Leese, 2017), reduced sequencing costs and mitogenome capture (Tang et al, 2014), both metabarcoding and metagenomics approaches are potentially affected by an additional factor: variable taxa biomass
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