Data from: Sorting things out: assessing effects of unequal specimen biomass on DNA metabarcoding

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.