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Sorting things out: Assessing effects of unequal specimen biomass on DNA metabarcoding.

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.

SUBMITTER: Elbrecht V

PROVIDER: S-EPMC5587478 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature

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Sorting things out: Assessing effects of unequal specimen biomass on DNA metabarcoding.

Elbrecht Vasco V Peinert Bianca B Leese Florian F

Ecology and evolution 20170728 17

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 amo ...[more]

PMID: 28904771

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Project description:DNA-based identification through the use of metabarcoding has been proposed as the next step in the monitoring of biological communities, such as those assessed under the Water Framework Directive (WFD). Advances have been made in the field of metabarcoding, but challenges remain when using complex samples. Uneven biomass distributions, preferential amplification and reference database deficiencies can all lead to discrepancies between morphological and DNA-based taxa lists. The effects of different taxonomic groups on these issues remain understudied. By metabarcoding WFD monitoring samples, we analyzed six different taxonomic groups of freshwater organisms, both separately and combined. Identifications based on metabarcoding data were compared directly to morphological assessments performed under the WFD. The diversity of taxa for both morphological and DNA-based assessments was similar, although large differences were observed in some samples. The overlap between the two taxon lists was 56.8% on average across all taxa, and was highest for Crustacea, Heteroptera, and Coleoptera, and lowest for Annelida and Mollusca. Taxonomic sorting in six basic groups before DNA extraction and amplification improved taxon recovery by 46.5%. The impact on ecological quality ratio (EQR) scoring was considerable when replacing morphology with DNA-based identifications, but there was a high correlation when only replacing a single taxonomic group with molecular data. Different taxonomic groups provide their own challenges and benefits. Some groups might benefit from a more consistent and robust method of identification. Others present difficulties in molecular processing, due to uneven biomass distributions, large genetic diversity or shortcomings of the reference database. Sorting samples into basic taxonomic groups that require little taxonomic knowledge greatly improves the recovery of taxa with metabarcoding. Current standards for EQR monitoring may not be easily replaced completely with molecular strategies, but the effectiveness of molecular methods opens up the way for a paradigm shift in biomonitoring.

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Dataset Information

Sorting things out: Assessing effects of unequal specimen biomass on DNA metabarcoding.

Publications

Sorting things out: Assessing effects of unequal specimen biomass on DNA metabarcoding.

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