Sources of uncertainty in DNA metabarcoding of whole communities: implications for its use in biomonitoring

These data were derived from a structured experiment to assess factors influencing the precision and accuracy of molecular methods for freshwater macroinvertebrate community assessment. Benthic macroinvertebrates were sorted, identified, and counted from nine individual kick-net samples using standard morphometric protocols, then reconstituted. These bulk specimen samples were then homogenised. From each bulk sample, aliquots of the homogenate were distributed among seven laboratories across Europe for DNA extraction, PCR amplification, library preparation, and sequencing. Additionally, each laboratory was provided with a DNA extract from the bulk homogenate to allow for amplification through to sequencing, in order to assess the influence of DNA extraction. The data consist of abundances of individual taxa identified using morphometric protocols and read counts of operational taxonomic units identified by the molecular methods. In addition, we provide a taxonomically harmonised matrix ..., Sample collection and processing
Field sampling
Nine samples of the benthic macroinvertebrate community were collected from seven river sites in southern England, UK (see Jones et al., 2025). Each had been the subject of at least three years study prior to the current sample collection, providing a comprehensive record of taxa likely to be present. Samples were collected using the UK standard macroinvertebrate biomonitoring protocol, comprising a 3-minute kick sample and 1-minute search with a 1 mm mesh size net sampling all habitats in proportion to their areal occurrence (BS EN 16150:2012). All material retained in the net was preserved on-site in 96% ethanol and returned to the laboratory.
Morphological identification
Macroinvertebrates were manually sorted from other material, identified to the lowest practicable taxonomic level (most to species or genus level, see Jones et al., 2025) and counted. Samples were processed by qualified, experienced freshwater biologists. All material a..., # Sources of uncertainty in DNA metabarcoding of whole communities: implications for its use in biomonitoring

Dataset DOI: [10.5061/dryad.dncjsxm9t](10.5061/dryad.dncjsxm9t)

## Description of the data and file structure

Advancements have been made in the use of DNA-based methods for detection of single species. However, the routine application of DNA-based methods to monitor whole communities using a metabarcoding approach and derive ecosystem status continues to be limited. We undertook a structured experiment to assess factors influencing the precision and accuracy of molecular methods for freshwater macroinvertebrate community assessment. To quantify the contributions of sequencing, laboratory, DNA extraction and sample composition to uncertainty and biases in macroinvertebrate taxonomic detection and identification through metabarcoding, we compared data generated by a well-established biomonitoring protocol used in the UK from a set of nine kick-net samples with data simultaneou...,