Scaling up DNA metabarcoding for freshwater macrozoobenthos monitoring

The viability of DNA metabarcoding for assessment of freshwater macrozoobenthos has been demonstrated over the recent years. The method has matured to a stage where it can be applied to monitoring at a large scale, keeping pace with increased high-throughput sequencing capacity. However, workflows and sample tagging need to be optimised to accommodate for hundreds of samples within a single sequencing run. Here, we conceptualise a streamlined metabarcoding workflow, in which samples are processed in 96-well plates. Each sample is replicated starting with tissue extraction. Negative and positive controls are included to ensure data reliability. With our newly developed fusion primer sets for the BF2 + BR2 primer pair up to three 96-well plates (288 wells) can be uniquely tagged for a single Illumina sequencing run. By including Illumina indices, tagging can be extended to thousands of samples. We hope that our metabarcoding workflow will be used as a practical guide for future large-scale biodiversity assessments involving freshwater invertebrates. However, as this is just one possible metabarcoding approach, we hope this article will stimulate discussion and publication of alternatives and extensions to this method.