Standardized high-throughput biomonitoring using DNA metabarcoding: Strategies for the adoption of automated liquid handlers

Reliable and comprehensive monitoring data are required to trace and counteract biodiversity loss. High-throughput metabarcoding using DNA extracted from community samples (bulk) or from water or sediment (environmental DNA) has revolutionized biomonitoring, given the capability to assess biodiversity across the tree of life rapidly with feasible effort and at a modest price. DNA metabarcoding can be upscaled to process hundreds of samples in parallel. However, while automated high-throughput analysis workflows are well-established in the medical sector, manual sample processing still predominates in biomonitoring laboratory workflows limiting the upscaling and standardization for routine monitoring applications. Here we present an automated, scalable, and reproducible metabarcoding workflow to extract DNA from bulk samples, perform PCR and library preparation on a liquid handler. Key features are the independent sample replication throughout the workflow and the use of many negative controls for quality assurance and quality control. We generated two datasets: i) a validation dataset consisting of 42 individual arthropod specimens of different species, and ii) a routine monitoring dataset consisting of 60 stream macroinvertebrate bulk samples. As a marker, we used the mitochondrial COI gene. Our results show that the developed single-deck workflow is free of laboratory-derived contamination and produces highly consistent results. Minor deviations between replicates are mostly due to stochastic differences for low abundant OTUs. Thus, we successfully demonstrated that robotic liquid handling can be used reliably from DNA extraction to final library preparation on a single deck, thereby substantially increasing throughput, reducing costs, and increasing data robustness for biodiversity assessments and monitoring.