Data and code from: Establishing DNA-based strategies for soil biodiversity assessment: Insights from carabid beetles

Molecular-based methods offer valuable opportunities for assessing soil biodiversity in different ecosystems. However, their reliability and large-scale applicability depend on developing, optimizing protocols, and establishing high-quality, curated local reference databases. This study aimed to evaluate key steps in the soil macroinvertebrate metabarcoding workflow, including the sample decontamination process and the efficiency of taxa recovery. Specifically, we sought to: (1) determine the impact of sample decontamination, (2) validate species-level recovery efficiency of the metabarcoding pipeline spiked with a curated mock community of morphologically identified and barcoded carabid beetles, and (3) compare traditional morphological identification and metabarcoding for specimens' taxonomic assignment and recovery. Our results showed that the commonly used decontamination process did not significantly impact OTU richness, suggesting it is not essential for this fauna. Compared to morphology, metabarcoding provided a more comprehensive taxonomic overview at higher-level taxa. However, validation with the mock community revealed discrepancies in species-level recovery, underscoring that its accuracy is highly contingent on the quality of the reference database. DNA metabarcoding is a currently used and promising technique for macroinvertebrate assessment regarding time, efficiency, and costs, yet reaching greater depth in taxonomic resolution. Yet, its species-level accuracy remains dependent on comprehensive and well-curated barcode reference databases. We recommend an integrative approach, combining molecular data with targeted validation, for the most robust outcomes. For this reason, we recommend the use of integrative methodologies for robust and rapid biodiversity assessments. We found that the common decontamination step is not crucial for soil macrofauna metabarcoding accuracy. Consequently, its removal streamlines sample processing. DNA metabarcoding revolutionizes soil biodiversity assessments by offering unparalleled taxonomic resolution and efficiency compared to traditional methods. Our study shows that the common decontamination step is unnecessary, streamlining workflows without compromising accuracy. However, the reliability of these molecular methods hinges on the development of curated local reference databases, underscoring the importance of integrative approaches for robust biodiversity monitoring.