Scalable isolation of soil genomic DNA from microbial eukaryotes to multicellular micro- and mesofauna

Global initiatives emphasise the need for harmonised soil biodiversity assessments. Efficient DNA extraction methods that accommodate larger soil volumes are essential for capturing organisms at higher trophic levels than bacteria and fungi and for supporting extensive sampling campaigns. We developed and evaluated a scalable, cost-efficient, and automation-ready soil DNA isolation protocol and compared its performance with two widely used commercial extraction methods: Qiagen DNeasy PowerSoil Pro (0.25 g soil input) and Qiagen DNeasy PowerMax Soil (2.5 g soil input). Four contrasting soil types representing agricultural and forest ecosystems (mull, sandy till, peat, and coarse mineral soil) were analysed. The new protocol combines bead-beating in sodium thiocyanate lysis buffer with either silica-membrane or carboxyl-coated magnetic bead purification and was tested using soil inputs of 2.5 g and 5 g for one soil type. DNA extracts were sequenced on an Illumina MiSeq platform using universal eukaryotic 18S rRNA gene primers to characterise soil metazoans and microbial eukaryotes. The developed protocol, combining a tenfold increase in soil input with commercial kit purification, produced the highest DNA yields in three of four soil types, increasing DNA recovery by up to 2.3-fold compared with the commercial maxiprep method. It also consistently detected the greatest metazoan richness, recovering up to 103% more taxa than the conventional miniprep method and up to 82% more taxa than the commercial maxiprep method. The carboxyl-coated magnetic bead workflow also performed well, recovering up to 83% more metazoan taxa than the miniprep method and up to 55% more taxa than the maxiprep method, particularly in forest soils. For microbial eukaryotes and protists, the commercial maxiprep and combination of developed high lysis volume and commercial kit silica purification protocols generally yielded the highest richness in agricultural soils, whereas the developed protocol performed best in coarse xeric forest soils. These results indicate that at least a tenfold increase in soil input relative to conventional 0.25 g extractions is required for reliable characterisation of mesofauna diversity, while further increases may provide additional gains. The developed DNA isolation protocol offers a solution for large-scale soil biodiversity assessments, supporting the integration of higher trophic levels into eDNA-based monitoring frameworks and future soil health monitoring programmes.