Validation of standard operating procedures for DNA extraction and microbiome analyses of soil samples

The development of Standard Operating Procedures (SOPs) for sampling, extraction, and analysis of soil microbiomes is crucial to ensure reproducibility in characterizing and utilizing microbial communities. This study aimed to validate previously developed SOPs by analyzing rhizosphere and bulk soil collected from strawberry cultivars Clery and Elodie in an experimental field in Boves, northwestern Italy. Homogenized samples were distributed to six Italian research partners (UNITO, ENEA-CASACCIA, UNIPG, UNIVR, UNIMIB, and UNIPA) for DNA extraction and quality assessment using a standardized commercial kit and protocol. While all teams successfully extracted DNA, statistical analyses revealed differences in the quality (absorbance ratios 260/280 and 260/230) and quantity (ng/mL) of DNA across groups. Sequencing results amplified these discrepancies, with UNITO obtaining the highest number of reads and features compared to other teams. Bacterial community analysis highlighted an overwhelming "operator effect" (91% of variance), whereas matrix type (4%) and cultivar (<0.1%) contributed minimally. Fungal community composition showed a more balanced distribution of variance, with operator effect (38%) and matrix (39%) being comparable, while cultivar influence remained low (4%). Despite these differences, the taxonomic identity of fungal and bacterial communities remained consistent across groups, with variations observed only in relative abundance. This study emphasizes DNA extraction as a critical step in soil microbiome research, independent of sampling or sequencing steps, and underscores the need for standardized procedures regarding sample homogenization, shipping conditions, and storage to minimize variability and enhance reproducibility.