Effect of DNA extraction method in gut fungal community assessment.

Abstract

Understanding the gut mycobiota composition and its impact on health requires reliable methods for fungal community assessment. This study explores the influence of DNA extraction methods in GM analysis. Three protocols were evaluated: Qiagen DNeasy blood and tissue kit with mechanical glass bead lysis (DNgb), Thermofisher MagMax Microbiome ultra-nucleic isolation kit automated method (MM), and MM combined with glass beads lysis (MMgb). Fecal samples from healthy volunteers were collected, DNA extracted and ITS2 amplicon library preparation and sequencing performed. Results showed that DNA yields did not significantly differ among methods and the addition of glass bead beating favored the recovery of DNA more appropriate for fungal analysis. Beta diversity revealed distinct clusters, with MMgb showing the most pronounced variation in mycobiota composition, exposing particularly the low abundance taxa. LEfSe analysis identified significant differences in the abundance of fungal species among the extraction methods. Samples extracted with bead beating were enriched in filamentous species, while those without this step showed higher relative abundance of yeast fungi. This study underscores the importance of selecting appropriate DNA extraction methods for accurate characterization of the gut mycobiota, emphasizing the need for standardized methodologies to ensure reproducibility and reliability in microbial data acquisition.