Comparing different DNA extraction techniques for microbiome analysis of swab samples taken from diabetic foot ulcers

Abstract

Diabetic foot ulcers (DFUs) are chronic wounds resulting from Diabetes Mellitus (DM)-related neuropathy and vascular impairment, creating favourable conditions for microbial growth. This study compared two DNA extraction methods for DNA isolation and microbiome analysis of DFU swab samples: An Enzyme Lytic mix (ELM) combined with the MagMAXTM Viral/Pathogen Nucleic Acid Isolation Kit (ELM-MagMAX), and the ZymoBIOMICSTM 96 Magbead DNA/RNA kit (ZymoBIOMICS), which relies on chemical lysis. Both kits operate on the principle of magnetic bead-based purification. DNA was extracted from 43 participant samples using both techniques. DNA yield was quantified in triplicate using fluorometry, and data was log-transformed to meet normality assumptions. Statistical comparisons of DNA concentrations were performed using a Linear Mixed Model (LMM) and paired t-test, each revealing a significant increase in yield from ZymoBIOMICS, 2.38x and 2.36x respectively. Subsequently, metagenomic analysis via next-generation sequencing (NGS) enabled taxonomic profiling of the microbial communities. The Wilcoxon signed-rank test assessed differences in species-specific counts between methods. None of the main taxa of interest showed statistically significant differences and were, therefore, explored further using bar chart visualisations. These indicated that ZymoBIOMICS was more effective at identifying Gram-positive bacteria, while results for Gram-negative bacteria were mixed. Practical considerations that may influence the choice of extraction method were also evaluated. Overall, the findings support ZymoBIOMICS as a more effective approach for DNA extraction in DFU microbiome analysis. Potential sources of error were considered, along with suggestions for methodological refinement. In the future, the findings may be used both for further research and potentially for diagnostic use.