Extracting cell-free extracellular DNA from wastewater: Evaluation of extraction protocols for use in complex matrices

Cell-free extracellular DNA (exDNA) plays important roles in DNA-based surveillance of wastewater treatment processes and reclaimed water quality. Several techniques have been applied to extract cell-free exDNA in wastewater. However, the applicability and feasibility of these techniques has not been evaluated. Five different cell-free exDNA extraction techniques were evaluated in this study. By spiking multiple DNA standards in buffer system, it was shown that recovery efficiencies for the five extraction methods were dependent on the size of the DNA. ExDNA recovery was also influenced differently among the five methods by the water quality parameters including conductivity, pH and dissolved organic carbon. By sequencing exDNA samples, it was shown that extraction techniques affect the downstream PCR and functional diversity derived from exDNA. Overall, anion exchange method presents the most optimal performance to extract cell-free exDNA in wastewater due to highest recovery efficiency, reproducibility and success rate of PCR for downstream molecular analysis among the five evaluated methods.