The characterization of microbial community dynamics using genomic methods is rapidly expanding, impacting many fields including medical, ecological, and environmental research and applications. One of the biggest challenges for such studies is the isolation of environmental DNA (eDNA) from a variety of samples, diverse microbes, and widely variable community compositions. The current study developed environmentally friendly, user safe, economical, and high throughput eDNA extraction methods for mixed aquatic microbial communities and tested them using 16 s rRNA gene meta-barcoding. Five different lysis buffers including (1) cetyltrimethylammonium bromide (CTAB), (2) digestion buffer (DB), (3) guanidinium isothiocyanate (GITC), (4) sucrose lysis (SL), and (5) SL-CTAB, coupled with four different purification methods: (1) phenol-chloroform-isoamyl alcohol (PCI), (2) magnetic Bead-Robotic, (3) magnetic Bead-Manual, and (4) membrane-filtration were tested for their efficacy in extracting eDNA from recreational freshwater samples. Results indicated that the CTAB-PCI and SL-Bead-Robotic methods yielded the highest genomic eDNA concentrations and succeeded in detecting the core microbial community including the rare microbes. However, our study recommends the SL-Bead-Robotic eDNA extraction protocol because this method is safe, environmentally friendly, rapid, high-throughput and inexpensive.

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