Efficient DNA Extraction from Water Samples

For DNA extraction, aliquots of 250 mL or 500 mL of water were filtered through 0.22 µm nitrocellulose filter membranes (GSWP, Whatman). Filters were stored at − 20 °C (or − 80 °C for longer storage) until use. DNA extraction was performed according to the protocol described in Kisand et al. [34 (link)] except for the lyticase incubation. Briefly, each frozen filter was thawed, incubated in 5 mL of 50 mM KH₂PO₄ buffer (pH 7.5) and shaken overnight (160 rpm) at 4 °C. The following day, the KH₂PO₄ buffer was recovered and filters were transferred to a tube with 3 mL of fresh 50 mM KH₂PO₄ to be sonicated at 60 °C for a total of 15 min. Filters were discarded, and sonicated and not sonicated buffers were pulled together and then shaken for 3 h at 30 °C with 533 µL lysozyme (100 mg/mL in DEPC water, Sigma Aldrich) and 7.7 µL β-mercaptoethanol (Sigma-Aldrich). Samples were frozen at − 20 °C, thawed and centrifuged at 4 °C for 20 min at 14.000 rpm. The DNA was extracted using the DNeasy blood and tissue kit (Qiagen) following the manufacturer’s instructions. DNA was quantified by measurements with both Nanodrop and Qubit (Thermofisher). DNA concentrations measured with the two instruments ranged from 18.6 to 48.6 ng/µL (Nanodrop) and from 13.7 to 48.8 ng/µL (Qubit). Purified DNA samples were subjected to 16S sequencing and shotgun analysis.