DNA extraction using the DNeasy® PowerFood® Microbial Kit

The DNeasy^® PowerFood^® Microbial Kit (Qiagen, Hilden, Germany) relies on mechanical lysis by microbeads and column extraction. There are no enzymes mentioned to be contained in the supplied lysis buffer, as the lytic reagent a detergent is given. The protocol was therefore adapted based on modifications by Quigley et al. (2012) and was used for all extractions throughout the optimization process of the sample preparation and library-PCR cycle number experiments (Supplementary Fig. S1A, C): 1.0 μL (25 μg mL⁻¹) lysozyme (Roth, Karlsruhe, Germany) and 100 U mutanolysin (Sigma-Aldrich, St. Louis, USA) were added together with 450 μL of the kit’s corresponding MBL buffer to the bacterial suspension and incubated for 60 min at 37 °C and 350 rpm. This was followed by the addition of 10 μL (12.5 mg mL⁻¹) proteinase K solution and incubation of 60 min at 55 °C and 350 rpm. Samples were then heated for 10 min at 70 °C, and the lysis suspension was transferred to the kit’s PowerBead tube. The vortexing step (10 min at maximum speed) of the manufacturer’s protocol was replaced by shaking the samples 4×6.5 m/s for 30 s using a FastPrep-24™ instrument. Extraction was further carried out according to the manufacturer’s instructions. To maximize the amount of extracted DNA, the elution volume was reduced to 50 μL, and for the kit comparisons, a final volume of 35 μL PCR-grade water was used. DNA was extracted after 1 min of incubation at RT and 1 min centrifugation (13,000×g).