Comparing DNA Extraction Protocols for Freshwater Prokaryotic Communities: Impacts on Yield and Microbial Profiling

Abstract DNA extraction from aquatic samples is a critical process that influences the quantity and purity of the DNA obtained. This can have profound effects on the accuracy of the community depiction. In this study, DNA extraction workflows of seven commercial kits (Qiagen: DNeasy PowerLyzer PowerSoil, DNeasy PowerSoil Pro, DNeasy PowerSoil, DNeasy PowerMax Soil; Macherey-Nagel: NucleoSpin Soil; Zymo: ZymoBIOMICS DNA; MP Biomedicals: FastDNA SPIN), along with several modifications of manufacturer’s protocols focusing on the lysis and elution steps, were tested, accounting for a total of 18 different protocols. For each protocol, DNA yield (quantity, replicability and quality), richness and compositional reproducibility based on 16S rRNA gene sequencing, as well as processing time and cost were assessed. The standard protocols recommended by the manufacturer showed comparable DNA yield results. Shared ASVs between all protocols accounted for >90% of the reads and were mostly abundant ASVs, indicating consistent detection of dominant taxa across all protocols. Adding supplementary lysis and elution steps to the manufacturer’s protocols yielded up to ∼4× more DNA. However, total read counts and ASV richness were lower as total DNA increased. Manufacturer’s protocols therefore showed higher values than their modified versions, although these effects were not significant on community composition. We conclude that the choice of a protocol is the balance between recovering sufficient DNA of good quality versus potential effects on downstream sequencing output (reads and ASVs).