Enhancing DNA recovery in low-biomass snow algae samples: a comparative study of extraction methods and their effect on community composition.

High-throughput sequencing is a powerful tool for environmental microbiology and can be particularly important for examining community structure and function for organisms that are difficult to culture or environments that are difficult to mimic, like snow. Nucleic acid extraction significantly impacts these analyses, often introducing more variation between samples than PCR or sequencing. Snow algae are widespread on mountain and polar snowfields, where they contribute to biogeochemical cycling and accelerate melt. Despite increasing research on snow algae, DNA extraction remains challenging, as the thick, resilient walls of snow algal cysts can limit cell lysis, and differences among extraction methods may therefore affect the estimates of community composition and richness. Here, we compared three common extraction methods (Qiagen DNeasy PowerSoil Pro, Qiagen DNeasy PowerWater, and phenol-chloroform) alongside ultrasonication in samples with varying snow algae abundance. The extraction method strongly influenced the resulting microbial profiles assessed by amplicon sequencing of rRNA genes. Ultrasonication improved DNA yield in low-biomass samples and enhanced the recovery of DNA from resilient cells, including mature-phase snow algae, likely due to improved cell lysis. Our findings provide insights to improve standardization and facilitate comparison among studies in snow and ice environments.IMPORTANCEHigh-throughput sequencing has transformed environmental microbiology, allowing for detailed, culture-independent analyses of microbial communities. However, multiple methodological factors, including DNA extraction, can introduce variability in results, making cross-study comparisons challenging. This research contributes to improving our understanding of snow algae, which play a role in alpine and polar ecosystems by influencing biogeochemical cycles and snow reflectivity. By evaluating common DNA extraction techniques for snow algae, this study helps improve the reliability and reproducibility of sequencing data, supporting broader efforts toward methodological standardization in microbial ecology.