Differential efficiency of sampling devices in the measurement of microbial diversity of Yellowstone National Park hot springs

Metagenomic characterization of low-biomass Yellowstone National Park (YNP) hot spring waters remains challenging because microbial recovery is influenced by filtration methodology, sample preservation, DNA extraction, and sequencing strategy. We characterized thermophilic microbial communities in alkaline YNP hot spring waters (62-90.5{degrees}C) using three high-temperature-compatible filtration systems (Sterivex, Supor, and polycarbonate membranes), automated onsite DNA extraction ({micro}Titan), and shotgun metagenomic sequencing with Illumina short-read and Oxford Nanopore Technologies (ONT) long-read platforms. Across all filtration systems and sequencing workflows, microbial communities were consistently dominated by Bacteria ([~]90% of reads), whereas Archaea represented <10% of recovered sequences. Dominant microbial populations were reproducibly recovered across all approaches; however, recovery of lower-abundance taxa varied among methods. This variability was most evident in polycarbonate-filtered samples, which exhibited greater replicate-to-replicate variation and less consistent detection of microbial species. Thermocrinis ruber and related Aquificae-associated thermophiles dominated the hottest waters (78.5-90.5{degrees}C), whereas warmer effluent-channel waters (63.5-66.5{degrees}C) contained T. ruber together with photosynthetic taxa, including Synechococcus spp. and Candidatus Thermochlorobacter aerophilum. Archaeal communities were primarily represented by Pyrobaculum- and Thermoproteus-related taxa. Non-metric multidimensional scaling analyses indicated that overall community structure was largely unaffected by filtration or sequencing methodology, whereas alpha-diversity metrics showed that filter selection influenced richness and diversity estimates. These findings identify field-deployable workflows for metagenomic characterization of low-biomass thermophilic aquatic systems and demonstrate the importance of integrating filtration and sequencing strategies for studying extremophile microbiomes under remote sampling conditions. IMPORTANCEAccurate characterization of low-biomass geothermal water microbiomes remains challenging because microbial recovery is strongly influenced by sample handling, filtration efficiency, DNA extraction chemistry, and sequencing methodology. This study demonstrated that Yellowstone National Park alkaline hot spring water microbiomes were consistently dominated by Bacteria (>90% of recovered reads), whereas Archaea represented <10% of community abundance across all filtration systems. Although dominant microbial populations were reproducibly recovered, filtration-device selection influenced the recovery of microbial diversity and low-abundance taxa. By integrating field-deployable onsite DNA extraction with ONT shotgun metagenomic sequencing, this work evaluates practical workflows for studying thermophilic planktonic microbial communities under remote field conditions. These findings are relevant, not only to geothermal microbiology, but also to low-biomass environments in medical, pharmaceutical, and aerospace industries, where rapid onsite processing and contamination-aware workflows are essential for preserving authentic microbial signatures in extreme environments.