From Microscale to Microbial Insights: Validating High-Throughput Microvolume Extraction Methods (HiMEx) for Microbial Ecology

Extracting and directly amplifying DNA from small-volume, low-biomass samples would enable rapid, ultra-high-throughput analyses, facilitate studying microbial communities where large-volume sample collection is challenging. This can aid where ‘conventional’ filtration-based methods miss capturing smaller microbes, or where microscale variability matters, such as the ocean. Here, we develop and validate physical and chemical-based DNA extractions from microvolumes with universal rRNA gene amplicons and metagenomic sequencing of all domains and viruses, on natural seawater and experimentally manipulated marine waters. Compared to 500-mL filter-based extraction, direct PCR of 3 μL of lysed seawater samples consistently captured comparable microbial community composition and diversity, with reliable amplification and little to no contamination. Metagenomic results of 10 μL of lysed microvolume samples captured high-quality assemblies, bacterial genomes, and diversity on par or better than our conventional extraction (48 vs 44 representative MAGs), and substantially more putative complete circular viral genomes (38 vs 1). Our approach enables scaling of rRNA gene sequencing and metagenomic library prep tremendously for a fraction of the cost of conventional methods and builds upon existing microvolume approaches by removing unnecessary expenses, like excess plasticware and expensive bead clean-up. The method expands opportunities for more comprehensive microbial community monitoring and controlled laboratory experiments.