Improved detection of fungi and uncultivated microorganisms in soil metagenomes using a comprehensive genome database

Soils harbor diverse microbial communities crucial for ecosystem functioning, but poor genomic representation of many uncultured soil microorganisms limits the utility of existing databases to address some of the most pressing questions in environmental microbiology. To address this, we developed the SoilMicrobeDB, a comprehensive, genome-based reference database to enhance metagenomic classification for soil ecosystems, with a focus on previously underrepresented fungal taxa and uncultured organisms. We evaluated the database using a large soil metagenome dataset, comparing classification rates, analyzing fungal-bacterial ratios against phospholipid fatty acid (PLFA) estimates, and validating lineage abundances with rRNA amplicon sequencing data. Mock community analysis was also conducted to test the precision of community classification and the prevalence of false positives. The SoilMicrobeDB workflow improved metagenomic read classification by over 20% and provided more accurate fungal abundance estimates, particularly for nutrient cycling groups such as ectomycorrhizal fungi. Metagenomic-derived fungal-bacterial ratios were correlated with PLFA and qPCR estimates, and lineage proportions were aligned with relative abundances estimates from rRNA amplicon sequencing. Uncultured taxa represented up to 50% of classifiable soil microbial communities in certain biomes. SoilMicrobeDB offers robust taxonomic and functional profiling of soil communities and provides a scalable and updatable tool for soil microbial ecology research. SoilMicrobeDB is accessible through an interactive platform linking genomes to environmental factors, enabling researchers to explore microbial distributions across soil conditions and potentially leading to new insights into soil ecology and management practices.