Ultra-deep long-read metagenomics captures diverse taxonomic and biosynthetic potential of soil microbes

Soil ecosystems have long been recognized as hotspots of microbial diversity, but most estimates of their microbial and functional complexity remain speculative despite decades of study, in part because conventional sequencing campaigns lack the depth and contiguity required to recover low-abundance and repetitive genomes. Here, we revisit this question using one of the deepest metagenomic sequencing efforts to date, applying 148 billion basepairs of Nanopore long-read data and 122 billion basepairs of Illumina short-read data to a single forest soil sample.

Our hybrid assembly reconstructed 837 metagenome-assembled genomes, including 466 that meet high- and medium-quality standards, nearly all lacking close relatives among cultivated taxa. Rarefaction and k-mer analyses reveal that, even at this depth, we capture only a fraction of the extant diversity: nonparametric models project that more than 10 trillion basepairs of sequencing data would be required to approach saturation. These findings offer a quantitative, technology-enabled update to long-standing diversity estimates and demonstrate that conventional metagenomic sequencing efforts likely miss most microbial and biosynthetic potential in soil. We further identify more than 11,000 biosynthetic gene clusters, over 99% of which have no match in current databases, underscoring the breadth of unexplored metabolic capacity.

Taken together, our results emphasize both the power and the present limitations of metagenomics in resolving natural microbial complexity, and they provide a new baseline for evaluating future advances in microbial genome recovery, taxonomic classification, and natural product discovery.