Bioactive molecules unearthed by terabase-scale long-read sequencing of a soil metagenome

Metagenomics provides access to the genetic diversity of uncultured bacteria through analysis of DNA extracted from whole microbial communities. Long-read sequencing is advancing metagenomic discovery by generating larger DNA assemblies than previously possible. However, harnessing the potential of long-read sequencing to access the vast diversity within soil microbiomes is hampered by the challenge of isolating high-quality DNA. Here we introduce a method that can liberate large, high-quality metagenomic DNA fragments from soil bacteria and pair them with optimized nanopore long-read sequencing to generate megabase-sized assemblies. Using this method, we uncover hundreds of complete circular metagenomic genomes from a single soil sample. Through a combination of bioinformatic prediction and chemical synthesis, we convert nonribosomal peptide biosynthetic gene clusters directly into bioactive molecules, identifying antibiotics with rare modes of action and activity against multidrug-resistant pathogens. Our approach advances metagenomic access to the vast genetic diversity of the uncultured bacterial majority and provides a means to convert it to bioactive molecules.