Oxford Nanopore sequencing enables strain-level metagenomics and reveals the structure and function of a mature compost microbiome

Abstract Characterizing the incredibly diverse microbial communities in compost is key to understanding the organic waste transformation process and the effects of compost amendment on soil health, fertility, and resilience. Here, we perform deep Oxford Nanopore sequencing of mature compost from a food and yard scrap-fed compost pile (one PromethION flow cell yielding155 Gb of sequencing), followed by metagenomic assembly and binning with state-of-the-art tools for highly accurate long reads, including metaMDBG and SemiBin2. This resulted in exceptionally contiguous assemblies (808 contigs >1Mb) and reconstruction of 598 high quality and 626 medium quality metagenome-assembled genomes (MAGs), 372 of which were comprised of a single contig. Out of 1,224 total MAGs of medium quality or higher, we found that 98.5% belonged to novel species, and 27% belonged to novel genera (no species- or genus-level classification against GTDB release 2.2.0, respectively). The high contiguity of and degree of genome recovery from these assemblies allowed us to characterize the structure and metabolic potential of the compost microbiome. We predict the compost community’s capacity for carbon and nitrogen cycling, identify antimicrobial resistance (AMR) genes and place them within genomic/plasmid context, and profile a diverse array of biosynthetic gene clusters (BGCs). Additionally, we investigated intra-species genomic variation by resolving linked strain mutations (i.e. strain haplotypes) and identifying recombination blocks in AMR genes with long read-specific tools Strainy and devider. These results illustrate the potential for long read sequencing to resolve genomes from complex microbiomes at the strain level, a longstanding goal of the field with implications for understanding microbiome evolution, characterizing associations between microbiomes and human disease, and designing microbiome therapeutics.