Oxford Nanopore User Group Meeting, Brisbane

Methylartist is an integrated suite of tools for working with modified bases including but not limited to 5mC. Since first publishing methylartist in 2022, we have steadily added new features as it has seen increased adoption and application across the ONT community. In this talk I will run through some of our use cases in scaling methylartist to study haplotype-aware methylation in larger cohorts, as well as new features including variant-specific analysis and support for comparison with methylation calls based on C/T substitution assays. This talk presents two case studies demonstrating the approach. In the first, samples from the gut microbiome of 51 urban pet dogs in Shanghai (sequenced to at least 20 Gbp of Nanopore plus 20 Gbp of Illumina data) yielded 2,676 MAGs spanning 320 bacterial species, roughly 72% of them near-finished. Many improved on the corresponding public reference genome for the same species. The catalog proved broadly representative of pet dogs worldwide (>90% median read mapping to external cohorts), and crucially recovered 185 circular extrachromosomal elements, including plasmids carrying antimicrobial resistance genes that also circulate in dog gut datasets globally. Living environment, not geography, emerged as the dominant structuring factor. We were also able to observe one interesting instance of structural genome rearrangements with a Fusobacterium sp. alternatively presenting as having two chromosomes or a single, fused, one.

The second case study applies the same approach to 58 urban soil samples from two Chinese cities. Here, long-read assembly recovered 7,949 medium- and high-quality MAGs representing 4,171 species-level bins, over 97% previously undescribed. The contiguity unlocked secondary metabolism >30,000 biosynthetic gene clusters, substantially more complete than short-read equivalents, alongside over 2 million small protein families enriched near defense systems and mobile elements, and an extensive repertoire of latent antimicrobial resistance genes.

Reference genomes have traditionally come from pure isolate cultures, yet most microbial diversity resists cultivation. Metagenome-assembled genomes (MAGs) have filled this gap but at the cost of fragmentation. Additionally, short-read assembly and binning routinely miss or mis-bin ribosomal proteins, mobile genetic elements, biosynthetic gene clusters, and antimicrobial resistance genes. Deep long-read metagenomics bypasses this trade-off: genomes reconstructed directly from complex communities can rival, and often surpass, the contiguity and completeness of existing isolate-derived references.

This talk presents two case studies demonstrating the approach. In the first, samples from the gut microbiome of 51 urban pet dogs in Shanghai (sequenced to at least 20 Gbp of Nanopore plus 20 Gbp of Illumina data) yielded 2,676 MAGs spanning 320 bacterial species, roughly 72% of them near-finished. Many improved on the corresponding public reference genome for the same species. The catalog proved broadly representative of pet dogs worldwide (>90% median read mapping to external cohorts), and crucially recovered 185 circular extrachromosomal elements, including plasmids carrying antimicrobial resistance genes that also circulate in dog gut datasets globally. Living environment, not geography, emerged as the dominant structuring factor. We were also able to observe one interesting instance of structural genome rearrangements with a Fusobacterium sp. alternatively presenting as having two chromosomes or a single, fused, one.

The second case study applies the same approach to 58 urban soil samples from two Chinese cities. Here, long-read assembly recovered 7,949 medium- and high-quality MAGs representing 4,171 species-level bins, over 97% previously undescribed. The contiguity unlocked secondary metabolism >30,000 biosynthetic gene clusters, substantially more complete than short-read equivalents, alongside over 2 million small protein families enriched near defense systems and mobile elements, and an extensive repertoire of latent antimicrobial resistance genes.