A-G.C.05: From fragmented MAGs to complete chromosomes: refining functional diversity in uncultured Patescibacteria
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Most metagenome-assembled genomes (MAGs) derived from short-read sequencing data remain fragmented, limiting structural and functional interpretation. This challenge is particularly evident in Patescibacteria, an abundant and uncultured bacterial phylum with ultra-small genomes, reduced metabolic capacity, and putative symbiotic lifestyles. Fragmentation frequently results in incomplete or misinterpreted genomic features, obscuring genome architecture, replication dynamics, and metabolic potential. In contrast, long-read sequencing approaches remain cost-prohibitive and difficult to implement at scale.
JORG, an iterative short-read assembly and binning framework, was applied to circularize Patescibacterial MAGs from marine and terrestrial environments. Based on data-driven prioritization criteria (≤10 contigs; 25–300× coverage), 128 high-quality MAGs were selected, and 60 (46.9%) were successfully circularized into single-chromosome assemblies.
Circularization significantly increased genome completeness in 76.6% of cases (paired t-test, p < 0.001) without increasing contamination, demonstrating robust structural refinement. Circularized genomes exhibited improved structural coherence and interpretability. Structural alignments revealed insertions and deletions in 86% of genomes, consistent with resolution of low-confidence regions and assembly gaps. Circularization refined near-threshold functional assignments, including KEGG modules linked to central carbon metabolism and metabolite transport, strengthening inference of metabolic dependencies and potential host interactions, and enabled unambiguous identification and chromosomal contextualization of replication origins.
These improvements were observed in genomes already classified as high quality, indicating that circularization enhances biological resolution beyond standard completeness metrics. Together, these findings demonstrate that automated genome circularization substantially improves ecological and evolutionary interpretation of uncultured microbial lineages such as Patescibacteria across diverse environments.
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