From contiguity to accuracy: Validation-centered perspectives on bacterial genome assembly

Recent advances in sequencing technologies, particularly long-read platforms, have substantially improved contiguity of bacterial genome assemblies and enabled the routine generation of near-complete or circular genomes. However, achieving a contiguous assembly does not necessarily guarantee accuracy. Assembly errors, including structural misassemblies, collapsed repeats, incorrect circularization, plasmid reconstruction errors, and nucleotide-level inaccuracies, remain prevalent and may lead to misleading biological interpretations if not properly identified. In this review, we provide a comprehensive overview of bacterial genome assembly from a validation-centered perspective and examine the underlying causes of draft genome formation and assembly uncertainty, highlighting the roles of repetitive genomic structures, platform-specific error profiles, and algorithmic limitations. We further emphasize that the central challenge in contemporary bacterial genomics is no longer simply to maximize assembly contiguity, but to determine whether apparently complete genomes are truly correct and sufficiently reliable for their intended downstream applications. We propose a practical decision-making framework that links sequencing strategy, assembly workflow, polishing, and validation rigor, and introduce a tiered confidence classification to guide the interpretation of genome assembly reliability. As bacterial genome sequencing becomes increasingly routine and large-scale, future efforts should prioritize accuracy, reproducibility, transparent reporting, and evidence-supported validation over completeness alone.