This study evaluates the accuracy of bacterial genome reconstruction using Oxford Nanopore Technologies' (ONT) R10.4.1 long-read sequencing. The authors compared the performance of R10.4.1 long-read assemblies with those of hybrid Illumina-ONT assemblies, which are currently the gold standard for high-quality bacterial genome reconstruction. They sequenced DNA from four common bacterial pathogens (Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Staphylococcus aureus) and 12 clinical E. coli isolates, both with and without the use of Bovine Serum Albumin (BSA) during library preparation. The results showed that R10.4.1 long-read assemblies, when combined with updated basecallers trained on native bacterial DNA and BSA, produced accurate assemblies from 40x depth or higher, comparable to hybrid Illumina-ONT assemblies. The study also found that BSA improved sequencing yield and read accuracy, making R10.4.1 long-read sequencing a cost-effective and efficient alternative to hybrid sequencing. The findings suggest that R10.4.1 long-read sequencing, with appropriate modifications, can be a viable single-platform solution for bacterial genome assembly, simplifying and accelerating pathogen genomics studies.This study evaluates the accuracy of bacterial genome reconstruction using Oxford Nanopore Technologies' (ONT) R10.4.1 long-read sequencing. The authors compared the performance of R10.4.1 long-read assemblies with those of hybrid Illumina-ONT assemblies, which are currently the gold standard for high-quality bacterial genome reconstruction. They sequenced DNA from four common bacterial pathogens (Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Staphylococcus aureus) and 12 clinical E. coli isolates, both with and without the use of Bovine Serum Albumin (BSA) during library preparation. The results showed that R10.4.1 long-read assemblies, when combined with updated basecallers trained on native bacterial DNA and BSA, produced accurate assemblies from 40x depth or higher, comparable to hybrid Illumina-ONT assemblies. The study also found that BSA improved sequencing yield and read accuracy, making R10.4.1 long-read sequencing a cost-effective and efficient alternative to hybrid sequencing. The findings suggest that R10.4.1 long-read sequencing, with appropriate modifications, can be a viable single-platform solution for bacterial genome assembly, simplifying and accelerating pathogen genomics studies.