Performance of mechanically sheared DNA in multiplexed Oxford Nanopore sequencing for Salmonella Typhi genomic surveillance.

Pathogen genomic surveillance is essential for monitoring high-risk lineages and antimicrobial resistance in Salmonella enterica serovar Typhi (S. Typhi), yet implementing whole-genome sequencing in sentinel hospital laboratories remains challenging. We evaluated whether mechanically sheared DNA, paired with high accuracy or super accuracy (SUP) basecalling, enables reliable Oxford Nanopore Technologies (ONT)-only sequencing for multiplexed S. Typhi genomic surveillance. Genomic DNA from three benchmark strains (Ty2, Ty42 and Ty43) was either mechanically sheared to ~15 kb or left unsheared and sequenced in a controlled 6-plex run. The optimized workflow was subsequently evaluated in a 24-plex field run that included 3 benchmark strains and 21 clinical isolates from a sentinel hospital in Manila, Philippines. Both runs were performed using the ONT Ligation Sequencing Kit with Native Barcoding 24 V14 (SQK-NBD114.24) and R10.4.1 flow cells (FLO-MIN114). Assembly metrics, single-nucleotide variation (SNV) concordance, core-genome MLST and downstream functional outputs were compared across DNA preparations and basecalling modes. In the 6-plex run, sheared libraries consistently achieved >1,000× pre-filtered coverage across all strains, whereas unsheared libraries showed highly variable depths and reduced stability after downsampling. When paired with SUP basecalling, sheared datasets produced assemblies comparable to Illumina and hybrid references across both structural accuracy and functional outputs. In the 24-plex run, all samples achieved >100× coverage, and 18 of the 22 retained S. Typhi genomes (81.8%) yielded fully circularized assemblies after exclusion of 2 non-S. Typhi isolates. Serotype prediction, genotyping and in silico antimicrobial resistance predictions remained concordant with the corresponding 6-plex benchmark results. These findings demonstrate that mechanical DNA shearing improves coverage uniformity under multiplexed conditions and, together with SUP basecalling, supports ONT-only S. Typhi genome reconstruction for routine genomic surveillance and broad phylogenetic contextualization. However, for fine-scale transmission analysis and high-resolution SNV interpretation, short-read or hybrid sequencing remains important. Overall, this approach provides a scalable framework for high-multiplex S. Typhi genomic surveillance in sentinel laboratory settings.