Improving Nanopore sequencing-based core genome MLST for global infection control: a strategy for GC-rich pathogens like <i>Burkholderia pseudomallei</i>

This study highlights a significant advancement in genomic surveillance of bacterial pathogens, specifically addressing the challenges posed by the GC-rich species <i>Burkholderia pseudomallei</i>. Core genome multilocus sequence typing (cgMLST) is widely used for bacterial typing as it combines high resolution with simple implementation and standardization. To improve cost efficiency and thus accessibility, we changed the sequencing approach from Illumina short-read (SR) to Oxford Nanopore long-read sequencing (ONT-LR). ONT-LR-based cgMLST showed a very high error rate compared with SR-based cgMLST, most likely due to methylation-associated errors. PCR-based library preparation, which is proposed to correct these errors, did not achieve the required accuracy. In contrast, native barcoding with advanced basecalling and polishing strategies massively reduces allelic differences. This optimized ONT-LR cgMLST workflow provides a transformative solution for cost-efficient, high-resolution typing of <i>B. pseudomallei</i>. Furthermore, this study can serve as a guide for similarly challenging bacteria.