Sequencing and analysis approaches of S. pneumoniae

Whole genome sequencing (WGS) is an invaluable tool that enables high-resolution genotyping for the precise identification of bacterial strains. It is particularly significant for highly pathogenic bacteria such as Streptococcus pneumoniae, a leading cause of mortality and morbidity worldwide. Illumina sequencing is highly established for S. pneumoniae, while Oxford Nanopore Technologies (ONT) data is limited. Hence, evaluating ONT-only data is needed. We aimed to compare the Illumina and ONT systems for S. pneumoniae sequencing. Moreover, we aimed to explore if the newer chemistry from ONT with R10.4.1 flow cells improves the data outputs from long-read sequencing. S. pneumoniae bacteria were isolated from hospitalized patients with invasive pneumococcal disease (IPD) and serotyped by multiplex PCR. Resistance profiles were determined with anti-microbial susceptibility testing. A total of 27 isolates were sequenced using ONT Mk1c with R9.4.1 flow cells and Kit10 chemistry (ONT_V10) and the Illumina Miseq system. Illumina and ONT data were compared, and hybrid assembly was assessed. ONT sequencing was additionally performed with R10.4.1 flow cells and Kit14 chemistry (ONT_V14) in 12 isolates. S. pneumoniae identification, serotyping, AMR and GPSC prediction were successfully achieved using ONT sequencing. The ONT_V14 chemistry significantly improved both MLST and pbp prediction in long-read sequencing. Overall, hybrid assembly produced circular and contiguous genomes with high N50 parameters. Moreover, long-read assembly followed by short-read polishing is a fast and reliable approach for hybrid assembly at ONT sequencing depth > 100X. For ONT sequencing depth <50X, tools that perform short-read-first assembly such as Unicycler are recommended.