Optimizing nanopore adaptive sampling for pneumococcal serotype surveillance in complex samples using the graph-based...

Serotype surveillance of Streptococcus pneumoniae (the pneumococcus) is critical for understanding the effectiveness of current vaccination strategies. However, existing methods for serotyping are limited in their ability to identify co-carriage of multiple pneumococci and detect novel serotypes. To develop a scalable and portable serotyping method that overcomes these challenges, we employed nanopore adaptive sampling (NAS), an on-sequencer enrichment method that selects for target DNA in real-time, for direct detection of S. pneumoniae in complex samples. Whereas NAS targeting the whole S. pneumoniae genome was ineffective in the presence of nonpathogenic streptococci, the method was both specific and sensitive when targeting the capsular biosynthetic locus (CBL), the operon that determines S. pneumoniae serotype. NAS significantly improved coverage and yield of the CBL relative to sequencing without NAS and accurately quantified the relative prevalence of serotypes in samples representing co-carriage. To maximize the sensitivity of NAS to detect novel serotypes, we developed and benchmarked a new pangenome-graph algorithm, named GNASTy. We show that GNASTy outperforms the current NAS implementation, which is based on linear genome alignment, when a sample contains a serotype absent from the database of targeted sequences. The methods developed in this work provide an improved approach for novel serotype discovery and routine S. pneumoniae surveillance that is fast, accurate, and feasible in low-resource settings. Although NAS facilitates whole-genome enrichment under ideal circumstances, GNASTy enables targeted enrichment to optimize serotype surveillance in complex samples.