Graph-based Nanopore Adaptive Sampling with GNASTy enables sensitive pneumococcal serotyping in complex samples

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 which 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 non-pathogenic 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 maximise 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. GNASTy therefore has the potential to increase the density and coverage of global pneumococcal surveillance. One sentence summaryPangenome graph-based Nanopore Adaptive Sampling, presented in our tool GNASTy, is a sensitive, portable and cost-effective method for Streptococcus pneumoniae surveillance.