Comparison of gene-by-gene and genome-wide short nucleotide sequence based approaches to define the global population structure of Streptococcus pneumoniae

Defining the population structure of a pathogen is a key part of epidemiology, as genomically related isolates are likely to share key clinical features such as antimicrobial resistance profiles and invasiveness. Multiple different methods are currently used to cluster together closely- related genomes, potentially leading to inconsistency between studies. Here, we use a global dataset of 26,306 S. pneumoniae genomes to compare four clustering methods: gene-by- gene seven-locus multi-locus sequencing typing (MLST), core genome MLST (cgMLST)- based hierarchical clustering (HierCC) assignments, Life Identification Number (LIN) barcoding, and k-mer-based PopPUNK clustering (known as GPSCs in this species). We compare the clustering results with phylogenetic and pan-genome analyses to assess their relationship with genome diversity and evolution, as we would expect a good clustering method to form a single monophyletic cluster that has high within-cluster similarity of genomic content. We show that the four methods are generally able to accurately reflect the population structure based on these metrics, and that the methods were broadly consistent with each other. We investigated further to study the discrepancies in clusters. The greatest concordance was seen between LIN barcoding and HierCC (Adjusted Mutual Information Score = 0.950), which was expected given that both methods utilise cgMLST, but have different methods for defining an individual cluster and different core genome schema. However, the existence of differences between the two methods show that the selection of a core genome schema can introduce inconsistencies between studies. GPSC and HierCC assignments were also highly concordant (AMI = 0.946), showing that k-mer based methods which use the whole genome and do not require the careful selection of a core genome schema are just as effective at representing the population structure. Additionally, where there were differences in clustering between these methods, this could be explained by differences in the accessory genome that were not identified in cgMLST. We conclude that for S. pneumoniae , standardised and stable nomenclature is important as the number of genomes available expands. Furthermore, the research community should transition away from seven- locus MLST, and cgMLST, GPSC, and LIN assignments should be used more widely. However, to allow for easy comparison between studies and to make previous literature relevant, the reporting of multiple clustering names should be standardised within research.

Data summary

Genome sequences are deposited in the European Nucleotide Archive (ENA); accession numbers. Metadata of the pneumococcal isolates in this study have been submitted as a supplementary file and are also available on the Monocle Database available at https://data.monocle.sanger.ac.uk/ . The authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.

Impact Statement

Using a global dataset of S. pneumoniae genomes allows us to thoroughly observe and analyse discrepancies between different clustering methods. Whilst all methods in this study are used to cluster S. pneumoniae genomes, no study has yet thoroughly compared the clustering results and discrepancies. This work summarises the strengths and weaknesses of the different methods and highlights the need for consistency between studies.