Rapid identification, capsular typing and molecular characterization of Streptococcus pneumoniae by using whole genome nanopore sequencing

S Garcia-Garcia,D Henares,C Muñoz-Almagro,N Timoneda,A Perez-Arguello

doi:10.1186/s12866-020-02032-x

Abstract

BackgroundWhole genome sequencing has emerged as a useful tool for identification and molecular characterization of pathogens. MinION (Oxford Nanopore) is a real-time third generation sequencer whose portability, affordability and speed in data production make of it an attractive device for whole genome sequencing. The objective of this study is to evaluate MinION sequencer for pathogen identification and molecular characterization of Streptococcus pneumoniae isolated at a children’s Hospital. Whole genome sequencing of 32 Streptococcus pneumoniae invasive isolates, previously characterized by standard methods (Quellung reaction, Multiplex PCR and Sanger-MLST), were performed. DNA was extracted using ZymoBIOMICS DNA Microprep kit. Quantification and purity of DNA was assessed by Qubit and Nanodrop, respectively. Library preparation was performed using the Rapid Barcoding Kit. Real-time workflow EPI2ME platform “What’s it in my pot” was used for species identification. Fast5 sequences were converted into FASTQ by Albacore software. Reads were assembled using CANU software. PathogenWatch, genomic epidemiology and pubmlst online tools were used for capsular typing and/or whole genome-MLST profile.ResultsRapid identification of Streptococcus pneumoniae was achieved by “What’s in my pot”. Capsular typing was correctly assigned with PathogenWatch in all 32 isolates at serogroup level and 24 at serotype level. Whole genome-MLST results obtained by genomic epidemiology and pubmlst were consistent with double locus variant clonal complex obtained by Sanger-MLST in 31 isolates.ConclusionMinION sequencer provides a rapid, cost-effective and promising pathway for performing WGS by a pocked-sized device for epidemiological purposes but improving its sequencing accuracy will make it more appealing to be used in clinical microbiology laboratories.

Highlights

Whole genome sequencing has emerged as a useful tool for identification and molecular characterization of pathogens
Comparison between Nanopore technology and conventional methods A rapid and correct identification of all 32 S.pneumoniae invasive strains was achieved by What’s in my pot?” (WIMP) workflow in only 5 min, at least two days are required by traditional bacterial identification
Regarding prices of both workflows, the cost per sample characterized by MinION is about 65.11€, by Sanger is roughly 53.40€

Summary

Introduction

Whole genome sequencing has emerged as a useful tool for identification and molecular characterization of pathogens. The objective of this study is to evaluate MinION sequencer for pathogen identification and molecular characterization of Streptococcus pneumoniae isolated at a children’s Hospital. Whole genome sequencing of 32 Streptococcus pneumoniae invasive isolates, previously characterized by standard methods (Quellung reaction, Multiplex PCR and Sanger-MLST), were performed. Streptococcus pneumoniae is responsible for causing disease especially in children younger than 5 years and the elderly. It is associated with high morbidity and mortality worldwide [1]. A sequence type (ST) is assigned by comparing each gene against other isolate profiles in the public MLST reference database (https://pubmlst.org/). Using conventional Sanger sequencing approaches, MLST turn out a laborious technique, expensive and difficult to perform at scale

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