Abstract
Whole genome sequencing (WGS) has become the reference standard for bacterial outbreak investigation and pathogen typing, providing a resolution unattainable with conventional molecular methods. Data generated with Illumina sequencers can however only be analysed after the sequencing run has finished, thereby losing valuable time during emergency situations. We evaluated both the effect of decreasing overall run time, and also a protocol to transfer and convert intermediary files generated by Illumina sequencers enabling real-time data analysis for multiple samples part of the same ongoing sequencing run, as soon as the forward reads have been sequenced. To facilitate implementation for laboratories operating under strict quality systems, extensive validation of several bioinformatics assays (16S rRNA species confirmation, gene detection against virulence factor and antimicrobial resistance databases, SNP-based antimicrobial resistance detection, serotype determination, and core genome multilocus sequence typing) for three bacterial pathogens ( Mycobacterium tuberculosis , Neisseria meningitidis , and Shiga-toxin producing Escherichia coli ) was performed by evaluating performance in function of the two most critical sequencing parameters, i.e. read length and coverage. For the majority of evaluated bioinformatics assays, actionable results could be obtained between 14 and 22 h of sequencing, decreasing the overall sequencing-to-results time by more than half. This study aids in reducing the turn-around time of WGS analysis by facilitating a faster response in time-critical scenarios and provides recommendations for time-optimized WGS with respect to required read length and coverage to achieve a minimum level of performance for the considered bioinformatics assay(s), which can also be used to maximize the cost-effectiveness of routine surveillance sequencing when response time is not essential.
Highlights
Whole genome sequencing (WGS) has become the reference standard for bacterial outbreak investigation and pathogen typing
Most national reference laboratories (NRLs) and centres (NRCs), as well as other laboratories working under a quality system, require extensive validation to demonstrate that employed methods are ‘fit-for- purpose’ and provide high-q uality results through a process of rigorous validation [14,15,16]
Similar trends were observed for N. meningitidis and M. tuberculosis, and are illustrated for all tested combinations in Figs S2–S25, except for the N50 value which was close to the maximum for the shortest read lengths for M. tuberculosis in contrast to E. coli and N. meningitidis, potentially explained by a lower number of repeat regions in the Mycobacterium genome [41]
Summary
Whole genome sequencing (WGS) has become the reference standard for bacterial outbreak investigation and pathogen typing. Information on the presence of genomic features associated with antimicrobial resistance (AMR) or virulence of relevance for rapid outbreak management or clinical intervention can Public health authorities are still actively facing the challenge of validating bioinformatics workflows for short-read technologies, for which a consensus is emerging but many issues still need to be addressed [17]. This renders the implementation and validation of the ONT technology, which is still constantly evolving resulting in quickly changing protocols and data analytical approaches [18], not necessarily the highest priority
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