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.
