Abstract:
The use of whole-genome sequencing (WGS) for routine typing of bacterial
isolates has increased substantially in recent years. For Mycobacterium tuberculosis (MTB), in
particular, WGS has the benefit of drastically reducing the time required to generate results
compared to most conventional phenotypic methods. Consequently, a multitude of solutions
for analyzing WGS MTB data have been developed, but their successful integration in
clinical and national reference laboratories is hindered by the requirement for their validation,
for which a consensus framework is still largely absent. We developed a bioinformatics
workflow for (Illumina) WGS-based routine typing of MTB complex (MTBC) member isolates
allowing complete characterization, including (sub)species confirmation and identification
(16S, csb/RD, hsp65), single nucleotide polymorphism (SNP)-based antimicrobial resistance
(AMR) prediction, and pathogen typing (spoligotyping, SNP barcoding, and core genome
multilocus sequence typing). Workflow performance was validated on a per-assay basis
using a collection of 238 in-house-sequenced MTBC isolates, extensively characterized with
conventional molecular biology-based approaches supplemented with public data. For SNPbased
AMR prediction, results from molecular genotyping methods were supplemented
with in silico modified data sets, allowing us to greatly increase the set of evaluated mutations.
The workflow demonstrated very high performance with performance metrics of
.99% for all assays, except for spoligotyping, where sensitivity dropped to ;90%. The validation
framework for our WGS-based bioinformatics workflow can aid in the standardization
of bioinformatics tools by the MTB community and other SNP-based applications regardless
of the targeted pathogen(s).
