Abstract
ABSTRACTThe 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 SNP-based 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). The bioinformatics workflow is available for academic and nonprofit use through the Galaxy instance of our institute at https://galaxy.sciensano.be.
Highlights
The use of whole-genome sequencing (WGS) for routine typing of bacterial isolates has increased substantially in recent years
We present an extensive validation of a bioinformatics workflow (Fig. 1) for characterization of MTB complex (MTBC) isolates using WGS data generated with Illumina technology
Several validation strategies have been proposed recently [35, 67,68,69] but are all commonly focused on the correct identification of genes or specific alleles and are not directly applicable for MTBC members, for which accurate detection of single nucleotide polymorphism (SNP) is of major importance [10]
Summary
The use of whole-genome sequencing (WGS) for routine typing of bacterial isolates has increased substantially in recent years. 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). Many solutions for analyzing Mycobacterium tuberculosis (MTB) WGS data have been developed to replace traditionally employed molecular approaches [5,6,7,8] This is facilitated by the well-characterized MTB genome, rendering it well suited for WGS analyses [9]. Species-agnostic methods such as 16S rRNA sequencing are generally applicable to bacterial pathogens, including MTB [16] Information for all these assays of interest can be obtained through a single WGS run, and WGS has proved itself a viable alternative for species confirmation and identification [12, 17]. Integration into a Journal of Clinical Microbiology jcm.asm.org 2
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