66 BIOTECHNOLOGY
Whole genome MLST analysis N
A universal typing approach for bacteria with many advantages. By Katrien De Bruyne, Bruno Pot & Hannes Pouseele.
ext-generation sequencing (NGS) technologies are rapidly
replacing classical Sanger sequencing. Te positive speed and cost evolution of NGS makes whole genome sequencing (WGS) a very attractive alternative not only for research but also for routine analyses, such as in the field of epidemiology and outbreak surveillance. Te WGS approach provides the possibility to obtain from one single assay many traditional typing results, such as MLST, rMLST, SPA- typing, spoligotyping, SNP-set based typing, etc. Tis allows for a seamless link between classical and new knowledge bases, without additional cost and effort of retrospective sequencing. Moreover, as the available sequencing data increases, functional prediction such as resistance or virulence prediction based on the presence or absence of genes involved, is becoming more reliable, which yields critical information for surveillance or for making therapeutic decisions.
Starting from WGS data, there are two main methodologies to obtain interpretable information:
Fig. 1. The wgMLST principle.
whole genome Single Nucleotide Polymorphism (wgSNP) analysis or whole genome Multi Locus Sequence Typing (wgMLST) analysis. As the latter is more readily providing functional information and is more stable for long-term applications, wgMLST is increasingly being considered for subtyping purposes at any desired taxonomic level.
wgMLST uses WGS data (assembled or not) to perform MLST [1]
on a genome-wide scale.
For each sample, locus presence is analysed and, when present, allele variants are determined. For each locus, new sequences are assigned new consecutive allele numbers (see Fig. 1).
In contrast to a wgSNP analysis, wgMLST is based on the concept of allelic variation, as the identity or non-identity of complete coding regions between strains is considered. Tis implies that recombination and deletions or insertions of multiple positions are counted as single evolutionary events. Tis approach might be biologically more relevant than approaches that consider only point mutations.
The advantages of wgMLST over wgSNP As loci generally correspond to functional genes, a wgMLST profile can be used as the basis for preliminary genotypic and (to some extent) phenotypic interpretations: starting from the complete wgMLST profile, different subschemes can be defined, composed of loci that have known functionality or mimic traditional typing schemes.
In contrast to wgSNP, where reference sequence(s) used to analyse a particular set of samples should be phylogenetically as close as possible to the samples, wgMLST is based on a single reference set that captures the complete known diversity of a taxon. Te wgMLST scheme, that is, the set of loci used in a wgMLST analysis, can be extended as more genomes are analysed and new genes (or loci) are detected (Fig. 2).
Te upfront determination of the wgMLST scheme leads to stability, in the sense that, in contrast to wgSNP, adding new samples does not have an influence on the existing information. Te ‘pan-genome’ approach has the advantage of maximising resolution in any sample comparison. When comparing closely related isolates, the pan-genome scheme aims at covering over 95% of the genes in each isolate, whereas for taxon-wide comparisons the pan- genome scheme naturally reduces to the ‘core genome’ subset, that is, a set of loci common to over, for instance, 95% of the strains belonging to the taxon considered.
Te use of a core scheme has been shown to have a high epidemiological relevance and is extremely stable over time.
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