BIOTECHNOLOGY 67
Fig. 2. The wgMLST workflow.
Terefore it is becoming the preferred tool for long-term monitoring, surveillance and outbreak investigation [2, 3]
.
REFERENCES 1
Jolley K.A., Maiden M.C.,
BIGSdb: Scalable analysis of bacterial genome variation at the population level, BMC Bioinformatics. 2010 Dec 10;11:595. doi: 10.1186/1471-
2105-11-595. 2
Leopold S. R., Goering
R. V., et al. 2014. Bacterial whole-genome sequencing revisited: portable, scalable, and standardized analysis for typing and detection of virulence and antibiotic resistance genes. J Clin Microbiol. 2014 Jul;52(7):2365-
70. 3
S. Roisin, C. Gaudin, et
al. (2015). Abstract O252, Session Staphylococcus – ESCMID, April 29 2015, Copenhagen. https://www.
escmid.org/escmid_library/ online_lecture_library/
material/?mid=22445 4
Pouseele H., Method of
typing nucleic acid or amino acid sequences based on sequence analysis, European patent 2502593 (pending).
The possible pitfalls As was the case with classical MLST, the choice and definition of the loci contained in the scheme is of paramount importance to obtain a noiseless analysis of WGS data. While the wgMLST scheme itself is based on the genes present in the taxon, the WG perspective extends the number of available loci far beyond the point of what is manually manageable, and thus there is a need for an automated tool able to create and evaluate a reliable wgMLST scheme[3] (Fig. 2).
Moreover, any scheme creation procedure not only needs to reliably define loci, but also needs to be able to predict and avoid possible locus convergence, as it is likely that, upon addition of new sequences, biological variation might lead to allelic variants with high similarity, albeit derived from different loci.
Te locus detection procedures are also of great importance (Fig. 2). We therefore propose a two-tier approach. Assembly- based methodologies identify alleles from de novo assembled genomes using BLAST[1]
. Te
assembly procedure in itself is computationally intensive, but is especially useful for extrinsic validation of the allele calls, such as in silico PCR, in silico hybridisation or synteny-based validation. However, a de novo approach implies that some loci can and will be missed due to draft assemblies. Moreover, de novo assembly has undefined behaviour for the reconstruction of multi-copy loci, and therefore multi-copy loci are not very well detected from de novo contigs. Terefore an additional assembly-free method[4]
should
be used to compensate for the assembly artefacts (Fig. 2). Tis is computationally less intensive, provides a more clear definition of missing loci (as they now are missing from the reads rather
than from the de novo assembly), and is designed to be exhaustive (as multi-copy loci are picked up as separate allele calls). It also provides invaluable quality control metrics for detecting contamination.
Once locus definitions are standardised, wgMLST is a highly unambiguous and portable method. Materials required for sequence typing can easily be exchanged between laboratories, as the method is reproducible and scalable. wgMLST can be automated, combining advances in high throughput sequencing and bioinformatics with established population genetics techniques. Most importantly, wgMLST data can be used to investigate evolutionary relationships among bacteria and provides close-to-ultimate discriminatory power to differentiate isolates.
For more information ✔ at
www.scientistlive.com/eurolab
Katrien De Bruyne, Bruno Pot & Hannes Pouseele are with Applied Maths in Belgium.
www.applied-maths.com
www.scientistlive.com
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