Backward compatibility of whole genome sequencing data with MLVA typing using a newMLVAtypeshiny application: the example ofVibrio cholerae
Authors: Ambroise Jérôme , Irenge Léonid M. , Durant Jean-François , Bearzatto Bertrand , Bwire Godfrey , Stine O. Colin , Gala Jean-Luc 
Published: June 2019

Multiple-Locus Variable Number of Tandem Repeats (VNTR) Analysis (MLVA) is widely used by laboratory-based surveillance networks for subtyping pathogens causing foodborne and water-borne disease outbreaks. However, Whole Genome Sequencing (WGS) has recently emerged as the new more powerful reference for pathogen subtyping, making a data conversion method necessary which enables the users to compare the MLVA identified by either method. TheMLVATypeshiny application was designed to extract MLVA profiles from WGS data while ensuring backward compatibility with traditional MLVA typing methods.

To test and validate theMLVATypealgorithm, WGS-derived MLVA profiles of nineteenVibrio choleraeisolates from Democratic Republic of the Congo (n=9) and Uganda (n=10) were compared to MLVA profiles generated by microchip electrophoresis (Bioanalyzer Agilent 2100), GeneScan analysis, and Sanger sequencing as the reference method. Unlike amplicon-size derived MLVA profiles, results obtained by Sanger sequencing and WGS were totally concordant. However, the latter were affected by censored estimations whose percentage was inversely proportional to the k-mer parameter used during genome assembly. With a k-mer of 127, less than 15% estimation ofV. choleraeVNTR was censored. Preventing censored estimation was only achievable when using a longer k-mer size (i.e. 175), which is not proposed in the SPAdes v.3.13.0 software.

In silicoanalysis showed that this limitation does not apply to other microbial species (e.g. Mycobacterium, Streptococcus, Staphylococcus, andPseudomonas) characterized by smaller lengths of motif repeats. As NGS read lengths and qualities tend to increase with time, one may expect the increase of k-mer size in a near future. UsingMLVATypeapplication with a longer k-mer size will then efficiently retrieve MLVA profiles from WGS data while avoiding censored estimation irrespective of the microbial species.

Author summary

Next Generation Sequencing (NGS) has emerged as a powerful high throughput genomic approach enabling the Whole Genome Sequence (WGS) of pathogens to be assembled in a relatively short time. A major advantage of WGS, compared to traditional genotypic identification and typing methods, is its ability to generate data that can be exploitedin silicofor multiple bacterial tests including accurate subtyping, determination of genetic relatedness, and characterization of virulence and antimicrobial resistance determinants. Accordingly, WGS is now rapidly replacing traditional methods like Multi-Locus Variable Number of Tandem Repeats Analysis (MLVA) that has long been used in the public health sector for laboratory-based surveillance of pathogens and outbreak response. While these missions require maintenance of data comparability within networks, the lack of backward compatibility between WGS-derived and traditional MLVA methods is a well-recognized issue. As illustrated here withVibrio choleraeisolates from DRC and Uganda, theMLVATypesoftware application analyzes WGS data to generate MLVA profiles that are identical to those determined with traditional typing. Interestingly, this tool has also the potential to extract MLVA profiles from any bacterial genome that are characterized by a small number of tandem repeats,e.g. Streptococcus, Staphylococcus, Pseudomonas, andMycobacteriumspecies. This restriction can be lifted if subsequences of length k, called k-mers, are longer than what is currently proposed by genome assembly algorithm like SPAdes v.3.13.0.