A loop-mediated isothermal amplification (LAMP) assay to identify isotype 1 β-tubulin locus SNPs in synthetic double-stranded Haemonchus contortus DNA

Development of sustainable gastrointestinal nematode (GIN) control strategies depends on the ability to identify the frequencies of drug-susceptible and resistant genotypes in GIN populations arising from management practices undertaken on individual farms. Resistance to BZ drugs in GINs has been shown to be conferred by the presence of defined SNPs in the isotype 1 β-tubulin locus. Loop-mediated isothermal amplification (LAMP) assays are amenable to use on a range of DNA templates and are potentially adaptable to use in practical, cost-effective, pen-side diagnostic platforms that are needed to detect anthelmintic resistance in the field. In this study, we designed primers and examined LAMP assays to detect each of the three major isotype 1 β-tubulin SNPs conferring genetic susceptibility to BZ drugs. We used artificial pools of synthetic DNA, containing different proportions of susceptible and resistant SNPs to determine reproducibility of the assays. We demonstrated the detection of each of the isotype 1 β-tubulin SNPs conferring susceptibility to BZ drugs using the optimal LAMP assay. Isotype 1 β-tubulin SNP typing was effective in detecting BZ susceptibility, but the accuracy was reduced in samples with less than 60 % susceptible DNA. Our results show the potential for LAMP SNP typing to detect genetic susceptibility or resistance to anthelmintic drugs in livestock GINs, and some of the limitations in our approach that will need to be overcome in order to evaluate this assay using field samples.

Improvement of the technique of SNP-typing of Vibrio cholerae strains on the basis of the analysis of the primary data of whole genome sequencing

Aim. To improve the method of the quality assessment of single nucleotide polymorphisms, which are used for SNP-typing, based on the analysis of their distribution in the primary data of whole genome sequencing (reads).Materials and methods. Data of the whole genome sequencing of 56 Vibrio cholerae strains obtained using different types of sequencers were used. The software was developed using Java programming language. Cluster analysis and construction of the dendrogram were performed with the author's software using the UPGMA method.Results and discussion. The «instability» of detection the number of SNP in the genome of cholera causative agent was shown. The method of selection of the SNP list for phylogenetic analysis based on the analysis of the primary data of whole genome sequencing (reads), has been developed. The method of using «control genomes» for cluster analysis of whole genome sequencing data has been proposed.Conclusion. The list of 3198 «stable SNP» for phylogenetic analysis has been composed. Genetic affinity between the non-toxigenic strains that contain the tcpA gene (ctxAB–tcpA+) and preCTX-strains of V. cholerae was shown.

Genotyping of Francisella tularensis subsp. holarctica from Hares in Germany

Francisella tularensis is the causative agent of the zoonotic disease tularemia. In Germany, most human infections are caused by contact with infected hares. The aim of this study was to characterize Francisella tularensis subsp. holarctica strains isolated from hares in Germany and to develop bioinformatics tools to analyze their genetic relatedness. In total, 257 German isolates—obtained mainly from hares (n = 233), other vertebrate animals, and ticks, but also from humans (n = 3)—were analyzed within this study. Publically available sequence data from 49 isolates were used to put our isolates into an epidemiological context and to compare isolates from natural foci and humans. Whole-genome sequences were analyzed using core-genome Multi-Locus-Sequence-Typing, canonical Single Nucleotide Polymorphism (SNP) typing and whole-genome SNP typing. An overall conformity of genotype clustering between the typing methods was found, albeit with a lower resolution for canonical single SNP typing. The subclade distribution, both on local and national levels, among strains from humans and hares was similar, suggesting circulation of the same genotypes both in animals and humans. Whilst close to identical isolates of the same subclade were found distributed over large areas, small geographical foci often harbored members of different subclades. In conclusion, although genomic high-resolution typing was shown to be robust, reproducible and allowed the identification of highly closely related strains, genetic profiling alone is not always conclusive for epidemiological linkage of F. tularensis strains.

Evaluation of Nanopore sequencing technology to differentiate Salmonella serotypes and serotype variants with the same or closely related antigenic formulae

ABSTRACTOur previous study demonstrated that whole genome sequencing (WGS) data generated by Oxford Nanopore Technologies (ONT) can be used for rapid and accurate prediction of Salmnonella serotypes. However, one limitation is that established methods for WGS-based serotype prediction cannot differentiate certain serotypes and serotype variants with the same or closely related antigenic formulae. This study aimed to evaluate Nanopore sequencing and corresponding data analysis for differentiation of these serotypes and serotype variants, thus overcoming this limitation. Five workflows that combined different flow cells, library construction methods and basecaller models were evaluated and compared. The workflow that consisted of the R9 flow cell, rapid sequencing library construction kit and guppy basecaller with base modified model performed best for Single Nucleotide Polymorphism (SNP) analysis. With this workflow, as high as 99.98% matched the identity of the assembled genomes and only less than five high quality SNPs (hqSNPs) between ONT and Illumina sequencing data were achieved. SNP typing allowed differentiation of Choleraesuissensu stricto, Choleraesuis var. Kunzendorf, Choleraesuis var. Decatur, Paratyphi C, and Typhisuis that share the same antigenic formula 6,7:c:1,5. Prophage prediction further distinguished Orion var. 15+ and Orion var. 15+, 34+. Our study improves the readiness of ONT as a Salmonella subtyping and source tracking tool for food industry applications.HighlightsSalmonella serotypes or serotype variants with the same antigenic formula were differentiated by SNP typing.Nanopore sequencing followed by phage prediction identified the Salmonella serotype variants caused by phage conversion.The latest ONT technology is capable of high fidelity SNP typing of Salmonella.