Typing of Echinococcus multilocularis by Region-Specific Extraction and Next-Generation Sequencing of the mitogenome
Background Infection by the fox tapeworm Echinococcus multilocularis may lead to a severe zoonosis in humans, alveolar echinococcosis, which may be fatal if left untreated. Typing is important to understand the epidemiology of this parasite, yet, there is limited knowledge on the microdiversity of E. multilocularis on the local scale, since the typing resolution of established methods is restricted. Methods The mitogenome of E. multilocularis was used as the target regions to modify, apply and validate Region-Specific Extraction (RSE) method in combination with Next-Generation Sequencing (NGS). Single Nucleotide Polymorphisms (SNPs) were detected in the mitochondrial DNA (mtDNA) and bioinformatically analysed. To validate the success and the accuracy of the RSE protocol, the mitogenomes of some E. multilocularis isolates were also analysed by the Whole-Genome Sequencing (WGS). Results With the chosen combination of methods, the entire mitogenome (~13 kb) of E. multilocularis could be captured and amplified. The read depth (median =156X) was sufficient to detect existing SNPs. The comparison of mitogenomes extracted by RSE data with mitogenome data obtained by WGS showed that the accuracy of the RSE method was consistently comparable to direct Whole-Genome Sequencing.  Conclusion The results demonstrate that the RSE method in combination with NGS is suitable to analyse the microdiversity of E. multilocularis on the whole mitogenome level. It may be less costly and less labour-intensive than conventional typing methods, for example like PCRs in combinations with Sanger sequencing for capturing and sequencing of large (several kb) regions in the nucleotide sequencing of E. multilocularis.
Background Infection by the fox tapeworm Echinococcus multilocularis may lead to a severe zoonosis in humans, alveolar echinococcosis, which may be fatal if left untreated. Typing is important to understand the epidemiology of this parasite, yet, there is limited knowledge on the microdiversity of E. multilocularis on the local scale, since the typing resolution of established methods is restricted. Methods The mitogenome of E. multilocularis was used as the target regions to modify, apply and validate Region-Specific Extraction (RSE) method in combination with Next-Generation Sequencing (NGS). Single Nucleotide Polymorphisms (SNPs) were detected in the mitochondrial DNA (mtDNA) and bioinformatically analysed. To validate the success and the accuracy of the RSE protocol, the mitogenomes of some E. multilocularis isolates were also analysed by the Whole-Genome Sequencing (WGS). Results With the chosen combination of methods, the entire mitogenome (~13 kb) of E. multilocularis could be captured and amplified. The read depth (median =156X) was sufficient to detect existing SNPs. The comparison of mitogenomes extracted by RSE data with mitogenome data obtained by WGS showed that the accuracy of the RSE method was consistently comparable to direct Whole-Genome Sequencing.  Conclusion The results demonstrate that the RSE method in combination with NGS is suitable to analyse the microdiversity of E. multilocularis on the whole mitogenome level. It may be less costly and less labour-intensive than conventional typing methods, for example like PCRs in combinations with Sanger sequencing for capturing and sequencing of large (several kb) regions in the nucleotide sequencing of E. multilocularis.
fli institute of epidemiology