Development of a next-generation sequencing-based typing method to detect within-genotype variation in Toxoplasma gondii

Kód výsledku v IS VaVaI

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Nalezeny alternativní kódy

RIV/62157124:16170/21:43879373

Výsledek na webu

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DOI - Digital Object Identifier

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Jazyk výsledku

angličtina

Název v původním jazyce

Development of a next-generation sequencing-based typing method to detect within-genotype variation in Toxoplasma gondii

Popis výsledku v původním jazyce

Introduction. Toxoplasma gondii is a protozoan parasite with a largely clonal population structure in Europe and North America. (Howe and Sibley 1995). The clonal lineage type II prevails in Europe (Fernández-Escobar et al. 2020, Herrmann et al. 2010, Jokelainen et al. 2018, Shwab et al. 2014). The currently available PCR-based typing methods have a limited ability to discriminate between different type II isolates. Therefore, it is our aim to establish a next-generation sequencing (NGS)-based typing method with a higher typing resolution among T. gondiitype II isolates. Methods. A large number of T. gondii isolates were collected from different parts of Europe and genotyped using PCR-RFLP (Su et al. 2010) and microsatellite typing (Ajzenberg et al. 2010). Based on this analysis, T. gondii type II isolates were chosen to establish a new typing method that allows a standardised and sensitive differentiation of T. gondii type II strains. For more than 60 cell-cultured T. gondii isolates, a whole genome sequencing (WGS) analysis was conducted. In comparison to ME49 (a clonal type II reference strain), highly polymorphic regions of the sequenced T. gondii type II field genomes were identified. These regions showed a considerable number of single nucleotide polymorphisms (SNPs), insertions and deletions (INDELS). In a second step, the existence of these highly polymorphic regions identified by WGS was further confirmed by Sanger sequencing using novel primer pairs. Results. For many, but not all genome regions, identified as highly polymorphic by WGS, Sanger sequencing was able to confirm their suitability for typing. In particular, regions with fewer polymorphisms detected (&lt;10-20 SNPs/333 bp relative to the reference genome) revealed good sequence quality by Sanger sequencing and the results were in accord with the WGS data. By contrast, most regions, that had a high number of polymorphisms in the WGS (&gt; 20 SNPs/333 bp relative to the reference genome), showed low sequence quality by the Sanger sequencing procedure. Actually, more than half of the chromosomes of the T. gondii igenome are covered, with regions showing notable typing power. Conclusion. The results of the study suggest that several polymorphic regions on different chromosomes of T. gondii exist, seeming to be good candidates for establishing a novel typing method. In a next step, an amplicon-based enrichment of these loci will be established. Amplified typing-loci will subsequently be assessed by NGS. A method based on the set of loci can be expected to be able to differentiate isolates from different settings (individual farms) or to identify a point source (outbreaks).

Název v anglickém jazyce

Development of a next-generation sequencing-based typing method to detect within-genotype variation in Toxoplasma gondii

Popis výsledku anglicky

Introduction. Toxoplasma gondii is a protozoan parasite with a largely clonal population structure in Europe and North America. (Howe and Sibley 1995). The clonal lineage type II prevails in Europe (Fernández-Escobar et al. 2020, Herrmann et al. 2010, Jokelainen et al. 2018, Shwab et al. 2014). The currently available PCR-based typing methods have a limited ability to discriminate between different type II isolates. Therefore, it is our aim to establish a next-generation sequencing (NGS)-based typing method with a higher typing resolution among T. gondiitype II isolates. Methods. A large number of T. gondii isolates were collected from different parts of Europe and genotyped using PCR-RFLP (Su et al. 2010) and microsatellite typing (Ajzenberg et al. 2010). Based on this analysis, T. gondii type II isolates were chosen to establish a new typing method that allows a standardised and sensitive differentiation of T. gondii type II strains. For more than 60 cell-cultured T. gondii isolates, a whole genome sequencing (WGS) analysis was conducted. In comparison to ME49 (a clonal type II reference strain), highly polymorphic regions of the sequenced T. gondii type II field genomes were identified. These regions showed a considerable number of single nucleotide polymorphisms (SNPs), insertions and deletions (INDELS). In a second step, the existence of these highly polymorphic regions identified by WGS was further confirmed by Sanger sequencing using novel primer pairs. Results. For many, but not all genome regions, identified as highly polymorphic by WGS, Sanger sequencing was able to confirm their suitability for typing. In particular, regions with fewer polymorphisms detected (&lt;10-20 SNPs/333 bp relative to the reference genome) revealed good sequence quality by Sanger sequencing and the results were in accord with the WGS data. By contrast, most regions, that had a high number of polymorphisms in the WGS (&gt; 20 SNPs/333 bp relative to the reference genome), showed low sequence quality by the Sanger sequencing procedure. Actually, more than half of the chromosomes of the T. gondii igenome are covered, with regions showing notable typing power. Conclusion. The results of the study suggest that several polymorphic regions on different chromosomes of T. gondii exist, seeming to be good candidates for establishing a novel typing method. In a next step, an amplicon-based enrichment of these loci will be established. Amplified typing-loci will subsequently be assessed by NGS. A method based on the set of loci can be expected to be able to differentiate isolates from different settings (individual farms) or to identify a point source (outbreaks).

Druh

D - Stať ve sborníku

CEP obor

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OECD FORD obor

40301 - Veterinary science

Projekt

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Návaznosti

I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Rok uplatnění

2021

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Název statě ve sborníku

Parasiten: alte und neue Herausforderungen

ISBN

978-3-86345-577-4

ISSN

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e-ISSN

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Počet stran výsledku

3

Strana od-do

29-31

Název nakladatele

DVG Service GmbH

Místo vydání

Leipzig

Místo konání akce

Giesen

Datum konání akce

28. 6. 2021

Typ akce podle státní příslušnosti

WRD - Celosvětová akce

Kód UT WoS článku

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