Multi-Locus Sequencing for Strain Typing of Toxoplasma gondii

Summary

Here, we present a protocol to rapidly identify and genotype Toxoplasma gondii strains using portable nanopore sequencing of selected genetic markers, enabling scalable, targeted strain identification in laboratory-maintained isolates with potential future application to surveillance settings.

Abstract

Toxoplasma gondii is a globally distributed apicomplexan parasite characterized by substantial genetic diversity, necessitating accurate molecular approaches for strain discrimination. The goal of this protocol is to establish a reproducible workflow for targeted multi-locus genotyping of cultured T. gondii isolates. The procedure involves in vitro propagation of representative Type I (RH) and Type II (ME49) strains, extraction of genomic DNA, locus-specific amplification of the SAG2 and SAG3 genes, and sequencing-based analyses to detect single-nucleotide polymorphisms (SNPs) and insertion/deletion (INDEL) events. Sequencing data are processed to generate high-confidence consensus sequences and to perform comparative alignment for the identification of strain-specific polymorphisms. Application of this workflow to archetypal strains demonstrated high mapping efficiency, consistent read depth across target loci, and accurate recovery of expected amplicon sizes, confirming reliable detection of locus-level genetic variation. Although comprehensive genotype assignment requires inclusion of additional standardized markers, this protocol provides a scalable and reproducible framework for targeted genetic characterization of laboratory-maintained T. gondii isolates and supports consistent multi-locus sequence analysis.

Introduction

Toxoplasma gondii is a globally distributed apicomplexan parasite and a major food- and waterborne zoonotic threat, capable of infecting virtually all warm-blooded animals, with felids serving as the definitive hosts¹. Infection in humans and animals commonly occurs through ingestion of tissue cysts or sporulated oocysts, and the parasite’s ability to alternate between tachyzoite and bradyzoite stages enables long-term persistence in neural and muscular tissues, contributing to both meat-borne and environmental transmission^2,3.

Although three c....

Protocol

This study used established laboratory parasite strains and commercially obtained human fibroblast cell lines. No human or animal subjects were directly involved.

NOTE: Unless otherwise specified, buffers and reagents used in this protocol are supplied within the commercial kits listed in the Table of Materials.

1. Biomarker selection

1. Download genomic sequences of SAG2 and SAG3 in FASTA format from ToxoDB (https://toxodb.org/toxo/app) genome database.
2. Perform pairwise sequence alignments using EMBOSS Water (https://www.ebi.ac.uk/jdispatcher....

Results

Genomic DNA of Toxoplasma gondii was successfully amplified by polymerase chain reaction (PCR) targeting two genetic markers: SAG2 and SAG3 (Figure 2). Following purification, the PCR products yielded distinct bands of the expected sizes on agarose gel electrophoresis, corresponding to 639 bp (SAG2) and 1158 bp (SAG3). The clarity and consistency of these bands confirmed successful amplification and integrity of the target gene fragments for subse.......

Discussion

This study demonstrates the utility of Oxford Nanopore long-read sequencing combined with a targeted multi-locus approach for rapid and reliable genotyping of Toxoplasma gondii. By focusing on two well-characterized surface antigen loci, SAG2 and SAG3, we establish proof-of-concept for Oxford Nanopore-based multi-locus sequencing through comparative analyses of the RH (Type I) and ME49 (Type II) strains, demonstrating the platform’s capacity to resolve strain-specific genetic variation.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Agarose powder	Thermo Fisher Scientific	16500100	For gel electrophoresis
Dulbecco’s Modified Eagle Medium (DMEM)	Thermo Fisher Scientific	11995065	Cell culture medium
epi2me-labs/wf-amplicon Nextflow workflow (v1.2.1)	Oxford Nanopore Technologies, UK	N/A	Nextflow workflow for processing and analyzing nanopore amplicon sequencing data; version 1.2.1 was used for reproducible analysis of MinION data.
epi2me-labs/wf-alignment Nextflow workflow (v1.2.3)	Oxford Nanopore Technologies, UK	N/A	Nextflow workflow for alignment of nanopore sequencing reads to reference sequences and integrated visualization using IGV; version 1.2.3 was used for reproducible analysis of MinION data.
Ethyl alcohol, pure	Merck Life Science	51976-500ML-F	For bead cleanup
Fetal bovine serum (FBS)	Sigma-Aldrich	MFCD00132239	Supplement for cell culture
Flow Cell Wash Kit	Oxford Nanopore Technologies	EXP-WSH004	Flow cell washing
GeneRuler 1 kb Plus DNA Ladder (10 kb)	Thermo Fisher Scientific	SM1331	Size confirmation
HFF-1 human foreskin fibroblast cell line	American Type Culture Collection (ATCC)	SCRC-1041	Human dermal fibroblasts isolated from neonatal foreskin; used for parasite propagation
MinION Flow Cell (R10.4.1)	Oxford Nanopore Technologies	FLO-MIN114	Flow cell for sequencing
MinION Mk1B Sequencing Device	Oxford Nanopore Technologies, UK	MIN-101B	Nanopore sequencer; sequencing performed using MinKNOW software (v25.09.16) for device control and real-time base calling.
MinKNOW Software	Oxford Nanopore Technologies, UK	N/A	Software used for MinION Mk1B device control, real-time sequencing, and base calling; version 25.09.16.
Native Barcoding Kit 24 V14	Oxford Nanopore Technologies	SQK-NBD114.24	Barcoding for multiplexed samples
NEB Blunt/TA Ligase Master Mix	New England Biolabs	M0367	DNA ligation
NEBNext Quick Ligation Module	New England Biolabs	E6056	Adapter ligation
NEBNext Quick Ligation Reaction Buffer (5×)	New England Biolabs	B6058	Used in DNA ligation
NEBNext Ultra II End Repair/dA-Tailing Module	New England Biolabs	E7546	Library preparation
Nuclease-free water	Thermo Fisher Scientific	R0581	Molecular biology grade
NucleoSpin Gel and PCR Clean-up Kit	Macherey-Nagel	740609.5	PCR product purification
Penicillin–Streptomycin (1%)	Thermo Fisher Scientific (Gibco)	15140-122	Antibiotic mixture
Phosphate-buffered saline (PBS)	Thermo Fisher Scientific (Gibco)	10010023	Washing buffer
Q5 High-Fidelity 2× Master Mix	New England Biolabs	M0492S	High-accuracy polymerase
QIAamp DNA Blood Mini Kit	Qiagen	69504	DNA extraction
Quick T4 DNA Ligase	New England Biolabs	M2200	Adapter ligation enzyme
SYBR Safe DNA Gel Stain	Thermo Fisher Scientific	S33102	DNA staining dye
TriTrack DNA Loading Dye (6×)	Thermo Fisher Scientific	R1161	Loading samples into gel
Trypsin–EDTA (0.25%)	Thermo Fisher Scientific (Gibco)	25200056	For passaging HFFs
1× TAE or 1× TBE buffer	—	—	Running buffer
2× PCR premix	—	—	Ready mix for PCR