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 clonal lineages (Types I, II, and III) dominate in Europe and North America, extensive genetic diversity, including atypical and recombinant strains, has been documented globally, especially in regions with rich wildlife reservoirs⁴. Accurate strain discrimination is therefore essential for elucidating transmission dynamics, investigating outbreaks, and understanding regional epidemiology. Traditional genotyping approaches such as PCR-RFLP, Sanger-based multi-locus sequence typing (MLST), and microsatellite analysis have contributed substantially to our understanding of T. gondii population structure but are limited in resolution, throughput, and their ability to detect intra-genotype variation. These approaches often require multiple separate reactions and may fail to detect insertions, deletions, or complex haplotypes within polymorphic loci¹. Short-read next-generation sequencing (NGS) has improved the detection of rare variants yet remains constrained in identifying complex structural changes⁴.

Long-read sequencing platforms, particularly Oxford Nanopore Technologies, now offer the ability to generate continuous high-accuracy reads suitable for resolving structural variants and highly polymorphic loci⁵. Their portability, real-time analysis capability, and compatibility with amplicon-based workflows make them promising tools for field applications and outbreak investigations⁶. Despite these advantages, standardized multi-locus long-read workflows for routine T. gondii genotyping remain limited.

To address this gap, we established a scalable long-read amplicon sequencing workflow targeting two polymorphic loci (Figure 1). Combined with a consensus polishing and variant-calling pipeline, this approach enables accurate detection of SNPs and INDELs, supporting strain-level discrimination. The protocol is suitable for laboratories equipped for PCR amplification and long-read sequencing and has been validated using cultured isolates. While adaptation to primary clinical or environmental samples may be feasible, such applications require additional optimization and validation. Overall, this workflow provides a structured framework for targeted molecular characterization and may support epidemiological investigations.

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/psa/emboss_water).
3. Select amplicon regions ≤1,600 base-pairs (bp) to minimize sequencing time and ensure efficient PCR amplification.
4. Ensure each region contains informative SNPs or INDEL polymorphisms for strain differentiation.

2. Culture and harvesting of Toxoplasma gondii tachyzoites

1. Maintenance of host cell cultures
   1. Obtain human foreskin fibroblasts (HFFs) from a certified commercial source and culture them in complete growth medium composed of Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin.
   2. Incubate cultures at 37 °C with 5% CO₂ in a humidified incubator.
2. Maintenance and expansion of parasite lines.
   1. Infect fibroblast monolayers at 80–90% confluence using tachyzoites at a multiplicity of infection of 1–3 parasites per host cell. For a T25 flask containing approximately 1 × 10⁶ fibroblasts, add 1–3 × 10⁶ tachyzoites.
   2. Examine cultures daily using an inverted light microscope at 20× or 40× magnification. Identify host cell lysis by observing widespread monolayer disruption.
      NOTE: Do not use fibroblast cultures beyond passage 20 to avoid culture-related artifacts.
   3. Harvesting of tachyzoites.
      1. Collect cultures when ≥90% host cell lysis is visible.
      2. Centrifuge at 1,300 × g for 10 min at 4 °C.
      3. Resuspend the pellet in 10 mL sterile phosphate-buffered saline by gentle pipetting, and centrifuge again at 1,300 × g for 10 min at 4 °C. Repeat once more.

3. Extraction of genomic DNA and amplicon generation

1. Extract genomic DNA using a silica column-based genomic DNA purification kit⁷. Elute DNA in 50 µL elution buffer.
2. Measure DNA concentration using a microvolume spectrophotometer.
3. Store DNA at 4 °C for short-term or −20 °C for long-term storage.
4. Prepare PCR reactions using a high-fidelity DNA polymerase master mix, locus-specific primers (Supplementary Table 1), and nuclease-free water.
5. Perform PCR amplification using a calibrated thermal cycler under the following cycling conditions: (a) initial denaturation at 98 °C for 30 s, (b) 35 cycles of 98 °C for 10 s; locus-specific annealing: 60 °C for SAG2, 68 °C for SAG3, 30 s; 72 °C for 20 s, (c) Final extension: 72 °C for 2 min.
6. Verify PCR products by electrophoresis on a 1.5% agarose gel.
7. Purify PCR products using a column-based PCR and gel extraction purification system according to the manufacturer’s instructions⁸. Elute DNA in 15–20 µL of elution buffer.
   NOTE: PCR efficiency and yield may be reduced by low DNA template quality, concentration, or suboptimal reaction conditions. If the amplicon yield is low, verify the DNA template integrity using agarose gel electrophoresis and concentration and purity using a spectrophotometer. PCR annealing temperatures and primer concentrations should also be optimized for the primer pairs used, and the PCR cycle number should be increased as necessary.

4. Library preparation for long-read sequencing

1. End-repair and dA-tailing
   1. Thaw all reagents and mix thoroughly by vortex. Dilute the DNA control sample with 10.5 µL of elution buffer during the first use only and keep it on ice.
      NOTE: Always vortex magnetic beads thoroughly before each use.
      ​CAUTION: Do not vortex the End-Prep Enzyme Mix; mix gently by pipetting.
   2. Pipette 200 fmol (~130 ng for a 1 kb fragment) of each DNA sample into 0.2 mL PCR tubes and adjust volume to 11.5 µL with nuclease-free water.
   3. Add 1 µL of diluted DNA control sample per tube and mix by pipetting 10–20 times.
   4. Prepare an end-prep master mix per sample containing 1.75 µL Reaction Buffer and 0.75 µL Enzyme Mix (including 5% excess).
   5. Add 2.5 µL of master mix to each sample, mix gently and spin down briefly.
   6. Incubate in a thermal cycler at 20 °C for 5 min followed by 65 °C for 5 min.
2. Purification of end-prepared DNA
   1. Transfer the 15 µL reaction into a 1.5 mL low DNA-binding microcentrifuge tube.
   2. Add 15 µL resuspended magnetic beads, mix gently and spin briefly.
   3. Incubate samples on a rotator mixer for 5 min at room temperature.
   4. Prepare fresh 80% ethanol for washing.
   5. Place tubes on a magnetic rack until the beads pellet and remove the supernatant carefully.
   6. Wash beads twice with 200 µL of 80% ethanol.
      NOTE: Allow the beads to fully pellet, then carefully remove the ethanol.
   7. Air-dry beads for ~30 s; avoid over-drying to prevent cracking.
   8. Resuspend beads in 10 µL nuclease-free water and incubate for 2 min at room temperature.
   9. Place tubes on a magnetic rack and transfer 10 µL eluate to a clean low DNA-binding microcentrifuge tube.
   10. Measure DNA concentration using 1 µL on a spectrophotometer.
3. Native barcoding
   1. Thaw Blunt/TA Ligase Master Mix, EDTA, Short Fragment Buffer (SFB), and Native Barcodes (NB01–NB24).
   2. Assign individual barcodes to samples 1–24.
   3. Assemble the barcoding reaction with 7.5 µL end-prepped DNA, 2.5 µL Native Barcode, and 10 µL Blunt/TA Ligase Master Mix
   4. Mix thoroughly by pipetting 10–20 times.
   5. Incubate for 20 min at room temperature and terminate the reaction with 4 µL EDTA, mix thoroughly by pipetting, and spin down briefly.
   6. Pool barcoded samples into a 1.5 mL low DNA-binding microcentrifuge tube. Add magnetic beads at 0.4× the total pooled sample volume (e.g., add 230 µL beads to 575 µL pooled DNA). Mix thoroughly by pipetting, then incubate on a rotator mixer at room temperature for 10 min.
   7. Wash the beads with 700 µL of Short Fragment Buffer (SFB). Flick the beads to resuspend, spin down, then return the sample to the magnetic rack and allow the beads to pellet. Remove the buffer using a pipette, then discard it.
   8. Repeat the previous step.
   9. Spin down and place the tube back on the magnetic rack. Pipette off any residual buffer.
   10. Remove the tube from the magnetic rack and resuspend the pellet in 35 µL nuclease-free water by gently flicking.
   11. Incubate at 37 °C for 10 min with intermittent mixing, then transfer the eluate to a clean low-DNA-binding microcentrifuge tube.
4. Adapter ligation and library clean-up
   1. Assemble ligation reaction with 30 µL pooled barcoded DNA, 5 µL Native Adapter, 10 µL Ligation Buffer (5×), and 5 µL T4 DNA Ligase.
   2. Thoroughly mix the reaction by gently pipetting and briefly spinning down. Incubate the reaction for 20 min at room temperature.
   3. Add 20 µL of resuspended magnetic beads to the reaction and mix by pipetting. Incubate on a rotator mixer for 10 min at room temperature.
   4. Spin down the sample and pellet on the magnetic rack. Keep the tube on the magnet and pipette off the supernatant.
   5. Wash the beads by adding 125 µL Short Fragment Buffer (SFB). Flick the beads to resuspend, spin down, then return the tube to the magnetic rack and allow the beads to pellet. Remove the supernatant using a pipette, then discard it.
   6. Repeat the previous step.
   7. Spin down and place the tube back on the magnet. Pipette off any residual supernatant. Remove the tube from the magnetic rack and resuspend the pellet in 15 µL Elution Buffer (EB).
   8. Spin down and incubate for 10 min at 37°C. Agitate the sample every 2 min by gently flicking for 10 s to encourage DNA elution.
   9. Pellet the beads on a magnet until the eluate is clear and colourless, for at least 1 min.
   10. Remove and retain 15 µL of eluate containing the DNA library into a clean 1.5 mL low-DNA-binding microcentrifuge tube.
   11. Quantify the final library using a spectrophotometer.

5. Flow cell preparation and nanopore sequencing

1. Flow cell preparation
   1. Inspect the flow cell and confirm ≥80 active pores.
   2. Prepare the priming mixture by combining 117 µL of flush solution with 3 µL of tether solution in a microcentrifuge tube for a total volume of 120 µL. Mix gently by pipetting to avoid introducing air bubbles. Slowly load the entire 120 µL priming mixture into the flow cell priming port, ensuring that no air bubbles are introduced during loading.
2. Library loading and sequencing
   1. Prepare the sequencing mix by combining 15 µL of Sequencing Buffer (SB), 10 µL of Library Beads (LIB), and 5 µL of DNA library. Mix gently by pipetting up and down 3–5 times to avoid introducing bubbles. The resulting 30 µL sequencing mix is ready to load or can be adjusted to 45 µL if needed.
   2. Slowly load 30–45 µL of the sequencing mix into the flow cell sample port by dispensing the solution dropwise.
      CAUTION: Avoid introducing air bubbles and do not allow the pipette tip to touch the membrane. Close the loading port securely after dispensing.
   3. Start sequencing with real-time base calling enabled and monitor pore activity.
   4. Continue sequencing until ≥500× coverage per amplicon is achieved (recommended ≥1000×), to ensure sufficient read depth for accurate base calling, reliable consensus sequence generation, and confident detection of low-frequency variants.
      NOTE: If low sequencing coverage is apparent, ensure high-quality amplicon input at the required concentration, sufficient active flow cell pores, and extend the sequencing to achieve the recommended coverage.

6. Data processing and variant analysis

1. Base-calling and demultiplexing
   1. Perform high-accuracy base-calling using platform-associated base-calling software with default high-accuracy settings to generate per-sample FASTQ files. Demultiplex reads by barcode and retain only reads passing quality control filters.
2. Read mapping and BAM processing
   1. Download the Toxoplasma gondii reference genome assembly GCF_000006565.2 (TGA4) from the National Center for Biotechnology Information (NCBI) genome database. Extract chromosome VIII (NC_031476.1) for SAG2 analysis and chromosome XII (NC_031480.1) for SAG3 analysis. Index the reference sequences using SAMtools and generate an alignment index using a long-read alignment index builder.
   2. Map reads to the indexed reference genome using a long-read alignment algorithm optimized for nanopore sequencing data (e.g., preset for long-read mapping; -ax map-ont).
   3. Convert alignment output to BAM format, sort, and index BAM files using SAMtools. Add read group information to each BAM file using Picard tools. Calculate average coverage per locus using depth statistics and generate mapping statistics using flagstat metrics.
3. Consensus sequence generation
   1. Generate per-amplicon consensus sequences using a neural network–based consensus polishing algorithm with default parameters. Export consensus sequences in FASTA format for downstream analysis.
   2. Perform reference-based alignment and comparative sequence analysis using publicly available genomic databases. Visualize read alignments and polymorphic positions using genome visualization software.
4. Variant calling
   1. Perform variant detection using a consensus-based variant-calling algorithm. Separate single-nucleotide polymorphisms (SNPs) and insertion/deletion events (INDELs) using variant selection tools (Supplementary File 1).
   2. Apply quality filtering thresholds. For SNPs, filter variants with e.g. QD < 2.0, FS > 60.0, or MQ < 40.0. For INDELs, filter variants with e.g. QD < 2.0 or FS > 200.0.
   3. Compress and index filtered VCF files using standard VCF processing tools. Generate variant statistics using VCF summary utilities.
5. Genotype assignment
   1. Extract locus-specific variants from the SAG2 and SAG3 regions and compare polymorphic profiles to reference strain sequences to assign T. gondii genotypes.

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