Name: Multi-Locus Sequencing for Strain Typing of Toxoplasma gondii (Video) | JoVE Journal
Uploaded: 2026-05-26T13:00:00.000+00:00
Duration: 11 min 9 s
Description: Discover multi-locus sequencing for strain typing of Toxoplasma gondii using nanopore technology. This computational protocol enables rapid detection of SNPs and INDELs for accurate genetic characterization.

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5 minutes

This work was funded by the Egyptian Ministry of Higher Education &#38; Scientific Research, represented by the Egyptian Bureau for Cultural &#38; Educational Affairs in London, and UKRI MRC (MR/X502947/1). Figure 1 was created with BioRender.com

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
<ol>
	<li>Download genomic sequences of SAG2 and SAG3 in FASTA format from ToxoDB (https://toxodb.org/toxo/app) genome database.</li>
	<li>Perform pairwise sequence alignments using EMBOSS Water (https://www.ebi.ac.uk/jdispatcher/psa/emboss_water).</li>
	<li>Select amplicon regions &#8804;1,600 base-pairs (bp) to minimize sequencing time and ensure efficient PCR amplification.</li>
	<li>Ensure each region contains informative SNPs or INDEL polymorphisms for strain differentiation.</li>
</ol>
2. Culture and harvesting of Toxoplasma gondii tachyzoites
<ol>
	<li>Maintenance of host cell cultures
	<ol>
		<li>Obtain human foreskin fibroblasts (HFFs) from a certified commercial source and culture them in complete growth medium composed of Dulbecco&#8217;s Modified Eagle Medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin.</li>
		<li>Incubate cultures at 37 &#176;C with 5% CO&#8322; in a humidified incubator.</li>
	</ol>
	</li>
	<li>Maintenance and expansion of parasite lines.
	<ol>
		<li>Infect fibroblast monolayers at 80&#8211;90% confluence using tachyzoites at a multiplicity of infection of 1&#8211;3 parasites per host cell. For a T25 flask containing approximately 1 &#215; 106 fibroblasts, add 1&#8211;3 &#215; 106 tachyzoites.</li>
		<li>Examine cultures daily using an inverted light microscope at 20&#215; or 40&#215; magnification. Identify host cell lysis by observing widespread monolayer disruption. 
		NOTE: Do not use fibroblast cultures beyond passage 20 to avoid culture-related artifacts.</li>
		<li>Harvesting of tachyzoites.
		<ol>
			<li>Collect cultures when &#8805;90% host cell lysis is visible.</li>
			<li>Centrifuge at 1,300 &#215; g for 10 min at 4 &#176;C.</li>
			<li>Resuspend the pellet in 10 mL sterile phosphate-buffered saline by gentle pipetting, and centrifuge again at 1,300 &#215; g for 10 min at 4 &#176;C. Repeat once more.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. Extraction of genomic DNA and amplicon generation
<ol>
	<li>Extract genomic DNA using a silica column-based genomic DNA purification kit7. Elute DNA in 50 &#181;L elution buffer.</li>
	<li>Measure DNA concentration using a microvolume spectrophotometer.</li>
	<li>Store DNA at 4 &#176;C for short-term or &#8722;20 &#176;C for long-term storage.</li>
	<li>Prepare PCR reactions using a high-fidelity DNA polymerase master mix, locus-specific primers (Supplementary Table 1), and nuclease-free water.</li>
	<li>Perform PCR amplification using a calibrated thermal cycler under the following cycling conditions: (a) initial denaturation at 98 &#176;C for 30 s, (b) 35 cycles of 98 &#176;C for 10 s; locus-specific annealing: 60 &#176;C for SAG2, 68 &#176;C for SAG3, 30 s; 72 &#176;C for 20 s, (c) Final extension: 72 &#176;C for 2 min.</li>
	<li>Verify PCR products by electrophoresis on a 1.5% agarose gel.</li>
	<li>Purify PCR products using a column-based PCR and gel extraction purification system according to the manufacturer&#8217;s instructions8. Elute DNA in 15&#8211;20 &#181;L 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.</li>
</ol>
4. Library preparation for long-read sequencing 
<ol>
	<li>End-repair and dA-tailing
	<ol>
		<li>Thaw all reagents and mix thoroughly by vortex. Dilute the DNA control sample with 10.5 &#181;L of elution buffer during the first use only and keep it on ice. 
		NOTE: Always vortex magnetic beads thoroughly before each use. 
		&#8203;CAUTION: Do not vortex the End-Prep Enzyme Mix; mix gently by pipetting.</li>
		<li>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 &#181;L with nuclease-free water.</li>
		<li>Add 1 &#181;L of diluted DNA control sample per tube and mix by pipetting 10&#8211;20 times.</li>
		<li>Prepare an end-prep master mix per sample containing 1.75 &#181;L Reaction Buffer and 0.75 &#181;L Enzyme Mix (including 5% excess).</li>
		<li>Add 2.5 &#181;L of master mix to each sample, mix gently and spin down briefly.</li>
		<li>Incubate in a thermal cycler at 20 &#176;C for 5 min followed by 65 &#176;C for 5 min.</li>
	</ol>
	</li>
	<li>Purification of end-prepared DNA
	<ol>
		<li>Transfer the 15 &#181;L reaction into a 1.5 mL low DNA-binding microcentrifuge tube.</li>
		<li>Add 15 &#181;L resuspended magnetic beads, mix gently and spin briefly.</li>
		<li>Incubate samples on a rotator mixer for 5 min at room temperature.</li>
		<li>Prepare fresh 80% ethanol for washing.</li>
		<li>Place tubes on a magnetic rack until the beads pellet and remove the supernatant carefully.</li>
		<li>Wash beads twice with 200 &#181;L of 80% ethanol. 
		NOTE: Allow the beads to fully pellet, then carefully remove the ethanol.</li>
		<li>Air-dry beads for ~30 s; avoid over-drying to prevent cracking.</li>
		<li>Resuspend beads in 10 &#181;L nuclease-free water and incubate for 2 min at room temperature.</li>
		<li>Place tubes on a magnetic rack and transfer 10 &#181;L eluate to a clean low DNA-binding microcentrifuge tube.</li>
		<li>Measure DNA concentration using 1 &#181;L on a spectrophotometer.</li>
	</ol>
	</li>
	<li>Native barcoding
	<ol>
		<li>Thaw Blunt/TA Ligase Master Mix, EDTA, Short Fragment Buffer (SFB), and Native Barcodes (NB01&#8211;NB24).</li>
		<li>Assign individual barcodes to samples 1&#8211;24.</li>
		<li>Assemble the barcoding reaction with 7.5 &#181;L end-prepped DNA, 2.5 &#181;L Native Barcode, and 10 &#181;L Blunt/TA Ligase Master Mix</li>
		<li>Mix thoroughly by pipetting 10&#8211;20 times.</li>
		<li>Incubate for 20 min at room temperature and terminate the reaction with 4 &#181;L EDTA, mix thoroughly by pipetting, and&#160;spin down briefly.</li>
		<li>Pool barcoded samples into a 1.5 mL low DNA-binding microcentrifuge tube. Add magnetic beads at 0.4&#215; the total pooled sample volume (e.g., add 230 &#181;L beads to 575 &#181;L pooled DNA). Mix thoroughly by pipetting, then incubate on a rotator mixer at room temperature for 10 min.</li>
		<li>Wash the beads with 700 &#181;L of Short Fragment Buffer (SFB). Flick the beads to resuspend,&#160;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.</li>
		<li>Repeat the previous step.</li>
		<li>Spin down and place the tube back on the magnetic rack. Pipette off any residual buffer.</li>
		<li>Remove the tube from the magnetic rack and resuspend the pellet in 35 &#181;L nuclease-free water by gently flicking.</li>
		<li>Incubate at 37 &#176;C for 10 min with intermittent mixing, then transfer the eluate to a clean low-DNA-binding microcentrifuge tube.</li>
	</ol>
	</li>
	<li>Adapter ligation and library clean-up
	<ol>
		<li>Assemble ligation reaction with 30 &#181;L pooled barcoded DNA, 5 &#181;L Native Adapter, 10 &#181;L Ligation Buffer (5&#215;), and 5 &#181;L T4 DNA Ligase.</li>
		<li>Thoroughly mix the reaction by gently pipetting and briefly spinning down. Incubate the reaction for 20 min at&#160;room temperature.</li>
		<li>Add 20 &#181;L of resuspended magnetic beads to the reaction and mix by pipetting. Incubate on a&#160;rotator mixer for 10 min at&#160;room temperature.</li>
		<li>Spin down&#160;the sample and pellet on the magnetic rack. Keep the tube on the magnet and pipette off the supernatant.</li>
		<li>Wash the beads by adding 125 &#181;L Short Fragment Buffer (SFB). Flick the beads to resuspend,&#160;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.</li>
		<li>Repeat the previous step.</li>
		<li>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 &#181;L Elution Buffer (EB).</li>
		<li>Spin down&#160;and incubate for 10 min at 37&#176;C. Agitate the sample every 2 min by gently flicking for 10 s to encourage DNA elution.</li>
		<li>Pellet the beads on a magnet until the eluate is clear and colourless, for at least 1 min.</li>
		<li>Remove and retain 15 &#181;L of eluate containing the DNA library into a clean 1.5 mL low-DNA-binding microcentrifuge tube.</li>
		<li>Quantify the final library using a spectrophotometer.</li>
	</ol>
	</li>
</ol>
5. Flow cell preparation and nanopore sequencing
<ol>
	<li>Flow cell preparation
	<ol>
		<li>Inspect the flow cell and confirm &#8805;80 active pores.</li>
		<li>Prepare the priming mixture by combining 117 &#181;L of flush solution with 3 &#181;L of tether solution in a microcentrifuge tube for a total volume of 120 &#181;L. Mix gently by pipetting to avoid introducing air bubbles. Slowly load the entire 120 &#181;L priming mixture into the flow cell priming port, ensuring that no air bubbles are introduced during loading.</li>
	</ol>
	</li>
	<li>Library loading and sequencing
	<ol>
		<li>Prepare the sequencing mix by combining 15 &#181;L of Sequencing Buffer (SB), 10 &#181;L of Library Beads (LIB), and 5 &#181;L of DNA library. Mix gently by pipetting up and down 3&#8211;5 times to avoid introducing bubbles. The resulting 30 &#181;L sequencing mix is ready to load or can be adjusted to 45 &#181;L if needed.</li>
		<li>Slowly load 30&#8211;45 &#181;L of the sequencing mix into the flow cell sample port by dispensing the solution dropwise. 
		CAUTION:&#160;Avoid introducing air bubbles and do not allow the pipette tip to touch the membrane. Close the loading port securely after dispensing.</li>
		<li>Start sequencing with real-time base calling enabled and monitor pore activity.</li>
		<li>Continue sequencing until &#8805;500&#215; coverage per amplicon is achieved (recommended &#8805;1000&#215;), 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.</li>
	</ol>
	</li>
</ol>
6. Data processing and variant analysis
<ol>
	<li>Base-calling and demultiplexing
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>Read mapping and BAM processing
	<ol>
		<li>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.</li>
		<li>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).</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Consensus sequence generation
	<ol>
		<li>Generate per-amplicon consensus sequences using a neural network&#8211;based consensus polishing algorithm with default parameters. Export consensus sequences in FASTA format for downstream analysis.</li>
		<li>Perform reference-based alignment and comparative sequence analysis using publicly available genomic databases. Visualize read alignments and polymorphic positions using genome visualization software.</li>
	</ol>
	</li>
	<li>Variant calling
	<ol>
		<li>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).</li>
		<li>Apply quality filtering thresholds. For SNPs, filter variants with e.g. QD &#60; 2.0, FS &#62; 60.0, or MQ &#60; 40.0. For INDELs, filter variants with e.g. QD &#60; 2.0 or FS &#62; 200.0.</li>
		<li>Compress and index filtered VCF files using standard VCF processing tools. Generate variant statistics using VCF summary utilities.</li>
	</ol>
	</li>
	<li>Genotype assignment
	<ol>
		<li>Extract locus-specific variants from the SAG2 and SAG3 regions and compare polymorphic profiles to reference strain sequences to assign T. gondii genotypes.</li>
	</ol>
	</li>
</ol>

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The authors have no conflicts of interest to declare.

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&#8217;s capacity to resolve strain-specific genetic variation.</...

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&#8217;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 transmission2,3.
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.

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

multi-locus sequencing for strain typing of toxoplasma gondii

Multi-Locus Sequencing for Strain Typing of Toxoplasma gondii

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.

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...

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.

Toxoplasma gondii is a globally distributed apicomplexan parasite characterized by substantial genetic diversity, necessitating accurate molecular ...

We focus on genetic variations in the foodborne parasite Toxoplasma to identify uncharacterized strains posing increased risk, especially during pregnancy. Rapid genetic characterization of strains of interest is key and this protocol outlines a rapid nanopore sequencing methodology. To begin, thaw the required reagents and the purified PCR amplicons generated from Toxoplasma gondii genomic DNA.Pipette each DNA sample into 0.2 milliliter polymerase chain reaction tubes and adjust the volume to 11.5 microliters with nuclease-free water. Add on microliter of the diluted DNA control sample to each tube and mix by pipetting 10 to 20 times. Prepare an NPREP master mix for each sample containing 1.75 microliters of reaction buffer and 0.75 microliters of enzyme mix, including 5%excess.Add 2.5 microliters of the master mix to each sample and mix gently. After a brief spin, incubate the samples in a thermal cycler at 20 degrees Celsius for five minutes, followed by 65 degrees Celsius for five minutes. Then purify the N-prepared DNA using DNA cleanup magnetic beads.After eluting the purified DNA, measure its concentration for downstream use. Add 7.5 microliters of N-prepared DNA to each tube, followed by 2.5 microliters of the native DNA barcode and 10 microliters of Blunt/TA ligase master mix to assemble the barcoding reaction. Incubate at room temperature for 20 minutes.After incubation, terminate the reaction with four microliters of EDTA and mix thoroughly by pipetting. Transfer the barcoded samples into a 1.5 milliliter low DNA binding microcentrifuge tube to pool them and purify the DNA using magnetic beads. Add 30 microliters of purified pooled barcoded DNA to a tube and mix it with five microliters each of native adapter and T4 DNA ligase along with 10 microliters of 5X ligation buffer.After a brief spin, incubate the sample at room temperature for 20 minutes. Next, add 20 microliters of resuspended magnetic beads to the reaction and mix by pipetting. Incubate the sample on a rotator mixer at room temperature for 10 minutes.Spin down the sample and place the tube on a magnetic rack to pellet the beads. Pipette off the supernatant while keeping the tube on the magnet. Next, add 125 microliters of Short Fragment Buffer to wash the beads.Flick the tube to resuspend the beads. Spin down briefly. Place the tube on a magnetic rack and allow the beads to settle.Remove the supernatant using a pipette and repeat the wash step with Short Fragment Buffer. After a brief spin, place the tube back on the magnetic rack and pipette off any residual supernatant. Remove the tube from the magnetic rack and resuspend the pellet in 15 microliters of elution buffer.Incubate the tube at 37 degrees Celsius for 10 minutes. Agitate the tube every two minutes by gently flicking for 10 seconds to encourage DNA elution. Then place the tube on a magnetic rack to let the bead settle until the eluent is clear and colorless for at least one minute.Transfer 15 microliters of eluent containing the DNA library into a clean 1.5 milliliter low DNA binding microcentrifuge tube. Measure the final library concentration using a spectrophotometer. Inspect the flow cell and confirm at least 80 active pores.Add 117 microliters of flush solution to a microcentrifuge tube, followed by three microliters of tether solution to reach a total volume of 120 microliters. Mix gently by pipetting to avoid introducing air bubbles. Slowly load the entire 120 microliters of priming mixture into the flow cell priming port without introducing air bubbles.Next, add 15 microliters of sequencing buffer to a tube. Add 10 microliters of library beads along with five microliters of DNA library to prepare the sequencing mix. Mix gently by pipetting up and down three to five times to avoid introducing bubbles.Slowly load a 30 to 45 microliter reaction volume of the sequencing mix into the flow cell sample port by dispensing dropwise. Avoid introducing air bubbles and ensure the pipette tip does not touch the membrane. Close the loading port securely after dispensing.Start sequencing after enabling real-time base calling and monitor the pore activity. Continue sequencing until at least 500 times coverage per amplicon is achieved with a recommended coverage of at least 1, 000 times for sufficient read depth and reliable analysis. Generate per amplicon consensus sequences using a neural network-based consensus polishing algorithm with default parameters.Export the consensus sequences in FASTA format for downstream analysis. Perform a reference-based alignment and comparative sequence analysis using publicly available genomic databases. Visualize the read alignments and polymorphic positions using a genome visualization software.Next, perform high accuracy base calling using the platform-associated base calling software with default high accuracy settings to generate per sample FASTQ files. Demultiplex the reads by barcode and retain the ones that pass the quality control filters. Finally, detect the variance using a consensus-based variant-calling algorithm.Separate the single nucleotide polymorphisms and the insertion or deletion events using variant selection tools. Genomic DNA of Toxoplasma gondii was successfully amplified targeting the SAG2 and SAG3 genetic markers. Agarose gel electrophoresis showed that the purified PCR products from RH and ME49 strains produced distinct bands corresponding to 639 base pairs for SAG2 and 1, 158 base pairs for SAG3.High-throughput nanopore sequencing generated high-quality datasets across examined loci in both RH and ME49 strains. The ME49 strain showed good relatively uniform sequencing coverage across the SAG2 locus with very few mismatches, whereas the RH strain showed multiple nucleotide substitutions across the same region. The ME49 strain showed limited sequence variation across the SAG3 locus with most reads matching the reference sequence, whereas the RH strain showed a markedly higher density of mismatches and clustered variants.At the SAG2 locus, only one single nucleotide polymorphism was detected in ME49, whereas six single nucleotide polymorphisms were detected in RH.At the SAG3 locus, greater genetic variation was observed in RH with 61 single nucleotide polymorphisms and 14 deletions detected, while ME49 showed 34 single nucleotide polymorphisms, six insertions, and 13 deletions. At the SAG3 locus, elevated transition-to-transversion ratios were observed in both ME49 and RH strains. And the insertion-to-deletion ratios showed slightly more deletions than insertions in both strains.This study has the potential to transform the detection monitoring of foodborne protozoan parasites, particularly in low resource settings. Reference strains were utilized in this study. Expansion to field strains may require whole genome sequence data in addition to high-quality genomic DNA.This procedure could be expanded and multiplexed to include other foodborne pathogens of interest, such as sarcocystis.

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Discover multi-locus sequencing for strain typing of Toxoplasma gondii using nanopore technology. This computational protocol enables rapid detection of SNPs and INDELs for accurate genetic characterization.

Multi-Locus Sequencing for Strain Typing of Toxoplasma gondii

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