Multi-Locus Sequencing for Strain Typing of Toxoplasma gondii

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.