Pyrosequencing is a sequencing by synthesis technique which can be used to genotype single nucleotide polymorphisms in mitochondrial DNA. This method is advantageous in its ease of implementation and cost-effectiveness when compared to other sequencing methods. It can easily be used as a diagnostic method for certain mitochondrial diseases by determining heteroplasmy values of pathogenic variants in mitochondrial DNA.
To begin, obtain a mitochondrial DNA sequence file for the species being genotyped and identify the position of the single nucleotide polymorphism, or SNP. Ensure that the reference sequence used employs the appropriate base numbering for the mutation studied so that the position of the SNP can be easily identified. Copy 1, 000 base pairs upstream and downstream of the SNP site and paste the truncated sequence into the pyrosequencing primer design software.
Highlight the polymorphic base, right-click on it, and then select Set Target Region to set the analyzed SNP base as the target of the pyrosequencing assay. Press the Play icon in the top right-hand corner of the interface to launch the primer selection, and wait for the software to automatically generate the primer trios, two primers for preamplification of the template DNA, and the third primer for sequencing by the synthesis in the pyrosequencing machine. Right-click and select Copy All Primer Sets to retain all the primer sets.
Open the run software provided with the pyrosequencer, select New Assay, then select the allele quantification assay template. Input the sequence to be analyzed into the corresponding box by typing in the nucleotides directly downstream of the sequencing primer. For the variable position, denote the two possible bases, separated by a slash.
Press Generate Dispensation Order, and let the software automatically determine a suitable order for the nucleotides to be dispensed in the sequencing by synthesis reaction. Save, and provide a name for the assay. Next, execute the run by diluting the DNA to be analyzed to 5 nanograms per microliter.
In the first run, ensure to include samples of known heteroplasmy as a reference and wild-type samples. Then perform a presequencing PCR of 5 microliters of diluted DNA and 25 microliters reactions, using the amplification primers and parameters. To run the file setup, select New Run in the pyrosequencer software.
The pyrosequencer can simultaneously sequence 48 separate preamplified reactions with an empty square representing a single sequencing well on a pyrosequencing disc. Load the assays by right-clicking on a square and selecting Load Assay. If required, sequence up to four separate assays with different sequencing primers.
Set the primer dispensation mode to automatic to automatically assign an injection chamber for each sequencing primer used for the run. Set run mode to standard, unless running four different types of assays on one sequencing disc. Ensure the number of assays on the run template file matches the number of PCR reactions being amplified.
Then save the run files to a USB drive. To prime the pyrosequencer, press the cleaning button on the main touchscreen of the device and clean all injectors with high-purity water, following the instructions on the screen. When inserting the absorber strip into the machine, ensure that the ends meet in the 9 o'clock position.
After plugging in the USB stick, press the Sequence button to load the run files prepared earlier. Follow the instructions on the device to load and prime the reagents, as required, into the corresponding injectors on the machine. Load 3 microliters of beads into the wells, and then load 10 microliters of each PCR reaction to be sequenced into the corresponding sample.
Pipette up and down to mix the sample with the magnetic beads. Look for the indication in the primed pyrosequencer that the disc can be loaded into the machine. Unscrew the plate holding nut, and align the loaded sample plate with the metal pin in the disc compartment.
Once the plate is firmly screwed into the plate compartment, launch the sequencing run by pressing the start button on the touchscreen interface. Once the run is complete, remove the USB drive from the machine, and plug it back into the computer running the pyrosequencer software. After the newly generated run file appear on the USB drive, double-click on the Run Result file.
This automatically analyzes the luciferase output of each well upon nucleotide incorporation, and quantifies the mitochondrial allele of interest. Look for the color scores shown by the software based on the quality of the reads. To save the results, select Reports in the top window, followed by Full Report to generate a PDF file containing the pyrograms and results from each well of the run.
Alternatively, download the results in other formats under the Reports tab. Agarose gel electrophoresis of a PCR amplification using the amplification primers from both sets selected for the m. 3243A>G mutation as shown in this figure.
Here, Het denotes the nearly homoplasmic m. 3243A>G sample to be analyzed. A comparison of the performance of both primer sets using molar standards for calibration is shown in this figure.
The measured values are denoted by the vertical dotted lines. An X=Y linear function is displayed to illustrate how close each of the two tested assays is to an ideal measurement. The names of the cell lines on the X-axis are the names of the various cybrid clones that were genotyped using this assay.
Genotyping results on four cybrid clones of unknown m. 3243A>G heteroplasmy using both assays are shown here. Sequencing was performed in technical triplicate.
Genotyping results of mitochondrial zinc finger nuclease-treated cells, along with untreated and mock-treated controls, and the pyrograms from two MEF cell sample PCR replicates used to generate the results as well as the wild-type genotyping control are shown here. Pyrosequencing can be used as a readout for gene therapy experiments for assessing various gene therapy technologies in mitochondrial DNA.
